ABSTRACT

The incidence of Type 1 Diabetes continues to increase around the world. Advances in technology of insulin delivery systems including closed loop and continuous glucose monitoring are improving the possibilities of maintaining desirable glucose control. Type 2 Diabetes is increasing in the adolescent age groups across the world, in certain populations especially including Native Americans, Pacific Islanders, Hispanics, African Americans, and South East Asians. For Type 2 Diabetes, the pharmacological armamentarium has markedly increased by the addition of GLP-1 agonists, DPP4 antagonists, and SGLT2 inhibitors, each of which has improved metabolic control and cardiovascular outcomes. To date, these newer modalities are being tested in adolescents with T2DM but several are not yet officially approved for this age group. Diabetic Ketoacidosis (DKA) remains the initial presentation of some 30%-40% of pediatric patients, and DKA remains the leading cause of death, sometimes associated with Cerebral Edema; complications are also very high in children/adolescents presenting with Hyperglycemia/Hyperosmolar syndrome in the context of a T2DM clinical picture. Appropriate treatment in medical centers with trained personnel and modern laboratory facilities has markedly reduced the mortality and morbidity associated with DKA and Hyperglycemic-Hyperosmolar Syndrome.

DIABETIC KETOACIDOSIS

Pathophysiology

Diabetic ketoacidosis (DKA) is a life-threatening metabolic decompensation considered to be a medical emergency and caused by a combination of insulin deficiency and the action of counter-regulatory hormones(1). The biochemical, metabolic and acid-base abnormalities that occur have been extensively documented at a physiologic level and to some extent at a molecular level (2-4).  Briefly, deficiency of insulin prevents the entry of glucose into insulin-sensitive cells in tissues such as liver, muscle, and fat and its appropriate metabolism. Sensing intra-cellular glucopenia, the organism responds by increased secretion of the 4 counter-regulatory hormones, glucagon, cortisol, growth hormone, and catecholamines. Acting synergistically, these hormones increase glucose production via glycogen breakdown and gluconeogenesis, induce lipolysis and ketogenesis and result in hyperglycemia, osmotic polyuria, dehydration, increased thirst, and acidosis from the accumulation of ketoacids, principally β-hydroxybutyrate, (B-OHB) which exceed buffering capacity, as well as lactic acidosis from the ensuing dehydration and limited tissue perfusion. Hence, the symptoms and signs are polyuria, polydipsia, dehydration, tachycardia, deep sighing respiration (Kussmaul breathing), and the smell of acetone on the breath, and abdominal pain and nausea imitating an acute abdominal condition; paradoxically, despite dehydration, blood pressure may be normal or elevated reflecting the effects of catecholamines (Table 1). These manifestations develop over hours or days, in contrast to hypoglycemia which can occur suddenly. In cases of new diabetes, weight loss, increased appetite, and nocturia, or enuresis in previously toilet-trained child, are almost universally present if a careful history is elicited. Left untreated, clouding of consciousness due decreased cerebral oxygen perfusion, acidosis, and neural biochemical changes lead to coma and eventually death. Absolute insulin deficiency occurs most often at onset of evolving T1DM, but it may also occur after deliberate or inadvertent omission of insulin in a child or adolescent responsible for their own care, or with kinking or obstruction of tubing in insulin pumps. Relative insulin deficiency occurs with major physiological stressors such as sepsis, infection, or severe trauma that result in profound increased secretion of the counter-regulatory hormones which overwhelm the actions of insulin. Recurrent episodes of DKA are almost the result of psycho-social mal-adjustment. These concepts are summarized in figure 1.

Figure 1. Pathophysiology of diabetic ketoacidosis(7). Copyright © 2006 American Diabetes Association. From Diabetes Care, Vol. 29, 2006:1150-1159. Reprinted with permission of The American Diabetes Association

Criteria for Defining DKA

The criteria for a diagnosis of DKA are hyperglycemia with glucose ≥200mg/dl (≥11mmol/l), pH ≤7.30 or bicarbonate (HCO3) ≤15mmol/l, and ketonuria or B-OHB ≥2.0mmol/l. Severity of DKA is defined by the degree of acidosis; mild=pH 7.20-7.30, HCO3 10-15mmol/l; moderate=pH 7.1-7.2, HCO3 5-10mmol/l; severe =pH<7.1, HCO3<5 mmol/l. Hyperglycemia usually ranges between 200-1000mg/dl (16.6-50.5mmol/l);values >1000mg/dl should raise the possibility of hyperosmolar hyperglycemia, separately discussed below. The reported frequency of DKA varies from about 13% to 80% in various countries and is generally higher in less developed countries; it is inversely related to socio-economic development, level of education of the family, and to the incidence of diabetes mellitus in the location. In the United States, about 30-40% of newly diagnosed patients with DM present in DKA, reflecting delay in establishing the diagnosis of diabetes in a child, particularly in children <5years of age (8-11).

Table 2 illustrates the average losses of fluids and electrolytes in diabetic ketoacidosis and maintenance requirements in normal children adapted from references (4, 7, 9).

Principles of Treatment  

The principles of treatment enunciated here are based on those of the International Society for Pediatric and Adolescent Diabetes, the American Diabetes Association, and the Pediatric Endocrine Societies of Europe and the USA (4, 7, 11).

Mild cases of DKA such as might occur in a patient using an insulin pump in which the tubing has become obstructed, or mild upper respiratory or mild abdominal infection without significant vomiting or diarrhea in an educated patient and strong family support might be managed via telephone instructions. When fluids are tolerated, 3-4 ounces of clear fluids (approximately 100ml) can be offered hourly. In addition, rapid acting insulin 0.1-0.2U/kg is given every 2-4 hours, glucose levels are checked via home meters and urinary ketones are checked via strips. Resolution of hyperglycemia and ketonuria, and tolerance to oral fluid intake indicates successful management and return to customary regimens including pump settings and/or subcutaneous basal-bolus insulin regimens.

For new onset patients, those that cannot tolerate oral fluid intake, and those with moderate to severe DKA, we recommend admission to a unit with capabilities similar to those of an ICU, possessing written guidelines on management, physicians and nursing staff trained in the management of DKA, bedside glucose and blood gas monitoring, vital sign monitoring (pulse, blood pressure, respiration), and laboratory back-up of acid-base and electrolyte status. A thorough physical examination including level of consciousness should determine the overall clinical status, degree of dehydration, and consider the need to evaluate for infection. Supplemental oxygen may be provided via mask or nasal cannula, and a gastric tube passed if the patient is vomiting. Urine output should be measured via bag collection and catheterization avoided if possible.  A blood sample should be obtained for measurement of glucose, electrolytes, β-OHB, acid-base status, hematocrit, and complete blood count; blood cultures and imaging studies should be considered in cases of suspected sepsis as the precipitating cause and appropriate antibiotics given. A modest increase in WBC with neutrophil predominance may reflect an acute phase response rather than sepsis; even if an acute abdominal condition is suspected, surgery should be deferred until several hours of resuscitation with fluids and electrolytes has occurred. Access for IV infusion should be established; this or preferably a separate IV site can serve as source of blood sampling.

The initial resuscitation consists of intravenous saline bolus infusion at 10-20 ml/kg over 1-2 hours depending on the degree of dehydration. Clinical assessment of dehydration is based on physical findings such as heart rate, blood pressure, speed of capillary refill, tissue turgor, and dry coated tongue and is generally rated as 5% (mild),10% (moderate) or greater than 10% (severe). Urine output is not a reliable sign as it usually continues due to the osmotic diuresis of hyperglycemia; diminished urine output may reflect evolving renal failure. Clinical judgment of degree of dehydration is notoriously inaccurate and can result in over or under estimation; hematocrit or a very recent weight may aid in assessment of the degree of dehydration so as to more accurately guide the amount and composition of fluids to be infused and in turn reduce the risk of the occurrence of cerebral edema (12, 13). Hence initial estimates of dehydration and osmolality of plasma based on glucose and electrolyte status, as well as acid base resolution, require re-assessment as treatment progresses. The initial resuscitation period with normal (0.9%) sodium chloride solution provides an opportunity to elicit a careful history and formulate the plan of management focused on provision of fluid, electrolytes, insulin, and monitoring to anticipate and correct complications.

FLUID

The amount of fluid to be administered is based on the estimated degree of dehydration, e.g., 5% of body weight in Kg, plus daily maintenance (see table 2) evenly infused over 24-36 hours, subtracting the fluid administered as resuscitation. For example, a 30kg child with estimated dehydration of 5% would require 1500 ml for deficit plus 1700 ml for daily maintenance, yields a total of 3200 ml (table2); subtracting the 20ml/kg bolus of normal 0.9% saline from this total (600ml), leaves 2600 ml, or approximately 100 ml/hour over the initial 24 hours with adjustments made according to response. The total daily fluid infused should rarely exceed 1.5-2.0 times daily maintenance.

COMPOSITION OF FLUID

Sodium Chloride

Normal isotonic saline is the initial crystalloid of choice to be given over the initial 4-6 hours. This fluid is hypotonic relative to the osmolality of the patient’s plasma which can be calculated as 2(Na meq/dl + K meq/dl) plus glucose in mmol, or mg/dl divided by 18. Assuming a Na of 140meq, K 4.0meq and glucose of 450 mg/dl, the osmolality is 284+25 = 309 mosm. Normal (0.9%) saline has an osmolality of 286meq/l; the difference between the osmolality of the infused saline and patient’s plasma becomes greater as the glucose concentration in plasma rises. But it is important to note that the infusate remains hypotonic relative to plasma as long as the hyperglycemia persists; decline in plasma osmolality must be carefully monitored to avoid rapid osmotic shifts that facilitate entry of water to the intracellular /intracerebral compartment. After the first 4-6 hours, 0.5-0.75 N saline plus added potassium(K) as the phosphate, acetate or chloride maintains an osmolality of the infusate close to that of the patient’s plasma. Because of the concerns regarding use of chloride in worsening acidosis, some have recommended the use of lactated Ringers solution or sodium acetate in lieu of normal saline (7).

Potassium

During acidosis K moves from the intra cellular to the extracellular compartment and considerable K is then lost in urine. As a result, total body K stores are almost always depleted and with correction of acidosis, K returns to the intracellular compartment resulting in hypokalemia, which may precipitate cardiac arrhythmia. Hyperkalemia is less common and may reflect impaired renal function. Hence, after initial resuscitation, as soon as urine output is documented, K should be added to the infusate at a concentration of 20-40 meq/l. The potassium may be in the form of potassium chloride, but this adds to the hyperchloremia which may result in persistent hyperchloremic acidosis. Hence, some recommend that the K may be administered, at least in part, as the acetate or phosphate, which may have additional benefit as described below. Total amounts of potassium replacement should not exceed 0.5mm/kg/hour. Potassium replacement should continue throughout the period of IV therapy to assist in the repletion of potassium stores. This may not be fully accomplished during IV therapy and continues when oral intake is resumed. The measurement of K concentration is an essential component of biochemical monitoring as described below; additional rapid monitoring of K concentration in plasma is in the evaluation of the EKG which may show high peaked T waves with hyperkalemia and low amplitude of T waves, T wave inversion, prolonged PR interval and prominent U waves with hypokalemia. 

Phosphate

As with potassium, phosphate stores are depleted in ketoacidosis and further losses occur with ongoing diuresis during treatment and the effects of insulin in promoting intracellular entry. Severe hypophosphatemia (<1mg/dl) may be associated with depletion of ATP and the resultant deleterious effects on any energy requiring processes, including muscle function, CNS disturbances, hemolysis, and rhabdomyolysis. In addition, phosphate participates in the regulation of the oxygen dissociation curve, so depletion impairs oxygen release to tissues and further exacerbates acidosis by promoting lactate accumulation. On the other hand, infusing phosphate is associated with hypocalcemia and limited trials have not shown consistent beneficial effects in the treatment of DKA. An advantage however, is its cautious use in limiting hyperchloremia and hence acidosis by providing some of the potassium requirement as phosphate rather than chloride, alternating KCl with KPO4 and monitoring calcium concentration to avoid or treat hypocalcemia. We use this approach in our practice recommendations.

INSULIN THERAPY

Fluid therapy alone incompletely corrects many of the biochemical features of DKA, but full resolution of DKA requires insulin to switch off ketogenesis, restore acid-base balance, and resume anabolic processes. For moderate to severe acidosis, we recommend a starting dose of insulin(regular) at 0.1 U/Kg/hour, infused intravenously until acidosis is curtailed; insulin should be continued even if the blood glucose concentration has declined to ~300mg/dl or less and additional glucose provided as 5%-10% solution to maintain glucose at ~300mg/dl. Temporarily switching off the insulin infusion may result in rebound or persistence of acidosis, as insulin is essential to curtail keto-acid production and enable metabolism of keto-acids to bicarbonate. It is permissible to reduce the insulin dose to 0.05U/Kg/hr if there is difficulty in maintaining glucose at ~300mg/dl, even with additional glucose infusion, but insulin infusion should not stop until acidosis is resolved and pH is 7.3 or higher. In those admitted with mild acidosis, or those who administered basal insulin prior to admission, the starting dose of insulin should be 0.05U/Kg/hr, in order to avoid too rapid decline in the glucose concentration. Monitoring of blood glucose decline may require upward adjustment of the insulin dose if glucose is not declining at least 50 mg/dl/hour. An intravenous insulin bolus of insulin is not recommended to be given at the start of therapy and may not be effective as acidosis promotes dissociation of hormone binding to its cognate receptor. Where venous access is not possible, IM or SQ fast acting insulin (aspart or lispro) may be given at a starting dose of 0.2-0.3U/Kg and doses of 0.1-0.2 U/kg repeated 1-2 hours apart depending on response in terms of decline in glucose and correction of acidosis.

BICARBONATE THERAPY

In controlled trials in adults, bicarbonate therapy has not been effective in shortening the time of acidosis; bicarbonate actually may cause harm. Harm may occur because HCO3^- combines with the H⁺ to form H2CO3 which dissociates to H2O and CO2.Whereas HCO3^- does not cross the blood-brain barrier, CO2 diffuses readily across the blood-brain barrier and may exacerbate acidosis. In addition, large doses of bicarbonate may induce alkalosis and promote hypokalemia. Although controlled trials have not been performed in children, observational outcomes in pediatric studies show resolution of acidosis with provision of fluids and insulin; bicarbonate therapy is not recommended in published guideline (4, 7, 12). In severe acidosis, with pH <7.0, myocardial contractility may be impaired and here bicarbonate may be helpful. In these circumstances, bicarbonate may be infused at 1-2mmol/Kg over 60 minutes and acid -base status reassessed thereafter. Bicarbonate therapy may be useful in treatment of severe hyperkalemia. Bicarbonate must not be given as a bolus in treating DKA.

An example of the losses and management of DKA in a child with weight 30kg (Surface area 1M²) are shown in Tables 3 and 4.

*Weight 30 kg; surface area 1 M²; See tables 2 and 3, references (4, 7, 12) and text for source of losses of water and electrolytes       

*Weight 30 kg; surface area 1 M²; In this formulation, calculated fluid deficit has been corrected by about 12 hours and basal requirement over the ensuing 24 hours; total fluid over the 36 hours has not exceeded 2 times daily maintenance. Total sodium infused only modestly exceeds the calculated deficit, but total chloride excess is considerable and may be associated with persistent (hyperchloremic) acidosis. Potassium and phosphate repletion is incomplete and continues after transition to oral intake of nutrition and subcutaneous insulin therapy. This example is for illustrative purposes only; the actual amount and composition of infused fluids is dictated by the biochemical responses monitored and recorded during therapy. Detailed discussion of electrolyte replacement can be found in references (12) and (13).

MONITORING

A flow sheet to record clinical and biochemical progress is an essential component of therapy. Actual real-time monitoring of vital signs should be complemented by hourly recordings. Initial chemical laboratory tests must include blood glucose, serum electrolytes with emphasis on sodium, chloride, and potassium, as well as phosphate, calcium, pH, pCO2, HCO3, base excess, BUN and creatinine as indices of renal function and β-hydroxybutyrate(B-OHB) as a measure of ketosis. Measurement of urine output, urine glucose and ketones also must be recorded. The urine ketone measurement uses the sodium nitroprusside reaction which measures aceto-acetic acid and weakly acetone, but not B-OHB, the predominant ketone in blood. Hence, the major contributor to ketoacidosis is not reflected in the urinary ketone measurement. Bedside blood glucose, electrolyte, and acid base, and ketone meters are very useful but must be verified by periodic formal laboratory measurements. Initially, hourly measurement of glucose, electrolytes, and acid base status are recommended for the first 4 hours and 2-4 hourly thereafter depending on indices of improvement and resolution of acidosis, defined as pH≥7.3 or bicarbonate ≥15 mm/l. At this time transition to oral intake and discontinuation of IV therapy can be undertaken; absence of ketonuria should not be a criterion as this may continue for some time due to conversion of B-OHB to aceto-acetate as ketosis resolves. After the first day, once daily measurement of electrolytes, acid base and renal function should be performed until restoration of normal function is confirmed.

TRANSITION TO ORAL INTAKE

Oral intake may be begun when clinical recovery has occurred even if the acid base status and ketonuria have not completely resolved. Oral sips of clear liquids precede the introduction of oral fluids to gradually supplant the IV provision and total daily fluid restricted to no more than 1.5 times calculated daily maintenance. The first dose of regular or fast acting insulin is given subcutaneously approximately 1-2 hours before discontinuing the IV insulin to allow for absorption. For patients on a basal-bolus insulin regimen, the first dose of basal insulin may be administered in the evening while the IV insulin is maintained till the morning and then discontinued.

Mortality and Morbidity of DKA

Mortality of DKA has declined markedly in the past 2 decades largely due to greater referral of patients to specialized centers (5, 6, 14). Cerebral edema (CE) is responsible for the majority of deaths and survivors of CE may have severe or mild residual impairment of CNS function including memory impairment (15-17). Several other causes of mortality and morbidity occur, but each is individually rare and include venous and arterial CNS thromboses, pulmonary embolus, rhabdomyolysis, pancreatitis, ARDS, and infections such as rhino-cerebral mucormycosis and other rare entities. These rarer complications are more fully described in prior reviews (4, 7, 16).

CEREBRAL EDEMA

Cerebral edema is the most feared complication of DKA occurring either early (cerebral ischemia/reperfusion injury) or later during the course of therapy; mechanisms have not been clearly defined and whether the composition of IV fluids and their rate of administration contribute to or may prevent this complication is hotly debated (12-19). New onset, younger age and indices of severity have been associated with greater risk of this complication (20). Symptoms and signs include severe headache and development of bradycardia and hypertension as evidence of raised intracranial pressure, restlessness and irritability, localizing neurological features such as nystagmus and incontinence or polyuria without glucosuria as indicators of evolving diabetes insipidus, as well as evidence of papilledema.  Clinical diagnosis based on bedside evaluation of neurological state as shown below has been proposed (16). In this formulation, one diagnostic criterion, two major criteria, or one major and two minor criteria have a sensitivity of 92% and a false positive rate of only 4% (16).  Signs that occur before treatment should not be included in the diagnosis of cerebral edema. Diagnostic criteria include abnormal motor or verbal response to pain; decorticate or decerebrate posture; cranial nerve palsy (especially III, IV, and VI), and abnormal neurogenic respiratory pattern such as grunting, or Cheyne-Stokes respiration. Major criteria include altered mentation/fluctuating level of consciousness; sustained heart rate deceleration (decrease more than 20 beats per minute) not attributable to improved intravascular volume or sleep state; and age-inappropriate incontinence with a rise in serum sodium indicative of loss of free water(diabetes insipidus).Minor criteria include vomiting, headache; lethargy or not easily arousable; diastolic BP >90 mm Hg; young age( <5 years) (16). The mechanisms responsible for the development of cerebral edema in DKA appear to be both osmotic (12-14, 18, 20) and vasogenic (21, 22), and the timing of appearance as early or late in the course of treatment may depend in part on the major contribution of the mechanism involved. Treatment should begin with reduction in the rate of fluid administration, elevating the head of the bed, administration of mannitol, 0.5-1 g/kg IV over 10-15 minutes, and repeating the dose of mannitol if there is no initial response in 30 minutes to 2 hours. Hypertonic saline (3%), at a dose 2.5-5 mL/kg over 10-15 minutes, may be used as an alternative to mannitol, especially if there is no initial response to mannitol. After these measures have begun, imaging of the CNS should be arranged to identify intracranial pathology such as thrombosis and treat as appropriate.

Caveats

1. Ketone bodies measured in urine grossly underestimate the degree of ketosis, because the common method uses sodium nitroprusside which reacts strongly with aceto-acetate, weakly with acetone, and not at all with βOHB. Yet the actual amount of βOHB may be 5 times or more than aceto-acetate, especially in the presence of acidosis. As acidosis is corrected and more of the βOHB is converted to aceto-acetate, it appears as if the ketosis is getting worse, when in fact acidosis and clinical parameters are improving. Measurements of βOHB via bedside meters or formal laboratory methods are better means to monitor “ketone” status.
2. After commencing treatment, acidosis may appear to worsen initially for 3 reasons (4). First, dilution of the total bicarbonate in the expanding fluid volume lowers the apparent bicarbonate concentration because the HCO₃ is expressed as mmol/L, and while the total mmols may not have changed, they are distributed in a greater volume. Second, with initially rapid rehydration, accumulated lactic acid enters into the circulation. Third, the βOHB acid is excreted in urine after it is converted to Na Butyrate; the Na derives from NaHCO3, leaving bicarbonate which combines with H⁺ to yield CO2 and H2O and permits loss of the CO2 in respiration. In these processes, bicarbonate (HCO3) and Na are lost, further depleting the bicarbonate content of plasma. With rehydration and insulin, which together curtail ketogenesis, acid base balance gradually returns to normal (4).
3. The use of phosphate as potassium phosphate or acetate rather than KCl, may reduce the large amount of chloride used and hence reduce hyperchloremic acidosis, as well as improving oxygen dissociation to enable lactate to be converted to pyruvate. However, this is not accepted by all authorities and some claim no additional benefit from using phosphate. In addition, the use of phosphate may result in hypocalcemia. However, with severe phosphate depletion, the use of phosphate is indicated and likely to be beneficial.

Recurring Episodes of DKA

A small subset of patients experience repeat episodes of DKA, and with each episode the prognosis for short-term and long-term outcome worsens. Recent data confirm that the majority of such recurrences reflect psycho-social maladjustments that require careful attention via medical and social support to avoid disastrous consequences (5, 6).

HYPERGLYCEMIA HYPEROSMOLAR SYNDROME (HHS)

The hyperglycemia-hyperosmolar syndrome (HHS) is characterized by blood glucose concentrations >600mg/dl (>33.3 mmol/l), serum hyper-osmolality ≥330mmol/l, and minor acidosis and ketosis; serum bicarbonate remains >15meq/l and urinary “ketones” (aceto-acetate) are usually negative or only trace positive on testing urine via dipstick (7, 21, 22). Hospital admissions for HHS are increasing in incidence, have high morbidity, and though classically considered to occur in obese patients with T2DM it may occur in T1DM as frequently as in T2DM (23). Although there are similarities to diabetic keto-acidosis, the fundamental difference is a greater degree of dehydration and less acidosis, so that treatment should focus on fluid and electrolyte replacement, and less on provision of insulin; indeed, insulin should be withheld initially to prevent a too rapid fall in serum glucose and lowering of serum osmolality which might result in fluid shifts into the cerebral compartment and cerebral edema (CE). However, CE is rarer in HHS than in DKA. The degree of insulin deficiency and the magnitude of counter-regulatory response appear to be less severe, so that the symptoms and signs of DKA are absent or less pronounced; abdominal pain and Kussmaul respiration are absent, and vomiting is less severe. These milder features also lead to greater time in evolution, greater degrees of dehydration and electrolyte losses resulting from the polyuria, and are often compounded by intake of highly glucose-enriched carbonated soft drinks consumed due to thirst. Glucose concentrations commonly exceed 1000mg/dl, dehydration may be as much as twice that occurring in DKA and may be difficult to estimate due to co-existing obesity and hypertonicity which retains fluid in the intra-vascular compartment. Persistence of the polyuria due to the persistence of glucose concentrations exceeding renal threshold of ~200mg/dl during treatment, requires careful monitoring of clinical status and fluid replacement to avoid dehydration and vascular collapse. The risk of thrombosis is greater in HHS than in DKA, possibly as a result of osmotic disruption of endothelial cells, with release of thromboplastins facilitating coagulation.

Treatment should assume dehydration of 10%-15% and initially isotonic (normal) saline should be provided at 20ml/kg bolus infusions to restore fluid deficits and maintain vascular volume with assessment of serum chemistries every 1-2 hours; subsequently, 0.5-0.75 N saline, with added potassium and phosphate should be infused to replace calculated losses over 24-48 hours, guided by laboratory chemistry every 2-4 hours and ongoing clinical monitoring performed in an ICU or equivalent setting. The aim should be to control the decline in blood glucose to 100 mg/dl per hour; if glucose is not declining at a rate of at least 50 mg/dl, or ketosis is more than mild, insulin at a rate of only 0.025-0.05U/kg/hour may be used with caution and careful clinical and laboratory monitoring. Potassium, phosphate and magnesium losses may be considerable; potassium should be infused at 40meq/l added to each liter of saline, with balanced mixtures of potassium chloride and potassium phosphate, the latter to replete phosphate depletion which may predispose to rhabdomyolysis and hemolytic anemia. As in DKA, use of bicarbonate is not recommended. Magnesium also may be severely depleted in HHS and predispose to hypocalcemia; the recommended doses of magnesium replacement are 25-50mg/kg/dose given every 4-6 hours at a maximum infusion rate of 150mg/min(2gm/hr.) for 3-4 doses. In addition to cerebral edema, thrombosis, and rhabdomyolysis, malignant hyperthermia is reported as a complication. Monitoring for these complications is based in part on clinical anticipation e.g., hyperthermia, and supplemented by appropriate biochemical testing e.g., serum creatinine kinase for rhabdomyolysis. Some patients have features that combine DKA and HHS that reflect the degree of insulin deficiency; clinical acumen, earlier use of insulin, and careful monitoring of the patient’s vital signs and chemistries guide treatment, especially the earlier use of insulin in appropriate doses. This syndrome of HHS in adolescents and young adults was classically considered a feature of T2DM (24), an entity that is increasing at an annual rate of 4.8% in the obese population of the USA (25). Hence, the frequency of HHS as a presenting feature is also likely to increase, so that physicians caring for these patients in an ICU or equivalent setting must be alert to the differences in management with the greater focus on fluid and electrolyte replacement in HHS rather than the use of insulin as in DKA.  

ROUTINE MANAGEMENT OF DIABETES

The goals of treating diabetes mellitus in children are to maintain metabolism as near to normal by the appropriate provision of insulin, maintaining nutrition by meeting caloric requirements and balanced composition of food choices within the cultural preferences of the family, and to balance both insulin and nutrition with recommended exercise and activity to allow normal growth and development. In order to prevent diabetes related complications, especially long-term microvascular disease, glycemic control is crucial.  This optimal diabetes regimen requires intensive management by patients and their families along with a multidisciplinary approach with psychosocial support.  Glycemic control is assessed by periodic measurement of hemoglobin A1C levels.

TYPE 1 DIABETES

Insulin Therapy

The management of diabetes can be cumbersome.  In caring for children and adolescents with Type 1 diabetes, providers must take into account unique factors such as a child’s pubertal stage and growth, ability to provide self-care, supervision of care, school environment, and neurological vulnerability of hypoglycemia in young children. 

However, it is crucial to normalize glucose levels in order to prevent long-term consequences of diabetes especially from microvasculopathies, leading to neuropathy, renal failure, and blindness. In 1993, The Diabetes Control and Complications Trial (DCCT) reported results demonstrating that the intensive therapy of T1DM reduces the risk of development and progression of microvascular complications. Furthermore, these benefits outweighed the increased risk of hypoglycemia that accompanied intensive diabetes therapy (27). Thereafter, The Epidemiology of Diabetes Interventions and Complications (EDIC) study assessed whether these benefits persisted after the end of DCCT. The findings of this study provide further support for the DCCT recommendation that most adolescents with T1D receive intensive therapy aimed at achieving glycemic control as close to normal as possible to reduce the risk of microvascular complications (28). This goal is not easily achieved even with a multi-dose insulin regimen of basal and short acting insulin that attempt to mimic normal patterns (Figure 2).

Figure 2. Normal Glucose and Insulin Profiles

This pattern is difficult to achieve because the perfect alignment of glucose and insulin in the normal person depends on a complex interaction of neural, hormonal, and nutritional signals that are absent in type 1 DM. Moreover, the “first pass” of endogenous insulin is via the portal vein to the liver, whereas SQ insulin injections first reach the liver via the systemic circulation. Hence the common problem of post-prandial hyperglycemia due to delay in the action of insulin during and immediately after a meal, and rebound hypoglycemia sometime after the meal. The following describes the insulin regimens recommended as standard of care attempting to reproduce the normal situation.

Figure 3. Time Course of Rapid and Long-Acting Insulin

Insulin Glargine and Detemir provide day-long basal insulin without significant peaks of action. In some children, however, Glargine may not be fully effective for a whole 24 hours and for this reason is usually given at night. This synthetic insulin cannot be safety mixed with other insulins in the same syringe due to pH incompatibilities. Throughout the day, short acting insulin preparations such as Insulin Aspart, Lispro, and Glulisine are given to normalize blood glucose levels and cover calories consumed during meals and as possible snacks. As 3 meals are eaten by most on a daily basis, short acting insulins should be given at least 3 times daily to prevent excessive hyperglycemic excursions.

Prepubertal children typically require a total daily insulin dose of ~ 0.7 -1 U/kg/day whereas pubertal children may require total daily insulin doses up to 1.2-1.5 U/kg/day; greater than 2.0 U/kg/day suggest extreme insulin resistance or non-compliance (29).  Of the total daily insulin dose, 40-60% should be given as basal insulin. The actual dose depends upon the level of glycemia and the quantity of calories and carbohydrates consumed. A fast-acting insulin bolus is given to cover meals, calculated from an estimation of the carbohydrate content in grams and an individual factor (insulin to carbohydrate ratio) relating insulin dosage to the amount of carbohydrate to be consumed. When using carbohydrate counting, the ‘500-rule’ can be used to obtain an initial carbohydrate ratio by dividing 500 by the total daily insulin dose.(29) The range is from 1 unit per 10-50 grams.  In addition to the meal bolus, the difference between the blood glucose recorded immediately before the meal and the target glucose concentration (120 mg/dl for older children; 150 mg/dl for younger children) is used to calculate a correction bolus, based on a theoretical Insulin Sensitivity Factor or ISF. This may range from 1 unit for 10- 200 mg/dl blood glucose depending upon age and body size. The ‘1800-rule’ can be used to obtain an initial ISF by dividing 1800 by the total daily insulin dose.

The intermittent, short acting insulin given as a bolus should be injected 15 minutes before the meal in order to have its full effect when glucose rises during and after the meal. For infants and toddlers, these short acting insulin doses may be given after the meal if food consumption has been found to be unpredictable. In addition to the 3 meals, additional amounts of short acting insulin may be taken to cover snacks, and to reduce blood glucose concentration as appropriate at bedtime. An insulin "pen" is a convenient way of carrying multiple doses in a single dispenser but this pen device does not reduce the burden of multiple insulin injections.  Super long-acting insulin preparations e.g. Degludec or super-fast acting insulin e.g. FIAsp(Ultra-Fast acting insulin Aspart) are available and may have advantages in specific situations. In particular, a super-fast acting insulin would allow more rapid equilibration between the systemic and portal circulations, so offering advantages to prevent excessive rise in glucose after a meal and avoiding later development of post-prandial hypoglycemia (31, 32).  The half-life of insulin degludec exceeds the dosing interval resulting in a very low peak:trough ratio at steady state.

An alternative and better method of insulin delivery is the use of continuous subcutaneous insulin infusion (CSII) through use of an insulin pump. In this case, only short acting insulin e.g., lispro, glulisine), aspart, or FiAsp is taken as a continuous basal infusion and multiple boluses of insulin are given as above. The newer generation of CSII pumps automatically calculate meal and/or correction boluses based on input insulin-to carbohydrate ratios and insulin sensitivity factors plus estimates of the amount of carbohydrate consumed. The infusion site is best changed every 2 days to avoid skin infections. The advantages of CSII when used correctly are that insulin is delivered only as needed, and not in an anticipatory fashion as with long-acting insulin. Insulin boluses are also delivered only as needed, nutritional intake may be more liberal, glycemic excursions both as hyperglycemic and hypoglycemic episodes can be reduced, and the system is convenient and portable. Patients receiving insulin via CSII and their parents have generally reported improved treatment satisfaction. The one disadvantage of CSII is that since only short acting insulin is used (effects of rapid acting insulins are dissipated within 3 hours), any blockage in the tubing or pump failure can lead to rapid onset of hyperglycemia, accumulation of serum ketones, and an uncontrolled diabetic state. Thus, patients and their care-givers must be educated on treatment of hyperglycemia with an insulin pen or syringe in case of suspected pump malfunction which can be a common cause of DKA. 

With advanced technology, insulin therapy is becoming more physiologic. Continuous subcutaneous insulin infusion (CSII) therapy is transforming care of T1DM while continuous glucose monitoring (CGM) of interstitial fluid has become widely available and increasingly used in the US. Without continuous glucose monitoring, manual adjustment of insulin doses in response to changes in blood glucose are based only upon intermittent blood glucose testing and corrections.  Moreover, as previously mentioned, insulin injections or infusions are given subcutaneously and initially enter the systemic circulation, whereas endogenous insulin is secreted into the portal vein and act directly and immediately on the liver.

Currently, we are entering a new era of diabetes care for children, with the adoption of closed loop systems (33). All systems in development rely on glucose measurements via CGM transmitted to an insulin delivery system (pump) which uses a computerized algorithm to adjust insulin infusion based on upper and lower limits and the rate of change in increase or decrease of glucose values (34-36).  Modified versions of the closed-loop system that are semi-automated are FDA approved for use in children.  Semi-automated closed-loop systems, also known as hybrid closed loop systems, are characterized by the combination of automated insulin delivery via an algorithm for basal requirement and user-initiated insulin delivery for meals.  A setting to suspend insulin delivery when glucose is low or rapidly downward trending can be utilized to avoid hypoglycemia. To improve ease of mobility, a tubeless insulin pump that can be operated by a third party through a wireless receiver is available and useful for very young children with control exercised by a parent.    

Bi-hormonal systems (insulin and glucagon) can rapidly alter high or low glucose values and be used with safety during normal activity and exercise (37, 38).This system has not yet been approved for commercial use. However, the next decade should see application of these tools to an increasing number of patients with T1DM.

The clinical management for patients with T1DM is based on adequate insulin replacement matched to food intake and modified by exercise. Insulin is required throughout the whole day to prevent development of a starvation state and ketosis i.e., the basal insulin requirement.

Recurrent hypoglycemia represents a mismatch between insulin provided and caloric expenditure, which occurs as a result of not covering the basal amount or a bolus with appropriate food intake, malabsorption of food e.g., celiac disease, or exercise without adjustment in the insulin dose or omission of additional calories before or after the exercise. unexplained episodes of hypoglycemia require re-evaluation of the insulin regimen, exclusion of concurrent conditions such as acute illness with diminished food intake, and testing for celiac disease, hypothyroidism, and Addison’s disease. 

In special circumstances where adherence to recommended regimens is not being followed due to various psycho-social limitations, a twice daily regimen of pre-mixed insulin (NPH/Reg70/30) may be prescribed, though it is known not to be ideal. In developing countries with limited resources to treat diabetes, this twice daily dosing regimen of NPH and regular continues to be vital to avoid DKA but is associated with less optimal glycemic control. 

Glucose Monitoring

Patients on insulin regimens are encouraged to check glucose levels prior to meals and snacks, at bedtime, prior to exercise, and when they suspect low blood glucose.  This typically amounts to 4-6 glucose levels per day.  Blood glucose monitoring allows patients and families to evaluate their individual response to therapy, assess whether achieving glycemic targets, and help guide treatment decisions.

Continuous glucose monitors and intermittently scanned monitors measures interstitial glucose which correlates well with plasma glucose.  Time in range is defined as percentage of the day with blood glucose levels between 70 and 180 mg/dL.  A time in range target of more than 70% is recommended (39).  Time in range is becoming a key glycemic metric, in addition to glycosylated hemoglobin (HbA1c) (39).  A correlation between improved HbA1c levels and fewer diabetes complications has been demonstrated (40).  Percent time in range provides real-time insights into glycemic variability and frequency of hypoglycemia and hyperglycemia. Time in range also correlates with complications of diabetes and HbA1c values and is being used to compare diabetes technology (40).

Nutrition

The goal of nutrition is to support normal growth and development, improve diabetes outcomes, and reduce cardiovascular risk factors.  Dietary recommendations should be based on healthy eating principles appropriate for all children and their families. Typically, distribution of energy sources recommended is 50-55% carbohydrates, <35% fat, and 15-20% protein (41).  Regular meals are recommended.  A commonly prescribed meal plan consists of 20% of calories at breakfast, 30% at lunch and 30% at dinner with 2 snacks of 10% each one of which is at bedtime to avoid nocturnal hypoglycemia. In the basal-bolus insulin regimen, insulin doses are matched to the amount of carbohydrates consumed during each meal. 

Protein and fat are not typically accounted for in the meal time insulin dose calculation though this issue is controversial and some authorities recommend that these protein-fat derived calories must be included. The predominant effect of dietary fats and protein is late postprandial hyperglycemia. Bolus corrections for insulin pump use when eating fatty meals have been devised and recommended (42-44).  Studies have found that lower glycemic index diets improved glycemic control compared to traditional higher glycemic index diets (45, 46).  Low glycemic index foods include whole-grain breads, pasta, temperate fruits, and dairy products

Dietary advice should be given in the context of cultural, ethnic and family traditions to be successful. Continuous nutritional education regarding a healthy diet and carbohydrate counting is recommended. Food labeling requirements have simplified the process, as many foods are clearly labeled with the amount of carbohydrate grams per serving. Additionally, apps, such as Calorie King, provide information on carbohydrate content of foods provided by many large restaurants.

Exercise

Establishing and maintaining an active lifestyle should be the goal for all children. Increased physical activity is associated with better glucose utilization and increased insulin sensitivity leading to lower insulin requirements. However, blood glucose levels can be difficult to regulate during these intervals of exercise. Hypoglycemia is common during exercise and can possibly last up to 24 hours afterwards, due to increased insulin sensitivity (47). This increases the risk of nocturnal hypoglycemia. Factors during exercise frequently associated with hypoglycemia are excessive insulin dosing prior to exercise, prolonged duration, and higher intensity aerobic exercise (47).

To reduce the risk of hypoglycemia during prolonged exercise, reductions in bolus and basal insulin are typically needed. In children using CSII pumps, simply suspending or reducing the basal infusion rate can markedly reduce the risk of hypoglycemia during exercise. If insulin doses prior to exercise are not reduced, a snack of 1-1.5 grams of carbohydrates per kilogram is recommended (47). Meals with high carbohydrate content should be consumed shortly after exercise. As the effects of exercise can be prolonged, blood glucose should be measured before bed and a decrease in basal insulin (either long acting or overnight basal) should be considered after exercise later in the day. 

Any exercise should be avoided if blood glucose prior to exercise are high (>250 mg/dl) and associated with ketonuria. Exercise during such insulinopenic states is dangerous owing to the effects of uninhibited counterregulatory hormones and may precipitate diabetic ketoacidosis. 

Sick Day Management

Children with intercurrent illnesses such as fever or vomiting, should be closely monitored for the development of hyperglycemia and ketonuria. On sick days, blood glucose levels should be checked every 2-3 hours when not tolerating food and urine should be checked for the presence of ketones with every void. Correction doses with rapid-acting insulin should be given approximately every 3 hours. Persistent vomiting and/or ketonuria are signs of diabetic ketoacidosis; patients with such signs and symptoms should be evaluated in an emergency department immediately. 

Adequate fluid intake is crucial to preventing dehydration and accumulation of ketones. For blood glucose >200 mg/dl, rehydration with sugar free fluids is recommended. Sugar containing fluids such as flat soda or diluted juice may be necessary to maintain normoglycemia if blood glucose is <140 mg/dl. 

Management of Co-Morbid Conditions

Besides insulin replacement therapy for T1DM, co-existing hypertension and dyslipidemia should be aggressively treated; it is important to use age-appropriate references for determining the presence of hypertension and upper levels of acceptable lipid values of LDL and triglycerides. 

Increased urinary protein excretion is the earliest clinical finding of diabetic nephropathy. Measurement of the urine albumin-to-creatinine ratio in an untimed urinary sample is the preferred screening strategy for moderately increased albuminuria in all patients with diabetes and should be repeated yearly. Screening for increased urinary albumin excretion can be deferred for five years after the onset of disease in patients with type 1 diabetes because increased albumin excretion is uncommon before this time; screening should begin at diagnosis in patients with type 2 diabetes because many have had diabetes for several years before diagnosis. Abnormal results should be confirmed by repeat testing before establishing a diagnosis because of the large number of false positives that can occur. The normal ratio of microalbumin to creatinine is less than 30 mg/g.  Thus, a persistently elevated ratio of 30-300 mg/g signifies microalbuminuria. Microalbuminuria and/or hypertension should be a call for use of angiotensin converting enzyme (ACE) inhibitors to minimize progression to chronic glomerulosclerotic damage. ACE inhibitors may induce angio-edema and can produce a troublesome dry cough.

Poorly controlled diabetes induces an increase in VLDL and triglyceride levels, when acute or chronic, pancreatitis may be induced. Diet reduced in animal fat and administration of fibrates (e.g., gemfibrozil or fenofibrate) may be used to combat hypertriglyceridemia. Co- existing Hashimoto's thyroiditis should be periodically sought through thyroid autoantibody analyses, and hypothyroidism when identified by elevated TSH levels, treated by thyroid hormone replacement. Celiac disease also should be regularly checked via titers of tissue transglutaminase antibody titers, and treated via gluten exclusion when diagnosis is confirmed by endoscopically obtained biopsy specimens. Addison's disease and atrophic gastritis/pernicious anemia should always be considered in patients with T1DM, especially with unexplained frequent episode of hypoglycemia, and if found, treated accordingly.

TYPE 2 DIABETES

The increased incidence of T2DM is attributed to the increase in obesity worldwide. Approximately 3700 youths are diagnosed with T2DM every year in the US (25) and it is estimated that the number of youths with T2DM will almost quadruple from 22,820 in 2010 to be approximately 85,000 adolescents with T2DM by 2050 (48). Similar rates of increases in youths with T2DM are reported from the UK, India, China and Japan (48). The child with T2DM as part of the insulin resistance syndrome (IRS) should be aggressively treated to prevent the burgeoning complications of the condition. The development of complications associated with T2DM is accelerated in youth, with reported rates of 6% with renal failure within 5 years of diagnosis, and 2.3% end stage renal disease by 10 years.  

Initial education for T2DM should focus on dietary and lifestyle modifications and this education should continue to be reinforced with the goal being to decrease insulin resistance. The approaches should include an exercise program such as walking or swimming for 30-40 minutes most days of the week, since at the level of the muscle, exercise provokes glucose entry into muscle without the involvement of insulin. Sedentary time including homework, computer and phone related activities, and video games should be assessed and established for appropriateness in each family setting. Caloric restriction, particularly of carbohydrates, is the key to reducing weight, a task that has proven resistant to success in many instances.  Elimination of sugar containing sodas and juices has been shown to result in significant weight loss (49). Barriers include older age at diagnosis, difference in socioeconomic status, and poor diet within the household (50). Also, clinicians should understand the health beliefs and behaviors of the family and community and take into account cultural food preferences and the use of food during celebrations and cultural festivals in order to collaborate with the family on diabetes management.  

The use of metformin as first-line therapy is based on its glucose-lowering efficacy, safety profile, weight neutrality, and reasonable cost. In most countries, metformin is currently approved for use in children. Metformin is approved for the treatment of T2DM in children, but is also the drug of choice for insulin resistance syndrome, also known as metabolic syndrome (IRS) and impaired glucose tolerance because of its property in improving insulin sensitivity. Monotherapy with metformin was associated with durable glycemic control in approximately half of children and adolescents with T2DM (51). Some suggest that it is the gastro- intestinal side effects of the drug that accounts for much of its beneficial effects. However, the drug is effective in T2DM even without weight loss, an action attributed to reduced hepatic glucose output.

The guidelines from the ADA and EASD indicate that any FDA-approved second agent can be used in combination with metformin to improve glycemic control, whereas the American Association of Clinical Endocrinologists recommends either incretin-based therapy or sodium glucose transporter 2 (SGLT2) inhibition agents (52). Sulfonylureas are approved for use in adolescents in some countries; these agents bind to receptors on the K+/ATP channel complex resulting in insulin secretion. The PPAR-γ agonists are effective at insulin sensitization but are less useful in supporting weight loss. Further, they promote salt retention and a tendency for edema. A new class of drugs which inhibit the sodium co-transporter 2 (SGLT2) resulting in glycosuria at a lower blood glucose threshold than normal have become available, though not currently approved for use in children (canagliflozin, dapagliflozin, empagliflozin). Use of SGLT2 inhibitors has been associated with an increase in fungal infections of the genital areas and missed symptoms of evolving keto-acidosis.

Two drug classes were developed that target the incretin system and increase endogenous insulin secretion: glucagon-like peptide (GLP)-1 receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors. GLP-1 receptor agonists (e.g., liraglutide and exenatide) resist degradation by DPP-4 resulting in increased circulating levels of the administered drug (53). DDP-4 inhibitors (e.g., sitagliptin, vildagliptin and saxagliptin) reduce endogenous GLP-1 degradation, thereby maintaining circulating levels of GLP-1 with biological effect. Both these classes of drugs improve glycemic control with a low incidence of hypoglycemia because of their glucose-dependent mechanism of action. In addition to their effects on improving insulin secretion, these drugs lower glucagon and delay gastric emptying, and potentiate weight loss, in part through decreased appetite.

Whereas the glucagon like peptide one (GLP-1) analogue exenatide given by subcutaneous injection will lower blood glucose levels and complement metformin in provoking weight loss, it should be reserved for more severely diabetic adults and teenagers who have become unresponsive to diet and exercise programs.  Some formulations of GLP-1 analogues can be given once weekly.  In 2019, liraglutide was approved for adjunct use to improve glycemic control in pediatric patients 10 years and older with T2DM. (54, 55) Sitagliptin blocks the dipeptidyl peptidase-4 (DPP-4) enzyme preventing it from inactivating GLP-1, thus prolonging the action of GLP-1 once induced by a meal. Whereas the latter agent is in general weight neutral, it can be of adjunctive help in lowering hyperglycemic excursions. When these additional agents also fail to maintain near normoglycemia, then insulin should be given instead of the secretagogues.

*Saxagliptin is a DPP4 inhibitor associated with heart failure. Other DPP4 inhibitors have not been shown to cause heart failure. Whereas studies of these agents are under investigation, only a GLP-1 agonist is currently approved by the FDA for use in children and adolescents

One of the main goals of therapy in IRS/T2DM should be to achieve an ideal body mass index (kg/m2) for age and gender. This is not readily achievable with lifestyle modification and medical therapy in many subjects; bariatric surgery is emerging as a successful and durable treatment in adults and adolescents with IRS, obesity, and their associated complications (56-58). In adults the ADA recommends bariatric surgery in those with BMI of 30 kg/m2 or greater and poorly controlled DM (59). Bariatric surgery is an effective treatment for severe obesity that results in the improvement or remission of many obesity-related comorbid conditions, as well as sustained weight loss and improvement in quality of life. Mortality owing to cardiovascular diseases, diabetes, and respiratory conditions is reduced after bariatric surgery (60). A prospective follow up studies of bariatric surgery in adolescents with severe obesity showed a substantial and durable weight reduction and cardio-metabolic benefits (57, 58). Changes in glucoregulatory hormones produced by the gastrointestinal tract, bile acid metabolism, and GI tract nutrient sensing and glucose utilization are proposed mechanism for improvement in glycemic control after bariatric surgery (61). Currently, bariatric surgery is considered only in children with BMI ≥ 40 kg/m² with comorbidities or BMI ≥ 50 kg/m²regardless of comorbidities (62, 63). Indications in adults are much less stringent; adults with BMI ≥ 35 kg/m² with comorbidities are candidates for these procedures. Updated recommendations for adolescents provide more aggressive recommendations similar to those for adults (64). Bariatric surgery is now safe, with mortality comparable to common elective general surgical operations. Level 1 evidence show that bariatric surgery provides superior short-term and long-term weight loss and improvement of T2DM compared with conventional medical therapy. However, patients require life-long follow up and monitoring for nutritional deficiencies and abdominal issues, and to date, results in adolescents are relatively short term.  Pediatric patients who are being considered for bariatric surgery should be evaluated by a multidisciplinary team dedicated to providing long-term follow- up care postoperatively. In addition, selection criteria often exclude the population most in need of this proven procedure.

Treatment of Associated Comorbidities

The typical dyslipidemia associated with IRS and T2DM should be treated by reduced intake of animal fat and a fibrate such as gemfibrozil or fenofibrate. Where there is an increased level of triglycerides, restriction of animal fats and simple sugars should be recommended. However, those patients who have prominent elevations in LDL-cholesterol should be treated with a statin. The mixed use of a statin and a fibrate should be undertaken cautiously since the risks of muscle necrosis (rhabdomyolysis) with renal failure has been. In patients taking a statin gemfibrozil should not be used and fenofibrate is the fibrate of choice as the risk of myositis is less. Hypertension and microalbuminuria, when present, should be aggressively treated, preferably with angiotensin converting enzyme inhibitors (ACE) and angiotensin receptor blockers (ARBs), at least initially. 

Oral contraceptive agents are often prescribed in IRS when there is evidence of hyper-androgenization, where they may counteract the effects of androgens. However, oral contraceptives also increase the level of hormonal binding globulins, including sex hormone binding globulin that binds testosterone, thereby lowering the level of free and bio-available testosterone. Estrogen containing therapies in a prepubertal patient increases the risk of premature closure of the epiphyses and hence risks loss of adult height; they also promote thrombosis and mitigate against weight loss.

TREATMENT OF MONOGENIC FORMS OF DIABETES

Monogenic forms of diabetes constitute a heterogeneous group of disorders classified according to clinical features that suggest possible type 1 diabetes, type 2 diabetes, and neonatal diabetes, all in the absence of markers of autoimmunity such as circulating antibodies to various islet antigens. The genes responsible for these forms share a role in the formation or function of the pancreatic β-cell, limiting normal insulin secretion that depending on severity, and under certain conditions, results in clinical diabetes. Increasingly, it is being recognized that there is a continuum in the spectrum of these disorders such that the severity of the genetic defect responsible for insulin secretion or action determines the clinical pattern (65-68). This is perhaps best exemplified in the genetic defects of the ATP-regulated potassium channel (KATP) involving the ABCC8 gene coding for the sufonylurea receptor SUR1, and KCNJ11 coding for the subunits of Kir, the inward rectifying potassium channel itself. Severe activating mutations in these genes maintain the KATP in an open state and result in permanent neonatal diabetes, sometimes associated with developmental delay and epilepsy (DEND). Progressively less severe functional mutations may result in transient neonatal diabetes, or in a form of maturity onset diabetes of youth (MODY), or in T2DM.These activating mutations typically respond to sulfonylurea therapy, high dose for the severe mutations and lower doses for the less severe mutations, inducing endogenous insulin secretion mediated in part by GLP-1, and improved metabolic control superior to that obtained by exogenous insulin injection. Similar considerations apply to transcription factors such as hepatocyte nuclear factor1α (HNF1A) and hepatocyte nuclear factor 4α (HNF4A), respectively responsible for MODY3 and MODY1, which may respond to oral sulfonylurea drugs, avoiding the need for injected insulin, at least initially. Heterozygous inactivating mutations in the glucokinase gene responsible for MODY2 delay insulin secretion and result in a mild diabetes that is not associated with an increased risk of macrovascular or microvascular complications, so that treatment with exogenous insulin or other drug therapies is not indicated (69). Hence, knowledge of the genetic mutation drives therapy, permits more precise genetic counselling, and may indicate prognosis. In an era of precision medicine and progressive decline in the cost of sequencing, genetic testing should be considered in those with a strong family history of diabetes, early onset diabetes, and in children or adolescents who present with features suggestive of T1DM, are negative for islet auto-antibodies, and have residual c-peptide secretion as determined by measurement or reflected in persistently low insulin requirements extending beyond 1 year after diagnosis. These concepts are discussed in greater detail in several recent publications (65-69).      

Maturity Onset Diabetes of Youth (MODY)

The term MODY refers to Maturity Onset Diabetes of Youth, a term coined by Fajans and Tattersall for a mild type of diabetes with autosomal dominant inheritance and varying degrees of impaired insulin secretion (70). The molecular basis for these entities was discovered initially to be due to transcription factors or the enzyme glucokinase responsible for phosphorylating glucose to enable its metabolism to yield ATP; numbering followed the timing of discovery of the genetic-molecular basis (70). There are now 14 entities considered to be MODY as listed in the table in our chapter on the “Etiology and Pathogenesis of Diabetes Mellitus in Children and Adolescents” in Endotext. Of the original 6 “classical” MODY entities, MODY3 (HNF1A mutation), MOY2 (GCK (glucokinase) mutation) and MODY1 (HNF4A mutation) constitute about 85% of all MODY cases (65-68). With rare exceptions, these patients present as milder types of diabetes before age 30-35 years, with a positive family history involving at least 2-3 generations, and negative for islet cell antibodies; a daily dose of insulin less than 0.5U/Kg /day after 1 year of diagnosis should raise suspicion for MODY. MODY affects both sexes and is found in all races, with a prevalence of ~2%-4% of patients diagnosed with diabetes ≤ 30 years (71, 72). The majority are misdiagnosed as type1 or type 2 diabetes and incorrectly and unnecessarily treated with insulin or ineffective drugs such as metformin. MODY 2 affects about 1:1000 people and in females may be noted for the first time during oral glucose tolerance testing in pregnancy, again resulting in inappropriate classification and treatment (73). Biomarkers such as the urinary C-peptide to creatinine ratio (≥0.2nmol/mmol), and negative islet cell antibodies (GAD and IA2) should lead to molecular genetic testing (72). Using this approach, the minimum prevalence of monogenic diabetes was found to be 3.6% of patients diagnosed ≤30years of age with diabetes (72). In a study screening for MODY in all antibody negative children with diabetes in a national population-based registry in Norway, the prevalence of MODY was found to be 6.5%, and in a study from Japan, 11/89 children with insulin requiring diabetes but negative for islet cell antibodies were found to have monogenic forms of diabetes involving mutations in INS, the insulin gene, and in HNF1A or HNF4A (74, 75). Mutations in HNF4A may be associated with large size at birth and neonatal hypoglycemia with hyperinsulinemia that resolves spontaneously, only later becoming manifest as diabetes. Family history is helpful but not essential; de novo mutations occur.

In summary, there should be a high index of suspicion for MODY in milder forms of diabetes and in those children who are islet cell negative; using biomarkers followed by molecular diagnostics, the yield becomes quite high for discovering a form of MODY. As the cost of molecular diagnostics declines, and newer algorithms to apply these tools to differentiate apparent type1 from monogenic forms of diabetes are being developed (76, 77), it is becoming apparent that some of these mutations also contribute to the genetics of apparent type 2 diabetes (78, 79). For MODY3 and MODY1, oral sufonylurea medication (Glipizide) is likely to be effective inducing endogenous insulin secretion; MODY2 does not require treatment. Genetic counselling should inform patients of the 50% likelihood of each of their offspring having MODY, so that inappropriate diagnosis and treatment is avoided. In addition, the prognosis for vascular complications is improved especially in MODY2, though not absolute in MODY3. MODY12 (ABCC8) and MODY13 (KCNJ11) are also responsive to oral sufonylurea medication, but may require careful upward titration. For the remaining forms of MODY, insulin is likely to be necessary to control diabetes. In particular, these less frequent forms of MODY may have involvement of other organ systems, e.g., kidney cysts and dysfunction in MODY5(HNF1B), gastrointestinal involvement in MODY8 (CEL), exocrine pancreatic disturbances in MODY4 (PDX-1), blood abnormalities in MODY11(BLK), as well as other abnormalities (see references (65-68) for details). Thus, establishing a diagnosis for a form of MODY has several important consequences. First, it guides treatment, obviating the need for insulin with its costs and discomforts in several forms of MODY, as well as anticipation for possible associated abnormalities. Second, it permits a more accurate prediction of the course and prognosis for complications, e.g., MODY2, which in turn has ramifications on the cost and ability to obtain life insurance policy, or the choice of occupations which may be restricted to a person with T1DM. Third, it permits precise genetic counselling for risk of occurrence in offspring, and targeted molecular screening for the existence of the mutation in suspected family members.    

NEONATAL DIABETES MELLITUS 

Figure 4. Causes of Neonatal Diabetes

Neonatal diabetes mellitus (NDM) is defined as diabetes occurring in the first 6 months of life; for some authorities, the window extends to 9 months of age, but several of the mutations may manifest only later (65, 80, 81). For convenience, NDM is classified into 3 categories; transient NDM which constitutes about 45%, permanent NDM also constituting about 45%, and NDM associated with various other syndromic features, about 10% (Figure 4).

Transient Neonatal Diabetes

The transient forms are characterized by a period of remission during which glucose tolerance is normal, but diabetes usually recurs later in life. Of these transient forms, about 2/3^rd involve methylation abnormalities in chromosome 6q24 which lead to malfunction of imprinted genes PLAGL, also known as ZAC, and HYMAI that arise by the mechanisms listed in the Figure. These infants display small size at birth due to inadequate in utero secretion of insulin, a major regulator of anabolic growth; there is rapid catch-up growth when insulin is provided by sub-cutaneous injection or via pump therapy with insulin diluted 1:10 so that 1 ml contains 10 U rather than the standard 100U/ml. Hyperglycemia and glucosuria are present but may be missed if not sought. Rare variants of these methylation defects may have initial hypoglycemia and devolve into hyperglycemia. Most are sporadic, but duplication of paternal chromosome 6 leads to dominant transmission (see figure). Of the remaining 1/3^rd of TNDM, the majority harbor mutations in the KATP genes ABCC8 and KCNJ11 which respond to therapy via oral hypoglycemic agents such as glipizide; dosage requires titration to individual responsiveness. Approximately 5% of transient cases involve mutation in the insulin gene INS, the β-cell glucose transporter SLCA2A, or other genes as listed in Figure 4.

Permanent Neonatal Diabetes

Permanent NDM primarily involves 3 genes; severe mutations in KCNJ11 or ABCC8, and the insulin gene INS. Because the KATP channel and its’ genes are also expressed in the CNS, severe mutations also may affect neural function and development. Developmental delay, Epilepsy, and Neonatal Diabetes constitute the DEND syndrome, with associated physical and neuropsychological features; early treatment with oral sulfonylurea medication benefits neuropsychological function and timing of treatment influences outcome, i.e., the earlier the better (82-84). There is debate whether treatment with oral sulfonylurea should be started before confirmation of the genetic defect, but we recommend that it not be started, as the transition to oral agents with concomitant reduction in the injected insulin dose, essential to control the severe hyperglycemia and sometimes associated with DKA, is potentially dangerous and the dose of sufonylurea needed is much higher than that used in adults with T2DM. If successful, transition to oral therapy is associated with remarkable improvement of metabolic control due to stimulation of endogenous insulin secretion and neuropsychological improvement; it is also easier and less traumatic to the patient than insulin injection (80). We therefore recommend that such transitions be performed in a hospital setting according to a published protocol (85). When insulin therapy is used, either in mutations of the KATP channel or where it must be in mutations of INS which do not respond to sulfonylurea, using continuous subcutaneous infusion via a pump and diluted insulin, appears to be the best option (86).

Neonatal Diabetes and Associated Syndromes

About 10% of cases of NDM are associated with a spectrum of syndromic disorders; the more common ones are listed in the figure 4 and greater details can be found in the references (65-68, 77, 80, 81).  In all forms of neonatal diabetes, children are born small for gestational age; the smaller the child, the more severe the defect in insulin synthesis, secretion or action is likely to be. The associated abnormalities provide clinical clues, and it was the clinical associations that often defined the syndrome, before the genetic mutation was known. Next generation sequencing with a panel specifically designed for NDM can provide a rapid diagnosis and guide therapy, predict associated abnormalities, and infer possible interventions before some of the classical features have evolved (77, 81). Indeed, this is the approach now recommended, i.e., non-selective genetic testing in any case of neonatal diabetes. In addition, exome sequencing of unusual cases not covered by the panel may uncover new entities, as recently described for a form of autoimmunity associated with NDM that is responsive to a CTL4 mimetic (87). For most of the syndromic forms, insulin is the required therapy to control diabetes; an exception may be thiamine responsive megaloblastic anemia and diabetes which is due to mutation in the thiamine transporter SLCA29 and initially responsive to thiamine replacement (77, 80).

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ABSTRACT

In this chapter, we review the etiology and pathogenesis of Type 1 diabetes mellitus (T1DM), with particular emphasis on the most common immune mediated form. Whereas Type 2 diabetes (T2DM) appears to be an increasing price paid for worldwide societal affluence, there is also evidence worldwide of a rising tide of T1DM. The increase in understanding of the pathogenesis of T1DM has made it possible to consider interventions to slow the autoimmune disease process in an attempt to delay or even prevent the onset or slow the progression of hyperglycemia. Although the prevention of T1DM is still at the stage of research trials, the trials are often mentioned in the lay press.  Current investigations will determine if antigen-based therapies can in fact abrogate ongoing autoimmunity via immuno-stimulation and ultimately prevent diabetes in humans without the risks of general immunosuppression.  We also review the etiology and pathogenesis of T2DM and monogenic forms of diabetes that may be confused with T1DM or T2DM. 

INTRODUCTION

Diabetes Mellitus (DM) is a syndrome of disturbed metabolism involving carbohydrate, protein, and fat which results from the degree of insulin deficiency (absolute or relative) and tissue sensitivity to its actions. The combination(s) of insulin deficiency and sensitivity to its actions bring about distinct clinical phenotypes with varying severity of disturbed metabolism, most conveniently monitored by the degree of hyperglycemia. Absolute insulin deficiency (Type 1 DM) occurs with autoimmune destruction of insulin secreting β-cells (Type 1A DM) and other congenital (genetic defects in the formation or function of the endocrine pancreas), or acquired (relapsing pancreatitis and pancreatectomy) conditions. Absolute deficiency of insulin action also can occur in the total absence of insulin receptors, a rare event. Relative insulin deficiency occurs with genetic or acquired defects in insulin synthesis or secretion that are inadequate to overcome the resistance caused by fewer functioning insulin receptors, or resistance to insulin action induced by stress, drugs, and most commonly obesity (Type 2 DM).The acute clinical manifestations are those related to hyperglycemia which exceeds renal threshold to result in polyuria, increased thirst, dehydration, electrolyte disturbances, weight loss, and metabolic decompensation, in extreme degree known as diabetic ketoacidosis and non-ketotic hyperosmolar coma. The chronic complications include macrovascular (CAD, CVD, amputations) and microvascular (retinopathy, nephropathy, neuropathy) lesions.  Both the acute and chronic complications are inversely related to the degree of metabolic control achieved.  These brief introductory comments form the basis for the etiology, pathogenesis, classification and diagnosis of diabetes mellitus.

Classification and Diagnosis of Diabetes

The American Diabetes Association Standards of Medical Care for Diabetes 2021(1) proposes the following classification (Table 1).

Criteria for the Diagnosis of Diabetes Mellitus

The Expert Committee on the Diagnosis and Classification of Diabetes Mellitus recommends the following criteria for diagnosing DM (1).  Two replicate fasting glucose levels that exceed 126 mg/dl (>7 mmol/L) is consistent with diabetes even in the absence of symptoms. Normal fasting blood glucose levels of 100 mg/dl or above are considered impaired fasting glucose (IFG). Persons with IFG levels (FPG= 100-125 mg/dl (5.66.9 mmol/l) and/or with impaired glucose tolerance test (IGT) (2hour post-load glucose 140-199 mg/dl (78.8 mmol/L-11.1 mmol/L) are at risk of diabetes and should be observed periodically to detect hyperglycemic progression. Replicate, two-hour glycemic responses >200 mg/dl (>11.1 mmol/L) after a standard oral glucose tolerance test also indicate diabetes. This stage is often reached before the fasting glucose levels rise in T2DM and post-prandial hyperglycemia may precede fasting hyperglycemia by months to years. The reliance on only fasting glucose levels is generally more useful for identification of impending T1D but not for T2D.

The ADA now recommends that measurement of HbA1c levels can be used in clinical practice for the diagnosis of diabetes, since the onset is seldom so acute that it will not be reflected in elevated HbA1c levels Table 2 (1).

*The OGTT should be performed as described by the World Health Organization (1.75 gm/kg up to 75 gm, using a glucose load containing anhydrous glucose dissolved in water).

ETIOLOGIC CLASSIFICATION

Type 1 Diabetes Mellitus

Type 1 diabetes mellitus (T1DM) comprises several diseases of the pancreatic ß cells which lead to an absolute insulin deficiency. This is usually considered to be the result of an autoimmune destruction of the pancreatic ß cells (type 1A). Some patients with T1DM with no evidence of ß cell autoimmunity have underlying defects in insulin secretion often from inherited defects in pancreatic ß cell glucose sensing and from other genetic or acquired diseases.

Type 2 Diabetes Mellitus

Type 2 diabetes mellitus (T2DM) is by far the more common type of diabetes and is characterized by insulin resistance resulting from defects in the action of insulin on its target tissues (muscle, liver, and fat), but complicated by varying and usually progressive failure of beta cells’ insulin secretary capacity. Most patients with T2DM in the US and Europe are overweight or obese, however in India and China, most T2DM patients have a lean body mass index (BMI), albeit with increased visceral and hepatic fat.

Monogenic Diabetes

Monogenic forms of diabetes are characterized by impaired secretion of insulin from pancreatic β cells caused by a single gene mutation. These forms comprise a genetically heterogenous group of diabetes including, maturity onset diabetes of the young (MODY), permanent or transient neonatal diabetes, and mitochondrial diabetes. MODY is the most common form of monogenic diabetes, with autosomal dominant transmission of one of several genes encoding a primary defect in insulin secretion.

TYPE 1 DIABETES MELLITUS

Epidemiology of Type 1 Diabetes

T1DM is one of the most common chronic diseases of childhood and is classified as an autoimmune disease. Most common autoimmune disorders predominantly affect females, but, T1DM equally affects males and females with a slight male predominance in younger children. This and other inconsistencies have raised questions as to whether T1DM is a “pure” auto-immune disease or whether the auto-immune component is a marker of a separate primary trigger (3,4).  We discuss these issues later in this chapter. 

The incidence and prevalence of T1DM vary by age, season, geographic location, and within different racial and ethnic groups. Of cases diagnosed before the age of 20, however, two peaks of T1DM presentation are observed; one between 5 and 7 years of age, and the other during puberty at the mid-teens (5). However, first presentation of T1DM actually is as common in adulthood as it is in childhood and is characterized by a milder course in adults; the term LADA, (Latent, Auto-immune, Diabetes of Adults) is used to describe this entity. A seasonal variation in the incidence of T1DM is also observed; the majority of new cases of T1DM are diagnosed mostly in autumn and winter (6).  Findings from large T1DM registry studies such as the World Health Organization Multinational Project for Childhood Diabetes, known as the DIAMOND Project, EURODIAB   and others monitor incidence and other epidemiological markers.

The World Health Organization Multinational Project for Childhood Diabetes, known as the DIAMOND Project (in 50 countries), EURODIAB (in Europe), and SEARCH for Diabetes in Youth (in the USA) were established to address the implications of diabetes in youth and describe the incidence of T1DM. Wide variations in incidence of T1DM exist throughout the world, lowest in China and Venezuela (0.1 per 100,000 per year) and highest in Finland and Sardinia (50-60 per 100,000 per year) (7). A multicenter study focusing on identifying the prevalence and incidence of diabetes by type, age, gender, and ethnicity found a 1.8% annual increase in the prevalence of T1DM among American youth from 2002-2003 to 2011-2012, whereas T2DM had increased 4.8% annually from 2002-2003 to 2011-2012 (Table 3) (8).  The greatest increase was seen in youth of minority racial/ethnic groups (8).  Similar rates of increase in T2DM in teens are reported from the UK, India, China and Japan.

Although, there is a wide variance in the incidence and prevalence of diabetes throughout the world, the number of youths who are being diagnosed with T1DM has been growing at an annual rate of about 3 percent (9) and a similar increased annual rate was also observed among U.S. youth (10). This rising incidence of T1DM in children across the world in a short period of time clearly cannot be explained by genetic factors. Analytical epidemiological studies suggest that environmental risk factors, operating early in life, might be contributing to the increasing trend in incidence of T1DM (11,12).

On the basis of estimates for the number of people with diabetes in 2014, the cost of health care of diabetes in the US is estimated to be $105 billion per annum and the direct annual cost of diabetes in the world is $825 billion (13). However studies indicate that many more diabetic adults diagnosed as having T2DM phenotype actually have T1DM  as defined by the presence of antibodies to islet cell components (14,15); the term LADA, Latent Autoimmune Diabetes of Adults, is often used to describe this group (16).

Natural History of Type 1 Diabetes 

After immune activation in the setting of genetic susceptibility, the disease progresses through pre-symptomatic stages identified by presence of autoantibodies and impaired glucose intolerance, arising from further loss of β-cell function and ultimately resulting in clinical diabetes. (Figure 1)

Figure 1. Type 1 diabetes disease progression (17)

Pancreatic ß cells secrete insulin and are found in the islets of Langerhans. These islets are specialized groups of a few hundred to a few thousand endocrine cells that are anatomically and functionally discrete from pancreatic exocrine tissue, the primary function of which is to secrete pancreatic enzymes into the duodenum. Normal subjects have about one million islets, which in total weigh only 1-2 grams and constitute less than 1% of the mass of the pancreas. Furthermore, islets are composed of various types of cells that are interconnected as a regulatory network to regulate the disposition of nutrients and their utilization for energy use and tissue growth and repair. At least 70% are ß cells localized in the core of the islets, surrounded by α-cells that secrete glucagon, δ-cells that secrete somatostatin, and PP cells that secrete pancreatic polypeptide. All the cells communicate with each other through their extracellular spaces and through gap junctions; communication is further modulated by a rich network of sympathetic and para sympathetic innervation.

Insulin, a peptide hormone composed of 51 amino acids is synthesized, packaged and secreted in pancreatic ß cells. Insulin is synthesized as preproinsulin in the ribosomes of rough endoplasmic reticulum. The preproinsulin is then cleaved to proinsulin that is transported to the Golgi apparatus where it is packaged into secretory granules. Most of the proinsulin is cleaved into equimolar amounts of insulin and connecting (or C)-peptide in the secretory granules. Because the C-peptide sequence differs from that of insulin, and because, unlike insulin, it is not extracted by the liver, it is possible to estimate β-cell insulin secretion by measuring C-peptide, even in the presence of insulin antibodies resulting from insulin replacement therapy that impair the ability to measure insulin directly. Similarly, because C-peptide is an index of endogenous insulin secretion, and because C-peptide is not extracted by the liver, the ratio of C-peptide: insulin should exceed 1; when it is less than 1, implying a high insulin value, exogenous insulin may have been used. This has diagnostic and forensic utility in diagnosing causes of hypoglycemia.

Glucose is a major regulator of insulin secretion (Figure 2). When extracellular fluid glucose concentrations rise after a meal, glucose is taken up by the ß cells via glucose transporters, GLUT2 and GLUT1. Glucose is then phosphorylated into glucose-6-phosphate by islet specific glucokinase and metabolized, thereby increasing cellular ATP concentrations. The rise in ATP raises the resting ratio of ATP:ADP, that closes ATP dependent potassium channels (K-ATP) in the β-cell membrane, resulting in accumulation of intracellular potassium, causing membrane depolarization and influx of calcium via a voltage gated calcium channel. The rise in intracellular free calcium in ß-cells promotes margination of the secretory granules, their fusion with the cell membrane, and release of cell contents which include insulin into the extracellular space. An immediately releasable pool of insulin granules adjacent to the plasma membrane is responsible for an acute (first phase) insulin response; with ongoing stimulation, a pool of granules in the interior of the cell is mobilized and released as the “second phase” response. Amino acids also stimulate insulin release by a similar mechanism that involves the enzyme glutamate dehydrogenase which enables metabolism and ATP production by certain amino acids. Defects in the genes regulating these processes may result in diabetes if the K-ATP channel is prevented from closing normally (activating mutations) or syndromes of hyperinsulinemic hypoglycemia if the K-ATP channel is prevented from opening (inactivating mutations).  These aspects are discussed in greater detail in the section on Monogenic forms of diabetes (see below).

Figure 2. Insulin secretion by Pancreatic β cells. In the stimulated state, glucose is transported into the β cell by the GLUT2 transporter which undergoes phosphorylation by glucokinase and glucose is then metabolized. This results in an increase in the ATP/ADP ratio and initiation of a cascade of events that is characterized by closure of the K-ATP channel, decreased flux of potassium across the membrane, membrane depolarization, and calcium influx. This cascade ultimately results in insulin release from storage granules. The K-ATP channel shown is composed of four small subunits, Kir6.2, that surround a central pore and four larger regulatory subunits constituting SUR1. In the resting state, the potassium channel is open, modulated by the ratio of ATP to ADP. Leucine also stimulates insulin secretion by allosterically activating GDH and by increasing the oxidation of glutamate; this then increases the ATPADP ratio leading to the cascade of events beginning with closure of the KATP channel.
MCT-1: Monocarboxylate transporter-1, SCHAD: Short chain 3-hydroxyacyl-CoA dehydrogenase, SUR1: Sulfonylurea receptor 1, Kir 6.2: Potassium Inward Rectifying Channel 6.2, UCP-2: Uncoupling protein 2, HNF4α: Hepatocyte Nuclear Factor 4α, HNF1 α: Hepatocyte Nuclear Factor 4α, K+: Potassium, ATP: Adenosine Triphosphate, GDH: Glutamate Dehydrogenase, GLUT-2: Glucose Transporter 2

Metabolic Derangements of Type 1 Diabetes

As the pancreatic ß cell mass declines in an islet cell antibody (ICA) positive person, the first metabolic abnormality discernable is a decline in the first phase of insulin release (FPIR) to an IVGTT (18). The insulin level after a 3-4 minute infusion of glucose at 0.5Gms/kg rises abruptly in normal children at about 8 years of age, perhaps coincident with the onset of adrenarche (19). In the relatives and children from the general population with positive ICA, a decline in the FPIR is a strong predictive marker of evolving diabetes (19-21).

Subsequently, in evolving T1DM there is a rise in the fasting glucose level followed by an inability to keep the two-hour, post-OGTT glucose level below 200mg/dl (11.1mM). Transient insulin resistance also occurs in untreated T1DM and is due to raised levels of free fatty acids (FFAs) from uncontrolled lipolysis (22), as well as decreased levels of hepatic glucokinase and insulin regulated GLUT 4 glucose transporters in adipocytes which contribute to  the onset of symptomatic diabetes (23-25). Prolonged hyperglycemia itself likely impairs the ability to secrete insulin and when insulin replacement therapy begins, there is usually some recovery in the patient's ability to secrete insulin (the "honeymoon" period). However, within months to years, this partial recovery in endogenous insulin secretion ultimately fails. If it does not fail after 2 years, another form of diabetes, such as MODY should be suspected. Initially, the glucagon secreting cells within the pancreatic islets remain relatively preserved, resulting in excessive secretion of glucagon relative to insulin after protein meals (26). These elevated glucagon levels exacerbate the effects of the insulin deficiency, and promote lipolysis and ketogenesis, effects that can be partially reversed by an infusion of somatostatin (27). As the mass of islet cells decline, there is also loss of amylin, an islet cell hormone that down-regulates glucagon secretion. Thus, an analogue of amylin (pramlintide- marketed under the trade name Symlin) can be used as adjunctive therapy with insulin replacement. In time, with continued loss of islets, glucagon deficiency develops in established long standing T1DM, rendering patients more susceptible to insulin-induced hypoglycemia (26,28).  

Insulin is the hormone of "feasting", promoting utilization and deposition of ingested nutrients into body stores, as well as having multiple anabolic effects in many tissues. Progressive insulin deficiency thus induces a starvation like state, associated with excessive hepatic and renal gluconeogenesis, decreased peripheral utilization of glucose, hyperglycemia with resultant glycosuria, loss of water and sodium salts, and proteolysis in muscle liberating amino acids such as alanine and glutamine as substrates for gluconeogenesis (29-31). Uncontrolled lipolysis leads to the rapid mobilization of fatty acids from adipose tissue and the increased delivery of fatty acids to the liver leading to the increased synthesis of triglycerides and secretion of very low-density lipoprotein (VLDL).

With severe insulin deficiency the fatty acids delivered to the liver are metabolized to yield beta hydroxybutyric and aceto-acetic acids (ketone bodies) and contribute to keto-acidosis. Ketoacidosis is a life-threatening metabolic decompensation that is characterized by hyperglycemia, dehydration, metabolic acidosis and ketosis, all the result of the effects of severe insulin deficiency as well as the counter-regulatory stress hormones, cortisol, growth hormone, catecholamines and glucagon. Specifically, hepatic glucokinase levels fall with insulinopenia, synthesis of hepatic triglyceride and glycogen levels decline, malonyl CoA falls and thereby carnitine palmitoyl transferase-I levels rise promoting the transport of fatty acyl-CoA into mitochondria with the formation of acetyl-CoA (32-34).  In the liver, acetyl-CoA is converted into ß-hydroxybutyrate and acetoacetate in a proportion that depends upon the prevailing redox state, which provide an additional fuel substrates for muscle and brain (31,35,36). Lipoprotein lipases are also inactivated, leading to reduced hydrolysis of triglycerides that, if severe, may turn the serum milky with increased VLDL characteristic of the type 4 lipemic phenotype (37-39).

Genetic Susceptibility to Type 1 Diabetes

Individuals with autoimmune T1DM have inherited a number of quantitative trait loci (QTL) that encode protective and predisposing alleles which have exceeded the net genetic threshold required to predispose them to the disease (40). However, this genetic threshold (penetrance) is dependent in turn on chance interactions with greater predisposing than protective environmental forces. The multiple genetic influences in T1DM comprise a major effect from DR/DQ genotypes of the HLA complex (some 50% of the genetic effect), coupled to several other QTLs with minor influences (Table 4). All of the latter QTLs are not obligatory genetic elements themselves since they are of minor-influence, but they collectively interact to create additive influences on the genetic threshold. Siblings of a diabetic patient develop T1DM at about 15-fold greater frequency than persons in the general population (prevalence 1:250-300), vs. a value of 15. The HLA predisposition to T1DM is encoded by cis- and trans complementation DQA1*/DQB1* heterodimers which have an arginine at residue 52 of the A chain and a neutral amino acid (DQB1*0302, *0201) rather than a charged aspartic acid at residue 57 of the B chain (DQB1*0602/3 and DQB1*0301) (40), as modified by DRB1*04 subtypes (*0401 and *0405 are susceptible and *0403 and 6 are resistant types) (41) in the HLA genotype. Further, HLA-DP alleles have also been implicated, even though they are at a considerable recombination frequency away from the closely linked DR/DQ loci (42). Other genes involved include the variable number of tandem repeat (VNTR) alleles 5' to the insulin (INS) gene on chromosome 11p15, where the protective class III alleles (>200 repeats) are associated with increased expression of insulin in the thymus, leading to a more efficient eradication of insulin autoreactive T cells than class I alleles (26-63 repeats) that confer susceptibility to develop diabetes (43,44). There are also CTLA-4 gene polymorphisms on chromosome 2q that are associated with T1DM. CTLA-4 is an induced accessory molecule that is expressed on activated T cells. CTLA-4 interacts with B7.2 expressed by antigen presenting cells (APC), signaling apoptosis of T cells that become activated as part of an immune response, thereby confining the immune response. The non-obese diabetic (NOD) mouse, a model for autoimmune diabetes, has an enlarged lymphoid mass because of resistance of their T cells to undergo apoptosis, as do CTLA-4 knockout mice, which readily develop lymphocytic organ infiltrates like NOD mice. These genes thus collectively affect the general ability to be tolerant to "self" antigens. Another susceptibility locus, (the IDDM 4) in the genomic interval on chromosome 11q13harbors the high affinity IgE Fc receptor gene that has been linked to atopy and asthma, which are characterized byTh2 responses that may protect individuals against the development of anti- islet Th1 responses, and thereby protect against T1DM. There are other genomic intervals associated with or linked to T1DM that have been putatively mapped, but these mostly lack plausible candidate genes in the DNA region, and pathogenic mechanisms for them cannot yet be offered. The NOD mouse however has been subjected to extensive genetic mapping studies, in the hopes that genomic intervals harboring susceptibility or protective genes which are syntenic to humans will be discovered, thus hastening the identification of equivalent defective genes.

In summary, T1DM is a complex, multifactorial disease involving genetic predisposition and an environmental triggering event, of which viral causes have been proposed. Although more than 50 loci have been identified, genes involved in immune regulation including HLA subtypes, VNTR in insulin itself, CTLA4, PTPN22, AIRE, and IL2R remain most prominent (65,66). The HLA association, especially class II, remains the strongest predictor of T1DM risk. The heterozygous DR3/DR4 genotype carries the highest genetic risk for T1DM in non-Hispanic whites (45-70).  In conclusion, insulin expressing islets from recent-onset T1D subjects show overexpression of interferon stimulated genes (ISGs), with an expression pattern similar to that seen in islets infected with virus or exposed to IFN-γ/interleukin-1β or IFN-α.

Autoantigens and Autoantibodies in Type 1 Diabetes

The Doniach group in London, first reported islet cell autoantibodies in patients with autoimmune polyglandular syndromes (APSs) (71), especially in those with APS type-1 (APS-1) (72), even though such patients did not often develop diabetes. Lendrum and colleagues, having failed to find serological evidence for an autoimmune basis for chronic pancreatitis, did succeed in finding Islet Cell Antibodies (ICA) detectable by indirect immunofluorescence in patients with T1DM. Islet cell surface reactive autoantibodies and autoreactive peripheral blood T cells were also reported (73,74). Over the years that followed, the presence of ICA in US patients was confirmed but with distinctly lower frequencies of ICA among African American diabetic patients (75). Insulin autoantibodies (IAA) were discovered in patients with T1DM before their first dose of insulin replacement had been received (76). The presence of IAA together with ICA identified a group of non-diabetic relatives of probands with T1DM, that were at high risk for T1DM themselves (77). Insulin itself is not an ICA antigen that can be detected by the indirect immunofluorescent technique. Subsequently, much of the antigenic nature of the ICA reactivity has become clearer. It was recognized that many patients with "stiff" man syndrome who were prone to develop diabetes, also had ICA and autoantibodies to glutamic acid decarboxylase (GAD65). These GAD autoantibodies penetrated the blood brain barrier. High concentrations of GAD in the cerebellum reduce brain levels of the inhibitory neurotransmitter gamma aminobutyric acid (GABA), thereby causing the appearance of temporal lobe epilepsy, depressed cognition, muscle spasms, cerebellar incoordination and motor dysfunctions. That GAD65 was the antigen that accounted for the 64 KDa islet cell protein previously discovered by Baekkeskov to react with autoantibodies in T1DM, was later confirmed by the same investigator (78). Antibodies to recombinant GAD65 and GAD67 in T1DM patients were soon reported (79). The autoantibodies reacted to the antigens by conformational rather than linear epitopes, and thus with native rather than denatured antigens. Therefore, they were best detected by liquid phase assays such as radioimmunoassay, rather than by an ELISA technique. In stiff-man syndrome, the predominant GAD autoantibodies reacted with linear epitopes. It became known that besides islet cell 64 KDa sized proteins, autoantibodies in the sera of T1DM patients also precipitated islet cell proteins of 50, 40 and 37 KDa as well (80).

The next islet cell antigen discovered was one of the two-dozen tyrosine phosphatases expressed in islet cells, insulinoma antigen-2 (IA-2) (81). This antigen shared structural homologies with the ICA-512 antigen (82). A second tyrosine phosphatase named IA-2ß was discovered next (83). These additional tyrosine phosphatase antigens allowed for the matching of the islet cell proteins previously identifiable only by their molecular weights. Thus, GAD65 and its tryptic fragment explained the 64 and 50 KDa proteins, while tryptic fragments of IA-2 and IA- 2ß were identical with the 40 KDa and the 37 KDa islet precipitable proteins respectively (84). The tyrosine phosphatases are a family of transmembrane enzymes of which only these two are expressed by the pancreatic islets and react with T1DM autoantibodies. The reactivity is almost exclusively with the internal domains of these molecules, suggesting that they arise as a consequence of islet cell damage from autoimmunity. Antibodies to IA-2 cross-react with those of IA-2ß in about 50% of the patient sera. Some unusual patient sera however react exclusively with IA-2ß. The question of why only these two members of the tyrosine phosphatase family are targets of islet cell autoimmunity has been answered by the finding that they are relatively resistant to proteolytic enzymatic digestion, and once released from islet cells after their lysis, are insoluble and thus become better antigens for auto-immunization, than those that remain soluble and are more rapidly digested (85).

Recently, another antigen of 38KDa size (GLIMA) was added to the islet cell group, albeit only a minority of patient's sera reacts to it (86). Still more islet cell autoantigens are likely to be discovered. The detection of islet cell autoantibodies is useful for differentiating T1DM from diabetes of other causes, and can be used to predict onset of diabetes months to years before onset of the clinical disease (20,21,87,88) in non-diabetic relatives of probands with T1DM.  Importantly, the clinical onset of the disease is often long preceded by the appearance of autoantibodies reactive to islet cells (ICA) (88) and to insulin (77), as independent age-related variables in predicting a diabetic outcome (89). Islet cell autoantibodies (ICA) also show a strong tendency to disappear after diabetes onset when all ß cells are destroyed (90,91).

Studies in mice demonstrated a critical role of autoantibodies to GAD65 in the induction of autoimmune diabetes in NOD mice. In humans, the German BABY-DIAB study and the Finnish TRIGR study showed that islet autoantibodies which are mostly IgG class can be transferred through the placenta from islet antibody-positive mothers to their offspring (92,93). Most of the antibodies, however, disappeared from the circulation of the infant within the first year of life, indicating that they represent maternal antibodies and unlikely that they are markers of fetal induction of B-cell autoimmunity (93). In the German BABY-DIAB study, it was demonstrated that 729 offspring of mothers with T1DM had significantly lower risk of developing multiple islet autoantibodies (5 year risk 1.3%) and diabetes (8-year risk 1.1%) when they were GAD or IA-2 positive, than offspring who were islet autoantibody negative at birth (94). These findings suggest that fetal exposure to islet autoantibodies may protect from future diabetes. Furthermore, the German BABY-DIAB study finding is consistent with the overall decreased risk of development of diabetes in offspring of mother with T1DM compared with that of offspring of fathers with T1DM and nondiabetic mothers (95).

The timing of the appearance of the autoantibodies seems to be important. It was found that progression to multiple islet autoantibodies was fastest in children who were antibody positive by age 2 years and that progression to diabetes was inversely related to the age of first positivity for multiple autoantibodies (96).

The presence of multiple autoantibodies strikingly increases the risk of diabetes, whereas one of the above autoantibodies in the absence of all of the others when tested for, denotes only a modestly increased risk (20,21). This suggests that antigenic epitope spreading is involved in a sustained or accelerated autoimmune attack (72) (97). Besides autoimmunity to islet cell autoantigens, patients with T1DM are subject to other autoimmunities. Thus T1DM is a component part of the autoimmune polyglandular syndromes, commonly in APS-2  (Diabetes Mellitus, Addison Disease, Hypothyroidism) and with less frequency in APS-1(AIRE gene mutations) (72). Accordingly, patients with T1DM have high rates of thyroid autoimmunity, especially if they are females (98) (99), and are at increased risk for Addison's disease (99), atrophic gastritis (100), pernicious anemia (98), celiac disease (101), and vitiligo (102).

Antigen Specific Cellular Immunity in Type1 Diabetes

Autoreactive T cells that develop in impending T1DM, localize to the pancreatic islets where they become a component part of the evolving insulitis lesions. Thus, circulating autoreactive T cells are relatively sparse in impending T1DM. Nevertheless, antigen specific T cells are identifiable through prolonged in-vitro cultures in the presence of purified or recombinant islet cell autoantigens such as GAD (103) (104) and IA-2 (105). In fact, autoreactivity to a large number of autoantigens have been reported in both human and murine diabetes (106). T cell proliferative responses to insulin and GAD65, and more generally to islet extracts, have been repeatedly reported in both patients with T1DM (107,108) and NOD mice. However, both in humans and NOD mice, reports of spontaneous proliferative responses have been difficult to reproduce and validate, probably because of the relative paucity of autoreactive T cells in peripheral blood samples, and the ready contamination of recombinant "test" antigens by lymphotoxin or lipopolysaccharide (LPS), that by itself, can produce proliferative responses even when present in trace amounts. Furthermore, significant T cell responses to insulin, proinsulin or GAD65 antigen were reported, in some normal controls as well as in T1DM patients (109-111). Numerous laboratories have reported T cell reactivity in diabetic patients against GAD65 and IA-2 and their peptides with variable results (105,107,112-117). However, in established diabetes, the loss of the majority of ß cell mass resulting in associated loss of GAD65 and other ß cell antigens, in turn leads to the inactivation of T cells due to the loss of the peptide antigens that were driving the response. Thus, antigenic/epitopic spreading is an undesirable phenomenon associated with progression in autoimmune diseases like T1DM to a clinically significant outcome.

Pathogenesis of Type 1 Diabetes

The availability of Biobreeding (BB) rats and nonobese diabetic (NOD) mice, the rodent models of T1DM, has greatly enhanced our understanding of the possible pathogenic mechanisms involved (Fig. 3). Recently, it has become possible to compare these findings with findings in human islets, obtained from post mortem specimens of the pancreas through the network of Pancreatic Organ Donors (nPOD) and from patients with recent onset DM via endoscopic pancreatic biopsy (DiViD study, Norway) (86,118,119). In addition, epidemiological studies aimed at the prediction and prevention of T1DM permit a picture of the natural history to emerge. The process of destruction of β-cells is chronic in nature, often beginning during infancy and continuing over the many months or years that follow. At the time of clinical diagnosis of T1DM, about +80% of the β- cells have been destroyed, the islets are infiltrated with chronic inflammatory mononuclear cells (insulitis), including CD8+ cytotoxic T cells. Once islet cell autoimmunity has begun, progression to islet cell destruction is quite variable, with some patients rapidly progressing to clinical diabetes, while others remain in a non-progressive state.

Figure 3. The pathogenesis of islet cell destruction. Islet cell proteins are presented by antigen presenting cells (APCs) to naïve Th0 type CD4+ T cells in association with MHC class II molecules. Interleukin (IL)-12 is thus secreted by APCs that promotes the differentiation of Th0 cells to Th1 type cells. Th1 cells secrete IL-2 and IFN-γ that further stimulate CD8+ cytotoxic T cells or macrophages to release free radicals (super-oxides) or perforin/granzymes, leading to ß cell apoptosis or death. CD8+ cytotoxic T cells further mediate ß cell death by Fas mediated mechanisms. Interleukin (IL)-4, on the other hand, secreted mainly by natural killer T (NKT) cells drives Th0 cell to Th2 pathway leading to benign insulitis.

Diabetes risk and time to diabetes in relatives of patients directly correlates with the number of different autoantibodies present. The pathogenesis of T1DM has been extensively studied, but the exact mechanism involved in the initiation and progression of β-cell destruction is still unclear. The presentation of beta cell-specific autoantigens by antigen- presenting cells (APC) [macrophages or dendritic cells (DC)] to CD4+ helper T cells in association with MHC class II molecules is considered to be the first step in the initiation of the disease process. Macrophages secrete interleukin (IL)-12, stimulating CD4 + T cells to secrete interferon (IFN)-γ and IL-2. IFN-γ stimulates other resting macrophages to release other cytokines such as IL-1β, tumor necrosis factor (TNF-α) and free radicals, which are toxic to pancreatic β-cells. During this process, cytokines induce the migration of β-cell autoantigen specific CD8+ cytotoxic T cells. On recognizing specific autoantigen on ß cells in association with class I molecules, these CD8+ cytotoxic T cells cause ß cell damage by releasing perforin and granzyme and by Fas-mediated apoptosis of the beta cells. Continued destruction of beta cells eventually results in the clinical onset of diabetes.

Recently, these concepts derived from studies in the rodent models have been challenged as having the same pathologic process that occur in humans. Analysis of variations in histopathology observed from these organ donors provide mechanistic differences related to etiological agents and serve an important function in terms of identifying the heterogeneity of T1D (120). The findings are not always consistent with those of the rodent models. For example, the dense infiltration of islets by T-cells is evident in the pancreas of those who succumb to DKA at onset, but more chronic cases show a patchy distribution of destroyed and functioning islets containing beta cells with insulin suggesting a defect in secretion rather than synthesis. In the DiViD (Diabetes Virus Detection) study, expression of inflammatory markers, predominance of Class I antigens (rather than expression of Class 2 antigens) in islets, and actual viral isolations suggest a more acute process. Taken together, the studies suggest that T1DM may be a heterogeneous group of conditions in which auto-immunity may be a consequence or companion rather than the initiating mechanism. These findings begin to explain why prediction of developing T1DM in those from affected families considered at risk has become quite accurate, whereas prevention or reversal of DM by immune intervention or modulation has failed repeatedly (3,4,121).

The Indian uctive Event in Type 1 Diabetes

Various mechanisms have been proposed:

MOLECULAR MIMCRY

In antigenic molecular mimicry, cross-reactive immune responses occur due to significant structural homologies shared by molecules encoded by dissimilar genes.

The incidence of T1DM has increased over the last three to four decades in Europe, and the clinical disease exhibits preferential seasonal onset (122). These observations emphasize the role of environmental factors in the disease process. It has long been suggested that T1DM in humans is caused by viral infections (123-125). However, despite a vast increase in the information regarding the various genetic factors controlling the disease, little is known about the role of the putative environmental factors that might provide a more direct approach to therapy (8). Specifically, allegations that childhood vaccines could be causal have not been upheld by more extensive controlled studies.

The disease pathogenesis may involve multiple factors including the genetics of the host, strain of the virus, activation status of the autoreactive T cells, upregulation of pancreatic MHC class I antigens, molecular mimicry between viral and ß cell epitopes and direct islet cell destruction by viral cytolysis. Viruses, as one of the environmental factors affecting the induction of T1DM, may act as triggering agents of autoimmunity or as primary injurious agents, which directly damage pancreatic ß cells. Immune responses against a determinant shared by host cells and a virus could cause a tissue-specific immune response by generation of cytotoxic cross-reactive effector lymphocytes or antibodies that recognize self-proteins located on the target cells.

Monoclonal antibodies against viruses have been observed to be capable of cross-reacting with host determinants (126).

Several studies in humans also point to viruses as triggers of the disease (127). Coxsackie B4 virus and rubella virus have been linked with T1DM. In a few instances, Coxsackie B4 virus has even been directly isolated from pancreatic tissues of individuals with acute T1DM. Inoculation of this virus into mice, in one report, produced diabetes (128). The possibility that viruses might cause some cases of T1DM by infecting and destroying pancreatic ß-cells has received considerable attention. However, it is difficult to demonstrate in-vivo that viruses replicate in human ß-cells and/or produce diabetes in man. An in-vitro system was therefore developed to determine whether viruses are capable of destroying human β-cells in culture (129,130). By this method, it was clearly shown that several common human viruses, including mumps virus (131), Coxsackie B3 virus(132), Coxsackie B4 virus (128), reovirus type 3 (133), could infect human ß-cells. In addition, by radioimmunoassay, it was shown that the infection markedly decreased the insulin content of the ß-cells.

A strong correlation was found between the CMV genome in the immunocytes and the islet cell autoantibodies in the sera from diabetic patients (134). About 15% of newly diagnosed autoimmune T1DM patients have been reported to have persistent CMV infections.

Furthermore, it has been proposed that a molecular mimicry between protein 2C (p2C) of Coxsackie virus B4 and the autoantigen GAD65 may play a role in pathogenesis of T1DM. Kaufman et al (135) and Vreugdenhil et al (125), showed that the amino acid sequence of p2C shares a striking homology with a sequence in GAD65 (PEVKEK) and is highly conserved in Coxsackie virus B4 isolates as well as in different viruses of the subgroup of Coxsackie B-like viruses. These are the most prevalent enteroviruses and therefore the exposure to the mimicry motif should be a frequent event throughout the life. Furthermore, they suggested that molecular mimicry might be limited to the HLA-DR3 subpopulation of the T1D patients.

Although numerous sequence similarities between viral proteins and ß-cell autoantigens are plausible, the relationship between Coxsackie virus infection and GAD65 autoimmunity has received the most attention.

Glutamate Decarboxylase (GAD)

The finding by Kauffman et al (135), of a striking sequence homology of 18 amino acid peptide between human GAD65 and the Coxsackie virus p2-C protein, enhanced the evidence of a specific molecular mimicry model involving GAD. In addition, this specific region of GAD65 contains a T cell epitope involved in the GAD cellular autoimmunity in humans with immune mediated diseases (103)  and this region is an early target of the cellular immunity in NOD mice (136,137). GAD catalyzes the formation of the inhibitory neurotransmitter γ-amino butyric acid (GABA) from glutamine (104). Two forms of GAD exist (GAD65 and GAD67). GAD65 is the predominant form within the human pancreatic islet cells, while GAD67 predominates in mouse islets. Within the islets, GAD is predominantly observed within the ß-cells, while its roles in the inhibition of somatostatin and glucagon secretion and in the regulation of proinsulin synthesis and insulin secretion, have also been suggested (138).

Another study further supports a link between Coxsackie virus and T1DM, associating IgM antibodies to Coxsackie B virus as a marker of recent exposure to the virus in newly diagnosed IMD patients and age/sex-matched controls (139). In that report, humoral immunity to Coxsackie virus and GAD appeared to cluster, even in people without diabetes. A series of overlapping synthetic GAD65 peptides were used to study the most reactive T cell determinants in individuals at increased risk for T1DM, i.e., autoantibody positive, first degree relatives of T1DM patients. Elevated in vitro T cell responses were observed to GAD65 peptides (amino acids 247-266 and 260-279) in newly diagnosed T1DM patients and autoantibody positive at- risk individuals (140). The sequence of this region of GAD65 (amino acids 250-273) is significantly similar to the p2-C protein of Coxsackie B virus (123). However, not all published reports have demonstrated a linkage between immunity to GAD and Coxsackie virus. For example, one study identified a non-Coxsackie-homologous region of GAD65 as a predominant cellular immune epitope while studying the polyclonal human T cell responses (115).

Insulinoma Antigen Two (IA-2)

Tyrosine phosphatase IA-2 is another molecular target of pancreatic islet autoimmunity in T1DM. In one recent study, the epitope spanning 805-820 amino acid elicited maximum T-cell responses in all at-risk relatives, out of a total of 68 overlapping, synthetic peptides encompassing the intracytoplasmic domain of IA-2 (141). This epitope was found to have 56% identity and 100% similarity over 9 amino acids with a sequence in VP7, a major immunogenic protein of human rotavirus. This dominant epitope also has 75-45% identity and 88-64% similarity over 8-14 amino acids to sequences in Dengue, cytomegalovirus, measles, hepatitis C and canine distemper viruses and the bacterium Haemophilus influenzae.

Furthermore, three other IA-2 epitope peptides have 71-100% similarity over 7-12 amino acid stretch to herpes, rhino-, hanta- and flavi-viruses. Two others have 80-82% similarity with dietary proteins of milk, wheat and bean proteins. These molecular mimicries could lead to triggering or exacerbation of ß-cell autoimmunity.

SUPERANTIGENS

Besides molecular mimicry, retroviral expression of superantigens (Sags) may be able to activate clonal expansion of autoreactive T cell clones. Superantigens have been implicated in the pathogenesis of the various autoimmune diseases (142,143). Originally described as minor-lymphocyte stimulating antigens, retroviral Sags expressed by B cells interact with the development of T helper cells of both Th1 and Th2 subtypes in mice. A study in patients with T1DM demonstrated that two thirds of IAA positive sera also reacted with p73 (144). Conrad et al (145)  isolated a novel mouse mammary tumor virus-related human endogenous retrovirus (HERV), in patients suffering from acute onset T1DM. He termed them the HERV IDDMK1,2 22 subtype. They further showed that the N-terminal moiety of the envelope (env) gene encoded an MHC class II-dependent superantigen. He proposed that expression of this Sag, induced extra-pancreatically and by professional antigen-presenting cells, could lead to ß-cell destruction via the systemic activation of autoreactive T cells. He further reported the selective expansion of Vß7+ T cells in the islet cell infiltrates from two patients with recent onset IMD was associated with extensive junctional diversity of Vß7+ T cell clones. These investigators demonstrated that islet cell membrane preparations preferentially expanded Vß7+ T cells from non-diabetic peripheral blood mononuclear cells (146). However, other investigators were unable to confirm T1DM specificity of the IDDMK1,2 22, since it was equally recoverable as viremia from controls as well as patients (147). Furthermore, both patients and controls made antibodies to env proteins.

In order to establish molecular mimicry as a mechanism responsible for the autoimmune diseases it is important to identify the precise epitope that initiates the putative cross-reactive immune response. Additional complexity that has come to various animal studies is that of

epitope spreading (148). An increasing array of autoantigens or autoantigenic peptides reactive with autoantibodies develop over time. Both intramolecular and intermolecular epitope spreading has been described in NOD mice (136,149). These studies demonstrated that T- cell responses in NOD mice expand in vivo against a defined group of islet cell antigens in an orderly sequential manner. These responses in the young NOD mice first show a strong reactivity to GAD enzyme and not to other islet cell antigens. Furthermore, the initial response to GAD is first limited to one region of the protein only. Gradually, this response spreads intramolecularly to involve other regions of the protein. Eventually, after the destructive islet cell inflammation (insulitis) as a result of autoimmunity to ß-cells, the T-cell responses spread intermolecularly to involve other islet cell proteins (e.g., heat shock protein 60, carboxypeptidase H and insulin) as well (150). This epitope spreading makes it difficult to predict which putative cross-reactions, if any, are important in terms of disease induction, and which do not give rise to autoimmune pathology, particularly in humans who are exposed to many infections.

Deficiencies in immunoregulation in Type 1 Diabetes

There is both evidence for and speculation about defective central and peripheral mechanisms of immunoregulation in the autoimmune form of T1DM. Deletion of autoreactive T cells in the thymus, is one mechanism for the induction of tolerance to self-antigens (central deletion). This may involve diminished expression of insulin in the thymus of susceptible individuals due to the presence of class I VNTR alleles 5' to the insulin gene as already discussed. Others have suggested that it is the ineffective antigenic binding of the T1DM-prone HLA-DQ or -DR that promotes islet cell autoimmunity, since this permits autoreactive T cells to escape thymic ablation and pass into circulation.

In addition to clonal T cell deletion and anergy in thymus, peripheral regulatory T (Treg) cells are essential for the down regulation of T cell responses to both foreign and self-antigens, and for the prevention of autoimmunity. Various studies have identified defects in the peripheral Treg cells in T1DM patients (151,152) as well as in NOD mice affecting both NKT cells (153,154) as well as CD4+CD25+ suppressor T cells (155). Since these Treg cells are not absent in either species, ways to stimulate them should be actively sought to provide novel therapies for the future. The possibility of future therapeutic use of Treg cells in human autoimmune diseases lies heavily on basic studies that are designed to elucidate the mechanisms of induction and function of these cells. Therapy with immunomodulatory compounds that specifically target endogenous pools of Treg cells can be envisioned (156). This approach requires a more detailed investigation into the intracellular and extracellular events that regulate the differentiation and expansion of these cells in-vivo.

Of great interest has been the emergence of immune mediated T1DM in patients treated with checkpoint inhibitors for various cancers (157).  Unlocking the immune response via drugs that block the molecules programmed death (PD1) or its ligand, PDL1, as well as CTL4, may result in immunotoxicity with emergence of autoimmunity affecting various organs, including endocrine tissues such as the thyroid, adrenal and pancreas causing a form of T1DM (158). Indeed, autoimmunity has been called the “Achilles’ Heel” of immunotherapy, with increasing reports of its association with T1DM (159).

Environmental Factors in Type 1 Diabetes

Besides the familial predispositions, much evidence points to a major role of environmental factors in the disease pathogenesis. More than 60% of identical twins affected by T1DM are discordant for the disease and most of the non-diabetic twins lack islet cell autoantibodies. Over the past 3 decades, the disease frequency is on a steep rise in Western countries that cannot be explained by the accumulation of the susceptible genes. Africans, who dominate the tropics, and Chinese, both have low frequencies of the susceptible genes and low incidence rates of T1DM (75), except where there has been a high rate of Caucasian genetic admixture.

More persuasively, migrants from countries with low hygiene and low incidence rates of T1DM to countries with high hygiene and high incidence become as susceptible as the natives within a generation (160). Animals reared in sterile environments have early onsets and increased frequencies of diabetes while those infected with a variety of micro-organisms and parasites become protected (161-165). The hygiene hypothesis was proposed.  A strong causal relationship between prevailing level of community hygiene, especially with respect to drinking water and the dramatic increase in the incidence of autoimmune diseases such as T1DM in the modern world, has been referred to as the hygiene hypothesis.

ROLE OF DIET

Despite persuasive epidemiological evidence for environmental factors that precipitate T1DM in genetically susceptible individuals, their identity remains elusive. This may be due to long period between exposure and the onset of hyperglycemia, the complex genetics of the disease, and the likely multiple insults of perhaps different derivation involved in the initiation of the insulitis and subsequent ß cell destruction. Dietary habits such as consumption of dairy products and early weaning of infants, and dietary toxins such as nitrates and nitrites have been associated with this autoimmune disease (166,167).

Close correlations between per capita consumption of unfermented milk proteins and the incidence of diabetes between countries(168-170) and within a country have been reported (171). The claimed negative association between diabetes incidence and a high frequency and long duration of breast-feeding is more controversial (166) and has not been confirmed by reports from Germany (172) and the United States. Several studies have found associations between the consumption of foods rich in nitrates (or nitrites), which is reduced to nitrite in the gut, and the occurrence of T1DM (173,174). The active species is believed to be N-Nitroso compounds that can be formed from the reaction of nitrite with amines (175). Most recently, the gut microbiome and its modulation by dietary factors, has been implicated in the causality of T1DM (176).

The incidence of T1DM varies worldwide according to dietary patterns. In-depth exploration of dietary risk factors during pregnancy and early neonatal life is warranted to confirm whether and to what extent diet cooperates with genetic susceptibility in the early onset of T1DM.

Screening Methods for Type 1 Diabetes

T1DM is by far the most common chronic metabolic disease of childhood and adolescence and its prevalence and incidence has been increasing worldwide (96). This increase of incidence is the highest among the children under 5 years of age (177). Prevention of T1DM would constitute a major advance in the lives of pre-diabetic individuals and significantly relieve a major current and predicted burden on both the individual and the health care system. Identifying individuals at risk developing the disease and the prevention of the disease progression are two important steps before the onset of disease. The presence of islet autoantibodies, as well as the genetic predisposition with specific HLA haplotypes are known risk factors associated with the development of diabetes. Most studies have been carried out on first-degree relatives of T1DM patients who have 15-fold increased risk of the developing diabetes in comparison to the general population. However, more than 90% of all patients developing T1DM do not have an affected family member. Therefore, it is crucial to establish a standardized screening method which will efficiently identify individuals at high risk in a general population. School children between 5-18 years of age were screened to evaluate the predictive value of autoantibodies over a period of 6-12 years (178). This study indicated that the risk of developing T1DM when ICA is detected in the absence of other autoantibodies is low, whereas with more than one autoantibody (either GAD65A, IAA, IA-2A or IA-2ßA) the risk of developing T1DM in a general population is high. Similar findings were also reported in other studies (179-181). These results support the value of multiple autoantibodies as good predictive markers for T1DM not only in first degree relatives but also in the generalpopulation.  Consequently, the American Diabetes Association now considers the presence of 2 or more autoantibodies as form of early presymptomatic diabetes (182).

Prevention Trials in Type 1 Diabetes

The elucidation of the natural history of pre-diabetes has allowed for the characterization of those individuals at greatest risk for developing autoimmune T1DM, through the use of genetic, immunologic and metabolic markers. This predictive ability has become possible in both high- risk relatives and the general population as mentioned above. The subclinical autoimmune destruction of ß-cells in the pancreas may last from a few months to several years. This pre- diabetic period has allowed investigators to test prevention strategies, which mainly have focused in modulation of autoimmune process (183). A number of studies initiated with general immunosuppressive agents, such as cyclosporin-A, azathioprine and prednisone in patients with new clinical onset T1DM, positive results in that insulin free remission rates were increased and endogenous insulin (C-peptide) reserves were improved (121). However, despite continued immunotherapy with the attendant risks of renal damage and lymphomas at higher doses, relapses proved to be the rule and such treatments were abandoned. Cyclosporin given at a prediabetic phase of the disease delayed but did not prevent diabetes (184,185).

With the observation that nicotinamide prevents pancreatic ß cell destruction from streptozotocin by raising otherwise depleted levels of islet cell NAD as a result of superoxide induced DNA breaks and repair, the vitamin was subjected to a large European and Canadian trial called The European Nicotinamide Diabetes Intervention Trial (ENDIT). However, nicotinamide failed to prevent progression to diabetes (186). In addition, a  study in Germany (DENIS)   was completed without any effect of nicotinamide on prevention of T1DM.(187).More recent studies have used Anti CD21(Rituximab), Anti CD3, Anti CTLA-4, oral insulin,GAD65 peptides, and infusions of Treg cells  with early encouraging results in preserving insulin secretion, but without durable effects (188). These results in humans were often based on animal studies in NOD mice (189-191). In stark contrast to these encouraging studies in NOD mice, where a variety of interventions induce long lasting remissions, none of the studies in humans has so far yielded long-lasting remissions in humans (183,188).

TYPE 2 DIABETES MELLITUS

As the US passed into the 21st century, the epidemic of obesity and T2DM continues unabated, affecting more younger adults and children than in the past.  They will spend longer periods of their life with the disease. Perhaps in part under pressure of commercial interests, we as a nation have learned to eat too fast, too much, and the wrong foods.  However, the problem of obesity and its consequences is pervasive globally, affecting developing as well as economically developed countries.  For those with the energy conserving "thrifty" genes of insulin resistance syndrome (IRS), this excess of food and especially of the insulin provoking carbohydrates, leads to obesity, an IRS phenotype and T2DM. Nearly half of the new cases of diabetes in teens can be termed T2DM (204).  Currently, in some US states where there are large numbers of ethnic groups prone to IRS and T2DM (Hispanics, American Indians, Asian Indians, African Americans), the number of children with T2DM is beginning to rival if not surpass the number with T1DM. It is estimated that 1 in 3 people born in the US in the year of 2000 will develop T2DM sometime in their lifetime (205).

The increased incidence of T2DM is attributed to the increase in obesity worldwide. Approximately 3700 youths are diagnosed with T2DM every year in the US (206) and it is estimated that the number of youth with T2DM will almost quadruple from 22,820 in 2010 to  approximately 85,000 adolescents with T2DM by 2050 (10). Similar rates of increased in youths with T2DM are reported from the UK, India, China and Japan (10).

Pathophysiology of Type 2 Diabetes

T2DM is characterized by insulin resistance in peripheral tissues (muscle, fat, and liver) with progressive β cell failure, ,especially manifest with defective insulin secretion in response to a glucose stimulus, increased glucose production by the liver, and no markers of pancreatic autoimmunity (207). The progressive decline in β cell function is more rapid in youths at 20-30% decline per year versus 7-11% decline per year in adults, even with aggressive medical therapy.

The natural history of progression to T2DM is that a person with IRS begins to decompensate, with a fall in the disposition index (the amount of insulin produced for the degree of insulin resistance). Subsequently levels of blood glucose rise after feeding; elevations in fasting blood glucose levels occur later. At this early stage, diet, exercise and insulin sensitizers are indicated.

INSULIN RESISTANCE SYNDROME (IRS)

This syndrome complex is centered upon genetic predispositions to insulin resistance and the hyperinsulinemia that results from it. This medical state is also named syndrome X and the metabolic syndrome, however the descriptive term insulin resistance syndrome (IRS) is the one increasingly used in the literature (207,212). In IRS, there are poorly understood genetic lesions that lead to insulin resistance from early life if not during embryogenesis. In many affected families, the disease occurrences suggest a dominant mode of transmission. In rare families, mutations affecting insulin receptors, or peroxisome proliferators-gamma (PPAR- gamma) expression may be the cause of it (213). IRS is the association of insulin and leptin resistance with obesity (typically with increased visceral fat), functional adrenal hyper-androgenism, functional ovarian hyperandrogenism, hypersecretion of pituitary LH, dyslipidemia, hypertension, and features of hyperinsulinemia such as late reactive hypoglycemia and acanthosis nigricans. When the compensation by increased insulin secretion fails, glucose intolerance and T2DM result.

Natural History of Insulin Resistance Syndrome

Several studies indicate that many children and adults with T2DM were born small for gestational age. This suggests that the insulin resistant state existed in-utero since it is insulin rather than pituitary growth hormone that is the principal growth-promoting hormone of the unborn child, and decreased insulin action might be anticipated to impair embryonic growth. After birth, premature pubarche resulting from excessive adrenal androgens such as dihydroepiandrosterone (DHEA) may occur, even before obesity has appeared. Thus, it has been proposed by some that obesity may be the result of insulin resistance, and not its cause. Excessive DHEA may be seen best after ACTH injection leading to a clinical suspicion that the 3ß hydroxysteroid dehydrogenase enzyme is underactive. Obesity can begin from infancy but often dates from about 8 years of age when physiological pubarche occurs. Early onset obesity raises the possibility of a genetic satiety causation such as the Prader-Willi Syndrome or deficiency of MC4R. Acanthosis nigricans resulting from increased keratinocytes in certain areas of skin is thought to result from insulin stimulation of insulin-like growth factor 1 (IGF-1) receptors and often manifests during puberty Menarche may be delayed in age at onset or menses may be missed after menarche, or else there can be dysfunctional bleeding resulting from anovulatory cycles.

Hirsutism often becomes bothersome during adolescence, as may male pattern hair thinning, persistent acne and development of polycystic ovaries. An increase in very low-density lipoprotein (VLDL) secretion by the liver is observed with increasing age, associated with diminished, atherogenesis protective, high density lipoprotein cholesterol (HDL-C), a dyslipidemic profile that promotes early and progressive onset of atherosclerosis, predisposing to coronary heart disease (CHD), stroke, and peripheral vascular diseases in later life. The latter problems are compounded by the appearance of hypertension and type-2 diabetes. The glucose intolerance that precedes type-2 diabetes often first involves post-prandial glucose levels or the two-hour time point of the OGTT as discussed above, but later induces a rise in fasting glucose (impaired fasting glucose) levels as well. The mechanism is thought to be ß cell exhaustion or more likely a glucosamine and lipid mediated islet cell toxicity. Once this stage is reached, damage to the islets can become irreversible, resulting in the dual problems of insulin resistance and insulinopenia, both of which need to be addressed in therapeutic strategies.  In children and adolescents, the progression of impaired insulin secretion and its complications including the appearance of albuminuria, exhibits a faster tempo than that of adults presenting later in life. Hence, these adolescents may more rapidly progress to requiring insulin therapy.

Underlying Mechanisms of Insulin Resistance

OBESITY

Affected patients commonly show polyphagia, and may have voracious appetites that are characteristically resistant to dietary advice. When leptin deficiency was discovered in Ob/Ob mice and leptin receptor deficiency discovered in Db/Db mice, the adipocyte became to be appreciated as an endocrine cell rather than one that was an inert repository of triglycerides. However, the promise of a breakthrough in the understanding of human obesity was quickly dissipated when such lesions proved to be rare in humans. Obese patients with their greater degrees of adiposity also have the highest levels of leptin as expected, however these high levels do not reduce the appetites of IRS patients (215). Thus, such patients are also leptin resistant. Early trials of leptin therapy have not affected weight loss. However, patients with lipodystrophy who have leptin deficiency develop insulin resistance, hyper-insulinemia, dyslipidemia and T2DM, all of which respond dramatically to leptin given as therapy (216,217).   Deficiencies in other appetite suppressing hormones such as resistin have more recently been implicated but not yet shown to have therapeutic relevance. Hyperinsulinemia itself is a compounding variable, in that excessive carbohydrate containing diets stimulate the highest levels of insulin and the greatest degrees of adiposity. Therapies such as metformin that improve insulin sensitivity when combined with a diet restricted in low amounts of simple carbohydrates and exercise, can dramatically lower weight in children with IRS when they adhere to therapeutic guidelines. However, failure to adhere to instructions is a common problem in adolescents (218,219).

HYPERANDROGENISM

It is uncertain as to the degree to which the pituitary abnormality of increased LH secretion leads to the androgenic excess or vice versa. Probably, both are responses to the insulin resistance and hyperinsulinemia of IRS by mechanisms that have yet to be clearly understood. Androgens of ovarian origins usually predominate over those of the adrenal gland, albeit both are often found to be elevated. Sex hormone binding globulins in the circulation are often low, resulting in increased free androgens with their increased bio-availability (220). This is often seen with testosterone, which can be raised or normal in hirsute girls whereas increased free testosterone levels are common.

Interestingly, we hold that there is a clinical overlap between Cushing's syndrome and IRS (221). Both tend to have visceral (central) obesity and striae suggestive of glucocorticoid excess. However, whereas the patient with Cushing's syndrome has high levels of serum cortisol, the patient with IRS has low normal levels, albeit both have increased levels of urinary free cortisol. Again, the explanation may lie in the low levels of corticosteroid binding globulins found in IRS where circulating cortisol is disproportionately free. Some investigators have suggested that there is an impaired conversion of cortisol to the metabolically inactive cortisone in IRS. Further, the child with Cushing's syndrome is invariably growth retarded in contrast to the child with IRS whose linear growth tends to be excessive. In IRS and obesity, the GH levels during stimulation tests are suppressed implying a diagnosis of GH deficiency which likely is not the case as these children tend to be tall. IGFBP levels in serum are depressed, resulting in an excessive free IGF-1 level, albeit the total IGF-1 concentration is usually normal. The pseudo-acromegaly observed in severely affected children with IRS may be occurring via this mechanism. In addition, high concentrations of insulin interact with the IGF-I receptor, thereby promoting growth (222).

ACANTHOSIS NIGRICANS

Stimulation of the IGF-1 receptors of skin keratinocytes by high levels of circulating insulin is thought to explain their hyperplasia and excessive laying down of keratin in the skin of the neck, axillae, elbows and knees, skin creases and indeed most areas of skin (223). In addition, excessive free IGF-1 may have the same effect, albeit the greater the degree of insulin resistance, the higher the insulin levels, the more striking the acanthosis nigricans. Increased bioavailability of IGF-1 (high IGF-1 and low IGFBP-1) are directly correlated with the severity of acanthosis nigricans

GLUCOSE INTOLERANCE AND T2DM

Children and young adults affected by IRS are often hyperinsulinemic. In such persons, stimulation of insulin secretion by carbohydrates alone or with protein can induce an excessive but delayed rise in insulin secretion, reflected in an early excessive rise in glucose, followed by an excessive fall in glucose levels 3-5 hours afterwards, of sufficient severity to provoke symptoms of hypoglycemia. As the ability to secrete insulin declines, impaired glucose intolerance appears first. Later in the evolution of T2DM, the 2-hour criteria for diabetes during OGTT become apparent, followed later by impaired fasting hyperglycemia and finally by fasting hyperglycemia that meets the criteria for the diagnosis of diabetes. An HbA1c level can be used to screen diabetes as recommended by the American Diabetes Association.

NON-ALCOHOLIC STEATOHEPATITIS (NASH)

It is also known as fatty liver or hepatic steatosis. The incidence of fatty liver among obese children was 2.6% in one study (224), and hyperinsulinemia was found to be the major contributor for its’ development (225). A number of factors may play a role in the development of fatty liver including, induction of cytochrome P4502E1 during obesity, which is capable of generating free radicals, while the high level of dietary intake of polyunsaturated fatty acids or low intake of nutritional antioxidants contributes to the oxidative stress. Fatty liver alone appears to be a relatively benign disease, and can be reversible. However, it may progress over years to hepatic cirrhosis, liver failure, or hepatocellular carcinoma. The onset of disease is usually insidious. Laboratory evaluation indicates mild to moderate elevation of serum aminotransferases in most children and serum alanine aminotransferase (ALT) levels had been shown a useful screening for fatty liver in obese children (226). The ratio of aspartate aminotransferase (AST) to ALT is usually less than 1, but this ratio increases as fibrosis advances. Serum aminotransferases, alkaline phosphatase and gamma glutamyl transferase (GGT) levels are proposed surrogate markers of fatty liver (227,228).

RENAL INVOLVEMENT

A form of focal glomerulosclerosis (often with IgA deposition) appears to be associated with IRS, leading to microalbuminuria. Hypertension becomes increasingly common through adolescence and beyond. The mechanisms responsible have not been elucidated.

INFLAMMATION

IRS and T2DM have increased markers of inflammation. This takes the form of increased levels of C-reactive protein, raised erythrocyte sedimentation rates (ESR) and increased cytokine (TNF-α) levels.  Obese patients also have abnormalities of thyroid function suggestive of primary thyroid deficiency with modestly elevated TSH but normal or slightly elevated fT4 and fT3.These abnormalities resolve with weight loss and have therefore been interpreted as representing an adaptive response to obesity i.e., by raising TSH and free T3, caloric expenditure would increase (229-231). Obese patients are thus often unnecessarily treated for hypothyroidism they do not have. They may however develop true hypothyroidism on the basis of associated Hashimoto's disease.

ATYPICAL DIABETES

Genetic Defects of ß-cell Function (Monogenic Diabetes)

Monogenic forms of diabetes are characterized by impaired secretion of insulin from pancreatic β cells caused by a single gene mutation. These forms comprise a genetically heterogenous group of diabetes including, maturity onset diabetes of the young (MODY), permanent or transient neonatal diabetes (NDM), and mitochondrial diabetes. MODY is the most common form of monogenic diabetes, with autosomal dominant transmission of a gene encoding a primary defect in insulin secretion (232-235).

Approximately 1 to 2 % of diabetes in Europe is MODY (236). The clinical characteristics of these patients are heterogeneous, and not reliable in predicting the underlying pathogenesis (237,238). It is often misdiagnosed as T1DM or T2DM. Several genetic abnormalities have been found that account for the disorder. Some members of an affected family may have the genetic defect but not develop the diabetes phenotype. Whether this is due to modifying genes or environmental factors is unclear. MODY differs from the classical immunological T1DM in several ways. With MODY, a dominant family history of diabetes (if known) is always present.  However, de novo mutations can occur.  Hyperglycemia is mostly mild with a minimal tendency to ketosis before the age of 25 years, the insulin secretion in response to oral (OGTT) or intravenous (IVGTT) glucose administration is modestly decreased, and evidence of islet cell autoimmunity is absent. It is estimated that more than 80% of patients with monogenic diabetes are either not diagnosed or are misclassified as type 1 or type 2 DM (239).

The underlying genetic defects of the many MODY subtypes have been identified, as indicated below (Table 11). To date, fourteen genetic forms of MODY are recognized. MODY resulting from defects in the glucokinase gene (GCK) and hepatocyte nuclear factor-1-alpha (HNF-1α) are the most common types seen during childhood (MODY-2) and post puberty (MODY-3), respectively.  MODY Types 2 and 3 together constitute 80% of all cases of MODY syndromes.

MODY 2 is the most common form of MODY with a prevalence of about 1:1000 people. It is caused by a dominant heterozygous inactivating mutation in glucokinase, the enzyme that phosphorylates glucose to permit its oxidation to ATP and hence insulin release. Insulin is released but at higher glucose concentration-the curve is right shifted but otherwise normal. Thus, fasting glucose is in the range of ~95-110 mg/dl and may remain above 140 mg/dl at 2 hours post prandial but returns to normal thereafter. HbA1c is in the range of 5.8-7.6% and generally remains in the low- mid 6% range. Patients are rarely symptomatic and may be discovered by chance when a blood glucose is obtained. Treatment is not necessary except during pregnancy in some cases; there is a very low prevalence of micro-macrovascular disease even after almost 50 years of follow-up. Young women are often discovered to have mid hyperglycemia when tested during pregnancy and erroneously labeled as having gestational diabetes. The non-affected fetus of an affected Mother may have some macrosomia in utero-the result of extra insulin secretion by the fetus in response to the maternal hyperglycemia (240).

MODY3 is the next most common form of MODY caused by a heterozygous mutation in HNF-1α, necessary for normal insulin secretion. Onset is usually in the teen years and glucose is in the mid-200s with mild to moderate symptoms. Patients may respond to sulfonylurea drugs initially, but later may go on to insulin dependence and more severe hyperglycemia. As with other MODY forms, a family history of diabetes is often obtained, with a diagnosis of T2DM common for older patients and T1DM in younger patients. Confirmation of the diagnosis by molecular testing is essential for recommending treatment and family counseling (241).

Defects in four pancreatic ß cell-specific transcription factor genes, HNF-1β (MODY5), HNF-4α (MODY1), pancreatic and duodenal homeobox 1 gene (PDX1) [previously termed insulin promoter factor-1 (IPF-1)] (MODY4) and neurogenic differentiation 1 gene (NeuroD1) and BETA2 (MODY6) are responsible for others. In contrast to MODY-2, patients with heterozygous mutations in the HNF1A, HNF4A, or HNF1B and more rarely in PDX1 or NEUROD1 have progressive deterioration in glucose tolerance and are at risk for developing complications of diabetes (242).

More recently, mutations in the tumor suppressor protein KLF-11 (MODY7), the carboxyl ester lipase CEL (MODY8), the transcription factor, paired box gene 4, PAX-4 (MODY9), the insulin gene, INS (MODY10), and tyrosine kinase, B-lymphocyte specific gene, BLK (MODY11) have been described.  MODY 12 and MODY 13 are due to mutations in the ABCC8 and KCNJ11 genes, respectively. Mutations in these 2 genes also have been reported in neonatal diabetes.  They are very rare and represent fewer than 1% of all MODY cases.

Neonatal Diabetes

Neonatal diabetes is a rare disorder with an incidence of 1:100,000-1:200,000 live births (232,258).  It presents in first 6 months of life and its’ severity depends on the underlying mutation in that it is either transient or permanent. Almost 50% of cases with neonatal diabetes are permanent (PND) while the remainder are “transient” (TNDM) in that they remit, but may reappear and become apparent later in life or at times of stress. Heterozygous activating mutations in KCNJ11 and ABCC8 —which encode the Kir6.2 and SUR1 subunits, respectively, of the ATP-sensitive potassium channel, are the most common causes of PND. Missense mutations in the INS gene are also identified in patients with PND and they may have an autosomal dominant or recessive inheritance pattern (232,254,258). Genetic diagnosis is important since the KCNJ11 and ABCC8 mutations respond to treatment by sulfonylureas, possibly without need for additional insulin therapy because these drugs can close the β cell potassium channel by an ATP-independent route (259). It is increasingly apparent that the same mutations can become manifest for the first time well beyond infancy and diagnosed as T2DM or rarely T1DM. Severe mutations in the KATP genes, especially KCNJ11 also may present with a neurological component in a syndrome known as DEND (Developmental delay, Epilepsy, Neonatal Diabetes); early diagnosis and treatment with sulfonylurea drugs is reported to ameliorate the neurological manifestations as the KATP channels are expressed in the brain. The major form of   transient neonatal diabetes results from anomalies of the imprinted region on chromosome 6q24,but mutations in KCNJ11 or ABCC8 can also cause TNDM (232).  Various rare forms of syndromic disease which include NDM are described; early diagnosis may diminish or delay the hitherto described natural history and consequences (258).

Mitochondrial Diabetes

Point mutations in mitochondrial m.3243A→G cause another form of diabetes with an insulin secretory defect that is commonly associated with neuro-sensory hearing impairment and a strict maternal mode of inheritance (260). In addition, genetic abnormalities that result in the inability to convert pro-insulin to insulin (261), or the production of mutant insulin molecules (262), are other examples of specific genetic defects in ß cell function which are rare causes of diabetes.

Chronic Illnesses

Hemochromatosis is a progressively more common recognized cause of diabetes with aging, and does not present in a pediatric age group. However repeated blood transfusions for conditions such as thalassemia major can lead to diabetes associated with hemosiderosis.

Many patients with cystic fibrosis develop a form of T1DM often during their teenage years which may require insulin replacement and is labeled “cystic fibrosis related diabetes (CFRD)” (263).  Most CF patients now live long enough for this to have become a more common problem with impact on overall well-being and severity of symptoms ascribed to CF and partially responsive to insulin therapy. DKA is rare in CFRD, perhaps because of the concurrent effects on the α-cell secreting glucagon as well as the β-cell secreting insulin. Patients with Gitelman’s syndrome develop diabetes which resolves when they are adequately replaced with magnesium, excessively lost through the kidneys in this syndrome. Gitelman syndrome is a recessively inherited genetic entity, but the presentation of DM is usually not until later midlife (264).

Genetic Defects in Insulin Action

There are a series of rare genetic abnormalities in the insulin receptor, or in the signal transduction events which follow insulin docking to its receptor resulting in diabetes. The recessive DNA breakage disease (Bloom’s syndrome) is associated with mild diabetes due to severe insulin resistance, with very high levels of circulating insulin and insulin like growth factor one (IGF-1). Progeria and lipodystrophy are other such causes (232). In the latter case, the absolute deficiency of leptin leads to uncontrolled lipolysis resulting in severe insulin resistance, which is partially reversible by leptin administration (232)/

Endocrinopathies Associated with Hyperglycemia

Several hormones, such as epinephrine, glucagon, cortisol, and growth hormone, antagonize the action of insulin. Whereas release of these hormones constitutes the protective counter regulatory response to hypoglycemia, primary over secretion of these hormones can result in glucose intolerance or overt diabetes.

• Cushing's syndrome, due to pituitary and ACTH secreting adenomas or adrenal hyperplastic disease or to exogenous glucocorticoid administration, can lead to diabetes (265). Steroid-induced diabetes is most often seen when there is pre- existing insulin resistance or a defect in insulin synthesis/secretion unmasked by the inability to increase insulin secretion to overcome the resistance to its actions induced by glucocorticoids.
• Acromegaly is associated with overt diabetes in 10 to 15% of cases, and impaired glucose tolerance in a further 50% (266,267). In acromegaly, there is marked insulin resistance and hyperinsulinemic responses; DM occurs only when the hyperinsulinemic response cannot match the requirement to overcome the degree of resistance.
• Pheochromocytomas are associated with both inhibition of insulin secretion and an increase in hepatic glucose output (268). These changes lead to impaired glucose tolerance, the severity of which is directly related to the magnitude of catecholamine production (269). When seen in children, these are usually a component of the Von Hippel-Lindau syndrome, MEN2, and NF1.
• Glucagon-secreting tumors (glucagonoma) are associated with an unusual constellation of clinical features, including skin rash, weight loss, anemia, and thromboembolic problems. Approximately 80% of these patients have either impaired glucose tolerance or diabetes (270).
• Somatostatin-secreting tumors (somatostatinomas) are typically associated with the triad of diabetes mellitus, cholelithiasis, and diarrhea with steatorrhea (271).
• Although thyroxine is not a counter regulatory hormone, hyperthyroidism can interfere with glucose metabolism. It is associated with both increased sensitivity of pancreatic ß cells to glucose, resulting in increased insulin secretion, and antagonism to the peripheral action of insulin. The latter effect usually predominates, leading to impaired glucose tolerance in some untreated patients (272).

Drug- or Chemical-induced Diabetes 

A large number of drugs can impair glucose tolerance; they may act by decreasing insulin secretion, increasing hepatic glucose production, and/or by causing resistance to the action of insulin (273). Included in this list are several classes of antihypertensive drugs, such as beta blockers (274), protease inhibitors used for the treatment of HIV infection (275), and tacrolimus and cyclosporine used primarily to prevent transplant rejection (276,277). Drugs of the serotonin re-uptake inhibitor (SSRIs) class can lead to obesity, impaired glucose intolerance and T2DM, especially if individuals were already insulin resistant before they started such medications.

There is a common association between obesity, insulin resistance, hypertension, and dyslipidemia, which has been called syndrome X or the metabolic syndrome (207,212,278,279). The administration of a thiazide diuretic or a ß-blocker to such patients can exacerbate the insulin resistance and may bring on hyperglycemia (274). In comparison, angiotensin-converting enzyme (ACE) inhibitors and alpha-adrenergic antagonists (such as doxazosin) may improve insulin sensitivity. Because the former also protect against renal disease, they are the drugs of choice for diabetic patients with hypertension.

Viral Infections

Certain viruses e.g., Coxsackie B4, have been implicated to cause diabetes, either through direct ß cell destruction or possibly by inducing autoimmune damage. The direct proof of this however remains tenuous. Chronic hepatitis C virus infection is associated with an increased incidence of diabetes, but it remains uncertain as yet if there is a cause-and-effect relationship.

Uncommon Forms of Immune-Mediated Diabetes

Several uncommon forms of immune-mediated diabetes have been identified.

• The stiff-man syndrome is an autoimmune disorder of the central nervous system, which is characterized by progressive muscle stiffness, rigidity, and spasms involving the axial muscles, with impairment of ambulation (280). Patients characteristically have high titers of glutamic acid decarboxylase (GAD65) autoantibodies and diabetes occurs in at least one-third of cases. Graves’ disease is also common in the syndrome. Presentation is usually in early
• Anti-insulin receptor antibodies can bind to insulin receptors and either act as an agonist, leading to hypoglycemia, or block the binding of insulin and cause diabetes (281). This so-called type B insulin resistance is more common in females who show other signs of autoimmunity including systemic lupus erythematosus (SLE). However one study found that almost 10% of young patients with insulin resistance in the absence of autoimmune stigmata were also positive for insulin receptor autoantibodies (282).

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