Glucagon Receptor Signaling Research Articles

Long-Term Inhibition of the Glucagon Receptor with a Monoclonal Antibody in Mice Causes Sustained Improvement in Glycemic Control, with Reversible α-Cell Hyperplasia and Hyperglucagonemia

Uncontrolled hepatic glucose output (HGO) contributes significantly to the pathological hyperglycemic state of patients with type 2 diabetes. Glucagon, through action on its receptor, stimulates HGO, thereby leading to increased glycemia. Antagonizing the glucagon signaling pathway represents an attractive therapeutic approach for the treatment of type 2 diabetes. We previously reported the generation and characterization of several high-affinity monoclonal antibodies (mAbs) targeting the glucagon receptor (GCGR). In the present study, we demonstrate that a 5-week treatment of diet-induced obese mice with mAb effectively normalized nonfasting blood glucose. Similar treatment also reduced fasting blood glucose without inducing hypoglycemia or other undesirable metabolic perturbations. In addition, no hypoglycemia was found in db/db mice that were treated with a combination of insulin and mAb. Long-term treatment with the mAb caused dose-dependent hyperglucagonemia and minimal to mild alpha-cell hyperplasia in lean mice. There was no evidence of pancreatic alpha-cell neoplastic transformation in mice treated with mAb for as long as 18 weeks. Treatment-induced hyperglucagonemia and alpha-cell hyperplasia were reversible after treatment withdrawal for periods of 4 and 10 weeks, respectively. It is noteworthy that pancreatic beta-cell function was preserved, as demonstrated by improved glucose tolerance throughout the 18-week treatment period. Our studies further support the concept that long-term inhibition of GCGR signaling by a mAb could be an effective approach for controlling diabetic hyperglycemia.

Hepatic energy state is regulated by glucagon receptor signaling in mice

The hepatic energy state, defined by adenine nucleotide levels, couples metabolic pathways with energy requirements. This coupling is fundamental in the adaptive response to many conditions and is impaired in metabolic disease. We have found that the hepatic energy state is substantially reduced following exercise, fasting, and exposure to other metabolic stressors in C57BL/6 mice. Glucagon receptor signaling was hypothesized to mediate this reduction because increased plasma levels of glucagon are characteristic of metabolic stress and because this hormone stimulates energy consumption linked to increased gluconeogenic flux through cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C) and associated pathways. We developed what we believe to be a novel hyperglucagonemic-euglycemic clamp to isolate an increment in glucagon levels while maintaining fasting glucose and insulin. Metabolic stress and a physiological rise in glucagon lowered the hepatic energy state and amplified AMP-activated protein kinase signaling in control mice, but these changes were abolished in glucagon receptor- null mice and mice with liver-specific PEPCK-C deletion. 129X1/Sv mice, which do not mount a glucagon response to hypoglycemia, displayed an increased hepatic energy state compared with C57BL/6 mice in which glucagon was elevated. Taken together, these data demonstrate in vivo that the hepatic energy state is sensitive to glucagon receptor activation and requires PEPCK-C, thus providing new insights into liver metabolism.

This Month in Gastroenterology

This Month in Gastroenterology

The Glucagon Receptor Is Required for the Adaptive Metabolic Response to Fasting

Glucagon receptor (Gcgr) signaling maintains hepatic glucose production during the fasting state; however, the importance of the Gcgr for lipid metabolism is unclear. We show here that fasted Gcgr-/- mice exhibit a significant increase in hepatic triglyceride secretion and fasting increases fatty acid oxidation (FAO) in wild-type (WT) but not in Gcgr-/- mice. Moreover fasting upregulated the expression of FAO-related hepatic mRNA transcripts in Gcgr+/+ but not in Gcgr-/- mice. Exogenous glucagon administration reduced plasma triglycerides in WT mice, inhibited TG synthesis and secretion, and stimulated FA beta oxidation in Gcgr+/+ hepatocytes. The actions of glucagon on TG synthesis and FAO were abolished in PPARalpha-/- hepatocytes. These findings demonstrate that the Gcgr receptor is required for control of lipid metabolism during the adaptive metabolic response to fasting.

Glucagon Receptor Signaling Is Essential for Control of Murine Hepatocyte Survival

Glucagon action in the liver is essential for control of glucose homeostasis and the counterregulatory response to hypoglycemia. Because receptors for the related peptides glucagon-like peptide-1 and glucagon-like peptide-2 regulate beta-cell and enterocyte apoptosis, respectively, we examined whether glucagon receptor (Gcgr) signaling modulates hepatocyte survival. The importance of the Gcgr for hepatocyte cell survival was examined using Gcgr+/+ and Gcgr-/- mice in vivo, and murine hepatocyte cultures in vitro. Gcgr-/- mice showed enhanced susceptibility to experimental liver injury induced by either Fas Ligord activation or a methionine- and choline-deficient diet. Restoration of hepatic Gcgr expression in Gcgr-/- mice attenuated the development of hepatocellular injury. Furthermore, exogenous glucagon administration reduced Jo2-induced apoptosis in wild-type mice and decreased caspase activation in fibroblasts expressing a heterologous Gcgr and in primary murine hepatocyte cultures. The anti-apoptotic actions of glucagon were independent of protein kinase A, phosphatidylinositol-3K, and mitogen-activated protein kinase, and were mimicked by the exchange protein directly activated by the cyclic AMP agonist 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3', 5'-cyclic monophosphate-cAMP. These findings extend the essential actions of the Gcgr beyond the metabolic control of glucose homeostasis to encompass the regulation of hepatocyte survival.

Activation of glucagon receptor signaling stimulates regulators of hepatic fat oxidation in vivo

Evidence suggests that glucagon (GN) has persistent effects on the liver that we hypothesize are related to AMPK, PPARα, and FGF‐21. These proteins are known to promote hepatic oxidative processes. We aimed to define signaling pathways downstream of the hepatic GN receptor in vivo. The role of GN stimulation was tested using wild type (WT) and glucagon receptor null (KO) mice catheterized in a carotid artery and jugular vein 5d prior to study. In 5h fasted mice, GN (10ng/kg/min) or saline (SAL) was infused for 2h and the interaction of GN with circulating fat levels was tested in separate studies in which intralipid (IL) was also infused. Pfloridzin was infused in all studies to prevent high glucose and insulin levels. Euglycemia (150mg/dL) was maintained using a variable glucose infusion. Elevated GN (∼5‐fold) in WT increased glucose production and caused increases in AMP:ATP (7.7±0.5 fold), p‐AMPK (2.1±0.3 fold), and PPARα and FGF‐21 mRNA (2.0±0.3 and 2.0±0.4 fold) in liver compared to SAL. IL alone had no effects. However, IL led to marked potentiation of GN‐induced stimulation of FGF‐21 mRNA (12.3±4.2‐fold) in WT mice. Effects of GN with and without IL were eliminated in KO mice. We show in vivo that glucagon drops the liver to a low energy state which corresponds to increased activation of AMPK and induction of PPARα and FGF‐21 mRNA. Effects of GN on FGF‐21 are potentiated by IL. In conclusion, GN not only acutely regulates glucose homeostasis, but also causes adaptations in processes that improve the ability of the liver to oxidize fat. Supported by DK 50277.

Glucagon Receptor Knockout Mice Display Increased Insulin Sensitivity and Impaired β-Cell Function

In previous studies, glucagon receptor knockout mice (Gcgr(-/-)) display reduced blood glucose and increased glucose tolerance, with hyperglucagonemia and increased levels of glucagon-like peptide (GLP)-1. However, the role of glucagon receptor signaling for the regulation of islet function and insulin sensitivity is unknown. We therefore explored beta-cell function and insulin sensitivity in Gcgr(-/-) and wild-type mice. The steady-state glucose infusion rate during hyperinsulinemic-euglycemic clamp was elevated in Gcgr(-/-) mice, indicating enhanced insulin sensitivity. Furthermore, the acute insulin response (AIR) to intravenous glucose was higher in Gcgr(-/-) mice. The augmented AIR to glucose was blunted by the GLP-1 receptor antagonist, exendin-3. In contrast, AIR to intravenous administration of other secretagogues was either not affected (carbachol) or significantly reduced (arginine, cholecystokinin octapeptide) in Gcgr(-/-) mice. In islets isolated from Gcgr(-/-) mice, the insulin responses to glucose and several insulin secretagogues were all significantly blunted compared with wild-type mice. Furthermore, glucose oxidation was reduced in islets from Gcgr(-/-) mice. In conclusion, the present study shows that glucagon signaling is required for normal beta-cell function and that insulin action is improved when disrupting the signal. In vivo, augmented GLP-1 levels compensate for the impaired beta-cell function in Gcgr(-/-) mice.

Cross-talk between angiotensin II and glucagon receptor signaling mediates phosphorylation of mitogen-activated protein kinases ERK 1/2 in rat glomerular mesangial cells

We have recently shown that the pancreatic hormone glucagon-induced phosphorylation of mitogen-activated protein (MAP) kinase ERK 1/2 as well as growth and proliferation of rat glomerular mesangial cells (MCs) via activation of cAMP-dependent protein kinase A (PKA)-and phospholipase C (PLC)/Ca 2+-mediated signaling pathways. Since circulating glucagon and tissue angiotensin II (Ang II) levels are inappropriately elevated in type 2 diabetes, we tested the hypothesis that glucagon induces phosphorylation of ERK 1/2 in MCs by interacting with Ang II receptor signaling. Stimulation of MCs by glucagon (10 nM) induced a marked increase in intracellular [Ca 2+] i that was abolished by [Des-His 1, Glu 9]-glucagon (1 μM), a selective glucagon receptor antagonist. Both glucagon and Ang II-induced ERK 1/2 phosphorylation (glucagon: 214 ± 14%; Ang II: 174 ± 16%; p < 0.001 versus control), and these responses were inhibited by the AT 1 receptor blocker losartan (glucagon + losartan: 77 ± 14%; Ang II + losartan: 84 ± 18%; p < 0.01 versus glucagon or Ang II) and the AT 2 receptor blocker PD 123319 (glucagon + PD: 78 ± 7%; Ang II + PD: 87 ± 7%; p < 0.01 versus glucagon or Ang II). Inhibition of cAMP-dependent PKA with H89 (1 μM) or PLC with U73122 (1 μM) also markedly attenuated the phosphorylation of ERK 1/2 induced by glucagon (glucagon + U73122: 109 ± 15%; glucagon + H89: 113 ± 16%; p < 0.01 versus glucagon) or Ang II (Ang II + U73122: 111 ± 13%; Ang II + H89: 86 ± 10%; p < 0.01 versus Ang II). Wortmannin (1 μM), a selective PI 3-kinase inhibitor, also blocked glucagon- or Ang II-induced ERK 1/2 phosphorylation. These results suggest that AT 1 receptor-activated cAMP-dependent PKA, PLC and PI 3-kinase signaling is involved in glucagon-induced MAP kinase ERK 1/2 phosphorylation in MCs. The inhibitory effect of PD 123319 on glucagon-induced ERK 1/2 phosphorylation further suggests that AT 2 receptors also play a similar role in this response.

Role of protein phosphatase and of proten kinase C (PKC) on the attenuated glucagon receptor signaling response by bile acids: Tadashi Ikegami, Alice Choe, Sumanth Kaza, Susan Ceryak, Bernard Bouscarel, The George Washington University, Washington, DC

Role of protein phosphatase and of proten kinase C (PKC) on the attenuated glucagon receptor signaling response by bile acids: Tadashi Ikegami, Alice Choe, Sumanth Kaza, Susan Ceryak, Bernard Bouscarel, The George Washington University, Washington, DC

Assessment of the Role of Interstitial Glucagon in the Acute Glucose Secretory Responsiveness of In Situ Pancreatic β-Cells

Glucagon is a potent stimulator of insulin release in the presence of a permissive glucose concentration, activating beta-cells in vitro via both glucagon- and glucagon-like peptide-1 (GLP-1)-receptors. It is still unclear whether locally released glucagon amplifies the secretory responsiveness of neighboring beta-cells in the intact pancreas. The present study investigates this question in the perfused pancreas by examining the effects of antagonists for glucagon receptors ([des-His(1),des-Phe(6),Glu(9)]glucagon-NH(2), 10 micromol/l) and GLP-1-receptors [exendin-(9-39)-NH(2), 1 micromol/l] on the insulin secretory response to glucose. The specificity of both antagonists was demonstrated by their selective interaction with glucagon-receptor signaling in rat hepatocytes and GLP-1-receptor signaling in Chinese hamster lung (CHL) fibroblasts. In purified rat beta-cells, the glucagon-receptor antagonist (10 micromol/l) inhibited the effect of 1 nmol/l glucagon upon glucose-induced insulin release by 78 plus minus 6%. In the perfused rat pancreas, neither of these antagonists inhibited the potent secretory response to 20 mmol/l glucose, although they effectively suppressed the potentiating effect of, respectively, an infusion of glucagon (1 nmol/l) or GLP-1 (1 nmol/l) on insulin release. When endogenous glucagon release was enhanced by isoproterenol (100 nmol/l), no amplification was seen in the simultaneous or subsequent insulin secretory response to glucose. It is concluded that, at least under the present selected conditions, the glucose-induced insulin release by the perfused rat pancreas seems to occur independent of an amplifying glucagon signal from neighboring alpha-cells.

Molecular determinants of glucagon receptor signaling.

A 29-amino acid polypeptide hormone, glucagon has been one of the most prolific models in the study of hormone action. The key biologic function of glucagon is to counterbalance the actions of insulin and maintain a normal level of serum glucose. Diabetes mellitus can thus be considered a bihormonal disorder with an excess of glucagon contributing to the hyperglycemic state. The effects of glucagon are mediated by the glucagon receptor, which is itself a prototypical member of a distinct category called family B receptors within the G protein-coupled superfamily of seven-helical transmembrane receptors (GPCRs). At the structural level, the peptide ligands of family B receptors are highly homologous, in particular in the N-terminal region of the molecules. The mechanism by which highly homologous peptide ligands selectively recognize their receptors involves distinct molecular interactions that are gradually being elucidated. This review focuses on structural determinants of the glucagon receptor that are important for its activity with respect to interaction with its ligand and G proteins. Information about the glucagon receptor is presented within the context of what is known about other members of the family B GPCRs.

Glucagon and vasopressin V1a receptor signaling in hepatocytes from aging rats

Glucose tolerance is reduced with age. The relationship between this change in glucose homeostasis and signaling of glucagon and vasopressin V1a receptors was investigated in hepatocytes isolated from 10- and 30-month-old female WAG/Rij rats. Binding capacity of hepatocytes for 125I glucagon and 3H vasopressin increased 2- and 1.8-fold, respectively, between 10 and 30 months. Intracellular cAMP accumulation induced by glucagon was 40% greater in hepatocytes of aging rats than of adults, although EC 50 were similar in the two groups. Conversely, phosphodiesterases activity and nucleotides leakage out of the cells were unchanged with age. The rise in intracellular calcium consecutive to the stimulation of V1a receptor was comparable in adult and senescent animals. Finally, glucose release by hepatocyte suspensions was greater in senescent than in adult animals in absence as in presence of glucagon. These experiments suggest that increase in glucagon receptor expression and cAMP generation would contribute to the impaired glucose tolerance characteristic of the aging process.

Two Cytoplasmic Loops of the Glucagon Receptor Are Required to Elevate cAMP or Intracellular Calcium

The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.