Truncated and full-length glucagon-like peptide-1 (GLP-1) differentially stimulate intestinal somatostatin release

Glucagon-like peptide-1^7–36NH2 (GLP-1^7–36NH2) is a potent stimulator of insulin secretion, as well as of somatostatin-14 (SS-14) release from the pancreatic and gastric D-cells. To investigate the possible effects of this peptide on release of intestinal somatostatin (SS-28 and SS-14), rat intestinal cultures were treated with 10⁻¹²–10⁻⁶ M GLP-1^7–36NH2, as well as with the structurally related peptides, GLP-1^1–36NH2 and GLP-2. Both forms of GLP-1 stimulated dose-dependent increases in intestinal somatostatin; secretion reached 643±126% of controls (p<0.001) after treatment with 10⁻⁶ M GLP-1^7–36NH2, and 398±76% of controls (p<0.001) after 10⁻⁶ M GLP-1^1–36NH2. Thus, GLP-1^7–36NH2 was more effective than GLP-1^1–36NH2 in stimulating secretion of intestinal somatostatin-like immunoreactivity (SLI) (p<0.05). GLP-2 did not affect intestinal somatostatin release. Gel permeation analysis demonstrated that 10⁻⁶ M GLP-1^7–36NH2 stimulated SS-28 by 2.9±0.4-fold and SS-14 by 9.1±3.7-fold, whereas GLP-1^1–36NH2 exerted equivalent effects (2.8±0.9-fold) on both forms of somatostatin. These findings define a novel biological role for GLP-1^7–36NH2 in the regulation of intestinal somatostatin secretion, and demonstrate that GLP-1^1–36NH2 exerts unique biological activities in this system.     

Synergistic effect of portal glucose and glucagon-like peptide-1 to lower systemic glucose and stimulate counter-regulatory hormones

Aims Glucagon-like peptide-1 (GLP-1) is an insulinotropic hormone released from the gut in response to nutrients. Besides its well-established direct effect on pancreatic beta cells, GLP-1 may also act by activating sensors in the hepatoportal area. We therefore studied the impact of putative GLP-1 sensors in the splanchnic circulation.Methods We infused GLP-1 into the portal vein of conscious dogs, while also infusing glucose intraportally or systemically. In the first experiment, we infused glucose intraportally, simulating portal glucose values obtained during a previous mixed-meal test, with or without co-infusion of intraportal GLP-1. In the second experiment, by infusing glucose systemically, with or without intraportal GLP-1, we investigated whether the effects of systemic glucose with or without portal GLP-1 infusion are similar to those observed in the first experiment.Results Intraportal infusion of GLP-1 and glucose significantly raised peripheral GLP-1 levels, but did not produce an insulin response different from intraportal glucose alone. However, the resulting peripheral glycaemia was significantly lower compared to glucose infusion alone, and there were elevations in glucagon, cortisol and lactate. In contrast to the portal glucose infusions, there were no significant differences in glucose, insulin, glucagon, cortisol or lactate levels between systemic glucose infusion with or without GLP-1.Conclusions/Interpretation Portal GLP-1 and portal glucose, but not systemic glucose, can produce decreased peripheral glucose levels independently of hyperinsulinaemia. This suggests that portal GLP-1 and glucose receptors mediate insulin-independent changes in peripheral glycaemia and determine a strong counter-regulatory response, as reflected by changes in glucagon and cortisol.     

Oligopeptides stimulate glucagon-like peptide-1 secretion in mice through proton-coupled uptake and the calcium-sensing receptor

Aims/hypothesis

Ingested 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.

Methods

GLP-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.

Results

GLP-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/interpretation

Oligopeptides 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.     

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.     

Addition of metformin to exogenous glucagon-like peptide-1 results in increased serum glucagon-like peptide-1 concentrations and greater glucose lowering in type 2 diabetes mellitus

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that lowers blood glucose after meals in type 2 diabetes mellitus. The therapeutic potential of GLP-1 in diabetes is limited by rapid inactivation by the enzyme dipeptidylpeptidase-4 (DPP-4). Metformin has been reported to inhibit DPP-4. Here we investigated the acute effects of metformin and GLP-1 alone or in combination on plasma DPP-4 activity, active GLP-1 concentrations, and glucose lowering in type 2 diabetes mellitus. Ten subjects with type 2 diabetes mellitus (8 male and 2 female; age, 68.7 ± 2.6 years [mean ± SEM]; body mass index, 29.6 ± 1.7 kg/m²; hemoglobin A_1c, 7.0% ± 0.1%) received 1 of 3 combinations after an overnight fast in a randomized crossover design: metformin 1 g orally plus subcutaneous injection saline (Metformin), GLP-1 (1.5 nmol/kg body weight subcutaneously) plus placebo tablet (GLP-1), or metformin 1 g plus GLP-1(Metformin + GLP-1). At 15 minutes, glucose was raised to 15 mmol/L by rapid intravenous infusion of glucose; and responses were assessed over the next 3 hours. This stimulus does not activate the enteroinsular axis and secretion of endogenous GLP-1, enabling the effect of exogenously administered GLP-1 to be examined. Mean area under curve (AUC) _{0-180 minutes} plasma glucose responses were lowest after Metformin + GLP-1 (mean ± SEM, 1629 ± 90 mmol/[L min]) compared with GLP-1 (1885 ± 86 mmol/[L min], P < .002) and Metformin (2045 ± 115 mmol/[L min], P < .001). Mean AUC serum insulin responses were similar after either Metformin + GLP-1 (5426 ± 498 mU/[L min]) or GLP-1 (5655 ± 854 mU/[L min]) treatment, and both were higher than Metformin (3521 ± 410 mU/[L min]; P < .001 and P < .05, respectively). Mean AUC for plasma DPP-4 activity was lower after Metformin + GLP-1 (1505 ± 2 μmol/[mL min], P < .001) and Metformin (1508 ± 2 μmol/[mL min], P < .002) compared with GLP-1 (1587 ± 3 μmol/[mL min]). Mean AUC measures for plasma active GLP-1 concentrations were higher after Metformin + GLP-1 (820 × 10⁴ ± 51 × 10⁴ pmol/[L min]) compared with GLP-1 (484 × 10⁴ ± 31 × 10⁴ pmol/[L min], P < .001) and Metformin (419 × 10⁴ ± 34 × 10⁴ pmol/[L min], P < .001), respectively. In patients with type 2 diabetes mellitus, metformin inhibits DPP-4 activity and thus increases active GLP-1 concentrations after subcutaneous injection. In combination with GLP-1, metformin significantly lowers plasma glucose concentrations in type 2 diabetes mellitus subjects compared with GLP-1 alone, whereas insulin responses were similar. Metformin enhances serum concentrations of injected active GLP-1(7-36)amide, and the combination results in added glucose-lowering potency.     

Mechanisms underlying metformin-induced secretion of glucagon-like peptide-1 from the intestinal L cell

Glucagon-like peptide-1(7-36NH2) (GLP-1) is secreted by the intestinal L cell in response to both nutrient and neural stimulation, resulting in enhanced glucose-dependent insulin secretion. GLP-1 is therefore an attractive therapeutic for the treatment of type 2 diabetes. The antidiabetic drug, metformin, is known to increase circulating GLP-1 levels, although its mechanism of action is unknown. Direct effects of metformin (5-2000 μm) or another AMP kinase activator, aminoimidazole carboxamide ribonucleotide (100-1000 μm) on GLP-1 secretion were assessed in murine human NCI-H716, and rat FRIC L cells. Neither agent stimulated GLP-1 secretion in any model, despite increasing AMP kinase phosphorylation (P < 0.05-0.01). Treatment of rats with metformin (300 mg/kg, per os) or aminoimidazole carboxamide ribonucleotide (250 mg/kg, sc) increased plasma total GLP-1 over 2 h, reaching 37 ± 9 and 29 ± 9 pg/ml (P < 0.001), respectively, compared with basal (7 ± 1 pg/ml). Plasma activity of the GLP-1-degrading enzyme, dipeptidylpeptidase-IV, was not affected by metformin treatment. Pretreatment with the nonspecific muscarinic antagonist, atropine (1 mg/kg, iv), decreased metformin-induced GLP-1 secretion by 55 ± 11% (P < 0.05). Pretreatment with the muscarinic (M) 3 receptor antagonist, 1-1-dimethyl-4-diphenylacetoxypiperidinium iodide (500 μg/kg, iv), also decreased the GLP-1 area under curve, by 48 ± 8% (P < 0.05), whereas the antagonists pirenzepine (M1) and gallamine (M2) had no effect. Furthermore, chronic bilateral subdiaphragmatic vagotomy decreased basal secretion compared with sham-operated animals (7 ± 1 vs. 13 ± 1 pg/ml, P < 0.001) but did not alter the GLP-1 response to metformin. In contrast, pretreatment with the gastrin-releasing peptide antagonist, RC-3095 (100 μg/kg, sc), reduced the GLP-1 response to metformin, by 55 ± 6% (P < 0.01) at 30 min. These studies elucidate the mechanism underlying metformin-induced GLP-1 secretion and highlight the benefits of using metformin with dipeptidylpeptidase-IV inhibitors in patients with type 2 diabetes.     

Comparison of the effects of glucagon-like peptide-1-(1-37) and -(7-37) and glucagon on islet hormone release from isolated perfused canine and rat pancreases

Recently, it has been demonstrated that glucagon-like peptide-1 (GLP-1)-(7-37) possesses a potent insulinotropic activity. In this paper, we compared the effects of GLP-1-(1-37) and -(7-37) and glucagon on insulin, glucagon, and somatostatin release from isolated perfused canine and rat pancreases under the perfusate condition of 5.5 mM glucose plus arginine. With canine pancreas perfusion, 1 nM GLP-1-(7-37) was more potent in stimulating insulin and somatostatin release than was the same dose of glucagon [stimulation to 375 +/- 36% vs. 302 +/- 28% of the basal level for insulin (P less than 0.05); 724 +/- 129% vs. 311 +/- 33% of the basal level for somatostatin (P less than 0.01)]. GLP-1-(1-37) (1 nM) did not stimulate either insulin or somatostatin release. GLP-1-(7-37) (1 nM) decreased the glucagon level of the effluent perfusate to 67.2 +/- 3.4% of its basal level; but 1 nM GLP-1-(1-37) did not. Glucagon (1 nM) decreased GLP-1-like immunoreactivity to 64.0 +/- 5.2% of its basal level. With rat pancreatic perfusion, the minimal dose for stimulation of insulin release was 100 nM for GLP-1-(1-37), 0.1 nM for GLP-1-(7-37), and 1 nM for glucagon, respectively. Glucagon release was partially inhibited by 100 nM GLP-1-(1-37) and 1 and 10 nM GLP-1-(7-37). The present results indicate that 1) since GLP-1-(7-37) is released from the intestine, it might be an important incretin candidate along with gastric inhibitory peptide; and 2) the release of proglucagon-derived peptides from pancreatic A-cells is regulated by autofeedback through glucagon and GLP-1.     

Enhanced glucose-dependent glucagon-like peptide-1 and insulin secretion in Crohn patients with terminal ileum disease is unrelated to disease activity or ileal resection

BACKGROUND: Enhanced secretion of glucagon-like peptide-1 (GLP-1) has been reported in patients with Crohn disease (CD). However, the correlation between the enteropancreatic axis and the activity of CD remains unclear. METHODS: Plasma glucose, insulin, GLP-1 levels and insulin sensitivity were determined before and after oral glucose tolerance tests in 13 patients with CD of the terminal ileum, in 13 patients after resection of the terminal ileum and in 7 healthy controls. Basal and stimulated insulin sensitivities were determined using the homeostasis model assessment (HOMA) and the insulin sensitivity index (ISI) methods, respectively. RESULTS: Basal and stimulated glucose levels were comparable in patients and controls. The peak stimulated GLP-1 secretion was significantly higher in the patient group compared to controls: 12.2 +/- 1.24 pM/L and 8.1 +/- 1.72 pM/L, respectively, P=0.03. This was associated with 52% increased overall insulin secretion in the patients' group as compared to controls (P=0.007) and a higher peak insulin response: 63.5 +/- 9.69 mU/L and 41.5 +/- 6.85 mU/L for patients and controls, respectively, P=0.04. Operated patients had similar GLP-1 levels but higher peak and overall insulin secretions compared with those in non-operated patients (P=0.01). Fasting and stimulated insulin sensitivities were reduced only in patients with ileal resection as compared to controls: P=0.01 and P=0.05, respectively. No correlation was found between the CD activity index and GLP-1 or insulin secretion. CONCLUSIONS: CD of the terminal ileum is associated with enhanced glucose-dependent GLP-1 secretion, which is unrelated to disease activity or ileal resection.     

Effects of truncated glucagon-like peptide-1 on the responses of starved sheep to glucose

The effects of i.v. glucagon-like peptide-1-(7-36)amide (GLP-1; 10 micrograms) on starved sheep given an i.v. glucose load (5 g) were studied. Plasma insulin concentrations rose significantly more after glucose administration in fed than in starved sheep. Giving GLP-1 to starved sheep increased the insulin response to the glucose load. The rise in plasma insulin concentrations in starved sheep given GLP-1 was similar to that observed in fed sheep. Plasma glucose concentrations returned to normal values more quickly in the starved sheep given GLP-1 than in starved sheep not given gut hormone. Plasma concentrations of free fatty acid, urea and alpha-amino nitrogen decreased more quickly following glucose administration in starved sheep given GLP-1 than in those not given GLP-1. The data suggest a role for GLP-1 in regulating plasma insulin concentrations and hence metabolism in ruminant animals. The possible role of gut hormones in ruminants is discussed.     

Two free isoforms of ovine glycoprotein hormone alpha-subunit strongly differ in their ability to stimulate prolactin release from foetal pituitaries

alpha-Subunit dissociated from glycoprotein hormones has been previously shown to stimulate rat pituitary lactotroph differentiation and proliferation. However, whether the free form of the alpha-subunit (free alpha) can also play such a role is not known. To test whether free alpha may act on prolactin (PRL) release from ovine foetal pituitaries, this molecule was purified and two major isoforms, alphaA and alphaB were isolated. Free alphaA was found to be more acidic and more hydrophobic than both free alphaB and ovine LH alpha-subunit (oLHalpha). Free alphaA and oLHalpha exhibited a molecular mass of 14 kDa as determined by mass spectrometry, whereas free alphaB displayed a molecular mass of only 13.5 kDa because of its truncated N-terminus. All three alpha molecules bear mature-type N-linked saccharide chains including Nacetyl galactosamine residues but none of them contains O-linked oligosaccharide. The free alphaA isoform, more than the oLHalpha, was able to stimulate PRL release from ovine foetal pituitary explants in culture, whereas the free alphaB isoform displayed no activity. Moreover, the free alphaA and alphaB isoforms were able to recombine with the ovine LH beta-subunit (oLHbeta). The free alphaB/oLHbeta, and the oLHalpha/oLHbeta dimer were 4-fold more active than the free alphaA/oLHbeta dimer in a specific LH radioreceptor assay and in the stimulation of testosterone release from rat Leydig cells. The present study demonstrates that the two free alpha isoforms of ovine glycoprotein hormones exhibit distinct efficiencies in stimulating PRL release from ovine foetal pituitaries. Moreover, despite their identical ability to recombine with the oLHbeta, the free alpha isoform, which is the most efficient on PRL release, is the least efficient in conferring LH activity on the alpha/beta dimer.     

Muscarinic receptors control postprandial release of glucagon-like peptide-1: in vivo and in vitro studies in rats

Plasma levels of glucagon-like peptide-1 (GLP-1) rise rapidly after nutrient ingestion through an indirect mechanism triggered from the proximal intestine and involving the vagus nerve that stimulates the L cell in the distal gut. The role of muscarinic receptors in this pathway was thus investigated using the anesthetized rat and fetal rat intestinal cells (FRIC) in culture. GLP-1 secretion from the distal gut increased 5-fold after 3 ml corn oil were placed into the proximal duodenum (P < 0.001). Atropine (a nonspecific muscarinic receptor antagonist) completely inhibited fat-induced GLP-1 secretion in vivo (P < 0.01). Pirenzepine (an M1 muscarinic receptor antagonist) also inhibited fat-induced GLP-1 secretion in vivo, by 91 +/- 6% (P < 0.01). Gallamine (an M2 muscarinic receptor antagonist) and 4-diphenylacetoxy-N-methylpiperidine (an M3 muscarinic receptor antagonist) had no effect. Incubating FRIC cultures with bethanechol (a muscarinic receptor agonist) stimulated GLP-1 secretion to 200 +/- 22% of control (P < 0.01). Pirenzepine and gallamine significantly inhibited bethanechol-stimulated GLP-1 secretion, by 96 +/- 12% and 98 +/- 8%, respectively (P < 0.01). Unexpectedly, 4-diphenylacetoxy-N-methylpiperidine stimulated GLP-1 secretion by FRIC cells, to 324 +/- 52% of the control value (P < 0.01). Double immunofluorescent staining using GLP-1 and M1, M2, and M3 muscarinic receptor antibodies showed expression of the three subtypes of muscarinic receptors by the L cells in rat ileal sections and FRIC cultures. These results demonstrate the role of M1 muscarinic receptors expressed by L cells in the control of postprandial secretion of GLP-1. M2 muscarinic receptors also seem to play a role in controlling GLP-1 secretion by fetal, but not adult, L cells.     

Differences in weight loss and safety between the glucagon-like peptide-1 receptor agonists: A non-randomized multicenter study from the titration phase

IntroductionObesity increases the risk of type 2 diabetes mellitus and cardiovascular disease (CVD). Weight loss (≥5 %) reduces the risk of CVD. Glucagon-like peptide-1 receptor agonists (GLP1 RA) have shown clinically weight loss. Objectives: 1) To assess differences in the efficacy of weight loss and HbA1c; 2) to evaluate the safety and adherence during the titration phase.MethodsIt is a multicenter, prospective, and observational study on GLP1 RA naïve patients. The primary end point was the weight loss (≥5 %). Changes in weight, BMI and HbA1c were also calculated as co-primary endpoints. Secondary endpoints were safety, adherence, and tolerance.ResultsAmong 94 subjects, 42.4 % received dulaglutide, 29,3 % subcutaneous semaglutide, 22,8 % oral semaglutide. 45 % female and the mean age was 62. Baseline characteristics were body weight 99.3 kg, BMI 36.7 kg/m² and Hba1c 8.2 %. Oral semaglutide achieved the highest reduction: 61.1 % of patients achieving ≥ 5 %, subcutaneous semaglutide 45.8 % and dulaglutide 40.6 %. GLP1 RA significantly reduced body weight (−4.95 kg, p < 0.001) and BMI (−1.86 kg/m², p < 0.001), without significant differences between groups. Gastrointestinal disorders were the most frequently reported events (74.5 %). 62 % of patients on dulaglutide, 25 % on oral semaglutide and 22 % on subcutaneous semaglutide.ConclusionsOral semaglutide achieved the highest proportion of patients that lost ≥ 5 %. GLP1 RA significantly reduced BMI and HbA1c. Most of the reported adverse events were gastrointestinal disorders and they were reported in a major frequency in the dulaglutide group. Oral semaglutide would be a reasonable switch in case of future shortages.     

Comparison of the effects of various C-terminal and N-terminal fragment peptides of glucagon-like peptide-1 on insulin and glucagon release from the isolated perfused rat pancreas

Truncated glucagon-like peptide-1 (GLP-1) possesses a potent stimulatory activity for insulin secretion and a slight inhibiting activity for glucagon secretion. The aim of this paper is to examine the activities of N- and C-terminal fragments of GLP-1 using a rat pancreas perfusion system. Concerning the N-terminal portion, GLP-1(7-37) amide elicited a clear insulinotropic activity at 0.1 or 1 nM with the perfusate containing 5.5 mM glucose and 5 mM arginine, while 10 nM GLP-1-(1-37) amide, -(6-37) amide, and -(8-37) amide did not. Concerning the C-terminal portion, GLP-1-(7-37) amide, -(7-37), and -(7-36) amide had a similar potency of insulinotropic activity, and GLP-1-(7-35) was less potent; 0.1 nM GLP-1-(7-35) did not stimulate insulin release, nor did 10 nM GLP-1-(7-20). Glucagon release was significantly suppressed by 1 and 10 nM GLP-1-(7-37) amide, 10 nM GLP-1-(7-37), and 1 nM GLP-1-(7-36) amide. Other fragment peptides of GLP-1, including GLP-1-(7-35), had no effect. From these results it is concluded that histidine at position 7 of GLP-1 as a free N-terminal amino acid is very important in GLP-1's insulinotropic activity and probably in glucagon-inhibiting activity, and that C-terminal amidation and three C-terminal amino acids are less important for these activities.     

Effect of glucagon-like peptide-1 on gastric somatostatin and gastrin secretion in the rat

The effect of glucagon-like peptide-1 (GLP-1) amide on gastric somatostatin and gastrin secretion was investigated in the isolated, vascularly perfused rat stomach preparation. GLP-1 (7-36) amide, 10(-12) to 10(-7)M, dose-dependently increased gastric somatostatin release, achieving maximal stimulation (314 +/- 15% above basal) at the highest dose. The somatostatin response to 10(-8)M GLP-1 (7-36) amide was not affected by concomitant perfusion with tetrodotoxin. GLP-1 (1-36) amide did not affect somatostatin release. Both basal and acetylcholine-stimulated gastrin were inhibited by GLP-1 (7-36) amide but were not influenced by GLP-1 (1-36) amide. In is concluded that GLP-1 (7-36) amide is the biologically effective peptide that stimulates gastric somatostatin and inhibits gastrin secretion, probably via non-neural pathways. GLP-1 (7-36) amide-induced inhibition of gastric acid secretion may, at least in part, be due to enhanced somatostatin and/or decreased gastrin release.     

Exaggerated release and preserved insulinotropic action of glucagon-like peptide-1 underlie insulin hypersecretion in glucose-tolerant individuals after Roux-en-Y gastric bypass

Aims/hypothesis

Roux-en-Y gastric bypass (RYGB) improves glycaemic control in part by increasing postprandial insulin secretion through exaggerated glucagon-like peptide (GLP)-1 release. However, it is unknown whether islet cell responsiveness to i.v. glucose, non-glucose (arginine) and incretin hormones, including GLP-1, is altered.

Methods

Eleven severely obese glucose-tolerant individuals underwent three hyperglycaemic clamps with arginine bolus and co-infusion of either GLP-1, glucose-dependent insulinotropic polypeptide (GIP) or saline before, and at 1 week and 3 months after RYGB. In addition, an OGTT was performed before and 3 months after surgery.

Results

After RYGB, insulin sensitivity improved at 1 week and 3 months, while insulin stimulation and glucagon suppression in response to the clamp with saline co-infusion were largely unaltered. The influence of i.v. GLP-1 and GIP on insulin and glucagon secretion was also unchanged postoperatively. In response to the postoperative OGTT at 3 months, insulin and GLP-1, but not GIP, secretion increased. Furthermore, the glucose profile during the OGTT was altered, with a substantial reduction in 2 h plasma glucose and a paradoxical hypersecretion of glucagon.

Conclusions/interpretation

After RYGB, insulin hypersecretion is linked to the oral, but not the i.v., route of administration and is associated with exaggerated release and preserved insulinotropic action of GLP-1, while both the secretion and action of GIP are unchanged. The results highlight the importance of increased GLP-1 secretion for improving postoperative glucose metabolism.

Trial registration

ClinicalTrials.gov NCT01559779.     

Role of vesicle-associated membrane protein 2 in exocytosis of glucagon-like peptide-1 from the murine intestinal L cell

Aims/hypothesis

Glucagon-like peptide-1 (GLP-1), secreted by the enteroendocrine L cell, is an incretin hormone that potently stimulates insulin secretion. Although signalling pathways promoting GLP-1 release are well characterised, the mechanisms by which GLP-1-containing granules fuse to the L cell membrane are unknown. As soluble NSF attachment proteins (SNAREs) are known to mediate granule–membrane fusion, the role of vesicle-associated membrane proteins (VAMPs) in GLP-1 exocytosis was examined.

Methods

SNARE expression was determined in murine GLUTag L cells by RT-PCR and immunoblot and in primary murine L cells by immunofluorescence. Co-immunoprecipitation was used to examine SNARE interactions, while tetanus toxin (TetX)-mediated cleavage of VAMP was used with a GLP-1 secretion assay and total internal reflection fluorescence microscopy to determine the role of VAMP2 in exocytosis.

Results

VAMP2 was expressed in murine L cells and localised to secretory granules in GLUTag cells. VAMP1/3 and the core membrane proteins syntaxin1a and synaptosomal-associated protein 25 kDa (SNAP25) were also detected. TetX cleaved VAMPs in GLUTag cells. However, only VAMP2 interacted with syntaxin1a, as did SNAP25 and Munc18-1. TetX treatment of GLUTag cells prevented glucose-dependent insulinotrophic peptide- and oleic-acid-stimulated GLP-1 secretion (p?<?0.05–0.01), as well as K⁺-stimulated single-cell exocytosis (p?<?0.05–0.001), while TetX-resistant VAMP2 expression rescued GLP-1 secretion (p?<?0.01–0.001).

Conclusions/interpretation

Together, these findings indicate an essential role for VAMP2 in GLP-1 exocytosis from the GLUTag L cell in response to a variety of established secretagogues. An improved understanding of the mechanisms governing the release of GLP-1 may lead to new therapeutic approaches to enhance the levels of this incretin hormone in patients with type 2 diabetes.     

Neuroendocrine function and response to stress in mice with complete disruption of glucagon-like peptide-1 receptor signaling

Glucagon-like peptide-1 (GLP-1), a potent regulator of glucose homeostasis, is also produced in the central nervous system, where GLP-1 has been implicated in the neuroendocrine control of hypothalamic-pituitary function, food intake, and the response to stress. The finding that intracerebroventricular GLP-1 stimulates LH, TSH, corticosterone, and vasopressin secretion in rats prompted us to assess the neuroendocrine consequences of disrupting GLP-1 signaling in mice in vivo. Male GLP-1 receptor knockout (GLP-1R-/-) mice exhibit reduced gonadal weights, and females exhibit a slight delay in the onset of puberty; however, male and female GLP-1R-/- animals reproduce successfully and respond appropriately to fluid restriction. Although adrenal weights are reduced in GLP-1R-/- mice, hypothalamic CRH gene expression and circulating levels of corticosterone, thyroid hormone, testosterone, estradiol, and progesterone are normal in the absence of GLP-1R-/- signaling. Intriguingly, GLP-1R-/- mice exhibit paradoxically increased corticosterone responses to stress as well as abnormal responses to acoustic startle that are corrected by glucocorticoid treatment. These findings suggest that although GLP-1R signaling is not essential for development and basal function of the murine hypothalamic-pituitary-adrenal axis, abrogation of GLP-1 signaling is associated with impairment of the behavioral and neuroendocrine responses to stress.