Antidiabetic properties of the histamine H3 receptor protean agonist proxyfan.

Proxyfan is a histamine H3 receptor protean agonist that can produce a spectrum of pharmacological effects including agonist, inverse agonist, and antagonist. We have discovered that proxyfan (10 mg/kg orally) significantly improved glucose excursion after an ip glucose tolerance test in either lean or high-fat/cholesterol diet-induced obese mice. It also reduced plasma glucose levels comparable to that of metformin (300 mg/kg orally) in a nongenetic type 2 diabetes mouse model. The dose-dependent decrease in glucose excursion correlated with inhibition of ex vivo H3 receptor binding in the cerebral cortex. In addition, glucose levels were significantly reduced compared with vehicle-treated mice after intracerebroventricular administration of proxyfan, suggesting the involvement of central H3 receptors. Proxyfan-induced reduction of glucose excursion was not observed in the H3 receptor knockout mice, suggesting that proxyfan mediates this effect through H3 receptors. Proxyfan reduced glucose excursion by significantly increasing plasma insulin levels in a glucose-independent manner. However, no difference in insulin sensitivity was observed in proxyfan-treated mice. The H1 receptor antagonist chlorpheniramine and the H2 receptor antagonist zolantidine had modest effects on glucose excursion, and neither inhibited the glucose excursion reduced by proxyfan. The H3 receptor antagonist/inverse agonist, thioperamide, had weaker effects on glucose excursion compared with proxyfan, whereas the H3 receptor agonist imetit did not affect glucose excursion. In conclusion, these findings demonstrate, for the first time, that manipulation of central histamine H3 receptor by proxyfan can significantly improve glucose excursion by increasing plasma insulin levels via a glucose-independent mechanism.

Ultra-performance hydrophilic interaction LC-MS/MS for the determination of metformin in mouse plasma.

INTRODUCTION: Ultra-performance (UP) hydrophilic interaction LC (UPHILIC) interfaced with an ESI source and MS/MS was developed for the determination of metformin in mouse plasma samples. MATERIALS & METHODS: Several silica stationary phases under HILIC conditions were adapted to evaluate the retention mechanism profiles of the analyte. The influences of experimental factors such as the compositions of mobile phases on the chromatographic performance and the ionization efficiency of the test compounds in positive ion mode were investigated. The applicability of the proposed UPHILIC-MS/MS approach following a protein precipitation procedure for the quantitative determination of metformin at nanomole levels was examined with respect to assay specificity and linearity. RESULTS: The analytical results obtained by the described UPHILIC-MS/MS approach were found to be in good agreement with those obtained by an ion-pair UPLC-MS/MS method in terms of assay sample throughput, sensitivity and accuracy. CONCLUSION: The results suggested that it is feasible to convert a reversed-phase UPLC-MS/MS method to a UPHILIC-MS/MS method by simply switching the analytical columns while maintaining the rest of the experimental conditions for polar pharmaceutical analyses with enhanced retention and sensitivity.

Ezetimibe improves high fat and cholesterol diet-induced non-alcoholic fatty liver disease in mice.

Ezetimibe is a novel cholesterol and plant sterol absorption inhibitor that reduces plasma low-density lipoprotein-cholesterol by selectively binding to the intestinal cholesterol transporter, Niemann-Pick C1-Like 1. Mice deficient in Niemann-Pick C1-Like 1 are protected from high fat/cholesterol diet-induced fatty liver as well as hypercholesterolemia. The object of the present study was to determine whether ezetimibe treatment could reduce hepatic steatosis in diet-induced obese mice. C57BL/6J mice were fed a high fat/cholesterol containing semi-purified diet (45% Kcal fat and 0.12% cholesterol) for 7 months after weaning. These mice were not only obese, but also developed hepatomegaly and hepatic steatosis, with varying degrees of liver fibrosis and steatohepatitis. About 87% of the mice on the high fat/cholesterol diet for 7 months had elevated plasma alanine aminotransferase activity, a biomarker for non-alcoholic fatty liver disease. Chronic administration of ezetimibe for 4 weeks significantly reduced hepatomegaly by decreasing hepatic triglyceride, cholesteryl ester and free cholesterol in diet-induced obese mice fed high fat/cholesterol diet for 7 months. Chronic ezetimibe treatment also significantly decreased plasma alanine aminotransferase activity. These results suggest that ezetimibe may be a novel treatment for high fat/cholesterol-induced non-alcoholic fatty liver disease.

Discovery of novel orally active ureido NPY Y5 receptor antagonists.

We have derived a novel series of neuropeptide Y (NPY) Y5 receptor antagonists from the biphenylurea 3. Cyclohexylurea 21c, a member of the series, is a potent NPY Y5 receptor antagonist that exhibits excellent pharmacokinetic parameters in rats and dogs. On chronic oral administration to diet-induced obese rats, 21c displayed an anti-obesity profile, causing a modest reduction in food intake, a significant decrease in body weight gain, a decrease in adipose mass, and an increase in lean tissue mass.

Energy metabolic profile of mice after chronic activation of central NPY Y1, Y2, or Y5 receptors.

OBJECTIVE: Neuropeptide Y (NPY), a 36-amino acid peptide with orexigenic properties, is expressed abundantly in the central nervous system and binds to several NPY receptor subtypes. This study examines the roles of the NPY Y1, Y2, and Y5 receptor(s) in energy homeostasis. RESEARCH METHODS AND PROCEDURES: We administered intracerebroventricular NPY (3 microg/d) or selective peptide agonists for the Y1, Y2, and Y5 receptor subtypes to C57Bl/6 mice for 6 days by mini-osmotic pumps to assess the role of each receptor subtype in NPY-induced obesity. Energy expenditure (EE) and respiratory quotient (RQ) were studied using indirect calorimetry. Adiposity was measured by DXA scanning and fat pad dissection. Insulin sensitivity was tested by whole-blood glucose measurement after an insulin challenge. RESULTS: Central administration of the selective Y1 agonist, Y5 agonist, or NPY for 6 days in mice significantly increased body weight, adiposity, and RQ, with significant hyperphagia in the Y5 agonist- and NPY-treated groups but not in the Y1 agonist-treated group. The NPY, Y1, or Y5 agonist-treated mice had little change in total EE during ad libitum and pair-feeding conditions. Conversely, selective activation of the Y2 receptor reduced feeding and resulted in a significant, but transient, weight loss. DISCUSSION: Central activation of both Y1 and Y5 receptors increases RQ and adiposity, whereas only Y5 receptor activation reduces energy expended per energy ingested. Selective activation of Y2 autoreceptors leads to hypophagia and transient weight loss, with little effect on total EE. Our study indicates that all three NPY receptor subtypes may play a role in regulating energy homeostasis in mice.

Selective activation of central NPY Y1 vs. Y5 receptor elicits hyperinsulinemia via distinct mechanisms.

Central administration of neuropeptide Y (NPY) stimulates hyperphagia and hyperinsulinemia. Recent evidence has suggested that the Y1 and Y5 receptor subtypes may both mediate NPY-stimulated feeding. The present study attempts to further characterize the role of central NPY receptor subtypes involved in hyperinsulinemia. NPY and peptide analogs of NPY that selectively activated the NPY Y1 or Y5 receptor subtype induced feeding and hyperinsulinemia in satiated Long Evans rats, whereas NPY analogs that selectively activated the NPY Y2 or Y4 receptor subtype did not. To determine whether NPY-induced hyperinsulinemia is secondary to its hyperphagic effect, we compared the plasma insulin levels in the presence and absence of food after a 1-min central infusion of NPY and its analogs at 15, 60, and 120 min postinfusion. Our data suggest that selective activation of central NPY Y1 receptor subtype induced hyperinsulinemia independent of food ingestion, whereas the NPY Y5 receptor-induced hyperinsulinemia was dependent on food ingestion. Central administration of the selective Y1 receptor agonist D-Arg25 NPY eventually decreased plasma glucose levels 2 h postinfusion in Long Evans rats.

Fat intake affects adiposity, comorbidity factors, and energy metabolism of sprague-dawley rats.

OBJECTIVE: Childhood obesity is an emerging health problem. This study assesses the effects of three levels of dietary fat (10%, 32%, and 45% measured by kilocalories) on weight gain, body composition, energy metabolism, and comorbidity factors in rats from weaning through maturation. RESEARCH METHODS AND PROCEDURES: The role of dietary fat on the susceptibility to obesity was assessed by feeding diets containing three levels of dietary fat to rats from weaning through 7 months of age. Body composition was analyzed by DXA after 6 and 12 weeks of dietary treatment. Energy metabolism was measured by indirect calorimetry. RESULTS: Energy intake, weight gain, fat mass, and plasma glucose, cholesterol, triglyceride, free fatty acid, leptin, and insulin levels increased dose-dependently with increased dietary fat. No difference in absolute lean mass among the three groups was observed. Therefore, the differences in weight gain are accounted for primarily by increased fat accretion. Compared with rats that were relatively resistant to obesity when on a 45% fat diet, diet-induced obesity-prone rats were in positive energy balance and had an elevated respiratory quotient, indicating a switch in energy substrate use from fat to carbohydrate, which promotes body-fat accretion. DISCUSSION: Our data support the hypothesis that administration of increasing amount of dietary fat to very young rats enhances susceptibility to diet-induced obesity and its comorbidities.

Characterization of diet-induced obese rats that develop persistent obesity after 6 months of high-fat followed by 1 month of low-fat diet.

A subset of Sprague-Dawley rats developed persistent obesity when maintained on a high-fat diet for 6 months followed by a low-fat diet for 1 month, while another subset from the same cohort of rats remained lean on the same diet regimens. The diet-induced obese (DIO) rats had higher energy intake than expenditure, while diet-resistant (DR) rats maintained energy balance. DIO rats also had an increased respiratory quotient and higher levels of plasma leptin, insulin and cholesterol. In the hypothalamic areas, DIO rats had elevated NPY and AGRP mRNA, but not MCH mRNA. Our data suggest that the increase in hypothalamic expression of NPY and AGRP may contribute to the development of persistent obesity in DIO rats.