Type 1 and Type 2

Diabetes Mellitus

Function of Insulin

Definition of DiabetesTypes of Diabetes MellitusType 1: Insulin-Dependent Diabetes Mellitus (IDDM)Genetics of Type 1 DiabetesDiabetic KetoacidosisType 2: Non-Insulin-Dependent Diabetes Mellitus (NIDDM)Measurement of HbA1c Levels

Genetics of Type 2 DiabetesNeonatal DiabetesDiabetes and the Metabolic Syndrome: MetSMitochondrial Dysfunction in Type 2 Diabetes and ObesityTherapeutic Intervention for HyperglycemiaNew Frontiers in Diabetes Treatment

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Definition of Diabetes

Diabetes is any disorder characterized by excessive urine excretion. The most common form of diabetes isdiabetes mellitus, a metabolic disorder in which there is an inability to oxidize carbohydrate due to disturbancesin insulin function. Diabetes mellitus is characterized by elevated glucose in the plasma and episodicketoacidosis. Additional symptoms of diabetes mellitus include excessive thirst, glucosuria, polyuria, lipemia andhunger. If left untreated the disease can lead to fatal ketoacidosis. Other forms of diabetes include diabetesinsipidus and brittle diabetes. Diabetes insipidus is the result of a deficiency of antidiuretic hormone (ADH, alsoreferred to as vasopressin or arginine vasopressin, AVP). The major symptom of diabetes insipidus (excessiveoutput of dilute urine) results from an inability of the kidneys to resorb water. Brittle diabetes is a form that is verydifficult to control. It is characterized by unexplained oscillations between hypoglycemia and acidosis.

Criteria, which clinically establish an individual as suffering from diabetes mellitus, include:

1. having a fasting plasma glucose level in excess of 126mg/dL (7mmol/L). Normal levels should be lessthan 100mg/dL (5.6mmol/L) or:

2. having plasma glucose levels in excess of 200mg/dL (11mmol/L) at two times points during an oral

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glucose tolerance test, OGTT, one of which must be within 2 hrs of ingestion of glucose.

Different clinical labs may use different units for the measurement of serum glucose concentrations, either inmmol/L or mg/dL. One can easily interconvert these values using the following formulas:

mg/dL x 0.0555 = mmol/L (or simply divide mg/dL by 18)

mmol/L x 18.0182 = mg/dL (or simply multiply mmol/L by 18)

The earlier a person is diagnosed with diabetes (principally type 2) the better chance the person has ofstaving off the primary negative consequences which are renal failure, blindness and limb amputations due tocirculatory problems. The American Diabetes Association is planning to recommend that physicians considerpatients to be pre-diabetic if their fasting blood glucose level is above 100mg/dL but less than 125mg/dL andwhose glucose levels are at least 140mg/dL but less than 200mg/dL following an oral glucose tolerance test(OGTT).

Glucose tolerance curve for a normal person and one with non-insulin-dependent diabetes mellitus(NIDDM, Type 2 diabetes). The dotted lines indicate the range of glucose concentration expected in a normalindividual.

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Types of Diabetes Mellitus

Diabetes mellitus is a heterogeneous clinical disorder with numerous causes. Two main classifications ofdiabetes mellitus exist, idiopathic and secondary.

Idiopathic diabetes is divided into two main types; insulin dependent and non-insulin-dependent. Insulin-dependent diabetes mellitus, IDDM (more commonly referred to as type 1 diabetes) is defined by thedevelopment of ketoacidosis in the absence of insulin therapy. See the Diabetic Ketoacidosis diagnosis andtreatment page. Type 1 diabetes most often manifests in childhood (hence, also called juvenile onset diabetes)and is the result of an autoimmune destruction of the -cells of the pancreas. Non-insulin-dependent diabetesmellitus, NIDDM (more commonly referred to as type 2 diabetes) is characterized by persistent hyperglycemiabut rarely leads to ketoacidosis. Type 2 diabetes generally manifests after age 40 and therefore has the obsoletename of adult onset-type diabetes. Type 2 diabetes can result from genetics defects that cause both insulinresistance and insulin deficiency. There are two main forms of type 2 diabetes:

1. Late onset associated with obesity.

2. Late onset not associated with obesity.


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There is a strong correlation between obesity and the onset of type 2 diabetes with itsassociated insulin resistance. It should be pointed out that in the United States theproportion of the population under 40 that can be clinically defined as obese now exceeds25%. Many children are obese and are developing type 2 diabetes at an alarmingepidemic rate. The dramatic rise in obesity in the US has lead to an equally alarmingincrease in the percentage of the population who suffer from the metabolic syndrome.The metabolic syndrome is a clustering of atherosclerotic cardiovascular disease riskfactors, one of which involves insulin resistance characteristic in type 2 diabetes. It shouldbe pointed out that obesity alone does not always lead to insulin resistance as someindividuals who are obese do not experience insulin resistance and conversely, someindividuals who manifest insulin resistance are not obese. These latter observations pointto the added role of genetics in the acquisition of insulin resistance.

Secondary, or other specific types of diabetes mellitus are the result of many causesincluding:

1. Maturity onset type diabetes of the young (MODY) was previously considered to bea third form of type 2 diabetes. However, with the discovery of specific mutations leadingto MODY, it is now classified under secondary or other specific types of diabetes. MODYis characterized by onset prior to age 25. All cases to date have shown impaired -cellfunction. Patients may also exhibit insulin resistance and late -cell failure. Evidenceindicates that mutations in 10-12 different genes have been correlated with thedevelopment of MODY. Mutations in the 8 genes described here are all clearly correlatedto MODY:

MODY1: the transcription factor identified as hepatic nuclear factor-4 (HNF-4;gene symbol = HNF4A). This gene is also called transcription factor-14 (TCF14).Expression of HNF-4 is associated with the growth and normal functioning of thepancreas. Many genes are known to be regulated by HNF-4 including thoseencoding HNF-1, PPAR, insulin, glucose-6-phosphatase, GLUT2, the liverpyruvate kinase isoform (L-PK) which is also expressed in the pancreas,glyceraldehyde-3-phosphate dehydrogenase (G3PDH), aldolase B and uncouplingprotein 2, UCP2.

MODY2: pancreatic glucokinase

MODY3: the transcription factor HNF-1 (gene symbol = HNF1A). This gene is also called hepatocytetranscription factor-1 (TCF1). HNF-1 is involved in a regulatory loop with HNF-4 controlling many genesinvolved in liver function such as the GLUT2 and L-PK genes.

MODY4: the homeodomain transcription factor insulin promoter factor-1 (IPF-1). This gene is morecommonly called PDX1 which is derived from pancreas duodenum homeobox-1.

MODY5: the transcription factor HNF-1. This gene is also called hepatocyte transcription factor-2 (TCF2).HNF-1 is a critical regulator of a transcriptional network that controls the specification, growth, anddifferentiation of the embryonic pancreas. In humans, mutations in the HNF-1 gene (symbol = HNF1B) areassociated with pancreatic hypoplasia, defective kidney development and genital malformations.

MODY6: the bHLH transcription factor NeuroD1. NeuroD1 was first identified as a neural fate-inducinggene. The hamster 2 gene, shown to regulate insulin transcription is identical to NeuroD1 so the gene isoften called NeuroD/2. MODY6 is a rare form of MODY

MODY7: the Krupple-like factor 11 (KLF11) protein is a zinc-finger transcription factor that is involved inactivation of the insulin promoter. KLF11 is a TGF--inducible transcription factor.

MODY8: the carboxyl-ester lipase gene (CEL) which is involved in lipid metabolism. Frameshift deletions inthe variable number tandem repeats (VNTR) of the CEL gene are associated with MODY8 which ischaracterized by pancreatic exocrine and -cell dysfunction. MODY8 is a rare form of MODY.

2. Pancreatic disease: Pancreatectomy leads to the clearest example of secondary diabetes. Cystic fibrosisand pancreatitis can also lead to destruction of the pancreas.

3. Endocrine disease: Some tumors can produce counter-regulatory hormones that oppose the action ofinsulin or inhibit insulin secretion. These counter-regulatory hormones are glucagon, epinephrine, growthhormone and cortisol.

a. Glucagonomas are pancreatic cancers that secrete glucagon.

b. Pheochromocytomas secrete epinephrine.

c. Cushing syndrome results from excess cortisol secretion.

d. Acromegaly results in excess growth hormone production.

4. Drug-induced diabetes; treatment with glucocorticoids and diuretics can interfere with insulin function.

5. Anti-insulin receptor autoantibodies (Type B insulin resistance).

6. Mutations in the insulin gene.

7. Mutations in insulin receptor gene which lead to the syndromes listed below. Two clinical features are

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common in all syndromes that result from mutations in the insulin receptor gene: acanthosis nigricans andhyperandrogenism (the latter being observed only in females).

a. Donohue syndrome (also referred to as Leprachaunism)

b. Rabson-Mendenhall syndrome

c. Type A insulin resistance

8. Gestational diabetes; this syndrome sets in during pregnancy and usually resolves itself followingchildbirth.

9. Many other genetic syndromes have either diabetes or impaired glucose tolerance associated with them;lipoatrophic diabetes, Wolfram syndrome, Down syndrome, Klinefelter syndrome (XXY males), Turner syndrome,myotonic dystrophy, muscular dystrophy, Huntington disease, Friedreich ataxia, Prader-Willi syndrome, Wernersyndrome, Cockayne syndrome, and others such as those indicated above.

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Insulin-Dependent Diabetes Mellitus (IDDM) Type 1

Etiology of Type 1 Diabetes

Type 1 diabetes has been shown to be the result of an autoimmune reaction to antigens of the islet cells ofthe pancreas. There is a strong association between IDDM and other endocrine autoimmunities (e.g. Addisondisease). Additionally, there is an increased prevalence of autoimmune disease in family members of IDDMpatients.

Types of Autoantibodies

1. Islet cell cytoplasmic antibodies: The primary antibodies found in 90% of type 1 diabetics are against isletcell cytoplasmic proteins (termed ICCA, islet cell cytoplasmic antibodies). In non-diabetics ICCA frequency is only0.5%4%. The presence of ICCA is a highly accurate predictor of future development of IDDM. ICCA are notspecific for the -cells and recognize antigens in other cell types in the islet. However, the autoimmune attackappears to selectively destroy -cells. Therefore, the antibodies may play a primary role in the destruction of isletcells. It is an equally likely possibility that the production of anti-islet antibodies occurs as a result of thedestruction of -cells. Whether a direct cause or an effect of islet cell destruction, the titer of the ICCA tends todecline over time.

2. Islet cell surface antibodies: Autoantibodies directed against cell-surface antigens (ICSA) have also beendescribed in as many as 80% of type 1 diabetics. Similar to ICCA, the titer of ICSA declines over time. Somepatients with type 2 diabetes have been identified that are ICSA positive.

3. Specific antigenic targets of islet cells: Antibodies to glutamic acid decarboxylase (GAD) have beenidentified in over 80% of patients newly diagnosed with IDDM. Like ICCA, anti-GAD antibodies decline over timein type 1 diabetics. There are two GAD genes in humans identified as GAD1 and GAD2. The GAD isoformsproduced by these two genes are identified as GAD67 (GAD1 gene: GAD67) and GAD65 (GAD2 gene: GAD65)

which is reflective of their molecular weights. Both the GAD1 and GAD2 genes are expressed in the brain andGAD2 expression also occurs in the pancreas. The presence of anti-GAD antibodies (both anti-GAD65 andanti-GAD67) is a strong predictor of the future development of IDDM in high-risk populations. Anti-insulinantibodies (IAA) have been identified in IDDM patients and in relatives at risk to develop IDDM. These IAA aredetectable even before the onset of insulin therapy in type 1 diabetics. IAA are detectable in around 40% ofyoung children with IDDM.

Pathophysiology of Type 1 Diabetes

The autoimmune destruction of pancreatic -cells leads to a deficiency of insulin secretion. It is this loss ofinsulin secretion that leads to the metabolic derangements associated with IDDM. In addition to the loss of insulinsecretion, the function of pancreatic -cells is also abnormal. There is excessive secretion of glucagon in IDDMpatients. Normally, hyperglycemia leads to reduced glucagon secretion. However, in patients with IDDM,glucagon secretion is not suppressed by hyperglycemia. The resultant inappropriately elevated glucagon levelsexacerbates the metabolic defects due to insulin deficiency (see below). The most pronounced example of thismetabolic disruption is that patients with IDDM rapidly develop diabetic ketoacidosis in the absence of insulinadministration. If somatostatin is administered to suppress glucagon secretion, there is a concomitantsuppression in the rise of glucose and ketone bodies. Particularly problematic for long term IDDM patients is animpaired ability to secrete glucagon in response to hypoglycemia. This leads to potentially fatal hypoglycemia inresponse to insulin treatment in these patients.

Although insulin deficiency is the primary defect in IDDM, in patients with poorly controlled IDDM there is alsoa defect in the ability of target tissues to respond to the administration of insulin. There are multiple biochemicalmechanisms that account for this impairment of tissues to respond to insulin. Deficiency in insulin leads toelevated levels of free fatty acids in the plasma as a result of uncontrolled lipolysis in adipose tissue. Free fattyacids suppress glucose metabolism in peripheral tissues such as skeletal muscle. This impairs the action ofinsulin in these tissues, i.e. the promotion of glucose utilization. Additionally, insulin deficiency decreases theexpression of a number of genes necessary for target tissues to respond normally to insulin such as glucokinasein liver and the GLUT 4 class of glucose transporters in adipose tissue. The major metabolic derangements which


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result from insulin deficiency in IDDM are impaired glucose, lipid and protein metabolism.

Glucose Metabolism: Uncontrolled IDDM leads to increased hepatic glucose output. First, liver glycogenstores are mobilized then hepatic gluconeogenesis is used to produce glucose. Insulin deficiency also impairsnon-hepatic tissue utilization of glucose. In particular in adipose tissue and skeletal muscle, insulin stimulatesglucose uptake. This is accomplished by insulin-mediated movement of glucose transporter proteins to theplasma membrane of these tissues. Reduced glucose uptake by peripheral tissues in turn leads to a reduced rateof glucose metabolism. In addition, the level of hepatic glucokinase is regulated by insulin. Therefore, a reducedrate of glucose phosphorylation in hepatocytes leads to increased delivery to the blood. Other enzymes involvedin anabolic metabolism of glucose are affected by insulin (primarily through covalent modifications). Thecombination of increased hepatic glucose production and reduced peripheral tissues metabolism leads toelevated plasma glucose levels. When the capacity of the kidneys to absorb glucose is surpassed, glucosuriaensues. Glucose is an osmotic diuretic and an increase in renal loss of glucose is accompanied by loss of waterand electrolytes, termed polyuria. The result of the loss of water (and overall volume) leads to the activation ofthe thirst mechanism (polydipsia). The negative caloric balance which results from the glucosuria and tissuecatabolism leads to an increase in appetite and food intake (polyphagia).

Lipid Metabolism: One major role of insulin is to stimulate the storage of food energy following theconsumption of a meal. This energy storage is in the form of glycogen in hepatocytes and skeletal muscle.Additionally, insulin stimulates hepatocytes to synthesize triglycerides and storage of triglycerides in adiposetissue. In opposition to increased adipocyte storage of triglycerides is insulin-mediated inhibition of lipolysis. Inuncontrolled IDDM there is a rapid mobilization of triglycerides leading to increased levels of plasma free fattyacids. The free fatty acids are taken up by numerous tissues (however, not the brain) and metabolized to provideenergy. Free fatty acids are also taken up by the liver.

Normally, the levels of malonyl-CoA are high in the presence of insulin. These high levels of malonyl-CoAinhibit carnitine palmitoyltransferase I, the enzyme required for the transport of fatty acyl-CoA's into themitochondria where they are subject to oxidation for energy production. Thus, in the absence of insulin,malonyl-CoA levels fall and transport of fatty acyl-CoA's into the mitochondria increases. Mitochondrial oxidationof fatty acids generates acetyl-CoA which can be further oxidized in the TCA cycle. However, in hepatocytes themajority of the acetyl-CoA is not oxidized by the TCA cycle but is metabolized into the ketone bodies,acetoacetate and -hydroxybutyrate. These ketone bodies leave the liver and are used for energy production bythe brain, heart and skeletal muscle. In IDDM, the increased availability of free fatty acids and ketone bodiesexacerbates the reduced utilization of glucose furthering the ensuing hyperglycemia. Production of ketone bodies,in excess of the organisms ability to utilize them leads to ketoacidosis. In diabetics, this can be easily diagnosedby smelling the breath. A spontaneous breakdown product of acetoacetate is acetone which is volatilized by thelungs producing a distinctive odor.

Normally, plasma triglycerides are acted upon by lipoprotein lipase (LPL), an enzyme on the surface of theendothelial cells lining the vessels. In particular, LPL activity allows fatty acids to be taken from circulatingtriglycerides for storage in adipocytes. The activity of LPL requires insulin and in its absence ahypertriglyceridemia results.

Protein Metabolism: Insulin regulates the synthesis of many genes, either positively or negatively that thenaffect overall metabolism. Insulin has a global effect on protein metabolism, increasing the rate of proteinsynthesis and decreasing the rate of protein degradation. Thus, insulin deficiency will lead to increasedcatabolism of protein. The increased rate of proteolysis leads to elevated concentrations in plasma amino acids.These amino acids serve as precursors for hepatic and renal gluconeogensis. In liver, the increasedgluconeogenesis further contributes to the hyperglycemia seen in IDDM.

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Genetics of Type 1 Diabetes

The majority of genetic loci associated with the development of type 1 diabetes (T1D) map to the humanleukocyte antigen (HLA) class II proteins which are encoded for by genes in the major histocompatibility complex(MHC) which is located on chromosome 6p21. The Figure below diagrams a simplified view of the MHC clusterwhich spans 3.5 megabases of chromosome 6 and encompasses over 200 genes divided into three subregionstermed class I, class II and class III.


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Simplified view of the MHC cluster genes. The class I genes encode peptide chains, which associate with2 microglobulin to form the class I molecules. Class I molecules are expressed on the surface of all nucleatedcells where they are involved in the restriction of cytotoxic T cell activity. The class II (HLA-D) loci aresubdivided into at least one A and one B gene which encode the and peptide chains, respectively. Theclass II proteins combine to form heterodimeric molecules that are expressed on antigen presenting cells, Bcells, and activated T cells. The HLA-DP, HLA-DQ, and HLA-DR molecules are involved in the activation ofhelper T cells. There are nine B genes in the DR cluster identified as DRB1DRB9. There are five distinct DRhaplotypes in humans identified as DR1 (composed of the DRB1, DRB6, and DRB9 genes), DR51 (composedof the DRB1, DRB6, DRB5, and DRB9 genes), DR52 (composed of the DRB1, DRB2, DRB3, and DRB9genes), DR8 (composed of the DRB1 and DRB9 genes), and DR53 (composed of the DRB1, DRB7, DRB8,DRB4, and DRB9 genes). The current MHC nomenclature arranges the DR sequences into different allelicgroups. DRB1 sequences are arranged into 13 different allelic groups that through phylogenetic analysescluster within the five haplotypes outlined above. These allelic groups are denoted: *01 and *10 (the DR1group), *08 (the DR8 group), *15 and *16 (the DR51 group), *03, *11, *12, *13, and *14 (the DR52 group), and*04, *07, and *09 (the DR53 group). The second expressed DRB loci (DRB3, DRB4, and DRB5) exhibit limitedpolymorphisms in the human genome. The class III genes encode a range of molecules with a variety offunctions, including complement components, tumor necrosis factor (TNF), and heat shock protein, Hsp70.

This is not to say that all genetic associations in T1D are due to mutations in HLA genes as more than 40additional T1D susceptibility loci have been identified that are not HLA genes. The most frequently observednon-HLA genes associated with T1D are the insulin (INS), protein tyrosine phosphatase, non-receptor type 22(PTPN22), cytotoxic T-lymphocyte-associated protein 4 (CTLA4), interleukin-2 receptor alpha (IL2RA), andinterferon-induced with helicase C domain 1 (IFIH1) genes. The INS gene is on chromosome 11p15.5, thePTPN22 gene is on chromosome 1p13, the CTLA4 gene is on chromosome 2q33, the IL2RA gene is onchromosome 10p15.1, and the IFIH1 gene is on chromosome 2q24.

Polymorphisms in the INS gene account for approximately 10% of genetic susceptibilities to T1D. All of theINS gene polymorphisms reside outside the coding region of the gene indicating that susceptibility to T1D isrelated to modulation of expression of the INS gene. The PTPN22 gene encodes a protein identified as lymphoid-specific phosphatase (LYP) which is involved in the prevention of spontaneous T cell activation. One of thepolymorphisms in the PTPN22 gene that is associated with T1D susceptibility is also associated with otherautoimmune diseases such as systemic lupus erythematosus (SLE), Graves disease, and rheumatoid arthritis(RA). The protein encoded by the CTLA4 gene is also involved in regulating T cell activation and likepolymorphisms in the PTPN22 gene, polymorphisms in CTLA4 are associated with other autoimmune disorderssuch as Addison disease and Graves disease.

The highest risk population for the development of T1D are children born with the HLA DR3/4DQ8 serotypeallele which accounts for almost 50% of all children who develop antibodies against pancreatic islet cells and thusdevelop T1D by the age of 5. HLA DR serotype alleles are molecules that recognize different DR gene products.The DR3 serotype recognizes the DRB1*03 gene products and the DR4 serotype recognizes the DRB1*04 geneproducts. Children with the high risk HLA alleles DR3/4DRQ or DR4/DR4 and who have a family history of T1Dhave a nearly 1 in 5 chance of developing islet cell autoantibodies resulting in T1D. These same children borninto a family with no history of T1D still have a 1 in 20 chance of developing T1D. It should be pointed out thatalthough there are these strong genetic associations to T1D over 85% of all children who develop the disease donot have a family history associated with T1D. The class II HLA molecules that are associated with increased riskof T1D have been shown to bind peptides derived from the currently identified autoantigens described above and

present these peptides to CD4+ T cells which then activate CD8+ cytotoxic T cells resulting in killing of islet cells.

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Non-Insulin-Dependent Diabetes Mellitus (NIDDM): Type 2

Etiology of Type 2 Diabetes

Type 2 diabetes is characterized by a lack of the need for insulin to prevent ketoacidosis. Type 2 diabetesrefers to the common form of idiopathic NIDDM. Type 2 diabetes is not an autoimmune disorder, however, thereis a strong genetic correlation to the susceptibility to this form of diabetes. The susceptibility genes thatpredispose one to NIDDM have not been identified in most patients. This is due in part to the heterogeneity of thegenes responsible for the susceptibility to type 2 diabetes. Obesity is a major risk factor that predisposes one totype 2 diabetes. Genetic studies in mice and rats have demonstrated a link between genes responsible forobesity and those that cause diabetes mellitus.

Pathophysiology of Type 2 Diabetes

Unlike patients with type 1 diabetes, those with type 2 diabetes have detectable levels of circulating insulin.On the basis of oral glucose tolerance testing the essential elements of type 2 diabetes can be divided into 4distinct groups; those with normal glucose tolerance, chemical diabetes (called impaired glucose tolerance),diabetes with minimal fasting hyperglycemia (fasting plasma glucose 140 mg/dL). In patients with the highestlevels of plasma insulin (impaired glucose tolerance group) there was also elevated plasma glucose. Thisindicates that these individuals are resistant to the action of insulin. In the progression from impaired glucose


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tolerance to diabetes mellitus the level of insulin declines indicating that patients with type 2 diabetes havedecreased insulin secretion.

Additional studies have subsequently demonstrated that both insulin resistance and insulin deficiency iscommon in the average type 2 diabetic patient. Many experts conclude that insulin resistance is the primarycause of type 2 diabetes, however, others contend that insulin deficiency is the primary cause because amoderate degree of insulin resistance is not sufficient to cause type 2 diabetes. As indicated above, most patientswith the common form of type 2 diabetes have both defects.

The major clinical complications of type 2 diabetes are the result of persistent hyperglycemia which leads tonumerous pathophysiological consequences. As the glucose level rises in the blood the blood becomes moreviscous which makes circulation of the blood in the small capillaries difficult. The reduced circulation results inprogressive vascular complications leading to diabetic retinopathy (referred to as diabetic blindness), peripheralneuropathy (resulting in numbness in the extremities and tingling in fingers and toes), poor wound healing, anderectile dysfunction. In addition to these major clinical complications, the body reacts by increasing the level ofglucose excretion by the kidneys leading to frequent urination which is called polyuria. As the glucose is excretedthere is a concomitant loss of water to maintain normal osmolarity of the urine. The water loss leads to excessivethirst called polydypsia.

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Measurement of HbA1c Levels

The development of hypoglycemia inducing drugs is the major pharmacological focus of type 2 diabetestherapies. Assessment of therapeutic efficacy in the treatment of the hyperglycemia in type 2 diabetes isaccomplished by routine measurement of the circulating levels of glycosylated hemoglobin, designated as thelevel of HbA1c, often designated as just A1C. HbA1 is the major form of adult hemoglobin in the blood and the "c"

refers to the glycosylated form of the protein. Since hemoglobin is present in red blood cells and these cells havea limited life span of 120 days in the circulation, measurement of HbA1c levels is a relatively accurate measure of

the amount of glucose in the blood and the length of time the level has been elevated. Typical values for HbA1cmeasurement (using the previous standard Diabetes Control and Complications Trial, DCCT units of %) areshown in the Table below. Beginning in 2011 a new international standard (International Federation of ClinicalChemistry, IFCC units) for the measurement of HbA1c levels will be utilized. This new standard equates the

mmole of HbA1c per mole of total measured hemoglobin, Hb (mmol/mol). The method for calculating the

relationship between these two measurement values is to use the following formula:

IFCC-HbA1c (mmol/mol) = [DCCT-HbA1c (%) - 2.15] 10.929.

To calculate the estimated average glucose (eAG) level in the blood using the DCCT (%) values one woulduse the following formula:

eAG(mg/dl) = 28.7 A1C 46.7 (for glucose level in mM use: eAG(mM) = 1.59 A1C 2.59

With new IFCC standard the target range of HbA1c for healthy levels is 4859mmol/mol.

HbA1cHbA1c/Hb

mmol/moleAG (mg/dl) eAG (mM)

4% 20 68 3.8

5% 31 97 5.4

6% 42 125 7

7% 53 154 8.5

8% 64 183 10

9% 75 212 11.7

10% 86 240 13.3

11% 97 270 15

12% 108 298 16.5

13% 119 326 18

14% 130 355 19.7


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Genetics of Type 2 Diabetes

Development of type 2 diabetes is the result of multifactorial influences that include lifestyle, environment andgenetics. The disease arises when insulin resistance-induced compensatory insulin secretion is exhausted. Ahigh-caloric diet coupled with a sedentary lifestyle are the major contributing factors in the development of theinsulin resistance and pancreatic -cell dysfunction. However, a predisposing genetic background has long beensuspected in playing a contributing role in the development of type 2 diabetes. By using whole-genome linkageanalysis the entire genome of affected family members can be scanned and the family members monitored overseveral generations. In addition, large numbers of affected sibling-pairs can also be studied. Using thesegenome-wide linkage methods the first major susceptibility locus for type 2 diabetes was located on chromosome2 in 1996. This locus was designated NIDDM1. The first gene identified in the NIDDM1 locus with polymorphismscorrelated to type 2 diabetes susceptibility was calpain 10 (CAPN10).

CAPN10 is a calcium-activated neutral protease that is a member of the calpain-like cysteine protease family.The CAPN10 gene is located on chromosome 2q37.3 and spans 31 kb composed of 15 exons encoding a 672amino acid protein. Variation in the non-coding region of the CAPN10 gene is associated with a threefoldincreased risk of type 2 diabetes in Mexican Americans. However, in European populations polymorphisms inCAPN10 are less contributory to type 2 diabetes than other recently discovered genes. Genetic variants inCAPN10 may alter insulin secretion or insulin action as well as the production of glucose by the liver. Recentstudies indicate that CAPN10 may have a critical role in the survival of pancreatic -cells.

Another early genetic marker for type 2 diabetes was hepatocyte nuclear factor 4- (HNF4A). Note thatHNF4A is also known to be associated with the development of MODY1 (see above). The hepatocyte nuclearfactor family of proteins was first identified as an abundant class of transcription factors in the liver. In addition tothe liver, HNF4A is expressed in pancreatic -cells, kidneys and intestines. As indicated above, mutations inHNF4A can cause MODY1 which is characterized by a normal response to insulin but an impaired insulinsecretory response in the presence of glucose. The HNF4A gene maps to a region of chromosome 20 that hasbeen linked to type 2 diabetes. Specifically the HNF4A gene is located at 20q12q13.1 and is encoded in 12exons. Single nucleotide polymorphisms (SNPs) in the HNF4A gene have an impact on pancreatic -cell functionleading to altered insulin secretion and result in the development of MODY1. The SNPs in the HNF4A gene thatare related to development of type 2 diabetes lie in a promoter element called P2. The P2 promoter is usedprimarily in pancreatic -cells, whereas, both the P1 and P2 promoters are used in liver cells. The P2 promoter isa binding site for the transcription factors HNF-1 (HNF1A), HNF-1 (HNF1B), and insulin promoter factor-1(IPF1). As described above, alteration in the function of each of these latter three transcription factors isassociated with various forms of MODY.

Recent evidence has demonstrated a role for a member of the nuclear hormone receptor superfamily ofproteins in the etiology of type 2 diabetes. The thiazolidinedione (TZD) class of drugs, used to increase thesensitivity of the body to insulin (see below), bind to and alter the function of the peroxisome proliferator-activatedreceptor-, PPAR. PPAR is also a transcription factor and, when activated, binds to another transcription factorknown as the retinoid X receptor, RXR. When these two proteins interact they bind to specific PPAR responseelements (termed PPREs) in target genes thereby regulating their expression. PPAR is a key regulator ofadipocyte differentiation; it can induce the differentiation of fibroblasts or other undifferentiated cells into maturefat cells. PPAR is also involved in the synthesis of biologically active compounds from vascular endothelial cellsand immune cells. Mutations in the gene for PPAR (gene symbol = PPARG) have been correlated with insulinresistance.

More recent genome-wide screens for polymorphisms (in particular single nucleotide polymorphisms, SNPs)in type 2 diabetes have identified several new candidate genes. The Table below lists several genes that either,reside within chromosomal loci that are highly correlated to the development of type 2 diabetes, or that have hadpolymorphisms identified in the gene itself that correlate to development of type 2 diabetes. Included in the Tableare PPARG and CAPN10 described above as well as the gene potassium inwardly-rectifying channel, subfamilyJ, member 11 (KCNJ11) which is described in the Insulin Function page.

The transcription factor TCF7L2 (transcription factor 7-like 2, T-cell specific HMG-box) is one of four TCFproteins involved in the signaling pathways initiated by the Wnt family of secreted growth factors. Two SNPsidentified in the TCF7L2 gene are the most highly correlated polymorphisms with type 2 diabetes. Given thatevidence is accumulating that Wnt and insulin signaling pathways exhibit cross-talk at the level of both the gutand the pancreas, it is likely that new targets in the treatment of type 2 diabetes will involve the interrelationshipsbetween these two factors.

In addition to the genes described in the following Table, and those described for permanent neonataldiabetes mellitus (next section), at least 25 additional genes have been shown by genome wide associationstudies (GWAS) to be associated with type 2 diabetes and/or elevated fasting plasma glucose levels.

Genes Associated with Type 2 Diabetes Susceptibility

Gene NameGene

SymbolGene Function, Comments

DiseaseMechanism


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a disintegrin-like andmetalloproteinase(ADAM) withthrombospondin type 1motif, 9

ADAMTS9

demonstrated to proteolytically cleaved bovineversican (a large extracellular matrixproteoglycan) and aggrecan (large aggregatedproteoglycan)

unknown

Ca2+/calmodulin-dependent protein kinase1-

CAMK1Dleads to activation of extracellular signal-regulated protein kinase 1 (ERK1) activity

-celldysfunction

calpain 10 CAPN10calcium-activated neutral protease, member ofthe calpain-like cysteine protease family

glucosetransport

cell division cycle 123homolog

CDC123CDC123 is in the same chromosomal regionas the CAMK1D gene

-celldysfunction

cyclin-dependentkinase-5 regulatorysubunit associatedprotein 1-like 1

CDKAL1 inhibitor of cyclin-dependent kinase 5 (CDK5)

-celldysfunction,impaired insulinsecretion

cyclin-dependent kinaseinhibitor 2A

CDKN2A/B

the CDKN2A gene produces 2 major proteins:p16(INK4), which is a cyclin-dependent kinaseinhibitor, and p14(ARF), which binds thep53-stabilizing protein MDM2, p14 is alsocalled CDKN2B

-celldysfunction

fat mass- and obesity-associated gene

FTO

catalyzes the iron- and 2-oxoglutarate-dependent demethylation of 3-methylthyminein single-stranded DNA, with concomitantproduction of succinate, formaldehyde, andCO2

obesity

hematopoieticallyexpressed homeobox

HHEX

is a transcriptional repressor in liver cells, maybe involved in the differentiation and/ormaintenance of the differentiated state inhepatocytes, is a target of the Wnt signalingpathway


hepatocyte nuclearfactor-1: hepatocytetranscription factor-2

HNF1Balso calledTCF2

mutations in gene associated with MODY5 unknown

insulin degrading enzyme IDE

is an extracellular thiol metalloprotease withpreference for insulin, also degradesamyloid- protein; the IDE gene resides withinthe same chromosomal locus as HHEX

-celldysfunction

insulin-like growthfactor-2 mRNA bindingprotein 2

IGF2BP2 binds to the IGF2 mRNA-cell

dysfunction

juxtaposed with anotherzinc-finger gene 1:TAK1(TGF-activatedkinase-1)-interactingprotein 27

JAZF1also calledTIP27

functions as a transcriptional repressor,exhibits antiapoptotic activity

-celldysfunction

potassium inwardly-rectifying channel,subfamily J, member 11

KCNJ11forms the core of the ATP-sensitive potassium(KATP) channel involved in insulin secretion,protein is also called Kir6.2

-celldysfunction

potassium channel,voltage-gated, KQT-likesubfamily, member 1

KCNQ1

pore-forming -subunit of a cardiac delayedrectifier potassium channel; also referred to asKvLQT because the gene resides in a criticalregion for the cardiac long QT syndrome-1disorder which is a region that is also in theimprinted locus associated with Beckwith-

-celldysfunction


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Weidemann syndrome; gene also expressed inepithelial cells of the exocrine and endocrinepancreas

Krppel-like factor 14 KLF14

Krppel-like transcription factors all related toDrosophila Krppel gene; are a family ofzinc-finger transcription factors; KLF14 is amaster trans regulator of adipose geneexpression

leucine-rich repeatcontaining G-proteincoupled receptor 5

LGR5

gene is expressed exclusively in the cyclingcrypt base of the columnar cells of the gut andhair follicle, protein is a glycoprotein thatassociates with integrins, the gene is a markerfor intestinal stem cells, expression isregulated by Wnt signaling

-celldysfunction

melanocortin 4 receptor MC4R

is a single exon (intronless) gene, mutations inthis gene are the most frequent genetic causeof severe obesity, receptor binds -melanocytestimulating hormone (-MSH)

obesity

melatonin receptor 1B MTNR1Bhigh affinity G-protein coupled receptor,expressed primarily in pancreatic -cells


Notch homolog 2 NOTCH2one of three mammalian homologues of theNotch gene of fruit flies which regulatescellular differentiation

unknown

peroxisome proliferator-activated receptor-(PPAR)

PPARG

transcriptional co-activator with retinoid Xreceptors (RXRs), master regulator ofadipogenesis, activation of adipocytes leads toincreased fat storage and secretion of insulin-sensitizing adipocytokines such as adiponectin

insulinsensitivity

solute carrier family 30(zinc transporter),member 8

SCL30A8 permits cellular efflux of zinc-cell

dysfunction

transcription factor 7-like2 (T-cell specificHMG-box)

TCF7L2

one of four TCF transcription factor proteinsinvolved in the signaling pathways initiated bythe Wnt family of secreted growth factors,polymorphisms in this gene have the highestcorrelation to type 2 diabetes


thyroid adenoma-associated gene

THADA

protein contains an ARM repeat (ARM =armadillo which is a fruit fly gene involved insegment polarity), the ARM repeat is involvedin protein-protein interactions

unknown

tetraspanin 8 TSPAN8tetraspanins are proteins that contain 4transmembrane domains, this gene and LGR5are found in the same chromosomal region

-celldysfunction

Wolfram syndrome gene;also called diabetesinsipidus, diabetesmellitus, optic atrophy,and deafness(DIDMOAD)

WFS1

is an integral ER membrane glycoprotein,associates with the C-terminal domain of the

ER-localized Na+/K+ATPase -1 subunit(ATP1B1)

-celldysfunction

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Neonatal Diabetes

Neonatal diabetes refers to a circumstance in which hyperglycemia results from dysfunction in insulin action


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within the first 6 months of life. This form of diabetes is not typical type 1 diabetes (T1D, or juvenile onsetdiabetes) since T1D involves immune destruction of the pancreatic -cells and thus, requires several years tofully develop. Neonatal diabetes can be transient or permanent. If an infant suffers from the transient form theyare at increased risk for developing full-blown later in life.

The advent of genetic studies to identify HLA haplotypes associated with the risk of development of T1D aswell as the description of several T1D-associated autoantibodies provided the foundation for characterization ofthe clinical features of the disease in newborns. Evidence is clear that the etiology of diabetes in the first year oflife is different from that of the autoimmune forms of T1D more classically diagnosed when children are older. Asindicated, the presentation of diabetes in infants prior to 6-months of age can be transient or permanent. Thepermanent form of the disease is termed Permanent Neonatal Diabetes Mellitus (PNDM). PNDM is a rare eventoccurring with a frequency of approximately 2 cases per 100,000 births.

Definitive determination of PNDM requires early gene screening as soon as symptoms manifest. This allowsfor a differential diagnosis to be made as to whether or not the symptoms can be expected to be transient orpermanent. Very low birth weight is highly correlated to PNDM and is associated with fetal lack of insulin. Themost prominent of symptoms is the onset of hyperglycemia within the first 6 months after birth. Affected infants donot secrete insulin in response to glucose or glucagon but will secrete insulin in response to tolbutamideadministration. Tolbutamide is a drug of the sulfonylurea class used to treat type 2 diabetes. Many infants willexhibit similar neurologic abnormalities, including developmental delay, muscle weakness, and epilepsy. Inpatients manifesting with neurologic abnormalities there are often associated dysmorphic features, includingprominent metopic suture (persistence of the space between the frontal bones of the skull), a downturned mouth,bilateral ptosis (drooping eyelid), and limb contractures.

Early on it was thought that the underlying defect resulting in neonatal diabetes was pancreatic -celldysfunction or a defect in -cell maturation. However, genetic evidence now indicates that neonatal diabetes, inparticular PNDM, is the result of single-gene defects. This make PNDM a monogenic disorder. The disorder canbe inherited although it is most often the result of a sporadic mutation in one of the parental gametes. Over thepast decade at least 12 genes have been identified as being associated with the development of PNDM. Themost commonly mutated genes are the potassium inwardly-rectifying channel, subfamily J, member 11 (KCNJ11),ATP-binding cassette transporter, subfamily C, member 8 (ABCC8), and insulin (INS) genes. The proteins of theKCNJ11 and ABCC8 genes form the ATP-sensitive potassium channel (KATP channel) that is involved in insulinsecretion (see the Insulin Function page). Mutations in the KCNJ11 gene are also associated with an increasedrisk for the development of T2D as described in the Genetics of Type 2 Diabetes section above. The insulin geneis one of the non-HLA genes that is mutated in T1D as indicated above in the Genetics of Type 1 Diabetessection.

Genes Associated with Permanent Neonatal Diabetes Mellitus

Gene NameGene

SymbolComments

ATP-binding cassettetransporter, subfamily C,member 8

ABCC8

along with KCNJ11 encoded proteins ABCC8 forms theATP-sensitive potassium (KATP) channel involved in insulinsecretion; gene is also known as the sulfonylurea receptor: SUR;mutations in the ABCC8 gene found in 13% of PNDM cases

eukaryotic translationinitiation factor 2- kinase3

EIF2AK3

also associated with skeletal dysplasia, mental retardation, andhepatic failure; gene also known as RNA-dependent proteinkinase-like endoplasmic reticulum kinase, PERK; this particularform of PNDM is also known as Wolcott-Rallinson syndrome(WRS)

forkhead box familymember P3

FOXP3

is a member of the fork-winged helix family of transcription factors,;plays an important role in development and function ofCD4-positive/CD25-positive regulatory T cells (Tregs); Tregs areinvolved in active suppression of inappropriate immune responses

pancreatic glucokinase GCK same gene found associated with MODY2

Gli similar (GLIS family)Krppel-like zinc fingertranscription 3

GLIS3also associated with severe congenital hypothyroidism,cholestasis, congenital glaucoma, and polycystic kidneys

insulin INS mutations in the INS gene represent 16% of PNDM cases

potassium inwardly-rectifying channel,subfamily J, member 11

KCNJ11forms the core of the ATP-sensitive potassium (KATP) channelinvolved in insulin secretion, protein is also called Kir6.2; mutationsin this gene found in 30%50% of PNDM cases


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pancreatic and duodenalhomeobox 1

PDX1

regulates transcription of the insulin gene; also is a key regulator ofthe development of the pancreas, most probably by determiningmaturation and differentiation of common pancreatic precursorcells in the developing gut

pancreas transcriptionfactor 1A

PTF1A

gene is essential to normal pancreas formation; mutations in genealso associated with cerebellar hypoplasia/agenesis, anddysmorphism; similar phenotypes to those resulting from PDX1mutations

regulatory factor x-boxbinding familytranscription factormember 6

RFX6involved in pancreatic islet cell differentiation; also associated withintestinal atresia and gall bladder hyoplasia

solute carrier family,facilitated glucose (GLUT)transporter subfamily,member 2

SLC2A2also associated with Fanconi-Bickel syndrome (was once calledglycogen storage disease XI, GSD11 but term is no longer valid)

solute carrier family,folate/thiaminetransporters subfamily,member 2

SLC19A2

mutations in gene result in thiamine-responsive megaloblasticanemia syndrome (also known as Rogers syndrome), defined bythe occurrence of megaloblastic anemia, diabetes mellitus, andsensorineural deafness; thiamine treatment results in varyingdegrees of positive response

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Diabetes and the Metabolic Syndrome: MetS

Although the metabolic syndrome (also called syndrome X) is not exclusively associated with type 2diabetes and the associated insulin resistance, the increasing prevalence of obesity and associated developmentof type 2 diabetes places insulin resistance as a major contributor to the syndrome. The metabolic syndrome isdefined as a clustering of atherosclerotic cardiovascular disease risk factors that include visceral adiposity(obesity), insulin resistance, low levels of HDLs and a systemic proinflammatory state. There are key componentsto the metabolic syndrome which include in addition to insulin resistance (the hallmark feature of the syndrome),hypertension, dyslipidemia, chronic inflammation, impaired fibrinolysis, procoagulation and most telling centralobesity. For more information on the biochemical and clinical aspects of MetS visit the Metabolic Syndrome page.

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Mitochondrial Dysfunction in Type 2 Diabetes and Obesity

Well established data demonstrate that mitochondrial dysfunction, particularly as it relates to the processes ofoxidative phosphorylation (oxphos), is contributory to the development of encephalomyopathy, mitochondrialmyopathy, and several age-related disorders that include neurodegenerative diseases, the metabolic syndrome,and diabetes. Indeed, with respect to diabetes, several mitochondrial diseases manifest with diabeticcomplications such as mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes(MELAS) and maternally inherited diabetes and deafness (MIDD).

Normal biogenesis of mitochondria is triggered in response to changes in the ATP/ADP ratio and to activationof AMPK which in turn results in increased expression of PPAR co-activator 1 (PGC-1) and nuclearrespiratory factor-1 (NRF1). PGC-1 is a master transcriptional co-activator of numerous genes involved inmitochondrial biogenesis. NRF1 is a transcription factor that regulates the expression of mitochondrialtranscription factor A (TFAM, for transcription factor A, mitochondrial; also designated mtTFA) which is a nucleartranscription factor essential for replication, maintenance, and transcription of mitochondrial DNA. NRF1 alsocontrols the expression of nuclear genes required for mitochondrial respiration and heme biosynthesis. Evidencehas shown that both PGC-1 and NRF1 expression levels are lower in diabetic patients as well as in non-diabeticsubjects from families with type 2 diabetes. The expression of NRF1 is highest in skeletal muscle which is alsothe tissue that accounts for the largest percentage of glucose disposal in the body and, therefore, is the tissuethat is most responsible for the hyperglycemia resulting from impaired insulin signaling.

Mitochondrial dysfunction results in increased production of ROS which activates stress responses leading toincreased activity of MAPK and JNK. Both of these serine/threonine kinases phosphorylate IRS1 and IRS2resulting in decreased signaling downstream of the insulin receptor. Inhibited IRS1 and IRS2 activity results indecreased activation of PI3K. PI3K activation is involved in the translocation of GLUT4 to the plasma membraneresulting in increased glucose uptake. Therefore, inhibition of PI3K results in reduced glucose uptake in skeletalmuscle and adipose tissue. Mitochondrial dysfunction results in a reduction in the level of enzymes involved in


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-oxidation leading to increases in intramyocellular lipid content. Indeed, skeletal muscle metabolism of lipids hasbeen shown to be impaired in type 2 diabetics. An increased delivery of fatty acids to skeletal muscle, as well asdiminished mitochondrial oxidation, results in increased intracellular content of fatty acid metabolites such asdiacylglycerol (DAG), fatty acyl-CoAs, and ceramides. These metabolites of fatty acids are all known to inducethe activity of protein kinase C isoforms (PKC and PKC) that phosphorylate IRS1 and IRS2 on serine residuesresulting in impaired insulin signaling downstream of the insulin receptor.

Because skeletal muscle consumes the largest amount of serum glucose, mitochondrial dysfunction in thistissue will have the greatest impact on glucose disposal. However, adipose tissue also plays an important role inglucose homeostasis and mitochondrial dysfunction in this tissue has been shown to result in impaired glucosehomeostasis resulting in diabetes. For example, when animals are treated with inhibitors of mitochondrialoxidation insulin-stimulated glucose uptake in adipose tissue is significantly impaired. Adipose tissue secretes anumber of proteins classified as adipokines. Adiponectin is an adipokine that promotes insulin-sensitivity ininsulin-responsive tissues, such as skeletal muscle. When plasma levels of adiponectin are measured in obese ortype 2 diabetic subjects it is found to be significantly lower than in age and sex matched control subjects that areof normal weight or that do not have diabetes. In animal studies, the enhancement of adipocyte mitochondrialbiogenesis results in increased adiponectin release from adipose tissue. Conversely, expression of adiponectinexpression is decreased in adipocytes with mitochondrial dysfunction.

Given that impaired mitochondrial function is clearly associated with obesity and type 2 diabetes, it is notsurprising that there is great interest in the use of pharmacology to augment mitochondrial function in thetreatment of these disorders. Of significance is the fact that the thiazolidinedione (TZD) class of drugs used totreat the hyperglycemia of type 2 diabetes (see the next section) activate PPAR which in turn increases the levelof activity of PGC-1. Although the TZDs were first marketed due to their ability to improve insulin sensitivity, theyhave since been shown to increase mitochondrial functions both in vitro and in vivo. Antioxidants have also beenshown to enhance mitochondrial function by reducing the production of ROS. Resveratrol (found in grape skinsand red wine) is a potent antioxidant whose activity is, in part, due to its ability to activate the deacetylase SIRT1(see below). Activated SIRT1 deacetylates PGC-1 resulting in increased transcriptional activity and thus,enhanced mitochondrial biogenesis.

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Therapeutic Intervention for Hyperglycemia

Many, if not all, of the vascular consequences of insulin resistance are due to the persistent hyperglycemiaseen in type 2 diabetes. For this reason a major goal of therapeutic intervention in type 2 diabetes is to reducecirculating glucose levels. There are many pharmacologic strategies to accomplish these goals.

1. The Thiazolidinediones (TZDs): The TZDs, such as rosiglitazone (Avandia) and pioglitazone (Actos)have proven useful in treating the hyperglycemia associated with insulin-resistance in both type 2 diabetes andnon-diabetic conditions. The TZDs function as agonists for the transcription factor, PPAR. PPAR is a member ofthe superfamily of nuclear receptor transcription factors. In addition to PPAR there are the closely relatedmembers, PPAR and PPAR/. PPAR exists as a heterodimer with the nuclear retinoid X receptors, RXRs. Theheterodimer binds to PPAR response elements (PPREs) in a number of target genes. Without ligand bound theheterodimer is associated with a co-repressor complex that includes a histone deacetylase. Deacetylated histonekeeps DNA in a transcriptionally repressed state. When ligand binds to PPAR the co-repressor complexdissociates and a co-activator complex containing histone acetylase associates resulting in chromatin structuralchanges and transcriptional activation. The net effect of the TZDs is a potentiation of the actions of insulin in liver,adipose tissue and skeletal muscle, increased peripheral glucose disposal and a decrease in glucose output bythe liver. Genes shown to be affected by PPAR action include those encoding glucokinase, GLUT4, malicenzyme, lipoprotein lipase, fatty acyl-CoA synthase and adipocyte fatty acid binding protein. PPAR is primarilyexpressed in adipose tissue and thus it was at first difficult to reconcile how a drug that was apparently actingonly in adipose tissue could lead to improved insulin sensitivity of other tissues. The answer to this question camewhen it was discovered that the TZDs stimulated the expression and release of the adipocyte hormone(adipokine), adiponectin. Adiponectin stimulates glucose uptake and fatty acid oxidation in skeletal muscle. Inaddition, adiponectin stimulates fatty acid oxidation in liver while inhibiting expression of gluconeogenic enzymesin this tissue. These responses to adiponectin are exerted via activation of AMPK. The significance of PPAR asa diabetes target is apparent not only from the observed effects of drugs that activate the receptor but also fromgenome wide screens showing that mutations in the PPAR gene are correlated to familial insulin resistance.

Recent studies have identified a critical role for an enzyme (phosphatidic acid phosphatase, PAP1) involvedin overall triacyglyceride and phospholipid homeostasis as a critical target of the PPAR signaling pathway. In theyeast Saccharomyces cerevisiae, the PAP1 gene was identified as Smp2p and the encoded protein was shownto be the yeast ortholog of the mammalian protein called lipin-1. The fission yeast lipin-1 ortholog is identified asNed1p. Lipin-1 is only one of four lipin proteins identified in mammals. The lipin-1 gene (symbol = LPIN1) wasoriginally identified in a mutant mouse called the fatty liver dystrophy (fld) mouse. The mutation causing thisdisorder was found to reside in the LPIN1 gene. There are three lipin genes with the LPIN1 gene encoding twoisoforms derived through alternative splicing. These two lipin-1 isoforms are identified as lipin-1A and lipin-1B.Mutations in the LPIN2 gene have recently been associated with Majeed syndrome which is characterized bychronic recurrent osteomyelitis, cutaneous inflammation, recurrent fever, and congenital dyserythropoieticanemia. In addition to the obvious role of lipin-1 in TAG synthesis, evidence indicates that the protein is alsorequired for the development of mature adipocytes, coordination of peripheral tissue glucose and fatty acidstorage and utilization, and serves as a transcriptional co-activator. The latter function has significance to


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diabetes as it has been shown that some of the effects of the TZDs are exerted via the effects of lipin-1. Lipin-1has been shown to interact with PPAR co-activator 1 (PGC-1) and PPAR. The interactions of lipin-1 withthese other transcription factors leads to increased expression of fatty acid oxidizing genes such as carnitinepalmitoyl transferase-1, acyl CoA oxidase, and medium-chain acylCoA dehydrogenase (MCAD).

2. Targeting glucagon-like peptide-1 (GLP-1): The synthesis and activities associated with GLP-1 aredescribed in detail in the Gut-Brain Interactions page. As review, the primary metabolic responses to GLP-1release from the enteroendocrine L-cells of the gut are inhibition of glucagon secretion and enhancement ofglucose-dependent insulin release from the pancreas, both effects lead to decreased glycemic excursion. Thehormonal action of GLP-1 is rapidly terminated as a consequence of enzymatic cleavage by dipeptidylpeptidaseIV (DPP IV or DPP4). Recent clinical evidence has shown that either infusion of GLP-1 or inhibition of DPP4 canresult in dramatic reductions in plasma glucose concentrations, reductions in HbA1c and improvement in

pancreatic -cell function. Thus, both represent potential targets for the prevention of the hyperglycemiaassociated with diabetes and impaired insulin function. For more information on the activities of DPP4 go to theDPP4 page.

There are advantages and disadvantages with the current therapeutic approaches to targeting GLP-1 actionin diabetic patients. Current use of GLP-1 mimetics and/or GLP-1 receptor (GLP-1R) agonists focus on peptidesor modified peptides and these must be injected. The need for chronic injection as a means of therapy alwaysruns into the problem of patient compliance. One of the most promising GLP-1R agonists that has recently beenapproved for use is YETTA (also written as BYETTA) developed by Amylin Pharmaceuticals and Eli Lilly andCo. BYETTA is composed of exenatide which is the lizard salivary peptide called exendin-4. Exenatide is 53%identical to GLP-1 at the level of amino acids and binds to and activates the GLP-1R. The advantage of exenatideas a therapeutic is that it is resistant to cleavage and inactivation by DPP4. In a recent trial in patients with type 2diabetes, BYETTA was shown not only to lower blood glucose levels and HbA1c, but patients also had an

associated weight loss.

Another GLP-1R agonist is Victoza (liraglutide) which was developed by Novo Nordisk. Victoza is aonce-a-day injectable recombinant DNA produced modified GLP-1 protein complex. The protein is a fattyacid-linked derivative of human GLP-1 that is resistant to DPP4 cleavage. The 16-carbon fatty acyl-chain(palmitic acid) addition to the protein allows liraglutide to bind to albumin in the blood which prevents its excretionvia the kidneys. Liraglutide has been shown to have a half-life of 11-13 hours making it ideal for once-a-dayinjection. Results of clinical studies demonstrated significant reductions in HbA1c levels in liraglutide treated

patients. Victoza was approved for use in the United States in January 2010. One problematic side effect ofVictoza treatment is pancreatitis which occurs in patients with a higher frequency than with other diabetestreatments.

Although targeting compounds that can inhibit the enzymatic action of DPP4 would seem like idealcandidates for treating the hyperglycemia of uncontrolled diabetes, there are several unknowns associated withDPP4 inhibition. One of these issues is the fact that GLP-1 and GIP are only two of the many known substratesfor DPP4 cleavage. Thus, prolonged inhibition of DPP4 enzymatic activity may have unexpected consequencesunrelated to control of hyperglycemia. Despite the potential for as yet unknown effects, the DDP4 inhibitordeveloped by Merck, Januvia (sitagliptin), has recently been approved for use alone or in combination witheither metformin or the thiazolidinediones. Treatment of patients with Januvia as the only therapeutic agent for 18weeks produced significant reductions of HbA1c, along with an improvement of -cell function and no change in

body weight.

A second generation DPP4 inhibitor developed by Novartis called Glavus (vildagliptin) has recentlyreceived approvable status from the US FDA. Glavus administration is associated with significantly increasedpancreatic -cell function and reduced HbA1c levels without hypoglycemia or other adverse events. Another drug

in the DPP4 inhibitor class to receive US FDA approval is Onglyza (saxagliptin) made by AstraZeneca andBristol-Myers Squibb. Onglyza is designed as a once-daily orally administered tablet.

DPP4 was originally identified as the lymphocyte cell surface antigen CD26. In humans CD26 functions inmany pathways that are not directly related to its peptidase activity. It harbors adenosine deaminase-binding(ADA-binding) properties and is involved in extracellular matrix binding. Of importance to the immune system,CD26 expression and activity are enhanced upon T-cell activation. CD26 interacts with other lymphocyte cellsurface antigens including ADA, CD45 and the chemokine receptor CXCR4 (notable is the fact that CXCR4 is aT-cell attachment site for HIV). Currently available data indicates that the peptidase activity of DPP4 isindependent of the T-cell activating and co-stimulatory functions assigned to CD26. Of significance, however, isthat in gene knock-out mice lacking CD26 there is enhanced insulin secretion and improved glucose tolerance.

The major clinical advantages to the use of DPP4 inhibitors is that the ones in use or in current trials areorally delivered. Compliance in patients is much higher with orally delivered drugs than with those that requireinjection.

3. The Biguanides: The biguanides are a class of drugs that function to lower serum glucose levels byenhancing insulin-mediated suppression of hepatic glucose production and enhancing insulin-stimulated glucoseuptake by skeletal muscle. Metformin (Glucophage) is a member of this class and is currently the most widelyprescribed insulin-sensitizing drug in current clinical use. Metformin administration does not lead to increasedinsulin release from the pancreas and as such the risk of hypoglycemia is minimal. Because the major site ofaction for metformin is the liver its use can be contraindicated in patients with liver dysfunction. The drug is idealfor obese patients and for younger type 2 diabetics.

Evidence on the mode of action of metformin shows that it improves insulin sensitivity by increasing insulinreceptor tyrosine kinase activity, enhancing glycogen synthesis and increasing recruitment and transport of


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GLUT4 transporters to the plasma membrane. Additionally, it has been shown that metformin affectsmitochondrial activities dependent upon the model system studied. Metformin has a mild inhibitory effect oncomplex I of oxidative phosphorylation, has antioxidant properties, and activates both glucose 6-phosphatedehydrogenase, G6PDH and AMP-activated protein kinase, AMPK. The importance of AMPK in the actions ofmetformin stems from the role of AMPK in the regulation of both lipid and carbohydrate metabolism (see AMPK:Master Metabolic Regulator for more details). In adipose tissue, metformin inhibits lipolysis while enhancingre-esterification of fatty acids. The activation of AMPK by metformin is likely related to the inhibitory effects of thedrug on complex I of oxidative phosphorylation. This would lead to a reduction in ATP production and, therefore,an increase in the level of AMP and as a result activation of AMPK. In fact, since the cells of the gut will see thehighest doses of metformin they will experience the greatest level of inhibited complex I which may explain thegastrointestinal side effects (nausea, diarrhea, anorexia) of the drug that limit its utility in many patients.

In adolescent females with type 2 diabetes, the use of metformin is highly recommended to reduce theincidence as well as the potential for polycystic ovarian syndrome, PCOS. PCOS is brought on by thehyperinsulinemia of type 2 diabetes. Insulin effects on the ovary drive conversion of progesterone to testosteroneand a reduction in serum hormone binding globulin (SHBG). Taken together, the effects of hyperinsulinemia leadto a hyperandrogenic state in the ovary resulting in follicular atresis and ovulatory dysfunction.

4. The Sulfonylureas: The sulfonylurea and meglitinide classes of oral hypoglycemic drugs are referred toas endogenous insulin secretagogues because they induce the pancreatic release of endogenous insulin.

The sulfonylureas have been used in the US for nearly 50 years. The first generation sulfonylureas(tolbutamide, acetohexamide, chlorpropramide and tolazamide) are not routinely prescribed any longer in the US.The second generation sulfonylureas include glipizide (Glucotrol), glimepiride (Amaryl) and glyburide(DiaBeta, Micronase, Glynase). Because all of these drugs can induce pronounced hypoglycemia, treatmentis initiated with the lowest possible dose and carefully monitored until the dose is found that results in a FPG of110-140mg/dL. Sulfonylureas function by binding to and inhibiting the pancreatic ATP-dependent potassiumchannel that is normally involved in glucose-mediated insulin secretion (see above under insulin function).Sulfonylureas have no significant effects on circulating triglycerides, lipoproteins or cholesterol.

5. The Meglitinides: As indicated, the meglitinides repaglinide (Prandin) and nateglinide (Starlix) arenon-sulfonylurea insulin secretagogues that are both fast acting and of short duration. Like the sulfonylureas,meglitinides therapy results in significant reduction in FPG as well as HbA1c. The mechanism of action of the

meglitinides is initiated by binding to a receptor on the pancreatic -cell that is distinct from the receptors for thesulfonylureas. However, meglitinides do exert effects on potassium conductance. Like the sulfonylureas, themeglitinides have no direct effects on the circulating levels of plasma lipids.

6. The -Glucosidase inhibitors: -glucosidase inhibitors such as acarbose (Precose) and miglitol(Glyset) function by interfering with the action of the -glucosidases present in the small intestinal brush border.The consequence of this inhibition is a reduction in digestion and the consequent absorption of glucose into thesystemic circulation. The reduction in glucose uptake allows the pancreatic -cells to more effectively regulateinsulin secretion. The advantage to the use of the -glucosidase inhibitors is that they function locally in theintestine and have no major systemic action. Hypoglycemia does not usually occur with the use of -glucosidaseinhibitors but they are effective at reducing fasting plasma glucose (FPG) levels and levels of glycosylatedhemoglobin (HbA1c). The common adverse side effects of these inhibitors are abdominal bloating and discomfort,

diarrhea and flatulence.

6. SGLT2 Antagonists: A new class of orally administered compounds that targets renal glucose transportand inducers of glucosuria are currently being tested for efficacy in type 2 diabetes treatment. In the kidney,glucose is filtered at the glomerulus and then reabsorbed via active transport in the proximal convoluted tubule.Two sodium-glucose co-transporters (SGLT1 and SGLT2) have been identified as responsible for this renalglucose reabsorption. The SGLT proteins are members of the solute carrier 5 family of membrane transporters,and thus, SGLT1 is SLC5A1 and SGLT2 is SLC5A2. SGLT1 is found in other tissues and accounts forapproximately 10% of the renal glucose reabsorption. SGLT2 is expressed exclusively in the S1 segment of theproximal tubule and is responsible for 90% of the renal glucose reabsorption (see Figure below). Therefore, it ispostulated that selective inhibition of the renal SGLT2 activity should result in greatly enhanced glucose release inthe urine. Several drugs (all of which carry the suffix gliflozin: empagliflozin, canagliflozin, dapagliflozin,ipragliflozin) that inhibit SGLT2 are currently under investigation. Canagliflozin (Invokana), dapagliflozin(Farxiga), and empagliflozin (Jardiance) are the only drugs in this class currently approved by the FDA forthe treatment of type 2 diabetes. The advantage of these drugs is that they are taken orally so compliance will behigher than injected type 2 diabetes drugs. In addition, these drugs are formulated as once-a-day tablets.


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Diagrammatic representation of the re-uptake of glucose in the S1 segment of the proximal tubule of the

kidney by the Na+-glucose co-transporter SGLT2. Following re-uptake the glucose is transported back into the

blood via the action of GLUT2 transporters. The Na+ that is reabsorbed with the glucose is transported into the

blood via a (Na+-K+)-ATPase.

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New Frontiers in Diabetes Therapy

Several new approaches are being taken in the search for treatments for diabetes. These include thedevelopment of newer drugs that target the same pathways as described in the sections above including, but notlimited to, new classes of DPP4 antagonists and GLP-1 receptor agonists. Additional, potentially exciting targets,include the hepatic-derived fibroblast growth factor 21 (FGF21), the renal sodium-glucose transporter-2 (SGLT2),

the NAD+-dependent deacetylase SIRT1 or sirtuin 1, and the lipid binding G-protein coupled receptor GPR119.

FGF21 Agonists: Hepatic lipid homeostasis is tightly controlled through the influences of insulin, free fattyacids, sterol regulated element-binding protein (SREBP), and nuclear receptors and associated regulatorymolecules such as the liver X receptors (LXRs) and peroxisome proliferator-activated receptor (PPAR),espectively. The LXRs are members of the steroid/thyroid hormone superfamily of cytosolic ligand bindingreceptors that migrate to the nucleus upon ligand binding and regulate gene expression by binding to specifictarget sequences. There are two forms of the LXRs, LXR and LXR. The LXRs form heterodimers with theretinoid X receptors (RXRs) and as such can regulate gene expression either upon binding oxysterols (e.g.22R-hydroxycholesterol) or 9-cis-retinoic acid.

Recent evidence demonstrated that expression of the fibroblast growth factor family member, FGF21, wassignificantly elevated in mice fed a high-fat, low-carbohydrate ketogenic diet. Additionally, in mice withexperimentally induced reduction in FGF21 expression there was an associated lipemia, reduced ketogenesisand a resultant fatty liver. Conversely, administration of FGF21 to diabetic animals resulted in reductions in thelevels of fasting glucose and serum lipids. These results indicate that FGF21 plays a key role in regulating theexpression of genes involved in hepatic lipid homeostasis and that activation of FGF21 activity could prove to bea significant tool in the treatment of the disrupted metabolic status in diabetic individuals.

SIRT1 Activators: SIRT1 or sirtuin 1 is the homolog of the yeast (S. cerevisiae) Sir2 gene (Sir refers toSilent mating type Information Regulator). SIRT1 is a member of the sirtuin family of proteins (seven members;SIRT1 through SIRT7) that are characterized by a sirtuin core domain and grouped into four classes. The yeastsirtuin proteins are known to regulate life-span extension, epigenetic gene silencing and suppress recombination

of ribosomal DNA (rDNA). SIRT1 is an NAD+-dependent deacetylase that modulates the activities of proteins thatare in pathways downstream of the beneficial effects of calorie restriction. SIRT1 catalyzes a reaction where

hydrolysis of NAD+ is coupled to the deacetylation of acetylated lysines in target proteins. These target proteins

include histones, transcription factors and transcription factor co-regulators. The NAD+ is hydrolyzed tonicotinamide (which is a strong inhibitor of SIRT1 activity) and O-acetyl-ADP ribose. Principal pathways involvedin glucose homeostasis and insulin sensitivity are affected by SIRT1 activity. In skeletal muscle, a major site ofinsulin-induced glucose uptake, SIRT1 and AMPK work in concert to increase the rate of fatty acid oxidation inperiods of decreased nutrient availability.

The plant-derived compound, resveratrol (a polyphenolic compound), is a known activator of SIRT1 function.The effects of resveratrol have been shown to increase mitochondrial content, ameliorate insulin resistance andprolong survival in laboratory mice fed a high-fat diet. Recent studies on the action of SIRT1 agonists havedemonstrated that compounds that activate SIRT1, but that are structurally unrelated to resveratrol, also improveinsulin sensitivity in adipose tissue, liver and skeletal muscle resulting in lower plasma glucose. The actions ofthese compounds in laboratory studies indicate the potential efficacy of a therapeutic approach to type 2 diabetes


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that includes activators of SIRT1 activity.

GPR119 Agonists: The fatty acid-sensing receptor, GPR119, is a Gs-type G-protein coupled receptor.

GPR119 is expressed at the highest levels in the pancreas and fetal liver with expression also seen in thegastrointestinal tract, specifically the ileum and colon. GPR119 is a member of the class A family (rhodopsin-type)of GPCRs. GPR119 binds long-chain fatty acids including oleoylethanolamide (OEA), lysophosphatidylcholine(LPC), various lipid amides, and retinoic acid. OEA is the most potent ligand and likely represents theendogenous ligand for GPR119. The demonstration that OEA is the most active endogenous ligand for GPR119is of particular interest since previous work has demonstrated that OEA, when administered to laboratoryanimals, causes a significant reduction in food intake and body weight gain. These effects of OEA are the resultof the activation of the nuclear receptor PPAR, increased expression of fatty acid translocase, and modificationof feeding behavior and motor activity. In addition, activation of GPR119 in the pancreas is correlated withenhanced glucose-stimulated insulin secretion (GSIS) and activation of the receptor in the gut results inincreased secretion of the incretin hormones GLP-1 and GIP. These observations indicate that GPR119 activationis associated with a dual mechanism of reducing blood glucose: acting directly through pancreatic -cells topromote GSIS and in the gut via the stimulation of the incretins GLP1 and GIP both of which increase insulinrelease from the pancreas in response to food intake. Currently there are several small molecule agonists ofGPR119 in clinical trials being tested for their efficacy in treating the hyperglycemia of type 2 diabetes as well asfor their efficacy in treating obesity.

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Michael W King, PhD | 19962014 themedicalbiochemistrypage.org, LLC | info @ themedicalbiochemistrypage.org

Last modified: April 5, 2015


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