Agonists at GPR119 mediate secretion of GLP‐1 from mouse enteroendocrine cells through glucose‐independent pathways

Glucagon-like peptide-1 (GLP-1) is a well-known incretin hormone secreted from enteroendocrinal L cells in response to nutrients, such as glucose and dietary fat, and controls glycemic homeostasis. However, the detailed intracellular mechanisms of how L cells control GLP-1 secretion in response to nutrients still remain unclear. Here, we report that bone morphogenetic protein (BMP) signaling pathway plays a pivotal role to control GLP-1 secretion in response to nutrient replenishment in well-established mouse enteroendocrinal L cells (GLUTag cells). Nutrient starvation dramatically reduced cellular respiration and GLP-1 secretion in GLUTag cells. Transcriptome analysis revealed that nutrient starvation remarkably reduced gene expressions involved in BMP signaling pathway, whereas nutrient replenishment rescued BMP signaling to potentiate GLP-1 secretion. Transient knockdown of inhibitor of DNA binding (ID)1, a well-known target gene of BMP signaling, remarkably reduced GLP-1 secretion. Consistently, LDN193189, an inhibitor of BMP signaling, markedly reduced GLP-1 secretion in L cells. In contrast, BMP4 treatment activated BMP signaling pathway and potentiated GLP-1 secretion in response to nutrient replenishment. Altogether, we demonstrated that BMP signaling pathway is a novel molecular mechanism to control GLP-1 secretion in response to cellular nutrient status. Selective activation of BMP signaling would be a potent therapeutic strategy to stimulate GLP-1 secretion in order to restore glycemic homeostasis.

Pasireotide frequently causes severe hyperglycemia; however, its detailed mechanism remains unknown. There are no published guidelines regarding the optimal management of pasireotide-induced hyperglycemia based on its pathophysiology. Herein, we successfully switched a patient with acromegaly from a dipeptidyl peptidase-4 (DPP-4) inhibitor to a glucagon-like peptide-1 (GLP-1) analog due to pasireotide-induced deterioration of glycemic control, and we examined the underlying mechanism for glycemic control. An in vitro study was conducted using pancreatic β-cell line, MIN-6, stably expressing GLP-1R (GLP-1R-MIN-6 cells) and intestinal L-cell line, GLUTag. High glucose levels and Gs-coupled receptor stimulation synergistically triggered insulin and GLP-1 secretion. Gs-coupled receptor stimulation primarily triggered GLP-1 secretion, which was amplified by high glucose levels in GLUTag cells. Pasireotide drastically inhibited GLP-1 secretion induced by Gs-coupled receptor stimulation through SSTR5-Gi-dependent inhibition of cAMP levels, suggesting that the main pathway was completely blocked. Furthermore, administering GLP-1 partially overcame the inhibitory effect of pasireotide in GLP-1R-MIN-6 cells, leading to a partial recovery of insulin secretion. The drastic inhibition of GLP-1 secretion via shutdown of the main pathway is the primary cause of pasireotide-induced hyperglycemia. GLP-1 analogs, rather than DPP-4 inhibitors, can spare pasireotide-induced depletion of endogenous GLP-1 and restore insulin secretion.

Dipeptidyl peptidase-4 and GLP-1 interplay in STC-1 and GLUTag cell lines

Biology of Incretins: GLP-1 and GIP

The gut hormones ghrelin, glucagon-like peptide 1 (GLP-1), and glucose-dependent insulinotropic peptide (GIP) have been intensively studied for their role in metabolism. It is, however, not well known whether the hormones interplay and regulate the secretion of each other. In this study, we studied the effect of ghrelin on GLP-1, GIP, and insulin secretion during an oral glucose tolerance test (OGTT) in mice. Intravenous administration of ghrelin caused increased GLP-1 secretion during the OGTT. On the other hand, ghrelin had no effect on circulating levels of glucose, insulin, and GIP. Furthermore, ghrelin treatment reduced proglucagon mRNA expression in GLUTag cells. The effect of ghrelin on GLP-1 secretion and proglucagon transcription was reinforced by the presence of GHS-R1a in human and mouse ileal L-cells, as well as in GLUTag cells. In summary, ghrelin is a regulator of GLP-1 secretion and transcription, and interfering with GHS-R1a signaling may be a way forward to enhance endogenous GLP-1 secretion in subjects with type 2 diabetes.

Palatinose (isomaltulose), a slowly digested disaccharide, is used as a non-cariogenic sugar and as a sucrose substitute in several foods. Because of its ability to lower postprandial glycemia, palatinose may be beneficial as a treatment for impaired glucose metabolism. Glucagon-like peptide-1 (GLP-1) improves glycemia via enhancing pancreatic beta-cell functions. The secretion of GLP-1 is stimulated by sugars, including glucose and artificial sweeteners. In this study, we examined whether palatinose induced GLP-1 secretion in vivo and in vitro. Firstly, portal GLP-1 and glucose were measured after oral administration of palatinose or sucrose in conscious rats. Secondly, portal GLP-1 and glucose were measured after jejunal or ileal administration of each sugar in anesthetized rats. Finally, GLUTag, a murine GLP-1-producing cell line, was exposed to several sugars, including palatinose and sucrose, to observe the direct effect of these sugars on GLP-1 secretion. Compared with sucrose, palatinose enhanced portal GLP-1 level when administered orally in conscious rats. Both palatinose and sucrose induced a significant increase in portal GLP-1 after jejunal or ileal administration of each sugar in anesthetized rats. Ileal administration triggered a greater response than did jejunal administration. Glycemic responses were higher in sucrose-treated rats than in palatinose-treated rats in every experiment. In GLUTag cells, glucose induced a significant increase in GLP-1 secretion, but neither sucrose nor palatinose had an effect. These data demonstrate that luminal palatinose induces GLP-1 secretion in rats. However, it is likely that GLP-1 secretion is triggered mainly by glucose released in the lumen rather than by palatinose itself.

(-)-Epicatechin and Anthocyanins Modulate GLP-1 Metabolism: Evidence from C57BL/6JMice and GLUTag Cells.

The lysophosphatidylinositol (LPI) has crucial roles in multiple physiological processes, including insulin exocytosis from pancreatic islets. However, the role of LPI in secretion of glucagon-like peptide-1 (GLP-1), a hormone that enhances glucose-induced insulin secretion, is unclear. Here, we used the murine enteroendocrine L cell line GLUTag and primary murine small intestinal cells to elucidate the mechanism of LPI-induced GLP-1 secretion. Exogenous LPI addition increased intracellular Ca2+ concentrations ([Ca2+] i ) in GLUTag cells and induced GLP-1 secretion from both GLUTag and acutely prepared primary intestinal cells. The [Ca2+] i increase was suppressed by an antagonist for G protein-coupled receptor 55 (GPR55) and by silencing of GPR55 expression, indicating involvement of Gq and G12/13 signaling pathways in the LPI-induced increased [Ca2+] i levels and GLP-1 secretion. However, GPR55 agonists did not mimic many of the effects of LPI. We also found that phospholipase C inhibitor and Rho-associated kinase inhibitor suppressed the [Ca2+] i increase and that LPI increased the number of focal adhesions, indicating actin reorganization. Of note, blockage or silencing of transient receptor potential cation channel subfamily V member 2 (TRPV2) channels suppressed both the LPI-induced [Ca2+] i increase and GLP-1 secretion. Furthermore, LPI accelerated TRPV2 translocation to the plasma membrane, which was significantly suppressed by a GPR55 antagonist. These findings suggest that TRPV2 activation via actin reorganization induced by Gq and G12/13 signaling is involved in LPI-stimulated GLP-1 secretion in enteroendocrine L cells. Because GPR55 agonists largely failed to mimic the effects of LPI, its actions on L cells are at least partially independent of GPR55 activation.

Although amino acids are dietary nutrients that evoke the secretion of glucagon-like peptide 1 (GLP-1) from intestinal L cells, the precise molecular mechanism(s) by which amino acids regulate GLP-1 secretion from intestinal L cells remains unknown. Here, we show that the G protein-coupled receptor (GPCR), family C group 6 subtype A (GPRC6A), is involved in amino acid-induced GLP-1 secretion from the intestinal L cell line GLUTag. Application of l-ornithine caused an increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) in GLUTag cells. Application of a GPRC6A receptor antagonist, a phospholipase C inhibitor, or an IP(3) receptor antagonist significantly suppressed the l-ornithine-induced [Ca(2+)](i) increase. We found that the increase in [Ca(2+)](i) stimulated by l-ornithine correlated with GLP-1 secretion and that l-ornithine stimulation increased exocytosis in a dose-dependent manner. Furthermore, depletion of endogenous GPRC6A by a specific small interfering RNA (siRNA) inhibited the l-ornithine-induced [Ca(2+)](i) increase and GLP-1 secretion. Taken together, these findings suggest that the GPRC6A receptor functions as an amino acid sensor in GLUTag cells that promotes GLP-1 secretion.

The GPR40/FFA1 receptor is a G-protein-coupled receptor expressed in the pancreatic islets and enteroendocrine cells. Here, we report the pharmacological profiles of (3S)-3-cyclopropyl-3-{2-[(1-{2-[(2,2-dimethylpropyl)(6-methylpyridin-2-yl)carbamoyl]-5-methoxyphenyl}piperidin-4-yl)methoxy]pyridin-4-yl}propanoic acid (SCO-267), a novel full agonist of GPR40. Ca2+ signaling and insulin and glucagon-like peptide-1 (GLP-1) secretion were evaluated in GPR40-expressing CHO, MIN6, and GLUTag cells. Hormone secretions and effects on fasting glucose were tested in rats. Single or repeated dosing effects were evaluated in neonatally streptozotocin-induced diabetic rats (N-STZ-1.5 rats), diet-induced obese (DIO) rats, and GPR40-knockout (Ffar1-/- ) mice. Treatment with SCO-267 activated Gq signaling in both high- and low-FFAR1-expressing CHO cells, stimulated insulin secretion in MIN6 cells, and induced GLP-1 release in GLUTag cells. When administered to normal rats, SCO-267 increased insulin, glucagon, GLP-1, glucose-dependent insulinotropic peptide, and peptide YY (PYY) secretions under nonfasting conditions. These results show the full agonistic property of SCO-267 against GPR40. Hypoglycemia was not induced in SCO-267-treated rats during the fasting condition. In diabetic N-STZ-1.5 rats, SCO-267 was highly effective in improving glucose tolerance in single and 2-week dosing studies. DIO rats treated with SCO-267 for 2 weeks showed elevated plasma GLP-1 and PYY levels, reduced food intake, and decreased body weight. In wild-type mice, SCO-267 induced GLP-1 secretion, food intake inhibition, and body weight reduction; however, these effects were abolished in Ffar1-/- mice, indicating a GPR40-dependent mechanism. In conclusion, SCO-267 stimulated islet and gut hormone secretion, improved glycemic control in diabetic rats, and decreased body weight in obese rats. These data suggest the therapeutic potential of SCO-267 for the treatment of diabetes and obesity.

Integrative function of adrenaline receptors for glucagon-like peptide-1 exocytosis in enteroendocrine L cell line GLUTag

G-protein coupled receptor 119 (GPR119) expression in pancreatic β-cells and intestinal L cells is a potential therapeutic target for treating type 2 diabetes. A natural GPR119 agonist oleoylethanolamide is well known to enhance a glucose-stimulated insulin secretion (GSIS) and glucagon-like peptide-1 (GLP-1) secretion by elevating intracellular cAMP levels. In the present study, a glucose lowering effect of the GPR119 agonist, JTP-109192 leading to improvement of insulin sensitivity was examined in Zucker Fatty (ZF) rats. We investigated the in vitro effects of JTP-109192 on GSIS in the rat pancreatic β-cell line (INS1E) cells and on GLP-1 secretion in the murine enteroendocrine cell line (GLUTag) cells. We also investigated the effect of JTP-109192 on GSIS in Sprague-Dawley (SD) rats with single administration and its effect on glucose metabolism in ZF rats with repeated administration once daily for about 6weeks. After repeated administration, the hyperinsulinaemic euglycaemic glucose clamp test was performed to evaluate insulin sensitivity. JTP-109192 increased intracellular cAMP levels (EC50 value: 3.6nmol/L) and enhanced GSIS in the INS1E cells and GLP-1 secretion in GLUTag cells. In SD rats, a single administration of JTP-109192 enhanced GSIS at high blood glucose levels. The repeated administrations in ZF rats improved glucose metabolism without lack of drug efficacy (tachyphylaxis) and increased glucose infusion rates due to improvement of insulin sensitivity.

DPP-4 inhibitors, used for treatment of type 2 diabetes, act by increasing the concentrations of intact glucagon-like peptide-1 (GLP-1), but at the same time, they inhibit secretion of GLP-1, perhaps by a negative feedback mechanism. We hypothesized that GLP-1 secretion is feedback regulated by somatostatin (SS) from neighboring D-cells, and blocking this feedback circuit results in increased GLP-1 secretion. We used a wide range of experimental techniques, including gene expression analysis, immunohistochemical approaches, and the perfused mouse intestine to characterize the paracrine circuit controlling GLP-1 and SS. We show that 1) antagonizing the SS receptor (SSTr) 2 and SSTr5 led to increased GLP-1 and SS secretion in the mouse, 2) SS exhibits strong tonic inhibition of GLP-1 secretion preferentially through SSTr5, and 3) the secretion of S was GLP-1 receptor dependent. We conclude that SS is a tonic inhibitor of GLP-1 secretion, and interventions in the somatostain-GLP-1 paracrine loop lead to increased GLP-1 secretion.

Aims/hypothesisTargeting the secretion of gut peptides such as glucagon-like peptide 1 (GLP-1) and peptide YY (PYY) is a strategy under development for the treatment of diabetes and obesity, aiming to mimic the beneficial alterations in intestinal physiology that follow gastric bypass surgery. In vitro systems are now well established for studying the mouse enteroendocrine system, but whether these accurately model the human gut remains unclear. The aim of this study was to establish and characterise human primary intestinal cultures as a model for assessing GLP-1 and PYY secretion in vitro.MethodsFresh surgical biopsies of human colon were digested with collagenase to generate primary cultures from which GLP-1 and PYY secretion were assayed in response to test stimuli. GLP-1 and PYY co-localisation were assessed by flow cytometry and immunofluorescence microscopy.ResultsGLP-1 and PYY were found localised in the same cells and the same secretory vesicles in human colonic tissue samples. GLP-1 release was increased to 2.6-fold the control value by forskolin + isobutylmethylxanthine (10 μmol/l each), 2.8-fold by phorbol myristate acetate (1 μmol/l) and 1.4-fold by linoleic acid (100 μmol/l). PYY release was increased to 2.0-, 1.8- and 1.3-fold by the same stimuli, respectively. Agonists of G-protein-coupled receptor (GPR)40/120 and G-protein-coupled bile acid receptor 1 (GPBAR1) each increased GLP-1 release to 1.5-fold, but a GPR119 agonist did not significantly stimulate secretion.Conclusions/interpretationPrimary human colonic cultures provide an in vitro model for interrogating the human enteroendocrine system, and co-secrete GLP-1 and PYY. We found no evidence of PYY-specific cells not producing GLP-1. GLP-1 secretion was enhanced by small molecule agonists of GPR40/120 and GPBAR1.

3-Deoxyglucosone (3DG) is derived from D-glucose during food processing and storage and under physiological conditions. We reported that glucagon-like peptide-1 (GLP-1) secretion in response to an oral glucose load in vivo and high-glucose stimulation in vitro was decreased by acute 3DG administration. In this study, we determined the acute effect of 3DG on GLP-1 secretion under basal conditions and investigated the possible mechanisms. Normal fasting rats were given a single acute intragastric administration of 50 mg/kg 3DG. Plasma basal GLP-1 levels and duodenum 3DG content and sweet taste receptor expression were measured. STC-1 cells were acutely exposed to 3DG (80, 300, and 1000 ng/ml) for 1 h under basal conditions (5.6 mM glucose), and GLP-1 secretion, intracellular concentrations of cyclic adenosine monophosphate (cAMP) and Ca2+, and molecular expression of STR signaling pathway were measured. Under the fasted state, plasma GLP-1 levels, duodenum 3DG content, and duodenum STR expression were elevated in 3DG-treated rats. GLP-1 secretion was increased in 3DG-treated cells under either 5.6 mM glucose or glucose-free conditions. 3DG-induced acute GLP-1 secretion from STC-1 cells under 5.6 mM glucose was inhibited in the presence of the STR inhibitor lactisole, which was consistent with the observation under glucose-free conditions. Moreover, acute exposure to 3DG increased the protein expression of TAS1R2 and TAS1R3 under either 5.6 mM glucose or glucose-free conditions, with affecting other components of STR signaling pathway, including the upregulation of transient receptor potential channel type M5 TRPM5 and the increment of intracellular Ca2+ concentration. In summary, the glucose-free condition was used to first demonstrate the involvement of STR in 3DG-induced acute GLP-1 secretion. These results first showed that acute 3DG administration induces basal GLP-1 secretion in part through upregulation of STR expression.