Opposing roles of the entero-pancreatic hormone urocortin-3 in glucose metabolism in rats.

AIM/HYPOTHESIS: Urocortin-3 (UCN3) is a glucoregulatory peptide produced in the gut and pancreatic islets. The aim of this study was to clarify the acute effects of UCN3 on glucose regulation following an oral glucose challenge and to investigate the mechanisms involved. METHODS: We studied the effect of UCN3 on blood glucose, gastric emptying, glucose absorption and secretion of gut and pancreatic hormones in male rats. To supplement these physiological studies, we mapped the expression of UCN3 and the UCN3-sensitive receptor, type 2 corticotropin-releasing factor receptor (CRHR2), by means of fluorescence in situ hybridisation and by gene expression analysis. RESULTS: In rats, s.c. administration of UCN3 strongly inhibited gastric emptying and glucose absorption after oral administration of glucose. Direct inhibition of gastrointestinal motility may be responsible because UCN3's cognate receptor, CRHR2, was detected in gastric submucosal plexus and in interstitial cells of Cajal. Despite inhibited glucose absorption, post-challenge blood glucose levels matched those of rats given vehicle in the low-dose UCN3 group, because UCN3 concomitantly inhibited insulin secretion. Higher UCN3 doses did not further inhibit gastric emptying, but the insulin inhibition progressed resulting in elevated post-challenge glucose and lipolysis. Incretin hormones and somatostatin (SST) secretion from isolated perfused rat small intestine was unaffected by UCN3 infusion; however, UCN3 infusion stimulated secretion of somatostatin from delta cells in the isolated perfused rat pancreas which, unlike alpha cells and beta cells, expressed Crhr2. Conversely, acute antagonism of CRHR2 signalling increased insulin secretion by reducing SST signalling. Consistent with these observations, acute drug-induced inhibition of CRHR2 signalling improved glucose tolerance in rats to a similar degree as administration of glucagon-like peptide-1. UCN3 also powerfully inhibited glucagon secretion from isolated perfused rat pancreas (perfused with 3.5 mmol/l glucose) in a SST-dependent manner, suggesting that UCN3 may be involved in glucose-induced inhibition of glucagon secretion. CONCLUSIONS/INTERPRETATION: Our combined data indicate that UCN3 is an important glucoregulatory hormone that acts through regulation of gastrointestinal and pancreatic functions.

Secretin release after Roux-en-Y gastric bypass reveals a population of glucose-sensitive S cells in distal small intestine.

OBJECTIVES: Gastrointestinal hormones contribute to the beneficial effects of Roux-en-Y gastric bypass surgery (RYGB) on glycemic control. Secretin is secreted from duodenal S cells in response to low luminal pH, but it is unknown whether its secretion is altered after RYGB and if secretin contributes to the postoperative improvement in glycemic control. We hypothesized that secretin secretion increases after RYGB as a result of the diversion of nutrients to more distal parts of the small intestine, and thereby affects islet hormone release. METHODS: A specific secretin radioimmunoassay was developed, evaluated biochemically, and used to quantify plasma concentrations of secretin in 13 obese individuals before, 1 week after, and 3 months after RYGB. Distribution of secretin and its receptor was assessed by RNA sequencing, mass-spectrometry and in situ hybridization in human and rat tissues. Isolated, perfused rat intestine and pancreas were used to explore the molecular mechanism underlying glucose-induced secretin secretion and to study direct effects of secretin on glucagon, insulin, and somatostatin secretion. Secretin was administered alone or in combination with GLP-1 to non-sedated rats to evaluate effects on glucose regulation. RESULTS: Plasma postprandial secretin was more than doubled in humans after RYGB (P < 0.001). The distal small intestine harbored secretin expressing cells in both rats and humans. Glucose increased the secretion of secretin in a sodium-glucose cotransporter dependent manner when administered to the distal part but not into the proximal part of the rat small intestine. Secretin stimulated somatostatin secretion (fold change: 1.59, P < 0.05) from the perfused rat pancreas but affected neither insulin (P = 0.2) nor glucagon (P = 0.97) secretion. When administered to rats in vivo, insulin secretion was attenuated and glucagon secretion increased (P = 0.04), while blood glucose peak time was delayed (from 15 to 45 min) and gastric emptying time prolonged (P = 0.004). CONCLUSIONS: Glucose-sensing secretin cells located in the distal part of the small intestine may contribute to increased plasma concentrations observed after RYGB. The metabolic role of the distal S cells warrants further studies.

No direct effect of SGLT2 activity on glucagon secretion.

AIMS/HYPOTHESIS: Sodium-glucose cotransporter (SGLT) 2 inhibitors constitute a new class of glucose-lowering drugs, but they increase glucagon secretion, which may counteract their glucose-lowering effect. Previous studies using static incubation of isolated human islets or the glucagon-secreting cell line α-TC1 suggested that this results from direct inhibition of alpha cell SGLT1/2-activity. The aim of this study was to test whether the effects of SGLT2 on glucagon secretion demonstrated in vitro could be reproduced in a more physiological setting. METHODS: We explored the effect of SGLT2 activity on glucagon secretion using isolated perfused rat pancreas, a physiological model for glucagon secretion. Furthermore, we investigated Slc5a2 (the gene encoding SGLT2) expression in rat islets as well as in mouse and human islets and in mouse and human alpha, beta and delta cells to test for potential inter-species variations. SGLT2 protein content was also investigated in mouse, rat and human islets. RESULTS: Glucagon output decreased three- to fivefold within minutes of shifting from low (3.5 mmol/l) to high (10 mmol/l) glucose (4.0 ± 0.5 pmol/15 min vs 1.3 ± 0.3 pmol/15 min, p < 0.05). The output was unaffected by inhibition of SGLT1/2 with dapagliflozin or phloridzin or by addition of the SGLT1/2 substrate α-methylglucopyranoside, whether at low or high glucose concentrations (p = 0.29-0.99). Insulin and somatostatin secretion (potential paracrine regulators) was also unaffected. Slc5a2 expression and SGLT2 protein were marginal or below detection limit in rat, mouse and human islets and in mouse and human alpha, beta and delta cells. CONCLUSIONS/INTERPRETATION: Our combined data show that increased plasma glucagon during SGLT2 inhibitor treatment is unlikely to result from direct inhibition of SGLT2 in alpha cells, but instead may occur downstream of their blood glucose-lowering effects.

Weight loss by calorie restriction does not alter appetite-regulating gut hormone responses from perfused rat small intestine.

AIM: Postprandial secretion of the appetite-inhibiting hormones, glucagon-like peptide-1 (GLP-1), and peptide YY are reduced with obesity. It is unclear if the reduced secretion persists following weight loss (WL), if other appetite-inhibiting hormones are also reduced, and if so whether reduced secretion results from intrinsic changes in the gut. METHODS: To address whether WL may restore secretion of GLP-1 and other appetite-inhibiting hormones, we performed a gut perfusion study of the small intestine in diet-induced obese (DIO) rats after WL. A 20% weight loss (means ± SEM (g): 916 ± 53 vs. 703 ± 35, p < 0.01, n = 7) was induced by calorie restriction, and maintained stable for ≥7 days prior to gut perfusion to allow for complete renewal of enteroendocrine cells. Age-matched DIO rats were used as comparator. Several gut hormones were analyzed from the venous effluent, and gene expression was performed on gut tissue along the entire length of the intestine. RESULTS: Secretion of cholecystokinin, gastrin, glucose-dependent insulinotropic peptide, GLP-1, neurotensin, and somatostatin was not affected by WL during basal conditions (p ≥ 0.25) or in response to macronutrients and bile acids (p ≥ 0.14). Glucose absorption was indistinguishable following WL. The expression of genes encoding the studied peptides, macronutrient transporters (glucose, fructose, and di-/tripeptides) and bile acid receptors did also not differ between DIO and WL groups. CONCLUSIONS: These data suggest that the attenuated postprandial responses of GLP-1, as well as reduced responses of other appetite-inhibiting gut hormones, in people living with obesity may persist after weight loss and may contribute to their susceptibility for weight regain.

Peptides in the regulation of glucagon secretion.

Glucose homeostasis is maintained by the glucoregulatory hormones, glucagon, insulin and somatostatin, secreted from the islets of Langerhans. Glucagon is the body's most important anti-hypoglycemic hormone, mobilizing glucose from glycogen stores in the liver in response to fasting, thus maintaining plasma glucose levels within healthy limits. Glucagon secretion is regulated by both circulating nutrients, hormones and neuronal inputs. Hormones that may regulate glucagon secretion include locally produced insulin and somatostatin, but also urocortin-3, amylin and pancreatic polypeptide, and from outside the pancreas glucagon-like peptide-1 and 2, peptide tyrosine tyrosine and oxyntomodulin, glucose-dependent insulinotropic polypeptide, neurotensin and ghrelin, as well as the hypothalamic hormones arginine-vasopressin and oxytocin, and calcitonin from the thyroid. Each of these hormones have distinct effects, ranging from regulating blood glucose, to regulating appetite, stomach emptying rate and intestinal motility, which makes them interesting targets for treating metabolic diseases. Awareness regarding the potential effects of the hormones on glucagon secretion is important since secretory abnormalities could manifest as hyperglycemia or even lethal hypoglycemia. Here, we review the effects of each individual hormone on glucagon secretion, their interplay, and how treatments aimed at modulating the plasma levels of these hormones may also influence glucagon secretion and glycemic control.

Expression Profile of the GLP-1 Receptor in the Gastrointestinal Tract and Pancreas in Adult Female Mice.

Therapies based on glucagon-like peptide-1 receptor (GLP-1R) agonism are highly effective in treating type 2 diabetes and obesity, but the localization of GLP-1Rs mediating the antidiabetic and other possible actions of GLP-1 is still debated. The purpose with this study was to identify sites of GLP-1R mRNA and protein expression in the mouse gastrointestinal system by means of GLP-1R antibody immunohistochemistry, Glp1r mRNA fluorescence in situ hybridization, and 125I-exendin (9-39) autoradiography. As expected, GLP-1R staining was observed in almost all ß-cells in the pancreatic islets, but more rarely in α- and δ-cells. In the stomach, GLP-1R staining was found exclusively in the gastric corpus mucous neck cells, known to protect the stomach mucosa. The Brunner glands were strongly stained for GLP-1R, and pretreatment with GLP-1 agonist exendin-4 caused internalization of the receptor and mucin secretion, while pretreatment with phosphate-buffered saline or antagonist exendin (9-39) did not. In the intestinal mucosa, GLP-1R staining was observed in intraepithelial lymphocytes, lamina propria lymphocytes, and enteroendocrine cells containing secretin, peptide YY, and somatostatin, but not cholecystokinin. GLP-1R staining was seen in nerve fibers within the choline acetyl transferase- and nitric oxide-positive myenteric plexuses from the gastric corpus to the distal large intestine being strongest in the mid- and hindgut area. Finally, intraperitoneal administration of radiolabeled exendin (9-39) strongly labeled myenteric fibers. In conclusion, this study expands our knowledge of GLP-1R localization and suggests that GLP-1 may serve an important role in modulating gastrointestinal health and mucosal protection.

Using a Reporter Mouse to Map Known and Novel Sites of GLP-1 Receptor Expression in Peripheral Tissues of Male Mice.

Glucagon-like peptide-1 receptor (GLP-1R) activation is used in the treatment of diabetes and obesity; however, GLP-1 induces many other physiological effects with unclear mechanisms of action. To identify the cellular targets of GLP-1 and GLP-1 analogues, we generated a Glp1r.tdTomato reporter mouse expressing the reporter protein, tdTomato, in Glp1r-expressing cells. The reporter signal is expressed in all cells where GLP-1R promoter was ever active. To complement this, we histologically mapped tdTomato-fluorescence, and performed Glp-1r mRNA in situ hybridization and GLP-1R immunohistochemistry on the same tissues. In male mice, we found tdTomato signal in mucus neck, chief, and parietal cells of the stomach; Brunner's glands; small intestinal enteroendocrine cells and intraepithelial lymphocytes; and myenteric plexus nerve fibers throughout the gastrointestinal tract. Pancreatic acinar-, ß-, and δ cells, but rarely α cells, were tdTomato-positive, as were renal arteriolar smooth muscle cells; endothelial cells of the liver, portal vein, and endocardium; aortal tunica media; and lung type 1 and type 2 pneumocytes. Some thyroid follicular and parafollicular cells displayed tdTomato expression, as did tracheal cartilage chondrocytes, skin fibroblasts, and sublingual gland mucus cells. In conclusion, our reporter mouse is a powerful tool for mapping known and novel sites of GLP-1R expression in the mouse, thus enhancing our understanding of the many target cells and effects of GLP-1 and GLP-1R agonists.

In the rat pancreas, somatostatin tonically inhibits glucagon secretion and is required for glucose-induced inhibition of glucagon secretion.

AIM: It is debated whether the inhibition of glucagon secretion by glucose results from direct effects of glucose on the α-cell (intrinsic regulation) or by paracrine effects exerted by beta- or delta-cell products. METHODS: To study this in a more physiological model than isolated islets, we perfused isolated rat pancreases and measured glucagon, insulin and somatostatin secretion in response to graded increases in perfusate glucose concentration (from 3.5 to 4, 5, 6, 7, 8, 10, 12 mmol/L) as well as glucagon responses to blockage/activation of insulin/GABA/somatostatin signalling with or without addition of glucose. RESULTS: Glucagon secretion was reduced by about 50% (compared to baseline secretion at 3.5 mmol/L) within minutes after increasing glucose from 4 to 5 mmol/L (P < .01, n = 13). Insulin secretion was increased minimally, but significantly, compared to baseline (3.5 mmol/L) at 4 mmol/L, whereas somatostatin secretion was not significantly increased from baseline until 7 mmol/L. Hereafter secretion of both increased gradually up to 12 mmol/L glucose. Neither recombinant insulin (1 µmol/L), GABA (300 µmol/L) or the insulin-receptor antagonist S961 (at 1 µmol/L) affected basal (3.5 mmol/L) or glucose-induced (5.0 mmol/L) attenuation of glucagon secretion (n = 7-8). Somatostatin-14 attenuated glucagon secretion by ~ 95%, and blockage of somatostatin-receptor (SSTR)-2 or combined blockage of SSTR-2, -3 and -5 by specific antagonists increased glucagon output (at 3.5 mmol/L glucose) and prevented glucose-induced (from 3.5 to 5.0 mmol/L) suppression of secretion. CONCLUSION: Somatostatin is a powerful and tonic inhibitor of glucagon secretion from the rat pancreas and is required for glucose to inhibit glucagon secretion.

An atlas of O-linked glycosylation on peptide hormones reveals diverse biological roles.

Peptide hormones and neuropeptides encompass a large class of bioactive peptides that regulate physiological processes like anxiety, blood glucose, appetite, inflammation and blood pressure. Here, we execute a focused discovery strategy to provide an extensive map of O-glycans on peptide hormones. We find that almost one third of the 279 classified peptide hormones carry O-glycans. Many of the identified O-glycosites are conserved and are predicted to serve roles in proprotein processing, receptor interaction, biodistribution and biostability. We demonstrate that O-glycans positioned within the receptor binding motifs of members of the neuropeptide Y and glucagon families modulate receptor activation properties and substantially extend peptide half-lives. Our study highlights the importance of O-glycosylation in the biology of peptide hormones, and our map of O-glycosites in this large class of biomolecules serves as a discovery platform for an important class of molecules with potential opportunities for drug designs.

Glucagon acutely regulates hepatic amino acid catabolism and the effect may be disturbed by steatosis.

OBJECTIVE: Glucagon is well known to regulate blood glucose but may be equally important for amino acid metabolism. Plasma levels of amino acids are regulated by glucagon-dependent mechanism(s), while amino acids stimulate glucagon secretion from alpha cells, completing the recently described liver-alpha cell axis. The mechanisms underlying the cycle and the possible impact of hepatic steatosis are unclear. METHODS: We assessed amino acid clearance in vivo in mice treated with a glucagon receptor antagonist (GRA), transgenic mice with 95% reduction in alpha cells, and mice with hepatic steatosis. In addition, we evaluated urea formation in primary hepatocytes from ob/ob mice and humans, and we studied acute metabolic effects of glucagon in perfused rat livers. We also performed RNA sequencing on livers from glucagon receptor knock-out mice and mice with hepatic steatosis. Finally, we measured individual plasma amino acids and glucagon in healthy controls and in two independent cohorts of patients with biopsy-verified non-alcoholic fatty liver disease (NAFLD). RESULTS: Amino acid clearance was reduced in mice treated with GRA and mice lacking endogenous glucagon (loss of alpha cells) concomitantly with reduced production of urea. Glucagon administration markedly changed the secretion of rat liver metabolites and within minutes increased urea formation in mice, in perfused rat liver, and in primary human hepatocytes. Transcriptomic analyses revealed that three genes responsible for amino acid catabolism (Cps1, Slc7a2, and Slc38a2) were downregulated both in mice with hepatic steatosis and in mice with deletion of the glucagon receptor. Cultured ob/ob hepatocytes produced less urea upon stimulation with mixed amino acids, and amino acid clearance was lower in mice with hepatic steatosis. Glucagon-induced ureagenesis was impaired in perfused rat livers with hepatic steatosis. Patients with NAFLD had hyperglucagonemia and increased levels of glucagonotropic amino acids, including alanine in particular. Both glucagon and alanine levels were reduced after diet-induced reduction in Homeostatic Model Assessment for Insulin Resistance (HOMA-IR, a marker of hepatic steatosis). CONCLUSIONS: Glucagon regulates amino acid metabolism both non-transcriptionally and transcriptionally. Hepatic steatosis may impair glucagon-dependent enhancement of amino acid catabolism.

Bile acids are important direct and indirect regulators of the secretion of appetite- and metabolism-regulating hormones from the gut and pancreas.

OBJECTIVE: Bile acids (BAs) facilitate fat absorption and may play a role in glucose and metabolism regulation, stimulating the secretion of gut hormones. The relative importance and mechanisms involved in BA-stimulated secretion of appetite and metabolism regulating hormones from the gut and pancreas is not well described and was the purpose of this study. METHODS: The effects of bile acids on the secretion of gut and pancreatic hormones was studied in rats and compared to the most well described nutritional secretagogue: glucose. The molecular mechanisms that underlie the secretion was studied by isolated perfused rat and mouse small intestine and pancreas preparations and supported by immunohistochemistry, expression analysis, and pharmacological studies. RESULTS: Bile acids robustly stimulate secretion of not only the incretin hormones, glucose-dependent insulinotropic peptide (GIP), and glucagon-like peptide-1 (GLP-1), but also glucagon and insulin in vivo, to levels comparable to those resulting from glucose stimulation. The mechanisms of GLP-1, neurotensin, and peptide YY (PYY) secretion was secondary to intestinal absorption and depended on activation of basolateral membrane Takeda G-protein receptor 5 (TGR5) receptors on the L-cells in the following order of potency: Lithocholic acid (LCA) >Deoxycholicacid (DCA)>Chenodeoxycholicacid (CDCA)> Cholic acid (CA). Thus BAs did not stimulate secretion of GLP-1 and PYY from perfused small intestine in TGR5 KO mice but stimulated robust responses in wild type littermates. TGR5 is not expressed on α-cells or ß-cells, and BAs had no direct effects on glucagon or insulin secretion from the perfused pancreas. CONCLUSION: BAs should be considered not only as fat emulsifiers but also as important regulators of appetite- and metabolism-regulating hormones by activation of basolateral intestinal TGR5.