exenatide and exendin-(9-39)

To test the hypothesis that food intake reduction after glucagon-like peptide-1 (GLP-1) receptor activation is mediated through brain areas regulating anticipatory and consummatory food reward.. As part of a larger study, we determined the effects of GLP-1 receptor activation on brain responses to anticipation and receipt of chocolate milk versus a tasteless solution, using functional MRI (fMRI). Obese subjects with type 2 diabetes, and obese and lean subjects with normoglycaemia (n = 48) underwent three fMRI sessions at separate visits with intravenous infusion of the GLP-1 receptor agonist exenatide, exenatide with prior GLP-1 receptor blockade by exendin-9-39 or placebo, during somatostatin pituitary-pancreatic clamps.. Body mass index negatively correlated with brain responses to receipt of chocolate milk and positively correlated with anticipation of receipt of chocolate milk in brain areas regulating reward, appetite and motivation. Exenatide increased brain responses to receipt of chocolate milk and decreased anticipation of receipt of chocolate milk compared with placebo, paralleled by reductions in food intake. Exendin-9-39 largely prevented these effects.. Our findings show that GLP-1 receptor activation decreases anticipatory food reward, which may reduce cravings for food and increases consummatory food reward, which may prevent overeating.

Topics: Adult; Aged; Animals; Anticipation, Psychological; Appetite; Brain; Cacao; Diabetes Mellitus, Type 2; Exenatide; Feeding Behavior; Female; Glucagon-Like Peptide-1 Receptor; Humans; Hypoglycemic Agents; Magnetic Resonance Imaging; Male; Middle Aged; Milk; Motivation; Obesity; Peptide Fragments; Peptides; Reward; Venoms

Gut-derived hormones, such as GLP-1, have been proposed to relay information to the brain to regulate appetite. GLP-1 receptor agonists, currently used for the treatment of type 2 diabetes (T2DM), improve glycemic control and stimulate satiety, leading to decreases in food intake and body weight. We hypothesized that food intake reduction after GLP-1 receptor activation is mediated through appetite- and reward-related brain areas. Obese T2DM patients and normoglycemic obese and lean individuals (n = 48) were studied in a randomized, crossover, placebo-controlled trial. Using functional MRI, we determined the acute effects of intravenous administration of the GLP-1 receptor agonist exenatide, with or without prior GLP-1 receptor blockade using exendin 9-39, on brain responses to food pictures during a somatostatin pancreatic-pituitary clamp. Obese T2DM patients and normoglycemic obese versus lean subjects showed increased brain responses to food pictures in appetite- and reward-related brain regions (insula and amygdala). Exenatide versus placebo decreased food intake and food-related brain responses in T2DM patients and obese subjects (in insula, amygdala, putamen, and orbitofrontal cortex). These effects were largely blocked by prior GLP-1 receptor blockade using exendin 9-39. Our findings provide novel insights into the mechanisms by which GLP-1 regulates food intake and how GLP-1 receptor agonists cause weight loss.

Topics: Adult; Aged; Amygdala; Appetite; Brain; Case-Control Studies; Cerebral Cortex; Cross-Over Studies; Diabetes Mellitus, Type 2; Exenatide; Feeding Behavior; Female; Functional Neuroimaging; Glucagon-Like Peptide-1 Receptor; Humans; Hypoglycemic Agents; Magnetic Resonance Imaging; Male; Middle Aged; Obesity; Peptide Fragments; Peptides; Photic Stimulation; Prefrontal Cortex; Putamen; Receptors, Glucagon; Reward; Venoms

Compared to traditional weight loss strategies, the compensatory decrease in energy expenditure in response to body weight loss is markedly attenuated after Roux-en-Y gastric bypass surgery (RYGB). Because basal and postprandial levels of glucagon-like peptide-1 (GLP-1) are increased after RYGB surgery, and because GLP-1 has been shown to increase energy expenditure, we investigated if increased GLP-1 levels are involved in the alterations in energy expenditure after RYGB. Adult male Wistar rats were randomized for RYGB (n=8) or sham surgery (n=17). Part of the sham-operated rats were food restricted and body weight-matched (n=8) to the RYGB animals. The effects of acute subcutaneous administration of the GLP-1 antagonist Exendin (9-39) (Ex-9, 30μg/kg) or the GLP-1 agonist Exendin-4 (Ex-4, 5μg/kg), respectively, on energy expenditure were tested using indirect calorimetry. We found that Ex-9 increased food intake in RYGB, but not in sham-operated rats. Energy expenditure was lower in RYGB and sham-operated body weight-matched rats compared to sham-operated ad libitum fed rats, but significantly higher in RYGB rats compared to sham-operated body weight-matched rats. There was no effect of Ex-9 treatment on energy expenditure in either group of animals. Similarly, Ex-4 decreased food intake more in RYGB than in sham-operated rats, but Ex-4 did not modulate energy expenditure in any surgical group. We conclude that acute modulation of GLP-1 signaling is not directly involved in altered energy expenditure after RYGB surgery in rats.

Topics: Activity Cycles; Animals; Body Weight; Calorimetry; Eating; Energy Metabolism; Exenatide; Food Deprivation; Gastric Bypass; Glucagon-Like Peptide-1 Receptor; Male; Motor Activity; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Respiration; Time Factors; Venoms

Growing evidences indicate that neuropathic pain is frequently accompanied with cognitive impairments, which aggravate the decrease in the quality of life of chronic pain patients. Furthermore, it has been shown that the activation of Glucagon-like-peptide-1receptor (GLP-1R) improved memory deficit in multiple diseases, including Alzheimer's disease (AD), stroke. However, whether GLP-1R activation could improve memory impairment induced by neuropathic pain and the mechanisms underlying the effect of the activation of GLP-1R on memory protection have not yet been established. The spared nerve injury (SNI) model was established as a kind of neuropathic pain. And novel-object recognition memory (hippocampus-dependent memory) was tested by the novel object recognition test (NORT). The expression levels of GLP-1, GLP-1R, adenosine monophosphate-activated protein kinase (AMPK), p-AMPKThr172, nuclear factor κ B p65 (NF-κB p65), interleukin-1beta (IL-1β), IL-1β p17 (mature IL-1β), tumor necrosis factor-alpha (TNF-α) and the synaptic proteins were tested in the murine hippocampus with memory deficits caused by neuropathic pain. Then, exenatide acetate (Ex-4, a GLP-1R agonist), exendin (9-39) (Ex(9-39), a GLP-1R antagonist) and Compound C dihydrochloride (CC, an AMPK inhibitor) were used to test the effects of the activation of GLP-1R in the mice with neuropathic pain. First, we uncovered that neuropathic pain could inhibit GLP-1/GLP-R axis, disturb inflammatory signaling pathway, increase the expression of IL-1β, IL-1β p17 and TNF-α, downregulate the synaptic proteins (postsynaptic density protein 95 (PSD95) and Arc). Subsequently, we reported that Ex-4 treatment could improve recognition memory impairment, increase the ratio of p-AMPKThr172/AMPK, inhibit the phosphorylation NF-κB p65 and decrease the expression of IL-1β, IL-1β p17 and TNF-α, upregulate the levels of PSD95 and Arc. Moreover, we found that Ex(9-39) and CC treatment could abrogate the memory protection of activation of GLP-1R in mice with neuropathic pain. The results indicated that the activation of GLP-1R could improve recognition memory impairment via regulating AMPK/NF-κB pathway, improving neuroinflammation, reversing the decreased level of synaptic proteins in neuropathic pain mice.

Topics: AMP-Activated Protein Kinase Kinases; Animals; Chronic Pain; Disease Models, Animal; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hippocampus; Interleukin-1beta; Memory Disorders; Mice; Neuralgia; Neuroinflammatory Diseases; Open Field Test; Peptide Fragments; Peripheral Nerve Injuries; Recognition, Psychology; Sciatic Nerve; Transcription Factor RelA; Tumor Necrosis Factor-alpha

Neuropeptides and peptide hormones affect food-directed motivation, in part, through actions on brain regions associated with reward processing. For instance, previous reports have shown that stimulating glucagon-like peptide-1 (GLP-1) receptors in the nucleus accumbens (NAc), an area that directs motivational processes towards food and drugs of abuse, has an anorectic effect. In contrast, µ-opioid receptor activation of the NAc increases feeding, particularly on highly palatable diets. While both neurotransmitters act within the NAc to impact food intake, it is not clear if and how they might interact to affect feeding. Therefore, these experiments tested the effects of NAc injections of the GLP-1 receptor agonist Exendin 4 (EX4) or antagonist Exendin 9 (EX9) on the consumption of a sweetened fat diet, with and without simultaneous µ-opioid receptor stimulation. Male Sprague-Dawley rats (n = 8/group, EX4 or EX9) underwent surgery to place bilateral cannula above the NAc core. After recovery, animals were tested following NAc injections of saline or the µ-opioid agonist [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO) (0.025 µg/side), combined with varying doses of EX4 (0, 0.05, or 0.10 µg/side) or EX9 (0, 2.5, 5.0 µg/side), counterbalanced across 6 testing days. Food and water intake, along with locomotor activity, was monitored for 2 h. Mu-opioid receptor stimulation significantly increased feeding, and this effect was reduced by GLP-1 receptor stimulation. In contrast, GLP-1 antagonism with EX9 altered the dynamics of DAMGO-induced binge-like feeding, extending µ-opioid-induced binging, and increasing food consumption. These findings are the first to demonstrate an interaction between NAc µ-opioid and GLP-1 receptors on palatable food intake.

Topics: Animals; Bulimia; Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; Exenatide; Feeding Behavior; Glucagon-Like Peptide-1 Receptor; Male; Neurotransmitter Agents; Nucleus Accumbens; Peptide Fragments; Rats; Receptors, Opioid, mu

The neurobiological substrates that mediate the anorectic effects of both endogenous glucagon-like peptide-1 (GLP-1) and exogenous GLP-1 receptor (GLP-1R) agonists are an active area of investigation. As the lateral dorsal tegmental nucleus (LDTg) expresses the GLP-1R and represents a potential neuroanatomical hub connecting the nucleus tractus solitarius (NTS), the major central source of GLP-1, with the other nuclei in the midbrain and forebrain, we tested the hypothesis that GLP-1R signaling in the LDTg controls food intake. Direct activation of LDTg GLP-1R suppresses food intake through a reduction in average meal size and independent of nausea/malaise. Immunohistochemical data show that GLP-1-producing neurons in the NTS project to the LDTg, providing anatomical evidence of endogenous central GLP-1 in the LDTg. Pharmacological blockade of LDTg GLP-1Rs with exendin-(9-39) dose-dependently increases food intake and attenuates the hypophagic effects of gastric distension. As GLP-1 mimetics are administered systemically in humans, we evaluated whether peripherally administered GLP-1R agonists access the LDTg to affect feeding. Immunohistochemical data show that a systemically administered fluorescent GLP-1R agonist accesses the LDTg and is juxtaposed with neurons. Additionally, blockade of LDTg GLP-1Rs attenuates the hypophagic effects of a systemic GLP-1R agonist. Together, these data indicate that LDTg GLP-1R signaling controls energy balance and underscores the role of the LDTg in integrating energy balance-relevant signals to modulate feeding.

Topics: Animals; Central Nervous System Agents; Dose-Response Relationship, Drug; Eating; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Male; Neural Pathways; Neurons; Peptide Fragments; Peptides; Rats, Sprague-Dawley; Solitary Nucleus; Tegmentum Mesencephali; Venoms

Cocaine addiction continues to be a significant public health problem for which there are currently no effective FDA-approved treatments. Thus, there is a clear need to identify and develop novel pharmacotherapies for cocaine addiction. Recent evidence indicates that activation of glucagon-like peptide-1 (GLP-1) receptors in the ventral tegmental area (VTA) reduces intake of highly palatable food. As the neural circuits and neurobiological mechanisms underlying drug taking overlap to some degree with those regulating food intake, these findings suggest that activation of central GLP-1 receptors may also attenuate cocaine taking. Here, we show that intra-VTA administration of the GLP-1 receptor agonist exendin-4 (0.05 μg) significantly reduced cocaine, but not sucrose, self-administration in rats. We also demonstrate that cocaine taking is associated with elevated plasma corticosterone levels and that systemic infusion of cocaine activates GLP-1-expressing neurons in the nucleus tractus solitarius (NTS), a hindbrain nucleus that projects monosynaptically to the VTA. To determine the potential mechanisms by which cocaine activates NTS GLP-1-expressing neurons, we microinjected corticosterone (0.5 μg) directly into the hindbrain fourth ventricle. Intraventricular corticosterone attenuated cocaine self-administration and this effect was blocked in animals pretreated with the GLP-1 receptor antagonist exendin-(9-39) (10 μg) in the VTA. Finally, AAV-shRNA-mediated knockdown of VTA GLP-1 receptors was sufficient to augment cocaine self-administration. Taken together, these findings indicate that increased activation of NTS GLP-1-expressing neurons by corticosterone may represent a homeostatic response to cocaine taking, thereby reducing the reinforcing efficacy of cocaine. Therefore, central GLP-1 receptors may represent a novel target for cocaine addiction pharmacotherapies.

Topics: Anesthetics, Local; Animals; Anti-Inflammatory Agents; Cocaine; Conditioning, Operant; Corticosterone; Exenatide; Fourth Ventricle; Glucagon-Like Peptide-1 Receptor; Green Fluorescent Proteins; Hypoglycemic Agents; Male; Neurons; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Reinforcement, Psychology; RNA, Small Interfering; Self Administration; Venoms; Ventral Tegmental Area

The glucagon-like peptide receptor (GLP-1R), which is a G-protein coupled receptor (GPCR), signals through both Gαs and Gαq coupled pathways and ERK phosphorylation to stimulate insulin secretion. The aim of this study was to determine molecular details of the effect of small molecule agonists, compounds 2 and B, on GLP-1R mediated cAMP production, intracellular Ca2+ accumulation, ERK phosphorylation and its internalisation. In human GLP-1R (hGLP-1R) expressing cells, compounds 2 and B induced cAMP production but caused no intracellular Ca2+ accumulation, ERK phosphorylation or hGLP-1R internalisation. GLP-1 antagonists Ex(9-39) and JANT-4 and the orthosteric binding site mutation (V36A) in hGLP-1R failed to inhibit compounds 2 and B induced cAMP production, confirming that their binding site distinct from the GLP-1 binding site on GLP-1R. However, K334A mutation of hGLP-1R, which affects Gαs coupling, inhibited GLP-1 as well as compounds 2 and B induced cAMP production, indicating that GLP-1, compounds 2 and B binding induce similar conformational changes in the GLP-1R for Gαs coupling. Additionally, compound 2 or B binding to the hGLP-1R had significantly reduced GLP-1 induced intracellular Ca2+ accumulation, ERK phosphorylation and hGLP-1R internalisation. This study illustrates pharmacology of differential activation of GLP-1R by GLP-1 and compounds 2 and B.

Topics: Binding Sites; Calcium; Cyclic AMP; Endocytosis; Exenatide; Extracellular Signal-Regulated MAP Kinases; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; HEK293 Cells; Humans; Immunoblotting; Intracellular Space; Microscopy, Confocal; Mutation; Peptide Fragments; Peptides; Phosphorylation; Signal Transduction; Small Molecule Libraries; Venoms

Hindbrain glucagon-like peptide 1 (GLP-1) neurons project to numerous forebrain areas, including the lateral septum (LS). Using a fluorescently labeled GLP-1 receptor (GLP-1R) agonist, Exendin 4 (Ex4), we demonstrated GLP-1 receptor binding throughout the rat LS. We examined the feeding effects of Ex4 and the GLP-1R antagonist Exendin (9-39) (Ex9) at doses subthreshold for effect when delivered to the lateral ventricle. Intra-LS Ex4 suppressed overnight chow and high-fat diet (HFD) intake, and Ex9 increased chow and HFD intake relative to vehicle. During 2-h tests, intra-LS Ex9 significantly increased 0.25 M sucrose and 4% corn oil. Ex4 can cause nausea, but intra-LS administration of Ex4 did not induce pica. Furthermore, intra-LS Ex4 had no effect on anxiety-like behavior in the elevated plus maze. We investigated the role of LS GLP-1R in motivation for food by examining operant responding for sucrose on a progressive ratio (PR) schedule, with and without a nutrient preload to maximize GLP-1 neuron activation. The preload strongly suppressed PR responding, but blockade of GLP-1R in the intermediate subdivision of the LS did not affect motivation for sucrose under either load condition. The ability of the nutrient load to suppress subsequent chow intake was significantly attenuated by intermediate LS Ex9 treatment. By contrast, blockade of GLP-1R in the dorsal subdivision of the LS increased both PR responding and overnight chow intake. Together, these studies suggest that endogenous activity of GLP-1R in the LS influence feeding, and dLS GLP-1Rs, in particular, play a role in motivation.

Topics: Animals; Anxiety; Conditioning, Operant; Diet, High-Fat; Eating; Exenatide; Food; Glucagon-Like Peptide-1 Receptor; Injections, Intraventricular; Male; Motivation; Peptide Fragments; Peptides; Pica; Rats; Rats, Wistar; Septum of Brain; Venoms

Although the nature of the humoral factor which mediates cardioprotection established by remote ischaemic conditioning (RIc) remains unknown, parasympathetic (vagal) mechanisms appear to play a critical role. As the production and release of many gut hormones is modulated by the vagus nerve, here we tested the hypothesis that RIc cardioprotection is mediated by the actions of glucagon-like peptide-1 (GLP-1).. A rat model of myocardial infarction (coronary artery occlusion followed by reperfusion) was used. Remote ischaemic pre- (RIPre) or perconditioning (RIPer) was induced by 15 min occlusion of femoral arteries applied prior to or during the myocardial ischaemia. The degree of RIPre and RIPer cardioprotection was determined in conditions of cervical or subdiaphragmatic vagotomy, or following blockade of GLP-1 receptors (GLP-1R) using specific antagonist Exendin(9-39). Phosphorylation of PI3K/AKT and STAT3 was assessed. RIPre and RIPer reduced infarct size by ∼50%. In conditions of bilateral cervical or subdiaphragmatic vagotomy RIPer failed to establish cardioprotection. GLP-1R blockade abolished cardioprotection induced by either RIPre or RIPer. Exendin(9-39) also prevented RIPre-induced AKT phosphorylation. Cardioprotection induced by GLP-1R agonist Exendin-4 was preserved following cervical vagotomy, but was abolished in conditions of M3 muscarinic receptor blockade.. These data strongly suggest that GLP-1 functions as a humoral factor of remote ischaemic conditioning cardioprotection. This phenomenon requires intact vagal innervation of the visceral organs and recruitment of GLP-1R-mediated signalling. Cardioprotection induced by GLP-1R activation is mediated by a mechanism involving M3 muscarinic receptors.

Topics: Animals; Disease Models, Animal; Exenatide; Femoral Artery; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Hormone Antagonists; Ischemic Preconditioning; Ligation; Male; Muscarinic Antagonists; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peptide Fragments; Peptides; Phosphatidylinositol 3-Kinase; Phosphorylation; Proto-Oncogene Proteins c-akt; Rats, Sprague-Dawley; Receptor, Muscarinic M3; Signal Transduction; STAT3 Transcription Factor; Vagotomy; Vagus Nerve; Venoms

Experimental evidence suggested that Exendin-4 (Exe4), an agonist at glucagon like receptor-1 (GLP-1R), promoted tissue regeneration. We aimed to verify the effect of Exe4, in the absence or in the presence of Exendin-4(9-39), an antagonist at GLP-1R, on the healing of abraded skin. Two wounds (approximately 1.1×1.1 cm(2); namely "upper" and "lower" in respect of the head) were produced by abrasion on the back of 12 mice, which were then randomly assigned to receive an intradermal injection (20 μl) of Group 1: saline (NT) or Exe4 (62 ng) in the upper and lower wound respectively; Group 2: Exendin-4(9-39) (70 ng) in the upper and Exendin-4(9-39) (70 ng) and, after 15 min, Exe4 (62 ng) in the lower wound. Wounds were measured at the time of abrasion (T0) and 144 h (T3) afterward taking pictures with a ruler and by using a software. The inflammatory cell infiltrate, fibroblasts/myofibroblasts, endothelial cells and GLP-1R expression, were each labeled by immunofluorescence in each wound, pERK1/2 was evaluated by Western-blot in wound lysates. At T3, the percentage of healing surface was 53% and 92% for NT and Exe4 wounds respectively and 68% and 79% for those treated with Exendin-4(9-39) and Exendin-4(9-39)+Exe4 respectively. Exe4, but not Exendin-4(9-39) induced quantitative increase in fibroblasts/myofibroblasts and vessel density when compared to NT wounds. This increase was not evident in wounds treated with Exendin-4(9-39)+Exe4. Exe4 promotes wound healing opening to the possible dermatological use of this incretin analogue.

Topics: Animals; Cell Proliferation; Cell Transdifferentiation; Dermatologic Agents; Disease Models, Animal; Endothelial Cells; Enzyme Activation; Exenatide; Fibroblasts; Glucagon-Like Peptide-1 Receptor; Incretins; Male; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Myofibroblasts; Peptide Fragments; Peptides; Phosphorylation; Skin; Time Factors; Venoms; Wound Healing; Wounds and Injuries

To extend preliminary studies on the effects on food intake of the combined use of cannabinoid (CB) 1 and glucagon-like peptide-1 (GLP-1) receptor agonists and antagonists, the effect of these drugs on the feeding behavior in rats maintained on a free-choice, high-carbohydrate diet was investigated over a longer period of time. Rats were fed a standard diet for 3 days and then fed with both the standard and the high-sucrose chow. After 4 days of the high-calorie diet, the following combination treatments were administered daily by an intraperitoneal injection for the next 3 days: 1 mg/kg AM 251 (a CB1 receptor antagonist) or 1 mg/kg WIN 55,212-2 (a CB1 receptor agonist) together with 3 µg/kg exendin-4 (Ex-4, a GLP-1 receptor agonist) or 160 µg/kg exendin (9-39) [Ex (9-39), a GLP-1 receptor antagonist]. The total daily caloric intake and body weight were significantly reduced in rats treated with Ex-4 and AM 251 or WIN 55,212-2 compared with either of the drugs injected alone and the saline-injected controls. Both drug combinations selectively inhibited ingestion of the high-sucrose chow. Although Ex (9-39) administration did not significantly affect food consumption, it resulted in a marked body weight gain, indicating that the GLP-1 receptor antagonist caused a positive energy balance. It is concluded that AM 251 or WIN 55,212-2 and Ex-4, injected together, exert additive, inhibitory effects on the consumption of high-sugar food.

Topics: Analysis of Variance; Animals; Benzoxazines; Body Weight; Dietary Carbohydrates; Drug Interactions; Exenatide; Feeding Behavior; Glucagon-Like Peptide-1 Receptor; Male; Morpholines; Naphthalenes; Peptide Fragments; Peptides; Piperidines; Pyrazoles; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Glucagon; Venoms

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Preconditioning of exendin-4 (Ex), a glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial I/R injury. The current study investigated whether Ex postconditioning also attenuated myocardial I/R injury and the potential mechanisms. Anesthetized male rats were subjected to ischemia for 30 min and treated with Ex (5 μg/kg, i.v.) 5 min before reperfusion, in the absence and/or presence of exendin (9-39) (an antagonist of glucagon-like peptide-1 receptor, 5 μg/kg, i.v.), followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), tumor necrosis factor-α, interleukin-6, and infarct size were measured. HMGB1 expression was assessed by immunoblotting. Postconditioning with Ex significantly decreased infarct size and levels of LDH and CK after 4 h reperfusion (all p < 0.05). Ex also significantly inhibited the increase in malondialdehyde level and decreased the level of superoxide dismutase (both p < 0.05). In addition, the increase in HMGB1 expression induced by I/R was significantly attenuated by Ex postconditioning. Administration of exendin (9-39) abolished the protective effect of Ex postconditioning (all p < 0.05). The present study suggests that Ex postconditioning may attenuate myocardial I/R injury, which may in turn be associated with inhibiting inflammation.

Topics: Animals; Creatine Kinase; Exenatide; Gene Expression Regulation; Glucagon-Like Peptide-1 Receptor; HMGB1 Protein; Humans; Inflammation; Interleukin-6; L-Lactate Dehydrogenase; Myocardial Reperfusion Injury; Peptide Fragments; Peptides; Rats; Receptors, Glucagon; Tumor Necrosis Factor-alpha; Venoms

The use of glucagon-like peptide-1 (7-36) amide (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus is commonly associated with nausea and vomiting. Previous studies using Suncus murinus revealed that the GLP-1 receptor agonist, exendin-4, induces emesis via the brainstem and/or hypothalamus. The present study investigated the mechanism of exendin-4-induced emesis in more detail. Ondansetron (1 mg/kg, s.c.) and CP-99,994 (10 mg/kg, s.c) failed to reduce emesis induced by exendin-4 (3 nmol, i.c.v.), suggesting that 5-HT3 and NK1 receptors are not involved in the mechanism. In other studies, the GLP-1 receptor antagonist, exendin (9-39), antagonised emesis and c-Fos expression in the brainstem and the paraventricular hypothalamus induced by the chemotherapeutic drug cisplatin (30 mg/kg, i.p.; p < 0.05), but not the emesis induced by nicotine (5 mg/kg, s.c.; p > 0.05), or copper sulphate pentahydrate (120 mg/kg, p.o.; p > 0.05). GLP-1 receptors may therefore represent a potential target for drugs to prevent chemotherapy-induced emesis in situations where 5-HT3 and NK1 receptor antagonists fail.

Topics: Animals; Antiemetics; Brain; Exenatide; Glucagon-Like Peptide-1 Receptor; Male; Ondansetron; Peptide Fragments; Peptides; Piperidines; Proto-Oncogene Proteins c-fos; Receptors, Glucagon; Shrews; Venoms; Vomiting

We recently discovered that the activation of the spinal glucagon-like peptide-1 receptors (GLP-1Rs) by the peptidic agonist exenatide produced antinociception in chronic pain. We suggested that the spinal GLP-1Rs are a potential target molecule for the management of chronic pain. This study evaluated the antinociceptive activities of geniposide, a presumed small molecule GLP-1R agonist. Geniposide produced concentration-dependent, complete protection against hydrogen peroxide-induced oxidative damage in PC12 and HEK293 cells expressing rat and human GLP-1Rs, but not in HEK293T cells that do not express GLP-1Rs. The orthosteric GLP-1R antagonist exendin(9-39) right-shifted the concentration-response curve of geniposide without changing the maximal protection, with identical pA2 values in both cell lines. Subcutaneous and oral geniposide dose-dependently blocked the formalin-induced tonic response but not the acute flinching response. Subcutaneous and oral geniposide had maximum inhibition of 72% and 68%, and ED50s of 13.1 and 52.7 mg/kg, respectively. Seven days of multidaily subcutaneous geniposide and exenatide injections did not induce antinociceptive tolerance. Intrathecal geniposide induced dose-dependent antinociception, which was completely prevented by spinal exendin(9-39), siRNA/GLP-1R and cyclic AMP/PKA pathway inhibitors. The geniposide iridoid analogs geniposidic acid, genipin methyl ether, 1,10-anhydrogenipin, loganin and catalpol effectively inhibited hydrogen peroxide-induced oxidative damage and formalin pain in an exendin(9-39)-reversible manner. Our results suggest that geniposide and its iridoid analogs produce antinociception during persistent pain by activating the spinal GLP-1Rs and that the iridoids represented by geniposide are orthosteric agonists of GLP-1Rs that function similarly in humans and rats and presumably act at the same binding site as exendin(9-39).

Topics: Analgesics; Animals; Central Nervous System Agents; Exenatide; Formaldehyde; Glucagon-Like Peptide-1 Receptor; HEK293 Cells; Heterocyclic Compounds, 3-Ring; Humans; Iridoid Glucosides; Iridoids; Male; Mice; Nociception; PC12 Cells; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Spinal Cord; Venoms

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.

Topics: Animals; Enterocytes; Exenatide; Gene Expression Regulation; Glucagon-Like Peptide-1 Receptor; Ileum; Intestinal Mucosa; Mice; Paneth Cells; Peptide Fragments; Peptides; Proto-Oncogene Proteins c-fos; Receptors, Glucagon; Tissue Distribution; Venoms

Circadian clocks in peripheral tissues are powerfully entrained by feeding. The mechanisms underlying this food entrainment remain unclear, although various humoral and neural factors have been reported to affect peripheral clocks. Because glucagon-like peptide-1 (GLP-1), which is rapidly secreted in response to food ingestion, influences multiple humoral and neural signaling pathways, we suggest that GLP-1 plays a role in the food entrainment of peripheral clocks. To test this, we compared the effects of exendin-4, a GLP-1 receptor agonist, on mRNA expression of the clock genes (Clock, Bmal1, Nr1d1, Per1, Per2, and Cry1) with those of refeeding. In addition, we investigated whether exendin-4 could affect the rhythms of the peripheral clocks. In male C57BL/6J mice, although refeeding rapidly (within 2 h) altered mRNA levels of Per1 and Per2 in the liver and that of Per1 in adipose tissue, a single i.p. injection of exendin-4 did not cause such changes. However, unlike the GLP-1 receptor antagonist exendin-(9-39), exendin-4 significantly influenced Per1 mRNA levels in the liver at 12 h after injection. Moreover, pretreatment with exendin-4 affected the rapid-feeding-induced change in Per1 not only in the liver, but also in adipose tissue, without effect on food intake. Furthermore, during light-phase restricted feeding, repeated dosing of exendin-4 at the beginning of the dark phase profoundly influenced both the food intake and daily rhythms of clock gene expression in peripheral tissues. Thus, these results suggest that exendin-4 modulates peripheral clocks via multiple mechanisms different from those of refeeding.

Topics: Adipose Tissue; Animals; Circadian Clocks; Circadian Rhythm; CLOCK Proteins; Eating; Exenatide; Gene Expression Regulation; Glucagon-Like Peptide 1; Incretins; Liver; Male; Mice; Mice, Inbred C57BL; Peptide Fragments; Peptides; RNA, Messenger; Signal Transduction; Venoms

High mobility group box 1 protein (HMGB1) plays an important role in myocardial ischemia and reperfusion (I/R) injury. Exendin-4 (Ex-4), glucagon-like peptide-1 receptor agonist, has been reported to attenuate myocardial I/R injury. This study was to investigate the potential mechanism by which Ex-4 attenuates myocardial I/R injury in rats.. Anesthetized male rats were once treated with Ex-4 (5 μg/kg, i.v.) 1 h before ischemiain the absence and/or presence of exendin (9-39) (an antagonist for glucagon-like peptide-1 receptor, 5 μg/kg, i.v.), and then subjected to ischemia for 30 min followed by reperfusion for 4 h. Lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD) activity and infarct size were measured. HMGB1 expression was assessed by immunoblotting.. The results showed that pretreatment of Ex-4 could significantly decrease the infarct size and the levels of LDH and CK after 4 h reperfusion (all p < 0.05). Ex-4 could also significantly inhibit the increase of the MDA level, the decrease of the SOD level (both p < 0.05). Meanwhile, Ex-4 could significantly inhibit HMGB1 expression induced by I/R. Administration of exendin (9-39) could abolish the protective effect of Ex-4 (all p < 0.05).. The present study suggested that Ex-4 could attenuate myocardial I/R injury which may be associated with inhibiting HMGB1 expression.

Topics: Animals; Creatine Kinase; Cytoprotection; Disease Models, Animal; Down-Regulation; Exenatide; Glucagon-Like Peptide-1 Receptor; HMGB1 Protein; L-Lactate Dehydrogenase; Male; Malondialdehyde; Myocardial Infarction; Myocardial Reperfusion Injury; Myocardium; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Superoxide Dismutase; Time Factors; Venoms

Glucagon-like peptide-1 (GLP-1) and endocannabinoids are involved in appetite control. Recently we have demonstrated that cannabinoid (CB)1 receptor antagonist and GLP-1 receptor agonist synergistically suppress food intake in the rat. The aim of the present study was to determine the effects of GLP-1 receptor stimulation or blockade on feeding behavior in rats treated with WIN 55,212-2, a CB1 receptor agonist.. Experiments were performed on adult male Wistar rats. In the first experiment the effects of increasing doses (0.5-4.0 mg/kg) of WIN 55,212-2 injected intraperitoneally on 24-hour food consumption were tested. In further experiments a GLP-1 receptor antagonist, exendin (9-39), and WIN 55,212-2 or a GLP-1 receptor agonist, exendin-4, and WIN 55,212-2 were injected intraperitoneally at subthreshold doses (that alone did not change food intake and body weight) to investigate whether these agents may interact to affect food intake in rats.. WIN 55,212-2 administered at low doses (0.5-2 mg/kg) did not markedly change 24-hour food consumption; however, at the highest dose, daily food intake was inhibited. Combined administration of WIN 55,212-2 and exendin (9-39) did not change the amount of food consumed compared to either the control group or to each agent injected alone. Combined injection of WIN 55,212-2 and exendin-4 at subthreshold doses resulted in a significant decrease in food intake and body weight in rats.. Stimulation of the peripheral CB1 receptor by its agonist WIN 55,212-2 can induce anorexigenic effects or potentiate, even at a subthreshold dose, the effects of exendin-4, a known anorectic agent. Hence, this dual action of the cannabinoid system should be considered in the medical use of CB1 agonists.

Topics: Animals; Benzoxazines; Body Weight; Dose-Response Relationship, Drug; Exenatide; Feeding Behavior; Glucagon-Like Peptide-1 Receptor; Injections, Intraperitoneal; Male; Morpholines; Naphthalenes; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptor, Cannabinoid, CB1; Receptors, Glucagon; Venoms

Hyperglycemia-induced cardiomyocyte apoptosis contributes to diabetic cardiomyopathy. Glucagon-like peptide-1 (Glp1) receptor (Glp1r) agonists improve cardiac function and survival in response to ischemia-reperfusion and myocardial infarction. The present studies assessed whether Glp1r activation exerts direct cardioprotective effects in response to hyperglycemia. Treatment with the Glp1r agonist Exendin-4 attenuated apoptosis in neonatal rat ventricular cardiomyocytes cultured in high (33 mM) glucose. This protective effect was mimicked by the cAMP inducer forskolin. The Exendin-4 protective effect was blocked by the Glp1r antagonist Exendin(9-39) or the PKA antagonist H-89. Exendin-4 also protected cardiomyocytes from hydrogen peroxide (H2O2)-induced cell death. Cardiomyocyte protection by Exendin-4 was not due to reduced reactive oxygen species levels. Instead, Exendin-4 treatment reduced endoplasmic reticulum (ER) stress, demonstrated by decreased expression of glucose-regulated protein-78 (GRP78) and CCAT/enhancer-binding homologous protein (CHOP). Reduced ER stress was not due to activation of the unfolded protein response, indicating that Exendin-4 directly prevents ER stress. Exendin-4 treatment selectively protected cardiomyocytes from thapsigargin- but not tunicamycin-induced death. This suggests that Exendin-4 attenuates thapsigargin-mediated inhibition of the sarco/endoplasmic reticulum Ca(2+) ATPase-2a (SERCA2a). High glucose attenuates SERCA2a function by reducing SERCA2a mRNA and protein levels, but Exendin-4 treatment prevented this reduction. Exendin-4 treatment also enhanced phosphorylation of the SERCA2a regulator phospholamban (PLN), which would be expected to stimulate SERCA2a activity. In sum, Glp1r activation attenuates high glucose-induced cardiomyocyte apoptosis in association with decreased ER stress and markers of enhanced SERCA2a activity. These findings identify a novel mechanism whereby Glp1-based therapies could be used as treatments for diabetic cardiomyopathy.

Topics: Animals; Apoptosis; Calcium-Binding Proteins; Cells, Cultured; Colforsin; Diabetic Cardiomyopathies; Endoplasmic Reticulum Stress; Enzyme Activation; Exenatide; Glucagon-Like Peptide-1 Receptor; Glucose; Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Hydrogen Peroxide; Hyperglycemia; Hypoglycemic Agents; Isoquinolines; Membrane Proteins; Myocytes, Cardiac; Oxidative Stress; Peptide Fragments; Peptides; Phosphorylation; Protein Kinase Inhibitors; Rats; Receptors, Glucagon; RNA, Messenger; Sarcoplasmic Reticulum Calcium-Transporting ATPases; Sulfonamides; Thapsigargin; Transcription Factor CHOP; Tunicamycin; Unfolded Protein Response; Venoms

The use of glucagon-like peptide-1 (7-36) amide (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus is commonly associated with nausea and vomiting. Therefore, the present studies investigated the potential of GLP-1 receptor ligands to modulate emesis and feeding in Suncus murinus. Exendin-4, a selective GLP-1 receptor agonist, was administered subcutaneously (1-30 nmol/kg) or intracerebroventricularly (0.03-3 nmol) after 12-h of fasting. In other studies, animals were pretreated with the GLP-1 receptor antagonist, exendin (9-39), or saline (5 μl) 15 min prior to exendin-4 (3 nmol, i.c.v.). Behaviour of animals and food and water intake were then recorded for 1-2 h; c-Fos expression was also assessed in the brains of animals in the i.c.v. studies. The subcutaneous administration of exendin-4 reduced food and water intake (p < 0.001) and induced emesis in 40% of animals (p > 0.05). The intracerebroventricular administration of exendin-4 also prevented feeding, and induced emesis (p < 0.01). In these studies, exendin (9-39) (30 nmol, i.c.v.) antagonised emesis induced by exendin-4 and the increased c-Fos expressions in the brainstem and hypothalamus (p < 0.05), but it was ineffective in reversing the exendin-4-induced inhibition of food and water intake (p > 0.05). These data suggest that exendin-4 exerts its emetic effects in the brainstem and/or hypothalamus via GLP-1 receptors. The action of exendin-4 to suppress feeding may involve non-classical GLP-1 receptors or other mechanisms.

Topics: Animals; Appetite Depressants; Brain Stem; Dose-Response Relationship, Drug; Drinking; Drug Interactions; Eating; Emetics; Exenatide; Female; Glucagon-Like Peptide-1 Receptor; Hypothalamus; Injections, Intraventricular; Injections, Subcutaneous; Motor Activity; Peptide Fragments; Peptides; Proto-Oncogene Proteins c-fos; Receptors, Glucagon; Shrews; Venoms

Based on the hypothesis that MIN6 cells could produce glucagon-like peptide-1 (GLP-1) to maintain cell survival, we analyzed the effects of GLP-1 receptor agonist, exendin-4 (Ex4), and antagonist, exendin-(9-39) (Ex9) on cell function and cell differentiation. MIN6 cells expressed proglucagon mRNAs and produced GLP-1, which was accelerated by Ex4 and suppressed by Ex9. Moreover, Ex4 further enhanced glucose-stimulated GLP-1 secretion, suggesting autocrine loop-contributed amplification of the GLP-1 signal. Ex4 up-regulated cell differentiation- and cell function-related CREBBP, Pdx-1, Pax6, proglucagon, and PC1/3 gene expressions. The confocal laser scanning images revealed that GLP-1 positive cells were dominant in the early stage of cells, but positive for insulin were more prominent in the mature stage of cells. Ex4 accelerated cell viability, while Ex9 and anti-GLP-1 receptor antibody enhanced cell apoptosis. MIN6 cells possess a mechanism of GLP-1 signal amplification in an autocrine fashion, by which the cells maintained insulin production and cell survival.

Topics: Antibodies, Neutralizing; Apoptosis; Autocrine Communication; Cell Differentiation; Cell Line; Cell Survival; CREB-Binding Protein; Exenatide; Eye Proteins; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Homeodomain Proteins; Humans; Insulin; Insulin Secretion; Microscopy, Confocal; Paired Box Transcription Factors; PAX6 Transcription Factor; Peptide Fragments; Peptides; Proglucagon; Receptors, Glucagon; Repressor Proteins; RNA, Messenger; Signal Transduction; Trans-Activators; Venoms

Roux-en-Y gastric bypass (RYGB) surgery has been shown to decrease consummatory responsiveness of rats to high sucrose concentrations, and genetic deletion of glucagon-like peptide-1 receptors (GLP-1R) has been shown to decrease consummatory responsiveness of mice to low-sucrose concentrations. Here we assessed the effects of RYGB and pharmacological GLP-1R modulation on sucrose licking by chow-fed rats in a brief-access test that assessed consummatory and appetitive behaviors. Rats were tested while fasted presurgically and postsurgically and while nondeprived postsurgically and 5 h after intraperitoneal injections with the GLP-1R antagonist exendin-3(9-39) (30 μg/kg), agonist exendin-4 (1 μg/kg), and vehicle in 30-min sessions during which a sucrose concentration series (0.01-1.0 M) was presented in 10-s trials. Other rats were tested postsurgically or 15 min after peptide or vehicle injection while fasted and while nondeprived. Independent of food-deprivation state, sucrose experience, or GLP-1R modulation, RYGB rats took 1.5-3× as many trials as sham-operated rats, indicating increased appetitive behavior. Under nondeprived conditions, RYGB rats with presurgical sucrose experience licked more to sucrose relative to water compared with sham-operated rats. Exendin-4 and exendin-3(9-39) impacted 0.3 M sucrose intake in a one-bottle test, but never interacted with surgical group to affect brief-access responding. Unlike prior reports in both clearly obese and relatively leaner rats given RYGB and in GLP-1R knockout mice, we found that neither RYGB nor GLP-1R blockade decreased consummatory responsiveness to sucrose in our less obese chow-fed rats. Collectively, these results highlight the fact that changes in taste-driven motivated behavior to sucrose after RYGB and/or GLP-1R modulation are very model and measure dependent.

Topics: Animals; Appetite; Behavior, Animal; Dose-Response Relationship, Drug; Drinking; Eating; Exenatide; Gastric Bypass; Glucagon-Like Peptide-1 Receptor; Injections, Intraperitoneal; Male; Models, Animal; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Sucrose; Time Factors; Venoms

The study aimed to explore exenatide-induced damage of pancreatic tissue in rats.. At first stage, 30 male rats were randomly divided into exenatide and control groups. At second stage, 10 male and 10 female rats were treated according to sex, exenatide dose and time, and with or without inhibitor. Exenatide was injected subcutaneously twice a day, and body weights were measured once a week. At approximately 10 weeks, blood and pancreatic tissue samples were harvested. Amylase, lipase, interleukin 1, interleukin 6, and tumor necrosis factor α in serums were determined. Pancreatic tissues were divided for dry-wet ratio, myeloperoxidase, hematoxylin-eosin staining, and electric microscope imaging.. Compared with the control group, myeloperoxidase in pancreatic tissue of rats administered with exenatide exhibited a significantly high level; dry-wet ratio of pancreatic tissue in rats administered with exenatide exhibited a significantly low level. Chronic pancreatic damage was observed in 30% of rats from exenatide group for both sexes and showed pycnosis of acinar cells, increased cytoplasmic vacuoles, widened cellular gap, and inflammatory cell infiltration in pancreatic tissue. No pancreatic damage was observed in the control and the inhibitor groups. Histopathological evaluation scores in exenatide group were significantly higher than those of the control group.. Long-term administration of exenatide in rats can result in chronic pancreatic damage.

Topics: Amylases; Animals; Cytokines; Exenatide; Female; Hypoglycemic Agents; Lipase; Male; Pancreas; Pancreatitis, Chronic; Peptide Fragments; Peptides; Peroxidase; Rats; Rats, Sprague-Dawley; Severity of Illness Index; Venoms

Previous data suggests that the adiposity signal leptin reduces food intake in part by enhancing sensitivity to short-term signals that promote meal termination, including glucagon-like peptide 1 (GLP-1). We hypothesized that maintenance on a high-fat (HF) diet, which causes resistance to leptin, would impair GLP-1's ability to reduce food intake. To test this hypothesis, we examined the anorexic responses to intraperitoneal injection of 100 μg/kg GLP-1 and 1 μg/kg exendin-4 (Ex-4), the potent, degradation resistant GLP-1 receptor agonist, in Wistar rats maintained on a low-fat (10%; LF) or HF (60%) diet for 4-6 weeks. Rats maintained on each of these diets were tested twice, once while consuming LF food and once while consuming HF food, to distinguish between effects of acute vs. chronic consumption of HF food. LF-maintained rats tested on LF diet reduced 60-min dark phase intake in response to GLP-1, but HF-maintained rats failed to respond to GLP-1 whether they were tested on HF or LF diet. LF-maintained rats tested on HF diet also showed no response, suggesting that even brief exposure to HF diet can impair sensitivity to GLP-1 receptor activation. Both LF- and HF-maintained rats showed significant anorexic responses to Ex4 at 4h post-treatment, but only LF-maintained rats had significantly reduced intake and body weight 24h after injections. To determine whether the ability of endogenous GLP-1 to promote satiation is impaired by HF maintenance, we examined the response to exendin 3 (9-39) (Ex9), a GLP-1 receptor antagonist. In LF-maintained rats, Ex9 increased intake significantly, but HF-maintained rats reduced food intake in response to Ex9. These data support the suggestion that maintenance on HF diet reduces the anorexic effects of GLP-1 receptor activation, and this phenomenon may contribute to overconsumption of high-fat foods.

Topics: Animals; Appetite Depressants; Dietary Fats; Drug Interactions; Eating; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Male; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Satiation; Venoms

The long-acting glucagon-like peptide-1 receptor (GLP-1R) agonists, exendin-4 and liraglutide, suppress food intake and body weight. The mediating site(s) of action for the anorectic effects produced by peripheral administration of these GLP-1R agonists are not known. Experiments addressed whether food intake suppression after i.p. delivery of exendin-4 and liraglutide is mediated exclusively by peripheral GLP-1R or also involves direct central nervous system (CNS) GLP-1R activation. Results showed that CNS delivery [third intracerebroventricular (3(rd) ICV)] of the GLP-1R antagonist exendin-(9-39) (100 μg), attenuated the intake suppression by i.p. liraglutide (10 μg) and exendin-4 (3 μg), particularly at 6 h and 24 h. Control experiments show that these findings appear to be based neither on the GLP-1R antagonist acting as a nonspecific competing orexigenic signal nor on blockade of peripheral GLP-1R via efflux of exendin-(9-39) to the periphery. To assess the contribution of GLP-1R expressed on subdiaphragmatic vagal afferents to the anorectic effects of liraglutide and exendin-4, food intake was compared in rats with complete subdiaphragmatic vagal deafferentation and surgical controls after i.p. delivery of the agonists. Both liraglutide and exendin-4 suppressed food intake at 3 h, 6 h, and 24 h for controls; for subdiaphragmatic vagal deafferentation rats higher doses of the GLP-1R agonists were needed for significant food intake suppression, which was observed at 6 h and 24 h after liraglutide and at 24 h after exendin-4.. Food intake suppression after peripheral administration of exendin-4 and liraglutide is mediated by activation of GLP-1R expressed on vagal afferents as well as direct CNS GLP-1R activation.

Topics: Animals; Appetite Depressants; Body Weight; Brain; Calcitonin; Eating; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Liraglutide; Male; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Receptors, Glucagon; Vagus Nerve; Venoms

Glucagon-like peptide-1 (7-36) amide (GLP-1) is released from the gut as an incretin hormone to stimulate glucose-stimulated insulin secretion. GLP-1 is also produced in the central nervous system (CNS) as a neurotransmitter that regulates feeding behaviour. By using polyclonal antiserum against GLP-1 and GLP-1 receptors, we identified the distribution of GLP-1 immunoreactive fibres and GLP-1 receptor immunoreactivity in the ventromedial hypothalamus of Suncus murinus (house musk shrew). In functional studies, subcutaneous administration of exendin-4 (1 - 30 nmol/kg) reduced blood glucose levels dose-dependently by up to 49% during an intraperitoneal glucose tolerance test (P<0.001). The glucose-lowering effects were also observed after an intracerebroventricular (i.c.v.; 0.3 - 3 nmol) or intracerebral ventromedial hypothalamic microinfusion (iVMH; 0.3 - 3 pmol) of exendin-4. The area under the curve values for glucose after i.c.v. and iVMH administrations of exendin-4 were reduced by up to 53% (P<0.01) and 46% (P<0.01), respectively. Exendin-4 (i.c.v.; 3 nmol) also increased glucose-stimulated insulin secretion by 20% compared to controls (P<0.05). The GLP-1 receptor antagonist, exendin (9-39) (10 nmol, i.c.v.) did not modify blood glucose levels but it antagonized the glucose-lowering effect of exendin-4 (1 nmol, i.c.v.; P<0.05). The data suggests that the central GLP-1 system may regulate glucose homeostasis by increasing insulin secretion. Further, GLP-1 receptors in the ventromedial hypothalamus appear to play an important role in the regulation of glucose homeostasis in S. murinus.

Topics: Animals; Brain; Eating; Energy Metabolism; Exenatide; Female; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Glucose Tolerance Test; Homeostasis; Immunohistochemistry; Insulin; Insulin Secretion; Male; Neurons; Peptide Fragments; Peptides; Receptors, Glucagon; Shrews; Venoms; Ventromedial Hypothalamic Nucleus

The widely expressed dipeptidyl peptidase-4 enzyme rapidly cleaves the gut hormone glucagon-like peptide-1 [GLP-1(7-36)amide] at the N terminus to generate GLP-1(9-36)amide. Both intact GLP-1(7-36)amide and GLP-1(9-36)amide exert cardioprotective actions in rodent hearts; however, the mechanisms underlying the actions of GLP-1(9-36)amide remain poorly understood. We used mass spectrometry of coronary effluents to demonstrate that isolated mouse hearts rapidly convert infused GLP-1(7-36)amide to GLP-1(9-36)amide. After ischemia-reperfusion (I/R) injury of isolated mouse hearts, administration of GLP-1(9-36)amide or exendin-4 improved functional recovery and reduced infarct size. The direct actions of these peptides were studied in cultured neonatal mouse cardiomyocytes. Both GLP-1(9-36)amide and exendin-4 increased levels of cAMP and phosphorylation of ERK1/2 and the phosphoinositide 3-kinase target protein kinase B/Akt. In I/R injury models in vitro, both peptides improved mouse cardiomyocyte viability and reduced lactate dehydrogenase release and caspase-3 activation. These effects were attenuated by inhibitors of ERK1/2 and phosphoinositide 3-kinase. Unexpectedly, the cardioprotective actions of GLP-1(9-36)amide were blocked by exendin(9-39) yet preserved in Glp1r(-/-) cardiomyocytes. Furthermore, GLP-1(9-36)amide, but not exendin-4, improved the survival of human aortic endothelial cells undergoing I/R injury, actions sensitive to the nitric oxide synthase inhibitor, N(G)-nitro-l-arginine methyl ester (L-NAME). In summary, our findings demonstrate separate actions for GLP-1(9-36)amide vs. the GLP-1R agonist exendin-4 and reveal the existence of a GLP-1(9-36)amide-responsive, exendin(9-39)-sensitive, cardioprotective signaling pathway distinct from that associated with the classical GLP-1 receptor.

Topics: Analysis of Variance; Animals; Blotting, Western; Cells, Cultured; Cytoprotection; Dose-Response Relationship, Drug; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Heart; Male; Mass Spectrometry; Mice; Mice, Knockout; Myocardium; Peptide Fragments; Peptides; Receptors, Glucagon; Recovery of Function; Reperfusion Injury; Signal Transduction; Time Factors; Venoms

The aim of this project is to establish a GLP-1 signaling pathway targeted cell model, for screening the new class of GLP-1 receptor agonists as anti-diabetic candidates. Firstly construct a recombined plasmid with multi-copied specific response element (RIP-CRE) regulated by GLP-1 signaling pathway and E-GFP reporter gene. Transient transfect this recombined plasmid into islet cell NIT-1, then detect the responsibility of transfected cell to GLP-1 analogue, Exendin 4. For secondly, use stable transfection and monocloning cell culture to obtain a GLP-1 signaling-specific cell line. It indicates that this cell model can response to Exendin 4, which response can be completely inhibited by GLP-1 receptor antagonist, Exendin 9-39, further showing GLP-1 receptor specific activity with a cAMP-PKA-independently mechanism. Establishment of this novel cell model can be used in high-throughput drug screening of peptides or small molecular GLP-1 analogues.

Topics: Animals; Cell Line; Cyclic AMP Response Element Modulator; Cyclic AMP-Dependent Protein Kinases; Drug Delivery Systems; Drug Evaluation, Preclinical; Exenatide; Genes, Reporter; Glucagon-Like Peptide-1 Receptor; Green Fluorescent Proteins; Hypoglycemic Agents; Islets of Langerhans; Isoquinolines; Peptide Fragments; Peptides; Plasmids; Rats; Receptors, Glucagon; Recombinant Proteins; Signal Transduction; Sulfonamides; Transfection; Venoms

Exenatide is a long-acting glucagon-like peptide-1 (GLP-1) mimetic used in the treatment of type 2 diabetes. There is increasing evidence that GLP-1 can influence glycemia not only via pancreatic (insulinotropic and glucagon suppression) and gastric-emptying effects, but also via an independent mechanism mediated by portal vein receptors. The aim of our study was to investigate whether exenatide has an islet- and gastric-independent glycemia-reducing effect, similar to GLP-1. First, we administered mixed meals, with or without exenatide (20 microg sc) to dogs. Second, to determine whether exenatide-induced reduction in glycemia is independent of slower gastric emptying, in the same animals we infused glucose intraportally (to simulate meal test glucose appearance) with exenatide, exenatide + the intraportal GLP-1 receptor antagonist exendin-(9-39), or saline. Exenatide markedly decreased postprandial glucose: net 0- to 135-min area under the curve = +526 +/- 315 and -536 +/- 197 mg.dl(-1).min(-1) with saline and exenatide, respectively (P < 0.05). Importantly, the decrease in plasma glucose occurred without a corresponding increase in postprandial insulin but was accompanied by delayed gastric emptying and lower glucagon. Significantly lower glycemia was induced by intraportal glucose infusion with exenatide than with saline (92 +/- 1 vs. 97 +/- 1 mg/dl, P < 0.001) in the absence of hyperinsulinemia or glucagon suppression. The exenatide-induced lower glycemia was partly reversed by intraportal exendin-(9-39): 95 +/- 3 and 92 +/- 3 mg/dl with exenatide + antagonist and exenatide, respectively (P < 0.01). Our results suggest that, similar to GLP-1, exenatide lowers glycemia via a novel mechanism independent of islet hormones and slowing of gastric emptying. We hypothesize that receptors in the portal vein, via a neural mechanism, increase glucose clearance independent of islet hormones.

Topics: Animals; Blood Glucose; Dogs; Exenatide; Gastric Emptying; Glucagon; Glucagon-Like Peptide 1; Glucose; Hypoglycemic Agents; Injections, Subcutaneous; Insulin; Islets of Langerhans; Peptide Fragments; Peptides; Postprandial Period; Venoms

Glucagon-like peptide-1 (GLP-1) increases pancreatic insulin secretion via a direct action on pancreatic beta-cells. A high density of GLP-1-containing neurons and receptors is also present in brain stem vagal circuits; therefore, the aims of the present study were to investigate 1) whether identified pancreas-projecting neurons of the dorsal motor nucleus of the vagus (DMV) respond to exogenously applied GLP-1, 2) the mechanism(s) of action of GLP-1, and 3) whether the GLP-1-responsive neurons (putative modulators of endocrine secretion) could be distinguished from DMV neurons responsive to peptides that modulate pancreatic exocrine secretion, specifically pancreatic polypeptide (PP). Whole cell recordings were made from identified pancreas-projecting DMV neurons. Perfusion with GLP-1 induced a concentration-dependent depolarization in approximately 50% of pancreas-projecting DMV neurons. The GLP-1 effects were mimicked by exendin-4 and antagonized by exendin-(9-39). In approximately 60% of the responsive neurons, the GLP-1-induced depolarization was reduced by tetrodotoxin (1 microM), suggesting both pre- and postsynaptic sites of action. Indeed, the GLP-1 effects were mediated by actions on potassium currents, GABA-induced currents, or both. Importantly, neurons excited by GLP-1 were unresponsive to PP and vice versa. These data indicate that 1) GLP-1 may act on DMV neurons to control pancreatic endocrine secretion, 2) the effects of GLP-1 on pancreas-projecting DMV neurons are mediated both via a direct excitation of their membrane as well as via an effect on local circuits, and 3) the GLP-1-responsive neurons (i.e., putative endocrine secretion-controlling neurons) could be distinguished from neurons responsive to PP (i.e., putative exocrine secretion-controlling neurons).

Topics: Action Potentials; Anesthetics, Local; Animals; Autonomic Fibers, Preganglionic; Dose-Response Relationship, Drug; Exenatide; gamma-Aminobutyric Acid; Glucagon-Like Peptide 1; In Vitro Techniques; Insulin; Insulin Secretion; Motor Neurons; Pancreas; Pancreatic Polypeptide; Peptide Fragments; Peptides; Potassium; Rats; Rats, Sprague-Dawley; Synaptic Transmission; Tetrodotoxin; Vagus Nerve; Venoms

Glucagon-like peptide-1 (GLP-1) receptor agonists have been reported to modulate gastrointestinal motility but the mechanism is essentially unknown. In the present studies, we investigated the potency and mechanism of action of GLP-1 receptor ligands on the isolated ileum of Suncus murinus, an insectivore used in anti-emetic research. Ileal segments were mounted in organ baths containing Kreb's solution. Cumulative concentration-response curves to GLP-1 (7-36) amide (0.1-300 nM) and exendin-4 (0.1-100 nM) were constructed in the absence and presence of exendin (9-39) amide (0.3-3 nM). GLP-1 (7-36) amide and exendin-4 induced concentration-dependent contractions yielding pEC50 values of 8.4+/-0.2 and 8.4+/-0.4, respectively. Exendin (9-39) antagonized the action of both agonists in a non-competitive reversible manner, with apparent pKB values of 9.5 and 9.7, respectively. Tetrodotoxin (1 microM), atropine (1 microM) and hexamethonium (500 microM) were used to determine the contractile mechanism of action of exendin-4. Tetrodotoxin and atropine significantly antagonized (P<0.01) the contractile action of exendin-4 (10 nM); hexamethonium (500 microM) had no action. These studies suggest that GLP-1 receptor agonists contract the ileum indirectly via postganglionic enteric neurones and an involvement of muscarinic receptors. These studies provide information relevant to the use of this species to estimate the therapeutic indexes of GLP-1 receptor agonists.

Topics: Animals; Exenatide; Glucagon-Like Peptide 1; Ileum; In Vitro Techniques; Male; Muscle Contraction; Peptide Fragments; Peptides; Shrews; Venoms

A role of GLP-1 (glucagon-like peptide-1) in the recovery of the metabolic conditions of morbidly obese patients after bariatric surgery has been proposed. Exendin 4 (Ex-4) and exendin 9 (Ex-9) both have GLP-1-like effects upon glucose metabolism in human myocytes. We investigated in normal human adipocytes the effect of GLP-1, Ex-4 and Ex-9, compared with insulin upon the activity of PI3K, PKB, MAPKs and p70s6 kinases, and the participation of these enzymes in their action upon 2-deoxy-D-glucose transport by using potential inhibitors. The study was extended to morbidly obese patients. In normal subjects, GLP-1, Ex-4 and insulin, but not Ex-9, increased glucose uptake. In addition, GLP-1 and Ex-4 stimulated PI3K and MAPKs, similar to insulin, but not PKB. Ex-9 only enhanced PI3K, while none affected p70s6k. Inhibition of both PI3K and MAPKs blocked the stimulatory action of GLP-1, Ex-4 and insulin upon glucose transport. In obese patients, basal PI3K, PKB and MAPK activity was, as a rule, lower than that in normal subjects, while cells maintained their normal incremental response to GLP-1, Ex-4 or insulin; Ex-9 induced a clear stimulation of p42 MAPK. In summary, in normal human adipocytes, GLP-1 and Ex-4 have a protein kinase-dependent increasing effect upon glucose transport, which is impaired in obese patients. The participation of GLP-1 in the normalization of the metabolic conditions of the obese may occur through its effects on lipid metabolism or through effects upon glucose transport and/or metabolism in the liver and muscle, which in human obesity remain to be investigated.

Topics: Adipocytes; Adult; Biological Transport; Cells, Cultured; Deoxyglucose; Exenatide; Female; Glucagon-Like Peptide 1; Glucose; Humans; Male; Middle Aged; Obesity, Morbid; Peptide Fragments; Peptides; Phosphotransferases; Venoms

Glucagon-like peptide-1 (GLP-1) controls glucose metabolism in extrapancreatic tissues participating in glucose homeostasis, through receptors not associated to cAMP. In rat hepatocytes, activation of PI3K/PKB, PKC and PP-1 mediates the GLP-1-induced stimulation of glycogen synthase. We have investigated the effect of GLP-1 in normal human myocytes, and that of its structurally related peptides exendin-4 (Ex-4) and its truncated form 9-39 (Ex-9) upon glucose uptake, and the participation of cellular enzymes proposed to mediate insulin actions. GLP-1 and both exendins activated, like insulin, PI3K/PKB and p42/44 MAPK enzymes, but p70s6k was activated only by GLP-1 and insulin. GLP-1, Ex-4 and Ex-9, like insulin, stimulated glucose uptake; wortmannin blocked the action of GLP-1, insulin and Ex-9, and reduced that of Ex-4; PD98059 abolished the effect of all peptides/hormones, while rapamycin blocked that of insulin and partially prevented that of GLP-1. H-7 abolished the action of GLP-1, insulin and Ex-4, while Ro 31-8220 prevented only the Ex-4 and Ex-9 effect. In conclusion, GLP-1, like insulin, stimulates glucose uptake, and this involves activation of PI3K/PKB, p44/42 MAPKs, partially p70s6k, and possibly PKC; Ex-4 and Ex-9 both have GLP-1-like effect upon glucose transport, in which both share with GLP-1 an activation of PI3K/PKB--partially in the case of Ex-4--and p44/42 MAPKs but not p70s6k.

Topics: 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine; Aged; Aged, 80 and over; Androstadienes; Biological Transport; Cells, Cultured; Enzyme Activation; Exenatide; Female; Flavonoids; Glucagon; Glucagon-Like Peptide 1; Glucose; Humans; Indoles; Insulin; Male; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle Cells; Peptide Fragments; Peptides; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Protein Kinase C; Protein Precursors; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-akt; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Sirolimus; Venoms; Wortmannin

Several kinases have been implicated in the metabolic response of human and rat myocytes to glucagon-like peptide-1 (GLP-1), exendin-4 (Ex-4) and exendin-9 (Ex-9). We have investigated, in isolated rat adipocytes, the changes caused by GLP-1, Ex-4 and Ex-9 compared with those provoked by insulin or glucagon, upon the activity of phosphatidylinositol-3-kinase (PI3K), protein kinase B (PKB), p42/44 MAP kinases (MAPKs) and p70s6 kinase (p70s6k), and the participation of these kinases and protein kinase C (PKC) in their action upon 2-deoxy-d-glucose uptake, lipolysis and lipogenesis. The study was conducted in normal rats, and extended to a streptozotocin-induced type-2 diabetic model (STZ-rats). The participation of distinct kinases was estimated by using potential kinase inhibitors, including wortmannin, PD98059, rapamycin, H-7 and RO31-8220. In normal rat adipocytes, GLP-1 and both exendins share with insulin an increasing action upon the activity of all kinases studied (except PKB), PI3K, p44 and p42 MAPKs and possibly PKC, all being required for their stimulating effect upon glucose uptake. Ex-4 and Ex-9, like GLP-1 and insulin, have lipogenic action, while only Ex-4 shares with GLP-1 its lipolytic effect which is antagonized by Ex-9. MAP kinases and PKC seem to have an essential role in the GLP-1 and Ex-4 lipolytic action, as does PI3K in that of Ex-4. An increase in PI3K and MAPKs activity for the lipogenic effect of Ex-4, Ex-9 and GLP-1 are required, and in the case of Ex-4 and Ex-9, a stimulation of p70s6k activity is also needed. In cells from STZ-rats the magnitude of the above parameters was, in general, comparable to that in normal animals, with some exceptions: basal PI3K activity and lipogenesis were higher, GLP-1, Ex-4 and Ex-9 failed to modify basal lipogenesis but increased PKB activity, insulin failed to affect the activity of MAPKs and the insulin-induced glucose uptake was impaired. The impaired insulin effects upon some of the variables in the STZ-rat, distinct from those of GLP-1 and exendins, adds knowledge to the mechanism of the beneficial action of GLP-1 and Ex-4 in diabetic states.

Topics: Adipocytes; Animals; Biological Transport, Active; Deoxyglucose; Diabetes Mellitus, Experimental; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucose; Humans; In Vitro Techniques; Lipid Metabolism; Lipids; Lipolysis; Male; Peptide Fragments; Peptides; Protein Kinases; Protein Precursors; Rats; Rats, Wistar; Venoms

Changes in the activity of glycogen synthase a and related kinases (phosphatidylinositol-3-kinase, protein kinase B, p44/42 MAP kinases and p70s6 kinase) evoked by GLP-1 in human myocytes from normal subjects were recently implied in the effect of this hormone upon D-glucose transport and glycogen synthesis in the same cells. The major aims of the present study were i) to investigate the possible extension of this knowledge to myocytes obtained from type 2 diabetic patients, ii) to compare in these patients the response to GLP-1, insulin or the structurally related GLP-1 peptides, exendin (1-39)amide and exendin(9-39)amide, and iii) to explore possible differences in the responsiveness to these agents between normal and diabetic subjects. Apart from the much higher basal PI3K activity and impaired response to insulin of p44/42 MAP kinases in the diabetic patients, the changes in enzyme activity caused by either hormone or peptide, although not identical, were essentially comparable. Nevertheless, significant differences in glucose transport and metabolism parameters were observed in the diabetic patients vs. normal subjects: in the diabetic patients, basal 2-deoxy-glucose uptake and glycogen synthase a activity were lower, accompanied by a similar increasing effect of GLP-1 or insulin; yet, the basal value for glycogen synthesis was higher, coinciding with a lesser relative increment in response to GLP-1 or insulin.

Topics: Aged; Aged, 80 and over; Cells, Cultured; Deoxyglucose; Diabetes Mellitus, Type 2; Exenatide; Female; Glucose; Glycogen; Glycogen Synthase; Humans; Immunoblotting; Insulin; Male; Middle Aged; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Muscle Cells; Peptide Fragments; Peptides; Phosphatidylinositol 3-Kinases; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction; Venoms

Gut-derived peptides including ghrelin, cholecystokinin (CCK), peptide YY (PYY), glucagon-like peptide (GLP-1), and GLP-2 exert overlapping actions on energy homeostasis through defined G-protein-coupled receptors (GPCRs). The proglucagon-derived peptide (PGDP) oxyntomodulin (OXM) is cosecreted with GLP-1 and inhibits feeding in rodents and humans; however, a distinct receptor for OXM has not been identified.. We examined the mechanisms mediating oxyntomodulin action using stable cell lines expressing specific PGDP receptors in vitro and both wild-type and knockout mice in vivo.. OXM activates signaling pathways in cells through glucagon or GLP-1 receptors (GLP-1R) but transiently inhibits food intake in vivo exclusively through the GLP-1R. Both OXM and the GLP-1R agonist exendin-4 (Ex-4) activated neuronal c-fos expression in the paraventricular nucleus of the hypothalamus, the area postrema, and the nucleus of the solitary tract following intraperitoneal (i.p.) injection. However, OXM transiently inhibited food intake in wild-type mice following intracerebroventricular (i.c.v.) but not i.p. administration, whereas Ex-4 produced a more potent and sustained inhibition of food intake following both i.c.v. and i.p. administration. The anorectic effects of OXM were preserved in Gcgr(-/-) mice but abolished in GLP-1R(-/-) mice. Although central Ex-4 and OXM inhibited feeding via a GLP-1R-dependent mechanism, Ex-4 but not OXM reduced VO2 and respiratory quotient in wild-type mice.. These findings demonstrate that structurally distinct PGDPs differentially regulate food intake and energy expenditure by interacting with a GLP-1R-dependent pathway. Hence ligand-specific activation of a common GLP-1R increases the complexity of gut-central nervous system pathways regulating energy homeostasis and metabolic expenditure.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; Brain; Cells, Cultured; Cricetinae; Dose-Response Relationship, Drug; Eating; Energy Metabolism; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide 2; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Injections, Intraperitoneal; Injections, Intraventricular; Iodine Radioisotopes; Mice; Mice, Inbred C57BL; Mice, Knockout; Molecular Sequence Data; Oxyntomodulin; Peptide Fragments; Peptides; Proglucagon; Protein Precursors; Proto-Oncogene Proteins c-fos; Rats; Receptors, G-Protein-Coupled; Receptors, Glucagon; Venoms

Glucagon-like peptide 1 (GLP-1) is produced by neurons in the caudal brainstem that receive sensory information from the gut and project to several hypothalamic regions involved in arousal, interoceptive stress, and energy homeostasis. GLP-1 axons and receptors have been detected in the lateral hypothalamus, where hypocretin neurons are found. The electrophysiological actions of GLP-1 in the CNS have not been studied. Here, we explored the GLP-1 effects on GFP (green fluorescent protein)-expressing hypocretin neurons in mouse hypothalamic slices. GLP-1 receptor agonists depolarized hypocretin neurons and increased their spike frequency; the antagonist exendin (9-39) blocked this depolarization. Direct GLP-1 agonist actions on membrane potential were abolished by choline substitution for extracellular Na+, and dependent on intracellular GDP, suggesting that they were mediated by sodium-dependent conductances in a G-protein-dependent manner. In voltage clamp, the GLP-1 agonist Exn4 (exendin-4) induced an inward current that reversed near -28 mV and persisted in nominally Ca2+-free extracellular solution, consistent with a nonselective cationic conductance. GLP-1 decreased afterhyperpolarization currents. GLP-1 agonists enhanced the frequency of miniature and spontaneous EPSCs with no effect on their amplitude, suggesting presynaptic modulation of glutamate axons innervating hypocretin neurons. Paraventricular hypothalamic neurons were also directly excited by GLP-1 agonists. In contrast, GLP-1 agonists had no detectable effect on neurons that synthesize melanin-concentrating hormone (MCH). Together, our results show that GLP-1 agonists modulate the activity of hypocretin, but not MCH, neurons in the lateral hypothalamus, suggesting a role for GLP-1 in the excitation of the hypothalamic arousal system possibly initiated by activation by viscera sensory input.

Topics: Action Potentials; Afferent Pathways; Animals; Arousal; Choline; Digestive System; Eating; Exenatide; Genes, Reporter; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glutamic Acid; Hypothalamic Hormones; Hypothalamus; Intracellular Signaling Peptides and Proteins; Melanins; Mice; Mice, Transgenic; Neurons; Neuropeptides; Orexin Receptors; Orexins; Paraventricular Hypothalamic Nucleus; Patch-Clamp Techniques; Peptide Fragments; Peptides; Pituitary Hormones; Protein Precursors; Receptors, G-Protein-Coupled; Receptors, Glucagon; Receptors, Neuropeptide; Sodium Channel Blockers; Solitary Nucleus; Synaptic Transmission; Tetrodotoxin; Venoms; Viscera

The effects of the glucagon-like peptide 1 (GLP-1) receptor agonist exendin-4 (EX4) and antagonist EX4(9-39) EX4-A on entero-insular axis have been investigated in normoglycemic and streptozotocin (STZ)-induced diabetic rats. Rats were administered daily subcutaneous injections of 1 nmol/kg EX4 and/or EX4-A for 7 days, and were decapitated 3 h after the last injection. In STZ-untreated rats, EX4 reduced body-weight (BW) gain and raised glycemia, and the effects were prevented by EX4-A; conversely, EX4 did not alter plasma concentrations of insulin, glucagon and leptin. STZ-treated rats displayed body and hematochemical alterations typical of experimental diabetes: decrease in BW and insulin blood level, coupled with normal glucagon plasma concentration and marked hyperglycemia. In diabetic rats, both EX4 and EX4-A decreased BW gain, thereby suggesting a mechanism at least in part independent of GLP-1 receptors. EX4 did not alter glucagon blood level, but decreased glycemia and raised insulin and leptin plasma levels. These effects were annulled by EX4-A, which indicates that they occur through the activation of GLP-1 receptors. Collectively, our findings add support to the view that EX4 can be considered an important therapeutical tool to improve glucose metabolism in diabetes.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Exenatide; Female; Glucagon; Glucagon-Like Peptide-1 Receptor; Insulin; Insulin Secretion; Islets of Langerhans; Leptin; Liver Glycogen; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Venoms

Lysine-288 in the glucagon-like peptide-1 receptor was predicted to be ideally positioned to play a role in hormone binding. Subsequent mutation of Lys-288 to Ala or Leu greatly reduced hormone affinity, while substitution with Arg had minimal effect. Compared to wild type, the Lys288-Ala receptor had a reduced affinity for three peptide ligands with complete N-terminal sequences but not for their N-truncated analogues. Hence, the role of this positively charged residue, which is conserved at the equivalent position in all other Family B receptors, was determined to be important for receptor interaction with the N-terminal eight residues of peptide agonists.

Topics: Amino Acid Sequence; Amino Acid Substitution; Binding, Competitive; Cells, Cultured; Cyclic AMP; Exenatide; Glucagon-Like Peptide-1 Receptor; Humans; Inhibitory Concentration 50; Kidney; Ligands; Lysine; Molecular Sequence Data; Peptide Fragments; Peptides; Radioligand Assay; Receptors, Glucagon; Recombinant Proteins; Sequence Homology, Amino Acid; Transfection; Venoms

High concentrations of glucagon-like peptide-1 (7-36) amide (GLP-1) and its specific receptor (GLP-1R) have been found in the rat hypothalamus. In this study the actions of GLP-1 and its related peptides, exendin-4 (GLP-1R agonist), exendin (9-39) (GLP-1R antagonist) and GLP-1 (9-36) amide (the major GLP-1 metabolite) on levels of serotonin (5-HT), 5-hydroxyindolacetic acid (5-HIAA) and amino acids (Glu, Asp, Gln, Gly, Tyr, Trp, GABA) in the hypothalamus were investigated. Intracerebroventricular (ICV) injection of GLP-1 (4 nmol) produced a significant reduction in levels of 5-HT (54%) and all measured amino acids (34 to 56%) compared with saline injected controls, whereas exendin (9-39) (4 nmol) was ineffective. ICV injection of exendin-4 produced a significant reduction in the levels of 5-HT, 5-HIAA, Trp, Glu, and Tyr. ICV injection of GLP-1(9-36) amide showed a statistically significant increase in the level of 5-HT, 5-HIAA and all the amino acids tested in this study. Prior administration of exendin (9-39) or GLP-1 (9-36) amide blocked the effects of GLP-1 on the levels of 5-HT and the amino acids. These data are consistent with exendin-4 being a GLP-1R agonist and exendin (9-39) being a specific GLP-1R antagonist. GLP-1 (9-36) amide, a primary metabolite of GLP-1, appears to act as an endogenous antagonist at the GLP-1R.

Topics: Animals; Down-Regulation; Exenatide; Glucagon; Glucagon-Like Peptide 1; Hypothalamus; Injections, Intraventricular; Male; Neurons; Neurotransmitter Agents; Peptide Fragments; Peptides; Protein Precursors; Rats; Rats, Sprague-Dawley; Serotonin; Venoms

Glucagon-like peptide-1 (GLP-1) has been shown to have insulin-like effects upon the metabolism of glucose in rat liver, muscle and fat, and on that of lipids in rat and human adipocytes. These actions seem to be exerted through specific receptors which, unlike that of the pancreas, are not - at least in liver and muscle - cAMP-associated. Here we have investigated the effect, its characteristics, and possible second messengers of GLP-1 on the glucose metabolism of human skeletal muscle, in tissue strips and primary cultured myocytes. In muscle strips, GLP-1, like insulin, stimulated glycogen synthesis, glycogen synthase a activity, and glucose oxidation and utilization, and inhibited glycogen phosphorylase a activity, all of this at physiological concentrations of the peptide. In cultured myotubes, GLP-1 exerted, from 10(-13) mol/l, a dose-related increase of the D-[U-(14)C]glucose incorporation into glycogen, with the same potency as insulin, together with an activation of glycogen synthase a; the effect of 10(-11) mol/l GLP-1 on both parameters was additive to that induced by the equimolar amount of insulin. Synthase a was still activated in cells after 2 days of exposure to GLP-1, as compared with myotubes maintained in the absence of peptide. In human muscle cells, exendin-4 and its truncated form 9-39 amide (Ex-9) are both agonists of the GLP-1 effect on glycogen synthesis and synthase a activity; but while neither GLP-1 nor exendin-4 affected the cellular cAMP content after 5-min incubation in the absence of 3-isobutyl-1-methylxantine (IBMX), an increase was detected with Ex-9. GLP-1, exendin-4, Ex-9 and insulin all induced the prompt hydrolysis of glycosylphosphatidylinositols (GPIs). This work shows a potent stimulatory effect of GLP-1 on the glucose metabolism of human skeletal muscle, and supports the long-term therapeutic value of the peptide. Further evidence for a GLP-1 receptor in this tissue, different from that of the pancreas, is also illustrated, suggesting a role for an inositolphosphoglycan (IPG) as at least one of the possible second messengers of the GLP-1 action in human muscle.

Topics: Analysis of Variance; Cells, Cultured; Culture Techniques; Dose-Response Relationship, Drug; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucose; Glycogen Phosphorylase; Glycogen Synthase; Humans; Isoenzymes; Muscle, Skeletal; Myocardium; Peptide Fragments; Peptides; Protein Precursors; Receptors, Glucagon; Second Messenger Systems; Venoms

The hypothesis that peripheral glucagon-like peptide-1 (GLP-1) is a regulator of both food intake and macronutrient selection in rats was tested by administration of its antagonist, exendin 9-39, and its agonist, exendin 4. The effect of exendin 9-39 given intraperitoneally (i.p.) on food intake was measured after carbohydrate, protein or fat preloads, and on choice between a protein-free, high carbohydrate (CHO) diet and a high protein, low carbohydrate (PRO) diet. The effect of exendin 4 on selection between the CHO and PRO diets was also investigated. Exendin 9-39 significantly enhanced food intake suppression occurring after glucose, but not after corn oil or albumin preloads. In diet selection studies, exendin 9-39 selectively decreased intake of only the CHO diet. In contrast, exendin 4 decreased intake of only the PRO diet. Thus, we suggest that peripheral GLP-1 plays a role in the regulation of macronutrient selection as well as food intake in rats.

Topics: Animals; Appetite Regulation; Dietary Carbohydrates; Dietary Fats; Dietary Proteins; Energy Intake; Exenatide; Glucagon; Glucagon-Like Peptide-1 Receptor; Injections, Intraperitoneal; Male; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Time Factors; Venoms

Glucagon-like peptide-1 (7-36)amide (tGLP-1), a representative humoral incretin, released into the portal circulation in response to a meal ingestion, exerts insulinotropic action through binding to the tGLP-1 receptor known to be a single molecular form thus far. We previously reported that the hepatic vagal nerve is receptive to intraportal tGLP-1, but not to non-insulinotropic full-length GLP-1-(1-37), through a mechanism mediated by specific receptor to the hormone. In the present study, we aimed to examine how modification of the receptor function alters this neural reception of tGLP-1, by using the specific agonist, exendin-4, and the specific antagonist, exendin (9-39)amide, of the receptor at doses known to exert their effects on the insulinotropic action of tGLP-1. Intraportal injection of 0.2 or 4.0 pmol tGLP-1, a periphysiological and pharmacological dose, respectively, facilitated significantly the afferent impulse discharge rate of the hepatic vagus in anesthetized rats, as reported previously. However, unexpectedly, intraportal injection of exendin-4 at a dose of 0.2 or 4.0 pmol, or of even 40.0 pmol, did not facilitate the afferents at all. Moreover, intraportal injection of exendin (9-39)amide at 100 times or more molar dose to that of tGLP-1, either 5 min before or 10 min after injection of 0.2 or 4.0 pmol tGLP-1, failed to modify the tGLP-1-induced facilitation of the afferents. The present results suggest that the neural reception of tGLP-1 involves a receptor mechanism distinct from that in the well-known humoral insulinotropic action.

Topics: Animals; Electrophysiology; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Injections, Intravenous; Liver; Male; Neurons, Afferent; Pancreas; Peptide Fragments; Peptides; Portal Vein; Rats; Rats, Wistar; Receptors, Glucagon; Vagus Nerve; Venoms

Glucagon-like peptide-1-(7---36) amide (GLP-1) is a potent incretin hormone secreted from distal gut. It stimulates basal and glucose-induced insulin secretion and proinsulin gene expression. The present study tested the hypothesis that GLP-1 may modulate insulin receptor binding. RINm5F rat insulinoma cells were incubated with GLP-1 (0.01-100 nM) for different periods (1 min-24 h). Insulin receptor binding was assessed by competitive ligand binding studies. In addition, we investigated the effect of GLP-1 on insulin receptor binding on monocytes isolated from type 1 and type 2 diabetes patients and healthy volunteers. In RINm5F cells, GLP-1 increased the capacity and affinity of insulin binding in a time- and concentration-dependent manner. The GLP-1 receptor agonist exendin-4 showed similar effects, whereas the receptor antagonist exendin-(9---39) amide inhibited the GLP-1-induced increase in insulin receptor binding. The GLP-1 effect was potentiated by the adenylyl cyclase activator forskolin and the stable cAMP analog Sp-5, 6-dichloro-1-beta-D-ribofuranosyl-benzimidazole-3', 5'-monophosphorothioate but was antagonized by the intracellular Ca(2+) chelator 1,2-bis(0-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM. Glucagon, gastric inhibitory peptide (GIP), and GIP-(1---30) did not affect insulin binding. In isolated monocytes, 24 h incubation with 100 nM GLP-1 significantly (P<0.05) increased the diminished number of high-capacity/low-affinity insulin binding sites per cell in type 1 diabetics (9,000+/-3,200 vs. 18,500+/-3,600) and in type 2 diabetics (15,700+/-2,100 vs. 28,900+/-1,800) compared with nondiabetic control subjects (25,100+/-2,700 vs. 26,200+/-4,200). Based on our previous experiments in IEC-6 cells and IM-9 lymphoblasts indicating that the low-affinity/high-capacity insulin binding sites may be more specific for proinsulin (Jehle, PM, Fussgaenger RD, Angelus NK, Jungwirth RJ, Saile B, and Lutz MP. Am J Physiol Endocrinol Metab 276: E262-E268, 1999 and Jehle, PM, Lutz MP, and Fussgaenger RD. Diabetologia 39: 421-432, 1996), we further investigated the effect of GLP-1 on proinsulin binding in RINm5F cells and monocytes. In both cell types, GLP-1 induced a significant increase in proinsulin binding. We conclude that, in RINm5F cells and in isolated human monocytes, GLP-1 specifically increases the number of high-capacity insulin binding sites that may be functional proinsulin receptors.

Topics: Adult; Animals; Exenatide; Female; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Humans; Insulin; Insulinoma; Male; Monocytes; Pancreatic Neoplasms; Peptide Fragments; Peptides; Proinsulin; Protein Precursors; Rats; Receptors, Glucagon; Tumor Cells, Cultured; Venoms

To examine the influence of the putative satiety factor (GLP-1) on the hypothalamo-pituitary-gonadal axis, we used GT1-7 cells as a model of neuronal luteinizing hormone- releasing hormone (LHRH) release. GLP-1 caused a concentration-dependent increase in LHRH release from GT1-7 cells. Specific, saturable GLP-1 binding sites were demonstrated on these cells. The binding of [125I]GLP-1 was time-dependent and consistent with a single binding site (Kd = 0.07+/-0.016 nM; binding capacity = 160+/-11 fmol/mg protein). The specific GLP-1 receptor agonists, exendin-3 and exendin-4, also showed high affinity (Ki = 0.3+/-0.05 and 0.32+/-0.06 nM, respectively) as did the antagonist exendin-(9-39) (Ki = 0.98+/-0.24 nM). At concentrations that increased LHRH release, GLP-1 (0.5-10 nM) also caused an increase in intracellular cAMP in GT1-7 cells (10 nM GLP-1: 7.66+/-0.4 vs. control: 0.23+/-0.02 nmol/mg protein; P < 0.001). Intracerebroventricular injection of GLP-1 at a single concentration (10 microg) produced a prompt increase in the plasma luteinizing hormone concentration in male rats (GLP-1: 1.09+/-0.11 vs. saline: 0.69+/-0.06 ng/ml; P < 0.005). GLP-1 levels in the hypothalami of 48-h-fasted male rats showed a decrease, indicating a possible association of the satiety factor with the low luteinizing hormone levels in animals with a negative energy balance.

Topics: Animals; Calcium; Cyclic AMP; Cytoplasm; Dose-Response Relationship, Drug; Exenatide; Food Deprivation; Glucagon; Glucagon-Like Peptide 1; Gonadotropin-Releasing Hormone; Hypothalamus; Luteinizing Hormone; Male; Mice; Mice, Transgenic; Neurons; Peptide Fragments; Peptides; Protein Precursors; Rats; Rats, Wistar; Receptors, Cell Surface; Time Factors; Tumor Cells, Cultured; Venoms

The GLP-1 structurally related peptides exendin-4 and exendin(9-39)amide were found to act, in rat liver and skeletal muscle, as agonist and antagonist, respectively, of the GLP-1(7-36)amide effects on glucose metabolism. Thus, like GLP-1(7-36)amide, exendin-4 increased glycogen synthase a activity and glucose incorporation into glycogen in both tissues and also stimulated exogenous D-glucose utilization and oxidation in muscle. These effects of GLP-1(7-36)amide and exendin-4 were inhibited by exendin(9-39)amide. Our findings provide further support to the proposed use of GLP-1, or exendin-4, as a tool in the treatment of diabetes mellitus. Thus, in addition to the well-known insulinotropic action of the peptides, they act both in liver and in muscle in a manner most suitable for restoration of glucose homeostasis, with emphasis on their positive effects upon glycogen synthesis in the two tissues and on the stimulation of exogenous glucose catabolism in muscle.

Topics: Animals; Cyclic AMP; Energy Metabolism; Enzyme Activation; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Glucose; Glycogen; Glycogen Synthase; Insulin; Liver; Muscle, Skeletal; Peptide Fragments; Peptides; Phosphorylase a; Rats; Rats, Wistar; Venoms

GLP-1-(7-36)-amide and exendin-4-(1-39) are glucagon-like peptide-1 (GLP-1) receptor agonists, whereas exendin-(9-39) is the only known antagonist. To analyze the transition from agonist to antagonist and to identify the amino acid residues involved in ligand activation of the GLP-1 receptor, we used exendin analogs with successive N-terminal truncations. Chinese hamster ovary cells stably transfected with the rat GLP-1 receptor were assayed for changes in intracellular cAMP caused by the test peptides in the absence or presence of half-maximal stimulatory doses of GLP-1. N-terminal truncation of a single amino acid reduced the agonist activity of the exendin peptide, whereas N-terminal truncation of 3-7 amino acids produced antagonists that were 4-10-fold more potent than exendin-(9-39). N-terminal truncation of GLP-1 by 2 amino acids resulted in weak agonist activity, but an 8-amino acid N-terminal truncation inactivated the peptide. Binding studies performed using 125I-labeled GLP-1 confirmed that all bioactive peptides specifically displaced tracer with high potency. In a set of exendin/GLP-1 chimeric peptides, substitution of GLP-1 sequences into exendin-(3-39) produced loss of antagonist activity with conversion to a weak agonist. The results show that receptor binding and activation occur in separate domains of exendin, but they are more closely coupled in GLP-1.

Topics: Amino Acid Sequence; Animals; Binding, Competitive; CHO Cells; Cricetinae; Cyclic AMP; Exenatide; Gastrointestinal Hormones; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Molecular Sequence Data; Peptide Fragments; Peptides; Rats; Receptors, Glucagon; Recombinant Fusion Proteins; Sequence Deletion; Structure-Activity Relationship; Transfection; Venoms

Glucagon-like peptide-1 (GLP-1) and glucagon are peptide hormones involved in glucose homeostasis. The ligands are closely related (48% identical) and bind with different affinities to distinct, although highly homologous (47% identical), G protein coupled receptors on the surface of cells. By these criteria, glucagon and GLP-1 receptors can be considered receptor subtypes. A series of chimeric receptors in which 4-6 amino acids in the N-terminal extracellular domain of the human GLP-1 receptor were replaced with the analogous region of the human glucagon receptor were constructed and expressed in COS-7 cells. One of these chimeric receptors, C29-32 displays a 7 to 10-fold decrease in affinity for GLP-1 and the GLP-1 antagonist exendin 9-39 amide and a concomitant 7 to 9-fold increase in its affinity for glucagon. This change in affinity results in a 50-fold decrease in the selectivity of this receptor for GLP-1 over glucagon. Thus, the substitution of as few as four residues of the GLP-1 receptor profoundly affects its selectivity for the homologous peptide agonists GLP-1 and glucagon. These results suggest the extracellular N terminal domain of the GLP-1 receptor harbours molecular determinants for both agonist binding affinity and selectivity.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cell Line; Chlorocebus aethiops; Cyclic AMP; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Humans; Molecular Sequence Data; Mutagenesis, Site-Directed; Peptide Fragments; Peptides; Protein Precursors; Rats; Receptors, Glucagon; Recombinant Fusion Proteins; Venoms

This study was designed to determine the interactions of peptide exendin-(9-39) with the effect of glucagon-like peptide-1-(7-36) (GLP-1 (7-36)) amide and of exendin-4 on arterial blood pressure and heart rate in the rat. Both GLP-1 (7-36) amide and exendin-4 produced a dose-dependent increase in systolic, diastolic and mean arterial blood pressure, as well as in heart rate, although the effect of exendin-4 was more prolonged. These data indicate a longer functional half-life in vivo for exendin-4 as compared to GLP-1 (7-36) amide, which may have therapeutical applications. The antagonist effect of exendin-(9-39) on these cardiovascular parameters was also tested with 3000 ng of exendin-(9-39) intravenously administered 5 min before i.v. injection of 10 ng of either GLP-1 (7-36) amide or exendin-4. Under these experimental conditions the effect of the latter two peptides on arterial blood pressure and heart rate was blocked. By contrast, single administration of exendin-(9-39) did not modify cardiovascular parameters. These findings indicate that exendin-4 is an agonist and that exendin-(9-39) is an antagonist of the action of GLP-1 (7-36) amide on arterial blood pressure and heart rate. Therefore, the action of GLP-1 (7-36) amide on these parameters seems to be mediated through its own receptors.

Topics: Animals; Blood Pressure; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Heart Rate; Male; Peptide Fragments; Peptides; Rats; Rats, Sprague-Dawley; Venoms

Glucagon-like peptide 1 (7-37)/(7-36) amide (GLP-1) is derived from the intestinal proglucagon processing. It is considered an important insulin-releasing gut hormone. This study uses exendin (9-39) amide as a GLP-1 receptor antagonist to evaluate the contribution of GLP-1 to the incretin effect. Anesthetized rats were challenged by an intraduodenal glucose infusion to evaluate maximally occurring GLP-1 and gastric inhibitory polypeptide (GIP) plasma levels. Maximal immunoreactive (IR) GLP-1 plasma levels amounted to 10 pmol/l (IR-GIP 11 pmol/l). Exendin (9-39) amide abolished the insulin-stimulatory effect of 60 pmol of GLP-1 or of the GLP-1 agonist exendin-4 (0.5 nmol) injected as bolus, respectively. An intravenous bolus injection of 5.94 nmol of exendin (9-39) amide 3 min before enteral glucose infusion grossly reduced the total insulin secretory response (by 60%) and significantly increased circulating blood glucose levels (P < 0.05). In contrast, the GLP-1 antagonist left the insulin response after an intravenous glucose or glucose plus GIP (60 pmol) load unaltered. Our data support the concept that GLP-1 is an important incretin factor. Exendin (9-39) amide is a useful GLP-1 antagonist for in vivo studies.

Topics: Animals; Drug Interactions; Exenatide; Gastric Inhibitory Polypeptide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Glucose; Insulin; Male; Peptide Fragments; Peptides; Protein Precursors; Radioimmunoassay; Rats; Rats, Wistar; Receptors, Glucagon; Venoms

Exendin-4 is a novel peptide from Heloderma suspectum venom which is 53% homologous with glucagon-like peptide-1 GLP-1-(7-36)NH2, a stimulant of cAMP-dependent H+ production in rat parietal cells. It was the aim of the present study to determine whether this effect of GLP-1-(7-36)NH2 is shared by exendin-4, and whether the responses to either peptide are blocked by exendin-(9-39)NH2, a competitive specific exendin receptor antagonist. In enriched rat parietal cells H+ production was measured indirectly by [14C]aminopyrine accumulation. cAMP production was determined by radioimmunoassay. [125I]GLP-1-(7-36)NH2 was prepared using chloramine T followed by high pressure liquid chromatography (HPLC) purification. Exendin-4 (10(-12) - 10(-8) M) stimulated [14C]aminopyrine accumulation in a concentration-dependent manner (EC50 = 7.6 x 10(-11) M). At the maximally effective concentration (10(-9) M) exendin-4 was as effective as GLP-1-(7-36)NH2 reaching 70-80% of the response to 10(-4) M histamine. Likewise, exendin-4 (10(-11) - 10(-7) M) stimulated parietal cell cAMP production up to 2.8-fold. Maximal stimulation by exendin-4 of [14C]aminopyrine accumulation was not affected by ranitidine (10(-4) M), but was concentration-dependently reduced by exendin-(9-39)NH2 (10(-11) - 10(-7) M). At the maximal concentration, exendin-(9-39)NH2 completely abolished the responses to 10(-9) M exendin-4 and to 10(-9) M GLP-1-(7-36)NH2 while not altering stimulation by 10(-4) M histamine. Binding of [125I]GLP-1-(7-36)NH2 to enriched parietal cells was displaced by exendin-4 (Ki = 4.6 x 10(-10) M) as well as by exendin-(9-39)NH2 (Ki = 4.0 x 10(-9) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Aminopyrine; Animals; Binding, Competitive; Cell Membrane; Cross-Linking Reagents; Cyclic AMP; Exenatide; Female; Gastric Acid; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptides; Histamine; In Vitro Techniques; Lizards; Parietal Cells, Gastric; Peptide Fragments; Peptides; Rats; Rats, Wistar; Receptors, Glucagon; Venoms

Glucagon-like peptide-I (GLP-I) is a potent insulinotropic peptide that mediates its actions at pancreatic B-cells via specific receptors. In the present study we stably expressed the rat B-cell GLP-I receptor in CHO cells and studied binding characteristics and receptor activation utilizing the naturally occurring receptor agonist GLP-I(7-36)-amide (GLP-I), the proglucagon-derived GLP-I-related peptide oxyntomodulin, the GLP-I receptor agonist exendin-4, and the specific antagonist exendin(9-39). The potencies to displace [125I]GLP-I from the receptor were GLP-I > exendin-4 > exendin(9-39) > oxyntomodulin, and to displace [125I]exendin-4 GLP-I = exendin-4 > exendin(9-39) > oxyntomodulin. cAMP production was stimulated equally by GLP-I and exendin-4. Oxyntomodulin was less potent to stimulate cAMP generation. Exendin(9-39) blocked the stimulatory action of GLP-I and exendin-4 on cAMP production, but not that of oxyntomodulin. This study shows that GLP-I and exendin-4 are potent agonists at the transfected rat B-cell GLP-I receptor whereas oxyntomodulin is only a weak GLP-I receptor agonist. Furthermore, exendin(9-39) is a potent GLP-I receptor antagonist. This peptide is a valuable tool to further study the physiological actions of GLP-I.

Topics: Animals; CHO Cells; Cricetinae; Evaluation Studies as Topic; Exenatide; Glucagon; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Glucagon-Like Peptides; Lizards; Neurotransmitter Agents; Oxyntomodulin; Peptide Fragments; Peptides; Rats; Receptors, Cell Surface; Receptors, Glucagon; Venoms

A complementary DNA for a glucagon-like peptide-1 receptor was isolated from a human pancreatic islet cDNA library. The isolated clone encoded a protein with 90% identity to the rat receptor. In stably transfected fibroblasts, the receptor bound [125I]GLP-1 with high affinity (Kd = 0.5 nM) and was coupled to adenylate cyclase as detected by a GLP-1-dependent increase in cAMP production (EC50 = 93 pM). Two peptides from the venom of the lizard Heloderma suspectum, exendin-4 and exendin-(9-39), displayed similar ligand binding affinities to the human GLP-1 receptor. Whereas exendin-4 acted as an agonist of the receptor, inducing cAMP formation, exendin-(9-39) was an antagonist of the receptor, inhibiting GLP-1-induced cAMP production. Because GLP-1 has been proposed as a potential agent for treatment of NIDDM, our present data will contribute to the characterization of the receptor binding site and the development of new agonists of this receptor.

Topics: Amino Acid Sequence; Amino Acids; Base Sequence; Cloning, Molecular; Cyclic AMP; Diabetes Mellitus, Type 2; DNA; Exenatide; Gene Expression; Glucagon-Like Peptide-1 Receptor; Humans; Islets of Langerhans; Ligands; Molecular Sequence Data; Peptide Fragments; Peptides; Receptors, Cell Surface; Receptors, Glucagon; Venoms