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Diabetes in People Living with HIV

ABSTRACT

 

People living with HIV (PLWH) are living longer and also have unique risk factors for developing several metabolic diseases, including diabetes. This has been observed in both high income and low to middle income countries. Risk factors for diabetes in PLWH consist of specific antiretroviral therapies (ART), including older generation protease inhibitors and nucleoside reverse transcriptase inhibitors, lipodystrophy, and hepatitis C co-infection. In addition, obese and overweight states are common in PLWH, and an increased risk of incident diabetes has been noted with weight gain after ART initiation in PLWH, compared to individuals without HIV. Inflammation associated with HIV has also been linked to incident diabetes. This chapter reviews points to consider in the diagnosis and monitoring of diabetes in PLWH and discusses interactions that may occur between specific ART agents and glucose-lowering medications. Moreover, PLWH have risk factors for complications involving organ systems that are also affected by microvascular disease in diabetes. Because PLWH have a greater risk for cardiovascular disease (CVD) than individuals without HIV, modifiable risk factors of CVD should be addressed in the care of PLWH, considering that dyslipidemia, hypertension, and cigarette smoking are all highly prevalent in PLWH.

 

BACKGROUND

 

HIV infection is prevalent in 36.9 million people worldwide as of 2017 [1]and 1.1 million people in the United States as of 2015. The incidence of HIV infection has decreased from 2010 to 2013 in the United States but has increased or remained stable in some demographic groups, including African-American and Latino gay and bisexual men [2]. Untreated, HIV infection can lead to opportunistic infections including cytomegalovirus disease and pneumocystis [3]associated with AIDS, which is defined as a CD4+ cell count < 200 cells/mL. With advances in antiretroviral therapy (ART), however, HIV infection has been transformed from a disease strongly linked to AIDS and opportunistic infections to a chronic disease that is associated with several cardiometabolic consequences, including diabetes [4], heart disease [5], and other non-AIDS related illnesses such as osteoporosis [6]. In part, this phenomenon is secondary to the fact that people living with HIV (PLWH) are living longer and are more susceptible to diseases of aging.

 

An important aspect to consider in the understanding of HIV associated cardiometabolic diseases is their growing global presence. The same factors that are associated with cardiometabolic disease prevalence in PLWH in high-income countries are also present in low-income countries, in addition to unique factors including industrialization, with resultant decreased physical activity and increased energy consumption [7][8]. As such, the care of PLWH needs to focus not only on ART but also on the management of chronic co-morbidities. This chapter will focus primarily on diabetes in PLWH.

 

EPIDEMIOLOGY AND RISK FACTORS FOR DIABETES IN PLWH

 

Epidemiology           

 

PLWH have a unique set of risk factors that increases their likelihood of developing diabetes. Illustrating the greater burden of diabetes in PLWH on ART, a study from 2005 on the Multicenter AIDS Cohort Study (MACS) of gay and bisexual men with (HIV+) and without HIV (HIV-) found that the incidence of diabetes was found to be more than four-fold higher in HIV+ men and that the prevalence of diabetes was 14% in HIV+ men on ART and 7% in HIV+ men not on ART, compared to 5% in HIV- men. These differences were significant even after adjusting for age and body mass index (BMI). Of note, the majority of HIV+ men on ART in the study were on first generation protease inhibitor (PI) therapy (discussed further in the section Protease Inhibitors) [9]. Other studies have described incidence rates of diabetes in PLWH of 4.4 cases per 1000 person-years of follow-up in the Swiss HIV Cohort Study [10]and 5.72 cases per 1000 person-years of follow-up in the Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) Study of participants from Europe, the US, Australia, and Argentina [11].

 

The effect of HIV disease on developing diabetes is also growing in low and middle income countries (LMIC), where the majority of the people with diabetes live [12]. Prevalence estimates of diabetes in PLWH in LMIC range from 6.8% in Chile [13]to 26% in Cameroon [14]. PLWH and diabetes in LMIC are younger than those in high income countries [15]. The wide range of prevalence estimates of diabetes in PLWH in LMIC may be in part because of differences in the definitions used to identifying diabetes. In addition to factors common to PLWH globally, urbanization may be a contributing factor to the development of diabetes in PLWH in LMIC [16].

 

Antiretroviral Therapies (ART)

 

PROTEASE INHIBITORS (PI)

 

One risk factor for developing diabetes in PLWH is PI use. In 1995, saquinavir became the first FDA approved PI [17]. Incidence rates of hyperglycemia as high as five-fold have been reported in the setting of PI use [18]. In addition, PIs, including ritonavir, have been associated with hypertriglyceridemia [19,20]and lipodystrophy [21]. Mechanisms that may account for these effects are multifold. In one study, insulin sensitivity, as measured by glucose infusion rate during hyperglycemic clamp, decreased significantly after 12 weeks of PI therapy compared to baseline. A defect in beta cell function, in particular, a decrease in the disposition index, was also observed after PI therapy [22]. An in vitrostudy demonstrated that indinavir may lower the function of the glucose transporter GLUT4 [23]. Additional mechanisms may include changes in the hormone adiponectin, which is associated with improved insulin sensitivity, although in vivoand in vitroeffects of PIs on adiponectin have differed [24,25].

 

The prescribing patterns of PI use have evolved. One study found that in the Veterans Affairs system, the prevalence of PI based regimen use decreased from 1997 to 2004 [26]. In addition, the effects of all PIs, namely older generation versus newer generation PIs, including atazanavir and darunavir, on glucose homeostasis are not equal. In an in vitro study, atazanavir, which received FDA approval in 2003 [27], did not inhibit either GLUT1 or GLUT4 glucose transporters [28]. In a clinical study of HIV- participants, atazanavir in combination with ritonavir had a significantly lower effect on insulin sensitivity compared to lopinavir/ritonavir, as measured by glucose disposal rate during hyperinsulinemic euglycemic clamp [29]. Moreover, the newer generation PIs have not been associated with an increased incidence of diabetes, with one study showing a lower risk of diabetes in individuals on these medications [30].

 

NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS (NRTIs)

 

In addition to PIs, certain nucleoside reverse transcriptase inhibitors (NRTIs) have been associated with diabetes. In the D:A:D study, incident diabetes was associated with exposure to the NRTIs stavudine and zidovudine, both thymidine analogs, and didanosine, after adjusting for age, sex, race, and BMI [11]. NRTIs have also been implicated in mitochondrial dysfunction, which may be a mechanism by which NRTIs are associated with diabetes. One study found that one month of treatment with stavudine was associated with  significant decreases in mitochondrial DNA from muscle biopsies and insulin sensitivity, as measured by the glucose infusion rate during a hyperinsulinemic euglycemic clamp [31]. Because of serious adverse effects linked to the use of these NRTIs, stavudine, zidovudine, and didanosine are not advised for use in the United States [32].

 

Similar to the newer generation of PIs, the use of newer NRTIs, including emtricitabine, abacavir, and tenofovir, have been associated with a lower risk of diabetes [30]. Supporting this finding, tenofovir, in a study of HIV- participants without diabetes, did not significantly affect insulin sensitivity, as measured by hyperinsulinemic euglycemic clamp [33].

 

INTEGRASE STRAND TRANSFER INHIBITORS (INSTIs)

 

Recent Department of Health and Human Service guidelines on ART have focused more on integrase strand transfer inhibitor (INSTI) based regimens as initial treatment[32][34]. Reasons for this include the effectiveness of and fewer adverse effects associated with INSTIsin studies of treatment-naïve patients [34-37]. However, newer studies have discovered some metabolic effects of INSTIs. These include weight gain on average of 2.9 kilograms over 18 months, as observed in a retrospective study of patients who switched to an INSTI based regimen from previously being on a non-NRTI (NNRTI)-based regimen. This was in contrast to an average 0.7 kg weight gain in those participants who switched to a PI-based regimen and 0.9 kg weight gain in those who continued on a NNRTI-based regimen [38]. In addition to weight gain, case reports of new onset diabetes have been reported in conjunction with INSTI use [39,40]. One clinical trial compared the INSTI raltegravir to 2 boosted PI regimens and found that the increases in homeostasis model assessment-insulin resistance (HOMA-IR) in all 3 arms were not significantly different from one another, were noted by 4 weeks, and appeared to be independent of changes in visceral adipose tissue [41].

 

The exact mechanisms by which INSTIs may be related to weight gain and diabetes are not known. One proposed mechanism by which INSTIs may cause hyperglycemia is their effect on magnesium, which is a necessary cation for insulin action [40].

 

Lipodystrophy

 

Lipodystrophy associated with ART is characterized by either lipoatrophy in the face, arms, and legs, lipoaccumulation that can lead to gynecomastia, dorsocervical fat tissue, and increased intra-abdominal fat,  [42,43], or mixed lipodystrophy, in which lipoatrophy and lipoaccumulation occur together. As noted above, lipodystrophy has been associated with PI use [21]and with older generation NRTI use, including stavudine [44], but less commonly with the newer generation NRTI tenofovir [45]. In a cross-sectional study, HIV+ men with lipodystrophy had greater insulin resistance than men without lipodystrophy [21]. Lipodystrophy has also been associated with incident diabetes [46,47].

 

In one study that measured body fat changes longitudinally using DEXA in PLWH on ART (specifically zidovudine and lamivudine or didanosine and stavudine in combination with nelfinavir, efavirenz, or both), 32% of participants had discordant changes in trunk and limb fat [48].

 

Lipodystrophy may also be persistent in patients with exposure to thymidine analogs. Although improvements in limb fat mass were reported in patients who switched from the thymidine analog zidovudine to the NRTI abacavir, no significant improvement was noted in self-assessment of dorsocervical fat [49].

 

Hepatitis C Co-Infection

 

25% of PLWH in the U.S. are co-infected with hepatitis C [50]. Hepatitis C infection is associated with a higher risk of developing diabetes. Treatments for hepatitis C, including direct-acting antiviral treatment and pegylated interferon/ribivirin, have been found to lower the incidence of diabetes, with a larger effect in those patients with advanced fibrosis/cirrhosis. In addition among individuals who received treatment for hepatitis C, those with a sustained virologic response were less likely to develop diabetes than those without a sustained virologic response [51].

 

Weight Gain, Overweight, and Obesity

 

Weight gain that is observed after the initiation of ART in those patients with wasting associated with untreated HIV has been characterized as a “return to health” phenomenon. However, because of an emphasis on early ART initiation, the wasting that was previously seen with advanced HIV infection is less common in countries with access to ART [52]. In one 2012 study conducted in Alabama, more than 40% of patients were overweight or obese at the time of starting ART. After 2 years on ART, the percentages of underweight and normal weight participants decreased significantly, and significant increases in the percentages of overweight and obese participants were observed. Having a lower baseline CD4 count and the use of a PI as a third drug were risk factors associated with a greater increase in BMI [53].

 

The consequences of weight gain in PLWH may be different from those in the general population. In a study of U.S. Veterans, the risk of incident diabetes with 10 or more pounds of weight gain during the first year after ART initiation in HIV+ Veterans was significantly greater than in HIV- Veterans [54].

 

Among cohorts of PLWH from different countries, prevalence estimates of an overweight state or obesity range from 25% to 68% [55,56]. In one study of PLWH, risk factors for being overweight or obese in PLWH included increasing age and either no evidence of hepatitis C infection or evidence of cleared hepatitis C infection [57].

 

Inflammation

 

In addition to the risk factors for diabetes in PLWH outlined above, inflammation may play a role in the development of diabetes in PLWH. Systemic inflammation results from several factors: viral replication, immune activation, which leads to T cell depletion, as well as T cell loss specifically in the gastrointestinal tract, with associated translocation of microbial factors including lipopolysaccharide [58]. Co-infection with viruses, including hepatitis C virus (as discussed above), hepatitis B virus, Epstein-Barr virus, and cytomegalovirus, perpetuate systemic inflammation [59].

 

Although ART reduces some inflammatory biomarkers in PLWH [60], there is evidence of residual inflammation, with levels of other inflammatory biomarkers in PLWH on ART that do not decrease to levels seen in HIV- individuals [61]. Persistent inflammation measured months after ART initiation has been associated with incident diabetes in PLWH [62].

 

DIAGNOSIS OF AND MONITORING DIABETES IN PLWH

 

The American Diabetes Association (ADA) recommends the use of a fasting plasma glucose, hemoglobin A1c, or a 2-hour plasma glucose after a 75-gram oral glucose tolerance test to establish the diagnosis of diabetes in the general population. However, there is a caveat that for certain populations of patients, including PLWH, fasting plasma glucose is preferable to hemoglobin A1c [63]. Hemoglobin A1c has been found to underestimate glycemia in PLWH [64,65]. One reason for this is the use of medications that cause hemolysis, including dapsone and trimethoprim-sulfamethoxazole, which are used for prophylaxis of Pneumocystis jiroveci, an opportunistic infection [65]. However, another reason for the discrepancy between glucose levels and hemoglobin A1c is NRTI use. NRTIs, especially the thymidine analogs, are associated with an increased risk of macrocytosis [66], which can lower hemoglobin A1c. Finally, lower CD4 cell count (< 500 cells/mm3) was associated with a significant discordance between expected and measured hemoglobin A1c in HIV- men in the MACS [67]. As such, self-monitoring of blood glucose in PLWH may be preferable to using hemoglobin A1c for monitoring glycemic control [64].

 

The most recent Infectious Diseases Society of America guidelines on the primary care of PLWH recommends obtaining either a fasting plasma glucose and/or hemoglobin A1c at baseline and at 1 to 3 months after starting ART. In addition, hemoglobin A1c is preferred to fasting glucose for diagnosing diabetes because of the relative ease of obtaining a hemoglobin A1c over a fasting glucose. However, a hemoglobin A1c level ≥ 5.8% can be considered to diagnose diabetes in PLWH, instead of the ADA recommendation of ≥ 6.5%, as this improves the sensitivity of the test [68,69]. Moreover, a hemoglobin A1c is recommended to be obtained every 6 months in PLWH and diabetes, with a goal hemoglobin A1c of < 7% [68].

 

TREATMENT OF DIABETES IN PLWH

 

Special considerations should be taken into account in the treatment of diabetes in PLWH. PLWH may not respond to treatment for diabetes in the same manner as HIV- individuals. Part of this observation may be because treatment responses were measured using hemoglobin A1c, which may be an inaccurate indicator of glycemia in PLWH [70], as noted above.

 

Interactions between several diabetes medications and ART are known. In addition, some diabetes medications may present advantages or disadvantages in PLWH. These conditions are described in further detail below and are organized by diabetes medication.

 

Metformin

 

The 2019 ADA Standards of Care on Pharmacologic Approaches to Glycemic Treatment recommend metformin as the first-line therapy in patients in the general population with type 2 diabetes [71].

 

In PLWH with lipodystrophy and insulin resistance but without diabetes, 3 months of metformin 1000 mg twice daily was found to significantly decrease insulin area under the curve after an oral glucose tolerance test. Although no increased incidence in lactic acidosis was noted in the participants who received metformin, the study was not powered for this outcome, and liver and kidney dysfunction were exclusion criteria [72].

 

The commonly used integrase transfer strand inhibitor, dolutegravir,  increases plasma levels of metformin, and thus adjusting the dose of metformin upon dolutegravir initiation has been recommended[73,74]. However, one retrospective study found that there was no significant difference in glycemic control before and after starting dolutegravir in PLWH and diabetes on metformin [75].

 

In summary, there are no guidelines to suggest that metformin should not be the first-line treatment of type 2 diabetes in PLWH, after consideration of liver and kidney function and dolutegravir treatment.

 

Sulfonylureas

 

Sulfonylureas are a substrate of the CYP2C9 enzyme. The PIs ritonavir and nelfinavir are CYP2C9 inducers and can decrease sulfonylurea levels [76,77]. In comparison with initial use of metformin in PLWH, no significant difference in glycemia after one year of therapy was noted with initial use of a sulfonylurea [78].

 

The 2019 ADA guidelines recommend the use of a sulfonylurea as a second-line treatment in patients for whom cost of medication is prohibitive [71]. In PLWH, consideration should be made if a patient is on ritonavir or nelfinavir and the possible loss of efficacy of sulfonylurea treatment.

 

Thiazolidinediones

 

The effect of thiazolidinediones on glycemic control in PLWH with diabetes specifically has not been studied, although some trials found that rosiglitazone lowered serum insulin levels in PLWH without diabetes [79]and improved insulin sensitivity in PLWH with hyperinsulinemia [80]. Several studies have focused on the effect of thiazolidinediones (TZDs) on body fat in PLWH with lipodystrophy, with equivocal findings. One 24 week study on the effect of rosiglitazone on PLWH, all of whom were on a PI, found no significant difference in arm fat between the treatment and placebo groups [81]. However, the study did not have enough power to detect a difference in this outcome [82]. On the other hand, other studies demonstrated that rosiglitazone increased visceral and subcutaneous abdominal fat in HIV+ men with lipodystrophy over 6 months [83]and subcutaneous leg fat in PLWH with lipodystrophy over 3 months [80].

 

Adverse side effects of TZDs should be considered and discussed prior to initiation. These include fluid retention, edema, osteoporosis, and potential liver injury [84].

 

Dipeptidyl Peptidase 4 Inhibitors (DPP4 Inhibitors)

 

Some clinical studies have demonstrated that dipeptidyl peptidase 4 (DPP4) inhibitors exert an anti-inflammatory effect in patients with type 2 diabetes [85,86]. This idea was further examined in PLWH in a pilot study of 20 PLWH on ART randomized to either the DPP4 inhibitor sitagliptin or placebo for 24 weeks. A significant decrease was noted in the chemokine SDF-1α in the treatment group. In addition, an improvement in glucose tolerance was noted at week 8 in the treatment group, but the difference in glucose tolerance between the two groups was no longer significant at the end of the study [87]. In another study of PLWH with impaired glucose tolerance, sitagliptin resulted in a significant improvement in glucose tolerance and decreases in the inflammatory markers hsCRP and CXCL10 from baseline after 8 weeks of treatment, compared to placebo [88]. Similarly, in a larger study of 84 PLWH on ART with viral suppression and without diabetes who were randomized to 16 weeks of sitagliptin versus placebo, a significant decrease from baseline was seen at week 15 in CXCL10 in participants in the treatment group [89]. The dose of saxagliptin should be decreased to 2.5mg per day when used with a strong CYP3A4 inhibitor (for example atazanavir, indinavir, nelfinavir, ritonavir, and saquinavir).

 

Glucagon-Like Peptide-1 (GLP-1) Receptor Agonists

 

Several glucagon-like peptide-1 (GLP-1) receptor agonists have been shown to improve weight and cardiovascular outcomes, albeit in patient populations not specific to PLWH[90,91]. Liraglutide and semaglutide have each been shown to lower the risk of a composite outcome of nonfatal stroke, nonfatal myocardial infarction, and cardiovascular death, in a study population of patients with diabetes and a mean hemoglobin A1c of 8.7 [90,91]. Liraglutide also has an indication from the Food and Drug Administration for lowering cardiovascular event risk in patients with cardiovascular disease and type 2 diabetes. The 2019 ADA guidelines recommend the use of a GLP-1 receptor agonist as a second-line treatment after metformin in patients with cardiovascular disease and type 2 diabetes.

 

One case report described a single HIV+ patient who was able to discontinue insulin treatment (insulin glargine 60 units daily) and who experienced improvements in weight and hemoglobin A1c after starting liraglutide therapy [92]. Relatively scant literature exists on the effects of GLP-1 receptors agonists in PLWH and diabetes [93,94].

 

Sodium-Glucose Co-Transporter-2 (SGLT-2) Inhibitors

 

Similar to the GLP-1 receptor agonists, sodium-glucose co-transporter-2 (SGLT2) inhibitors have demonstrated reductions in adverse cardiovascular outcomes, including cardiovascular death and hospitalization for heart failure in general population studies [95,96]. The 2019 ADA guidelines recommend the use of a SGLT2 inhibitor as a second-line treatment after metformin in patients with cardiovascular disease and type 2 diabetes. One small trial studied canagliflozin for 24 weeks in 8 obese PLWH with type 2 diabetes and hemoglobin A1c > 7% and observed improvements in weight and hemoglobin A1c at the end of the study compared to baseline [97].

 

Adverse side effects of SGLT2 inhibitors include genital mycotic and urinary tract infections. In addition, 55 post-marketing cases of Fournier’s gangrene have been reported between March 2013 to January 2019 [98]. A risk factor for Fournier’s gangrene is HIV infection [99-101], especially a CD4 cell count < 200 cells/μL [102].

 

Insulin

 

The 2019 ADA guidelines recommend considering insulin initiation in the patient 1) with a hemoglobin A1c > 11%, 2) with signs and symptoms of catabolism, 3) who has a presentation concerning for type 1 diabetes, and/or 4) whose hemoglobin A1c is not at goal despite taking 2 to 3 medications for diabetes, in addition to a GLP-1 receptor agonist.

 

As with all patients with diabetes, side effects of insulin therapy to consider in a discussion with a patient with HIV disease and diabetes include the risks of hypoglycemia and weight gain.

 

Sequence of Initiating Diabetes Medications in PLWH

 

There are no specific guidelines for the treatment of diabetes in PLWH. The 2019 ADA guidelines recommend metformin as a first-line treatment for type 2 diabetes, followed by add-on treatments to be chosen based on the patient’s history of cardiovascular disease, need for weight loss, and cost of medications for the patient [71]. In PLWH, an additional factor to consider is the possible interaction of a diabetes medication with specific ART.

 

MICROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

 

Peripheral Neuropathy

 

HIV is associated with multiple peripheral neuropathies (PNs), including a distal, symmetric polyneuropathy (DSPN) [103]. The prevalence of HIV-associated PN among PLWH varies < 10% to upwards of 50%, with the wide variability in prevalence estimates secondary in part to differences in methods used to assess PN [104]. Risk factors for HIV-associated DSPN include use of the NRTIs zalcitabine, didanosine, and stavudine [105]. In addition, HIV-associated DSPN has been observed in PLWH within 1 year of HIV transmission and is significantly associated with evidence of immune activation present in the central nervous system [106].

 

HIV-associated PN, in addition to the risk of developing PN secondary to diabetes, may place PLWH and diabetes at increased risk of sequelae including falls, particularly in those PLWH with a detectable viral load [107], foot ulceration, and amputations.

 

In summary, in addition to considering the risk of diabetic peripheral neuropathy, the risk of HIV-associated PNs should be kept in mind in the assessment and treatment of PLWH with diabetes.

 

Nephropathy

 

The prevalence estimates of chronic kidney disease (CKD) in PLWH vary depending on geographic region, with 7.9% of PLWH affected in Africa (with the highest prevalence among African regions in West Africa at 22%), compared to 3.7% in Europe. In addition, prevalence estimates of CKD in PLWH are significantly greater among those individuals with co-morbid diabetes [108]. In the MACS, two independent risk factors associated with greater odds of proteinuria were HIV+ serostatus with ART use, compared to HIV- serostatus, and a history of diabetes [109]. Moreover, glomerular hyperfiltration, or a supranormal estimated glomerular filtration rate, is more prevalent among HIV+ men without CKD than HIV- men without CKD [110]. Glomerular hyperfiltration has been reported to be an initial state of dysfunction seen in patients with diabetic kidney disease and proteinuria [111].

 

Other risk factors for CKD in PLWH include the following: recurrent acute kidney injury, African-American race, in part because of risk variants of the APOL1 gene, and persistent inflammation, even in the setting of ART [112-114]. In addition, an HIV associated nephropathy (HIVAN), which is characterized by several insults to the kidney, including focal and segmental glomerulosclerosis, exists. Certain ART drugs within the NRTI, NNRTI, and PI classes are also associated with renal injury [114]. Among these are the NRTI tenofovir, which is associated with adverse kidney disease outcomes independent of diabetes [115], and the PI indinavir [116].

 

Retinopathy

 

Ocular opportunistic infections in PLWH include CMV retinitis and ischemic HIV retinopathy [117]. In addition, patients with a history of CMV retinitis should be monitored periodically for retinitis recurrence by an ophthalmologist [118]. However, these ocular opportunistic infections are less common in the setting of ART. However, retinal disease has been noted in PLWH on ART as part of a larger syndrome of the HIV-associated neuroretinal disorder [117], which has an incidence of more than 50% at 20 years after a diagnosis of AIDS [119]. One study found that among HIV+ men with a median duration of ART use of 12 years and suppressed viremia did have a significant difference in total peripheral retinal thickness from HIV- men, although the long-term clinical relevance of this is unknown [117].

 

There is limited literature on the effect of co-morbid diabetes on HIV-associated retinal disease.

 

MODIFIABLE FACTORS OF MACROVASCULAR COMPLICATIONS ASSOCIATED WITH DIABETES IN PLWH

 

PLWH are at increased risk of developing atherosclerotic cardiovascular disease (ASCVD) compared to individuals without HIV, despite controlling for traditional cardiovascular (CV) risk factors such as diabetes [5,120]. In addition, PLWH have a greater burden of traditional CV risk factors [120]. Other non-traditional risk factors include some ART such as older generation PIs [121]and inflammation [122]. As such, calculators developed for the general population to calculate ASCVD risk may not accurately capture risk in PLWH [123].

 

Aspirin

 

Recent American College of Cardiology/American Heart Association (ACC/AHA) recommends the use of aspirin for primary prevention of ASCVD in general population patients age 40 to 70 years with high ASCVD risk [124], and similarly the American Diabetes Association (ADA) guidelines also recommend aspirin use in select patients with diabetes < 70 years of age with high ASCVD risk and low bleeding risk [125]. However, evidence demonstrates that aspirin use is lower in PLWH with CV risk factors than in HIV- individuals [126], and PLWH are less likely to be prescribed aspirin for primary prevention than HIV- individuals [127].

 

Blood pressure

 

The prevalence of hypertension globally in PLWH is substantial. PLWH have several risk factors for developing hypertension, including a greater prevalence of smoking (as noted below) [128,129]and ART use [130]. The relationship of ART to hypertension is thought to be in part from its association with weight gain. Moreover, in the MACS, ART use was associated with greater systolic hypertension but not diastolic hypertension. A mechanism for this could be a change in arterial compliance as a consequence of ART use [131].

 

The 2019 ADA guidelines recommend a goal blood pressure of < 140/90 in people with diabetes, although a goal blood pressure of < 130/80 may be considered in individuals at higher cardiovascular risk [132].

 

Cholesterol

 

Dyslipidemia is seen in both untreated PLWH and PLWH on ART [133,134]. The decision to initiate a statin in a patient living with HIV for primary prevention should take into account the patient’s HIV serostatus, especially in those patients with a 10-year ASCVD risk of 5 to < 20% [124].

 

Cigarette Smoking

 

Cigarettesmoking is more prevalent in PLWH than in individuals without HIV [129], and life expectancy is estimated to be lower in HIV+ men and women who are current smokers at the time of HIV care initiation than in HIV+ men and women who are former or never smokers, with a 2.9 year gain in life expectancy at 10 years after HIV care initiation [135]. The ADA guidelines recommend that people with diabetes not smoke cigarettes and that smoking cessation treatment be offered for those patients with diabetes who do smoke [136].

 

SUMMARY

 

In summary, PLWH have unique risk factors that increase their risk of diabetes. Factors to take into consideration in the treatment of PLWH include the following: type of ART, evidence of lipodystrophy, co-infection with other viruses, and overweight or obese state. A caveat is that HbA1c may be inaccurate in diagnosing and monitoring diabetes in PLWH. PLWH have additional risk factors for developing the microvascular complications of diabetes. Some glycemic agents may interact with ART, and other glycemic agents may have unwanted effects, including weight gain, that should be addressed in a patient-provider discussion. Finally, additional modifiable cardiovascular risk factors, including hypertension and smoking, should be addressed in the comprehensive treatment of diabetes in PLWH.

 

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Emergencies Related To Pheochromocytoma/ Paraganglioma Syndrome

INTRODUCTION

Pheochromocytomas (PCCs) and paragangliomas (PGLs) are rare chromaffin cells tumors (PPGLs) characterized by the production, storage, metabolism, and secretion of catecholamines and their metabolites, metanephrines and methoxytyramine. These tumors can raise significant challenges in clinical recognition, diagnosis, and therapy and when undiagnosed can result in severe morbidity as well as mortality, especially due to cardiovascular system toxicity. Despite anatomical dissimilarity – PCC arising from adrenal medulla and PGLs from extra-adrenal sympathetic or parasympathetic paraganglia – these tumors display common embryonic origin, enzymatic milieu, and the ability to produce catecholamines and their metabolites. While once thought as mostly benign and biochemically active, these tumors can show a wide spectrum of cellular and biochemical dedifferentiation, including an aggressive metastatic course and biochemical silence.

The PPGL field has undergone a significant transformation in recent years. We now know that PPGLs represent the highest hereditary-driven endocrine condition with up to 40% of cases related to mutations in 15 well-established driver genes and a growing number of disease-modifying genes (now about 25). We now also appreciate that a significant proportion of what previously was thought to be almost exclusively benign disease is actually malignant and can display biochemical silence and a specific secretory profile with mainly elevated norepinephrine and/or dopamine.

Unfortunately, and despite tremendous advances in our understanding of the biology of PPGLs, the severity of disease-associated morbidity still remains significant since most of these tumors are not well recognized and diagnosis is delayed.

Definition

What would define actual urgency or emergency in PPGL? Is it a biochemical phenotype closely associated with a particular catecholamine secretion or various biomarkers suggestive of dedifferentiation and thus a malignant course, disease symptomatology, or the rapidity of disease progression? Is it an overall basic patient’s health status that can rapidly deteriorate or the expected complications from surgery? Is it the level of comfort and experience of the managing endocrinologist or abilities of the operating surgeon or possible lack of these? Or maybe it is the ability of the patient to follow-up frequently enough for appropriate management or the affordability of tests and medications or lack of those (some due to their price) which would eventually associate with a grim outcome. At the end of the day, it is probably all of the above and even more including some psychological aspects and fear to develop metastatic disease. Any disease that can potentially deteriorate to severely morbid outcomes needs to be seen as urgent and emergent most of the time. Obviously, in the case of a severe hypertensive crisis in the operating room that developed shortly after a previously undiagnosed/misdiagnosed abdominal mass was manipulated, the diagnosis that will drive appropriate therapy will be acute catecholamine crisis and there is a universal awareness of such a situation. Same should be true in case of severe therapy-resistant hypertension which rapidly deteriorates with use of β-adrenoceptor blockers. Unfortunately, there are many other possible scenarios that could start relatively slow and rapidly deteriorate to true medical emergencies. An example is our recent case that shows all aspects of PPGL management, including emergencies. A young female in late pregnancy was admitted for increased BP, thought to be related to noncompliance with BP medications and possible development of pre-eclampsia. Because of resistance to therapy while hospitalized the patient had an assessment of plasma catecholamines, which came back markedly elevated. Her imaging studies showed a 12 cm abdominal PGL with the fetus’ head laying directly on it. The tumor was massively vascularized and engulfed major abdominal vessels. The team that discussed her management could fill a lecture hall and included obstetrics, gynecologic surgery, endocrine surgery, general surgery, vascular surgery, anesthesiology, neonatology ICU, medical ICU, endocrinology, nursing and many more. All of the above worked hard on the day of combined caesarian section delivery and open abdominal surgery, which was complicated, but resulted in full recovery for both mother and baby.

CLINICAL ASPECTS

Clinical course and outcomes of excessive catecholamine secretion by PPGLs closely correlate with multiple factors related to the biochemistry of catecholamine action, secretory profile, acuity, and severity of actual hypercatecholaminemia. This gets very complicated by the fact that clinical symptomatology of hypercatecholaminemia lacks specificity and often presents as much more prevalent conditions, like hypertension, anxiety, or cardiac arrhythmias. If there is a single most important factor to define the overall outcome of the disease, we personally would pick timely suspicion and initiation of appropriate workup. While hypertension – paroxysmal or sustained – usually represents the initial or most common symptom, the overall clinical symptomatology varies widely and is summarized in Table 1.

Table 1. Clinical Syndromes Related to PPGL

Organ

Syndrome

Mechanism

Receptor action

Heart

§ Angina

§ Heart attack

§ Cardiomyopathies

§ Myocarditis

§ Arrythmias

§ Heart failure

§ Coronary spasm

§ Positive inotropy

§ Positive chronotropy

§ Unmatched O2 demand

§ Hypoperfusion

§ Coronary α1, β2

§ Conducting system β1, β2

§ Conducting β1, β2

Brain

§ Stroke

§ Encephalopathy

§ Vasoconstriction

§ Unmatched O2 demand

§ Hypoperfusion

§ Cerebral arterioles α1

§ Effect of systemic HTN

Vascular

§ Shock

§ Postural hypotension

§ Aortic dissection

§ Organ ischemia

§ Limb ischemia

§ Arteriolar vasoconstriction

§ Arteriolar vasodilation

§ Vasodilation

§ Unmatched O2 demand

§ Hypoperfusion

§ Vascular α1, α2, β2

Kidneys

§ ARF

§ Hematuria

§ Vasoconstriction

§ Vasodilation

§ Increased renin secretion

§ Unmatched O2 demand

§ Hypoperfusion

§ Vascular α1, α2, β1, β2

Lungs

§ Pulmonary edema

§ ARDS

§ Fibrosis

§ Pulmonary HTN

§ Vasoconstriction

§ Vasodilation

§ Bronchodilation

§ Vascular α1, α2, β2

GI

§ Intestinal ischemia

§ Vasoconstriction

§ Unmatched O2 demand

§ Hypoperfusion

§ Visceral arterioles α1, β2

Physiology of Catecholamine Action

Catecholamine production takes place in both adrenals, as well as sympathetic paraganglia. The synthetic pathway is specific for the abovementioned organs, defined by the unique set of intracellular enzymes able to convert the amino acid tyrosine to end product epinephrine or norepinephrine, dependent on the site of the synthesis. The actual catecholamine is related to the site/type of the cell, as well as degree or differentiation or lack of such (Figure 1). The transport of tyrosine through the cell membrane is active process and carried out by a member of the amino acid transporter family – large neutral amino acid transporters of the L family, mostly LAT1. These transporters can be induced and show overexpression, especially in some cancers. Dedifferentiated/malignant PPGL can produce a phenomenon of massive or predominantly norepinephrine production/secretion profile driven by the both overexpressed LAT1 and the lack of phenylethanolamine N-methyltransferase (PNMT) – the enzyme that converts norepinephrine to epinephrine.

Figure 1. Catecholamine Synthesis

Although currently we manage both conditions as part of a single syndrome, the physiology of catecholamine production and secretion from both systems is relatively distinct. Adrenals – the mastermind of the fight and flight response – are designed to produce significant amounts of epinephrine/adrenaline, to be secreted in response to stress. The secretion pattern is episodic/paroxysmal and relatively short lived. Epinephrine, the end-product of the synthetic pathway, is stored in secretory granules and secreted on an as-needed basis. Norepinephrine in this case is a co-secretory catecholamine, somewhat different in affinity to adrenergic receptors – through which both substances are signaling (Figure 2 and 3). In cases of catecholamine-producing tumors, the pattern of secretion can vary between paroxysmal or sustained, while the ratio between two catecholamines relates to some degree to the level of differentiation of the adenomatous tissue. Sympathetic paraganglia, on the other hand, is mostly involved in production of norepinephrine as a major sympathetic neurotransmitter rather than as a systemic hormone. The product is mostly secreted into the synaptic space, which than spills over into the systemic circulation. In the physiologic state, a significant amount of norepinephrine re-uptake back into the pre-synapse for repeated use occurs. In the case of PGLs, the increased amount of product reaches the systemic circulation to produce symptoms and signs indistinguishable from adrenally secreted norepinephric clinical picture of adrenergic overactivity.

Figure 2. Adrenergic Receptors and Ligands

Figure 3. Catecholamine Secretion

Catecholamines, both epinephrine and norepinephrine act through activation of the G protein coupled adrenergic receptors (GPCRs), both α1 and 2 and β1 and 2 with minor difference in the fact that norepinephrine has lower affinity to β2-adrenoceptors and thus norepinephric hypercatecholaminemia lack a mild component of peripheral vasodilation and could have slightly different clinical appearance compared to purely epinephric hypercatecholaminemia Table 1 and Figure 2). As with other GPCRs, adrenoceptors can undergo desensitization, which could explain the different clinical presentations in relatively mild long standing disease compared to more rapidly developing hypercatecholaminemia. One also needs to remember that in massive biochemical hypercatecholaminemia, competitive α- and β-adrenoceptor blockers could be overwhelmed by the concentration of the ligand and safe preoperative adrenoceptor blockade can take longer to achieve and can be partial rather than complete.

Clinical Features

Hypercatecholaminemia-related endocrine emergencies define rare, but truly severe and potentially deadly end of the clinical spectrum of the PPGL syndrome. While it is called a great masquerader, this is misleading because it is not that the disease that masquerades, but rather because of the fact that clinical symptomology is completely non-specific and lacks any definitive symptom or signs that would point towards PPGL as a sole contender. It rather presents with symptoms and signs of much more prevalent conditions – like hypertension, benign cardiac arrhythmias, anxiety – and thus, progresses towards acute or chronic complications without being suspected. Needless to say, that unless the disease is severe or acute, it could be treated as a mainstay symptom-driven state – like hypertension – with at least some success. Clinical emergencies, related to the PPGL represent a completely different scenario – these are usually either unsuspected or only partially treated cases with severe short-term morbidity and significant mortality. In these cases, clinical suspicion is an absolute cornerstone of the management and the delay in diagnosis is adversely proportional to the overall outcome. Clinical scenarios with resultant PPGL-related emergencies usually include unrelated surgeries, where overall stress or tumor manipulation results in massive and acute hypercatecholaminemia with fully sensitized adrenergic receptors and lack of any adrenoceptor blockade, which precipitates acute and severe hypertensive crisis and potentially multiorgan failure. Less dramatic, but still a potentially severe condition includes treating progressive hypertension in the general or obstetric population with a medication that predisposes to unopposed α-adrenoceptor stimulation and thus precipitates severe peripheral vasoconstriction and either worsening of hypertension or heart failure. Obviously, patients with pre-existing heart or renal failure will be much more susceptible to severe outcomes. Because of the fact that some tumors express slow biochemical progression, we need to keep a high index of suspicion not only for patients with resistant HTN, familial HTN, or young age of onset, but for any patient who might have potential to have this disease.

ACUTE VS CHRONIC HYPERCATECHOLAMINEMIA

While an acute increase in catecholamine levels is directly responsible for precipitation of a hypertensive crisis through vascular vasoconstriction and positive inotropy, a long-lasting increase in catecholamine levels, especially of relatively mild degree, can be completely asymptomatic. This can probably be explained to some degree by several physiologic processes, including desensitization of the adrenergic receptors. Slowly progressive disease will mask, at least partially, clinical symptomatology, as well as allow sometime for the patient to try antihypertensive, antiarrhythmic, or antianxiety therapies as part of the therapy for aforementioned nonspecific conditions, as well as clinically desensitize the patient to mild hypertensive symptoms.

As mentioned above, clinical scenarios will mostly associate with unrelated surgeries, obstetric conditions like delivery and pre-eclampsia, as well as sudden or rapidly progressive deterioration of a previously stable person with significant conditions that would be sensitive to rapid increases in BP, pulse rate, or overall oxygen requirement. Currently, there is a well-accepted awareness, especially in the operating/delivery rooms, that sudden and rapid increases in systolic BP must be treated immediately by medications capable to act in the hypercatecholaminemic state. There is also a sufficient awareness of predominant β-adrenoceptor activity of labetalol, which could provide only partial α-blockage and be insufficient alone in full hypercatecholaminemic crisis. On the other hand, IV phentolamine is not a readily available operating room medication and this leaves nitroprusside as a medication mostly available in the operating room settings. Its use for prolonged and complicated surgery or delivery could possibly be associated with generation of methemoglobin and thiocyanite in the patient or the newborn. Because of the fact that majority of acute and severe hypercatecholaminemic states will have either mixed adrenergic/noradrenergic or noradrenergic biochemical phenotype, there is less expectation of β2 -adrenoceptor driven vasodilation and orthostasis.

SEVERE VS MILD HYPERCATECHOLAMINEMIA

It is accepted that patients with mild hypercatecholaminemia can be relatively asymptomatic or mildly symptomatic with some response to the usual antihypertensive therapy, thus disease can be present for a significant length of time undiagnosed. Severe hypercatecholaminemia, on the other hand, is markedly symptomatic and should be suspected right away. Clinical problems arise in cases when this happens during unrelated surgeries, as stated above, especially when an unrecognized abdominal mass, which in this case will be a PGL, is manipulated and releases massive amounts of pre-synthesized catecholamines. These cases are rare and close to impossible to predict, but in cases of severe intra-operative or intra-labor hypertension, should be immediately suspected and treated. Another scenario represents rapidly progressive disease in a younger patient – these are usually familial paragangliomas that can rapidly progress and metastasize. In this case, younger patients present with what suggest anxiety, especially in patients with an episodic secretory profile. Appropriate diagnosis can be significantly delayed when these patients enter the “outpatient workup mode” with infrequent appointments to assess the efficacy of anti-anxiety medications. This delay in diagnosis can associate with development of significant complications in patients with other pre-existing conditions. Obviously, acute concomitant illnesses will precipitate acute hypertensive crisis. Although over-suspicion could result in significant number of questionably necessary tests, it seems reasonable to test keeping in mind potentially morbid outcomes of severe untreated hypercatecholaminemia.

EPISODIC VS CONTINUOUS SECRETION

Both episodic and sustained secretion of catecholamines can produce hypertension as well as an acute crisis. One can argue that the episodic form is more symptomatic owing to the nature of an on and off symptomatology that can be easier to detect for both the patient and the physician. We are not aware of differential adrenoceptor desensitization of episodic hypercatecholaminemia when compared to a persistent secretory state. Both forms are capable of rapid secretion of massive amounts of catecholamines in case of stress or manipulation, so the actual presentation or management of acute hypertensive emergency will not differ.

LARGE VS SMALL MASS

Historically, the size of the mass was thought to be proportional to the biochemical activity of PCC, with the exception of larger tumors, which were thought to overgrow their vascular supply, become necrotic and decrease the ability to be significantly active biochemically. Current knowledge complicates this to a significant degree because of several added details. The degree of differentiation of PPGL can massively affect both the actual profile of the secreted catecholamines (the higher the differentiation, the more probable the synthetic catecholamine pathway leads to epinephrine), as well as the amount of secreted catecholamines, where lesser differentiation could associate with a significant decrease in the amount of the synthetic catecholamine.

One should also remember that larger intra-abdominal masses can also result in local tissue invasion, including large or multiple vessels, adjacent organs etc. In this case, knowing the anatomical relationship between the tumor and the adjacent tissues can help avoid a potentially prolonged and complicated surgery.

We are also historically aware of the fact that the actual size of the adrenal tumor correlates with possible metastatic/malignant state/course. In PPGL this postulate is also relative, making genetic milieu more important factor for the prediction of malignancy (like SDHB mutation or younger age), then the actual size of the initial adrenal mass. It worth mentioning that multiple masses and PGLs per se will have a higher predisposition to malignancy as compared to a single adrenal pheochromocytoma.

In any case, finding a small PPGL and assuming that there would be no significant hypercatecholaminemia during stress or surgery is as wrong as finding a large mass and assuming that it had outgrown the vascular supply and thus is necrotic and incapable of acute delivery of massive hypercatecholaminemia.

PHEOCHROMOCYTOMA VS PARAGANGLIOMA

The division of PPGL tumors into PCC and PGL is mostly anatomical rather than functional. The only major difference is that PCCs express significantly higher content of PNMT and thus higher probability of predominantly the adrenergic or mixed biochemical phenotype, as compared to predominantly noradrenergic phenotype of PGLs. With that said, the actual profile will strongly depend on the degree of tumor differentiation, as well as possibility of mixed PPGL cases.

Another possible cause of differences in the acute conditions associated with different PPGL tumors is the fact that adrenal incidentalomas are readily diagnosed on unrelated imaging studies, especially in recent years when both chest and abdominal CT scans, which both image adrenal glands, are done for progressively increasing number of conditions. PGL are frequently missed, especially in cases where clinical symptomatology is less severe or the patient is young and is otherwise seen as “healthy”. Acute and severe hypercatecholaminemic crisis can occur when a previously unknown abdominal or chest mass is seen during unrelated surgery or invasive procedure and is manipulated, causing release of massive amount of pre-synthesized catecholamines. In these cases, surgical awareness of uncommon locations and anesthesiology readiness for appropriate therapy of potentially life-threatening crisis is the true cornerstone of the management of this endocrine emergency.

SINGLE TUMOR VS METASTATIC DISEASE

The main difference in the approach to the possibility of metastatic disease is based on the expectation that rapid postoperative withdrawal of adrenoceptor blockade will associate with rebound hypertensive crisis. In addition to this, possibility of distant metastatic disease with significant morbidity associated with involvement of affected organs needs to be kept in mind.

SPONTANEOUS VS FAMILIAL/SYNDROMAL CASE

Recent years have tremendously changed many aspects of our understanding of the biology and management of the PPGL particularly the progress in understanding the genetics of the disease. While possibility of a genetically driven condition should be increased in younger patients or ones with a positive family history, finding a predominantly noradrenergic biochemical phenotype, multiple masses on imaging studies, or additional clinical findings – thyroid nodules happen to be medullary thyroid cancer (MTC), renal tumors etc. – should strongly suggest a genetic condition. The opposite is even more important – like sending patient with thyroid nodule of unclear pathology to surgery and ending up with it being MTC and patient having a hypertensive crisis during the surgery. Possible syndromal association with SDHB mutation should prompt assessment of multiple tumors, as well as early recurrence and metastatic disease to prevent early post-operative discontinuation of medical therapy and rebound hypertension or discontinuation of long term follow up. In addition, the head and neck PGLs, rarely seen by endocrinologists in the past, are associated with a SDHD gene mutation and can metastasize and locally invade, while being secretory silent. Establishment of a genetic disorder requires institution of testing and biochemical screening of relatives.

DOPAMINE VS NOREPINEPHRINE VS EPINEPHRINE SECRETING TUMORS

Based on the differences in the affinity of epinephrine and norepinephrine to adrenoceptors, with norepinephrine having lesser action on the β2-adrenoceptor, one can expect a pure “vasoconstrictive” clinical presentation in cases with pure norepinephric secretory profile, while with epinephrine and dopamine-secreting tumors, orthostatic or episodic hypotension will be much more frequent.

PPGL IN PREGNANCY

PPGL during pregnancy is a rare clinical entity. In the case of pregnancy, there are 2 patients at the same time, both the mother and the fetus. Both can be severely affected by the disease, although in a somewhat different manner. PPGL is difficult to suspect during pregnancy because of pre-eclampsia-driven management attitude. Diagnosis can be significantly delayed causing fetal morbidity and affecting both the pregnancy and delivery. Several physiologic phenomena drive the unique behavior of PPGL in pregnancy. These include high placental expression of catechol-O-methyltransferase (COMT) and monoamine oxidase (MAO) and lack of autoregulation in uteroplacental circulation. While both enzymes are responsible for production of inactive catecholamine metabolites, they provide some kind of “fetal barrier”, shielding the fetus from exposure to increased catecholamine levels. Lack of uteroplacental vascular autoregulation, on the other hand, directly affects placental blood flow and fetal blood supply in the hypertensive vasoconstricted mother and can associate with rapid development of uteroplacental insufficiency. As far as management – MRI will be the preferred imaging modality, medical therapy will be started with same medications as in non-pregnant patients, and the management of acute severe hypercatecholaminemia will be similar to non-pregnant cases, with exception for the need to avoid methyldopa and more prevalent use of intravenous magnesium sulfate, which will be effective in both PPGL and pre-eclampsia. Surgery still remains the treatment of choice and there is continuous debate about the sequence of delivery versus surgery.

PPGL IN PEDIATRIC POPULATION

Hypertension in the pediatric population is mostly secondary and is mostly related to renal disease with endocrine causes happening much less frequently. With this said, the possibility of both genetically-driven as well as a malignant course is much higher and needs to be assessed in every pediatric case. Overall management is similar to adult PPGL. On the other hand, the patient will need an extended follow up to assure that any possibility of recurrence is monitored.

CO-SECRETORY SUBSTANCES

The unique enzymatic machinery of PPGL cells provides a series of steps that transforms an amino acid to an amine. In this case, the amino acid is tyramine and the end product are catecholamines. One should appreciate that PPGLs can co-secrete multiple active substances, most clinically relevant of which will probably be ACTH/CRH, which can cause frank and at times severe Cushing syndrome. This needs to be kept in mind, especially when the patient presents with suspicious symptoms or biochemical findings. PPGL as part of MEN2 will associate with overproduction of calcitonin and disseminated metastatic disease, which needs to be diagnosed, hopefully prior to PPGL surgery.

SYMPTOMATIC VS SILENT

While we had discussed in length symptomatic PPGL, parasympathetic PGL could associate with silent tumors, which could be associated with SDHB/D mutation and might have a malignant/metastatic course with local involvement of carotid sinus, as well as major neck vessels, in times associated with different – “silent/local” urgencies/emergencies.

TREATMENT-ASSOCIATED SEVERE HYPERCATECHOLAMINEMIA

Inoperable or recurrent metastatic disease can be treated through multiple modalities, which usually cause different degrees of tumor destruction. These include older therapies (radiofrequency ablation, cryotherapy, external beam radiation, transarterial chemoembolization, ethanol injection), as well as newly rediscovered 131I-MIBG and somatostatin receptor-driven peptide receptor radionuclide therapy (PRRT) with 90Y-DOTATOC/DOTATATE and 177Lu-DOTATATE. This tumor destruction is associated with the potential of massive release of the pre-synthesized catecholamines and could generate severe hypercatecholaminemia for a prolonged period of time. In preparation for therapy, patients need to undergo a protocol, identical to surgical preparation and their biochemical response needs to be followed for weeks after therapy. Overtly secreting or very large tumors should probably generate post-procedural admission for closer monitoring to make sure that the patient will not develop a hypertensive emergency. As we had discussed above, pre-treatment with a competitive α-adrenoceptor antagonist must be used in almost all patients but may provide insufficient α-adrenoceptor blockade with massive hypercatecholaminemia. On the other hand, use of phenoxybenzamine in a full dose, can potentially result in prolonged hypotension but is less problematic than a severe hypertensive crisis and its consequences.

CLINICAL SYNDROMES ASSOCIATED WITH PPGL

Multisystem Failure

This is by far most feared complication because of the high morbidity and mortality associated with a rapid and at times unexpected and unpredicted development, resembling an avalanche starting small but rapidly leaping into a clinical disaster. While it could be preceded by a hypertensive crisis, patients who are sicker and fragile at baseline can develop it with little or no warning symptoms. The blood pressure pattern can show either hypertension or hypotension in case of progressive shock and cardiac failure. It can associate with fever, encephalopathy, as well as renal failure, pulmonary edema and even disseminated intravascular coagulation. Clinical outcomes mostly depend on delays in diagnosis and initiation of appropriate therapy.

Cardiovascular Emergencies

HYPERTENSIVE CRISIS

While hypertension in patients with PPGL can be both paroxysmal and sustained, a severe hypertensive crisis is usually precipitated by stress, postural changes, food containing large amounts of catecholamine precursors, as well as local manipulation of an unsuspected tumor. Medications can also induce hypertensive crisis through direct stimulation of release of stored catecholamines – which could be of a massive quantity. These medications include ACTH, tricyclic antidepressants, phenothiazine, nasal decongestants containing sympathomimetic or histamine, and metoclopramide. Treatment needs to be initiated immediately, intravenously and one needs to remember that α-adrenoceptor blockade is the drug of choice, as well as the fact that β-adrenoceptor medication can both cause and precipitate acute deterioration of the hypertensive crisis.

HYPOTENSIVE SHOCK

Hypotension in PPGL is usually perceived as exclusively related to dopamine or epinephrine-secreting tumors. While this is true, hypovolemia and acute heart failure due to an acute coronary event, myocarditis, or pulmonary edema can produce profound hypotension and shock in norepinephrine-secreting tumors too.

CARDIAC ARRHYTHMIAS

Tachyarrhythmias are frequently associated with PPGL and are related to β-adrenergic stimulation-driven positive inotropy. These are mostly supraventricular including atrial fibrillation and flutter, as well as wide complex ventricular tachycardia. One needs to remember that in case of myocarditis, cardiomyopathy, or an acute coronary event, myocardial susceptibility to any type of rhythm disturbances is significantly increased and can manifest with bradyarrhythmia’s.

MYOCARDITIS AND CARDIOMYOPATHY

Development of myocarditis and cardiomyopathy in PPGL is well known and described, but still remains poorly understood as far as the actual mechanistic process. It could relate to direct myocardial toxicity of significant and prolonged hypercatecholaminemia, as well as prolonged hypertension or a coronary event. It could be of any type – either hypertrophic or dilated – as well as asymmetric (tako-tsubo type). It could improve to some degree after successful treatment.

ACUTE MYOCARDIAL OR PERIPHERAL ISCHEMIA

Both could be caused by prolonged hypertension, resulting in intimal hypertrophy, as well as local spasm in the naïve or already sclerotic vessel. It can also result from increased and uncompensated oxygen demand.

Pulmonary Emergencies

Pulmonary edema can be both cardiac and non-cardiac of origin. The first is discussed above, while the last is mostly related to increased capillary pressure together with vasoconstriction related stasis and an increase in vascular permeability.

Gastrointestinal Emergencies

Clinically, acute GI emergencies are usually associated with abdominal pain and vomiting. These could be related to mesenteric ischemia, which consequently can result in bowel perforation, ileus, and GI bleeding. Ileus in PPGL can be both paralytic and pseudo-obstructive and can also associate with megacolon. Although rarely thought to be related to PPGL, these disorders need to be diagnosed early and treated to prevent rapid deterioration and the need for urgent surgery. Severe hypertension can also associate with an aneurysm of the aorta that can undergo dissection with a hypertensive spike.

Renal Emergencies

Acute vasoconstriction of the renal arteries can result in acute renal failure, while prolonged hypertension can cause progressive deterioration of renal function over a relatively short period of time, especially in patients with underlying hypertension or susceptibility to significant vascular changes.

Neurologic Emergencies

Strokes are known to occur with both paroxysmal and sustained hypertension, but other neurologic emergencies could include hypertensive encephalopathy, subarachnoid hemorrhage, and seizures. Neurologic deficiencies related to brain or spinal metastases, as well as local neurologic deficits caused by paragangliomas are also seen.

MANAGEMENT

The diagnostic approach and treatment of PPGL are discussed in detail in the PPGL section of Endotext and shown in Table 2, but what we will discuss here is the approach to PPGL-related medical emergencies.

Table 2. Treatment of PPGL

Stage

Goal

Primary

Alternative

Initial oral

Normalization of BP

Minimal organ effect

In the following order:

α-blocker

β blocker

Metyrosine

Calcium channel blocker

Labetalol

Pre-operative

Normal BP

Normovolemia

Optimized cardiac performance

As Initial

Fluids to normovolemia

As Initial

Intra-operative

Prevention of the following:

Severe hypercatecholaminemia

Severe hypertension

Severe hypotension

Phentolamine IV

Nitroprusside IV

Aggressive fluid replacement

Labetalol IV

Post-operative

Prevention of hypotension

Prevention of hypoglycemia

Aggressive fluid replacement

Glucose supplementation

 

Inoperable disease

Maintenance of normal BP

Treatment of metastatic disease

Chemotherapy

Radiotherapy

Debulking

Experimental Therapy

As with non-urgent PPGL, the main part of successful management and prevention of its deterioration into a medical emergency is timely suspicion and diagnosis. Obviously, this cannot happen in each and every case and we will continue to see acute severe hypertensive crises and poor outcomes that could not be prevented. But otherwise, the overall suspicion should be relatively high, even if it will generate some unnecessary workups, while preventing avoidable death. We also feel that the common flamboyancy of PPGL being a great and friendly masquerader should probably be revised to some degree. It should include a quite real possibility of metamorphosis of this apparent benignity into behavioral tendencies of a Grim Reaper; just to make sure that the reality of severely morbid outcomes is known and respected.

As was discussed above, in case of acute intra-operative hypertensive crisis with or without identifiable mass, therapy should be initiated assuming PPGL-related severe hypercatecholaminemia. Medications are to be administered IV and should include phentolamine or nitroprusside. Nitroprusside is more readily available in ORs compared to phentolamine, which needs to be prepared by the pharmacy. Nitroprusside can cause adverse effects when administered over an extended period during complicated surgery (discussed above). If existence of a PPGL is established prior to surgery, it would definitely be advisable to procure phentolamine to be available in OR/ICU. Phentolamine, an α-adrenoceptor antagonist, is given as an i.v. bolus of 2.5 mg to 5 mg at 1 mg/min, which can be repeated every 5 min for adequate control of hypertension. Alternatively, it can be given as a continuous infusion (100 mg of phentolamine in 500 mL of 5% dextrose in water, not available in USA) with an infusion rate adjusted to the patient’s blood pressure during continuous blood pressure monitoring. Sodium nitroprusside can be administered at 0.5 to 10.0 mcg/kg per minute (stop if no results are seen after 10 minutes). Magnesium sulfate acts as vasodilator and antiarrhythmic and is administered as a 1-2 gm bolus and then continuously at 1 to 3 gm/h. Esmolol, a short acting β1-adrenoceptor antagonist can improve uncontrolled tachycardia. Continuous infusion of Nicardipine, that is usually a very good initial choice, can prevent catecholamine-induced coronary vasospasm, hypertension, and tachycardia and it is given intravenously at 1 to 2.5 mg for 2 min, then at 5 to 15 mg/h. If the patient was not on a α-adrenoceptor blocker prior to the surgery, use of Labetalol could precipitate deterioration in blood pressure because of 1:7 α:β-specific blocking effect. If the patient is on a short-acting competitive α-adrenoceptor blocker, using i.v. Labetalol bolus could be beneficial for better control of blood pressure.

The same approach should be carried out in cases of a severe hypertensive crisis if it happens acutely in a patient with known and insufficiently treated PPGL (recurrence, lack of compliance), acute deterioration of hypertension, or resistant to initial therapy including pre-eclampsia. There will be little time to sufficiently and efficiently pre-load patient with oral therapy, which should be carried out after resolution of the crisis if surgery was not performed.

If, on the other hand, there is time for oral therapy in less urgent situations or while awaiting upcoming surgery, α-adrenoceptor blockade should be initiated as soon as possible and the patient should be clinically evaluated on a frequent basis to adjust therapy as tolerated. The choice of medication should be dictated by several factors. In cases of severe hypercatecholaminemia or relatively recent onset, where one should expect less time available for significant desensitization of adrenoceptors, competitive α-adrenoceptor blockers could provide lesser control of symptoms just by the virtue of pharmacokinetics against massive concentration of catecholamines. In such cases, use of a non-competitive agonist – phenoxybenzamine - will make more sense. Otherwise, competitive adrenoceptor blockers seem to be efficient and safe and could result in shorter hospitalization due to shorter action and lesser postoperative hypotension. Also, doxazosin (as well as others (prazosin and terazosin, which seem to be used less frequently) seems to be both safe and efficient in PPGL management. Physician’s preferences and experience play a major role in the selection of prescribed medication. In addition to this, the cost and availability affects the choice of medication. Endocrinologist need to be comfortable with multiple different classes of medications used for therapy. Calcium channel blockers (nicardipine/amlodipine) proved to be also safe and efficient, but, again, we would suggest avoiding them alone in cases of severe hypercatecholaminemia, especially with concomitant congestive heart failure. A competitive inhibitor of tyrosine hydroxylase - α-methyl-para-tyrosine (metyrosine, Demser) can be used to both compete with tyrosine (the substrate for catecholamine synthesis), as well as a direct inhibitor of tyrosine hydroxylase.

Hypotension is managed by fluid administration and/or vasopressors including phenylephrine. Hypoglycemia can occur after removal of PPGL-related catecholamine excess as a result of rebound release of insulin secretion and is treated with intravenous glucose.

GUIDELINES

Lenders JW, Duh QY, Eisenhofer G et al.; Endocrine Society. Pheochromocytoma and paraganglioma: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. 2014;99(6):1915–1942.

REFERENCES

Pacak K, Tella SH. Pheochromocytoma and Paraganglioma. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000-2018 Jan 4.

Crona J, Taieb D, Pacak K. New Perspectives on Pheochromocytoma and Paraganglioma: Toward a Molecular Classification. Endocrine Reviews. 2017;38(6):489-515.

Fishbein L, Leshchiner I, Walter V, et al. Cancer Genome Atlas Research Network. Comprehensive molecular characterization of pheochromocytoma and paraganglioma. Cancer Cell. 2017;31(2):181–193

Prete A, Paragliola RA, Salvatori R, Salvatore MC. Management of catecholamine-secreting tumors in pregnancy: a review. Endocrine Practice. 2015;22(3):357-70.

Dahia PL. Pheochromocytoma and paraganglioma pathogenesis: learning from genetic heterogeneity. Nat Rev Cancer. 2014;14(2):108–119.

Tischler AS, Pacak K, Eisenhofer G.The Adrenal Medulla and Extra-adrenal Paraganglia: Then and Now. Endocr Pathol. 2013 Dec 24

Pacak K Preoperative management of the pheochromocytoma patient. J Clin Endocrinol Metab. 2007:92(11):4069-79.

Congenital Hypothyroidism

INTRODUCTION

 

Thyroid hormones are essential for normal development and growth of many target tissues, including the brain and the skeleton. Thyroid hormone (TH) action on critical genes for neurodevelopment is limited to a specific time window, and even a short period of deficiency of TH can cause irreversible brain damage. During the first trimester of pregnancy fetal brain development is totally dependent on maternal thyroid function. Congenital hypothyroidism (CH) is one of the most preventable causes of mental retardation, but early diagnosis is needed in order to prevent irreversible damage. Today more than 70% of the babies worldwide are born in areas without an organized screening program. Screening for CH has enabled the virtual eradication of the devastating effects of mental retardation due to sporadic CH in most developed countries of the world. The survival of increasingly small and premature fetuses has resulted in a growing number of neonates with abnormalities in thyroid function and a continuing controversy as to which of these infants require therapy.

 

Non endemic CH is one of the commonest treatable causes of mental retardation. The importance of early treatment in diminishing the neuro-psychological abnormalities of CH was demonstrated convincingly in the 1970’s. The development of a sensitive and specific radioimmunoassay for the measurement of T4 in dried whole blood and later tests for T4 and TSH using 1/8″ discs provided the technical means to screen all newborns for CH prior to the development of clinical manifestations. Thus, CH includes all the characteristics of a disease for which screening is justified: 1) it is common (4-5 times more common than phenylketonuria for which screening programs were initially developed); 2) to prevent mental retardation, the diagnosis must be made early, preferably within the first few days of life; 3) at that age, clinical recognition is difficult if not impossible; 4) sensitive, specific screening tests are available; 5) simple, cheap effective treatment is available; and 6) the cost-benefit ratio is highly favorable. Newborn screening programs have been introduced throughout the industrialized nations and are under development in many other parts of the world. Although there continues to be some disagreement as to whether minor neuro-intellectual sequelae remain in the most severely affected infants, accumulating evidence suggests that a normal outcome is possible even in the latter group of babies as long as treatment is started sufficiently early and is adequate.

 

National screening programs are well organized in many developed countries. However, it must be emphasized that approximately 71% of babies worldwide are not born in an area with an established national screening program for CH. The economic burden of disability owing to CH is still a significant public health challenge.

 

CLINICAL RECOGNITION

 

Clinical findings of CH are usually difficult to appreciate in the newborn period except in the unusual situation of combined maternal-fetal hypothyroidism. Many of the classic features (large tongue, hoarse cry, facial puffiness, umbilical hernia, hypotonia, mottling, cold hands and feet and lethargy), when present, are subtle and develop only with the passage of time. In addition to the aforementioned findings, nonspecific signs that suggest the diagnosis of neonatal hypothyroidism include: prolonged, unconjugated hyperbilirubinemia, gestation longer than 42 weeks, feeding difficulties, delayed passage of stools, hypothermia, or respiratory distress in an infant weighing over 2.5 kg. A large anterior fontanelle and/or a posterior fontanelle > 0.5 cm is frequently present in affected infants but may not be appreciated. In general, the extent of the clinical findings depends on the cause, severity, and duration of hypothyroidism. Babies in whom severe feto-maternal hypothyroidism was present in utero tend to be the most symptomatic at birth. Similarly, babies with athyreosis or a complete block in thyroid hormonogenesis tend to have more signs and symptoms at birth than infants with an ectopic thyroid, the most common cause of CH. Unlike acquired hypothyroidism, babies with CH are of normal size. However, if diagnosis is delayed, subsequent linear growth is impaired. The finding of palpable thyroid tissue suggests that the hypothyroidism is due to an abnormality in thyroid hormonogenesis or in thyroid hormone action.

 

Bone maturation reflects the duration and the severity of hypothyroidism. Signs of delayed epiphyseal maturation on knee x-rays, persistence of the posterior fontanelle, a large anterior fontanelle, and a wide sagittal suture all reflect delayed bone maturation. The absence of one or both knee epiphyses has been shown to be related to T4 concentration at diagnosis and to IQ outcome, and is thus a reliable index of intrauterine hypothyroidism.

 

PATHOPHYSIOLOGY

 

For a detailed discussion of the cause of CH and hypothyroidism in infants see the chapter in Endotext entitled Disorders of the Thyroid Gland in Infancy, Childhood and Adolescence by Segni in the Thyroid section.

 

Permanent Primary Congenital Hypothyroidism

 

Permanent primary CH can be the consequence of a disorder in thyroid development and/or migration (thyroid dysgenesis), or due to defects at every step-in thyroid hormone synthesis (thyroid dyshormonogenesis). Although CH is in the great majority of cases a sporadic disease, the recent guidelines for CH recommend genetic counseling in targeted cases. Positive family history for CH, association with cardiac or kidney malformation, midline malformations, deafness, neurological sigs (i.e., choreoathetosis, hypotonia), any sign of Albright hereditary osteodystrophy, lung disorders, suggest genetic counseling, in order to assess the risk of recurrence and to provide further information about a possible genetic etiology of CH. Genetic causes of CH are described in table 1.

 

TABLE 1. GENETIC CAUSES OF CONGENITAL HYPOTHYROIDISM

 

Gene locus

Inheritance

PRIMARY HYPOTHYROIDISM

 

 

Monogenic forms of thyroid dysgenesis

 

 

Thyroid stimulating hormone receptor (TSHR)

 

AR

NK2 1 (NK2-1, TTF1) brain-lung thyroid syndrome

14q13

AD

Paired box gene 8 (PAX8)

2q11.2

AD

Forkhead boxE1 (FOXE1, TTF2) (Bambforth-Lazarus syndrome)

9q22

AR

NK2 homeobox 5 (NKX2-5)

 

 

New candidate genes

 

 

Nertrin 1 (NTN-1)

 

 

JAG1

20p.12.2

 

Glis3

9p24.2

AR

Inborn errors of thyroid hormonogenesis

 

 

Sodium/Iodide symporter (SLC5A5, NIS)

19p13.2

AR

Thyroid peroxidase (TPO)

2p25

AR

Pendred syndrome (SLC26A4, PDS)

7q31

AR

Thyroglobulin (TG)

8q24

AR

Iodothyrosine deiodinase (IYD, DEHAL1)

6q24-25

AR

Dual oxidase 2 (DUOX2)

15q15.3

AR/AD

Dual oxidase maturation factor 2 (DUOXA2)

 

AR/AD

CENTRAL HYPOTHYROIDISM

 

 

Isolated TSH deficiency

 

 

TRHR

14q31

AR

TSHB

1p13

AR

Isolated TSH deficiency or combined pituitary hormone deficiency

 

 

Immunoglobulin superfamily member1 (IGSF1) gene defects

Xq26.1

X-Linked

Combined pituitary hormone deficiency

 

 

POU1F1

3p11

AR, AD

PROP1

5q

AR

HESX1

3p21.2-21.2

AR/AD

LHX3

9q.34

AR

LHX4

1q25

AD

SOX3

 

X-linked

OTX2

 

AD

 

Thyroid Dysgenesis

 

The majority (85 to 90%) of cases of permanent CH in North America, Western Europe, and Japan are due to an abnormality of thyroid gland development (thyroid dysgenesis). Thyroid dysgenesis may result in the complete absence of thyroid tissue (agenesis, 20-30%) owing to a defect in survival of the thyroid follicular cells precursors) or it may be partial (hypoplasia); the latter often is accompanied by a failure to descend into the neck (ectopy) mostly located in a sublingual position as a result of a premature arrest of its migratory process. Lowering of cut off TSH values for newborn screening increases the percentage of CH with thyroid in situ. Females are affected twice as often as males. In the United States, thyroid dysgenesis, is less frequent among African Americans and more common among Hispanics and Asians. Babies with CH have an increased incidence of cardiac anomalies, particularly atrial and ventricular septal defects. An increased prevalence of renal and urinary tract anomalies has also been reported. Most cases of thyroid dysgenesis are sporadic. Familial cases represent approximately 2% of cases.

 

Genetic causes of congenital hypothyroidism are described in table 1. Thyroid transcription factors (TTF) such as NKX2-1 (or formerly TTF1/TITF1), FOXE1 (Forkhread Box E1, formerly TTF2/TITF2), PAX8 (Paired box gene 8), and NKX2-5, are expressed during early phases of thyroid organogenesis (budding and migration), and thyroid stimulating hormone receptor gene (TSHR) is expressed during the later phases of thyroid development. All these genes are involved in normal thyroid development and in thyroid dysgenesis, however, abnormalities in these genes have been found in only a small proportion of affected patients, usually in association with other developmental abnormalities. Alternately, epigenetic modifications, early somatic mutations, or stochastic developmental events may play a role. Five monogenic forms due to mutations in TSHR, NXK2-1, PAX8, FOXE-1. NXK2-5 have been reported. Monogenic forms represent less than 10% in thyroid dysgenesis. Inactivating TSHR mutations are the most frequent cause of monogenic thyroid dysgenesis and non-syndromic CH, with prevalence in CH cohorts around 4 %.

 

Inborn Errors of Thyroid Hormonogenesis

 

Inborn errors of thyroid hormonogenesis (thyroid dyshormonogenesis) are responsible for most of the remaining cases (15%) of neonatal thyroidal hypothyroidism. Unlike thyroid dysgenesis, most are sporadic condition. These inborn errors of thyroid hormonogenesis are commonly associated with an autosomal recessive form of inheritance, consistent with a single gene abnormality. DUOX2 mutations can be transmitted in autosomal dominant way. Thyroid dyshormonogenesis is caused by genetic defects in proteins involved in all steps of thyroid hormone synthesis and often associated with goiter formation. Goiter can be present in utero or at birth. .A number of different defects have been characterized based on radioiodine uptake and perchlorate test and include: 1) Iodide transport defect that shows failure to concentrate iodide, with low or absent radioiodine uptake; 2) Iodide organification defects due to thyroid peroxidase mutations (TPO), Dual Oxidase 2 (DUOX2), Dual Oxidase Maturation Factor 2 mutations (DUOX2A), SLC26A4, and pendrin defects that have normal radioiodine uptake and altered perchlorate discharge test; and 3) Forms with normal radioiodine uptake and a normal perchlorate test due to thyroglobulin TG mutations, iodide recycling defects, and iodothyrosine deiodinase mutations.

 

Pendred Syndrome

 

Pendred syndrome is defined by the association of familial profound deafness with multinodular goiter. It is caused by biallelic mutation in the pendrin gene. Pendred syndrome is the only form of thyroid dyshormonogenesis associated with a malformation. The inner ear presents a characteristic malformation of the cochlea. Congenital hypothyroidism is present in only 30% of cases, goiter occurs often in childhood. Perchlorate test shows a partial organification defect. Pendred syndrome is the most frequent etiology of familial deafness.

 

Central Congenital Hypothyroidism (CCH)

 

CCH is caused by an insufficient thyroid hormone biosynthesis due to a defective stimulation by TSH, in the presence of an otherwise normal thyroid. This condition includes all causes of CH due to a pituitary or hypothalamic pathology (secondary or tertiary hypothyroidism). CCH was previously considered a very rare disease with a prevalence initially estimated to be 1:100,000 in newborns. In more recent data, CCH had an incidence that could reach 1:16,000, as shown from results from screening for CH in the Netherlands.

 

CCH is sometime not identified at birth, because the limiting step is “how low is a low T4”, low enough to be considered an effective cutoff value and allow the determination of TSH and TBG. Many cases are diagnosed in infancy or childhood, if not later in adulthood. The majority of screening programs are based on TSH determination and a high index of suspicion is needed to identify CCH in the preclinical phase. Delayed diagnosis may result in neurodevelopment delay. More than 50% of children with CCH have moderate or severe hypothyroidism, so, if not treated, the risk of neurodevelopmental delay should not be underestimated.

 

In the majority of cases identified early, TSH deficiency is a part of combined pituitary hormone deficiency. A timely correction of ACTH and cortisol deficiency, and/or GH deficiency may avoid life threatening emergencies. CCH can be transient (mostly due to drugs or maternal hyperthyroidism), or permanent. Genetic causes are listed in Table 1.

 

Defects of Thyroid Hormone Transport in Serum

 

For complete coverage of this and related areas visit the chapter entitled “Defects of thyroid

hormone transport in serum” in the thyroid section of Endotext by Samuel Refetoff. Inherited abnormalities of the iodothyronine-binding serum proteins include TBG deficiency (partial or complete), TBG excess, transrethyretin (TTR) (prealbumin) variants, and familial

dysalbuminemic hyperthyroxinemia (FDH). In these conditions the concentration of free hormones is unaltered, but the abnormal total thyroxine concentrations can be misleading during neonatal screening and in the evaluation of thyroid function.

 

Impaired Sensitivity to Thyroid Hormone

 

For complete coverage of this and related areas visit the chapter entitled: “Impaired sensitivity to thyroid hormone: defects of transport, metabolism and action” in the thyroid section of Endotext by Alexandra M. Dumitrescu and Samuel Refetoff. Impaired sensitivity to thyroid hormone includes defects in thyroid hormone action, transport, and metabolism. They are classified as a) thyroid hormone cell membrane transport defects, b) thyroid hormone metabolism defect, and c) thyroid hormone action defect that include resistance to thyroid hormone.

 

Causes of Transient Neonatal Hypothyroidism

 

Transient neonatal hypothyroidism should be distinguished from a ‘false positive’ result in which the screening result is abnormal but the confirmatory serum sample is normal. Causes of transient abnormalities of thyroid function in the newborn period are listed in Table 2. While iodine deficiency, iodine excess, drugs and maternal TSH receptor blocking antibodies are the most common causes of transient hypothyroidism, in some cases the cause is unknown.

 

TABLE 2.  CAUSES OF TRANSIENT HYPOTHYROIDISM IN THE NEWBORN

PRIMARY HYPOTHYROIDISM

Prenatal or postnatal iodine deficiency or excess

Maternal antithyroid medication

Maternal TSH receptor blocking antibodies

Mild gene mutations (i.e. DUOX2, TSH-R)

Maternal hypothyroidism

Prematurity, VLBW

Drugs, (i.e. Dopamine, steroids)

Hypothyroxinemia (low T4, normal TSH)

CENTRAL HYPOTHYROIDISM

Prenatal exposure to maternal hyperthyroidism

Prematurity (particularly <27 weeks gestation)

Drugs

 

Iodine Deficiency or Excess

 

In addition to iodine deficiency, both the fetus and newborn infant are sensitive to the thyroid- suppressive effects of excess iodine, whether administered to the mother during pregnancy, lactation, or directly to the baby. This occurs because recovery from the thyroid-suppressive effect of iodine does not mature before 36 weeks gestation. Reported sources of iodine include drugs (e.g., potassium iodide, amiodarone), radiocontrast agents, and antiseptic solutions (e.g., povidone-iodine) used for skin cleansing or vaginal douches. In contrast to Europe, iodine-induced transient hypothyroidism has not been documented frequently in North America.

 

Maternal Antithyroid Medication

 

Transient neonatal hypothyroidism may develop in babies whose mothers are being treated with antithyroid medication (either propylthiouracil, PTU or methimazole) for the treatment of Graves’ disease. Even maternal PTU doses of 200 mg or less have been associated with an effect on neonatal thyroid function, illustrating the increased fetal sensitivity to these drugs. Babies with PTU- or methimazole-induced hypothyroidism characteristically develop an enlarged thyroid gland and if the dose is sufficiently large, respiratory embarrassment may occur. Both the hypothyroidism and goiter resolve spontaneously with clearance of the drug from the baby’s circulation. Usually replacement therapy is not required.

 

Maternal TSH Receptor Antibodies

 

Maternal TSH receptor blocking antibodies, a population of antibodies closely related to the TSH receptor stimulating antibodies in Graves’ disease, may be transmitted to the fetus in sufficient titer to cause transient neonatal hypothyroidism. The incidence of this disorder has been estimated to be 1 in 180,000. TSH receptor blocking antibodies most often are found in mothers who have been treated previously for Graves’ disease or who have the non-goitrous form of chronic lymphocytic thyroiditis (primary myxedema). Occasionally these mothers are not aware that they are hypothyroid and the diagnosis is made in them only after CH has been recognized in their infants. Unlike TSH receptor stimulating antibodies that mimic the action of TSH, TSH receptor blocking antibodies inhibit both the binding and action of TSH. Because TSH-induced growth is blocked, these babies do not have a goiter. Similarly, inhibition of TSH-induced radioactive iodine uptake may result in a misdiagnosis of thyroid agenesis. Usually the hypothyroidism resolves in 3 or 4 months. Babies with TSH receptor blocking-antibody induced hypothyroidism are difficult to distinguish at birth from the more common thyroid dysgenesis but they differ from the latter in a number of important ways (Table 3). They do not require lifelong therapy, and there is a high recurrence rate in subsequent offspring due to the tendency of these antibodies to persist for many years in the maternal circulation. Unlike babies with thyroid dysgenesis in whom a normal cognitive outcome is found if postnatal therapy is early and adequate, babies with maternal blocking-antibody induced hypothyroidism may have a permanent deficit in intellectual development if feto-maternal hypothyroidism was present in utero.

 

TABLE 3. CLINICAL FEATURES OF THYROID DYSGENESIS VERSUS TSH RECEPTOR BLOCKING ANTIBODY INDUCED CONGENITAL HYPOTHYROIDISM

 

Dysgenesis

Blocking Ab

Severity of CH

+ to ++++

+ to ++++

Palpable thyroid

No

No

123I uptake

None to low

None to normal

Clinical Course

Permanent

Transient

Familial risk

No

Yes

TPO Abs

Variable

Variable

TSH Receptor Abs

Absent

Potent

 

Transient Central Hypothyroidism Due to Maternal Hyperthyroidism

 

Occasionally, babies born to mothers who were hyperthyroid during pregnancy develop transient hypothalamic-pituitary suppression. This hypothyroxinemia is usually self-limited, but in some cases, it may last for years and require replacement therapy. In general, the titer of TSH receptor stimulating antibodies in this population of infants is lower than in those who develop transient neonatal hyperthyroidism.

 

Prematurity

 

Hypothyroxinemia in the presence of a ‘normal’ TSH occurs most commonly in premature infants in whom it is found in 50% of babies of less than 30 weeks gestation. Often the free T4 when measured by equilibrium dialysis is less affected than the total T4. The reasons for the hypothyroxinemia of prematurity are complex. As well as hypothalamic-pituitary immaturity, premature infants frequently have TBG deficiency due to both immature liver function and undernutrition, and they may have “sick euthyroid syndrome”. They may also be treated with drugs that suppress the hypothalamic-pituitary-thyroid axis. Hypothyroxinemia of prematurity may be associated with adverse neurodevelopmental outcomes.  L-T4 treatment overall has no proven benefit and can be harmful.  Long term outcome evaluation in young adults did not find an association between transient hypothyroxinemia of prematurity and neurodevelopmental outcome. Whether or not premature infants with hypothyroxinemia should be treated remains controversial at the present time. Although several retrospective, cohort studies have documented a relationship between severe hypothyroxinemia and both developmental delay and disabling cerebral palsy in preterm infants <32 weeks gestation a causal relationship could not be determined since the serum T4 in premature infants, as in adults, has been shown to reflect the severity of illness and risk of death.

 

Drugs

 

Drugs that suppress the hypothalamic-pituitary axis include known agents such as steroids and dopamine, but also aminophylline, caffeine and diamorphine, which are commonly used in sick premature infants.

 

Other Causes of Hypothyroidism in Infancy

 

Chronic lymphocytic thyroiditis

 

Chronic lymphocytic thyroiditis (CLT) is a rare disease in infancy, but if not recognized and treated, can cause severe hypothyroidism with permanent brain damage. CLT can be associated with other autoimmune disease such as type 1 diabetes or as a manifestation of the IPEX syndrome. Clinical manifestations and biochemical hypothyroidism (TSH ranged from >42 to 928 mU/L) were severe and very high levels of antibodies were detectable.

 

 

Lymphocytic thyroiditis has also been described in newborns with severe defects in tolerance and autoimmunity with immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, a polyglandular disorder characterized by early-onset diabetes and colitis. IPEX disorders are an expanding spectrum of disease with mutations in FOXP3, CD25 deficiency, STAT5 deficiency, and others.

 

Hepatic hemangiomas: consumptive hypothyroidism

 

Hepatic hemangioendothelioma is a rare tumor typically presenting in infancy. Hypothyroidism is caused by a production of type 3 deiodinase by the vascular tumor. D3 deiodinase increases inactivation of T4 and T3 to reverse T3 and T2 and a large amount of LT4 (up to 94/ µg/kg/day) is needed to compensate for this inactivation. Frequent monitoring is required, adapting the LT4 treatment to the growing proliferative phase of the tumor. Today hemangioendotheliomas in infancy may successfully being treated with steroids and propranolol and may undergo spontaneous regression. Some babies underwent liver transplantation.

 

SCREENING STRATEGIES

 

The aim of neonatal screening is the earliest identification of any form of CH, but particularly those patients with severe hypothyroidism in whom disability is greatest if not treated. The identification of Central CH by screening programs is under debate. Two screening strategies for the detection of CH have evolved. In the primary T4/backup TSH method, still favored in much of North America and the Netherlands, T4 is measured initially while TSH is checked on the same blood spot in those specimens in which the T4 concentration is low. In the primary TSH approach, favored in most parts of Europe and Japan, blood TSH is measured initially.

 

A primary T4/backup TSH program will detect overt primary hypothyroidism, secondary or tertiary hypothyroidism, babies with a low serum T4 level but delayed rise in the TSH concentration, TBG deficiency and hypothyroxinemia; this approach may, however, miss subclinical hypothyroidism.  A primary TSH strategy, on the other hand, will detect both overt and subclinical hypothyroidism, but will miss secondary or tertiary hypothyroidism, a delayed TSH rise, TBG deficiency and hypothyroxinemia. There are fewer false positives with a primary TSH strategy. Both programs will miss the rare infant whose T4 level on initial screening is normal but who later develops low T4 and elevated TSH concentrations. This pattern has been termed “atypical” CH or “delayed TSH” and is observed most commonly in premature babies with transient hypothyroidism or infants with less severe forms of permanent disease.

 

According to the European Society for Pediatric Endocrinology (ESPE) guidelines, the most sensitive test for detecting primary CH is the determination of TSH concentration that detects primary CH more effectively than primary T4 screening  Primary T4 screening with confirmatory TSH testing can detect some cases of central CH, but some cases of mild CH can be missed, depending on the cutoff T4 value used.

 

Measurement of T4 and/or TSH is performed on an eluate of dried whole blood (DBS) collected on filter paper by skin puncture on day 1-4 of life. Primary CH screening has been shown to be effective for the testing of cord blood or the blood collected on filter paper after the age of 24 hours. Blood is applied directly to the filter paper and after drying the card is sent to the laboratory. The best time to collect blood for TSH screening is 48 to 72 hours of age. The practice of early discharge from the hospital of otherwise healthy full-term infants has resulted in a greater proportion of babies being tested before this time. For example, it has been estimated that in North America 25% or more of newborns are now discharged within 24 hours of delivery and 40% in the second 24 hours of life. Because of the neonatal TSH surge and the dynamic changes in serum T4 and T3 concentrations that occur within the first few days of life, early discharge increases the number of false positive results. It is important that in the screening laboratory the results of TSH are interpreted in relation to time of sampling.

 

Physicians caring for infants need to appreciate that there is always the possibility for human error in failing to identify affected infants, whichever screening program is utilized. This can occur due to poor communication, lack of receipt of requested specimens, or the failure to test an infant who is transferred between hospitals during the neonatal period. Therefore, if the diagnosis of hypothyroidism is suspected clinically, the infant should always be tested. Adult normative values, provided by many general hospital laboratories, differ from those in the newborn period and should never be employed.

 

Special categories of neonates with CH can be missed at screening performed at the usual time, particularly preterm babies and neonates with serious illnesses and multiple births.  Drugs used in neonatal intensive care (i.e., dopamine, glucocorticoids that suppresses TSH), immaturity of hypothalamic-pituitary thyroid axis, decreased hepatic production of thyroid binding globulin, reduced transfer of maternal T4, reduced intake of iodine or excess iodine exposure, fetal blood mixing in multiple births can affect the first sample, and in many centers a second specimen is required to rule out CH. Preterm babies have a higher incidence of a unique form of hypothyroidism, characterized by a delayed elevation of TSH. These babies can later develop low T4 and elevated TSH concentrations. This pattern has been termed “atypical” CH or “delayed TSH”. Preterm babies with a birth weight of less than 1500 gr. have an incidence of CH of 1:300. Survival of even extremely premature babies (<28 weeks of gestation) is around 90% in developed countries, and the incidence of prematurity is around 11.5 % in US and 11.8 % worldwide. So, an increasing subpopulation of preterm babies and high-risk newborns deserves a special screening and follow up for CH.

 

In these categories a second specimen 2-6 weeks from the first (ESPE guidelines suggested at about 15 days, or after 15 days from the first) may be indicated in a) preterm neonates with a gestational age of less than 37 weeks, b) Low Birth Weight and Very Low Birth Weight neonates, c) ill and preterm neonates admitted to neonatal intensive care unit, d) if specimen collection was within the first 24 hours of life, and e) multiple births, particularly in the case of same sex twins. The interpretation of the screening results should consider the results of a multiple sampling strategy, the age of sampling, and the maturity (GA/birth weight) of the neonate. A second screen (using a lower TSH cutoff) is able to detect the delayed elevation of TSH that occurs in these babies.

 

CH is defined on the basis of serum FT4 levels as severe when FT4 is <5 pmol/l, moderate when FT4 is 5 to 10 pmol/l, and mild when FT4 is 10 to 15 pmol/l, respectively. Determination of serum thyroglobulin (Tg) is useful, if below the detection threshold, to suggest athyreosis or a complete thyroglobulin synthesis defect. Measurement of Tg is most helpful when a defect in Tg synthesis or secretion is being considered. In the latter condition the serum Tg concentration is low or undetectable despite the presence of a normal or enlarged, eutopic thyroid gland. Serum Tg concentration also reflects the amount of thyroid tissue present and the degree of stimulation. For example, Tg is undetectable in most patients with thyroid agenesis, intermediate in babies with an ectopic thyroid gland, and may be elevated in patients with abnormalities of thyroid hormonogenesis not involving Tg synthesis and secretion. Considerable overlap exists, and so, the Tg value needs to be considered in association with the findings on imaging. In patients with inactivating mutations of the TSH receptor discordance between findings on thyroid imaging and the serum Tg concentration has been described in some but not all studies.

 

IMAGING TECHNIQUES IN CH

 

Imaging studies are helpful to determine the specific etiology of CH. Both scintigraphy and ultrasound (US) should be considered in neonates with high TSH concentrations. Ideally, the association of US and scintigraphy gives the best information in a child with primary hypothyroidism. Scintigraphy shows the presence/absence (athyreosis), position (ectopic gland, in any point from the foramen caecum at the base of the tongue to the anterior mediastinum) and rough anatomic structure of the thyroid gland. US, is a useful tool in defining size and morphology of a eutopic thyroid gland, however, US alone is less effective in detecting ectopic glands. Color Doppler US improves the effectiveness of US. It is important to remember that an attempt to obtain imaging in a newborn should never delay the initiation of treatment. Scintigraphy should be carried out within 7 days of starting LT4 treatment. Scintigraphy may be carried out with either 10-20 MBq of technetium 99m (99mTc) or 1-2 MBq of iodine123 (I123). Tc is more widely available, less expensive, and quicker to use than I123. Scintigraphy with I123, if available, is usually preferred because of the greater sensitivity and because, I123, unlike of technetium99, is organified. Therefore, imaging with this isotope allows quantitative uptake measurements and tests for both iodine transport defects and abnormalities in thyroid oxidation. An enrichment of the tracer within the salivary gland can lead to misinterpretation, especially on lateral views, but this can be avoided by allowing the infant to feed before scintigraphy, thus empting the salivary glands and keeping the child calm under the camera. The perchlorate discharge test is considered indicative of an organification defect when a discharge of > 10% of the administered I123 dose occurs in a thyroid in normal position (when perchlorate is given at 2 hours).

 

Excess iodine intake through exposure, maternal TSH receptor blocking antibodies, inactivating mutation in the TSH receptor and in the sodium/iodide symporter (NIS), and TSH suppression from LT4 treatment can interfere with the I123 uptake, showing no uptake in the presence of a thyroid in situ (apparent athyreosis).

 

Thyroid ultrasonography is performed with a high frequency linear array transducer (10-15 MHz) and allows a resolution of 0.7 to 1mm. Thyroid tissue is more echogenic than muscle and less echogenic than fat. In the case of absence of the thyroid, fat tissue can be misdiagnosed as dysplastic thyroid gland in situ. Distinguishing between thyroid hypoplasia and dysplastic non-thyroidal tissue in a newborn requires an experience and reevaluation at a later age can result in a different diagnosis.

 

Combining scintigraphy and thyroid ultrasound improves diagnostic accuracy and helps to address further investigations, including molecular genetic studies. Infants found to have a normal sized gland in situ in the absence of a clear diagnosis should undergo further reassessment of the thyroid axis and imaging at a later age.

 

THERAPY

 

Timing of normalization of thyroid hormones is critical for brain development and therefore replacement therapy with L-thyroxine (L-T4) should be begun as soon as the diagnosis of CH is confirmed. Treatment should be started immediately if DBS TSH concentration is >40 mUI/l because this value strongly suggests decompensated hypothyroidism. If TSH is < 40 mUI/l the clinician may postpone treatment, pending the serum results, for 1-2 days. ESPE guidelines suggest treatment should be started if venous TSH concentration is persistently >20 mUI/l, even if serum FT4 is normal. Severe hypothyroidism is defined by T4 <5 mcg/dL (64 nmol/L) and/or TSH >40 mU. According to ESPE guidelines, CH is defined on the basis of serum FT4 levels as severe when FT4 is <5 pmol/l, moderate when FT4 is 5 to 10 pmol/l, and mild when FT4 is 10 to 15 pmol/l. As noted above, treatment need not be delayed in anticipation of performing thyroid imaging studies as long as the latter are done within 5-7 days of initiating treatment (before suppression of the serum TSH). Parents should be counseled regarding the causes of CH, the importance of compliance, and the excellent prognosis in most babies if therapy is initiated sufficiently early and is adequate. Educational materials should be provided. An initial dosage of 10-15 mcg/kg/day of L-T4 is generally recommended to normalize the T4 as soon as possible. The highest dose is indicated in infants with severe disease, and the lower dose in those with a mild to moderate CH. L-T4 tablets can be crushed and given via a small spoon, with suspension, if necessary in a few milliliters of water or breast milk or formula or juice, but care should be taken that all of the medicine has been swallowed. Thyroid hormone should not be given with substances that interfere with its absorption, such as iron, calcium, soy, or fiber. Drugs such as antacids (aluminum hydroxide) or infantile colic drops (simethicone) can interfere with L-thyroxine absorption. Many babies will swallow the pills whole or will chew the tablets with their gums even before they have teeth. Reliable liquid preparations are not available commercially in the US, although they have been used successfully in Europe. A brand name rather a generic formulation of L-T4 is recommended because they are not bioequivalent.

 

It is still a matter of debate if treatment can be beneficial in otherwise healthy babies with venous TSH concentration between 6-20 mUI/l and FT4 concentration within the normal limits for age. In these cases, diagnostic imaging is recommended to try to establish a definitive diagnosis. If TSH concentration remains high for more than 3 or 4 weeks, it is possible (in discussion with the family) to either start LT4 supplementation immediately and then retest off treatment at a later stage or retest two weeks later without treatment. Given the irreversibility of possible harm, treating during early childhood and revaluating thyroid function after myelination of the central nervous system is completed (by 36 to 40 months of age) can be a prudent approach. LT4 treatment must be started immediately if FT4 or TT4 levels are low, given the known adverse effect of untreated decompensated CH on neurodevelopment and somatic growth.

 

The aims of therapy are to normalize the T4 as soon as possible, to avoid hyperthyroidism where possible, and to promote normal growth and development. When an initial dosage of 10-15 mcg/kg is used, the T4 will normalize in most infants within 1 week and the TSH will normalize within 1-month. Subsequent adjustments in the dosage of medication are made according to the results of thyroid function tests and the clinical picture. Often small increments or decrements of L-thyroxine (12.5 mcg) are needed. This can be accomplished by 1/2 tablet changes, by giving an alternating dosage on subsequent days, or by giving an extra tablet once a week.

 

As stated in ESPE guidelines: “L-T4 alone is recommended as the medication of choice and should be started as soon as possible, no later than two weeks of life or immediately after confirmatory test results in infants identified in a second routine screening test. L-T4 should be given orally. If intravenous administration is necessary, the dose should be no more than 80% of the oral dose”. Serum or plasma FT4 (or TT4) and TSH concentration should be determined at least 4 hours after the last L-T4 administration. TSH should be maintained in the age-specific reference range and FT4 in the upper half of the age- specific reference range. “The first follow up examination is indicated after 1-2 weeks after the start of LT4 treatment and then every 2 weeks until TSH levels are completely normalized and then every 1- 3 months until 12 months of age. Between the age of one and three years, children should undergo frequent clinical and laboratory evaluations (every 2 to 4 months).” Thereafter, evaluations should be carried out every 3 to 12 months until growth is completed. “More frequent evaluations should be carried out if compliance is questioned or abnormal values are obtained. Any reduction of L-T4 dose should not be based on a single increase of FT4 concentration during treatment. “Measurements should be performed after 4-6 weeks any change in the dosage or in the L-T4 formulation”.

 

RE-EVALUATION AND TRIAL OFF THERAPY

 

In hypothyroid babies in whom an organic basis was not established at birth and in whom transient disease is suspected, a trial off replacement therapy can be initiated after the age of 3 years when most thyroxine-dependent brain maturation has occurred, as shown by MRI studies. Re-evaluation is recommended if the treatment was started in a sick child (i.e. preterm), if thyroid antibodies were detectable, if no diagnostic assessment was completed, and in children who have required no increase in L-T4 dosage since infancy. Re-evaluation is recommended also in the case of a eutopic gland with or without goiter, if no enzyme defects have been detected, or if any other cause of transient hypothyroidism is suspected.

 

Re-evaluation is not necessary if venous TSH concentration has risen during the first year of life, due to either LT4 underdosage or poor compliance. To perform a precise diagnosis, LT4 treatment is suspended for 4-6 weeks, and biochemical testing and thyroid imaging are carried out. To establish the presence of primary hypothyroidism, without defining the cause, L-T4 dose may be decreased by 20-30% for 2 to 3 weeks. If TSH serum levels rise to > 10 mU/L during this period, the hypothyroidism can be confirmed.

 

PROGNOSIS

 

Although all agree that the mental retardation associated with untreated CH has been largely eradicated by newborn screening, controversy persists as to whether subtle cognitive and behavioral deficits remain, particularly in the most severely affected infants. Both the initial treatment dose and early onset of treatment (before 2 weeks) are important. Time to normalization of circulating thyroid hormone levels, the initial free T4 concentration, maternal IQ, socioeconomic status, and ethnic status have also been related to outcome. The long-term problems for these babies appear to be in the areas of memory, language, fine motor, attention, and visual spatial. Inattentiveness can occur both in patients who are overtreated and those in whom treatment was initiated late or was inadequate. In addition to adequate dosage, assurance of compliance and careful long-term monitoring are essential for an optimal developmental outcome. More details about long term follow up are reported in ESPE guidelines. Progressive hearing loss in CH should be recognized and corrected, because they strongly influenced the outcome.

 

ACKNOWLEDGEMENTS

 

This chapter is, in part, based on the previous version written by Prof. Rosalind Brown.

 

GUIDELINES

 

Lazarus JH, Mandel SJ, Peeters RP, Sullivan S. 2017 Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease During Pregnancy and the Postpartum. Thyroid. 2017 Mar;27(3):315-389.

 

Leger J, Olivieri A, Donaldson M, Torresani T, Krude H, van Vliet G, Polak M, Butler G on behalf of ESPE-PES-SLEP-JSPE-APEG-APPE-ISPAE, and the Congenital Hypothyroidism Consensus Conference Group. European Society for Pediatric Endocrinology Consensus Guidelines on Screening, Diagnosis, and management of congenital hypothyroidism. J Clin Endocrinol Metab. 2014; 99:363-384.

 

REFERENCES

 

Segni M. Disorders of the Thyroid Gland in Infancy, Childhood and Adolescence. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2017 Mar 18

 

Dumitrescu AM, Refetoff S. Impaired Sensitivity to Thyroid Hormone: Defects of Transport, Metabolism and Action. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2015 Aug 20

 

Refetoff S. Abnormal Thyroid Hormone Transport. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2015 Jul 15

 

Bernal J. Thyroid Hormones in Brain Development and Function. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, Dungan K, Grossman A, Hershman JM, Kaltsas G, Koch C, Kopp P, Korbonits M, McLachlan R, Morley JE, New M, Perreault L, Purnell J, Rebar R, Singer F, Trence DL, Vinik A, Wilson DP, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2015 Sep 2.

 

Kaluarachchi DC, Allen DB, Eickhoff JC, Dawe SJ, Baker MW. Thyroid-Stimulating Hormone Reference Ranges for Preterm Infants. Pediatrics. 2019 Aug;144(2).

 

Ford G, LaFranchi SH. Screening for congenital hypothyroidism: a worldwide view of strategies. Best Practice & Research Clinical Endocrinology & Metabolism. 2014; 28:175-187.

 

Bauer AJ, Wassner AJ. Thyroid hormone therapy in congenital hypothyroidism and pediatric hypothyroidism. Endocrine. 2019 Jul 26. doi: 10.1007/s12020-019-02024-6. [Epub ahead of print]

 

 

 

 

Hypercalcemia

CLINICAL RECOGNITION

 

Hypercalcemia can be defined as a serum calcium greater than 2 standard deviations above the normal mean in a reference laboratory. Calcium in the blood is normally transported:

partly bound to plasma proteins (about 45%), notably to albumin; partly bound to small anions such as phosphate and citrate (about 10%); partly in the free or ionized state (about 45%).

 

Only the ionized calcium is metabolically active i.e. subject to transport into cells, but most laboratories report total serum calcium concentrations. Hypercalcemia is therefore often defined as a total serum calcium (bound plus ionized) greater than 10.6 mg/dl (2.65 mM) or an ionized serum calcium greater than 5.3 mg/dl (1.3 mM) but values may vary between laboratories.

Dehydration, or hemoconcentration during venipuncture, may elevate total serum albumin whereas ionized calcium may remain normal. Consequently, a falsely elevated total serum calcium may be reported. Conversely when serum albumin levels are low, total serum calcium may be falsely low. To correct for an abnormally high or low serum albumin the following formula can be used:

Corrected calcium (mg/dL) = measured total serum calcium (mg/dL) + [4.0-serum albumin (g/dL) X 0.8] or Corrected calcium (mM) = measured total serum Ca (mM) + [40 - serum albumin (g/L) X 0.02]

 

Changes in blood pH can also alter the equilibrium constant of the albumin-calcium complex: Acidosis reduces binding and alkalosis enhances binding. Consequently, when major shifts in serum protein or pH are present it is prudent to directly measure the ionized calcium level in order to determine the presence of hypercalcemia.

 

Clinical Manifestations may be due to hypercalcemia or may be due to the causal disorder or may be due to both. Hypercalcemic manifestations will vary depending on whether the hypercalcemia is of acute onset and severe (greater than 12 mg/dL or 3 mM) or whether it is chronic and relatively mild. Patients may also tolerate higher serum calcium levels more readily if the onset is relatively gradual, but at concentrations above 14 mg/dL (3.5 mM) most patients are symptomatic. In both acute and chronic cases, the major manifestations affect gastrointestinal, renal and neuromuscular function (Table 1).

 

Table 1. Manifestations of Hypercalcemia

 

Acute

Chronic

Gastrointestinal

Anorexia, nausea, vomiting

Dyspepsia, constipation, pancreatitis

Renal

Polyuria, polydipsia

Nephrolithiasis, nephrocalcinosis

Neuro-muscular

Depression, confusion,
stupor, coma

Weakness

Cardiac

Short Q-T interval
bradycardia, first degree
atrioventricular block,
digitalis sensitivity

Hypertension

 

PATHOPHYSIOLOGY

 

Fluxes of calcium across the skeleton, the gut, and the kidney play a major role in maintaining calcium homeostasis. When the extracellular fluid (ECF) calcium is raised above the normal range, the calcium ion per se, by stimulating the G-protein coupled calcium sensing receptor (CaSR), can inhibit parathyroid hormone (PTH) release. Decreased PTH and CaSR stimulation will both facilitate reduced renal calcium reabsorption, and decreased PTH will result in reduced bone resorption and diminished release of calcium from bone. Decreased PTH and hypercalcemia will also reduce renal production of the active form of vitamin D, 1,25-dihydroxyvitamin D [1,25(OH)2D], and decrease gut absorption of calcium. The net effect of the diminished renal calcium reabsorption, intestinal calcium absorption, and skeletal calcium resorption will be to reduce the elevated ECF calcium to normal. Consequently, decreased levels of PTH and decreased levels of 1,25(OH)2D should accompany hypercalcemia unless the PTH or 1,25(OH)2D is the cause of the hypercalcemia. The converse sequence of events occurs when the ECF calcium is reduced below the normal range.

 

A genetic relative of PTH, PTH-related peptide (PTHrP), can also resorb bone, when released from certain tumors. Both PTH and PTHrP act on osteoblastic cells to increase production of cytokines, notably receptor activator of nuclear factor kappa B ligand (RANKL) which increases production and activation of multinucleated osteoclasts which then resorb mineralized bone.

DIAGNOSIS AND DIFFERENTIAL (FIGURE 1)

Figure 1 Algorithm for Diagnosing the Cause of Hypercalcemia

 

Hypercalcemic disorders can be broadly grouped into Endocrine Disorders, Malignant Disorders, Inflammatory Disorders, Medication-Induced Hypercalcemia, and Immobilization as given in Tables 2-8. Primary hyperparathyroidism (HPTH) and malignancy-associated hypercalcemia (MAH) account for the vast majority of hypercalcemic disorders. (For a more complete discussion of hypercalcemic disorders and the underlying pathophysiology, see reference 1)

 

Table 2. Endocrine Disorders Associated with Hypercalcemia

1. Endocrine Disorders with Excess PTH Production

Primary Sporadic Hyperparathyroidism (HPTH)

Adenoma (85-95%)

Hyperplasia (10-15%)

Carcinoma (<1%)

(80% of primary hyperparathyroidism is “asymptomatic”)

Primary Familial HPTH (Syndromic HPTH)

Multiple Endocrine Neoplasia, Type I (MEN1)- Autosomal dominant, MEN1 mutation (encodes menin)

Multiple Endocrine Neoplasia, Type II (also called MENIIA)- Autosomal dominant, RET mutation (encodes c-Ret)

Multiple Endocrine Neoplasia, Type IV (MENIV)- Autosomal dominant, CDKN1B mutation (encodes P27(Kip1))

Hyperparathyroidism – Jaw Tumor Syndrome-

   Autosomal dominant, CDC73/HRPT2 mutation (encodes parafibromin)

Non-Syndromic HPTH

Familial Hypocalciuric Hypercalcemia (FHH)
Heterozygotes and Neonatal Severe Primary Hyperparathyroidism (NSHPT) (homozygotes)
   FHH1:  CaSR mutation (encodes calcium sensing receptor)

   FHH2: GNA11 mutation (encodes G protein subunit α11)

   FHH3: AP2S1 mutation (encodes adaptor protein-2 sigma subunit)

Familial Isolated HPTH(Non-Syndromic)

  Mutations inf MEN1, CDC73/HRPT2 or CASR may account for a minority of kindreds with the FIHP phenotype upon initial ascertainment. Activating variants in GCM2 (encodes the transcription factor GCM2) have also been described.  

Tertiary HPTH

Chronic Kidney Disease

Phosphate Treatment of Hypophosphatemic Rickets/Osteomalacia

2. Endocrine Disorders without Excess PTH Production

Hyperthyroidism

Pheochromocytoma

VIPoma

Hypoadrenalism

Jansen’s Metaphyseal Chondrodysplasia- Due to activating mutation of PTHR1, the gene encoding the type1 PTH/PTHrP receptor

 

 

Table 3. Malignancy-Associated Hypercalcemia (MAH)

Accounts for about 90% of hypercalcemia in hospitalized patients.
Hypercalcemia is often acute and severe and usually a late manifestation of malignancy

1. MAH with Elevated PTHrP

Solid tumors (e.g. breast, lung, kidney, GI)

Hematologic malignancies (e.g. Non-Hodgkin’s lymphoma, adult T cell leukemia/lymphoma, chronic myelogenous, leukemia, chronic lymphocytic leukemia)

2. MAH with Elevation of Other Systemic Factors

1,25(OH)2D (e.g. Hodgkin’s Disease), cytokines (Multiple Myeloma and malignancies metastatic to bone), and rarely ectopic PTH production (e.g. ovarian, lung, thyroid and thymus)

 

 

Table 4. Granulomatous Disorders Causing Hypercalcemia

Due to extra-renal mononuclear cell 1,25(OH)2D production

1 Non-infectious (e.g. Sarcoidosis, Wegener’s granulomatosis, berylliosis)

2 Infectious (e.g. TB, histoplasmosis)

 

Table 5. Pediatric Syndromes

1. Williams Syndrome

2. Idiopathic Infantile Hypercalcemia
Due to loss-of-function of CYP24A1, encoding CYP24A1, the enzyme metabolizing 1,25(OH)2D, or due to loss-of-function of SLC34A1, encoding the renal proximal tubular sodium-phosphate cotransporter, Na/Pi-IIa.

 

  Table 6. Viral Syndromes

1. Human Immunodeficiency Virus (HIV) infections

2. Cytomegalovirus (CMV) infections

 

 

Table 7. Medication-Induced

1. Thiazides

2. Lithium

3. Vitamin D

4. Vitamin A

5. Tamoxifen (during treatment of skeletal breast cancer metastases)

6. Aminophylline/theophylline

7. Aluminum Intoxication

8. Milk-Alkali Syndrome

 

Table 8. Immobilization

Immobilized patients continue to resorb bone whereas bone formation is inhibited. Consequently, immobilization may precipitate hypercalcemia and hypercalciuria in individuals with high bone turnover such as growing children, patients with Paget’s Disease or patients with primary HPTH or MAH.

 

DIAGNOSTIC TESTS NEEDED AND SUGGESTED

 

Laboratory testing should be guided by the results of a careful history and a detailed physical examination and should be geared toward assessing the extent of the alteration in calcium homeostasis and toward establishing the underlying diagnosis and determining its severity. Most patients with primary HPTH, the most common cause of hypercalcemia in the clinic, present with mild hypercalcemia discovered on a routine biochemical assessment. There may be a history of a recent or remote renal stone. Bone pain and fractures are rare although the patient may carry a diagnosis of osteoporosis based on a previous bone mineral density (BMD) measurement. A history of a documented peptic ulcer is rare in primary sporadic HPTH and should raise concern about MEN1. Although cardiovascular and neuropsychiatric manifestations have been described they appear to require more validation. Documentation of at least two elevated corrected (or ionized) serum calcium levels with concomitant elevated (or at least normal) serum PTH levels is required to establish the diagnosis (Figure 1). Lithium treatment has been associated with hypercalcemia, elevated or normal serum PTH, and increased renal calcium reabsorption. The presence of a family history of hypercalcemia or of kidney stones should raise suspicion of MEN1 or MEN2a (reference 3 and 4). If, in addition to primary HPTH in the proband, one or more first-degree relatives are found to have at least one of the three tumors characterizing MEN1 (parathyroid, pituitary, pancreas) or MEN2a (parathyroid, medullary thyroid carcinoma, pheochromocytoma) then it is highly likely that the disease is familial. The presence of ossifying fibromas of the mandible and maxilla, and renal lesions such as cysts and hamartomas in addition to HPTH would suggest HPTH-jaw tumor syndrome. In all patients with documented primary HPTH, a 24-hour urine calcium and creatinine level should be obtained to exclude familial hypocalciuric hypercalcemia (FHH). If the urine calcium to creatinine ratio is less than 0.01 and if testing serum and urine calcium in three relatives discloses hypercalcemia and relative hypocalciuria in other family members, then this diagnosis is likely and parathyroid surgery is to be avoided. If the urine calcium to creatinine ratio is greater than 0.01 then estimated glomerular filtration rate (eGFR) and a BMD test should be performed and guidelines for treatment of primary HPTH should be considered (see below).

 

Tertiary hyperparathyroidism with hypercalcemia and elevated PTH has been described in chronic kidney disease patients on hemodialysis, or in patients with hypophosphatemic syndromes (e.g. x-linked hypophosphatemic rickets) receiving long-term oral phosphate therapy without concomitant calcitriol.

 

If hypercalcemia is associated with very low or suppressed serum PTH levels, then malignancy would be an important consideration, either in association with elevated serum PTHrP or in its absence, in which case it is generally as a result of the production of other cytokines, often with osteolytic metastases. When malignancy-associated hypercalcemia is suspected then an appropriate malignancy screen should be done including skeletal imaging to identify skeletal metastases. As well appropriate general biochemical assessment such as a complete blood count and serum creatinine and specific biochemical assessment such as serum and urine protein electrophoresis to exclude multiple myeloma would be appropriate.

 

Detection of elevated serum 1,25(OH)2D levels in the absence of elevated serum PTH levels, suggests the need for a search for lymphoma or for non-infectious (e.g. sarcoidosis) or infectious granulomatous disease.

 

Hypercalcemia may also occur with thyrotoxicosis, pheochromocytoma, VIPoma, and hypoadrenalism. Increased PTHrP may be associated with neuroendocrine tumors. Serum PTH levels are suppressed in these disorders and 1,25(OH)2D levels are not elevated. Although these conditions may be suspected from clinical examination, detailed biochemical evaluation of these non-PTH associated endocrine disorders is required for confirmation.

 

Detection of elevated serum 25-hydroxyvitamin D [25(OH)D], should lead to a search for vitamin D intoxication. Vitamin A intoxication may also lead to hypercalcemia, but in the absence of elevated serum 25(OH)D, 1,25(OH)2D, or PTH. Hypercalcemia has been reported in association with human immunodeficiency virus (HIV), HTLV-III or cytomegalovirus (CMV) infections of the skeleton, presumably due to direct skeletal resorption. Use of foscarnet as an antiviral agent has also been associated with hypercalcemia. Transient hypercalcemia may accompany thiazide diuretic ingestion, possibly associated with dehydration, but prolonged hypercalcemia with thiazides requires a search for other causes. Hypercalcemia may be seen in patients with advanced breast cancer with skeletal metastases, at the initiation of treatment with tamoxifen. Aminophylline and theophylline used as bronchodilators have (rarely) been reported to be associated with hypercalcemia. The use of aluminum-containing phosphate binders in patients on chronic hemodialysis was associated with hypercalcemia in the past but, with the advent of other modes of therapy, this is rarely seen today. Similarly, the use of absorbable alkali (NaHCO3) along with large quantities of milk for ulcer treatment was a cause of hypercalcemia in the past but this therapy has been superseded today.

 

In the pediatric age group, hypercalcemia may include Jansens’s Metaphyseal Chondrodysplasia due to an activating mutation of the type 1 PTH/PTHrP receptor; neonatal severe hyperparathyroidism (NSHPTH) which may present with life-threatening hypercalcemia in neonates that are homozygous for inactivating mutations in CaSR; William’s Syndrome, an autosomal dominant disorder with hemizygous submicroscopic deletions of chromosome 7q11.23, characterized phenotypically by multiple congenital abnormalities, and in which hypercalcemia may occur possibly due to aberrant vitamin D metabolism; and idiopathic infantile hypercalcemia (IIH) in which hypercalcemia may be associated with increased 1,25(OH)2D.due to loss-of-function mutations in CYP24A1, the gene encoding the enzyme responsible for the first step in inactivation of 1,25(OH)2D. IIH may also be caused by loss-of-function mutations in SLC34A1, encoding the renal proximal tubular sodium-phosphate cotransporter, Na/Pi-IIa, leading to phosphaturia, phosphate depletion, suppression of the hormone fibroblast growth factor-23 (FGF-23), decreased CYP24A1,and increased 1,25(OH)2D production.

 

THERAPY

 

If the patient's serum calcium concentration is less than 12 mg/dL (3 mM) then treatment of the hypercalcemia can be aimed solely at treatment of the underlying disorder. If the patient has symptoms and signs of acute hypercalcemia as described above and serum calcium is greater than 12 mg/dL (3 mM), then a series of urgent measures should be instituted (Table 9). These measures are almost always required with a serum calcium above 14 mg/dL (3.5 mM).

 

Table 9. Management of Acute Hypercalcemia

1. Hydration to Restore Euvolemia

0.9% saline (e.g. an initial rate of 200-300 mL/h subsequently adjusted to maintain a urine output at 100-150 mL/h). Use caution in patients with compromised cardiovascular or renal function.

2. Inhibition of Bone Resorption

Zoledronate 4 mg intravenously in 5 ml over 15 min or Pamidronate, 90 mg, intravenously in 500 ml of 0.9% saline or 5% dextrose in water over 4 hours.
Peak decrease in serum calcium after 4 days but may last for 8 weeks.
Flu-like syndrome or myalgias may occur

If bisphosphonates are contraindicated due to severe renal impairment, denosumab can be given instead (e.g. 0.3 mg/kg sc), with a second dose administered if the calcium is not lowered within approximately one week. Low serum 25(OH)D, if present, should be corrected before administering denosumab

Calcitonin, 4 to 8 IU/Kg im or sc, every 6-12 hours may be used with a bisphosphonate or denosumab because of its more rapid onset of action.
Peak decrease in serum calcium within 2-6 hours
Tachyphylaxis may occur after 24-48 hours
May use with a parenteral bisphosphonate for severe hypercalcemia because onset of calcium reduction is earlier

3. Calciuresis (when decreased renal excretion is suspected e.g. with excess PTH or PTHrP)

Loop diuretic e.g. furosemide, 10 to 20 mg IV
Administer only after rehydration

4. Glucocorticoids (when indicated)

e.g. hydrocortisone 200 to 300 mg intravenously over 24 hours for 3 to 5 days
For patients with responsive hematologic malignancies such as lymphoma or myeloma
For patients with vitamin D intoxication or granulomatous disease with increased 1,25(OH)2D

5. Dialysis

Patients refractory to other therapies
Patients with renal insufficiency
Either peritoneal dialysis or hemodialysis can be effective

6. Calcimimetics

The calcimimetic, cinacalcet, may be used in doses starting from 30 mg twice daily orally to as high as 90 mg 4 times daily for the treatment of hypercalcemia due to severe primary HPTH (especially if caused by a parathyroid carcinoma)

7. Mobilization

Mobilize as rapidly as possible after the acute episode

 

FOLLOW-UP

 

In the patient with primary sporadic HPTH who presents with kidney stones, fractures, or a low BMD (T-score less than -2.5) surgery would be indicated. In the patient with documented asymptomatic primary HPTH, follow-up should be done annually with measurement of serum calcium and serum creatinine (to determine estimated GFR). BMD should be repeated every one to two years. Guidelines below should be considered for recommending surgery (reference 2). The diagnosis of familial disease raises issues of management of HPTH in the proband and affected family members in view of the fact that familial HPTH generally is generally associated with multigland disease, whereas the sporadic disease is usually due to an adenoma. In HPTH jaw tumor syndrome there should be recognition of the high frequency of parathyroid carcinoma.

 

 

Table 10. Guidelines for Surgery in Primary HPTH

Serum calcium

>1mg/dL(0.25mM) above the upper limit of normal

Renal function

Estimated GFR: <60ml/min/1.73m2 [24 h urine calcium excretion >400 mg (>10mmol) is still regarded as an indication for surgery by some.]

Skeletal function

Fragility fracture or BMD T-score <–2.5 at the lumbar spine, total hip, femoral neck, or distal third of the radius and fracture fragility in postmenopausal women and men ≥50 yr.

Age

<50 years

Other

Operate when medical surveillance is neither desired nor possible

 

Management of other etiologies of hypercalcemia are generally directed toward the specific entity involved.

 

REFERENCES

 

  1. Goltzman D. Approach to Hypercalcemia. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2016 Aug 8.
  2. Bilezikian JP. Primary Hyperparathyroidism. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2017 Jan 15.
  3. Vinik A, Perry RR, Hughes MS, Feliberti E. Multiple Endocrine Neoplasia Type 1. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2017 Oct 7.
  4. Hughes MS, Feliberti E, Perry RR, Vinik A. Multiple Endocrine Neoplasia Type 2A (including Familial Medullary Carcinoma) and Type 2B. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2017 Oct 8.
  5. Kaltsas G, Dimitriadis GK, Androulakis II, Grossman A. Paraneoplastic Syndromes related to Neuroendocrine Tumours. In: De Groot LJ, Chrousos G, Dungan K, Feingold KR, Grossman A, Hershman JM, Koch C, Korbonits M, McLachlan R, New M, Purnell J, Rebar R, Singer F, Vinik A, editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000- 2017 Feb 16.

 

Control of Energy Expenditure in Humans

ABSTRACT

 

Resting and meal-related energy requirements can be assessed by measuring energy expenditure with indirect calorimetry. The indicated method to assess free-living energy expenditure is the doubly labelled water technique. Variation in energy expenditure is mainly a function of body size and composition (resting energy expenditure) and of physical activity (activity energy expenditure). Thus, energy expenditure can be calculated with a prediction equation for resting energy expenditure, based on height, age, weight and gender, in combination with the measurement of the physical activity level of a subject with a doubly labelled water validated accelerometer for movement registration. Energy balance in humans is maintained by adjusting energy intake to energy expenditure. Over- and underfeeding induces changes in activity-induced energy expenditure and maintenance expenditure as a function of changes in body weight and body composition. Additionally, underfeeding causes a metabolic adaptation as reflected in a reduction of maintenance expenditure below predicted values and defined as adaptive thermogenesis. When intake exceeds energy requirements, the excess is primarily stored as body fat. As a substrate for energy metabolism, fat is less likely than carbohydrate or protein to be oxidized for fuel. Consumed fat is mostly stored before oxidation, especially in heavier people, increasing the likelihood of creating a positive energy balance. An activity-induced increase in energy requirement is typically followed by an increase in energy intake, whereas a reduction in physical activity does not result in an equivalent reduction of energy intake. Thus, preventing weight gain is more effectively reached by eating less than by moving more.

 

MEASURING ENERGY EXPENDITURE

 

Living can be regarded as a combustion process. The metabolism of an organism requires energy production by the combustion of fuel in the form of carbohydrate, protein, fat, or alcohol. In this process oxygen is consumed and carbon dioxide produced. Measuring energy expenditure means measuring heat production or heat loss, and this is known as direct calorimetry. The measurement of heat production by measuring oxygen consumption and/or carbon dioxide production is called indirect calorimetry.

 

Early calorimeters for the measurement of energy expenditure were direct calorimeters. In the end of the 18th century Lavoisier constructed one of the first calorimeters, measuring energy expenditure in a guinea pig. The animal was placed in a wire cage, which occupied the center of an apparatus. The surrounding space was filled with chunks of ice. As the ice melted from the animal's body heat, the water was collected in a container, and weighed. The ice cavity was surrounded by a space filled with snow to maintain a constant temperature. Thus, no heat could dissipate from the surroundings to the inner ice jacket. Figure 1 shows Lavoisier's calorimeter schematically. Today, heat loss is measured in a calorimeter by removing the heat with a cooling stream of air or water or measuring the heat flow through the wall. In the first case, heat conduction through the wall of the calorimeter is prevented and the flow of heat is measured by the product of temperature difference between inflow and outflow and the rate of flow of the cooling medium. In the latter case instead of preventing heat flow through the wall, the rate of this flow is measured from differences in temperature over the wall. This method is known as gradient layer calorimetry.

 

Figure 1: Lavoisier’s calorimeter. Heat expended by the animal melts the ice in the inner jacket. Snow in the outer jacket prevents heat exchange with the surrounding environment (From reference 1).

 

In indirect calorimetry, heat production is calculated from chemical processes. Knowing, for example, that the oxidation of 1 mol glucose requires 6 mol oxygen and produces 6 mol water, 6 mol carbon dioxide and 2.8 MJ heat, the heat production can be calculated from oxygen consumption or carbon dioxide production. The energy equivalent of oxygen and carbon dioxide varies with the nutrient oxidized (Tables 1 and 2).

 

Table 1: Gaseous Exchange and Heat Production of Metabolized Nutrients.

Nutrient

Consumption oxygen

(l/g)

Production carbon dioxide

(l/g)

Heat

(kJ/g)

Carbohydrate

0.829

0.829

17.5

Protein

0.967

0.775

18.1

Fat

2.019

1.427

39.6

 

 

Table 2: Energy Equivalents of Oxygen and Carbon Dioxide.

Nutrient

Oxygen

(kJ/l)

Carbon dioxide

(kJ/l)

Carbohydrate

21.1

21.1

Protein

18.7

23.4

Fat

19.6

27.8

 

Brouwer (2) drew up simple formulae for calculating the heat production and the quantities of carbohydrate (C), protein (P) and fat (F) oxidized from oxygen consumption, carbon dioxide production, and urine-nitrogen loss. The principle of the calculation consists of three equations with the mentioned three measured variables:

 

Oxygen consumption              = 0.829 C + 0.967 P + 2.019 F

Carbon dioxide production      = 0.829 C + 0.775 P + 1.427 F

Heat production                       = 21.1 C + 18.7 P + 19.6 F

 

Protein oxidation (g) is calculated as 6.25 x urine-nitrogen (g), and subsequently oxygen consumption and carbon dioxide production can be corrected for protein oxidation to allow calculation of carbohydrate and fat oxidation. The general formula for the calculation of energy production (E) derived from these figures is:

E = 16.20 * oxygen consumption + 5.00 * carbon dioxide production - 0.95 P

 

In this formula the contribution of P to E, the so-called protein correction, is only small. In the case of a normal protein oxidation of 10-15 per cent of the daily energy production, the protein correction for the calculation of E is about 1 per cent. Usually only urine nitrogen is measured when information on the contribution of C, P, and F to energy production is needed. For calculation of energy production, the protein correction is often neglected.

 

Metabolizable energy is available for energy production in the form of heat and for external work. At present, the state of the art for assessing total energy expenditure is with indirect calorimetry. With indirect calorimetry, the energy expenditure is calculated from gaseous exchange of oxygen and carbon dioxide. The result is the total energy expenditure of the body for heat production and work output. With direct calorimetry, only heat loss is measured. At rest, total energy expenditure is converted to heat. During physical activity, there is work output as well. The proportion of energy expenditure for external work is the work efficiency. At rest, indirect calorimetry-assessed energy expenditure matches heat loss as measured with direct calorimetry. During physical activity, heat loss is systematically lower than indirect calorimetry-assessed energy expenditure and can be up to 25% lower than total energy expenditure during endurance exercise. The difference increases with exercise intensity. For example, during cycling, indirect calorimetry assessed energy expenditure matches the sum of heat loss and power output (3). The work efficiency during cycling, power output divided by energy expenditure is in the range of 15 to 25%.

 

Current techniques utilizing indirect calorimetry for the measurement of energy expenditure in man include a facemask or ventilated hood, respiration chamber (whole room calorimeter), and the doubly labelled water method. A facemask is typically used to measure energy expenditure during standardized activities on a treadmill or a cycle ergometer. A ventilated hood is used to measure resting energy expenditure and energy expenditure for food processing (diet-induced energy expenditure). A respiration chamber is an airtight room, which is ventilated with fresh air. Basically, the difference between a respiration chamber and a ventilated hood system is size. In a respiration chamber the subject is fully enclosed instead of enclosing the head only, allowing physical activity depending on the size of the chamber. For measurements under a hood or in a respiration chamber, air is sucked through the system with a pump and blown into a mixing chamber where a sample is taken for analysis. Measurements taken are those of the airflow and of the oxygen and carbon dioxide concentrations of the air flowing in and out. The most common device to measure the airflow is a dry gas meter comparable to that used to measure natural gas consumption at home. The oxygen and carbon dioxide concentrations are commonly measured with a paramagnetic oxygen analyzer and an infrared carbon dioxide analyzer respectively. The airflow is adjusted to keep differences in oxygen and carbon dioxide concentrations between inlet and outlet within a range of 0.5 to 1.0%. For adults, this means airflow rates around 50 l/min at rest under a hood, 50-100 l/min when sedentary in a respiration chamber, while in exercising subjects the flow has to be increased to over 100 l/min. In the latter situation, one has to choose a compromise for the flow rate when measurements are to be continued over 24 hours that include active and inactive intervals. During exercise bouts, the 1% carbon dioxide level should not be surpassed for long periods. During resting bouts, like an overnight sleep, the level should not fall too far below the optimal measuring range of 0.5-1.0%. Changing the flow rate during an observation interval reduces the accuracy of the measurements due to the response time of the system. Though the flow rate of a hood and a chamber system is comparable, the volume of a respiration chamber is more than 20 times the volume of a ventilated hood. Consequently, the minimum length of an observation period under a hood is about 0.5 hours and in a respiration chamber in the order of 5-10 hours.

 

The doubly labelled water method is an innovative variant on indirect calorimetry based on the discovery that oxygen in the respiratory carbon dioxide is in isotopic equilibrium with the oxygen in body water. This technique involves enriching the body water with an isotope of oxygen and an isotope of hydrogen and then determining the washout kinetics of both isotopes. Doubly labelled water provides an excellent method to measure total energy expenditure in unrestrained humans in their normal surroundings over a time period of 1-4 weeks. After enriching the body water with labelled oxygen and hydrogen by drinking doubly labelled water, most of the oxygen isotope is lost as water, but some is also lost as carbon dioxide because CO2 in body fluids is in isotopic equilibrium with body water due to exchange in the bicarbonate pools (4). The hydrogen isotope is lost as water only. Thus, the washout for the oxygen isotope is faster than for the hydrogen isotope, and the difference represents the CO2 production. The isotopes of choice are the stable, heavy, isotopes of oxygen and hydrogen, oxygen-18 (18O) and deuterium (2H), since these avoid the need to use radioactivity and can be used safely. Both isotopes naturally occur in drinking water and thus in body water. The CO2 production, calculated from the difference in elimination between the two isotopes, is a measure of metabolism. In practice, the observation duration is set by the biological half-life of the isotopes as a function of the level of the energy expenditure. The minimum observation duration is about 3 days in subjects with high energy turnover like premature infants or endurance athletes. The maximum duration is 30 days or about 4 weeks in elderly (sedentary) subjects. An observation period begins with collection of a baseline sample. Then, a weighed isotope dose is administered, usually a mixture of 10% 18O and 5% 2H in water. For a 70 kg adult, between 100-150ml water would be used. Subsequently, the isotopes equilibrate with the body water and the initial sample is collected. The equilibration time is dependent on body size and metabolic rate. For an adult the equilibration would take between 4-8 hours. During equilibration, the subject usually does not consume any food or drink. After collecting the initial sample, the subject performs routines according to the instructions of the experimenter. Body water samples (blood, saliva or urine) are collected at regular intervals until the end of the observation period. The doubly labelled water method gives precise and accurate information on carbon dioxide production. Converting carbon dioxide production to energy expenditure needs information on the energy equivalent of CO2 (Table 2), which can be calculated with additional information on the substrate mixture being oxidized. One option is the calculation of the energy equivalent from the macronutrient composition of the diet. In energy balance, substrate intake and substrate utilization are assumed to be identical.

 

ENERGY EXPENDITURE AND COMPONENTS

 

Daily energy expenditure consists of four components: sleeping metabolic rate, the energy cost of arousal, the thermic effect of food (or diet induced energy expenditure (DEE)), and the energy cost of physical activity or activity-induced energy expenditure (AEE). Usually, sleeping metabolic rate and the energy cost of arousal are combined and referred to as resting energy expenditure (REE). Overnight when one sleeps quietly, food intake and physical activity are generally low or absent and energy expenditure gradually decreases to a daily minimum before increasing upon awakening (Figure 2). Then, increases in energy expenditure during arousal are primarily caused by activity-induced energy expenditure as well as diet-induced energy expenditure. Thus, energy expenditure varies throughout a day as a function of body size and body composition (the major components determining REE), physical activity as determinant of AEE, and food intake as determinant of DEE.

Figure 2: Average energy expenditure (upper line) and physical activity (lower line) as measured over a 24-h interval in a respiration chamber. Arrows denote meal times. Data are the average of 37 subjects, 17 women and 20 men, age 20-35 y and body mass index 20-30 kg/m2 (5).

 

Resting energy expenditure is defined as the metabolic rate required to maintain vital physiological functions of an individual that is in rest, awake, in a fasted state, and in a thermoneutral environment. To perform an accurate measurement of REE, a subject is instructed not to exercise the day before, to fast overnight, transported to a laboratory after waking up in the morning and habituated for 15-30 min to the testing procedure under a ventilated hood, before the actual measurement of 20-30 min, at a comfortable room temperature of 22-24 0C (6).

 

Standardizing to fat-free mass as an estimate of metabolic body size is most commonly used in the literature to compare REE between individuals. However, although fat-free body mass is a strong predictor of REE, energy expenditure should not be solely divided by the absolute fat-free mass value as the relationship between energy expenditure and fat-free mass has a y and x intercept significantly different from zero (Figure 3). For example, fat-free adjusted REE is significantly different between women and men (Figure 3, 0.143±0.012 and 0.128±0.080 MJ/kg for women and men, respectively, P < 0.0001). The smaller the fat-free mass, the higher the REE/ fat-free mass ratio and thus the REE per kg fat-free mass is on average higher in women with on average a lower fat-free mass compared with men. Instead, a more accurate approach for comparing REE data is by regression analysis that includes both fat-free mass and fat mass as covariates.

 

REE (MJ/d) = 1.39 + 0.93 fat-free mass (kg) + 0.039 fat mass (kg), r2 = 0.93.

Using this equation, gender no longer comes out as a significant contributor to the explained variation in the group of women and men as presented in Figure 3.

Figure 3: Resting energy expenditure (REE) plotted as a function of fat-free mass for the subjects from reference 5 as described in Figure 2 (17 women: closed symbols; 20 men: open symbols) with the calculated linear regression line (REE (MJ/d) = 2.27 + 0.091 fat-free mass (kg), r2 = 0.78).

 

Diet-induced energy expenditure is defined as the energy-required for intestinal absorption of nutrients, the initial steps of their metabolism, and the storage of the absorbed but not immediately oxidized nutrients during the post-prandial period. As such, the amount of food ingested quantified as the energy content of the food is a determinant of DEE. The most common way to express DEE is derived from the difference between energy expenditure after food consumption and REE, divided by the rate of nutrient energy administration. Theoretically, based on the amount of ATP required for the initial steps of metabolism and storage, the DEE is different for each nutrient. Reported DEE values for separate nutrients are 0 to 3% for fat, 5 to 10% for carbohydrate, and 20 to 30% for protein (7). In healthy subjects in energy balance with a mixed diet, DEE represents about 10% of the total amount of energy ingested over 24 hours.

A typical mean pattern of DEE throughout the day is presented in Figure 4. Data are from a study where DEE was calculated by plotting the residual of the individual relationship between energy expenditure and physical activity in time, as measured over 30-min intervals from a 24-h observation in a respiration chamber. The level of REE after waking up in the morning, and directly before the first meal, was defined as basal metabolic rate. Resting metabolic rate did not return to basal metabolic rate before lunch at 4 hours after breakfast, or before dinner at 5 hours after lunch. Instead, basal metabolic rate was restored overnight, approximately 8 hours after dinner consumption.

Figure 4: The mean pattern of resting energy expenditure throughout the day, where arrows denote meal times (adapted from reference 8).

 

Activity-induced energy expenditure, the most variable component of daily energy expenditure, is derived from total energy expenditure (TEE) minus resting energy expenditure and diet-induced energy expenditure.

 

AEE = TEE – REE – DEE.

 

Total energy expenditure is measured with doubly labelled water as described in the section on measuring energy expenditure. Diet induced energy expenditure is assumed to be 10% of TEE in subjects consuming the average mixed diet and being in energy balance. Thus, AEE can be calculated as: AEE = 0.9 TEE – REE.

 

A frequently used method to quantify the physical activity level (PAL) of a subject is to express TEE as a multiple of REE:

 

PAL = TEE/REE.

 

This assumes that the variation in total energy expenditure is due to body size and physical activity. The effect of body size is corrected for by expressing TEE as a multiple of REE. Data on daily energy expenditure, as measured with doubly labelled water, permit the evaluation of limits to the physical activity level. In our site, data were compiled for more than 500 subjects, where energy expenditure was measured over an interval of two weeks with the same protocol. The sample excludes individuals aged less than 18 years, and those involved in interventions in energy intake, physical activity including athletic performance, or who were pregnant, lactating or with an acute or chronic illness. The sample includes similar numbers of women and men, with a wide range for age, height, weight, and body mass index. Despite the wide variation in subject characteristics, a narrow range of the physical activity level (between 1.1 and 2.75) amongst the subjects was found (Figure 5) with no sex differences (9).

 

The physical activity level of a subject can be classified in three categories as defined by the last FAO/WHO/UNU expert consultation on human energy requirements (10). The physical activity for sedentary and light activity lifestyles ranges between 1.40 and 1.69, for moderately active or active lifestyles between 1.70 and 1.99, and for vigorously active lifestyles between 2.00 and 2.40. An active lifestyle improves heath parameters like insulin sensitivity (11). Higher PAL values are difficult to maintain over a long period and generally result in weight loss.

Figure 5: Frequency distribution of the value of the physical activity level (PAL, calculated as the total energy expenditure / resting energy expenditure), in a group of 556 healthy adults, women closed bars and men open bars (data from reference 9).

 

An alternative for the measurement of energy expenditure with indirect calorimetry is a prediction equation for resting energy expenditure, in combination with an estimation of activity energy expenditure from measurement of body movement with an accelerometer. Typically, prediction equations for resting energy expenditure can explain 70-80% of the variation from race, height, age, weight and gender of a subject (12). Doubly labelled water studies show the best accelerometers for movement registration so far can explain 50-70% of variation in activity energy expenditure (13).

 

DETERMINANTS OF ENERGY EXPENDITURE

 

The main determinants of energy expenditure are body size and body composition, food intake, and physical activity. Additional determinants are ambient temperature and health. As most people are able to live in a thermoneutral environment or prevent heat loss with appropriate clothing, energy expenditure is not affected by ambient temperature for longer time intervals.

Body size and body composition determine REE, the largest component of daily energy expenditure, as depicted in Figure 6. Energy expenditure is generally higher in men than in women because men generally have a larger metabolic body size. They are on average heavier than women and for the same weight men have relatively more fat-free mass. For similar reasons, gaining weight implicates gaining fat mass and fat-free mass, and daily energy expenditure is generally higher in overweight and obese people, compared with lean people matched for age, height and gender. This higher energy expenditure in obese people is mainly a consequence of higher resting energy expenditure than lean people (Figure 6).

Figure 6: The three components of energy expenditure: resting energy expenditure (closed bar), diet-induced energy expenditure (stippled bar), and activity-induced energy expenditure (open bar) as observed in lean and obese subjects. In the lean group, women and men weighed 61 kg and 74 kg with 29% and 17% body fat, respectively. In the obese group, subjects were on average 40 kg heavier, where 70% of the additional weight was fat mass and 30% fat-free mass. The figure clearly illustrates the higher energy expenditure (primarily in resting energy expenditure) in men than in women and in obese than in lean subjects. (After reference 14).

 

Food intake affects all three components of daily (total) energy expenditure: REE, DEE and AEE. The most obvious effect is on DEE, which represents about 10% of the amount of daily energy ingested. Thus, changing energy intake changes total energy expenditure accordingly. Overeating induces an additional increase for storage of excess energy, estimated at about 10 % of the energy surplus (15). When overfeeding is lower than twice the maintenance requirements, there does not seem to be an effect of this overfeeding on physical activity (16). Undereating induces a decrease in REE, DEE and AEE. Undereating induces weight loss accompanied by adaptive thermogenesis, a disproportional or greater than expected reduction of REE. The reduction in REE seems to be sustained if weight loss is maintained (17). Weight loss due to a negative energy balance is accompanied by a decrease in AEE as well. Here, the decrease is due to less body movement and a lower cost to move a smaller body mass. The reduction in body movement recovers to baseline values or higher when weight loss in maintained (18). A classic example of the effect of undereating on energy expenditure is the Minnesota Experiment from the 1950’s (19). Energy intake of normal-weight men was reduced for 24 weeks from 14.6 MJ/d to 6.6 MJ/d. The subjects reached a new energy balance by saving 8 MJ/d (Table 3). Of the total saving of 8 MJ/d the main part stemmed from reduced AEE. The reduction of AEE was mainly due to moving less.

 

Table 3: Energy Saved by 24 weeks Undereating in the Minnesota Experiment (19).

 

MJ/d

% of saving

explanation

Resting energy expenditure

2.6

32

65% for a decreased bodyweight

35% for a lowered tissue metabolism

Diet-induced expenditure

0.8

10

 

Activity-induced expenditure

4.7

58

40% for a decreased bodyweight

60% for less body movement

Total

8.0

   

 

Activity-induced energy expenditure is the most variable component of daily expenditure and can be increased with exercise participation. The variation in energy expenditure between subjects is a function of body size and physical activity, where AEE is an important contributor. Most of the variation in AEE is accounted for by genetic factors. Genes determine for a large part whether a person is prone to engage in activities and how much energy is expended for these activities (20). Exercise training can increase AEE. However, under some conditions the added exercise expenditure is compensated with a reduction of non-training activity. Examples are non-ad libitum food intake and old age (Figure 7).

Figure 7: The physical activity level, total energy expenditure as a multiple of resting energy expenditure, before (open bar) and at the end of a training program (closed bar), for eight studies displayed in a sequence of age of the participants as displayed on the horizontal axis (After reference 21).

 

Activity-induced energy expenditure does not increase linearly with increasing physical activity. Novice runners, training to run a half marathon, could increase the training volume without a change in AEE (22). In the selected group of sedentary subjects, the initial training induced increase in AEE was twice as high as predicted from the training load. Subsequent training allowed a doubling of the training load for the same AEE, probably through an improvement of exercise economy. Similarly, exercise training was shown to decrease the energetic cost of walking in older adults (23).

 

Physical activity level reaches a maximum value of 2.0-2.4 (Figure 7). Higher values can be reached over shorter time intervals. Runners in a 140-day transcontinental Race Across the USA showed an initial increase in PAL from a Pre-Race value of 1.76 to 3.76 over the first five days of marathon running (24). In the last week (week 20) of marathon running, PAL had decreased to a mean value of 2.81. The authors explained the decrease in PAL during sustained physical activity from an alimentary energy supply limit.

 

During negative energy balance, additional exercise is compensated by a reduction of non-training activity. In elderly subjects, exercise training has a similar compensatory effect on spontaneous physical activity, even under ad-libitum food conditions. Despite the absence of an effect of exercise training on total energy expenditure in elderly people, there are many beneficial effects of exercise training like aerobic capacity, endurance, flexibility, and range of motion.

 

ENERGY BALANCE

 

Adult humans maintain weight stability through a balance between energy intake and energy expenditure. When weight is stable, the energy store of the body does not fluctuate much, as evident by constancy in body weight and body composition. This weight constancy can be achieved through the balanced control of energy intake and expenditure. This balance does not, however, take place on an immediate basis. For example, on days with high energy expenditure, energy intake is usually normal or even below normal. The 'matching' increase in energy intake comes a couple of days afterwards (25). Energy intake can change by at least a factor of three when adapting to changes in energy expenditure. Under sedentary living conditions the energy balance is maintained at about 1.5 times basal metabolic rate (BMR), while during sustained exercise levels of 4.5 times BMR are reached (26).

 

Humans are discontinuous eaters and continuous metabolizers. An animal that takes its food in meals, such as a human, periodically consumes more than their physiological needs even when in (daily) energy balance. During meal-related hyperphagia, metabolites are initially stored then mobilized during inter-meal intervals of energy deficiency. This pattern of intermittent feeding and fasting has consequences for energy expenditure (Figure 4). During and after a meal, expended energy increases to process the ingested food, while energy deficiency before a new meal is started can lead to a reduction of energy expenditure. The latter probably does not occur normally when the energy deficiency is only short. However, people tend to be less energetic when skipping breakfast prolongs inter-meal intervals or during more extended fasts.

Disturbances of energy balance result in energy mobilization from, or energy storage in, body reserves. Energy intake occurs via macronutrients consumed in meals in the form of carbohydrate, protein, fat and alcohol. During positive energy balance, excess energy is stored as carbohydrate in glycogen, primarily in the liver, and as fat in adipose depots. The storage capacity for carbohydrate is small, typically covering energy needs during the overnight fast that accompanies sleep. Longer-term shortages are mainly covered by mobilization of the larger energy stores in fat. On days with a positive energy balance, protein and carbohydrate intake match protein and carbohydrate oxidation and the difference between energy intake and energy expenditure shows up in a positive fat balance (27). In the early morning, at arousal, carbohydrate oxidation goes up and continues to increase at the first food intake of the day (28). After getting up, initial energy (‘fast’) requirements are met by glycogen reserves. Subsequently, carbohydrate requirement is higher at breakfast, and one eats relatively more fat at the evening dinner (29, 30).

 

Energy balance does not equate to substrate balance, and when in substrate balance one does not produce energy just from the foods consumed. Fat, as a substrate for energy metabolism is at the bottom of the oxidation hierarchy that determines fuel selection and studies show a direct link between macronutrient balance for fat and energy balance. Changes in alcohol, protein, and carbohydrate intake elicit auto regulatory adjustments in oxidation whereas a change in fat intake fails to elicit such a response, or only in the long term (31). One of the explanations is the routing of dietary fat.

 

Fat metabolism can be traced with isotope-labelled fatty acids. Oxidation and adipose tissue uptake of dietary fat can be measured by adding fatty acid labelled with heavy hydrogen (2H) to meals. Upon oxidation, these deuterated fatty acids enrich the body water with deuterium, which is subsequently detectable in urine. Therefore, the urine enrichment for deuterium is a measure for dietary fat oxidation. The first label appears in the urine in about two hours and the peak concentration is reached after 12-24h (Figure 8). After 24 hours, 5-30% of the fat from a meal is oxidized and the remaining part partitioned to the reserves. The percentage of dietary fat oxidation is independent of the composition of the meal with respect to protein, carbohydrate and fat. However, there is a clear relation of dietary fat oxidation with the body fat content. The larger the fat mass, the lower the fractional oxidation of the fat consumed on the same day (32). The observed reduction in dietary fat oxidation in subjects with greater body fat may therefore play a role in expression and maintenance of human obesity. Low dietary fat oxidation makes subjects prone to weight gain.

 

Figure 8: Cumulative oxidation (mean ± standard deviation) of dietary fat as a percentage of intake, over time after ingestion, as calculated from tracer recovery in urine produced at two-hour intervals (From reference 32).

 

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  30. Almoosawi S, Prynne CJ, Hardy R, Stephen AM. (2013) Time-of-day and nutrient composition of eating occasions: prospective association with the metabolic syndrome in the 1946 British birth cohort. Int J Obes 37:725-731.
  31. Schrauwen P, Van Marken Lichtenbelt WD, Saris WHM, Westerterp KR. (1997) Changes in fat oxidation in response to a high-fat diet. Am J Clin Nutr 66:276-282.
  32. Westerterp KR, Smeets A, Lejeune MP, Wouters-Adriaens MPE, Westerterp-Plantenga MS. (2008) Dietary fat oxidation as a function of body fat. Am J Clin Nutr 87: 132-135.

 

 

 

 

 

 

 

Evaluation and Treatment of Gender-Dysphoric/Gender Incongruent Adults

ABSTRACT

 

Gender dysphoria refers to the suffering due to an incongruence between one’s sex assigned at birth and one’s self-perceived gender. Primary care physicians often play an important role in diagnosis and initiation of treatment of gender dysphoria. However, gender dysphoria is preferentially diagnosed by a specialized psychologist or psychiatrist. This does not imply that gender dysphoria in itself is a mental disorder, but co-morbidity needing attention, is frequently present. The prevalence of transgender people who receive medical treatment has steeply increased in the last decades. The current prevalence is estimated at 1:2,800 for transwomen (male sex assigned at birth, female gender identity) and 1:5,200 for transmen (female sex assigned at birth, male gender identity). Treatment of transgender people often includes gender-affirming hormonal therapy and/or surgery, and is optimally provided by a multidisciplinary team consisting of psychologists, endocrinologists, plastic surgeons, gynecologists, urologists, otorhinolaryngologists, and/or dermatologists. Medical treatment usually improves the quality of life of transgender people, but might also have side-effects such as increased risk for cardiovascular disease or hormone-sensitive tumors. There is still little known about the optimal therapy (for specific transgender subpopulations) and its long-term side-effects. Nowadays, guidelines are mainly based on clinical experience instead of evidence. However, transgender medicine is a growing field and the increasing number of good quality studies are helping to improve care of transgender people. In this contribution we mainly focus on what is known about the side-effects of hormonal therapy. In addition, we provide information about surgical and fertility preservation options for transgender people. We conclude this contribution with remarks about special conditions such as older age and unsupervised hormone use.

 

INTRODUCTION

 

Gender identity is the personal sense of one's own gender. A significant incongruence between one’s physical phenotype and one’s gender identity is defined as gender dysphoria. While care for transgender people has long been based on a binary understanding of gender (male versus female), the existence of non-binary or gender queer genders is getting increasing attention. Non-binary or gender queer peopleidentify with a gender that is neither exclusively male nor female, but is composed of both, oscillates between genders, is situated beyond the binary, or rejects the binary (1). Gender dysphoria is usually diagnosed by a specialized psychologist, which does not imply that it is a mental disorder per se. People who have a male sex assigned at birth but have developed a female gender identity are called transwomen and people who have a female sex assigned at birth but experience a male gender identity are called transmen.The prevalence of transgender people who seek medical treatment has dramatically increased in the last years, and a recent Dutch study estimated a prevalence of 1:2,800 for transwomen and 1:5,200 for transmen (2). While the etiology is complex and probably multifactorial, the most widely believed hypothesis is that transgender people have experienced a sex-atypical differentiation of the brain during fetal development (3,4).

 

To many physicians, transgender medicine is a novel area of medicine. Most physicians need intensive interaction with a transgender individual to empathize the suffering, and to arrive at the insight that hormonal and surgical treatment might alleviate gender dysphoria-related distress. It is usually assumed that medical interventions can only be justified when objective, identifiable pathological processes account for human suffering. The role that biological factors play in the development of gender identity is still not solidly established but an increasingly number of reports provide evidence that androgens play a role in the development of gender identity. The association is not absolute, and the information is not robust enough to draw solid conclusions. Nevertheless, it has been demonstrated that gender-affirming hormonal therapy and/or surgery, which is only recommended in people with well-documented gender dysphoria (5), contributes to an improved well-being (6). Medical treatment is preferably provided by a multidisciplinary team consisting of psychologists, endocrinologists, plastic surgeons, gynecologists, urologists, otorhinolaryngologists, and/or dermatologists.

 

When providing transgender care, physicians are confronted with a lack of systematically and accurately collected data. Transgender people are not a well-defined group, and show considerable heterogeneity, for instance in age of onset, in the degree of suffering, and in the wish for treatment. The attention that transgender people receive in medicine is certainly improving, however transgender people still experience health disparities. There is a dearth of research and evidence-based guidelines for treatment, and the specific health needs for transgender people are understudied. It is problematic to include sufficient numbers of participants to perform statistically robust studies with regard to the best transgender hormonal therapy, and the long-term side-effects of treatment. Nowadays, physicians with extensive clinical experience have drafted guidelines based primarily on empiric observation. Fortunately, the number of good quality studies is currently increasing.

 

HORMONAL THERAPY

 

In transwomen, hormonal therapy usually consists of estrogens ± antiandrogens (e.g. cyproterone acetate, spironolactone, or GnRH analogues) (9,10). Estrogen in the form of estradiol is recommended to minimalize the risk of venous thrombosis and cardiovascular disease. Since progestogens, other than progestogenic antiandrogens, have not been proven to have an additional effect on feminization in transwomen, they are usually not recommended (5,9,10), although they might have beneficial effects in cisgender women (11). In transmen, hormonal therapy usually consists of testosterone only (10). See Table 1 for an overview of the hormonal options for transwomen, and Table 2 for the hormonal options for transmen. For details on the hormonal regimens for transwomen and transmen the Endocrine Society Clinical Practice Guideline is an excellent source of information (10).

 

Table 1. Hormonal Regimens Regularly Used in Transwomen

Estrogens

Recommended Dose

Oral estradiol

2-6 mg daily

Intramuscular estradiol valerate/cypionate

10-20 mg/1-2 weeks

Estradiol patch*

50-100 mcg/24 hours

Estradiol gel*

0.75 mg-2.25 mcg daily

Antiandrogens**

 

Cyproterone acetate

10-50 mg daily

Spironolactone

50-200 mg daily

GnRH analogue

Varies per preparation

* Transdermal preparations are recommended in transwomen
aged ≥40 years or in those with an increased cardiovascular risk
** Antiandrogens are discontinued after orchiectomy

 

Table 2. Hormonal Regimens Regularly Used in Transmen

Testosterone

Recommended dose

Intramuscular enanthate or cypionate*

Intramuscular mixed esters (Sustanon®) Intramuscular undecanoate   

100–200 mg/2 weeks

250 mg/2-3 weeks
1000 mg/12 weeks OR  
 750 mg / 8 weeks**

Testosterone gel

 25-100 mg/day

Testosterone patch

 2 or 4 mg/day

Preparation availability will differ between countries

* Due to associated serum testosterone peaks, these injections may not the best option for transmen who develop polycythemia (12)
** The above are the conventional dosages for cismen. To virilize transmen 50-75% of these dosages are usually sufficient

 

Effects of Hormonal Therapy

 

TRANSWOMEN

 

In transwomen, hormonaltherapy induces feminization such as breast growth, skin softness, fat redistribution, and a decrease of body hair (9,10). Figure 1 shows the expected effects of hormonal therapy in transwomen, and the period over which achievement of maximum effects could be expected (9,10,13). Most of the hormone-induced effects start within the first months of treatment. It is important to realize that transwomen might not achieve the desired breast size; one year of hormonal therapy in transwomen usually results in less than an AAA cup size (13). As a result, many transwomen choose for breast augmentation surgery (14). It is also important to note that for complete permanent removal of (facial) hair, additional laser skin treatments are required. Furthermore, hormonal therapy does not induce voice changes. Therefore, transwomen who desire raising of their voice pitch may benefit from referral to a speech therapist (10).

Figure 1. Hormone Effects and the Term of Maximum Expected Effects in Transwomen

TRANSMEN

 

In transmen, hormonal therapy induces masculinization such as an increase in facial and body hair, an increase in muscle mass and strength, a masculinized voice, and a cessation of the menstruation. Figure 2 shows the expected effects of hormonal therapy in transmen, and the term that maximum effects could be expected (10). Most of the hormone-induced effects start within the first months of treatment. In most cases the menses cease during testosterone therapy. However, some transmen who experience persistent vaginal bleeding need additional therapy such as the progestin lynesterol or GnRH analogues, which suppress gonadotropin secretion.

Figure 2. Hormone Effects and Term of Maximum Expected Effects in Transmen

Hormonal Effects on Bone Health

 

Before puberty, the size and volume of the skeleton are similar in the two sexes. But upon the rise of androgens during puberty, a higher peak bone mass is attained in boys than in girls. Bone mass accrual, bone growth and maintenance of skeletal integrity in adulthood are critically determined by sex hormone production. In both sexes, hypogonadism leads to loss of bone. In men a significant role of estrogens in bone metabolism has been demonstrated. It is of note that testosterone is partially aromatized to estradiol, and it is well established that estradiol also plays a pivotal role in the bone health of men (15,16). These mechanisms underscore that hormonal therapy in transgender people will affect bone metabolism.

 

TRANSWOMEN

 

Unexpectedly, transwomenhave a lower bone mineral density than controls before starting with hormonal therapy. This might be explained by a less active lifestyle and/or a lower vitamin D status (17,18). As remarked above, in both sexes’ estrogens are important in the maintenance of bone health in adulthood. Initial studies examining the effect of long‐term hormonal therapy on bone mineral density showed contradictory results. However, most of these studies were small cross-sectional studies in which a baseline difference in bone mineral density was not taken into account (19–21). The most recent cohort study in transwomen showed that hormonal therapy does not have negative effects on bone mineral density, and that the lower bone mineral density in transwomen found in previous studies is solely based on a baseline lower bone mineral density (18). Therefore, it would be worthwhile to give lifestyle advice regarding physical exercise, adequate vitamin D status, and calcium intake to transwomen.

 

In postmenopausal women, bone mineral density depends on estrogens derived from aromatization of ovarian androgen production (22). Many transwomen have undergone orchiectomy in the process of their transition. Their status could probably be compared with a surgically-induced menopause in a ciswoman. Women with a surgically-induced menopause experience rapid bone loss during the first five years after oophorectomy. Based on this information, it has become clear that complete discontinuation of hormonal therapy in transwomen above the age of 50 leads to a profound loss of bone strength. Therefore, it is advisable to not discontinue estrogen therapy in older transwomen (23).

 

TRANSMEN

 

In contrast to transwomen, transmen show no lower bone mineral density before starting hormonal therapy (24,25). Furthermore, no negative effects of testosterone therapy on bone mineral density have been found (18,20,24).

 

Hormonal Effects on Cardiovascular Health

 

Cardiovascular disease is a prominent cause of morbidity and mortality in both women and men. Sex is known to affect one’s risk for cardiovascular disease. Men have a higher (age-adjusted) risk of strokes and acute coronary events than women (26–28). Strokes are 33% more incident in men than in women (28), and acute coronary events 172% (29). In addition, during reproductive age, women have a 100% higher risk of venous thromboembolic events than men (30,31). These data suggest that sex hormones play a role in the occurrence of cardiovascular events. Based on this information, it is surprising that recent studies found that estrogen therapy (without progestogen) increases the risk for developing strokes in menopausal women (32). There is also evidence that suggests a relationship between testosterone therapy and an increased risk of cardiovascular events (33–35). If exogenous sex hormones indeed have impact on the cardiovascular system, this might have consequences for transgender people receiving hormonal therapy. At the Amsterdam University Medical Center, the Netherlands, we have analyzed the development of cardiovascular disease in the population of the Gender Clinic. The Gender Clinic started in 1972 and there is now a large cohort of transgender people being followed-up including a growing number of older transgender people. The findings are summarized below.

 

TRANSWOMEN

 

Upon analysis of our transgender population in 1989 and in 1997 (36,37), cardiovascular disease, other than venous thromboembolism, was not increased in transwomen compared to cismen. However, the most recent evidence from 2011 and 2018 shows that transwomen receiving hormonal therapy have an increased risk for strokes and venous thromboembolism (but not acute coronary events) compared to cismen (38,39). The current estimated incidence rate for strokes in transwomen on hormonal therapy is 127 per 100,000 person-years, which is 80% higher than in cismen. The current estimated incidence rate for venous thromboembolic events is 320 per 100,000 person-years, which is 355% higher than in cismen (38). While the increased cardiovascular risk in transwomen was initially attributed to the usage of ethinylestradiol, recent studies found that transwomen who use other types of estrogens also have an increased risk for strokes and venous thromboembolism (38–40). The hypercoagulable effect of hormonal therapy (41)may be one of the mediators of the increased cardiovascular risk in transwomen.

 

Possibly, venous and arterial cardiovascular side-effects become more prominent past the age of 40-50 years, and in people with cardiovascular risk factors. While strong evidence is currently lacking, transdermal estradiol might be preferred over oral estrogens in these transwomen (9,42,43). In addition, one should be aware that progestogenic antiandrogens (e.g. cyproterone acetate) may further increase one’s risk for venous thromboembolism (44), and should therefore be continued no longer than necessary. Modifiable cardiovascular risk factors such as lipid concentrations, glucose concentrations, and blood pressure should be regularly monitored and treated in accordance with guidelines for ciswomen.

 

TRANSMEN

 

As in transwomen, first analyses from our center did not show an increased risk of cardiovascular disease in transmen using testosterone (36,37,45). However, the most recent analysis shows an increased risk for acute coronary events in transmen receiving testosterone, with a current estimated incidence rate of 100 per 100,000 person-years, which is 269% higher than the rate in ciswomen (38). The increased risk of acute coronary events in transmen receiving testosterone may be (partly) explained by the testosterone-induced combination of increases in hematocrit, thromboxane, triglycerides, and low-density lipoprotein cholesterol, and a decrease in plasma high-density lipoprotein cholesterol concentrations (35,46,47). Although, the design of the study made it impossible to draw any conclusions about a causal relationship we recommend to regularly monitor cardiovascular risk factors in transmen on testosterone therapy.

 

Hormonal Effects on Tumor Risk

 

Malignant neoplasms are the second leading cause of death worldwide (48). The risk for certain types of tumors differs between men and women. While this is obvious for neoplasms that develop in sex-specific organs such as the ovaries or the prostate, it is also the case for other types of tumors such as those of the meninges (49)and thyroid gland (50). Some of these differences are attributed to the exposure of sex hormones. Combined hormonal therapy in postmenopausal women has been found to increase the risk of breast cancer and death from lung cancer (51). In women with polycystic ovary syndrome a higher risk for endometrial cancer has been described, which is probably explained by the prolonged endometrial exposure to unopposed estrogen that results from anovulation (52). Testosterone therapy in hypogonadal men is not clearly associated with an increased cancer risk, but breast cancer risk in prostate cancer patients who receive estrogen therapy seems 3.91 times higher than in prostate cancer patients not receiving estrogen therapy (53). If exogenous sex hormones indeed are able to induce cell proliferation, this might have consequences for transgender people receiving hormonal therapy. It is good to keep in mind that transwomen and transmen remain susceptible to cancers of reproductive organs that are no longer in alignment with their gender. For example, postsurgical transwomen, and attending physicians, might not recognize their persisting risk of prostate cancer (the prostate is not removed during vaginoplasty). In addition, transmen who have not undergone removal of the uterus still have risk for cervical cancer. It is also important to realize that transgender people may opt out of cancer screening and examinations because of emotional or physical distress associated with the discordance between their experienced gender and their birth assigned sex.

 

To date, large-scale studies investigating neoplasms in transgender people are scarce and the literature mainly consists of case reports. One of the first reviews was presented in 2008 (54), and an extensive, high quality review appeared in 2017 (55). A cautious comparison of the two reports helps us to provide insight into the neoplasm-related morbidity and mortality in transgender people.

 

TRANSWOMEN

 

Breast Cancer

 

Estrogen in combination with antiandrogen therapy in transwomen stimulate the development of breast lobules, ducts, and acini which are histologically identical to those of ciswomen (56). While for a long time it was believed that the risk of breast cancer in transwomen receiving hormonal therapy was not higher than those of men (57,58), most recent evidence show that transwomen receiving hormonal therapy do have a 46-fold higher risk for breast cancer compared to men (59). As became clear in the Women’s Health Initiative study, addition of progestin to estrogen leads to an increase of the risk of breast cancer in women (60). Although evidence regarding breast cancer and the usage of the progestogenic cyproterone acetate is lacking, the above described data suggest that cyproterone acetate should be continued no longer than necessary. In addition, based on the most recent study that shows a much higher risk of breast cancer in transwomen compared to men, it is reasonable to recommend transwomen on hormonal therapy to participate in population-based breast cancer screening programs (9).

 

Prostate Cancer

While in the past, estrogens have been used to treat prostate cancer, estrogen and its related compounds have also been suggested as potential causative agents (61). Current literature, suggests that prostate cancer is very rare among transwomen. The few cases that have been reported in transwomen were in those who had not been screened for prostate cancer before starting hormonal therapy. Consequently, it remained unclear whether the prostate cancer was already present before hormonal therapy had been initiated (62). While prostate cancer has been rarely reported, underdiagnosis is possible due to lack of close prostate monitoring. Based on available evidence it does not seem necessary to screen transwomen in a different way to cismen, for which population-based screening is not recommended. But similarly, a transwoman with a first-degree male relative with prostate cancer should be made aware of her increased risk and prostate cancer [PSA] testing should be discussed to allow informed decision making. However, when interpreting PSA values in this context, it has to be kept in mind that suppression of testosterone by antiandrogens or due to gonadectomy lowers PSA values. A cross-sectional study of Wierckx et al. (63)found median PSA levels of 0.003 ng/mL with an interquartile range of 0.03 to 0.09 in a group of 50 postoperative transwomen using hormonal therapy on an average of 10 years. Therefore a serum level of PSA >1.0 ng/mL may already be a reason for suspicion in transwomen (64).

 

Prolactinoma

Serum prolactin concentrations usually rise slightly in response to estrogen administration and more so by cyproterone acetate (65,66). Based on case reports, it was initially believed that prolactin concentrations in transwomen had to be regularly monitored because of their increased risk for prolactinomas. Surprisingly, a very recent cohort study suggests that the occurrence of prolactinomas in transwomen using hormonal therapy is not higher than that in ciswomen, and that regular prolactin checks are not necessary (67). However, cyproterone acetate should be continued no longer than necessary.

 

Meningioma

Several meningiomas have been reported in transwomen. The current estimated incidence rate of this type of tumor is 33 per 100,000 person-years. This incidence rate is 4 times higher than the incidence rate in ciswomen and 12 times higher than the incidence rate in cismen (67,68). It has been suggested that the occurrence of meningiomas in transwomen is mainly related to cyproterone acetate usage as progesterone receptors are abundantly expressed in human meningiomas (67). Since the occurrence of meningiomas is still rare in transwomen, regular screening for this type of tumor seems not necessary. It is recommended to continue cyproterone acetate no longer than necessary.

 

Other Types of Cancer

As sexually transmitted infections may be more prevalent in transwomen, tumors related to sexually transmitted infections, such as Kaposi sarcoma or anal cancer, may also occur more often. Indeed, disproportionately high incidences of these types of tumors have been found in the transgender population (55,69). Some case reports have been published on cancer in surgically constructed organs like the neo-vagina in transwomen (70,71). While the incidence of these types of tumors seem to be very low it is important to be aware of this possibility.

 

TRANSMEN

 

Breast Cancer

Cases of breast cancer have been reported in transmen before mastectomy (59,72,73). It is important to know that because of cosmetic reasons not all glandular tissue is removed during a mastectomy in transmen. Indeed, several cases of breast cancer have been reported in transmen who already had received mastectomy (59,73–75). The incidence of breast cancer in transmen who have received mastectomy seems higher than in cismen, but much lower than in ciswomen (59). While physicians and transmen have to be aware of their risk of breast carcinoma after mastectomy, it seems unnecessary for transmen to participate in the screening programs for women. However, for transmen with a genetic predisposition for breast cancer, more radical forms of mastectomy could be considered.

 

Endometrial Cancer

Not all transmen choose to remove their uterus. Menstruation usually ceases in transmen receiving testosterone therapy. Testosterone can be converted into estradiol, which may induce proliferation of the endometrium. These mechanisms may induce a higher risk of endometrial cancer in transmen. Women with polycystic ovary syndrome who do not menstruate and suffer from hyperestrogenism, have a thicker endometrium and a higher risk of endometrial cancer (76). It is also possible that the risk for endometrial cancer in transmen using testosterone is lower due to complete atrophy of the endometrium (55). There is currently only 1 case of endometrial cancer reported in a transman using testosterone (77). But it is important to know that, until recently, many countries required removal of female sex organs before transmen could change their sex on the birth certificate. Therefore, long-term follow-up data about testosterone receiving transmen with a uterus are lacking. This makes it impossible to draw hard conclusions. Nevertheless, in transmen with non-cyclic vaginal blood loss, we recommend to perform a vaginal ultrasound.

 

Cervical Cancer

Transmen in whom the uterus has not been removed have a risk of cervical carcinoma. Human papilloma virus is the most important risk factor for developing cervix carcinoma. Studies in ciswomen show that testosterone may also be a risk factor (78). To date, only 2 cases of cervical carcinoma in transmen have been described (77,79). Again, it is important to keep in mind that, until recently, many countries required removal of female sex organs before a transman could change the sex on the birth certificate, which makes the data available limited. As there is no evidence for a decreased risk of cervical carcinoma in transmen, it seems reasonable for transmen with a uterus to participate in screening/HPV vaccination programs for ciswomen. It is important to inform transmen about the need for this screening as they probably do not receive invitations from screening organizations.

 

Ovarian Cancer

Endometrial epidermal growth factor receptor, which is stimulated by testosterone, is commonly found in ovarian cancer cells, and its expression has been associated with poor prognosis (80). However, whether testosterone therapy increases the risk for ovarian cancer in transmen has not been elucidated yet. To date, 3 cases of ovarian cancer have been reported in transmen using testosterone (81,82). Future studies need to provide more evidence about the risk of gynecological cancers in transmen. Until then, screening for ovarian cancer seems unnecessary.

 

EVALUATION OF TRANSGENDER PEOPLE RECEIVING HORMONAL THERAPY

 

Since hormonal therapy is associated with several side-effects it is recommended that medical conditions which can be exacerbated by hormonal therapy are addressed before the start of therapy. During hormonal therapy it is advisable to regularly measure hormone concentrations and maintain them in the normal physiological range. For transwomen estradiol levels between 100 to 200 pg/mL (367 pmol/L to 734 pmol/L) and testosterone levels of <50 ng/dL (<2 nmol/L) are recommended. For transmen, the testosterone level is dependent on the specific assay, but is typically 320 to 1000 ng/dL (11 nmol/L to 35 nmol/L) (10). However, the peak testosterone level after a short acting testosterone injection is often (much) higher than 1200 ng/dL (42 nmol/L). It is also recommended to regularly measure glucose concentrations, lipid panel, and blood pressure during hormonal therapy in both transwomen and transmen, hematocrit in transmen, and electrolytes in transwomen receiving spironolactone (in the first year at baseline and 3 and 12 months, hereafter every 6 months to 2 years).

 

SURGERY

 

Many transgender people choose to have surgery in addition to hormonal therapy. There are several surgical options. While some types of surgery affect fertility, such as vaginoplasty in transwomen or oophorectomy/hysterectomy in transmen, others do not, such as breast surgery in both transwomen and transmen. For surgery which affects fertility, most guidelines recommend the usage of gender-affirming hormones for at least 12 months, which is based on expert consensus that 12 continuous months of living in the experienced gender role is needed for transgender individuals to experience and socially adjust in the desired gender role (5,10).

 

Surgical Options in Transwomen

 

ORCHIECTOMY

Orchiectomy can be performed independently or as part of a vaginoplasty. Orchiectomy is a relatively low-risk procedure (83). After orchiectomy the antiandrogens are no longer necessary and can discontinued.

 

VAGINOPLASTY

 

During a vaginoplasty an orchiectomy (if not previously performed) is performed, in combination with an amputation of the penis, the creation of a neovaginal cavity with lining, the reconstruction of a urethral meatus, and the creation of labia and clitoris. The most frequent procedure is penile inversion vaginoplasty during which the penile skin is used a pedicled flap for the vaginal lining. Since the amount of penile skin is limited, the penile skin flap is often combined with a scrotal skin flap. To prevent hair in the posterior lining of the vagina, hair removal therapy is desirable before penile inversion vaginoplasty. An alternative for penile inversion vaginoplasty is intestinal vaginoplasty, which is a good option in cases in which insufficient skin is available (for example in transwomen who have received puberty blockers during adolescence (83)).

 

BREAST AUGMENTATION

 

Many transwomen choose for breast augmentation since they are not satisfied with their hormone-induced breast growth. The breast augmentation procedure does not differ from an breast augmentation in ciswomen (83).

 

FACIAL FEMINIZATION SURGERY

 

Facial feminization surgery includes a wide range of craniomaxillofacial surgical procedures which are designed to create more feminine facial features. Overall, facial feminization surgery seems a highly efficacious and beneficial procedure for transwomen (84).

 

VOCAL CORD SURGERY

 

Surgically shortening of the vibrating vocal cords or increasing the vocal cord tension can raise the voice pitch in cases that voice therapy does not achieve the desired effect (83).

 

CHONDROLARYNGOPLASTY

 

During chondrolaryngoplasty (tracheal shaving) the prominence of the thyroid cartilage is reduced. Chondroplasty can be performed alone or in combination with vocal cord surgery. Reduction of the Adam’s apple can have positive effects on the psychological well-being of transwomen (85).

 

Surgical Options in Transmen

 

SUBCUTANEOUS MASTECTOMY

 

Most transmen choose mastectomy. A mastectomy in transmen which is performed for aesthetic reasons differs from a mastectomy in ciswomen which is performed because of breast cancer. During the mastectomy in transmen not all glandular tissue is removed. In addition, (more) attention has to be paid to reduction and adequate positioning of the nipple areola complex, destruction of the inframammary fold, and minimization of scars (83).

 

HYSTERECTOMY

 

Many transmen desire uterus extirpation with or without a salpingo-oophorectomy for gender affirmation, pelvic pain, persistent vaginal blood loss, or cancer-risk reduction. It is preferred to use a vaginal approach instead of a transabdominal approach although this could be technically challenging as many transmen have not experienced penetrative intercourse and are on testosterone therapy (86). Transmen who also want a colpectomy can also choose to have a robot-assisted laparoscopic hysterectomy (with salpingo-oophorectomy) in combination with a robot-assisted laparoscopic colpectomy (87).

 

COLPECTOMY

 

Transmen may choose for a colpectomy (removal of the vaginal epithelium) for several reasons, such as unwanted vaginal discharge in general or as result of sexual arousal, or the wish for phalloplasty with urethral extension. There are two options for colpectomy, the vaginal approach and the robot-assisted laparoscopic colpectomy in combination with hysterectomy and salpingo-oophorectomy. The robot-assisted procedure seems to be safer than the vaginal approach (87). 

 

PHALLOPLASTY

 

Phalloplasty is the surgical creation of a full-size penis. It is a difficult surgical procedure with high rates of complications such as urethral stenosis. An ideal phallus has sufficient length for vaginal penetration, has sensibility, and, if desired, enables the transman to urinate in standing position. Multiple flaps have been used to create the phallus, but for penile reconstruction the free radial forearm flap remains the gold standard (83,88).

 

METADOIDIOPLASTY

 

During a metoidioplasty a microphallus is created by using the testosterone-induced hypertrophied clitoris. While a metadoidioplasty gives a lower risk for complications than a phalloplasty, it cannot be guaranteed that voiding in the standing position is possible. In addition, vaginal penetration will not be possible (83,88).

 

FERTILITY PRESERVATION

 

It is estimated that about 47% of transgender individuals would like to have a child to whom they are genetically related (89). Gender affirmation therapy, both hormonal and surgical, is an indication for fertility preservation since hormonal therapy adversely affects fertility, and surgery may include gonadal removal. While the adverse effects of hormonal therapy may be reversible when the therapy is ceased, it is important to discuss fertility preservation options with a transgender individual before the start of hormonal therapy (90). In transwomen the percentage that would have frozen sperm if this option was offered, varied from 13% in asexual or heterosexual (being attracted to men) transwomen to 56% in homosexual (being attracted to women) and bisexual transwomen (91). It is estimated that about 37% (92)of the transmen wish to have their gametes preserved before any gender affirming therapy. For transgender adolescents it is important to involve parents in the fertility preservation counseling as they play an important role in exploring options for their children and usually have to give their consent to interventions. A recent study found that parents overall did not emphasize the importance of their child having children to whom they are genetically related but they agreed that that fertility preservation counseling is relevant (93).

 

Transwomen

 

Semen cryopreservation using specimens obtained from masturbation or penile vibratory stimulation is technically the most easy, reliable and inexpensive method for fertility preservation in transwomen. However, for some transwomen this option may not be possible because of the psychologically distress induced by this procedure, or the difficulties in erection and ejaculation. Alternatives are electro-stimulation or surgical sperm retrieval, or in case of azoospermia, testicular sperm extraction (90). The obtained sperm can be used to fertilize the partner of the transwoman if this partner is female. In case of a male partner a gestational surrogate is needed for fertilization.

 

Transmen

 

For transmen fertility options are embryo cryopreservation, oocyte cryopreservation, and ovarian tissue cryopreservation. As long as the ovaries and uterus are in situ, it is also possible for a transgender man to become pregnant spontaneously. Since testosterone therapy may be dangerous for fetal development it is important that testosterone therapy be discontinued before the transman becomes pregnant. In contrast to the other options, ovarian tissue cryopreservation is more experimental and not widely available. For embryo creation or to fertilize a preserved oocyte, sperm from an intimate partner or an anonymous donor, is needed. When a transgender man does not want to carry the child, a gestational surrogate is also needed. However, surrogacy for transgender individuals is still not widely available due to ethical and legal issues. Furthermore it is important to note that all fertility options in transmen are sooner or later accompanied with controlled ovarian hyperstimulation, which could be very distressing for a transman (90).

 

SPECIAL TOPICS

 

Hormonal Therapy in Older Transgender People

 

Some transgender people start the transition to their experienced gender at an older age (often after a long time of struggling), even past the age of 50 or 60 years (23,94). The majority of these people are currently transwomen (94), but the number of transgender individuals, and especially transmen is currently rapidly growing, possibly due to a greater tolerance and acceptability in society (2). There is no evidence that the manifestations of biological effects of sex hormones will be less in the elderly than in younger people (13,95,96). Age itself should not be regarded as a contraindication to start with hormonal therapy, but the risks of side-effects may be higher at an older age (97).

 

Unsupervised Use of Hormonal Therapy

 

Ideally, the indication for hormonal therapy is the result of psychological assessment that concludes that medical treatment will bring relief to an individual suffering from gender dysphoria (98). However, it is not uncommon that transgender people self-medicate. The use of health care facilities specialized in gender care may be unaffordable, difficult to assess due to long waiting lists, or people are unwilling to undergo psychodiagnostic assessment of their gender problems. Hormones are relatively easy to obtain, and peer groups and the internet provide (sometimes misguided) information on their use (99–101). Frequently, contraceptive pills containing ethinylestradiol with its associated risks (45,102)are used in high doses. Our knowledge about self-medication in the transgender population is very limited, but a study has indicated increased side-effects with illicit use of hormones (101). Another study found 23% of applicants for treatment had used sex hormones already. Remarkably, 32% were transwomen and 6% transmen. The individuals who had used self-prescribed hormones had much less knowledge about appropriate use and potential side-effects (103). Physicians should be aware of illicit hormone use and intervene when necessary.

 

CONCLUSIONS

 

Transgender care is a challenging, multidisciplinary, and developing field in medicine. The transgender population is rapidly growing and the existence of non-binary or gender queer genders gets increasingly more attention.Before the start of any type of therapy, the physician needs to discuss the pros and cons of the several treatment options so that the transgender individual can make a well-considered decision. Due to the increase of high-quality studies, hormonal and surgical therapy will probably be further optimized in the (near) future. Therefore, it is important for physicians who provide transgender care to stay up-to-date with the latest literature. In addition, as the waiting lists may be growing due to the rapidly the increasing number of new applications to gender clinics, physicians should be aware of a growing number of transgender individuals who use self-prescribed hormones.

 

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Lipid and Lipoprotein Metabolism in Liver Disease

ABSTRACT

 

The liver plays a central role in lipid metabolism, serving as the center for lipoprotein uptake, formation, and export to the circulation. Alterations in hepatic lipid metabolism can contribute to the development of chronic liver disease, such as nonalcoholic fatty liver disease (NAFLD) and add to the progression of other chronic liver disease, as occurs in hepatitis C. Moreover, chronic liver disease can impact hepatic lipid metabolism leading to alterations in circulating lipid levels contributing to dyslipidemia. This chapter discusses the interplay between lipid metabolism and chronic liver diseases focusing on NAFLD, alcoholic liver disease, hepatitis C, hepatitis B, cholestatic liver disease, and cirrhosis.

NONALCOHOLIC FATTY LIVER DISEASE

 

Case Presentation

 

A 60-year-old woman with a past medical history significant for hypertension, dyslipidemia and diabetes mellitus presents for management of newly diagnosed nonalcoholic steatohepatitis (NASH). She has a strong family history of coronary artery disease and a personal history of dyslipidemia characterized by a serum triglyceride level of 220 mg/dl, low-density lipoprotein (LDL) cholesterol of 180 mg/dl, high-density lipoprotein (HDL) cholesterol of 50 mg/dl and total cholesterol of 274 mg/dl. Based on these values, her primary physician has recommended she start a lipid lowering medication. However, with her history of liver disease she is uncertain whether she can safely take lipid-lowering medications.

 

Introduction

 

Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the United States, affecting up to a third of adults (1,2). NASH is the progressive form of NAFLD and can lead to cirrhosis, hepatocellular carcinoma, and the need for liver transplantation. In addition to significant morbidity and mortality from end-stage liver disease, NAFLD confers an increased risk of cardiovascular disease (CVD) (3). CVD is the leading cause of mortality among individuals with NAFLD (4). The dyslipidemia of NAFLD may be one of several important and modifiable CVD risk factors.

 

Changes in Lipoprotein Metabolism and Clinical Manifestations

 

DEVELOPMENT OF STEATOSIS

 

NAFLD is characterized in part by steatosis, excess lipid deposition as lipid droplets within hepatocytes. These lipid droplets consist largely of triglycerides and are the result of an imbalance of hepatic lipid handling. Steatosis can occur when one or more of the following conditions is present; 1) excess delivery of free fatty acids (FFA) to the liver from adipose tissue, 2) increased de novo lipogenesis (DNL) within the liver, 3) decreased oxidation of fatty acids within hepatocytes and 4) impaired export of triglycerides from the liver in the form of very-low density lipoproteins (VLDL).

 

Excess FFA Delivery to the Liver

 

When excess adiposity and insulin resistance are present, FFA release from adipocytes is increased (5). Upon release FFA are then delivered via the circulation to the liver and may overwhelm the liver’s capacity to oxidize or export lipids, contributing to the development of steatosis. The fatty acid translocase FAT/CD36 mediates uptake of FFA into the liver and is upregulated in human and experimental NAFLD, which may contribute to steatosis (6,7,8).

 

Increased DNL

 

Hyperinsulinemia, often seen in the setting of obesity and the metabolic syndrome, can also contribute to DNL as the result of increased transcriptional activities of sterol regulatory element binding protein (SREBP) 1c- and peroxisome proliferator-activated receptor (PPAR)-γ (5,9,10). Increased circulating glucose levels also mediate lipogenesis via cholesterol regulatory element binding protein (ChREBP) activation (11). The increased synthesis of lipids within the liver can lead to accumulation within hepatocytes and can promote the development of steatosis.

 

Insufficient Export of Hepatic Triglycerides

 

Export of triglycerides from the liver requires the formation of VLDL and when VLDL formation is impaired steatosis can develop. VLDL are formed when triglycerides are complexed to apolipoprotein B100 (apoB100) via the action of microsomal triglyceride transfer protein (MTP). Steatosis can develop when any of the components of VLDL formation are missing or impaired. Genetic or pharmacologic alteration of MTP or the truncation or absence of ApoB100 can lead to steatosis (12-16). In addition, ApoB100 levels can be decreased by FFA accumulation. FFA accumulation within the liver can lead to chronic stress of the hepatocyte endoplasmic reticulum (ER). Increased ER stress results in increased ApoB100 degradation, decreasing the ability of the liver to export triglycerides and potentially worsen steatosis.

 

Complete VLDL assembly and secretion relies on several additional steps. Following the formation of nascent VLDL particles, further lipidation is needed to create mature VLDL particles. The process of this lipidation is not well understood but may rely on fusion with lipid droplets (17). Interruption of this process of lipid mobilization from lipids droplets to VLDL may also contribute to the development of steatosis (18). Recent genetic studies have shown a strong link between a polymorphism in the gene patatin-like phospholipase domain-containing 3 (PNPLA3) and NAFLD. This coding region polymorphism (I148M) reduces hepatic VLDL secretion, possibly by interfering with triglyceride mobilization and results in hepatic steatosis (19-21). However, conflicting data indicates there may be a compensatory increase in VLDL export in some NAFLD patients, although this increase is insufficient to counterbalance the elevated hepatic triglyceride content (22). The transmembrane 6 superfamily 2 (TM6SF2) E167K variant results in decreased hepatic VLDL secretion and is associated with NAFLD, fibrosis and cirrhosis in the setting of decreased LDL and triglyceride levels. This variant is associated with progressive liver disease but a decreased risk of cardiovascular disease (23,24). Familial hypobetalipoproteinemia (FHBL) is a condition characterized by diminished levels of functional ApoB100, resulting in impaired VLDL export and the development of hepatic steatosis. Magnetic resonance spectroscopy studies have shown liver fat content in individuals with FHBL to be five times greater than in controls (25,26). Progress to steatohepatitis, cirrhosis and hepatocellular carcinoma (HCC) has been noted in this population (27,28,29,30).

 

Hepatic Accumulation of Free Cholesterol

 

The degree of hepatic free cholesterol accumulation in NAFLD correlates with presence and severity of cytologic ballooning (31). Decreased expression of ATP-binding cassette (ABC) A1 and ABCG8 cholesterol efflux proteins, may disrupt transfer of cholesterol from hepatocytes, driving up hepatocyte cholesterol (32,33). There is conflicting evidence regarding changes to hepatic uptake of LDL in individuals with NAFLD, with some studies indicating upregulation of LDL receptors resulting in cholesterol overloading (34).

 

CHANGES IN LIPID METABOLISM

 

Dyslipidemia is frequent in adults with radiographic and biopsy-proven NAFLD and is characterized by hypertriglyceridemia, increased LDL particle concentrations, decreased LDL particle size, and decreased HDL levels (35). High ratios of total cholesterol or triglyceride to HDL-cholesterol are associated with NAFLD (36). In addition, non-HDL-cholesterol (non-HDL-C), a composite measure of apolipoprotein-B containing lipoproteins and an important marker of CVD risk, is elevated in individuals with NASH (19). NASH is also characterized by alterations in lipoprotein subfractions. Lipoprotein subfraction assays measure lipoprotein particle size, density and composition. NASH is characterized by large VLDL particle size and decreased LDL and HDL particle size (35). However, there is conflicting data on the association between NASH and VLD particle size (17,18). Furthermore, increased levels of LDL-III and IV particles, atherogenic forms of LDL, and reduced HDL2b levels, a cardioprotective lipoprotein, are observed in NASH (36,37). Fortunately, resolution of NASH is associated with increases in HDL, decreases in triglycerides, and increases in mean LDL particle diameter and the frequency of LDL phenotype A (39).

 

Insulin Resistance

 

Insulin resistance is a fundamental aspect of NAFLD and can result in many of the alterations in lipid metabolism and circulating lipid levels seen in NAFLD.

 

INSULIN RESISTANCE INCREASES CIRCULATING LDL, VLDL AND TRIGLYCERIDES LEVELS

 

Insulin resistance can increase circulating VLDL and triglyceride levels via several mechanisms. Insulin resistance leads to a loss of suppression of MTP transcription, which increases the efficiency of VLDL assembly (40,41). Insulin resistance also impacts VLDL levels by decreasing lipoprotein lipase (LPL) levels. LPL is an enzyme found on the endothelial cells within muscle and adipose tissue. LPL hydrolyzes triglycerides from circulating VLDL and facilitates triglyceride delivery to muscle and adipose tissues. In the setting of insulin resistance, LPL is downregulated decreasing the clearance of VLDL from the circulation and increasing circulating VLDL levels (42).

 

Insulin resistance can also act via ApoCIII levels to increase circulating VLDL and triglyceride levels. ApoCIII, a lipoprotein found on VLDL, inhibits LPL and can decrease VLDL clearance from the circulation (43). In the setting of insulin resistance, ApoCIII levels are increased, leading to decreased VLDL/triglyceride clearance and resulting in hypertriglyceridemia and increased VLDL levels. ApoCIII also appears to modulate plasma triglyceride levels via LPL-independent mechanisms. In patients with LPL deficiency due to familial chylomicronemia syndrome, administration of an ApoCIII mRNA inhibitor for 13 weeks reduced plasma triglycerides by 56-86% (44).

 

Insulin resistance also impacts LDL metabolism via upregulation of hepatic lipase and increased LDL receptor degradation. Hepatic lipase is an enzyme that remove triglycerides from intermediate-density lipoproteins (IDL) leading to the development of smaller, denser low-density lipoproteins. In NAFLD and insulin resistance, hepatic lipase levels are upregulated leading to increased levels of small, dense LDL (sdLDL) (45). Insulin can also increase circulating LDL levels via its effects on the LDL receptor. Insulin upregulates proprotein convertase subtilisin/kexin type 9 (PCSK9), a protein that can bind and degrade the LDL receptor (46). Upregulation of PCSK9 leads to decreased LDL receptor availability on hepatocytes and increased circulating LDL levels.

 

INSULIN RESISTANCE DECREASES CIRCULATING HDL LEVELS

 

Insulin resistance decreases circulating HDL levels by interfering with HDL particle assembly. HDL is formed within plasma at the surface of the hepatocyte and requires the interaction of ApoA-1 and ABCA1 (47). Nascent HDL particles are formed when ApoA-1, secreted by the liver or released from other lipoproteins, is lipidated by ABCA1 with phospholipids and free cholesterol. Insulin resistance hampers HDL formation by promoting the phosphorylation and degradation of ABCA1 and by reducing ABCA1 activity (48). In addition to hampering HDL production, insulin resistance may interfere with reverse cholesterol transport. Insulin resistance can result in the formation of particularly triglyceride-rich HDL particles via the action of cholesterol ester transfer protein (CETP) (49). Triglyceride-rich HDL are taken up more rapidly by the liver and may result in lower circulating HDL levels.

 

Management

 

Diet and exercise are the foundations of the management of both NAFLD and the dyslipidemia of NAFLD. Small studies have indicated that both a low carbohydrate diet as well as the Mediterranean diet may improve serum lipid levels and NAFLD (50-52). Further, adherence to a Mediterranean diet reduces the development of CVD (53). As CVD is a cause of considerable morbidity and mortality in NAFLD patients, adherence to a Mediterranean diet may have multiple benefits.

 

Routine aerobic exercise, defined as 30 minutes of moderate exercise most days of the week, can result in significant improvements in lipid levels and may improve hepatic lipid content (54,55). Individuals with NAFLD should be advised to participate in regular, aerobic exercise.

 

LIPID LOWERING MEDICATIONS

 

HMG-CoA Reductase Inhibitors

 

When diet and exercise are insufficient in individuals with NAFLD, HMG-CoA reductase inhibitors or “statins” are recommended. Statins play an important role in both the primary and secondary prevention of CVD and should be used in patients with NAFLD and dyslipidemia. Compared to placebo, statins have been shown, in a post-hoc analysis of the Greek Atorvastatin and Coronary Heart Disease Evaluation (GREACE) study, to significantly reduce cardiovascular events in individuals with NAFLD (56). Statins have also been shown to exert a protective effect on liver histology in patients with NAFLD/NASH, with dose-dependent reduction in steatosis, steatohepatitis and fibrosis stages F2-F4, although protection against steatohepatitis in the presence of the I148M PNPLA3 risk variant did not reach statistical significance (57).

 

It is important to note that while there remains a concern among physicians about statin hepatotoxicity, the incidence of statin-induced hepatotoxicity in the general population is extremely low and is not increased in individuals with NAFLD or NASH (58-60). Apprehension among physicians may partly account for the current under prescribing of statins in patients with NAFLD (61,62).

 

Omega-3 Fatty Acids

 

Omega-3 fatty acids can be used in patients with NAFLD for the treatment of isolated hypertriglyceridemia or when statins alone are insufficient to control triglyceride levels. Omega-3 fatty acids act to reduce hepatic VLDL secretion and lower serum triglyceride levels. Doses of up to 4 grams daily can decrease triglycerides by 25-35% (63). Omega-3 fatty acids may reduce radiographic steatosis and several randomized controlled trials (RCTs) of omega-3 fatty acids are ongoing to determine their impact on NASH histology (64-66).

 

Cholesterol Absorption Inhibitors

 

A further class of drugs which may hold promise are the cholesterol absorption inhibitors, of which ezetimibe has been most extensively studied. A recently conducted RCT involving 32 NAFLD patients found that ezetimibe use led to significant improvement in fibrosis stage and ballooning score (67). Of note, Loomba et al. reported no significant impact of ezetimibe on liver fat content, as assessed by magnetic resonance imaging proton density-fat fraction and liver biopsy (68). The influence of ezetimibe on the various stages of NAFLD pathogenesis remains to be fully characterized. Further large-scale RCTs are warranted to explore ezetimibe’s potential as a component of NAFLD/NASH therapy alongside statins.

 

LIPID TREATMENT GOALS

 

We recommend that patients with NAFLD adhere to the Cholesterol Clinical Practice Guidelines from the American Heart Association and American College of Cardiology released in 2018. The guidelines recommend that all adults with any form of CVD or an LDL ≥ 190 mg/dL should be treated with high intensity statins for a goal 50% reduction in LDL. Patients aged 45-70 years with diabetes with LDL < 189 mg/dL or patients with > 7.5% global 10-year CVD-risk should receive moderate intensity statins for a goal 30-50% reduction in LDL. A specific target LDL is no longer formally recommended.

 

Return to Case

 

For our patient with NAFLD it would be both safe and important for her to take lipid-lowering medication to manage her dyslipidemia and reduce her risk of a CVD development. She would benefit from administration of a statin of either moderate or high intensity, based on the outcome of risk assessment.

 

Table 1. Key Points- Non-Alcoholic Fatty Liver Disease

NAFLD is associated with insulin resistance which results in atherogenic dyslipidemia characterized by increased small dense LDL and triglyceride levels and decreased HDL levels.

The dyslipidemia of NAFLD may contribute to the increased risk of CVD observed in individuals with NAFLD

Patients with NAFLD and NASH should be treated for their dyslipidemia to reduce their CVD risk.

Individuals with NAFLD can be treated with statins without increased risk of hepatotoxicity.

 

ALCOHOLIC LIVER DISEASE

 

Case Presentation

 

An obese 48-year-old man with a past medical history significant for coronary heart disease, hypertension, and diabetes mellitus presents for management of newly diagnosed hepatic steatosis. He has a family history of coronary artery disease. He admits to consuming 3 glasses of wine per night during the week and an additional two per evening on weekends. His fasting plasma triglyceride concentration is 350 mg/dl, his LDL cholesterol is 130 mg/dl, and HDL cholesterol is 55 mg/dl. The alanine aminotransferase level (ALT) is modestly elevated at 55 IU/ml. He would like to know whether he has NAFLD and whether you recommend continuing his current alcohol intake to protect against CVD, especially since he was told that his good cholesterol was elevated.

 

Introduction

 

Alcoholic liver disease (ALD) accounts for nearly half of cirrhosis-related mortality in the United States (69). A hallmark feature of ALD is hepatic steatosis, which develops in more than 90% of heavy drinkers. However, less than one third of these individuals develop complications that include alcoholic hepatitis, cirrhosis and HCC (69). Risk factors for disease progression include female sex, obesity, drinking patterns, dietary factors, non–sex-linked genetic factors, and cigarette smoking (70,71). Alcohol also synergizes with other etiologies of chronic liver disease, including NAFLD and viral hepatitis to accelerate progression (69). Hypertriglyceridemia is the primary dyslipidemia associated with alcohol ingestion (72), and a J-shaped association exists between alcohol intake and CVD (73), which may reflect a parallel effect of plasma triglycerides (72). Although its contribution to metabolic syndrome is unclear, alcohol intake appears to interact with obesity to further increase plasma triglyceride concentrations (72).

 

Changes in Lipoprotein Metabolism and Clinical Manifestations

 

DEVELOPMENT OF STEATOSIS

 

As with NALFD, the development of steatosis in response to alcohol is multifactorial. Alcohol impairs the β-oxidation of fatty acids by mitochondria, promotes de novolipogenesis in the liver, and increases fatty acid uptake. As is the case in NALFD, VLDL secretion is also increased due to alcohol.

 

Excess FFA Delivery to the Liver

 

As is the case for NAFLD, fatty acids from extrahepatic sources appear to contribute to hepatic steatosis. In addition to increasing mobilization of fatty acids from adipose tissue (74), alcohol intake augments the supply of lipids to the liver from the small intestine in the form of chylomicron remnants (75).

 

Increased DNL

 

Increased DNL contributes to alcohol-related steatosis by direct and indirect mechanisms (69). The alcohol metabolite acetaldehyde increases transcription of SREBP1c, which upregulates transcription of lipogenic genes. Alcohol-induced endoplasmic reticulum stress and inflammation leads to increased processing of the SREBP1c protein within hepatocytes. Alcohol also inhibits proteins that suppress lipogenesis. The protein deacetylase Sirtuin 1 (SIRT1), plays a central role (76). Suppression of SIRT1 by alcohol leads to hyperacetylation of a group of molecules, including those that promote lipogenesis. Inhibition of adenosine monophosphate kinase (AMPK) contributes, because AMPK-mediated phosphorylation of SREBP1c reduces transcriptional activity. AMPK also phosphorylates and inhibits acetyl-CoA carboxylate (ACC), the rate-limiting step in lipogenesis.

 

Impaired Oxidation and Degradation of Fatty Acids

 

Alcohol decreases mitochondrial fatty acid oxidation principally by decreasing activity of the transcription factor peroxisome proliferator activated receptor (PPAR) α. This occurs in response to increased NADH/NAD+ ratios and decreased AMPK activity, among other factors (69). PPARα promotes the transcription of genes that mediate fatty acid oxidation. Alcohol intake may also inhibit autophagy (69), which plays an important role removing lipids from the liver (77).

 

Insufficient Export of Hepatic Triglycerides

 

Alcohol increases VLDL secretion (72,78), apparently by increasing the transcription of MTP (74). The increased in export of hepatic triglycerides is insufficient to offset the accumulation due to increases in fatty acid uptake and synthesis in the setting of decreased oxidation.

 

HYPERTRIGLYCERIDEMIA

 

Increased VLDL secretion contributes to hypertriglyceridemia that is observed in the setting of alcohol consumption. This is exacerbated by decreased expression of LPL (79), which promotes clearance of VLDL triglycerides into muscle and fat tissue. There is also an interaction between alcohol consumption and genetic polymorphisms in apoCIII, which circulates in the plasma and functions to inhibit lipoprotein lipase activity (80).

 

CIRCULATING HDL LEVELS

 

Alcohol increases HDL lipids and apolipoproteins in patterns that depend upon amount of consumption: Moderate consumption tends to increase plasma concentrations of smaller HDL particles, whereas heavier consumption favors larger HDL particles (81). Alcohol interacts with HDL metabolism in multiple steps, which can ultimately lead to increased reverse cholesterol transport, the process by which cellular cholesterol is transported to the liver for elimination into bile (81,82). Heavier alcohol consumption impairs CETP activity, so the typical inverse relationship observed under circumstances associate with NAFLD is not necessarily observed in the setting of alcohol use and HDL may be increased as well (72,83). Moderate alcohol consumption also appears to enhance the anti-inflammatory and anti-oxidant properties of HDL particles (81).

 

CIRCULATING LDL LEVELS

 

The effects of alcohol on plasma LDL cholesterol concentrations is less consistent than observed for HDL, with different patterns observed in different populations, which may be attributable to genetic polymorphisms with these populations (81).

 

Management

 

Although considerable anecdotal evidence exists to support a CVD benefit of moderate alcohol consumption, insufficient data are available to translate this concept into a clinical recommendation. In the setting of alcohol-related hepatic steatosis, cessation of drinking, along with therapeutic lifestyle modifications, are the mainstays of therapy.

 

Return to Case

 

The diagnosis of NAFLD is based on the absence of significant alcohol consumption. For a man, the upper limit of alcohol intake is 2 drinks per day. This means that this patient cannot be categorized simply as NAFLD, although the coexistence of alcoholic liver disease and NAFLD is likely in this patient. He is at high risk for CVD, so should be managed accordingly, including lipid lowering therapy with statins. His alcohol consumption should be reduced to less than 2 drinks per day, which may help reduce his fasting triglyceride concentrations. He should not be falsely reassured by his elevated HDL cholesterol concentration.

 

Table 2. Key Points- Alcoholic Liver Disease

The consumption of alcohol is a common cause of excess fat accumulation in the liver.

There are multiple mechanisms by which alcohol promotes hepatic steatosis.

Alcohol can increase plasma HDL cholesterol concentrations and fasting triglyceride concentrations.

Although modest alcohol consumption is associated with reduced CVD risk, this cannot be recommended due to other potential adverse effects, including alcoholic liver disease.

 

VIRAL HEPATITIS-- C

 

Case Presentation

 

A 65-year-old woman with a past medical history of CVD and untreated genotype 1 chronic hepatitis C presents for management of CVD. Her lipid levels are notable for an LDL of 99. She has read that since her LDL is below the recommended level for patients with CVD she would not benefit from lipid lowering therapy. What would you advise her?

 

Introduction

 

Hepatitis C virus (HCV) is a positive-strand RNA virus of the family Flaviviridaethat can lead to chronic infection as well as the development of cirrhosis, HCC, and the need for liver transplantation. Chronic HCV (CHC) infection impacts between 130 and 170 million individuals worldwide (84).

 

Changes in Lipoprotein Metabolism

 

HCV replication is intricately linked with host cell lipids and impacts host lipid metabolism. Circulating HCV virions complex with host lipoproteins and form lipoviroparticles (85). This lipid composition is a prerequisite for maintenance of viral particle morphology and HCV infectivity (86,87,88,89). For example, lipids on the virion surface shield viral envelope epitopes, protecting them from antibody engagement (90). Lipoviroparticles can enter hepatocytes via multiple receptors including the hepatocyte LDL receptor (which may also facilitate the replication step of the HCV cycle (91)) and utilizes cell surface molecules including Niemann-Pick C1-like 1 (NPC1L1), a receptor for cholesterol resorption, and scavenger receptor class B member 1 (SRB1), which acts to promote cholesterol uptake from lipoproteins, and interacts with HCV envelope glycoprotein E2 to promote HCV entry (92,93,94). LDL receptor and SRB1 appear to have a redundant role in HCV entry (95). Several apolipoproteins influence HCV uptake: apoC1 interacts with HCV glycoproteins to promote infection, and apoE mediates initial attachment between virus and hepatocyte. Hepatocyte VLDL receptor mediates an additional HCV entry mechanism, involving E2 and apoE, with increased VLDL receptor expression conferring greater susceptibility to infection (96). Formation of the HCV core protein involves interaction with host cytosolic lipid droplets and interaction with diacylglycerol O-acetyltransferase 1, a host enzyme involved in triglyceride synthesis. HCV replication also interacts with host cholesterol synthesis within hepatocytes. The host protein FBL2 undergoes geranylgeranylation, an intermediate of the cholesterol synthesis pathway (97). When this pathway is interrupted, the HCV replication complex is extinguished (98). Finally, HCV secretion from hepatocytes involves complexing with apoE-containing host lipoproteins in the form of VLDL or HDL (99).

 

Clinical Manifestations

 

Like NAFLD, HCV infection is associated with the development of hepatic steatosis. However, unlike NAFLD, HCV is also associated with hypolipidemia. CHC infection is associated with significantly lower host LDL and total cholesterol levels than in uninfected controls (100). Treatment is associated with increases in both LDL and cholesterol levels in patients with HCV who achieve a cure, defined as a sustained virologic response (SVR). Changes in host serum lipids are also seen in patients with acute HCV. Acute HCV infection is associated with a decrease in total cholesterol, LDL and non-HDL-cholesterol from pre-infection levels. In addition, total cholesterol, LDL, triglycerides and non-HDL-C progressively decline over a 10-year period following HCV seroconversion, after adjusting for BMI and FIB-4 score (101). In patients who achieved viral clearance, either spontaneous or treatment-induced, total cholesterol, LDL and non-HDL-C increased significantly from infection levels. In an important proportion of patients with both acute and chronic infection, post-viral clearance lipid levels exceed pre-infection levels (102).

 

While HCV infection is associated with a decrease in LDL and non-HDL-C, important CVD risk factors, HCV infection is associated with an increased overall risk of CVD (103,104). When non-HCV infected individuals with similar lipid levels are compared to those with CHC, HCV infection independently confers an increased risk of acute myocardial infarction (AMI), with a more pronounced increase seen in younger individuals (105). Further, lipid-lowering therapy among individuals with CHC was associated with a greater reduction in AMI risk than uninfected persons with similar lipid levels. Therefore, lipid levels may not accurately reflect CVD risk in patients with CHC.

 

Management

 

Lipid treatment goals for individuals with CHC are not well established. We recommend that patients with CHC adhere to the Cholesterol Clinical Practice Guidelines from the American Heart Association and American College of Cardiology released in 2018 (106). Retrospectively-collected data links statin use to improved liver-related outcomes, with higher likelihood of achieving SVR, and lower rates of fibrosis progression, cirrhosis development, HCC incidence, and mortality amongst patients with CHC (107,108,109,110). Simon et al. identified that atorvastatin and fluvastatin have the most significant anti-fibrotic benefit, compared with simvastatin, pravastatin, lovastatin or no statin use (111). It is important to note that for individuals who have achieved an SVR after HCV treatment, lipid levels often increased to or above pre-infection levels. Induction of SVR using DAA therapy led to pro-atherogenic lipid changes (increased total cholesterol, LDL, LDL/HDL ratio, and non-HDL-C), irrespective of DAA regimen or fibrotic stage, with a parallel reduction in insulin resistance. The balance of these effects with respect to CVD risk remains to be determined (112). Hashimoto et al. found greater increases in serum LDL-cholesterol (LDL-C) levels in patients undergoing therapy with ledipasvir/sofosbuvir compared to daclatasvir/asunaprevir. Decline in HCV core protein was also independently associated with rises in LDL-C (113). Thus, practitioners should be mindful to monitor post-treatment lipid levels and treat appropriately.

 

Return to Case

 

For our patient with CHC and CVD it would be important for her to take a lipid-lowering medication to reduce her risk of a second CVD event. Based on the guidelines, she would benefit from high intensity statin therapy, with a goal of decreasing LDL cholesterol by >50%.

 

Table 3. Key Points- Hepatitis C

The hepatitis C virus interacts with host lipids for hepatocyte entry, viral replication and secretion.

HCV infection decreases host serum LDL and total cholesterol levels.

HCV infection is still associated with an increased risk of AMI and treatment with statins reduces this risk.

Treatment of HCV results in increase in serum lipid levels to at least pre-infection levels.

 

VIRAL HEPATITIS –HEPATITIS B

 

Introduction

 

Approximately 240 million individuals are chronically infected with the hepatitis B virus (HBV) (114). Like HCV, chronic HBV infection can lead to cirrhosis and hepatocellular carcinoma.

 

Lipoprotein Metabolism in Hepatitis B

 

HBV interacts with host lipid metabolism in several important ways including during viral cell entry and formation of a vital viral protein, the HBV surface antigen. HBV uses the Na+-taurocholate cotransporting polypeptide (NTCP), a peptide that normally allows for hepatocyte uptake of host bile acids, to gain access to hepatocytes (115). HBV binding to NTCP impairs the ability of NTCP to promote hepatocyte uptake of bile acids. This results in an increase in conversion of cholesterol to bile acids.

 

The formation of the HBV surface antigen within hepatocytes relies in part on host cell cholesterol (116). The surface antigen particle is synthesized in the membrane of the hepatocyte endoplasmic reticulum (ER) and is associated with the host ER lipid bilayer. Association with the lipid bilayer helps make the particle resistant to degradation by cellular proteases. The surface antigen is then transported to the ER lumen and exported from the hepatocyte as a lipoprotein particle. Approximately 25% of the surface antigen complex is composed of host lipids including phosphatidylcholine, triglycerides, cholesterol and cholesterol esters (116).

 

HBV infection may also alter lipogenic gene expression. Two studies have demonstrated increased in lipogenic gene expression in HBV-infected transgenic mice compared to uninfected mice. HBV-infected transgenic mice have increased gene expression of SREBP2, 3-hydroxy-3-methylglutaryl-coenzyme A reductase, LDL receptor, fatty acid synthase, and ATP citrate lyase, all of which play a role in either cholesterol metabolism or fatty acid synthesis (117,118). Oehler et al also found that in HBV infected humanized mice, gene expression of human apolipoprotein A1, a lipoprotein found in HDL which plays a role in reverse cholesterol transport and PPAR-gamma which regulates adipocyte differentiation and fatty acid storage, was significantly enhanced.

 

HBV-infected transgenic mice also demonstrate elevated levels of 7α-hydroxylase (hCYP7A1), which promotes bile acid formation from cholesterol. In liver biopsy samples from patients with chronic HBV infection, hCYP7A1 was significantly induced when compared to uninfected controls. These findings suggest that HBV replication may impact cholesterol metabolism.

 

Clinical Manifestations

 

Data on the impact of HBV infection on circulating lipid levels in humans is limited. HBV infection may be associated with lower triglyceride levels than in uninfected patients (119), however its influence on HDL remains ambiguous. Hsu et al performed a case control study comparing 322 individuals with chronic HBV infection to 870 age-matched, uninfected controls. Individuals with HBV infection were found to have significantly lower triglyceride and HDL levels when compared to controls. In a second retrospective cohort of 122 individuals with chronic HBV, HBV DNA levels was inversely proportional to serum triglyceride levels but no relationship was seen with HDL levels (119). Amongst a cohort of non-diabetic patients, HBsAg-seropositivity was inversely correlated with hypertriglyceridemia and low serum HDL cholesterol. Hence, chronic HBV infection may favorably impact lipid profiles, which could partly account for the inverse relationship between HBsAg-seropositivity and metabolic syndrome seen in this cohort (120). Similarly, Joo et al. demonstrated that in patients who were initially free of dyslipidemia, HBsAg-positivity was associated with lower risk of developing dyslipidemia during an average follow up of 4.46 years (121).

 

Circulating lipid levels may be predictive of clinical outcomes in HBV-infected patients. Chen et al. found that average plasma apolipoprotein A-V level was decreased amongst 209 non-survivors of HBV-acute on chronic liver failure versus 121 survivors (122).

 

Like HCV, chronic HBV infection is frequently associated with hepatic steatosis. Between 25% and 51% of patients with HBV are found to have steatosis on imaging or biopsy (123). However, while concurrent steatosis is common in HBV infections, steatohepatitis is not frequently described. Further, the pathogenesis of steatosis in HBV is not well understood and may be related to co-existing metabolic factors such as body mass index (BMI) and insulin resistance rather than the viral infection itself (124).

 

Management

 

As data on the impact of HBV infection on circulating lipids is limited there are no formal guidelines for dyslipidemia management in this population. Clinicians should be mindful of a possible decrease in HDL in this population and follow standard guidelines from the American Heart Association and American College of Cardiology on lipid management. Recent studies have shown reduced risk of cirrhosis development (125), decompensation (125, 126, 127, 128), mortality (126, 127) and portal hypertension (126) amongst statin users compared to non-users with chronic HBV- and HCV-related hepatitis. Furthermore, statin use was associated with a 32% reduced HCC risk. Concomitant use of statin and nucleos(t)ide analogue led to an additive chemopreventive effect (129). Large-scale RCTs to comprehensively evaluate statins as a means of protection against disease progression in patients with viral hepatitis are warranted.

 

Table 4. Key Points- Hepatitis B

HBV infection may interact with host lipids and enhance lipogenic gene expression

The clinical manifestations of HBV on host lipids are not well studied but HBV infection may decrease serum triglyceride and HDL levels.

Management of patients with HBV and dyslipidemia should be guided by standard recommendations for the treatment of dyslipidemia.

 

CHOLESTATIC DISEASES

 

Case Presentation

 

A 58-year-old woman is referred with primary biliary cirrhosis (PBC) for the management of an elevated plasma total cholesterol of 450. She reports symptoms only consistent with mild and intermittent pruritis. She is currently taking ursodeoxycholic acid. Her physical is notable for xanthelasma under the eyes.

 

Introduction

 

Bile is the route for cholesterol elimination from the body. Plasma cholesterol is taken up by the liver in the form of apolipoprotein B-containing lipoproteins (i.e. remnant lipoproteins and LDL) by receptor-mediated endocytosis or by selective uptake of HDL cholesterol (130). Cholesterol is eliminated by conversion to bile salts and by biliary secretion. Biliary obstruction, notable when due to cholestatic diseases, can interfere with cholesterol elimination leading to hypercholesterolemia. This occurs most commonly in the setting of primary biliary cirrhosis (PBC), which is an autoimmune-mediated destruction of intrahepatic bile ducts. It can also occur in primary sclerosing cholangitis (PSC), in which there is inflammatory stricturing of larger bile ducts by poorly understood mechanisms.

 

Lipoprotein Metabolism in Cholestasis

 

Hypercholesterolemia associated with cholestasis is largely attributable to the formation of lipoprotein X, an atypical lipoprotein particle. Lipoprotein X comprises principally unesterified cholesterol and phospholipids (131), resembling the cholesterol-phospholipid vesicles that are secreted by the liver into bile (132). The principal proteins associated with lipoprotein-X are apoC and albumin contained within the core (133,134). The lipids of the particle comprise a sphere, with an aqueous core. Lipoprotein-X is devoid of apoB. It appears to be formed due to the secretion of biliary-type particles into plasma in the setting of obstruction to bile flow (135), although defects in plasma cholesterol esterification may also contribute (131). Lipoprotein X has similar characteristics as LDL including density, so that its presence in plasma requires electrophoretic separation (136).

 

Plasma total cholesterol concentrations are increased in PBC in proportion to disease severity, with elevations that can be striking and exceed 1,000 mg/dl, and can be a rare cause of pseudohyponatremia (133). Where these elevations are primarily attributable to lipoprotein-X, apolipoprotein B concentrations may also be elevated due abnormal lipoprotein metabolism associated with liver disease (131,133). Serum metabolomics analysis of patients with PBC revealed elevated levels of VLDL and LDL compared to controls (137). HDL cholesterol concentrations are elevated in the early stages of PBC and tend to decline as the disease progresses (138), apparently because of increased circulating hepatic lipase activity that promotes HDL catabolism (131). Patients with more advanced PBC exhibit increased plasma triglycerides (131), presumably attributable to decreased hepatic lipase activity (138).

 

Plasma lipids in PSC have been less well characterized than in PBC. In a small series (139), the hypercholesterolemia was more modest than generally observed in PBC, but did increase in concert with disease severity. HDL cholesterol levels tended to be high, and triglyceride elevations were uncommon.

 

Clinical Manifestations

 

An important consideration has been whether the lipid abnormalities associated with cholestatic diseases confer increased CVD risk. This has been studied more extensively in PSC in the form of prospective trials (138,140). Although each had limitations, collectively there was no suggestion of increased atherosclerotic events, which is in keeping with the relative absence of elevations in atherogenic particles There is also evidence in vitroto suggest that lipoprotein-X may be atheroprotective by reducing oxidation of LDL (136). In patients with PBC, the presence of xanthelasma does not appear to connote an increased CVD burden (138).

 

As with PBC, the cholesterol elevations associated with PSC do not tend to confer CVD risk. None was observed in the small series cited previously, but it was acknowledged that patients were young enough that excess CVD complications would not have been expected (139). Lipid levels ultimately fell in patients who had progressed to cirrhosis and hepatic failure.

 

Management

 

Due to the overall lack of clinical evidence, the management of hypercholesterolemia associated with cholestasis lacks formal recommendations. In PBC, ursodeoxycholic acid (UDCA) slows the progression of disease and prolongs survival (141).Chronic UDCA administration also reduces plasma LDL concentrations. In PBC patients, statin therapy is generally safe and is effective at lowering LDL cholesterol in PBC patients (58,142-144). At present, UDCA is generally not recommended in the management of PSC (145), and data are lacking regarding lipid-lowering therapies in these patients. Of note, some patients with obstructive jaundice are treated with bile acid binders to reduce pruritus and not primarily to reduce plasma cholesterol concentrations.

 

Return to Case

 

For our patient with PBC, the presence of lipoprotein-X may be confirmed by lipoprotein electrophoresis. The possible contribution of atherogenic particles may be estimated by the measurement of the plasma apoB concentration. The institution of statin therapy should be based on standard estimates of CVD risk.

 

Table 5. Key Points- Cholestatic Disease

Plasma total cholesterol concentrations are commonly elevated in the setting of cholestasis.

Lipoprotein-X is an abnormal lipoprotein that circulates in patients with cholestasis and is primarily responsible for the elevations in plasma total cholesterol concentrations.

Elevations in plasma cholesterol concentrations due to cholestasis do not appear to confer excess CVD risk.

Patients with cholestatic disorders may be candidates for lipid lowering therapy if they are otherwise at risk for CVD.

 

CIRRHOSIS

 

Introduction

 

Cirrhosis is the common advanced histologic endpoint for chronic liver diseases in which the formation of fibrotic nodules in the liver often obscured the etiology of the responsible disease process. The clinical correlates range widely from well-compensated liver function with no apparent clinical manifestations to advanced decompensated liver disease with portal hypertension, with complications that include hepatic encephalopathy, esophageal varices, and ascites. Moreover, the development of cirrhosis confers increased risk of HCC.

 

Changes in Lipoprotein Metabolism and Clinical Manifestations

 

The changes in lipoprotein metabolism associated with cirrhosis generally reflect the degree of impairment of hepatic function. In one study (146), plasma concentrations of total cholesterol, HDL cholesterol, LDL cholesterol, and VLDL cholesterol varied with increases in prothrombin time and decreases in albumin, which reflect hepatic synthetic function.These findings are in general agreement with other studies (147). Lipoprotein compositions are also altered in the setting of cirrhosis, with LDL particles enriched with triglycerides and deficient in cholesteryl esters, and HDL particles enriched with triglycerides, free cholesterol and phospholipids (147). These changes are secondary to characteristic abnormalities in plasma enzymes that remodel lipoproteins, including lecithin-cholesterol acyl transferase (LCAT), hepatic lipase, and phospholipid transfer protein (PLTP) (147). HDL-C and enzymes involved in HDL maturation and metabolism are decreased in patients with cirrhosis. There is a shift in the composition of HDL in those with cirrhosis towards the larger HDL2 subclass, with a reduction in small HDL3 particles. The latter is associated with diminished cholesterol efflux capacity which in turn independently predicts 1-year mortality (148).

 

Hepatocellular carcinoma can occur in the setting of cirrhosis and may be associated with alterations in plasma lipids (149-151). In instances of hypercholesterolemia, the increase may be driven by elevated rates of cholesterol synthesis and cellular levels of 3-hydroxy-3-methylglutarylcoenzyme A. It is unclear whether this hypercholesterolemia confers increased CVD risk (152,153).

 

CVD risk is dependent upon the etiology of cirrhosis, at least in part due to the association of type 2 diabetes. Cirrhosis due to NASH, HCV, and alcoholic liver disease increases the risk of type 2 diabetes, which is not observed in cholestatic liver diseases and presumably contributes to CVD risk (147,154). Statin therapy may be safely administered in patients with compensated cirrhosis and increased CVD risk (58). In patients with non-cholestatic cirrhosis, low HDL cholesterol serves as a liver function test that is an indicator of poor prognosis, increasing the risk of cirrhotic death (155).

 

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Pediatric implications of normal insulin-GH-IGF-axis physiology

Abstract

 

Understanding the involvement of the insulin-GH-IGF-axis in the different phases of human growth, development, and metabolism is the key to understanding human pathophysiology. The normal physiological actions of the axis optimize human growth and metabolism to impact adult height by approximately one third. IGF binding proteins modulate access of circulating IGF-I to the tissues and modulate IGF-I and -II access to the type 1 IGF receptor (IGF1R). Complete lack of IGF1R signaling is most likely not compatible with a viable human fetus, while allelic haploinsufficiency impairs brain development and causes severe short stature. Lack of insulin receptor signaling in Leprechaunism may result in the rare event of an alive but severely small for gestational age baby that will only survive if treated with recombinant-IGF-1 to substitute inulin receptor signaling for IGF1R signaling via their common intracellular pathways. IGF-I gene defects result in mental retardation and severe fetal and postnatal growth failure with GH hypersecretion and marked insulin resistance. Likewise, IGF2 gene defects or imprinting defects cause severe fetal growth failure but somewhat less adverse effects on postnatal growth, more variable effects on brain development, and an absence of marked metabolic effects. GH fine-tunes insulin and IGF-I signaling with no impact on IGF-II expression and has a minor impact on fetal development and growth. GH effects on lipolysis are established in the newborn and ensure gluconeogenesis preventing hypoglycemia after birth. The complete absence of GH such as in GHRHR or GH1 gene defects or absence of GH signaling in GHR or STAT5B gene defect leads to an adult height of 120-130cm if untreated, and has severe metabolic consequences. Excess insulin, GH, IGF-I, and IGF-II signaling are associated with severe metabolic disease and excess growth and/or obesity. Malnutrition or malabsorption causes decreased insulin signaling which reduces GHR expression blocking the GH signaling pathway leading to IGF-I expression (GHR uncoupling), while GH’s metabolic actions on lipolysis and gluconeogenesis are unaffected. GH signaling attenuate insulin actions on glucose metabolism which causes insulin resistance, hyperinsulinemia, and may precipitate diabetes. However, insulin signaling pathways that enhances GHR function or suppress IGFBP-1 or SHBG production are still intact and promote anabolism, optimize growth, enhance androgen actions and play a mechanistic role in premature adrenarche and PCOS. Long-term nutritional deprivation compromises growth, while from a developmental perspective, decreased insulin signaling (leading to GHR uncoupling) prolongs life (in some animal models) which ensures that fertile age is reached, and survival of the species is ensured. For the health of the general population, the subtle changes in insulin, GH and IGF-I signaling associated with gene polymorphisms or epigenetic changes programmed during fetal and early postnatal life that affect gene expression are important. They determine growth and pubertal development in childhood and predispose the individual for developing the metabolic diseases and malignancies in adult life, as predicted by the Barker hypothesis. The roles of the insulin-GH-IGF-axis in growth and metabolism are intimately linked and will be described jointly here.

 

Early work defining the insulin-GH-IGF-axis

 

Daughaday realized that the mitogenic effect of GH in the growth plate was not direct but mediated by Insulin-like Growth Factor-I (IGF-I), at that time named sulfation factor or somatomedin C (1). Another effect of IGF-I was insulin-like and not inhibited by insulin specific anti-bodies (2,3,4) and therefore it was named non-suppressible Insulin-like activity (NSILA). Hall and Van Wyk purified IGF-I from human muscle extracts (5,6) and realized that these biological activities originated from the same molecule. They also identified significant quantities in blood (7). The primary structure of IGF-I and Insulin-like Growth Factor-II (IGF-II) was discovered by Froesch and coworkers as a result of their persistent work to characterize the metabolic activity of NSILA (8,9). Soon after, the mitogenic activity of the sulfation factor or somatomedin C as well as somatomedin A was shown to be identical to IGF-I (10). Rechler and Nissley demonstrated that IGF-II was identical to multiplication stimulating activity, a factor known to stimulate DNA synthesis in chick embryo fibroblasts (11).

 

The concept that binding proteins existed for peptide hormones like the IGFs, similar to those for steroid and thyroid hormones, were suggested by studies from Zapf and Froesch (12) and by Hintz (13), demonstrating that NSILA was present in high molecular weight complexes in serum. The binding was exclusive to IGFs and did not apply to insulin or proinsulin despite their structural similarities. High molecular weight IGF-I complexes with IGFBPs were GH dependent (14) and formed a ternary complex composed of IGFBP-3 (15), the Acid Labile Subunit (ALS) (16), and IGF-I or IGF-II. Low molecular weight complexes contained IGF-I or IGF-II bound to an insulin regulated liver derived protein IGFBP-1 (17, 18), at first called the 28 kDa binding protein or PP12 (19). The existence of other IGF binding proteins, six in total, became clear when Hossenloop (20) developed Western ligand blotting as a technique to quantify these proteins. The components of the IGF-IGFBP-system are outlined in Figure 1.

Figure 1. The IGF-IGFBP-System

 

The primary structures of IGF-I, IGF-II and insulin are similar. IGFs are produced by many differentiated cell types, and their bioactivity in the extracellular fluids or in the circulation are coordinated by six IGF binding proteins (IGFBP-1 through -6). IGFBP-3, the major binding protein in serum is stimulated by GH and it forms a large 150 kDa ternary complex with IGF-I or -II and the GH regulated acid labile subunit (ALS). IGFBP-5, an important supporter of bone tissue formation, also forms ternary complexes with IGF-I or -II and ALS. IGFBP-1, suppressed by insulin, is one of several binding proteins in the smaller 50 kDa binary complexes with IGF-I or –II. IGFBP-2 has inverse association with insulin under many physiological conditions. In contrast, IGFBP-4, -5 and -6 do not appear to be directly regulated by GH or insulin and are important local regulators of IGF activity in bone and the CNS. The type 1 IGF receptor (IGF1R) is the mediator of the mitogenic, anti-apoptotic, differentiating and metabolic effects of both IGF-I and -II. The structural similarity of the IGF1R with the insulin receptor (IR) explains the formation of hybrid receptors in cells that expresses both receptors such as myocytes and pre-adipocytes. Cross reactivity among the ligands and the receptors have been demonstrated, although it has minor importance under physiological conditions but may cause non-islet-cell tumor hypoglycemia due to unprocessed pro-IGFs with markedly decreased binding affinity to IGFBPs. A second receptor, exclusively binding IGF-II, work as a scavenger receptor and is identical to the mannose-6-phosphate receptor, known to direct proteins for degradation in the lysosomes. A second level of control of IGF bioactivity is exerted by IGFBP proteases which release IGF-I activity after fragmentation of IGFBPs. Specific production and regulation of IGFBP proteases at the tissue level controls processes such as ovulation and atherosclerosis. Furthermore, interaction of IGFBPs and IGFBP proteases with the extracellular matrix modify the binding affinity for the IGFs and are involved in prolonging the actions of IGFs at the tissue level. Extracellular matrix also signals though integrin receptors on the cell surface and modifies IGF-1R signaling. Figure 1 also shows the existence of IGFBP-related proteins with markedly lower affinity for the IGFs and with physiological roles not related to their IGF binding.

 

Animal experiments WERE essential for the understanding of human insulin-GH-IGF axis physiology

 

Insulin, IGF-I, and IGF-II and Their Receptors

 

Efstratiadis’ series of knock-outs of the insulin-GH-IGF-axis in mice in the early 1990s clearly confirmed its importance in fetal and postnatal growth and metabolism (21). It also predicted the phenotype of experiments of nature in humans with gene defects in the axis yet to be discovered. The studies opened new insights, not least the equal importance of IGF-I and IGF-II in fetal growth, reducing birth weight by about 60 % in both Igf1 knock-out (Igf1ko) and Igf2ko animals and demonstrating that the previous perceived concept that there was a fetal (IGF-II) and a postnatal (IGF-I) form of IGF was incorrect. IGF-I and -II had actions through the type 1 IGF receptor (IGF1R) demonstrated by Igf1rko animals with 45% of wild type birth weight and no further effect when crossed with Igf1ko animals. While Igf1ko animals were viable depending on genetic background and were non-fertile, the Igf1rko animals died from respiratory failure but with an absence of apparent malformations. Interestingly, crossing Igf2ko with Igf1rko resulted in further growth retardation indicating that IGF-II had actions through an additional receptor. Another new insight came from knock-out of the ‘mysterious type 2 IGF receptor’, identical to the mannose-6 phosphate receptor (M6P-R), specifically binding IGF-II and involved in internalization of proteins for lysosomal degradation. Knock-out of the Igf2r/M6pr resulted in increased serum and tissue levels of IGF-II and fetal overgrowth (140% of wild-type birth weight) (22). This receptor works to clear IGF-II and its presence in endothelial cells may, at least partly, explain the lack of endocrine actions of IGF-II due to its proteolytic lysosomal degradation (23). Thus, IGF-II effects on fetal growth are paracrine/autocrine actions mediated by the IGF1R. Knock-out of the Igf2r/M6pr gene combined with Igf2ko/Igf1rko could partly rescue growth retardation, a finding that was explained by IGF-II actions via the insulin receptor (INSR). The formation of heterodimers, more commonly named hybrid receptors, between type A or B isoforms of the insulin receptors (INSRA or INSRB) and the IGF1R of which IRA-IGF1R are highly expressed in the fetus (and in malignant cells) and activated by IGF-II, may further point to the importance of IGF-II during the fetal period. INSRB-IGF1R hybrids comprises up to 30 % of INSR and IGF-I receptors in muscle due to high expression of both and this hybrid predominantly responds to IGF-I (less to insulin) and explains the important role of IGF-I in growth and metabolism in skeletal muscle.

 

Postnatally, the Igf1ko mice continued to grow poorly, resulting in an adult weight 30% of wild-type and with poor gonadal function and delayed bone development. Knock-out of GH or its receptor (GHR), both expressed in the mouse fetus, did not affect birth size, indicating that the Igf1 gene is not under GH control during the fetal period. The actions of GH and its receptor on growth in mice were obvious from postnatal day 15 and largely slowed growth resulting in a 50% reduction of wild-type adult weight. On the other hand, double Ghrko/Igf1ko resulted in further postnatal growth retardation relative to Igf1ko mice completely obstructing further weight gain after postnatal day 15 and supporting previous studies suggesting that progenitor cells in the growth plate require direct GH actions (24).

 

IGFBPs and IGFBP Proteases

 

Similar to the above attempts to pinpoint the role of important ligands and receptors in the axis, steps to assess the role of IGF binding proteins involved in modulating IGF-I and IGF-II bioactivity were taken (reviewed by Pintar) (25). In contrast to the pronounced phenotypes caused by mutations in receptors and their ligands, the growth phenotypes of the various IGFBP knock-out animals were far less pronounced as were the metabolic changes observed (26,27,28). It was argued that there is a large degree of redundancy among the functions of the IGFBPs which to some extend contradicts their specialized functions in various tissues (29). However, this idea was to some extent supported by the finding of somewhat more pronounced phenotypes in double and triple knock-out animals (30). This is largely in accordance with the absence of reports of IGFBP gene defects causing growth retardation in humans. The most affected phenotype identified was that of Igfbp4ko mice who were growth retarded at birth and displayed poor postnatal growth (30). No such mutation has been identified in humans. IGFBP-4 is specifically degraded by the metalloproteinase PAPP-A (Pregnancy Associated Plasma Protease -A) produced by the placenta as well as bone and ovary. In Pappa knock-out animals a 20-30% reduction in body weight was reported (31). Interestingly, the growth restriction phenotype of mice null for Pappa could be rescued by disruption of IGF-II imprinting during embryonic development (32).

 

Endocrine Versus Paracrine Autocrine IGF-I

 

One of the controversies of the area has been the relative contribution to linear growth of circulating endocrine IGF-I largely produced by the liver versus peripherally produced IGF-I with major paracrine/autocrine actions on local tissues. The major importance of paracrine/autocrine IGF-I was demonstrated by liver specific Igf1ko mice (Ligf1ko) with largely unaffected longitudinal growth (33). Circulating levels were 20% of wild-type with compensatory elevation of GH, insulin resistance, and hyperinsulinemia. With age the animals developed type 2 diabetes, underlining the metabolic consequences of largely elevated GH combined with circulating IGF-I deficiency (34). Somewhat unexpectedly, this animal model closely resembles children and adolescents with type 1 diabetes, as further elaborated on below.

 

Another model to assess the relative importance of endocrine versus paracrine/autocrine IGF-I is the liver-specific Ghrko mouse. It produces a similar phenotype but with more specific hepatic consequences of absent GH signaling (35).

 

Insulin-GH-IGF-axis physiology in a pediatric perspective

 

Insulin-GH-IGF-Axis and Human Fetal Growth

 

IGF-I controls the pace of the cell cycle from early on in human embryogenesis. INSR and IGF1R is expressed in human pre-implantation blastocysts already from the 8-cell stage, while IGF-II is expressed already in the oocyte (36). After implantation, IGF-I is produced in the human embryo (37), but until then the source of IGF-I is thought to be the female reproductive tract, and it is known that the availability of the IGF-I ligand is important for blastocyst growth in human in vitro fertilization - IVF (38). IGF-I production is controlled by nutritional factors in the early embryo and even later during human fetal development (39). Circulating endocrine IGF-I increases with gestational age (40) and is strongly correlated with fetal growth in the second part of gestation (41,42). However, little has been reported concerning the regulatory control of the IGF1 gene in the human fetus. IGFBPs can be identified in the human fetal circulation (40), and particularly IGFBP-4 and IGFBP-4 specific proteases, such as PPAP-A2, have an impact on human growth (43). Insulin continues to be permissive for IGF-I production even after GH is established as the major pituitary stimuli controlling endocrine as well as paracrine/autocrine IGF-I, as described below.

 

Fetal Growth Restriction and Programming of the Insulin-GH-IGF-Axis Setpoint

 

Insulin resistance has been developmentally advantageous for mankind until very recent decades of excess food and sedentary life style. It was proposed by Barker et al (44) in his ‘fetal and infant origin of adult disease’ hypothesis that intrauterine restriction of growth compensated by excessive postnatal catch-up growth results in an increased risk of developing disease entities of the metabolic syndrome later in life. In his early epidemiological studies, he demonstrated that there is a U-shaped relationship between birth weight and risk of obesity, insulin resistance, type 1 diabetes, hypertension, dyslipidemia, and ischemic heart disease, with lower birth weight (within the normal range) imposing a risk. Notably, at higher birth weights this risk rises again which may represent genetic risks of obesity and type 2 diabetes. The concept was that poor fetal nutrition would lower fetal IGF-I and program the child to a low IGF-I setpoint and slower postnatal growth, an epigenetic phenomenon that could be preserved over a few generations (45). At the same time, small for gestational age (SGA) babies becomes insulin resistant (46) and this trait is enforced by a low endocrine IGF-I setpoint (47), creating the best physiological circumstances for the storage of fat during short times of food availability in a world with limited access to food. However, in a world of plenty, this advantage would turn into a disadvantage and give fast increases in body weight, hyperinsulinemia, and the development of metabolic syndrome problems early on (reviewed by Dunger et al (48). New information even suggests that the parental nutritional state can impose epigenetic metabolic changes in the fetus (49).

Figure 2. Serum IGF-1 Levels Throughout Life. In the fetus (insert) IGF-I increases with gestational age toward birth. Endocrine circulating IGF-I is strongly nutritionally dependent and correlated with birth size.  Pituitary GH control of IGF-I production is not fully established during the first year of human life. The ability of serum IGF-I levels to increase during childhood is dependent on the shift from binary complexes of IGF-I with short half-life to a complete dominance of IGF-I bound in a stable ternary complex with the GH dependent proteins IGFBP-3 and ALS. Both these proteins increase, when pituitary GH control of the axis is established. During pubertal development, sex steroids changes the set-point of negative IGF-I feedback and allow a peak of IGF-I in mid-puberty. Total IGF-I levels decline to low levels in senescence. Serum IGF-I reference values based on Juul (50).

 

Postnatal Establishment of Pituitary GH Control of IGF-I Production

 

In humans, full GH control of the IGFI gene, as well as the IGFBP3 and Acid Labile Subunit (ALS) genes, is developmentally regulated and established not until the first year of life. IGF-I and IGFBP-3 levels increase slowly from birth until a more rapid increase and peak during puberty, which is followed by a decline toward low levels in senescence (50, 51) (Figure 2). The late establishment of pituitary GH control of the axis is strongly supported by animal data from GHR KO mice reviewed above, and by a new model of Laron syndrome (GHR defect) in pigs (52). In accordance, newborns with mutations in the GH Releasing Hormone Receptor (GHRHR) gene resulting in an isolated GH deficiency (GHD) phenotype was associated with normal birth weight in one cohort (53) and slightly subnormal birth weight in another (54). GH1 mutations appear to be slightly more affected with mean birth weights of −1.0 ± 0.9 (54). Studies of common polymorphisms in GH1 demonstrate dose effects of 150 and 100 grams in term newborns of normal and low birth weight, respectively (55). 

 

Somewhat contradictory to the observations in animals, Savage et al (56) reported 27 prepubertal children with severe GH insensitivity syndrome (GHIS or Severe Primary IGF Deficiency (SPIGFD)) to have a median (range) birth weight SDS of -0.72 (1.75 - (-3.29)) and birth length SDS of -1.59 (0.63 - (-3.63)). SPIGFD in these patients were defined by phenotypic and biochemical characteristics and they were treated with recombinant human (rh)IGF-1 in one of the clinical trials leading to approval of this therapy, as described later. There was no complete genetic characterization of these patients and 7 patients had a normal serum GH Binding Protein (GHBP) suggesting that the extracellular part of GHR was not affected. In a monograph, Professor Zvi Laron (57), who gave his name to this syndrome, reported that birth weight is unaffected while birth length is slightly on the shorter side. In summary, human fetal growth is only marginally affected by GH. GH is detectable and the GHR is expressed in the human fetus and the metabolic effects of GH on lipolysis are essential to maintain normal levels of glucose in the newborn. Given this critical role of GH in adjusting metabolism to the fasting condition, it is plausible that the metabolic effects of GH are required for optimal linear growth of the human fetus and that this explains marginal effects on linear growth of the human fetus. However, strict GH control of IGF-I in the human fetus would predict severe growth retardation of the above-mentioned genetic defects comparable with the birth size observed in defects in the IGF-I gene. And that is not observed: Children with IGF1 defects suffer from far more severe fetal growth retardation with birth weight SDS around -4 and birth length SDS of -5 to -6 (reviewed in (58).

 

GHR Signaling Pathway to IGF1 Gene Transcription

 

The important cell signaling steps associated with GH stimulated IGF1 gene activation, transcription and IGF-I production are detailed in the Endotext chapter ‘Normal Physiology of Growth Hormone in Adults´. Briefly, GH binding to preformed dimeric GHR - JAK2 complexes introduces structural changes in the receptor complex that separates JAK2 inhibitory and kinase active sites and enables trans-phosphorylation of the two JAK2 molecules (reviewed in (59). The GHR belongs to the class 1 cytokine receptors which uses STAT as one of its principal secondary messengers, and the subsequent phosphorylation of two STAT5b molecules results in a phospo-STAT5b homodimer which translocate to the cell nucleus, binds to STAT5b recognition sites on the IGF1 gene promoter, and initiates transcription (Figure 3).

 

Insulin Enhancement of GHR Signaling to IGF1 Gene Transcription

 

Insulin signaling enhances the GH signaling pathway to enable IGF-I production in the fed state and promotes linear growth and other anabolic responses (60, 61, 62). Moreover, GH signaling to elicit IGF1 gene transcription is blocked in the absence of appropriate insulin signaling, a phenomenon also known as un-coupling, and resulting in growth arrest (60, 61, 63, 64) (Figure 3). This is partly a result of insulin’s effects on hepatic GHR expression, and partly a post-receptor signaling effect as unraveled by extensive animal studies (reviewed in (60). In obese individuals with hyperinsulinemia, hepatic GHR expression is enhanced as indicated by elevated GHBP levels reflecting proteolytic cleavage of highly expressed surface GHR and release of the extracellular part to the circulation (65). This allows obese individuals to maintain normal serum IGF-I levels despite markedly diminished GH secretion (66, 67). Consequently, obese individuals have attenuated GH responses to GH secretagogues (68).

Figure 3. Multiple, partly identical, pathways have been described to be activated by the GHR, the INSR and many other hormone kinase receptors not shown on the slide. Limiting this cartoon to the GHR and INSR, still the complexity is very high and the potential candidate hubs for crosstalk are numerous. The crosstalk that, following activation of the GHR, leads to resistance to specific signaling events from the INSR (related to glucose metabolism) is more well established and describe in detail in the Endotext chapter ‘Normal Physiology of Growth Hormone in Adults´. In the current review the focus is on the crosstalk that is executed by activation of the INSR and results in enhanced signaling from the GHR leading to gene activation of IGF1 and other GH dependent genes such as IGFBP3 and ALS. There are basically no studies addressing this crosstalk on the cellular level despite the strong evidence for INSR signaling being permissive for IGF1 transcription. Given that mTORC1 and mTORC2, downstream the INSR, are essential hubs for substrate and energy sensing and thus controlling the switch between cell anabolism and catabolism, they appear to be strong candidates to determine whether IGF1 should be on or off. A further argument for their candidate role is the so far limited evidence of mTORC1 and mTORC2 involvement in branched chain amino acid sensing directly enhancing IGF1 transcription. The unique role of the Jak2, STAT5b pathway in connecting the GHR with IGF1 gene activation has not been challenged and is thus the major candidate pathway to be affected by INSR signaling crosstalk. It is less clear which of the signaling pathways from the GHR that results in enhanced lipolysis although the STAT5b pathways has been implicated. This is particularly interesting given that GHR induced lipolysis does not require INSR signaling crosstalk. The reader is encouraged to seek specific information regarding other GHR and INSR pathways depicted in the cartoon but not further discussed in this review.

 

While short term fasting decreases serum IGF-I but does not affect GHBP (69), the triad of IGF-I deficiency, poor growth and pubertal delay/arrest in long term nutritional deficiency such as in anorexia nervosa is associated with low GHBP that is partly restored with weight gain (63). Also, circulating IGF-I deficiency due to hepatic under-insulinization in type 1 diabetes is associated with low GHBP levels. Normal circulating IGF-I and GHBP are fully restored only after experimental intra-peritoneal (70) or intraportal (71) insulin delivery.

 

Insulin Deficiency - Uncoupling of GHR Signaling to IGF1 Gene Transcription and Maintained GHR Metabolic Signaling

 

Increased GHR signaling in obese children does not generally result in elevated IGF-I, due to negative feedback inhibition of GH. In contrast, impairment of GH signaling due to insulin deficiency cannot generally be compensated by GH hypersecretion. This is true in fasting children (63) and adults (62). The exact mechanisms by which insulin and GH signaling crosstalk on the post-receptor level is not yet understood (Figure 3). More recent data suggesting that FGF21 plays a role in mediating these events needs further confirmation (72). Interestingly, GH signaling leading to activation of lipolysis in adipose tissue and increased hepatic glucose production via both glycogenolysis and gluconeogenesis in the liver, is not affected by the absence of insulin crosstalk (reviewed in (72)). This has important implications in securing substrate mobilization and gluconeogenesis during fasting and explains the cardinal hypoglycemic symptoms in GHD and GHIS newborns in the absence of intrauterine growth retardation (IUGR). GH signaling leading to lipolysis is thought to involve STAT5b. Most information comes from animal models and involves GH signaling in the liver, but in mouse adipose tissue GHR KO downregulates beta-3 adrenergic receptor expression and inhibits lipolysis (73). GH effects are lost if STAT5b signaling is blocked (74), gene transcript profiles of GHR KO and STAT5b KO animals overlap largely, and STAT5b controls key regulator enzymes involved in lipid metabolism (75). However, if STAT5b mediates both metabolic signaling and IGF-I production it still needs to be understood where the two pathways diverge, and why GH metabolic signaling is not blocked in the absence of insulin crosstalk. In humans, recent studies have identified new GH signaling responses involving GH downregulation of fat-specific protein (FSP27), a negative regulator of lipolysis. MEK/ERK activation and inhibition of peroxisome proliferator-activated receptor-γ (PPARγ) are involved, and this offers an alternative signaling pathway from the GHR (76).

 

Interactions Among Endocrine Axes

 

The activity of the insulin-GH-IGF-axis is dependent on the other endocrine axes which have permissive actions on GH stimulated IGF-I expression and affect IGFBPs and proteases (Figure 4). For example, thyroxine is needed to enhance GH effects on endocrine IGF-I expression and a normal GH-IGF-IGFBP-axis is needed for optimal thyroid hormone production (77). Sex steroids further enhance the function of the GH-IGF-axis, most likely by attenuating pituitary and hypothalamic sensitivity to IGF-I negative feedback (78). The pivotal role of sex steroids on the setpoint of the axis is reflected by the peak circulating levels of IGF-I and IGFBP-3 reached in mid-puberty (50,51). On the other hand, GH via its stimulation of local IGF-I is important for testicular production of testosterone and spermatogenesis (79), and the local IGF-IGFBP-axis is involved in selection and growth of the primary follicle in the ovary, estradiol production and ovulation (80, 81). Finally, cortisol has impact on the actions of the GH-IGF-axis on growth by blocking IGF1R signaling leading to apoptosis (82) despite normal endocrine IGF-I levels (83).

Figure 4. Hypothalamic GH releasing hormone and somatostatin establish the pulsatile pituitary GH secretion that is established as the main regulator of endocrine and paracrine/autocrine IGF-I production during the first year of life in humans. Insulin is permissive for this regulation by modulating GHR signaling, and normal beta-cell release of insulin is required for normal liver derived endocrine IGF-I levels measured in serum (blue insert) that in most cases is a good marker of the local production and actions of IGF-I. During fasting the GH regulation of IGF-I is uncoupled, resulting in decreased IGF-I (and catabolism) and elevated GH secretion and maintained lipolytic signals securing gluconeogenesis and preventing hypoglycemia. Apart from insulin, the endocrine thyroid axis is important for normal GH induced IGF-I production and during pubertal development sex steroids from the gonads enhance the performance of the GH-IGF-axis presumable by relaxation of the negative IGF-I feedback on GH secretion allowing a higher set-point of the axis. Whether this is an estradiol effect is not fully elucidated but it is suggested by the fact that non-aromatizable androgens such as oxandrolone do not affect IGF-I levels. The actions of the adrenal axis are most likely local and involve actions on IGF1R signaling leading to apoptosis of stem cells in the growth plate and thus irreversible loss of height. Cortisol excess leaves endocrine IGF-I and GH levels largely unaffected. 

 

Discordance Between Endocrine Versus Paracrine/Autocrine IGF-I

 

An important example of metabolic and mitogenic consequences of an unbalanced endocrine versus autocrine/paracrine insulin-GH-IGF-axis comes from observations in children and adolescents with type 1 diabetes (Figure 5). They suffer specifically from insulin deficiency in the hepatic portal circulation as a result of the subcutaneous delivery of insulin (reviewed by Dunger (64)). This attenuates their endocrine production of circulating IGF-I despite excessive GH secretion (84). Circulating IGF-I deficiency and GH hypersecretion induce insulin resistance which is further augmented by insufficient suppression of hepatic glucose output. To overcome this, higher subcutaneous insulin doses are needed to maintain glycemic control, and this results in aggravated systemic hyperinsulinemia. The importance of local tissue hyperinsulinemia and GH hypersecretion in generating high paracrine/autocrine IGF-I production and promoting mitogenic vascular events leading to diabetic long-term complications should not be underestimated. Based on this insight, a promising new drug targeting the alphaVbeta3 integrin affecting IGF-I signaling in smooth muscle cells has been found to inhibit the development of atherosclerotic lesions in diabetic pigs (85). Another consequence of a compensatory increased in local IGF-I activity is the finding of normal childhood and pubertal linear growth despite endocrine IGF-I deficiency in type 1 diabetes (86). It is interesting that the endocrine and paracrine/autocrine changes in the insulin-GH-IGF-axis observed in children with type 1 diabetes closely resembles those observed in liver IGF-I KO mice which eventually leads to diabetes in the KO mice. Given that portal delivery of insulin, which has the potential to completely restore IGF-I levels in type 1 diabetes (70, 71, 87), remains an experimental treatment, rhIGF-1 treatment to restore circulating IGF-I and suppress GH and decrease insulin needs appears to be the most feasible approach to take (88). In a 6-month clinical trial of a single daily injection of rhIGF-1 improved glycemic control in adolescents with type 1 diabetes were found (89). Long-term studies on diabetic vascular complications have yet to be performed.

 

If paracrine/autocrine IGF-I production is lost in addition to liver-derived IGF-I, the metabolic consequences becomes obvious. This situation was first reported in a boy with a deletion of exon 4 of the IGF-I gene (90) resulting in severe fetal and post-natal growth arrest, poor brain development and extreme insulin resistance with compensatory hyperinsulinemia and acanthosis nigricans. A short trial of treatment with rhIGF-1 resulted in normalization of circulating IGF-I, suppression of GH hypersecretion and a markedly decreased insulin response to a meal tolerance test (91). In this example and in type 1 diabetes, it has been discussed whether the normalization of glucose metabolism following rhIGF-I therapy is most importantly associated with insulin-like effects of IGF-I on glucose uptake in muscle or suppression of GH hypersecretion? Although most studies support the importance of GH suppression, prolonged actions of IGF-I similar to that of long acting insulin analogs in type 1 diabetic patients are important. IGF-I is equipotent with insulin in stimulating glucose uptake in human muscle but has less effects in fat and liver (92). Reports that newborns with a complete lack of insulin effects due to inactivating defects in the INSR gene can now survive for extended time into adolescence when treated with recombinant IGF-I, that stimulate glucose uptake via the IGF1R sharing common signaling pathways with the INSR, support an important direct role of IGF-1 signaling on metabolism (93).

Figure 5. Changes in liver derived endocrine IGF-I measured in the circulation and paracrine/autocrine IGF-I are in most cases concordant. In the absence of practical and validated methods to measure IGF-I at the tissue site of action, paracrine/autocrine IGF-I activity is assessed by determining known physiological actions of IGF-I such as growth or glucose metabolism. Type 1 diabetes is a condition with discordant changes in endocrine vs. paracrine/autocrine changes in IGF-I that in many ways resembles those reported in a mouse model of conditional knock-out of IGF-1 expression in the liver. In type 1 diabetes, insulin deficiency in the liver, caused by a systemic rather than a portal insulin replacement therapy, results in a functional GHR signaling defect to IGF-I transcription (uncoupling). Low endocrine IGF-I production decreases circulating IGF-I and results in decreased negative pituitary feedback and GH hypersecretion. The lack of direct IGF-I effect on glucose uptake in muscle and the diabetogenic effects of GH (including maintained signaling to lipolysis) decreases insulin actions on glucose metabolism (known as insulin resistance). The portal insulin deficiency also fails to suppress hepatic glucose production. In other to maintain glycemic control, the increased insulin requirement can only be met by more subcutaneous insulin leading to systemic hyperinsulinemia. There is no direct information about local paracrine/autocrine IGF-I activity, but there are several indications that tissue hyperinsulinemia and GH hypersecretion results in a compensatory increase of tissue IGF-I activity. Firstly, linear growth is not impaired in children and adolescents with Type 1 Diabetes despite of their low endocrine IGF-I (comparable to levels in short stature children), indicating a compensatory upregulation of local IGF-I activity (IGF-I being the most important stimulator of longitudinal growth). Secondly, it is plausible that increased local IGF-I activity contributes to diabetes complications known to be tightly associated with increased rather than decreased IGF-I activity. While type 1 diabetes is not generally associated with increased risk of cancer, the increase in local production of IGF-I in obesity and type 2 diabetes may contribute.

 

Liver Disease and Endocrine Versus Paracrine/Autocrine IGF-1 Production

 

In children with severe liver disease, there may be similar discrepancies between circulating endocrine levels of IGF-I and IGF-I activity in peripheral levels contributed by paracrine/autocrine secretion of IGF-I (94). However, less is known about peripheral IGF-I activity and it is possible that there are more secondary metabolic and nutritional issues that could lower local IGF-I production and impact on linear growth. Particularly in liver cirrhosis associated with thalassemia major, which concomitantly can impair pituitary GH secretion, there is no secondary upregulation of local IGF-I and linear growth failure is common (95). Recently, increased IGF-I expression was reported in obese children with non-alcoholic fatty liver disease (NAFLD) and it was combined with upregulation of IGF1R (96), not expressed in the normal liver but involved in liver repair, such as after liver resection in a mouse model (97).

 

GH and Cytokine Crosstalk

 

STAT5b phosphorylation is also mediated by activation of other members of the cytokine receptor family and has an impact on immunological function: this is evident from the finding of immune deficient symptoms in children with STAT5b genetic defects (98) but these are not found in GHD and GHIS children.

 

Negative Control of GHR Signaling

 

Control of the GH signaling cascade is also under inhibitory control, principally by two mechanisms. Firstly, tyrosine phosphatases including PTP dephosphorylate GHR associated molecules. In Noonan’s syndrome genetic defects in the PTPN11 gene may affect this pathway and has been implicated in poor growth and poor response to GH therapy, although reports are conflicting (99, 100). In addition, the SOCS gene is activated by GH signaling and works as a short intracellular negative feedback loop which rapidly down-regulate GHR activity by internalization and receptor ubiquitination resulting in lysosomal and proteasomal degradation (101).

 

Other Nutritional Signals to the GH-IGF-axis

 

Nutritional supplementation increasing dietary protein intake from cow’s milk increases endocrine IGF-I, while an equal intake of animal protein from meat does not (102). It is possible that the amino-acid composition that differs depending on the dietary source may contribute, and there are other constituents in skimmed milk not present in meat. More likely, however, it is explained by a higher carbohydrate intake in the milk group (while fat content was higher with meat supplementation) and the finding that fasting insulin levels doubled (103), in accordance with the essential regulatory role of insulin on GHR signaling discussed above. There is, however, direct evidence for a regulatory role of amino-acids on IGF-I production that is independent of insulin. Branched chained amino-acids (BCAA) are known to stimulate cell growth by the activation of mTORC2, a protein complex that controls protein synthesis in cells by sensing nutrient and energy availability and is also one of the main signaling pathways of IGF-I and insulin (Figure 3). The role of BCAA has been studied in rats given a restricted diet containing high levels of BCAA, compared to a group given low levels of BCAA (104).

 

The availability of nutrients in the circulation might also have a direct effect on the production of IGF-I. Human breast adipocytes cultured in high glucose levels have been found to produce more IGF-I compared to adipocytes cultured in low glucose levels (105).

 

IGF-II

 

IGF-II is a paracrine/autocrine hormone which is as essential for fetal growth as IGF-I (21). IGF-II may also contribute considerable to postnatal growth although the absence of endocrine effects makes it difficult to study in humans. Less is known about the metabolic actions of IGF-II. Growth promoting actions of IGF-II is via the IGF1R and IGF1R-INSR hybrid receptors which has preference for IGF-II actions if the A isoform of the INSR receptor – expressed in the fetus and in malignantly transformed cells - pairs with the IGF1R in the hybrid (106). The human IGF2 gene is an imprinted gene (107) exclusively expressed from the paternal allele in certain tissues (reviewed by Rossignol (108). The imprinted promoter region is found in a complex configuration with the H19 gene on chromosome 11 and shares two important imprinting regions with this gene. Methylation of the imprinting region ICR1 results in expression of the paternal IGF2 allele, while H19 gene expression is suppressed. Correct imprinting should lead to expression of the paternal allele only and sufficient expression of IGF-II for normal growth. Loss of methylation of the ICR1 on the paternal allele results in the phenotype of Silver Russell syndrome (SRS) which may also arise from other genetic aberrations that have not yet been linked to the IGF-II production or signaling cascade including maternal uniparental dyssomnia of chromosome 7.

 

SRS is characterized by proportional IUGR with severe SGA at birth, relative sparing of the brain with close to normal head circumference at birth, severe feeding difficulties in infancy and childhood (which in contrast to Prader-Willi syndrome does not rebound into feeding obsession later in life), postnatal growth retardation and body asymmetry. As indicated by the SRS phenotype, IGF2 gene transcription of some organs are not controlled by imprinting. The relative normal development of the brain and the relative macrocephaly of SRS is explained by the lack of imprinting control of IGF-II expression in the brain. Children with genetic mutations in the expressed paternal allele of the IGF2 gene, were reported to have an SRS phenotype. They had somewhat more pronounced psychomotor developmental problems compared with the SRS phenotype, which has increased risk of autism spectrum defects including Attention deficit hyperactivity disorder. In SRS, there is a normal postnatal expression of the IGF2 gene in the liver leading to normal levels of circulating IGF-II. Interestingly, the normal endocrine levels of IGF-II do not overcome the postnatal growth restriction. A similar lack of endocrine IGF-II effects on growth and metabolism was reported in the IGF-I deficient child mentioned previously with a loss of exon 4 of the IGF1 gene. He had compensatory increased GH, IGFBP-3, ALS as a result of lack of negative IGF-I feedback and secondary to the increased IGF binding capacity, increased IGF-II (see also the chapter on IGF-binding proteins below). Another observation in favor of this view is the lack of a correlation between newborn cord levels of IGF-II and birth size (41). This contrasts with a strong positive correlation between cord blood IGF-I concentrations and birth size. The role of IGF-II should be viewed in the light of Efstradiadis series of knock-out experiments in mice (21) where the Igf2ko mice had the same degree of fetal growth retardation as the igf1ko which demonstrates that IGF-II is a paracrine/autocrine and not an endocrine hormone.

 

It is possible that circulating IGF-II after release from the ternary complex is cleared from the circulation by binding to the IGF2R – identical to the mannose-6-phosphate receptor – which is associated with lysosomes and results in degradation of IGF-II in endothelial cells (23). In the elegant mouse KO experiments by Efstradiadis et al (21), KO of the IGF2R resulted in fetal overgrowth. However, the largely elevated IGF-II serum levels in that model are more likely a secondary finding, while the lack of clearance of paracrine/autocrine IGF-II is the explanation for the excessive growth.

 

IGF Binding Proteins

 

Six IGFBPs bind IGF-I and IGF-II inside and outside the circulation and has impact on IGF bioactivity (reviewed by Clemmons (109). The IGFBP-related proteins share some structural similarities with the six IGFBPs but have no relevant impact on IGF bioactivity. IGF-I passes the endothelium intact primarily via IGF1R mediated transcytosis and this process is essential for endocrine actions of liver derived IGF-I (110). Limited experimental evidence from animal and tissue cultures suggest that IGF-I complexed with IGFBP-1 and -2 may leave the circulation, although the extent and importance is unclear.  After endothelial passage IGF-I redistributes to soluble IGFBPs in the extravascular fluids or IGFBPs bound on extracellular matrix or cell surfaces (111). The concentration of unbound IGF-I in the circulation is likely to be proportional to unbound IGF-I concentrations in the tissues, but they are not equal and may have different relationship in different target tissues with differentially-expressed IGFBPs.

 

Free IGF-I Assays

 

Assays claiming to measure free circulating IGF-I have been developed, but it is unclear to what extent different techniques are influenced by redistribution of IGF-I among IGFBPs associated with the assay procedure (112). Anyway, the fact that IGF-I redistribute among extravascular IGFBPs after passing the endothelium is likely to affect the local tissue bioavailability even more. Moreover, the fact that most data in the literature originate from one assay technique established in one single laboratory has resulted in a lack of confirmatory reports. In a few cases, measurements with different free IGF-I assays have been reported from the same study/experiment with large differences in results (113). The bottom line is that measurements of free IGF-I have not been demonstrated to better predict different physiological or pathophysiological conditions in humans, and do therefore not provide any clinically important contributions (114, 112). Techniques to assess IGF-I at the tissue site of action pose practical and methodological challenges. Attempts to establish and validate a method to determine local tissue levels by microdialysis have been reported in adolescents with type 1 diabetes, where endocrine levels are a poor marker of local IGF-I activity (115).

 

Ternary Complex Formation

 

The developmental establishment of GH control over the IGF1, IGFBP3 and IGFALS genes in early childhood initiates the dominance of the ternary complex formed by IGF-I or IGF-II and IGFBP-3 (or IGFBP-5) and ALS as the quantitatively most important circulating form of IGF-I and IGF-II (reviewed by Baxter (116). In the human fetus and newborn, serum IGFBP-3 and ALS concentrations are low and ternary complex formation is absent (117). Although IGF2 gene expression is not under GH control, the circulating levels of IGF-II are largely influenced by GH status since IGF-II (as well as IGF-I and IGFBP-3) is rapidly cleared from the circulation if not bound in the ternary complex. This can be observed in children with SPIGFD, who are deficient in IGF-I as well as IGFBP-3 and ALS, and in whom sc injected rhIGF-I displays a very fast serum clearance rate (118). As mentioned, formation of the ternary complex also governs the circulating levels of IGFBP-3 which under physiological conditions is present in a 1:1 molar relationship with IGF-I plus IGF-II. ALS is a large glucoprotein that under physiological conditions are present in a two-fold molar excess (16).

Immunometric IGFBP-3 assays have been claimed to be more predictive of GH status in very young children; however, the support for that is weak. It is rather a misconception related to problems of commercial IGF-I assays at the lower end of IGF-I detection. Moreover, IGFBP-3 has been claimed to provide information about IGF-I bioavailability from calculating the molar ratio of total IGF-I to IGFBP-3. Given that both IGF-I and IGFBP-3 are rapidly cleared from the circulation if unbound, using the IGF-I/IGFBP-3 ratio and disregarding IGF-II concentrations (that are 2-3-fold those of IGF-I on a molar basis) does not make any sense. During puberty, for example, IGF-I bioactivity is increased (114). This is dependent on the 3-4-fold increase in total IGF-I (50), which consequently results in an increase in unbound IGF-I, even if the increase is matched with the same absolute molar increase in IGFBP-3 (and complexed with ALS in a ternary complex). A common view is that increased IGF-I bioactivity depends on a higher IGF-I/IGFBP-3 molar ratio during puberty. However, the increase in molar ratio is entirely explained by the fact that IGF-I and IGFBP-3 increase with the same number of moles per liter, but with a larger relative increase in IGF-I than IGFBP-3 and with IGF-II molar concentrations being unchanged (112).

 

IGFBP Proteolysis and Physiological Consequences

 

The fact that proteolytic cleavage of IGFBP-3 is common, and may result in falsely elevated IGFBP-3 immunoactivity, is the most likely reason for observing a low IGF-I/ IGFBP-3 ratio. Under certain physiological conditions first described in pregnancy (119, 120), specific proteases cleave IGFBP-3 into several proteolytic fragments of which each may retain immunoactivity and thus give rise to signals in an immunometric assay (121). This will lead to overestimations of the IGFBP-3 immunoreactivity in pregnancy, which is already truly increased due to increased placental GH tonus. It may also lead to the erroneous conclusion that IGF-I bioactivity is decreased. On the contrary, IGF-I bioactivity is increased in the maternal circulation resulting from increased total serum IGF-I and decreased binding affinity of fragmented IGFBP-3 (122). There is strong experimental evidence that IGFBP proteolysis results in lower IGF binding affinity. The finding that partial IGFBP-3 proteolysis, such as in pregnancy, does not disrupt the ternary complex, has questioned its significance. However, evidence for increased IGF-I bioactivity in a ternary complex with fragmented IGFBP-3 exists (123). IGFBP-3 proteolysis has also been described in insulin resistant states such as fasting, obesity and type 1 and 2 diabetes (62, 124, 125). While several known proteolytic enzymes such as those involved in blood clotting (126) and cancer metastasis (127, 128) have been identified as IGFBP-3 proteases, the identity of the pregnancy protease is still not resolved.

 

Recently, a human mutation of PAPP-A2, a circulating IGFBP-4 protease corresponding to matrix associated PAPP-A, was reported to cause fetal and post-natal growth retardation in children in a consanguineous family (36). It is beyond the scope of this paper to review the overwhelming evidence from cell biology experiments demonstrating the important role of IGFBPs in modulating IGF bioactivity and the role of IGFBP proteases and their actions at the cellular level are beyond the scope of this review. Furthermore, IGFBPs other than IGFBP-3 may play a role in the access of IGF-I to various tissues (129).

 

Summary

 

In the present review the pivotal role of nutrition and insulin in determining the regulation and actions of the GH-IGF-axis is reviewed. For the pediatrician, caring for patients in a phase of rapid growth and development, it is important to refer to normality and understand the requirements for a normal insulin-GH-IGF-axis in order to succeed in this task. In the complex work-up, treatment and management of growth disorders a thorough understanding of the normal physiology of the axis is essential in taking the right actions (130, 131). From the normal physiology of this axis, it is possible to understand the consequences of various genetic defects and disorders that affect its regulation and function. The most severe conditions associated with defects in the axis may cause a loss of adult height of approximately 1/3 and may cause severe developmental and neurological deficits and compromise pubertal maturation and fertility. Minor changes in the setpoint of the axis caused by programming of the fetus exposed to intra-uterine growth retardation may predispose the individual for poor linear growth and later metabolic disease, insights that the pediatrician should be aware of and consider in order to improve health and prevent later disease.

 

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Islet Transplantation

Abstract

 

Transplanting islets of Langerhans consists of implantation in the recipient’s hepatic portal system of endocrine pancreatic tissue, with a variable degree of purification. The field of islet transplantation has evolved significantly since the initial attempts by doctors Minkowski and von Mering in 1882, with remarkable acceleration over the last three decades, thanks to the incredible efforts of the research community worldwide, with continuous improvements in cell processing and transplantation techniques, patient management, and development of specific immunotherapy protocols. Restoration of beta-cell function can be obtained by transplantation of allogeneic islets in both non-uremic (Islet Transplant Alone, ITA) and uremic (Simultaneous Islet and Kidney, SIK and Islet After Kidney, IAK) patients with diabetes, providing long-term sustained function and improved metabolic control even when requiring exogenous insulin (i.e., suboptimal islet mass transplanted or development of graft dysfunction). Preservation of beta-cell function is now attained in virtually all recipients of islet autografts, a therapeutic option that should be considered for individuals undergoing total pancreatectomy for non-malignant conditions and, as recently reported for selected cases with malignant conditions. In addition, islet transplantation represents an excellent platform toward the development of cellular therapies aimed at the restoration of beta-cell function using stem cells in the near future.  In this chapter, we will review the state-of-the art of clinical islet transplantation.

 

Introduction

Diabetes affects 425 million people throughout the world (2017) and this number will rise to 629 million by 2045 (IDF Diabetes Atlas Eight edition 2017, http://www.idf.org/diabetesatlas/introduction). Many cases of diabetes are successfully treated with life-long multiple daily injections of exogenous insulin and monitoring of blood glucose levels. In the last decades significant improvements in insulin therapy thanks to new  preparations (i.e., ultrafast and long-lasting insulin analogues) and the adoption of intensive diabetes management (infusion pumps and continuous glucose monitoring system) have resulted in an overall improvement of patients’ glycemic control and a decreased incidence of chronic complications of diabetes (1, 2). However, exogenous insulin administration cannot attain the desirable tight control in the majority of diabetics (3-5), cannot avoid the long-term complications of diabetes in all patients, and the life expectancy of patients with diabetes is still shorter compared to that of the general population (6-8).  Diabetes is one of the leading causes of end-stage renal disease, blindness, and amputation (9). In principle, the treatment for type 1 diabetes, type 3c diabetes and many cases of type 2 diabetes lies in the possibility of replacing destroyed or exhausted beta cell mass in order to restore two essential functions: sensing blood sugar levels and secreting appropriate amounts of insulin in the vascular bed, ideally into the portal system. Currently, the only available clinical approach of restoring beta cell mass in patients with diabetes is the allogenic/autologous transplantation of beta cells (i.e., pancreas or islet transplantation). Clinical trials performed in the last three decades have shown that restoration of beta-cell function via transplantation of isolated islet cells or vascularized pancreas allows reproducibly achievement of a more physiological release of endocrine hormones than exogenous insulin in subjects with diabetes (10). Transplanting islets of Langerhans consists of implantation in the recipient’s hepatic portal system of endocrine pancreatic tissue, with a variable degree of purification. Isolated islets are transplanted using minimally invasive techniques with lower morbidity than vascularized pancreas transplantation, which requires major surgery. The field of islet transplantation has evolved significantly since the initial attempts by doctors Minkowski and von Mering in 1882 (11), with remarkable acceleration over the last three decades, thanks to the incredible efforts of the research community worldwide, with continuous improvements in cell processing and transplantation techniques, patient management, and development of specific immunotherapy protocols. In addition, islet transplantation represents an excellent platform toward the development of cellular therapies aimed at the restoration of beta-cell function using stem cells in the near future.  In this chapter, we will review the state-of-the art of clinical islet transplantation.

 

When to Consider Islet Transplantation?

 

Transplantation of pancreatic islet may be considered as a therapeutic option in several conditions associated with loss of beta-cell function (Table 1).  The procedure may be performed as Islet Transplant Alone (ITA) in non-uremic subjects, an option generally indicated for the treatment of iatrogenic (surgery-induced) diabetes and for non-uremic patients with Type 1 diabetes.  Subjects with end-stage renal disease (ESRD) may be considered for Simultaneous Islet-Kidney (SIK) or, if already undergone renal transplantation, Islet After Kidney (IAK) transplantation, respectively. In special situations, transplantation of islets may be considered in combination with other organs (i.e., in the context of multi-visceral transplantation following exenteration comprising the pancreas) (12). 

 

The source of the islets for transplantation may be the patient’s own pancreas (autologous or auto-transplant) mainly when surgical removal of the gland is required due to different conditions.  After total pancreatectomy, the subject develops surgery-induced (iatrogenic) insulin-requiring diabetes.  Introduced in the early 1970’s (13), islet auto-transplantation allows achieving optimal metabolic control without the need for exogenous insulin in approximately 70% of the cases when adequate islet numbers can be recovered from the pancreas (generally >250,000 islet equivalents).  More than 500 auto-transplant in patients with near-total or total pancreatectomy have been performed to date (14). The largest series were published by the University of Minnesota (15-18), the University of Cincinnati (19, 20), and Leicester (21-24). Even when an inadequate islet mass to attain insulin-independence has been recovered, stable metabolic control and excellent management can be achieved in most subjects undergoing autologous islet transplantation (17, 25-30).  Islet auto-transplantation is currently reimbursed by health insurance in the United States.  In the past auto-transplant has been performed almost exclusively in patients undergoing pancreatectomy because of chronic pancreatitis, successfully preserving β-cell mass and preventing diabetes after major pancreatic resections (14, 15, 31, 32). Additional indications for auto-transplant other than chronic pancreatitis are still controversial (33), and have been limited to the procedure performed only in small case series (34-39) of benign enucleable tumors or pancreatic trauma. Recently, broader selection criteria for  auto-transplant were  published (38, 40), exploring the possibility of extending auto-transplant to patients with known malignancy, either having completion pancreatectomy as treatment for severe pancreatic fistulae or extensive distal pancreatectomy for neoplasms of the pancreatic neck or pancreatoduodenectomy because at high risk of pancreatic fistula (Table 1).

 

In the case of subjects who lost islet function (mainly patients with Type 1 Diabetes or, more rarely, previous total pancreatectomy) the only option currently available for transplantable islet cells is allogeneic donor pancreata. These are generally obtained through multi-organ donation after cerebral death, following conventional donor: recipient ABO blood type matching.  The use of a segment of the pancreas from living-related donors is technically feasible (41, 42), but at the present time not preferred for islet transplantation due to the limited duration of graft function after transplantation of suboptimal islet numbers under standard immunosuppressive protocols, as well as the intrinsic risks for the donor (i.e., morbidity and risk to develop diabetes) (43). 

 

Table 1.  Indication for Islet Transplantation

Condition

Procedure

Type of Transplant

Diabetes Mellitus

Type 1

ITA, SIK, IAK

Allogeneic

Type 2

ITA, SIK, IAK

Allogeneic

Surgery-Induced Diabetes (Iatrogenic)

Chronic pancreatitis

ITA

Autologous/Allogeneic

Trauma

ITA

Autologous/Allogeneic

Multi-visceral transplantation

Different combinations: Liver-Islet Transplantation, Bowel-Liver-Islet Transplantation, etc.

Allogeneic

Cystic Fibrosis

ITA

Lung-Islet Transplantation

Autologous/Allogeneic

Allogeneic

Benign enucleable tumors

ITA

Autologous

Borderline/malignant pancreatic neoplasms

ITA

Autologous/Allogeneic

 

Grade C pancreatic fistula requiring completion pancreatectomy

ITA

Autologous/Allogeneic

 

 

The current main indication for an allogeneic islet transplant is Type 1 Diabetes, which is characterized by the selective destruction of islet beta cells due to an autoimmune process.  Ongoing clinical trials of allogeneic islet transplantation are recruiting subjects with unstable Type 1 Diabetes 18-65 years of age, either sex, with frequent metabolic instability requiring medical treatment (hypo-, hyperglycemia, ketoacidosis) despite intensive insulin therapy; hypoglycemia unawareness (<54mg/dL); severe metabolic lability (mean amplitude of glycemic excursion >11,1 mmol/L or 200 mg/dl).  The inadequate efficacy of medical therapy to attain the desirable metabolic control in this specific patient population with unstable diabetes justifies the use of transplantation of pancreatic islets (either isolated cellular graft or vascularized whole pancreas) (44).  The main objective of the transplant is to correct the high susceptibility to severe hypoglycemia and glycemic imbalance that are associated with high mortality (8% in nonuremic subjects in the waiting list for 4 years to receive pancreas transplantation).  Further indications for an islet transplant are presence of progressive complications of diabetes and psychological problems with insulin therapy that may compromise adherence to the therapeutic regimen.  Islet transplant is indicated also for cases of subcutaneous insulin resistance requiring intraperitoneal or intravenous infusions, which are associated with substantial management hurdles and morbidity.

 

Table 2.  Inclusion and Exclusion Criteria for Allogeneic Islet Transplantation in T1DM*

Inclusion Criteria:

·       Mentally stable and able to comply with study procedures

·       Clinical history compatible with type 1 diabetes with onset of disease at <40 years of age, insulin dependence for at least 5 years at study entry, and a sum of age and insulin dependent diabetes duration of at least 28

Absent stimulated C-peptide (<0.3 ng/ml) 60 and 90 minutes post-mixed-meal tolerance test

·       Involvement of intensive diabetes management, defined as:

o   - Self-monitoring of glucose values no less than a mean of three times each day averaged over each week

o   - Administration of three or more insulin injections each day or insulin pump therapy

o   - Under the direction of an endocrinologist, diabetologist, or diabetes specialist with at least three clinical evaluations during the past 12 months prior to study enrollment

·       At least one episode of severe hypoglycemia in the past 12 months, defined as an event with one of the following symptoms: memory loss; confusion; uncontrollable behavior; irrational behavior; unusual difficulty in awakening; suspected seizure; seizure; loss of consciousness; or visual symptoms, compatible with hypoglycemia in which the individual required assistance of another subject was unable to treat him/herself person and which was associated with either a blood glucose level <54 mg/dl or prompt recovery after oral carbohydrate, intravenous glucose, or glucagon administration in the 12 months prior to study enrollment

·       Reduced awareness of hypoglycemia

Exclusion Criteria:

·       - Body mass index (BMI) >30 kg/m2 or weight ≤50 kg

·       - Insulin requirement of >1.0 IU/kg/day or <15 U/day

·       - HbA1c >10%

·       - Untreated proliferative diabetic retinopathy

·       - Systolic blood pressure >160 mmHg or diastolic blood pressure >100 mmHg

·       - Measured glomerular filtration rate using iohexol of <80 ml/min/1.73mm2.

·       - Presence or history of macroalbuminuria (>300 mg/g creatinine)

·       - Presence or history of panel-reactive anti-HLA antibody levels greater than background by flow cytometry.

·       - Pregnant, breastfeeding, or unwilling to use effective contraception throughout the study and 4 months after study completion

·       - Presence or history of active infection, including hepatitis B, hepatitis C, HIV, or tuberculosis.

·       - Negative for Epstein-Barr virus by IgG determination

·       - Invasive aspergillus, histoplasmosis, or coccidioidomycosis infection in the past year

·       - History of malignancy except for completely resected squamous or basal cell carcinoma of the skin

·       - Known active alcohol or substance abuse

·       - Baseline Hgb below the lower limits of normal, lymphopenia, neutropenia, or thrombocytopenia

·       - History of Factor V deficiency

·       - Any coagulopathy or medical condition requiring long-term anticoagulant therapy after transplantation or individuals with an INR greater than 1.5

·       - Severe coexisting cardiac disease, characterized by any one of the following conditions:

o   - Heart attack within the last 6 months

o   - Evidence of ischemia on functional heart exam within the year prior to study entry

o   - Left ventricular ejection fraction <30%

·       - Persistent elevation of liver function tests at the time of study entry

·       - Symptomatic cholecystolithiasis

·       - Acute or chronic pancreatitis

·       - Symptomatic peptic ulcer disease

·       - Severe unremitting diarrhea, vomiting, or other gastrointestinal disorders that could interfere with the ability to absorb oral medications

·       - Hyperlipidemia despite medical therapy, defined as fasting LDL cholesterol >130 mg/dl (treated or untreated) and/or fasting triglycerides >200 mg/dl

·       - Currently receiving treatment for a medical condition that requires chronic use of systemic steroids except for the use of 5 mg or less of prednisone daily, or an equivalent dose of hydrocortisone, for physiological replacement only

·       - Treatment with any antidiabetic medication other than insulin within the past 4 weeks

·       - Use of any study medications within the past 4 weeks

·       - Received a live attenuated vaccine(s) within the past 2 months

·       - Any medical condition that, in the opinion of the investigator, might interfere with safe participation in the trial

o   - Treatment with any immunosuppressive regimen at the time of enrollment.

o   - A previous islet transplant.

·       - A previous pancreas transplant, unless the graft failed within the first week due to thrombosis, followed by pancreatectomy and the transplant occurred more than 6 months prior to enrollment.

 

*Modified from the information relative to active trials from the Clinical Islet Transplant Consortium (www.citisletstudy.org/) as listed at http://clinicaltrials.gov/ct2/show/NCT00434811.

 

Multidisciplinary Team

 

Islet Transplant Programs require the integration of multidisciplinary expertise.  The endocrinologist expert in diabetes diagnosis and management is an essential member of the team, and can identifying subjects who may benefit from beta-cell replacement therapy, and help with the evaluation of metabolic control during all phases of the follow-up.  The psychologist is involved in the evaluation of islet transplant candidates to assess their motivation, mental fit to enroll in the trial, and ability to adhere to the therapy.  Psychometric and psychological evaluations are performed during the follow-up period after transplantation.  Transplant surgeons provide the expertise in organ procurement, with transplant procedures, overall management of patients, and immunosuppression.  A dedicated Cell Transplant Center with specialized experts in pancreatic cell isolation, purification, culture, potency assessment, and quality assurance warrant that islet cell products are manufactured for clinical transplantation following cGMP standards and FDA regulations.  The interventional radiologist performs the noninvasive cannulation of the portal vein and participates in the post-transplant monitoring of the liver using noninvasive imaging techniques.  The organ procurement organizations and organ distribution networks (UNOS in the U.S.) contribute to the identification and allocation of donor organs matching the recipient’s characteristics.  The ophthalmologist and nephrologist are involved to monitor and treat progressive diabetic complications (i.e., retinopathy and renal function, respectively).

 

Islet Isolation and transplantation

 

Islets are highly vascularized cell clusters ranging <50um to ~800um in diameter that constitute the endocrine component of the pancreas.  It has been estimated that a healthy pancreas may contain approximately 106 islets scattered throughout the gland, and accounting for only ~1% of total pancreatic tissue.  Each cluster comprises several thousands of endocrine cell subsets that are closely in touch with capillaries and with each other.  Complex cell-cell interactions between different cell subsets, innervation, incretins, and metabolites (sugar and amino acids, amongst other) in the blood and interstitial space all contribute to the proper control of glucose homeostasis (45).  Preservation of the integrity of islet cell cluster is a prerequisite for their optimal function.  The procedure currently used to extract islets from human pancreas is the so called automated method for isolation of the islets of Langerhans, established in 1987 by Ricordi and colleagues (46). Before the beginning of the isolation procedure, the spleen and the duodenum are removed from the pancreas and an accurate dissection and removal of the peri-pancretic fat, lymph nodes, and vessels is performed. Then, the pancreas is divided at the neck and two 16-20-gauge angiocatheters are inserted into the main pancreatic ducts. The organ is then perfused with cooled perfusion solution containing collagenase and serine – protease inhibitor – dissolved in buffer at a pressure of 140-180 mmHg. After 10 minutes of cold perfusion, the distended pancreas is further cut into smaller sections, and placed into the Ricordi chamber. This chamber is composed of a superior and an inferior part, separated by a filter that has pores of about 700μm. Seven to nine stainless steel balls and the fragments of the pancreas are placed into the inferior part of the chamber, which is then filled with the digestion solution and closed together with the superior part of the chamber. A peristaltic pump connected to the system is activated creating a flow of 40 ml/min. The digestion runs in a closed circuit where warm Hank’s solution is pumped in the inferior chamber and the tissue released as the solution passes into the superior chamber through the filter. The collagenase is re-circulated at a temperature not exceeding 37°C and the chamber is agitated. When most of the islets are free of the surrounding acinar tissue, and intact islets are observed, the heating circuit is bypassed. The temperature is progressively decreased to 10°C and the collagenase diluted with cold RPMI. The free islets are then collected in containers, washed several times, re-suspended in cold organ preservation solution and purified with a continuous ficoll gradient using a Cobe 2991 cell separator. At the end of the procedure samples of the islet preparation are collected and evaluated through staining with dithizone (DTZ) which marks zinc in the insulin granules, resulting in a characteristic red stain. Adding few drops of DTZ solution to a sample allows easy evaluation of the morphology and number of isolated islets through computerized digital analysis.

 

The islet manufacturing processes must be controlled by different assays and the islet batch product validated and characterized. Then safety testing is carried out for sterility and pyrogenicity, identity (insulin content), cell number (amount of tissue, counting of islets), purity (percentage of ductal, acinar, beta, and other cells), viability (islet nucleotide content), potency (insulin secretory response), and finally stability (storage in culture). Specific features of the final islet preparation are a required for islet preparations used in islet transplantation, in particular purity (> 20% of the preparation being islets), adequate number of islets (>5,000 islet equivalent recipient body weight for the first infusion, >3,000 for further infusions) and total tissue volume (< 5 ml).

 

The infusion of the islets can be performed a few hours after the end of the isolation process or up to 72 hours thereafter. The implantation site is usually the hepatic parenchyma through the portal system of the recipient. Recently other implantation sites have been proposed (47) in the clinical setting including the bone marrow (48, 49), subcutaneous sites (50), the gastric submucosa (51), the omentum (52, 53) and striated muscle (54, 55), which in the future, may prove to be valid alternative sites for islet transplantation. An adequate amount of islets obtained is calculated with respect to the body weight of the recipient and re-suspended immediately before intrahepatic transplantation in 40-60 mL of a solution suitable for injection (Ringer Lactate, 1% Human Albumin and 2000 IE of heparin). Percutaneous trans hepatic catheterization is the most common access route, as well as a mini-laparotomy and cannulation of an omental or mesenteric vein, or recanalization of the umbilical vein. Access to the portal vein is usually provided by interventional radiologists. If the portal pressure is documented to be below 20 mmHg, the islet infusion bag is connected with the portal vein catheter and infused over a period of 15 to 60 minutes. Islet infusion is halted if the portal pressure exceeds 22 mmHg. After completion of the islet infusion, the catheter is withdrawn; coils and gelatin-sponges are deployed in the puncture tract to prevent bleeding. A schematic animation of the islet isolation and transplant procedures is available online (http://www.youtube.com/watch?v=aMNKu-ZVUls).

 

The Consortium Concept

 

A major development in the field of islet transplantation is the combination of individual centers into larger groups such as the GRAGIL network in France and Switzerland, the Nordic Network for Clinical Islet Transplantation (NNCIT) in the Scandinavian countries, and the Clinical Islet Transplant Consortium (CITC) internationally but concentrated in North America. The need for dedicated infrastructures and personnel specialized in islet cell processing, quality assessment, and cGMP standards impose an enormous financial burden on any Clinical Islet Transplant Program.  Acquiring and maintaining the specialized expertise in islet cell processing requires a steep learning curve and continuous refinements and training that add to the costly procedure.  Recent data have shown that the experience of the clinical islet transplant team in cell processing and management of immunosuppression are critically important in determining the success of a clinical trial (56).  Based on these premises, the development of regional cell processing centers that are part of consortia that are integrated with distant transplant centers is increasingly being considered as a practical and cost-effective strategy (Figure 1).  Initial reports of successful clinical trials carried out in the context of Consortia both in Europe and North America (57-60) support the feasibility of such an approach, which may be of assistance in reducing the operational costs while enhancing the success rate of clinical trials (i.e., better utilization of donor pancreata, more reproducible success in obtaining adequate numbers of functional islets from a donor pancreata, etc.).

Figure 1.  Islet Transplant Consortium Models.  A.  The centralized (or ‘regional’) Cell Processing Facility receives the donor pancreas from a distant Transplant Center and isolates islet cell products that are sent back for implant.  B.  The centralized Cell Processing facility receives the donor pancreas from one of the Transplant Centers and distributes the isolated islets to any of the Transplant Center in the Consortium according to the best match of the cell product for the transplant candidate on the waiting list for transplant (that is, the islet cell product is not necessary returned to the center recovering the pancreas). 

 

Islet Transplant Activity: The Collaborative Islet Transplant Registry (CITR)      

 

In 2001, the National Institute of Diabetes & Digestive & Kidney Diseases established the Collaborative Islet Transplant Registry (CITR) to compile data from all islet transplant programs in North America from 1999 to the present. The Juvenile Diabetes Research Foundation (JDRF) granted additional funding to include the participation of JDRF-funded European and Australian centers from 2006 through 2015. The cumulated North American, European and Australian data are pooled for analyses included in the annual report. CITR Annual Reports are publicly available as open access and can be downloaded or requested in hard copy at www.citregistry.org. From 1999 through 2015 – the cut-off for the last 10th Annual Report – CITR has collected data on the following groups of study subjects:

  1. a) Allogeneic islet transplantation (typically cadaveric donor), performed as either islet transplant alone (ITA) or islet-after-kidney (IAK). A small number of cases have been performed as islet simultaneous with kidney (SIK) or kidney-after-islet (KAI).
  2. b) Autologous islet transplantation, performed after total pancreatectomy (N=819) are also reported to CITR.

 

As of September 30, 2015, the CITR Registry included data on 1,086 allogeneic islet transplant recipients (877 islet transplants alone, ITA, and 183 islets after kidney, IAK, 24 simultaneous islet kidney, SIK, and 2 kidneys after islet, KAI), who received 2,150 infusions from 2,619 donors (Exhibit A). The North American sites contributed 55%, while the European and Australian sites contributed 45% of the data. Combining the ITA and IAK recipients, 29% received a single islet infusion, 49% received two, 19% received three, and 3% received 4-6 infusions. Of 23 North American sites performing Auto-ITx during this period, 11 reported data to CITR, along with 4 European and Australian islet transplant centers. These sites registered 819 auto islet transplant recipients. Of these, 754 recipients were in North America, 63 in Europe, and 2 in Australia. Ninety-six (96) were aged less than 18, and 723 were 18 or older at the time of their transplant.

 

Clinical Management of Islet Transplant Recipients

 

The clinical management of islet transplant recipients requires the concerted effort of endocrinologists and transplant teams. 

 

Immunosuppression

 

Preexisting and transplant-induced auto- and allo-specific cellular immune responses play a crucial role in the loss of islets and islet function infused in the liver (61-63) along with non-specific immune responses predominantly mediated by innate inflammatory processes related to mechanics and site (64-67). Islet graft rejection occurs without clinical symptoms. Neither guidelines nor formal consensus on the “best” or “standard” immunosuppressive strategy for human islet transplantation are currently available. Multiple induction and maintenance agents are administered peri- and post- every infusion in the same recipient. According to CITR data (68), a substantial shift in immunosuppression strategies has been documented during the last years.

 

Induction with interleukin-2 receptor antagonist (e.g., daclizumab) only, which comprised about 54% of all initial infusions in 1999-2002, was replaced or supplemented with regimens that included T-cell depletion with/without TNF antagonists in about 68% of the new infusions performed by 2011-2014 (10, 69-76). In 1999-2002, maintenance immunosuppression was predominantly (65%) calcineurin (CNI) +mTOR inhibitors (56). It was increasingly replaced or supplemented throughout the eras by a CNI and IMPDH-inhibitor combination (70, 77-79).; in the most recent era, CNI+mTOR inhibitors were used in 15% of new infusions while CNI+IMPDH inhibitors were used in about 56%. Moreover, the use of alemtuzumab-induction therapy was recently reported and associated with encouraging longer-term function (80, 81). New biologic agents with potentially lower islet cell and organ toxicity profiles are currently being evaluated in ongoing clinical trials.  Amongst these are agents that target co-stimulation pathways in immune cells and/or adhesion molecules (CTLA4-Ig, LFA-1 PD-1/PD-L1 CD40 ) (82-88) or chemokine receptors (CXCR1/2) (64, 89) Finally, a calcineurin inhibitor-free immunosuppressive regimen was reported (90).

 

Antibiotic and Antiviral Prophylaxis 

 

Subjects receiving immunosuppression therapy are more susceptible to opportunistic infections, as well as reactivation or de novo occurrence of viral infections.  Antibiotic prophylaxis for Pneumocystis carinii consists in trimethoprim and sulfamethoxazole three times a week.  Antiviral prophylaxis is aimed are reducing the risk, or treating the occurrence, of cytomegalovirus infections (which have been recognized to increase the risk of graft loss in solid organ transplantation) and of reactivation of Epstein - Barr virus infection (which has been associated with the dreadful post-transplant lymphoproliferative disease, PTLD).  Current protocols utilize antiviral therapy with vanglancyclovir daily for the first trimester post-transplant.  Monitoring of viremia in peripheral blood samples by PCR is becoming a routine during follow-up as it allowed for the early detection of reactivation or de novo infections that may be treatable without compromising graft outcome (91, 92). 

 

Thromboembolism Prophylaxis 

 

It has been recognized that isolated islets produce tissue factor and other pro-inflammatory molecules that may trigger an instant blood-mediated inflammatory reaction upon infusion into the blood-stream.  This, in turn, may enhance the generation of noxious stimuli after embolization in hepatic sinusoids of the liver, significantly reducing the mass of functional islets engrafting.  Aggressive heparin treatment is generally implemented in the early period after transplant.  Heparin is added to the transplant medium used during the islet infusion, while low molecular weight heparin injections are administered in the post-transplant period.  This is aimed at enhancing islet engraftment in the hepatic portal system while reducing the risk of portal thrombosis. 

 

Peri-Transplant Insulin Management 

 

Islet engraftment may take up to a few weeks to allow for neovascularization of the clusters in the transplant microenvironment.  The monitoring of glycemic control in the immediate post-transplant period should be intense to attain tight glycemic values in order to avoid excessive workload for the newly transplanted islets as well as to prevent hypoglycemic episodes.  This is generally done by providing basal exogenous insulin that is then progressively reduced and withdrawn according to the glycemic values measured.

 

Post-Transplant Clinical Monitoring

 

Monitoring of cell blood counts (erythrocytes, white blood cells and differential), hemoglobin, platelets and coagulation markers is routinely performed in the post-transplant period.  These tests allow assessing the myelosuppressive effects of anti-rejection drugs.  In the case of severe anemia iron supplementation may be indicated, while for more severe cases erythropoietin treatment is implemented.  In the case of severe neutropenia, marrow stimulation with granulocyte-colony stimulating factor (G-CSF) is promptly implemented.

 

Renal function is monitored periodically in the follow-up of islet transplant recipients to assess the impact of restoring beta-cell function on the progression of diabetic nephropathy, and also to identify and timely correct potential nephrotoxicity of anti-rejection drugs (i.e., CNI and mTOR inhibitors).  Standard tests (serum creatinine, azotemia), urine tests (spot and 24-hr collections) are frequently performed during the follow-up and glomerular filtration rates (GFR) estimated using different algorithms (i.e., MDRD).  The nephroprotective effect of ACE inhibitors and of antagonists of angiotensin-receptor (ARB) in subjects with diabetes has been recognized, and their use is particularly indicated in transplant recipients treated with immunosuppressive drugs known for their negative effects on renal function (93-97).  Elevations of blood pressure from the range 130/80 mmHg are promptly treated pharmacologically.

 

Monitoring of lipid levels and prompt treatment of dyslipidemia are important in transplanted patients.  Some of the anti-rejection drugs (i.e., mTOR inhibitors) are prone to induce dyslipidemia, which in turn may have toxic effects on beta-cells or contribute to creating an unfavorable environment (i.e., steatosis) in the liver (98).  Prophylactic use of statins targeting LDL cholesterol levels <100mg/dL can be contemplated for islet transplant recipients. 

 

Liver function is monitored in the post-transplant period.  It is common to observe a transient and self-limited elevation of liver enzymes (transaminitis) because of the embolization of islets into the liver sinusoids (99, 100).  This is often associated with hyper-echoic pattern of the liver parenchyma at ultrasound evaluation in the early days post-transplant.  This phenomenon resolves spontaneously without the need for medical treatment.  Ultrasound evaluation of the liver and abdominal cavity in the days post-transplant also allows identifying timely possible procedural complications of the transplant, such as portal thrombosis, peritoneal hemorrhage and alterations of echogenicity of hepatic parenchyma (101).

 

Immune monitoring after islet transplantation does not differ much from that of any other organ transplant (102).  Basal and serial evaluation of Panel Reactive Antibodies (PRA) is performed to determine possible allosensitization against Human Leukocyte Antigens (HLA) class I and II of the Histocompatibility complexes of transplanted tissue.  Generally, maintenance of an adequate immunosuppressive regimen can prevent the development of alloantibodies, thereby preventing their deleterious effects on graft survival and function (i.e., chronic rejection leading to graft loss) (103-105).  Nonetheless, development of alloreactivity against donor or non-specific antigens may develop whenever reduction (i.e., during infections, toxicity and drug conversion, amongst other causes) or suspension (i.e., after complete graft loss) of immunosuppression is needed (105, 106). 

 

The autoimmune process underlying Type 1 Diabetes is associated with the appearance of antibodies against self-antigens (autoantibodies; i.e., towards GAD, IA-1 and insulin).  Serial titration of autoantibody levels during the follow-up period may enable detecting a reactivation of the autoimmune process, measured as conversion to positive values in previously negative subjects, or increase of antibody titers.  These have been associated with a lower rate of insulin independence and shorter duration of graft function after islet transplantation (56, 63).  New assays for additional autoantibodies (i.e., ZnT8) and for autoreactive T cells are under evaluation to help enhance the sensitivity of immune monitoring for early detection of recurrence of autoimmunity, which may enable implementation of timely immune interventions to rescue the transplanted cells (107-109).  

 

Monitoring Islet Graft Function

 

Several metabolic parameters allow for monitoring the function of transplanted islets (Table 3).  Since only subjects with Type 1 Diabetes who have undetectable stimulated c-peptide (<0.3 ng/dl) before transplant are recruited for an islet transplant, monitoring of basal and stimulated c-peptide levels offers an excellent biomarker of graft function, even when exogenous insulin is required.  There is no consensus on which approach is most suited to accurately assess functional islet mass.  Algorithms and indices that combine multiple parameters have been developed and proposed to help simplify obtaining objective functional assessment of islet transplant recipients (110-119).  The main goal is to identify early changes that indicate propensity to graft dysfunction (i.e., functional impairment during an infection, drug-induced toxicity).  Stimulation tests are performed before (at enrollment) and during the follow-up after transplant to evaluate the functional potency of the transplanted islets in response to different secretagogues (i.e., glucose, arginine, or mixed meal test).  Insulin therapy is generally implemented when random glycemic sampling demonstrates on three subsequent occasions within the same week fasting values >140 mg/dl (7.8 mmol/L) and postprandial values >180 mg/dl (10.0 mmol/L), or after recording two consecutive A1c values >6.5%.

 

Table 3.  Monitoring of Islet Graft Function

Standard

Stimulation

Indices

Glycosylated Hb (A1c)

Fasting glycemia

Postprandial glycemia

MAGE*

CGMS*

Basal C-peptide

Daily insulin requirement

 

Mixed Meal

Intravenous glucose

Intravenous arginine

Hypo score

Liability index

Βeta score

Beta 2 score

Basal C-peptide/Glucose ratio

HOMA-B*

HOMA-IR*

TEF*

*Abbreviations.  CGMS: Continuous Glucose Monitoring System. MAGE: Mean Amplitude of Glucose Excursions. HOMA-B: Homeostasis Model Assessment – functional beta cell mass.  HOMA-IR: Homeostasis Model Assessment – Insulin-Resistance. TEF: Transplant Estimated Function

 

The IGLS Score

 

The lack of standardized definition of graft functional and clinical outcomes remains a source of concern in β-cell replacement influencing its recognition as a valid clinical option from the endocrinology community. In order to address this issue, the International Pancreas & Islet Transplant Association (IPITA) joined with the European Pancreas & Islet Transplant Association (EPITA) for a two-day workshop on “Defining Outcomes for β-Cell Replacement Therapy in the Treatment of Diabetes” in January 2017 in Igls, Austria. The main objective was to develop consensus on the definition of function and failure of current and future forms of β-cell replacement therapies.  As result of the workshop, an IPITA/EPITA Statement was recently published (120, 121). This Statement introduces some relevant innovations in the field including a new classification for the definition of clinically successful outcome. The functional status and clinical success of a β-cell graft should be defined separately using the same components of assessment: the HbA1c, severe hypoglycemic events, insulin requirements, and C-peptide. Concordantly, a four-tiered system was proposed to classify the functional outcomes of β-cell replacement:

 

  • optimal β-cell graft function: HbA1c ≤6.5%, the absence of any severe hypoglycemia, the absence of any requirement for exogenous insulin or other anti-diabetic drugs, and documentation of an increase over pre-transplant measurement of C-peptide.
  • good β-cell graft function: HbA1c <7.0%, the absence of any severe hypoglycemia, a reduction by more than 50% from baseline in insulin requirements or the use of non-insulin anti-diabetic drugs, and documentation of an increase over pre-transplant measurement of C-peptide.
  • marginal β-cell graft function: no modification of HbA1c, the reduction of severe hypoglycemia, a reduction by less than 50% from baseline in insulin requirements, and documentation of an increase over pre-transplant measurement of C-peptide.
  • failure β-cell graft function: absence of any evidence for a clinical impact (no modification of HbA1c, incidence of severe hypoglycemia and insulin requirement) and clinically insignificant levels of C-peptide.

 

Clinically successful outcomes include both optimal and good functional outcomes, implying that the use of exogenous insulin or other anti-diabetic drugs is not synonymous with graft loss or failure. Neither a marginal β-cell graft nor a failed β-cell graft is considered clinically successful.  However, if documented impairment in hypoglycemia awareness, frequent occurrence or exposure to severe hypoglycemia, or marked glycemic variability/lability is convincingly improved, then it may be appropriate to consider that the β-cell graft is clinically impactful also in marginal function and the benefit of maintaining β-cell graft function may outweigh risks of maintaining immunosuppression. This implies that hypoglycemia awareness, serious hypoglycemia, and glycemic variability/lability must be evaluated at baseline for monitoring whether a marginally functioning graft is continuing to provide any clinical impact.

 

IPITA / EPITA Statement has the merit of having introduced a defined concept of clinical success based on easily measurable parameters over time and with a wide consensus of international experts. Implementation of this new β-cell replacement outcome definition and its use in publication will  be critical to improve the performance and to reliably compare the different β-cell replacement  strategies(122).

 

Impact of Islet Transplantation on Metabolic Control

 

Two successful large-scale Phase 3 clinical trials in islet transplantation have been published recently: CIT-07 (multicenter, single-arm) and TRIMECO (multicenter, open-label, randomized).(123, 124) Both these studies were designed to demonstrate that human islet transplanted in T1D subjects with impaired awareness of hypoglycemia and severe hypoglycemic events can safely and efficaciously maintain glycemic balance and eliminate one of the most severe complications associated with insulin therapy. The main goal of islet transplantation has historically been insulin independence; however, investigators are currently considering additional relevant outcomes, such as the reduction in the frequency of hypoglycemic episodes and the positive effects on complications and quality of life (QoL) (125).

 

Recent clinical trials demonstrated that the effects of islet transplantation on metabolic control are quite reproducible in subjects with unstable Type 1 Diabetes (Table 4) (68).  Exogenous insulin requirements to attain optimal metabolic control are dramatically reduced immediately after islet transplantation, with a reduction of mean amplitude of glycemic excursions (MAGE) throughout the day and normalization of A1c <6.5% (77, 78). 

 

Since the main indications for islet transplantation in subjects with Type 1 Diabetes are unstable control and frequent severe hypoglycemic episodes, the most remarkable effect of the transplant is the abrogation of severe hypoglycemia (126-129).  Using Hypo Score and Liability index to assess longitudinally islet transplant recipients, a significant reduction in the incidence of severe hypoglycemia over a four-year follow-up period was demonstrated, a finding suggesting that the intervention can support a better and more physiological metabolic control than medical therapy (123, 124, 130, 131)  It is noteworthy that the prevention of severe hypoglycemia persists long-term and even in subjects requiring exogenous insulin to maintain optimal glycemic control (such as after implantation of a suboptimal islet mass or after development of graft dysfunction) as far as c-peptide is measurable (69, 75). 

 

Following islet transplantation, the restoration of beta-cell responses to secretagogue stimulation is observed, with improved insulin secretion (‘first phase’) in response to intravenous glucose, as well as increased c-peptide secretion in response to oral glucose.  Normalization of glycemic threshold triggering the release of counter-regulatory hormones can be demonstrated during hypoglycemic clamp studies, albeit without reaching normalization of the magnitude of the vegetative response.  Furthermore, quasi-normal glucagon secretion in response to hypoglycemia can be observed (132-136).  These observations may, at least in part, explain the significant improvement in metabolic control and recovery of hypoglycemia awareness observed after islet transplantation, which persists after development of graft dysfunction and even several months after graft failure (and loss of detectable c-peptide)  (68, 127).

 

Evaluation of quality of life (QOL) using standardized psychometric instruments and interviews carrier on by psychologists has demonstrated a significant improvement during the follow-up of islet transplant recipients (126-129, 137-141)(39, 128-131). The absence of severe hypoglycemic events is most likely the reason for significant positive effects on the fear score and a gained sense of independence from insulin therapy.

 

After transplantation of an adequate islet mass obtained from one or more donor pancreata (estimated ≥10.000 islet equivalents (IEq)/kg of recipient’s body weight), insulin independence can be reproducibly achieved.  By combining donor selection criteria with improved isolation techniques and adequate immunomodulation of the recipient, insulin independence after single donor islet preparation is becoming more reproducibly possible to achieve.  Islet preparations obtained from more than one donor pancreas can be transplanted at once after pooling them, or sequentially based on the metabolic needs of each subject.  Data from the Clinical Islet Transplant Registry and independent trial reports have shown that insulin independence at one year from completion of the transplant is up to 70% with virtually 100% of the subjects maintaining graft function (c-peptide) if adequately immunosuppressed (68, 75).  A progressive loss of insulin independence with approximately 90% of subjects requiring reintroduction of exogenous insulin (most of them with detectable c-peptide) has been reported in recent clinical trials based on the ‘Edmonton protocol’ (induction with anti-IL2R antibody; maintenance with sirolimus and tacrolimus) and some variants of it (56, 70, 77, 79, 131).  More recent trials using more potent lymphodepletion (i.e., thymoglobulin, anti-CD3 or anti-CD52 antibodies) and/or biologics (anti-IL2R, anti-TNF, anti-LFA-1 antibody or CTLA4Ig) have shown great promise with approximately 50% of insulin independence at 5 years after islet transplantation (79, 142-146)., which is comparable to some of the data in whole pancreas transplantation in subjects with Type 1 Diabetes (73, 76, 79, 84, 85). In light of the results of the last decade of clinical islet transplant trials, achievement of insulin independence, although desirable, no longer should be considered the main goal of islet transplantation.  The sizable improvement of metabolic control in the absence of severe hypoglycemic events, the amelioration of diabetes complications and the achievement of sustained better quality of life, which are quite cumbersome to reproduce by the means of medical treatment, justify the risks associated with the islet transplant procedure and immunosuppression in this high-risk population of subjects with unstable diabetes.

 

Regarding auto transplantation the largest published series are from the University of Minnesota (15-18), University of Cincinnati (19, 20), and Leicester (21-24, 147). Overall, one-third of patients in the Minnesota series achieves insulin independence, and the majority have islet graft function, as documented by C-peptide positivity (15, 21). Cincinnati, Leicester, and other centers have published similar results, with 22-40% of the patients being insulin independent after islet transplant (20, 148, 149). A significant association between insulin independence and the IEQ/kg transplanted (i.e., islet mass standardized by patient’s weight) was described. Bellin et al. (18) and White et al. (23) reported that insulin independence is related to the number of transplanted islet cells (>2,000 IEQ/kg and >3,000 IEQ/kg, respectively). Similarly, Sutherland et al. (150) reported that insulin independence at 1 year was observed in 63% of the patients who received greater than 5,000 IE/kg. Moreover, pancreatectomy recipients benefit from an islet autograft in ways apart from insulin independence. In fact, the major goal of IAT in these patients is a good glycemic control without brittle diabetes. Ninety percent of patients in the Minnesota series and 100% of those in the Leicester series were C-peptide positive after the procedure (15, 21).  The majority of patients receiving an islet auto transplant maintained good glycemic control, with 82% of all recipients having average HbA1c levels <7.0% (15).

 

Impact of Islet Transplantation on Diabetes Complications

 

Encouraging results have been reported in recent years on the multiple beneficial effects of islet transplantation on the progression of diabetes complications (reviewed in (151)).  Although based on nonrandomized pilot studies, which should be cautiously evaluated, they provide the proof of concept of the importance of restoring beta-cell function in patients with diabetes.  In particular, improvement of micro- and macro-angiopathy (main causes of diabetic nephropathy) (72, 94, 95, 140, 152-158) and stabilization/reduced progression of retinopathy (97, 152, 159, 160) and neuropathy (159, 161-163) have been described.  Amelioration of cardiovascular and endothelial function, reduction of atherothrombotic profile paralleled by reduced incidence of cardiovascular accidents and higher survival rates were reported In IAK recipients (164-167) (155, 163, 164, 166, 168).  Furthermore, significantly improved longevity of a renal transplant was observed after islet transplantation (155).  It is likely that these benefits are the consequence of improve metabolic control conferred by the islet transplant.  In addition, it has been proposed a contribution of restored c-peptide secretion and its effects on multiple targets (169).

 

Table 4.  Benefits of Islet Transplantation

Metabolic control

-        Reduction of exogenous insulin requirements or insulin independence

-        Reduction of MAGE

-        Reduction or normalization of A1c

-        Absence of severe hypoglycemia

Quality of Life

-        Reduced fear of hypoglycemia

-        Improvement of Diabetes Quality of Life

Diabetes complications

-        Improvement of micro- and micro-angiopathy

-        Improvement of cardiovascular and endothelial function

-        Reduced incidence of acute cardiovascular events

-        Reduced nephropathy progression

-        Stabilization/slower neuropathy progression

-        Stabilization/slower retinopathy progression

 

Common Adverse Events and Their Management (Table 5)

The procedure of islet transplantation has proven to be very safe, especially when compared with whole pancreas transplant (144, 170, 171).  Although islet allotransplantation is a relatively safe procedure, adverse events and serious adverse events are not infrequent.

 

For allogenic islet transplantation bleeding, either intraperitoneal or liver subcapsular, is the most common procedure-related complication, occurring with an incidence as high as 13%  (172). The use of fibrin tissue sealant and embolization coils in the hepatic catheter tract seems to effectively minimize the bleeding risk (172, 173). Partial portal vein thrombosis complicates fewer than 5% of islet infusion procedures (131), and complete portal venous thrombosis is rare. The use of purer islet preparations, greater expertise in portal vein catheterization, and new radiological devices (catheters medicated with anticoagulation) will continue reducing the risk of portal vein thrombosis, although the risk is unlikely be completely eliminated. Other complications of islet cell transplantation include transient liver enzyme elevation (50% incidence) (99), abdominal pain (50% incidence), focal hepatic steatosis (20% incidence) (174, 175), and severe hypoglycemia (< 3% incidence). Another complication related to the intrahepatic islet transplantation procedure is portal hypertension that can occur acutely during the islet infusion, especially in the case of infusions other than the first one (176). Finally, severe hypoglycemia is a risk associated with the infusion of islets. Iatrogenic hypoglycemia in the immediate post-transplant period is a rare event.  Frequent blood glucose monitoring immediately following islet transplantation is recommended to avoid severe unrecognized hypoglycemia in the early post-transplant period. The risk of transmission of CMV disease from donor to recipient has been surprisingly low in recipients of islet allografts, particularly in the most recent period with routine use of purified islet preparations (140-144). As with any allogeneic transplant, islet transplant recipients may become sensitized to islet donor histocompatibility antigens (HLA), leading to the development of panel reactive alloantibodies (PRA).Data on the development of cytotoxic antibodies against donor HLA in islet allotransplant recipients with failing grafts have been reported from several islet transplant centers (148-152). A potential consequence of high PRA levels in recipients of a failed islet transplants is that if these individuals develop diabetic nephropathy in the future, a high PRA may increase their time on a transplant list for a suitable kidney graft.

 

The need to implement anti-rejection therapy exposes transplant recipients to an increased risk of untoward side effects expected in any immunosuppressed subjects (Table 5) (100).Opportunistic infections of urinary tract, upper respiratory tract, and skin are frequent, along with myelosuppressive and gastrointestinal effects of the immunosuppressive drugs.  In the majority of the cases, these effects are not severe and resolve without sequel with medical treatment.

 

Elevation of viremic titers for cytomegalovirus (CMV) or Epstein-Barr virus (EBV) in the presence of overt clinical symptoms (i.e., de novo infection or reactivation in seropositive subjects) imposes the implementation of anti-viral therapy and reduction of immunosuppressive drug dose (91).  Timely intervention may result in faster resolution of the symptoms without compromising graft survival. Direct organ toxicity of immunosuppressive drugs has been recognized.  Symptoms associated with neuro- and/or nephro-toxicity are relatively frequent in subjects receiving chronic immunosuppressive agents currently in use in the clinical arena.   In these cases, modification of the anti-rejection regimen is indicated, with dose reduction or conversion to a different combination of drugs.  In the majority of cases, these changes resolve the symptoms without compromising graft survival (177, 178). Nephrotoxicity from sirolimus and/or tacrolimus has been described in patients with T1D undergoing islet transplantation, particularly when kidney function is already impaired because of pre-existing diabetic nephropathy (179, 180).

 

The decline in eGFR (CKD-Epi) after islet transplantation is both statistically significant and clinically important. IAK had much lower pre-transplant levels than ITA, which then declined at a slower rate. Initial levels were also lower in recipients age 35 and older and declined at a slower rate compared to younger recipients. Levels were generally higher among recipients managed with both mTOR inhibitors and calcineurin inhibitors compared to other maintenance immunosuppression regimens. Compared with an age unadjusted cohort of 1,141 T1D followed by the Diabetes Control and Complications Trial and then by the Epidemiology of Diabetes Interventions and Complications (EDIC) (The DCCT/EDIC Research Group, 2011) who started with mean eGFR levels of 126 ml/min/1.73m3 , CITR allograft recipients had much lower mean eGFR (88.1±0.9SE for ITA and 63.1±1.8 for IAK) at their first transplant. CITR ITA recipients exhibited a decline in eGFR of 14.2 ml/min/1.73m3 and IAK experienced a mean decline of 5.3 ml/min/1.73m3 in 5 years from last infusion, compared to a mean decline of about 9 ml/min/1.73m3 over the first 5 years in the DCCT.

 

The Tenth Annual Report of the Collaborative Islet Transplantation Registry (CITR) reported 1,854 adverse events (AEs) on 877 ITA, 364 AEs on 183 IAK, and 53 AEs on 24 SIK. For ITA, the adverse events most frequently deemed “possibly or definitely related” to the infusion procedure included: peritoneal hemorrhage (n=33), hepatic hematoma, hepatic hemorrhage, or portal vein thrombosis (n=23), increased AST (n=7), blood alkaline phosphatase (n=19), gammaglutamyl transferase (n=12), abnormal liver function tests (n=121), hematoma/hemorrhage (n=18), and GI/peritoneal hemorrhage (n=35), while those deemed “possibly or definitely related” to immunosuppression included leukopenia/lymphopenia/neutropenia/granulocytes (n=339), diarrhea/GI disorder (n=68), fatigue (n=13), mucosal inflammation (n=16), graft vs host disease (n=2), infection (n=65), pneumonia (n=10), increased blood creatinine (n=18), neoplasm (n=39), renal disorder/failure (n=14), lung disorder/infiltration (n=9), skin disorder/exfoliative rash (n=12), and hypertension (n=8). For IAK, the adverse events most frequently deemed “possibly or definitely related” to the infusion procedure included: blood disorder/leukopenia/lymphopenia/neutropenia (n=14), gastrointestinal/peritoneal hemorrhage (n=16), infection (n=36), and renal disorder/failure/pyelonephritis (n=15). For SIK, the adverse events most frequently deemed “possibly or definitely related” to immunosuppression included lymphopenia (n=4) and pneumonia (n=3). The adverse event most frequently deemed “possibly or definitely related” to the infusion procedure among SIK was peritoneal hemorrhage (n=3).

 

The Tenth Annual Report of CITR reported that in the first 30 days following islet transplantation, about 26% of recipient experienced a reportable adverse event. The majority (70%) were adjudicated by the local investigator as possibly or definitely related to either the infusion procedure or the immunosuppression (IS). The majority were not unexpected, such as abnormal lymphocyte count and increased liver function. Very few were infections. The instances of peritoneal hemorrhage seen in the early era 1999-2002 have been drastically reduced in the recent eras. About 14% of allo-islet recipients experienced a serious adverse event in the first 30 days, which occurred about equally in IAK as in ITA, and have declined somewhat over the eras.  In the first year after islet transplantation, which includes a majority of the re-infusions that were performed, about 43% of all recipients experienced a reportable adverse event, with a decline in the most recent eras. About one-third have experienced a serious adverse event within the first year, with a significant decline in the most recent era. This pattern is also seen for all adverse events in all follow-up after islet transplantation. The outcomes of the reported adverse events have improved over the decade, with fewer patients experiencing long-term sequelae of their adverse events in the most recent era. Many adverse events seen in this population are unrelated to islet transplantation but not unexpected in a cohort of older T1D with significant co-morbidity. Overall, 17% of all recipients failed to recover completely from an adverse event. This is the worst outcome of all adverse events, including those not related to the islet infusion or immunosuppression. Among related adverse events only 11% failed to recover completely. Life-threatening events have occurred in 13.5% of islet-alone, in 18.0% of IAK recipients, and in 29.2% of SIK recipients (p<0.0001). Recent eras have seen a substantial decline in the incidence of life-threatening events. Most involved neutropenia and abnormal liver function. The vast majority recovered, 3% died, 3% did not recover, and 8% recovered with sequelae.

 

The total cohort of 1086 allo-islet recipients were followed for a mean of 4.2±3.5 SD years, comprising 4,583 person-years of follow-up from first infusion. A total of 51 instances of neoplasm have been diagnosed in 34 of the 1,086 islet recipients. This equates to about 0.01 neoplasms per person-year. Of the total 51 events, 73% were deemed possibly related to immunosuppression, and 6% definitely related. Of the total events, 65% recovered, 12% did not recover, and 6% recovered with sequelae. There were 35 instances in 22 patients (1 in 18 recipients and multiple in 4 recipients) of basal or squamous cell carcinoma of the skin. Of the 18 patients with a single instance, 16 recovered (1 with sequelae) and 2 have an unknown recovery status. Of recipients with multiple instances, 3 have recovered from all instances (2 with sequelae) and 1 has not recovered. There were 4 instances of breast cancer (2 instances in 1 recipient), 4 instances of thyroid cancer (2 instances in 1 recipient), 2 instances of PTLD and 1 instance of CNS lymphoma, 2 instances of lung cancer, and 1 instance of mucinous adenocarcinoma of the appendix. Of the recipients with non-skin cancers, 6 recovered, 1 was still recovering, 4 had not recovered, and 1 died (lung cancer). For 2 instances of cancer, the type of neoplasm was not specified, but the recipients were both reported to have recovered without sequelae.

 

There have been 33 reports of death to the Registry for islet allograft recipients, for 3.0% crude mortality over a mean of 4.4 years elapsed follow-up per patient (including periods after complete graft failure and loss to observed follow-up). Causes of death were (# cases): cardiovascular (8), hemorrhage (3), pneumonia (2), renal failure (2), respiratory arrest (2), acute toxicity (1), cerebrovascular event (1), diabetic ketoacidosis (1), infection (1), lung cancer (1), multi-organ failure of unknown etiology (1), necrosis (1), pneumopathy (1), and viral meningitis (1). The remaining 7 deaths did not have a cause specified. Cumulative mortality rates differed significantly both by era and transplant type. Mortality has declined steadily over the eras. SIK transplant recipients were disproportionately represented among fatalities comprising only 2% of the allo-islet recipient population, but 18% percent of deaths. Of the reported deaths, three were deemed possibly related and three were deemed definitely related to islet transplantation or immunosuppression.

 

An assessment of the surgical complication of islet auto transplantation was recently reported for the entire Minnesota series (n=413) (15). Surgical complications requiring reoperation during the initial admission occurred in 15.9% of the patients. The most common reason for reoperation was bleeding, occurring in 9.5% of the procedures. Anastomotic leaks occurred in 4.2 % of the patients, biliary in 1.4%, and enteric in 2.8%. Intra-abdominal infection requiring reoperation occurred in 1.9% of patients, wound infections requiring operative debridement in 2.2%. Gastrointestinal issues, such as bowel obstruction, omental infarction, bowel ischemia, delayed reconstruction because of bowel edema, tube perforation, requiring reoperation in 4.7% of the patients. Two patients (<1%) required reoperation to remove an ischemic or bleeding spleen after spleen sparing pancreatectomy (done in 30% of patients).

 

Table 5.  Most Frequent Complications in Islet Transplant Recipients

Procedure-related

-        Hemorrhage

-        Portal thrombosis

-        Transient transaminitis

Immunosuppression-related

Hematological

-        Anemia

-        Leucopenia

-        Neutropenia

Metabolic

-        Dyslipidemia

Gastrointestinal

-        Oral ulcers (Sirolimus)

-        Diarrhea (Mycophenolic acid)

-        CMV colitis

Respiratory tract

-        Upper respiratory infections

-        Interstitial pneumonitis (Sirolimus)

Neurological

-        Neurotoxicity (Tacrolimus)

Genitourinary

-        Urinary infections

-        Ovarian cysts

-        Dysmenorrhea

-        Nephropathy

-        Proteinuria

Cutaneous

-        Infections

-        Cancer

 

Current Challenges

 

There are many challenges that are currently limiting islet cell transplantation (Table 6) (181-183)  While significant progress has been made in the islet transplantation field, several obstacles remain precluding its widespread use. The clinical experience of islet transplantation has been developed almost exclusively using the intra-hepatic infusion through the portal vein (56). It has been suggested that the loss of as many as 50-75% of islets during engraftment is the reason why a very large number of islets are needed to achieve normoglycemia (47, 65). Moreover, two additional important limitations are the currently inadequate immunosuppression for preventing islet rejection (63) and the limited oxygen supply to islet in the engraftment site (184, 185). Current immunosuppressive regimens are capable of preventing islet failure for months to years, but the agents used in these treatments may increase the risk for specific malignancies and opportunistic infections. In addition, the most commonly used agents (like calcineurin inhibitors and rapamycin) are also known to impair normal islet function and/or insulin action. Furthermore, like all medications, these agents have other associated toxicities, including the harmful effect of certain widely employed immunosuppressive agents on renal function. The second very significant factor for early and late loss of islet mass is the critical lack of immediate vascularization and chronic hypo-oxygenation. Physiological supply of oxygen and nutrients in native islets is maintained by a tight capillary network, destroyed by the islet isolation procedure, restricting supply to diffusion from the portal vein and hepatic arterial capillaries until the revascularization process is completed. Oxygen tension in the liver parenchyma decreases from approximately 40 to 5 mmHg, eight-fold lower compared to the intra-pancreatic levels, leading to severe hypoxia, and β-cell death. Revascularization of the islet graft in rodent transplant requires 10-14 days and much longer in non-human primates and human recipients. Even after the revascularization of the islets is completed, the capillary’ density is significantly lower compared to the physiological intra-pancreatic situation. Finally, yet importantly, one of the main challenges is the cost of the procedure that, with the exception of islet auto-transplantation, is currently not covered by health care providers in the United States.  A validation multicenter trial aimed at obtaining a Biological License Application approved by the Food and Drug Administration is currently ongoing, and there is great hope this will propel forward the efforts in the field of cellular therapies for the treatment of diabetes.  In several European Countries and Canada, where the Health System covers the costs of the procedure, clinical islet programs have thrived at a faster pace than in the U.S.A.

 

Table 6. Current Challenges Facing Islet Transplantation

Challenge

Possible impact

Potential solutions

Progressive graft dysfunction

Reintroduction of exogenous insulin;

Destabilization of metabolic control;

Supplemental islet transplant.

Incretin mimetics;

Alternative islet implantation sites;

Novel immunosuppressive protocols.

Multiple islet donors

Increased operational costs;

Shortage of deceased donor pancreata for transplantation;

Risk of allosensitization.

Improved donor selection criteria;

Optimized cell processing;

Alternative sources of transplantable tissue (i.e., stem cells-derived or xenogeneic islets;

Alternative implantation sites.

Chronic immunosuppression

Systemic toxicity;

Increased risk of opportunistic infections;

Islet cell toxicity.

Use of biologics;

Immune isolation techniques; Development of immune tolerance inducing protocols.

Allosensitization

Reduced graft survival;

Preclude/worsen outcome of subsequent transplantation (i.e., islet or renal)

Maximizing the success rate of single donor islet transplantation;

Alternative sources of transplantable tissue;

Immune isolation;

Plasmapheresis / depletion of alloantibodies;

Novel immunosuppressive protocols;

Development of immune tolerance inducing protocols.

Cumbersome graft monitoring

Mainly rely on metabolic function tests, but cannot discriminate between immunological and metabolic causes of dysfunction;

Liver needle biopsies do not provide adequate graft tissue;

MRI and PET lack the resolution to detect islets scattered throughout the liver.

Improved simple metabolic measures predictive of graft dysfunction;

Improved sensitivity of noninvasive imaging techniques (functional MRI?);

Improved immune monitoring techniques for early detection of immune events able to discriminate between rejection and autoimmunity.

 

Future Developments in Beta-Cell Replacement Therapies

 

The field of cellular therapies for the treatment of diabetes is rapidly evolving.  Several new developments emerging in recent years may push to a new and broader dimension this interesting technology. While a wide range of improvements may be implemented in the donor selection and organ allocation scheme to increase pancreas utilization for transplantation, there is increasing new excitement for the use of unlimited alternative sources of transplantable islets, such as xenogeneic (i.e., obtained from other species such as porcine islets) (reviewed in (186)) or derived from human stem cells (187-192).  Pig islets may be available in plentiful amounts.  Importantly, the ability to obtain genetically modified pigs that lack or overexpress specific molecules may be of assistance in developing cellular products with reduced immunogenicity for transplantation into humans.  In turn, this technology may allow achieving long-term function under immunosuppressive regimens that are used for allogeneic cells or facilitating the induction of long-term acceptance of xenogeneic islet cells. 

 

Another area reporting great progress is that of regenerative medicine using human stem cells from embryonic or adult sources.  Encouraging experimental data suggests that insulin producing cells can be obtained from human multipotent stem cells, and great efforts are currently concentrated on developing cellular products with consistent potency and safety profile (ability to generate tumors) for future clinical application (187-190, 193, 194). A current limitation for islet transplantation is the inability to use non- or minimally-invasive predictive tests as well as biomarkers of early graft dysfunction to guide timely interventions aimed at preserving functional islet cell mass. 

 

Metabolic tests (i.e., glycemic control, insulin requirement, HbA1c, basal and stimulated c-peptide) remain the main indicators of graft function, the alteration of which may indicate underlying distress of the graft but cannot discriminate possible causes such as metabolic overload, immunity, or drug toxicity.  In some cases, graft dysfunction may be reversible (i.e., transient metabolic overload due to an infection episode), but in many other cases at the time graft dysfunction is detected, a considerable mass of functional beta cells might already be irreversibly lost. Monitoring of transplanted islets by noninvasive imaging techniques (such as MRI, PET-CT, and US) is cumbersome, as they lack the resolution for the detection of cellular clusters the size of islets (~50-900um) that are scattered throughout the recipient’s liver (reviewed in (195)).  While encouraging preliminary studies have shown that preloading of aliquots of the islet graft with iron nanoparticles (for MRI) (196-200) or labeled glucose (for PET-CT) (201-204) can be used safely, these techniques do not allow assessing the whole mass of transplanted clusters and provide only passive and transient information on islet distribution in the transplant site.  The progress in the field of functional MRI (fMRI) and toward the development of more sensitive beta-cell specific imaging techniques may allow a more objective assessment of islet cell mass over time in a near future.

 

Detection of biomarkers (reviewed in (102)) in blood samples to determine immune cell function (i.e., cell surface expression of specific markers by flow cytometry and cytotoxic lymphocyte gene expression profiles, amongst other) (205-207) and autoimmunity reactivation (namely, autoantibody titers) is evaluated in ongoing clinical trials to identify means of assessing the efficacy of the immunomodulation strategies, detecting rejection episodes and reactivation of autoimmunity early enough to implement timely immune interventions to prevent graft loss (62, 63, 208-210).  Unfortunately, some of the current tests lack adequate specificity as they may be affected also with underlying infections.  With the rapid evolution of high throughput arrays, it is likely that new and more specific molecular biomarkers of islet cell distress and immune cell function will become available in the near future.

 

Alternative transplantation sites (reviewed in (47)) are currently being explored that may contribute enhancing islet engraftment and attain sustained graft function long-term (48).  Importantly, alternative sites may be modified using bioengineering approaches that could enable creating an ideal bio-artificial endocrine pancreas (reviewed in (211)).  The use of immune-isolation techniques, such as using hydrogel polymers that shield islet cell clusters from immune cell attack, may contribute to achieve sustained function of transplanted cells without the need for life-long immunosuppression (reviewed in (212) and (211)).

 

Conclusions

Restoration of physiologic metabolic control in patients with diabetes is highly desirable.  Transplantation of islets of Langerhans allows the achievement of stable metabolic control in the most severe manifestations that cannot be matched with conventional medical therapies.  The steady progress of clinical islet transplantation and the promising emerging new approaches that address immunity and beta cell sources justifies cautious optimism for the potential applicable of beta-cell replacement to all cases of insulin-dependent diabetes in the near future.

 

Acknowledgments

This work was partially supported by the Italian Minister of Health (Ricercar Finalizzata RF-2009-1483387, RF-2009-1469691), Ministry of EducationUniversity and Research (PRIN 2008, prot. 2008AFA7LC), Associazione Italiana per la Ricerca sul Cancro (AIRC, bando 5 x 1,000 N_12182 and Progetto IGN_ 11783), EU (HEALTH-F5-2009-241883-BetaCellTherapy) and the Juvenile Diabetes Research Foundation International.

 

The author alone is responsible for reporting and interpreting these data; the views expressed herein are those of the author and not necessarily those of the funding agencies.

 

Online resources on the subject

 

Clinical Islet Transplant Consortium; Collaborative Islet Transplant Registry; Diabetes Research Institute Foundation; Health Resources and Services Administration; International Pancreas & Islet Transplant Association; The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK); Organ Procurement and Transplantation Network; The Cell Transplant Society; Scientific Registry of Transplant Recipients; United States Department of Health and Human Services; United Network For Organ Sharing (UNOS).

 

 

American Diabetes Association; American Society of Transplantation; American Society of Transplant Surgeons; Beta Cell Biology Consortium; European Pancreas Club; European Society for Organ Transplantation; International Pancreas Transplant Registry; International Xenotransplantation Association; Juvenile Diabetes Research Foundation.

 

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(151) Bassi R, Fiorina P (2011) Impact of islet transplantation on diabetes complications and quality of life. Curr Diab Rep 11: 355-363

(152) Thompson DM, Meloche M, Ao Z, et al. (2011) Reduced progression of diabetic microvascular complications with islet cell transplantation compared with intensive medical therapy. Transplantation 91: 373-378

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(154) Fiorina P, Folli F, Zerbini G, et al. (2003) Islet transplantation is associated with improvement of renal function among uremic patients with type I diabetes mellitus and kidney transplants. J Am Soc Nephrol 14: 2150-2158

(155) Fiorina P, Venturini M, Folli F, et al. (2005) Natural history of kidney graft survival, hypertrophy, and vascular function in end-stage renal disease type 1 diabetic kidney-transplanted patients: beneficial impact of pancreas and successful islet cotransplantation. Diabetes Care 28: 1303-1310

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(159) Lee TC, Barshes NR, O'Mahony CA, et al. (2005) The effect of pancreatic islet transplantation on progression of diabetic retinopathy and neuropathy. Transplant Proc 37: 2263-2265

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(161) Del Carro U, Fiorina P, Amadio S, et al. (2007) Evaluation of polyneuropathy markers in type 1 diabetic kidney transplant patients and effects of islet transplantation: neurophysiological and skin biopsy longitudinal analysis. Diabetes Care 30: 3063-3069

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(163) D'Addio F, Maffi P, Vezzulli P, et al. (2014) Islet transplantation stabilizes hemostatic abnormalities and cerebral metabolism in individuals with type 1 diabetes. Diabetes Care 37: 267-276

(164) Fiorina P, Folli F, Bertuzzi F, et al. (2003) Long-term beneficial effect of islet transplantation on diabetic macro-/microangiopathy in type 1 diabetic kidney-transplanted patients. Diabetes Care 26: 1129-1136

(165) Fiorina P, Folli F, Maffi P, et al. (2003) Islet transplantation improves vascular diabetic complications in patients with diabetes who underwent kidney transplantation: a comparison between kidney-pancreas and kidney-alone transplantation. Transplantation 75: 1296-1301

(166) Fiorina P, Gremizzi C, Maffi P, et al. (2005) Islet transplantation is associated with an improvement of cardiovascular function in type 1 diabetic kidney transplant patients. Diabetes Care 28: 1358-1365

(167) Del Carro U, Fiorina P, Amadio S, et al. (2007) Evaluation of polyneuropathy markers in type 1 diabetic kidney transplant patients and effects of islet transplantation: neurophysiological and skin biopsy longitudinal analysis. Diabetes Care 30: 3063-3069

(168) Danielson KK, Hatipoglu B, Kinzer K, et al. (2013) Reduction in carotid intima-media thickness after pancreatic islet transplantation in patients with type 1 diabetes. Diabetes Care 36: 450-456

(169) Hansen A, Johansson BL, Wahren J, von Bibra H (2002) C-peptide exerts beneficial effects on myocardial blood flow and function in patients with type 1 diabetes. Diabetes 51: 3077-3082

(170) Ryan EA, Paty BW, Senior PA, Shapiro AM (2004) Risks and side effects of islet transplantation. Curr Diab Rep 4: 304-309

(171) Gaba RC, Garcia-Roca R, Oberholzer J (2012) Pancreatic islet cell transplantation: an update for interventional radiologists. J Vasc Interv Radiol 23: 583-594; quiz 594

(172) Villiger P, Ryan EA, Owen R, et al. (2005) Prevention of bleeding after islet transplantation: lessons learned from a multivariate analysis of 132 cases at a single institution. Am J Transplant 5: 2992-2998

(173) Froud T, Yrizarry JM, Alejandro R, Ricordi C (2004) Use of D-STAT to prevent bleeding following percutaneous transhepatic intraportal islet transplantation. Cell Transplant 13: 55-59

(174) Bhargava R, Senior PA, Ackerman TE, et al. (2004) Prevalence of hepatic steatosis after islet transplantation and its relation to graft function. Diabetes 53: 1311-1317

(175) Maffi P, Angeli E, Bertuzzi F, et al. (2005) Minimal focal steatosis of liver after islet transplantation in humans: a long-term study. Cell Transplant 14: 727-733

(176) Casey JJ, Lakey JR, Ryan EA, et al. (2002) Portal venous pressure changes after sequential clinical islet transplantation. Transplantation 74: 913-915

(177) Molinari M, Al-Saif F, Ryan EA, et al. (2005) Sirolimus-induced ulceration of the small bowel in islet transplant recipients: report of two cases. Am J Transplant 5: 2799-2804

(178) Ponte GM, Baidal DA, Romanelli P, et al. (2007) Resolution of severe atopic dermatitis after tacrolimus withdrawal. Cell transplantation 16: 23-30

(179) Andres A, Toso C, Morel P, et al. (2005) Impairment of renal function after islet transplant alone or islet-after-kidney transplantation using a sirolimus/tacrolimus-based immunosuppressive regimen. Transpl Int 18: 1226-1230

(180) Gala-Lopez BL, Senior PA, Koh A, et al. (2011) Late cytomegalovirus transmission and impact of T-depletion in clinical islet transplantation. Am J Transplant 11: 2708-2714

(181) Pileggi A, Cobianchi L, Inverardi L, Ricordi C (2006) Overcoming the challenges now limiting islet transplantation: a sequential, integrated approach. Ann N Y Acad Sci 1079: 383-398

(182) Mineo D, Pileggi A, Alejandro R, Ricordi C (2009) Point: steady progress and current challenges in clinical islet transplantation. Diabetes Care 32: 1563-1569

(183) Pellegrini S, Cantarelli E, Sordi V, Nano R, Piemonti L (2016) The state of the art of islet transplantation and cell therapy in type 1 diabetes. Acta Diabetol

(184) Lo JF, Wang Y, Li Z, et al. (2013) Quantitative and temporal control of oxygen microenvironment at the single islet level. J Vis Exp: e50616

(185) Barkai U, Weir GC, Colton CK, et al. (2013) Enhanced oxygen supply improves islet viability in a new bioartificial pancreas. Cell Transplant 22: 1463-1476

(186) Marigliano M, Bertera S, Grupillo M, Trucco M, Bottino R (2011) Pig-to-nonhuman primates pancreatic islet xenotransplantation: an overview. Curr Diab Rep 11: 402-412

(187) Pagliuca FW, Millman JR, Gurtler M, et al. (2014) Generation of Functional Human Pancreatic beta Cells In Vitro. Cell 159: 428-439

(188) Rezania A, Bruin JE, Arora P, et al. (2014) Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol 32: 1121-1133

(189) Kroon E, Martinson LA, Kadoya K, et al. (2008) Pancreatic endoderm derived from human embryonic stem cells generates glucose-responsive insulin-secreting cells in vivo. Nat Biotechnol 26: 443-452

(190) Schulz TC, Young HY, Agulnick AD, et al. (2012) A scalable system for production of functional pancreatic progenitors from human embryonic stem cells. PLoS One 7: e37004

(191) Pellegrini S, Piemonti L, Sordi V (2018) Pluripotent stem cell replacement approaches to treat type 1 diabetes. Curr Opin Pharmacol 43: 20-26

(192) Odorico J, Markmann J, Melton D, et al. (2018) Report of the Key Opinion Leaders Meeting on Stem Cell-derived Beta Cells. Transplantation 102: 1223-1229

(193) Pellegrini S, Ungaro F, Mercalli A, et al. (2015) Human induced pluripotent stem cells differentiate into insulin-producing cells able to engraft in vivo. Acta Diabetol

(194) Sordi V, Pellegrini S, Krampera M, et al. (2017) Stem cells to restore insulin production and cure diabetes. Nutrition, Metabolism and Cardiovascular Diseases 27: 583-600

(195) Borot S, Crowe LA, Toso C, Vallee JP, Berney T (2011) Noninvasive imaging techniques in islet transplantation. Curr Diab Rep 11: 375-383

(196) Wang P, Schuetz C, Vallabhajosyula P, et al. (2015) Monitoring of Allogeneic Islet Grafts in Nonhuman Primates Using MRI. Transplantation 99: 1574-1581

(197) Malosio ML, Esposito A, Brigatti C, et al. (2014) Mr Imaging Monitoring of Iron Labeled Pancreatic Islets in a Small Series of Patients: Islets Fate in Successful, Unsuccessful and Auto-Transplantation. Cell Transplant

(198) Esposito A, Palmisano A, Maffi P, et al. (2014) Liver perfusion changes occurring during pancreatic islet engraftment: a dynamic contrast-enhanced magnetic resonance study. Am J Transplant 14: 202-209

(199) Saudek F, Jirak D, Girman P, et al. (2010) Magnetic resonance imaging of pancreatic islets transplanted into the liver in humans. Transplantation 90: 1602-1606

(200) Toso C, Vallee JP, Morel P, et al. (2008) Clinical magnetic resonance imaging of pancreatic islet grafts after iron nanoparticle labeling. Am J Transplant 8: 701-706

(201) Toso C, Zaidi H, Morel P, et al. (2005) Positron-emission tomography imaging of early events after transplantation of islets of Langerhans. Transplantation 79: 353-355

(202) Eriksson O, Alavi A (2012) Imaging the islet graft by positron emission tomography. Eur J Nucl Med Mol Imaging 39: 533-542

(203) Kriz J, Jirak D, Berkova Z, et al. (2012) Detection of pancreatic islet allograft impairment in advance of functional failure using magnetic resonance imaging. Transpl Int 25: 250-260

(204) Eich T, Eriksson O, Lundgren T (2007) Visualization of early engraftment in clinical islet transplantation by positron-emission tomography. N Engl J Med 356: 2754-2755

(205) Han D, Leith J, Alejandro R, Bolton W, Ricordi C, Kenyon NS (2005) Peripheral blood cytotoxic lymphocyte gene transcript levels differ in patients with long-term type 1 diabetes compared to normal controls. Cell Transplant 14: 403-409

(206) Han D, Xu X, Baidal D, et al. (2004) Assessment of cytotoxic lymphocyte gene expression in the peripheral blood of human islet allograft recipients: elevation precedes clinical evidence of rejection. Diabetes 53: 2281-2290

(207) Han D, Xu X, Pastori RL, Ricordi C, Kenyon NS (2002) Elevation of cytotoxic lymphocyte gene expression is predictive of islet allograft rejection in nonhuman primates. Diabetes 51: 562-566

(208) Huurman VA, Velthuis JH, Hilbrands R, et al. (2009) Allograft-specific cytokine profiles associate with clinical outcome after islet cell transplantation. Am J Transplant 9: 382-388

(209) Roep BO, Stobbe I, Duinkerken G, et al. (1999) Auto- and alloimmune reactivity to human islet allografts transplanted into type 1 diabetic patients. Diabetes 48: 484-490

(210) Stobbe I, Duinkerken G, van Rood JJ, et al. (1999) Tolerance to kidney allograft transplanted into Type I diabetic patients persists after in vivo challenge with pancreatic islet allografts that express repeated mismatches. Diabetologia 42: 1379-1380

(211) Hallé JP, deVos P, Rosenberg L (2009) The bioartificial pancreas and other biohybrid therapies. Transworld Research Network, Kerala, India

(212) Basta G, Calafiore R (2011) Immunoisolation of pancreatic islet grafts with no recipient's immunosuppression: actual and future perspectives. Curr Diab Rep 11: 384-391

 

Nonalcoholic Fatty Liver Disease: The Overlooked Complication of Type 2 Diabetes

* Denotes equal effort

ABSTRACT

 

Nonalcoholic fatty liver disease (NAFLD) is a common complication of obesity and type 2 diabetes mellitus (T2DM).  Most times it is an unrecognized comorbidity to the primary care provider and endocrinologist. Today it is the most common chronic liver disease in developed countries. It is characterized by insulin resistance and hepatic triglyceride accumulation in the absence of co-existing etiologies, such as excessive alcohol consumption, viral hepatitis, medications or other etiologies for hepatic steatosis.  Its more severe form of the disease with steatohepatitis (NASH) is associated with hepatocyte injury (necrosis and inflammation) and frequently with fibrosis. Although it appears to be an indolent condition, with few symptoms and often normal plasma aminotransferases, NASH is a leading cause of end-stage liver disease and hepatocellular carcinoma (HCC), and significantly increases the risk of developing cardiovascular disease (CVD) and T2DM. The pathogenesis of NASH remains poorly understood, and likely to be multifactorial, but insulin-resistant adipose tissue plays an important role. The natural history of NAFLD is incompletely understood, but risk factors for disease progression include weight gain, obesity and T2DM, as well as the severity of fibrosis stage at diagnosis.  Diagnostic algorithms are evolving but we offer an approach that integrates for the non-hepatologist plasma biomarkers, imaging, and the role of liver biopsy for the management of these complex patients.  At the present time, early screening -with biomarker panels or a liver ultrasound, ideally with transient elastography- is reserved for high-risk patients (i.e., obese patients with T2DM or elevated plasma AST/ALT levels or evidence of steatosis at a random liver exam) until more accurate non-invasive methods are available.  A liver biopsy should be considered on a case-by-case basis, to identify those at risk of NASH-cirrhosis, working in close collaboration with a hepatologist. Treatment should include a comprehensive approach with lifestyle modification and therapeutic agents tested in RCTs, such as vitamin E (in patients without diabetes) or pioglitazone for patients with or without diabetes.   Pioglitazone, given its low-cost as a generic medication, long-standing track record of efficacy in NASH, and cardiometabolic benefits, is likely to be for NASH what metformin has become for the management of T2DM. However, proper patient selection and close monitoring is needed.  In addition, a number of new pharmacological agents are being studied in phase II/III trials and future management will involve the use of combination therapy, as for other chronic metabolic conditions. In summary, endocrinologists need to be aware of the severe metabolic and liver-specific complications of NASH and establish early-on a long-term management plan. Screening will likely take place in the same way as for diabetic retinopathy or nephropathy.  A better understanding of its natural history and pathogenesis of NASH, combined with improved diagnostic and treatment options, will likely place endocrinologists at the forefront of the management efforts to prevent end-stage liver disease in patients with NASH. 

 

INTRODUCTION

 

Nonalcoholic fatty liver disease (NAFLD) is a chronic liver condition that is on the rise. It has become the most common chronic liver condition in many parts of the world. It encompasses a wide spectrum of disease with different clinical implications. NAFLD means that there is evidence of liver steatosis, either by imaging or histology, in the absence of secondary causes of hepatic fat accumulation such as significant alcohol consumption, chronic use of steatogenic medication, or another established chronic liver disease.  Between 40 to 50% of patients that are obese have NAFLD and this rises to about 70% if they have type 2 diabetes mellitus (T2DM). In its simplest form, known as isolated steatosis or NAFL (nonalcoholic fatty liver), there is triglyceride accumulation of ≥5% without evidence of hepatocellular injury in the form of hepatocyte ballooning or evidence of fibrosis.  Although the natural history of this condition remains uncertain, and may possibly progress to more severe disease, at the present moment NAFL is considered to be associated with limited risk of liver morbidity. However, it is associated with insulin resistance so that the liver can be seen as a “mirror” of metabolic health (i.e., in obesity steatosis being a reflection of insulin resistance, and in particular, of adipose tissue dysfunction) and with an increased risk of cardiovascular disease (CVD). In its more severe form, known as nonalcoholic steatohepatitis or NASH, steatosis ≥5% is associated with hepatocyte injury with necrosis (“ballooning”) and lobular inflammation, with or without fibrosis.

 

Steatohepatitis is often a progressive disorder in T2DM associated with the development of fibrosis that can eventually lead to cirrhosis. Liver fibrosis is defined by its severity in stages ranging from absence of fibrosis (stage F0) to mild (stage F1), moderate (stage F2, with zone 3 sinusoidal fibrosis plus periportal fibrosis), and “advanced” fibrosis referring specifically to stages 3 (bridging fibrosis) or 4 (cirrhosis). Having fibrosis is the most important histological feature of NAFLD associated with long-term mortality. Fibrosis also predisposes patients to hepatocellular carcinoma (HCC).

 

Type 2 diabetes mellitus has been a well-established factor in the progression of NAFLD to more severe forms, including a higher incidence of HCC (1-3). However, in clinical practice NAFLD still remains an under-recognized complication of T2DM, unlike the other microvascular and macrovascular complications.

 

As discussed later, isolated steatosis and NASH may carry an increased risk of CVD and being the most common cause of death in patients with NAFLD, independent of other metabolic comorbidities. It is important for endocrinologists and primary care physicians to recognize that NAFLD in T2DM has been shown to be associated with adverse metabolic changes resulting in increased atherosclerotic disease and cardiovascular consequences (4,5).

 

NAFLD: KEY CONCEPTS

 

The incidence of NAFLD is rising, paralleling that of obesity and diabetes mellitus. There has been extensive research in the area of NAFLD, especially over the past two decades. However, given the lack of highly reliable noninvasive diagnostic methods, the burden of NAFLD probably remains overlooked. By liver ultrasound, studies have demonstrated the prevalence of NAFLD to be 24% in United States, whereas using blood testing alone this is underestimated at just 13% (6). By the gold-standard magnetic resonance imaging and spectroscopy (1H-MRS), the prevalence of NAFLD in the general population is estimated to be 34% (7).

 

Unfortunately, imaging techniques cannot adequately evaluate for hepatocellular necrosis or inflammation (i.e. NASH). Studies that have utilized a liver biopsy to confirm the diagnosis of NAFLD have shown that 59% of patients with NAFLD have NASH, this being much higher in obese individuals (6). Moreover, recent studies have reported that about 18% of unselected patients with T2DM have moderate-to-severe (F2-F4) fibrosis (6,8).

 

NAFLD often progresses to steatohepatitis (NASH), especially in patients with T2DM. NASH is hallmarked by hepatocellular necrosis, lobular inflammation and often fibrosis. Many studies have now documented that patients with NASH and fibrosis have the worst mortality (9). As fibrosis progresses, cirrhosis develops. This rate of progression to cirrhosis is highly variable and dependent on age, BMI, blood pressure control, presence of T2DM, and degree of steatohepatitis (10). The three most relevant risk factors are obesity (excessive BMI or visceral obesity), T2DM, and presence of moderate to severe fibrosis (11). However, given the high heterogeneity in disease progression one must admit that the precise factors leading to cirrhosis remain unclear.

 

NASH is currently the second most common indication for liver transplantation, after hepatitis C. It is predicted to be the leading indication for liver transplantation in the next decade given the rise in incidence (8). The annual incidence of HCC in NAFLD – related cirrhosis is about 1% (1,8,12,13). Nonalcoholic steatohepatitis related cirrhosis is currently the third leading cause for HCC, after HCV and alcohol-related liver disease. Importantly, those with NASH- related HCC that undergo liver transplantation are more likely to have a higher BMI and higher rate of T2DM (13). In one study, it has also been demonstrated that HCC can develop in NASH in the absence of cirrhosis (14).

 

NAFLD and Cardiovascular Disease

 

Many factors lead to cardiovascular disease in patients with T2DM and NAFLD. For instance, they have increased intrahepatic triglyceride accumulation and insulin resistance. This is associated with increased hepatic VLDL secretion and a decrease in the peripheral clearance of triglyceride-rich lipoproteins. This results in a proatherogenic profile, which includes hypertriglyceridemia, low HDL-C, and an increase in small, dense LDL particles, plus a state of subclinical inflammation (8).

 

These patients also often have more severe hepatic insulin resistance leading to progressive deterioration of glycemic control (9). Hepatic insulin resistance is associated with hyperinsulinemia from increased insulin secretion and decreased insulin clearance (3,15). Hyperinsulinemia per sehas been associated with atherogenesis in animal models of disease and in epidemiological studies.  Chronic hyperinsulinemia also causes downregulation of insulin signaling pathways and acquired insulin resistance in short-term clinical studies in humans (11). In this context, hyperglycemia is more severe and also appears to contribute to CVD. Endothelial dysfunction also has been shown to cause increased cardiovascular risk in patients with NAFLD (16). Early left ventricular “diastolic dysfunction” (or heart failure with preserved ejection fraction or HFpEF) has been noted in patients with NAFLD and well controlled T2DM independent of other risk factors (17). Patients with NAFLD are often found to have a significantly worse carotid intima-media thickness with increased atherosclerotic disease when compared with clinically matched patients without NAFLD. This has been correlated in some studies with an advancing degree of steatosis, inflammation, and/or fibrosis (18,19). In NASH with cirrhosis, CVD is the leading cause of mortality (1,8,20).

 

Thus, it is not unexpected that in NAFLD many studies have reported a higher rate of CVD (Tables 1 and 2).  In addition to insulin resistance and hyperinsulinemia, NAFLD and CVD cluster with other common risk factors, including hypertension, hyperlipidemia, T2DM, obesity and inflammation (8). The evidence of the association between NAFLD and increased CVD often persists even after adjusting for traditional cardiovascular risk factors (Tables 1 and 2) (9,21).This suggests that the presence of NAFLD may independently increase an individual’s cardiovascular risk, but whether this is worse in patients with steatohepatitis compared to those just having isolated steatosis remains controversial. It should also be noted that many investigatorshave failed to see the association of NAFLD with CVD after adjusting for traditional cardiovascular risk factors, as detailed in Tables 1 and 2.

 

Table 1. Cardiovascular Disease in Patients with NAFLD without Type 2 Diabetes

Author (year)

NAFLD vs controls

(n)

Diagnosis of NAFLD

Primary endpoint

Increased CVD

Adjusted CV risk

Study design

Villanova et al (22)

80

Liver biopsy

Endothelial function 

Yes

Yes

Prospective case-control, Cross-sectional

Brea et al (23)

80

Ultrasound (US)

Carotid intima-media thickness test (CIMT)

Yes

No

Case-control, Cross-sectional

Adams et al (24)

420

US, CT, MRI or Liver biopsy

All cause and CV mortality

Yes

No

Retrospective cohort, Longitudinal

Volzke et al (25)

4222

Ultrasound

CIMT

Yes

No

Case-control, Cross-sectional

Ekstedt et al (26)

129

Liver biopsy

All cause and CV mortality

Yes *

Yes

Retrospective cohort, Longitudinal

Mirbagheri et al (27)

171

Ultrasound

Coronary angiography

Yes

Yes

Cross-sectional

Hamaguchi et al (28)

1221

Ultrasound

CV events

Yes

Yes

Prospective cohort, Longitudinal

Schindhelm et al (29)

1439

ALT

CV events

Yes

Yes

Retrospective cohort, Longitudinal

Fracanzani et al (30)

375

Ultrasound

CIMT

Yes

Yes

Case-control, Cross-sectional

Goessling et al (31)

2812

AST, ALT

CV events

Yes

No

Retrospective cohort, Longitudinal

Aygun et al (32)

80

Liver biopsy

CIMT

Yes

Yes

Prospective case-control, cross-sectional

Haring et al (33)

4160

Ultrasound

All cause and CV mortality

Yes**

Yes

Retrospective cohort, Longitudinal

Rafiq et al (34)

173

Liver biopsy

All cause and CV mortality

No***

No

Retrospective cohort, Longitudinal

Salvi et al (35)

220

Ultrasound

Arterial stiffness by carotid-femoral pulse wave velocity

Yes

Yes

Case-control, Cross-sectional

Soderberg et al (36)

118

Liver biopsy

All cause and CV mortality

Yes

Yes

Retrospective cohort, Longitudinal

Zhou et al (37)

3324

Ultrasound

All cause and CV mortality

Yes

Yes

Retrospective cohort, Longitudinal

Stepanova et al (38)

11,613

Ultrasound

All cause and CV mortality

Yes

No

Retrospective cohort, Longitudinal

Lee et al (39)

1442

Ultrasound

Arterial stiffness by brachial-ankle pulse wave velocity

Yes

Yes

Case-control, Cross-sectional

Kozakova et al (40)

1012

Fatty Liver Index

CIMT

Yes

Yes

Cross-sectional

Kim et al (41)

4023

Ultrasound

Coronary artery calcification score by CT

Yes

Yes 

Cross-sectional

Hallsworth et al (42)

38

MR spectroscopy

LV dysfunction by cardiac MRI

Yes

Yes

Case-control, Cross-sectional

Colak et al (43)

72

Liver biopsy

Endothelial function by flow mediated dilation (FMD) and CIMT

Yes

Yes

Observational case-control, cross-sectional

Pisto et al (44)

988

Ultrasound

CV events

Yes

No

Retrospective cohort, Longitudinal

Ekstedt et al (45)

2515

Liver biopsy

CV events

Yes

No****

Retrospective cohort, Longitudinal

Zeb et al (46)

4119

CT

CV events

Yes

Yes

Prospective cohort, Longitudinal

Fracanzani et al (47)

273

Ultrasound

CIMT

Yes

Yes

Prospective cohort, Longitudinal

Wong et al (48)

612

Ultrasound

CV events, Coronary artery stenosis by angiogram

Yes*****

No

Prospective cohort, Longitudinal

*Patients with NASH but not with only steatosis had increased cardiovascular mortality.

       

** NAFLD was associated with increased all cause and cardiovascular mortality in men only.

       

***Compared NASH vs non-NASH NAFLD patients, no difference in overall mortality was found,

but liver mortality was significantly different, with higher rates in NASH patients. Overall, most

common causes of death reported were cardiovascular disease, malignancy and liver related deaths.

****No increased CV risk when diabetics were excluded.

         

***** Patients with NAFLD were more likely to have significant coronary artery stenosis at baseline,

and more likely to undergo percutaneous coronary intervention; however, no increased association

of NAFLD with CV events during follow up.

 

 

           
               

Table 2. Cardiovascular Disease in Patients with NAFLD with Type 2 Diabetes

Author

NAFLD vs controls

(n)

Diagnosis of NAFLD

Primary Endpoint

Increased CVD

Adjusted CV Risk

Study Design

Targher et al (49)

200

Ultrasound

CIMT

Yes

Yes*

Cross-sectional

Targher et al (50)

2103

Ultrasound

CV events

Yes

Yes

Longitudinal

McKimmie et al (51)

623

CT

CIMT and coronary artery calcium score

No

No

Cross-sectional

Petit et al (52)

101

MR spectroscopy

CIMT

No

No

Prospective, Cross-sectional

Adams et al (53)

337

Liver US, CT or biopsy

All-cause mortality and CVD

No

No

Longitudinal

Poanta et al (54)

56

Ultrasound

CIMT

No

No

Case-control, Cross-sectional

Bonapace et al (55)

50

Ultrasound

LV diastolic dysfunction

Yes

Yes

Cross-sectional, Prospective

Dunn et al (56)

2343

CT

CV mortaility

No

No

Retrospective cohort, Longitudinal

Khashper et al (57)

93

CT

Coronary artery calcium score

No

No

Prospective, Cross-sectional

Kim et al (58)

4437

Ultrasound

CIMT

Yes

Yes

Cross-sectional

Idilman et al (59)

273

CT

Coronary artery calcium score

Yes**

Yes

Prospective, Cross-sectional

Silaghi et al (60)

336

Ultrasound

CIMT

No

No

Cross-sectional

Kwak et al (61)

213

Ultrasound

Coronary artery calcium score

Yes***

Yes

Cross-sectional

Mantovani et al (62)

222

Ultrasound

LV diastolic dysfunction

Yes

Yes

Cross-sectional

*CV risk remained significant after adjustment for other traditional cardiovascular risk factors, however did not remain significant after adjustment for HOMA-IR.                                     

**Only significant association was between NAFLD and significant CAD (defined as more than or equal to 50% stenosis in at least one coronary artery).                                   

***Only significant association in patients with NAFLD and A1C > 7% but not in lower A1C.                                                                     

NAFLD and Chronic Kidney Disease

  

The presence of NAFLD and NASH with fibrosis have been recently associated with chronic kidney disease (CKD), and more severe forms of fatty liver disease correlate with worse and progressive stages of CKD. In most studies, CKD has been defined as having an estimated glomerular filtration rate (eGFR) < 60 ml/min/1.73m2or increased albuminuria/proteinuria (20,63,64). In a case control study by Targher et al, the severity of liver histology in patients with biopsy-proven NASH was found to be independently associated with the degree of worsening eGFR (65).

 

A cross-sectional study of Japanese patients with biopsy-proven NAFLD showed an increased prevalence of CKD with worsening liver histology. They found that overall, 14% of patients with NAFLD had evidence of CKD. Of the patients with biopsy proven NASH, 21% had the presence of CKD; and of the patients with NAFLD with no evidence of NASH, only 6% had CKD (64). This was higher than in patients without NAFLD or NASH.  The pathophysiology of this association is not well understood, but the increased atherogenicity associated with NAFLD is likely a contributing factor (20). A more recent meta-analysis also showed a higher prevalence of CKD in patients with NASH when compared with patients with NAFLD without NASH, and a higher prevalence of CKD in patients with advanced fibrosis when compared with patients with lower degree of fibrosis (63).

 

NAFLD and Polycystic Ovarian Syndrome

 

Women with polycystic ovarian syndrome (PCOS) have been found to have an increased prevalence of NAFLD. This association has been present even after adjusting for other factors associated with the metabolic syndrome, such as BMI, hypertension, and type 2 diabetes mellitus (66,67). Evidence of hyperandrogenism, especially with testosterone level > 3 nmol/L has been associated with increased risk of NAFLD in women with PCOS (66,68).  

 

PATHOGENESIS

 

Of note, the pathogenesis of NASH is poorly understood in humans. Most proposed mechanisms at the molecular level have only been observed in cell systems or animal models, but not confirmed in humans. Animal models of NASH are far from ideal in resembling human disease (69). Often treatments that are promising in animal models are in discordance with results in humans – indeed, most treatments that have resolved NASH, and even fibrosis, in mice have failed so far in large RCTs.  A detailed description of the potential pathways leading to steatohepatitis exceeds the scope of this review, therefore we refer the reader to recent in-depth reviews involving a broad spectrum of mechanisms involved in the development of NASH and liver fibrosis (11,69-72).  In Figure 1(below) we propose a schematic representation of the factors and many pathways leading to NASH and fibrosis.

Figure 1: Pathogenies of NAFLD, adapted from Cusi K (11).
PNPLA3=patatin-like phospholipase domain-containing protein 3. TM6SF2=transmembrane-6 superfamily member 2. GCKR=glucokinase regulator. HSD17B13=hydroxysteroid 17-beta dehydrogenase 13. NAFLD=non-alcoholic fatty liver disease. HDL-C=high-density lipoprotein cholesterol. LDL-C=low-density lipoprotein cholesterol. VLDL=very low-density lipoprotein. CETP=Cholesteryl ester transfer protein.

Development of Steatosis

 

Clinical studies have shown that the source of intrahepatic triglycerides in NAFLD is about two-thirds from free fatty acids originating from adipose tissue. However, higher rates of de novolipogenesis (DNL) are also observed in obesity and T2DM (73). In obesity, adipocytes adapt to chronic excess energy supply by undergoing hypertrophy and hyperplasia. This is likely a protective adaptation to allow for an increase in adipocyte storage capacity and ameliorate the potential for ectopic triglyceride accumulation in tissues with limited ability to do so such as the liver, skeletal muscle, pancreas and others. When these adaptive mechanisms are overwhelmed by a chronic excess in nutrient supply, the chronic flux of FFAs promotes a state of “lipotoxicity” across different tissues (11). Adaptation to chronic overnutrition occurs at the expense of developing adipose tissue insulin resistance and triggering mechanisms that attract macrophage accumulation and activation in fat and systemic subclinical inflammation. Moreover, it has been shown that hypertrophic adipocytes share a gene expression pattern that is similar to macrophages and produce adipocytokines similar to those produces by foam cells (74). Adipocytokines have a key role to play in the pathogenesis of insulin resistance by inhibiting insulin signaling pathways via action of insulin receptor substrate (IRS)-1 and c-Jun N terminal kinase (JNK) pathways. Insulin resistance and inflammation is also triggered by the generation of reactive oxygen species, and lipid intermediates such as diacylglycerol (DAG) (75), ceramides (76, 77) and acylcarnitines (77).

 

Normally, insulin decreases gluconeogenesis and increases hepatic synthesis of fatty acids and triglycerides.  Based on animal models of T2DM, it has been postulated that there may be a selective hepatic insulin resistance to glucose metabolism pathways (i.e., inhibition of gluconeogenesis) while preservation of insulin sensitivity at lipid synthetic pathways (78, 79). Selective insulin resistance in the gluconeogenic pathway would explain (at least in part) how hyperinsulinemia may attempt to normalize glucose metabolism at the expense of driving triglyceride synthesis, as hepatic lipid synthetic pathways retain a normal insulin sensitivity, explaining the etiology of both hyperglycemia and hypertriglyceridemia in diabetes.  More recently, Perry et al (80) reported that the major mechanism by which insulin suppresses hepatic glucose production appears to be through a reduction in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT). This is associated with a reduction in pyruvate carboxylase flux. Of interest, insulin’s ability to inhibit hepatic acetyl CoA and lipolysis is lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion (80).

 

However, the above relationship between hyperinsulinemia and steatosis does not completely explain the role of both factors in patients with NASH. In subjects with hepatic steatosis, increasing insulin levels only have a modest correlation with the severity of intrahepatic triglyceride accumulation (81) and there is no relationship between hyperinsulinemia or hepatic steatosis with the severity of inflammation, hepatocyte ballooning (injury), or fibrosis (15, 81).This is despite patients with NASH having worse hyperinsulinemia compared to patients with isolated steatosis (NAFL). This suggests that other mechanisms play a role in human disease.

 

Lipotoxicity has been extensively studied in skeletal muscle, where accumulation of ectopic triglycerides promotes the formation of toxic lipid metabolites (i.e., such as DAGs) that are closely associated with impairment in insulin signaling. Lipid infusions in healthy subjects have shown that at levels of plasma FFAs typically seen in obesity and NAFLD, there is suppression of insulin signaling and hence development of insulin resistance (82). Lipotoxicity has also reported in pancreatic beta-cells in humans.  Normally, FFAs are the main energy source in the fasting state, with a switch to using glucose as the primary fuel after a meal. However, chronically elevated plasma FFA concentrations impair insulin secretion in subjects that are genetically prone to T2DM (83).

 

NAFLD has been shown to also be a heritable disease (72, 84-90). Nuclear receptors such as peroxisome proliferator-activated nuclear receptors (PPAR) play a key role in hepatic lipid metabolism, however results on association of PPAR and severity of NAFLD have been variable (75). Studies have shown that first-degree relatives of subjects with NAFLD are more susceptible to develop chronic liver disease as compared to the general population (72, 84). Patatin-like phospholipase domain-containing protein 3 (PNPLA3) gene polymorphism has been shown to be associated with worse hepatic steatosis and a worse long-term prognosis in patients with NASH (85). PNPLA3 is usually involved in hydrolysis of hepatocyte triglycerides. This polymorphism results in a loss of function mutation resulting in accumulation of intrahepatic triglycerides. Recently, it has been described that accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis observed with this polymorphism (86).

 

Another commonly described polymorphism involves transmembrane 6 superfamily member 2 (TM6SF2) which normally plays a role in interaction between triglycerides and Apolipoprotein B during the extrahepatic secretion of very low-density lipoprotein (87). This polymorphism results in increased hepatic triglyceride deposition, and lower circulating lipoproteins. Recent studies show this polymorphism is associated with higher risk of NAFLD but lower cardiovascular risk (87). A loss of function mutation in the glucokinase regulator (GCKR) gene locus has been implicated in the accumulation of hepatic fat (88,89).   Normally, GCKR is involved in controlling the glucose influx into hepatocytes and hence regulating DNL. A protective splice variant HSD17B13 has also been identified. HSD17B13 encodes the lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13 (HSD17B13) (90). This allele was associated with a reduced risk for progression from steatosis to steatohepatitis and fibrosis. Interestingly, it also seems to mitigate the effects of PNPLA3 polymorphism.  Finally, an interesting observation is that individuals with familial hypobetalipoproteinemia (FHBL) are prone to NAFL but are characterized by very low levels of plasma low-density lipoprotein (LDL) cholesterol that is protective against CVD (91).

 

However, at the present time, genetic testing is not recommended in clinical practice as it remains unclear how the presence of a given mutation should modify current management of NASH (92).

 

Development of Hepatocyte Injury and Steatohepatitis: Role of Mitochondrial Dysfunction

 

It should be emphasized that the mechanisms leading to steatohepatitis in humans remain unknown. With limited exceptions that point to subtle defects in mitochondrial function in the liver of subjects with NAFLD and/or T2DM (reviewed in ref. 71), almost all of the available information has been extrapolated from cell culture studies or animal models of NASH.  It is also unclear if NASH is always heralded by isolated steatosis, and what are the drivers of disease.  While there is an increasing recognition that NASH is an heterogeneous disease affecting obese and non-obese individuals, disease progression is often with associated with obesity/weight gain and T2DM. Obvious limitations in obtaining sufficient liver tissue for molecular studies, as well as ethical challenges for performing paired liver biopsies before and after a given intervention, have greatly hampered our ability to make significant progress in understanding the pathogenesis of NASH in humans.  However, factors associated with overnutrition and insulin resistance likely play a role in the maladaptation of mitochondrial oxidative function that leads to inefficient oxidative flux, accumulation of lipotoxic intermediates and the progression from isolated steatosis to NASH (71,93). As mentioned above, genetic factors may also regulate lipid droplet accumulation that may exacerbate disease progression.  Many other trigger factors associated with endoplasmic reticulum (ER) stress, oxidative stress and inflammasome activation have been described.  However, the exact temporal relationship and sequence of events remains elusive.

 

Normally, there is a close regulation between beta-oxidation, hepatic tricarboxylic acid (TCA) cycle activity, ketogenesis and ATP synthesis. Normally, FFAs influx is efficiently dealt through beta-oxidation. However, in states of chronic overfeeding, beta-oxidation can over time become relatively ineffective, resulting in the accumulation of hepatocyte ceramides and DAGs (as well as acylcarnitines), as seen in states of hepatic steatosis (71,75-77). As summarized in Figure 2, the current working hypothesis in NASH is that overactive hepatic TCA cycle carries the risk of overloading the mitochondrial electron transport chain and hence promoting not only the formation of toxic metabolites but the production of reactive oxygen species (ROS) and other inflammatory mediators. In this setting, it is believed that inflammatory pathways are triggered which then lead to hepatocyte necrosis and chronic inflammation, Kupffer cell activation and recruitment, as well as hepatic stellate cell activation. This disruption of the normal equilibrium between hepatocyte and its microenvironment (i.e., in particular with Kupffer cells and hepatic stellate cells, the latter promoting fibrogenesis) seems to determine the degree of hepatocyte injury and the triggering of downstream pathways that lead to cirrhosis, as reviewed in-depth elsewhere (70).  However, while many recent interventions successful in animal models have failed in humans, it is of interest that there is a  correlation between successful treatment for NASH in humans (with GLP-1RA or pioglitazone [8]) with studies in vivowith such interventions that restore hepatocyte TCA function and reduce intracellular toxic lipids (94, 95), giving support to the hypothesis of increased mitochondrial FFA flux as a potential therapeutic target for patients with NASH. 

Figure 2. Hepatic Mitochondrial Oxidative Dysfunction during NASH (71). Adipose tissue insulin resistance results in increased lipolysis and higher flux of FFAs into the liver (1), resulting in high rates of hepatic triglyceride accretion (2). Initial breakdown of FFA in the liver proceeds through b-oxidation, generating two-carbon units of acetyl-CoA (3). During hepatic insulin resistance, disposal of acetylCoA units through ketogenesis undergoes an early compensatory induction in simple steatosis, but is impaired in NASH (4). In spite of FFA overload, hepatic insulin resistance and steatosis result in beta-oxidation being inefficient and incomplete as evident from accumulating levels of hepatic ceramides, DAGs, and long-chain acylcarnitine (5). However, complete oxidation of acetyl-CoA units through the mitochondrial TCA cycle continues unabated during simple steatosis and NASH (6), potentially to meet the energetic demands of maintaining high rates of gluconeogenesis (7). The chronically elevated oxidative flux through TCA cycle during NASH has the potential to uncouple hepatic TCA cycle activity from mitochondrial respiration (8) by disrupting the mitochondrial electrochemical gradient and to impair ATP synthesis (9). This mitochondrial milieu could be a chronic source of ROS generation (10) and cellular inflammation, and could be a target for therapeutic manipulations. Abbreviations: Cer, ceramides; CoA, coenzyme A; DAGs, diacylglycerols; FFAs, free fatty acid; NASH, nonalcoholicsteatohepatitis; PEP, phosphoenolpyruvate; ROS, reactive oxygen species; TCA, tricarboxylic acid.

However, linking NASH only to altered mitochondrial flux is obviously an oversimplification of a complex web of many factors at play.  Other pathways that have been implicated in hepatocyte injury and the development of NASH, although rather broadly, include cholesterol accumulation in hepatocytes (96) and a tangled web involving activation of apoptotic pathways with ER stress and abnormal unfolded protein response (97), as well as defects in autophagy (98). Recently, inflammasome activation has gained attention as it integrates many cytoplasmic signals into danger-associated molecular patterns (DAMPs) from diverse sources such as intracellular lipids to the gut microbiome (97, 98).

 

Diet and gut microbiota have been repeatedly implicated to play a role in the pathogenesis of NAFLD.  In particular, fructose appears to play a role in NASH by stimulating DNL and suppressing -oxidation of FFAs, hence leading to hepatocyte injury (99). Many studies have shown that excess fructose consumption, usually as sugar-sweetened beverages with sucrose (converted to fructose and glucose after ingestion), is associated with development of NAFLD and NASH.   Obesity is also associated with a change in gut microbiota that produce more reactive oxygen species and are involved in triggering a variety of inflammatory pathways (100). However, the causative role of the gut microbiome in the development of T2DM or NAFLD remains overall poorly understood (101).  

 

Development of Liver Fibrosis

 

Here too the data in humans is scarce and largely limited to in vitroand in vivoevidence. Potential mechanisms linked to the development of NASH have focused on hepatocyte apoptosis with the release of a broad spectrum of cytokines (e.g., interleukins [-1, -2, -18], hedgehog ligands, TNF-, TGF-, and many others) (11, 97, 98). Wang et al (102) have identified one such pathway (the transcriptional activator TAZ) that appears to play an important profibrogenic role in NASH in a mouse model of NASH. Taken together, this extensive signaling network, triggered by injured hepatocytes, activates nearby Kupffer cells that induce hepatic stellate cells to become myofibroblasts and increase the production of matrix proteins that result in cirrhosis over time. Genetics also appear to play a role as the PNPLA3-I148M variant may not only modify lipid droplet metabolism but have a direct role on stellate cell function in NASH (103). Recently, Lindén et al (104) reported a reduction in liver inflammation and fibrosis in a Pnpla3 knock-in 148M/M mutant mice (with a human PNPLA3 I148M mutation) with a liver-targeted GalNAc3-conjugated antisense oligonucleotide (ASO)-mediated that silenced Pnpla3 expression.

 

At a clinical level, a recent study examined factors associated with disease progression in a large (n = 475 patients) clinical trial (105).  The main factor associated with clinical disease progression is severity of fibrosis at baseline and greater increases in hepatic collagen content, level of alpha-smooth muscle actin, and Enhanced Liver Fibrosis score overtime.  Over a follow-up period of 96 weeks, progression occurred in 22% of patients with bridging fibrosis (F3), while liver-related clinical events occurred in 19% of patients with cirrhosis.  

 

Beyond liver histology, from a clinical perspective, practitioners must keep in mind that obesity and T2DM remain the two major risk factors for liver disease progression which calls for screening and early intervention.

 

DIAGNOSIS

 

Having T2DM is associated with a much greater risk of NAFLD with approximately 70% of patients with T2DM having NAFLD when MRI-based techniques are used, as well as higher risk of having more advanced forms of the disease, such as fibrosis and cirrhosis (6,8,106). Despite all the current evidence, there is lack of awareness in primary care physicians and endocrinologists to evaluate patients with prediabetes or type 2 diabetes mellitus for NAFLD. Even if suspected to have NAFLD based on clinical characteristics, there is currently a lack of further investigations being undertaken as non-invasive biomarkers of the disease and even imaging, are not as reliable as wished and not available at every clinic. The widely accepted thought by primary care physicians, as well as many endocrinologists, is to not pursue any confirmatory testing to assess for the presence or degree of fibrosis, as it is believed to seldom change their management, except to re-emphasize lifestyle modifications and weight loss.  However, few healthcare providers are aware about the efficacy of lifestyle changes and some currently available pharmacological agents to revert NASH, and even fibrosis, if done early and before the development of end-stage liver disease.

 

Early detection and treatment of NAFLD can lead to better histological and metabolic outcomes, including CVD, and improve overall morbidity and mortality. NAFLD is a diagnosis of exclusion, so it is imperative to eliminate all other causes of liver disease (such as, alcoholic liver disease, medication induced toxicity, viral or autoimmune hepatitis, hemochromatosis, alpha 1 antitrypsin deficiency, Wilson’s disease, other) prior to the diagnosis of NAFLD. Often management may require referral to hepatology and developing multidisciplinary teams (107, 108). Once NAFLD is diagnosed, there needs to be further testing to evaluate for the presence and severity of fibrosis (8).

 

Blood Tests

 

Plasma aminotransferases are considered an insensitive marker for the presence of NAFLD. It has been shown that the prevalence of NAFLD may be as high as 50% in patients with T2DM and “normal” (≤40 IU/L) plasma aminotransferases using 1H-MRS for the detection of hepatic steatosis (109). Of note, 56% of these patients had a diagnosis of NASH on liver biopsy, highlighting that reliance on ALT/AST alone may be an inadequate approach for the systematic detection of NASH in endocrinology or primary care clinics (50). Maximos et al (110) have reported a comparable degree of NASH in patients with normal vs. abnormal levels of plasma aminotransferases, emphasizing the non-reliability of plasma aminotransferases as clinical biomarkers for presence or severity of disease, a finding consistent with the literature by others (6,9,111).  Factors affecting elevation of plasma aminotransferases included adipose tissue insulin resistance and intra-hepatic triglyceride content, rather than hepatic insulin resistance (110). There is some evidence to suggest lowering the optimal threshold for considering plasma alanine transferase (ALT) as normal to be ≤30 U/L in men and ≤19 U/L in women (112). This increases the sensitivity of this screening method. Plasma ALT is usually more elevated than AST in the presence of NAFLD and NASH, unless there is advanced disease or cirrhosis, when AST usually increases.

 

Significant efforts have been made in finding the ideal biomarker panel for the diagnosis of NAFLD/NASH. Simple metabolic algorithms such as fatty liver index (using measures, such as BMI, waist circumference, triglyceride levels, and GGT) used for diagnosis of NAFLD have not been shown to be very reliable when compared with more accurate and advanced techniques, such as 1H-MRS (113). It is not a test for the diagnosis of inflammation or fibrosis (114).

 

Several biomarker clinical scores (using different measures, such as AST, ALT, BMI, platelets, albumin, T2DM) have been developed to evaluate for the presence and degree of liver fibrosis (8). These tests are listed in the Table 3. Among these, only the NAFLD fibrosis score and FIB-4 have been confirmed across a broad spectrum of populations and considered the most reliable for the exclusion of advanced fibrosis (115). It is apparent that these scores are only able to distinguish relatively well between the two extremes – a population without evidence of NAFLD and a population with advanced fibrosis (F3-4). Most times, results fall in an intermediate or undetermined range, thus are not able to accurately classify patients in the spectrum of mild (F1) to moderate (F2) disease (9). These scores are also limited for use in population without T2DM. They have not been shown to be very reliable in this specific high-risk population of patients with T2DM (9).

 

Table 3. Biomarkers Available for use in Diagnosis of Advanced Fibrosis (Stages 3 or 4). Modified from reference (8)

Test

Parameters included

number

PPV

NPV

Patients unable to be classified “grey zone”

NAFLD fibrosis score

Age, BMI, diabetes, AST/ALT ratio, platelets, albumin

733

82%

88%

24%

Fibrotest

Age, sex, total bilirubin, GGT,

a2-macroglobulin, apolipoprotein A1, haptoglobin

267

60%

98%

32%

FIB-4 index

Age, AST and ALT, platelets

541

80%

90%

30%

BARD score

BMI, diabetes, AST/ALT ratio

827

43%

96%

N/A

NAFIC score

Ferritin, type IV collagen, insulin

619

36%

99%

15%

Hepascore

Age, sex, total bilirubin, GGT,

a2-macroglobulin, hyaluronic acid

242

57%

92%

11%

N/A, not applicable; NPV, negative predictive value; PPV, positive predictive value.

No independent validation cohort included in the study.

 

Some commercially available tests based on a metabolomic profile have been tested as a novel means to evaluate for NAFLD or NASH and recently tested in patients with type 2 diabetes mellitus. These tests have shown some promise to distinguish between normal liver and NAFLD and also able to detect NASH in people without diabetes. However, when applied to a population with T2DM, these tests have not been as accurate as expected to predict presence of NAFLD, NASH or fibrosis (115, 116). There is an increasing interest in assessing the utility of novel biomarkers, such as plasma fragments of propeptide of type III procollagen (PROC3) for the detection of liver fibrosis in patients with T2DM.  A recent study reported that PRO-C3 performed well (overall similarly to APRI or FIB-4) but with the added value of predicting histological changes in fibrosis stage with treatment (117). However, more studies are needed to determine its real value to monitor therapy.  At the present time, available genetic tests include PNPLA3 and TM6SF2 and a few others (as described above), but they are not routinely performed at this time and limited to academic centers for research only. This is likely to change in the near future as more sophisticated genetic testing becomes available.

 

In summary, clinicians may use plasma aminotransferases or simple panels such as FIB-4 or NAFLD fibrosis score to identify patients at the highest risk of having NASH with advanced fibrosis (F3-4) in the clinic, but knowing that while the specificity may be acceptable (“rule out” advanced fibrosis or cirrhosis) the sensitivity is rather low.  A screening strategy should include the above and imaging as described below as ultrasound and/or controlled attenuation parameter (CAP) have better sensitivity for the diagnosis of steatosis. The 2019 American Diabetes Association (ADA) guidelines for the first time recommend screening to identify liver fibrosis in patients with prediabetes or T2DM with elevated plasma aminotransferases and/or steatosis (118).

 

Imaging Modalities

 

MEASUREMENT OF INTRAHEPATIC TRIGLYCERIDES

 

Liver Ultrasound

 

Ultrasound is a relatively low-cost technique that is widely availability. Because of this it is routinely used for the diagnosis of NAFLD. However, it should be noted that the sensitivity of the test can be widely variable due to differences in operator technique and devices available, definition of steatosis, use of different echographic parameters to define steatosis, as well as the heterogeneity of the liver disease. While in one meta-analysis liver ultrasound was found to have a pooled sensitivity of 84.8% and specificity of 93.6% to detect hepatic steatosis of more than 20-30% (119), this literature is largely from liver clinics where disease severity is greater (i.e., more steatosis and better performance) but may not reflect the setting of primary care physicians or endocrinologists.  More relevant was also the fact that the investigators calculated the sensitivity to diagnose moderate-to-severe fatty liver from the absence of steatosis, without considering mild-to-moderate NAFLD. However, clinicians are faced with many patients with NAFLD that have only mild-to-moderate intrahepatic triglycerides, emphasizing the importance of having simple imaging tools that can make the correct diagnosis in the clinic.

 

In a study by Bril et al (120), the authors compared in 146 patients the performance of ultrasound using a score from five echographic parameters for steatosis or liver fat quantified by1H-MRS. They used as the gold-standard histology (liver biopsy). They reported that the performance of liver ultrasound (parenchymal echo alone) was relatively poor but improved to an acceptable level when compared to 1H-MRS when enhanced by the five echographic parameter score for steatosis was utilized. The greatest sensitivity of the ultrasound test was reached at a hepatic steatosis content of at least 12.5%. Below this threshold, the test was unreliable. Technological improvements may enhance in the near future the performance of liver ultrasound and its value in the management of patients with NAFLD.

 

Controlled Attenuation Parameter (CAP)

 

CAPis a relatively new imaging methodology to quantify steatosis. It is based on the principle that intrahepatic triglycerides delay ultrasound waves, so that when travelling through tissue with steatosis they will be attenuated when compared to normal liver tissue.  The diagnostic range of CAP is from 100 to 400 dB/m. The higher the value the more suggestive of the presence of steatosis. The sensitivity of the test to diagnose hepatic steatosis was 68.8% and specificity was 82.2% in a meta-analysis of patients with biopsy-proven steatosis (121). Usually the cut-off of ≥280 dB/m is used to establish the diagnosis of steatosis. As discussed below, one advantage of CAP is that in addition to being a simple and useful point-of-care tool (often available in liver clinics), the estimation of CAP can be performed simultaneously with that of the liver stiffness measurement (LSM; FibroscanÒ) and from the same liver region of interest, significantly facilitating clinical management although the test has its limitations when liver fat is only mildly elevated.

 

MR Spectroscopy

 

MR-based techniques have been the most accurate procedure to quantify liver triglyceride content (122). The use of 1H-MRS has proven to be very accurate for quantification of intrahepatic triglyceride content, with the results correlating well with steatosis on histology (120). MR spectroscopy derived proton density fat fraction (MR-PDFF) has recently evolved into a simpler and easier to standardize method for multicenter studies examining the effect of liver steatosis of new agents for the treatment of NASH (9,108,114). It has shown better diagnostic and grading capabilities for liver steatosis when compared with controlled attenuation parameter modality using transient elastography (122). However, MR spectroscopy remains an expensive test available mostly in academic centers, and requires special expertise for performance and analysis of the test (108).

 

MEASUREMENT OF LIVER FIBROSIS

 

Liver Stiffness Measurement (LSM; FibroscanÒ)

 

Liver fibrosis can be assessed in the clinic or bedside by measuring the “stiffness” of the liver.  The LSM is estimated by using vibration controlled transient elastography or VCTE (FibroscanÒ) to assess presence and severity of fibrosis. This modality also allows for a reasonably accurate quantification of the degree of fibrosis and hence, prognosis (108,92).  It is a quick (10 minutes), easy, and economical tool for assessment, however the test requires a 3-hour fast, and in obese people liver fibrosis cannot be always estimated and performance is worse (particularly when BMI ≥40 kg/m2) (108). At present, this test is not FDA-approved to be performed in patients with a pacemaker or during pregnancy.

 

MR Elastography

 

This modality is based on the same principle of liver “stiffness” as VCTE but it is a MR-based technique that has a sensitivity of 86% and specificity of 91% for assessment of degree of fibrosis (108). It is shown to be superior to VCTE, especially in diagnosis of early as well as advanced stages of fibrosis and cirrhosis. It is however much more expensive, requires special expertise to perform, and the current availability is limited. It also needs to take into account patient’s size and weight, any metal implants, as well as anxiety and claustrophobia during the procedure (92,108).

 

Liver Biopsy

 

Liver biopsy remains the gold-standard for diagnosis of NASH and for assessing the degree/severity of fibrosis (94). It is the only modality to reliably distinguish between steatosis alone from NASH and advanced fibrosis and to eliminate other etiologies of liver disease 108,123-125).

 

The degree of liver disease on histopathology is graded on a score that has been developed, called the NAFLD activity score (NAS). NAS score ranges from 0-8 and includes three parameters that are graded separately – steatosis (0-3), hepatocellular ballooning (0-2), and lobular inflammation (0-3). The degree or stage of fibrosis is graded separately from 0-3. These scores and staging ranges allow for a more accurate and reproducible way of monitoring of disease (108,123,125). However, there are limitations involving liver biopsies as well due to the inter-pathologist variability in interpretation of grades and degree of steatosis, inflammation and fibrosis (92,124).

 

Despite all the current advances, there remains an urgent need for development of more cost-effective and reliable methods for non-invasive screening of NAFLD to ensure early and prompt diagnosis for the best treatment outcomes.

 

TREATMENT

 

The aim of treatment for patients with NASH is to delay or reverse the progression of fibrosis and improve NASH-related morbidity/mortality due to hepatic (cirrhosis and HCC) and extra-hepatic complications, mainly cardiovascular disease (CVD). Currently there is no pharmacotherapy approved by regulatory agencies for the treatment of NAFLD, although pioglitazone is recommended by the current guidelines as a choice for patients with or without T2DM, and vitamin E for patients without diabetes (92,124). The FDA has accepted 2 endpoints as valid ones for drug approval in clinical trials: a) Resolution of the histologicalfindings that define NASH (necroinflammation) without worsening of fibrosis, and b) Reversal of ≥1 fibrosis stage without worsening of steatohepatitis/NASH (124,126,127). Despite the many ongoing efforts to find novel pharmacological agents the first-line of treatment will always be lifestyle modification including diet, exercise and weight loss (92,124), to combat insulin resistance and the relatedconditions like diabetes and obesity so closely related to NAFLD (128-130). 

 

Weight loss: Lifestyle, Bariatric Surgery and Weight Loss Agents

 

Numerous studies have shown the beneficial effect of weight loss to improve hepatic steatosis. It has been reported that weight loss not only improvesliver steatosis and other histological features of NASH (including fibrosis) but can decrease insulin resistance and blood pressure as well as improve atherogenic dyslipidemia (elevated LDL-C and triglycerides, low HDL-C) (92,131). In a meta-analysis of eight trials including 373 patients, improvement in hepatic steatosis was seen in patients who lost ≥5% of body weight, while NAFLD activity score (NAS) improvement was associated with weight loss of ≥7% body weight (132). In another randomized well-controlled trial paired with liver biopsy, weight loss and exercise program resulted in improvement of NASH. Moreover, this study showed that the magnitude of weight loss correlated strongly with improvement in histology (133). However, even with intensive multidisciplinary lifestyle interventions, more than half of patients were unable to achieve the weight loss target (weight loss of ≥7% body weight) which makes patient compliance the mainconcern (132). Despite the presence of multiple studies that correlates weight loss with the improvementof histological disease in NASH, little is known about the long-termeffect (i.e. beyond 1 year) of weight loss on liver histology (8).

 

Weight reduction of 10% by lifestyle modification may cause a significant regression of fibrosis (133,134). A greater and a more sustained over time decrease in weight loss with improvement in steatohepatitis, and even fibrosis, can be achieved by bariatric surgery (92,135,136). In a systematic review that included 21 observational studies of bariatric surgery in patients with NASH, an improvement in steatosis was reported in 18 studies, decreased inflammation was reported in 11 studies and improvement in fibrosis was reported in 6 studies (137). Only four studies reported some (minor) worsening of fibrosis (137). However, most bariatricsurgery studies have some limitations: these include small size, lack of proper standardization of preoperative low-caloric diet, frequent dropouts, and often no standardized time after the repeat postoperative liver biopsy. Finally, there are no randomized clinical trials (RCT) that compare bariatric surgery versus conservative management in patients with NASH with liver histology as the primary endpoint (137,138). Weight loss agents had no specific liver benefit (131), but can help with weight control and cause improvement in plasma aminotransferases and liver histology (139,140).

 

Adding regular moderate-intensity aerobic exercise/resistance training is highly encouraged as a lifestyle intervention for NAFLD. Exercise not only improves steatosis but the high cardio-metabolic risk profile, even in the absence of significant weight loss (92,124,141). In an uncontrolled study of 293 patients paired with liver biopsies, one year of structured exercise (walking 200 min/week) combined with ahypocaloricdiet improved hepatic steatosis and necroinflammation (133). In order to sustain weight loss, most dietary recommendations for NAFLD reflect a combination of hypocaloric diet (500–1000 kcal/day energy deficit) with exercise (92,134).

 

Heavy alcohol consumption should be avoided by patients with NAFLD and NASH.  Heavy drinking is defined as four standard drinks on any day or more than 14 drinks per week in men, or more than three drinks on any day or seven drinks per week in women (92).  There are no longitudinal studies reporting the effect of ongoing alcohol consumption on disease progression or the natural history of NAFLD or NASH.

 

Pharmacological Agents with Evidence from RCTs for the Treatment of NASH

 

Pharmacologic treatment has been extensively studied for patients with NASH with or without diabetes mellitus. For patients with NASH and T2DM, the typical initial therapy is with metformin. However, randomized controlled trials did not show improvement in liver histology (92,142).

 

Given that insulin resistance is a core feature in the pathogenesis of NAFLD/NASH, thiazolidinediones (TZDs), targeting the transcription factor PPAR gamma in adipose tissue and other tissues, has been tested in several RCTs in patients with NASH (3).  Pioglitazone at the molecular level modulates glucose and lipid metabolism and improves adipose tissue and hepatic insulin signaling and insulin sensitivity, collectively leading to improved liver histology in patients with NASH (143-149).However, the exact mechanism of action in humans is unknown and likely involves other pathways, for instance, activation of a mitochondrial pyruvate carrier (MPC) and/or PPAR alpha effects that may enhance mitochondrial fatty acid oxidation.  A recent study in vitro and in vivosuggested effects independent of activation of MPC (150). Of note, when rosiglitazone was comparedto placebo in patients with NASH it did not show any improvement beyond a reduction in steatosis as hepatocyte necrosis, lobular inflammation and fibrosis were unchanged (151). This suggests that improvement in fibrosis is not necessarily due to PPAR gamma as rosiglitazone is strictly a PPAR gamma agonist while pioglitazone is a considered a weaker agonist that also has PPAR alpha activity. Of note, different PPAR gamma activators do not modulate function or increase the expression of identical genes. The expression profiles can vary, which can explain differential effects via PPAR gamma activation.

 

Pioglitazone has been the agent most studied to date in patients with and without diabetes and biopsy-proven NASH (143-149), as recently reviewed in-depth along with other medications to treat diabetes regarding their effect in NAFLD (152). Resolution of NASH with pioglitazone treatment has been fairly consistent across studies of 6 to 36 month duration and ranges from ~47% (or 29% placebo-subtracted) in patients without diabetes with pioglitazone 30 mg/day for 24 months (94), to ~60% (or ~40% placebo-subtracted) with pioglitazone 45 mg/day in those with prediabetes or T2DM treated for 6 to 36 months (143,148, 149).  Taken together, these results suggest that pioglitazone might play a role in modifying disease progression and its natural history in patients with or without diabetes.

 

In addition, pioglitazone may improve the cardiometabolic profile of patients with NASH by reducing progression to diabetes and CVD.  Many patients with obesity and NAFLD/NASH have (often undiagnosed) prediabetes. Pioglitazone has proven effective for the prevention of diabetes in subjects with prediabetes (153) and shown to ameliorate cardiovascular events in patients with metabolic syndrome or prediabetes with a history of a stroke.Recently, the IRIS study reported the effect of pioglitazone in patients that had taken ≥80% of the prescribed medication reduced stroke by 36%, acute coronary syndromes by 53%, and the combined endpoint of stroke/MI/hospitalization for heart failure by 39% (154).

 

However, it remains puzzling that for a population with such a high cardiovascular risk from having obesity, T2DM and NASH, the cardiometabolic benefits of pioglitazone are frequently dismissed because of potential side effects that can be mitigated with close monitoring: bone loss, weight gain (3-5%) (most usually associated with improved insulin action on adipose tissue, not edema),or lower extremity edema in ~5% but higher if on amlodipine or high-dose insulin (152,155). Consistent with diabetes prevention and CVD reduction (156-160), patients become more metabolically healthy despite weight gain (143,149). While pioglitazone improves left ventricular function in healthy patients with T2DM (161), it may trigger heart failure in patients who have fluid retention and subclinical (undiagnosed) heart failure with preserved left ejection fraction (HFpEF), also known as “diastolic dysfunction” (≤1%) (155).  Obese patients with T2DM and NASH are more prone to HFpEF (162). Therefore, in our experience, this can be avoided if pioglitazone is not prescribed to poor candidates, such as those with long-standing history of severe CVD that could be associated with heart failure, baseline presence of unexplained shortness of breath or lower extremity edema, severe obesity (BMI ≥40 kg/m2), or longstanding diabetes on high-dose insulin.  Concomitant use of amlodipine, that is often already associated with lower extremity edema, should also be avoided.  The clinician suspecting HFpEF may consider ruling this condition out before initiating therapy.  Options to this end are ordering a transthoracic echocardiogram or plasma N-terminal (NT)-pro hormone B-type natriuretic peptide (NT-proBNP), the non-active prohormone from BNP. Both BNP and NT-proBNP are released in response to changes in cardiac pressure with plasma levels increasing when heart failure develops or worsens (162).

 

There is significant controversy about the risk of bladder cancer with pioglitazone and unlikely ever to be resolved given the overall low frequency of bladder cancer in the general population.  A recent 10-year prospective study was negative for bladder cancer (163) and there was no association found in a recent meta-analysis comparing patients who had been ever vs. never users of pioglitazone, but there was a small but significant association with 1–2 years (HR = 1·28 [1·08–1·55]) and >2 years (HR = 1·42 [1·14–1·77]) of exposure (164). In absolute terms, bladder cancer developed in <0.3% of patients both exposed and not exposed to pioglitazone. The numbers needed to treat for one additional case of bladder cancer ranged from 899 to 6380 (median of 2540), while the benefit for CVD and NASH ranged from 4–256 and 2–12, respectively.

 

Taken together, pioglitazone is an evidence-based treatment option for patients with and without diabetes and NASH (92). It is also a generic medication recommended by the current ADA and EASD guidelines as a low-cost option, along with sulfonylureas, for the management of T2DM.  Pioglitazone is likely to become for patients with NASH what metformin is for the management of T2DM, an inexpensive and effective option offering liver histological and cardiometabolic benefit and likely to be combined with novel therapeutic agents under development.

 

Glucagon-like peptide 1 (GLP-1) receptor agonists are another group of pharmacologic agents widely used for the treatment of diabetes that also have significant cardiometabolic benefits. A recent review summarized the many studies that have tested GLP-1RAs in patients with NAFLD (152). Typically, treatment is associated with weight loss and a decrease in plasma aminotransferases and hepatic steatosis.  In the only study to date examining their role in NASH, Armstrong et al (165) randomized 52 patients with NASH to receive either liraglutide or placebo for 48 weeks. NASH resolved in nine patients (39%) who received liraglutide compared to two patients (9%) in the placebo group (RR 4.3; 95% CI 1.0-17). Patients who received liraglutide were less likely to have progression of fibrosis (9 versus 36 percent; RR 0.2; 95% CI 0.1-1.0). These results are consistent with most other controlled and uncontrolled trials with liraglutide and other GLP-1RAs that have consistently led to weight loss and a reduction hepatic steatosis on imaging and in plasma aminotransferases in patients with NAFLD (166). In contrast, DPP-IV agents have largely been ineffective in RCTs in NAFLD (166).

 

The sodium–glucose cotransporter 2 (SGLT2) inhibitors have a significant role in the management of patients with T2DM (167). They promote weight loss, reduce the risk of CKD and of heart failure, and decrease overall rates of cardiovascular events in patients with T2DM (168). Several studies in animal models of NAFLD have reported that this class of agents reverses hepatic steatosis and necroinflammation. Early studies reported improvements in plasma aminotransferases and hepatic steatosis (152).Recent controlled RCTs have reported a (modest) reduction in hepatic steatosis on imaging with canagliflozin (169) and dapagliflozin (170) in patients with T2DM and NAFLD.  These findings combined with their attractive properties of weight loss and decreasing diabetic comorbidities would make them potentially valuable for combination therapy (i.e., pioglitazone) for patients with NAFLD, as shown from combination therapy trials in patients with T2DM (171-173).

 

Finally, it is important to mention vitamin E as it has been examined in RCTs for the treatment of NASH in patients with (149) and without (147) T2DM.   In a study in patients with NASH but without diabetes, vitamin E showed improvement in the primary outcome, but had borderline efficacy for resolution of NASH (considered today a more relevant outcome) compared to placebo (36% vs. 21%; p = 0.05) and numerically appeared as less significant compared to pioglitazone (47%; p = 0.001 vs. placebo) (147).Recently, Bril et al (149) found that vitamin E alone appeared to not be as effective in patients with T2DM, as it failed to meet the primary outcome of a two-point reduction in the NAFLD activity score from two different parameters, without worsening of fibrosis.  However, when vitamin E was combined with pioglitazone more patients on combination therapy achieved the primary outcome versus placebo (54% vs. 19%, P = 0.003) although the efficacy did not seem to be greater than that with pioglitazone alone in previous trials (143, 148). Resolution of NASH occurred in both groups compared with placebo (combination group: 43% vs. 12%, P = 0.005; vitamin E alone: 33% vs. 12%, P = 0.04).

 

Other relevant group of agents tested in NASH include the lipid-loweringdrugs (e.g. statins, colesevelam, omega 3 fatty acids, fibrates and niacin), whichhave not shown much success when studied in clinical trials in patients with NASH (174-179).

 

The Future: Many Agents on the Horizon for NASH

 

Given the rapid evolution of the field, with constant new drugs entering the arena of trials and others failing, we to refer the reader to recent in-dept reviews on the topic (180,181). Many pharmacological agents are being tested in phase 2 and phase 3 trials targeting a broad spectrum of pathways involved in the pathogenesis of NASH. Therapeutic targets of significant interest include farnesoid X receptor (FXRs), which regulate hepatic glucose and lipid metabolism (182). In the FLINT trial (183), in which obeticholic acid (manufactured by Intercept) was compared to placebo there was some evidence of histological improvement, including a mild effect on fibrosis that was recently confirmed in the Interim Analysis of the Phase 3 REGENERATE trial but showed no improvement in resolution of NASH (184). Unfortunately, a significant number of patients complain of pruritus and there was a worsening of dyslipidemia that can be mitigated by co-administration of statins (183).  Several novel FXR compounds are in development (180,181).

 

As discussed, PPAR nuclear receptors play a key role in insulin sensitivity. In the light of their roles in NAFLD and NASH several combined PPAR agonists have been studied. Elafibranor (manufactured by Genfit), is a dual receptor PPAR-α/δagonist that improves in insulin resistance and glucose/lipid metabolism (185). In the GOLDEN trial, a phase study 2b study, elafibranor 120 mg/day for a year led to a modest improvement in resolution of NASH compared to placebo in the subgroup with worse steatohepatitis (186).Another PPAR agonist is lanifibranor, a panPPAR agonist (PPAR-α/δ/ γ), is currently undergoing phase 2 clinical trials in NASH (187).Saroglitazar (by Zydus), is a dual PPAR-α/γ agonist with a predominant PPAR-α activity, reverses steatohepatitis in experimental NASH models (188)and is undergoing clinical trials. A phase 2b RCT of MSDC-0602K by Cirius is expected to report results in late 2019 for the treatment of NASH. It is a compound designed to minimize PPAR gamma binding activity but to maintain binding affinity to a second cellular target of all TZDs that has been identified as the mitochondrial target of the TZDs (mTOT) or mitochondrial pyruvate carrier (MPC) (189). Other insulin-sensitizers in earlier stages of development include PXL-065 (by Poxel), an enantiomer of pioglitazone, and CHS-131 (by Coherus) a compound with PPARγ activity tested earlier in patients with T2DM.

 

Other pharmaceutical compounds being tested for the treatment of NASH aim at a variety of potential pathways. We will mention only a few examples for the reader to appreciate the broad spectrum of targets being studied. Aramchol (by Galmed) is a novel compound that downregulates stearoyl-CoA desaturase 1 (SCD1), a key enzyme involved in triglyceride biosynthesis (190). Inhibition of de novolipogenesis (increased in NASH) by an inhibitor of acetyl-CoA carboxylase (ACC), the rate limiting enzyme in this pathway, is also being studied in RCTs in patients with NASH (GS-0976, Gilead) (191,192). Fibroblast growth factor (FGF)-19 functions as a hormone that regulates bile acid metabolism with effects on glucose and lipid metabolism (193).NGM282 (NGM Biopharmaceuticals) is an engineered analogue of FGF-19 for the treatment of NASH with promising early results (194). Several companies are testing analogues of FGF21 that have significant metabolic effects on glucose and lipid metabolism as well as hepatic fat (180, 181). Thyroid hormone receptor (THR) β-selective agonists, appear to specifically target the liver and improve steatohepatitis in animal models and early clinical trials in patients with NASH (195,196). Many other agents are being tested at this time.

 

CONCLUSION

 

Endocrinologists must be aware that NAFLD is a potentially severe disease in patients with T2DM, due to both its hepatic and extrahepatic complications.  In 2019 the ADA included for the first time in its recommendations to implement regular screening for advanced fibrosis in all patients with prediabetes or T2DM with evidence of elevated plasma aminotransferases or steatosis, so an early diagnosis can prevent long-term complications (118).This is the first step of management while being aware of the significant need for accurate and cost-effective diagnostic modalities and for continued research efforts for new treatments.

Figure 3 is a suggested algorithm to be used for endocrinologists and primary care settings when evaluating a patient with prediabetes or T2DM for the possibility of having NASH. 

In the future, we anticipate that patients with T2DM will be routinely screened for NASH in the same way they are today for diabetic retinopathy or nephropathy.

Figure 3. Management of patients with prediabetes or type 2 diabetes mellitus and suspected NAFLD. Based on figure from reference (8). *High risk patients include patients with type 2 diabetes > 10 years, A1c > 8.5%, triglycerides > 250mg/dl, evidence of steatosis based on MR imaging or controlled attenuation parameter (CAP), or genetic testing (PNPLA3 and/or TM6SF2).

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