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.

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.

L-Cell Expression of Melanocortin-4-Receptor Is Marginal in Most of the Small Intestine in Mice and Humans and Direct Stimulation of Small Intestinal Melanocortin-4-Receptors in Mice and Rats Does Not Affect GLP-1 Secretion.

The molecular sensors underlying nutrient-stimulated GLP-1 secretion are currently being investigated. Peripheral administration of melanocortin-4 receptor (MC4R) agonists have been reported to increase GLP-1 plasma concentrations in mice and humans but it is unknown whether this effect results from a direct effect on the GLP-1 secreting L-cells in the intestine, from other effects in the intestine or from extra-intestinal effects. We investigated L-cell expression of MC4R in mouse and human L-cells by reanalyzing publicly available RNA sequencing databases (mouse and human) and by RT-qPCR (mouse), and assessed whether administration of MC4R agonists to a physiologically relevant gut model, isolated perfused mouse and rat small intestine, would stimulate GLP-1 secretion or potentiate glucose-stimulated secretion. L-cell MC4R expression was low in mouse duodenum and hardly detectable in the ileum and MC4R expression was hardly detectable in human L-cells. In isolated perfused mouse and rat intestine, neither intra-luminal nor intra-arterial administration of NDP-alpha-MSH, a potent MC4R agonist, had any effect on GLP-1 secretion (P ≥0.98, n = 5-6) from the upper or lower-half of the small intestine in mice or in the lower half in rats. Furthermore, HS014-an often used MC4R antagonist, which we found to be a partial agonist-did not affect the glucose-induced GLP-1 response in the rat, P = 0.62, n = 6). Studies on transfected COS7-cells confirmed bioactivity of the used compounds and that concentrations employed were well within in the effective range. Our combined data therefore suggest that MC4R-activated GLP-1 secretion in rodents either exclusively occurs in the colon or involves extra-intestinal signaling.

Effects of Manipulating Circulating Bile Acid Concentrations on Postprandial GLP-1 Secretion and Glucose Metabolism After Roux-en-Y Gastric Bypass.

Background: Altered bile acid (BA) turnover has been suggested to be involved in the improved glucose regulation after Roux-en-Y gastric bypass (RYGB), possibly via stimulation of GLP-1 secretion. We investigated the role of exogenous as well as endogenous BAs for GLP-1 secretion after RYGB by administering chenodeoxycholic acid (CDCA) and the BA sequestrant colesevelam (COL) both in the presence and the absence of a meal stimulus. Methods: Two single-blinded randomized cross-over studies were performed. In study 1, eight RYGB operated participants ingested 200 ml water with 1) CDCA 1.25 g or 2) CDCA 1.25 g + colesevelam 3.75 g on separate days. In study 2, twelve RYGB participants ingested on separate days a mixed meal with addition of 1) CDCA 1.25 g, 2) COL 3.75 g or 3) COL 3.75 g × 2, or 4) no additions. Results: In study 1, oral intake of CDCA increased circulating BAs, GLP-1, C-peptide, glucagon, and neurotensin. Addition of colesevelam reduced all responses. In study 2, addition of CDCA enhanced meal-induced increases in plasma GLP-1, glucagon and FGF-19 and lowered plasma glucose and C-peptide concentrations, while adding colesevelam lowered circulating BAs but did not affect meal-induced changes in plasma glucose or measured gastrointestinal hormones. Conclusion: In RYGB-operated persons, exogenous CDCA enhanced meal-stimulated GLP-1 and glucagon secretion but not insulin secretion, while the BA sequestrant colesevelam decreased CDCA-stimulated GLP-1 secretion but did not affect meal-stimulated GLP-1, C-peptide or glucagon secretion, or glucose tolerance. These findings suggest a limited role for endogenous bile acids in the acute regulation of postprandial gut hormone secretion or glucose metabolism after RYGB.

What Is an L-Cell and How Do We Study the Secretory Mechanisms of the L-Cell?

Synthetic glucagon-like peptide-1 (GLP-1) analogues are effective anti-obesity and anti-diabetes drugs. The beneficial actions of GLP-1 go far beyond insulin secretion and appetite, and include cardiovascular benefits and possibly also beneficial effects in neurodegenerative diseases. Considerable reserves of GLP-1 are stored in intestinal endocrine cells that potentially might be mobilized by pharmacological means to improve the body's metabolic state. In recognition of this, the interest in understanding basic L-cell physiology and the mechanisms controlling GLP-1 secretion, has increased considerably. With a view to home in on what an L-cell is, we here present an overview of available data on L-cell development, L-cell peptide expression profiles, peptide production and secretory patterns of L-cells from different parts of the gut. We conclude that L-cells differ markedly depending on their anatomical location, and that the traditional definition of L-cells as a homogeneous population of cells that only produce GLP-1, GLP-2, glicentin and oxyntomodulin is no longer tenable. We suggest to sub-classify L-cells based on their differential peptide contents as well as their differential expression of nutrient sensors, which ultimately determine the secretory responses to different stimuli. A second purpose of this review is to describe and discuss the most frequently used experimental models for functional L-cell studies, highlighting their benefits and limitations. We conclude that no experimental model is perfect and that a comprehensive understanding must be built on results from a combination of models.

Do sodium-glucose co-transporter-2 inhibitors increase plasma glucagon by direct actions on the alpha cell? And does the increase matter for the associated increase in endogenous glucose production?

Sodium-glucose co-transporter-2 inhibitors (SGLT2is) lower blood glucose and are used for treatment of type 2 diabetes. However, SGLT2is have been associated with increases in endogenous glucose production (EGP) by mechanisms that have been proposed to result from SGLT2i-mediated increases in circulating glucagon concentrations, but the relative importance of this effect is debated, and mechanisms possibly coupling SGLT2is to increased plasma glucagon are unclear. A direct effect on alpha-cell activity has been proposed, but data on alpha-cell SGLT2 expression are inconsistent, and studies investigating the direct effects of SGLT2 inhibition on glucagon secretion are conflicting. By contrast, alpha-cell sodium-glucose co-transporter-1 (SGLT1) expression has been found more consistently and appears to be more prominent, pointing to an underappreciated role for this transporter. Nevertheless, the selectivity of most SGLT2is does not support interference with SGLT1 during therapy. Paracrine effects mediated by secretion of glucagonotropic/static molecules from beta and/or delta cells have also been suggested to be involved in SGLT2i-induced increase in plasma glucagon, but studies are few and arrive at different conclusions. It is also possible that the effect on glucagon is secondary to drug-induced increases in urinary glucose excretion and lowering of blood glucose, as shown in experiments with glucose clamping where SGLT2i-associated increases in plasma glucagon are prevented. However, regardless of the mechanisms involved, the current balance of evidence does not support that SGLT2 plays a crucial role for alpha-cell physiology or that SGLT2i-induced glucagon secretion is important for the associated increased EGP, particularly because the increase in EGP occurs before any rise in plasma glucagon.

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.

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.

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.

Hepatic Bile Acid Reuptake in the Rat Depends on Bile Acid Conjugation but Not on Agonistic Properties towards FXR and TGR5.

Farnesoid X receptor (FXR) and Takeda G-protein coupled receptor 5 (TGR5) are the two known bile acid (BA) sensitive receptors and are expressed in the intestine and liver as well as in extra-enterohepatic tissues. The physiological effects of extra-enterohepatic FXR/TRG5 remain unclear. Further, the extent BAs escape liver reabsorption and how they interact with extra-enterohepatic FXR/TGR5 is understudied. We investigated if hepatic BA reuptake differed between BAs agonistic for FXR and TGR5 compared to non-agonists in the rat. Blood was collected from the portal vein and inferior caval vein from anesthetized rats before and 5, 20, 30, and 40 min post stimulation with sulfated cholecystokinin-8. Plasma concentrations of 20 different BAs were assessed by liquid chromatography coupled to mass spectrometry. Total portal vein BA AUC was 3-4 times greater than in the vena cava inferior (2.7 ± 0.6 vs. 0.7 ± 0.2 mM x min, p < 0.01, n = 8) with total unconjugated BAs being 2-3-fold higher than total conjugated BAs (AUC 8-10 higher p < 0.05 for both). However, in both cases, absolute ratios varied greatly among different BAs. The average hepatic reuptake of BAs agonistic for FXR/TGR5 was similar to non-agonists. However, as the sum of non-agonist BAs in vena portae was 2-3-fold higher than the sum agonist (p < 0.05), the peripheral BA pool was composed mostly of non-agonist BAs. We conclude that hepatic BA reuptake varies substantially by type and does not favor FXR/TGR5 BAs agonists.

Bilio-enteric flow and plasma concentrations of bile acids after gastric bypass and sleeve gastrectomy.

BACKGROUND/OBJECTIVES: Bile acids in plasma are elevated after bariatric surgery and may contribute to metabolic improvements, but underlying changes in bile flow are poorly understood. We assessed bilio-enteric flow of bile and plasma bile concentrations in individuals with Roux-en-Y gastric bypass (RYGB) or sleeve gastrectomy (SG) surgery compared with matched non-surgical controls (CON). SUBJECTS/METHODS: Fifteen RYGB, 10 SG and 15 CON underwent 99Tc-mebrofenin cholescintigraphy combined with intake of a high-fat 111In-DTPA-labelled meal and frequent blood sampling. A 75Se-HCAT test was used to assess bile acid retention. RESULTS: After RYGB, gallbladder filling was decreased (p = 0.045 versus CON), basal flow of bile into the small intestine increased (p = 0.005), bile acid retention augmented (p = 0.021) and basal bile acid plasma concentrations elevated (p = 0.009). During the meal, foods passed unimpeded through the gastric pouch resulting in almost instant postprandial mixing of bile and foods, but the postprandial rise in plasma bile acids was brief and associated with decreased overall release of fibroblast growth factor-19 (FGF-19) compared with CON (p = 0.033). After SG, bile flow and retention were largely unaltered (p > 0.05 versus CON), but gastric emptying was accelerated (p < 0.001) causing earlier mixture of bile and foods also in this group. Neither basal nor postprandial bile acid concentrations differed between SG and CON. CONCLUSIONS: Bilio-enteric bile flow is markedly altered after RYGB resulting in changes in plasma concentrations of bile acids and FGF-19, whereas bile flow and plasma concentrations are largely unaltered after SG.

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.

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.

Peptone-mediated glucagon-like peptide-1 secretion depends on intestinal absorption and activation of basolaterally located Calcium-Sensing Receptors.

Protein intake robustly stimulates the secretion of the incretin hormone, glucagon-like peptide-1 (GLP-1) but the molecular mechanisms involved are not well understood. In particular, it is unknown whether proteins stimulate secretion by activation of luminal or basolateral sensors. We characterized the mechanisms using a physiologically relevant model - the isolated perfused proximal rat small intestine. Intraluminal protein hydrolysates derived from meat (peptone; 50 mg/mL) increased GLP-1 secretion 2.3-fold (from a basal secretion of 110 ± 28 fmol/min). The sensory mechanisms underlying the response depended on di/tripeptide uptake through Peptide Transporter 1 (PepT1) and subsequent basolateral activation of the amino acid sensing receptor, Calcium-Sensing Receptor (CaSR), since inhibition of PepT1 as well as CaSR both attenuated the peptone-induced GLP-1 response. Supporting this, intraluminal peptones were absorbed efficiently by the perfused intestine (resulting in increased amino acid concentrations in the venous effluent) and infusion of amino acids robustly stimulated GLP-1 secretion. Inhibitors of voltage-gated L-type Ca2+ channels had no effect on secretion suggesting that peptone-mediated GLP-1 secretion is not mediated by L-cell depolarization with subsequent opening of these channels. Specific targeting of CaSR could serve as a target to stimulate the endogenous secretion of GLP-1.

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.

Mechanisms Underlying Gut Hormone Secretion Using the Isolated Perfused Rat Small Intestine.

The gut is the largest endocrine organ of the body, producing more than 15 different peptide hormones that regulate appetite and food intake, digestion, nutrient absorption and distribution, and post-prandial glucose excursions. Understanding the molecular mechanisms that regulate gut hormone secretion is fundamental for understanding and translating gut hormone physiology. Traditionally, the mechanisms underlying gut hormone secretion are either studied in vivo (in experimental animals or humans) or using gut hormone-secreting primary mucosal cell cultures or cell lines. Here, we introduce an isolated perfused rat small intestine as an alternative method for studying gut hormone secretion. The virtues of this model are that it relies on the intact gut, meaning that it recapitulates most of the physiologically important parameters responsible for the secretion in in vivo studies, including mucosal polarization, paracrine relationships and routes of perfusion/stimulus exposure. In addition, and unlike in vivo studies, the isolated perfused rat small intestine allows for almost complete experimental control and direct assessment of secretion. In contrast to in vitro studies, it is possible to study both the magnitude and the dynamics of secretion and to address important questions, such as what stimuli cause secretion of different gut hormones, from which side of the gut (luminal or vascular) is secretion stimulated, and to analyze in detail molecular sensors underlying the secretory response. In addition, the preparation is a powerful model for the study of intestinal absorption and details regarding the dynamics of intestinal absorption including the responsible transporters.

Acipimox Acutely Increases GLP-1 Concentrations in Overweight Subjects and Hypopituitary Patients.

CONTEXT: Glucagon-like peptide-1 (GLP-1) is an incretin hormone used therapeutically in type 2 diabetes and obesity. The interplay between ambient free fatty acids (FFAs) and GLP-1 remains unclear. Acipimox suppresses adipose tissue lipolysis via activation of the PUMA-G (also known as HCA2 and GPR109a) receptor. OBJECTIVE: To investigate whether lowering of serum FFA level with acipimox affects GLP-1 secretion. DESIGN: Two randomized crossover studies were performed in human subjects. Rat intestine was perfused intra-arterially and intraluminally, and l-cells were incubated with acipimox. PARTICIPANTS: The participants were healthy overweight subjects and hypopituitary adult patients. INTERVENTIONS: The overweight participants received acipimox 250 mg 60 minutes before an oral glucose test. The hypopituitary patients received acipimox 250 mg 12, 9, and 2 hours before and during the metabolic study day, when they were studied in the basal state and during a hyperinsulinemic euglycemic clamp. RESULTS: Acipimox suppressed FFA but did not affect insulin in the clinical trials. In overweight subjects, the GLP-1 increase after the oral glucose tolerance test (area under the curve) was more than doubled [4119 ± 607 pmol/L × min (Acipimox) vs 1973 ± 375 pmol/L × min (control), P = 0.004]. In hypopituitary patients, acipimox improved insulin sensitivity (4.7 ± 0.8 mg glucose/kg/min (Acipimox) vs 3.1 ± 0.5 mg glucose/kg/min (control), P = 0.005], and GLP-1 concentrations increased ~40%. An inverse correlation between FFA and GLP-1 concentrations existed in both trials. In rat intestine, acipimox did not affect GLP-1 secretion, and l-cells did not consistently express the putative receptor for acipimox. CONCLUSIONS: Acipimox treatment increases systemic GLP-1 levels in both obese subjects and hypopituitary patients. Our in vitro data indicate that the underlying mechanisms are indirect.

Acute administration of interleukin-6 does not increase secretion of glucagon-like peptide-1 in mice.

Interleukin 6 (IL-6) is a cytokine secreted from skeletal muscle in response to exercise which, based on animal and cell studies, has been suggested to contribute to glucose metabolism by increasing secretion of the incretin hormone glucagon-like peptide-1 (GLP-1) and affecting secretion of insulin and glucagon from the pancreatic islets. We investigated the effect of IL-6 on GLP-1 secretion in GLP-1 producing cells (GLUTag) and using the perfused mouse small intestine (harboring GLP-1 producing cells). Furthermore, the direct effect of IL-6 on insulin and glucagon secretion was studied using isolated perfused mouse pancreas. Incubating GLUTag cells with 1000 ng/mL of IL-6 for 2 h did not significantly increase secretion of GLP-1 whereas 10 mmol/L glucose (positive control) did. Similarly, IL-6 (100 ng/mL) had no effect on GLP-1 secretion from perfused mouse small intestine whereas bombesin (positive control) increased secretion. Finally, administering IL-6 (100 ng/mL) to perfused mouse pancreases did not significantly increase insulin or glucagon secretion regardless of perfusate glucose levels (3.5 vs. 12 mmol/L glucose). Acute effects of IL-6 therefore do not seem to include a stimulatory effect on GLP-1 secretion in mice.

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.

On the relationship between glucose absorption and glucose-stimulated secretion of GLP-1, neurotensin, and PYY from different intestinal segments in the rat.

Ingested glucose powerfully stimulates the secretion of appetite- and metabolism-regulating peptide hormones from the gut - including glucagon-like peptide-1 (GLP-1), neurotensin (NT), and polypeptide YY (PYY). However, the regional origin of these secretions after glucose stimulation is not well characterized, and it remains uncertain how their secretion is related to glucose absorption. We isolated and perfused either the upper (USI) or the lower (LSI) small intestine or the colon from rats and investigated concomitant glucose absorption and secretory profiles of GLP-1, NT, and PYY In the USI and LSI luminal glucose (20%, w/v) increased GLP-1 and NT secretion five to eightfold compared to basal secretion. Compared to the USI, basal and stimulated GLP-1 secretion from the colon was 8-10 times lower and no NT secretion was detected. Luminal glucose stimulated secretion of PYY four to fivefold from the LSI and from the USI and colon, but the responses in the USI and colon were 5- to 15-fold lower than in the LSI Glucose was absorbed to a comparable extent in the USI and LSI by mechanisms that partly depended on both SGLT1 and GLUT2 activity, whereas the absorption in the colon was 80-90% lower. The absorption rates were, however, similar when adjusted for segmental length. Glucose absorption rates and NT, PYY and in particular GLP-1 secretion were strongly correlated (P < 0.05). Our results indicate that the rate of secretion of GLP-1, NT, and PYY in response to glucose, regardless of the involved molecular machinery, is predominantly regulated by the rate of glucose absorption.