Part 4

Etiology of Type 2 Diabetes Insulin Resistance and -Cell Dysfunction 2

Etiology of Type 2 Diabetes Primary Predisposing Factors Genes Adverse intrauterine environment Tertiary Accelerating Glucose and lipid toxicity Secondary Precipitating Factors Obesity Low physical activity Age Smoking Sleep disturbance Other Multiple factors contribute to the etiology of type 2 diabetes. These include Primary predisposing factors (ie, genetic and/or intrauterine). Modifiable and nonmodifiable secondary precipitating factors, such as obesity, physical inactivity, age, smoking, sleep disturbances. Tertiary factors, such as glucose and lipid toxicity, which can accelerate disease progression.

Type 2 Diabetes: A Heterogeneous Disorder Functional -cell Failing -cell Insulin resistance Insulin resistance Current evidence suggests that type 2 diabetes is a heterogeneous disorder involving both genetic and acquired defects.1 Genetics play an important role in determining whether an individual’s β-cell function remains normal or becomes impaired over time.1 Insulin resistance is a major acquired factor and can occur in the presence of functional or failing β-cells.1 Although not depicted on this slide, intrauterine exposure to maternal glucose levels also increases the risk for future diabetes development. Specifically, it has been demonstrated that infants who are either small or large for gestational age are at risk for future type 2 diabetes.2 Decreases in β-cell functionality lead ultimately to hyperglycemia, while insulin resistance in obese patients is associated with the metabolic syndrome (eg, dyslipidemia, hypertension, IGT). The next step in disease progression is often the development of both micro- and macrovascular complications, such as retinopathy, nephropathy, neuropathy, MI, and stroke.1 The occurrence of microvascular events is also a predictor of macrovascular events in patients with type 2 diabetes.   Gerich JE. Contributions of insulin-resistance and insulin-secretory defects to the pathogenesis of type 2 diabetes mellitus. Mayo Clin Proc. 2003;78:447-456. Jovanovic L. A tincture of time does not turn the tide: type 2 diabetes trends in offspring of diabetic mothers. Diabetes Care. 2000:23:1219-1220. Metabolic syndrome Hyperglycemia Heine RJ, Spijkerman AM. 2006. 4 4

Type 2 Diabetes: Insulin Resistance Plus Impaired -Cell Function Both insulin resistance and b-cell dysfunction are present at the time of diagnosis of type 2 diabetes Insulin resistance Normal -cell function Compensatory hyperinsulinemia Normoglycemia (Metabolic syndrome) Abnormal -cell function Relative insulin deficiency Hyperglycemia Type 2 diabetes Insulin resistance and β-cell dysfunction are key pathophysiologic triggers in the development of type 2 diabetes; both of these deficits tend to be present at the time of diagnosis. In individuals with normal β-cell function, insulin resistance leads to compensatory hyperinsulinemia and the development of metabolic syndrome, but glucose levels will remain normal. However, in individuals with impaired β-cell function, insulin resistance gives rise to relative insulin deficiency, hyperglycemia, and type 2 diabetes.    Gerich JE. Contributions of insulin-resistance and insulin-secretory defects to the pathogenesis of type 2 diabetes mellitus. Mayo Clin Proc. 2003;78:447-456. 5

Natural History of Type 2 Diabetes Insulin- Mediated Glucose Uptake (mg/m2 • min) 300 250 200 150 100 Mean Plasma Insulin During OGTT (µU/mL) Mean Plasma Glucose During OGTT (mg/dL) 140 60 20 400 OB- DM Lo INS Lean NGT Hi INS IGT OB The natural history of type 2 diabetes is reflected in the results of this study performed in lean and obese patients with varying levels of glucose tolerance. Patients received an OGTT followed by a euglycemic insulin clamp. In lean subjects with normal glucose tolerance (NGT), OGTT results included a mean plasma glucose level of 110 mg/dL (6.1 mmol/L) and a mean plasma insulin concentration of 60 U/mL (430.5 pmol/L), while insulin-mediated glucose uptake was approximately 300 mg/m2 per minute.1 Compared with lean controls, obese nondiabetic individuals experienced dramatic decreases in insulin sensitivity but retained NGT due to compensatory increases in insulin secretion.2,3 Obese patients with mild glucose intolerance demonstrated further decreases in insulin-mediated glucose uptake but only slight increases in mean plasma glucose; this was a result of compensatory augmentation of β-cell insulin release.1-3 In obese hyperinsulinemic type 2 diabetes patients, OGTT led to further increases in mean plasma glucose levels and a substantial decline in mean plasma insulin levels as β-cell secretory capacity deteriorated; no marked decreases in insulin sensitivity were observed at this stage.1-3 Finally, in obese hypoinsulinemic type 2 diabetes patients, mean plasma insulin levels during OGTT were dramatically decreased as glucose tolerance became severely impaired; at the same time, insulin resistance remained largely unchanged.2,3 These data support the need for type 2 diabetes medications that act to improve β-cell function and increase insulin sensitivity.   DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes. Diabetes. 2009;58:773-795. DeFronzo RA. The triumvirate: β-cell, muscle, liver. A collusion responsible for NIDDM. Diabetes. 1988;37:667-687. Jallut D, Golay A, Munger R, et al. Impaired glucose tolerance and diabetes in obesity: a 6-year followup study of glucose metabolism. Metabolism. 1990;39:1068-1075. DM=diabetes mellitus; IGT=impaired glucose tolerance; INS=insulin; NGT=normal glucose tolerance; OB=obesity. DeFronzo RA. Diabetes. 1988;37:667-687; Jallut D, et al. Metabolism. 1990;39:1068-1075. 6 6

Etiology of -Cell Dysfunction in Type 2 Diabetes Age Genetics (TCF 7L2) ↓ Incretin Effect β-Cell failure in type 2 diabetes has a number of potential causes. First, studies have shown a progressive age-related decline in β-cell function. In addition, research has led to the identification of specific genes, such as transcription factor TCF 7L2, that are linked to β-cell dysfunction in patients with type 2 diabetes. Insulin resistance plays a major role in progressive β-cell failure by creating an ongoing demand for insulin hypersecretion. Evidence indicates that lipotoxicity, defined as an increase in plasma free fatty acid (FFA) concentrations, can impair β-cell function in genetically predisposed subjects. Research has shown that glucose toxicity, which refers to chronically elevated plasma glucose levels, also leads to reduced insulin secretion. Deposition of amyloid and islet amyloid-like polypeptide may play a role in progressive β-cell failure in type 2 diabetes as well. Finally, decreases in the incretin effect have been shown to be a major contributor to β-cell dysfunction.    DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes. Diabetes. 2009;58:773-795. Amyloid (Islet Amyloid Polypeptide) Deposition -Cell Dysfunction Insulin Resistance Lipotoxicity ↑ Free Fatty Acids Glucose Toxicity 7 7

Natural History of -Cell Dysfunction in Type 2 Diabetes β-Cell failure occurs much earlier in the natural history of type 2 diabetes and is more severe than previously appreciated 8

San Antonio Metabolism and VAGES Studies Normal glucose tolerance 318 Impaired glucose tolerance 259 Type 2 diabetes 201 Subjects Number Subjects were classified as Nonobese if BMI <30 kg/m2 Obese if BMI ≥30 kg/m2 The San Antonio metabolism study and Veterans Administration Genetic Epidemiology Study (VAGES) utilized OGTT and euglycemic insulin clamps to assess changes in insulin response and insulin sensitivity in lean and obese patients with NGT or IGT or overt type 2 diabetes.1-3 Subjects with a body mass index ≥30 kg/m2 were classified as obese; study results are depicted on the following slides.    Gastaldelli A, Ferrannini E, Miyazaki Y, Matsuda M, DeFronzo RA for the San Antonio metabolism study. Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia. 2004;47:31-39. Ferrannini E, Gastaldelli A, Miyazaki Y, Matsuda M, Mari A, DeFronzo RA. beta-Cell function in subjects spanning the range from normal glucose tolerance to overt diabetes: a new analysis. J Clin Endocrinol Metab. 2005;90:493-500. Abdul-Ghani MA, Jenkinson CP, Richardson DK, Tripathy D, DeFronzo RA. Insulin secretion and action in subjects with impaired fasting glucose and impaired glucose tolerance: results from the Veterans Administration Genetic Epidemiology Study. Diabetes. 2006;55:1430-1435.  Methods: OGTT and insulin clamp VAGES=Veterans Administration Genetic Epidemiology Study. Abdul-Ghani MA, et al. Diabetes. 2006;55:1430-1435; Ferrannini E, et al. J Endocrinol Metab. 2005;90:493-500; Gastaldelli A, et al. Diabetologia. 2004;47:31-39. 9 9

Plasma Glucose and Insulin AUC 12 12 Q1 T2DM Q2 Q3 Q4 <160 <180 <200 IGT 8 8 NGT (mmol/L  120 min) Glucose AUC (pmol/L  120 min) Insulin AUC This slide depicts selected results from the San Antonio metabolism and VAGES studies. The San Antonio metabolism study was designed to examine the major determinants of glucose homeostasis, while VAGES evaluated changes in insulin secretion and resistance in subjects with impaired fasting glucose (IFG) and/or IGT.1, 2 Plasma glucose and insulin areas under the curve (AUC) during OGTT for individuals with NGT are shown in yellow, followed by subjects with IGT in green; the IGT group is subdivided into tertiles based on 2-hour plasma glucose levels.1,3 For patients with type 2 diabetes, results are divided into 4 equivalently distributed quartiles and appear in orange.3 Glucose clearance was reduced in subjects with IGT or type 2 diabetes; this led to increased glucose AUC in these subjects.1 The plasma insulin response has the typical inverted U-shape seen in Starling’s curve of the pancreas (blue arrow).1,3 The increase in plasma insulin concentration exhibited by subjects with IGT is not an indication that β-cell function is normal, but rather of the β-cell’s response to increases in plasma glucose levels in the presence of insulin resistance. Plasma insulin levels undergo a marked decline in the type 2 diabetes population as β-cells become incapable of secreting sufficient amounts of insulin to compensate for the effects of decreased insulin sensitivity.1,3   Gastaldelli A, Ferrannini E, Miyazaki Y, Matsuda M, DeFronzo RA for the San Antonio metabolism study. Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia. 2004;47:31-39. Abdul-Ghani MA, Jenkinson CP, Richardson DK, Tripathy D, DeFronzo RA. Insulin secretion and action in subjects with impaired fasting glucose and impaired glucose tolerance: results from the Veterans Administration Genetic Epidemiology Study. Diabetes. 2006;55:1430-1435.  DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes. Diabetes. 2009;58:773-795. 4 4 Gastaldelli A, et al. Diabetologia. 2004;47:31-39. 10 10

Insulin Secretion / Insulin Resistance (Disposition) Index During OGTT 40 NGT Lean <100 <120 <140 Obese 30 ∆ I / ∆ G ÷IR 20 This slide depicts selected results from the San Antonio metabolism study.1 The graph shows the gold standard index for β-cell function, which consists of insulin secretion divided by insulin resistance (or disposition) on the Y-axis shown as a function of 2-hour plasma glucose concentration on the X-axis.1,2 Individuals with NGT are shown in yellow, while those with IGT are in green. Subjects in both of these categories were divided into 3 groups based on 2-hour plasma glucose levels during OGTT. Patients in the upper tertile of the NGT group have lost approximately two-thirds of their β-cell function.2 Patients with overt type 2 diabetes, shown in orange, have experienced and continue to experience substantial deterioration of β-cell function. By the time of diabetes diagnosis, >80% of β-cell functionality has been lost.2  Gastaldelli A, Ferrannini E, Miyazaki Y, Matsuda M, DeFronzo RA for the San Antonio metabolism study. Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia. 2004;47:31-39. DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes. 2009;58:773-795. 10 <180 IGT <200 <160 <240 <280 <360 <320 >400 <400 T2DM 2-Hour Plasma Glucose (mg/dL) G=glucose; I=insulin; IR=insulin resistance. Gastaldelli A, et al. Diabetologia. 2004;47:31-39. 11 11

Ln ∆I / ∆G ÷ IR (mL/min • kgFFM) Ln 2-Hour Plasma Glucose (mg/dL) Log Normalization of the Relationship Between 2-Hour Plasma Glucose and Insulin Secretion / Insulin Resistance Index 6 NGT IGT 4 T2DM 2 Ln ∆I / ∆G ÷ IR (mL/min • kgFFM) This slide depicts selected results from the San Antonio metabolism study.1 A graph of the natural log of the insulin secretion / insulin resistance index versus the natural log of the 2-hour plasma glucose concentration reveals that these parameters exhibit a strong inverse linear association (r=0.91).1,2 These data provide evidence of the correlation between the progressive decline in β-cell function and glucose tolerance and indicate that this relationship moves along a continuum from NGT to IGT to overt type 2 diabetes.1,2  Gastaldelli A, Ferrannini E, Miyazaki Y, Matsuda M, DeFronzo RA for the San Antonio metabolism study. Beta-cell dysfunction and glucose intolerance: results from the San Antonio metabolism (SAM) study. Diabetologia. 2004;47:31-39. DeFronzo RA. Banting Lecture. From the triumvirate to the ominous octet: a new paradigm for the treatment of type 2 diabetes mellitus. Diabetes. 2009;58:773-795. -2 r=0.91 P<0.00001 -4 4.0 4.5 5.0 5.5 6.0 6.5 Ln 2-Hour Plasma Glucose (mg/dL) Ln=log normalization. Gastaldelli A, et al. Diabetologia. 2004;47:31-39. 12 12

GENFIEV: Insulin Secretion as a Function of Insulin Sensitivity Trend test P<0.001 Δ AUC C-peptide / Δ AUC Glucose ÷ HOMA-R The Genetics, Physiopathology and Evolution of Type 2 Diabetes (GENFIEV) study was a multicenter investigation designed to identify pheno- and genotypic features for subjects (N=1017) with a high risk of type 2 diabetes. Plasma glucose and C-peptide levels were assessed using OGTT. Fasting insulin values were also evaluated.1 Of study participants, 50% had NGT, 4% had IFG, 23% had IGT, 8% had IFG and IGT, and 15% were diagnosed with type 2 diabetes.1 Study results revealed that the C-peptide AUC to glucose AUC ratio decreased across the range of 2-hour plasma glucose values (r=-0.37, P<0.001), while the homeostasis model assessment index ratio (HOMA-IR), a measure of insulin sensitivity, increased (r=0.28; P<0.001, data not shown).1,2 As shown in this bar graph, the C-peptide AUC/glucose AUC to HOMA-IR ratio, a disposition index surrogate, decreased linearly (arrow) as a function of increasing 2-hour plasma glucose values (r=-0.44, P<0.001).1,2 This investigation provided evidence that changes in insulin secretion and sensitivity occur in a linear fashion as a function of decreasing glucose tolerance.1  Del Prato S for the GENFIEV Study Group. Insulin secretion and insulin action in individuals at high risk for type 2 diabetes. The GENFIEV Study of the Italian Society of Diabetology [abstract]. 66th Scientific Sessions of the American Diabetes Association; June 9-13, 2006; Washington, DC. Abstract 1381-P. GENFIEV. Genetics, Physiopathology and Evolution of Type 2 Diabetes. Available at: http://host.genfiev.it/genfiev/immagini/Results06.pdf. Accessed March 8, 2009. 2-Hour Plasma Glucose (mg/dL) HOMA-R=homeostasis model assessment index ratio. Diabetes. 2006;55(suppl 2):A322. 13

Insulin Secretion Rate GeNFIEV: Stimulus-response Curve (Proportional Control) of Insulin Secretion GENFIEV: Stimulus-Response Curve (Proportional Control) of Insulin Secretion * Insulin Secretion Rate (pmol . min-1 . m-2) The GENFIEV Study Group also investigated β-cell response to the rate of increased glucose concentration, referred to as derivative control, and response to actual glucose concentration, or proportional control, following oral glucose administration in subjects with a range of FPG values and glucose tolerance levels.1 Two cohorts were selected from the GENFIEV database. Cohort A (n=134) was comprised of subjects with NGT and low-normal, high-normal, or impaired FPG. Cohort B (n=159) was comprised of subjects with normal FPG and low-normal, high-normal, impaired, or diabetic glucose tolerance.1  All subjects received OGTT, with derivative control and proportional control calculated based on glucose and C-peptide curves.1 As this graph of proportional control data shows, the insulin secretion rate as a function of plasma glucose concentration was highest in subjects with NFG/NGT, with decreasing values observed for those with IFG/NGT, NFG/IGT, IFG/IGT, NFG/DGT, DFG/IGT, IFG/DGT, and DFG/DGT. Proportional control values for both cohorts were lower in high-normal than in low-normal subjects (P<0.01) but were similar between impaired FPG and high-normal FPG, and between IGT and high NGT.1,2 Based on proportional control results, FPG and glucose tolerance were both found to be independent predictors of the decline in β-cell response to oral glucose administration in nondiabetic subjects.1 Bonadonna RC for the GENFIEV Study Group. Selective decline of β-cell function within the normal range of glucose concentration in humans [abstract]. 66th Scientific Sessions of the American Diabetes Association; June 9-13, 2006; Washington, DC. Abstract 2472-PO. GENFIEV. Genetics, Physiopathology and Evolution of Type 2 Diabetes. Available at: http://host.genfiev.it/genfiev/immagini/Results06.pdf. Accessed March 8, 2009. § # Plasma Glucose (mmol/L) *P<0.01 vs NFG/NGT; §P<0.05 vs NFG/IGT and IFG/NGT; #P<0.05 vs IFG/IGT and NFG/DGT. Diabetes. 2006;55(suppl 2):A2472. 14

Decrease in AIR Necessary to Convert From NGT to IGT Insulin Secretion and Insulin Resistance in Different Ethnic Populations With IGT Decrease in AIR Necessary to Convert From NGT to IGT Pima Indian Latino/Hispanic White Δ AIR (%) A number of studies have provided evidence of a relationship between ethnicity and insulin sensitivity. For example, Pima Indian, Latino/Hispanic, and white populations are characterized by varying degrees of insulin resistance, with Pima Indians being most severe and whites being least severe.1 Research has shown that in Pima Indian, Latino/Hispanic, and white subjects with IGT, acute insulin response, an index of insulin secretion, is decreased by 8%, 18%, and 32%, respectively, relative to individuals with NGT.1 As such, populations with higher levels of insulin resistance require smaller decreases in β-cell function to progress from NGT to IGT.1,2   Abdul-Ghani MA, Tripathy D, DeFronzo RA. Contributions of β-cell dysfunction and insulin resistance to the pathogenesis of impaired glucose tolerance and impaired fasting glucose. Diabetes Care. 2006;29:1130-1139. Jensen CC, Cnop M, Hull RL, Fujimoto WY, Kahn SE for the American Diabetes Association GENNID Study Group. β-cell function is a major contributor to oral glucose tolerance in high-risk relatives of four ethnic groups in the U.S. Diabetes. 2002;51:2170-2178. Insulin resistance ↑↑↑ ↑↑ ↑ AIR=acute insulin response to glucose. Abdul-Ghani MA, et al. Diabetes Care. 2006;29:1130-1139.