Evaluation of Commercially Available Glucagon Receptor Antibodies and Glucagon Receptor Expression

Abstract

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.