Interest in how the gut microbiome can influence the metabolic state of the host has recently heightened. One postulated link is bacterial fermentation of “indigestible” prebiotics to short-chain fatty acids (SCFAs), which in turn modulate the release of gut hormones controlling insulin release and appetite. We show here that SCFAs trigger secretion of the incretin hormone glucagon-like peptide (GLP)-1 from mixed colonic cultures in vitro. Quantitative PCR revealed enriched expression of the SCFA receptors ffar2 (grp43) and ffar3 (gpr41) in GLP-1–secreting L cells, and consistent with the reported coupling of GPR43 to Gq signaling pathways, SCFAs raised cytosolic Ca2+ in L cells in primary culture. Mice lacking ffar2 or ffar3 exhibited reduced SCFA-triggered GLP-1 secretion in vitro and in vivo and a parallel impairment of glucose tolerance. These results highlight SCFAs and their receptors as potential targets for the treatment of diabetes.

Aims/hypothesisIngested protein is a well-recognised stimulus for glucagon-like peptide-1 (GLP-1) release from intestinal L cells. This study aimed to characterise the molecular mechanisms employed by L cells to detect oligopeptides.MethodsGLP-1 secretion from murine primary colonic cultures and Ca2+ dynamics in L cells were monitored in response to peptones and dipeptides. L cells were identified and purified based on their cell-specific expression of the fluorescent protein Venus, using GLU-Venus transgenic mice. Pharmacological tools and knockout mice were used to characterise candidate sensory pathways identified by expression analysis.ResultsGLP-1 secretion was triggered by peptones and di-/tripeptides, including the non-metabolisable glycine-sarcosine (Gly-Sar). Two sensory mechanisms involving peptide transporter-1 (PEPT1) and the calcium-sensing receptor (CaSR) were distinguishable. Responses to Gly-Sar (10 mmol/l) were abolished in the absence of extracellular Ca2+ or by the L-type calcium-channel blocker nifedipine (10 μmol/l) and were PEPT1-dependent, as demonstrated by their sensitivity to pH and 4-aminomethylbenzoic acid and the finding of impaired responses in tissue from Pept1 (also known as Slc15a1) knockout mice. Peptone (5 mg/ml)-stimulated Ca2+ responses were insensitive to nifedipine but were blocked by antagonists of CaSR. Peptone-stimulated GLP-1 secretion was not impaired in mice lacking the putative peptide-responsive receptor lysophosphatidic acid receptor 5 (LPAR5; also known as GPR92/93).Conclusions/interpretationOligopeptides stimulate GLP-1 secretion through PEPT1-dependent electrogenic uptake and activation of CaSR. Both pathways are highly expressed in native L cells, and likely contribute to the ability of ingested protein to elevate plasma GLP-1 levels. Targeting nutrient-sensing pathways in L cells could be used to mobilise endogenous GLP-1 stores in humans, and could mimic some of the metabolic benefits of bariatric surgery.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-013-3037-3) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

Exposure of pancreatic b-cells to long-chain fatty acids leads to the activation of some components of the endoplasmic reticulum (ER) stress pathway and this mechanism may underlie the ability of certain fatty acids to promote b-cell death. We have studied ER stress in BRIN-BD11 b-cells exposed to either the saturated fatty acid palmitate (C16:0) or the monounsaturated palmitoleate (C16:1). Palmitate (0 . 025-0 . 25 mM) induced the expression of various markers of the RNA-dependent protein kinase-like ER eukaryotic initiation factor 2a (eIF2a) kinase (PERK)-dependent pathway of ER stress (phospho-eIF2a; ATF4, activating transcription factor 4 and C/EBP homologous protein (CHOP-10)) although it failed to promote the expression of the ER chaperone GRP78. By contrast, palmitoleate did not induce any markers of the ER stress pathway even at concentrations as high as 1 mM. When palmitate and palmitoleate were added in combination, a marked attenuation of the ER stress response occurred. Under these conditions, the levels of phospho-eIF2a, ATF4 and CHOP-10 were reduced to less than those found in control cells. Palmitoleate also attenuated the ER stress response to the protein glycosylation inhibitor, tunicamycin, and improved the viability of the cells exposed to this agent. Exposure of the BRIN-BD11 cells to the protein phosphatase inhibitor, salubrinal, in the absence of fatty acids resulted in increased eIF2a phosphorylation but this was abolished by co-incubation with palmitoleate. We conclude that saturated fatty acids activate components of the PERK-dependent ER stress pathway in b-cells, ultimately leading to increased apoptosis. This effect is antagonised by monounsaturates that may exert their anti-apoptotic actions by regulating the activity of one or more kinase enzymes involved in mediating the phosphorylation of eIF2a.

Long-chain saturated and monounsaturated fatty acids differ in their propensity to induce β-cell death in vitro with palmitate (C16:0) being cytotoxic, whereas palmitoleate (C16:1n-7) is cytoprotective. We now show that this cytoprotective capacity extends to a poorly metabolised C16:1n-7 derivative, methyl-palmitoleate (0·25 mM palmitate alone: 92±4% death after 18 h; palmitate plus 0·25 mM methyl-palmitoleate: 12±2%; P<0·001). Palmitoleate and its methylated derivative also acted as mitogens in cultured β-cells (5-bromo-2-deoxyuridine incorporation – control: 0·15±0·01 units; 0·25 mM palmitoleate: 0·22±0·01 units; P<0·05). It has been proposed that alterations in neutral lipid synthesis (particularly triacylglycerol (TAG) formation) might mediate the differential responses to saturated and unsaturated fatty acids and we have examined this proposition. Palmitate and palmitoleate both promoted β-cell phospholipid remodelling and increased TAG formation (control: 0·9±0·1 nmol TAG/106 cells; 0·25 mM palmitate: 1·55±0·07; 0·25 mM palmitoleate: 1·4±0·05; palmitate plus palmitoleate: 2·3±0·1). By contrast, methyl-palmitoleate failed to influence TAG levels (0·25 mM methyl-palmitoleate alone: 0·95±0·06 nmol TAG/106 cells; methyl-palmitoleate plus palmitate: 1·5±0·05) or its fatty acid composition in β-cells exposed to palmitate. The results suggest that monounsaturated fatty acids can promote cell viability and mitogenesis by a mechanism that does not require their metabolism and is independent of alterations in TAG formation.

The hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulintropic polypeptide (GIP) are secreted after a meal. Like other enteroendocrine hormones they help to orchestrate the bodies' response to the availability of newly absorbable nutrients and are noteworthy as they stimulate postprandial insulin secretion, underlying what is known as the incretin effect. GLP-1-mimetics are now widely used in the treatment of type 2 diabetes and advantages over older insulinotropic therapies include weight loss. An alternative treatment regime might be the recruitment of endogenous GLP-1, however, very little is known about the physiological control of enteroendocrine responses. This review focuses on the molecular mechanisms to detect nutrient arrival in the gut that have been implicated within the incretin secreting cells.

Stimulus-coupled incretin secretion from enteroendocrine cells plays a fundamental role in glucose homeostasis and could be targeted for the treatment of type 2 diabetes. Here, we investigated the expression and function of transient receptor potential (TRP) ion channels in enteroendocrine L cells producing GLP-1. By microarray and quantitative PCR analysis, we identified trpa1 as an L cell-enriched transcript in the small intestine. Calcium imaging of primary L cells and the model cell line GLUTag revealed responses triggered by the TRPA1 agonists allyl-isothiocyanate (mustard oil), carvacrol, and polyunsaturated fatty acids, which were blocked by TRPA1 antagonists. Electrophysiology in GLUTag cells showed that carvacrol induced a current with characteristics typical of TRPA1 and triggered the firing of action potentials. TRPA1 activation caused an increase in GLP-1 secretion from primary murine intestinal cultures and GLUTag cells, an effect that was abolished in cultures from trpa1 2/2 mice or by pharmacological TRPA1inhibition. These findings present TRPA1 as a novel sensory mechanism in enteroendocrine L cells, coupled to the facilitation of GLP-1 release, which may be exploitable as a target for treating diabetes.The global rise in the prevalence of type 2 diabetes makes it one of the biggest health and socioeconomic burdens of the 21st century. One of the newest and most successful strategies for the treatment of type 2 diabetes is based on mimicking, or enhancing, the endogenous action of incretin hormones. Glucose-dependent insulinotropic peptide (GIP) and GLP-1 are incretin peptides released from K and L cells, respectively, located in the epithelial layer of the gastrointestinal tract. GIP and GLP-1 potentiate glucose-dependent insulin secretion, whereas GLP-1 additionally suppresses appetite, glucagon secretion, and gastric emptying (1). These effects have been exploited pharmacologically through the use of long-acting GLP-1 mimetics or inhibitors of incretin inactivation by dipeptidyl peptidase-4 (DPP4) (2). Incretin therapy has significant benefits over older insulinotropic medications like sulphonylureas, as it does not bypass the glucosesensing machinery of the pancreatic b-cell and thus promotes insulin secretion in relation to ambient plasma glucose levels, minimizing the risk of hypoglycemia. In addition, GLP-1 mimetics are associated with sustained weight loss (3,4), whereas DPP4 inhibitors appear to be weight neutral (5), a difference that might reflect the higher effective concentrations of active GLP-1 reached with the former. Importantly, the substantially increased release of endogenous GLP-1 seen after Roux-en-Y gastric bypass surgery is likely to contribute toward the observed high rates of resolution of type 2 diabetes (6,7). Understanding the mechanisms that underlie endogenous GLP-1 secretion could therefore facilitate the development of novel therapies based on potentiating the postprandial incretin effect. Over the past decade, we and others have investigated the mechanisms underl...

Chronic exposure of pancreatic beta-cells to long-chain fatty acids can cause loss of secretory function and enhanced apoptosis by a process of 'lipotoxicity', which may be a contributory factor to the rising incidence of Type 2 diabetes in humans. However, when incubated in vitro, beta-cells respond differentially to long-chain saturated and mono-unsaturated fatty acids, suggesting that these molecules may regulate cell functionality by different mechanisms. In particular, it is clear that, whereas saturated fatty acids [e.g. palmitate (C16:0)] exert detrimental effects on beta-cells, the equivalent mono-unsaturated species [e.g. palmitoleate (C16:1)] are well tolerated. Indeed, mono-unsaturated species are potently cytoprotective. The present review explores the differential effects of these various fatty acids on beta-cell viability and considers the possible mechanisms involved in cytoprotection by mono-unsaturates.

Recent evidence indicates that treatment of pancreatic beta-cells with long chain fatty acids can lead to the development of an ER (endoplasmic reticulum) stress response. This is manifest as the activation of some components of the PERK [RNA-dependent protein kinase-like ER eIF2alpha (eukaryotic initiation factor 2alpha) kinase]-dependent arm of ER stress and is seen most dramatically when cells are treated with long-chain saturated fatty acids (e.g. palmitate). By contrast, the equivalent mono-unsaturates (e.g. palmitoleate) are much less effective and they can even attenuate the ER stress response to palmitate. This may be due to the regulation of eIF2alpha phosphorylation in cells exposed to mono-unsaturates. The present review discusses the differential effects of saturated and mono-unsaturated fatty acids on ER stress in beta-cells and considers the extent to which regulation of this pathway may be involved in mediating their effects on viability.