A neutral CB1 receptor antagonist reduces weight gain in rat

Cannabinoid (CB)1 receptor inverse agonists inhibit food intake in animals and humans but also potentiate emesis. It is not clear whether these effects result from inverse agonist properties or from the blockade of endogenous cannabinoid signaling. Here, we examine the effect of a neutral CB1 antagonist, AM4113, on food intake, weight gain, and emesis. Neutral antagonist and binding properties were confirmed in HEK-293 cells transfected with human CB1 or CB2 receptors. AM4113 had no effect on forskolin-stimulated cAMP production at concentrations up to 630 nM. The Ki value of AM4113 (0.80 +/- 0.44 nM) in competitive binding assays with the CB1/2 agonist [3H]CP55,940 was 100-fold more selective for CB1 over CB2 receptors. We determined that AM4113 antagonized CB1 receptors in brain by blocking hypothermia induced by CP55,940. AM4113 (0-20 mg/kg) significantly reduced food intake and weight gain in rat. Compared with AM251, higher doses of AM4113 were needed to produce similar effects on food intake and body weight. Unlike AM251 (5 mg/kg), a highly anorectic dose of AM4113 (10 mg/kg) did not significantly potentiate vomiting induced by the emetic morphine-6-glucoronide. We show that a centrally active neutral CB1 receptor antagonist shares the appetite suppressant and weight loss effects of inverse agonists. If these compounds display similar properties in humans, they could be developed into a new class of antiobesity agents.     

Comparative effects of the long-acting GLP-1 receptor ligands, liraglutide and exendin-4, on food intake and body weight suppression in rats

The glucagon-like-peptide-1 receptor (GLP-1R) agonists, liraglutide (Victoza) and the synthetic product of exendin-4 (Byetta), are approved for type II diabetes mellitus (T2DM) treatment and may be efficacious in obesity treatment as well, in part, due to the drugs' resistance to enzymatic degradation and prolonged half-life relative to endogenous GLP-1. To address the need to directly compare the food intake- and body weight-suppressive effects of these two GLP-1R ligands, acute and chronic dosing experiments were performed. Once-daily (q.d.) exendin-4 (0, 0.33, 1.5, and 3.0 μg/kg) and liraglutide (0, 50, 100, and 300 μg/kg, q.d.) both reduced the chow intake in nonobese rats in a dose-dependent fashion following either intraperitoneal (IP) or subcutaneous (SC) administration, whereas only liraglutide reduced 24 and 48 h body weight in nonobese, chow-maintained rats. Chow intake and body weight suppression by liraglutide were of greater magnitude and shorter latency following IP compared to SC delivery, whereas for exendin-4, the magnitude of intake-suppression was similar for IP and SC administration. The effects of chronic delivery (7 consecutive days; IP) of liraglutide (25 and 50 μg/kg; q.d.) and exendin-4 (3 μg/kg; q.d. and twice-daily (b.i.d.)) on food intake and body weight were also examined in diet-induced obese (DIO) rats. Liraglutide (50 μg/kg q.d.) and exendin-4 (3 μg/kg b.i.d.) were comparable in suppressing overall high fat/sucrose diet (HFS; 60% kcal from fat) intake. Both drugs regimens yielded marked weight loss over the 7-day period. The weight loss effect of liraglutide was achieved in the first 2 days and remained stable for the duration of the experiment; weight loss with exendin-4 appeared more linear over the 7-day period. In conclusion, administration of the GLP-1R ligands, exendin-4 (b.i.d.) and liraglutide (q.d.), lead to comparable and pronounced suppression of food intake and body weight in DIO rats, suggesting a potential role for these drugs as a clinical tool for obesity treatment.     

Cannabinoid-1 receptor antagonists reduce caloric intake by decreasing palatable diet selection in a novel dessert protocol in female rats

Although many feeding protocols induce obesity, few use multiple foods to analyze diet selection within a single group of animals. To this end, we describe a protocol using time-limited access to a dessert that induces hyperphagia and body weight gain while allowing simple analysis of diet selection. Female retired breeder Sprague-Dawley rats were provided with ad libitum access to standard moist chow (1.67 kcal/g) and daily 8-h nocturnal access to either a sugar gel (SG; 0.31 kcal/g) or sugar fat whip (SFW; 7.35 kcal/g) for 15 days, and food intake and body weight were measured daily. Rats given SFW reduced moist chow intake but not enough to compensate for the large amount of calories consumed from SFW, and thus gained weight. We use this SFW overconsumption protocol to investigate the hypothesis that cannabinoid (CB)1 receptor antagonists reduce caloric intake by selectively decreasing consumption of palatable foods. In two experiments, female retired breeder Sprague-Dawley rats were injected with either Rimonabant (1 mg/kg ip) or vehicle (equal parts polyethylene glycol and saline, 1 ml/kg ip) for 7 days, or one of three doses of AM251 (0.3, 1.0, or 3.0 mg/kg ip), or vehicle for 15 days; food intake and body weight were measured daily. Both Rimonabant and AM251 decreased 24-h caloric intake, but the reduction was specific to a decrease in SFW consumption. This supports the hypothesis that these CB1 receptor antagonists impact feeding by modulating the perception of palatability.     

UHRF1 depletion causes a G2/M arrest, activation of DNA damage response and apoptosis

The ECS (endocannabinoid system) plays an important role in the onset of obesity and metabolic disorders, implicating central and peripheral mechanisms predominantly via CB1 (cannabinoid type 1) receptors. CB1 receptor antagonist/inverse agonist treatment improves cardiometabolic risk factors and insulin resistance. However, the relative contribution of peripheral organs to the net beneficial metabolic effects remains unclear. In the present study, we have identified the presence of the endocannabinoid signalling machinery in skeletal muscle and also investigated the impact of an HFD (high-fat diet) on lipid-metabolism-related genes and endocannabinoid-related proteins. Finally, we tested whether administration of the CB1 inverse agonist AM251 restored the alterations induced by the HFD. Rats were fed on either an STD (standard/low-fat diet) or an HFD for 10 weeks and then treated with AM251 (3 mg/kg of body weight per day) for 14 days. The accumulated caloric intake was progressively higher in rats fed on the HFD than the STD, resulting in a divergence in body weight gain. AM251 treatment reduced accumulated food/caloric intake and body weight gain, being more marked in rats fed on the HFD. CB2 (cannabinoid type 2) receptor and PPARα (peroxisome-proliferator-activated receptor α) gene expression was decreased in HFD-fed rats, whereas MAGL (monoglyceride lipase) gene expression was up-regulated. These data suggest an altered endocannabinoid signalling as a result of the HFD. AM251 treatment reduced CB2 receptor, PPARγ and AdipoR1 (adiponectin receptor 1) gene expression in STD-fed rats, but only partially normalized the CB2 receptor in HFD-fed rats. Protein levels corroborated gene expression results, but also showed a decrease in DAGL (diacylglycerol) β and DAGLα after AM251 treatment in STD- and HFD-fed rats respectively. In conclusion, the results of the present study indicate a diet-sensitive ECS in skeletal muscle, suggesting that blockade of CB1 receptors could work towards restoration of the metabolic adaption imposed by diet.     

Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice

GIP receptor antagonism with (Pro³)GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabinoid CB₁ receptor antagonism with AM251 reduces appetite and body weight gain in mice. The present study has examined and compared the effects of chronic daily administrations of (Pro³)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of both drugs in high-fat fed mice. Daily i.p. injection of (Pro³)GIP, AM251 or combined drug administration over 22 days significantly (P < 0.05 to <0.01) decreased body weight compared with saline-treated controls. This was associated with a significant (P < 0.05 to <0.01) reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose tolerance were significantly (P < 0.05) lowered by 22 days (Pro³)GIP, AM251 or combined drug treatment. These changes were accompanied by a significant (P < 0.05) improvement of insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when administered acutely. These data indicate that chemical blockade of GIP- or CB₁-receptor signaling using (Pro³)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro³)GIP.     

The contribution of serotonin 5-HT2C and melanocortin-4 receptors to the satiety signaling of glucagon-like peptide 1 and liragultide, a glucagon-like peptide 1 receptor agonist, in mice

Glucagon-like peptide 1 (GLP-1), an insulinotropic gastrointestinal peptide produced mainly from intestinal endocrine L-cells, and liraglutide, a GLP-1 receptor (GLP-1R) agonist, induce satiety. The serotonin 5-HT2C receptor (5-HT2CR) and melanoroctin-4 receptor (MC4R) are involved in the regulation of food intake. Here we show that systemic administration of GLP-1 (50 and 200 μg/kg)-induced anorexia was blunted in mice with a 5HT2CR null mutation, and was attenuated in mice with a heterozygous MC4R mutation. On the other hand, systemic administration of liraglutide (50 and 100 μg/kg) suppressed food intake in mice lacking 5-HT2CR, mice with a heterozygous mutation of MC4R and wild-type mice matched for age. Moreover, once-daily consecutive intraperitoneal administration of liraglutide (100 μg/kg) over 3 days significantly suppressed daily food intake and body weight in mice with a heterozygous mutation of MC4R as well as wild-type mice. These findings suggest that GLP-1 and liraglutide induce anorexia via different central pathways.     

Peptides that regulate food intake: glucagon-like peptide 1-(7-36) amide acts at lateral and medial hypothalamic sites to suppress feeding in rats

Glucagon-like peptide 1-(7-36) amide (GLP-1) potently inhibits rat feeding behavior after central administration. Because third ventricular injection of GLP-1 appeared to be less effective than lateral ventricular injection, we have reexamined this issue. In addition, we attempted to identify brain regions other than the paraventricular nucleus of the hypothalamus that are sensitive toward GLP-1-induced feeding suppression. Finally, we examined the local role of endogenous GLP-1 by specific GLP-1 receptor blockade. After lateral ventricular injection, GLP-1 significantly inhibited food intake of 24-h-fasted rats in a dose-dependent fashion with a minimal effective dose of 1 microg. After third ventricular injection, GLP-1 (1 microg) was similarly effective in suppressing food intake, which extends previous findings. Intracerebral microinjections of GLP-1 significantly suppressed food intake in the lateral (LH), dorsomedial (DMH), and ventromedial hypothalamus (VMH), but not in the medial nucleus of the amygdala. The minimal effective dose of GLP-1 was 0.3 microg at LH sites and 1 microg at DMH or VMH sites. LH microinjections of exendin-(9-39) amide, a GLP-1 receptor antagonist, at 1 or 2.5 microg did not alter feeding behavior in 24-h-fasted rats. In satiated animals, however, a single LH injection of 1 microg exendin-(9-39) amide significantly augmented food intake, but only during the first 20 min (0.6 vs. 0.1 g). With three repeated injections of 2.5 microg exendin-(9-39) amide every 20 min, 1-h food intake was significantly increased by 300%. These data strongly support and extend the concept of GLP-1 as a physiological regulator of food intake in the hypothalamus.     

Colocalization of Glucagon-Like Peptide-1 (GLP-1) Receptors, Glucose Transporter GLUT-2, and Glucokinase mRNAs in Rat Hypothalamic Cells: Evidence for a Role of GLP-1 Receptor Agonists as an Inhibitory Signal for Food and Water Intake

Abstract: This study was designed to determine the possible role of brain glucagon-like peptide-1 (GLP-1) receptors in feeding behavior. In situ hybridization showed colocalization of the mRNAs for GLP-1 receptors, glucokinase, and GLUT-2 in the third ventricle wall and adjacent arcuate nucleus, median eminence, and supraoptic nucleus. These brain areas are considered to contain glucose-sensitive neurons mediating feeding behavior. Because GLP-1 receptors, GLUT-2, and glucokinase are proteins involved in the multistep process of glucose sensing in pancreatic β cells, the colocalization of specific GLP-1 receptors and glucose sensing-related proteins in hypothalamic neurons supports a role of this peptide in the hypothalamic regulation of macronutrient and water intake. This hypothesis was confirmed by analyzing the effects of both systemic and central administration of GLP-1 receptor ligands. Acute or subchronic intraperitoneal administration of GLP-1 (7–36) amide did not modify food and water intake, although a dose-dependent loss of body weight gain was observed 24 h after acute administration of the higher dose of the peptide. By contrast, the intracerebroventricular (i.c.v.) administration of GLP-1 (7–36) amide produced a biphasic effect on food intake characterized by an increase in the amount of food intake after acute i.c.v. delivery of 100 ng of the peptide. There was a marked reduction of food ingestion with the 1,000 and 2,000 ng doses of the peptide, which also produced a significant decrease of water intake. These effects seemed to be specific because i.c.v. administration of GLP-1 (1–37), a peptide with lower biological activity than GLP-1 (7–36) amide, did not change feeding behavior in food-deprived animals. Exendin-4, when given by i.c.v. administration in a broad range of doses (0.2, 1, 5, 25, 100, and 500 ng), proved to be a potent agonist of GLP-1 (7–36) amide. It decreased, in a dose-dependent manner, both food and water intake, starting at the dose of 25 ng per injection. Pretreatment with an i.c.v. dose of a GLP-1 receptor antagonist [exendin (9–39); 2,500 ng] reversed the inhibitory effects of GLP-1 (7–36) amide (1,000 ng dose) and exendin-4 (25 ng dose) on food and water ingestion. These findings suggest that GLP-1 (7–36) amide may modulate both food and drink intake in the rat through a central mechanism.     

The contribution of serotonin 5-HT2C and melanocortin-4 receptors to the satiety signaling of glucagon-like peptide 1 and liraglutide, a glucagon-like peptide 1 receptor agonist, in mice

Glucagon-like peptide 1 (GLP-1), an insulinotropic gastrointestinal peptide produced mainly from intestinal endocrine L-cells, and liraglutide, a GLP-1 receptor (GLP-1R) agonist, induce satiety. The serotonin 5-HT2C receptor (5-HT2CR) and melanoroctin-4 receptor (MC4R) are involved in the regulation of food intake. Here we show that systemic administration of GLP-1 (50 and 200μg/kg)-induced anorexia was blunted in mice with a 5HT2CR null mutation, and was attenuated in mice with a heterozygous MC4R mutation. On the other hand, systemic administration of liraglutide (50 and 100μg/kg) suppressed food intake in mice lacking 5-HT2CR, mice with a heterozygous mutation of MC4R and wild-type mice matched for age. Moreover, once-daily consecutive intraperitoneal administration of liraglutide (100μg/kg) over 3days significantly suppressed daily food intake and body weight in mice with a heterozygous mutation of MC4R as well as wild-type mice. These findings suggest that GLP-1 and liraglutide induce anorexia via different central pathways.     

Increase in hypothalamic AMPK phosphorylation induced by prolonged exposure to LPS involves ghrelin and CB1R signaling

Acute administration of lipopolysaccharide (LPS) from Gram-negative bacteria induces hypophagia. However, the repeated administration of LPS leads to desensitization of hypophagia, which is associated with increased hypothalamic p-AMPK expression. Because ghrelin and endocannabinoids modulate AMPK activity in the hypothalamus, we hypothesized that these neuromodulators play a role in the reversal of tolerance to hypophagia in rats under long-term exposure to LPS. Male Wistar rats were treated with single (1 LPS, 100 μg/kg body weight, ip) or repeated injections of LPS over 6 days (6 LPS). Food intake was reduced in the 1 LPS, but not in the 6 LPS group. 6 LPS rats showed an increased serum concentration of acylated ghrelin and reduced ghrelin receptor mRNA expression in the hypothalamus. Ghrelin injection (40 μg/kg body weight, ip) increased food intake, body weight gain, p-AMPK hypothalamic expression, neuropeptide Y (NPY) and Agouti related peptide (AgRP) mRNA expression in control animals (Saline). However, in 6 LPS rats, ghrelin did not alter these parameters. Central administration of a CB1R antagonist (AM251, 200 ng/μl in 5 μl/rat) induced hypophagia in 6 LPS animals, suggesting that the endocannabinoid system contributes to preserved food intake during LPS tolerance. In the presence of AM251, the ability of ghrelin to phosphorylate AMPK in the hypothalamus of 6 LPS group was restored, but not its orexigenic effect. Our data highlight that the orexigenic effects of ghrelin require CB1R signaling downstream of AMPK activation. Moreover, CB1R-mediated pathways contribute to the absence of hypophagia during repeated exposure to endotoxin.     

Jejunal linoleic acid infusions require GLP-1 receptor signaling to inhibit food intake: implications for the effectiveness of Roux-en-Y gastric bypass

Roux-en-Y gastric bypass surgery results in sustained decreases in food intake and weight loss. A key component is likely the direct delivery of nutrients to the jejunum and resulting changes in levels of gut peptide secretion. Prior work modeling this aspect of the surgery has shown that small-volume, prolonged jejunal infusions of linoleic acid (LA) produce sustained decreases in food intake and weight loss. LA infusions also significantly elevate plasma glucagon-like peptide-1 (GLP-1) levels. To assess a role for the increased circulating GLP-1 in the feeding suppression, we examined the effect of prolonged peripheral minipump administration of the GLP-1 receptor antagonist exendin 9-39 (Ex 9) on the feeding suppression produced by jejunal LA. Using a 2 × 2 design, we infused either saline or LA in the jejunum (7 h/day, 11.4 kcal) for 5 days with a subset of animals from each group receiving either saline or Ex 9 (25 pmol·kg(-1)·min(-1)) continuously via a minipump. The antagonist alone had no effect on food intake. LA reduced daily food intake greatly in excess of the kilocalories infused. Ex 9 completely blocked the feeding suppression produced by the jejunal LA infusion. Ex 9 also attenuated the increase in plasma GLP-1 induced by jejunal LA infusions. These data demonstrate that endogenous GLP-1 receptor signaling is necessary for the reduction in food intake produced by jejunal LA infusions. Whether increased secretion of additional gut peptides is also necessary for such suppressions remains to be determined.     

Effects of leptin replacement alone and with exendin-4 on food intake and weight regain in weight-reduced diet-induced obese rats

Weight loss in obese humans produces a relative leptin deficiency, which is postulated to activate potent orexigenic and energy conservation mechanisms to restrict weight loss and promote weight regain. Here we determined whether leptin replacement alone or with GLP-1 receptor agonist exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced diet-induced obese (DIO) rats. Forty percent restriction in daily intake of a high-fat diet in DIO rats for 4 wk reduced body weight by 12%, body fat by 29%, and plasma leptin by 67% and normalized leptin sensitivity. When food restriction ended, body weight, body fat, and plasma leptin increased rapidly. Daily administration of leptin [3-h intraperitoneal (ip) infusions (4 nmol·kg(-1)·h(-1))] at onset and end of dark period for 3 wk did not attenuate hyperphagia and weight regain, nor did it affect mean daily meal sizes or meal numbers. Exendin-4 (50 pmol·kg(-1)·h(-1)) infusions during the same intervals prevented postrestriction hyperphagia and weight regain by normalizing meal size. Coadministration of leptin and exendin-4 did not reduce body weight more than exendin-4 alone. Instead, leptin began to attenuate the inhibitory effects of exendin-4 on food intake, meal size, and weight regain by the end of the second week of administration. Plasma leptin in rats receiving leptin was sevenfold greater than in rats receiving vehicle and 17-fold greater than in rats receiving exendin-4. Together, these results do not support the hypothesis that leptin replacement alone or with exendin-4 attenuates weight regain or promotes greater weight loss in weight-reduced DIO rats.     

Exendin-4 inhibits structural remodeling and improves Ca2+ homeostasis in rats with heart failure via the GLP-1 receptor through the eNOS/cGMP/PKG pathway

The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 is a long-acting analog of GLP-1, which stimulates insulin secretion and is clinically used in the treatment of type 2 diabetes. Previous studies have demonstrated that GLP-1 agonists and analogs serve as cardioprotective factors in various conditions. Disturbances in calcium cycling are characteristic of heart failure (HF); therefore, the aim of this study was to investigate the effect of exendin-4 (a GLP-1 mimetic) on the regulation of calcium handling and to identify the underlying mechanisms in an HF rat model after myocardial infarction (MI). Rats underwent surgical ligation of the left anterior descending coronary artery or sham surgery prior to infusion with vehicle, exendin-4, or exendin-4 and exendin9-39 for 4 weeks. Exendin-4 treatment decreased MI size, suppressed chamber dilation, myocyte hypertrophy, and fibrosis and improved in vivo heart function in the rats subjected to MI. Exendin-4 resulted in an increase in circulating GLP-1 and GLP-1R in ventricular tissues. Additionally, exendin-4 activated the eNOS/cGMP/PKG signaling pathway and inhibited the Ca²⁺/calmodulin-dependent kinase II (CaMKII) pathways. Myocytes isolated from exendin-4-treated hearts displayed higher Ca²⁺ transients, higher sarcoplasmic reticulum Ca²⁺ content, and higher l-type Ca²⁺ current densities than MI hearts. Exendin-4 treatment restored the protein expression of sarcoplasmic reticulum Ca²⁺ uptake ATPase (SERCA2a), phosphorylated phospholamban (PLB) and Cav1.2 and decreased the levels of phosphorylated ryanodine receptor (RyR). Moreover, the favorable effects of exendin-4 were significantly inhibited by exendin9-39 (a GLP-1 receptor antagonist). Exendin-4 treatment of an HF rat model after MI inhibited cardiac and cardiomyocytes progressive remodeling. In addition, Ca²⁺ handling and its molecular modulation were also improved by exendin-4 treatment. The beneficial effects of exendin-4 on cardiac remodeling may be mediated through activation of the eNOS/cGMP/PKG pathway.     

The common hepatic branch of the vagus is not required to mediate the glycemic and food intake suppressive effects of glucagon-like-peptide-1

The incretin and food intake suppressive effects of intraperitoneally administered glucagon-like peptide-1 (GLP-1) involve activation of GLP-1 receptors (GLP-1R) expressed on vagal afferent fiber terminals. Central nervous system processing of GLP-1R-driven vagal afferents results in satiation signaling and enhanced insulin secretion from pancreatic-projecting vagal efferents. As the vast majority of endogenous GLP-1 is released from intestinal l-cells following ingestion, it stands to reason that paracrine GLP-1 signaling, activating adjacent GLP-1R expressed on vagal afferent fibers of gastrointestinal origin, contributes to glycemic and food intake control. However, systemic GLP-1R-mediated control of glycemia is currently attributed to endocrine action involving GLP-1R expressed in the hepatoportal bed on terminals of the common hepatic branch of the vagus (CHB). Here, we examine the hypothesis that activation of GLP-1R expressed on the CHB is not required for GLP-1's glycemic and intake suppressive effects, but rather paracrine signaling on non-CHB vagal afferents is required to mediate GLP-1's effects. Selective CHB ablation (CHBX), complete subdiaphragmatic vagal deafferentation (SDA), and surgical control rats received an oral glucose tolerance test (2.0 g glucose/kg) 10 min after an intraperitoneal injection of the GLP-1R antagonist, exendin-(9-39) (Ex-9; 0.5 mg/kg) or vehicle. CHBX and control rats showed comparable increases in blood glucose following blockade of GLP-1R by Ex-9, whereas SDA rats failed to show a GLP-1R-mediated incretin response. Furthermore, GLP-1(7-36) (0.5 mg/kg ip) produced a comparable suppression of 1-h 25% glucose intake in both CHBX and control rats, whereas intake suppression in SDA rats was blunted. These findings support the hypothesis that systemic GLP-1R mediation of glycemic control and food intake suppression involves paracrine-like signaling on GLP-1R expressed on vagal afferent fibers of gastrointestinal origin but does not require the CHB.     

Effects of Selenium and Exendin-4 on Glucagon-Like Peptide-1 Receptor, IRS-1, and Raf-1 in the Liver of Diabetic Rats

Selenium and exendin-4 exert antidiabetic effects by unknown mechanisms. Herein, we investigated their effects on the expression of glucagon-like peptide-1 receptor (GLP-1R), insulin receptor substrate-1 (IRS-1), and Raf-1 in the livers of rats with streptozotocin-induced diabetes. Diabetic rats were injected intraperitoneally with exendin-4 (0.03?μg/kg body weight) twice daily or treated with 5?ppm selenium as sodium selenite in drinking water for 4?weeks. Both selenium and exendin-4 reduced the hyperglycemia in diabetic rats. Induction of diabetes mellitus resulted in decreased level of GLP-1R and increased levels of IRS-1 and Raf-1 in the liver. Treatment of diabetic rats with selenium or exendin-4 resulted in increased level of GLP-1R and decreased levels of IRS-1 and Raf-1 in the liver, compared with the levels in diabetic rats. Therefore, the antidiabetic actions of selenium and exendin-4 involve their effects on GLP-1R, IRS-1, and Raf-1 levels in the liver.     

Functional activity of murine intestinal mucosal cells is regulated by the glucagon-like peptide-1 receptor

To determine whether the glucagon-like peptide-1 receptor (GLP-1r) plays a role in the regulation of intestinal functional activity, we analyzed the distribution of the GLP-1r in mouse tissues and tested if tissues expressing the receptor respond to exendin-4 and exendin (9–39) amide, a GLP-1r agonist and antagonist respectively. In ileum, Glp1r mRNA level was two fold higher in extracts from epithelial cells than non-epithelial tissues. By immunohistochemistry, the receptor was localized to the mucosal cell layer of villi of ileum and colon, to the myenteric and submucosal plexus and to Paneth cells. Intravenous administration of exendin-4 to CD-1 mice induced expression of the immediate early gene c-fos in mucosal cells but not in cells of the enteric plexuses or in L cells of ileum. The induction of c-fos was inhibited by the voltage-gated sodium channel blocker tetrodotoxin. Exendin-4 also increased c-fos expression in ileal segments in vitro, suggesting that this action of the analog was independent of an extrinsic input. The induction of c-fos expression by exendin-4 was inhibited by exendin (9–39) amide, indicating that the action of exendin-4 was mediated by activation of the receptor. Our findings indicate that the GLP-1r is involved in ileal enterocyte and Paneth cell function, that the GLP-1 analog activates c-fos expression in the absence of an extrinsic input and that some of the actions of the receptor is/are mediated by voltage-gated Na channels.     

Effects of Exendin‐4 Alone and With Peptide YY3–36 on Food Intake and Body Weight in Diet‐Induced Obese Rats

Significant weight loss following Roux‐en‐Y gastric bypass surgery (RYGB) in obese humans correlates with enhanced secretion of anorexigenic gut hormones glucagon‐like peptide‐1 (GLP‐1) and peptide YY_3–36 (PYY_3–36). Our aim here was to identify a dosing strategy for intraperitoneal (IP) infusion of GLP‐1 homologue exendin‐4 alone and with PYY_3–36 that produces a sustained reduction in daily food intake and body weight in diet‐induced obese (DIO) rats. We tested 12 exendin‐4 strategies over 10 weeks. Exendin‐4 infused during the first and last 3 h of the dark period at 15–20 pmol/h (0.15 nmol/kg/day) produced a sustained 24 ± 1% reduction in daily food intake for 17 days, and decreased body weight by 7%. In a separate group of DIO rats, none of seven dosing strategies combining exendin‐4 and PYY_3–36 produced a similar reduction in daily food intake for >10 days. The subsequent decline in efficacies of exendin‐4 alone and with PYY_3–36 on food intake and body weight in each experiment suggested possible receptor downregulation and tolerance to treatments. However, when treatments were discontinued for 1 day following losses in efficacies, daily food intake significantly increased. Together, these results demonstrate that (i) intermittent IP infusion of a low dose of exendin‐4 can produce a relatively prolonged reduction in daily food intake and body weight in DIO rats, (ii) co‐infusion of exendin‐4 and PYY_3–36 does not further prolong this response, and (iii) activation of an orexigenic mechanism gradually occurs to counteract the inhibitory effects of exendin‐4 alone and with PYY_3–36 on food intake and body weight.     

Glucagon-like peptide-1 receptor agonists suppress water intake independent of effects on food intake

Glucagon-like peptide-1 (GLP-1) is produced by and released from the small intestine following ingestion of nutrients. GLP-1 receptor (GLP-1R) agonists applied peripherally or centrally decrease food intake and increase glucose-stimulated insulin secretion. These effects make the GLP-1 system an attractive target for the treatment of type 2 diabetes mellitus and obesity. In addition to these more frequently studied effects of GLP-1R stimulation, previous reports indicate that GLP-1R agonists suppress water intake. The present experiments were designed to provide greater temporal resolution and site specificity for the effect of GLP-1 and the long-acting GLP-1R agonists, exendin-4 and liraglutide, on unstimulated water intake when food was and was not available. All three GLP-1R ligands suppressed water intake after peripheral intraperitoneal administration, both in the presence of and the absence of food; however, the magnitude and time frame of water intake suppression varied by drug. GLP-1 had an immediate, but transient, hypodipsic effect when administered peripherally, whereas the water intake suppression by IP exendin-4 and liraglutide was much more persistent. Additionally, intracerebroventricular administration of GLP-1R agonists suppressed water intake when food was absent, but the suppression of intake showed modest differences depending on whether the drug was administered to the lateral or fourth ventricle. To the best of our knowledge, this is the first demonstration of GLP-1 receptor agonists affecting unstimulated, overnight intake in the absence of food, the first test for antidipsogenic effects of hindbrain application of GLP-1 receptor agonists, and the first test of a central effect (forebrain or hindbrain) of liraglutide on water intake. Overall, these results show that GLP-1R agonists have a hypodipsic effect that is independent of GLP-1R-mediated effects on food intake, and this occurs, in part, through central nervous system GLP-1R activation.     

Residues within the transmembrane domain of the glucagon-like peptide-1 receptor involved in ligand binding and receptor activation: modelling the ligand-bound receptor

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9-39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9-39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues.     

Truncated glucagon-like peptide-1 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Identification of a mammalian analogue of the reptilian peptide exendin-4.

To find mammalian analogues of exendin-4, a peptide from Helodermatidae venoms that interacts with newly discovered exendin receptors on dispersed acini from guinea pig pancreas, we examined the actions of recent additions to the vasoactive intestinal peptide/secretin/glucagon family of regulatory peptides. In every respect tested, the truncated form of glucagon-like peptide-1, GLP-1(7-36)NH2, mimicked the actions of exendin-4. Like exendin-4, GLP-1(7-36)NH2 caused an increase in acinar cAMP without stimulating amylase release. GLP-1(7-36)NH2-induced increases in cAMP were inhibited progressively by increasing concentrations of the specific exendin-receptor antagonist, exendin(9-39)NH2. In dispersed acini from guinea pig and rat pancreas, concentrations of GLP-1(7-36)NH2 that stimulated increases in cAMP caused potentiation of cholecystokinin-induced amylase release. Binding of 125I-[Y39]exendin-4 or 125I-GLP-1(7-36)NH2 to dispersed acini from guinea pig pancreas was inhibited by adding increasing concentrations of unlabeled exendin-4 or GLP-1(7-36)NH2. We conclude that the mammalian peptide GLP-1(7-36)NH2 interacts with exendin receptors on dispersed acini from guinea pig pancreas. Exendin(9-39)NH2, a competitive antagonist of the actions of GLP-1(7-36)NH2 in pancreatic acini, may be a useful tool for examining the physiological actions of this peptide.