GLP-1 Receptor Knockout Mice Research Articles

Impaired brain glucose metabolism in glucagon-like peptide-1 receptor knockout mice

BackgroundQuantitative mapping of the brain’s metabolism is a critical tool in studying and diagnosing many conditions, from obesity to neurodegenerative diseases. In particular, noninvasive approaches are urgently required. Recently, there have been promising drug development approaches for the treatment of disorders related to glucose metabolism in the brain and, therefore, against obesity-associated diseases. One of the most important drug targets to emerge has been the Glucagon-like peptide-1 (GLP-1) and its receptor (GLP-1R). GLP and GLP-1R play an important role in regulating blood sugar and maintaining energy homeostasis. However, the macroscopic effects on brain metabolism and function due to the presence of GLP-1R are unclear.MethodsTo explore the physiological role of GLP-1R in mouse brain glucose metabolism, and its relationship to brain function, we used three methods. We used deuterium magnetic resonance spectroscopy (DMRS) to provide quantitative information about metabolic flux, fluorodeoxyglucose positron emission tomography (FDG-PET) to measure brain glucose metabolism, and resting state-functional MRI (rs-fMRI) to measure brain functional connectivity. We used these methods in both mice with complete GLP-1R knockout (GLP-1R KO) and wild-type C57BL/6N (WT) mice.ResultsThe metabolic rate of GLP-1R KO mice was significantly slower than that of WT mice (p = 0.0345, WT mice 0.02335 ± 0.057 mM/min, GLP-1R KO mice 0.01998 ± 0.07 mM/min). Quantification of the mean [18F]FDG signal in the whole brain also showed significantly reduced glucose uptake in GLP-1R KO mice versus control mice (p = 0.0314). Observing rs-fMRI, the functional brain connectivity in GLP-1R KO mice was significantly lower than that in the WT group (p = 0.0032 for gFCD, p = 0.0002 for whole-brain correlation, p < 0.0001 for ALFF).ConclusionsGLP-1R KO mice exhibit impaired brain glucose metabolism to high doses of exogenous glucose, and they also have reduced functional connectivity. This suggests that the GLP-1R KO mouse model may serve as a model for correlated metabolic and functional connectivity loss.

Consumption of carotenoid-rich Momordica cochinchinensis (Gac) aril improves glycemic control in type 2 diabetic mice partially through taste receptor type 1 mediated glucagon-like peptide 1 secretion.

Incretin-based therapies are widely used to improve glycemic control and β cell dysfunction in the treatment of type 2 diabetes. Momordica cochinchinensis (Gac fruit), a nutritious melon cultivated in many regions, has underexplored health benefits, particular its edible aril. This study comprehensively investigates the stimulatory effect of Gac aril on glucagon-like peptide 1 (GLP-1) secretion, identifies the responsible active constituents, and explores the underlying mechanisms related to its anti-diabetic effects. GLP-1-secreting STC-1 intestinal L cells were used to assess bioactivity and molecular mechanisms. Additionally, the in vivo anti-diabetic effects of Gac aril consumption were evaluated using type 2 diabetic mice induced by a high fat diet and streptozotocin injection, with or without GLP-1 receptor expression. The results demonstrated that Gac pulp and aril stimulated GLP-1 secretion, while Gac seeds did not. β-Carotene, a major constituent of Gac aril, was identified as the key mediator of GLP-1 secretion via sweet taste receptor-mediated signaling in STC-1 cells. Dietary intake of Gac aril significantly improved fasting blood glucose, glucose tolerance, insulin sensitivity, β-cell function, and hemoglobin A1c in type 2 diabetic mice. GLP-1 levels increased 2-fold, and decreased levels of ghrelin and adiponectin were restored. The anti-diabetic effects were partially diminished in GLP-1 receptor knockout mice, suggesting Gac aril's effects are mediated, in part, through GLP-1. In conclusion, Gac aril consumption may provide health benefits for managing type 2 diabetes, partially by enhancing endogenous GLP-1 levels.

SAT037 Glucagon-like Peptide-1 Receptor Knockout Mice Do Not Compensate For The Lack Of Incretin Action By Increasing Glucose-dependent Insulinotropic Polypeptide Secretion

Abstract Disclosure: K.D. Galsgaard: None. H. Kissow: None. M.M. Smits: None. J.J. Juul: None. Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), secreted from the intestinal L-cells and K-cells, respectively, both enhance glucose-stimulated insulin secretion. This enhancement is referred to as the incretin effect. In the pancreatic islets of GIP receptor knockout mice, GLP-1 sensitivity may be increased. Conversely, deletion of the GLP-1 receptor has been shown to be compensated for by increased GIP secretion and action. This suggests a compensatory adaptation to loss of incretin signaling, via increased action/secretion of the remaining incretin hormone. To investigate this further, we subjected GLP-1 receptor knockout (Glp-1r-/-) mice and their wild-type littermates (Glp-1r+/+ mice) to an oral glucose tolerance test (2 g/kg body weight of glucose via oral gavage; OGTT) with subsequent measurement of plasma GIP concentrations. In separate experiments we measured pancreatic insulin, glucagon, somatostatin, and total GLP-1 concentrations in Glp-1r-/- and Glp-1r+/+ mice, and total GLP-1 and GIP concentrations in the duodenum and distal ileum were also measured. During the OGTT, we found a similar GIP secretion (total area under the curve (tAUC0-30min)) in Glp-1r-/- and Glp-1r+/+ mice (15,432±2,148 vs. 13,683±1,316 pg/mL×min, P=0.5, n≥7). Blood glucose concentrations (tAUC0-30min) were increased in Glp-1r-/- mice during the OGTT (523.9±27.4 vs. 429.1±21.2 mmol/L×min, P=0.02, n≥6). Extractable pancreatic hormone concentrations were similar in Glp-1r+/+ and Glp-1r-/- mice; insulin (284.3±11.4 vs. 303.6±32.4 pmol/g tissue, P=0.6, n≥8); glucagon (203.6±24.3 vs. 270.8±36.9 pmol/g tissue, P=0.2, n≥8); somatostatin (299.9±94.97 vs. 321.4±78.79 pmol/g tissue, P=0.9, n≥9); and total GLP-1 (30.9±8.5 vs. 20.2±4.0 pmol/g tissue, P=0.2, n≤8). In the duodenum, extractable GIP (175.0±29.6 vs. 177.3±10.8 pmol/g tissue, P&amp;gt;0.9, n≥8) and GLP-1 (57.5±35.6 vs. 28.9±3.2 pmol/g tissue, P=0.4, n≥9). In the distal ileum, extractable GIP (2.2±0.5 vs. 2.1±0.4 pmol/g tissue, P=0.8, n≥9) and GLP-1 (266.6±57.1 vs. 236.0±43.1, P=0.7, n≥9) concentrations were also similar in Glp-1r+/+ and Glp-1r-/- mice. In conclusion, our results do not support the notion that Glp-1r-/- mice compensate for the lack of GLP-1 receptor signaling by increasing glucose stimulated GIP secretion or by increasing the intestinal GIP content. Presentation: Saturday, June 17, 2023

Incretins play an important role in FFA4/GPR120 regulation of glucose metabolism by GW-9508

AimsTo assess the role of GPR120 in glucose metabolism and incretin regulation from enteroendocrine L- and K-cells with determination of the cellular localisation of GPR120 in intestinal tissue and clonal Glucagon-Like Peptide-1 (GLP-1)/Gastric Inhibitory Polypeptide (GIP) cell lines. Main methodsAnti-hyperglycaemic, insulinotropic and incretin secreting properties of the GPR120 agonist, GW-9508 were explored in combination with oral and intraperitoneal glucose tolerance tests (GTT) in lean, diabetic and incretin receptor knockout mice. Cellular localisation of GPR120 was assessed by double immunofluorescence. Key findingsCompared to intraperitoneal injection, oral administration of GW-9508 (0.1 μmol/kg body weight) together with glucose reduced the glycaemic excursion by 22–31 % (p < 0.05-p < 0.01) and enhanced glucose-induced insulin release by 30 % (p < 0.01) in normal mice. In high fat fed diabetic mice, orally administered GW-9508 lowered plasma glucose by 17–27 % (p < 0.05-p < 0.01) and augmented insulin release by 22–39 % (p < 0.05-p < 0.001). GW-9508 had no effect on the responses of GLP-1 receptor knockout mice and GIP receptor knockout mice. Consistent with this, oral GW-9508 increased circulating total GLP-1 release by 39–44 % (p < 0.01) and total GIP by 37–47 % (p < 0.01-p < 0.001) after 15 and 30 min in lean NIH Swiss mice. Immunocytochemistry demonstrated GPR120 expression on mouse enteroendocrine L- and K-cells, GLUTag cells and pGIP/Neo STC-1 cells. SignificanceGPR120 is expressed on intestinal L- and K-cells and stimulates GLP-1/GIP secretory pathways involved in mediating enhanced insulin secretion and improved glucose tolerance, following oral GW-9508. These novel data strongly support the development of potent and selective GPR120 agonists as an effective therapeutic approach for diabetes.

Liraglutide abrogates nephrotoxic effects of chemotherapies

Acute kidney injury (AKI) is a common clinical complication. Cisplatin (Cis) is an effective chemotherapeutic drug; however, its acute nephrotoxicity often limits its application. The role of liraglutide (Lir), an agonist of the glucagon-like peptide-1 receptor (GLP-1R), has recently attracted increasing attention beyond glycemic regulation. This study showed that Lir significantly ameliorated Cis-induced kidney dysfunction and renal damage. However, this renoprotective effect was partially abolished in GLP-1R knockout (GLP-1R-/-) mice. Furthermore, we synthesized Lir metabolites, GLP-1 (9−37) and GLP-1 (28−37), and found that they also exerted reno-protective effects that were not impaired in GLP-1R-/- mice. We also demonstrated that Lir and its metabolites reduced cisplatin-induced apoptosis in human renal tubular epithelial cells (HK-2). After silencing GLP-1R expression in HK-2 cells with small interfering ribose nucleic acid (siRNA), the protective effect of Lir on HK-2 cells was inhibited, while the protective effects of GLP-1 (9−37) and GLP-1 (28−37) were not affected. Additionally, we demonstrated that Lir and its metabolites inhibited Cis-induced high-mobility group box 1 (HMGB1) nuclear-cytoplasmic translocation and release, and reduced inflammatory cytokines and HMGB1 receptor expression. The exogenous use of recombinant HMGB1 (rHMGB1) dramatically weakened the protective effects of Lir and its metabolites. In conclusion, our study shows that Lir significantly attenuated Cis-induced AKI through GLP-1R dependent and independent pathways, mediated by inhibiting nuclear-cytoplasmic translocation and release of HMGB1. Lir and its metabolites may be effective drugs for treating cisplatin-induced nephrotoxicity.

BI 456906: Discovery and preclinical pharmacology of a novel GCGR/GLP-1R dual agonist with robust anti-obesity efficacy

ObjectiveObesity and its associated comorbidities represent a global health challenge with a need for well-tolerated, effective, and mechanistically diverse pharmaceutical interventions. Oxyntomodulin is a gut peptide that activates the glucagon receptor (GCGR) and glucagon-like peptide-1 receptor (GLP-1R) and reduces bodyweight by increasing energy expenditure and reducing energy intake in humans. Here we describe the pharmacological profile of the novel glucagon receptor (GCGR)/GLP-1 receptor (GLP-1R) dual agonist BI 456906. MethodsBI 456906 was characterized using cell-based in vitro assays to determine functional agonism. In vivo pharmacological studies were performed using acute and subchronic dosing regimens to demonstrate target engagement for the GCGR and GLP-1R, and weight lowering efficacy. ResultsBI 456906 is a potent, acylated peptide containing a C18 fatty acid as a half-life extending principle to support once-weekly dosing in humans. Pharmacological doses of BI 456906 provided greater bodyweight reductions in mice compared with maximally effective doses of the GLP-1R agonist semaglutide. BI 456906’s superior efficacy is the consequence of increased energy expenditure and reduced food intake. Engagement of both receptors in vivo was demonstrated via glucose tolerance, food intake, and gastric emptying tests for the GLP-1R, and liver nicotinamide N-methyltransferase mRNA expression and circulating biomarkers (amino acids, fibroblast growth factor-21) for the GCGR. The dual activity of BI 456906 at the GLP-1R and GCGR was supported using GLP-1R knockout and transgenic reporter mice, and an ex vivo bioactivity assay. ConclusionsBI 456906 is a potent GCGR/GLP-1R dual agonist with robust anti-obesity efficacy achieved by increasing energy expenditure and decreasing food intake.

Tirzepatide suppresses palatable food intake by selectively reducing preference for fat in rodents.

To investigate the role of glucose-dependent insulinotropic polypeptide receptor (GIPR) agonists alone or combined with glucagon-like peptide-1 receptor (GLP-1R) agonists to regulate palatable food intake and the role of specific macronutrients in these preferences. To understand this regulation, we treated mice and rats on several choice diet paradigms of chow and a palatable food option with individual or dual GIPR and GLP-1R agonists. In mice, the dual agonist tirzepatide suppressed total caloric intake, while promoting the intake of chow over a high fat/sucrose diet. Surprisingly, GIPR agonism alone did not alter food choice. The food intake shift observed with tirzepatide in wild-type mice was completely absent in GLP-1R knockout mice, suggesting that GIPR signalling does not regulate food preference. Tirzepatide also selectively suppressed the intake of palatable food but not chow in a rat two-diet choice model. This suppression was specific to lipids, as GLP-1R agonist and dual agonist treatment in rats on a choice paradigm assessing individual palatable macronutrients robustly inhibited the intake of Crisco (lipid) without decreasing the intake of a sucrose (carbohydrate) solution. Decreasing preference for high-caloric, high-fat foods is a powerful action of GLP-1R and dual GIPR/GLP-1R agonist therapeutics, which may contribute to the weight loss success of these drugs.

The alpha-7 nicotinic acetylcholine receptor agonist GTS-21 engages the glucagon-like peptide-1 incretin hormone axis to lower levels of blood glucose in db/db mice.

To establish if alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a blood glucose-lowering action in db/db mice, and to test if this action requires coordinate α7nAChR and GLP-1 receptor (GLP-1R) stimulation by GTS-21 and endogenous GLP-1, respectively. Blood glucose levels were measured during an oral glucose tolerance test (OGTT) using db/db mice administered intraperitoneal GTS-21. Plasma GLP-1, peptide tyrosine tyrosine 1-36(PYY1-36), glucose-dependent insulinotropic peptide (GIP), glucagon, and insulin levels were measured by ELISA. A GLP-1R-mediated action of GTS-21 that is secondary to α7nAChR stimulation was evaluated using α7nAChR and GLP-1R knockout (KO) mice, or by co-administration of GTS-21 with the dipeptidyl peptidase-4 inhibitor, sitagliptin, or the GLP-1R antagonist, exendin (9-39). Insulin sensitivity was assessed in an insulin tolerance test. Single or multiple dose GTS-21 (0.5-8.0mg/kg) acted in a dose-dependent manner to lower levels of blood glucose in the OGTT using 10-14 week-old male and female db/db mice. This action of GTS-21 was reproduced by the α7nAChR agonist, PNU-282987, was enhanced by sitagliptin, was counteracted by exendin (9-39), and was absent in α7nAChR and GLP-1R KO mice. Plasma GLP-1, PYY1-36, GIP, glucagon, and insulin levels increased in response to GTS-21, but insulin sensitivity, body weight, and food intake were unchanged. α7nAChR agonists improve oral glucose tolerance in db/db mice. This action is contingent to coordinate α7nAChR and GLP-1R stimulation. Thus α7nAChR agonists administered in combination with sitagliptin might serve as a new treatment for type 2 diabetes.

Liraglutide Attenuates Hepatic Ischemia-Reperfusion Injury by Modulating Macrophage Polarization.

Ischemia-reperfusion injury (IRI) is a common complication associated with liver surgery, and macrophages play an important role in hepatic IRI. Liraglutide, a glucagon-like peptide-1 (GLP-1) analog primarily used to treat type 2 diabetes and obesity, regulates intracellular calcium homeostasis and protects the cardiomyocytes from injury; however, its role in hepatic IRI is not yet fully understood. This study aimed to investigate whether liraglutide can protect the liver from IRI and determine the possible underlying mechanisms. Our results showed that liraglutide pretreatment significantly alleviated the liver damage caused by ischemia-reperfusion (I/R), as evidenced by H&E staining, serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, and TUNEL staining. Furthermore, the levels of inflammatory cytokines elicited by I/R were distinctly suppressed by liraglutide pretreatment, accompanied by significant reduction in TNF-α, IL-1β, and IL-6 levels. Furthermore, pretreatment with liraglutide markedly inhibited macrophage type I (M1) polarization during hepatic IRI, as revealed by the significant reduction in CD68+ levels in Kupffer cells (KCs) detected via flow cytometry. However, the protective effects of liraglutide on hepatic IRI were partly diminished in GLP-1 receptor-knockout (GLP-1R-/-) mice. Furthermore, in an in vitro study, we assessed the role of liraglutide in macrophage polarization by examining the expression profiles of M1 in bone marrow-derived macrophages (BMDMs) from GLP-1R-/- and C57BL/6J mice. Consistent with the results of the in vivo study, liraglutide treatment attenuated the LPS-induced M1 polarization and reduced the expression of M1 markers. However, the inhibitory effect of liraglutide on LPS-induced M1 polarization was largely abolished in BMDMs from GLP-1R-/- mice. Collectively, our study indicates that liraglutide can ameliorate hepatic IRI by inhibiting macrophage polarization towards an inflammatory phenotype via GLP-1R. Its protective effect against liver IRI suggests that liraglutide may serve as a potential drug for the clinical treatment of liver IRI.

Glucagon-Like Peptide-1 Receptor Regulates Macrophage Migration in Monosodium Urate-Induced Peritoneal Inflammation.

Glucagon-like peptide-1 (GLP-1) is an insulinotropic peptide that signals through the GLP-1 receptor (GLP-1R). GLP-1R, therefore, plays a critical role in diabetes and cardiovascular disease. Whether GLP-1R is involved in inflammatory disease such as gout remains unclear. Macrophages are critical effector cells in the pathogenesis of gout, a common form of inflammatory arthritis caused by the deposition of uric acid in joints. The expression of GLP-1R at the protein level is controversial due to the lack of specificity of existing antibodies against GLP-1R. Using a transgenic mouse model expressing enhanced green fluorescent protein (EGFP) under the control of GLP-1R promoter, here we confirmed the expression of GLP-1R by macrophages. M2 type macrophages and Ly6C+ macrophages expressed higher levels of GLP-1R, compared to their counterparts. GLP-1R deficient macrophages displayed a reduced the migratory ability and an enhanced expression of interleukin (IL)-6, while the expression of IL-1β was not affected. In monosodium urate (MSU) crystal-induced peritonitis, an experimental model of gout, the recruitment of macrophages, especially M2 macrophages, was significantly suppressed in GLP-1R knockout mice compared to wild-type mice. In conclusion, our data suggests that GLP-1R plays a critical role in macrophage migration in MSU-induced inflammation.

Dietary supplementation of rutin and rutin-rich buckwheat elevates endogenous glucagon-like peptide 1 levels to facilitate glycemic control in type 2 diabetic mice

Rutin and rutin-rich buckwheat are commonly used as alternative medicines due to their wide range of health benefits. The present study aims to investigate their anti-diabetes effects and the association with glucagon-like peptide 1 (GLP-1) levels. A murine model of type 2 diabetes (T2D), induced by a high-fat diet combined with streptozocin injection, was utilized. T2D mice were fed 3% buckwheat, 10% buckwheat, or 0.1% rutin as part of their diet for 4 weeks. Dipeptidyl peptidase-4 assays, secretin tumor cell line (STC-1) cells, and GLP-1 receptor-knockout mice were employed for mechanistic studies. Dietary consumption of 10% buckwheat and 0.1% rutin provided beneficial outcomes including lowering blood glucose levels and HbA1c, enhancing HOMA-B%, improving glucose tolerance and pyruvate tolerance. Serum GLP-1, ghrelin, leptin, and adiponectin levels were also increased at the end of the treatment. Increased GLP-1 levels and the improvement of β-cell function were closely associated with the blood glucose-lowering effects observed. Hypoglycemic effects of buckwheat and rutin supplement were abolished in diabetic GLP-1 receptor knockout mice. In conclusion, rutin and related herbal materials are expected to provide health benefits due to their ability to modulate endogenous GLP-1 levels in T2D mice.

Glucagon-like peptide-1 and beta cell glucose sensitivity - a glucose ramp study in mice

The incretin glucagon-like peptide-1 (GLP-1) is a gut hormone but also locally produced in pancreatic islets. We evaluated effects of GLP-1 on the insulin response to a gradual increase in glucose in mice within physiological levels. We initially developed a glucose ramp technique in mice. Glucose levels were slowly increased by 0.2 mmol/l/min for 40 min under control conditions, during intravenous infusion of GLP-1 and in GLP-1 receptor knockout mice. In control mice, glucose levels increased from 8.5 ± 0.3 to 16.1 ± 0.3 mmol/l over the 40 min, i.e., by 0.22 ± 0.01 mmol/l/min. This resulted in a slow increase in insulin levels by 96 ± 38 pmol/l from the baseline of 319 ± 53 pmol/l. GLP-1 at 0.5 nmol/kg as bolus plus 0.3 nmol/kg/min over 40 min progressively increased this insulin response by 100-fold, to 9.5 ± 0.2 nmol/l (P < 0.001). Higher doses of GLP-1 enhanced the insulin response similarly (1.0 or 3.0 nmol/kg bolus followed by 0.4 or 1.2 nmol/kg/min), whereas a lower dose (0.3 nmol/kg bolus plus 0.15 nmol/kg/min) had no significant effect compared to controls. Moreover, there was no significant difference in insulin responses between controls and GLP-1 receptor knockout mice. Since the increase in glucose levels were standardized, there was no significant difference in glucose levels between the experimental groups. We conclude that the glucose ramp technique is a tool for studies on insulin responses to slow changes in circulating glucose levels in mice. We also conclude that GLP-1 is extraordinarily potent in enhancing the insulin response to a slow increase in glucose levels.

Impact of Incretin Hormone Receptors on Insulin-Independent Glucose Disposal in Model Experiments in Mice.

A large contribution to glucose elimination from the circulation is achieved by insulin-independent processes. We have previously shown that the two incretin hormones, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) increase this process and, therefore, seem to contribute to glucose disposal both through this effect and through the classical incretin effect resulting in enhanced insulin levels. We have now explored in more detail the potential contribution by incretin hormone receptors to insulin-independent processes for glucose elimination. To that end, we have performed intravenous glucose tests (0.35g/kg) in C57BL/6J mice and analyzed glucose elimination rate and glucose effectiveness (i.e., insulin-independent glucose disposal, SG) in wildtype mice and in mice with genetic deletion of GIP receptors or GLP-1 receptors. We performed studies with or without complete blockade of insulin secretion by the drug diazoxide (25 mg/kg). The mice were anesthetized with a novel fentanyl citrate/fluanisone formulation, called Fluafent, together with midazolam. Initially we demonstrated that glucose and insulin data after intravenous and oral glucose were not different using this anesthesia compared to the previously commonly used combination of HypnormR and midazolam. The results show that SG was reduced in GLP-1 receptor knockout mice, whereas there was no difference between GIP receptor knockout mice and wildtype mice, and this was evident both under normal conditions and after complete inhibition of insulin secretion. The study therefore indicates that insulin-independent glucose elimination requires active GLP-1 receptors and thus that the two incretin hormone receptor types show dissociated relevance for this process.

The Insulin Response to Oral Glucose in GIP and GLP-1 Receptor Knockout Mice: Review of the Literature and Stepwise Glucose Dose Response Studies in Female Mice.

A key factor for the insulin response to oral glucose is the pro-glucagon derived incretin hormone glucagon-like peptide-1 (GLP-1), together with the companion incretin hormone, glucose-dependent insulinotropic polypeptide (GIP). Studies in GIP and GLP-1 receptor knockout (KO) mice have been undertaken in several studies to examine this role of the incretin hormones. In the present study, we reviewed the literature on glucose and insulin responses to oral glucose in these mice. We found six publications with such studies reporting results of thirteen separate study arms. The results were not straightforward, since glucose intolerance in GIP or GLP-1 receptor KO mice were reported only in eight of the arms, whereas normal glucose tolerance was reported in five arms. A general potential weakness of the published study is that each of them have examined effects of only one single dose of glucose. In a previous study in mice with genetic deletion of both GLP-1 and GIP receptors we showed that these mice have impaired insulin response to oral glucose after large but not small glucose loads, suggesting that the relevance of the incretin hormones may be dependent on the glucose load. To further test this hypothesis, we have now performed a stepwise glucose administration through a gastric tube (from zero to 125mg) in model experiments in anesthetized female wildtype, GLP-1 receptor KO and GIP receptor KO mice. We show that GIP receptor KO mice exhibit glucose intolerance in the presence of impaired insulin response after 100 and 125 mg glucose, but not after lower doses of glucose. In contrast, GLP-1 receptor KO mice have normal glucose tolerance after all glucose loads, in the presence of a compensatory increase in the insulin response. Therefore, based on these results and the literature survey, we suggest that GIP and GLP-1 receptor KO mice retain normal glucose tolerance after oral glucose, except after large glucose loads in GIP receptor KO mice, and we also show an adaptive mechanism in GLP-1 receptor KO mice, which needs to be further examined.

Dietary Fiber Is Essential to Maintain Intestinal Size, L-Cell Secretion, and Intestinal Integrity in Mice.

Dietary fiber has been linked to improved gut health, yet the mechanisms behind this association remain poorly understood. One proposed mechanism is through its influence on the secretion of gut hormones, including glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2). We aimed to: 1) investigate the impact of a fiber deficient diet on the intestinal morphological homeostasis; 2) evaluate L-cell secretion; and 3) to ascertain the role of GLP-1, GLP-2 and Takeda G protein-receptor-5 (TGR5) signaling in the response using GLP-1 receptor, GLP-2 receptor and TGR5 knockout mice. Female C57BL/6JRj mice (n = 8) either received a standard chow diet or were switched to a crude fiber-deficient diet for a short (21 days) and long (112 days) study period. Subsequent identical experiments were performed in GLP-1 receptor, GLP-2 receptor and TGR5 knockout mice. The removal of fiber from the diet for 21 days resulted in a decrease in small intestinal weight (p < 0.01) and a corresponding decrease in intestinal crypt depth in the duodenum, jejunum and ileum (p < 0.001, p < 0.05, and p < 0.01, respectively). Additionally, colon weight was decreased (p < 0.01). These changes were associated with a decrease in extractable GLP-1, GLP-2 and PYY in the colon (p < 0.05, p < 0.01, and p < 0.01). However, we could not show that the fiber-dependent size decrease was dependent on GLP-1 receptor, GLP-2 receptor or TGR5 signaling. Intestinal permeability was increased following the removal of fiber for 112 days. In conclusion, our study highlights the importance of dietary fiber to maintain intestinal weight, colonic L-cell secretion and intestinal integrity.

The mediation by GLP-1 receptors of glucagon-induced insulin secretion revisited in GLP-1 receptor knockout mice

To study whether activation of GLP-1 receptors importantly contributes to the insulinotropic action of exogenously administered glucagon, we have performed whole animal experiments in normal mice and in mice with GLP-1 receptor knockout. Glucagon (1, 3 or 10 μg/kg), the GLP-1 receptor antagonist exendin 9-39 (30 nmol/kg), glucose (0.35 g/kg) or the incretin hormone glucose-dependent insulinotropic polypeptide (GIP; 3 nmol/kg) was injected intravenously or glucose (75 mg) was given orally through gavage. Furthermore, islets were isolated and incubated in the presence of glucose with or without glucagon. It was found that the insulin response to intravenous glucagon was preserved in GLP-1 receptor knockout mice but that glucagon-induced insulin secretion was markedly suppressed in islets from GLP-1 receptor knockout mice. Similarly, the GLP-1 receptor antagonist markedly suppressed glucagon-induced insulin secretion in wildtype mice. These data suggest that GLP-1 receptors contribute to the insulinotropic action of glucagon and that there is a compensatory mechanism in GLP-1 receptor knockout mice that counteracts a reduced effect of glucagon. Two potential compensatory mechanisms (glucose and GIP) were explored. However, neither of these seemed to explain why the insulin response to glucagon is not suppressed in GLP-1 receptor knockout mice. Based on these data we confirm the hypothesis that glucagon-induced insulin secretion is partially mediated by GLP-1 receptors on the beta cells and we propose that a compensatory mechanism, the nature of which remains to be established, is induced in GLP-1 receptor knockout mice to counteract the expected impaired insulin response to glucagon in these mice.

Amino acids are sensitive glucagon receptor-specific biomarkers for glucagon-like peptide-1 receptor/glucagon receptor dual agonists.

The aim of this study was to evaluate amino acids as glucagon receptor (GCGR)-specific biomarkers in rodents and cynomolgus monkeys in the presence of agonism of both glucagon-like peptide-1 receptor (GLP1R) and GCGR with a variety of dual agonist compounds. Primary hepatocytes, rodents (normal, diet-induced obese and GLP1R knockout) and cynomolgus monkeys were treated with insulin (hepatocytes only), glucagon (hepatocytes and cynomolgus monkeys), the GLP1R agonist, dulaglutide, or a variety of dual agonists with varying GCGR potencies. A long-acting dual agonist, Compound 2, significantly decreased amino acids in both wild-type and GLP1R knockout mice in the absence of changes in food intake, body weight, glucose or insulin, and increased expression of hepatic amino acid transporters. Dulaglutide, or a variant of Compound 2 lacking GCGR agonism, had no effect on amino acids. A third variant with ~31-fold less GCGR potency than Compound 2 significantly decreased amino acids, albeit to a significantly lesser extent than Compound 2. Dulaglutide (with saline infusion) had no effect on amino acids, but an infusion of glucagon dose-dependently decreased amino acids on the background of GLP1R engagement (dulaglutide) in cynomolgus monkeys, as did Compound 2. These results show that amino acids are sensitive and translatable GCGR-specific biomarkers.

Glucagon-like peptide-1 cleavage product GLP-1(9–36) reduces neuroinflammation from stroke via the activation of insulin-like growth factor 1 receptor in astrocytes

Glucagon-like peptide-1 (GLP-1) is an endogenous gut hormone and a key regulator in maintaining glucose homeostasis by stimulating insulin secretion. Its natural cleavage product GLP-1 (9–36), which was formerly considered a “bio-inactive” metabolite mainly due to its low affinity for GLP-1 receptor, possesses unique properties such as cardiovascular protection. Little is known about the effects and mechanisms of GLP-1 (9–36) in cerebral ischemia and reperfusion injury. Here, we report that systemic application of GLP-1 (9–36) in adult mice facilitated functional recovery and reduced infarct volume, astrogliosis, and neuronal apoptosis following middle cerebral artery occlusion and reperfusion. Interestingly, these effects were still observed in GLP-1 receptor knockout (Glp-1rKO) mice but were partially reversed in insulin-like growth factor 1 (IGF-1) receptor knockdown (Igf-1rKD) mice. Primary astrocytes were cultured and subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), and enzyme-linked immunosorbent assay indicated that GLP-1 (9–36) pretreatment reduces tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 levels. This effect was not diminished in Glp-1rKO astrocytes but was reversed in Igf-1rKO astrocytes, emphasizing that the anti-inflammatory effect of GLP-1 (9–36) in astrocytes is independent of GLP-1 receptor signaling and is instead mediated by IGF-1 receptor. Immunoprecipitation experiments showed that GLP-1 (9–36) directly interacts with IGF-1 receptor in astrocytes. Western blot data indicated that GLP-1 (9–36) activates IGF-1 receptor and downstream PI3K-AKT pathway in astrocytes upon OGD/R injury, which was abrogated by preincubation with IGF-1 receptor autophosphorylation inhibitor picropodophyllin. Thus, our findings suggest that GLP-1 (9–36) improved stroke outcome by reducing inflammation in astrocytes via interaction with IGF-1 receptor.

Glucagon-like peptide-1 receptor regulation of basal dopamine transporter activity is species-dependent

IntroductionA solid body of preclinical evidence shows that glucagon-like peptide-1 receptor (GLP-1R) agonists attenuate the effects of substance use disorder related behaviors. The mechanisms underlying these effects remain elusive. In the present study, we hypothesized that GLP-1R activation modulates dopaminetransporter (DAT) and thus dopamine (DA) homeostasis in striatum. This was evaluated in three different experiments: two preclinical and one clinical. MethodsRat striatal DA uptake, DA clearance and DAT cell surface expression was assessed following GLP-1 (7-36)-amide exposure in vitro. DA uptake in mice was assesed ex vivo following systemic treatment with the GLP-1R agonist exenatide. In addition, DA uptake was measured in GLP-1R knockout mice and compared with DA-uptake in wild type mice. In healthy humans, changes in DAT availability was assessed during infusion of exenatide measured by single-photon emission computed tomography imaging. ResultsIn rats, GLP-1 (7-36)-amide increased DA uptake, DA clearance and DAT cell surface expression in striatum. In mice, exenatide did not change striatal DA uptake. In GLP-1R knockout mice, DA uptake was similar to what was measured in wildtype mice. In humans, systemic infusion of exenatide did not result in acute changes in striatal DAT availability. ConclusionsThe GLP-1R agonist-induced modulation of striatal DAT activity in vitro in rats could not be replicated ex vivo in mice and in vivo in humans. Therefore, the underlying mechanisms of action for the GLP-1R agonists-induced efficacy in varios addiction-like behavioural models still remain.

GLP-1-induced renal vasodilation in rodents depends exclusively on the known GLP-1 receptor and is lost in prehypertensive rats.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone known to stimulate postprandial insulin release. However, GLP-1 also exerts extrapancreatic effects, including renal effects. Some of these renal effects are attenuated in hypertensive rats, where renal expression of GLP-1 receptors is reduced. Here, we assessed the expression and vascular function of GLP-1 receptors in kidneys from young prehypertensive rats. We also examined GLP-1-induced vasodilation in the renal vasculature in wild-type (WT) and GLP-1 receptor knockout mice using wire and pressure myography and the isolated perfused juxtamedullary nephron preparation. We investigated whether GLP-1 and the metabolite GLP-1(9-36)amide had renal vascular effects independent of the known GLP-1 receptor. We hypothesized that hypertension decreased expression of renal GLP-1 receptors. We also hypothesized that GLP-1-induced renal vasodilatation depended on expression of the known GLP-1 receptor. In contrast to normotensive rats, no immunohistochemical staining or vasodilatory function of GLP-1 receptors was found in kidneys from prehypertensive rats. In WT mice, GLP-1 induced renal vasodilation and reduced the renal autoregulatory response. The GLP-1 receptor antagonist exendin 9-39 inhibited relaxation, and GLP-1(9-36)amide had no vasodilatory effect. In GLP-1 receptor knockout mice, no relaxation induced by GLP-1 or GLP-1(9-36)amide was found, the autoregulatory response in afferent arterioles was normal, and no GLP-1-induced reduction of autoregulation was found. We conclude that in prehypertensive kidneys, expression and function of GLP-1 receptors is lost. The renal vasodilatory effect of GLP-1 is mediated exclusively by the known GLP-1 receptor. GLP-1(9-36)amide has no renal vasodilatory effect. GLP-1 attenuates renal autoregulation by reducing the myogenic response.