Peripherally administered amylin inhibits stress-like behaviors and enhances cognitive performance.

Amylin, a 37 amino acid peptide pancreatic hormone co-secreted with insulin, normalizes the altered eating patterns induced by chronic stress in the rat. Because these stress-induced changes are driven, in part, by brain corticotropin-releasing factor and corticosterone, and because alterations in the activity of these molecules and the stress system are commonly associated with neuropsychiatric diseases like anxiety, depression, and schizophrenia, we hypothesized that amylin might mitigate behavioral states associated with stress. Therefore, we tested the effects of rat amylin in rodent-based behavioral assays sensitive to neuropsychiatric drugs, including anxiolytic, antidepressant, antipsychotic, and cognitive enhancing drugs: stress-induced hyperthermia (SIH); marble burying; elevated plus maze (EPM)), forced swim test (FST), pre-pulse inhibition, and phencyclidine-induced locomotion. To assess the neural underpinnings of amylin's anxiolytic-like effects, we examined the effect of amylin on SIH after lesioning the area postrema (AP), which mediates amylin's metabolic effects. Amylin injection (IP, 0.1, 1.0, & 10 mg/kg) significantly (P < 0.05) decreased SIH (97% below vehicle) and AP lesions inhibited this effect. Amylin also reduced marble burying (72% below vehicle), but had no effect in the EPM. Together, these effects suggest anxiolytic-like activity or potential. Amylin injection also enhanced cognitive performance in the novel object recognition test. When administered continuously by implanted osmotic pumps, amylin (300 mg/kg/d) blocked SIH when tested at 1 and 4 weeks. Compared to vehicle, amylin infusion (1 and 3 mg/kg/d) reduced the time immobile in the FST (P < 0.05; 30% below vehicle), suggesting antidepressant-like potential. Although further testing is needed, our findings support a potential for peripherally administered amylin to access and benefit pathways that regulate memory, emotion, and mood.

A once-monthly GLP-1 receptor agonist for treatment of diabetic cats.

There is growing evidence that peptidic glucagon-like peptide-1 receptor agonists (GLP-1RA), such as exenatide, may provide useful therapeutic options for treatment of feline diabetes. However, because such drugs are administered subcutaneously, it is desirable that they be long-acting and not require frequent injections. We have developed a chemically controlled delivery system to support half-life extension of peptidic therapeutics. Here, the peptide is covalently attached to hydrogel microspheres by a self-cleaving ß-eliminative linker; after subcutaneous injection of the microspheres, the peptide is slowly released from the depot to the systemic circulation. Using this technology, we developed a delivery system that supports once-monthly administration of a stable exenatide analog, [Gln28]exenatide, in rodents (Schneider, et al, ACS Chem Biol 12, 2107 to 2116, 2017). The purposes of the present study were a) to demonstrate pharmacokinetic and pharmacodynamic similarities of the deamidation-sensitive GLP-1RA exenatide and the closely related, more stable [Gln28]exenatide and b) to develop a long-acting GLP-1RA in cats. The results show that exenatide and [Gln28]exenatide injected intravenously or subcutaneously at 10 µg/kg have nearly identical pharmacokinetics in the cat-both having elimination half-lives of ∼40 min-but subcutaneously administered [Gln28]exenatide has superior bioavailability-93% for [Gln28]exenatide vs 52% for exenatide. The results also show that exenatide and [Gln28]exenatide have similar insulinotropic activities in the cat during a high-dose intravenous glucose tolerance test; they increased the area under the curve (AUC) for insulin to a similar extent but had no effect on glucose AUC. Finally, subcutaneous injection of a microsphere-[Gln28]exenatide conjugate containing an appropriate self-cleaving linker in the cat provides plasma [Gln28]exenatide with a half-life of about 40 d vs 40 min with the injected free peptide. Hence, the large body of information available for exenatide can be used to facilitate clinical development of [Gln28]exenatide as a treatment for feline diabetes, and the microsphere-[Gln28]exenatide conjugate is quite suitable for once-monthly subcutaneous administration of the peptide in the cat.

Amylin/leptin synergy is absent in extreme obesity and not restored by calorie restriction-induced weight loss in rats.

OBJECTIVE: Co-administration of amylin and leptin induces synergistic and clinically meaningful (>10%) weight loss that is attenuated as the degree of obesity increases. We explored whether calorie restriction (CR) could restore amylin/leptin synergy in very obese rats. METHODS: Sprague Dawley rats on high-fat diet (696 ± 8 g, n = 72) were randomized to three cohorts (C1-C3). Rats in C1 were administered vehicle, rat amylin (50 µg kg-1 d-1), murine leptin (125 µg kg-1 d-1) or amylin and leptin for 28 days (n = 6 per group) via subcutaneous minipump. Simultaneously, C2 and C3 rats initiated CR. After moderate (12.4 ± 0.3%, 86.7 ± 2.8 g; C2) or severe (24.9 ± 0.3%, 172.7 ± 4.7 g; C3) weight loss, amylin and/or leptin was administered as described. RESULTS: In C1, leptin did not alter weight, and amylin induced 40.2 ± 6.1 g weight loss (-6.0 ± 0.9%), which was not enhanced by leptin (44.4 ± 4.9 g, -6.1 ± 0.8%). In C2, vehicle-treated (75.1 ± 7.8 g weight change from start of treatment, 1.1 ± 0.8% difference from start of pre-CR phase) and leptin-treated rats (68.6 ± 9.2 g, -1.3 ± 1.0%) rebounded to pre-restriction weight that was attenuated by amylin (29.2 ± 11.4 g, -6.2 ± 0.7%). Leptin did not enhance the effect of amylin (22.8 ± 11.7 g, -8.3 ± 1.5%). In C3, vehicle-treated and leptin-treated rats regained most of their weight (161.9 ± 11.8, -2.3 ± 0.8% and 144.6 ± 9.5 g, -2.3 ± 0.9%, respectively), which was attenuated by amylin (91.1 ± 16.8 g, -11.2 ± 0.7%), but not enhanced by leptin (83.0 ± 7.6 g, -10.7 ± 0.8%). CONCLUSIONS: Extreme obesity associated with leptin resistance perturbs amylin/leptin weight loss synergy in rats, which cannot be restored by pre-treatment weight loss.

Synergistic metabolic benefits of an exenatide analogue and cholecystokinin in diet-induced obese and leptin-deficient rodents.

AIM: To test the impact of cholecystokinin (CCK) plus either amylin or a glucagon-like peptide-1 receptor (GLP-1R) agonist on metabolic variables in diet-induced obese (DIO) rodents. METHODS: A stabilized acetylated version of CCK-8 (Ac-Y*-CCK-8), selective CCK1 receptor (CCK1R) or CCK2 receptor (CCK2R) agonists, amylin or the GLP-1R agonist and exenatide analogue AC3174 were administered in select combinations via continuous subcutaneous infusion to DIO rats for 14 days, or Lep(ob) /Lep(ob) mice for 28 days, and metabolic variables were assessed. RESULTS: Combined administration of Ac-Y*-CCK-8 with either amylin or AC3174 induced greater than additive weight loss in DIO rats, with the overall magnitude of effect being greater with AC3174 + Ac-Y*-CCK-8 treatment. Co-infusion of AC3174 with a specific CCK1R agonist, but not a CCK2R agonist, recapitulated the weight loss mediated by AC3174 + Ac-Y*-CCK-8 in DIO rats, suggesting that synergy is mediated by CCK1R activation. In a 4 × 4 full-factorial response surface methodology study in DIO rats, a synergistic interaction between AC3174 and the CCK1R-selective agonist on body weight and food intake was noted. Co-administration of AC3174 and the CCK1R-selective agonist to obese diabetic Lep(ob) /Lep(ob) mice elicited a significantly greater reduction in percentage of glycated haemoglobin and food intake relative to the sum effects of monotherapy groups. CONCLUSIONS: The anti-obesity and antidiabetic potential of combined GLP-1R and CCK1R agonism is an approach that warrants further investigation.

Combined antidiabetic benefits of exenatide and dapagliflozin in diabetic mice.

The combined glucose-lowering effect of exenatide and dapagliflozin has not yet been studied. We investigated this combination (single-dose or 4-week dosing) in diabetic ob/ob mice. Vehicle-corrected basal glucose showed greater reduction 1 h following exenatide + dapagliflozin than with exenatide or dapagliflozin alone, and stayed significantly lower for all groups versus vehicle over 3 h. During an oral glucose tolerance test, glucose excursion (30 min post-dose) was significantly lower for exenatide + dapagliflozin versus exenatide or dapagliflozin, or vehicle. Exenatide + dapagliflozin and exenatide, but not dapagliflozin alone, reduced glucose excretion over 24 h versus vehicle. After dosing for 4 weeks, exenatide, dapagliflozin and exenatide + dapagliflozin similarly decreased haemoglobin A1c (HbA1c). Body weight was reduced only with exenatide or exenatide + dapagliflozin. The glomerular filtration rate was similar with exenatide, dapagliflozin and vehicle, and increased with exenatide + dapagliflozin. Optimized combinatorial dosing of these antidiabetic agents may provide additive glucose lowering in type 2 diabetes mellitus.

A novel long-acting glucose-dependent insulinotropic peptide analogue: enhanced efficacy in normal and diabetic rodents.

AIM: Glucose-dependent insulinotropic peptide (GIP) is an incretin hormone that is released from intestinal K cells in response to nutrient ingestion. We aimed to investigate the therapeutic potential of the novel N- and C-terminally modified GIP analogue AC163794. METHODS: AC163794 was synthesized by solid-phase peptide synthesis. Design involved the substitution of the C-terminus tail region of the dipeptidyl peptidase IV (DPP-IV)-resistant GIP analogue [d-Ala(2) ]GIP(1-42) with the unique nine amino acid tail region of exenatide. The functional activity and binding of AC163794 to the GIP receptor were evaluated in RIN-m5F ß-cells. In vitro metabolic stability was tested in human plasma and kidney membrane preparations. Acute insulinotropic effects were investigated in isolated mouse islets and during an intravenous glucose tolerance test in normal and diabetic Zucker fatty diabetic (ZDF) rats. The biological actions of AC163794 were comprehensively assessed in normal, ob/ob and high-fat-fed streptozotocin (STZ)-induced diabetic mice. Acute glucoregulatory effects of AC163794 were tested in diet-induced obese mice treated subchronically with AC3174, the exendatide analogue [Leu(14) ] exenatide. Human GIP or [d-Ala(2) ]GIP(1-42) were used for comparison. RESULTS: AC163794 exhibited nanomolar functional GIP receptor potency in vitro similar to GIP and [d-Ala(2) ]GIP(1-42). AC163794 was metabolically more stable in vitro and displayed longer duration of insulinotropic action in vivo versus GIP and [d-Ala(2) ]GIP(1-42). In diabetic mice, AC163794 improved HbA1c through enhanced insulinotropic action, partial restoration of pancreatic insulin content and improved insulin sensitivity with no adverse effects on fat storage and metabolism. AC163794 provided additional baseline glucose-lowering when injected to mice treated with AC3174. CONCLUSIONS: These studies support the potential use of a novel GIP analogue AC163794 for the treatment of type 2 diabetes.

GLP-1 receptor activated insulin secretion from pancreatic ß-cells: mechanism and glucose dependence.

The major goal in the treatment of type 2 diabetes mellitus is to control the hyperglycaemia characteristic of the disease. However, treatment with common therapies such as insulin or insulinotrophic sulphonylureas (SU), while effective in reducing hyperglycaemia, may impose a greater risk of hypoglycaemia, as neither therapy is self-regulated by ambient blood glucose concentrations. Hypoglycaemia has been associated with adverse physical and psychological outcomes and may contribute to negative cardiovascular events; hence minimization of hypoglycaemia risk is clinically advantageous. Stimulation of insulin secretion from pancreatic ß-cells by glucagon-like peptide 1 receptor (GLP-1R) agonists is known to be glucose-dependent. GLP-1R agonists potentiate glucose-stimulated insulin secretion and have little or no activity on insulin secretion in the absence of elevated blood glucose concentrations. This 'glucose-regulated' activity of GLP-1R agonists makes them useful and potentially safer therapeutics for overall glucose control compared to non-regulated therapies; hyperglycaemia can be reduced with minimal hypoglycaemia. While the inherent mechanism of action of GLP-1R agonists mediates their glucose dependence, studies in rats suggest that SUs may uncouple this dependence. This hypothesis is supported by clinical studies showing that the majority of events of hypoglycaemia in patients treated with GLP-1R agonists occur in patients treated with a concomitant SU. This review aims to discuss the current understanding of the mechanisms by which GLP-1R signalling promotes insulin secretion from pancreatic ß-cells via a glucose-dependent process.

Interactions of amylinergic and melanocortinergic systems in the control of food intake and body weight in rodents.

AIMS: Amylinergic and melanocortinergic systems have each been implicated in energy balance regulation. We examined the interactive effects of both systems using gene knockout and pharmacological approaches. METHODS: Acute food consumption was measured in overnight fasted male wild-type (WT) and melanocortin-4 receptor (MC-4R) deficient rats and in male and female WT and amylin knockout mice (AmyKO). Changes in food intake, body weight and composition in male WT and MC-4R deficient rats and in male diet-induced obese (DIO) rats. Pharmacological treatments included either rat amylin, murine leptin and/or the MC-4R agonist, Ac-R[CEH-dF-RWC]-amide. RESULTS: Amylin (10 µg/kg, IP) decreased food intake in WT but not in MC-4R deficient rats (30 and 60 min post-injection). Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) suppressed food intake similarly in male WT and AmyKO, but was ineffective in female AmyKO. Amylin (50 µg/kg/day for 28 days) and leptin (125 µg/kg/day) synergistically reduced food intake and body weight in WT and MC-4R deficient rats to a similar extent. Amylin (100 µg/kg) combined with Ac-R[CEH-dF-RWC]-amide (100 µg/kg, IP) decreased acute food intake over 3 h to a greater extent than either agent alone in fasted mice. In DIO rats, additive anorexigenic, weight- and fat-lowering effects were observed over 12 days with the combination of rat amylin (50 µg/kg/day) and Ac-R[CEH-dF-RWC]-amide (2.3 mg/kg, SC injected daily). CONCLUSIONS: Although amylin's acute anorexigenic effects are somewhat blunted in MC-4R deficiency and those of MC-4R agonism in amylin deficiency, these effects are surmountable with pharmacological administration lending therapeutic potential to combined amylin/melanocortin agonism for obesity.

Glucoregulatory effects and prolonged duration of action of davalintide: a novel amylinomimetic peptide.

AIMS: Davalintide is a second-generation amylinomimetic peptide possessing enhanced pharmacological properties over rat amylin to reduce food intake in preclinical models. The current experiments in rats describe additional glucoregulatory actions of davalintide consistent with amylin agonism, and explore the duration of action of these effects. METHODS: Subcutaneous (SC) injection of davalintide slowed gastric emptying with equal potency to amylin (ED50's = 2.3 and 4.1 µg/kg). This effect was maintained for 8 h with davalintide, but not amylin. Intraperitoneal injection of davalintide also reduced food intake with a potency similar to amylin (ED50's = 5.0 and 11.3 µg/kg). Consistent with amylin agonism, davalintide (10 µg/kg, SC) suppressed the plasma glucagon response over 90 min following an intravenous arginine bolus in anaesthetized rats. The elimination t(½) of davalintide (200 µg/kg, SC) was 26 min, similar to the t(½) of amylin, suggesting that pharmacokinetic-independent mechanisms contribute to davalintide's enhanced duration of action. Binding kinetic studies using ¹²5I davalintide revealed no appreciable dissociation from the amylin nucleus accumbens receptor after 7 h while ¹²5I rat amylin did dissociate from this receptor (K(off) = 0.013/min). Sustained SC infusion of davalintide (275 µg/kg/day) or amylin (300) decreased plasma glucose after an oral glucose challenge at 2 weeks (by 27 and 31%) and suppressed gastric emptying at 3 weeks (by 29 and 47%), demonstrating durable glucoregulatory actions of both peptides. CONCLUSIONS: These data show glucoregulatory properties of davalintide consistent with amylin agonism and suggest that slowed receptor dissociation plays a role in davalintide's prolonged pharmacodynamic actions.

Davalintide (AC2307), a novel amylin-mimetic peptide: enhanced pharmacological properties over native amylin to reduce food intake and body weight.

OBJECTIVE: The current set of studies describe the in vivo metabolic actions of the novel amylin-mimetic peptide davalintide (AC2307) in rodents and compares these effects with those of the native peptide. RESEARCH DESIGN AND METHODS: The anti-obesity effects of davalintide were examined after intraperitoneal injection or sustained peripheral infusion through subcutaneously implanted osmotic pumps. The effect of davalintide on food intake after lesioning of the area postrema (AP) and neuronal activation as measured by c-Fos, were also investigated. RESULTS: Similar to amylin, davalintide bound with high affinity to amylin, calcitonin and calcitonin gene-related peptide receptors. Acutely, davalintide displayed greater suppression of dark-cycle feeding and an extended duration of action compared with amylin (23 versus 6 h). Davalintide had no effect on locomotor activity or kaolin consumption at doses that decreased food intake. Davalintide-induced weight loss through infusion was dose dependent, durable up to 8 weeks, fat-specific and lean-sparing, and was associated with a shift in food preference away from high-fat (palatable) chow. Metabolic rate was maintained during active weight loss. Both davalintide and amylin failed to suppress food intake after lesioning of the AP and activated similar brain nuclei, with davalintide displaying an extended duration of c-Fos expression compared with amylin (8 versus 2 h). CONCLUSION: Davalintide displayed enhanced in vivo metabolic activity over amylin while retaining the beneficial properties possessed by the native molecule. In vitro receptor binding, c-Fos expression and AP lesion studies suggest that the metabolic actions of davalintide and amylin occur through activation of similar neuronal pathways.

Antiobesity effects of the beta-cell hormone amylin in combination with phentermine or sibutramine in diet-induced obese rats.

OBJECTIVE: To characterize the interactive effects of amylin with phentermine or sibutramine on food intake, body weight/composition and gene expression in diet-induced obese (DIO) rats. DESIGN: DIO rats were intraperitoneally injected with a single dose of amylin (10 microg kg(-1)) and/or phentermine (1 mg kg(-1)) or chronically infused with amylin (100 microg kg(-1) d(-1)) or vehicle with or without phentermine (0.5-10 mg kg(-1) d(-1)) or sibutramine (3 mg kg(-1) d(-1)) using two surgically implanted subcutaneous osmotic mini-pumps. MEASUREMENTS: Twenty-four hour food intake, locomotor activity and components of meal microstructure (meal size, latency, duration and intermeal interval) were measured following acute administration (amylin, phentermine or amylin+phentermine). Body weight and composition (for amylin and/or sibutramine or phentermine) and metabolism-related gene mRNA expression in the liver (fatty acid synthase, stearoyl-CoA desaturase-1 and carnitine palmitoyltransferase-1) and brown fat (beta-adrenergic receptors and uncoupling protein-1) were measured (for amylin and/or phentermine) after sustained infusion (2 weeks). RESULTS: Acute co-administration of amylin (10 microg kg(-1)) and phentermine (1 mg kg(-1)) reduced acute food intake (up to 19 h) more than either monotherapy. In two studies, sustained subcutaneous infusion of amylin for 2 weeks decreased cumulative food intake (22%) and vehicle-corrected body weight gain ( approximately 4-8%). Phentermine's anorexigenic (10-17%) and weight-reducing effects ( approximately 0-5%) were only evident at the highest dose tested (10 mg kg(-1) d(-1)). Combination of amylin (100 microg kg(-1) d(-1)) and phentermine reduced food intake (30-43%), body weight (8-12%) and adiposity to a greater extent than either monotherapy. Amylin prevented phentermine-induced reductions in UCP-1 mRNA in brown adipose tissue. When amylin+sibutramine were infused, mathematically additive decreases in food intake (up to 45%) and body weight (up to 12%) were evident. Similar to amylin+phentermine treatment, amylin+sibutramine mediated weight loss was attributable to significant reductions in fat mass. CONCLUSIONS: Combined treatment of DIO rats with the pancreatic beta-cell hormone amylin and phentermine or sibutramine resulted in additive anorexigenic, weight- and fat-reducing effects.

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 microg/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 microg/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 microg/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 microg/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.

Dose-response for glycaemic and metabolic changes 28 days after single injection of long-acting release exenatide in diabetic fatty Zucker rats.

AIMS/HYPOTHESIS: Exenatide (exendin-4) injected subcutaneously twice daily reduces glycaemic deterioration in diabetic fatty Zucker (ZDF) rats and reduces HbA1c in humans with type 2 diabetes. Because tachyphylaxis may develop with continuous peptide exposure, we examined the activity of a long-acting-release (LAR) formulation of exenatide on HbA1c, insulin sensitivity and beta cell secretion in ZDF rats. METHODS: Single subcutaneous injections of a poly-lactide-glycolide microsphere suspension (3% peptide) containing 0, 1, 10, 100, 1,000, 3,000 or 9,000 mug exenatide were administered to 9-week-old ZDF rats with matched initial HbA1c values (n=7 rats/group). RESULTS: In contrast to the progressive 3.22+/-0.42% increase in HbA1c in control ZDF rats observed over 28 days, single exenatide-LAR injections dose-proportionally prevented such glycaemic deterioration (median effective dose 74 microg+/-0.1 log per rat; median effective concentration 52 pmol/l+/-0.06 log). Hyperinsulinaemic-euglycaemic clamp procedures incorporating an intraclamp glucose challenge performed 28 days after treatment revealed increases in beta cell response to the glucose challenge at lower exenatide-LAR doses, and up to a 2.1-fold increase in insulin sensitivity at higher exenatide-LAR doses. CONCLUSIONS/INTERPRETATION: The finding that a single dose of exenatide-LAR enhanced glucose control for 28 days in the ZDF rat model of type 2 diabetes suggests that tachyphylaxis is unlikely to be a feature of exenatide-LAR preparations, and supports further clinical exploration.

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 ED(50) of 12.5 microg/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 ED(50)'s (466, 297 and 201 microg/kg/day, respectively) and prevented a diet-induced increase in HbA1c. Infusion at 100 microg/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 (ED(50)'s 30 and 44 microg/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].

Cardiovascular actions of CRH and urocortin: an update.

Urocortin is a potent regulator of cardiac function, with actions that are prolonged in experimental animals. These changes are mediated via binding to CRH receptors found in peripheral tissues. The diversity of actions of urocortin on behaviour, appetite, inflammation and the cardiovascular system suggest that this peptide may be an endogenous factor mediating actions previously attributed to CRH. The present review will focus on the recent understanding of mechanisms mediating the cardiovascular actions of urocortin and CRH reported to date.

Insulinotropic actions of exendin-4 and glucagon-like peptide-1 in vivo and in vitro.

This study compares in vitro effects of exendin-4 and glucagon-like peptide (GLP)-1 on basal and glucose-stimulated insulin release from isolated rat islets and in vivo insulinotropic actions of exendin-4 and GLP-1 after an intravenous glucose challenge in rats. In static incubation of isolated islets, changing ambient glucose concentration from 3 mmol/L to 10 mmol/L stimulated insulin secretion 9.8 +/- 1.3-fold. The addition of exendin-4 or GLP-1 (1 nmol/L to 1 micromol/L) increased glucose-stimulated insulin secretion up to 5.8 +/- 1.6-fold and 3.3 +/- 1.0-fold, respectively, over basal rates (defined as no hormones added, 3 mmol/L glucose) and 19.6 +/- 2.3-fold and 15.0 +/- 3.1-fold at 10 mmol/L glucose. In dynamically perfused isolated islets exposed to 7.5 mmol/L glucose, insulin secretion increased 6.4 +/- 1.5-fold, and exendin-4 (20 nmol/L) or GLP-1 (20 nmol/L) increased this similarly by up to 13.5 +/- 2.8 and 12.7 +/- 3.9-fold,respectively. Anesthetized rats administered 5.7 mmol/kg intravenous glucose increased plasma insulin concentration 3.0-fold. Infusion of exendin-4 or GLP-1 increased this to a maximum of 7.6-fold and 5.3-fold, respectively. As with isolated islet studies, in vivo dose responses and concentration responses with exendin-4 and GLP-1 were bell-shaped. When insulinotropic effects were mapped onto the steady-state plasma concentrations associated with these infusion rates, both peptides exhibited bell-shaped concentration responses with peak insulinotropic effects occurring with plasma peptide concentrations of approximately 1 nmol/L in this model. In summary, exendin-4 and GLP-1 exhibited similar insulinotropic potencies (median effective dose [ED(50)]) when assessed on a concentration basis in in vitro and in vivo models, while exendin-4 exhibited greater efficacy (maximum response).

Selective amylin inhibition of the glucagon response to arginine is extrinsic to the pancreas.

Amylin, a peptide hormone from pancreatic beta-cells, is reported to inhibit insulin secretion in vitro and in vivo and to inhibit nutrient-stimulated glucagon secretion in vivo. However, it has been reported not to affect arginine-stimulated glucagon secretion in vitro. To resolve if the latter resulted from inactive peptide (a problem in the early literature), those experiments were repeated here with well-characterized peptide and found to be valid. In isolated perfused rat pancreas preparations, coperfusion with 1 nM amylin had no effect on arginine-, carbachol-, or vasoactive intestinal peptide-stimulated glucagon secretion. Amylin also had no effect on glucagon output stimulated by decreasing glucose concentration from 11 to 3.2 mM or on glucagon suppression caused by increasing glucose from 3.2 to 7 mM. Amylin at 100 nM had no effect in isolated islets in which glucagon secretion was stimulated by exposure to 10 mM arginine, even though glucagon secretion in the same preparation was inhibited by somatostatin. In anesthetized rats, amylin coinfusion had no effect on glucagon secretion stimulated by insulin-induced hypoglycemia. To reconcile reports of glucagon inhibition with the absence of effect in the experiments just described, anesthetized rats coinfused with rat amylin or with saline were exposed sequentially to intravenous L-arginine (during a euglycemic clamp) and then to hypoglycemia. Amylin inhibited arginine-induced, but not hypoglycemia-induced, glucagon secretion in the same animal. In conclusion, we newly identify a selective glucagonostatic effect of amylin that appears to be extrinsic to the isolated pancreas and may be centrally mediated.

Regulation of corticotropin-releasing factor (CRF) messenger ribonucleic acid and CRF peptide in the amygdala: studies in primary amygdalar cultures.

Amygdalar CRF has been implicated in the mediation of stress behaviors. The signal transduction pathways that regulate amygdalar CRF are not well understood. In this report, we have examined the effect of protein kinase A and C activators, dexamethasone, and interleukin 6 on CRF messenger RNA (mRNA) and CRF peptide expression in dissociated amygdalar cultures. The amygdala from E19 rat pups was dissected out bilaterally and dissociated in 0.25% trypsin for 10-15 min and plated. On day 17 in culture, CRF mRNA and peptide were measured following treatment with the following agents: forskolin, the phorbol ester-phorbol 12 myristate 13-acetate (TPA), dexamethasone, and interleukin-6 (IL6). Both forskolin and IL6, but not TPA, increased CRF mRNA in a time- and dose-dependent manner. Secretion and intracellular content of the CRF peptide also increased with both forskolin and IL6 treatment but not with TPA. Dexamethasone treatment did not alter the expression of CRF message or peptide. Transfection of the primary cultures with a rat CRF promoter-luciferase reporter construct followed by treatment with all four agents produced alterations in luciferase expression that were consistent with changes observed at the level of CRF mRNA and peptide. The results suggest that CRF regulation in the amygdala differs from that known to occur in the hypothalamus, and that elevation of IL6 levels within the central nervous system may directly act to stimulate CRF production and secretion from limbic structures such as the amygdala, to promote subsequent behavioral changes.

Cardiac inotropic actions of urocortin in conscious sheep.

Urocortin (Ucn) is a recently isolated peptide related to the corticotropin-releasing factor (CRF) family, which can produce hemodynamic and hormonal actions in conscious rats. This study examined in detail the cardiovascular actions of Ucn and CRF after intravenous injection in chronically instrumented, conscious sheep. Injection of Ucn produced dose-dependent changes in cardiac contractility [rate of increase of aortic flow (dF/dt)], maximum aortic flow (Fmax), mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), and coronary blood flow (CF). Ucn injected at 100 micrograms produced a potent increase in dF/dt, from 909 +/- 44 to a maximum of 1,849 +/- 901.min-1.s-1, and in Fmax, from 25.5 +/- 0.8 to 36.6 +/- 1.4 l/min. Cardiac contractility increased within 30 min of injection and remained significantly elevated for up to 24 h. MAP increased from 78 +/- 2 to 90 +/- 3 mmHg, and HR increased from 73 +/- 4 to 103 +/- 9 beats/min. CO rose from 5.0 +/- 0.1 to 5.8 +/- 0.2 l/min, whereas central venous pressure, total peripheral conductance, and stroke volume were unchanged. All Ucn-induced cardiovascular effects were inhibited by prior treatment with the CRF antagonist alpha-helical CRF-(9-41). Equimolar doses of CRF produced little change in any hemodynamic parameter. Both peptides increased plasma levels of adrenocorticotropin and cortisol, with Ucn having a more potent effect than CRF. We have shown for the first time that Ucn can produce potent and long-lasting actions to elevate cardiac contractility in conscious animals.

Direct cardiac and vascular actions of adrenomedullin in conscious sheep.

1. Adrenomedullin (ADM) is a recently characterized circulating hormone which affects haemodynamic, renal and pituitary function in mammals. We have shown previously that in sheep, ADM produces vasodilatation together with increases in cardiac output and contractility. However, whether these effects are direct or mediated by autonomic reflexes is unclear. The present study examined the cardiovascular actions of an intravenous infusion of ADM in conscious, chronically instrumented sheep with either sympathetic, parasympathetic or autonomic ganglion blockade, to determine the role of the autonomic nervous system in mediating these cardiovascular changes. 2. Human ADM (1-52) was infused for 60 min at 2 micrograms kg-1 h-1 following: (1) saline control, (2) combined alpha/beta-adrenoceptor (sympathetic) blockade (proporanolol 0.4 mg kg-1 h-1 + phentolamine 0.15 mg kg-1 h-1 for 20 h), (3) muscarinic (parasympathetic) blockade (methscopolamine 0.05 mg kg-1 h-1 for 20 h) or (4) ganglion blockade (hexamethonium 3 mg kg-1 h-1 for 4 h). Measurements were made of mean arterial pressure (MAP), heart rate (HR), cardiac output (CO), stroke volume (SV), total peripheral conductance (TPC), maximal aortic flow (Fmax) and maximal rate of change of aortic flow (dF/dt). 3. ADM reduced MAP by 3 +/- 1 mmHg, and increased CO (1.2 +/- 0.2 l min-1), HR (14 +/- 2 beats min-1), TPC (21 +/- 3 ml min-1 mmHg-1). Fmax (2.3 +/- 0.8 l min-1) and dF/dt (86 +/- 21 l min-1 s-1) in normal sheep. In animals with alpha/beta blockade, similar changes were observed with ADM. However, during muscarinic blockade, the increases in HR (32 +/- 4 beats min-1), CO (2.1 +/- 0.4 l min-1), TPC (31 +/- 4 ml min-1 mmHg-1). Fmax (4.0 +/- 0.6 l min-1), and dF/dt (150 +/- 12 l min-1 s-1) produced by ADM were enhanced. During ganglion blockade, ADM produced a greater reduction in MAP (-10 +/- 2 mmHg) compared to controls (-3 +/- 1 mmHg). However, there was no increase in HR. The changes in CO, TPC and contractility were similar to those observed in control animals. 4. These results suggest that the vasodilator effects of ADM on the periphery and its ability to increase CO and cardiac contractility are not mediated by the autonomic nervous system, but are probably the result of direct actions of ADM on the heart and vasculature.