Voltage-gated calcium channel α2δ-1 subunit is involved in the regulation of glucose-stimulated GLP-1 secretion in mice.

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.

The gut microbiota participates in the control of energy homeostasis partly through fermentation of dietary fibers hence producing short-chain fatty acids (SCFAs), which in turn promote the secretion of the incretin Glucagon-Like Peptide-1 (GLP-1) by binding to the SCFA receptors FFAR2 and FFAR3 on enteroendocrine L-cells. We have previously shown that activation of the nuclear Farnesoid X Receptor (FXR) decreases the L-cell response to glucose. Here, we investigated whether FXR also regulates the SCFA-induced GLP-1 secretion. GLP-1 secretion in response to SCFAs was evaluated ex vivo in murine colonic biopsies and in colonoids of wild-type (WT) and FXR knock-out (KO) mice, in vitro in GLUTag and NCI-H716 L-cells activated with the synthetic FXR agonist GW4064 and in vivo in WT and FXR KO mice after prebiotic supplementation. SCFA-induced GLP-1 secretion was blunted in colonic biopsies from GW4064-treated mice and enhanced in FXR KO colonoids. In vitro FXR activation inhibited GLP-1 secretion in response to SCFAs and FFAR2 synthetic ligands, mainly by decreasing FFAR2 expression and downstream Gαq-signaling. FXR KO mice displayed elevated colonic FFAR2 mRNA levels and increased plasma GLP-1 levels upon local supply of SCFAs with prebiotic supplementation. Our results demonstrate that FXR activation decreases L-cell GLP-1 secretion in response to inulin-derived SCFA by reducing FFAR2 expression and signaling. Inactivation of intestinal FXR using bile acid sequestrants or synthetic antagonists in combination with prebiotic supplementation may be a promising therapeutic approach to boost the incretin axis in type 2 diabetes.

We carried out a systematic review of clinical studies investigating glucagon-like peptide-1 (GLP-1) secretion in patients with type 2 diabetes and non-diabetic controls and performed meta-analyses of plasma total GLP-1 concentrations during an OGTT and/or meal test. Random effects models for the primary meta-analysis and random effects meta-regression, subgroup and regression analyses were applied. Random effects meta-analysis of GLP-1 responses in 22 trials during 29 different stimulation tests showed that patients with type 2 diabetes (n = 275) and controls without type 2 diabetes (n = 279) exhibited similar responses of total GLP-1 (p = NS) as evaluated from peak plasma concentrations (weighted mean difference [95% CI] 1.09 pmol/l [-2.50, 4.67]), total AUC (tAUC) (159 pmol/l × min [-270, 589]), time-corrected tAUC (tAUC min⁻¹) (0.99 pmol/l [-1.28, 3.27]), incremental AUC (iAUC) (-122 pmol/l × min [-410, 165]) and time-corrected iAUC (iAUC min⁻¹) (-0.49 pmol/l [-2.16, 1.17]). Fixed effects meta-analysis revealed higher peak plasma GLP-1 concentrations in patients with type 2 diabetes. Subgroup analysis showed increased responses after a liquid mixed meal test (peak, tAUC and tAUC min⁻¹) and after a 50 g OGTT (AUC and tAUC min⁻¹), and reduced responses after a solid mixed meal test (tAUC min⁻¹) among patients with type 2 diabetes. Meta-regression analyses showed that HbA1c and fasting plasma glucose predicted the outcomes iAUC and iAUC min⁻¹, respectively. The present analysis suggests that patients with type 2 diabetes, in general, do not exhibit reduced GLP-1 secretion in response to an OGTT or meal test, and that deteriorating glycaemic control may be associated with reduced GLP-1 secretion.

The glucose-dependent secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1) is a critical step in the regulation of glucose homeostasis. Two molecular mechanisms have separately been suggested as the primary mediator of intestinal glucose-stimulated GLP-1 secretion (GSGS): one is a metabotropic mechanism requiring the sweet taste receptor type 2 (T1R2) + type 3 (T1R3) while the second is a metabolic mechanism requiring ATP-sensitive K(+) (K(ATP)) channels. By quantifying sugar-stimulated hormone secretion in receptor knockout mice and in rats receiving Roux-en-Y gastric bypass (RYGB), we found that both of these mechanisms contribute to GSGS; however, the mechanisms exhibit different selectivity, regulation, and localization. T1R3(-/-) mice showed impaired glucose and insulin homeostasis during an oral glucose challenge as well as slowed insulin granule exocytosis from isolated pancreatic islets. Glucose, fructose, and sucralose evoked GLP-1 secretion from T1R3(+/+), but not T1R3(-/-), ileum explants; this secretion was not mimicked by the K(ATP) channel blocker glibenclamide. T1R2(-/-) mice showed normal glycemic control and partial small intestine GSGS, suggesting that T1R3 can mediate GSGS without T1R2. Robust GSGS that was K(ATP) channel-dependent and glucose-specific emerged in the large intestine of T1R3(-/-) mice and RYGB rats in association with elevated fecal carbohydrate throughout the distal gut. Our results demonstrate that the small and large intestines utilize distinct mechanisms for GSGS and suggest novel large intestine targets that could mimic the improved glycemic control seen after RYGB.

The G protein-coupled receptor 119 (GPR119) mediates insulin secretion from pancreatic β cells and glucagon-like peptide 1 (GLP-1) release from intestinal L cells. While GPR119-mediated insulin secretion is glucose dependent, it is not clear whether or not GPR119-mediated GLP-1 secretion similarly requires glucose. This study was designed to address the glucose-dependence of GPR119-mediated GLP-1 secretion, and to explore the cellular mechanisms of hormone secretion in L cells versus those in β cells. GLP-1 secretion in response to GPR119 agonists and ion channel modulators, with and without glucose, was analysed in the intestinal L cell line GLUTag, in primary intestinal cell cultures and in vivo. Insulin secretion from Min6 cells, a pancreatic β cell line, was analysed for comparison. In GLUTag cells, GPR119 agonists stimulated GLP-1 secretion both in the presence and in the absence of glucose. In primary mouse colon cultures, GPR119 agonists stimulated GLP-1 secretion under glucose-free conditions. Moreover, a GPR119 agonist increased plasma GLP-1 in mice without a glucose load. However, in Min6 cells, GPR119-mediated insulin secretion was glucose-dependent. Among the pharmacological agents tested in this study, nitrendipine, an L-type voltage-dependent calcium channel blocker, dose-dependently reduced GLP-1 secretion from GLUTag cells, but had no effect in Min6 cells in the absence of glucose. Unlike that in pancreatic β cells, GPR119-mediated GLP-1 secretion from intestinal L cells was glucose-independent in vitro and in vivo, probably because of a higher basal calcium tone in the L cells.

Glucose stimulates the secretion of the incretin hormones: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP). It is debated whether the sweet taste receptor (STR) triggers this secretion. We investigated the role of STR activation for glucose-stimulated incretin secretion from an isolated perfused rat small intestine and whether selective STR activation by artificial sweeteners stimulates secretion. Intra-luminal administration of the STR agonists, acesulfame K (3.85% w/v), but not sucralose (1.25% w/v) and stevioside (2.5% w/v), stimulated GLP-1 secretion (acesulfame K: 31 ± 3 pmol/L vs. 21 ± 2 pmol/L, p < 0.05, n = 6). In contrast, intra-arterial administration of sucralose (10 mM) and stevioside (10 mM), but not acesulfame K, stimulated GLP-1 secretion (sucralose: 51 ± 6 pmol/L vs. 34 ± 4 pmol/L, p < 0.05; stevioside: 54 ± 6 pmol/L vs. 32 ± 2 pmol/L, p < 0.05, n = 6), while 0.1 mM and 1 mM sucralose did not affect the secretion. Luminal glucose (20% w/v) doubled GLP-1 and GIP secretion, but basolateral STR inhibition by gurmarin (2.5 µg/mL) or the inhibition of the transient receptor potential cation channel 5 (TRPM5) by triphenylphosphine oxide (TPPO) (100 µM) did not attenuate the responses. In conclusion, STR activation does not drive GIP/GLP-1 secretion itself, nor does it have a role for glucose-stimulated GLP-1 or GIP secretion.

Molecular mechanisms of glucose-stimulated GLP-1 secretion from perfused rat small intestine.

The gastrointestinal tract constitutes a large interface with the inner body and is a crucial barrier against gut microbiota and other pathogens. As soon as this barrier is damaged, pathogen-associated molecular patterns (PAMPs) are recognized by immune system receptors, including toll-like receptors (TLRs). Glucagon-like peptide 1 (GLP-1) is an incretin that was originally involved in glucose metabolism and recently shown to be rapidly and strongly induced by luminal lipopolysaccharides (LPS) through TLR4 activation. In order to investigate whether the activation of TLRs other than TLR4 also increases GLP-1 secretion, we used a polymicrobial infection model through cecal ligation puncture (CLP) in wild-type and TLR4-deficient mice. TLR pathways were assessed by intraperitoneal injection of specific TLR agonists in mice. Our results show that CLP induces GLP-1 secretion both in wild-type and TLR4-deficient mice. CLP and TLR agonists increase gut and systemic inflammation. Thus, the activation of different TLRs increases GLP-1 secretion. This study highlights for the first time that, in addition to an increased inflammatory status, CLP and TLR agonists also strongly induce total GLP-1 secretion. Microbial-induced GLP-1 secretion is therefore not only a TLR4/LPS-cascade.

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.

Glucagon-like peptide-1 (GLP-1) is released from enteroendocrine cells (L cells) in response to food ingestion. The mechanism by which dietary peptides stimulate GLP-1 secretion in the gut is unknown. In the present study, we found that a hydrolysate prepared from zein, a major corn protein [zein hydrolysate (ZeinH)], strongly stimulates GLP-1 secretion in enteroendocrine GLUTag cells. Stimulatory mechanisms of GLP-1 secretion induced by ZeinH were investigated in the rat small intestine under anesthesia. Blood was collected through a portal catheter before and after ZeinH administration into different sites of the small intestine. The duodenal, jejunal, and ileal administration of ZeinH induced dose-dependent increases in portal GLP-1 concentration. GLP-1 secretion in response to the ileal administration of ZeinH was higher than that in the duodenal or jejunal administration. Capsaicin treatment on esophageal vagal trunks abolished the GLP-1 secretion induced by duodenal ZeinH but did not affect the secretion induced by jejunal or ileal ZeinH. These results suggest that ZeinH in the jejunum or ileum directly stimulates GLP-1 secretion but duodenal ZeinH indirectly stimulates GLP-1 secretion via the vagal afferent nerve. A direct blood sampling method from the duodenal vein and ileal mesenteric vein revealed that ZeinH administered into the ligated duodenal loop enhanced GLP-1 concentration in the ileal mesenteric vein but not in the duodenal vein. This confirmed that ZeinH in the duodenum induces GLP-1 secretion from L cells located in the ileum by an indirect mechanism. These results indicate that a potent GLP-1-releasing peptide, ZeinH, induces GLP-1 secretion by direct and indirect mechanisms in the rat intestine.

Glucagon-like peptide-1 (GLP-1), released from gut endocrine L cells in response to glucose, regulates appetite, insulin secretion, and gut motility. How glucose given orally, but not systemically, induces GLP-1 secretion is unknown. We show that human duodenal L cells express sweet taste receptors, the taste G protein gustducin, and several other taste transduction elements. Mouse intestinal L cells also express alpha-gustducin. Ingestion of glucose by alpha-gustducin null mice revealed deficiencies in secretion of GLP-1 and the regulation of plasma insulin and glucose. Isolated small bowel and intestinal villi from alpha-gustducin null mice showed markedly defective GLP-1 secretion in response to glucose. The human L cell line NCI-H716 expresses alpha-gustducin, taste receptors, and several other taste signaling elements. GLP-1 release from NCI-H716 cells was promoted by sugars and the noncaloric sweetener sucralose, and blocked by the sweet receptor antagonist lactisole or siRNA for alpha-gustducin. We conclude that L cells of the gut "taste" glucose through the same mechanisms used by taste cells of the tongue. Modulating GLP-1 secretion in gut "taste cells" may provide an important treatment for obesity, diabetes and abnormal gut motility.

The colonic epithelium harbors a large number of endocrine cells, but little is known about the endocrine functions of the colon. However, the high density of glucagon like peptide-1 (GLP-1)- and peptide-YY (PYY)-secreting L cells is of great interest because of the potential antidiabetic and antiobesity effects of GLP-1 and PYY. Short-chain fatty acids (SCFAs) produced by local bacterial fermentation are suggested to activate the colonic free fatty acid receptors FFAR2 (GPR43) and FFAR3 (GPR41), stimulating the colonic L cells. We used the isolated perfused rat colon as a model of colonic endocrine secretion and studied the effects of the predominant SCFAs formed: acetate, propionate, and butyrate. We show that luminal and especially vascular infusion of acetate and butyrate significantly increases colonic GLP-1 secretion, and to a minor extent also PYY secretion, but only after enhancement of intracellular cAMP. Propionate neither affected GLP-1 nor PYY secretion whether administered luminally or vascularly. A FFAR2- and FFAR3-specific agonist [( S)-2-(4-chlorophenyl)-3,3-dimethyl- N-(5-phenylthiazol-2-yl)butamide (CFMB)/ AR420626 ] had no effect on colonic GLP-1 output, and a FFAR3 antagonist ( AR399519 ) did not decrease the SCFA-induced GLP-1 response. However, the voltage-gated Ca2+-channel blocker nifedipine, the KATP-channel opener diazoxide, and the ATP synthesis inhibitor 2,4-dinitrophenol completely abolished the responses. FFAR2 receptor studies confirmed low-potent partial agonism of acetate, propionate, and butyrate, compared with CFMB, which is a full agonist with ~750-fold higher potency than the SCFAs. In conclusion, SCFAs may increase colonic GLP-1/PYY secretion, but FFAR2/FFAR3 do not seem to be involved. Rather, SCFAs are metabolized and appear to function as a colonocyte energy source. NEW & NOTEWORTHY By the use of in situ isolated perfused rat colon we show that short-chain fatty acids (SCFAs) primarily are used as a colonocyte energy source in the rat, subsequently triggering glucagon like peptide-1 (GLP-1) secretion independent of the free fatty acid receptors FFAR2 and FFAR3. Opposite many previous studies on SCFAs and FFAR2/FFAR3 and GLP-1 secretion, this experimental model allows investigation of the physiological interactions between luminal nutrients and secretion from cells whose function depend critically on their blood supply as well as nerve and paracrine interactions.

To investigate the role of chemerin reduction in mediating exercise-induced Glucagon-like peptide-1 (GLP-1) secretion and the amelioration of pancreatic β-cell function in obesity. Obesity models were established using wild-type and chemerin systemic knockout mice, followed by 8 weeks of moderate-intensity continuous aerobic exercise training. Serum chemerin levels, GLP-1 synthesis, glucose tolerance, pancreatic β-cell function, structure, and apoptosis were assessed. In vitro experiments were conducted on STC-1 cells, derived from murine intestinal endocrine cells, to evaluate GLP-1 secretion following exogenous chemerin treatment. Additionally, colonic tissue inflammation and apoptosis were analyzed using qPCR and TUNEL staining. In obese wild-type mice, moderate-intensity aerobic exercise significantly reduced serum chemerin levels, enhanced GLP-1 secretion, and improved glucose tolerance, pancreatic β-cell structure, function, and apoptosis. These effects were absent in obese chemerin knockout mice. Exogenous chemerin treatment reduced GLP-1 secretion in STC-1 cells. Furthermore, the beneficial effects of exercise on colonic inflammation and apoptosis observed in wild-type mice were abolished in chemerin knockout mice. Reduction of chemerin is crucial for the beneficial effects of aerobic exercise on GLP-1 secretion and pancreatic β-cell function in obesity. The mechanisms behind these effects may involve improvements in colonic inflammation and apoptosis. These findings offer new insights into the molecular mechanisms through which exercise improves obesity-related metabolic dysfunction.

Galanin is a widely expressed neuropeptide, which in the gut is thought to modulate gastrointestinal motility and secretion. We aimed to elucidate the poorly characterised mechanisms underlying the inhibitory effect of galanin and the potential involvement of G-protein coupled inwardly rectifying potassium, Kir 3, (GIRK) channels in glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) secretion. Purified murine L and K cells were analysed for expression of galanin receptors and GIRK subunits. Hormone secretion was measured from primary murine intestinal cultures. Intracellular cAMP was monitored in primary L cells derived from mice expressing the Epac2camps sensor under the control of the proglucagon promoter. Galanin receptor 1 (GAL1, Galr1) and GIRK channel 1 (Kir 3.1, Kcnj3) and 4 (Kir 3.4, Kcnj5) mRNA expression was highly enriched in K and L cells. Galanin and a selective GAL1 receptor agonist (M617) potently inhibited GLP-1 and GIP secretion from primary small intestinal cultures. In L cells, galanin significantly inhibited the forskolin-induced cAMP response. The GIRK1/4 activator ML297 significantly reduced glucose-stimulated and IBMX-stimulated GLP-1 secretion but had no effect on GIP. The GIRK blocker tertiapin-Q did not impair galanin-mediated GLP-1 inhibition. Galanin, acting via the GAL1 receptor and Gi -coupled signalling in L and K cells, is a potent inhibitor of GLP-1 and GIP secretion. Although GIRK1/4 channels are expressed in these cells, their activation does not appear to play a major role in galanin-mediated inhibition of incretin 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.