Islet Glucagon-like Peptide-1 Receptor Research Articles

Oleanolic acid and moderate drinking increase the pancreatic GLP-1R expression of the β-cell mass deficiency induced hyperglycemia.

Oleanolic acid (OA) and moderate drinking have been reported to attenuate diabetes. However, the underlying mechanism of OA and moderate drinking alone or in combination on the islet β-cell deficiency induced diabetes is not fully elucidated. Male Sprague Dawley (SD) rats were intraperitoneally injected with 55 mg/kg streptozotocin (STZ) to induce β-cell deficiency. OA, 5% ethanol (EtOH), or a mixture of OA in 5% ethanol (OA+EtOH) were applied to three treatment groups of hyperglycemia rats for 6 weeks. STZ caused the increase of fast blood glucose (FBG) level.OA and EtOH treatment alone or in combination decreased the STZ increased FBG level during the 6 weeks of treatment. In addition, OA treatment also significantly increased the β-cell to total islet cell ratio. Both EtOH and OA+EtOH treatments promoted the increase of total islet cell number and α-cell to β-cell ratio when compared to OA group. STZ induced hyperglycemia dramatically reduced the glucagon-like peptide-1 receptor (GLP-1R) positive cells in islets, all the three treatments significantly increased the pancreatic GLP-1R positive cell number. In the meantime, STZ induced hyperglycemia suppressed the insulin mRNA expression and boosted the glucagon mRNA expression. EtOH and OA+EtOH treatments increased the insulin mRNA expression, but none of the 3 treatments altered the elevated glucagon level. GLP-1R positive cell ratio in islets is crucial for the blood glucose level of diabetes. OA and 5% ethanol alone or in combination suppresses the blood glucose level of β-cell deficiency induced diabetes by increasing islet GLP-1R expression.

2113-P: Discrepancy between Single-Cell RNA Sequencing and Protein Expression Assessments of GLP-1Rr Heterogeneity in Beta Cells

The glucagon-like peptide 1 receptor (GLP-1R) is insulinotropic in β-cells and the target of multiple classes of diabetes drugs. People with T2D secrete normal amounts of GLP-1, but are less responsive to its actions; the mechanisms for this are unknown. Studies have shown that metabolic stress decreases GLP-1R in islets, however, it is unknown if this occurs in all, or just a subset, of β-cells. We hypothesized that GLP-1R expression is heterogeneous in β-cells, such that not all β-cells express the GLP-1R, and that metabolic stress would decrease the number of GLP-1R+ β-cells. To test this, we first performed single-cell RNA sequencing (scRNAseq) in mouse and human islet cells and found that the majority of β-cells were GLP-1R negative, supporting significant heterogeneity. To validate the scRNAseq results, we generated a GLP-1R reporter mouse by crossing GLP-1R-Cre with mTmG mice. Using FACS to separate and qPCR to characterize positive (GFP+) from negative (tdTomato+) dispersed islet cells, we found GLP-1R+ cells were enriched for Ins2 and Sst, while GLP-1R- cells were enriched for Gcg, suggesting GLP-1R promoter activity is localized to β- and d-cells. In addition, β-cell markers were not present in the tdTomato+ population, suggesting very few GLP-1R negative β-cells. Next, we stained dispersed islets from the reporter mice with a validated GLP-1R antibody and found >90% congruence with GFP+ cells and no staining in tdTomato+ cells. In dispersed islets from wild type mice, ∼90% of β-cells were GLP-1R+; very few of the α- or d-cells were GLP-1R+. Metabolic stress induced through chronic high-fat feeding or using multiparous models did not alter the number of GLP-1R+ β-cells. In conclusion, assessment of GLP-1R expression and GLP-1R protein revealed the majority of β-cells are GLP-1R+, a finding that conflicts with the interpretation of scRNAseq data. These findings suggest caution in interpreting the results of scRNAseq for low abundant transcripts such as the GLP-1R. Disclosure S.M. Gray: None. B.M. Chazotte: None. E.C. Ross: None. B. Svendsen: None. P. Ravn: Employee; Self; AstraZeneca. K. Sloop: None. J. Campbell: Research Support; Self; Eli Lilly and Company, Novo Nordisk Inc. Speaker’s Bureau; Self; Merck Sharp & Dohme Corp. D. D’Alessio: Advisory Panel; Self; Eli Lilly and Company. Consultant; Self; Intarcia Therapeutics. Research Support; Self; Ansh Labs, Eli Lilly and Company, Merck Sharp & Dohme Corp. Other Relationship; Self; Novo Nordisk A/S.

MiR-204 Antagomir Enhances GLP-1 Receptor Function and Glucose Control

Glucagon-like peptide 1 receptor (GLP-1R) is a G protein-coupled receptor that is highly expressed on pancreatic beta-cells and activated by endogenous incretins or antidiabetic GLP-1R agonist drugs. GLP-1R function is important in maintaining glucose homeostasis and involves stimulation of glucose-induced beta-cell insulin secretion via cAMP generation. While GLP-1R expression has been shown to be downregulated in diabetes, the molecular mechanisms have not been fully elucidated. In this study, we have discovered that miR-204, a highly beta-cell-enriched microRNA that is upregulated in diabetes, directly targets the 3’ UTR of GLP-1R and decreases its expression in INS-1 cells as well as primary mouse and human islets. Moreover, genetic knockout of miR-204 in vivo increased islet GLP-1R expression and enhanced GLP-1R agonist-induced cAMP production and insulin secretion, resulting in improved glucose tolerance as well as protection against diabetes. In addition, miR-204 antagomir administration was able to mimic the effects of genetic miR-204 deletion and resulted again in GLP-1R agonist-induced insulin secretion as well as improved glucose tolerance. Thus, these results indicate for the first time that GLP-1R is under the control of a micro RNA and suggest that antagomir-mediated inhibition of miR-204 represents a potential novel therapeutic approach to promote the actions of GLP-1R agonist drugs in a beta-cell-specific manner and thereby help treat diabetes. Disclosure S. Jo: None. J. Chen: None. G. Xu: None. T. Grayson: None. L. Thielen: None. A. Shalev: None.

MiR-204 Controls Glucagon-Like Peptide 1 Receptor Expression and Agonist Function.

Glucagon-like peptide 1 receptor (GLP1R) agonists are widely used to treat diabetes. However, their function is dependent on adequate GLP1R expression, which is downregulated in diabetes. GLP1R is highly expressed on pancreatic β-cells, and activation by endogenous incretin or GLP1R agonists increases cAMP generation, which stimulates glucose-induced β-cell insulin secretion and helps maintain glucose homeostasis. We now have discovered that the highly β-cell–enriched microRNA, miR-204, directly targets the 3′ UTR of GLP1R and thereby downregulates its expression in the β-cell–derived rat INS-1 cell line and primary mouse and human islets. Furthermore, in vivo deletion of miR-204 promoted islet GLP1R expression and enhanced responsiveness to GLP1R agonists, resulting in improved glucose tolerance, cAMP production, and insulin secretion as well as protection against diabetes. Since we recently identified thioredoxin-interacting protein (TXNIP) as an upstream regulator of miR-204, we also assessed whether in vivo deletion of TXNIP could mimic that of miR-204. Indeed, it also enhanced islet GLP1R expression and GLP1R agonist–induced insulin secretion and glucose tolerance. Thus, the present studies show for the first time that GLP1R is under the control of a microRNA, miR-204, and uncover a previously unappreciated link between TXNIP and incretin action.

Upregulated insulin secretion in insulin-resistant mice: evidence of increased islet GLP1 receptor levels and GPR119-activated GLP1 secretion

We previously demonstrated that the overall incretin effect and the β-cell responsiveness to glucagon-like peptide-1 (GLP1) are increased in insulin-resistant mice and may contribute to the upregulated β-cell function. Now we examined whether this could, first, be explained by increased islet GLP1 receptor (GLP1R) protein levels and, secondly, be leveraged by G-protein-coupled receptor 119 (GPR119) activation, which stimulates GLP1 secretion. Female C57BL/6J mice, fed a control (CD, 10% fat) or high-fat (HFD, 60% fat) diet for 8 weeks, were anesthetized and orally given a GPR119 receptor agonist (GSK706A; 10 mg/kg) or vehicle, followed after 10 min with gavage with a liquid mixed meal (0.285 kcal). Blood was sampled for determination of glucose, insulin, intact GLP1, and glucagon, and islets were isolated for studies on insulin and glucagon secretion and GLP1R protein levels. In HFD vs CD mice, GPR119 activation augmented the meal-induced increase in the release of both GLP1 (AUCGLP1 81±9.6 vs 37±6.9 pM×min, P=0.002) and insulin (AUCINS 253±29 vs 112±19 nM×min, P<0.001). GPR119 activation also significantly increased glucagon levels in both groups (P<0.01) with, however, no difference between the groups. By contrast, GPR119 activation did not affect islet hormone secretion from isolated islets. Glucose elimination after meal ingestion was significantly increased by GPR119 activation in HFD mice (0.57±0.04 vs 0.43±0.03% per min, P=0.014) but not in control mice. Islet GLP1R protein levels was higher in HFD vs CD mice (0.8±0.1 vs 0.5±0.1, P=0.035). In conclusion, insulin-resistant mice display increased islet GLP1R protein levels and augmented meal-induced GLP1 and insulin responses to GPR119 activation, which results in increased glucose elimination. We suggest that the increased islet GLP1R protein levels together with the increased GLP1 release may contribute to the upregulated β-cell function in insulin resistance.

Glucagon-like peptide-1 (GLP-1) receptors are not overexpressed in pancreatic islets from patients with severe hyperinsulinaemic hypoglycaemia following gastric bypass

Glucagon-like peptide-1 (GLP-1) receptors are highly overexpressed in benign insulinomas, permitting in vivo tumour visualisation with GLP-1 receptor scanning. The present study sought to evaluate the GLP-1 receptor status in vitro in other pancreatic disorders leading to hyperinsulinaemic hypoglycaemia, specifically after gastric bypass surgery. Fresh frozen pancreatic tissue samples (n=7) from six gastric bypass surgery patients suffering from hyperinsulinaemic hypoglycaemia were evaluated for GLP-1 receptor content using in vitro receptor autoradiography, and compared with normal pancreas and with pancreatic insulinoma tissues. GLP-1 receptor analysis of the pancreatic tissues, which histopathologically were compatible with nesidioblastosis and originated from post-bypass hypoglycaemic patients, revealed a mean density value of GLP-1 receptors in the islets of 1,483 ± 183 dpm/mg tissue. Pharmacological characterisation indicated the presence of specific GLP-1 receptors. The density of islet GLP-1 receptor in post-gastric bypass patients did not differ from that of normal pancreas (1,563 ± 104 dpm/mg tissue, n = 10). Receptor density in pancreatic acini was low in post-bypass and control conditions. In contrast, benign insulinomas showed a high density of GLP-1 receptors, with a mean value of 8,302 ± 1,073 dpm/mg tissue (n = 6). In contrast to insulinoma, hyperinsulinaemic hypoglycaemia after gastric bypass surgery is not accompanied by overexpression of GLP-1 receptor in individual islets. Thus, patients with post-gastric bypass hyperinsulinaemic hypoglycaemia are not candidates for GLP-1 receptor imaging in vivo using radiolabelled exendin. These GLP-1 receptor data support the notion that the islet pathobiology of post-gastric bypass hypoglycaemia is distinctly different from that of benign insulinomas.

Expression of the GLP-1 Receptor in Mouse, Rat, and Human Pancreas

We studied the intra-islet localization of the glucagon-like peptide 1 receptor (GLP-1R) by colocalization studies of the GLP-1R mRNA and protein with islet cell hormones in mice, rats, and humans. In contrast to previous reports, we show that the GLP-1R is selectively located on the beta cells. The localization of GLP-1R in islets and ducts was studied using ISH and double and triple fluorescence microscopy. In normal pancreatic tissue from mice and rats, GLP-1R mRNA was only detectable in the beta cells. Double and triple immunofluorescence using two different GLP-1R antisera and combinations of insulin, glucagon, pancreatic polypeptide, and somatostatin showed that GLP-1R protein is almost exclusively colocalized with insulin. The same pattern was observed in human pancreas, but the GLP-1R expression was more heterogeneous, with populations of insulin immunoreactive cells with high and low expression. This is the first time that the GLP-1R has been localized in human islets. Furthermore, GLP-1R immunoreactivity was found in the pancreatic ducts in mouse, rat, and human pancreas. As an important confirmation of the specificity of our methods, we found no signals for GLP-1R mRNA or protein in pancreatic tissue from gene-targeted GLP-1R-deficient mice. In conclusion, our data suggest that the GLP-1 receptor is restricted to the pancreatic beta cells and the lack of receptor immunoreactivity on delta cells cannot be explained suitably to correspond with published in vivo and in vitro data. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Bioactive GLP-1 in gut, receptor expression in pancreas, and insulin response to GLP-1 in diabetes-prone rats.

Glucagon-like peptide-1 (GLP-1) is the most insulinogenic of the glucagon-like peptides secreted mainly by L cells in the small and large intestine in response to the ingestion of nutrients. It binds to a specific GLP-1 receptor (GLP-1R) on beta-cells and can increase islet neogenesis and beta-cell mass. It is not clear whether the transmission of information from the gut to islet beta-cells by messengers such as GLP-1 is different in individuals who develop autoimmune diabetes. In the present study the expression of bioactive GLP-1 protein in the gut and its receptor in the pancreas was examined in diabetes-prone BioBreeding (BBdp) rats in the period before overt diabetes and in age-matched control, non-diabetes-prone BB (BBc) rats. An N-terminal directed antibody specific for the bioactive forms of GLP-1 (GLP-1(7-37) and GLP-1(7-36amide)) was used to mea-sure GLP-1 by radioimmunoassay in proximal, median, and distal gut. Pancreas GLP-1R area fraction, GLP-1R gene expression, and insulin content were analyzed, as were plasma GLP-1, glucose, and insulin. The concentration of GLP-1 protein in the jejunum and ileum of BBdp rats was lower than in BBc rats. Although these animals maintained normal blood glucose, there was impaired pancreatic endocrine function, characterized by low baseline insulin concentration in plasma and pancreas. GLP-1R mRNA expression was threefold less in islets isolated from BBdp rats, and GLP-1R+ islet area fraction in pancreas sections was decreased. When injected iv with GLP-1, BBdp rats displayed lower second-phase insulin response (and insulin/glucose ratios) compared with BBc rats. Thus, young BBdp rats displayed decreased concentrations of bioactive GLP-1 in jejunum and ileum, reduced GLP-1R in islets, and lower second-phase insulin response to iv GLP-1 than controls. The decrease in insulinogenic and islet beta-cell mass-promoting signal from GLP-1 in BBdp rats may contribute to impaired glucoregulation and ineffective maintenance of normal islet mass that shifts islet homeostasis in favor of development of diabetes.

Adaptive changes in insulin secretion by islets from neonatal rats raised on a high-carbohydrate formula.

Artificial rearing of neonatal rats on a high-carbohydrate (HC) milk formula resulted in the immediate onset of hyperinsulinemia. This study examines, in islets of 12-day-old HC rats, adaptive changes that support the hyperinsulinemic state. Increases in plasma glucagon-like peptide-1 (GLP-1) levels and islet GLP-1 receptor mRNA supported increased insulin secretion by HC islets. Isolated HC islets, but not mother-fed (MF) islets, secreted moderate amounts of insulin in a glucose- and Ca(2+)-independent manner. Under stringent Ca(2+)-free conditions and in the presence of glucose, GLP-1 plus acetylcholine augmented insulin release to a larger extent in HC islets. Levels of adenylyl cyclase type VI mRNA and activities of protein kinase A, protein kinase C, and calcium calmodulin kinase II were increased in HC islets. A tenfold increase in norepinephrine concentration was required to inhibit insulin secretion in HC islets compared with MF islets, indicating reduced sensitivity to adrenergic signals. This study shows that significant alterations at proximal and distal sites of the insulin secretory pathway in HC islets may support the hyperinsulinemic state of these rats.

Tissue distribution of messenger ribonucleic acid encoding the rat glucagon-like peptide-1 receptor.

The incretin hormone glucagon-like peptide-1 (GLP-1) is an important regulator of postprandial insulin secretion. In addition to its insulinotropic actions on pancreatic beta-cells, GLP-1 enhances glucose disposal by insulin-independent mechanisms, suggesting that GLP-1 receptors are located on extrapancreatic tissues. In this study, we examined the tissue distribution of GLP-1 receptor (GLP-lR) messenger RNA (mRNA) in rat by RNAse protection, RT-PCR, and in situ hybridization. We identified GLP-1R mRNA in the lung, pancreatic islets, stomach, and kidney by the RNAse protection assay. RT-PCR analysis also detected GLP-1R mRNA in the hypothalamus and heart. In situ hybridization experiments identified receptor mRNA in the gastric pits of the stomach, large nucleated cells in the lung, crypts of the duodenum, and pancreatic islets. No localized specific grains were found in kidney, skeletal muscle, heart, liver, or adipocytes. These results indicate that sequences corresponding to the cloned rat islet GLP-1 receptor are expressed in the pancreatic islets, lung, hypothalamus, stomach, heart, and kidney but not in adipose, liver, and skeletal muscle. Further, the GLP-1 receptor expressed in the kidney and heart may be structural variants of the known receptor. Therefore, the observed extrapancreatic actions of GLP-1 may not be strictly confined to interactions with the defined GLP-1 receptor.