Chronic AT1 blockade improves hyperglycemia by decreasing adipocyte inflammation and decreasing hepatic PCK1 and G6PC1 expression in obese rats.

Inappropriate activation of the renin-angiotensin system decreases glucose uptake in peripheral tissues. Chronic angiotensin receptor type 1 (AT1) blockade (ARB) increases glucose uptake in skeletal muscle and decreases the abundance of large adipocytes and macrophage infiltration in adipose. However, the contributions of each tissue to the improvement in hyperglycemia in response to AT1 blockade are not known. Therefore, we determined the static and dynamic responses of soleus muscle, liver, and adipose to an acute glucose challenge following the chronic blockade of AT1. We measured adipocyte morphology along with TNF-α expression, F4/80- and CD11c-positive cells in adipose and measured insulin receptor (IR) phosphorylation and AKT phosphorylation in soleus muscle, liver, and retroperitoneal fat before (T0), 60 (T60) and 120 (T120) min after an acute glucose challenge in the following groups of male rats: 1) Long-Evans Tokushima Otsuka (LETO; lean control; n = 5/time point), 2) obese Otsuka Long Evans Tokushima Fatty (OLETF; n = 7 or 8/time point), and 3) OLETF + ARB (ARB; 10 mg olmesartan/kg/day; n = 7 or 8/time point). AT1 blockade decreased adipocyte TNF-α expression and F4/80- and CD11c-positive cells. In retroperitoneal fat at T60, IR phosphorylation was 155% greater in ARB than in OLETF. Furthermore, in retroperitoneal fat AT1 blockade increased glucose transporter-4 (GLUT4) protein expression in ARB compared with OLETF. IR phosphorylation and AKT phosphorylation were not altered in the liver of OLETF, but AT1 blockade decreased hepatic Pck1 and G6pc1 mRNA expressions. Collectively, these results suggest that chronic AT1 blockade improves obesity-associated hyperglycemia in OLETF rats by improving adipocyte function and by decreasing hepatic glucose production via gluconeogenesis.NEW & NOTEWORTHY Inappropriate activation of the renin-angiotensin system increases adipocyte inflammation contributing to the impairment in adipocyte function and increases hepatic Pck1 and G6pc1 mRNA expression in response to a glucose challenge. Ultimately, these effects may contribute to the development of glucose intolerance.

Simultaneous angiotensin receptor blockade and glucagon-like peptide-1 receptor activation ameliorate albuminuria in obese insulin-resistant rats.

Insulin resistance increases renal oxidant production by upregulating NADPH oxidase 4 (Nox4) expression contributing to oxidative damage and ultimately albuminuria. Inhibition of the renin-angiotensin system (RAS) and activation of glucagon-like peptide-1 (GLP-1) receptor signalling may reverse this effect. However, whether angiotensin receptor type 1 (AT1) blockade and GLP-1 receptor activation improve oxidative damage and albuminuria through different mechanisms is not known. Using insulin-resistant Otsuka Long-Evans Tokushima Fatty (OLETF) rats, we tested the hypothesis that simultaneous blockade of AT1 and activation of GLP-1r additively decrease oxidative damage and urinary albumin excretion (Ualb V) in the following groups: (a) untreated, lean LETO (n = 7), (b) untreated, obese OLETF (n = 9), (c) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d; n = 9), (d) OLETF + GLP-1 mimetic (EXE; 10 µg exenatide/kg/d; n = 7) and (e) OLETF + ARB +exenatide (Combo; n = 6). Mean kidney Nox4 protein expression and nitrotyrosine (NT) levels were 30% and 46% greater, respectively, in OLETF compared with LETO. Conversely, Nox4 protein expression and NT were reduced to LETO levels in ARB and EXE, and Combo reduced Nox4, NT and 4-hydroxy-2-nonenal levels by 21%, 27% and 27%, respectively. At baseline, Ualb V was nearly double in OLETF compared with LETO and increased to nearly 10-fold greater levels by the end of the study. Whereas ARB (45%) and EXE (55%) individually reduced Ualb V, the combination completely ameliorated the albuminuria. Collectively, these data suggest that AT1 blockade and GLP-1 receptor activation reduce renal oxidative damage similarly during insulin resistance, whereas targeting both signalling pathways provides added benefit in restoring and/or further ameliorating albuminuria in a model of diet-induced obesity.

Amylin: Pharmacology, Physiology, and Clinical Potential.

Amylin is a pancreatic ß-cell hormone that produces effects in several different organ systems. Here, we review the literature in rodents and in humans on amylin research since its discovery as a hormone about 25 years ago. Amylin is a 37-amino-acid peptide that activates its specific receptors, which are multisubunit G protein-coupled receptors resulting from the coexpression of a core receptor protein with receptor activity-modifying proteins, resulting in multiple receptor subtypes. Amylin's major role is as a glucoregulatory hormone, and it is an important regulator of energy metabolism in health and disease. Other amylin actions have also been reported, such as on the cardiovascular system or on bone. Amylin acts principally in the circumventricular organs of the central nervous system and functionally interacts with other metabolically active hormones such as cholecystokinin, leptin, and estradiol. The amylin-based peptide, pramlintide, is used clinically to treat type 1 and type 2 diabetes. Clinical studies in obesity have shown that amylin agonists could also be useful for weight loss, especially in combination with other agents.

Long-term metabolic benefits of exenatide in mice are mediated solely via the known glucagon-like peptide 1 receptor.

Glucagon-like peptide 1 receptors (GLP-1R) are expressed in multiple tissues and activation results in metabolic benefits including enhanced insulin secretion, slowed gastric emptying, suppressed food intake, and improved hepatic steatosis. Limited and inconclusive knowledge exists regarding whether the effects of chronic exposure to a GLP-1R agonist are solely mediated via this receptor. Therefore, we examined 3-mo dosing of exenatide in mice lacking a functional GLP-1R (Glp1r(-/-)). Exenatide (30 nmol · kg(-1) · day(-1)) was infused subcutaneously for 12 wk in Glp1r(-/-) and wild-type (Glp1r(+/+)) control mice fed a high-fat diet. Glycated hemoglobin A1c (HbA1c), plasma glucose, insulin, amylase, lipase, alanine aminotransferase (ALT), aspartate aminotransferase (AST), body weight, food intake, terminal hepatic lipid content (HLC), and plasma exenatide levels were measured. At the end of the study, oral glucose tolerance test (OGTT) and rate of gastric emptying were assessed. Exenatide produced no significant changes in Glp1r(-/-) mice at study end. In contrast, exenatide decreased body weight, food intake, and glucose in Glp1r(+/+) mice. When compared with vehicle, exenatide reduced insulin, OGTT glucose AUC0-2h, ALT, and HLC in Glp1r(+/+) mice. Exenatide had no effect on plasma amylase or lipase levels. Exenatide concentrations were approximately eightfold higher in Glp1r(-/-) versus Glp1r(+/+) mice after 12 wk of infusion, whereas renal function was similar. These data support the concept that exenatide requires a functional GLP-1R to exert chronic metabolic effects in mice, and that novel "GLP-1" receptors may not substantially contribute to these changes. Differential exenatide plasma levels in Glp1r(+/+) versus Glp1r(-/-) mice suggest that GLP-1R may play an important role in plasma clearance of exenatide and potentially other GLP-1-related peptides.

Glucagon-like peptide-1 receptor agonism improves metabolic, biochemical, and histopathological indices of nonalcoholic steatohepatitis in mice.

These preclinical studies aimed to 1) increase our understanding the dietary induction of nonalcoholic steatohepatitis (NASH), and, 2) further explore the utility and mechanisms of glucagon-like peptide-1 receptor (GLP-1R) agonism in NASH. We compared the effects of a high trans-fat (HTF) or high lard fat (HLF) diet on key facets of nonalcoholic fatty liver disease (NAFLD)/NASH in Lep(ob)/Lep(ob) and C57BL6J (B6) mice. Although HLF-fed mice experienced overall greater gains in weight and adiposity, the addition of trans-fat better mirrored pathophysiological features of NASH (e.g., hepatomegaly, hepatic lipid, and fibrosis). Administration of AC3174, an exenatide analog, and GLP-1R agonist to Lep(ob)/Lep(ob) and B6 ameliorated hepatic endpoints in both dietary models. Next, we assessed whether AC3174-mediated improvements in diet-induced NASH were solely due to weight loss in HTF-fed mice. AC3174-treatment significantly reduced body weight (8.3%), liver mass (14.2%), liver lipid (12.9%), plasma alanine aminotransferase, and triglycerides, whereas a calorie-restricted, weight-matched group demonstrated only modest nonsignificant reductions in liver mass (9%) and liver lipid (5.1%) relative to controls. Treatment of GLP-1R-deficient (GLP-1RKO) mice with AC3174 had no effect on body weight, adiposity, liver or plasma indices pointing to the GLP-1R-dependence of AC3174's effects. Interestingly, the role of endogenous GLP-1Rs in NASH merits further exploration as the GLP-1RKO model was protected from the deleterious hepatic effects of HTF. Our pharmacological data further support the clinical evaluation of the utility of GLP-1R agonists for treatment of NASH.

Leptin responsiveness restored by amylin agonism in diet-induced obesity: evidence from nonclinical and clinical studies.

Body weight is regulated by complex neurohormonal interactions between endocrine signals of long-term adiposity (e.g., leptin, a hypothalamic signal) and short-term satiety (e.g., amylin, a hindbrain signal). We report that concurrent peripheral administration of amylin and leptin elicits synergistic, fat-specific weight loss in leptin-resistant, diet-induced obese rats. Weight loss synergy was specific to amylin treatment, compared with other anorexigenic peptides, and dissociable from amylin's effect on food intake. The addition of leptin after amylin pretreatment elicited further weight loss, compared with either monotherapy condition. In a 24-week randomized, double-blind, clinical proof-of-concept study in overweight/obese subjects, coadministration of recombinant human leptin and the amylin analog pramlintide elicited 12.7% mean weight loss, significantly more than was observed with either treatment alone (P < 0.01). In obese rats, amylin pretreatment partially restored hypothalamic leptin signaling (pSTAT3 immunoreactivity) within the ventromedial, but not the arcuate nucleus and up-regulated basal and leptin-stimulated signaling in the hindbrain area postrema. These findings provide both nonclinical and clinical evidence that amylin agonism restored leptin responsiveness in diet-induced obesity, suggesting that integrated neurohormonal approaches to obesity pharmacotherapy may facilitate greater weight loss by harnessing naturally occurring synergies.

Chronic peptide-based GIP receptor inhibition exhibits modest glucose metabolic changes in mice when administered either alone or combined with GLP-1 agonism.

Combinatorial gut hormone therapy is one of the more promising strategies for identifying improved treatments for metabolic disease. Many approaches combine the established benefits of glucagon-like peptide-1 (GLP-1) agonism with one or more additional molecules with the aim of improving metabolic outcomes. Recent attention has been drawn to the glucose-dependent insulinotropic polypeptide (GIP) system due to compelling pre-clinical evidence describing the metabolic benefits of antagonising the GIP receptor (GIPR). We rationalised that benefit might be accrued from combining GIPR antagonism with GLP-1 agonism. Two GIPR peptide antagonists, GIPA-1 (mouse GIP(3-30)NH2) and GIPA-2 (NαAc-K10[γEγE-C16]-Arg18-hGIP(5-42)), were pharmacologically characterised and both exhibited potent antagonist properties. Acute in vivo administration of GIPA-1 during an oral glucose tolerance test (OGTT) had negligible effects on glucose tolerance and insulin in lean mice. In contrast, GIPA-2 impaired glucose tolerance and attenuated circulating insulin levels. A mouse model of diet-induced obesity (DIO) was used to investigate the potential metabolic benefits of chronic dosing of each antagonist, alone or in combination with liraglutide. Chronic administration studies showed expected effects of liraglutide, lowering food intake, body weight, fasting blood glucose and plasma insulin concentrations while improving glucose sensitivity, whereas delivery of either GIPR antagonist alone had negligible effects on these parameters. Interestingly, chronic dual therapy augmented insulin sensitizing effects and lowered plasma triglycerides and free-fatty acids, with more notable effects observed with GIPA-1 compared to GIPA-2. Thus, the co-administration of both a GIPR antagonist with a GLP1 agonist uncovers interesting beneficial effects on measures of insulin sensitivity, circulating lipids and certain adipose stores that seem influenced by the degree or nature of GIP receptor antagonism.

Exenatide does not evoke pancreatitis and attenuates chemically induced pancreatitis in normal and diabetic rodents.

The risk of developing pancreatitis is elevated in type 2 diabetes and obesity. Cases of pancreatitis have been reported in type 2 diabetes patients treated with GLP-1 (GLP-1R) receptor agonists. To examine whether the GLP-1R agonist exenatide potentially induces or modulates pancreatitis, the effect of exenatide was evaluated in normal or diabetic rodents. Normal and diabetic rats received a single exenatide dose (0.072, 0.24, and 0.72 nmol/kg) or vehicle. Diabetic ob/ob or HF-STZ mice were infused with exenatide (1.2 and 7.2 nmol·kg(-1)·day(-1)) or vehicle for 4 wk. Post-exenatide treatment, pancreatitis was induced with caerulein (CRN) or sodium taurocholate (ST), and changes in plasma amylase and lipase were measured. In ob/ob mice, plasma cytokines (IL-1ß, IL-2, IL-6, MCP-1, IFNγ, and TNFα) and pancreatitis-associated genes were assessed. Pancreata were weighed and examined histologically. Exenatide treatment alone did not modify plasma amylase or lipase in any models tested. Exenatide attenuated CRN-induced release of amylase and lipase in normal rats and ob/ob mice but did not modify the response to ST infusion. Plasma cytokines and pancreatic weight were unaffected by exenatide. Exenatide upregulated Reg3b but not Il6, Ccl2, Nfkb1, or Vamp8 expression. Histological analysis revealed that the highest doses of exenatide decreased CRN- or ST-induced acute inflammation, vacuolation, and acinar single cell necrosis in mice and rats, respectively. Ductal cell proliferation rates were low and similar across all groups of ob/ob mice. In conclusion, exenatide did not modify plasma amylase and lipase concentrations in rodents without pancreatitis and improved chemically induced pancreatitis in normal and diabetic rodents.

The exenatide analogue AC3174 attenuates hypertension, insulin resistance, and renal dysfunction in Dahl salt-sensitive rats.

BACKGROUND: Activation of glucagon-like peptide-1 (GLP-1) receptors improves insulin sensitivity and induces vasodilatation and diuresis. AC3174 is a peptide analogue with pharmacologic properties similar to the GLP-1 receptor agonist, exenatide. Hypothetically, chronic AC3174 treatment could attenuate salt-induced hypertension, cardiac morbidity, insulin resistance, and renal dysfunction in Dahl salt-sensitive (DSS) rats. METHODS: DSS rats were fed low salt (LS, 0.3% NaCl) or high salt (HS, 8% NaCl) diets. HS rats were treated with vehicle, AC3174 (1.7 pmol/kg/min), or GLP-1 (25 pmol/kg/min) for 4 weeks via subcutaneous infusion. Other HS rats received captopril (150 mg/kg/day) or AC3174 plus captopril. RESULTS: HS rat survival was improved by all treatments except GLP-1. Systolic blood pressure (SBP) was lower in LS rats and in GLP-1, AC3174, captopril, or AC3174 plus captopril HS rats than in vehicle HS rats (p < 0.05). AC3174 plus captopril attenuated the deleterious effects of high salt on posterior wall thickness, LV mass, and the ratio of LV mass to body weight (P < or = 0.05). In contrast, GLP-1 had no effect on these cardiovascular parameters. All treatments reduced LV wall stress. GLP-1, AC3174, captopril, or AC3174 plus captopril normalized fasting insulin and HOMA-IR (P < or = 0.05). AC3174, captopril, or AC3174 plus captopril improved renal function (P < or = 0.05). Renal morphology in HS rats was associated with extensive sclerosis. Monotherapy with AC3174, captopril, or GLP-1 attenuated renal damage. However, AC3174 plus captopril produced the most effective improvement. CONCLUSIONS: Thus, AC3174 had antihypertensive, cardioprotective, insulin-sensitizing, and renoprotective effects in the DSS hypertensive rat model. Furthermore, AC3174 improved animal survival, an effect not observed with GLP-1.

Glucagon-like peptide-1 and the exenatide analogue AC3174 improve cardiac function, cardiac remodeling, and survival in rats with chronic heart failure.

BACKGROUND: Accumulating evidence suggests glucagon-like peptide-1 (GLP-1) exerts cardioprotective effects in animal models of myocardial infarction (MI). We hypothesized that chronic treatment with GLP-1 or the exenatide analog AC3174 would improve cardiac function, cardiac remodeling, insulin sensitivity, and exercise capacity (EC) in rats with MI-induced chronic heart failure (CHF) caused by coronary artery ligation. METHODS: Two weeks post-MI, male Sprague-Dawley rats were treated with GLP-1 (2.5 or 25 pmol/kg/min), AC3174 (1.7 or 5 pmol/kg/min) or vehicle via subcutaneous infusion for 11 weeks. Cardiac function and morphology were assessed by echocardiography during treatment. Metabolic, hemodynamic, exercise-capacity, and body composition measurements were made at study end. RESULTS: Compared with vehicle-treated rats with CHF, GLP-1 or AC3174 significantly improved cardiac function, including left ventricular (LV) ejection fraction, and end diastolic pressure. Cardiac dimensions also improved as evidenced by reduced LV end diastolic and systolic volumes and reduced left atrial volume. Vehicle-treated CHF rats exhibited fasting hyperglycemia and hyperinsulinemia. In contrast, GLP-1 or AC3174 normalized fasting plasma insulin and glucose levels. GLP-1 or AC3174 also significantly reduced body fat and fluid mass and improved exercise capacity and respiratory efficiency. Four of 16 vehicle control CHF rats died during the study compared with 1 of 44 rats treated with GLP-1 or AC3174. The cellular mechanism by which GLP-1 or AC3174 exert cardioprotective effects appears unrelated to changes in GLUT1 or GLUT4 translocation or expression. CONCLUSIONS: Chronic treatment with either GLP-1 or AC3174 showed promising cardioprotective effects in a rat model of CHF. Hence, GLP-1 receptor agonists may represent a novel approach for the treatment of patients with CHF or cardiovascular disease associated with type 2 diabetes.

Amylin-mediated restoration of leptin responsiveness in diet-induced obesity: magnitude and mechanisms.

Previously, we reported that combination treatment with rat amylin (100 microg/kg.d) and murine leptin (500 microg/kg.d) elicited greater inhibition of food intake and greater body weight loss in diet-induced obese rats than predicted by the sum of the monotherapy conditions, a finding consistent with amylin-induced restoration of leptin responsiveness. In the present study, a 3 x 4 factorial design was used to formally test for a synergistic interaction, using lower dose ranges of amylin (0, 10, and 50 microg/kg.d) and leptin (0, 5, 25, and 125 microg/kg.d), on food intake and body weight after 4 wk continuous infusion. Response surface methodology analysis revealed significant synergistic anorexigenic (P < 0.05) and body weight-lowering (P < 0.05) effects of amylin/leptin combination treatment, with up to 15% weight loss at doses considerably lower than previously reported. Pair-feeding (PF) experiments demonstrated that reduction of food intake was the predominant mechanism for amylin/leptin-mediated weight loss. However, fat loss was 2-fold greater in amylin/leptin-treated rats than PF controls. Furthermore, amylin/leptin-mediated weight loss was not accompanied by the counterregulatory decrease in energy expenditure and chronic shift toward carbohydrate (rather than fat) utilization observed with PF. Hepatic gene expression analyses revealed that 28 d treatment with amylin/leptin (but not PF) was associated with reduced expression of genes involved in hepatic lipogenesis (Scd1 and Fasn mRNA) and increased expression of genes involved in lipid utilization (Pck1 mRNA). We conclude that amylin/leptin interact synergistically to reduce body weight and adiposity in diet-induced obese rodents through a number of anorexigenic and metabolic effects.

Pharmacokinetics and pharmacodynamics of exenatide following alternate routes of administration.

Exenatide is a 39-amino acid peptide incretin mimetic approved for adjunctive treatment of type 2 diabetes. It shares several glucoregulatory activities with the mammalian hormone, glucagon-like peptide-1 (GLP-1). In clinical use, subcutaneous exenatide injections demonstrate glucoregulatory and weight loss effects with sustained plasma concentrations in the 50-100 pM range. We investigated the pharmacokinetics of exenatide in normoglycemic rats and biological activity in diabetic db/db mice after delivery to various epithelial surfaces of the intestinal and respiratory tracts. In rats, elimination kinetics were similar for all routes of administration (median k(e) 0.017 min(-1)). Bioavailability (versus intravenous administration) and C(max) per unit dose differed markedly. For gastrointestinal administration, sublingual administration invoked the highest bioavailability (0.37%); in db/db mice, potentially therapeutic concentrations were obtainable. In contrast, intraduodenal bioavailability was low (0.0053%). In regard to respiratory surfaces, bioavailability of intratracheal exenatide was up to 13.6%, and for nasal administration, 1.68%. Both routes of administration produced therapeutic plasma concentrations and glucose-lowering in db/db mice. At high doses, aerosolized exenatide also achieved effective concentrations and glucose-lowering. In summary, the intestinal tract seems to have limited potential as a route of exenatide administration, with sublingual being most promising. In contrast, the respiratory tract appears to be more viable, comparing favorably with the clinically approved subcutaneous route. Despite little optimization of the delivery formulation, exenatide bioavailability compared favorable to that of several commercially available bioactive peptides.

Chronic AT1 blockade improves glucose homeostasis in obese OLETF rats.

Obesity is associated with the inappropriate activation of the renin-angiotensin system (RAS), which increases arterial pressure, impairs insulin secretion and decreases peripheral tissue insulin sensitivity. RAS blockade reverses these detriments; however, it is not clear whether the disease state of the organism and treatment duration determine the beneficial effects of RAS inhibition on insulin secretion and insulin sensitivity. Therefore, the objective of this study was to compare the benefits of acute vs chronic angiotensin receptor type 1 (AT1) blockade started after the onset of obesity, hyperglycemia and hypertension on pancreatic function and peripheral insulin resistance. We assessed adipocyte morphology, glucose intolerance, pancreatic redox balance and insulin secretion after 2 and 11 weeks of AT1 blockade in the following groups of rats: (1) untreated Long-Evans Tokushima Otsuka (lean control; n = 10), (2) untreated Otsuka Long-Evans Tokushima Fatty (OLETF; n = 12) and (3) OLETF + ARB (ARB; 10 mg olmesartan/kg/day by oral gavage; n = 12). Regardless of treatment duration, AT1 blockade decreased systolic blood pressure and fasting plasma triglycerides, whereas chronic AT1 blockade decreased fasting plasma glucose, glucose intolerance and the relative abundance of large adipocytes by 22, 36 and 70%, respectively. AT1 blockade, however, did not improve pancreatic oxidative stress or reverse impaired insulin secretion. Collectively, these data show that AT1 blockade after the onset of obesity, hyperglycemia and hypertension improves peripheral tissue insulin sensitivity, but cannot completely reverse the metabolic derangement characterized by impaired insulin secretion once it has been compromised.

Combination therapy with amylin and peptide YY[3-36] in obese rodents: anorexigenic synergy and weight loss additivity.

Circulating levels of the pancreatic beta-cell peptide hormone amylin and the gut peptide PYY[3-36] increase after nutrient ingestion. Both have been implicated as short-term signals of meal termination with anorexigenic and weight-reducing effects. However, their combined effects are unknown. We report that the combination of amylin and PYY[3-36] elicited greater anorexigenic and weight-reducing effects than either peptide alone. In high-fat-fed rats, a single ip injection of amylin (10 microg/kg) plus PYY[3-36] (1000 microg/kg) reduced food intake for 24 h (P < 0.05 vs. vehicle), whereas the anorexigenic effects of either PYY[3-36] or amylin alone began to diminish 6 h after injection. These anorexigenic effects were dissociable from changes in locomotor activity. Subcutaneous infusion of amylin plus PYY[3-36] for 14 d suppressed food intake and body weight to a greater extent than either agent alone in both rat and mouse diet-induced obesity (DIO) models (P < 0.05). In DIO-prone rats, 24-h metabolic rate was maintained despite weight loss, and amylin plus PYY[3-36] (but not monotherapy) increased 24-h fat oxidation (P < 0.05 vs. vehicle). Finally, a 4 x 3 factorial design was used to formally describe the interaction between amylin and PYY[3-36]. DIO-prone rats were treated with amylin (0, 4, 20, and 100 microg/kg.d) and PYY[3-36] (0, 200, 400 microg/kg.d) alone and in combination for 14 d. Statistical analyses revealed that food intake suppression with amylin plus PYY[3-36] treatment was synergistic, whereas body weight reduction was additive. Collectively, these observations highlight the importance of studying peptide hormones in combination and suggest that integrated neurohormonal approaches may hold promise as treatments for obesity.

Biological activity of AC3174, a peptide analog of exendin-4.

Exenatide, the active ingredient of BYETTA (exenatide injection), is an incretin mimetic that has been developed for the treatment of patients with type 2 diabetes. Exenatide binds to and activates the known GLP-1 receptor with a potency comparable to that of the mammalian incretin GLP-1(7-36), thereby acting as a glucoregulatory agent. AC3174 is an analog of exenatide with leucine substituted for methionine at position 14, [Leu(14)]exendin-4. The purpose of these studies was to evaluate the glucoregulatory activity and pharmacokinetics of AC3174. In RINm5f cell membranes, the potency of AC3174 for the displacement of [(125)I]GLP-1 and activation of adenylate cyclase was similar to that of exenatide and GLP-1. In vivo, AC3174, administered as a single IP injection, significantly decreased plasma glucose concentration and glucose excursion following the administration of an oral glucose challenge in both non-diabetic (C57BL/6) and diabetic db/db mice (P<0.05 vs. vehicle-treated). The magnitude of glucose lowering of AC3174 was comparable to exenatide. The ED(50) values of AC3174 for glucose lowering (60 minute post-dose) were 1.2 microg/kg in db/db mice and 1.3 microg/kg in C57BL/6 mice. AC3174 has insulinotropic activity in vivo. Administration of AC3174 resulted in a 4-fold increase in insulin concentrations in normal mice following an IP glucose challenge. AC3174 was also shown to inhibit food intake and decrease gastric emptying in rodent models. AC3174 was stable in human plasma (>90% of parent peptide was present after 5 h of incubation). In rats, the in vivo half-life of AC3174 was 42-43 min following SC administration. In summary, AC3174 is an analog of exenatide that binds to the GLP-1 receptor in vitro and shares many of the biological and glucoregulatory activities of exenatide and GLP-1 in vivo.

A Hydrogel-Microsphere Drug Delivery System That Supports Once-Monthly Administration of a GLP-1 Receptor Agonist.

We have developed a chemically controlled very long-acting delivery system to support once-monthly administration of a peptidic GLP-1R agonist. Initially, the prototypical GLP-1R agonist exenatide was covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection in rats, the peptide was slowly released into the systemic circulation. However, the short serum exenatide half-life suggested its degradation in the subcutaneous depot. We found that exenatide undergoes deamidation at Asn28 with an in vitro and in vivo half-life of approximately 2 weeks. The [Gln28]exenatide variant and exenatide showed indistinguishable GLP-1R agonist activities as well as pharmacokinetic and pharmacodynamic effects in rodents; however, unlike exenatide, [Gln28]exenatide is stable for long periods. Two different hydrogel-[Gln28]exenatide conjugates were prepared using ß-eliminative linkers with different cleavage rates. After subcutaneous injection in rodents, the serum half-lives for the released [Gln28]exenatide from the two conjugates were about 2 weeks and one month. Two monthly injections of the latter in the Zucker diabetic fatty rat showed pharmacodynamic effects indistinguishable from two months of continuously infused exenatide. Pharmacokinetic simulations indicate that the delivery system should serve well as a once-monthly GLP-1R agonist for treatment of type 2 diabetes in humans.

Investigation of exenatide elimination and its in vivo and in vitro degradation.

Exenatide is a 39 amino acid incretin mimetic for the treatment of type 2 diabetes, with glucoregulatory activity similar to glucagon-like peptide-1 (GLP-1). Exenatide is a poor substrate for the major route of GLP-1 degradation by dipeptidyl peptidase-IV, and displays enhanced pharmacokinetics and in vivo potency in rats relative to GLP-1. The kidney appears to be the major route of exenatide elimination in the rat. We further investigated the putative sites of exenatide degradation and excretion, and identified primary degradants. Plasma exenatide concentrations were elevated and sustained in renal-ligated rats, when compared to sham-operated controls. By contrast, exenatide elimination and degradation was not affected in rat models of hepatic dysfunction. In vitro, four primary cleavage sites after amino acids (AA)-15, -21, -22 and -34 were identified when exenatide was degraded by mouse kidney membranes. The primary cleavage sites of exenatide degradation by rat kidney membranes were after AA-14, -15, -21, and -22. In rabbit, monkey, and human, the primary cleavage sites were after AA-21 and -22. Exenatide was almost completely degraded into peptide fragments <3 AA by the kidney membranes of the species tested. The rates of exenatide degradation by rabbit, monkey and human kidney membranes in vitro were at least 15-fold slower than mouse and rat membranes. Exenatide (1-14), (1-15), (1-22), and (23-39) were not active as either agonists or antagonists to exenatide in vitro. Exenatide (15-39) and (16-39) had moderate-to-weak antagonist activity compared with the known antagonist, exenatide (9-39). In conclusion, the kidney appears to be the primary route of elimination and degradation of exenatide.

Exenatide (exendin-4) improves insulin sensitivity and {beta}-cell mass in insulin-resistant obese fa/fa Zucker rats independent of glycemia and body weight.

The effects of the incretin mimetic exenatide (exendin-4) on metabolic parameters, insulin sensitivity, and beta-cell mass were examined in nondiabetic, insulin-resistant obese fa/fa Zucker rats. After 6 wk of treatment, ad libitum-fed exenatide-treated (EX) and pair-fed vehicle control (PF) rats had comparable food intake, body weight, hemoglobin A(1c) (HbA(1c)), and fasting plasma concentrations of glucose, insulin, and lipids. Concurrent decreases in food intake and weight gain were observed in EX and PF rats, compared with ad libitum-fed vehicle control (CON) rats (P < 0.001). The increases in HbA(1c) and fasting plasma insulin concentrations that occur during the normal progression of this disease model were significantly reduced in EX and PF rats, compared with CON rats (P < 0.001). The insulin sensitivity index (ISI; glucose infusion rate to plasma insulin concentration) measured during a hyperinsulinemic euglycemic clamp was 224% higher in EX rats than CON rats (P < 0.001) and 61% higher in EX rats than PF rats (P < 0.004). The latter difference was despite comparable HbA(1c), fasting glucose, fasting insulin, total cholesterol, high-density lipoprotein, and daily food consumption between EX and PF animals. In the absence of exenatide, beta-cell mass was hyperbolically related to ISI (beta-cell mass * ISI was constant). Analogous to the disposition index, the beta-cell mass * ISI product was 63% greater in EX than PF rats (P < 0.05). Thus, exenatide increased beta-cell mass to a greater extent than would be expected in animals of comparable insulin resistance, suggesting a direct trophic effect on islet neogenesis in obese fa/fa rats independent of body weight and glycemia.

Pharmacology of exenatide (synthetic exendin-4): a potential therapeutic for improved glycemic control of type 2 diabetes.

Exenatide (synthetic exendin-4), glucagon-like peptide-1 (GLP-1), and GLP-1 analogues have actions with the potential to significantly improve glycemic control in patients with diabetes. Evidence suggests that these agents use a combination of mechanisms which may include glucose-dependent stimulation of insulin secretion, suppression of glucagon secretion, enhancement of beta-cell mass, slowing of gastric emptying, inhibition of food intake, and modulation of glucose trafficking in peripheral tissues. The short in vivo half-life of GLP-1 has proven a significant barrier to continued clinical development, and the focus of current clinical studies has shifted to agents with longer and more potent in vivo activity. This review examines recent exendin-4 pharmacology in the context of several known mechanisms of action, and contrasts exendin-4 actions with those of GLP-1 and a GLP-1 analogue. One of the most provocative areas of recent research is the finding that exendin-4 enhances beta-cell mass, thereby impeding or even reversing disease progression. Therefore, a major focus of this is article an examination of the data supporting the concept that exendin-4 and GLP-1 may increase beta-cell mass via stimulation of beta-cell neogenesis, stimulation of beta-cell proliferation, and suppression of beta-cell apoptosis.

Novel exenatide analogs with peptidic albumin binding domains: potent anti-diabetic agents with extended duration of action.

The design, synthesis and pharmacology of novel long-acting exenatide analogs for the treatment of metabolic diseases are described. These molecules display enhanced pharmacokinetic profile and potent glucoregulatory and weight lowering actions compared to native exenatide. [Leu(14)]exenatide-ABD is an 88 residue peptide amide incorporating an Albumin Binding Domain (ABD) scaffold. [Leu(14)]exenatide-ABP is a 53 residue peptide incorporating a short Albumin Binding Peptide (ABP). [Leu(14)]exenatide-ABD and [Leu(14)]exenatide-ABP exhibited nanomolar functional GLP-1 receptor potency and were metabolically stable in vitro in human plasma and in a pancreatic digestive enzyme mixture. Both molecules displayed picomolar and nanomolar binding association with albumin across multiple species and circulating half lives of 16 and 11 hours, respectively, post a single IV dose in rats. Unlike exenatide, both molecules elicited robust glucose lowering when injected 1 day prior to an oral glucose tolerance test, indicative of their extended duration of action. [Leu(14)]exenatide-ABD was compared to exenatide in a Lep (ob/ob) mouse model of diabetes. Twice-weekly subcutaneously dosed [Leu(14)]exenatide-ABD displayed superior glucose lowering and weight loss in diabetic mice when compared to continuously infused exenatide at the same total weekly dose. A single oral administration of each molecule via an enteric coated capsule to cynomolgus monkeys showed superior pharmacokinetics for [Leu(14)]exenatide-ABD as compared to [Leu(14)]exenatide-ABP with detectable exposure longer than 14 days. These studies support the potential use of these novel long acting exenatide analogs with different routes of administration for the treatment of type 2 diabetes.