The Role of ICL1 and H8 in Class B1 GPCRs; Implications for Receptor Activlation

The first intracellular loop (ICL1) of G protein-coupled receptors (GPCRs) has received little attention, although there is evidence that, with the 8th helix (H8), it is involved in early conformational changes following receptor activation as well as contacting the G protein β subunit. In class B1 GPCRs, the distal part of ICL1 contains a conserved R12.48KLRCxR2.46b motif that extends into the base of the second transmembrane helix; this is weakly conserved as a [R/H]12.48KL[R/H] motif in class A GPCRs. In the current study, the role of ICL1 and H8 in signaling through cAMP, iCa2+ and ERK1/2 has been examined in two class B1 GPCRs, using mutagenesis and molecular dynamics. Mutations throughout ICL1 can either enhance or disrupt cAMP production by CGRP at the CGRP receptor. Alanine mutagenesis identified subtle differences with regard elevation of iCa2+, with the distal end of the loop being particularly sensitive. ERK1/2 activation displayed little sensitivity to ICL1 mutation. A broadly similar pattern was observed with the glucagon receptor, although there were differences in significance of individual residues. Extending the study revealed that at the CRF1 receptor, an insertion in ICL1 switched signaling bias between iCa2+ and cAMP. Molecular dynamics suggested that changes in ICL1 altered the conformation of ICL2 and the H8/TM7 junction (ICL4). For H8, alanine mutagenesis showed the importance of E3908.49b for all three signal transduction pathways, for the CGRP receptor, but mutations of other residues largely just altered ERK1/2 activation. Thus, ICL1 may modulate GPCR bias via interactions with ICL2, ICL4 and the Gβ subunit.

Evaluation of Commercially Available Glucagon Receptor Antibodies and Glucagon Receptor Expression

Glucagon is a key regulator of numerous metabolic functions including glucose, protein and lipid metabolism, and glucagon-based therapies are explored for diabetes, fatty liver disease and obesity. Insight into tissue and cell specific expression of the glucagon receptor (GCGR) is important to understand the biology of glucagon as well as to differentiate between direct and indirect actions of glucagon. However, it has been challenging to accurately localize the GCGR in tissue due to low expression levels and lack of specific methodologies. Immunohistochemistry has frequently been used for GCGR localization, but G-protein-coupled receptors (GPCRs) targeting antibodies are notoriously unreliable. In this study, we systematically evaluated all commercially available GCGR antibodies. Initially, twelve GCGR antibodies were evaluated using HEK293 cells transfected with mouse or human GCGR cDNA. Of the twelve antibodies tested, eleven showed positive staining of GCGR protein from both species. Human liver tissue was investigated using the same GCGR antibodies. Five antibodies failed to stain human liver biopsies (despite explicit claims to the contrary from the vendors). Immunohistochemical (IHC) staining demonstrated positive staining of liver tissue from glucagon receptor knockout (Gcgr-/-) mice and their wild-type littermates (Gcgr+/+) with only one out of the twelve available GCGR antibodies. Three antibodies were selected for further evaluation by western blotting and bands corresponding to the predicted size of the GCGR (62 kDa) were identified using two of these. Finally, a single antibody (no. 11) was selected for specific GCGR localization studies in various tissues. In mouse tissue the most intense immunostainings were found in lever, kidney, ileum, heart, and pancreas. Western blotting, performed on liver tissue from Gcgr+/+ and Gcgr-/- mice, confirmed the specificity of antibody no. 11, detecting a band at high intensity in material from Gcgr+/+and no bands in liver tissue from Gcgr-/-mice. Staining of human kidney tissue, with antibody no. 11, showed GCGR localization to the distal tubules. Autoradiography was used as an antibody-independent approach to support the antibody-based findings, revealing specific binding in liver, pancreas, and kidney. As a final approach, RNA-sequencing and single-cell RNA (scRNA)-sequencing were implemented. RNA-sequencing confirmed GCGR presence within liver and kidney tissue. The GCGR was specifically found to be expressed in hepatocytes by scRNA-sequencing and potentially also in collecting and distal tubule cells in the kidney. Our results clearly indicate the liver and the kidneys as the primary targets of glucagon action.

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.

The Glucagon Receptor Antagonist LY2409021 has No Effect on Postprandial Glucose in Type 2 Diabetes

Objective: Type 2 diabetes (T2D) pathophysiology includes fasting and postprandial hyperglucagonemia, which has been linked to hyperglycemia via increased endogenous glucose production (EGP). We used a glucagon receptor antagonist (LY2409021) and stable isotope tracer infusions to investigate consequences of hyperglucagonemia in type 2 diabetes. Design: A double-blinded, randomized, placebo-controlled crossover study was conducted. Methods: Ten patients with T2D and ten matched non-diabetic controls underwent two liquid mixed meal tests preceded by single-dose administration of LY2409021 (100 mg) or placebo. Double-tracer technique was used to quantify EGP. Antagonist selectivity towards related incretin receptors was determined in vitro. Results: Compared to placebo, LY2409021 lowered fasting plasma glucose from 9.1 to 7.1 mmol/L in patients and from 5.6 to 5.0 mmol/L in controls (both P<0.001) by mechanisms involving reduction of EGP. Postprandial plasma glucose excursions (baseline-subtracted area under the curve) were unaffected by LY2409021 in patients and increased in controls compared to placebo. Glucagon concentrations more than doubled during glucagon receptor antagonism. The antagonist interfered with both glucagon-like peptide 1 and glucose-dependent insulinotropic polypeptide receptors, complicating the interpretation of the postprandial data. Conclusions: LY2409021 lowered fasting plasma glucose concentrations but did not improve postprandial glucose tolerance after a meal in patients with T2D and controls. The metabolic consequences of postprandial hyperglucagonemia are difficult to evaluate using LY2409021 because of its antagonizing effects on the incretin receptors.

Moderate Calorie Restriction Enhances Hepatic Glucagon Sensitivity in Aged Mice

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&lt;.01) and decreases basal serum glucagon (P&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&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&lt;.05) and FGF21 mRNA expression (P&lt;.05). Additionally, CR reduces hepatic lipid accumulation (P&lt;.05), and decreases fasting respiratory quotient (P&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.

A glucagon analogue decreases body weight in mice via signalling in the liver

Glucagon receptor agonists show promise as components of next generation metabolic syndrome pharmacotherapies. However, the biology of glucagon action is complex, controversial, and likely context dependent. As such, a better understanding of chronic glucagon receptor (GCGR) agonism is essential to identify and mitigate potential clinical side-effects. Herein we present a novel, long-acting glucagon analogue (GCG104) with high receptor-specificity and potent in vivo action. It has allowed us to make two important observations about the biology of sustained GCGR agonism. First, it causes weight loss in mice by direct receptor signalling at the level of the liver. Second, subtle changes in GCG104-sensitivity, possibly due to interindividual variation, may be sufficient to alter its effects on metabolic parameters. Together, these findings confirm the liver as a principal target for glucagon-mediated weight loss and provide new insights into the biology of glucagon analogues.

Hepatocyte cholesterol content modulates glucagon receptor signalling

Glucagon decreases liver fat, and non-alcoholic fatty liver disease (NAFLD) is associated with hepatic glucagon resistance. Increasingly it is recognised that the function of G protein-coupled receptors can be regulated by their local plasma membrane lipid environment. The aim of this study was to evaluate the effects of experimentally modulating hepatocyte cholesterol content on the function of the glucagon receptor (GCGR). We found that glucagon-mediated cAMP production is inversely proportional to cholesterol content of human hepatoma and primary mouse hepatocytes after treatment with cholesterol-depleting and loading agents, with ligand internalisation showing the opposite trend. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Molecular dynamics simulations identified potential membrane-exposed cholesterol binding sites on the GCGR. Overall, our data suggest that increased hepatocyte membrane cholesterol could directly contribute to glucagon resistance in NAFLD.