Effect Of Glucagon Research Articles

Glucagon receptor antagonism impairs and glucagon receptor agonism enhances triglycerides metabolism in mice

ObjectiveTreatment with glucagon receptor antagonists (GRAs) reduces blood glucose but causes dyslipidemia and accumulation of fat in the liver. We investigated the acute and chronic effects of glucagon on lipid metabolism in mice. MethodsChronic effects of glucagon receptor signaling on lipid metabolism were studied using oral lipid tolerance tests (OLTTs) in overnight fasted glucagon receptor knockout (Gcgr−/−) mice, and in C57Bl/6JRj mice treated with a glucagon receptor antibody (GCGR Ab) or a long-acting glucagon analogue (GCGA) for eight weeks. Following treatment, liver tissue was harvested for RNA-sequencing and triglyceride measurements. Acute effects were studied in C57Bl/6JRj mice treated with a GRA or GCGA 1 h or immediately before OLTTs, respectively. Direct effects of glucagon on hepatic lipolysis were studied using isolated perfused mouse liver preparations. To investigate potential effects of GCGA and GRA on gastric emptying, paracetamol was, in separate experiments, administered immediately before OLTTs. ResultsPlasma triglyceride concentrations increased 2-fold in Gcgr−/− mice compared to their wild-type littermates during the OLTT (P = 0.001). Chronic treatment with GCGR Ab increased, whereas GCGA treatment decreased, plasma triglyceride concentrations during OLTTs (P < 0.05). Genes involved in lipid metabolism were upregulated upon GCGR Ab treatment while GCGA treatment had opposite effects. Acute GRA and GCGA treatment, respectively, increased (P = 0.02) and decreased (P = 0.003) plasma triglyceride concentrations during OLTTs. Glucagon stimulated hepatic lipolysis, evident by an increase in free fatty acid concentrations in the effluent from perfused mouse livers. In line with this, GCGR Ab treatment increased, while GCGA treatment decreased, liver triglyceride concentrations. The effects of glucagon appeared independent of changes in gastric emptying of paracetamol. ConclusionsGlucagon receptor signaling regulates triglyceride metabolism, both chronically and acutely, in mice. These data expand glucagon´s biological role and implicate that intact glucagon signaling is important for lipid metabolism. Glucagon agonism may have beneficial effects on hepatic and peripheral triglyceride metabolism.

Molecular Characterization of Grass Carp GIPR and Effect of Nutrition States, Insulin, and Glucagon on Its Expression.

GIP plays an important regulatory role in glucose and lipid metabolism. As the specific receptor, GIPR is involved in this physiological process. To assess the roles of GIPR in teleost, the GIPR gene was cloned from grass carp. The ORF of cloned GIPR gene was 1560 bp, encoding 519 amino acids. The grass carp GIPR was the G-protein-coupled receptor which contains seven predicted transmembrane domains. In addition, two predicted glycosylation sites were contained in the grass carp GIPR. The grass carp GIPR expression is in multiple tissues and is highly expressed in the kidney, brain regions, and visceral fat tissue. In the OGTT experiment, the GIPR expression is markedly decreased in the kidney, visceral fat, and brain by treatment with glucose for 1 and 3 h. In the fast and refeeding experiment, the GIPR expression in the kidney and visceral fat tissue was significantly induced in the fast groups. In addition, the GIPR expression levels were markedly decreased in the refeeding groups. In the present study, the visceral fat accumulation of grass carp was induced by overfed. The GIPR expression was significantly decreased in the brain, kidney, and visceral fat tissue of overfed grass carp. In primary hepatocytes, the GIPR expression was promoted by treatment with oleic acid and insulin. The GIPR mRNA levels were significantly reduced by treatment with glucose and glucagon in the grass carp primary hepatocytes. To our knowledge, this is the first time the biological role of GIPR is unveiled in teleost.

Vasodilatory effects of glucagon: A possible new approach to enhanced subcutaneous insulin absorption in artificial pancreas devices.

Patients with diabetes mellitus type 1 depend on exogenous insulin to keep their blood glucose concentrations within the desired range. Subcutaneous bihormonal artificial pancreas devices that can measure glucose concentrations continuously and autonomously calculate and deliver insulin and glucagon infusions is a promising new treatment option for these patients. The slow absorption rate of insulin from subcutaneous tissue is perhaps the most important factor preventing the development of a fully automated artificial pancreas using subcutaneous insulin delivery. Subcutaneous insulin absorption is influenced by several factors, among which local subcutaneous blood flow is one of the most prominent. We have discovered that micro-doses of glucagon may cause a substantial increase in local subcutaneous blood flow. This paper discusses how the local vasodilative effects of micro-doses of glucagon might be utilised to improve the performance of subcutaneous bihormonal artificial pancreas devices. We map out the early stages of our hypothesis as a disruptive novel approach, where we propose to use glucagon as a vasodilator to accelerate the absorption of meal boluses of insulin, besides using it conventionally to treat hypoglycaemia.

Glucagon receptor signaling at white adipose tissue does not regulate lipolysis.

Although the physiological role of glucagon receptor signaling in the liver is well defined, the impact of glucagon receptor (Gcgr) signaling on white adipose tissue (WAT) continues to be debated. Although numerous studies propose that glucagon stimulates WAT lipolysis, we lack evidence that physiological concentrations of glucagon regulate WAT lipolysis. In turn, we performed studies in both wild-type and WAT Gcgr knockout mice to determine if glucagon regulates lipolysis at WAT in the mouse. We assessed the effects of fasting and acute exogenous glucagon administration in wild-type C57BL/6J and GcgrAdipocyte+/+ versus GcgrAdipocyte-/- mice. Using an ex vivo lipolysis protocol, we further examined the direct effects of glucagon on physiologically (fasted) and pharmacologically stimulated lipolysis. We found that adipocyte Gcgr expression did not affect fasting-induced lipolysis or hepatic lipid accumulation in lean or diet-induced obese (DIO) mice. Acute glucagon administration did not affect serum nonesterified fatty acids (NEFA), leptin, or adiponectin concentration, but did increase serum glucose and FGF21, regardless of genotype. Glucagon did not affect ex vivo lipolysis in explants from either GcgrAdipocyte+/+ or GcgrAdipocyte-/- mice. Gcgr expression did not affect fasting-induced or isoproterenol-stimulated lipolysis from WAT explants. Moreover, glucagon receptor signaling at WAT did not affect body weight or glucose homeostasis in lean or DIO mice. Our studies have established that physiological levels of glucagon do not regulate WAT lipolysis, either directly or indirectly. Given that glucagon receptor agonism can improve dyslipidemia and decrease hepatic lipid accumulation, it is critical to understand the tissue-specific effects of glucagon receptor action. Unlike the crucial role of hepatic glucagon receptor signaling in maintaining glucose and lipid homeostasis, we observed no metabolic consequence of WAT glucagon receptor deletion.NEW & NOTEWORTHY It has been postulated that glucagon stimulates lipolysis and fatty acid release from white adipose tissue. We observed no metabolic effects of eliminating or activating glucagon receptor signaling at white adipose tissue.

Myonectin in grass carp (Ctenopharyngodon idella): Molecular cloning and effect of nutrition state, insulin and glucagon on myonectin expression

As an endocrine organ, skeletal muscle secretes multiple myokines. Myonectin, a myokine, participates in lipid and glucose metabolism. To explore the functions of myonectin in teleosts, the myonectin gene was cloned from the brain tissue of grass carp. The open reading frame (ORF) of isolated myonectin was 813 bp, encoding 270 amino acids. Grass carp myonectin has four predicted domains: signal peptide, N-terminal domain (NTD), collagen protein domain, and C1q/TNF-like domain. In addition, four predicted glycosylation sites and four conserved cysteine residues were located in grass carp myonectin. Myonectin mRNA was detected in multiple tissues of grass carp and was highly expressed in the brain region, heart and kidney. In the oral glucose tolerance test (OGTT), myonectin mRNA levels were significantly induced in the kidney, hypothalamus, muscle and brain after treatment with glucose. In the fast and refeeding experiment, the serum glucose level was decreased in the fast group. However, serum glucose levels were increased in the refeeding group. The myonectin mRNA levels in the kidney, heart, brain and liver were significantly reduced in the fast groups. Moreover, the myonectin expression was markedly increased in the refeeding groups. In grass carp primary hepatocytes, myonectin mRNA levels were induced by treatment with glucose and oleic acid for 12 or 24 h. Myonectin expression was dramatically promoted by treatment with insulin. Moreover, myonectin expression was markedly decreased with glucagon treatment in primary hepatocytes. The results of this study will provide a basis for exploring the roles of grass carp myonectin in the future.

The effect of glucagon like peptide-1 receptor agonist on behavioral despair and anxiety-like behavior in ovariectomized rats: Modulation of BDNF/CREB, Nrf2 and lipocalin 2

Ovariectomized (OVX) rodents show behavioral despair and anxiety-like behaviors. Glucagon-like peptide-1 receptor agonists (GLP-1RA) possess neuroprotective effects by reducing oxidative stress and neuroinflammation, thereby preventing synaptic loss. The objective of the present study is to evaluate the effect of GLP-1RA, namely liraglutide, on emotional behaviors, and to identify the level of oxidative stress, neuroinflammation, and BDNF signaling in the hippocampus of OVX rats. Forty female young Wistar rats were divided into 5 groups: Control, Control+liraglutide treated, OVX, OVX+fluoxetine, and OVX+liraglutide (150 µg/kg for 15 days, sc). Open field test and elevated plus-maze test were used to evaluate behaviors that are suggestive of anxiety. A forced swimming test was used to evaluate behavioral despair. At the end of the experiments, blood glucose level and body weight gain were measured. The levels of BDNF, CREB, Nrf2, and lipocalin 2 in the hippocampal tissue were measured by ELISA. Malondialdehyde (MDA) and glutathione levels were also evaluated. Statistical analysis was conducted through ANOVA and Bonferroni tests. Seven weeks post-OVX rats exhibited high anxiety related behavior and behavioral despair in comparison with the control groups. These behavioral changes were associated with increased lipocalin 2 and MDA levels in rats. Moreover, BDNF, CREB, and Nrf2 levels decreased significantly in the hippocampus of OVX rats. Liraglutide treatment limited the reduction of BDNF and Nrf2 levels in the hippocampus, maintaining them at the control levels. Liraglutide treatment also prevented the symptoms of behavioral despair and anxiety related behavior. As the main finding of the study GLP-1RA reduced behavioral despair and anxiety level and this may be related to the preservation of BDNF/Nrf2 levels and the decrease in oxidative stress and lipocalin 2 levels in the hippocampus.

The Effect of Glucagon on Protein Catabolism During Insulin Deficiency: Exchange of Amino Acids Across Skeletal Muscle and the Splanchnic Bed.

Transient insulin deprivation with concurrent hyperglucagonemia is a catabolic state that can occur in type 1 diabetes. To evaluate glucagon's catabolic effect in the setting of its glucogenic effect, we measured the regional exchanges of amino acid metabolites (amino-metabolites) across muscle and splanchnic beds in 16 healthy humans during either somatostatin followed by glucagon or saline infusion alone. Despite a twofold or greater increase in the regional exchange of amino-metabolites by glucagon, whole-body kinetics and concentrations of amino acids (AA) remained stable. Glucagon increased the splanchnic uptake of not only gluconeogenic but also essential (EAA) AA while increasing their release from the muscle bed. Regional tracer-based kinetics and 3-methylhistidine release indicate that EAA release from muscle is likely caused by reduced protein synthesis rather than increased protein degradation. Furthermore, many metabolites known to affect insulin action and metabolism were altered by hyperglucagonemia including increase in branched-chain AA and keto acids of leucine and isoleucine in arterial plasma. Further, an increase in arterial concentrations of α-aminoadipic acid arising from increased conversion from lysine in the splanchnic bed was noted. These results demonstrate that hyperglucagonemia during hypoinsulinemia increases net muscle protein catabolism and substantially increases the exchange of amino metabolites across splanchnic and muscle beds.

Next generation GLP-1/GIP/glucagon triple agonists normalize body weight in obese mice

ObjectivePharmacological strategies that engage multiple mechanisms-of-action have demonstrated synergistic benefits for metabolic disease in preclinical models. One approach, concurrent activation of the glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and glucagon (Gcg) receptors (i.e. triagonism), combines the anorectic and insulinotropic activities of GLP-1 and GIP with the energy expenditure effect of glucagon. While the efficacy of triagonism in preclinical models is known, the relative contribution of GcgR activation remains unassessed. This work aims to addresses that central question. MethodsHerein, we detail the design of unimolecular peptide triagonists with an empirically optimized receptor potency ratio. These optimized peptide triagonists employ a protraction strategy permitting once-weekly human dosing. Additionally, we assess the effects of these peptides on weight-reduction, food intake, glucose control, and energy expenditure in an established DIO mouse model compared to clinically relevant GLP-1R agonists (e.g. semaglutide) and dual GLP-1R/GIPR agonists (e.g. tirzepatide). ResultsOptimized triagonists normalize body weight in DIO mice and enhance energy expenditure in a manner superior to that of GLP-1R mono-agonists and GLP-1R/GIPR co-agonists. ConclusionsThese pre-clinical data suggest unimolecular poly-pharmacology as an effective means to target multiple mechanisms contributing to obesity and further implicate GcgR activation as the differentiating factor between incretin receptor mono- or dual-agonists and triagonists.

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

SAT104 - The effect of glucagon on rates of hepatic mitochondrial oxidation and pyruvate carboxylase flux in man assessed by positional isotopomer tracer analysis (PINTA)

FRI156 - Multi-target engagement effect of a novel long-acting Glucagon/ GIP/GLP-1 triple agonist, HM15211, in animal model of NASH/ fibrosis

FRI156 - Multi-target engagement effect of a novel long-acting Glucagon/ GIP/GLP-1 triple agonist, HM15211, in animal model of NASH/ fibrosis

Role of Glucagon and Its Receptor in the Pathogenesis of Diabetes.

Various theories for the hormonal basis of diabetes have been proposed and debated over the past few decades. Insulin insufficiency was previously regarded as the only hormone deficiency directly leading to metabolic disorders associated with diabetes. Although glucagon and its receptor are ignored in this framework, an increasing number of studies have shown that they play essential roles in the development and progression of diabetes. However, the molecular mechanisms underlying the effects of glucagon are still not clear. In this review, recent research on the mechanisms by which glucagon and its receptor contribute to the pathogenesis of diabetes as well as correlations between GCGR mutation rates in populations and the occurrence of diabetes are summarized. Furthermore, we summarize how recent research clearly establishes glucagon as a potential therapeutic target for diabetes.

Regulation of Hepatic Lipid and Glucose Metabolism by INSP3R1.

With the rising epidemics of obesity and nonalcoholic fatty liver disease (NAFLD) and its downstream consequences including steatohepatitis, cirrhosis, and type 2 diabetes in the U.S. and worldwide, new therapeutic approaches are urgently needed to treat these devastating conditions. Glucagon, known for a century to be a glucose-raising hormone and clearly demonstrated to contribute to fasting and postprandial hyperglycemia in both type 1 and type 2 diabetes, represents an unlikely target to improve health in those with metabolic syndrome. However, recent work from our group and others' identifies an unexpected role for glucagon as a potential means of treating NAFLD, improving insulin sensitivity, and improving the lipid profile. We propose a unifying, calcium-dependent mechanism for glucagon's effects both to stimulate hepatic gluconeogenesis and to enhance hepatic mitochondrial oxidation: signaling through the inositol 1,4,5-trisphosphate receptor type 1 (INSP3R1), glucagon activates phospholipase C (PKC)/protein kinase A (PKA) signaling to enhance adipose triglyceride lipase (ATGL)-dependent intrahepatic lipolysis and, in turn, increase cytosolic gluconeogenesis by allosteric activation of pyruvate carboxylase. Simultaneously in the mitochondria, calcium transferred through mitochondria-associated membranes activates several dehydrogenases in the tricarboxylic acid cycle, correlated with an increase in mitochondrial energy expenditure and reduction in ectopic lipid. This model suggests that short-term, cyclic treatment with glucagon or other INSP3R1 antagonists could hold promise as a means to reset lipid homeostasis in patients with NAFLD.

360-OR: Hyperglucagonemia Reduces De Novo Lipogenesis and Increases Gluconeogenesis in Healthy Subjects

Although the effect of glucagon on glucose metabolism has been well characterized, its effect on lipid metabolism, particularly lipolysis and de novo lipogenesis, in humans in vivo has been poorly studied with controversial results. We examined the effect of short-term hyperglucagonemia on de novo lipogenesis (DNL) and endogenous glucose production (EGP) in 8 healthy normal glucose tolerant subjects (5M/3F, age=35±5, BMI= 24±1) who received a 12-hour (6PM to 6AM) glucagon infusion (6ng/kg/min) infusion with measurement of EGP (3-3H-glucose) on the following morning 6-AM. Subjects received deuterated water (D2O, 5g/kg fat free mass) at PM on the night prior to study. 8 weeks later subjects returned for a repeat study with 12-hour infusion of normal saline. DNL was quantified by measuring the incorporation of deuterated water into circulating triglycerides. Gluconeogenic rate was measured as (EGP) x (plasma C5/C2 glucose ratio) (EGP) x (plasma C5/C2 glucose ratio) . Plasma esterified and non-esterified FFA levels were measured with gas chromatography mass spectrometry (GC-MS) . Plasma glucagon increased from 57±3 to 219±21 pg/ml within one hour and remained elevated throughout the study. DNL was significantly decreased (1.64 ± 0.58% vs. 3.± 0.98%, p&amp;lt;0.05) after glucagon versus saline infusion. Both plasma palmitate (0.73 ± 0.vs. 0.67 ± 0.mmol/L) and total FFA (1.35 ± 0.vs. 1.21 ±0.mmol/L) concentrations were elevated after glucagon infusion (p&amp;lt;0.05) . EGP increased following glucagon infusion (2.13 ± 0.14 vs. 1.97± 0.11mg/kg/min, p =0.006) . The increase in EGP was primarily due to increased gluconeogenesis (1.48 ± 0.14 vs. 1.27 ± 0.11, p=0.02) , while the rate of glycogenolysis did not change (0.65± 0.14 vs. 0.70± 0.11, p=ns) . Conclusion: Short-term (12-hour) physiologic hyperglucagonemia in healthy subjects reduces hepatic de novo lipogenesis, stimulates adipose tissue lipolysis, and increases hepatic glucose production by increasing gluconeogenesis. Disclosure X.Chen: None. F.Carli: None. S.Pezzica: None. G.Mocciaro: None. D.Ciociaro: None. A.Gastaldelli: Advisory Panel; Boehringer Ingelheim International GmbH, Novo Nordisk Global Business Services, Consultant; Boehringer Ingelheim International GmbH, Inventiva Pharma, Other Relationship; Eli Lilly and Company, Gilead Sciences, Inc., Pfizer Inc., Speaker's Bureau; Novo Nordisk. R.A.Defronzo: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Intarcia Therapeutics, Inc., Novo Nordisk, Research Support; AstraZeneca, Boehringer Ingelheim International GmbH, Merck &amp; Co., Inc., Speaker's Bureau; AstraZeneca. D.Tripathy: None. Funding EFSD Albert Renold Travel Fellowship; FAVHR

1343-P: The Acute Effects of Glucagon on Glucose Dynamics Are Not Impaired in Individuals with Nonalcoholic Fatty Liver Disease

Glucagon is essential for glucose control and increased levels of glucagon (hyperglucagonemia) observed in patients with type 2 diabetes contribute to their hyperglycemia. Recently, hyperglucagonemia has also been found in individuals with non-alcoholic fatty liver disease (NAFLD) as well as impaired actions of glucagon on amino acid catabolism. Whether glucagon actions on hepatic glucose production are impaired is unknown. We investigated the acute effects of a single bolus of glucagon (0.2mg) on glucose dynamics in 18 normoglycemic individuals (age: 51±3 years, BMI; 31± 0.8kg/m2, hepatic fat content: 20±2%, fasting glucose: 5.5±0.1mM) with magnetic resonance imaging verified NAFLD and 22 controls (age: 38±3 years, BMI; 24± 0.8kg/m2, hepatic fat content: 4±0.1%, fasting glucose: 5.0±0.1mM) . On a separate day, a mixture of amino acids (14 g/L; 331 mg/min/kg body weight) was infused intravenously for 45min to evaluate the actions of endogenous glucagon on glucose dynamics. Glucose levels (see figure) were significantly increased in individuals with NAFLD 60min after the glucagon bolus and during the amino acid infusion with a maximal difference of 0.5mM 30min into the infusion. These data suggest that the actions of glucagon on hepatic glucose production are not impaired by NAFLD. Therefore, the hyperglucagonemia in patients with NAFLD may constitute a diabetogenic risk factor. Disclosure S.Kjeldsen: None. H.Vilstrup: None. F.V.Schiødt: Advisory Panel; Novo Nordisk. A.Møller: None. E.B.Rashu: None. L.Gluud: Advisory Panel; Novo Nordisk, Consultant; Pfizer Inc., Research Support; Alexion Pharmaceuticals, Inc., Gilead Sciences, Inc., Novo Nordisk, Sobi. S.B.Haugaard: None. J.J.Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. J.Rungby: Advisory Panel; Abbott, Boehringer Ingelheim International GmbH, Speaker’s Bureau; AstraZeneca, Bayer AG, Novo Nordisk, Pfizer Inc. N.J.Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker’s Bureau; Merck &amp; Co., Inc., Mercodia AB. N.J.Jensen: None. M.Nilsson: None. N.Heinz: None. J.D.Nybing: None. F.H.Linden: None. E.Høgh-schmidt: n/a. M.P.Boesen: None. S.Madsbad: None. Funding NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude.

The effect of glucagon on local subcutaneous blood flow in non-diabetic volunteers; a proof-of-concept study

IntroductionGlucagon and insulin are the two most important hormones in glucose metabolism and have been incorporated in the dual-hormonal artificial pancreas, a device for automated glucose regulation for people with diabetes type 1. Currently the subcutis is the preferred site of hormone delivery for insulin-only as well as dual-hormonal artificial pancreas systems. The delay in glucose-lowering effect after subcutaneous injection of insulin is substantial, in contrast to the elevation of blood glucose values after subcutaneously injected glucagon which is occurs shortly after injection. We hypothesize that this is caused by properties of glucagon and have investigated the vasodilative effect of glucagon on subcutaneous blood flow in this proof-of-concept study. MethodsTwenty-two volunteers received subcutaneous injections of 0.1 mg and 0.01 mg glucagon, and saline on the abdomen. Blood flow was measured by a laser doppler blood flowmeter for 35 min after injections. ResultsInjection of 0.1 mg glucagon resulted in a significant increase in blood flow compared with baseline blood flow for all time intervals. Significant increase was also observed after the 0.01 mg glucagon injection, except between two- and five-min post injection. The inter-individual variance was large and a third of the subjects did not show an apparent increase in local subcutaneous blood flow after the 0.1 mg glucagon injection. ConclusionThis proof-of-concept study shows that micro-boluses of glucagon increases local subcutaneous blood flow on the abdomen of non-diabetic subjects. However, the vasodilative effect of glucagon is not observed in all subjects. The trial was not registered to protect intellectual property rights.

Mass Spectrometry‐based Proteomics in Deciphering Hormonal Regulation of Ubiquitin Linkages

Hormones such as insulin and glucagon regulate metabolic organ liver. Ubiquitin (Ub) is a 76‐amino acid protein that can be attached on a lysine of the substrate protein. Ub posttranslational modification (PTM) is unique in its diversity as many possible mono‐ and multiple Ub molecules can be attached to protein substrates leading to homotypic and mixed linkages. The type of linkages added determine the functional outcomes such as protein degradation or localization changes. Hormones regulate protein PTM and metabolic disorders such as diabetes involve dysregulation of hormonal signaling. We hypothesized that hormones regulate Ub linkage profile and dysfunction of this regulation is a part of diabetes pathophysiology. We studied the effect of insulin and glucagon on protein ubiquitination in HepG2 cells. We utilized Tandem Ubiquitin Binding Entities (TUBE) and immunoprecipitation strategies for linkage‐specific ubiquitin protein enrichment and further analyzed them by western blotting and proteomics. Our preliminary data indicates differential regulation by hormones of the protein Ub linkage profile. Significant leads obtained from proteomic studies will be further characterized. In addition, we aim to study the ubiquitin linkage profiles in tissues obtained from rodent diabetes models. Our study has potential to find key targets involved in hormonal action and their dysfunction in diabetes.

Pharmacokinetics of glucagon after intravenous, intraperitoneal and subcutaneous administration in a pig model.

IntroductionThere is increasing scientific evidence to substantiate using low‐dose glucagon as a supplement to insulin therapy in artificial pancreata for diabetes mellitus type 1. The delivery of both these hormones intraperitoneally would mimic normal physiology. However, our knowledge of the pharmacological properties of glucagon after intraperitoneal administration is limited. This study compared the pharmacokinetics of glucagon after intraperitoneal, subcutaneous and intravenous administration and the pharmacodynamic effects of glucagon on glucose metabolism after intraperitoneal and subcutaneous administration in a pig model.Materials and methodsTwelve pigs were included. Glucagon was administered intraperitoneally, subcutaneously and intravenously in a randomised order. Arterial samples were collected every 2–10 min for 150 min to determine plasma glucagon and blood glucose concentrations.ResultsThe bioavailability of glucagon was significantly lower after intraperitoneal compared with subcutaneous administration with a median difference (95% confidence interval) of 13% (4–22). The effect of glucagon on glucose metabolism was equal after intraperitoneal and subcutaneous administration.ConclusionsIntraperitoneal glucagon administration resulted in lower systemic glucagon exposure than subcutaneous administration without loss of efficiency. We interpret this as evidence of a major first‐pass metabolism of glucagon in the liver.

Preclinical evaluation of a protracted GLP-1/glucagon receptor co-agonist: Translational difficulties and pitfalls.

During recent years combining GLP-1 and glucagon receptor agonism with the purpose of achieving superior weight loss and metabolic control compared to GLP-1 alone has received much attention. The superior efficacy has been shown by several in preclinical models but has been difficult to reproduce in humans. In this paper, we present the pre-clinical evaluation of NN1177, a long-acting GLP-1/glucagon receptor co-agonist previously tested in clinical trials. To further investigate the contribution from the respective receptors, two other co-agonists (NN1151, NN1359) with different GLP-1-to-glucagon receptor ratios were evaluated in parallel. In the process of characterizing NN1177, species differences and pitfalls in traditional pre-clinical evaluation methods were identified, highlighting the translational challenges in predicting the optimal receptor balance in humans. In diet-induced obese (DIO) mice, NN1177 induced a dose-dependent body weight loss, primarily due to loss of fat mass, and improvement in glucose tolerance. In DIO rats, NN1177 induced a comparable total body weight reduction, which was in contrast mainly caused by loss of lean mass, and glucose tolerance was impaired. Furthermore, despite long half-lives of the three co-agonists, glucose control during steady state was seen to depend on compound exposure at time of evaluation. When evaluated at higher compound exposure, glucose tolerance was similarly improved for all three co-agonists, independent of receptor balance. However, at lower compound exposure, glucose tolerance was gradually impaired with higher glucagon receptor preference. In addition, glucose tolerance was found to depend on study duration where the effect of glucagon on glucose control became more evident with time. To conclude, the pharmacodynamic effects at a given GLP-1-to-glucagon ratio differs between species, depends on compound exposure and study length, complicating the identification of an optimally balanced clinical candidate. The present findings could partly explain the low number of clinical successes for this dual agonism.

Glucagon signaling via supraphysiologic GCGR can reduce cell viability without stimulating gluconeogenic gene expression in liver cancer cells

BackgroundDeregulated glucose metabolism is a critical component of cancer growth and survival, clinically evident via FDG-PET imaging of enhanced glucose uptake in tumor nodules. Tumor cells utilize glucose in a variety of interconnected biochemical pathways to generate energy, anabolic precursors, and other metabolites necessary for growth. Glucagon-stimulated gluconeogenesis opposes glycolysis, potentially representing a pathway-specific strategy for targeting glucose metabolism in tumor cells. Here, we test the hypothesis of whether glucagon signaling can activate gluconeogenesis to reduce tumor proliferation in models of liver cancer.MethodsThe glucagon receptor, GCGR, was overexpressed in liver cancer cell lines consisting of a range of etiologies and genetic backgrounds. Glucagon signaling transduction was measured by cAMP ELISAs, western blots of phosphorylated PKA substrates, and qPCRs of relative mRNA expression of multiple gluconeogenic enzymes. Lastly, cell proliferation and apoptosis assays were performed to quantify the biological effect of glucagon/GCGR stimulation.ResultsSignaling analyses in SNU398 GCGR cells treated with glucagon revealed an increase in cAMP abundance and phosphorylation of downstream PKA substrates, including CREB. qPCR data indicated that none of the three major gluconeogenic genes, G6PC, FBP1, or PCK1, exhibit significantly higher mRNA levels in SNU398 GCGR cells when treated with glucagon; however, this could be partially increased with epigenetic inhibitors. In glucagon-treated SNU398 GCGR cells, flow cytometry analyses of apoptotic markers and growth assays reproducibly measured statistically significant reductions in cell viability. Finally, proliferation experiments employing siCREB inhibition showed no reversal of cell death in SNU398 GCGR cells treated with glucagon, indicating the effects of glucagon in this setting are independent of CREB.ConclusionsFor the first time, we report a potential tumor suppressive role for glucagon/GCGR in liver cancer. Specifically, we identified a novel cell line-specific phenotype, whereby glucagon signaling can induce apoptosis via an undetermined mechanism. Future studies should explore the potential effects of glucagon in diabetic liver cancer patients.

Therapeutic Use of Glucagon: The Future of Diabetes Mellitus Therapy

The etiology of type 2 diabetes has long been centered on insulin. Recently, however, glucagon has been attracting attention. It has been reported that glucagon receptor or pancreatic α cell-deficient mice do not deteriorate glucose intolerance after pancreatic beta cells are destroyed with streptozotocin and insulin secretion is depleted. This suggests that glucagon and glucagon receptors are crucial for glucose intolerance in diabetes mellitus, and the elucidation of glucagon signaling is still awaited. This review will begin with an overview of the history of glucagon. In the nearly 100 years since the discovery of glucagon, it has become clear that glucagon plays a role in blood glucose in type 2 diabetes as much or more than insulin. This is due in large part to the advent of the sandwich Enzyme-Linked Immunosorbent Assay (ELISA) method, which allows accurate measurement of glucagon by using N- and C-terminal antibodies. Next, the mechanism of action of glucagon on blood glucose levels will be outlined. Glucagon increases blood glucose levels by enhancing gluconeogenesis and glycogenolysis. Glucagon also enhances lipolysis, which indirectly contributes to weight loss and improved blood glucose levels. Recently, mice with defective or decreased glucagon secretion have been developed, and the effects of glucagon are becoming clearer in vivo. Mice with elevated glucagon secretion have also appeared, and their glucose tolerance improvement through lipolysis and weight loss is also attracting attention. Research on glucagon is not limited to animal experiments but has already reached the stage of clinical application. Drugs that suppress glucagon have side effects such as liver damage, but they are effective in improving blood glucose. Drugs that increase glucagon also have hypoglycemic effects resulting from weight loss. Thus, glucagon has undergone a dizzying evolution over the past decade. The day may soon come when drugs that target glucagon will dominate diabetes treatment.