Acute nicotine intake increases feeding behavior through decreasing glucagon signaling in dependent male and female rats.

Chronic use of nicotine is known to dysregulate metabolic signaling through altering circulating levels of feeding-related hormones, contributing to the onset of disorders like type 2 diabetes. However, little is known about the acute effects of nicotine on hormonal signaling. We previously identified an acute increase in food intake following acute nicotine, and we sought to determine whether this behavior was due to a change in hormone levels. We first identified that acute nicotine injection produces an increase in feeding behavior in dependent rats, but not nondependent rats. We confirmed that chronic nicotine use increases circulating levels of insulin, leptin, and ghrelin, and these correlate with rats' body weight and food intake. Acute nicotine injection in dependent animals decreased circulating GLP-1 and glucagon levels, and administration of glucagon prior to acute nicotine injection prevented the acute increase in feeding behavior. Thus, acute nicotine injection increases feeding behavior in dependent rats by decreasing glucagon signaling.

Establishment of a long-term stable ß-cell line and its application to analyze the effect of Gcg expression on insulin secretion.

A pancreatic ß-cell line MIN6 was previously established in our lab from an insulinoma developed in an IT6 transgenic mouse expressing the SV40 T antigen in ß-cells. This cell line has been widely used for in vitro analysis of ß-cell function, but tends to lose the mature ß-cell features, including glucose-stimulated insulin secretion (GSIS), in long-term culture. The aim of this study was to develop a stable ß-cell line that retains the characteristics of mature ß-cells. Considering that mice derived from a cross between C3H and C57BL/6 strains are known to exhibit higher insulin secretory capacity than C57BL/6 mice, an IT6 male mouse of this hybrid background was used to isolate insulinomas, which were independently cultured. After 7 months of continuous culturing, we obtained the MIN6-CB4 ß-cell line, which stably maintains its GSIS. It has been noted that ß-cell lines express the glucagon (Gcg) gene at certain levels. MIN6-CB4 cells were utilized to assess the effects of differential Gcg expression on ß-cell function. Our data show the functional importance of Gcg expression and resulting basal activation of the GLP-1 receptor in ß-cells. MIN6-CB4 cells can serve as an invaluable tool for studying the regulatory mechanisms of insulin secretion, such as the GLP-1/cAMP signaling, in ß-cells.

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.

Rational design of a GLP-1/GIP/Gcg receptor triagonist to correct hyperglycemia, obesity and diabetic nephropathy in rodent animals.

AIMS: To design and screen a potent GLP-1/GIP/Gcg receptors triagonist with therapeutic potential in rodent animals with diabetes and obesity. MAIN METHODS: First, we obtained a 12-mer dual GIP/Gcg receptor agonist from a large combinatorial peptide library via high-throughput screening technique and then fused to the Exendin (9-39) to generate a potent GLP-1/GIP/Gcg triagonist. Further site fatty chain modification was performed to improve the druggability via enhancing in vivo stability and cyclic half-life. In vitro signaling and functional assays in cell lines expressing each receptor and in vivo efficacy evaluation in rodent model animals with hyperglycemia and obesity were all carefully performed. KEY FINDINGS: We screened and obtained a potent GLP-1/GIP/Gcg triagonist, termed XFL0, which promotes in vitro GLP-1, GIP, Gcg receptor activation comparable to native GLP-1, GIP and glucagon, respectively. Site-specific fatty acid modification significantly enhanced plasma stability of XFL0 and exhibited no obvious impact on receptor activation. The selected XFL0 conjugates termed XFL6, showed glucose-dependent insulin secretion and improved glucose tolerance by acting on all GLP-1, GIP and Gcg receptors in gene-deficient mice of which the effects were all significantly greater than any single receptor agonist. After chronic treatment in rodent animals with diabetes and obesity, XFL6 potently decreased body weight and food intake, ameliorated the hyperglycemia and hemoglobin A1c levels as well as the lipid metabolism and diabetic nephropathy related disorders. SIGNIFICANCE: XFL6, as a novel GLP-1/GIP/Gcg receptor triagonist, held potential to deliver outstanding improvement in correcting hyperglycemia, obesity and diabetic nephropathy.

A Potential Role for Endogenous Glucagon in Preventing Post-Bariatric Hypoglycemia.

Obesity and obesity-related diseases are major public health concerns that have been exponentially growing in the last decades. Bariatric surgery is an effective long-term treatment to achieve weight loss and obesity comorbidity remission. Post-bariatric hypoglycemia (PBH) is a late complication of bariatric surgery most commonly reported after Roux-en-Y gastric bypass (RYGB). PBH is the end result of postprandial hyperinsulinemia but additional endocrine mechanisms involved are still under debate. Our aim was to characterize entero-pancreatic hormone dynamics associated with postprandial hypoglycemia after RYGB. Individuals previously submitted to RYGB (N=23) in a single tertiary hospital presenting PBH symptoms (Sym, n=14) and asymptomatic weight-matched controls (Asy, n=9) were enrolled. Participants underwent a mixed-meal tolerance test (MMTT) to assess glucose, total amino acids (total AA), insulin, C-peptide, glucagon, glucose-dependent insulinotropic polypeptide (GIP), glucagon-like peptide-1 (GLP-1), and neurotensin (NT). We found that hypoglycemia during the MMTT was equally frequent in Sym and Asy groups (p=1.000). Re-grouped according to glucose nadir during the MMTT (Hypo n=11 vs NoHypo n=12; nadir <3.05 mmol/l vs ≥3.05 mmol/l), subjects presented no differences in anthropometric (BMI: p=0.527) or metabolic features (HbA1c: p=0.358), yet distinct meal-elicited hormone dynamics were identified. Postprandial glucose excursion and peak glucose levels were similar (p>0.05), despite distinct late glycemic outcomes (t=60 min and t=90 min: p<0.01), with overall greater glycemic variability in Hypo group (minimum-to-maximum glucose ratio: p<0.001). Hypo group meal-triggered hormone profile was characterized by lower early glucagon (t=15 min: p<0.01) and higher insulin (t=30 min: p<0.05, t=45 min: p<0.001), C-peptide (t=30 min: p<0.01, t=45 min: p<0.001, t=60 min: p<0.05), and GLP-1 (t=45 min: p<0.05) levels. Hyperinsulinemia was an independent risk factor for hypoglycemia (p<0.05). After adjusting for hyperinsulinemia, early glucagon correlated with glycemic nadir (p<0.01), and prevented postprandial hypoglycemia (p<0.05). A higher insulin to glucagon balance in Hypo was observed (p<0.05). No differences were observed in total AA, GIP or NT excursions (p>0.05). In sum, after RYGB, postprandial hyperinsulinemia is key in triggering PBH, but a parallel and earlier rise in endogenous glucagon might sustain the inter-individual variability in glycemic outcome beyond the effect of hyperinsulinism, advocating a potential pivotal role for glucagon in preventing hyperinsulinemic hypoglycemia.

Regulation of Hepatic Follistatin Expression at Rest and during Exercise in Mice.

INTRODUCTION: Follistatin (FST) is a protein with numerous biological roles and was recently identified as an exercise-inducible hepatokine; however, the signals that regulate this are not well understood. The purpose of this study was to delineate potential endocrine factors that may regulate hepatic FST at rest and during exercise. METHODS: This study used four experiments. First, male and female C57BL/6J mice remained sedentary or were subjected to a single bout of exercise at moderate or exhaustive intensity with liver collected immediately post. Second, mice were injected with glucagon (1 mg·kg, 60 min), epinephrine (2 mg·kg, 30 min), glucagon then epinephrine, or saline. Third, mice were pretreated with propranolol (20-60 mg·kg, 30 min) before epinephrine injection. Fourth, glucagon receptor wild type (Gcgr) or knockout (Gcgr) mice were pretreated with saline or propranolol (20 mg·kg, 30 min) and were subjected to a single bout of exhaustive exercise with liver collected immediately post or after 2 h recovery. In all experiments liver FST mRNA expression was measured, and in experiment four FST protein content was measured. RESULTS: A single bout of treadmill exercise performed at an exhaustive but not moderate-intensity increased FST expression, as did injection of glucagon or epinephrine alone and when combined. Pretreatment of mice with propranolol attenuated the epinephrine-induced increase in FST expression. The exercise-induced increase in FST expression was attenuated in Gcgr mice, with no effect of propranolol. Gcgr mice had higher protein content of FST, but there was no effect of exercise or propranolol. CONCLUSIONS: These data suggest that both glucagon and epinephrine regulate hepatic FST expression at rest; however, only glucagon is required for the exercise-induced increase.

Peptide-based multi-agonists: a new paradigm in metabolic pharmacology.

Obesity and its comorbidities, such as type 2 diabetes, are pressing worldwide health concerns. Available anti-obesity treatments include weight loss pharmacotherapies and bariatric surgery. Whilst surgical interventions typically result in significant and sustained weight loss, available pharmacotherapies are far less effective, typically decreasing body weight by no more than 5-10%. An emerging class of multi-agonist drugs may eventually bridge this gap. This new class of specially tailored drugs hybridizes the amino acid sequences of key metabolic hormones into one single entity with enhanced potency and sustained action. Successful examples of this strategy include multi-agonist drugs targeting the receptors for glucagon-like peptide-1 (GLP-1), glucagon and the glucose-dependent insulinotropic polypeptide (GIP). Due to the simultaneous activity at several metabolically relevant receptors, these multi-agonists offer improved body weight loss and glucose tolerance relative to their constituent monotherapies. Further advancing this concept, chimeras were generated that covalently link nuclear acting hormones such as oestrogen, thyroid hormone (T3 ) or dexamethasone to peptide hormones such as GLP-1 or glucagon. The benefit of this strategy is to restrict the nuclear hormone action exclusively to cells expressing the peptide hormone receptor, thereby maximizing combinatorial metabolic efficacy of both drug constituents in the target cells whilst preventing the nuclear hormone cargo from entering and acting on cells devoid of the peptide hormone receptor, in which the nuclear hormone might have unwanted effects. Many of these multi-agonists are in preclinical and clinical development and may represent new and effective tools in the fight against obesity and its comorbidities.

The nuclear bile acid receptor FXR is a PKA- and FOXA2-sensitive activator of fasting hepatic gluconeogenesis.

BACKGROUND & AIMS: Embedded into a complex signaling network that coordinates glucose uptake, usage and production, the nuclear bile acid receptor FXR is expressed in several glucose-processing organs including the liver. Hepatic gluconeogenesis is controlled through allosteric regulation of gluconeogenic enzymes and by glucagon/cAMP-dependent transcriptional regulatory pathways. We aimed to elucidate the role of FXR in the regulation of fasting hepatic gluconeogenesis. METHODS: The role of FXR in hepatic gluconeogenesis was assessed in vivo and in mouse primary hepatocytes. Gene expression patterns in response to glucagon and FXR agonists were characterized by quantitative reverse transcription PCR and microarray analysis. FXR phosphorylation by protein kinase A was determined by mass spectrometry. The interaction of FOXA2 with FXR was identified by cistromic approaches and in vitro protein-protein interaction assays. The functional impact of the crosstalk between FXR, the PKA and FOXA2 signaling pathways was assessed by site-directed mutagenesis, transactivation assays and restoration of FXR expression in FXR-deficient hepatocytes in which gene expression and glucose production were assessed. RESULTS: FXR positively regulates hepatic glucose production through two regulatory arms, the first one involving protein kinase A-mediated phosphorylation of FXR, which allowed for the synergistic activation of gluconeogenic genes by glucagon, agonist-activated FXR and CREB. The second arm involves the inhibition of FXR's ability to induce the anti-gluconeogenic nuclear receptor SHP by the glucagon-activated FOXA2 transcription factor, which physically interacts with FXR. Additionally, knockdown of Foxa2 did not alter glucagon-induced and FXR agonist enhanced expression of gluconeogenic genes, suggesting that the PKA and FOXA2 pathways regulate distinct subsets of FXR responsive genes. CONCLUSIONS: Thus, hepatic glucose production is regulated during physiological fasting by FXR, which integrates the glucagon/cAMP signal and the FOXA2 signal, by being post-translationally modified, and by engaging in protein-protein interactions, respectively. LAY SUMMARY: Activation of the nuclear bile acid receptor FXR regulates gene expression networks, controlling lipid, cholesterol and glucose metabolism, which are mostly effective after eating. Whether FXR exerts critical functions during fasting is unknown. The results of this study show that FXR transcriptional activity is regulated by the glucagon/protein kinase A and the FOXA2 signaling pathways, which act on FXR through phosphorylation and protein-protein interactions, respectively, to increase hepatic glucose synthesis.

Use of glucagon-like peptide-1 receptor agonists among individuals on basal insulin requiring treatment intensification.

As Type 2 diabetes progresses, treatment is intensified with additional therapies in an effort to manage hyperglycaemia effectively and therefore avoid complications. When greater efficacy is required, options for injectable treatments include glucagon-like peptide-1 receptor agonists and insulin, which may be added on to oral glucose-lowering treatments. Among individuals receiving long-acting basal insulin as their first injectable treatment, ~40-60% are unable to achieve or maintain their target HbA1c goals. For these people, treatment intensification options are relatively limited and include the addition of short-acting prandial insulin or a glucagon-like peptide-1 receptor agonist. Glucagon-like peptide-1 receptor agonists vary in their effects, with short- and long-acting agents having a greater impact on postprandial and fasting hyperglycaemia, respectively. Studies comparing treatment intensification options have found both glucagon-like peptide-1 receptor agonists and prandial insulin to be effective in reducing HbA1c concentrations; however, recipients of glucagon-like peptide-1 receptor agonists lost weight and had a greater frequency of gastrointestinal adverse events, whereas those receiving prandial insulin gained weight and had a greater incidence of hypoglycaemia. In addition to the separate administration of a glucagon-like peptide-1 receptor agonist and basal insulin, fixed-ratio combinations of a glucagon-like peptide-1 receptor agonist and basal insulin offer a single administration for both treatments but have less flexibility in dose titration than treatment with their individual components. For individuals who require treatment intensification beyond basal insulin, use of these various options allows physicians to target the individual needs of their patients for the achievement of optimal long-term glycaemic control.

The role of incretin hormones and glucagon in patients with liver disease.

Non-alcoholic fatty liver disease (NAFLD) is defined as hepatic steatosis exceeding 5% of hepatocytes with no other reason for hepatic fat accumulation. The association between NAFLD and type 2 diabetes is strong. Accordingly, up to 70% of obese patients with type 2 diabetes have NAFLD. The spectrum of NAFLD ranges from simple steatosis to non-alcoholic steatohepatitis with variable degrees of fibrosis and cirrhosis. Cirrhosis is the end-stage of chronic liver disease and is characterised by diffuse fibrosis and nodular regeneration of hepatocytes. Alcoholic liver disease and NAFLD are the most common aetiologies of cirrhosis. The WHO estimates that 70% of patients with cirrhosis have impaired glucose tolerance and 30% have manifest diabetes. The latter is termed hepatic diabetes and is associated with increased complications to cirrhosis and hepatocellular carcinoma. The objective of this thesis was to study the impact of liver dysfunction on incretin and glucagon (patho)physiology in relation to glucose metabolism. We hypothesised that NAFLD patients with normal glucose tolerance would develop reduced incretin effect and that NAFLD would worsen the incretin effect in patients with existing type 2 diabetes. Thus, in study I, we investigated the incretin effect and glucagon secretion in patients with NAFLD with and without type 2 diabetes compared to controls. We also hypothesised that the incretin effect would be disturbed in non-diabetic patients with more severe liver disease. Hence, the objective of study II was to investigate the incretin effect in patients with cirrhosis. Finally, the hypothesis in study III was that an impaired glucagonostatic effect of GLP-1 contributes to the hyperglucagonaemia of patients with liver disease. We therefore explored the glucagonostatic properties of GLP-1 in non-diabetic patients with NAFLD. The results of study I show that patients with NAFLD have normal secretion of GLP-1 and GIP and a reduced incretin effect. The groups with type 2 diabetes have the lowest incretion effect. We also find that NAFLD patients have high fasting glucagon concentrations regardless of their glucose (in)tolerance. We further demonstrated that patients with normal glucose tolerance and NAFLD have preserved glucagon suppression to both oral and intravenous glucose.  In study II, we find that non-diabetic patients with cirrhosis have elevated concentrations of GLP-1 and GIP and a reduced incretin effect. Patients with cirrhosis also have fasting hyperglucagonaemia, but show intact glucagon suppression during both oral and intravenous glucose administration. Finally, study III demonstrates that normal glucose tolerant NAFLD patients had preserved glucagonostatic effect of GLP-1. In conclusion, our studies offer important information regarding the pathophysiology of glucose intolerance in patients with liver disease. We demonstrate that patients with NAFLD, in spite of normal glucose tolerance, have reduced incretin effect that is further aggravated by type 2 diabetes. We also find fasting hyperglucagonaemia in NAFLD patients, independently of type 2 diabetes. We show that cirrhosis is associated with impaired handling of oral glucose and reduced incretin effect. Finally, we find a preserved glucagonostatic effect of GLP-1 in patients with NAFLD, in spite of their hyperglucagonaemia. In light of our findings, the insulinotropic and glucagonostatic effects of GLP-1 receptor agonists might prove beneficial in patients with liver disease.

Physiological and pathophysiological aspects of incretin hormones and glucagon.

Infusion of oxyntomodulin and the separate and combined infusion of GLP-1 and glucagon inhibited food intake similarly in healthy individuals, with no superior effect of combining GLP-1 and glucagon. We confirm the inhibitory effects of oxyntomodulin and GLP-1, respectively, on GE and appetite scores observed previously, but by adding glucagon to the infusion of GLP-1 we found no additive effects. Unexpectedly, glucagon alone had no effect on GE and appetite scores, but inhibited food intake to the same extent as oxyntomodulin, GLP-1 and GLP-1 + glucagon. Both the GLP-1, oxyntomodulin and GLP-1 + glucagon infusions appeared to increase O2 compared to saline but this observation is most likely confounded by a residual meal-induced thermogenesis because the calorimetry was performed relatively soon after the paracetamol peak indicating that a considerable volume still resided in the stomach and a high rate of nutrient absorption probably was still going on compared to the saline infusion. Flint et al previously concluded from a protocol very similar to ours using GLP-1 infusions, that the observed increases in energy expenditure most likely were linked to the meal. In contrast, we observed no significant changes in O2 from baseline in any of the experiments in our study. The lack of a clear effect on O2 is in contrast to recently reported findings regarding infusions of glucagon and GLP-1. But the dose of glucagon used in that particular study was more than 15-fold higher than ours and associated with large changes in glucose and insulin levels. Such levels are likely to influence REE and offer an explanation of the reported additive effect of combinations of GLP-1 and glucagon. Our conclusion is consistent with recent findings showing no increases after short-term native GLP-1 infusions. Long-term treatment with the GLP-1 analogue liraglutide using 24 h chamber calorimetry has so far shown no differences in energy expenditure following the treatment. Surprisingly, the infusion of glucagon did not change gastric emptying. This finding is controversial since glucagon previously has been used to inhibit bowel motility. However, the doses used to inhibit bowel motility were more than 3,000-fold higher than the dose used in the present study and as mentioned above, such doses might activate the GLP-1 receptor pathway. Interestingly, the glucagon infusion did result in decreased food intake to the same extent as the other peptide infusions despite having no impact on gastric emptying and appetite scores. We found a mean 180 kcal (120 g) difference in food intake following infusions of all the peptides compared to saline. This would roughly sum up to a body weight loss of 402 g of fat per week, which is in the range of what previously has been found in overweight and obese humans with the injection of oxyntomodulin.

Protein- and diabetes-induced glomerular hyperfiltration: role of glucagon, vasopressin, and urea.

A single protein-rich meal (or an infusion of amino acids) is known to increase the glomerular filtration rate (GFR) for a few hours, a phenomenon known as "hyperfiltration." It is important to understand the factors that initiate this upregulation because it becomes maladaptive in the long term. Several mediators and paracrine factors have been shown to participate in this upregulation, but they are not directly triggered by protein intake. Here, we explain how a rise in glucagon and in vasopressin secretion, directly induced by protein ingestion, might be the initial factors triggering the hepatic and renal events leading to an increase in the GFR. Their effects include metabolic actions in the liver and stimulation of sodium chloride reabsorption in the thick ascending limb. Glucagon is not only a glucoregulatory hormone. It is also important for the excretion of nitrogen end products by stimulating both urea synthesis in the liver (along with gluconeogenesis from amino acids) and urea excretion by the kidney. Vasopressin allows the concentration of nitrogenous end products (urea, ammonia, etc.) and other protein-associated wastes in a hyperosmotic urine, thus allowing a very significant water economy characteristic of all terrestrial mammals. No hyperfiltration occurs in the absence of one or the other hormone. Experimental results suggest that the combined actions of these two hormones, along with the complex intrarenal handling of urea, lead to alter the composition of the tubular fluid at the macula densa and to reduce the intensity of the signal activating the tubuloglomerular feedback control of GFR, thus allowing GFR to raise. Altogether, glucagon, vasopressin, and urea contribute to set up the best compromise between efficient urea excretion and water economy.

Evidence That in Uncontrolled Diabetes, Hyperglucagonemia Is Required for Ketosis but Not for Increased Hepatic Glucose Production or Hyperglycemia.

Several lines of evidence implicate excess glucagon secretion in the elevated rates of hepatic glucose production (HGP), hyperglycemia, and ketosis characteristic of uncontrolled insulin-deficient diabetes (uDM), but whether hyperglucagonemia is required for hyperglycemia in this setting is unknown. To address this question, adult male Wistar rats received either streptozotocin (STZ) to induce uDM (STZ-DM) or vehicle and remained nondiabetic. Four days later, animals received daily subcutaneous injections of either the synthetic GLP-1 receptor agonist liraglutide in a dose-escalating regimen to reverse hyperglucagonemia or its vehicle for 10 days. As expected, plasma glucagon levels were elevated in STZ-DM rats, and although liraglutide treatment lowered glucagon levels to those of nondiabetic controls, it failed to attenuate diabetic hyperglycemia, elevated rates of glucose appearance (Ra), or increased hepatic gluconeogenic gene expression. In contrast, it markedly reduced levels of both plasma ketone bodies and hepatic expression of the rate-limiting enzyme involved in ketone body production. To independently confirm this finding, in a separate study, treatment of STZ-DM rats with a glucagon-neutralizing antibody was sufficient to potently lower plasma ketone bodies but failed to normalize elevated levels of either blood glucose or Ra. These data suggest that in rats with uDM, hyperglucagonemia is required for ketosis but not for increased HGP or hyperglycemia.

A novel glucagon-like peptide-1 (GLP-1)/glucagon hybrid peptide with triple-acting agonist activity at glucose-dependent insulinotropic polypeptide, GLP-1, and glucagon receptors and therapeutic potential in high fat-fed mice.

Glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic polypeptide (GIP), and glucagon bind to related members of the same receptor superfamily and exert important effects on glucose homeostasis, insulin secretion, and energy regulation. The present study assessed the biological actions and therapeutic utility of novel GIP/glucagon/GLP-1 hybrid peptides. Nine novel peptides were synthesized and exhibited complete DPP-IV resistance and enhanced in vitro insulin secretion. The most promising peptide, [dA(2)]GLP-1/GcG, stimulated cAMP production in GIP, GLP-1, and glucagon receptor-transfected cells. Acute administration of [dA(2)]GLP-1/GcG in combination with glucose significantly lowered plasma glucose and increased plasma insulin in normal and obese diabetic (ob/ob) mice. Furthermore, [dA(2)]GLP-1/GcG elicited a protracted glucose-lowering and insulinotropic effect in high fat-fed mice. Twice daily administration of [dA(2)]GLP-1/GcG for 21 days decreased body weight and nonfasting plasma glucose and increased circulating plasma insulin concentrations in high fat-fed mice. Furthermore, [dA(2)]GLP-1/GcG significantly improved glucose tolerance and insulin sensitivity by day 21. Interestingly, locomotor activity was increased in [dA(2)]GLP-1/GcG mice, without appreciable changes in aspects of metabolic rate. Studies in knock-out mice confirmed the biological action of [dA(2)]GLP-1/GcG via multiple targets including GIP, GLP-1, and glucagon receptors. The data suggest significant promise for novel triple-acting hybrid peptides as therapeutic options for obesity and diabetes.

RNAi screening in primary human hepatocytes of genes implicated in genome-wide association studies for roles in type 2 diabetes identifies roles for CAMK1D and CDKAL1, among others, in hepatic glucose regulation.

Genome-wide association (GWA) studies have described a large number of new candidate genes that contribute to of Type 2 Diabetes (T2D). In some cases, small clusters of genes are implicated, rather than a single gene, and in all cases, the genetic contribution is not defined through the effects on a specific organ, such as the pancreas or liver. There is a significant need to develop and use human cell-based models to examine the effects these genes may have on glucose regulation. We describe the development of a primary human hepatocyte model that adjusts glucose disposition according to hormonal signals. This model was used to determine whether candidate genes identified in GWA studies regulate hepatic glucose disposition through siRNAs corresponding to the list of identified genes. We find that several genes affect the storage of glucose as glycogen (glycolytic response) and/or affect the utilization of pyruvate, the critical step in gluconeogenesis. Of the genes that affect both of these processes, CAMK1D, TSPAN8 and KIF11 affect the localization of a mediator of both gluconeogenesis and glycolysis regulation, CRTC2, to the nucleus in response to glucagon. In addition, the gene CDKAL1 was observed to affect glycogen storage, and molecular experiments using mutant forms of CDK5, a putative target of CDKAL1, in HepG2 cells show that this is mediated by coordinate regulation of CDK5 and PKA on MEK, which ultimately regulates the phosphorylation of ribosomal protein S6, a critical step in the insulin signaling pathway.

Hypothalamic glucagon signaling inhibits hepatic glucose production.

Glucagon activates hepatic protein kinase A (PKA) to increase glucose production, but the gluco-stimulatory effect is transient even in the presence of continuous intravenous glucagon infusion. Continuous intravenous infusion of insulin, however, inhibits glucose production through its sustained actions in both the liver and the mediobasal hypothalamus (MBH). In a pancreatic clamp setting, MBH infusion with glucagon activated MBH PKA and inhibited hepatic glucose production (HGP) in rats, as did central glucagon infusion in mice. Inhibition of glucagon receptor-PKA signaling in the MBH and hepatic vagotomy each negated the effect of MBH glucagon in rats, whereas the central effect of glucagon was diminished in glucagon receptor knockout mice. A sustained rise in plasma glucagon concentrations transiently increased HGP, and this transiency was abolished in rats with negated MBH glucagon action. In a nonclamp setting, MBH glucagon infusion improved glucose tolerance, and inhibition of glucagon receptor-PKA signaling in the MBH enhanced the ability of intravenous glucagon injection to increase plasma glucose concentrations. We also detected a similar enhancement of glucose concentrations that was associated with a disruption in MBH glucagon signaling in rats fed a high-fat diet. We show that hypothalamic glucagon signaling inhibits HGP and suggest that hypothalamic glucagon resistance contributes to hyperglycemia in diabetes and obesity.

Contribution of gastroenteropancreatic appetite hormones to protein-induced satiety.

BACKGROUND: Effects of protein intake on appetite-regulating hormones and their dynamics are unclear. OBJECTIVES: We investigated the satiating effects of meals with varying protein contents and whether there was an effect of dose on appetite-regulating hormones and appetite ratings. DESIGN: Twenty-five men [mean ± SD age: 30.0 ± 8.7 y; body mass index (BMI; in kg/m(2)): 25.9 ± 4.7] participated in the 3-way, randomized, double-blind crossover study. Test meals were isocaloric with 30% of energy from fat and protein content adjusted at the expense of carbohydrate. Test meals were normal protein (NP; 14% of energy from protein), medium-high protein (MHP; 25% of energy from protein), and high protein (HP, 50% of energy from protein). Appetite ratings and blood samples were assessed every 0.5 h for 4 h. An ad libitum lunch was served 4 h after the meal. RESULTS: Protein increased dose-dependently glucagon-like peptide-1 (GLP-1), peptide YY (PYY) 3-36, and glucagon; MHP produced 10%, 7%, and 47% greater responses, respectively; and HP produced 20%, 14%, and 116% greater responses, respectively, than did NP (P < 0.03). Compared with NP, HP increased insulin and cholecystokinin and decreased ghrelin and glucose-dependent insulinotropic polypeptide (P < 0.05). Satiety and fullness dose-dependently increased by 7% and 6% for MHP and 16% and 19% for HP compared with NP (P < 0.001). Hunger and prospective consumption dose-dependently decreased by 15% and 13% for MHP and by 25% and 26% for HP compared with NP (P < 0.0003). There was a combined effect of GLP-1 and PYY 3-36 (P = 0.03) next to the additive effect of GLP-1 (P = 0.006) on the composite appetite score. No difference was shown in ad libitum energy intake. CONCLUSION: Protein dose-dependently increased satiety and GLP-1, PYY 3-36, and glucagon, which may, at least in part, be responsible for the satiety-stimulating effect of protein. This trial was registered at clinicaltrials.gov as NCT01561235.

Choline supplementation promotes hepatic insulin resistance in phosphatidylethanolamine N-methyltransferase-deficient mice via increased glucagon action.

Biosynthesis of hepatic choline via phosphatidylethanolamine N-methyltransferase (PEMT) plays an important role in the development of type 2 diabetes and obesity. We investigated the mechanism(s) by which choline modulates insulin sensitivity. PEMT wild-type (Pemt(+/+)) and knock-out (Pemt(-/-)) mice received either a high fat diet (HF; 60% kcal of fat) or a high fat, high choline diet (HFHC; 4 g of choline/kg of HF diet) for 1 week. Hepatic insulin signaling and glucose and lipid homeostasis were investigated. Glucose and insulin intolerance occurred in Pemt(-/-) mice fed the HFHC diet, but not in their Pemt(-/-) littermates fed the HF diet. Plasma glucagon was elevated in Pemt(-/-) mice fed the HFHC diet compared with Pemt(-/-) mice fed the HF diet, concomitant with increased hepatic expression of glucagon receptor, phosphorylated AMP-activated protein kinase (AMPK), and phosphorylated insulin receptor substrate 1 at serine 307 (IRS1-s307). Gluconeogenesis and mitochondrial oxidative stress were markedly enhanced, whereas glucose oxidation and triacylglycerol biosynthesis were diminished in Pemt(-/-) mice fed the HFHC diet. A glucagon receptor antagonist (2-aminobenzimidazole) attenuated choline-induced hyperglycemia and insulin intolerance and blunted up-regulation of phosphorylated AMPK and IRS1-s307. Choline induces glucose and insulin intolerance in Pemt(-/-) mice through modulating plasma glucagon and its action in liver.

Glucagon receptor is required for long-term survival: a natural history study of the Mahvash disease in a murine model.

BACKGROUND AND AIM: We have described a novel Mahvash disease of hyperglucagonemia and pancreatic neuroendocrine tumors (PNETs) associated with an inactivating glucagon receptor mutation, and identified the glucagon receptor-deficient (Gcgr(-/-)) mice as its murine model. We aim to elucidate the natural history of the rare Mahvash disease by long-term observation of the Gcgr(-/-) mice. MATERIALS AND METHOD: Wild type (WT) (n=52), heterozygous (n=127), and Gcgr(-/-) (n=56) mice living under standard vivarium conditions were observed without specific treatments over 22 months. Autopsy was performed on dead animals. RESULTS: The WT and heterozygous mice did not exhibit any measurable differences. The Gcgr(-/-) mice became progressively lethargic and cachexic after 12 months. Random glucose levels were stable in WT and heterozygous mice but decreased with age in the Gcgr(-/-) mice. At the end of observation, 28/56 Gcgr(-/-), 7/52 WT, and 24/127 heterozygous mice died. The survival curve of Gcgr(-/-) mice began to separate from those of WT and heterozygous mice at 12 months and the survival difference widened with age. At 18 months, survival probability was 17% for Gcgr(-/-) mice but 77% for WT and 81% for heterozygous mice. Autopsy revealed numerous PNETs up to 15 mm in diameter in most well-preserved Gcgr(-/-) pancreata (17/20) but none in WT or heterozygous ones. Four Gcgr(-/-) mice developed liver or subcutaneous metastasis. CONCLUSION: The untreated Mahvash disease may cause cachexia, severe hypoglycemia, and early death. Patients with Mahvash disease need to undergo life-long surveillance for PNETs. Functional glucagon receptor is thus required for long-term survival.