Robust anti-obesity and metabolic effects of a dual GLP-1/glucagon receptor peptide agonist in rodents and non-human primates.

AIMS: To characterize the pharmacology of MEDI0382, a peptide dual agonist of glucagon-like peptide-1 (GLP-1) and glucagon receptors. MATERIALS AND METHODS: MEDI0382 was evaluated in vitro for its ability to stimulate cAMP accumulation in cell lines expressing transfected recombinant or endogenous GLP-1 or glucagon receptors, to potentiate glucose-stimulated insulin secretion (GSIS) in pancreatic ß-cell lines and stimulate hepatic glucose output (HGO) by primary hepatocytes. The ability of MEDI0382 to reduce body weight and improve energy balance (i.e. food intake and energy expenditure), as well as control blood glucose, was evaluated in mouse models of obesity and healthy cynomolgus monkeys following single and repeated daily subcutaneous administration for up to 2 months. RESULTS: MEDI0382 potently activated rodent, cynomolgus and human GLP-1 and glucagon receptors and exhibited a fivefold bias for activation of GLP-1 receptor versus the glucagon receptor. MEDI0382 produced superior weight loss and comparable glucose lowering to the GLP-1 peptide analogue liraglutide when administered daily at comparable doses in DIO mice. The additional fat mass reduction elicited by MEDI0382 probably results from a glucagon receptor-mediated increase in energy expenditure, whereas food intake suppression results from activation of the GLP-1 receptor. Notably, the significant weight loss elicited by MEDI0382 in DIO mice was recapitulated in cynomolgus monkeys. CONCLUSIONS: Repeated administration of MEDI0382 elicits profound weight loss in DIO mice and non-human primates, produces robust glucose control and reduces hepatic fat content and fasting insulin and glucose levels. The balance of activities at the GLP-1 and glucagon receptors is considered to be optimal for achieving weight and glucose control in overweight or obese Type 2 diabetic patients.

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.

Genetic variation in SH3-domain GRB2-like (endophilin)-interacting protein 1 has a major impact on fat mass.

OBJECTIVE: The SH3-domain GRB2-like (endophilin)-interacting protein 1 (SGIP1) gene has been shown to be differentially expressed in the hypothalamus of lean versus obese Israeli sand rats (Psammomys obesus), and is suspected of having a role in regulating food intake. The purpose of this study was to assess the role of genetic variation in SGIP1 in human disease. SUBJECTS: We performed single-nucleotide polymorphism (SNP) genotyping in a large family pedigree cohort from the island of Mauritius. The Mauritius Family Study (MFS) consists of 400 individuals from 24 Indo-Mauritian families recruited from the genetically homogeneous population of Mauritius. We measured markers of the metabolic syndrome, including diabetes and obesity-related phenotypes such as fasting plasma glucose, waist:hip ratio, body mass index and fat mass. RESULTS: Statistical genetic analysis revealed associations between SGIP1 polymorphisms and fat mass (in kilograms) as measured by bioimpedance. SNP genotyping identified associations between several genetic variants and fat mass, with the strongest association for rs2146905 (P=4.7 × 10(-5)). A strong allelic effect was noted for several SNPs where fat mass was reduced by up to 9.4% for individuals homozygous for the minor allele. CONCLUSIONS: Our results show association between genetic variants in SGIP1 and fat mass. We provide evidence that variation in SGIP1 is a potentially important determinant of obesity-related traits in humans.

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.

The mitochondrial rhomboid protease PSARL is a new candidate gene for type 2 diabetes.

AIMS/HYPOTHESIS: This study aimed to identify genes that are expressed in skeletal muscle, encode proteins with functional significance in mitochondria, and are associated with type 2 diabetes. METHODS: We screened for differentially expressed genes in skeletal muscle of Psammomys obesus (Israeli sand rats), and prioritised these on the basis of genomic localisation and bioinformatics analysis for proteins with likely mitochondrial functions. RESULTS: We identified a mitochondrial intramembrane protease, known as presenilins-associated rhomboid-like protein (PSARL) that is associated with insulin resistance and type 2 diabetes. Expression of PSARL was reduced in skeletal muscle of diabetic Psammomys obesus, and restored after exercise training to successfully treat the diabetes. PSARL gene expression in human skeletal muscle was correlated with insulin sensitivity as assessed by glucose disposal during a hyperinsulinaemic-euglycaemic clamp. In 1,031 human subjects, an amino acid substitution (Leu262Val) in PSARL was associated with increased plasma insulin concentration, a key risk factor for diabetes. Furthermore, this variant interacted strongly with age to affect insulin levels, accounting for 5% of the variation in plasma insulin in elderly subjects. CONCLUSIONS/INTERPRETATION: Variation in PSARL sequence and/or expression may be an important new risk factor for type 2 diabetes and other components of the metabolic syndrome.

Calpain 3 gene expression in skeletal muscle is associated with body fat content and measures of insulin resistance.

OBJECTIVE: To investigate whether skeletal muscle gene expression of calpain 3 is related to obesity and insulin resistance. DESIGN: Cross-sectional studies in 27 non-diabetic human subjects and in Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes. MEASUREMENTS: Expression of CAPN3 in skeletal muscle was measured using Taqman fluorogenic PCR. In the human subjects, body composition was assessed by DEXA and insulin sensitivity was measured by euglycemic-hyperinsulinemic clamp. In Psammomys obesus, body composition was determined by carcass analysis, and substrate oxidation rates, physical activity and energy expenditure were measured by whole-body indirect calorimetry. RESULTS: In human subjects, calpain 3 gene expression was negatively correlated with total (P = 0.022) and central abdominal fat mass (P = 0.034), and with blood glucose concentration in non-obese subjects (P = 0.017). In Psammomys obesus, calpain 3 gene expression was negatively correlated with circulating glucose (P = 0.013) and insulin (P = 0.034), and with body fat mass (P = 0.049). Indirect calorimetry revealed associations between calpain 3 gene expression and carbohydrate oxidation (P = 0.009) and energy expenditure (P = 0.013). CONCLUSION/INTERPRETATION: Lower levels of expression of calpain 3 in skeletal muscle were associated with reduced carbohydrate oxidation and elevated circulating glucose and insulin concentrations, and also with increased body fat and in particular abdominal fat. Therefore, reduced expression of calpain 3 in both humans and Psammomys obesus was associated with phenotypes related to obesity and insulin resistance.

Beacon: a novel gene involved in the regulation of energy balance.

The hypothalamus plays a major role in the control of energy balance via the coordination of several neuropeptides and their receptors. We used a unique polygenic animal model of obesity, Psammomys obesus, and performed differential display polymerase chain reaction on hypothalamic mRNA samples to identify novel genes involved in obesity. In this study, we describe a novel gene that encodes a small protein we have termed "beacon." Beacon mRNA gene expression in the hypothalamus was positively correlated with percentage of body fat. Intracerebroventricular infusion of beacon resulted in a dose-dependent increase in food intake and body weight and an increase in hypothalamic expression of neuropeptide Y (NPY). Simultaneous infusion of beacon and NPY significantly potentiated the orexigenic response and resulted in rapid body weight gain. These data suggest a role for beacon in the regulation of energy balance and body weight homeostasis that may be mediated, at least in part, through the NPY pathway.

Intracellular accumulation of detergent-soluble amyloidogenic A beta fragment of Alzheimer's disease precursor protein in the hippocampus of aged transgenic mice.

To study amyloid beta-protein (A beta) production and aggregation in vivo, we created two transgenic (Tg) mouse lines expressing the C-terminal 100 amino acids of human amyloid precursor protein (APP): Tg C100.V717F and Tg C100.WT. Western blot analysis showed that human APP-C100 and A beta were produced in brain and some peripheral tissues and A beta was produced in serum. Using antibodies specific for the A beta C terminus we found that Tg C100.V717F produced a 1.6-fold increase in A beta42/A beta40 compared with Tg C100.WT. Approximately 30% of total brain A beta (approximately 122 ng/g of wet tissue) was water-soluble. The remaining 70% of A beta partitioned into the particulate fraction and was completely sodium dodecyl sulfate-soluble. In contrast, human Alzheimer's disease brain has predominantly sodium dodecyl sulfate-insoluble A beta. Immunohistochemistry with an A beta(5-8) antibody showed that A beta or A beta-containing fragments accumulated intracellularly in the hippocampus of aged Tg C100.V717F mice. The soluble A beta levels in Tg brain are similar to those in normal human brain, and this may explain the lack of microscopic amyloid deposits in the Tg mice. However, this mouse model provides a system to study the intracellular processing and accumulation of A beta or A beta-containing fragments and to screen for compounds directed at the gamma-secretase activity.