Jennifer Athanacio

Diet-induced mouse model of fatty liver disease and nonalcoholic steatohepatitis reflecting clinical disease progression and methods of assessment

Shortcomings of previously reported preclinical models of nonalcoholic steatohepatitis (NASH) include inadequate methods used to induce disease and assess liver pathology. We have developed a dietary model of NASH displaying features observed clinically and methods for objectively assessing disease progression. Mice fed a diet containing 40% fat (of which ∼18% was trans fat), 22% fructose, and 2% cholesterol developed three stages of nonalcoholic fatty liver disease (steatosis, steatohepatitis with fibrosis, and cirrhosis) as assessed by histological and biochemical methods. Using digital pathology to reconstruct the left lateral and right medial lobes of the liver, we made comparisons between and within lobes to determine the uniformity of collagen deposition, which in turn informed experimental sampling methods for histological, biochemical, and gene expression analyses. Gene expression analyses conducted with animals stratified by disease severity led to the identification of several genes for which expression highly correlated with the histological assessment of fibrosis. Importantly, we have established a biopsy method allowing assessment of disease progression. Mice subjected to liver biopsy recovered well from the procedure compared with sham-operated controls with no apparent effect on liver function. Tissue obtained by biopsy was sufficient for gene and protein expression analyses, providing the opportunity to establish an objective method of assessing liver pathology before subjecting animals to treatment. The improved assessment techniques and the observation that mice fed the high-fat diet exhibit many clinically relevant characteristics of NASH establish a preclinical model for identifying pharmacological interventions with greater likelihood of translating to the clinic.

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 μg kg−1) and/or phentermine (1 mg kg−1) or chronically infused with amylin (100 μg 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 (β-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 μg 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 (∼4–8%). Phentermines anorexigenic (10–17%) and weight-reducing effects (∼0–5%) were only evident at the highest dose tested (10 mg kg−1 d−1). Combination of amylin (100 μg 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 β-cell hormone amylin and phentermine or sibutramine resulted in additive anorexigenic, weight- and fat-reducing effects.

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

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.

Effects of amylin and bupropion/naltrexone on food intake and body weight are interactive in rodent models.

Antagonism of opioid systems (e.g., with naltrexone) has been explored as an anti-obesity strategy, and is particularly effective when co-administered with dual inhibitors of dopamine and norepinephrine reuptake (e.g., bupropion). Previously, we demonstrated that amylin enhances the food intake lowering and weight loss effects of neurohormonal (e.g., leptin, cholecystokinin, melanocortins) and small molecule (e.g., phentermine, sibutramine) agents. Here, we sought to characterize the interaction of amylin with naltrexone/bupropion on energy balance. Wild-type and amylin knockout mice were similarly responsive to the food intake lowering effects of either naltrexone (1mg/kg, subcutaneous) or bupropion (50mg/kg, subcutaneous) suggesting that they act independently of amylinergic systems and could interact additively when given in combination with amylin. To test this, diet-induced obese rats were treated (for 11 days) with vehicle, rat amylin (50 μg/kg/d, infused subcutaneously), naltrexone/bupropion (1 and 20mg/kg, respectively by twice daily subcutaneous injection) or their combination. We found that amylin+naltrexone/bupropion combination therapy exerted additive effects to reduce cumulative food intake, body weight and fat mass. In a separate study, the effects of amylin and naltrexone/bupropion administered at the same doses (for 14 days) were compared to a pair-fed group. Although the combination and pair-fed groups lost a similar amount of body weight, rats treated with the combination lost 68% more fat and better maintained their lean mass. These findings support the strategy of combined amylin agonism with opioid and catecholaminergic signaling systems for the treatment of 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.

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.

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

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.