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Aberrant regulation of glucose production makes a critical contribution to the impaired glycemic control that is observed in type 2 diabetes. Although isotopic tracer methods have proven to be informative in quantifying the magnitude of such alterations, it is presumed that one must rely on venous access to administer glucose tracers which therein presents obstacles for the routine application of tracer methods in rodent models. Since intraperitoneal injections are readily used to deliver glucose challenges and/or dose potential therapeutics, we hypothesized that this route could also be used to administer a glucose tracer. The ability to then reliably estimate glucose flux would require attention toward setting a schedule for collecting samples and choosing a distribution volume. For example, glucose production can be calculated by multiplying the fractional turnover rate by the pool size. We have taken a step-wise approach to examine the potential of using an intraperitoneal tracer administration in rat and mouse models. First, we compared the kinetics of [U-13C]glucose following either an intravenous or an intraperitoneal injection. Second, we tested whether the intraperitoneal method could detect a pharmacological manipulation of glucose production. Finally, we contrasted a potential application of the intraperitoneal method against the glucose-insulin clamp. We conclude that it is possible to 1) quantify glucose production using an intraperitoneal injection of tracer and 2) derive a “glucose production index” by coupling estimates of basal glucose production with measurements of fasting insulin concentration; this yields a proxy for clamp-derived assessments of insulin sensitivity of endogenous production.

Aim: SGLT inhibitors was found to paradoxically enhance EGP in type 2 diabetes patients, while reduce EGP in diabetic mice during hyperinsulinemic-euglycemic clamp. In this study, we assess acute and 7-day effects of SGLT inhibitors on EGP in rats by 13C-glucose tracer dilution method to identify a translational rodent model. Method: SD rats aged 7 weeks were assigned to 3 groups (n=6 each), vehicle, LX4211 (10 mg/kg) and canagliflozin (10 mg/kg). On day 1, rats were fasted for 3 hours and dosed with SGLT inhibitors, LX4211 or canagliflozin before a 13C-glucose IV injection (25 mg/kg) 2 hour post drug administration. Blood were collected at 0, 5, 15, 30, 45, 60, 75, 90, 120, and 150 minutes time points. Plasma 13C-glucose and 12C-glucose levels were detected by LC/MS. EGP was calculated by multiplying fractional glucose turnover rate by pool size. After washout and the same 18 rats were dosed for 7 days, EGP were assessed on day 7. Results: 13C--glucose IV injection after acute LX4211 and canagliflozin dosing did not significantly changed plasma glucose concentration over time, but led to a rapid rise in plasma 13C-glucose enrichment followed by a time-dependent decrease of 13C-glucose enrichment. The fractional glucose turnover rate and EGP were 3.14±0.35 (SD) percent and 6.28±0.70 (SD) mg.kg-1.minutes-1 in vehicle group. Single dose of LX4211 and canagliflozin significantly increased EGP by 27.5% and 57.1%, respectively. After 7-days of treatment, the fractional glucose turnover and EGP were 3.30±0.28 (SD) percent and 6.59±0.56 (SD) mg.kg-1.minutes-1 in vehicle group, which was highly consistent to the previous EGP measurement. Moreover, 7 days treatment of LX4211 and canagliflozin significantly elevated EGP by 24.8% and 33.4%, respectively. Conclusion: This study demonstrated that SGLT inhibitors can increase EGP in animal model as in type 2 diabetes patients. Detecting EGP using 13C-glucose tracer dilution method may facilitate the discovery of novel SGLT inhibitors with differential effects on EGP. Disclosure S. Huang: None. D. Cai: None. Z. Yue: None. H. Yan: None. Z. Zhou: None. X. Fu: None. H. Wang: None. Y. Tao: None. J.J. Hwa: None.

A novel series of spiroimidazolone-based antagonists of the human glucagon receptor (hGCGR) has been developed. Our efforts have led to compound 1, N-((2H-tetrazol-5-yl)methyl)-4-((R)-1-((5r,8R)-8-(tert-butyl)-3-(3,5-dichlorophenyl)-2-oxo-1,4-diazaspiro[4.5]dec-3-en-1-yl)-4,4-dimethylpentyl)benzamide (SCH 900822), a potent hGCGR antagonist with exceptional selectivity over the human glucagon-like peptide-1 receptor. Oral administration of 1 lowered 24 h nonfasting glucose levels in imprinting control region mice on a high fat diet with diet-induced obesity following single oral doses of 3 and 10 mg/kg. Furthermore, compound 1, when dosed orally, was found to decrease fasting blood glucose at 30 mg/kg in a streptozotocin-treated, diet-induced obesity mouse pharmacodynamic assay and blunt exogenous glucagon-stimulated glucose excursion in prediabetic mice.

A structure–activity relationship study was undertaken to address the lack of oral exposure of the H3 antagonist 1 , which incorporated an arylketone. Among a number of sub-series, the 4 H -pyrido[1,2- a ]pyrimidin-4-one analog 21 showed an improved PK profile in rat and mouse and was active in an obesity model. The pyrimidin-4-one proved to be a novel and useful ketone bioisostere.

A series of novel 2-piperidinopiperidine thiadiazoles were synthesized and evaluated as new leads of histamine H3 receptor antagonists. The 4-(5-([1,4'-bipiperidin]-1'-yl)-1,3,4-thiadiazol-2-yl)-2-(pyridin-2-yl)morpholine (5u) displayed excellent potency and ex vivo receptor occupancy. Compound 5u was also evaluated in vivo for antidiabetic efficacy in STZ diet-induced obesity type 2 diabetic mice for 2 or 12 days. Non-fasting glucose levels were significantly reduced as compared with vehicle-treated mice. In addition, 5u dose dependently blocked the increase of HbA1c after 12 days of treatment.

Melanin concentrating hormone (MCH) is a cyclic neuropeptide expressed in the lateral hypothalamus that plays an important role in energy homeostasis. To investigate the pharmacological consequences of inhibiting MCH signaling in murine obesity models, we examined the effect of acute and chronic administration of a selective MCH1 receptor antagonist (SCH-A) in diet-induced obese (DIO) and Lepob/ob mice. Oral administration of SCH-A for 5 consecutive days (30 mg/kg q.d.) produced hypophagia, a loss of body weight and adiposity, and decreased plasma leptin levels in DIO mice, and hypophagia and reduced weight gain in Lepob/ob mice. Chronic administration of SCH-A to DIO mice decreased food intake, body weight and adiposity, and plasma leptin and free fatty acids. These effects were accompanied by increases in several hypothalamic neuropeptides. Acute administration of SCH-A (30 mg/kg) prevented the decrease in energy expenditure associated with food restriction. These results indicate that MCH1 receptor antagonists may be effective in the treatment of obesity.

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 μg/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.

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.

Melanocortins play a critical role in appetite and body weight regulation, because manipulations of this pathway can lead to the development of obesity in several animal models. The purpose of this study was to use a melanocortin receptor agonist and antagonist to evaluate the involvement of melanocortins in feeding, energy metabolism, and body weight regulation in lean and obese Zucker rats. Central administration of a melanocortin receptor antagonist (SHU9119) elevated food intake and body weight of lean Zucker rats but had little effect in obese Zucker rats. In contrast, the melanocortin receptor agonist MTII reduced food intake in both lean and obese rats but was more potent in the obese Zucker rats. These data indicate the existence of functional melanocortin receptors in both lean and obese Zucker rats but suggest that obese Zucker rats have reduced endogenous melanocortin tone. In addition to its effects on food intake, MTII infusion elevated oxygen consumption and decreased respiratory quotient dose dependently during the light cycle. Our data suggest that a melanocortin receptor agonist can induce weight loss by increasing energy expenditure and promoting body fat utilization in addition to its inhibitory effects on food intake in both obese and lean Zucker rats.

Galanin, a 29 - 30 amino acid neuropeptide, is widely distributed in both the central and peripheral nervous systems. The peptide is unique to other families of neuropeptides and is highly conserved in the N-terminal portion among several mammalian species. Cloning efforts have successfully yielded three galanin receptor (GalR) subtypes that have high affinities for galanin. A recently isolated galanin-like peptide has high affinity for at least one of the three receptors. Galanin modulates a variety of physiological processes via activation of these receptor subtypes. In the CNS, galanin alters the release of several neurotransmitters. For example, it has an inhibitory effect on the release of acetylcholine (ACh) in the hippocampus, suggesting a potential role in the modulation of memory and learning. When injected directly into the ventricles of the brain, galanin increases food intake and alters plasma levels of hormones involved in the maintenance of nutrient and body weight homeostasis. The peptide a...

The neuropeptide galanin mediates a diverse array of physiological functions through activation of specific receptors. Roles of the three recently cloned galanin receptors (GalRs) in rat intestinal contraction and food intake were examined using GalR-selective ligands and the results were compared with the pharmacological profiles of defined GalRs. The action profile of these ligands in jejunal contraction resembled only that of GalR2 and only a high level of GalR2 mRNA was detected in the tissue, supporting GalR2 as the receptor mediating jejunal contraction. The action profile for food intake in rats excluded GalR2, GalR3 and the putative pituitary galanin receptor as the `feeding receptor', suggesting that either GalR1 or an unidentified GalR is responsible for mediating this function.

Advances in our understanding of the mechanisms underlying the development of obesity have occurred over the past few years, prompted by the discovery of leptin and its role in modulating energy balance. The discovery that leptin modulates neurotransmitter pathways in the CNS and the growing body of evidence suggesting that obesity in humans and rodent models is associated with leptin resistance have focused attention on downstream CNS pathways. From these neurotransmitter systems, a series of novel potential targets for the development of antiobesity agents is emerging.

Intracerebroventricular (i.c.v.) administration of glucagon-like peptide-1-(7-37) amide (GLP-1) has been shown to modulate food and water intake. The present studies further characterize the effects of i.c.v. GLP-1 in the regulation of energy balance in lean and obese animals. In both obese and lean Zucker rats, a single i.c.v. infusion of GLP-1 (1-30 microg) resulted in a dose-dependent reduction of food intake and decrease in respiratory quotient relative to the saline control during the first 2 h of the nocturnal cycle. In obese Zucker rats, the food intake was reduced by 73 +/- 11% of the control at the 30 microg dose, whereas a modest 45 +/- 18% reduction was observed in lean rats. Despite the large reduction in food intake seen with GLP-1, there was no compensatory decrease in nocturnal oxygen consumption in the obese Zucker rats. Interestingly, low doses of GLP-1 (1 microg) in lean Zucker rats, which had minimal effects on food intake, caused a 19 +/- 7% increase in O2 consumption during the first 2 h of the nocturnal cycle. These data suggest that central GLP-1 may be an important factor controlling negative energy balance in both the lean and obese Zucker rats.

Obesity occurs whenever energy intake exceeds energy expenditure. The ob gene product leptin is a potent anorectic agent when administered to ob/ob mice, but its effects on energy expenditure have not been investigated in detail. The present study was designed to analyze the acute metabolic effects of leptin in vivo. Analysis of oxygen consumption in ob/ob mice demonstrated a reduction in energy expenditure compared with lean controls; this reduction showed a diurnal fluctuation and was most evident during the light cycle. A single intraperitoneal dose of leptin increased oxygen consumption during the light cycle in ob/ob mice, ablating the circadian fluctuation in this parameter. In addition, leptin had a profound effect on fuel selection: the respiratory quotient was markedly reduced, indicating a reduction in carbohydrate oxidation and an increase in fat oxidation. These acute effects of leptin on metabolic parameters are consistent with the selective loss of body fat observed on chronic leptin treatment and suggest that increased energy utilization plays an important role in the anti-obese actions of leptin.

Diacylglycerol acyltransferase 1 (DGAT1) presents itself as a potential therapeutic target for obesity and diabetes for its important role in triglyceride biosynthesis. Herein we report the rational design of a novel class of DGAT1 inhibitors featuring a benzomorpholine core (23n). SAR exploration yielded compounds with good potency and selectivity as well as reasonable physical and pharmacokinetic properties. This class of DGAT1 inhibitors was tested in rodent models to evaluate DGAT1 inhibition as a novel approach for the treatment of metabolic diseases. Compound 23n conferred weight loss and a reduction in liver triglycerides when dosed chronically in mice with diet-induced obesity and depleted serum triglycerides following a lipid challenge.

Phosphodiesterase 10A (PDE10A) is a novel therapeutic target for the treatment of schizophrenia. Here we report a novel role of PDE10A in the regulation of caloric intake and energy homeostasis. PDE10A-deficient mice are resistant to diet-induced obesity (DIO) and associated metabolic disturbances. Inhibition of weight gain is due to hypophagia after mice are fed a highly palatable diet rich in fats and sugar but not a standard diet. PDE10A deficiency produces a decrease in caloric intake without affecting meal frequency, daytime versus nighttime feeding behavior, or locomotor activity. We tested THPP-6, a small molecule PDE10A inhibitor, in DIO mice. THPP-6 treatment resulted in decreased food intake, body weight loss, and reduced adiposity at doses that produced antipsychotic efficacy in behavioral models. We show that PDE10A inhibition increased whole-body energy expenditure in DIO mice fed a Western-style diet, achieving weight loss and reducing adiposity beyond the extent seen with food restriction alone. Therefore, chronic THPP-6 treatment conferred improved insulin sensitivity and reversed hyperinsulinemia. These data demonstrate that PDE10A inhibition represents a novel antipsychotic target that may have additional metabolic benefits over current medications for schizophrenia by suppressing food intake, alleviating weight gain, and reducing the risk for the development of diabetes.

Inhibition of Diacylglycerol O-acyltransferase 1 (DGAT1) has been a mechanism of interest for metabolic disorders. DGAT1 inhibition has been shown to be a key regulator in an array of metabolic pathways; however, based on the DGAT1 KO mouse phenotype the anticipation is that pharmacological inhibition of DGAT1 could potentially lead to skin related adverse effects. One of the aims in developing small molecule DGAT1 inhibitors that target key metabolic tissues is to avoid activity on skin-localized DGAT1 enzyme. In this report we describe a modeling-based approach to identify molecules with physical properties leading to differential exposure distribution. In addition, we demonstrate histological and RNA based biomarker approaches that can detect sebaceous gland atrophy pre-clinically that could be used as potential biomarkers in a clinical setting.

A novel series of non-imidazole bicyclic and tricyclic histamine H3 receptor antagonists has been discovered. Compound 17 was identified as a centrally penetrant molecule with high receptor occupancy which demonstrates robust oral activity in rodent models of obesity. In addition compound 17 possesses clean CYP and hERG profiles and shows no behavioral changes in the Irwin test.

Abstract The thrombin receptor was the first cloned G protein–coupled receptor reported to be activated by proteolytic cleavage of its extracellular amino terminus. A second proteinase-activated receptor (PAR-2) was cloned recently and expressed in Xenopus laevis oocytes. PAR-2 was activated by trypsin and by a peptide (SLIGRL) derived from the new amino terminus. Since PAR-2 mRNA was detected in highly vascularized organs, we compared the physiological functions of the thrombin receptor and PAR-2 in vascular endothelium. Thrombin and trypsin both elicited endothelium-dependent relaxations in prostaglandin F 2α (PGF 2α )–contracted strips of porcine coronary artery. Whereas high doses of both thrombin or trypsin (10 U/mL) caused homologous desensitization, trypsin caused further relaxation of thrombin-desensitized tissues. Thrombin and PAR-2–derived peptides (SFLLRN and SLIGRL) both induced endothelium-dependent relaxations in PGF 2α -contracted porcine coronary arteries. SFLLRN or SLIGRL (30 μmol/L) also showed homologous desensitization but not cross desensitization. In the presence of the NO synthase inhibitor N G -monomethyl- l -arginine (1 mmol/L), both SFLLRN- and SLIGRL-induced relaxations were partially inhibited. SFLLRN elicited weak contraction in coronary arteries without endothelium, whereas SLIGRL had no effect. Intravenous injection of SFLLRN (1 mg/kg, bolus) into anesthetized rats elicited a transient depressor response followed by pronounced pressor response. In contrast, intravenous administration of SLIGRL (1 mg/kg, bolus) produced only a marked depressor response. Consistent with the in vivo data, SFLLRN contracted the endothelium-rubbed rat aortic rings and aggregated human platelets in vitro, whereas SLIGRL had no effect. The finding that both trypsin and SLIGRL induced endothelium-dependent relaxations indicates the presence of PAR-2 on endothelial cells. In addition, both trypsin and SLIGRL elicited relaxations in thrombin- or SFLLRN-desensitized tissue, suggesting that PAR-2 is distinct from thrombin receptor in vascular endothelium. The lack of PAR-2–mediated platelet aggregation or smooth muscle contraction suggested it might not share the pathogenic properties associated with the thrombin receptor in the vasculature.

Inhibitors based on a benzo-fused spirocyclic oxazepine scaffold were discovered for stearoyl-coenzyme A (CoA) desaturase 1 (SCD1) and subsequently optimized to potent compounds with favorable pharmacokinetic profiles and in vivo efficacy in reducing the desaturation index in a mouse model. Initial optimization revealed potency preferences for the oxazepine core and benzylic positions, while substituents on the piperidine portions were more tolerant and allowed for tuning of potency and PK properties. After preparation and testing of a range of functional groups on the piperidine nitrogen, three classes of analogs were identified with single digit nanomolar potency: glycine amides, heterocycle-linked amides, and thiazoles. Responding to concerns about target localization and potential mechanism-based side effects, an initial effort was also made to improve liver concentration in an available rat PK model. An advanced compound 17m with a 5-carboxy-2-thiazole substructure appended to the spirocyclic piperidine scaffold was developed which satisfied the in vitro and in vivo requirements for more detailed studies.

The synthesis and SAR of a novel series of 4-azabenzoxazole histamine H 3 antagonists is described. Introduction of substituted phenyl, pyridyl and fused heterocyclic groups to the 6-position of the 4-azabenzoxazole core gave a series of compounds with good H 3 antagonist activity in both ex vivo and in vivo assays.

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.

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.

A series of 2-piperidinopiperidine-5-arylthiadiazoles was synthesized and subjected to a structure-activity relationship (SAR) investigation. The potency of this series was improved to the single digit nanomolar range. The key analogs were shown to be free of P450 issues, and they also maintained good ex vivo activity and brain penetration.

Inbred strains of mice exhibit large genetic variations in hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity. A tissue-specific genetic variation between the strains BALB/c and C57BL/6, resulting in about 5-fold higher levels in hepatic reductase activity in strain C57BL/6, was examined in detail. The activity difference between these two strains could be explained entirely by differences in hepatic reductase mRNA levels. In genetic crosses, the variation segregated as a single major Mendelian element. Surprisingly, the mode of inheritance was recessive since F1 mice exhibited the BALB/c levels of enzyme activity. Despite the fact that the rates of hepatic sterol synthesis also differed between the strains by a factor of about five, the altered hepatic reductase expression did not significantly influence plasma lipoprotein levels. The response to a high cholesterol, high fat diet between the strains was remarkably different. Thus, in BALB/c mice, both hepatic reductase activity and mRNA levels were affected only slightly, if at all, by cholesterol feeding, while in strain C57BL/6 mice both were reduced more than 10-fold by cholesterol feeding. Several lines of evidence, including analysis of cis-acting regulatory elements, the nonadditive mode of inheritance, and genetic studies of the HMG-CoA reductase gene locus on mouse chromosome 13, support the possibility that the variation in reductase expression is not due to a mutation of the structural gene but, rather, is determined by a trans-acting factor controlling reductase mRNA levels. The variation provides a striking example, at the molecular level, of the importance of dietary-genetic interactions in the control of cholesterol metabolism.

A series of 2-(1,4′-bipiperidine-1′-yl)thiazolopyridines was synthesized and evaluated as a new lead of non-imidazole histamine H3 receptor antagonists. Introduction of diversity at the 6-position of the pyridine ring was designed to enhance in vitro potency and decrease hERG activity. The structure–activity relationships for these new thiazolopyridine antagonists are discussed.

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.

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.

Recent work has shown that neuromedin U (NmU), a peptide initially identified as a smooth muscle contractor, may play a role in regulating food intake and energy homeostasis. To further evaluate this putative function, we measured food intake, body weight, energy expenditure and glucose homeostasis in transgenic mice that ubiquitously overexpress murine proNmU. NmU transgenic mice were lighter and had less somatic and liver fat, were hypophagic, and had improved insulin sensitivity as judged by an intraperitoneal insulin tolerance test. Transgenic mice had higher levels of hypothalamic NPY, POMC and MCH mRNA. There was no difference in O2 consumption between genotypes; however, NmU transgenic mice displayed a modest increase in respiratory quotient during food deprivation and refeeding. There were no behavioral disturbances in the NmU transgenic mice that could account for the results (e.g. changes in locomotor activity). When placed on a high-fat diet, transgenic mice remained lighter than wild-type mice and ate less, but gained weight at a rate similar to wild-type mice. Despite the increased weight gain with high-fat feeding, glucose tolerance was significantly improved in the transgenic mice. These findings support the hypothesized role of NmU as an endogenous anorexigenic peptide.

Publisher Summary This chapter discusses recent developments made in neuropeptide Y (NPY) receptor modulators research. NPY is a 36 amino acid neuropeptide that is widely distributed in both the central and peripheral nervous systems and is one of the most abundant neuropeptides identified to date. NPY is a member of the pancreatic polypeptide family including structurally related peptides, peptide YY (PYY), and pancreatic polypeptide (PP). Scientific investigation in the past 20 years has generated a large body of knowledge about the biology of NPY, and it is now known that NPY is involved in a broad spectrum of physiological systems. The effects of NPY are mediated by several G protein-coupled receptors that have been designated as Y1, Y2, Y3, Y4, Y5, and Y6. These receptors share only modest primary sequence homology (30–50%) and have unique tissue localizations. The chapter briefly describes the role that NPY and its receptors play in these physiological systems. It summarizes the status of drug discovery in the NPY field and elaborates the potential therapeutic applications of NPY.

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.

The neuropeptide Y (NPY) Y(5) receptor has been proposed to mediate several physiological effects of NPY, including the potent orexigenic activity of the peptide. However, the lack of selective NPY Y(5) receptor ligands limits the characterization of the physiological roles of this receptor. Screening of several analogs of NPY revealed that [D-Trp(34)]NPY is a potent and selective NPY Y(5) receptor agonist. Unlike the prototype selective NPY Y(5) receptor agonist [D-Trp(32)]NPY, [D-Trp(34)]NPY markedly increases food intake in rats, an effect that is blocked by the selective NPY Y(5) receptor antagonist CGP 71683A. These data demonstrate that [D-Trp(34)]NPY is a useful tool for studies aimed at determining the physiological roles of the NPY Y(5) receptor.

Intracerebroventricular (ICV) administration of neuropeptide Y (NPY) has been shown to decrease energy expenditure, induce hypothermia, and stimulate food intake. Recent evidence has suggested that the Y5 receptor may be a significant mediator of NPY-stimulated feeding. The present study attempts to further characterize the role of NPY Y5-receptor subtypes in feeding and energy expenditure regulation. Satiated Long-Evans rats with temperature transponders implanted in the interscapular brown adipose tissue (BAT) displayed a dose-dependent decrease in BAT temperature and an increase in food intake after ICV infusion of NPY. Similar effects were induced by ICV administration of peptide analogs of NPY that activate the Y5 receptor, but not by analogs that activate Y1, Y2, or Y4 receptors. Furthermore, ICV infusion of the Y5 selective agonistd-[Trp32]-NPY significantly reduced oxygen consumption and energy expenditure of rats as measured by indirect calorimetry. These data suggest that the NPY Y5-receptor subtype not only mediates the feeding response of NPY but also contributes to brown fat temperature and energy expenditure regulation.

Genetically obese (ob/ob) mice display a number of metabolic alterations, including decreased thermogenesis, hyperphagia, hyperglycemia and increased body fat. A single intracerebroventricular (i.c.v.) injection of these mice with leptin (0.01 to 1 microg) lowered food intake and body weight within 24 h. In addition, i.c.v. administration of leptin increased 22 h energy expenditure while reducing the respiratory quotient (RQ) in a dose-dependent manner. The leptin-induced decrease in RQ suggests a reduction in the fraction of total energy derived from carbohydrate oxidation and a corresponding increase in energy derived from fat oxidation. Our data suggest that leptin controls energy homeostasis through activation of receptor(s) in the central nervous system (CNS) that regulate both food intake and energy metabolism.

The relative contributions of nitric oxide (NO) to in vitro relaxation responses elicited by acetylcholine (ACh) were compared in vessels of different sizes from the rat mesenteric vascular bed. ACh elicited an endothelium-dependent relaxation in phenylephrine-contracted superior mesenteric arteries (SMA, unstretched luminal diam 650 microns), which was blocked by compounds that inhibited NO, such as hemoglobin (10 microM), methylene blue (10 microM), and NG-monomethyl-L-arginine (1 mM). In contrast, the endothelium-dependent relaxation induced by ACh in phenylephrine-contracted mesenteric resistance arteries (MRA, unstretched luminal diam 200 microns) was not blocked by hemoglobin, methylene blue, or NG-monomethyl-L-arginine. KCl (25 mM) partially inhibited the ACh-dependent relaxation in MRA. Furthermore, the ACh-dependent relaxation in MRA was selectively inhibited by the Ca(2+)-activated K+ channel blocker charybdotoxin (0.1 microM). In contrast, the ATP-sensitive K+ channel blocker glibenclamide (50 microM) did not block the ACh-dependent relaxation in MRA. We conclude that 1) NO is a major component of the ACh-dependent relaxation in SMA and 2) the ACh-dependent relaxation of MRA is resistant to NO inhibitors but sensitive to a Ca(2+)-activated K+ channel blocker. This suggests that an endothelium-derived hyperpolarization factor may be involved in the relaxation of MRA.

The present experiment was planned to provide information about relations between behaviorally augmented tolerance and accompanying upregulation of muscarinic receptors (mAChR) (physiological tolerance) in the CNS during chronic administration of the cholinergic antagonist scopolamine. Analyses of the data on mAChR binding established significant upregulation (Bmax) had occurred in the cortex, hippocampus, and striatum of animals treated with scopolamine, but not of those in the saline or methylscopolamine groups. There were no treatment effects in affinity (KD). The effect of scopolamine administered before a behavioral test session was to cause an acute decrease in FR5 responding to water reinforcement, and hence in resulting water consumption. Animals immediately compensated for this decrement by higher response rates during a free drinking (FDR) period which followed. When scopolamine was injected between the FR5 and FDR periods, FR5 responding increased to compensate for the drug's effect on the FDR. There was evidence that physiological tolerance also occurred as indicated by a more slowly developing trend toward recovery of levels of behavioral responding related to mAChR upregulation, although full recovery to pretreatment baselines did not occur within the 25 days of chronic treatments. The results as a whole are consistent with a multifactorial model of tolerance development, to which both behavioral and neurochemical processes contribute.

Upon stretch in normal physiological salt solution (PSS), two dihydropyridine (DHP) Ca 2+ agonists (Bay k 8644 and CGP 28392) enhanced the level of stretch-dependent myogenic tone in rabbit ear resistance arteries. This potentiating effect was not blocked by phenoxybenzamine (1 μM) and was Ca 2+ -dependent. Bay k 8644 was more potent than CGP 28392. These agonists had a smaller potentiating effect on K + (20 mM)-induced contraction, and had little effect in the presence of high K + (50–134 mM) PSS. Stretch-dependent myogenic tone is resistant to the inhibitory effect of nimodipine, a selective DHP Ca 2+ antagonist which inhibited K + (50 mM)-induced contraction in the nanomolar range. The potentiating effects of Bay k 8644 and CGP 28392 on myogenic tone were reversed by nimodipine. Our data suggest that (1) DHP Ca 2+ agonists selectively enhance myogenic tone by a mechanism that is antagonized by nimodipine, although physiological activation of myogenic tone is nimodipine insensitive, and (2) the calcium related mechanisms of myogenic tone are distinct from those for high K + depolarization-induced tone.

Cerebral vessels of many mammalian species exhibit stretch-dependent or myogenic tone which would be expected to contribute to autoregulation. This tone is dependent on extracellular calcium and probably involves mechanisms other than those associated with agonist and potassium-induced tone. This tone may be contingent upon the redistribution of strategic substances within the cell membrane.