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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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Neonatal Hyperthyroidism

INTRODUCTION

 

Neonatal hyperthyroidism in most cases is transient and results from the transplacental passage of maternal stimulating TSH receptor antibodies (TRAb) known as neonatal Graves’ disease (GD).  Permanent non autoimmune neonatal hyperthyroidism is rare and is due to activating mutations of TSH receptor or due to somatic activating mutations in the stimulatory alpha subunit of the guanine nucleotide-binding protein (GNAS gene) in McCune-Albright syndrome. Exposure to topical iodine has also been reported as a rare cause of hyperthyroidism in newborns.

 

TRANSIENT NEONATAL HYPERTHYROIDISM

 

Neonatal Graves’ disease (GD) is usually a self-limited disease, but it can be life threatening and permanently damage the brain.  Neonatal GD is caused by transplacental passage of TSH receptor antibodies (TRAb) with stimulatory activity.

 

TRAb are Immunoglobulin of G class and freely cross the placenta. Different types of TRAb can be found: TRAb that bind to the TSH receptor and stimulates the production of thyroid hormones, (TSH receptor stimulating antibodies, TSI), TRAb that bind to the TSH receptor, do not stimulate the production of thyroid hormones and can block the binding of TSH (TSH receptor blocking antibodies TBI).

 

Hyperthyroidism develops only in babies born to mothers with the most potent stimulatory activity in serum. This corresponds to 1-2% of mothers with Graves’ disease, or 1 in 50,000 newborns, an incidence that is approximately four times higher than is that for transient neonatal hypothyroidism due to maternal TSH receptor blocking antibodies. Some mothers have mixtures of stimulating and blocking antibodies in their circulation, the relative proportion of which may change over time. Not surprisingly, the clinical picture in the fetus and neonate of these mothers is more complex and depends not only on the relative proportion of each activity in the maternal circulation at any one time but on the rate of their clearance from the neonatal circulation postpartum.

 

Occasionally, neonatal hyperthyroidism may even occur in infants born to hypothyroid mothers. A prospective study showed that 40% of patients treated for Graves’ disease with radioactive iodine had TRAb detectable after 5 years (13). In these situations, the maternal thyroid has been destroyed either by prior radioablation, surgery, or by coincident destructive autoimmune processes so that potent thyroid stimulating antibodies, present in the maternal circulation, are silent in contrast to the neonate whose thyroid gland is normal. Persistence of TRAb after thyroidectomy is higher in females with Graves’ ophthalmopathy or smokers. Fetal/neonatal thyrotoxicosis can occur also in newborn from hypothyroid mothers with chronic lymphocytic thyroiditis.

 

CLINICAL MANIFESTATIONS

 

TABLE 1. Situations That Should Prompt Consideration of Neonatal Hyperthyroidism

·       Unexplained tachycardia, goiter or stare

Unexplained petechiae, hyperbilirubinemia, or hepatosplenomegaly

·       History of persistently high TSH receptor antibody titer in mother during pregnancy

·       History of persistently high requirement for antithyroid medication in mother during pregnancy

·       History of thyroid ablation for hyperthyroidism in mother

·       History of previously affected sibling

 

Maternal TSH receptor antibody-mediated hyperthyroidism may present in utero. Fetal hyperthyroidism is suspected in the presence of fetal tachycardia (pulse greater than 160/min) especially if there is evidence of failure to thrive. Obstetric complications are common. Fetal goiter (fetal neck circumference >95%) can by monitored by ultrasound using nomograms for fetal thyroid growth. Fetal goiter can cause esophageal and/or tracheal obstructions and polyhydramnios. Fetal goiter can also be due to transplacental passage of antithyroid drugs that cause hypothyroidism in the fetus.

 

TABLE 2. Clinical Manifestations in the Fetus

Unexplained tachycardia,

Failure to thrive

Intrauterine growth retardation

Goiter

Advanced bone age

Prematurity

Craniosynostosis, microcephaly

Fetal death

 

In the neonate infant typically, the onset is during the first one two weeks of life but can occur by 45 days. This is due both to the clearance of maternally administered antithyroid drug (propylthiouracil- PTU, methimazole- MMI, or carbimazole) from the infant ’s circulation and to the increased conversion of T4 to the more metabolically active T3 after birth. Rarely, as noted earlier, the onset of neonatal hyperthyroidism may be delayed until later if higher affinity blocking antibodies are also present.

In the newborn infant, characteristic signs and symptoms include tachycardia, irritability, poor weight gain, and prominent eyes.  Goiter, when present, may be related to maternal antithyroid drug treatment as well as to the neonatal Graves’ disease itself.

 

Rarely, infants with neonatal Graves’ disease present with thrombocytopenia, jaundice, hepatosplenomegaly, and hypoprothrombinemia, a picture that may be confused with congenital infections such as toxoplasmosis, rubella, or cytomegalovirus. In addition, arrhythmias and cardiac failure may develop and may cause death, particularly if treatment is delayed or

inadequate. In addition to a significant mortality rate that approximates 20% in some older series, untreated fetal and neonatal hyperthyroidism is associated with deleterious long-term consequences, including premature closure of the cranial sutures (cranial synostosis), failure to

thrive, and developmental delay. The half-life of TSH receptor antibodies is 1 to 2 weeks. The duration of neonatal hyperthyroidism, a function of antibody potency and the rate of their metabolic clearance, is usually 2 to 3 months but may be longer.

 

TABLE 3. Clinical Manifestations in the Neonate

Irritability, hyperexcitability, sleep disorders

Tachycardia, hypertension, cardiac failure

Flushing, sweating

Respiratory distress, pulmonary hypertension

Goiter, stare

Feeding difficulties, increased appetite but no/poor weight gain

Diarrhea

Unexplained petechiae, hyperbilirubinemia, jaundice, or hepatosplenomegaly

Craniosynostosis, microcephaly,

Death

 

LABORATORY EVALUATION

 

The recent guidelines for management of hyperthyroidism and the updated guidelines for the management of thyroid disease during pregnancy released from the American Thyroid Association ATA both suggest determining TRAb levels in pregnant women with Graves’ disease at 18-22 weeks instead of 20-24 weeks of gestation because a severe case of fetal Graves’ disease has occurred at 18 weeks of pregnancy.

 

Because of the importance of early diagnosis and treatment, infants at risk for neonatal hyperthyroidism should undergo both clinical and biochemical assessment as soon as possible.

All neonates born from a woman with TRAb positivity in pregnancy should undergo determination of TRAb from cord blood at delivery. If TRAb is negative, the risk to neonatal hyperthyroidism is negligible (Sensitivity is around 100%). FT3, FT4 and TSH determination from cord blood did not predict neonatal hyperthyroidism. Increases in FT4 on day 3 to 5 seems to better indicate the onset of hyperthyroidism. Situations that should prompt consideration of neonatal hyperthyroidism are listed in Table 1. A high index of suspicion is necessary in babies of women who have had thyroid ablation because in them a high titer of TSH receptor antibodies would not be evident clinically. Similarly, women with persistently elevated TSH receptor antibodies and with a high requirement for antithyroid medication are at an increased risk of having an affected child.

 

The diagnosis of hyperthyroidism is confirmed by the demonstration of an increased concentration of circulating T4 (and free T4, and T3, if possible) accompanied by a suppressed TSH level in neonatal or fetal blood. Results should be compared with normal values during gestation. Fetal ultrasonography may be helpful in detecting the presence of a fetal goiter and in monitoring fetal growth. Demonstration in the baby or mother of a high titer of TSH receptor antibodies will confirm the etiology of the hyperthyroidism and, in babies whose thyroid function testing is normal initially, indicate the degree to which the baby is at risk.

 

 In general, babies likely to become hyperthyroid have the highest TSH receptor antibody titer but levels of maternal TRAb in the serum as low as 4.4 U/L has been associated with neonatal thyrotoxicosis.  If TSH receptor antibodies are not detectable, the baby is very unlikely to become hyperthyroid. In the latter case, it can be anticipated that the baby will be euthyroid, have transient hypothalamic-pituitary suppression, or have a transiently elevated TSH, depending on the relative contribution of maternal hyperthyroidism versus the effects of maternal antithyroid medication, respectively. Close follow up of all babies with abnormal thyroid function tests or detectable TSH receptor antibodies is mandatory.

 

THERAPY

 

In the fetus, treatment is accomplished by maternal administration of antithyroid medication. Until recently PTU was the preferred drug for pregnant women in North America, but current recommendations suggest the use of MMI rather than PTU after the first trimester because of concerns about potential PTU-induced hepatotoxicity. The goals of therapy are to utilize the minimal dosage necessary to normalize the fetal heart rate and render the mother euthyroid or slightly hyperthyroid. In the neonate MMI (0.25 to 1.0 mg/kg/day) has been used initially in 3 divided doses. If the hyperthyroidism is severe, a strong iodine solution (Lugol’s solution or SSKI, 1 drop every 8 hours) is added to block the release of thyroid hormone immediately. Often the effect of MMI is not as delayed in infants as it is in older children or adults, a consequence of decreased intrathyroidal thyroid hormone storage. Therapy with both antithyroid drug and iodine is adjusted subsequently, depending on the response. Propranolol (2 mg/kg/day in 2 or 3 divided doses) is added if sympathetic overstimulation is severe, particularly in the presence of pronounced tachycardia. If cardiac failure develops, treatment with digoxin should be initiated, and propranolol should be discontinued. Rarely, prednisone (2 mg/kg/day) is added for immediate inhibition of thyroid hormone secretion. Measurement of TSH receptor antibodies in treated babies may be helpful in predicting when antithyroid medication can be safely discontinued. Lactating mothers on antithyroid medication can continue nursing as long as the dosage of PTU or MMI does not exceed 400 mg or 40 mg, respectively. The milk/serum ratio of PTU is 1/10 that of MMI, a consequence of pH differences and increased protein binding, so one might anticipate less transmission to the infant, but concerns about potential PTU toxicity need to be considered. At higher dosages of antithyroid medication, close supervision of the infant is advisable. A review about management of neonates born to mothers with Graves’ disease has been recently published.

 

PERMANENT NEONATAL HYPERTHYROIDISM

 

Rarely, neonatal hyperthyroidism is permanent and is due to a germline mutation in the TSH receptor (TSH-R) resulting in its constitutive activation. A gain of function mutation of the TSH-R should be suspected if persistent neonatal hyperthyroidism occurs in the absence of detectable TSH-R antibodies in the maternal circulation. Prematurity, low birth weight, and advanced bone age are common. Most cases result from a mutation in exon 10 which encodes the transmembrane domain and intracytoplasmic tail of the TSH-R, a member of the G protein coupled receptor superfamily. Less frequently, a mutation encoding the extracellular domain has been described. An autosomal dominant inheritance has been noted in many of these infants; other cases have been sporadic, arising from a de novo mutation.

 

Early recognition is important because the thyroid function of affected infants is frequently difficult to manage medically and, when diagnosis and therapy is delayed, irreversible sequelae, such as cranial synostosis and developmental delay may result. For this reason, early, aggressive therapy with either thyroidectomy or even radioablation has been recommended.

 

Two clinical forms were described: the first one is the “familial non-autoimmune autosomal dominant hyperthyroidism” (FNAH). High variable age of manifestation from neonatal period to 60 years, with variability also within the same family is reported. Goiter is present in children, with nodules in older age. The second one is “Persistent sporadic congenital non autoimmune hyperthyroidism” (PSNAH) includes forms with sporadic (de novo) germline mutations in the TSH-R. PSNAH is characterized by fetal-neonatal onset or within 11 months and more severe hyperthyroidism requiring early aggressive therapy.

 

ThyroidfunctioninbabieswithagainoffunctionmutationoftheTSH receptormaybe difficult tomanagemedically and,whendiagnosis andtherapyisdelayed,irreversible sequelae,suchas cranial synostosis anddevelopmental delaymayresult.Thyroidablationmayberequired.Thyroidsurgeryis thepreferredapproachif anexperienced pediatric surgeonisavailable.Thetimingatwhichthyroidectomy canbeperformedwilldependoninstitutionalpreference.  Ifthis is notfeasible,thenradioablationmaybenecessary. Guidelines about this rare condition have recently been published.

 

MCCUNE ALBRIGHT SYNDROME

 

McCune Albright is a syndrome due to somatic activating mutations in Gsαgene and can rarely present with neonatal hyperthyroidism.

 

ACKNOWLEDGEMENTS

 

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

 

GUIDELINES

 

Ross DS, Burch HB, Cooper DS, Greenlee MC, Launberg P, Maia AL, Rivkees S, Samuels M, Sosa JA, Stan MN, Walter MA. 2016 American Thyroid Association Guidelines for Diagnosis and management of hyperthyroidism and other causes of thyrotoxicosis. Thyroid. 2016; 2:1343-1421.

 

Alexander EK, Pearce EN, Brent GA, Brown RS, Chen H, Dosiou C, Grobman WA, Launberg P, 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: 27:315.

 

REFERENCES

 

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van Dijk MM, Smits IH, Fliers E, Bisschop PH. Maternal thyrotropin receptor antibody concentration and the risk of fetal and neonatal thyrotoxicosis: a systematic review. Thyroid 2018: 28:257-

 

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Van der Kaay D, Wasserman JD, Palmert MR. Management of neonates born to mothers with Graves’ disease. Pediatrics. 2016;137:e20151878

 

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Kleinau G, Vassart G. TSH Receptor Mutations and Diseases. 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.; 2017 Jul 24.

 

 

 

Congenital Adrenal Hyperplasia: Diagnosis and Emergency Treatment

CLINICAL RECOGNITION


Congenital adrenal hyperplasia (CAH) is a group of autosomal recessive disorders that arise from defective steroidogenesis. The production of cortisol in the zona fasciculata of the adrenal cortex occurs in five major enzyme-mediated steps. CAH results from deficiency in any one of these enzymes. Impaired cortisol synthesis leads to chronic elevations of ACTH via the negative feedback system, causing overstimulation of the adrenal cortex and resulting in hyperplasia and over-secretion of the precursors to the enzymatic defect. The forms of CAH are summarized in Table 1. Impaired enzyme function at each step of adrenal cortisol biosynthesis leads to a unique combination of elevated precursors and deficient products. The most common enzyme deficiency that accounts for more than 90% of all CAH cases is 21-hydroxylase deficiency (21OHD).

 

Table 1. Types of Congenital Adrenal Hyperplasia

Condition

Onset

Abnormality

Genitalia

Mineralocorticoid Effect

Gene

Chromosomal Location

Typical Features

Lipoid CAH
Congenital
StAR Protein

Female, with no sexual development
Salt wasting

StAR 

8p11.2
All steroid products low

Lipoid CAH

Congenital

P450scc

Female, with no sexual development
Salt wasting

CYP11A

15q23-24
All steroid products low

3β-HSD deficiency Congenital
3β-HSD

Females virilized, males hypovirilized
Salt wasting

HSD3B2

1p13.1
Elevated DHEA, 17-pregnenolone, low androstenedione, testosterone, elevated K, low Na, CO2

17α-OH deficiency Congenital

P450c17

Males hypovirilized, Hyperkalemic low-renin hypertension

CYP17

CYP17

10q24.3
Decreased androgens and estrogen, elevated DOC, corticosterone

Classic 21-OH deficiency, salt wasting
Congenital

P450c21

Females prenatally virilized, males unchanged
Salt wasting occurs in ¾ of 21OHD patients

CYP21 

6p21.3
Elevated 17-OHP, DHEA, and androstenedione, elevated K, low Na, CO2, low aldosterone, high plasma renin

Classic 21-OH deficiency, simple virilizing
Congenital P450c21

Females prenatally virilized, males unchanged
No salt wasting

CYP21 

6p21.3
Elevated 17-OHP, DHEA, and androstenedione, normal electrolytes

Non-classic 21-OH deficiency

Postnatal
P450c21

All with normal genitalia at birth, hyperandrogenism postnatally
No salt wasting

CYP21 

6p21.3
Elevated 17-OHP, DHEA, and
androstenedione on ACTH stimulation

11β-OH deficiency Congenital

P450c11B1

Females virilized, males unchanged
Low-renin hypertension

CYP11B1  

8q24.3
Elevated DOC, 11-deoxycortisol (S); androgens, low K, elevated Na, CO2

P450 Oxidoreductase deficiency (POR), Congenital

P450 oxidoreductase

 

Males undervirilized, females unchanged

Variable degree of mineralocorticoid deficiency

P450 Oxidoreductase gene (POR)

7q11.2

Combined and variable enzymatic defects of P450c21, P450c17 and P450aro

Wide range of phenotypes: normal to genital ambiguity +/- skeletal abnormalities (Antley Bixler type)

 

 

Classical CAH occurs in 1:13,000 to 1:15,000 live births. It is estimated that 75% of patients have the salt-wasting (SW) phenotype and the rest have simple-virilizing (SV) phenotype. Non-classical 21-OHD CAH (NC-CAH) is more common, and is one of the most common disorders in the Ashkenazi Jewish population with 1 in 27 Jews affected.  CAH owing to 11β-hydroxylase deficiency (11β-OHD) is the second most common cause of CAH, accounting for 5-8% of all cases. The other forms of CAH are considered rare diseases and the incidence is unknown in the general population.

 

PATHOPHYSIOLOGY

Adrenal steroidogenesis occurs in three major pathways: glucocorticoids, mineralocorticoids, and sex steroids as shown in Figure 1. Glucocorticoids (particularly cortisol), androgens, and estrogens are synthesized in the zona fasciculata and reticularis; and aldosterone in the zona glomerulosa. The HPA feedback system is mediated through the circulating level of plasma cortisol by negative feedback of cortisol on CRF and ACTH secretion. Therefore, a decrease in cortisol secretion leads to increased ACTH production, which in turn stimulates (1) excessive synthesis of adrenal products in those pathways unimpaired by the enzyme deficiency and (2) an increase of precursor molecules in pathways blocked by the enzyme deficiency.

Figure 1. Pathways of Adrenal Steroidogenesis: Five enzymatic steps necessary for cortisol production are shown in numbers. 1= 20, 22 desmolase, 2= 17 hydroxylase (17-OH), 3=3ß-hydroxysteroid dehydrogenase (3ß HSD), 4=21 hydroxylase (21-OHD), 5=11ß hydroxylase (11-OH) In the first step of adrenal steroidogenesis, cholesterol enters mitochondria via a carrier protein called StAR. ACTH stimulates cholesterol cleavage, the rate limiting step of adrenal steroidogenesis.

 

The clinical symptoms of the five different forms of CAH result from the particular hormones that are deficient and those that are produced in excess as outlined in Table 1. In 21 OHD-CAH, there is an accumulation of 17-hydroxyprogesterone (17-OHP), a precursor to the 21-hydroxylation step, which is then shunted into the intact androgen pathway, where the 17,20-lyase enzyme converts the 17-OHP to D4-androstenedione, which is converted into androgens. Mineralocorticoid deficiency is a feature of SW-CAH, the most severe form of CAH. The enzyme defect in NC-CAH is only partial and salt wasting in this mild form of the disease does not occur. The analogy of all other enzyme deficiencies in terms of precursor retention and product deficiencies are shown in Table 1.

 

CLINICAL FEATURES

Genitalia

Females with Classical 21-OHD and 11β-hydroxylase deficiency CAH present at birth with virilization of their genitalia. Adrenocortical function begins around the 7th week of gestation; thus, a female fetus with classical CAH is exposed to adrenal androgens at the critical time of sexual differentiation (approximately 9 to 15 weeks gestational age). This leads to clitoral enlargement, fusion and scrotalization of the labial folds, and rostral migration of the urethral/vaginal perineal orifice, placing the phallus in the male position. Degrees of genital virilization are classified into five Prader stages (see Figure 2).

Figure 2. Different degrees of virilization according to the scale developed by Prader

Stage I: clitoromegaly without labial fusion

Stage II: clitoromegaly and posterior labial fusion

Stage III: greater degree of clitoromegaly, single perineal urogenital orifice, and almost complete labial fusion

Stage IV: increasingly phallic clitoris, urethra-like urogenital sinus at base of clitoris, and complete labial fusion

Stage V: penile clitoris, urethral meatus at tip of phallus, and scrotum-like labia (appear like males without palpable gonads)

Prader, A. Helv Paediatr Acta, 1954. 9:230-248.

 

Internal female genitalia, such as the uterus, fallopian tubes and ovaries, develop normally. Females with classical CAH maintain the internal genitalia potential for fertility.

 

Postnatal Effects, Growth and Puberty

Lack of appropriate postnatal treatment in boys and girls results in continued exposure to excessive androgens, causing progressive penile or clitoral enlargement, the development of premature pubic hair, axillary hair and acne. Advanced somatic and epiphyseal development occurs with exaggerated growth and is usually accompanied by premature epiphyseal maturation and closure, resulting in a final adult height that is typically significantly below that expected from parental heights. Excess glucocorticoid treatment can also lead to poor growth. The mean age at onset of puberty in both males and females is slightly younger than the general population. In those who are inadequately treated, central precocious puberty can occur. Following the onset of puberty, in a majority of successfully treated patients, the milestones of further development of secondary sex characteristics in general appear to be normal. In female adolescents and adults, signs of hyperandrogenism may include male-pattern alopecia (temporal balding), acne, hirsutism, menstrual irregularities, secondary PCOS and impaired fertility. Although the expected age of menarche may be delayed in females with classical CAH, when adequately treated many have regular menses after menarche.  In males, short stature and impaired fertility are observed.

 

Gender Role Behavior and Cognition

Prenatal androgen exposure in females affected with classical forms of CAH not only has a masculinizing effect on the development of the external genitalia, but also on childhood behavior. Both physical and behavioral masculinization are related to each other and to genotype, indicating that behavioral masculinization in childhood is a consequence of prenatal androgen exposure. The majority of genetic females with CAH retain the female gender identity even in the setting of prenatal androgen exposure and postnatal hyperandrogenism.

 

Fertility

Difficulty with fertility in females with CAH may be due to anovulation, secondary polycystic ovarian syndrome, irregular menses, non-suppressible serum progesterone levels, or an inadequate introitus. Fertility is reduced in SW-CAH with rare reports of pregnancy. Non-classical CAH is an important and frequently unrecognized form of infertility. Males with CAH, particularly if poorly treated, may have reduced sperm counts and low testosterone as a result of high androstenedione concentrations which suppress gonadotropins and testicular adrenal rest tumors. Testicular adrenal rest tumors (TART) are thought to arise from aberrant adrenal cells in the testes; TARTs are always benign and mostly bilateral. Microscopic examination shows that adrenal rest cells are present in the testicles of all male patients with CAH and often detected radiographically in those with longstanding poorly controlled disease. Regular testicular examination and periodic testicular ultrasonography are recommended for early detection of adrenal rest tumors of the testes. However, MRI studies have been increasingly used to diagnose TARTs.

 

Salt-Wasting 21-Hydroxylase Deficiency

When the loss of 21-hydroxylase function is severe, adrenal aldosterone secretion is not sufficient for sodium reabsorption by the distal renal tubules, and individuals suffer from salt wasting as well as cortisol deficiency and androgen excess. Infants with renal salt wasting have poor feeding, weight loss, failure to thrive, vomiting, dehydration, hypotension, hyponatremia, and hyperkalemic metabolic acidosis progressing to adrenal crisis (azotemia, vascular collapse, shock, and death). Adrenal crisis can occur as early as age one to four weeks. Affected males who are not detected in a newborn screening program are at high risk for a salt-wasting adrenal crisis because their normal male genitalia do not alert medical professionals to their condition. It is important to recognize that the extent of genital virilization may not differ among SV-CAH and SW-CAH.

 

Simple-Virilizing 21-Hydroxylase Deficiency

The salient features of classical SV-CAH are prenatal virilization and progressive postnatal masculinization with rapid somatic growth and advanced epiphyseal maturation leading to early epiphyseal closure and likely short stature. There is no evidence of mineralocorticoid deficiency in this disorder and serum electrolyte concentrations are normal. Diagnosis at birth of a female with SV-CAH is usually made immediately because of the apparent genital ambiguity. Since the external genitalia are not affected in newborn males, hyperpigmentation may be the only clue suggesting increased ACTH secretion and cortisol deficiency. Diagnosis at birth in males thus rests on prenatal or newborn screening.

 

Non-Classical 21-Hydroxylase Deficiency

Individuals with the non-classical (NC) form of 21-OHD have only mild to moderate enzyme deficiency and present postnatally, eventually developing signs of hyperandrogenism. Females with NC-CAH do not have virilized genitalia at birth. NC-CAH may present at any age after birth with a variety of hyperandrogenic symptoms. While serum cortisol concentration is typically low in patients with the classic form of the disease, it is usually normal in patients with NC 21-OHD. Similar to classical CAH, NC-CAH may cause premature development of pubic hair, acne, secondary PCOS, advanced bone age with accelerated linear growth velocity, and short stature. In adult males, early balding, acne, infertility or short stature may prompt the diagnosis of NC-CAH.

 

DIAGNOSIS

Diagnosis of CAH must be suspected in infants born with ambiguous genitalia. The physician is obliged to make the diagnosis as quickly as possible to initiate therapy. The diagnosis and rational decision of sex assignment must rely on the determination of genetic sex, the hormonal determination of the specific deficient enzyme, genotype, and an assessment of the patient's potential for future sexual activity and fertility. As indicated in Table 1, each form of CAH has its own unique hormonal profile, consisting of elevated levels of precursors and elevated or diminished levels of adrenal steroid products. Diagnosis of the 21-OHD CAH can also be confirmed biochemically by a hormonal evaluation. In a randomly timed blood sample, a very high concentration of 17-hydroxyprogesterone (17-OHP), the precursor of the defective enzyme, is diagnostic of classical 21-OHD. Such testing is the basis of the newborn-screening program developed to identify classically affected patients who are at risk for salt wasting crisis. False-positive results are, however, common with premature infants. Appropriate references based on weight and gestational age are therefore in place in many screening programs. False negative results may occur if samples are drawn late in the afternoon as adrenal hormones exhibit diurnal variation.  The gold standard for hormonal diagnosis is the corticotropin stimulation test (250 μg cosyntropin intravenously), measuring levels of 17-OHP and Δ4 androstenedione at baseline and 60 min. These values can then be plotted in the published nomogram (Figure 4) to ascertain disease severity. The corticotropin stimulation test should not be performed during the initial 24 hours of life as samples from this period are typically elevated in all infants and may yield false-positive results. Establishing a genetic diagnosis is not only important for the genotype-phenotype correlation, but also for genetic counseling for future pregnancies and for genetic counseling for the patient and his/her reproductive future.

 

For 21-OHD CAH, genetic analysis of the CYP21A2 gene may provide more clues to predict phenotypic severity. In about 50% of the causative genotypes, genotype-phenotype correlation can be found, although certain mutations can lead to variable phenotypes in different population groups especially in the simple virilizer group. Sequencing of the entire gene should be performed to detect rare mutations when genotype–phenotype non-concordance is observed in patients with CAH.

 

Newborn screening for CAH, which utilizes 17 hydroxyprogesterone levels, is a useful tool for early detection of CAH prior to the development of adrenal crisis in the affected neonate.   However, screening is associated with a high rate of false positive results as levels are affected by prematurity and birth weight. Molecular genetics, especially genotyping of the CYP21A2 gene should be considered as a second-tier screening test in the new born screening program.

 

Prenatal testing for CAH in utero has historically utilized invasive techniques like amniocentesis and chorionic villus sampling which cannot be done prior to 14 weeks of gestation. Prenatal dexamethasone treatment must begin prior to genital formation occurring at approximately 9 weeks, in order to avoid genital ambiguity in the affected female fetus. Massive parallel sequencing using hybridization probes on cell-free fetal DNA in maternal plasma indicated that the fetal CAH status was correctly deduced as early as 5 weeks 6 days of gestation. This is a noninvasive technique that accurately diagnoses CAH before the ninth week of gestation.

 

TREATMENT

Routine Treatment

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The goal of therapy in CAH is to both correct the deficiency in cortisol secretion and to suppress ACTH overproduction. Proper treatment with glucocorticoid reduces stimulation of the androgen pathway, thus preventing further virilization and allowing normal growth and development. The usual requirement of hydrocortisone (or its equivalent) for the treatment of classical 21-OHD form of CAH is about 10-15 mg/m2/day divided into 2 or 3 doses per day and for non-classical 21-OHD 5-8 mg/m2/day divided into 2 or 3 doses per day. Hydrocortisone is the glucocorticoid of choice in the pediatric age group. Prednisolone and dexamethasone are not used in growing children given growth suppressive effects. A small dose of dexamethasone at bedtime (0.25 to 0.5 mg) is usually adequate for androgen suppression in non-classical adult patients. Adequate biochemical control is assessed by measuring serum levels 17-OHP and androstenedione; serum testosterone can be used in females and prepubertal males (but not newborn males). We recommend that hormone levels are measured at a consistent time in relation to medication dosing, usually 1-2 hours after the morning corticosteroid. Titration of the dose should be aimed at maintaining 17-OHP concentrations below 1000 ng/dL and androstenedione concentrations below 200 ng/dl. Over-treatment should be avoided because it can lead to Cushing syndrome. Patients with salt wasting CAH have elevated plasma renin in response to the sodium-deficient state, and they require treatment with the salt-retaining 9α-fludrocortisone acetate. The average dose is 0.1 mg daily (0.05-0.2 mg daily). Infants should also be started on salt supplementation, as sodium chloride, at 1-2 g daily, divided into several feedings. Measurements of plasma renin and aldosterone are used to monitor the efficacy of mineralocorticoid therapy.   Advancement of bone age is monitored by bone age x-rays. Growth hormone therapy, in conjunction with a GnRH analogue, has been shown to be effective in improving final adult height. Patients may also experience peripheral precocious puberty, which requires treatment with gonadotropin-releasing hormone analogues. Aromatase inhibitors and growth hormone therapy should only be used in patients with a very short predicted final stature or in clinical trials. Use of aromatase inhibitors in CAH has been shown decrease bone maturation rates and some increase in adult height but the differences were not statistically significant.

 

Treatment During Illness and Emergency


Adrenal crisis can present as hypotension or shock and serum electrolyte abnormalities (hypoglycemia, hyponatremia, hyperkalemia, acidosis). During adrenal crisis, an immediate bolus of hydrocortisone 50-100 mg can be given intravenously or intramuscularly followed by hydrocortisone 100 mg/m2/day given as either continuous infusion or divided at least every 6 hours. Rehydration can be started with 20ml/kg isotonic saline with D5 as rapid bolus followed by repeat boluses or continuous infusion guided by level of dehydration. Hypoglycemia may require dextrose bolus and an initial bolus of 0.5-1 gram/kg of dextrose can be given intravenously at 2-3 ml per minute. If hyperkalemia is present, cardiac monitoring should be done to monitor for EKG changes. If changes are present, hyperkalemia should be treated using insulin with glucose infusion with or without other measures.

 

In non-life-threatening periods of illness or physiologic stress, the corticosteroid dose should be increased to 2 or 3 times the maintenance dose for the duration of that period, divided into 3 daily doses. Each family should be given injection kits of hydrocortisone, i.e. Solu-Cortef, for emergency use, and all family members should be trained in its intramuscular administration. The injectable dose of hydrocortisone in an emergency is 25 mg for infants, 50 mg for children under 40 kg, and 100 mg for children over 40 kg and for adults. In the event of a surgical procedure, 5-10 times the daily maintenance dose of hydrocortisone is needed, with 25-100 mg hydrocortisone IM/IV administered before and during a surgical procedure (as per infant, child, adult recommendations above), followed by high doses of hydrocortisone during the first 24-48 post-operative hours; the dose can then be tapered over the following days to the normal preoperative schedule. Stress doses of dexamethasone should not be given because of the delayed onset of action. It is not necessary for increased mineralocorticoid doses during these periods of stress. It is imperative for all patients who are receiving corticosteroid replacement therapy, such as patients with CAH, to wear a Medical Alert bracelet or medallion that will enable correct and appropriate therapy in case of emergencies. It is also crucial to re-educate parents at regular intervals on the life-threatening nature of this emergency.

 

GUIDELINES

 

Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, Meyer-Bahlburg HFL, Miller WL, Murad MH, Oberfield SE, White PC. Congenital Adrenal Hyperplasia Due to Steroid 21-Hydroxylase Deficiency: An Endocrine Society Clinical Practice Guideline.

J Clin Endocrinol Metab. 2018 Nov 1;103(11):4043-4088

 

Rodriguez A, Ezquieta B, Labarta JI, Clemente M, Espino R, Rodriguez A, et al. Recommendations for the diagnosis and treatment of classic forms of 21-hydroxylase-deficient congenital adrenal hyperplasia. An Pediatr (Barc). 2017;87(2):116 e1- e10.

 

REFERENCES

 

New M, Yau M, Lekarev O, Lin-Su K, Parsa A, Pina C, Yuen T, Khattab A. Congenital Adrenal Hyperplasia. 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 15

 

El-Maouche D, Arlt W, Merke DP. Congenital adrenal hyperplasia. Lancet. 2017 Nov 11;390(10108):2194-2210

 

Fluck CE, Miller WL. P450 oxidoreductase deficiency: a new form of congenital adrenal hyperplasia. Curr Opin Pediatr. 2006;18(4):435-41.

 

Yilmaz R, Sahin D, Aghayev A, Erol OB, Poyrazoglu S, Saka N, et al. Sonography and Magnetic Resonance Imaging Characteristics of Testicular Adrenal Rest Tumors. Pol J Radiol. 2017;82:583-8.

 

New MI, Abraham M, Gonzalez B, Dumic M, Razzaghy-Azar M, Chitayat D, et al. Genotype-phenotype correlation in 1,507 families with congenital adrenal hyperplasia owing to 21-hydroxylase deficiency. Proc Natl Acad Sci U S A. 2013;110(7):2611-6.

 

Balsamo A, Baldazzi L, Menabo S, Cicognani A. Impact of molecular genetics on congenital adrenal hyperplasia management. Sex Dev. 2010;4(4-5):233-48.

 

New MI, Tong YK, Yuen T, Jiang P, Pina C, Chan KC, et al. Noninvasive prenatal diagnosis of congenital adrenal hyperplasia using cell-free fetal DNA in maternal plasma. J Clin Endocrinol Metab. 2014;99(6):E1022-30.

 

Lin-Su K, Harbison MD, Lekarev O, Vogiatzi MG, New MI. Final adult height in children with congenital adrenal hyperplasia treated with growth hormone. J Clin Endocrinol Metab. 2011;96(6):1710-7.