Effects of a bitter substance, denatonium benzoate, on pancreatic hormone secretion.

There is increasing evidence linking bitter taste receptor (BTR) signaling to gut hormone secretion and glucose homeostasis. However, its effect on islet hormone secretion has been poorly characterized. This study investigated the effect of the bitter substance, denatonium benzoate (DB), on hormone secretion from mouse pancreatic islets and INS-1 832/13 cells. DB (0.5-1 mM) augmented insulin secretion at both 2.8 mM and 16.7 mM glucose. This effect was no longer present at 5 mM DB likely due to the greater levels of cellular apoptosis. DB-stimulated insulin secretion involved closure of the KATP channel, activation of T2R signaling in beta-cells, and intraislet glucagon-like peptide-1 (GLP-1) release. DB also enhanced glucagon and somatostatin secretion, but the underlying mechanism was less clear. Together, this study demonstrates that the bitter substance, DB, is a strong potentiator of islet hormone secretion independent of glucose. This observation highlights the potential for widespread off-target effects associated with the clinical use of bitter-tasting substances.NEW & NOTEWORTHY We show that the bitter substance, denatonium benzoate (DB), stimulates insulin, glucagon, somatostatin, and GLP-1 secretion from pancreatic islets, independent of glucose, and that DB augments insulin release via the KATP channel, bitter taste receptor signaling, and intraislet GLP-1 secretion. Exposure to a high dose of DB (5 mM) induces cellular apoptosis in pancreatic islets. Therefore, clinical use of bitter substances to improve glucose homeostasis may have unintended negative impacts beyond the gut.

Berberine Metabolites Stimulate GLP-1 Secretion by Alleviating Oxidative Stress and Mitochondrial Dysfunction.

Berberine (BBR) is a principal component of Rhizoma coptidis known for its therapeutic potential in treating diseases such as type 2 diabetes mellitus (T2DM) and obesity. Despite the trace levels of BBR in plasma, it's believed that its metabolites play a pivotal role in its biological activities. While BBR is recognized to promote GLP-1 production in intestinal L cells, the cytoprotective effects of its metabolites on these cells are yet to be explored. The present study investigates the effects of BBR metabolites on GLP-1 secretion and the underlying mechanisms. Our results revealed that, out of six BBR metabolites, berberrubine (BBB) and palmatine (PMT) significantly increased the production and glucose-stimulated secretion of GLP-1 in GLUTag cells. Notably, both BBB and PMT could facilitate GLP-1 and insulin secretion and enhance glucose tolerance in standard mice. Moreover, a single dose of PMT could markedly increase plasma GLP-1 and improve glucose tolerance in mice with obesity induced by a high-fat diet. In palmitic acid or TNF[Formula: see text]-treated GLUTag cells, BBB and PMT alleviated cell death, oxidative stress, and mitochondrial dysfunction. Furthermore, they could effectively reverse inflammation-induced inhibition of the Akt signaling pathway. In general, these insights suggest that the beneficial effects of orally administered BBR on GLP-1 secretion are largely attributed to the pharmacological activity of BBB and PMT by their above cytoprotective effects on L cells, which provide important ideas for stimulating GLP-1 secretion and the treatment of T2DM.

Elucidating the central anorexigenic mechanism of glucagon-like peptide 1 in Japanese quail (Coturnix japonica).

Glucagon-like peptide 1 (GLP-1) elicits a potent reduction in food intake, although the central mechanism mediating this appetite-suppressive effect is not fully understood in all species. To begin to elucidate the molecular mechanisms in quail, we administered GLP-1 via intracerebroventricular (ICV) injection to 7-day-old Japanese quail (Coturnix japonica) and determined effects on food and water intake, behavior, and brain nucleus activation. We observed a reduction in food and water intake, with the lowest effective dose being 0.01 nmol. Quail injected with GLP-1 displayed fewer steps, feeding pecks, exploratory pecks, and jumps, while time spent sitting increased. We quantified c-Fos immunoreactivity at 60 min post-injection in hypothalamic and brainstem nuclei that mediate food intake and determined that the hypothalamic paraventricular nucleus (PVN), and nucleus of the solitary tract and area postrema of the brainstem were activated in response to GLP-1. In conclusion, these results suggest that GLP-1 induces anorexigenic effects that are likely mediated at the level of the PVN and brainstem.

Administration of Exendin-4 but not CCK alters lick responses and trial initiation to sucrose and intralipid during brief-access tests.

Administration of cholecystokinin (CCK) or the glucagon-like peptide 1 (GLP-1) receptor agonist Exendin-4 (Ex-4) reduces food intake. Findings in the literature suggest CCK reduces intake primarily as a satiety signal whereas GLP-1 may play a role in both satiety and reward-related feeding signals. Compounds that humans describe as âsweetâ and âfattyâ are palatable yet are signaled via separate transduction pathways. Here, unconditioned lick responses to sucrose and intralipid were measured in a brief-access lick procedure in food-restricted male rats in response to i.p. administration of Ex-4 (3 h before test), CCK (30 min before test), or a combination of both. The current experimental design measures lick responses to water and varying concentrations of both sucrose (0.03, 0.1, and 0.5 M) and intralipid (0.2%, 2%, and 20%) during 10-s trials across a 30-min single test session. This design minimized postingestive influences. Compared with saline-injected controls, CCK (1.0, 3.0, or 6.0 µg/kg) did not change lick responses to sucrose or intralipid. Number of trials initiated and lick responses to both sucrose and intralipid were reduced in rats injected with 3.0 µg/kg, but not 1.0 µg/kg Ex-4. The supplement of CCK did not alter lick responses or trials initiated compared with Ex-4 administration alone. These findings support a role for GLP-1 but not CCK in the oral responsiveness to palatable stimuli. Furthermore, Ex-4-induced reductions were observed for both sucrose and intralipid, compounds representing âsweetâ and âfat,â respectively.

A novel DPP-4 inhibitor Gramcyclin A attenuates cognitive deficits in APP/PS1/tau triple transgenic mice via enhancing brain GLP-1-dependent glucose uptake.

Enhancing glucagon-like peptide 1 (GLP-1) signaling with a dipeptidyl peptidase IV (DPP-4) inhibitor might exert protective effects on Alzheimer's disease (AD). We found that intragastric administration of Gramcyclin A (10, 20 and 40 mg/kg), a novel DPP-4 inhibitor, for 3 months significantly reversed cognitive decline in APP/PS1/tau triple transgenic mice in a dose-dependent manner. Gramcyclin A treatment markedly reduced Aß plaques as well as the insoluble and soluble forms of Aß40 and Aß42 in the hippocampus of APP/PS1/tau mice. Treatment with Gramcyclin A remarkedly decreased the level of microglia and suppressed neuroinflammation in the hippocampus of APP/PS1/tau mice. Moreover, Gramcyclin A treatment could increase brain glucose uptake in APP/PS1/tau mice, as detected by 18-fluoro-2-deoxyglucose (18 F-FDG) micro-positron emission tomography (micro-PET) imaging. Furthermore, Gramcyclin A significantly increased expression of glucagon-like peptide-1 (GLP-1), GLP-1R, proliferator-activated receptor gamma coactivator (PGC)-1α and glucose transporter 4 (GLUT4), and inhibited insulin receptor (IRS)-1 phosphorylation and tau hyperphosphorylation in the hippocampus of APP/PS1/tau mice. Collectively, Gramcyclin A conferred protective effects against AD via enhancing brain GLP-1-dependent glucose uptake. The DPP-4 inhibitor Gramcyclin A might be a potential therapeutic drug for AD.

The glucose-dependent insulinotropic polypeptide (GIP) regulates body weight and food intake via CNS-GIPR signaling.

Uncertainty exists as to whether the glucose-dependent insulinotropic polypeptide receptor (GIPR) should be activated or inhibited for the treatment of obesity. Gipr was recently demonstrated in hypothalamic feeding centers, but the physiological relevance of CNS Gipr remains unknown. Here we show that HFD-fed CNS-Gipr KO mice and humanized (h)GIPR knockin mice with CNS-hGIPR deletion show decreased body weight and improved glucose metabolism. In DIO mice, acute central and peripheral administration of acyl-GIP increases cFos neuronal activity in hypothalamic feeding centers, and this coincides with decreased body weight and food intake and improved glucose handling. Chronic central and peripheral administration of acyl-GIP lowers body weight and food intake in wild-type mice, but shows blunted/absent efficacy in CNS-Gipr KO mice. Also, the superior metabolic effect of GLP-1/GIP co-agonism relative to GLP-1 is extinguished in CNS-Gipr KO mice. Our data hence establish a key role of CNS Gipr for control of energy metabolism.

Glucagon-Like Peptide-1 Analog Liraglutide Attenuates Pressure-Overload Induced Cardiac Hypertrophy and Apoptosis through Activating ATP Sensitive Potassium Channels.

PURPOSE: This study aimed to investigate whether inhibition of glucagon-like peptide-1 (GLP-1) on pressure overload induced cardiac hypertrophy and apoptosis is related to activation of ATP sensitive potassium (KATP) channels. METHODS: Male SD rats were randomly divided into five groups: sham, control (abdominal aortic constriction), GLP-1 analog liraglutide (0.3 mg/kg/twice day), KATP channel blocker glibenclamide (5 mg/kg/day), and liraglutide plus glibenclamide. RESULTS: Relative to the control on week 16, liraglutide upregulated protein and mRNA levels of KATP channel subunits Kir6.2/SUR2 and their expression in the myocardium, vascular smooth muscle, aortic endothelium, and cardiac microvasculature. Consistent with a reduction in aortic wall thickness (61.4 ± 7.6 vs. 75.0 ± 7.6 µm, p < 0.05), liraglutide enhanced maximal aortic endothelium-dependent relaxation in response to acetylcholine (71.9 ± 8.7 vs. 38.6 ± 4.8%, p < 0.05). Along with a reduction in heart to body weight ratio (2.6 ± 0.1 vs. 3.4 ± 0.4, mg/g, p < 0.05) by liraglutide, hypertrophied cardiomyocytes (371.0 ± 34.4 vs. 933.6 ± 156.6 µm2, p < 0.05) and apoptotic cells (17.5 ± 8.2 vs. 44.7 ± 7.9%, p < 0.05) were reduced. Expression of anti-apoptotic protein BCL-2 and contents of myocardial ATP were augmented, and expression of cleaved-caspase 3 and levels of serum Tn-I/-T were reduced. Echocardiography and hemodynamic measurement showed that cardiac systolic function was enhanced as evidenced by increased ejection fraction (88.4 ± 4.8 vs. 73.8 ± 5.1%, p < 0.05) and left ventricular systolic pressure (105.2 ± 10.8 vs. 82.7 ± 7.9 mmHg, p < 0.05), and diastolic function was preserved as shown by a reduction of ventricular end-diastolic pressure (-3.1 ± 2.9 vs. 6.7 ± 2.8 mmHg, p < 0.05). Furthermore, left ventricular internal diameter at end-diastole (5.8 ± 0.5 vs. 7.7 ± 0.6 mm, p < 0.05) and left ventricular internal diameter at end-systole (3.0 ± 0.6 vs. 4.7 ± 0.4 mm, p < 0.05) were improved. Dietary administration of glibenclamide alone did not alter all the parameters measured but significantly blocked liraglutide-exerted cardioprotection. CONCLUSION: Liraglutide ameliorates cardiac hypertrophy and apoptosis, potentially via activating KATP channel-mediated signaling pathway. These data suggest that liraglutide might be considered as an adjuvant therapy to treat patients with heart failure.

Inhibition of MGAT2 modulates fat-induced gut peptide release and fat intake in normal mice and ameliorates obesity and diabetes in ob/ob mice fed on a high-fat diet.

Monoacylglycerol O-acyltransferase 2 (MGAT2) is one of the key enzymes responsible for triglyceride (TG) re-synthesis in the small intestine. We have previously demonstrated that pharmacological inhibition of MGAT2 has beneficial effects on obesity and metabolic disorders in mice. Here, we further investigate the effects of MGAT2 inhibition on (a) fat-induced gut peptide release and fat intake in normal mice and (b) metabolic disorders in high-fat diet (HFD)-fed ob/ob mice, a model of severe obesity and type 2 diabetes mellitus, using an orally bioavailable MGAT2 inhibitor Compound B (CpdB). CpdB inhibited elevation of plasma TG in mice challenged with an oil-supplemented liquid meal. Oil challenge stimulated the secretion of two gut anorectic hormones (peptide tyrosine-tyrosine and glucagon-like peptide-1) into the bloodstream, and these responses were augmented in mice pretreated with CpdB. In a two-choice test using an HFD and a low-fat diet, CpdB selectively inhibited intake of the HFD in normal mice. Administration of CpdB to HFD-fed ob/ob mice for 5 weeks suppressed food intake and body weight gain and inhibited elevation of glycated hemoglobin. These results indicate that pharmacological MGAT2 inhibition modulates fat-induced gut peptide release and fat intake in normal mice and improves obesity and diabetes in HFD-fed ob/ob mice and thus may have potential for development into a treatment of obesity and its related metabolic diseases.

Screening GLP-1 Receptor Ligands from Natural Products in Herbs through High-Content Technique.

AIM AND OBJECTIVE: Screening of active components from a natural product, especially from a crude extract, is a great challenge. To avoid potential activity interference of the N-terminus modification in the most common constructs based on GCPRs labeled with GFP technology, a Cterminus tGFP-labeled hGLP-1 receptor containing recombinant cell line hGLP-1R-tGFP was constructed and tried to be used in the screening of natural products from Chinese herb. MATERIALS AND METHODS: The GLP1 receptor gene was amplified and the inserts pCMV6-AC-tGFP and tGFP were fused at the C-terminus of GLP1 receptor to construct a recombinant plasmid. The recombinant was transfected into U2OS cell and selected with antibiotics and flow cytometry. The constructed cell line was named as hGLP-1R-tGFP cell line. The expression levels of GLP-1R-tGFP protein were confirmed by western-blot. The fluorescence imaging of re-distribution from diffusing to aggregate spots inside the cells was quantitated and analyzed by High Content Screening (HCS) assay. Meanwhile, the specificity, stability and C-terminus function of hGLP-1R-tGFP cell line were characterized. In order to allow the recombinant cell line of hGLP-1R-tGFP to be suitable in highcontent system of Arrayscan-infinity-700 in screening mode, several conditions have also been optimized. In the end, a total of 100 crude extract samples provided by the Yunnan Institute of Materia Medica have been screened with this method. RESULTS: Upon the activation of GLP-1 receptors by Exendin 4, fluorescent patches appeared on the cell membrane and subsequently internalized to form fluorescent aggregates inside the cells under fluorescent microscopy examination. The agonistic activity, sensitivity and specificity of the formation of fluorescent aggregate spot in hGLP-1R-tGFP cells have been confirmed by the activation of GLP-1R using the GLP-1analogues. The agonistic effects of GLP-1 analogues are blocked by a GLP-1R antagonist, Exendin9-39. The downstream of GLP-1 pathway, the activation of adenylate cyclase and the raising of cellular cAMP levels, remained intact in these tGFP modified C-terminus GLP-1 receptor cells. Meanwhile, a total of 100 crude extract samples from Chinese herbs have been screened by this method to find new active ingredients. CONCLUSION: Combined with High Content Screening image and data automatic acquisition processing, a new screening assay based on a recombinant U2OS cell line which GFP labeled at the C terminus of GLP1 receptor has been developed. GLP-1R agonist activity in extracts of Astragalus propinquus and Panax notoginseng from Chinese herbs has been determined by this method.

Effects of CCK-8 and GLP-1 on fatty acid sensing and food intake regulation in trout.

We hypothesize that cholecystokinin (CCK) and glucagon-like peptide-1 (GLP-1) are involved in the modulation of metabolic regulation of food intake by fatty acids in fish. Therefore, we assessed in rainbow trout (Oncorhynchus mykiss) the effects of intracerebroventricular treatment with 1 ng/g of CCK-8 and with 2 ng/g of GLP-1 on food intake, expression of neuropeptides involved in food intake control and the activity of fatty acid-sensing systems in hypothalamus and hindbrain. Food intake decreased up to 24 h post-treatment to 49.8-72.3% and 3.1-17.8% for CCK-8 and GLP-1, respectively. These anorectic responses are associated with changes in fatty acid metabolism and an activation of fatty acid-sensing mechanisms in the hypothalamus and hindbrain. These changes occurred in parallel with those in the expression of anorexigenic and orexigenic peptides. Moreover, we observed that the activation of fatty acid sensing and the enhanced anorectic potential elicited by CCK-8 and GLP-1 treatments occurred in parallel with the activation of mTOR and FoxO1 and the inhibition of AMPKα, BSX and CREB. The results are discussed in the context of metabolic regulation of food intake in fish.

Primary study on the hypoglycemic mechanism of 5rolGLP-HV in STZ-induced type 2 diabetes mellitus mice.

5rolGLP-HV is a promising dual-function peptide for the treatment of diabetes and thrombosis simultaneously. For investigating the therapeutic mechanism of 5rolGLP-HV for type 2 diabetes mellitus (T2DM), STZ-induced diabetic mice were established and treated with 5rolGLP-HV. The results showed that daily water and food intake, blood glucose, serum and pancreatic insulin levels significantly decreased after 5rolGLP-HV treatment with various oral concentrations, and 16 mg/kg was the optimal dose for controlling diabetes. 5rolGLP-HV treatment decreased the MDA levels and the T-SOD activity in serum and pancreatic of diabetic mice (but not up to significant difference), and significantly increased the expression of signal pathways related genes of rolGLP-1, also the density of insulin expression and the numbers of apoptosis cells in islets of diabetic mice were significantly decreased in comparison to the negative diabetic mice. These effects above may be clarified the hypoglycemic mechanisms of 5rolGLP-HV, and 5rolGLP-HV may be as a potential drug for diabetes in future.

Lithocholic Acid-Based Peptide Delivery System for an Enhanced Pharmacological and Pharmacokinetic Profile of Xenopus GLP-1 Analogs.

GLP-1 analogs suffer from the main disadvantage of a short in vivo half-life. Lithocholic acid (LCA), one of the four main bile acids in the human body, possesses a high albumin binding rate. We therefore envisioned that a LCA-based peptide delivery system could extend the half-life of GLP-1 analogs by facilitating the noncovalent binding of peptides to human serum albumin. On the basis of our previously identified Xenopus GLP-1 analogs (1-3), a series of LCA-modified Xenopus GLP-1 conjugates were designed (4a-4r), and the bioactivity studies of these conjugates were performed to identify compounds with balanced in vitro receptor activation potency and plasma stability. 4c, 4i, and 4r were selected, and their LCA side chains were optimized to further increase their stability, affording 5a-5c. Compound 5b showed a more increased albumin affinity and prolonged in vitro stability than that of 4i and liraglutide. In db/ db mice, 5b exhibited comparable hypoglycemic and insulinotropic activity to liraglutide and semaglutide. Importantly, the enhanced albumin affinity of 5b resulted in a prolonged in vivo antidiabetic duration. Finally, chronic treatment investigations of 5b demonstrated the therapeutic effects of 5b on HbA1c, body weight, blood glucose, and pancreatic endocrine deficiencies on db/ db mice. Our studies revealed 5b as a promising antidiabetic candidate. Furthermore, our study suggests the derivatization of Xenopus GLP-1 analogs with LCA represents an effective strategy to develop potent long-acting GLP-1 receptor agonists for the treatment of type 2 diabetes.

GLP-1 receptor agonist liraglutide exerts central action to induce ß-cell proliferation through medulla to vagal pathway in mice.

Endogenous GLP-1 and GLP-1 receptor agonists (GLP-1RAs) regulate glucose metabolism via common and distinct mechanisms. Postprandial release of GLP-1 is modest and it is degraded by DPP-4 within 2 min, and hence it cannot enter the brain in substantial amount. In contrast, DPP-4-resistant GLP-1RAs are administered at 10 times higher concentration than endogenous GLP-1 level, which enables them to reach several brain regions including ARC and AP, the areas implicated in glucose metabolism. Hence, some of the effects of GLP-1RAs observed clinically and experimentally, including pancreatic ß-cell proliferation, are thought to involve the brain. However, the effects of centrally acting GLP-1/GLP-1RAs on glucose metabolism and underlying neural mechanism are unclear. This study aimed to establish the link of central GLP-1/GLP-1RA action to pancreatic ß-cell proliferation. Both subcutaneous (SC) and intracerebroventricular (ICV) injections of liraglutide increased the number of pancreatic ß-cells expressing Ki67 and PCNA, proliferation markers, in C57BL/6J mice. This effect was induced by single ICV administration of liraglutide at relatively low dose that was incapable of suppressing food intake. These SC and ICV liraglutide-induced effects were inhibited by 50% and 70%, respectively, by pretreatment with atropine, a muscarinic receptor blocker. ICV liraglutide induced c-Fos expression in the area postrema (AP), nucleus tractus solitaries (NTS), and dorsal motor nucleus of the vagus (DMX) of the brain stem. These results demonstrate that central action of liraglutide induces pancreatic ß-cell proliferation via the pathway involving the brain stem AP/NTS/DMX area and vagus nerve. This route is highly sensitive to GLP-1/GLP-1RA. Hence, this brain-pancreatic ß-cell pathway may operate in type 2 diabetic patients treated with GLP-RAs and serve to counteract the reduction of ß-cell mass.

GLP-1 increases Kiss-1 mRNA expression in kisspeptin-expressing neuronal cells.

Feeding-related metabolic factors exert regulatory influences on the hypothalamic-pituitary-gonadal axis. Glucagon-like peptide-1 (GLP-1) is an anorexigenic hormone synthesized from the ileum in response to food intake. The purpose of this study was to examine the direct effect of GLP-1 on hypothalamic kisspeptin and gonadotropin-releasing hormone (GnRH) expression using the rat clonal hypothalamic cell line rHypoE-8. GLP-1 significantly increased Kiss-1 mRNA expression in rHypoE-8 cells up to 1.94 ± 0.22-fold. This effect of GLP-1 on Kiss-1 gene expression was also observed in GT1-7 GnRH-producing neurons and in primary cultures of fetal rat brain. GLP-1 increased cAMP-mediated signaling, as determined by cAMP response element activity assays, but failed to activate extracellular signal-regulated kinase pathways. Another anorexigenic factor, leptin, similarly increased Kiss-1 mRNA levels up to 1.34 ± 0.08-fold in rHypoE-8 cells. However, combined treatment with GLP-1 and leptin failed to potentiate their individual effects on Kiss-1 mRNA expression. Gnrh mRNA expression was not significantly increased by GLP-1 stimulation in rHypoE-8, but kisspeptin significantly stimulated the expression of Gnrh mRNA in these cells. Our current observations suggest that the anorexigenic peptide GLP-1 directly regulates Kiss-1 mRNA expression in these hypothalamic cell lines and in neuronal cells of fetal rat brain and affects the expression of Gnrh mRNA.

Glucagon-like peptide-1 reduces pancreatic ß-cell mass through hypothalamic neural pathways in high-fat diet-induced obese rats.

We examined whether glucagon-like peptide-1 (GLP-1) affects ß-cell mass and proliferation through neural pathways, from hepatic afferent nerves to pancreatic efferent nerves via the central nervous system, in high-fat diet (HFD)-induced obese rats. The effects of chronic administration of GLP-1 (7-36) and liraglutide, a GLP-1 receptor agonist, on pancreatic morphological alterations, c-fos expression and brain-derived neurotrophic factor (BDNF) content in the hypothalamus, and glucose metabolism were investigated in HFD-induced obese rats that underwent hepatic afferent vagotomy (VgX) and/or pancreatic efferent sympathectomy (SpX). Chronic GLP-1 (7-36) administration to HFD-induced obese rats elevated c-fos expression and BDNF content in the hypothalamus, followed by a reduction in pancreatic ß-cell hyperplasia and insulin content, thus resulting in improved glucose tolerance. These responses were abolished by VgX and SpX. Moreover, administration of liraglutide similarly activated the hypothalamic neural pathways, thus resulting in a more profound amelioration of glucose tolerance than native GLP-1 (7-36). These data suggest that GLP-1 normalizes the obesity-induced compensatory increase in ß-cell mass and glucose intolerance through a neuronal relay system consisting of hepatic afferent nerves, the hypothalamus, and pancreatic efferent nerves.

Estradiol modulates the anorexic response to central glucagon-like peptide 1.

Estrogens suppress feeding in part by enhancing the response to satiation signals. Glucagon-like peptide 1 (GLP-1) acts on receptor populations both peripherally and centrally to affect food intake. We hypothesized that modulation of the central GLP-1 system is one of the mechanisms underlying the effects of estrogens on feeding. We assessed the anorexic effect of 0, 1, and 10µg doses of GLP-1 administered into the lateral ventricle of bilaterally ovariectomized (OVX) female rats on a cyclic regimen of either 2µg ß-estradiol-3-benzoate (EB) or oil vehicle 30min prior to dark onset on the day following hormone treatment. Central GLP-1 treatment significantly suppressed food intake in EB-treated rats at both doses compared to vehicle, whereas only the 10µg GLP-1 dose was effective in oil-treated rats. To follow up, we examined whether physiologic-dose cyclic estradiol treatment influences GLP-1-induced c-Fos in feeding-relevant brain areas of OVX females. GLP-1 significantly increased c-Fos expression in the area postrema (AP) and nucleus of the solitary tract (NTS), and the presence of estrogens may be required for this effect in the paraventricular nucleus of the hypothalamus (PVN). Together, these data suggest that modulation of the central GLP-1 system may be one of the mechanisms by which estrogens suppress food intake, and highlight the PVN as a region of interest for future investigation.

The Hypothalamic Glucagon-Like Peptide 1 Receptor Is Sufficient but Not Necessary for the Regulation of Energy Balance and Glucose Homeostasis in Mice.

Pharmacological activation of the hypothalamic glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) promotes weight loss and improves glucose tolerance. This demonstrates that the hypothalamic GLP-1R is sufficient but does not show whether it is necessary for the effects of exogenous GLP-1R agonists (GLP-1RA) or endogenous GLP-1 on these parameters. To address this, we crossed mice harboring floxed Glp1r alleles to mice expressing Nkx2.1-Cre to knock down Glp1r expression throughout the hypothalamus (GLP-1RKDΔNkx2.1cre). We also generated mice lacking Glp1r expression specifically in two GLP-1RA-responsive hypothalamic feeding nuclei/cell types, the paraventricular nucleus (GLP-1RKDΔSim1cre) and proopiomelanocortin neurons (GLP-1RKDΔPOMCcre). Chow-fed GLP-1RKDΔNkx2.1cre mice exhibited increased food intake and energy expenditure with no net effect on body weight. When fed a high-fat diet, these mice exhibited normal food intake but elevated energy expenditure, yielding reduced weight gain. None of these phenotypes were observed in GLP-1RKDΔSim1cre and GLP-1RKDΔPOMCcre mice. The acute anorectic and glucose tolerance effects of peripherally dosed GLP-1RA exendin-4 and liraglutide were preserved in all mouse lines. Chronic liraglutide treatment reduced body weight in chow-fed GLP-1RKDΔNkx2.1cre mice, but this effect was attenuated with high-fat diet feeding. In sum, classic homeostatic control regions are sufficient but not individually necessary for the effects of GLP-1RA on nutrient homeostasis.

A novel dual-glucagon-like peptide-1 and glucose-dependent insulinotropic polypeptide receptor agonist is neuroprotective in transient focal cerebral ischemia in the rat.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) receptor agonists have been shown to be neuroprotective in previous studies in animal models of Alzheimer's or Parkinson's disease. Recently, novel dual-GLP-1/GIP receptor agonists that activate both receptors (DA) were developed to treat diabetes. We tested the protective effects of a novel potent DA against middle cerebral artery occlusion injury in rats and compared it with a potent GLP-1 analog, Val(8)-GLP-1(glu-PAL). Animals were evaluated for neurologic deficit score, infarct volume, and immunohistochemical analyses of the brain at several time points after ischemia. The Val(8)-GLP-1(glu-PAL)-treated and DA-treated groups showed significantly reduced scores of neurological dysfunction, cerebral infarction size, and percentage of TUNEL-positive apoptotic neurons. Furthermore, the expression of the apoptosis marker Bax, the inflammation marker iNOS, and the survival marker Bcl-2 was significantly increased. The DA-treated group was better protected against neurodegeneration than the Val(8)-GLP-1(glu-PAL) group, and the scores of neurological dysfunction, cerebral infarction size, and expression of Bcl-2 were higher, whereas the percentage of TUNEL-positive neurons and the levels of Bax and iNOS were lower in the DA group. DA treatment reduced the infarct volume and improved the functional deficit. It also suppressed the inflammatory response and cell apoptosis after reperfusion. In conclusion, the novel GIP and GLP-1 dual-receptor agonist is more neuroprotective than a GLP-1 receptor agonist in key biomarkers of neuronal degeneration.

Acoustic Dispensing Preserves the Potency of Therapeutic Peptides throughout the Entire Drug Discovery Workflow.

Routine peptide structure-activity relationship screening requires the serial dilution of peptides to produce full concentration-response curves. Established tip-based protocols involve multiple tip changes and high exposure to plasticware. In the case of peptides, this becomes a challenge, since peptides can adsorb to plastic, resulting in an observed loss of potency. Various methods can be employed to prevent peptide loss during compound handling, such as the inclusion of bovine serum albumin or solvents in assay buffer and the siliconization of plasticware, yet protein binding remains unpredictable. The degree of variation by which peptides will adhere to plasticware can confuse results and cause inaccuracies in potency predictions. We evaluated acoustic noncontact methods for peptide serial dilution and compared it with traditional tip-based methods, on the effect on potency curves for glucagon-like peptide-1 and glucagon peptide analogues. The current study demonstrates the benefits of noncontact dispensing for high-density microplate assay preparation of peptides using nanoliter droplets across our entire drug discovery workflow, from in vitro high-throughput screening to drug exposure determinations from in vivo samples.

Does Glucagon-like Peptide-1 Ameliorate Oxidative Stress in Diabetes? Evidence Based on Experimental and Clinical Studies.

Glucagon-like peptide-1 (GLP-1) has shown to influence the oxidative stress status in a number of in vitro, in vivo and clinical studies. Well-known effects of GLP-1 including better glycemic control, decreased food intake, increased insulin release and increased insulin sensitivity may indirectly contribute to this phenomenon, but glucose-independent effects on ROS level, production and antioxidant capacity have been suggested to also play a role. The potential 'antioxidant' activity of GLP-1 along with other proposed glucose-independent modes of action related to ameliorating redox imbalance remains a controversial topic but could hold a therapeutic potential against micro- and macrovascular diabetic complications. This review discusses the presently available knowledge from experimental and clinical studies on the effects of GLP-1 on oxidative stress in diabetes and diabetes-related complications.