Joy E. Koda

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 amylins 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.

Enhanced Weight Loss With Pramlintide/ Metreleptin: An Integrated Neurohormonal Approach to Obesity Pharmacotherapy

The neurohormonal control of body weight involves a complex interplay between long‐term adiposity signals (e.g., leptin), and short‐term satiation signals (e.g., amylin). In diet‐induced obese (DIO) rodents, amylin/leptin combination treatment led to marked, synergistic, fat‐specific weight loss. To evaluate the weight‐lowering effect of combined amylin/leptin agonism (with pramlintide/metreleptin) in human obesity, a 24‐week, randomized, double‐blind, active‐drug‐controlled, proof‐of‐concept study was conducted in obese or overweight subjects (N = 177; 63% female; 39 ± 8 years; BMI 32.0 ± 2.1 kg/m2; 93.3 ± 13.2 kg; mean ± s.d.). After a 4‐week lead‐in period with pramlintide (180 µg b.i.d. for 2 weeks, 360 µg b.i.d. thereafter) and diet (40% calorie deficit), subjects achieving 2–8% weight loss were randomized 1:2:2 to 20 weeks of treatment with metreleptin (5 mg b.i.d.), pramlintide (360 µg b.i.d.), or pramlintide/metreleptin (360 µg/5 mg b.i.d.). Combination treatment with pramlintide/metreleptin led to significantly greater weight loss from enrollment to week 20 (−12.7 ± 0.9%; least squares mean ± s.e.) than treatment with pramlintide (−8.4 ± 0.9%; P < 0.001) or metreleptin (−8.2 ± 1.3%; P < 0.01) alone (evaluable, N = 93). The greater reduction in body weight was significant as early as week 4, and weight loss continued throughout the study, without evidence of a plateau. The most common adverse events with pramlintide/metreleptin were injection site events and nausea, which were mostly mild to moderate and decreased over time. These results support further development of pramlintide/metreleptin as a novel, integrated neurohormonal approach to obesity pharmacotherapy.

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.

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.

Interaction of Leptin and Amylin in the Long-term Maintenance of Weight Loss in Diet-induced Obese Rats

We have previously shown that combined amylin + leptin agonism elicits synergistic weight loss in diet‐induced obese (DIO) rats. Here, we assessed the comparative efficacy of amylin, leptin, or amylin + leptin in the maintenance of amylin + leptin–mediated weight loss. DIO rats pretreated with the combination of rat amylin (50 µg/kg/day) and murine leptin (125 µg/kg/day) for 4 weeks were subsequently infused with either vehicle, amylin, leptin, or amylin + leptin for an additional 4 weeks. Food intake, body weight, body composition, plasma parameters, and the expression of key metabolic genes in liver and white adipose tissue (WAT) were assessed. Amylin + leptin treatment (weeks 0–4) reduced body weight to 87.5% of baseline. Rats subsequently maintained on vehicle or leptin regained all weight (to 104.2 and 101.2% of baseline, respectively), those maintained on amylin had partial weight regain (97.0%). By contrast, weight loss was largely maintained with continued amylin + leptin treatment (91.4%), associated with a 10% decrease in adiposity. Cumulative food intake (weeks 5–8) was reduced by amylin and amylin + leptin, but not by leptin alone. Amylin + leptin, but not amylin or leptin alone, reduced plasma triglycerides (by 55%), total cholesterol (by 19%), and insulin (by 57%) compared to vehicle. Amylin + leptin also reduced hepatic stearoyl‐CoA desaturase‐1 (Scd1) mRNA, and increased WAT mRNA levels of adiponectin, fatty acid synthase (Fasn), and lipoprotein lipase (Lpl). We conclude that, in DIO rats, maintenance of amylin + leptin–mediated weight loss requires continued treatment with both agonists, and is accompanied by sustained improvements in body composition, and indices of lipid metabolism and insulin sensitivity.

Pramlintide: a human amylin analogue reduced postprandial plasma glucose, insulin, and C-peptide concentrations in patients with type 2 diabetes.

In order to determine the influence of a 5 h infusion of pramlintide compared to placebo on postprandial glucose, lactate, insulin, and C‐peptide concentrations in patients with Type 2 diabetes, a single‐blind, randomized, cross‐over study was conducted in 24 patients; 12 treated with exogenous insulin and 12 managed with diet and/or oral hypoglycaemic agents. One hour after initiation of infusion, patients consumed a Sustacal® test meal. The protocol was repeated on the following day with each patient receiving the alternate study medication. Pramlintide infusion in the insulin‐treated patients resulted in statistically significant reductions in mean glucose, insulin, C‐peptide, and lactate concentrations during the 4‐h period after the Sustacal® test meal. Pramlintide infusion also resulted in significant reductions of mean insulin, C‐peptide, and lactate concentrations, but not glucose concentrations, in the patients treated with diet and/or oral hypoglycaemic agents. Within this latter group, reduction in postprandial glucose concentrations in individual patients correlated with glycated haemoglobin values. These results suggest that administration of pramlintide may improve glycaemic control in patients with Type 2 diabetes treated with insulin or poorly controlled on diet and/or oral hypoglycaemic agents. © 1997 John Wiley & Sons, Ltd.

Pharmacokinetics and Pharmacodynamics of AC137 (25,28,29 Tripro‐Amylin, Human) After Intravenous Bolus and Infusion Doses in Patients with Insulin‐Dependent Diabetes

A study was conducted to evaluate the effect of 30‐μg, 100‐μg, and 300‐μg 2‐minute bolus doses and 2‐hour infusion doses of AC137 (25,28,29 tripro‐amylin, human) on plasma AC137 concentrations and plasma glucose and lactate responses in patients with insulin‐dependent diabetes mellitus (IDDM). The study design was an imbedded two‐way crossover wherein patients received placebo and active boluses in one period and placebo and active infusions in the other period. Two patients in each dose group received placebo throughout the two periods. Pharmacokinetics and pharmacodynamics (PK/PD) were determined during the 6‐hour period after initiation of dosing. Data were fitted with a linked PK/PD model. Pharmacokinetics were linear over the dose range studied, and attenuation of glucose and lactate responses to a mixed meal was dose and concentration dependent. The results of the PK/PD model indicate that the attenuation of glucose and lactate responses was greater after AC137 infusion doses than after the same doses given as a bolus. Glucose and lactate responses to a mixed meal were essentially negated by the 300‐μg infusion dose.

Effect of Race and Hypertension on Plasma Amylin Concentrations

Amylin is a recently discovered peptide hormone composed of 37 amino acids that is cosecreted with insulin by pancreatic beta cells. Amylin has been reported to be present in increased amounts in insulin-resistant subjects who are hyper-insulinemic. Because blacks and whites differ in the prevalence of both hypertension and diabetes, we examined amylin levels in 77 individuals; 42 were black (11 hypertensive and 31 normotensive) and 35 were white (10 hypertensive and 25 normotensive) individuals who were either healthy control subjects or hypertensive subjects not receiving antihypertensive medication. Plasma amylin concentrations were measured in two separate monoclonal antibody-based immunofluorescent sandwich-type assays. The F002-2 capture antibody binds amylin plus at least two additional amylin-like peptides, and the F024-4 capture antibody detectably binds only the amylin peptide. There was a significant race-by-diagnosis interaction for levels of amylin immunoreactivity during a 2-hour glucose tolerance test (P<.005 for F002-2 antibody and P<.05 for F024-4 antibody). Highest levels were found in black hypertensive subjects. The results appear to fit with previously observed differences in metabolic status between blacks and whites and with the association between hypertension and alterations in metabolic status.