Highlights d Paracrine glucagon actions are required for maintenance of normal insulin secretion d Glucagon signaling in islets involves activation of glucagon and GLP-1 receptors d Beta cell GLP-1 receptors are activated by local glucagon in a paracrine manner

Paracrine interactions between pancreatic islet cells have been proposed as a mechanism to regulate hormone secretion and glucose homeostasis. Here, we demonstrate the importance of proglucagon-derived peptides (PGDPs) for α to β cell communication and control of insulin secretion. Signaling through this system occurs through both the glucagon-like peptide receptor (Glp1r) and glucagon receptor (Gcgr). Loss of PGDPs, or blockade of their receptors, decreases insulin secretion in response to both metabolic and nonmetabolic stimulation of mouse and human islets. This effect is due to reduced β cell cAMP and affects the quantity but not dynamics of insulin release, indicating that PGDPs dictate the magnitude of insulin output in an isolated islet. In healthy mice, additional factors that stimulate cAMP can compensate for loss of PGDP signaling; however, input from α cells is essential to maintain glucose tolerance during the metabolic stress induced by high-fat feeding. These findings demonstrate an essential role for α cell regulation of β cells, raising the possibility that abnormal paracrine signaling contributes to impaired insulin secretion in diabetes. Moreover, these findings support reconsideration of the role for α cells in postprandial glucose control.

Aims/hypothesisIntra-islet and gut–islet crosstalk are critical in orchestrating basal and postprandial metabolism. The aim of this study was to identify regulatory proteins and receptors underlying somatostatin secretion though the use of transcriptomic comparison of purified murine alpha, beta and delta cells.MethodsSst-Cre mice crossed with fluorescent reporters were used to identify delta cells, while Glu-Venus (with Venus reported under the control of the Glu [also known as Gcg] promoter) mice were used to identify alpha and beta cells. Alpha, beta and delta cells were purified using flow cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases.ResultsA database was constructed of all genes expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused pancreases, correlating with a decrease in insulin and glucagon release. The inhibition of insulin secretion by ghrelin was prevented by somatostatin receptor antagonism.Conclusions/interpretationOur transcriptomic database of genes expressed in the principal islet cell populations will facilitate rational drug design to target specific islet cell types. The present study indicates that ghrelin acts specifically on delta cells within pancreatic islets to elicit somatostatin secretion, which in turn inhibits insulin and glucagon release. This highlights a potential role for ghrelin in the control of glucose metabolism.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-4033-1) contains peer-reviewed but unedited supplementary material, which is available to authorised users.

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 Ca-channel blocker nifedipine, the K-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.

Glucose is an important stimulus for glucagon-like peptide 1 (GLP-1) secretion, but the mechanisms of secretion have not been investigated in integrated physiological models. We studied glucose-stimulated GLP-1 secretion from isolated perfused rat small intestine. Luminal glucose (5% and 20% w/v) stimulated the secretion dose dependently, but vascular glucose was without significant effect at 5, 10, 15, and 25 mmol/L. GLP-1 stimulation by luminal glucose (20%) secretion was blocked by the voltage-gated Ca channel inhibitor, nifedipine, or by hyperpolarization with diazoxide. Luminal administration (20%) of the nonmetabolizable sodium-glucose transporter 1 (SGLT1) substrate, methyl-a-D-glucopyranoside (a-MGP), stimulated release, whereas the SGLT1 inhibitor phloridzin (luminally) abolished responses to a-MGP and glucose. Furthermore, in the absence of luminal NaCl, luminal glucose (20%) did not stimulate a response. Luminal glucose-stimulated GLP-1 secretion was also sensitive to luminal GLUT2 inhibition (phloretin), but in contrast to SGLT1 inhibition, phloretin did not eliminate the response, and luminal glucose (20%) stimulated larger GLP-1 responses than luminal a-MGP in matched concentrations. Glucose transported by GLUT2 may act after metabolization, closing K ATP channels similar to sulfonylureas, which also stimulated secretion. Our data indicate that SGLT1 activity is the driving force for glucose-stimulated GLP-1 secretion and that K ATP -channel closure is required to stimulate a full-blown glucoseinduced response.

SUMMARY The melanocortin-4 receptor (MC4R) is expressed in the brainstem and vagal afferent nerves, and regulates a number of aspects of gastrointestinal function. Here we show that the receptor is also diffusely expressed in cells of the gastrointestinal system, from stomach to descending colon. Furthermore, MC4R is the second most highly expressed GPCR in peptide YY (PYY) and glucagon-like peptide one (GLP-1) expressing enteroendocrine L cells. When vectorial ion transport is measured across mouse or human intestinal mucosa, administration of α-MSH induces a MC4R-specific PYY-dependent anti-secretory response consistent with a role for the MC4R in paracrine inhibition of electrolyte secretion. Finally, MC4R-dependent acute PYY and GLP-1 release from L cells can be stimulated in vivo by intraperitoneal administration of melanocortin peptides to mice. This suggests physiological significance for MC4R in L cells, and indicates a previously unrecognized peripheral role for the MC4R, complementing vagal and central receptor functions.

Gut endocrine cells are generally thought to have distinct localization and secretory products. Recent studies suggested that the cells are highly related and have potential to express more than one hormone. We studied the coexpression and cosecretion of gut hormones in separate segments of rat small intestine. We measured secretion of glucagon-like peptide-1 (GLP-1), peptide YY (PYY), neurotensin, glucose-dependent insulinotropic polypeptide (GIP), and cholecystokinin (CCK) from proximal and distal half of the small intestine, isolated from male rats and perfused ex vivo. Hormone secretion was stimulated by bombesin, the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, and peptones. Furthermore, tissue samples collected along the intestine were analyzed for expression, hormone content, and cell densities including colocalization. Most hormones responded to all three stimuli (but no GIP response to bombesin). GLP-1 secretion was similar from proximal and distal intestine, whereas PYY was secreted only from the distal half. CCK and GIP were mainly secreted proximally, whereas neurotensin was equally secreted from both parts. Cell densities, hormone concentrations, and expression patterns were generally parallel, with increasing values distally for GLP-1 and PYY, an exclusively proximal pattern for CCK, even distribution for neurotensin and GIP except for the most distal segments. PYY nearly always colocalized with GLP-1. Approximately 20% of GLP-1 cells colocalized with CCK and neurotensin, whereas GLP-1/GIP colocalization was rare. Our findings indicate that two L cell types exist, a proximal one secreting GLP-1 (and possibly CCK and neurotensin), and a distal one secreting GLP-1 and PYY. GIP seems to be secreted from cells that are not cosecreting other peptides.

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