James L. Trevaskis

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.

Identification of Adropin as a Secreted Factor Linking Dietary Macronutrient Intake with Energy Homeostasis and Lipid Metabolism

Obesity and nutrient homeostasis are linked by mechanisms that are not fully elucidated. Here we describe a secreted protein, adropin, encoded by a gene, Energy Homeostasis Associated (Enho), expressed in liver and brain. Liver Enho expression is regulated by nutrition: lean C57BL/6J mice fed high-fat diet (HFD) exhibited a rapid increase, while fasting reduced expression compared to controls. However, liver Enho expression declines with diet-induced obesity (DIO) associated with 3 months of HFD or with genetically induced obesity, suggesting an association with metabolic disorders in the obese state. In DIO mice, transgenic overexpression or systemic adropin treatment attenuated hepatosteatosis and insulin resistance independently of effects on adiposity or food intake. Adropin regulated expression of hepatic lipogenic genes and adipose tissue peroxisome proliferator-activated receptor gamma, a major regulator of lipogenesis. Adropin may therefore be a factor governing glucose and lipid homeostasis, which protects against hepatosteatosis and hyperinsulinemia associated with obesity.

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.

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 AC3174s 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.

Mechanisms of amylin/leptin synergy in rodent models.

The present studies aimed to identify mechanisms contributing to amylin/leptin synergy in reducing body weight and adiposity. We reasoned that if amylin/leptin harnessed complementary neuronal pathways, then in the leptin-sensitive state, amylin should augment leptin signaling/binding and that in the absence of endogenous amylin, leptin signaling should be diminished. Amylin (50 microg/kg, ip) amplified low-dose leptin-stimulated (15 microg/kg, ip) phosphorylated signal transducer and activator of transcription-3 signaling within the arcuate nucleus (ARC) in lean rats. Amylin (50 microg/kg x d) or leptin (125 microg/kg x d) infusion to lean rats decreased 28-d food intake (14 and 10%, respectively), body weight (amylin by 4.3%, leptin by 4.9%), and epididymal fat (amylin by 19%, leptin by 37%). Amylin/leptin co-infusion additively decreased food intake (by 26%) and reduced body weight (by 15%) and epididymal fat (by 78%; all P < 0.05 vs. all groups) in a greater than mathematically additive manner, consistent with synergy. Amylin increased leptin binding within the ventromedial hypothalamus (VMN) by 35% and dorsomedial hypothalamus by 47% (both P < 0.05 vs. vehicle). Amylin/leptin similarly increased leptin binding in the VMN by 40% and ARC by 70% (P < 0.05 vs. vehicle). In amylin-deficient mice, hypothalamic leptin receptor mRNA expression was reduced by 50%, leptin-stimulated phosphorylated signal transducer and activator of transcription-3 within ARC and VMN was reduced by 40%, and responsiveness to leptins (1 mg/kg x d for 28 d) weight-reducing effects was attenuated (all P < 0.05 vs. wild-type controls). We suggest that amylin/leptins marked weight- and fat-reducing effects are due to activation of intrinsic synergistic neuronal signaling pathways and further point to the integrated neurohormonal therapeutic potential of amylin/leptin agonism in obesity.

Melanocortin signaling in the CNS directly regulates circulating cholesterol

Cholesterol circulates in the blood in association with triglycerides and other lipids, and elevated blood low-density lipoprotein cholesterol carries a risk for metabolic and cardiovascular disorders, whereas high-density lipoprotein (HDL) cholesterol in the blood is thought to be beneficial. Circulating cholesterol is the balance among dietary cholesterol absorption, hepatic synthesis and secretion, and the metabolism of lipoproteins by various tissues. We found that the CNS is also an important regulator of cholesterol in rodents. Inhibiting the brains melanocortin system by pharmacological, genetic or endocrine mechanisms increased circulating HDL cholesterol by reducing its uptake by the liver independent of food intake or body weight. Our data suggest that a neural circuit in the brain is directly involved in the control of cholesterol metabolism by the liver.

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.

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.

Insights into amylin-leptin synergy.

Although the adipokine leptin is regarded as the prototypical long-term signal of energy balance, obese individuals are largely nonresponsive to exogenous leptin administration. Restoration of leptin responsiveness in obesity has been elusive despite a detailed understanding of the molecular mechanisms of leptin signaling. Recent translational research findings point to a potential therapeutic approach that incorporates amylin (a beta-cell hormone) and leptin agonism, with amylin restoring or enhancing leptin sensitivity. Here we hypothesize various physiological, neurobiological and molecular mechanisms that could mediate the interaction of these two neurohormonal signals and discuss several methodological challenges. Understanding how amylin agonism improves leptin function could point to general therapeutic strategies for combating leptin resistance and associated obesity.

Multi-hormonal weight loss combinations in diet-induced obese rats: therapeutic potential of cholecystokinin?

Cholecystokinin (CCK) acutely synergizes with amylin to suppress food intake in lean mice. To extend on these findings, the present studies sought to identify neural correlates for the interaction of amylin and CCK, as well as further understand the therapeutic potential of CCK-based combinations in obesity. First, c-Fos activation was assessed in various brain nuclei after a single intraperitoneal injection of amylin (5microg/kg) and/or CCK (5microg/kg). Amylin and CCK additively increased c-Fos within the area postrema (AP), predominantly in noradrenergic (e.g., dopamine-beta-hydroxylase-containing) cells. Next, amylin (100 or 300microg/kg/d) and/or CCK (100 or 300microg/kg/d) were subcutaneously infused for 7days in diet-induced obese (DIO) rats. Amylin treatment of DIO rats for 7days induced significant body weight loss. CCK, while ineffective alone, significantly enhanced body weight loss when co-administered with the higher dose of amylin. Finally, the addition of CCK (300microg/kg/d) to leptin (125microg/kg/d), and to the combination of amylin (50microg/kg/d) and leptin (125microg/kg/d), was also explored in DIO rats via sustained subcutaneous infusion for 14days. Infusion of amylin/leptin/CCK for 14days exerted significantly greater body weight loss, inhibition of food intake, and reduction in adiposity compared to amylin/leptin treatment alone in DIO rats. However, co-infusion of CCK and leptin was an ineffective weight loss regimen in this model. Whereas CCK agonism alone is ineffective at eliciting or maintaining weight loss, it durably augmented the food intake and body weight-lowering effects of amylin and amylin/leptin in a relevant disease model, and when combined with amylin, cooperatively activated neurons within the caudal brainstem.