Glucagon Receptor Signaling Research Articles

Glucagon receptor activation contributes to the development of kidney injury.

The underlying causes of diabetic kidney disease are still largely unknown. New insights into the contributing causes of diabetic nephropathy are important to prevent this complication. Hyperglycemia and hypertension are some of the risk factors for diabetic nephropathy. However, the incidence of diabetic nephropathy is increasing despite efforts to normalize blood glucose levels and blood pressure. Therefore, other factors should be investigated as causes of diabetic nephropathy. We investigated whether long-term increased plasma levels of glucagon contribute to the development of pathophysiological changes in kidney function as seen in patients with diabetic nephropathy. Using mouse models of chronic activation and inactivation of glucagon receptor signaling, we investigated whether glucagon is involved in changes in renal function, renal structure, and transcriptional changes. We found several histopathological changes in the kidney, such as thickening of the parietal layer of Bowman's capsule, glomerular mesangial cell expansion, and significant albuminuria in the mice with activated glucagon receptor signaling. Opposite effects on mesangial area expansion and the development of albuminuria were demonstrated in mice with glucagon receptor inactivation. RNA sequencing data revealed that transcription of genes related to fatty acid metabolism, podocytes, Na+-K+-ATPase, and sodium/glucose transport was significantly changed in mice with activated glucagon receptor signaling. These data implicate that glucagon receptor signaling is involved in the development of kidney injury, as seen in type 2 diabetes, and that glucagon receptor is a potential therapeutic target in the treatment of diabetes. NEW & NOTEWORTHY This study suggests that the glucagon receptor is a potential therapeutic target in the treatment of diabetic kidney disease. We show, in mice, that long-term treatment with a glucagon analog showed not only pathophysiological changes and changes in renal function but also transcriptional changes in the kidneys, whereas opposite effects were demonstrated in mice with glucagon receptor inactivation. Therefore, the use of glucagon in a treatment regimen requires investigation of possible metabolic and renal abnormalities.

Glucagon Receptor Antagonist for Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is an emerging major unmet need and one of the most significant clinic challenges in cardiology. The pathogenesis of HFpEF is associated with multiple risk factors. Hypertension and metabolic disorders associated with obesity are the 2 most prominent comorbidities observed in patients with HFpEF. Although hypertension-induced mechanical overload has long been recognized as a potent contributor to heart failure with reduced ejection fraction, the synergistic interaction between mechanical overload and metabolic disorders in the pathogenesis of HFpEF remains poorly characterized. We investigated the functional outcome and the underlying mechanisms from concurrent mechanic and metabolic stresses in the heart by applying transverse aortic constriction in lean C57Bl/6J or obese/diabetic B6.Cg-Lepob/J (ob/ob) mice, followed by single-nuclei RNA-seq and targeted manipulation of a top-ranked signaling pathway differentially affected in the 2 experimental cohorts. In contrast to the post-transverse aortic constriction C57Bl/6J lean mice, which developed pathological features of heart failure with reduced ejection fraction over time, the post-transverse aortic constriction ob/ob mice showed no significant changes in ejection fraction but developed characteristic pathological features of HFpEF, including diastolic dysfunction, worsened cardiac hypertrophy, and pathological remodeling, along with further deterioration of exercise intolerance. Single-nuclei RNA-seq analysis revealed significant transcriptome reprogramming in the cardiomyocytes stressed by both pressure overload and obesity/diabetes, markedly distinct from the cardiomyocytes singularly stressed by pressure overload or obesity/diabetes. Furthermore, glucagon signaling was identified as the top-ranked signaling pathway affected in the cardiomyocytes associated with HFpEF. Treatment with a glucagon receptor antagonist significantly ameliorated the progression of HFpEF-related pathological features in 2 independent preclinical models. Importantly, cardiomyocyte-specific genetic deletion of the glucagon receptor also significantly improved cardiac function in response to pressure overload and metabolic stress. These findings identify glucagon receptor signaling in cardiomyocytes as a critical determinant of HFpEF progression and provide proof-of-concept support for glucagon receptor antagonism as a potential therapy for the disease.

CD9 Counteracts Liver Steatosis and Mediates GCGR Agonist Hepatic Effects.

Glucagon receptor (GCGR) agonism offers potentially greater effects on the mitigation of hepatic steatosis. However, its underlying mechanism is not fully understood. Here, it screened tetraspanin CD9 might medicate hepatic effects of GCGR agonist. CD9 is decreased in the fatty livers of patients and upregulated upon GCGR activation. Deficiency of CD9 in the liver exacerbated diet-induced hepatic steatosis via complement factor D (CFD) regulated fatty acid metabolism. Specifically, CD9 modulated hepatic fatty acid synthesis and oxidation genes through regulating CFD expression via the ubiquitination-proteasomal degradation of FLI1. In addition, CD9 influenced body weight by modulating lipogenesis and thermogenesis of adipose tissue through CFD. Moreover, CD9 reinforcement in the liver alleviated hepatic steatosis, and blockage of CD9 abolished the remission of hepatic steatosis induced by cotadutide treatment. Thus, CD9 medicates the hepatic beneficial effects of GCGR signaling, and may server as a promising therapeutic target for hepatic steatosis.

Development of a glucagon sensitivity test in humans: Pilot data and the GLUSENTIC study protocol

A physiological feedback system exists between hepatocytes and the alpha cells, termed the liver-alpha cell axis and refers to the relationship between amino acid-stimulated glucagon secretion and glucagon-stimulated amino acid catabolism. Several reports indicate that non-alcoholic fatty liver disease (NAFLD) disrupts the liver-alpha cell axis, because of impaired glucagon receptor signaling (glucagon resistance). However, no experimental test exists to assess glucagon resistance in humans. The objective was to develop an experimental test to determine glucagon sensitivity with respect to amino acid and glucose metabolism in humans. The proposed glucagon sensitivity test (comprising two elements: 1) i.v. injection of 0.2 mg glucagon and 2) infusion of mixed amino acids 331 mg/hour/kg) is based on nine pilot studies which are presented. Calculation of a proposed glucagon sensitivity index with respect to amino acid catabolism is also described. Secondly, we describe a complete study protocol (GLUSENTIC) according to which the glucagon sensitivity test will be applied in a cross-sectional study currently taking place. 65 participants including 20 individuals with a BMI 18.6–25 kg/m2, 30 individuals with a BMI ≥ 25–40 kg/m2, and 15 individuals with type 1 diabetes with a BMI between 18.6 and 40 kg/m2 will be included. Participants will be grouped according to their degree of hepatic steatosis measured by whole-liver magnetic resonance imaging (MRI). The primary outcome measure will be differences in the glucagon sensitivity index between individuals with and without hepatic steatosis. Developing a glucagon sensitivity test and index may provide insight into the physiological and pathophysiological mechanism of glucagon action and glucagon-based therapies.

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.

Opposing effects of chronic glucagon receptor agonism and antagonism on amino acids, hepatic gene expression, and alpha cells

The pancreatic hormone, glucagon, is known to regulate hepatic glucose production, but recent studies suggest that its regulation of hepatic amino metabolism is equally important. Here, we show that chronic glucagon receptor activation with a long-acting glucagon analog increases amino acid catabolism and ureagenesis and causes alpha cell hypoplasia in female mice. Conversely, chronic glucagon receptor inhibition with a glucagon receptor antibody decreases amino acid catabolism and ureagenesis and causes alpha cell hyperplasia and beta cell loss. These effects were associated with the transcriptional regulation of hepatic genes related to amino acid uptake and catabolism and by the non-transcriptional modulation of the rate-limiting ureagenesis enzyme, carbamoyl phosphate synthetase-1. Our results support the importance of glucagon receptor signaling for amino acid homeostasis and pancreatic islet integrity in mice and provide knowledge regarding the long-term consequences of chronic glucagon receptor agonism and antagonism.

Alpha-cells and therapy of diabetes: Inhibition, antagonism or death?

Absolute or relative hyperglucagonaemia is a characteristic of both Type 1 and Type 2 diabetes, resulting in fasting hyperglycaemia due in part to increased hepatic glucose production and lack of postprandial suppression of circulating glucagon concentrations. Consequently, therapeutics that target glucagon secretion or biological action may be effective antidiabetic agents. In this regard, specific glucagon receptor (GCGR) antagonists have been developed that exhibit impressive glucose-lowering actions, but unfortunately may cause off-target adverse effects in humans. Further to this, several currently approved antidiabetic agents, including GLP-1 mimetics, DPP-4 inhibitors, metformin, sulphonylureas and pramlintide likely exert part of their glucose homeostatic actions through direct or indirect inhibition of GCGR signalling. In addition to agents that inhibit the release of glucagon, compounds that enhance the transdifferentiation of glucagon secreting alpha-cells towards an insulin positive beta-cell phenotype could also help curb excess glucagon secretion in diabetes. Use of alpha-cell toxins represents another possible strategy to address hyperglucagonaemia in diabetes. In that respect, research from the 1920 s with diguanides such as synthalin A demonstrated effective glucose-lowering with alpha-cell ablation in both animal models and humans with diabetes. However, further clinical use of synthalin A was curtailed due its adverse effects and the increased availability of insulin. Overall, these observations with therapeutics that directly target alpha-cells, or GCGR signaling, highlight a largely untapped potential for diabetes therapy that merits further detailed consideration.

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.

Insights into the Role of Glucagon Receptor Signaling in Metabolic Regulation from Pharmacological Inhibition and Tissue-Specific Knockout Models.

While glucagon has long been recognized as the primary counter hormone to insulin’s actions, it has recently gained recognition as a metabolic regulator with its effects extending beyond control of glycemia. Recently developed models of tissue-specific glucagon receptor knockouts have advanced our understanding of this hormone, providing novel insight into the role it plays within organs as well as its systemic effects. Studies where the pharmacological blockade of the glucagon receptor has been employed have proved similarly valuable in the study of organ-specific and systemic roles of glucagon signaling. Studies carried out employing these tools demonstrate that glucagon indeed plays a role in regulating glycemia, but also in amino acid and lipid metabolism, systemic endocrine, and paracrine function, and in the response to cardiovascular injury. Here, we briefly review recent progress in our understanding of glucagon’s role made through inhibition of glucagon receptor signaling utilizing glucagon receptor antagonists and tissue specific genetic knockout models.

Cross Talk Between Insulin and Glucagon Receptor Signaling in the Hepatocyte.

While the consumption of external energy (i.e., feeding) is essential to life, this action induces a temporary disturbance of homeostasis in an animal. A primary example of this effect is found in the regulation of glycemia. In the fasted state, stored energy is released to maintain physiological glycemic levels. Liver glycogen is liberated to glucose, glycerol and (glucogenic) amino acids are used to build new glucose molecules (i.e., gluconeogenesis), and fatty acids are oxidized to fuel long-term energetic demands. This regulation is driven primarily by the counterregulatory hormones epinephrine, growth hormone, cortisol, and glucagon. Conversely, feeding induces a rapid influx of diverse nutrients, including glucose, that disrupt homeostasis. Consistently, a host of hormonal and neural systems under the coordination of insulin are engaged in the transition from fasting to prandial states to reduce this disruption. The ultimate action of these systems is to appropriately store the newly acquired energy and to return to the homeostatic norm. Thus, at first glance it is tempting to assume that glucagon is solely antagonistic regarding the anabolic effects of insulin. We have been intrigued by the role of glucagon in the prandial transition and have attempted to delineate its role as beneficial or inhibitory to glycemic control. The following review highlights this long-known yet poorly understood hormone.

1332-P: Increased and Decreased Glucagon Receptor Signaling Respectively Enhances and Impairs Triglyceride Metabolism Acutely and Chronically

Glucagon receptor antagonists (GRAs) reduce blood glucose in subjects with type 2 diabetes (T2D) but may cause dyslipidemia. Increased plasma triglyceride (TG) levels in patients with T2D are thought to be caused partly by insulin resistance but it is unknown if altered glucagon receptor (GCGR) signaling contributes. We investigated the effects of chronic GCGR inhibition and activation on plasma TG and cholesterol levels during an 180 min oral lipid tolerance test (µL/g body weight olive oil; OLTT) in GCGR knockout (Gcgr-/-) mice, and in C57Bl/6JRj mice treated with a GCGR antibody (GCGR Ab, REGN1193, Regeneron) or a long-acting glucagon analogue (GCGA, NNC9204-0043, Novo Nordisk A/S) . Plasma TG levels increased 3-fold in Gcgr-/- mice compared to wild-type littermates (Gcgr+/+) during the OLTT. Eight weeks treatment with GCGR Ab increased plasma TG levels during OLTTs by 39% whereas GCGA reduced levels by 39%. Low-density lipoprotein levels, estimated using fast protein liquid chromatography, were increased in Gcgr-/- mice and GCGR Ab treated mice, whereas GCGA treatment lowered very-low density lipoprotein levels. We next investigated the effects of acute GCGR inhibition and activation; a single treatment with GRA (25-2648, Novo Nordisk A/S) and GCGA respectively increased and decreased TG levels by 55% and 32% during OLTTs. Our study suggests that impaired and increased GCGR signaling respectively impairs and accelerates TG metabolism, chronically and acutely, in female mice. We were unable to identify GCGR in adipocytes using autoradiography and immunohistochemistry and our data thus implicate that glucagon has acute and chronic effects on intestinal lipid uptake and/or acts to enhance hepatic lipid metabolism in mice. Thus, impaired actions of glucagon may cause hypertriglyceridemia, while enhancing GCGR signaling may benefit subjects with dyslipidemia. Disclosure K.D.Galsgaard: None. E.E.Christensen: None. A.B.Bomholt: None. J.Hunt: None. T.Kruse: Employee; Novo Nordisk. J.F.Lau: Employee; Novo Nordisk A/S, Stock/Shareholder; Novo Nordisk A/S. C.Christoffersen: None. J.J.Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. N.J.Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker’s Bureau; Merck &amp; Co., Inc., Mercodia AB. Funding This work was supported by the Novo Nordisk Foundation (NNF) Center for Basic Metabolic Research University of Copenhagen, (NNF Application Number: 13563) ; The A.P. Møller Foundation; NNF Tandem Programme (NNF Application Number: 31526) ; NNF Project Support in Endocrinology and Metabolism–Nordic Region (NNF Application Number: 34250) . Nicolai J. Wewer Albrechtsen were financed by NNF Excellence Emerging Investigator Grant – Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and DFF Sapere Aude. Novo Nordisk Foundation Center for Protein Research is supported financially by the Novo Nordisk Foundation (grant agreement NNF14CC0001) .

230-LB: Amino Acid–Induced Glucagon Secretion and Glucagon Enhancement of Amino Acid Catabolism Is Independent of Glucose

Amino acids (aa) stimulate the secretion of glucagon (gcg) and gcg receptor signaling controls aa catabolism, constituting the liver-alpha cell axis. Impairment of the liver-alpha cell axis is observed in diabetes, but how glucose (glc) levels affect this axis is unknown. We investigated the role of glc on the liver-alpha cell axis by 1) isolated perfused pancreas, 2) isolated perfused liver, and 3) a randomized in-vivo study in male mice. The isolated perfused pancreas was used to evaluate the direct effect of low (3.5mM) and high (15mM) glc levels, respectively on aa (10mM arginine) -induced gcg secretion. High glc levels significantly lowered basal gcg secretion, but aa-induced gcg responses were comparable to those at low glc levels (p=0.38) . The direct effect of glc on gcg induced ureagenesis was assessed by isolated single pass perfused liver stimulated with 10mM mixture of 18 aa + 10nM gcg for two periods of 15 min with glc infused at either 1 or 18 mmol/L. The order of glc addition was randomized. Aa and gcg significantly increased urea production compared to baseline, and this was independent of low or high glc levels (p=0.95) . Whole-body effects of glc on the liver-alpha cell axis were assessed in-vivo including four groups receiving an intraperitoneal injection of either Glc-Saline (2 g/kg, Glc/S, n=8) , Glc-Aa (2 g/kg, + 14 g/l, respectively Glc/Aa, n=10) , Saline-Aa (S/Aa, n=10) and saline-saline (volume-matched, S/S, n=10) . Blood samples were obtained at 0,2,4,10 and 20 minutes and levels of glc, gcg, insulin (ins) , and aa were measured. Glc levels did not differ significantly between Glc/S and Glc/Aa groups, but ins and gcg were significantly higher in the G/Aa group. The gcg:ins ratio did not differ between groups. Plasma levels of gcg and aa were not significantly different between the G/Aa and S/Aa groups (p=0.28) . These results suggest that aa overrule the inhibitory effect of glc on gcg secretion and the actions of glucagon on aa catabolism may be independent of glc in mice. Disclosure K. Maruszczak: None. C. Rasmussen: None. F. R. Ceutz: None. A. Ørgaard: Employee; Novo Nordisk A/S. E. E. Christensen: None. M. M. Richter: None. J. J. Holst: Advisory Panel; Novo Nordisk, Board Member; Antag Therapeutics, Bainan Biotech. M. Winther-soerensen: None. N. J. Wewer albrechtsen: Research Support; Mercodia AB, Novo Nordisk, Regeneron Pharmaceuticals Inc., Speaker's Bureau; Merck &amp; Co., Inc., Mercodia AB. Funding NNF Excellence Emerging Investigator Grant ? Endocrinology and Metabolism (Application No. NNF19OC0055001) , EFSD Future Leader Award (NNF21SA0072746) and Independent Research Fund Denmark Sapere Aude Program (1052-00003B) .

197-OR: Glucagon Receptor Agonism Stimulates Time-Dependent Glycemic Regulation

Historically, glucagon is proposed to be a key factor in the development of diabetes. However, we demonstrated that while chronic glucagon receptor (GCGR) agonism induces hyperglycemia and glucose intolerance, it also improves insulin secretion and insulin action. These paradoxical actions suggest the mechanisms regulating GCGR-mediated glucose metabolism have yet to be fully elucidated. Glucose metabolism and glucagon-stimulated glucose production are both regulated in a circadian manner. Thus, we hypothesized that chronic GCGR signaling regulates glycemia in a time-of-day dependent manner. We treated lean mice for 4d with vehicle or the GCGR agonist IUB288 administered either during the early (ZT 3) or late (ZT9) light period. Ad libitum glycemia was assessed every 4h over a 24h period after the last injection. Independent of the time injected, IUB288-treated mice displayed improved glycemia 4h after the last injection and hyperglycemia after 12h, compared to vehicle-treated controls. This suggests that chronic GCGR agonism is not exclusively hyperglycemic and variations in GCGR-stimulated glycemia are dependent on the time after treatment, rather than the time of day. Interestingly, ad libitum insulin levels were no different between IUB288 and vehicle-treated mice, suggesting differences in insulin sensitivity. Surprisingly, 4d IUB288 treatment in diet-induced obese (DIO) mice reduced ad libitum glycemia for a greater duration than chow IUB288-treated mice. We used hyperinsulinemic-euglycemic clamps to further dissect the mechanisms of these benefits. In DIO mice, IUB288 treatment increased glucose infusion rate, suppression of endogenous glucose production, and phosphorylation of AKT (S473 and T308) . Together, these data increase our understanding of GCGR pharmacology and its therapeutic role in diabetes and obesity treatment. Disclosure S.Nason: None. T.Kim: None. J.P.Antipenko: None. B.Finan: Employee; Novo Nordisk. R.Dimarchi: None. K.M.Habegger: Consultant; Glyscend Inc., Research Support; Eli Lilly and Company, Novo Nordisk, Stock/Shareholder; Glyscend Inc. Funding T32 AI007051

The Essential Role of Pancreatic α-Cells in Maternal Metabolic Adaptation to Pregnancy.

Pancreatic α-cells are important in maintaining metabolic homeostasis, but their role in regulating maternal metabolic adaptations to pregnancy has not been studied. The objective of this study was to determine whether pancreatic α-cells respond to pregnancy and their contribution to maternal metabolic adaptation. With use of C57BL/6 mice, the findings of our study showed that pregnancy induced a significant increase of α-cell mass by promoting α-cell proliferation that was associated with a transitory increase of maternal serum glucagon concentration in early pregnancy. Maternal pancreatic GLP-1 content also was significantly increased during pregnancy. Using the inducible Cre/loxp technique, we ablated the α-cells (α-null) before and during pregnancy while maintaining enteroendocrine L-cells and serum GLP-1 in the normal range. In contrast to an improved glucose tolerance test (GTT) before pregnancy, significantly impaired GTT and remarkably higher serum glucose concentrations in the fed state were observed in α-null dams. Glucagon receptor antagonism treatment, however, did not affect measures of maternal glucose metabolism, indicating a dispensable role of glucagon receptor signaling in maternal glucose homeostasis. However, the GLP-1 receptor agonist improved insulin production and glucose metabolism of α-null dams. Furthermore, GLP-1 receptor antagonist Exendin (9-39) attenuated pregnancy-enhanced insulin secretion and GLP-1 restored glucose-induced insulin secretion of cultured islets from α-null dams. Together, these results demonstrate that α-cells play an essential role in controlling maternal metabolic adaptation to pregnancy by enhancing insulin secretion.

Metabolic Adaptations to Aerobic Exercise in Aged Mice

Aerobic exercise training is a potent intervention for the treatment and prevention of age-related disease, such as heart disease, obesity, and Type 2 Diabetes. Insulin resistance, a hallmark of Type 2 Diabetes, is reversed in response to aerobic exercise training. However, the effect of aerobic exercise training on glucagon sensitivity is unclear. Glucagon signaling at the liver promotes fatty acid oxidation, inhibits De novo lipogenesis, and activates AMP Kinase, a key mediator of healthy aging. Like humans, aging in mice age leads to a decline in physical and metabolic function. To understand the role of glucagon signaling in exercise-induced improvements in physical and metabolic function in the mouse, we implemented a 16-week aerobic exercise training protocol in young and aged mice. 16 weeks of exercise training initiated at 6 months of age increased markers of physical function (P<0.01) and attenuated age-related weight gain (P<0.05) and fat mass (P<0.0001). Additionally, exercise training improved glucose clearance (P<0.01), enhanced glucose-stimulated insulin secretion (P<0.01) and decreased hepatic lipid accumulation (P<0.05). Importantly, exercise training decreased hypoglycemia stimulated glucagon secretion (P<0.01), with no effect on hepatic glucagon receptor mRNA expression or serum glucagon. Thus, we propose that aerobic exercise training enhances glucagon sensitivity at the liver, implicating glucagon as a potential mediator of exercise-induced improvements in aging. Studies initiating the same aerobic exercise training intervention at 18 months of age in the mouse are currently underway to establish the role of glucagon receptor signaling in exercise-induced improvements in aging.

Moderate Calorie Restriction Enhances Hepatic Glucagon Sensitivity in Aged Mice

Abstract Chronic calorie restriction (CR) without malnutrition delays the onset of aging, extends lifespan, and improves metabolic function in many species. These CR-induced benefits have largely concentrated on the role of insulin signaling, while ignoring its counter-regulatory hormone, glucagon. Like insulin, hyperglucagonemia and decreased glucagon sensitivity are associated with impaired glucose homeostasis and decreased longevity. Conversely, activation of target molecules downstream of glucagon signaling such as AMPK and FGF21 are known to ameliorate these age-related impairments in metabolic function. To investigate the potential role of glucagon receptor signaling in CR-induced improvements in aging, we have implemented a moderate 15% CR in the mouse. Our studies show that a 15% calorie restriction initiated at 4 months of age enhances hypoglycemia-stimulated glucagon secretion (P &amp;lt;.01) and decreases basal serum glucagon (P &amp;lt;.01), while having no effect on glucagon receptor expression at the liver in 26-month-old mice. Consistent with enhanced hepatic glucagon sensitivity, CR increases glucagon-stimulated hepatic cyclic AMP production (P &amp;lt;.05). Glucagon is a primary regulator of AMPK activation and FGF21 release, both of which have been proposed as key molecules to account for CR-induced benefits to aging. CR increases both hepatic AMPK activation (P &amp;lt;.05) and FGF21 mRNA expression (P &amp;lt;.05). Additionally, CR reduces hepatic lipid accumulation (P &amp;lt;.05), and decreases fasting respiratory quotient (P &amp;lt;.001), indicating an increase in lipid oxidation. Our studies demonstrate that a moderate (15%) CR regimen enhances glucagon sensitivity and decreases hepatic lipid accumulation in aged mice. Thus, we propose glucagon signaling as a mediator of CR-induced improvements in aging.

Intestinal Growth in Glucagon Receptor Knockout Mice Is Not Associated With the Formation of AOM/DSS-Induced Tumors.

Treatment with exogenous GLP-2 has been shown to accelerate the growth of intestinal adenomas and adenocarcinomas in experimental models of colonic neoplasia, however, the role of endogenous GLP-2 in tumor promotion is less well known. Mice with a global deletion of the glucagon receptor (Gcgr-/-) display an increase in circulating GLP-1 and GLP-2. Due to the intestinotrophic nature of GLP-2, we hypothesized that Gcgr-/- mice would be more susceptible to colonic dysplasia in a model of inflammation-induced colonic carcinogenesis. Female Gcgr-/- mice were first characterized for GLP-2 secretion and in a subsequent study they were given a single injection with the carcinogen azoxymethane (7.5 mg/kg) and treated with dextran sodium sulfate (DSS) (3%) for six days (n=19 and 9). A cohort of animals (n=4) received a colonoscopy 12 days following DSS treatment and all animals were sacrificed after six weeks. Disruption of glucagon receptor signaling led to increased GLP-2 secretion (p<0.0001) and an increased concentration of GLP-2 in the pancreas of Gcgr-/- mice, coinciding with an increase in small intestinal (p<0.0001) and colonic (p<0.05) weight. Increased villus height was recorded in the duodenum (p<0.001) and crypt depth was increased in the duodenum and jejunum (p<0.05 and p<0.05). Disruption of glucagon receptor signaling did not affect body weight during AOM/DSS treatment, neither did it affect the inflammatory score assessed during colonoscopy or the number of large and small adenomas present at the end of the study period. In conclusion, despite the increased endogenous GLP-2 secretion Gcgr-/- mice were not more susceptible to AOM/DSS-induced tumors.

Glucagon receptor signaling regulates weight loss via central KLB receptor complexes.

Glucagon regulates glucose and lipid metabolism and promotes weight loss. Thus, therapeutics stimulating glucagon receptor (GCGR) signaling are promising for obesity treatment; however, the underlying mechanism(s) have yet to be fully elucidated. We previously identified that hepatic GCGR signaling increases circulating fibroblast growth factor 21 (FGF21), a potent regulator of energy balance. We reported that mice deficient for liver Fgf21 are partially resistant to GCGR-mediated weight loss, implicating FGF21 as a regulator of glucagon’s weight loss effects. FGF21 signaling requires an obligate coreceptor (β-Klotho, KLB), with expression limited to adipose tissue, liver, pancreas, and brain. We hypothesized that the GCGR-FGF21 system mediates weight loss through a central mechanism. Mice deficient for neuronal Klb exhibited a partial reduction in body weight with chronic GCGR agonism (via IUB288) compared with controls, supporting a role for central FGF21 signaling in GCGR-mediated weight loss. Substantiating these results, mice with central KLB inhibition via a pharmacological KLB antagonist, 1153, also displayed partial weight loss. Central KLB, however, is dispensable for GCGR-mediated improvements in plasma cholesterol and liver triglycerides. Together, these data suggest GCGR agonism mediates part of its weight loss properties through central KLB and has implications for future treatments of obesity and metabolic syndrome.

Targeting the Glucagon Receptor Signaling Pathway As a Novel Strategy to Counteract PI3K Inhibitor Induced Hyperglycemia While Sustaining Tumor PI3K Inhibition

Pathologic activation of the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway is an oncogenic driver for many malignancies, including lymphomas1. Although validated as a therapeutic oncologic target, the PI3K signaling pathway is also implicated in normal glucose homeostasis. Specifically, since the PI3K subunit p110α is chiefly responsible for downstream insulin receptor (INSR) signaling, PI3K signaling inhibition that includes p110α leads to severe hyperglycemia. Both compensatory endogenous insulin release as well as treatment of hyperglycemia with exogenous insulin/insulin mimetics activate INSR-associated PI3K signaling, ultimately limiting blockade of tumor associated PI3K signaling2. Aggressive lymphomas driven by pathologic PI3K signaling respond to combined PI3Kα and δ/γ inhibition, but this is likely submaximal due to endogenous insulin feedback, and more intense dosing is difficult to achieve safely due to severe hyperglycemia3. Therefore, a novel strategy to maintain blockade of tumor associated PI3K signaling while reducing hyperglycemia is needed. We hypothesized that inhibition of glucagon receptor (GCGR) signaling, a pathway that does not depend on PI3K, may normalize PI3K inhibitor induced hyperglycemia without disrupting antitumor PI3K blockade. GCGR blockade with a GCGR specific monoclonal antibody (REMD-477, a human anti-GCGR antibody, or REMD2.59c, a murine equivalent) may allow for the safer use of more potent PI3K inhibitors or more intensive dosing schedules in the treatment of aggressive lymphomas. The effect of GCGR blockade on PI3K inhibitor induced hyperglycemia was first evaluated in a non-tumor bearing mouse model using REMD2.59c. The PI3K inhibitor copanlisib was administered by tail vein injection to CB17 SCID mice in the control group every other day at the reported MTD of 15 mg/kg. REMD2.59c was administered by intraperitoneal injection to the treatment group intermittently on the day prior to copanlisib injection. Blood glucose levels were measured 2 hours after copanlisib injection. Significant hyperglycemia was observed after copanlisib treatment, and this was corrected by REMD2.59c pretreatment, confirming the effectiveness of GCGR blockade (Figure A-B). Having demonstrated that GCGR blockade controls hyperglycemia caused by PI3K inhibition with copanlisib in a mouse model, we next treated a 54 year old non-diabetic woman with relapsed and refractory peripheral T cell lymphoma (PTCL) on an IRB-approved clinical pilot study of copanlisib in combination with REMD-477. Following copanlisib 60mg IV alone, glucose increased from a baseline of 100mg/dl to a maximum of 592 mg/dl at 5 hours. For the following weekly doses of copanlisib, she was pre-treated with 70mg REMD-477 SQ, and copanlisib induced hyperglycemia was significantly ameliorated (Figure C). The patient tolerated treatment with REMD-477 well without significant episodes of hypoglycemia. With improved glycemic control on REMD-477, our patient did not require dose reductions or treatment delays, and experienced clinical improvement in lymphadenopathy on copanlisib. She had previously progressed through 6 lines of therapy, including duvelisib, a selective PI3K δ/γ inhibitor which necessitated dose reduction due to gastrointestinal toxicity. Our study of the GCGR mAb REMD-477 as a novel strategy to counteract PI3K inhibitor induced hyperglycemia and insulin feedback is an important advance that may allow for more effective and safer use of potent PI3K inhibitors in the treatment of aggressive lymphomas.

Elevated Serum Amino Acids Induce a Subpopulation of Alpha Cells to Initiate Pancreatic Neuroendocrine Tumor Formation

SummaryThe cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing β cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.