The dual GCGR/GLP-1R agonist survodutide: Biomarkers and pharmacological profiling for clinical candidate selection.

AIM: To describe the biomarker strategy that was applied to select survodutide (BI 456906), BI 456908 and BI 456897 from 19 dual glucagon receptor (GCGR)/ glucagon-like peptide-1 receptor (GLP-1R) agonists for in-depth pharmacological profiling, which led to the qualification of survodutide as the clinical development candidate. MATERIALS AND METHODS: Potencies to increase cyclic adenosine monophosphate (cAMP) were determined in Chinese hamster ovary (CHO)-K1 cells stably expressing human GCGR and GLP-1R. Agonism for endogenously expressed receptors was investigated in insulinoma cells (MIN6) for mouse GLP-1R, and in rat primary hepatocytes for the GCGR. In vivo potencies to engage the GLP-1R or GCGR were determined, measuring improvement in oral glucose tolerance (30 nmol/kg) and increase in plasma fibroblast growth factor-21 (FGF21) and liver nicotinamide N-methyltransferase (NNMT) mRNA expression (100 nmol/kg), respectively. Body weight- and glucose-lowering efficacies were investigated in diet-induced obese (DIO) mice and diabetic db/db mice, respectively. RESULTS: Upon acute dosing in lean mice, target engagement biomarkers for the GCGR and GLP-1R demonstrated a significant correlation (Spearman correlation coefficient with p < 0.05) to the in vitro GCGR and GLP-1R potencies for the 19 dual agonists investigated. Survodutide, BI 456908 and BI 456897 were selected for in-depth pharmacological profiling based on the significant improvement in acute oral glucose tolerance achieved (area under the curve [AUC] of 54%, 57% and 60% vs. vehicle) that was comparable to semaglutide (AUC of 45% vs. vehicle), while showing different degrees of in vivo GCGR engagement, as determined by hepatic NNMT mRNA expression (increased by 15- to 17-fold vs. vehicle) and plasma FGF21 concentrations (increased by up to sevenfold vs. vehicle). In DIO mice, survodutide (30 nmol/kg/once daily), BI 456908 (30 nmol/kg/once daily) and BI 456897 (10 nmol/kg/once daily) achieved a body weight-lowering efficacy from baseline of 25%, 27% and 26%, respectively. In db/db mice, survodutide and BI 456908 (10 and 20 nmol/kg/once daily) significantly lowered glycated haemoglobin (0.4%-0.6%); no significant effect was observed for BI 456897 (3 and 7 nmol/kg/once daily). CONCLUSIONS: Survodutide was selected as the clinical candidate based on its balanced dual GCGR/GLP-1R pharmacology, engaging the GCGR for robust body weight-lowering efficacy exceeding that of selective GLP-1R agonists, while achieving antidiabetic efficacy that was comparable to selective GLP-1R agonism. Survodutide is currently being investigated in Phase 3 clinical trials in people living with obesity.

Evaluation of long acting GLP1R/GCGR agonist in a DIO and biopsy-confirmed mouse model of NASH suggest a beneficial role of GLP-1/glucagon agonism in NASH patients.

OBJECTIVE: The metabolic benefits of GLP-1 receptor (GLP-1R) agonists on glycemic and weight control are well established as therapy for type 2 diabetes and obesity. Glucagon's ability to increase energy expenditure is well described, and the combination of these mechanisms-of-actions has the potential to further lower hepatic steatosis in metabolic disorders and could therefore be attractive for the treatment for non-alcoholic steatohepatitis (NASH). Here, we have investigated the effects of a dual GLP-1/glucagon receptor agonist NN1177 on hepatic steatosis, fibrosis, and inflammation in a preclinical mouse model of NASH. Having observed strong effects on body weight loss in a pilot study with NN1177, we hypothesized that direct engagement of the hepatic glucagon receptor (GCGR) would result in a superior effect on steatosis and other liver related parameters as compared to the GLP-1R agonist semaglutide at equal body weight. METHODS: Male C57Bl/6 mice were fed a diet high in trans-fat, fructose, and cholesterol (Diet-Induced Obese (DIO)-NASH) for 36 weeks. Following randomization based on the degree of fibrosis at baseline, mice were treated once daily with subcutaneous administration of a vehicle or three different doses of NN1177 or semaglutide for 8 weeks. Hepatic steatosis, inflammation and fibrosis were assessed by immunohistochemistry and morphometric analyses. Plasma levels of lipids and liver enzymes were determined, and hepatic gene expression was analyzed by RNA sequencing. RESULTS: NN1177 dose-dependently reduced body weight up to 22% compared to vehicle treatment. Plasma levels of ALT, a measure of liver injury, were reduced in all treatment groups with body weight loss. The dual agonist reduced hepatic steatosis to a greater extent than semaglutide at equal body weight loss, as demonstrated by three independent methods. Both the co-agonist and semaglutide significantly decreased histological markers of inflammation such as CD11b and Galectin-3, in addition to markers of hepatic stellate activation (αSMA) and fibrosis (Collagen I). Interestingly, the maximal beneficial effects on above mentioned clinically relevant endpoints of NN1177 treatment on hepatic health appear to be achieved with the middle dose tested. Administering the highest dose resulted in a further reduction of liver fat and accompanied by a massive induction in genes involved in oxidative phosphorylation and resulted in exaggerated body weight loss and a downregulation of a module of co-expressed genes involved in steroid hormone biology, bile secretion, and retinol and linoleic acid metabolism that are also downregulated due to NASH itself. CONCLUSIONS: These results indicate that, in a setting of overnutrition, the liver health benefits of activating the fasting-related metabolic pathways controlled by the glucagon receptor displays a bell-shaped curve. This observation is of interest to the scientific community, due to the high number of ongoing clinical trials attempting to leverage the positive effects of glucagon biology to improve metabolic health.

Glucagon cell hyperplasia and neoplasia: a recently recognized endocrine receptor disease.

Glucagon cell hyperplasia and neoplasia (GCHN) is the name of an endocrine receptor disease, whose morphology was first described in 2006. Three years later, this rare disease was found to be to be caused by an inactivating mutation of the glucagon receptor (GCGR) gene. Functionally, the genetic defect mainly affects glucagon signaling in the liver with changes in the metabolism of glycogen, fatty acids and amino acids. Recent results of several studies in GCGR knockout mice suggested that elevated serum amino acid levels probably stimulate glucagon cell hyperplasia with subsequent transformation into glucagon cell neoplasia. This process leads over time to numerous small and some large pancreatic neuroendocrine tumors which are potentially malignant. Despite high glucagon serum levels, the patients develop no glucagonoma syndrome. In 2015, GCHN was identified as an autosomal recessive hereditary disorder.

Effects of site-directed mutagenesis of GLP-1 and glucagon receptors on signal transduction activated by dual and triple agonists.

The paradigm of one drug against multiple targets, known as unimolecular polypharmacology, offers the potential to improve efficacy while overcoming some adverse events associated with the treatment. This approach is best exemplified by targeting two or three class B1 G protein-coupled receptors, namely, glucagon-like peptide-1 receptor (GLP-1R), glucagon receptor (GCGR) and glucose-dependent insulinotropic polypeptide receptor for treatment of type 2 diabetes and obesity. Some of the dual and triple agonists have already shown initial successes in clinical trials, although the molecular mechanisms underlying their multiplexed pharmacology remain elusive. In this study we employed structure-based site-directed mutagenesis together with pharmacological assays to compare agonist efficacy across two key signaling pathways, cAMP accumulation and ERK1/2 phosphorylation (pERK1/2). Three dual agonists (peptide 15, MEDI0382 and SAR425899) and one triple agonist (peptide 20) were evaluated at GLP-1R and GCGR, relative to the native peptidic ligands (GLP-1 and glucagon). Our results reveal the existence of residue networks crucial for unimolecular agonist-mediated receptor activation and their distinct signaling patterns, which might be useful to the rational design of biased drug leads.

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.

The 14-3-3 protein YWHAB inhibits glucagon-induced hepatic gluconeogenesis through interacting with the glucagon receptor and FOXO1.

Glucagon antagonism has been reported as a new therapeutic approach to hyperglycaemia. As the 14-3-3 protein YWHAB has been identified as a regulator of the glucagon receptor (GCGR) by affinity purification and mass spectrometry, we examined the role of YWHAB in vivo. Ywhab knockout mice display impaired blood glucose homeostasis only under pyruvate stimulation. Deletion of Ywhab in mouse primary hepatocytes (MPHs) increases hepatocyte glucose production by magnifying the effect of glucagon. Mechanistic analysis indicates that YWHAB forms a phosphorylation-dependent complex with GCGR and directly interacts with forkhead box O1 (FOXO1). Together, these results reveal the inhibitory role of YWHAB in glucagon-mediated hepatic glucose production, which may be a potential target for the control of gluconeogenesis and associated metabolic diseases.

Design and preparation of the class B G protein-coupled receptors GLP-1R and GCGR for 19 F-NMR studies in solution.

The human glucagon-like peptide-1 receptor (GLP-1R) and the glucagon receptor (GCGR) are class B G protein-coupled receptors (GPCRs) that are activated by interactions with, respectively, the glucagon-like peptide-1 (GLP-1) and glucagon (GCG). These polypeptide hormones are involved in the regulation of lipid and cholic acid metabolism, and thus play an important role in the pathogenesis of glucose metabolism and diabetes mellitus, which attracts keen interest of these GPCRs as drug targets. GLP-1R and GCGR have therefore been extensively investigated by X-ray crystallography and cryo-electron microscopy (cryo-EM), so that their structures are well known. Here, we present the groundwork for using nuclear magnetic resonance (NMR) spectroscopy in solution to complement the molecular architectures with information on intramolecular dynamics and on the thermodynamics and kinetics of interactions with physiological ligands and extrinsic drug candidates. This includes the generation of novel, near-wild-type constructs of GLP-1R and GCGR, optimization of the solution conditions for NMR studies in detergent micelles and in nanodiscs, post-translational chemical introduction of fluorine-19 NMR probes, and sequence-specific assignments of the 19 F-labels attached to indigenous cysteines. Addition of the negative allosteric modulator (NAM) NNC0640 was critically important for obtaining the long-time stability needed for our NMR experiments, and we report on novel insights into the allosteric effects arising from binding of NNC0640 to the transmembrane domain of GLP-1R (GLP-1R[TMD]).

The V369M Gcgr knock-in mice are a precision medicine model of mild Mahvash disease.

The detailed metabolic characterization of the glucagon receptor (Gcgr)V369M+/+ mutant mice described in Lin et al. in the Biochemical Journal is of interest and resulting in the expected metabolic profile. We would like to point out that these mice might also be extremely useful as a precision medicine model of mild Mahvash disease, a rare hereditary pancreatic neuroendocrine tumor syndrome characterized by inactivating mutations in the glucagon receptor. Further characterization of pancreas morphology and histology in the GcgrV369M+/+ mice at more advanced ages will be critically important to understand mild Mahvash disease in humans.

Deficiency of glucagon gene-derived peptides induces peripheral polyneuropathy in mice.

Although diabetic polyneuropathy (DPN) is the commonest diabetic complication, its pathology remains to be clarified. As previous papers have suggested the neuroprotective effects of glucagon-like peptide-1 in DPN, the current study investigated the physiological indispensability of glucagon gene-derived peptides (GCGDPs) including glucagon-like peptide-1 in the peripheral nervous system (PNS). Neurological functions and neuropathological changes of GCGDP deficient (gcg-/-) mice were examined. The gcg-/- mice showed tactile allodynia and thermal hyperalgesia at 12-18 weeks old, followed by tactile and thermal hypoalgesia at 36 weeks old. Nerve conduction studies revealed a decrease in sensory nerve conduction velocity at 36 weeks old. Pathological findings showed a decrease in intraepidermal nerve fiber densities. Electron microscopy revealed a decrease in circularity and an increase in g-ratio of myelinated fibers and a decrease of unmyelinated fibers in the sural nerves of the gcg-/- mice. Effects of glucagon on neurite outgrowth were examined using an ex vivo culture of dorsal root ganglia. A supraphysiological concentration of glucagon promoted neurite outgrowth. In conclusion, the mice with deficiency of GCGDPs developed peripheral neuropathy with age. Furthermore, glucagon might have neuroprotective effects on the PNS of mice. GCGDPs might be involved in the pathology of DPN.

Glucagon receptor antagonism promotes the production of gut proglucagon-derived peptides in diabetic mice.

Glucagon is an essential regulator of glucose homeostasis, particularly in type 2 diabetes (T2D). Blocking the glucagon receptor (GCGR) in diabetic animals and humans has been shown to alleviate hyperglycemia and increase circulating glucagon-like peptide-1 (GLP-1) levels. However, the origin of the upregulated GLP-1 remains to be clarified. Here, we administered high-fat diet + streptozotocin-induced T2D mice and diabetic db/db mice with REMD 2.59, a fully competitive antagonistic human GCGR monoclonal antibody (mAb) for 12 weeks. GCGR mAb treatment decreased fasting blood glucose levels and increased plasma GLP-1 levels in the T2D mice. In addition, GCGR mAb upregulated preproglucagon gene expression and the contents of gut proglucagon-derived peptides, particularly GLP-1, in the small intestine and colon. Notably, T2D mice treated with GCGR mAb displayed a higher L-cell density in the small intestine and colon, which was associated with increased numbers of LK-cells coexpressing GLP-1 and glucose-dependent insulinotropic polypeptide and reduced L-cell apoptosis. Furthermore, GCGR mAb treatment upregulated GLP-1 production in the pancreas, which was detected at lower levels than in the intestine. Collectively, these results suggest that GCGR mAb can increase intestinal GLP-1 production and L-cell number by enhancing LK-cell expansion and inhibiting L-cell apoptosis in T2D.

Glucagon-Receptor Signaling Reverses Hepatic Steatosis Independent of Leptin Receptor Expression.

Glucagon (GCG) is an essential regulator of glucose and lipid metabolism that also promotes weight loss. We have shown that glucagon-receptor (GCGR) signaling increases fatty acid oxidation (FAOx) in primary hepatocytes and reduces liver triglycerides in diet-induced obese (DIO) mice; however, the mechanisms underlying this aspect of GCG biology remains unclear. Investigation of hepatic GCGR targets elucidated a potent and previously unknown induction of leptin receptor (Lepr) expression. Liver leptin signaling is known to increase FAOx and decrease liver triglycerides, similar to glucagon action. Therefore, we hypothesized that glucagon increases hepatic LEPR, which is necessary for glucagon-mediated reversal of hepatic steatosis. Eight-week-old control and liver-specific LEPR-deficient mice (LeprΔliver) were placed on a high-fat diet for 12 weeks and then treated with a selective GCGR agonist (IUB288) for 14 days. Liver triglycerides and gene expression were assessed in liver tissue homogenates. Administration of IUB288 in both lean and DIO mice increased hepatic Lepr isoforms a-e in acute (4 hours) and chronic (72 hours,16 days) (P < 0.05) settings. LeprΔliver mice displayed increased hepatic triglycerides on a chow diet alone (P < 0.05), which persisted in a DIO state (P < 0.001), with no differences in body weight or composition. Surprisingly, chronic administration of IUB288 in DIO control and LeprΔliver mice reduced liver triglycerides regardless of genotype (P < 0.05). Together, these data suggest that GCGR activation induces hepatic Lepr expression and, although hepatic glucagon and leptin signaling have similar liver lipid targets, these appear to be 2 distinct pathways.

GLP-1 signaling is required for improvement of glucose tolerance by osteocalcin.

Osteocalcin is a bone-derived hormone that in its uncarboxylated form (GluOC) plays an important role in glucose and energy metabolism by stimulating insulin secretion and pancreatic ß-cell proliferation through its putative receptor GPRC6A. We previously showed that the effect of GluOC on insulin secretion is mediated predominantly by glucagon-like peptide-1 (GLP-1) released from intestinal endocrine cells in response to GluOC stimulation. Moreover, oral administration of GluOC was found to reduce the fasting blood glucose level, to improve glucose tolerance, and to increase the fasting serum insulin concentration and ß-cell area in the pancreas in wild-type mice. We have now examined the effects of oral GluOC administration for at least 4 weeks in GLP-1 receptor-knockout mice. Such administration of GluOC in the mutant mice triggered glucose intolerance, enhanced gluconeogenesis and promoted both lipid accumulation in the liver as well as adipocyte hypertrophy and inflammation in adipose tissue. Furthermore, inactivation of GLP-1 receptor signaling in association with GluOC administration induced activation of the transcription factor FoxO1 and expression of its transcriptional coactivator PGC1α in the liver, likely accounting for the observed upregulation of gluconeogenic gene expression. Our results thus indicate that the beneficial metabolic effects of GluOC are dependent on GLP-1 receptor signaling.

Combined Amylin/GLP-1 pharmacotherapy to promote and sustain long-lasting weight loss.

A growing appreciation of the overlapping neuroendocrine mechanisms controlling energy balance has highlighted combination therapies as a promising strategy to enhance sustained weight loss. Here, we investigated whether amylin- and glucagon-like-peptide-1 (GLP-1)-based combination therapies produce greater food intake- and body weight-suppressive effects compared to monotherapies in both lean and diet-induced obese (DIO) rats. In chow-maintained rats, systemic amylin and GLP-1 combine to reduce meal size. Furthermore, the amylin and GLP-1 analogs salmon calcitonin (sCT) and liraglutide produce synergistic-like reductions in 24 hours energy intake and body weight. The administration of sCT with liraglutide also led to a significant enhancement in cFos-activation in the dorsal-vagal-complex (DVC) compared to mono-therapy, suggesting an activation of distinct, yet overlapping neural substrates in this critical energy balance hub. In DIO animals, long-term daily administration of this combination therapy, specifically in a stepwise manner, results in reduced energy intake and greater body weight loss over time when compared to chronic mono- and combined-treated groups, without affecting GLP-1 receptor, preproglucagon or amylin-receptor gene expression in the DVC.

Increased SLC38A4 Amino Acid Transporter Expression in Human Pancreatic α-Cells After Glucagon Receptor Inhibition.

Plasma amino acids and their transporters constitute an important part of the feedback loop between the liver and pancreatic α-cell function, and glucagon regulates hepatic amino acid turnover. Disruption of hepatic glucagon receptor action activates the loop and results in high plasma amino acids and hypersecretion of glucagon associated with α-cell hyperplasia. In the present study, we report a technique to rescue implanted human pancreatic islets from the mouse kidney capsule. Using this model, we have demonstrated that expression of the amino acid transporter SLC38A4 increases in α-cells after administration of a glucagon receptor blocking antibody. The increase in SLC38A4 expression and associated α-cell proliferation was dependent on mechanistic target of rapamycin pathway. We confirmed increased α-cell proliferation and expression of SLC38A4 in pancreas sections from patients with glucagon cell hyperplasia and neoplasia (GCHN) with loss-of-function mutations in the glucagon receptor. Collectively, using a technique to rescue implanted human islets from the kidney capsule in mice and pancreas sections from patients with GCHN, we found that expression of SLC38A4 was increased under conditions of disrupted glucagon receptor signaling. These data provide support for the existence of a liver-human α-cell endocrine feedback loop.

Fractionated whole body gamma irradiation modulates the hepatic response in type II diabetes of high fat diet model rats.

HFD animals were exposed to a low rate of different fractionated whole body gamma irradiation doses (0.5, 1 and 2 Gy, three fractions per week for two consecutive months) and the expression of certain genes involved in type 2 diabetes mellitus (T2DM) in livers and brains of HFD Wistar rats was investigated. Additionally, levels of diabetes-related proteins encoded by the studied genes were analyzed. Results indicated that mRNA level of incretin glucagon like peptite-1 receptor (GLP-1R) was augmented in livers and brains exposed to 1 and 2 Gy doses. Moreover, the mitochondrial uncoupling proteins 2 and 3 (UCP2/3) expressions in animals fed on HFD compared to those fed on normal chow diet were significantly increased at all applied doses. GLP-1R and UCP3 protein levels were up regulated in livers. Total protein content increased at 0.5 and 1 Gy gamma irradiation exposure and returned to its normal level at 2 Gy dose. Results could be an indicator of type 2 diabetes delayed development during irradiation exposure and support the importance of GLP-1R as a target gene in radiotherapy against T2DM and its chronic complications. A new hypothesis of brain-liver and intestine interface is speculated by which an increase in the hepatic GLP-1R is influenced by the effect of fractionated whole body gamma irradiation.

Hypercalcemia in Glucagon Cell Hyperplasia and Neoplasia (Mahvash Syndrome): A New Association.

Context: Hyperglucagonemia in the absence of glucagonomas is rare. Biallelic-inactivating mutations in the glucagon receptor gene (GCGR) cause glucagon cell hyperplasia and neoplasia (GCHN), also termed Mahvash syndrome. Here, we report the first case to our knowledge of GCHN presenting with hypercalcemia and demonstrate a unique relationship between calcium and α-cell hyperplasia. Case Description: A 47-year-old man presented with severe PTH-independent hypercalcemia, 13.95 mg/dL (3.48 mmol/L). Imaging and extensive pathology tests yielded no conclusive cause. Glucagon levels >300 times the upper limit of normal were discovered. Subtotal pancreatectomy identified α-cell hyperplasia and neoplasia with metastatic disease in lymph nodes. Genomic analysis confirmed a homozygous missense variant in GCGR (Asp63Asn). This is a previously described pathologic variant and has a known association with GCHN. Conclusions: Inactivating mutations of the glucagon receptor gene lead to nonfunctional hyperglucagonemia and are associated with GCHN. Homozygous or compound heterozygous GCGR mutations are associated with α-cell hyperplasia, a known precursor to pancreatic neuroendocrine tumors that can metastasize. Hypercalcemia is an unreported consequence of GCHN with an unclear mechanism.

Glucagon Receptor Signaling Regulates Energy Metabolism via Hepatic Farnesoid X Receptor and Fibroblast Growth Factor 21.

Glucagon, an essential regulator of glucose and lipid metabolism, also promotes weight loss, in part through potentiation of fibroblast growth factor 21 (FGF21) secretion. However, FGF21 is only a partial mediator of metabolic actions ensuing from glucagon receptor (GCGR) activation, prompting us to search for additional pathways. Intriguingly, chronic GCGR agonism increases plasma bile acid levels. We hypothesized that GCGR agonism regulates energy metabolism, at least in part, through farnesoid X receptor (FXR). To test this hypothesis, we studied whole-body and liver-specific FXR-knockout (Fxr∆liver) mice. Chronic GCGR agonist (IUB288) administration in diet-induced obese (DIO) Gcgr, Fgf21, and Fxr whole-body or liver-specific knockout (∆liver) mice failed to reduce body weight when compared with wild-type (WT) mice. IUB288 increased energy expenditure and respiration in DIO WT mice, but not Fxr∆liver mice. GCGR agonism increased [14C]palmitate oxidation in hepatocytes isolated from WT mice in a dose-dependent manner, an effect blunted in hepatocytes from Fxr∆liver mice. Our data clearly demonstrate that control of whole-body energy expenditure by GCGR agonism requires intact FXR signaling in the liver. This heretofore-unappreciated aspect of glucagon biology has implications for the use of GCGR agonism in the therapy of metabolic disorders.

GLP-2 receptor signaling controls circulating bile acid levels but not glucose homeostasis in Gcgr-/- mice and is dispensable for the metabolic benefits ensuing after vertical sleeve gastrectomy.

OBJECTIVE: Therapeutic interventions that improve glucose homeostasis such as attenuation of glucagon receptor (Gcgr) signaling and bariatric surgery share common metabolic features conserved in mice and humans. These include increased circulating levels of bile acids (BA) and the proglucagon-derived peptides (PGDPs), GLP-1 and GLP-2. Whether BA acting through TGR5 (Gpbar1) increases PGDP levels in these scenarios has not been examined. Furthermore, although the importance of GLP-1 action has been interrogated in Gcgr-/- mice and after bariatric surgery, whether GLP-2 contributes to the metabolic benefits of these interventions is not known. METHODS: To assess whether BA acting through Gpbar1 mediates improved glucose homeostasis in Gcgr-/- mice we generated and characterized Gcgr-/-:Gpbar1-/- mice. The contribution of GLP-2 receptor (GLP-2R) signaling to intestinal and metabolic adaptation arising following loss of the Gcgr was studied in Gcgr-/-:Glp2r-/- mice. The role of the GLP-2R in the metabolic improvements evident after bariatric surgery was studied in high fat-fed Glp2r-/- mice subjected to vertical sleeve gastrectomy (VSG). RESULTS: Circulating levels of BA were markedly elevated yet similar in Gcgr-/-:Gpbar1+/+ vs. Gcgr-/-:Gpbar1-/- mice. Loss of GLP-2R lowered levels of BA in Gcgr-/- mice. Gcgr-/-:Glp2r-/- mice also exhibited shifts in the proportion of circulating BA species. Loss of Gpbar1 did not impact body weight, intestinal mass, or glucose homeostasis in Gcgr-/- mice. In contrast, small bowel growth was attenuated in Gcgr-/-:Glp2r-/- mice. The improvement in glucose tolerance, elevated circulating levels of GLP-1, and glucose-stimulated insulin levels were not different in Gcgr-/-:Glp2r+/+ vs. Gcgr-/-:Glp2r-/- mice. Similarly, loss of the GLP-2R did not attenuate the extent of weight loss and improvement in glucose control after VSG. CONCLUSIONS: These findings reveal that GLP-2R controls BA levels and relative proportions of BA species in Gcgr-/- mice. Nevertheless, the GLP-2R is not essential for i) control of body weight or glucose homeostasis in Gcgr-/- mice or ii) metabolic improvements arising after VSG in high fat-fed mice. Furthermore, despite elevations of circulating levels of BA, Gpbar1 does not mediate elevated levels of PGDPs or major metabolic phenotypes in Gcgr-/- mice. Collectively these findings refine our understanding of the relationship between Gpbar1, elevated levels of BA, PGDPs, and the GLP-2R in amelioration of metabolic derangements arising following loss of Gcgr signaling or after vertical sleeve gastrectomy.