Evaluation of commercially available glucagon receptor antibodies and glucagon receptor expression.

Glucagon is a major regulator of metabolism and drugs targeting the glucagon receptor (GCGR) are being developed. Insight into tissue and cell-specific expression of the GCGR is important to understand the biology of glucagon and to differentiate between direct and indirect actions of glucagon. However, it has been challenging to localize the GCGR in tissue due to low expression levels and lack of specific methods. Immunohistochemistry has frequently been used for GCGR localization, but antibodies targeting G-protein-coupled-receptors may be inaccurate. We evaluated all currently commercially available GCGR antibodies. The antibody, ab75240 (Antibody no. 11) was found to perform best among the twelve antibodies tested and using this antibody we found expression of the GCGR in the kidney, liver, preadipocytes, pancreas, and heart. Three antibody-independent approaches all confirmed the presence of the GCGR within the pancreas, liver and the kidneys. GCGR expression should be evaluated by both antibody and antibody-independent approaches.

Nonalcoholic Fatty Liver Disease Impairs the Liver-Alpha Cell Axis Independent of Hepatic Inflammation and Fibrosis.

Nonalcoholic fatty liver disease (NAFLD) is associated with impaired hepatic actions of glucagon and insulin. Glucagon and amino acids are linked in an endocrine feedback circuit, the liver-alpha cell axis, that may be disrupted by NAFLD. We investigated how NAFLD severity affects glucagon and insulin resistance in individuals with obesity and whether bariatric surgery improves these parameters. Plasma and liver biopsies from 33 individuals with obesity (collectively, OBE) were obtained before and 12 months after bariatric surgery (Roux-en-Y gastric bypass [RYGB] or sleeve gastrectomy [SG]). Nine healthy control individuals (collectively, CON) undergoing cholecystectomy were used as a comparison group. The NAFLD activity score (NAS) was used to subdivide study participants into the following groups: OBE-no steatosis, OBE+steatosis, and nonalcoholic steatohepatitis (NASH) and/or grade 2 fibrosis (Fib) (OBE-NASH-Fib). Measurements of amino acids by targeted metabolomics and glucagon were performed. Glucagon, amino acids (P < 0.05), and the glucagon-alanine index, a validated surrogate marker of glucagon resistance, were increased in OBE by 60%, 56%, and 61%, respectively, when compared with CON but irrespective of NAFLD severity. In contrast, markers of hepatic insulin resistance increased concomitantly with NAS. Hyperglucagonemia resolved in OBE-no steatosis and OBE+steatosis but not in OBE-NASH-Fib (median, 7.0; interquartile range, 5.0-9.8 pmol/L), regardless of improvement in insulin resistance and NAS. The type of surgery that participants underwent had no effect on metabolic outcomes. Conclusion: Glucagon resistance to amino acid metabolism exists in individuals with NAFLD independent of NAS severity. Patients with NASH showed persistent hyperglucagonemia 12 months after bariatric surgery, indicating that a disrupted liver-alpha cell may remain in NAFLD despite major improvement in liver histology.

Secretion of parathyroid hormone may be coupled to insulin secretion in humans.

OBJECTIVE: Parathyroid hormone (PTH) is a key hormone in regulation of calcium homeostasis and its secretion is regulated by calcium. Secretion of PTH is attenuated during intake of nutrients, but the underlying mechanism(s) are unknown. We hypothesized that insulin acts as an acute regulator of PTH secretion. METHODS: Intact PTH was measured in plasma from patients with T1D and matched healthy individuals during 4-h oral glucose tolerance tests (OGTT) and isoglycemic i.v. glucose infusions on 2 separate days. In addition, expression of insulin receptors on surgical specimens of parathyroid glands was assessed by immunochemistry (IHC) and quantitative PCR (qPCR). RESULTS: The inhibition of PTH secretion was more pronounced in healthy individuals compared to patients with T1D during an OGTT (decrementalAUC0-240min: -5256 ± 3954 min × ng/L and -2408 ± 1435 min × ng/L, P = 0.030). Insulin levels correlated significantly and inversely with PTH levels, also after adjusting for levels of several gut hormones and BMI (P = 0.002). Expression of insulin receptors in human parathyroid glands was detected by both IHC and qPCR. CONCLUSION: Our study suggests that insulin may act as an acute regulator of PTH secretion in humans.

Sacubitril/valsartan increases postprandial gastrin and cholecystokinin in plasma.

AIMS: Neprilysin degrades natriuretic peptides in circulation and is also suggested to degrade the gut hormones gastrin and cholecystokinin. Neprilysin inhibition has become a therapeutic strategy and thus a regimen in need of further testing in terms of other hormonal axes besides natriuretic peptides. The aim of this study was to examine whether acute inhibition of neprilysin affects meal-induced responses in gastrin and cholecystokinin concentrations in healthy individuals. METHODS AND RESULTS: Nine healthy young men were included in an open-labelled, randomized cross-over clinical trial. The participants received a standardized meal (25 g fat, 26 g protein, 42 g carbohydrate) on two separate days with or without a one-time dosage of sacubitril ((194 mg)/valsartan (206 mg)). Blood pressure, heart rate and blood samples were measured and collected during the experiment. Statistical differences between groups were assessed using area under the curve together with an ANOVA with a Bonferroni post hoc test. Sacubitril/valsartan increased the postprandial plasma concentrations of both gastrin and cholecystokinin (80% (AUC0-270 min, P = 0.004) and 60% (AUC0-270 min, P = 0.003), respectively) compared with the control meal. No significant hemodynamic effects were noted (blood pressure, AUC0-270 min, P = 0.86, heart rate, AUC0-270 min, P = 0.96). CONCLUSION: Our study demonstrates that sacubitril/valsartan increases the postprandial plasma concentrations of gastrin and cholecystokinin in healthy individuals. The results thus suggest that neprilysin-mediated degradation of gastrin and cholecystokinin is physiologically relevant and may have a role in heart failure patients treated with sacubitril/valsartan.

Proglucagon peptide secretion profiles in type 2 diabetes before and after bariatric surgery: 1-year prospective study.

INTRODUCTION: Hyperglucagonemia is a key pathophysiological driver of type 2 diabetes. Although Roux-en-Y gastric bypass (RYGB) is a highly effective treatment for diabetes, it is presently unclear how surgery alters glucagon physiology. The aim of this study was to characterize the behavior of proglucagon-derived peptide (glucagon, glucagon-like peptide-1 (GLP-1), oxyntomodulin, glicentin) secretion after RYGB surgery. RESEARCH DESIGN AND METHODS: Prospective study of 19 patients with obesity and pre-diabetes/diabetes undergoing RYGB. We assessed the glucose, insulin, GLP-1, glucose-dependent insulinotropic peptide (GIP), oxyntomodulin, glicentin and glucagon responses to a mixed-meal test (MMT) before and 1, 3 and 12 months after surgery. Glucagon was measured using a Mercodia glucagon ELISA using the 'Alternative' improved specificity protocol, which was validated against a reference liquid chromatography combined with mass spectrometry method. RESULTS: After RYGB, there were early improvements in fasting glucose and glucose tolerance and the insulin response to MMT was accelerated and amplified, in parallel to significant increases in postprandial GLP-1, oxyntomodulin and glicentin secretion. There was a significant decrease in fasting glucagon levels at the later time points of 3 and 12 months after surgery. Glucagon was secreted in response to the MMT preoperatively and postoperatively in all patients and there was no significant change in this postprandial secretion. There was no significant change in GIP secretion. CONCLUSIONS: There is a clear difference in the dynamics of secretion of proglucagon peptides after RYGB. The reduction in fasting glucagon secretion may be one of the mechanisms driving later improvements in glycemia after RYGB. TRIAL REGISTRATION NUMBER: NCT01945840.

Responses of gut and pancreatic hormones, bile acids, and fibroblast growth factor-21 differ to glucose, protein, and fat ingestion after gastric bypass surgery.

Postprandial gut hormone responses change after Roux-en-Y gastric bypass (RYGB), and we investigated the impact of glucose, protein, and fat (with and without pancreas lipase inhibition) on plasma responses of gut and pancreas hormones, bile acids, and fibroblast growth factor 21 (FGF-21) after RYGB and in nonoperated control subjects. In a randomized, crossover study 10 RYGB operated and 8 healthy weight-matched control subjects were administered 4 different 4-h isocaloric (200 kcal) liquid meal tests containing >90 energy (E)% of either glucose, protein (whey protein), or fat (butter with and without orlistat). The primary outcome was glucagon-like peptide-1 (GLP-1) secretion (area under the curve above baseline). Secondary outcomes included responses of peptide YY (PYY), glucose-dependent insulinotropic polypeptide (GIP), cholecystokinin (CCK), glicentin, neurotensin, ghrelin, insulin, glucagon, bile acids, and FGF-21. In the RYGB group the responses of GLP-1, GIP, glicentin, FGF-21, and C-peptide were increased after glucose compared with the other meals. The neurotensin and bile acids responses were greater after fat, while the glucagon and CCK responses were greater after protein ingestion. Furthermore, compared with control subjects, RYGB subjects had greater responses of total PYY after glucose, glucagon after glucose and fat, glicentin after glucose and protein, and GLP-1 and neurotensin after all meals, while GIP and CCK responses were lower after fat. Ghrelin responses did not differ between meals or between groups. Orlistat reduced all hormone responses to fat ingestion, except for ghrelin in the RYGB group. In conclusion, after RYGB glucose is a more potent stimulator of most gut hormones, especially for the marked increased secretion of GLP-1 compared with fat and protein.NEW & NOTEWORTHY We investigated the impact of glucose, protein, and fat meals on intestinal and pancreatic hormones, bile acid, and fibroblast growth factor 21 (FGF-21) secretion in gastric bypass-operated patients compared with matched nonoperated individuals. The fat meal was administered with and without a pancreas lipase inhibitor. We found that the impact of the different meals on gut hormones, bile, and FGF 21 secretion differ and was different from the responses observed in nonoperated control subjects.

Effects of Exercise Training and IL-6 Receptor Blockade on Gastric Emptying and GLP-1 Secretion in Obese Humans: Secondary Analyses From a Double Blind Randomized Clinical Trial.

BACKGROUND: Interleukin-6 (IL-6) is released from skeletal muscle during exercise and systemic IL-6 levels therefore increase acutely in response to a single bout of exercise. We recently showed that an acute increase in IL-6 delayed gastric emptying rate and improved postprandial glycemia. Here we investigate whether repeated increases in IL-6, induced by exercise training, influence gastric emptying rate and moreover if IL-6 is required for exercise-induced adaptations in glycemic control including secretion of glucagon and glucagon-like peptide-1 (GLP-1). METHODS: A total of 52 abdominally obese non-diabetic men and women were randomly assigned into four groups performing 12 weeks of endurance exercise or no exercise with or without IL-6 receptor blockade (tocilizumab). The primary endpoint was change in gastric emptying rate in response to the intervention and other endpoints included changes in glycemic control, glucagon, and GLP-1 secretion. RESULTS: There was no change in gastric emptying rate in any of the four groups following the intervention and comparing differences in change between groups also revealed no difference. Postprandial glucose remained unchanged in all groups but the exercise + tocilizumab group, which improved postprandial glucose in response to the intervention. The area under the curve for meal-stimulated glucagon, active and total GLP-1 increased in response to IL-6 receptor blockade, this effect was independent of exercise. CONCLUSION: Exercise training and long-term IL-6 receptor blockade did not change gastric emptying rates in obese humans. IL-6 receptor blockade increased glucagon and GLP-1 secretion and implicate IL-6 in the regulation of the human alpha and L cells.

Bile acids drive colonic secretion of glucagon-like-peptide 1 and peptide-YY in rodents.

A large number of glucagon-like-peptide-1 (GLP-1)- and peptide-YY (PYY)-producing L cells are located in the colon, but little is known about their contribution to whole body metabolism. Since bile acids (BAs) increase GLP-1 and PYY release, and since BAs spill over from the ileum to the colon, we decided to investigate the ability of BAs to stimulate colonic GLP-1 and PYY secretion. Using isolated perfused rat/mouse colon as well as stimulation of the rat colon in vivo, we demonstrate that BAs significantly enhance secretion of GLP-1 and PYY from the colon with average increases of 3.5- and 2.9-fold, respectively. Furthermore, we find that responses depend on BA absorption followed by basolateral activation of the BA-receptor Takeda-G protein-coupled-receptor 5. Surprisingly, the apical sodium-dependent BA transporter, which serves to absorb conjugated BAs, was not required for colonic conjugated BA absorption or conjugated BA-induced peptide secretion. In conclusion, we demonstrate that BAs represent a major physiological stimulus for colonic L-cell secretion. NEW & NOTEWORTHY By the use of isolated perfused rodent colon preparations we show that bile acids are potent and direct promoters of colonic glucagon-like-peptide 1 and peptide-YY secretion. The study provides convincing evidence that basolateral Takeda-G protein-coupled-receptor 5 activation is mediating the effects of bile acids in the colon and thus add to the existing literature described for L cells in the ileum.

Neuromedin U Does Not Act as a Decretin in Rats.

Studies on isolated pancreatic islets suggest that neuromedin U (NMU), a brain and gastrointestinal peptide, acts as a decretin hormone, inhibiting glucose-stimulated insulin secretion. We investigated whether this effect could be reproduced in vivo and in isolated perfused rat pancreas. Unlike the incretin hormone, glucagon-like peptide 1 (GLP-1), intravenous NMU administration had no effects on blood glucose and plasma insulin and glucagon in vivo. Moreover, NMU neither changed insulin, glucagon, or somatostatin secretion from isolated perfused rat pancreas, nor affected GLP-1-stimulated insulin and somatostatin secretion. For NMU to act as a decretin hormone, its secretion should increase following glucose ingestion; however, glucose did not affect NMU secretion from isolated perfused rat small intestine, which contained extractable NMU. Furthermore, the two NMU receptors were not detected in endocrine rat or human pancreas. We conclude that NMU does not act as a decretin hormone in rats.

Interleukin-6 Delays Gastric Emptying in Humans with Direct Effects on Glycemic Control.

Gastric emptying is a critical regulator of postprandial glucose and delayed gastric emptying is an important mechanism of improved glycemic control achieved by short-acting glucagon-like peptide-1 (GLP-1) analogs in clinical practice. Here we report on a novel regulatory mechanism of gastric emptying in humans. We show that increasing interleukin (IL)-6 concentrations delays gastric emptying leading to reduced postprandial glycemia. IL-6 furthermore reduces insulin secretion in a GLP-1-dependent manner while effects on gastric emptying are GLP-1 independent. Inhibitory effects of IL-6 on gastric emptying were confirmed following exercise-induced increases in IL-6. Importantly, gastric- and insulin-reducing effects were maintained in individuals with type 2 diabetes. These data have clinical implications with respect to the use of IL-6 inhibition in autoimmune/inflammatory disease, and identify a novel target that could be exploited pharmacologically to delay gastric emptying and spare insulin, which may be beneficial for the beta cell in type 2 diabetes.

The Gut: A Key to the Pathogenesis of Type 2 Diabetes?

In this communication we discuss the role of the gut for the development of type 2 diabetes mellitus (T2DM). Gastric emptying rates importantly determine postprandial glucose excursions and regulate postprandial secretion of the incretin hormones, glucose-dependent insulinotropic polypeptide (GIP), and glucagon-like peptide-1 (GLP-1). It thereby also determines their powerful, amplifying effect on glucose-induced insulin secretion and thus the ability of the body to regulate glucose disposal. Although disturbances in gastric emptying are not consistent findings in type 2 diabetes, the incretin system is seriously impaired, probably associated with insulin resistance and obesity. Both of the incretin hormones lose (part of) their insulinotropic activity resulting, together with (genetically) defective beta cell function, in the impaired postprandial insulin secretion of T2DM. In addition, glucagon responses are inappropriately increased and importantly contribute to both fasting and postprandial hyperglycemia. This may involve stimulation by GIP, but evidence also points to a role of circulating amino acids, which are elevated due to steatosis-induced impaired glucagon-mediated hepatic clearance, in line with recent work suggesting that the alpha cells and the liver are linked in a close, amino acid-mediated feedback circuit. Thus, the gut plays an important role in the development of T2DM spurred by overeating and defective beta cells.

Peptide production and secretion in GLUTag, NCI-H716, and STC-1 cells: a comparison to native L-cells.

GLUTag, NCI-H716, and STC-1 are cell lines that are widely used to study mechanisms underlying secretion of glucagon-like peptide-1 (GLP-1), but the extent to which they resemble native L-cells is unknown. We used validated immunoassays for 14 different hormones to analyze peptide content (lysis samples; n = 9 from different passage numbers) or peptide secretion in response to buffer (baseline), and after stimulation with 50 mM KCl or 10 mM glucose + 10 µM forskolin/3-isobutyl-1-methylxanthine (n = 6 also different passage numbers). All cell lines produced and processed proglucagon into GLP-1, GLP-2, glicentin, and oxyntomodulin in a pattern (prohormone convertase (PC)1/3 dependent) similar to that described for human gut. All three cell lines showed basal secretion of GLP-1 and GLP-2, which increased after stimulation. In contrast to freshly isolated murine L-cells, all cell lines also expressed PC2 and secreted large amounts of pancreatic glucagon. Neurotensin and somatostatin storage was low and secretion was not consistently increased by stimulation. STC-1 cells released more glucose-dependent insulinotropic polypeptide than GLP-1 at baseline (P < 0.01) and KCl elevated its secretion (P < 0.05). Peptide YY, which normally co-localizes with GLP-1 in distal L-cells, was not detected in any of the cell lines. GLUTag and STC-1 cells also expressed vasoactive intestinal peptide, but none expressed pancreatic polypeptide or insulin. GLUTag contained and secreted large amounts of CCK, while NCI-H716 did not store this peptide and STC-1 contained low amounts. Our results show that hormone production in cell line models of the L-cell has limited similarity to the natural L-cells.

Do glucagonomas always produce glucagon?

Pancreatic islet α-cell tumours that overexpress proglucagon are typically associated with the glucagonoma syndrome, a rare disease entity characterised by necrolytic migratory erythema, impaired glucose tolerance, thromboembolic complications and psychiatric disturbances. Paraneoplastic phenomena associated with enteric overexpression of proglucagon-derived peptides are less well recognized and include gastrointestinal dysfunction and hyperinsulinaemic hypoglycaemia. The diverse clinical manifestations associated with glucagon-expressing tumours can be explained, in part, by the repertoire of tumorally secreted peptides liberated through differential post-translational processing of tumour-derived proglucagon. Proglucagon-expressing tumours may be divided into two broad biochemical subtypes defined by either secretion of glucagon or GLP-1, GLP-2 and the glucagon-containing peptides, glicentin and oxyntomodulin, due to an islet α-cell or enteroendocrine L-cell pattern of proglucagon processing, respectively. In the current review we provide an updated overview of the clinical presentation of proglucagon-expressing tumours in relation to known physiological actions of proglucagon-derived peptides and suggest that detailed biochemical characterisation of the peptide repertoire secreted from these tumours may provide new opportunities for diagnosis and clinical management.

Glucose stimulates neurotensin secretion from the rat small intestine by mechanisms involving SGLT1 and GLUT2, leading to cell depolarization and calcium influx.

Neurotensin (NT) is a neurohormone produced in the central nervous system and in the gut epithelium by the enteroendocrine N cell. NT may play a role in appetite regulation and may have potential in obesity treatment. Glucose ingestion stimulates NT secretion in healthy young humans, but the mechanisms involved are not well understood. Here, we show that rats express NT in the gut and that glucose gavage stimulates secretion similarly to oral glucose in humans. Therefore, we conducted experiments on isolated perfused rat small intestine with a view to characterize the cellular pathways of secretion. Luminal glucose (20% wt/vol) stimulated secretion but vascular glucose (5, 10, or 15 mmol/l) was without effect. The underlying mechanisms depend on membrane depolarization and calcium influx, since the voltage-gated calcium channel inhibitor nifedipine and the KATP channel opener diazoxide, which causes hyperpolarization, eliminated the response. Luminal inhibition of the sodium-glucose cotransporter 1 (SGLT1) (by phloridzin) eliminated glucose-stimulated release as well as secretion stimulated by luminal methyl-α-D-glucopyranoside (20% wt/vol), a metabolically inactive SGLT1 substrate, suggesting that glucose stimulates secretion by initial uptake by this transporter. However, secretion was also sensitive to GLUT2 inhibition (by phloretin) and blockage of oxidative phosphorylation (2-4-dinitrophenol). Direct KATP channel closure by sulfonylureas stimulated secretion. Therefore, glucose stimulates NT secretion by uptake through SGLT1 and GLUT2, both causing depolarization either as a consequence of sodium-coupled uptake (SGLT1) or by closure of KATP channels (GLUT2 and SGLT1) secondary to the ATP-generating metabolism of glucose.

Measurement of the incretin hormones: glucagon-like peptide-1 and glucose-dependent insulinotropic peptide.

The two incretin hormones, glucagon-like peptide 1 (GLP-1) and glucose-dependent insulinotropic peptide (GIP), are secreted from the gastrointestinal tract in response to meals and contribute to the regulation of glucose homeostasis by increasing insulin secretion. Assessment of plasma concentrations of GLP-1 and GIP is often an important endpoint in both clinical and preclinical studies and, therefore, accurate measurement of these hormones is important. Here, we provide an overview of current approaches for the measurement of the incretin hormones, with particular focus on immunological methods.