Incretins Daniel J. Drucker, MD Professor of Medicine Director, Banting and Best Diabetes Centre

A Fundamental Observation: Insulin Secretion is Greater with Oral versus IV Glucose Blood Glucose Plasma Insulin 300 50 100 150 200 250 300 -30 -15 15 30 45 60 75 90 105 120 135 Time (min) Plasma Insulin (pmol/L) Intravenous 250 IV/IJ glucose Intrajejunal 200 Blood Glucose (mg/dL) 150 100 These experimental findings illustrate the incretin effect, namely the potentiation of glucose-stimulated insulin secretion when glucose is given orally via the gut, versus intravenously. In this depicted experiment, healthy volunteers were given identical amounts of glucose (5% glucose infused at 15 ml/min) either intravenously or into the jejunum. Although higher plasma glucose concentrations were observed during the IV infusion, the insulin response was significantly greater during intrajejunal infusion. These observations strongly suggested the presence of a circulating factor that was released from the jejunum during enteral loading which potentiated glucose-stimulated insulin release. The putative gut factors or hormones were termed “incretins”1 and their actions to augment glucose-stimulated insulin release were termed “the incretin effect.”2 1Zunz E, LeBarre J. Arch Int Physiol Biochim. 1929:31:20-44. 2Creutzfeldt W. Diabetologia. 1979;16:75-85. 50 -30 -15 15 30 45 60 75 90 105 120 135 Time (min) McIntyre N. Lancet. 1964 Jul 4;41:20-21. Copyright © 1964.

Measurement of the Incretin Effect OGTT and Matched IV Infusion Glucose (mg/dL) Insulin (pmol/L) 200 400 Oral IV 150 300 100 200 50 100 To more precisely quantitate the incretin effect, glucose was infused intravenously (IV) at a rate necessary to reproduce the plasma glucose profile observed after a 50 g oral glucose load. This slide demonstrates that superimposed glucose profiles obtained during the IV vs. oral glucose administration studies were virtually indistinguishable (left panel) In contrast, a much greater increment in plasma insulin levels was observed following oral glucose loading (right panel). The difference between the insulin curves obtained during the oral vs. IV glucose loads is attributed to the gut-derived non-glycemic -cell stimulus and illustrates the “incretin effect.” -30 30 60 90 120 150 180 210 -30 30 60 90 120 150 180 210 Time (min) Time (min) Nauck MA, et al. J Clin Endocrinol Metab. 1986 Aug;63(2):492-498. Copyright © 1986. The Endocrine Society.

The Incretin Effect is Diminished in Type 2 Diabetes Oral glucose IV glucose Oral glucose IV glucose Oral glucose IV glucose Non-diabetic 30 60 90 120 1 2 3 Time (hours) Plasma insulin (µU/mL) Non-diabetic 30 60 90 120 1 2 3 Time (hours) Plasma insulin (µU/mL) Type 2 DM 30 60 90 120 1 2 3 Time (hours) Plasma insulin (µU/mL) Comparison of the relative difference in plasma insulin levels achieved following oral vs intravenous glucose loading in normal subjects (left panel) vs human subjects with type 2 diabetes (right panel) demonstrates a reduction in the magnitude of the incretin effect. The precise explanation for the reduced incretin effect in type 2 diabetes may reflect a combination of reduced incretin secretion (predominantly GLP-1) and defective incretin action (predominantly GIP). Adapted from Perley MJ et al. J Clin Investigation. 1967;46:1954–1962.

The Incretins GLP-1: Glucagon-like peptide-1 A G F S V S L G A H E D S Y Q A A K R K L F I G G V W E GIP: Glucose-dependent insulinotropic polypeptide A G F S Y I M K H Y E This slide depicts the amino acid sequences of the 2 major incretins, glucagon-like peptide–1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). The amino acid residues shown in gold are those shared with the structure of glucagon. GLP-1 is a 37 amino acid peptide derived from the proglucagon gene. GLP-1 undergoes a monobasic cleavage at position 6 to generate GLP-1(7-36)amide or GLP-1(7-37), which are both bioactive. The peptide shown here is GLP-1(7-37). GIP is encoded by a different gene and is liberated following posttranslational processing to generate a 42 amino acid peptide. The second and third amino acids shown in red represent alanine and glutamate. The enzyme dipeptidyl peptidase–IV (DPP-4) acts to cleave the N-terminal dipeptide following the position 2 alanine, thus inactivating the hormones. T I D S A D I Q K G D A L N F Q K W N K Q L W V D K N T H I Q

Synthesis and Secretion of GLP-1 and GIP K-Cell (jejunum) ProGIP GIP [1-42] L-Cell (ileum+ colon) Proglucagon GLP-1 [7-37] GLP-1 [7-36NH2] GIP is synthesized predominantly in the proximal small bowel, principally in gut endocrine K cells residing within the duodenum and jejunum. In contrast, GLP-1 is liberated following posttranslational processing of proglucagon in enteroendocrine L cells residing within the ileum and colon. Nevertheless, a small amount of GLP-1 may also be derived from more proximally located L cells in the upper small bowel. Both peptides are rapidly released within minutes of nutrient ingestion.

Incretin Secretion is Proportionate to the Amount of Food Ingested GLP-1 GIP 50 kcal 260 160 kcal 260 kcal 520 kcal 520 40 120 30 Total GLP-1 (pmol/L) Total GIP (pmol/L) 80 20 This slide depicts total plasma GLP-1 and GIP immunoreactivity in healthy human subjects with assays that recognize both the intact biologically active incretins and their largely inactive metabolites) following a small (260 kcal) or large (520 kcal) breakfast meal in healthy volunteers. It is evident that the circulating levels of both incretins rise promptly in response to nutrient ingestion in direct proportion to the size of the ingested meal. 40 10 -30 30 60 90 120 150 180 210 -30 30 60 90 120 150 180 210 Time (min) Time (min) Vilsbøll T, et al. J Clin Endocrinol Metab. 2003 Jun;88(6):2706-2713. Copyright © 2003. The Endocrine Society.

Meal-Stimulated Levels of GLP-1 are Decreased in Type 2 Diabetes 10 20 30 50 100 150 Time (min) GLP-1 (pmol/L) * P <0.05 Total GLP-1, Controls Total GLP-1, Patients Intact GLP-1, Controls Intact GLP-1, Patients This slide depicts the rise in plasma levels of GLP-1 following meal ingestion in normal human subjects (controls), and in patients with type 2 diabetes. The plasma measurements were obtained using 2 different assays; one that recognizes total GLP-1 (inactive and bioactive) and a second assay that only recognizes the intact biologically active peptide. The data shows a modest but statistically significant reduction in levels of GLP-1 following meal ingestion in diabetic patients Vilsbøll T, et al. Diabetes. 2001;50:609-613.

Overlapping and Contrasting Actions of GLP-1 and GIP Released from L cells in ileum and colon Released from K cells in duodenum Stimulates insulin release from -cell Potent inhibition of gastric emptying Modest effects on gastric emptying Potent inhibition of glucagon secretion No significant inhibition of glucagon secretion Reduction of food intake and body weight Significant effects on -cell growth and survival Insulinotropic actions preserved in type 2 diabetes No significant effects on satiety or body weight Potential effects on -cell growth and survival Defective insulinotropic action in type 2 diabetes Comparison of the properties and actions of GLP-1 vs. GIP. Although both peptides share overlapping actions on the islet β-cell, GLP-1 exerts additional actions, including inhibition of gastric emptying, glucagon secretion, and food intake, that contribute to the lowering of blood glucose. Drucker DJ. Diabetes Care. 2003;26:2929–2940.

Burcelin et al. Diabetes 50, 1720-1728 2001 Peripheral but not Portal Glucose Infusion Increases Blood Glucose in Mice 40 30 8 *# 20 6 AUC (mM) Blood glucose (mM) 10 * 4 This experiment illustrates the concept of the portal glucose sensor, via experiments in mice examining the differential clearance of glucose administered peripherally via the femoral vein, vs. glucose administered into the portal circulation via the portal vein. Although identical amounts of glucose were administered via the different routes, a much greater glycemic excursion is detected after the femoral glucose infusion. In contrast, portal infusion of the same amount of glucose does not produce a significant rise in glycemic excursion. Additional experiments using GLP-1 receptor antagonists and GLP-1 receptor knockout mice demonstrated that an intact GLP-1 receptor signaling system is an essential component of the portal glucose sensor. This concept is important for understanding the mechanism of action of drugs, such as the DPP-4 inhibitors, that potentiate the endogenous levels of portal GLP-1. Saline 2 Portal Glucose infusion -10 # Femoral glucose infusion -20 20 60 100 140 180 S P F min Burcelin et al. Diabetes 50, 1720-1728 2001

Burcelin et al. Diabetes 50, 1720-1728 2001 Enhanced Insulin Secretion Following Portal vs Peripheral GLP-1 Infusion # 120 50 100 40 80 *# 30 Insulin (µU/ml) 60 AUC (µU/ml) 20 This experiment illustrates the concept that the portal glucose sensor is already maximally activated following portal glucose infusion, likely by small amounts of endogenous GLP-1. Further pharmacological administration of exogenous GLP-1 is not sufficient to augment the levels of plasma insulin obtained with the femoral glucose infusion to the levels achieved by the portal glucose infusion. 40 Saline + GLP-1 * 10 Portal Glucose + GLP-1 Femoral Glucose + GLP-1 20 20 60 100 140 180 min S P F Burcelin et al. Diabetes 50, 1720-1728 2001

Adapted from Drucker D. J. Cell Metabolism 2006 Incretins Enhance Glucose Disposal via Activation of Neural and Endocrine Pathways Vagal Afferent Vagal Efferent Glucose Disposal Glucagon Secretion Insulin Secretion The available evidence, from studies done in rats, dogs, and mice, suggests that glucose, derived from ingested nutrients, triggers one or more portal neural sensors located in the portal vein, that signal, via vagal afferents to the brain, that glucose has arrived. The vagal afferents in turn communicate with specific regions of the CNS, and result in processing of the nutrient-derived signals, followed by additional efferent signals that communicate with islet β-cells, enhancing the secretion of insulin. The available evidence supports a role for GLP-1, secreted from gut endocrine cells, as a key component of the portal glucose sensor. It seems likely that additional vagal efferents also communicate with other tissues, such as the muscle and liver, to coordinate the uptake and storage of glucose. In contrast, circulating levels of GLP-1 and GIP also directly regulate insulin secretion via interaction with their receptors expressed on islet β-cells. D. J. Drucker Cell Metabolism 2006 Portal Vein Portal GLP - 1 Peripheral GLP - 1 Peripheral GIP Gut Endocrine Cell Adapted from Drucker D. J. Cell Metabolism 2006

GLP-1 but not GIP Stimulates Insulin Secretion in Patients with Type 2 Diabetes 8000 Hyperglycemic Clamp Saline or GIP or GLP-1 6000 GLP-1 GIP Saline C-peptide (pmol/L) 4000 2000 This study showed the differential effects of GIP vs GLP-1 in stimulating glucose-dependent C-peptide secretion in human subjects with type 2 diabetes. GIP is a potent stimulator of insulin secretion at elevated glucose concentrations in normal human subjects. However, as is evident from the results shown here, GIP frequently fails to significantly augment insulin secretion in subjects with type 2 diabetes. In contrast, GLP-1 retains its insulinotropic properties in patients with type 2 diabetes. -15 -10 0 5 10 15 20 30 45 60 75 90 105 120 150 Time (min) Adapted from Vilsbøll T, et al. Diabetologia. 2002;45:1111-1119.

GLP-1 Inhibits Gastric Emptying in Type 2 Diabetes Placebo SC-Injection GLP-1 500 GLP-1 Liquid Meal 400 300 Gastric Volume (mL) 200 Exogenous GLP-1 administration inhibits gastric emptying following ingestion of either a solid or liquid meal in patients with type 2 diabetes. 100 *P <.0001 -30 30 60 90 120 150 180 210 240 Time (min) Nauck MA, et al. Diabetologia. 1996;39:1546-1553.