Receptors In Pancreatic Islets Research Articles

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Acid-Resistant BODIPY Amino Acids for Peptide-Based Fluorescence Imaging of GPR54 Receptors in Pancreatic Islets.

The G protein-coupled kisspeptin receptor (GPR54 or KISS1R) is an important mediator in reproduction, metabolism and cancer biology; however, there are limited fluorescent probes or antibodies for direct imaging of these receptors in cells and intact tissues, which can help to interrogate their multiple biological roles. Herein, we describe the rational design and characterization of a new acid-resistant BODIPY-based amino acid (Trp-BODIPY PLUS), and its implementation for solid-phase synthesis of fluorescent bioactive peptides. Trp-BODIPY PLUS retains the binding capabilities of both short linear and cyclic peptides and displays notable turn-on fluorescence emission upon target binding for wash-free imaging. Finally, we employed Trp-BODIPY PLUS to prepare some of the first fluorogenic kisspeptin-based probes and visualized the expression and localization of GPR54 receptors in human cells and in whole mouse pancreatic islets by fluorescence imaging.

Potential Therapeutic Targeting Neurotransmitter Receptors in Diabetes.

Neurotransmitters are signaling molecules secreted by neurons to coordinate communication and proper function among different sections in the central neural system (CNS) by binding with different receptors. Some neurotransmitters as well as their receptors are found in pancreatic islets and are involved in the regulation of glucose homeostasis. Neurotransmitters can act with their receptors in pancreatic islets to stimulate or inhibit the secretion of insulin (β cell), glucagon (α cell) or somatostatin (δ cell). Neurotransmitter receptors are either G-protein coupled receptors or ligand-gated channels, their effects on blood glucose are mainly decided by the number and location of them in islets. Dysfunction of neurotransmitters receptors in islets is involved in the development of β cell dysfunction and type 2 diabetes (T2D).Therapies targeting different transmitter systems have great potential in the prevention and treatment of T2D and other metabolic diseases.

Expression of beta2 adrenergic receptors in pancreatic islets of metabolic syndrome induced C57BL/6J Mice

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Regulated expression and function of the GABAB receptor in human pancreatic beta cell line and islets

G protein-coupled receptors are seven transmembrane signaling molecules that are involved in a wide variety of physiological processes. They constitute a large protein family of receptors with almost 300 members detected in human pancreatic islet preparations. However, the functional role of these receptors in pancreatic islets is unknown in most cases. We generated a new stable human beta cell line from neonatal pancreas. This cell line, named ECN90 expresses both subunits (GABBR1 and GABBR2) of the metabotropic GABAB receptor compared to human islet. In ECN90 cells, baclofen, a specific GABAB receptor agonist, inhibits cAMP signaling causing decreased expression of beta cell-specific genes such as MAFA and PCSK1, and reduced insulin secretion. We next demonstrated that in primary human islets, GABBR2 mRNA expression is strongly induced under cAMP signaling, while GABBR1 mRNA is constitutively expressed. We also found that induction and activation of the GABAB receptor in human islets modulates insulin secretion.

Distinctive expression and cellular distribution of dopamine receptors in the pancreatic islets of rats.

Activation of the dopamine (DA) D2 receptor inhibits glucose-stimulated insulin secretion in isolated rodent islets in vitro; however, no information is available regarding the cellular localization of DA receptors (DRs, including D1-D5 receptors) in pancreatic islets in situ. We investigate the protein expression and cellular localization of five types of DRs in pancreatic islets by means of Western blotting and double-labeling immunofluorescence in both normal control and alloxan-induced type 1 diabetes model (T1DM) rats. In control rats, D1 immunoreactivity (-IR) was distributed in the core of the islet and co-localized with insulin-IR, D2-IR was peripherally distributed and found only in somatostatin-immunoreactive cells and D5-IR was co-localized with glucagon-IR and pancreatic polypeptide-IR. No IR for either the D3 or D4 receptor was observed in rat islets. The protein level of the D1 receptor was reduced in T1DM rats (D1/D-glyceraldehyde-3-phosphate dehydrogenase [GAPDH], 0.63 ± 0.05 in control rats compared with 0.16 ± 0.03 in T1DM rats, n = 8, P < 0.05) but no significant alteration was detected in the protein expression of either the D2 receptor (D2/GAPDH, 0.48 ± 0.04 compared with 0.43 ± 0.04, n = 8, P = 0.42) or the D5 receptor (D5/GAPDH, 0.50 ± 0.04 compared with 0.47 ± 0.04, n = 8, P = 0.58). The present study is the first clear demonstration of the protein expression and cellular localization of the D1, D2 and D5 receptors in rat pancreatic islets and provides crucial morphological evidence for further investigations of the underlying mechanism regarding the DA regulation of pancreatic endocrine function.

Angiotensin type 2 receptor in pancreatic islets of adult rats: a novel insulinotropic mediator

In the present study, we evaluated the relative abundance of angiotensin type 2 receptor (AT2R) protein in various tissues of adult rats. We found that pancreatic islets expressed the highest AT2R protein compared with all other tissues. Accordingly, we then determined the functional significance of AT2R in the endocrine pancreas in in vivo and in vitro experiments by using angiotensin II (ANG II) alone, losartan (Los; AT1R antagonist), compound 21 (C21; AT2R agonist), and PD-123319 (PD; AT2R antagonist). Experiments carried out in rats indicated that, 1) ANG II treatment significantly increased plasma insulin concentration (1.51 ± 0.20 vs. 0.82 ± 0.14 ng/ml, n = 7, P < 0.05) in the fed state. This insulinotropic effect was further augmented by combined treatment with ANG II + Los (2.31 ± 0.25 ng/ml, n = 7, P < 0.01). C21 also elevated insulin levels (2.13 ± 0.20 ng/ml, n = 7, P < 0.01), which was completely abolished by PD. 2) ANG II impaired glucose tolerance, whereas ANG II + Los or C21 improved this function. 3) All treated rats displayed an enhanced insulin secretory response to a glucose challenge. 4) All treated rats displayed upregulated proinsulin 2 mRNA and insulin protein expression in the pancreas. In in vitro experiments using INS-1E cells and isolated rat islets, we found that AT2R activation significantly improved insulin biosynthesis and secretion. These results suggest that the AT2R functions as an insulinotropic mediator. AT2R and its downstream signaling pathways may be potential therapeutic targets for diabetes.

The role of endogenous glucocorticoids in glucose metabolism and immune status of MIF-deficient mice

Macrophage migration inhibitory factor (MIF)-deficient mice develop glucose intolerance and hyperglycemia, but remain entirely responsive to exogenous insulin in adult age. Furthermore, as a consequence of MIF deficiency, the immune response in these mice is predominantly anti-inflammatory. Since MIF is a natural counter-regulator of glucocorticoid action, and it is known that excessive concentration of glucocorticoids contribute both to beta cell dysfunction and immunosuppression, we hypothesized that MIF absence enables elevation of glucocorticoids which in turn caused the observed condition. Our results confirm that MIF-knockout (MIF-KO) mice possess higher levels of circulating corticosterone, but lower expression of glucocorticoid receptor in pancreatic islets, liver and adipose tissue to the one observed in wild type (WT) mice. A significant up-regulation of glucocorticoid receptor expression was however noticed in MIF-deficient lymph node cells. The inhibition of glucocorticoid receptor by RU486 improved tolerance to glucose in MIF-KO mice and restored euglycemia. Although RU486 treatment did not alter the level of glucose receptor GLUT2, it enhanced insulin secretion and up-regulated insulin-triggered Akt phosphorylation within hepatic tissue. Finally, inhibition of glucocorticoid receptor changed anti-inflammatory phenotype of MIF-KO lymphocytes toward a physiological profile. Our results indicate that deregulated glucocorticoid secretion and glucocorticoid receptor expression in the absence of MIF possibly contributes to the development of glucose intolerance and immunosuppression in MIF-KO mice. However, since MIF-KO mice respond normally to insulin and their beta cell function is within physiological range, additional cause for glucose intolerance could be sought in the possible malfunction of their insulin.

Effect of the combination of metformin and fenofibrate on glucose homeostasis in diabetic Goto-Kakizaki rats

Metformin has been reported to increase the expression of the glucagon-like peptide-1 (GLP-1) receptor in pancreatic beta cells in a peroxisome proliferator-activated receptor (PPAR)-α-dependent manner. We investigated whether a PPARα agonist, fenofibrate, exhibits an additive or synergistic effect on glucose metabolism, independent of its lipid-lowering effect, when added to metformin. Non-obese diabetic Goto-Kakizaki (GK) rats were divided into four groups and treated for 28 days with metformin, fenofibrate, metformin plus fenofibrate or vehicle. The random blood glucose levels, body weights, food intake and serum lipid profiles were not significantly different among the groups. After 4 weeks, metformin, but not fenofibrate, markedly reduced the blood glucose levels during oral glucose tolerance tests, and this effect was attenuated by adding fenofibrate. Metformin increased the expression of the GLP-1 receptor in pancreatic islets, whereas fenofibrate did not. During the intraperitoneal glucose tolerance tests with the injection of a GLP-1 analog, metformin and/or fenofibrate did not alter the insulin secretory responses. In conclusion, fenofibrate did not confer any beneficial effect on glucose homeostasis but reduced metformin's glucose-lowering activity in GK rats, thus discouraging the addition of fenofibrate to metformin to improve glycemic control.

Altered Expression of Somatostatin Receptors in Pancreatic Islets from NOD Mice Cultured at Different Glucose ConcentrationsIn Vitroand in Islets Transplanted to Diabetic NOD MiceIn Vivo

Somatostatin acts via five receptors (sst1–5). We investigated if the changes in pancreatic islet sst expression in diabetic NOD mice compared to normoglycemic mice are a consequence of hyperglycemia or the ongoing immune reaction in the pancreas. Pancreatic islets were isolated from NOD mice precultured for 5 days and further cultured for 3 days at high or low glucose before examined. Islets were also isolated from NOD mice and transplanted to normal or diabetic mice in a number not sufficient to cure hyperglycemia. After three days, the transplants were removed and stained for sst1–5 and islet hormones. Overall, changes in sst islet cell expression were more common in islets cultured in high glucose concentration in vitro as compared to the islet transplantation in vivo to diabetic mice. The beta and PP cells exhibited more frequent changes in sst expression, while the alpha and delta cells were relatively unaffected by the high glucose condition. Our findings suggest that the glucose level may alter sst expressed in islets cells; however, immune mechanisms may counteract such changes in islet sst expression.

Melatonin receptors in pancreatic islets: good morning to a novel type 2 diabetes gene

Melatonin is a circulating hormone that is primarily released from the pineal gland. It is best known as a regulator of seasonal and circadian rhythms; its levels are high during the night and low during the day. Interestingly, insulin levels also exhibit a nocturnal drop, which has previously been suggested to be controlled, at least in part, by melatonin. This regulation can be explained by the proposed inhibitory action of melatonin on insulin release. Indeed, both melatonin receptor 1A (MTNR1A) and MTNR1B are expressed in pancreatic islets. The role of melatonin in the regulation of glucose homeostasis has been highlighted by three independent publications based on genome-wide association studies of traits connected with type 2 diabetes, such as elevated fasting glucose, and, subsequently, of the disease itself. The studies demonstrate a link between variations in the MTNR1B gene, hyperglycaemia, impaired early phase insulin secretion and beta cell function. The risk genotype predicts the future development of type 2 diabetes. Carriers of the risk genotype exhibit increased expression of MTNR1B in islets. This suggests that these individuals may be more sensitive to the actions of melatonin, leading to impaired insulin secretion. Blocking the inhibition of insulin secretion by melatonin may be a novel therapeutic avenue for type 2 diabetes.

Changes in expression of P2X7 receptors in NOD mouse pancreas during the development of diabetes

This study examined the expression of P2X7 receptors in pancreatic islets of the non-obese diabetic (NOD) mouse model of human autoimmune insulin-dependent diabetes mellitus, to determine whether they are involved in islet cell destruction during early- and late-developing diabetes. Pancreatic cells containing glucagon (α-cells), insulin (β-cells) and somatostatin (δ-cells) were co-localized with P2X7 receptors. We examined P2X7 receptor expression in normal and diabetic spleens using flow cytometry. In non-diabetic NOD controls, P2X7 receptors were expressed in glucagon-containing cells at the periphery of islets, being consistent with previous studies. In early NOD diabetes (12 weeks), there was migration of peripheral P2X7 receptor positive, glucagon-containing cells into the center of islets. In late NOD diabetes (34 weeks), P2X7 receptor- and glucagon-stained α-cells were gone from islets. Migration of macrophages and dendritic cells into islets took place, but they lacked P2X7 immunoreactivity. There was no significant difference in the percentage of splenic macrophages stained for P2X7 receptors from control and diabetic spleens. In conclusion, in the development of early to late diabetes, there is a down-regulation of P2X7 receptors on islet cells and a loss of α- and β-cell populations. P2X7 receptor signalling might be involved in α-cell clearance from late diabetic islets.

Neuromedin U receptor 1 expression in the rat endocrine pancreas and evidence suggesting neuromedin U suppressive effect on insulin secretion from isolated rat pancreatic islets

Neuromedin U (NmU) is a regulatory peptide found in significant concentrations in both the brain and gut of the rat and is named according to its ability to powerfully contract the uterus. Two types of NmU receptors were recently identified and subsequent studies evidenced NmU involvement in the regulation of energy homeostasis. Such a role of neuromedin U suggests that a polypeptide may also be involved in the regulation of adipoinsular axis function. Therefore in the present study we examined the expression of NmU receptors in pancreatic islets using RT-PCR and Western blotting analysis. We also investigated the role of NmU in regulation of insulin secretion in vitro using isolated pancreatic islets. We have confirmed that NmUR1 but not NmUR2 is specifically expressed in isolated rat pancreatic islets. In all tested doses (1, 10, 100 nmol/l) NmU dose- dependently decreased insulin output by isolated pancreatic islets. These inhibitory effects of NmU on insulin secretion may suggest the involvement of NmU in regulating the pancreatic branch of adipoinsular axis function. Thus, NmU can be included in that group of anorectic peptides, which are also involved in the regulation of insulin secretion.

Effects of leptin administration on the endocrine pancreas and liver in the lizard Podarcis sicula

In this study, we investigated the presence of leptin receptor in pancreatic islets and the effect of exogenous leptin administration in Podarcis sicula on glucose metabolism. Our data show the presence of leptin receptor immunoreactivity in the endocrine pancreas suggesting that leptin may act at a peripheral level as previously postulated in mammals. The effects of short- and long-term and dose-response treatment with supraphysiological concentrations of leptin on circulating levels of insulin, glucagon and glucose in the blood have been evaluated. Taken together, our results indicate that leptin treatment was followed by an increase in insulin, glucagon and glucose in the blood, depending on the dose of leptin. Moreover, leptin treatment brought about a decrease of glycogen and the appearance of tyrosine-phosphorylated proteins in the liver. This study shows that in the lizard P. sicula leptin is involved in glucose metabolism.

Activation of cannabinoid CB 1 receptors induces glucose intolerance in rats

Recent reports have described the presence of cannabinoid CB 1 receptors in pancreatic islets. Here we show that administration of the endogenous cannabinoid anandamide or the selective cannabinoid CB 1 receptor agonist Arachidonyl-2′-chloroethylamide (ACEA) results in glucose intolerance after a glucose load. This effect is reversed by the selective cannabinoid CB 1 receptor antagonist N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide (AM251). These results suggest that targeting cannabinoid CB 1 receptors may serve as new therapeutic alternatives for metabolic disorders such as diabetes.

Glucagon-like peptide 1 (GLP-1) and incretin mimetics for the treatment of diabetes

Glucagon-like peptide 1 (GLP-1) was discovered as an insulinotropic (‘incretin’) hormone, suggesting a physiological role in the potentiation of insulin secretory responses after oral glucose or meals. The incretin effect (i.e. the phenomenon that more insulin is secreted in response to oral as compared to intravenous glucose) is reduced/lost in patients with type 2 diabetes. GLP − 1(a) stimulates insulin secretion in a glucose-dependent manner, (b) suppresses glucagon, (c) reduces appetite and food intake, (d) decelerates gastric emptying, (e) stimulates pancreatic β cell neogenesis, growth and differentiation in animal and tissue culture experiments, and (f) inhibits β cell apoptosis in vitro. Intravenous GLP-1 normalises and subcutaneous GLP-1 significantly lowers plasma glucose in the majority of patients with type 2 diabetes. GLP-1 itself, however, is not useful for chronic antidiabetic treatment, because it is too rapidly inactivated by proteolysis (DPP-4) and eliminated from the circulation. Agents interacting with GLP-1 receptors in pancreatic islets or the (central) nervous system have been found (like exendin-4) or developed as ‘incretin mimetics’ (like liraglutide). Clinical trials with exenatide (two injections per day) and liraglutide (one injection per day) have shown reductions in glucose concentrations and HbA1c by approximately 1%, associated with moderate weight loss (2-3 kg), but also some nausea and, rarely, vomiting. This new class of antidiabetic agents will be available for clinical use pending completion of phase 3 trials and/or approval by regulatory agencies after 2005 (USA) and 2007 (Europe). Copyright © 2005 John Wiley & Sons, Ltd.

Endothelial differentiation gene receptors in pancreatic islets and INS-1 cells.

The endothelial differentiation gene (EDG) receptors are a class of G protein-coupled receptors. EDG-1, -3, -5, -6, and -8 bind the bioactive lipid sphingosine-1-phosphate (SPP) as the primary signaling ligand. EDG-2, -4, and -7 bind the ligand lysophosphatidic acid. EDG-1, -2, -3, -5, -6, and -7, but not -8, mRNAs were expressed in isolated rat pancreatic islets, whereas INS-1 insulinoma cells expressed only EDG-1, -2, -3, and -5 mRNAs. EDG-4 mRNA was expressed in mouse islets. EDG-1 mRNA but not EDG-3 mRNA was rapidly induced relative to 18S rRNA after stimulation of isolated islets with phorbol 12-myristate 13-acetate (PMA) or cholecystokinin-8S for 2 h. The protein kinase C inhibitor GF 109203X blocked the EDG-1 induction by PMA. Similarly, in islets stimulated for 2 h with 17 mmol/l glucose, the relative EDG-1 mRNA levels increased almost twofold compared with levels in control islets at 5.5 mmol/l glucose. In contrast, after 11 mmol/l glucose stimulation for 7 days, the relative levels of rat islet EDG-1 mRNA were significantly reduced to 54% below that of islets cultured at 5.5 mmol/l glucose. There was no change in relative EDG-3 mRNA levels. Stimulation of EDG receptors in islets and INS-1 cells with SPP inhibited glucagon-like peptide 1 (GLP-1)-stimulated cAMP production and insulin secretion in a concentration-dependent manner. Pertussis toxin antagonized the SPP effects on insulin release. Thus, EDG receptors are expressed in pancreatic islet beta-cells and G(i) seems to mediate the inhibition by SPP of adenylyl cyclase and cAMP formation and inhibition of the stimulation of insulin secretion by GLP-1.

Expression and function of the calcium-sensing receptor in pancreatic islets and insulinoma cells.

We previously demonstrated that human insulinoma cells express the calcium-sensing receptor (CaR). To investigate the expression of CaR in the human pancreas and to evaluate the differences in responses of human insulinoma cells and normal rat islets to extracellular calcium2+ [Ca2+]o. To evaluate CaR expression in the normal human pancreas, immunohistochemical and reverse transcription polymerase chain reaction studies were performed. To evaluate the response of normal islets and insulinoma cells to changes in the [Ca2+]o concentration, cytosolic free calcium levels were measured by microfluorometry. Because it is difficult to obtain viable normal human islets, we used normal rat islets instead. CaR is expressed in both human pancreatic islets and human insulinoma cells. Microfluorometry showed an increase in the [Ca2+]i level in response to changes in the [Ca2+]o concentration, with a more sensitive response in human insulinoma cells than in normal islets. When 1 micromol/L wortmannin (a selective phosphatidylinositol 3-kinase inhibitor) was added to the perfusion medium, the response disappeared in insulinoma cells but not in islets. Both insulinoma cells and islets expressed CaR; however, the reactivity to changes in the [Ca2+]o concentration was different between them. These findings suggest that the signaling pathways controlling the changes in [Ca2+]i differ between normal rat islets and human insulinoma cells.

Overexpression of leptin receptors in pancreatic islets of Zucker diabetic fatty rats restores GLUT-2, glucokinase, and glucose-stimulated insulin secretion.

The high-Km glucose transporter, GLUT-2, and the high-Km hexokinase of beta cells, glucokinase (GK), are required for glucose-stimulated insulin secretion (GSIS). GLUT-2 expression in beta cells of Zucker diabetic fatty (ZDF) rats is profoundly reduced at the onset of beta-cell dysfunction of diabetes. Because ZDF rats are homozygous for a mutation in their leptin receptor (OB-R) gene and are therefore leptin-insensitive, we expressed the wild-type OB-R gene in diabetic islets by infusing a recombinant adenovirus (AdCMV-OB-Rb) to determine whether this reversed the abnormalities. Leptin induced a rise in phosphorylated STAT3, indicating that the transferred wild-type OB-R was functional. GLUT-2 protein rose 17-fold in AdCMV-OB-Rb-treated ZDF islets without leptin, and leptin caused no further rise. GK protein rose 7-fold without and 12-fold with leptin. Preproinsulin mRNA increased 64% without leptin and rose no further with leptin, but leptin was required to restore GSIS. Clofibrate and 9-cis-retinoic acid, the partner ligands for binding to peroxisome proliferator-activator receptor alpha (PPARalpha) and retinoid X receptor, up-regulated GLUT-2 expression in islets of normal rats, but not in ZDF rats, in which PPARalpha is very low. Because the fat content of islets of diabetic ZDF rats remains high unless they are treated with leptin, it appears that restoration of GSIS requires normalization of intracellular nutrient homeostasis, whereas up-regulation of GLUT-2 and GK is leptin-independent, requiring only high expression of OB-Rb.

Adenine nucleotide-induced inhibition of binding of sulphonylureas to their receptor in pancreatic islets.

1. The effects of the Mg complex of adenosine 5'-triphosphate (MgATP) on binding of sulphonylureas to microsomes obtained from mouse pancreatic islets were examined. 2. MgATP inhibited the binding of both glibenclamide and tolbutamide to microsomes. 3. Binding of [3H]-glibenclamide inhibited by MgATP was not further diminished by Mg(2+)-bound adenosine 5'-(beta, gamma-imidotriphosphate) (AMP-PNP) or free adenosine 5'-diphosphate (ADP). Higher concentrations of MgAMP-PNP induced a partial reversal of the inhibitory effect of MgATP on [3H]-glibenclamide binding. 4. The apparent dissociation constant (K'D) for binding of [3H]- glibenclamide remained constant when 5. Extracellular ADP did not markedly stimulate insulin release from mouse pancreatic islets. 6. It is concluded that sulphonylureas and cytosolic nucleotides exert their inhibitory effects on the K-ATP-channels of beta-cells by binding to different sites. The binding properties of the sulphonylurea receptor seem to be modulated by protein phosphorylation.