Glucagon-like Peptide-1 Receptor Antagonist Research Articles

Histopathological Evaluation of the Teratogenic Potential of Lixisenatide on the Cerebral Cortex of Developing Cheek Embryo

Background and Objectives: Chick embryos are commonly used in research to investigate the negative impacts of various drugs. One drug, lixisenatide, is superior to other GLP1 receptor antagonists for treating type 2 diabetes mellitus. However, the specific impact of lixisenatide on the developing cerebral cortex of the chick embryo remains unclear. Therefore, further research is necessary to fully understand the potential adverse effects of lixisenatide on brain development. Materials and Methods: It’s an experimental study done at the Department of Anatomy, Santosh Medical College, Ghaziabad, Uttar Pradesh. The study aimed to investigate the effects on specific parameter in two groups, a control group, and an experimental group. The expected mean ± SD of the parameter for the control and experimental groups were found to be 27±5.32 and 32±5.30, respectively, based on a literature survey and pilot study. To achieve appropriate sample size, G*Power software was used, with an alpha level of 5% and a power of 95.36%, which resulted in an effect size of 1.961 and a sample size of 28 for each group, leading to a total sample size of 280.The chick embryos first sacrificed then dissected, and the brain was separated and kept in a 10% formaldehyde solution. To identify histological characteristics of the cerebral cortex, the light and compound microscope were used for sections. Results: During the experiment, Lixisenatide induced necrotic changes and cellular condensation in chick embryo’s cerebral cortex, specifically in groups C, D, and E. Additionally, there was mild distortion found in the cortex indicating degenerative changes experimental group E. There was a statistically significant increase in histopathological changes observed in the cerebral cortex as the dose of Lixisenatide increased in each experimental group (p<0.05). Conclusion: Insufficient research has been conducted on the development of resistance to lixisenatide. Despite its widespread use, observations have revealed that lixisenatide has adverse effects on the cerebral cortex of chick embryos, including cellular condensation and necrotic changes and mild distortion found in the cortex indicating degenerative changes in experimental groups C, D & E. Therefore, it is imperative that the administration of this vital medication is carefully considered to prevent neuronal complications.

Intestinal perfusion of unacylated ghrelin alleviated metabolically associated fatty liver disease in rats via a central glucagon-like peptide-1 pathway.

Unacylated ghrelin (UAG), the unacylated form of ghrelin, accounts for 80%-90% of its circulation. Accumulated studies have pointed out that UAG may be used to treat metabolic disorders. This study aimed to investigate the effect of intestinal perfusion of UAG on metabolically associated fatty liver disease (MAFLD) induced by a high-fat diet and its possible mechanisms. Neuronal retrograde tracking combined with immunofluorescence, central administration of a glucagon-like peptide-1 receptor (GLP-1R) antagonist, and hepatic vagotomy was performed to reveal its possible mechanism involving a central glucagon-like peptide-1 (GLP-1) pathway. The results showed that intestinal perfusion of UAG significantly reduced serum lipids, aminotransferases, and food intake in MAFLD rats. Steatosis and lipid accumulation in the liver were significantly alleviated, and lipid metabolism-related enzymes in the liver were regulated. UAG upregulated the expression of GLP-1 receptor (GLP-1R) in the paraventricular nucleus (PVN) and GLP-1 in the nucleus tractus solitarii (NTS), as well as activated GLP-1 neurons in the NTS. Furthermore, GLP-1 fibers projected from NTS to PVN were activated by the intestinal perfusion of UAG. However, hepatic vagotomy and GLP-1R antagonists delivered into PVN before intestinal perfusion of UAG partially attenuated its alleviation of MAFLD. In conclusion, intestinal perfusion of UAG showed a therapeutic effect on MAFLD, which might be related to its activation of the GLP-1 neuronal pathway from NTS to PVN. The present results provide a new strategy for the treatment of MAFLD.NEW & NOTEWORTHY Intestinal perfusion of UAG, the unacylated form of ghrelin, has shown promising potential for treating MAFLD. This study unveils a potential mechanism involving the central GLP-1 pathway, with UAG upregulating GLP-1R expression and activating GLP-1 neurons in specific brain regions. These findings propose a novel therapeutic strategy for MAFLD treatment through UAG and its modulation of the GLP-1 neuronal pathway.

Differential effect of endogenous glucagon-like peptide-1 on prandial glucose counterregulatory response to hypoglycaemia in humans with and without bariatric surgery.

To determine the effect of endogenous glucagon-like peptide 1 (GLP-1) on prandial counterregulatory response to hypoglycaemia after gastric bypass (GB). Glucose fluxes, and islet-cell and gut hormone responses before and after mixed-meal ingestion, were compared during a hyperinsulinaemic-hypoglycaemic (~3.2 mmol/L) clamp with and without a GLP-1 receptor (GLP-1R) antagonist exendin-(9-39) infusion in non-diabetic patients who had previously undergone GB compared to matched participants who had previously undergone sleeve gastrectomy (SG) and non-surgical controls. Exendin-(9-39) infusion raised prandial endogenous glucose production (EGP) response to insulin-induced hypoglycaemia in the GB group but had no consistent effect on EGP response among the SG group or non-surgical controls (p < 0.05 for interaction). The rates of systemic appearance of ingested glucose or prandial glucose utilization did not differ among the three groups or between studies with and without exendin-(9-39) infusion. Blockade of GLP-1R had no effect on insulin secretion or insulin action but enhanced prandial glucagon in all three groups. These results indicate that impaired post-meal glucose counterregulatory response to hypoglycaemia after GB is partly mediated by endogenous GLP-1, highlighting a novel pathogenic mechanism of GLP-1 in developing hypoglycaemia in this population.

Intestinal Acyl-CoA synthetase 5 (ACSL5) deficiency potentiates postprandial GLP-1 & PYY secretion, reduces food intake, and protects against diet-induced obesity

ObjectiveIn the small intestine, the products of digestion of dietary triacylglycerol (TAG), fatty acids (FA) and monoacylglycerol, are taken up by absorptive cells, enterocytes, for systemic energy delivery. These digestion products can also bind receptors on endocrine cells to stimulate the release of hormones capable of influencing systemic energy metabolism. The initial phase of intestinal FA absorption involves the acylation of FAs to acyl-CoA by the acyl-CoA long chain synthetase (ACSL) enzymes. ACSL5 is abundantly expressed in the small intestinal epithelium where it is the major ACSL isoform, contributing approximately 80% of total ACSL activity. In mice with whole body deficiency of ACSL5, the rate of dietary fat absorption is reduced and energy expenditure is increased. However, the mechanisms by which intestinal ACSL5 contributes to intestinal FA metabolism, enteroendocrine signaling, and regulation of energy expenditure remain undefined. Here, we test the hypothesis that intestinal ACSL5 regulates energy metabolism by influencing dietary fat absorption and enteroendocrine signaling. MethodsTo explore the role of intestinal ACSL5 in energy balance and intestinal dietary fat absorption, a novel mouse model of intestine specific ACSL5 deficiency (ACSL5IKO) was generated by breeding ACSL5 floxed (ACSL5loxP/loxP) to mice harboring the tamoxifen inducible, villin-Cre recombinase. ACSL5IKO and control, ACSL5loxP/loxP mice were fed chow (low in fat) or a 60% high fat diet (HFD), and metabolic phenotyping was performed including, body weight, body composition, insulin and glucose tolerance tests, energy expenditure, physical activity, and food intake studies. Pair-feeding studies were performed to determine the role of food intake in regulating development of obesity. Studies of dietary fat absorption, fecal lipid excretion, intestinal mucosal FA content, and circulating levels of glucagon like peptide 1 (GLP-1) and peptide YY (PYY) in response to a TAG challenge were performed. Treatment with a GLP-1 receptor antagonist was performed to determine the contribution of GLP-1 to acute regulation of food intake. ResultsWe found that ACSL5IKO mice experienced rapid and sustained protection from body weight and fat mass accumulation during HFD feeding. While intestine specific deficiency of ACSL5 delayed gastric emptying and reduced dietary fat secretion, it did not result in increased excretion of dietary lipid in feces. Energy expenditure and physical activity were not increased in ACSL5IKO mice. Mice deficient in intestinal ACSL5 display significantly reduced energy intake during HFD, but not chow feeding. When HFD intake of control mice was matched to ACSL5IKO during pair-feeding studies, no differences in body weight or fat mass gain were observed between groups. Postprandial GLP-1 and PYY were significantly elevated in ACSL5IKO mice secondary to increased FA content in the distal small intestine. Blockade of GLP-1 signaling by administration of a long-acting GLP-1 receptor antagonist partially restored HFD intake of ACSL5IKO. ConclusionsThese data indicate that intestinal ACSL5 serves as a critical regulator of energy balance, protecting mice from diet-induced obesity exclusively by increasing satiety and reducing food intake during HFD feeding. The reduction in food intake observed in ACSL5IKO mice is driven, in part, by increased postprandial GLP-1 and PYY secretion. These effects are only observed during HFD feeding, suggesting that altered processing of dietary fat following intestinal ACSL5 ablation contributes to GLP-1 and PYY mediated increases in satiety.

Gut Microbiota-Tryptophan Metabolism-GLP-1 Axis Participates in β-Cell Regeneration Induced by Dapagliflozin.

Sodium-glucose co-transporter 2 (SGLT2) inhibitor, an efficacious anti-diabetic agent, which has cardiovascular and renal benefits, can promote pancreatic β-cell regeneration in type 2 diabetic mice. However, the underlying mechanism remains unclear. In this study, we aimed to use multi-omics to identify the mediators involved in β-cell regeneration induced by dapagliflozin. We showed that dapagliflozin lowered blood glucose level, upregulated plasma insulin level, and increased islet area in db/db mice. Dapagliflozin reshaped gut microbiota, and modulated microbiotic and plasmatic metabolites related to tryptophan metabolism, especially L-tryptophan, in the diabetic mice. Notably, L-tryptophan upregulated the mRNA level of GLP-1 production-related genes (Gcg and Pcsk1) expression and promoted GLP-1 secretion in cultured mouse intestinal L-cells, and it increased supernatant insulin level in primary human islets, which was eliminated by GPR142 antagonist. Transplantation of fecal microbiota from dapagliflozin-treated mice, supplementation of L-tryptophan or treatment with dapagliflozin upregulated L-tryptophan, GLP-1, and insulin or C-peptide level, and promoted β-cell regeneration in db/db mice. Addition of exendin 9-39, a GLP-1 receptor (GLP-1R) antagonist, or pancreatic Glp1r knockout diminished these beneficial effects. In summary, treatment with dapagliflozin in type 2 diabetic mice promotes β-cell regeneration by upregulating GLP-1 production, which is mediated via gut microbiota and tryptophan metabolism.

GLP-1 and PYY for the treatment of obesity: a pilot study on the use of agonists and antagonists in diet-induced rats.

Combination therapies with gut hormone analogs represent promising treatment strategies for obesity. This pilot study investigates the therapeutic potential of modulators of the glucagon-like peptide 1 (GLP-1) and neuropeptide Y (NPY) system using GLP-1 receptor agonists (semaglutide) and antagonists (exendin 9-39), as well as non-selective and NPY-Y2-receptor selective peptide tyrosine tyrosine (PYY) analogs (PYY3-36/NNC0165-0020 and NNC0165-1273) and an NPY-Y2 receptor antagonist (JNJ31020028). High-fat diet (HFD)-induced obese rats were randomized into following treatment groups: group 1, nonselective PYY analog + semaglutide (n = 4); group 2, non-selective and NPY-Y2 receptor selective PYY analog + semaglutide (n = 2); group 3, GLP-1 receptor antagonist + NPY-Y2 receptor antagonist (n = 3); group 4, semaglutide (n = 5); and group 5, control (n = 5). Animals had free access to HFD and low-fat diet. Food intake, HFD preference and body weight were measured daily. A combinatory treatment with a non-selective PYY analog and semaglutide led to a maximum body weight loss of 14.0 ± 4.9% vs 9.9 ± 1.5% with semaglutide alone. Group 2 showed a maximum weight loss of 20.5 ± 2.4%. While HFD preference was decreased in group 2, a strong increase in HFD preference was detected in group 3. PYY analogs (especially NPY-Y2 selective receptor agonists) could represent a promising therapeutic approach for obesity in combination with GLP-1 receptor agonists. Additionally, combined GLP-1 and PYY3-36 receptor agonists might have beneficial effects on food preference.

The Key Role of Liraglutide in Preventing Autophagy of Vascular Smooth Muscle Cells in High Glucose Conditions

The glucagon-like peptide-1 (GLP-1) receptor agonist liraglutide (LIRA) is a potential hypoglycemic drug with anti-atherosclerosis (AS) effects. Autophagy in the vascular smooth muscle cells (VSMCs) facilitates AS. However, the role of autophagy in the anti-AS mechanism of LIRA remains unclear. To examine the role and mechanisms of autophagy in LIRA’s improvement of the biological characteristics of VSMCs in high glucose conditions. Experimental animal study. VSMCs isolated from the thoracic aorta of male SD rats were subjected to a high glucose (HG) condition (25 mM) in Dulbecco’s Modified Eagle’s Medium with or without LIRA, the GLP-1 receptor antagonist exendin9-39 (Exe9-39), a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY294002), and autophagy inhibitors (3-methyladenine [3-MA] and bafilomycin A1 [Baf A1]). Acridine orange staining, western blotting, transmission electron microscopy, and mCherry-GFP-LC3 transfection were performed to evaluate the autophagy flux. Additionally, VSMC migration, calcification, proliferation, and apoptosis in HG conditions were observed. Addition of LIRA alone or in combination with autophagy inhibitors significantly downregulated Beclin, increased the LC3-II/LC3-I ratio, and upregulated p62 in VSMCs in HG conditions. Furthermore, autophagolysosome formation was markedly curbed after treatment with LIRA and/or autophagy inhibitors. Inhibition of autophagy by LIRA and/or the autophagy inhibitors attenuated VSMC phenotype conversion, proliferation, migration, and calcification and promoted VSMC apoptosis in HG conditions. This protective role of LIRA was augmented by LY294002, but inhibited by Exe9-39. LIRA plays a significant role in the improvement of the biological features of VSMCs in HG conditions.

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.

Trends in the epidemiology of chronic kidney disease in patients with diabetes in Russian Federation according to the Federal diabetes register (2010–2022)

BACKGROUND: Chronic kidney disease (CKD) in diabetes mellitus (DM) is a supranosological concept that characterizes multifactorial kidney damage associated with increased cardiovascular and mortality risk, which determines the high medical and social significance of this problem in diabetic patients.AIMS: To assess the clinical and epidemiological characteristics of CKD in adult DM patients with type 1(T1) and type 2 (T2) in Russian Federation (RF) in 2010–2022 according to the Federal Register of Diabetes Mellitus (FDR) and to present the capabilities of the register’s analytical tools for assessing organ-protective therapy and predicting the risk of pathology.MATERIALS AND METHODS: We have used the database of FRD (http://diaregistry.ru), 85 regions of the RF. The data are presented as of 01.01.2023 and in dynamics for the period 2010–2022.RESULTS: The CKD prevalence in adult DM patients in RF in dynamics 2010→2022 showed in an increase in the rate for T1 from 21.5 to 27.1% (1.3 times), for T2 from 5.2 to 19.1% (3.7 times). The incidence of new CKD cases was 153.3→106.3/10 000 adult patients in T1, and 64.3→212.8/10 000 adult patients in T2. The analysis of the distribution by CKD stage indicates improved diagnosis of the complication. In the structure of new cases of CKD in the dynamics of 2010→2022. The proportions of patients with low and moderate combined risk of cardiovascular events and end-stage renal failure according to KDIGO criteria increased for T1 63.7→82.4%, for T2 64.5→77.4%. The proportions of patients with very high risk progressively decreased for T1 12.3→4.0%, for T2 13.1→1.6%. The average age of onset of CKD increased by an average of 6 years in persons with type 1 and type 2 diabetes (35.6→42 years, 63.3→69.3 years, respectively), with the dynamics of the average DM duration at the time of CKD development: in T1 11.5→14.8 years, in T2 7.4→7.8 years. The cause of terminal CKD in the structure of mortality in DM patients took only in T1 patients 5.6% and in T2D — 2.0%. A retrospective analysis of factors influencing the fatal outcome of DM patients with COVID-19 showed the significance of a history of CKD in T2DM patients, which increases the risk of death by 1.49 times (95% CI 1.01–2.04). Analysis of the structure of glucose-lowering therapy in T2DM patients and CKD indicates a more frequent prescription of drugs from the group of SGLT-2 inhibitors, DPP-4 inhibitors, and GLP-1 receptor antagonists compared to the general cohort of T2DM. There is the CKD prognosis calculator in the FRD, which allows assessing the risk of developing of pathology within 5 years in a particular patient based on a set of the most significant predictors, which included 6 factors for T1 and 11 factors for T2. CONCLUSIONS: Epidemiological trends in the prevalence of CKD over a 13-year period indicate the growing importance of this problem in DM patients who are at risk. Along with the positive trends in the development of pathology with a longer duration of diabetes, in clinical practice the problem remains of untimely detection of CKD with impaired renal function at advanced stages. The progressive nature of the course and the negative impact of kidney damage on the risks of premature mortality in patients determine the priority of preventive diagnostic and therapeutic strategies aimed at compliance with the standard of examination, detection of pathology in the early stages and a multifactorial approach to nephroprotection, according to clinical recommendations.

Microbial dietary protein metabolism regulates GLP-1 mediated intestinal transit.

Depletion of gut microbiota is associated with inefficient energy extraction and reduced production of short-chain fatty acids from dietary fibers, which regulates colonic proglucagon (Gcg) expression and small intestinal transit in mice. However, the mechanism by which the gut microbiota influences dietary protein metabolism and its corresponding effect on the host physiology is poorly understood. Enteropeptidase inhibitors block host protein digestion and reduce body weight gain in diet-induced obese rats and mice, and therefore they constitute a new class of drugs for targeting metabolic diseases. Enteroendocrine cells (EECs) are dispersed throughout the gut and possess the ability to sense dietary proteins and protein-derived metabolites. Despite this, it remains unclear if enteropeptidase inhibition affects EECs function. In this study, we fed conventional and antibiotic treated mice a western style diet (WSD) supplemented with an enteropeptidase inhibitor (WSD-ETPi), analyzed the expression of gut hormones along the length of the intestine, and measured small intestinal transit under different conditions. The ETPi-supplemented diet promoted higher Gcg expression in the colon and increased circulating Glucagon like peptide-1(GLP-1) levels, but only in the microbiota-depleted mice. The increase in GLP-1 levels resulted in slower small intestinal transit, which was subsequently reversed by administration of GLP-1 receptor antagonist. Interestingly, small intestinal transit was normalized when an amino acid-derived microbial metabolite, p-cresol, was supplemented along with WSD-ETPi diet, primarily attributed to the reduction of colonic Gcg expression. Collectively, our data suggest that microbial dietary protein metabolism plays an important role in host physiology by regulating GLP-1-mediated intestinal transit.

Effects of liraglutide on ANP secretion and cardiac dynamics.

To observe the effects of liraglutide (analog of glucagon-like peptide 1 (GLP-1)) on atrial natriuretic peptide (ANP) secretion and atrial dynamics, an ex vivo isolated rat atrial perfusion model was used to determine atrial ANP secretion and pulse pressure. DPP-4-/- mice were also established in vivo. ANP levels were determined by radioimmunoassay; GLP-1 content was determined by Elisa. The expression levels of GLP-1 receptor (GLP-1R), PI3K/AKT/mTOR, piezo 1, and cathepsin K were analyzed by Western blot. In the clinical study, patients with acute coronary syndrome (ACS) had low levels of plasma GLP-1 but relatively high levels of plasma ANP. In ex vivo (3.2 nmol/L) and in vivo (30 μg/kg) models, liraglutide significantly decreased ANP levels and atrial pulse pressure. Exendin9-39 alone (GLP-1R antagonist) reversibly significantly increased ANP secretion, and the reduction effect of liraglutide on the secretion of ANP was significantly alleviated by Exendin9-39. Exendin9-39 demonstrated slightly decreased atrial pulse pressure; however, combined liraglutide and Exendin9-39 significantly decreased atrial pulse pressure. Ly294002 (PI3K/AKT inhibitor) inhibited the increase of ANP secretion by liraglutide for a short time, while Ly294002 didn't counteract the decrease in pulse pressure by liraglutide in atrial dynamics studies. Liraglutide increased the expression of GLP-1R and PI3K/AKT/mTOR in isolated rat atria and the hearts of mice in vivo, whereas Exendin9-39 reversibly reduced the expression of GLP-1R and PI3K/AKT/mTOR. Piezo 1 was significantly decreased in wild type and DPP-4-/- mouse heart or isolated rat atria after being treated with liraglutide. Cathepsin K expression was only decreased in in vivo model hearts. Liraglutide can inhibit ANP secretion while decreasing atrial pulse pressure mediated by GLP-1R. Liraglutide probably plays a role in the reduction of ANP secretion via the PI3K/AKT/mTOR signaling pathway. Piezo 1 and cathepsin K may be involved in the liraglutide mechanism of reduction.

Exendin-4 increases the firing activity of hippocampal CA1 neurons through TRPC4/5 channels

The central neuropeptide GLP-1 is synthesized by preproglucagon (PPG) neurons in the brain. GLP-1 receptors are widely distributed in central nervous system. Hippocampus is a key component of the limbic system which is involved in learning, memory, and cognition. Previous studies have shown that overexpression of GLP-1 receptors in the hippocampus could improve the process of learning and memory. However, up to now, the direct electrophysiological effects and possible molecular mechanisms of GLP-1 in hippocampal CAl neurons remain unexplored. The present study aims to evaluate the effects and mechanisms of GLP-1 on the spontaneous firing activity of hippocampal CAl neurons. Employing multibarrel single-unit extracellular recordings, the present study showed that micro-pressure administration of GLP-1 receptor agonist, exendin-4, significantly increased the spontaneous firing rate of hippocampal CA1 neurons in rats. Furthermore, application of the specific GLP-1 receptor antagonist, exendin(9−39), alone significantly decreased the firing rate of CA1 neurons, suggesting that endogenous GLP-1 modulates the firing activity of CA1 neurons. Co-application of exendin(9−39) completely blocked exendin-4-induced excitation of hippocampal CA1 neurons. Finally, the present study demonstrated for the first time that the transient receptor potential canonical 4 (TRPC4)/TRPC5 channels may be involved in exendin-4-induced excitation. The present studies may provide a rationale for further investigation of the modulation of GLP-1 on learning and memory as well as its possible involvement in Alzheimer's disease.

Clinical utility of a novel test for assessing cardiovascular disease risk in type 2 diabetes: a randomized controlled trial

BackgroundThe risk for and treatment of cardiovascular disease (CVD) in type 2 diabetes (T2DM) is often incorrect and delayed. We wished to determine if a novel test improved physicians’ ability to risk stratify, diagnose, and treat patients with T2DM.MethodsIn a 2-phase randomized controlled trial comparing the clinical workup, diagnosis, and management of online, simulated patients with T2DM in a nationwide sample of cardiologists and primary care physicians, participants were randomly assigned to control or one of two intervention groups. Intervention participants had access to standard of care diagnostic tools plus a novel diagnostic CVD risk stratification test.ResultsIn control, there was no change in CV risk stratification of simulated patients between baseline and round 2 (37.1 to 38.3%, p = 0.778). Pre-post analysis showed significant improvements in risk stratification in both Intervention 1 (38.7 to 65.3%) and Intervention 2 (41.9 to 65.8%) (p < 0.01) compared to controls. Both intervention groups significantly increased prescribing SGLT2 inhibitors/GLP1 receptor agonists versus control, + 18.9% for Intervention 1 (p = 0.020) and 1 + 9.4% for Intervention 2 (p = 0.014). Non-pharmacologic treatment improved significantly compared to control (+ 30.0% in Intervention 1 (p < 0.001) and + 22.8% in Intervention 2 (p = 0.001). Finally, monitoring HgbA1C, blood pressure, and follow-up visit frequency improved by + 20.3% (p = 0.004) in Intervention 1 and + 29.8% (p < 0.001) in Intervention 2 compared with control.ConclusionUse of the novel test significantly improved CV risk stratification among T2DM patients. Statistically significant increases treatments were demonstrated, specifically SGLT2 inhibitors and GLP1 receptor antagonists and recommendations of evidence-based non-pharmacologic treatments.Trial registration ClinicalTrials.gov, NCT05237271

Mechanism of preventive effects of exendin-4 and des-fluoro-sitagliptin in a murine model of fructose-induced prediabetes

Protective effects of exendin-4 (glucagon-like peptide-1 -GLP-1- receptor agonist) and des-fluoro-sitagliptin (dipeptidyl peptidase-4 inhibitor) on fructose-induced hepatic disturbances were evaluated in prediabetic rats. Complementary, a possible direct effect of exendin-4 in human hepatoblastoma-derived cell line HepG2 incubated with fructose in presence/absence of exendin-9-39 (GLP-1 receptor antagonist) was investigated. In vivo, after 21 days of fructose rich diet, we determined: glycemia, insulinemia, and triglyceridemia; hepatic fructokinase, AMP-deaminase, and G-6-P dehydrogenase (G-6-P DH) activities; carbohydrate-responsive element-binding protein (ChREBP) expression; triglyceride content and lipogenic gene expression (glycerol-3-phosphate acyltransferase -GPAT-, fatty acid synthase -FAS-, sterol regulatory element-binding protein-1c -SREBP-1c); oxidative stress and inflammatory markers expression. In HepG2 cells we measured fructokinase activity and triglyceride content. Hypertriglyceridemia, hyperinsulinemia, enhanced liver fructokinase, AMP-deaminase, and G-6-P DH activities, increased ChREBP and lipogenic genes expression, enhanced triglyceride level, oxidative stress and inflammatory markers recorded in fructose fed animals, were prevented by co-administration of either exendin-4 or des-fluoro-sitagliptin. Exendin-4 prevented fructose-induced increase in fructokinase activity and triglyceride contain in HepG2 cells. These effects were blunted co-incubating with exendin-9-39. The results demonstrated for the first time that exendin-4/des-fluro-sitagliptin prevented fructose-induced endocrine-metabolic oxidative stress and inflammatory changes probably acting on the purine degradation pathway. Exendin 9–39 blunted in vitro protective exendin-4 effects, thereby suggesting a direct effect of this compound on hepatocytes through GLP-1 receptor. Direct effect on fructokinase and AMP-deaminase activities, with a key role in the pathogenesis of liver dysfunction induced by fructose, suggests purine degradation pathway constitute a potential therapeutic objective for GLP-1 receptor agonists.

Optimization of a glucagon-like peptide 1 receptor antagonist antibody for treatment of hyperinsulinism.

Congenital hyperinsulinism (HI) is a genetic disorder in which pancreatic beta cell insulin secretion is excessive and results in hypoglycemia that can cause brain damage or death without treatment. Most patients with loss-of-function mutations in ABCC8 and KCNJ11, the genes encoding the beta cell ATP sensitive potassium channel (KATP), are unresponsive to diazoxide, the only FDA approved medical therapy, and require a pancreatectomy. The glucagon-like peptide 1 receptor (GLP-1R) antagonist exendin-(9-39) is an effective therapeutic agent to inhibit insulin secretion in both congenital and acquired hyperinsulinism. Previously, we identified a highly potent antagonist antibody, TB-001-003, which was derived from our synthetic antibody libraries that were designed to target G protein-coupled receptors. Here, we designed a combinatorial variant antibody library to optimize the activity of TB-001-003 against GLP-1R and performed phage-display on cells overexpressing GLP-1R. One antagonist, TB-222-023, is more potent than exendin-(9-39), also known as Avexitide. TB-222-023 effectively decreased insulin secretion in primary isolated pancreatic islets from a mouse model of hyperinsulinism, Sur1-/- mice, and in islets from an infant with HI as well as increased plasma glucose and decreased the insulin to glucose ratio in Sur1-/- mice. These findings demonstrate that targeting GLP-1R with an antibody antagonist is an effective and innovative strategy for treatment of hyperinsulinism.

842-P: Development of Long-Acting GLP-1 Receptor Antagonist for the Treatment of Congenital Hyperinsulinism

Targeting beta-cell GLP-1 receptors has proven to be a viable strategy for the treatment of congenital hyperinsulinism (CHI), especially CHI due to loss of function mutations of KATP channels. Currently, Phase II clinical study of the GLP-1 receptor antagonist, Exendin-(9-39) has been successfully initiated. However, Exendin-(9-39) has a short half-life (less than 2 hrs), which limits its clinical applications. We are therefore trying to develop a long-acting GLP-1 receptor antagonist. AR-GLP1R-01 was synthesized based on chemical modification of Exendin-9-39. In order to test its efficacy in disease mouse models, SUR1 knockout mice was created by targeted deletion of abcc8 gene using CRISPR/Cas9. Exendin-9-39 and AR-GLP1R-01 were injected subcutaneously at doses of 216 nmol/kg. Plasma concentrations were determined by UPLC-MS at 30 min, 2 hrs and 10 hrs after injection. Plasma levels of Exendin-9-39 were 0.5 µM at 30 min after injection and were later undetectable at 2 and 10 hrs. In contrast, plasma levels of AR-GLP1R-01 were 2.0 µM at 30 min, peaked at 2 hrs, and remained at 2.9 µM after 10 hrs of injection. Compared to Exendin-(9-39), AR-GLP1R-01 has similar inhibitory effects in amino acid mixture-stimulated insulin secretion in perifused islets isolated from SUR1 knockout mice. In vivo experiments were performed by subcutaneous injection at a dosage of 216 nmol/kg twice a day at 10 am and 5 pm for 4 days. Similar to Exendin-9-39, AR-GLP1R-01 caused plasma glucose to increase 1 hr after injection. However, the glucose-increasing effects of AR-GLP1R-01 were maintained overnight before the 10 am injection for 4 days, while the effects of Exendin-9-39 failed to do so. In the separate experiments, the effects of AR-GLP1R-01 on glucose elevation can last for 24 hrs after a single dose injection. This early phase “proof of concept” study has shown that AR-GLP1R-01 is a long-acting GLP-1 antagonist and has potential to be developed into a once-a-day injection for the treatment of CHI. Disclosure Y.Yuan: Employee; Nanjing AscendRare Pharmaceutical Technology. H.Jiang: None. S.Meng: Employee; Nanjing AscendRare Pharmaceutical Technology. D.Han: Employee; Nanjing AscendRare Pharmaceutical Technology. Q.Zhu: Employee; Nanjing AscendRare Pharmaceutical Technology. M.Wang: Employee; Shandong Baixinyuan Pharmaceutical Co. LLC.. H.Li: Employee; Shandong Baixinyuan Pharmaceutical Co. LLC.. J.Li: None. C.Li: Employee; Hua Medicine, Nanjing AscendRare Pharmaceutical Technology. Funding Shandong Innovation Project (2019JZZY021012)

Liraglutide ameliorates oxidized LDL-induced endothelial dysfunction by GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation

ABSTRACTObjectiveTo investigate the effects of glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide on endothelial dysfunction in LDL receptor-deficient (LDLR-KO) mice and ox-LDL-challenged human umbilical vein endothelial cells (HUVECs) and its possible mechanism.MethodsLDLR-KO mice were randomly treated with normal saline, liraglutide, or liraglutide plus a GLP-1R antagonist exendin-9 for four weeks. In parallel, HUVECs were cultured with ox-LDL alone or combined with liraglutide, in the presence or absence of lectin-like ox-LDL receptor-1(LOX-1) overexpression or GLP-1R knockdown. Endothelial-dependent relaxation and LOX-1 protein expression of thoracic aorta, circulating levels of oxidative and inflammatory markers in mice, and cell survival, reactive oxygen species production, and expression of adhesion molecules and signal regulators in ox-LDL cultured endothelial cells were measured.Resultsliraglutide effectively enhanced acetylcholine-induced vasodilation, reduced LOX-1 expression in aortas, and decreased circulatory oxidative and inflammatory levels in LDLR-KO mice, which were abolished by cotreatment with exendin-9. HUVECs exposed to ox-LDL exhibited reduced cell viability, increased reactive oxygen species production and apoptosis, and elevated protein expression of ICAM-1, VCAM-1, LOX-1, NOX4, and NF-κB, which were markedly ameliorated by liraglutide treatment. The protective effects of liraglutide against ox-LDL-induced cell injury were abrogated in HUVECs overexpressing LOX-1 or silencing GLP-1R.ConclusionsLiraglutide improved oxidized LDL-induced endothelial dysfunction via GLP-1R-dependent downregulation of LOX-1-mediated oxidative stress and inflammation.

Glucagon-like peptide-1 facilitates cerebellar parallel fiber glutamate release through PKA signaling in mice in vitro

Glucagon-like peptide-1 (GLP-1) is mainly secreted by preproglucagon neurons; it plays important roles in modulating neuronal activity and synaptic transmission through its receptors. In the present study, we investigated the effects of GLP-1 on parallel fiber–Purkinje cell (PF-PC) synaptic transmission in mouse cerebellar slices using whole-cell patch-clamp recording and pharmacology methods. In the presence of a γ-aminobutyric acid type A receptor antagonist, bath application of GLP-1 (100 nM) enhanced PF-PC synaptic transmission, with an increased amplitude of evoked excitatory postsynaptic synaptic currents (EPSCs) and a decreased paired-pulse ratio. The GLP-1-induced enhancement of evoked EPSCs was abolished by a selective GLP-1 receptor antagonist, exendin 9–39, as well as by the extracellular application of a specific protein kinase A (PKA) inhibitor, KT5720. In contrast, inhibiting postsynaptic PKA with a protein kinase inhibitor peptide-containing internal solution failed to block the GLP-1-induced enhancement of evoked EPSCs. In the presence of a mixture of gabazine (20 μM) and tetrodotoxin (1 μM), application GLP-1 significantly increased frequency, but not amplitude of miniature EPSCs via PKA signaling pathway. The GLP-1-induced increase in miniature EPSC frequency was blocked by both exendin 9–39 and KT5720. Together, our results indicate that GLP-1 receptor activation enhances glutamate release at PF-PC synapses via the PKA signaling pathway, resulting in enhanced PF-PC synaptic transmission in mice in vitro. These findings suggest that, in living animals, GLP-1 has a critical role in the modulation of cerebellar function by regulating excitatory synaptic transmission at PF-PC synapses.

The local GLP-1 system in the olfactory bulb is required for odor-evoked cephalic phase of insulin release in mice

ObjectiveThe olfactory bulb (OB) codes for sensory information and contributes to the control of energy metabolism by regulating foraging and cephalic phase responses. Mitral cells are the main output neurons of the OB. The glucagon-like peptide-1 (GLP-1)/GLP-1 receptor (GLP-1R) system in the OB (GLP-1OB) has been shown to be a major regulator of mitral cell activity but its function in vivo is unclear. Therefore, we investigated the role of GLP-1OB in foraging behavior and odor-evoked Cephalic Phase Insulin Release (CPIR). Methods and resultsBy fluorescent labeling, we confirmed the presence of GLP-1 producing neurons and the expression of GLP-1R in the mouse OB. In response to food odor presentation, we collected blood, quantified plasma insulin by ELISA and showed the existence of an odor-evoked CPIR in lean mice but its absence in obese animals. Expression of shRNA against preproglucagon mRNA in the OB resulted in blunted CPIR in lean mice. Injecting Exendin (9-39), a GLP-1R antagonist, into the OB of lean mice also resulted in decreased CPIR. Since parasympathetic cholinergic input to the pancreas is known to be partly responsible for CPIR, we systemically administered the muscarinic M3 receptor antagonist 4-DAMP which resulted in a reduced odor-evoked CPIR. Finally, local injection of Exendin (9-39) in the OB extinguished olfactory foraging in lean mice whereas the injection of the GLP-1R agonist Exendin-4 rescued the loss of foraging behavior in obese mice. ConclusionsOur results demonstrate that GLP-1OB controls olfactory foraging and is required for odor-evoked CPIR. We describe a new crucial brain function for GLP-1 and GLP-1R expressed within the brain.

Acarbose diminishes postprandial suppression of bone resorption in patients with type 2 diabetes.

The alpha-glucosidase inhibitor acarbose is an antidiabetic drug delaying assimilation of carbohydrates and, thus, increasing the amount of carbohydrates in the distal parts of the intestines, which in turn increases circulating levels of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1). As GLP-1 may suppress bone resorption, acarbose has been proposed to potentiate meal-induced suppression of bone resorption. We investigated the effect of acarbose treatment on postprandial bone resorption in patients with type 2 diabetes and used the GLP-1 receptor antagonist exendin(9-39)NH2 to disclose contributory effect of acarbose-induced GLP-1 secretion. In a randomised, placebo-controlled, double-blind, crossover study, 15 participants with metformin-treated type 2 diabetes (2 women/13 men, age 71 (57-85years), BMI 29.7 (23.6-34.6kg/m2), HbA1c 48 (40-74mmol/mol)/6.5 (5.8-11.6%) (median and range)) were subjected to two 14-day treatment periods with acarbose and placebo, respectively, separated by a six-week wash-out period. At the end of each period, circulating bone formation and resorption markers were assessed during two randomised 4-h liquid mixed meal tests (MMT) with infusions of exendin(9-39)NH2 and saline, respectively. Glucagon-like peptide 2 (GLP-2) was also assessed. Compared to placebo, acarbose impaired the MMT-induced suppression of CTX as assessed by baseline-subtracted area under curve (P=0.0037) and nadir of CTX (P=0.0128). During acarbose treatment, exendin(9-39)NH2 infusion lowered nadir of CTX compared to saline (P=0.0344). Neither parathyroid hormone or the bone formation marker procollagen 1 intact N-terminal propeptide were affected by acarbose or GLP-1 receptor antagonism. Acarbose treatment induced a greater postprandial GLP-2 response than placebo treatment (P=0.0479) and exendin(9-39)NH2 infusion exacerbated this (P=0.0002). In patients with type 2 diabetes, treatment with acarbose reduced postprandial suppression of bone resorption. Acarbose-induced GLP-1 secretion may contribute to this phenomenon as the impairment was partially reversed by GLP-1 receptor antagonism. Also, acarbose-induced reductions in other factors reducing bone resorption, e.g. glucose-dependent insulinotropic polypeptide, may contribute.