Andrew A. Young

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.

Effects of PYY[3–36] in rodent models of diabetes and obesity

BACKGROUND: Peptide YY (PYY) is a 36 amino-acid peptide secreted from ileal L cells following meals. The cleaved subpeptide PYY[3–36] is biologically active and may constitute the majority of circulating PYY-like immunoreactivity. The peptide family that includes PYY, pancreatic peptide and neuropeptide Y is noted for its orexigenic effect following intracerebroventricular administration.OBJECTIVE: To investigate the effects of peripheral (intraperitoneal and chronic subcutaneous) infusions of PYY[3–36] on food intake, body weight and glycemic indices.DESIGN/RESULTS: Food intake was measured in normal mice and in several rodent models of obesity and type II diabetes. In marked contrast to the reported central orexigenic effects, in the present study, PYY[3–36] acutely inhibited food intake by up to 45%, with an ED50 of 12.5 μg/kg in fasted female NIH/Swiss mice. A 4-week infusion reduced weight gain in female ob/ob mice, without affecting the cumulative food intake. In diet-induced obese male mice, PYY[3–36] infusion reduced cumulative food intake, weight gain and epididymal fat weight (as a fraction of carcass) with similar ED50s (466, 297 and 201 μg/kg/day, respectively) and prevented a diet-induced increase in HbA1c. Infusion at 100 μg/kg/day for 8 weeks in male fa/fa rats reduced the weight gain (288±11 vs 326±12 g in saline-infused controls; P<0.05), similar to effects in a pair-fed group. In female ob/ob and db/db mice, there was no acute effect of PYY[3-36] on plasma glucose concentrations. In male diabetic fatty Zucker rats, PYY[3–36] infused for 4 weeks reduced HbA1c and fructosamine (ED50s 30 and 44 μg/kg/day).CONCLUSION: Peripheral PYY[3–36] administration reduced the food intake, body weight gain and glycemic indices in diverse rodent models of metabolic disease of both sexes. These findings justify further exploration of the potential physiologic and therapeutic roles of PYY[3–36].

Gastric emptying is accelerated in diabetic BB rats and is slowed by subcutaneous injections of amylin

SummaryGastric emptying was measured in normal and insulin-treated spontaneously diabetic BB rats using the retention of an acaloric methylcellulose gel containing phenol red delivered by gavage. Dye content in stomachs removed after killing 20 min later was determined spectroscopically, and was compared to that in rats killed immediately after gavage to assess emptying. Diabetic rats had a markedly greater gastric emptying (90.3±1.7% passed) compared to normal Harlan Sprague Dawley rats (49.1±4.7% passed; p<0.001) and non-diabetic BB rats (61.1±9.2% passed; p<0.001). The pancreatic beta-cell peptide, amylin, which is deficient in insulin-dependent diabetes mellitus, dose-dependently inhibited gastric emptying in both normal and diabetic rats. The ED50 of the response in normal rats measured by phenol red and novel [3-3H]glucose gavage techniques was approximately 0.4 Μg. This dose was estimated to increase plasma amylin concentration by a mean of approximately 20 pmol/l to concentrations within the range observed in vivo. It is proposed that amylin could participate in the physiological control of nutrient entry into the duodenum, and that the accelerated gastric emptying seen in BB rats could be related to their lack of amylin secretion.

Preliminary report Dose-responses for the slowing of gastric emptying in a rodent model by glucagon-like peptide (7–36)NH2, amylin, cholecystokinin, and other possible regulators of nutrient uptake

Several peptides have been proposed as regulators of nutrient release from the stomach and subsequent uptake from the gut. Using a phenol red gavage method, we compared the potencies of subcutaneously preinjected amylin, glucagon-like peptide-1 (7-36)amide (GLP-1), cholecystokinin octapeptide (CCK-8), gastric inhibitory peptide (GIP), glucagon, and pancreatic peptide on slowing the release of an acaloric gel from rat stomach. The latter three peptides did not fully inhibit gastric emptying at subcutaneous doses up to 100 micrograms. Amylin, GLP-1, and CCK-8 fully inhibited gastric emptying, with ED50s of 0.42 +/- 0.07, 6.1 +/- 0.12, and 8.5 +/- 0.20 nmol/kg +/- SE of log, respectively.

Dose-response for glucagonostatic effect of amylin in rats.

Glucagon secretion from pancreatic alpha cells is inhibited by insulin from beta cells. Amylin is a partner hormone to insulin cosecreted in response to nutrient stimuli, which, like insulin, inhibits beta-cell secretion. We investigated whether amylin also inhibits alpha-cell secretion of glucagon in response to infused L-arginine. Rat amylin (1.2, 3.6, 12, 36, or 120 pmol/kg/min; calculated plasma concentration, 13, 47, 195, 713, and 2,950 pmol/L, respectively; n = 7, 8, 6, 4, and 7) or saline (n = 23) was infused into anesthetized male Harlan-Sprague-Dawley rats during hyperinsulinemic-euglycemic clamps, which were used to equalize the influences of glucose and insulin on glucagon secretion. Plasma glucose and insulin concentrations and mean arterial pressures were not different between amylin- and saline-treated rats during a 10-minute 2-mmol L-arginine infusion delivered during the clamps. Plasma glucagon measurements taken during and after the arginine challenge showed that compared with saline infusions, amylin administration dose-dependently suppressed the glucagon response to arginine by a maximum of 62% (incremental area under the curve [AUC] 0 to 60 minutes) with a plasma amylin EC50 of 18 pmol/L +/- 0.3 log units. These data indicate that amylin potently inhibits arginine-stimulated glucagon secretion.

Antiobesity action of peripheral exenatide (exendin-4) in rodents: effects on food intake, body weight, metabolic status and side-effect measures.

Background:Exenatide (exendin-4) is an incretin mimetic currently marketed as an antidiabetic agent for patients with type 2 diabetes. In preclinical models, a reduction in body weight has also been shown in low-fat-fed, leptin receptor-deficient rodents.Objective:To more closely model the polygenic and environmental state of human obesity, we characterized the effect of exenatide on food intake and body weight in high-fat-fed, normal (those with an intact leptin signaling system) rodents. As glucagon-like peptide-1 receptor agonism has been found to elicit behaviors associated with visceral illness in rodents, we also examined the effect of peripheral exenatide on kaolin consumption and locomotor activity.Methods and results:High-fat-fed C57BL/6 mice and Sprague–Dawley rats were treated with exenatide (3, 10 and 30 μg/kg/day) for 4 weeks via subcutaneously implanted osmotic pumps. Food intake and body weight were assessed weekly. At 4 weeks, body composition and plasma metabolic profiles were measured. Kaolin consumption and locomotor activity were measured in fasted Sprague–Dawley rats following a single intraperitoneal injection of exenatide (0.1–10 μg/kg). Exenatide treatment in mice and rats dose-dependently decreased food intake and body weight; significant reductions in body weight gain were observed throughout treatment at 10 and 30 μg/kg/day (P<0.05). Decreased body weight gain was associated with a significant decrease in fat mass (P<0.05) with sparing of lean tissue. Plasma cholesterol, triglycerides and insulin were also significantly reduced (P<0.05). Exenatide at 10 μg/kg significantly reduced food intake (P<0.05) but failed to induce kaolin intake. In general, locomotor activity was reduced at doses of exenatide that decreased food intake, although a slightly higher dose was required to produce significant changes in activity.Conclusion:Systemic exenatide reduces body weight gain in normal, high-fat-fed rodents, a model that parallels human genetic variation and food consumption patterns, and may play a role in metabolic pathways mediating food intake.

Synergy between amylin and cholecystokinin for inhibition of food intake in mice

Several gastrointestinal peptides which are secreted in response to nutrients have been reported to suppress food intake. Amylin is a peptide hormone co-secreted with insulin from pancreatic beta-cells in response to nutrient stimuli. Cholesystokinin (CCK) is secreted from duodenal and jejunal mucosal cells in response to fat and protein. Amylin and CCK-8 have been reported to reduce food intake in rodents when given centrally as well as peripherally. Amylin injected intraperitoneally (i.p.) reduced food intake over the subsequent 30 min in overnight fasted mice by a maximum of 57 +/- 6% with an ED50 of 0.93 nmol/kg (3.63 microg/kg) +/- 0.34 log units. On a molar basis, this potency was similar to that of CCK-8 (ED50 0.85 nmol/kg (0.97 microg/kg) +/- 0.28 log units; p = 0.93) which inhibited food intake by a maximum of 71 +/- 7%. When amylin and CCK-8 were injected i.p. as an amylin:CCK-8 mixture, immediately before presentation of food in overnight fasted mice, food intake in the subsequent 30 min was reduced by a maximum of 91%, an amount that was greater than that producable by i.p. injection of amylin or CCK-8 alone. Isobolar analysis revealed a marked synergy between amylin and CCK-8 in reducing food intake, such that statistically ineffective doses of amylin and CCK, when combined, evoked near-maximal inhibition of food intake. Because the typical physiological event is for amylin and CCK both to be secreted in response to mixed meals, the synergy between them could indicate a shared role in physiological appetite control.

Preclinical pharmacology of pramlintide in the rat: Comparisons with human and rat amylin

The pancreatic β‐cell hormone, amylin, is absent or reduced in individuals with type I diabetes mellitus and in many insulin‐treated patients with type II diabetes. Amylin replacement therapy may be beneficial in these individuals, but the pharmaceutically inconvenient physicochemical properties of native human amylin led to the development instead of the amylin agonist, [Pro25,28,29]human amylin, or pramlintide (formerly designated AC137). Here we compare for rat amylin, human amylin and pramlintide, receptor binding and biological actions in rats in vivo and in rat soleus muscle. In the rat, the spectrum of actions and pharmacokinetic and pharmacodynamic properties of pramlintide are either very similar to, or indistinguishable from, those of rat or human amylin.

Structure and biology of amylin

Amylin is a recently discovered 37 amino acid peptide secreted into the bloodstream, along with insulin, from pancreatic beta-cells. It is about 50% identical to calcitonin gene-related peptides (CGRP alpha and CGRP beta) and structurally related to the calcitonins. Amylin can elicit the vasodilator effects of CGRP and the hypocalcaemic actions of calcitonin, while these peptides can mimic newly discovered actions of amylin on carbohydrate metabolism. The different relative potencies of these peptides suggest that they act with different selectivities at a family of receptors. Amylin is deficient in insulin-dependent diabetes mellitus, while plasma levels are elevated in insulin-resistant conditions such as obesity and impaired glucose tolerance. In this Viewpoint article, Tim Rink and colleagues propose that amylin is an endocrine partner to insulin and glucagon; deficiency or excess of amylin may therefore contribute to important metabolic diseases.

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.