GCGR encodes a glucagon receptor that is important in controlling blood glucose levels. Defects in this gene are a cause of non-insulin-dependent diabetes mellitus (NIDDM). Activation of the human glucagon receptor (GCGR) by its endogenous ligand glucagon triggers the release of glucose from the liver during fasting, making it a potential drug target for type 2 diabetes.
Species-specific analysis of GCGR gene expression in WT and B-hGCGR mice by RT-PCR and Western Blot. A. Mouse GCGR mRNA was detectable only in liver cells of wild type C57BL/6 mice (+/+). Human GCGR mRNA was detectable only in homozygous B-hGCGR mice (H/H) , but not in wild type C57BL/6 mice (+/+). B. Kidney tissue was collected from wild type C57BL/6 mice (+/+) and homozygous B-hGCGR mice (H/H), and analyzed by western blot with anti-GCGR antibody. Mouse GCGR was detectable in wild type mice and homozygous B-hGCGR mice, as the antibody is cross-reactive with GCGR in human and mice. Human GCGR was exclusively detected in homozygous B-hGCGR mice.
Analysis of leukocytes cell subpopulation in B-hGCGR mice
Analysis of leukocyte subpopulations by FACS.
Splenocytes and blood were isolated from female C57BL/6 and B-hGCGR mice (n=4, 7-week-old), and assessed by flow cytometry analysis was performed to assess leukocyte subpopulations. A. Representative FACS plots (spleen). Single live cells were gated for CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, dendritic cells, granulocytes, monocytes and macrophages in homozygous B-hGCGR mice were similar to those in the C57BL/6 mice, demonstrating that introduction of hGCGR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these cell types. Values are expressed as mean ± SEM.
Analysis of T cell subpopulation in B-hGCGR mice
Analysis of spleen T cell subpopulations by FACS.
Spleen, blood and lymph nodes were isolated from female C57BL/6 and B-hGCGR mice (n=4, 7-week-old). Flow cytometry analysis was performed to assess leukocyte subpopulations. A. Representative FACS plots (spleen). Single live CD45+ T cells were gated for CD3 and used for further analysis as indicated. B-D. Percent of CD4+ T cells, CD8+ T cells and Tregs in spleen, blood, and lymph nodes were similar between B-hGCGR and C57BL/6 mice, demonstrating that introduction of hGCGR in place of its mouse counterpart does not change the overall development, differentiation or distribution of these T cell subtypes. Values are expressed as mean ± SEM.
In vivo efficacy of anti-human GCGR antibody in B-hGCGR mice
Experimental schedule for in vivo efficacy assessment of anti-human GCGR antibody. Anti-human GCGR antibody crotedumab (in-house) or isotype control (IgG4k) was administered by intraperitoneal injection into C57BL/6J mice or B-hGCGR mice (n=6/condition) once a week on days 1 to 8 as shown. Blood glucose, insulin, glucagon and lipid profiles were assessed on the days shown in the chart.
Anti-human GCGR antibody reduces blood glucose in male B-hGCGR mice.
- Random blood glucose from male mice before and at multiple time points after injection of crotedumab (in house) or isotype control antibody (IgG4k; n = 6). B. Body weights. C. OGTT on day 4. D. Area under the curve for the OGTT shown in C. Serum levels of (E) insulin, (F) glucagon on Day 7. Anti-GCGR antibody reduced the random blood glucose, fasting blood glucose and OGTT compared to the isotype antibody in B-hGCGR mice. Serum levels of insulin was reduced slightly and glucagon level was significantly increased in the antibody treated group. Results indicated that the regulatory function on blood glucose in humanized B-hGCGR mice was similar to wild-type C57BL/6 mice, and that the anti-human GCGR antibody was efficacious in controlling blood glucose in B-hGCGR mice. Values are expressed as mean ± SEM. OGTT, oral glucose tolerance test.
Anti-human GCGR antibody improved lipid metabolism in male B-hGCGR mice.
Wild-type C57BL/6 and B-hGCGR mice were treated with crotedumab (in house) or isotype control antibody (male, n = 6). Blood was analyzed on Day 11 for triglycerides and cholesterol levels. Serum levels of TG (A) and TC (B). HDL-C (C) and LDL-C (D) were assessed. Serum levels of TG was reduced, while TC, HDL-C and LDL-C were increased in the anti-human GCGR antibody-treated group compared to the isotype control. Results indicated that lipid metabolism in humanized B-hGCGR (G2) mice was mostly similar to wild-type C57BL/6 mice (G1). Furthermore, the anti-human GCGR antibody was efficacious in controlling blood lipid in male B-hGCGR mice. Values are expressed as mean ± SEM. TG, triglycerides; TC, total cholesterol; HDL-C, high density lipoprotein cholesterol; LDL-C, low density lipoprotein cholesterol.