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.

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.