Glucagon-like Peptide-1 Receptor mRNA Research Articles

Development of Syringaldehyde as an Agonist of the GLP-1 Receptor to Alleviate Diabetic Disorders in Animal Models.

The phenolic aldehyde syringaldehyde (SA) has been shown to have an antihyperglycemic effect in diabetic rats due to increased glucose utilization and insulin sensitivity. To understand the direct effect of SA on the GLP-1 receptor, STZ-induced diabetic rats were used. The levels of pro-inflammatory cytokines, liver enzymes, and renal function were measured using specific ELISA kits. The mechanisms of SA effects were investigated using CHO-K1 cells, pancreatic Min-6 cells, and cardiomyocyte H9c2 cells. The results indicated that the antihyperglycemic effect of SA in diabetic rats was abolished by blocking the GLP-1 receptor with an antagonist. SA has a direct effect on the GLP-1 receptor when using CHO-K1 cells transfected with the exogenous GLP-1 receptor gene. In addition, SA stimulated insulin production in Min-6 cells by activating GLP-1 receptors. SA caused a dose-dependent rise in GLP-1 receptor mRNA levels in cardiac H9c2 cells. These in vitro results support the notion that SA has a direct effect on the GLP-1 receptor. Otherwise, SA inhibited the increase of pro-inflammatory cytokines, including interleukins and tumor TNF-α, in type 1 diabetic rats in a dose-dependent manner. Moreover, as with liraglutide, SA reduced plasma lipid profiles, including total cholesterol and triglyceride, in mixed diet-induced type 2 diabetic rats. Intriguingly, chronic treatment with SA (as with liraglutide) reversed the functions of both the liver and the kidney in these diabetic rats. SA displayed less efficiency in reducing body weight and food consumption compared to liraglutide. In conclusion, SA effectively activates GLP-1 receptors, resulting in a reduction in diabetic-related complications in rats. Therefore, it is beneficial to develop SA as a chemical agonist for clinical applications in the future.

Abstract PR003: The combination of the glucagon like peptide-1 receptor agonist semaglutide and the progestin levonorgestrel is highly effective in preclinical studies of endometrial cancer

Abstract Obesity is a major risk factor for endometrial cancer (EC), and glucagon like peptide-1 receptor (GLP-1R) agonists such as semaglutide may be helpful to achieve weight loss during conservative treatment or for EC prevention. Progestins such as levonorgestrel are effective in preventing or treating early-stage EC. However, better and more durable response rates could be achieved using combinatorial therapeutic regimens of well tolerated agents with the potential to enhance progestin effectiveness. We theorized that the combination of semaglutide and levonorgestrel would be useful as a novel treatment or prevention regimen and tested this hypothesis using EC cell lines, patient-derived organoids, and RNA-sequencing from patient samples. From the molecular perspective, EC cell lines, organoids, and tissues express GLP-1R as determined by both qPCR and Western blotting, and GLP-1R agonist treatment induces GLP-1R mRNA transcription through positive feedback mechanisms in EC cell models. Preclinical studies in 6 patient-derived organoid models of EC demonstrated the promising therapeutic effects of the combination of semaglutide and levonorgestrel on endometrial cell viability. Specifically, patient-derived organoids from grade 1 endometrial carcinomas were treated with progesterone, levonorgestrel, semaglutide, or levonorgestrel + semaglutide at drug concentrations of 100 nM for 72 hours. Multiple models demonstrated a significant reduction in viability in response to combinatorial treatment. Most interesting was the induction of not only the membrane GLP-1 receptor with treatment, but also the significant upregulation of nuclear and membrane progesterone receptors, PR and PGRMC1/2, respectively, indicating a positive feedback loop between semaglutide and progestins such as levonorgestrel. In addition, other steroid hormone receptor mRNAs such as the estrogen, androgen and mineralocorticoid receptors (ER, AR, and MR) were similarly upregulated. In these studies, we identify synergistic molecular crosstalk between the GLP-1R and steroid hormone receptor pathways with the potential to enhance the anti-cancer activity of semaglutide and levonorgestrel when combined. Future studies are aimed at testing this regimen in patients conservatively managed for atypical endometrial hyperplasia or low-grade endometrial cancers. Citation Format: Kimberly K. Leslie, Andrea Hagemann, Kristina Thiel, Ian Hagemann, David Mutch, Eric Devor, Paige Malmrose. The combination of the glucagon like peptide-1 receptor agonist semaglutide and the progestin levonorgestrel is highly effective in preclinical studies of endometrial cancer [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr PR003.

GLP1 receptor agonists exhibit antioxidant and anti-inflammatory properties in human heart tissue via the canonical GLP1 receptor expression through a AMPK-mediated pathway

Abstract Background Clinical research has demonstrated that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) can decrease the occurrence of major cardiovascular events in diabetic patients, regardless of their glycemic control. However, a significant challenge in understanding the positive effects of GLP1RA has been the lack of knowledge regarding the specific cellular types that express GLP1R. Despite the established cardiovascular benefits of GLP1RA, previous studies have been unable to confirm the expression of GLP1R in the heart. Aim The study aimed to investigate the expression and function of GLP1R in heart tissue. Methods We collected human cardiac tissue samples from 30 patients who underwent left ventricle (LV) biopsies (n=21) or right atrial (RA) appendage closure (n=9) at a university hospital. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results LV biopsies and RA appendages showed a substantial heterogeneity in GLP1R mRNA levels. GLP1R mRNA levels exhibited a positive correlation with IL1B, IL6, TNFα, CCL2, CD68, ACE1, AT1R, and VCAM1 mRNA levels, but a negative correlation with NOS3 mRNA levels. High GLP1R expression in both RA appendages and LV biopsies was associated with increased oxidative stress levels, which were mitigated by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50-60 %. Exendin, a GLP1R antagonist reversed the effect of liraglutide and semaglutide which accounted for a receptor-mediated antioxidant effect. H-89, a PKA inhibitor equally reversed liraglutide and semaglutide effect on ROS generation suggesting a canonical GLP1R mediated effect involving the AMPK pathway. Conclusion These findings indicate that human cardiac GLP1R expression levels are correlated to low-grade inflammation and endothelial dysfunction. Increased expression levels of GLP1R were associated with higher levels of oxidative stress sensitive to GLP1RA in a canonical GLP1R AMPK-mediated manner. Therefore, the cardiovascular beneficial effects of GLP1RA might be attributable to their antioxidant and anti-inflammatory properties, specifically within the cardiac system.

Role of glucagon-like peptide 1 (GLP-1) and its association with inflammatory markers in the pathogenesis of type 2 diabetes mellitus

Background: Diabetes mellitus is a multifactorial disease associated with hyperglycemia and increased risk of progression of vascular complications. Stimulation of insulin secretion by the incretin hormone glucagon-like peptide 1 (GLP-1) has been found to be diminished in hyperglycemia. We hypothesized that this impairment is due to defect at the receptor level induced by the diabetic state. Inflammatory markers like TNF-α and IL-6 plays a potential role in the pathogenesis of T2DM. Therefore, the present study aims to evaluate whether GLP-1 plays a role in the development of T2DM by modulating the balance between pro and anti-inflammatory markers. Material and methods: A total of 60 subjects were recruited in this study among them 30 were T2DM cases and 30 were healthy controls. m-RNA expression and protein level of GLP-1 receptor, TNF-α and IL-6 in peripheral blood lymphocytes were determined by real time PCR and ELISA respectively. Results: We observed plasma level of GLP-1 was significantly lower in diabetic subjects while serum level of IL-6 and TNF-α were significantly higher level in diabetic subjects (p < 0.05). We found significant down regulation of GLP-1 receptor m-RNA expression in diabetic subjects while expression level of IL-6 and TNF-α were 5.8 and 4 folds respectively higher in diabetic subjects. We found significant negative correlation of m-RNA expression of GLP-1 with protein level while IL-6 and TNF-α showed significant positive correlation. Conclusion: Inflammation plays an important role in the pathogenesis of diabetes mellitus and low GLP-1 levels may promote expression of inflammatory markers due to lack of anti-inflammatory effects of GLP-1

Investigation of fructose consumption on hippocampal insulin and glucagon-like peptide-1 receptors, and metabolic effects in rats.

The detrimental effects of high fructose consumption on metabolic health have been extensively studied. However, limited research has focused on the impact of fructose intake on neuroprotective mechanisms, specifically the expression of insulin receptor (INSR) and glucagon-like peptide-1 receptor (GLP-1R) in the hippocampus. Understanding the effects of fructose on these neuroprotective molecules can provide valuable insights into the potential role of fructose in hippocampal dysfunction. The goal of this study is to aim at the basal plasma levels of lipid profile, insulin, GLP-1, and HOMA-IR, as well as the mRNA and protein expression of neuroprotective molecules such as INSR and GLP-1R in Wistar rats fed a high fructose diet. Rats were separated into control (C) and high fructose (HF) groups. The HF group was given 20% fructose water to drink for 16 weeks. Fructose ingestion significantly increased abdominal fat (C=1.24±0.08 g, HF=1.79±0.19 g, P<0.05) and plasma triglyceride levels (C=179.22±22.85 µg/ml, HF=242.45±14.45 µg/ml, P<0.05), but had no statistically significant effect on body weight and plasma HDL, LDL, total cholesterol, insulin, and GLP-1 levels (P>0.05). Although INSR mRNA expression in the hippocampus was significantly lower in the HF group compared to the control group (P<0.05), GLP-1R mRNA expression did not differ significantly across the groups (P>0.05). Furthermore, whereas INSR and GLP-1R protein levels in the experimental group were on a declining trend, this trend was not substantially different (P>0.05). These data suggest that fructose consumption may be harmful to the hippocampus by lowering the expression of INSR.

Glucagon-like peptide-1 receptor regulates receptor of advanced glycation end products in high glucose-treated rat mesangial cells.

Hyperglycemia-induced advanced glycation end products (AGEs) and receptor for AGEs (RAGEs) play major roles in diabetic nephropathy progression. In previous study, both glucagon-like peptide-1 (GLP-1) and peroxisome proliferator-activated receptors delta (PPARδ) agonists were shown to have anti-inflammatory effect on AGE-treated rat mesangial cells (RMCs). The interaction among PPARδ agonists, GLP-1, and AGE-RAGE axis is, however, still unclear. In this study, the individual and synergic effect of PPARδ agonist (L-165 041) and siRNA of GLP-1 receptor (GLP-1R) on the expression of GLP-1, GLP-1R, RAGE, and cell viability in AGE-treated RMCs were investigated. L-165 041 enhanced GLP-1R mRNA and protein expression only in the presence of AGE. The expression of RAGE mRNA and protein was enhanced by AGE, attenuated by L-165 041, and siRNA of GLP-1R reversed L-165 041-induced inhibition. Cell viability was also inhibited by AGE. L-165 041 attenuated AGE-induced inhibition and siRNA GLP-1R diminished L-165 041 effect. PPARδ agonists increase GLP-1R expression on RMC in the presence of AGE. PPARδ agonists also attenuate AGE-induced upregulated RAGE expression and downregulated cell viability. The effect of PPARδ agonists needs the cooperation of GLP-1R activation.

GLP-1 receptor expression in human internal thoracic arteries is dependent on low-grade inflammation potentially explaining the cardiovascular protective effect of GLP1-R agonists

Abstract Background Clinical research showed that glucagon-like peptide-1 receptor (GLP1R) agonists (GLP1RA) reduced the incidence of major cardiovascular events in diabetic patients. The protective effect was more noteworthy in patients with established cardiovascular disease, associated with chronic low-grade inflammation resulting in endothelial dysfunction and arterial remodeling and fibrosis. Despite the clinically proven cardiovascular benefit of GLP1RA, the expression pattern of GLP1R and its function in the human vasculature remain poorly studied. Aim This study investigated the expression of GLP1R and its role in internal thoracic arteries (ITA) from patients with coronary artery disease. Methods Human ITA were collected from patients (N=30) undergoing coronary artery bypass surgery at the University Hospital of Strasbourg. Gene expression levels were assessed using RT-qPCR, protein levels by Western blot analysis, in situ tissue localization of proteins by immunofluorescence (IF) staining, and the level of oxidative stress using dihydroethidium. Results ITA showed a substantial difference in GLP1R mRNA levels reaching up to 7-fold among patients. GLP1R mRNA levels were positively correlated with those of SLC5A1, SLC5A2, F3, AT1R, IL1B, IL6, TNFα, VCAM1 and NCF1, and negatively with NOS3 mRNA levels. High GLP1R expressing ITA showed high levels of phosphorylated p65 NF-κB, cell adhesion molecule VCAM1, members of the angiotensin system (ACE1 and AT1R), remodeling and fibrotic markers (MMP9, MMP2, TGFβ) and a low level of eNOS, whereas the contrary was observed for low GLP1R expressing ITA. IF staining showed that GLP1R signals were predominantly observed in the endothelium of low GLP1R expressing ITA and in the vascular smooth muscle of high GLP1R expressing ITA. Prominent CD68 staining associated with the endothelium and the perivascular adipose tissue were observed in high but not low GLP1R expressing ITA. High GLP1R expressing ITA showed increased levels of oxidative stress, which were inhibited by the antioxidant N-acetylcysteine (NAC), NADPH oxidase inhibitor (VAS-2870), ACE inhibitor (perindoprilat), AT1R antagonist (losartan), TNF-α receptor neutralizing antibody (infliximab), selective SGLT2 inhibitor (empagliflozin), dual SGLT1/2 inhibitor (sotagliflozin) and by GLP1R agonists (liraglutide and semaglutide) with inhibitory effects amounting to about 50 to 75%. Conclusion These findings indicate that GLP1R expression levels in human ITA are dependent on low-grade inflammation and linked to endothelial dysfunction, pro-thrombotic, pro-remodeling and pro-fibrotic responses. Furthermore, they were associated with increased level of oxidative stress sensitive to inhibitors of either the local angiotensin system, SGLT2, TNFα and agonists of GLP1R. Thus, the cardiovascular beneficial effects of GLP1R agonists might be attributable to their ability to reduce the pro-oxidant stimulatory signal related to low-grade inflammation. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): Frency Society of Vascular Medicine (SFMV)

The glucagon-like peptide-1 system is modulated by acute and chronic alcohol exposure: Findings from human laboratory experiments and a post-mortem brain study.

Growing evidence suggests that the glucagon-like peptide-1 (GLP-1) system modulates alcohol seeking and consumption, and GLP-1 analogues may represent novel pharmacotherapies for alcohol use disorder (AUD). Accordingly, it is important to understand the potential effects of alcohol on the endogenous GLP-1 system. In a series of secondary analyses of previous human laboratory experiments, we first examined the effects of alcohol administration, with different doses and routes of administration, on peripheral active GLP-1 concentrations in heavy-drinking individuals with AUD enrolled in placebo-controlled pharmacological studies (only placebo conditions were analysed here). Alcohol administration resulted in a significant reduction of GLP-1 levels across the four experiments (oral alcohol, variable dose: F3,28 = 6.52, p = 0.002; oral alcohol, fixed dose: F7,75.94 = 5.08, p < 0.001; intravenous alcohol, variable dose: F4,37.03 = 20.72, p < 0.001; intravenous alcohol, fixed dose: F4,13.92 = 10.44, p < 0.001). Next, central expression of the GLP-1 receptor (GLP-1R) in post-mortem brain tissues (amygdala, ventral tegmental area, nucleus accumbens, hippocampus and prefrontal cortex) was compared between individuals with AUD and controls. Fold change of GLP-1R mRNA in the hippocampus was significantly higher in individuals with AUD, compared to controls (F1,21 = 6.80, p = 0.01). A trend-level effect with the same direction was also found in the prefrontal cortex (F1,20 = 3.07, p = 0.09). Exploratory analyses showed that GLP-1R gene expression levels were correlated with behavioural measures of alcohol drinking (hippocampus) and cigarette smoking (hippocampus and prefrontal cortex). Collectively, these data provide novel information on the crosstalk between alcohol and GLP-1 in a clinically relevant sample. Further studies are needed to understand the underlying mechanisms of this link.

Abstract 3: Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice

Abstract Glucagon-like peptide-1 (GLP1) is a pleiotropic hormone released from gut enteroendocrine cells following nutrient ingestion. In addition to promoting insulin secretion and inhibiting glucagon secretion, GLP1 acts via the glucagon-like peptide-1 receptor (GLP1R) to reduce gastric emptying and food intake. Therefore, development of GLP1R agonists appears to be a promising therapeutic option for type 2 diabetes. To evaluate the efficacy of GLP1 analogs in preclinical studies, Biocytogen has successfully generated a novel humanized GLP1R (B-hGLP1R) knock-in mouse line to support related drug development studies. In this mouse model, the full coding sequence of the human GLP1R gene was inserted into the mouse Glp1r gene locus. Human GLP1R mRNA expression was detected by RT-PCR in B-hGLP1R mice but not in wild-type mice. Protein expression of human GLP1R in B-hGLP1R mice was confirmed by western blot analysis and immunohistochemistry using pancreatic tissues. Furthermore, flow cytometry analysis of leukocyte subpopulations showed that B-hGLP1R mice do not exhibit a change in the overall development, differentiation, or distribution of immune cell types in the spleen, blood, and lymph nodes. Additionally, our in vivo efficacy study confirmed Dulaglutide, a GLP1 receptor agonist, reduced the non-fasting blood glucose, fasting blood glucose, glucagon, and food intake levels in B-hGLP1R obese mice and increased plasma insulin and GLP1 secretion. In B-hGLP1R mice, glucose tolerance (GTT) was also improved by Dulaglutide treatment, similar GTT results were observed after treatment with PF-06882961, a phase II agent specifically recognizing human GLP1R. Altogether, B-hGLP1R mice provide an efficacious preclinical animal model to evaluate novel GLP1 based therapeutic drugs. Keywords: Humanized mice, GLP1R, agonist Citation Format: Yuelei Shen, Jiawei Yao, Mostafa Kokabee, Yanan Guo, Yang BaI, Chengzhang Shang. Developing a translational tool for GLP-1-based therapeutic agonists in humanized GLP1R mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3.

Liraglutide preserves CD34+ stem cells from dysfunction Induced by high glucose exposure

BackgroundGlucagon like peptide-1 receptor agonists (GLP-1RAs) have shown to reduce mortality and cardiovascular events in patients with type 2 diabetes mellitus (T2DM). Since the impairment in number and function of vasculotrophic circulating CD34+ hematopoietic stem progenitor cells (HSPCs) in T2D has been reported to increase cardiovascular (CV) risk, we hypothesized that one of the mechanisms whereby GLP-1 RAs exert CV protective effects may be related to the ability to improve CD34+ HSPC function.MethodsIn cord blood (CB)-derived CD34+ HSPC, the expression of GLP-1 receptor (GLP-1R) mRNA, receptor protein and intracellular signaling was evaluated by RT-qPCR and Western Blot respectively. CD34+ HSPCs were exposed to high glucose (HG) condition and GLP-1RA liraglutide (LIRA) was added before as well as after functional impairment. Proliferation, CXCR4/SDF-1α axis activity and intracellular ROS production of CD34+ HSPC were evaluated.ResultsCD34+ HSPCs express GLP-1R at transcriptional and protein level. LIRA treatment prevented and rescued HSPC proliferation, CXCR4/SDF-1α axis activity and metabolic imbalance from HG-induced impairment. LIRA stimulation promoted intracellular cAMP accumulation as well as ERK1/2 and AKT signaling activation. The selective GLP-1R antagonist exendin (9–39) abrogated LIRA-dependent ERK1/2 and AKT phosphorylation along with the related protective effects.ConclusionWe provided the first evidence that CD34+ HSPC express GLP-1R and that LIRA can favorably impact on cell dysfunction due to HG exposure. These findings open new perspectives on the favorable CV effects of GLP-1 RAs in T2DM patients.

Sodium-glucose co-transporter 1 (SGLT1) differentially regulates gluconeogenesis and GLP-1 receptor (GLP-1R) expression in different diabetic rats: a preliminary validation of the hypothesis of "SGLT1 bridge" as an indication for "surgical diabetes".

BackgroundSodium-glucose co-transporter 1 (SGLT1) may play a synergistic role in gluconeogenesis (GNG) and glucagon-like peptide-1 (GLP-1) expression. We proposed the hypothesis of a “SGLT1 bridge” as an indication for “surgical diabetes” that was preliminary validated in the present study.MethodsWe selected nonobese diabetic Goto-Kakizaki (GK) rats and Zuker diabetic fat (ZDF) rats to represent advanced and early diabetes, respectively. Based on glucose gavage with or without SGLT1 inhibitor phlorizin, the rats were divided into 4 groups: Gk-Glu, GK-P, ZDF-Glu, and ZDF-P. The expressions of SGLT1, GLP-1 receptor (GLP-1R), glucose-6 phosphatase (G6Pase), and phosphoenolpyruvate carboxykinase-1 (Pck1) were determined by immunohistochemistry (IHC) or quantitative reverse transcription polymerase chain reaction (RT-qPCR), and the effects of phlorizin were analyzed.ResultsGlucose tolerance was worse in GK rats and the homeostasis model assessment-insulin resistance (HOMA-IR) was higher in ZDF rats, indicating different pathophysiological conditions between the different diabetic rats. GK rats showed higher activity of duodenal SGLT1 (P=0.022) and jejunal SGLT1 mRNA expression (P=0.000) and lower SGLT1 mRNA expression in the liver (P=0.000) and pancreas (P=0.000). Phlorizin effectively inhibited the activity of duodenal SGLT1 in both GK rats (P=0.000) and ZDF rats (P=0.000). In ZDF rats, the expression of GLP-1R mRNA was downregulated in the jejunum (P=0.001) and upregulated in the pancreas (P=0.021) by phlorizin, but there were no regulatory effects on GLP-1R mRNA in the jejunum and pancreas of GK rats. As for the regulatory effects on GNG, phlorizin upregulated Pck1 mRNA in the duodenum (P=0.000) and the jejunum (P=0.038), whereas it downregulated hepatic G6Pase mRNA in ZDF rats (P=0.005) and Pck1 mRNA expression in GK rats (P=0.001), suggesting that SGLT1 inhibitor may have upregulated intestinal GNG in ZDF rats and downregulated hepatic GNG in both ZDF and GK rats.ConclusionsSGLT1 showed synergistic regulatory effects on the entero-insular axis (EIA) and the gut-brain-liver axis (GBLA), preliminarily validating the hypothesis of a “SGLT1 bridge”. The distinct expression of SGLT1 and its differentially regulatory effects on diabetic rats with different pathophysiological conditions may provide probable potential indications involved in the “Surgical Diabetes” that is supposed as the inclusion for diabetic surgery.

Comparison of the effects of intracerebroventricular administration of glucagon-like peptides 1 and 2 on hypothalamic appetite regulating factors and sleep-like behavior in chicks

Glucagon-like peptide (GLP)-1 and GLP-2, proglucagon-derived brain-gut peptides, function as anorexigenic neuropeptides in mammals. We previously showed that central administration of GLP-1 and GLP-2 potently suppressed food intake in chicks. GLP-1 and GLP-2 specifically activate their receptors GLP-1 receptor (GLP1R) and GLP-2 receptor (GLP2R), respectively in chickens. In adult chickens, GLP1R and GLP2R are expressed in different brain regions. These findings raise the hypothesis that both GLP-1 and GLP-2 function as anorexigenic peptides in the chicken brain but the mechanisms underlying the anorexigenic effects are different between them. In the present study, we compared several aspects of GLP-1 and GLP-2 in chicks. GLP1R mRNA levels in the brain stem and optic lobes were significantly higher than in other parts of the brain, whereas GLP2R mRNA was densely expressed in the telencephalon. Intracerebroventricular administration of either GLP-1 or GLP-2 significantly reduced the mRNA levels of corticotrophin releasing factor and AMP-kinase (AMPK) α1. The mRNA level of proopiomelanocortin was significantly increased, and those of AMPKα2 and GLP2R were significantly decreased by GLP-2, whereas the mRNA level of pyruvate dehydrogenase kinase 4 was significantly increased, and that of GLP1R was significantly decreased by GLP-1. Intracerebroventricular administration of either GLP-1 or GLP-2 induced sleep-like behavior in chicks. Our findings suggest that the anorexigenic peptides GLP-1 and GLP-2 induce similar behavioral changes in chicks, but the mechanism may differ between them.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all β-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

The Effect of Verapamil on TXNIP Gene Expression, GLP1R mRNA, FBS, HbA1c, and Lipid Profile in T2DM Patients Receiving Metformin and Sitagliptin.

IntroductionType 2 diabetes mellitus (T2DM) is the most common type of diabetes. A decrease in the number of pancreatic beta cells is a pathological sign of diabetes, and to date there is no drug treatment that targets damage to these cells. Pancreatic beta cells have a weak antioxidant system and are highly sensitive to oxidative stress reactions that occur within cells. Thioredoxin interacting protein (TXNIP) inhibits thioredoxin, which is part of the intracellular antioxidant system, thereby accelerating oxidative stress and apoptosis of pancreatic beta cells. Verapamil is a non-dihydropyridine calcium channel blocker. The efficacy of this drug to improve beta cell survival and glucose homeostasis by inhibiting TXNIP expression has been demonstrated in in vitro studies. Although several retrospective studies have shown a lower incidence of T2DM with verapamil treatment, no prospective intervention studies have determined the efficacy of this drug in patients with T2DM.MethodsThe aim of this randomized, double-blind, placebo-controlled study was to evaluate the efficacy and safety of oral verapamil administration in T2DM patients. In this 90-day study, the effects of verapamil on fasting blood sugar (FBS), hemoglobin A1C (HbA1c), and the lipid profile were evaluated and compared with those of the placebo.ResultsThere was a significant decrease in HbA1c (about 0.5%) in the verapamil group at the end of the intervention period. The effects of verapamil on TXNIP gene expression and glucagon-like peptide-1 receptor (GLP1R) mRNA were compared with those of the placebo (at baseline, after 15 and 30 days, and at the end of the study). During the first month of the study, decreased TXNIP gene expression and increased GLP1R mRNA were associated with the administration of verapamil when compared with the placebo, although the differences were not significant.ConclusionVerapamil can lead to better control of T2DM by reducing TXNIP gene expression and increasing beta cell survival and, possibly, by other mechanisms.Clinical Trial RegistrationIRCT registration no.: IRCT20180417039339N1 (https://www.IRCT.ir).

Glucagon-like peptide 1 receptor-mediated stimulation of a GABAergic projection from the bed nucleus of the stria terminalis to the hypothalamic paraventricular nucleus

We previously reported that GABAergic neurons within the ventral anterior lateral bed nucleus of the stria terminalis (alBST) express glucagon-like peptide 1 receptor (GLP1R) in rats, and that virally-mediated “knock-down” of GLP1R expression in the alBST prolongs the hypothalamic-pituitary-adrenal axis response to acute stress. Given other evidence that a GABAergic projection pathway from ventral alBST serves to limit stress-induced activation of the HPA axis, we hypothesized that GLP1 signaling promotes activation of GABAergic ventral alBST neurons that project directly to the paraventricular nucleus of the hypothalamus (PVN). After PVN microinjection of fluorescent retrograde tracer followed by preparation of ex vivo rat brain slices, whole-cell patch clamp recordings were made in identified PVN-projecting neurons within the ventral alBST. Bath application of Exendin-4 (a specific GLP1R agonist) indirectly depolarized PVN-projecting neurons in the ventral alBST and adjacent hypothalamic parastrial nucleus (PS) through a network-dependent increase in excitatory synaptic inputs, coupled with a network-independent reduction in inhibitory inputs. Additional retrograde tracing experiments combined with in situ hybridization confirmed that PVN-projecting neurons within the ventral alBST/PS are GABAergic, and do not express GLP1R mRNA. Conversely, GLP1R mRNA is expressed by a subset of neurons that project into the ventral alBST and were likely contained within coronal ex vivo slices, including GABAergic neurons within the oval subnucleus of the dorsal alBST and glutamatergic neurons within the substantia innominata. Our novel findings reveal potential GLP1R-mediated mechanisms through which the alBST exerts inhibitory control over the endocrine HPA axis.

Long-Lasting Effects of GSPE on Ileal GLP-1R Gene Expression Are Associated with a Hypomethylation of the GLP-1R Promoter in Female Wistar Rats.

Flavonoids have been shown to modulate GLP-1 in obesity. GLP-1 induces some of its effects through the intestinal GLP-1 receptor (GLP-1R), though no data exist on how flavonoids affect this receptor. Here, we examine how a dose of grape seed proanthocyanidin extract (GSPE) with anti-obesity activity affects intestinal GLP-1R and analyze whether epigenetics play a role in the long-lasting effects of GSPE. We found that 10-day GSPE administration prior to the cafeteria diet upregulated GLP-1R mRNA in the ileum 17 weeks after the GSPE treatment. This was associated with a hypomethylation of the GLP-1R promoter near the region where the SP1 transcription factor binds. In the colon, the cafeteria diet upregulated GLP-1R without showing any GSPE effect. In conclusion, we have identified long-lasting GSPE effects on GLP-1R gene expression in the ileum that are partly mediated by hypomethylation at the gene promoter and may affect the SP1 binding factor.

Hyperglycemia induces NF-κB activation and MCP-1 expression via downregulating GLP-1R expression in rat mesangial cells: inhibition by metformin.

Hyperglycemia impairs glucagon-like peptide-1 receptor (GLP-1R) signaling in multiple cell types and thereby potentially attenuates the therapeutic effects of GLP-1R agonists. We hypothesized that the downregulation of GLP-1R by hyperglycemia might reduce the renal-protective effects of GLP-1R agonists in diabetic nephropathy (DN). In this study, we examined the effects of high glucose on the expression of GLP-1R and its signaling pathways in the HBZY-1 rat mesangial cell line. We found that high glucose reduced GLP-1R messenger RNA (mRNA) levels in HBZY-1 cells and in the renal cortex in db/db mice comparing with control groups. In consistence, GLP-1R agonist exendin-4 induced CREB phosphorylation was attenuated by high glucose but not low glucose treatment, which is paralleled with abrogated anti-inflammatory functions in HBZY-1 cells linked with nuclear factor-κB (NF-κB) activation. In consistence, GLP-1R inhibition aggravated the high glucose-induced activation of NF-κB and MCP-1 protein levels in cultured HBZY-1 cells while overexpression of GLP-1R opposite effects. We further proved that metformin restored high glucose-inhibited GLP-1R mRNA expression and decreased high glucose evoked inflammation in HBZY-1 cells. On the basis of these findings, we conclude that high glucose lowers GLP-1R expression and leads to inflammatory responses in mesangial cells, which can be reversed by metformin. These data support the rationale of combinative therapy of metformin with GLP-1R agonists in DN.

1969-P: Time-Based Internalized Characterization of Exendin-4-Based Glucagon-Like Peptide-1 Receptor-Targeting Imaging Probe

Aims: For the purpose of visualization and quantification of β cells, glucagon-like peptide 1 receptor (GLP-1R)-targeting imaging including single photon emission computed and positron emission tomography (SPECT/PET) has emerged recently. It is well known that internalization of the probes can affect visualization of regions of interest on SPECT/PET. However, the internalization and its effect on β-cell visualization of GLP-1R-targeting probes have not been fully examined. Therefore, we investigated time-based internalized activities of an exendin-4 (Ex4)-based probe, which was developed for clinical GLP-1R-targeting imaging. Method: [18F]FB(ePEG12)12-Ex4 (18F-Ex4) was synthesized. Rat insulinoma cell line, INS-1 and Human Embryonic Kidney cells 293 expressing human GLP-1R (HEK_GLP-1R) were used as GLP-1R-positive cells. These cells were incubated with 18F-Ex4 (1.48 MBq) for different periods of time (0-120 minutes). Whole cell (WC) and internalized (IC) radioisotope activities were measured by the gamma counter. Results: WC per incubated probe (IP) activities showed rapid rise within 30 minutes of incubation time and further gradual increase up to 120 minutes in INS-1 and HEK_GLP-1R cells, whereas wild type HEK293 showed no significant WC activity. The WC/IP differences between INS-1 and HEK_GLP-1R cells were significant at the 15-minutes mark (p=0.04) but reduced to insignificant after 30 minutes, although the two cell lines showed significantly different levels of GLP-1R mRNA expressions. IC/IP increased rapidly within 30 minutes and gradually up to 120 minutes. The IC/WC at the 120-minutes mark were significantly higher than those at the 30-minutes mark in both cell lines. Conclusion: The 18F-Ex4 probe showed rapid uptakes and internalization in GLP-1R-expressing cell lines, which indicates that dual or delayed phases of GLP-1R-targeting imaging may be beneficial. In addition, a time-based hampering effect of GLP-1R-expression levels for β-cell visualization can be suggested. Disclosure T. Murakami: None. H. Fujimoto: None. N. Fujita: None. K. Hamamatsu: None. J. Fujikura: None. N. Inagaki: Research Support; Self; Astellas Pharma Inc., Boehringer Ingelheim Pharmaceuticals, Inc., Daiichi Sankyo Company, Limited, Japan Tobacco Inc., Kyowa Hakko Kirin Co., Ltd., Merck Sharp &amp; Dohme Corp., Mitsubishi Tanabe Pharma Corporation, Novartis Pharmaceuticals Corporation, Ono Pharmaceutical Co., Ltd., Sanofi, Sumitomo Dainippon Pharma Co., Ltd., Takeda Pharmaceutical Company Limited.

Chronic Suppression of Glucagon-Like Peptide-1 Receptor (GLP1R) mRNA Translation in the Rat Bed Nucleus of the Stria Terminalis Reduces Anxiety-Like Behavior and Stress-Induced Hypophagia, But Prolongs Stress-Induced Elevation of Plasma Corticosterone.

The anterior lateral bed nucleus of the stria terminalis (alBST) expresses glucagon-like peptide-1 receptors (GLP1Rs) and receives input from caudal brainstem GLP1 neurons. GLP1 administered centrally reduces food intake and increases anxiety-like behavior and plasma corticosterone (cort) levels in rats, whereas central GLP1R antagonism has opposite effects. Anxiogenic threats and other stressors robustly activate c-fos expression in both GLP1-producing neurons and also in neurons within alBST subregions expressing GLP1R. To examine the functional role of GLP1R signaling within the alBST, adult male Sprague Dawley rats received bilateral alBST-targeted injections of an adeno-associated virus (AAV) vector expressing short hairpin RNA (shRNA) to knock down the translation of GLP1R mRNA (GLP1R-KD rats), or similar injections of a control AAV (CTRL rats). In situ hybridization revealed that GLP1R mRNA is expressed in a subset of GABAergic alBST neurons, and quantitative real-time PCR confirmed that GLP1R-KD rats displayed a significant 60% reduction in translatable GLP1R mRNA. Compared with CTRL rats, GLP1R-KD rats gained more body weight over time and displayed less anxiety-like behavior, including a loss of light-enhanced acoustic startle and less stress-induced hypophagia. Conversely, while baseline plasma cort levels were similar in GLP1R-KD and CTRL rats, GLP1R-KD rats displayed a prolonged stress-induced elevation of plasma cort levels. GLP1R-KD and CTRL rats displayed similar home cage food intake and a similar hypophagic response to systemic Exendin-4, a GLP1R agonist that crosses the blood-brain barrier. We conclude that GLP1R expressed within the alBST contributes to multiple behavioral responses to anxiogenic threats, yet also serves to limit the plasma cort response to acute stress.SIGNIFICANCE STATEMENT Anxiety is an affective and physiological state that supports threat avoidance. Identifying the neural bases of anxiety-like behaviors in animal models is essential for understanding mechanisms that contribute to normative and pathological anxiety in humans. In rats, anxiety/avoidance behaviors can be elicited or enhanced by visceral or cognitive threats that increase glucagon-like peptide-1 (GLP1) signaling from the caudal brainstem to the hypothalamus and limbic forebrain. Data reported here support a role for limbic GLP1 receptor signaling to enhance anxiety-like behavior and to attenuate stress-induced elevations in plasma cort levels in rats. Improved understanding of central GLP1 neural pathways that impact emotional responses to stress could expand potential therapeutic options for anxiety and other stress-related disorders in humans.

Simultaneous analyses of carbohydrate-mediated serum GLP-1 and GLP-2 and duodenal receptor expression in children with and without celiac disease.

Background:Variability in glucagon-like peptide (GLP)-1 and GLP-2 plasma concentrations has been suggested in Celiac disease (CD), with inconclusive results. We assessed the association between serum levels of GLP-1 and GLP-2 and their duodenal receptor expression in children with and without CD.Methods:This was a two-part, cross-sectional and prospective cohort study. Group assignment, performed after duodenal samples for mRNA expression of GLP-1 receptor (GLP1R) and GLP-2 receptor (GLP2R), were taken during esophagogastroduodenoscopy. The control group consisted of patients with normal endoscopy and negative serology. The CD group consisted of patients with positive serology and endoscopy suggestive of CD. All had an oral glucose-tolerance test (OGTT). CD patients underwent a second OGTT after 6 months of a gluten-free diet (GFD).Results:The CD group included 12 patients; 7 males with mean age 9.2 ± 2.5 years. The control group included 10 patients; 5 males with mean age 12 ± 4 years, (p = 0.14). No differences were detected in basal or peak levels of GLP-1 or GLP-2 between control, naïve CD (before GFD) and treated CD (after GFD) groups. Expression of GLP1R and GLP2R mRNA was similar. Significant positive correlations between glucose and C-peptide secretion (r = 0.9, p < 0.01) and GLP-1 and GLP-2 (r = 0.8, p = 0.01) were detected in the control group. Significant negative correlations were found in the naïve CD group between GLP2R expression and glucose secretion (r = −0.68, p = 0.015) and GLP1R expression and serum GLP-1 (r = −0.7, p = 0.016).Conclusions:Although no significant differences were detected in secretion patterns or gut receptor expression of GLP-1 and GLP-2 in healthy versus CD pediatric patients, the detected discrepancy between the ligand levels and their tissue receptors requires additional study.