mRNA and protein expression analysis

Protein expression analysis

Analysis of leukocytes cell subpopulation in B-hGLP1R mice

Analysis of leukocyte subpopulations by FACS. Splenocytes were isolated from female C57BL/6 and B-hGLP1R mice (n=3, 9-week-old). Flow cytometry analysis of the splenocytes were performed to assess leukocyte subpopulations. A. Representative FACS plots. Single live cells were gated for the CD45+ population and used for further analysis as indicated here. B. Results of FACS analysis. Percent of T cells, B cells, NK cells, monocytes, dendritic cells, granulocytes and macrophages in homozygous B-hGLP1R mice were similar to those in the C57BL/6 mice, demonstrating that GLP1R humanized does not change the overall development, differentiation or distribution of these cell types in spleen. The same results were observed in blood and lymph node, data was not shown. Values are expressed as mean ± SEM.

Analysis of T cell subpopulation in spleen by FACS. The lymphocytes were isolated from spleen in C57BL/6 and B-hGLP1R mice (n=3, 9-week-old). The proportion of T cells subpopulation was tested by flow cytometry. There were no differences between C57BL/6 and B-hGLP1R mice, demonstrating that humanized of GLP1R does not change the overall development, differentiation or distribution of these T cell subtypes. The same results were observed in blood and lymph node, data was not shown. Values are expressed as mean ± SEM.

Dulaglutide reduces blood glucose in B-hGLP1R mice. Blood was collected from C57BL/6 mice and B-hGLP1R mice (male, 7-8w, n=5) at multiple time points after injection of Dulaglutide (in house) or PBS for analysis. (A). Body weight. (B). Non-fasting blood glucose. (C). Fasting blood glucose. (D). IPGTT. (E). Area under the curve for the IPGTT. The body weight has no significant change during the experiment. Dulaglutide reduced the non-fasting blood glucose and fasting blood glucose compared to the PBS in C57BL/6 and B-hGLP1R mice. For the IPGTT results, B-hGLP1R mice and wild-type mice display similar physiological responses for glucose metabolism. Values are expressed as mean ± SEM. IPGTT, Intraperitoneal glucose tolerance test.

Dulaglutide decreased food intake in B-hGLP1R mice. Food intake was monitored in C57BL/6 and B-hGLP1R mice (male, 15w, n=5) after treatment with dulaglutide for 24 and 48 hours separately, then calculated for each cage and corrected for food intake per mouse. (A). Food intake in 24h. (B). Food intake in 48h. Dulaglutide decreased food intake both in C57BL/6 and B-hGLP1R mice. Values are expressed as mean ± SEM.

Model schematic and antibody evaluation scheme

Schedule for in vivo efficacy of Dulaglutide. B-hGLP1R (male, n=10) mice were fed with high-fat diet for 12 weeks to induce mice obesity. Then, the mice were randomized according to body weight. About 24 hours before or after injected drugs,  measured and calculated the food intake. The Dulaglutide (in house) was subcutaneous injection every two days from Day 1 to 8 after grouping. Blood was collected to measure blood glucose and IPGTT on the days shown in the schematic diagram and the table. Measure plasma insulin during the IPGTT.

Dulaglutide reduces blood glucose in B-hGLP1R mice with obesity. Blood was collected from B-hGLP1R mice (male, n=10) at multiple time points after injection of Dulaglutide (in house) or PBS for analysis. (A). Body weight. (B). Non-fasting blood glucose. (C). Fasting blood glucose. (D). Serum glucose levels following intraperitoneal glucose challenge. (E). Area under the curve (AUC) for the IPGTT. (F). Food intake. Dulaglutide resulted in no weight loss during the experiment, and reduced the non-fasting blood glucose and fasting blood glucose compared to PBS. For the IPGTT results, glucose tolerance was improved by dulaglutide compared to PBS. The results show that B-hGLP1R mice provide an efficacious preclinical animal model for evaluating related drugs. Values are expressed as mean ± SEM. IPGTT, Intraperitoneal glucose tolerance test.

Dulaglutide regulated blood glucose by increasing insulin and decreased glucagon secretion in B-hGLP1R obesity mice, and influenced the food intake. Food intake was measured in C57BL/6 and B-hGLP1R mice (male, n=10) after treatment with dulaglutide for 24h, then calculated for each cage and corrected for food intake per mice. The plasma insulin was measured during the IPGTT. And collected the serum for GLP-1 and glucagon analysis. (A) Food intake in 24h after treatment. (B). Plasma insulin secretion. (C). The area under the curve for plasma insulin during 0-30 minutes. (D). Serum GLP-1. (E). Serum Glucagon. Dulaglutide decreased food intake in B-hGLP1R mice. Meanwhile, Insulin and GLP-1 increased, glucagon secretion decreased after treatment with Dulaglutide, which is consistent with the mechanisms of control blood glucose. Values expressed as mean ± SEM. IPGTT, Intraperitoneal glucose tolerance test.

PF-06882961 improved glucose tolerance in B-hGLP1R mice with obesity. (A). Serum glucose levels following intraperitoneal glucose challenge (IPGTT). (B). Area under the curve (AUC) for the IPGTT. Glucose tolerance was improved by PF-06882961 compared to vehicle in B-hGLP1R mice but not in wild-type C57BL/6 mice. The results show that B-hGLP1R mice provide an efficacious preclinical animal model for evaluating species-specific new 'biological' medicines. Values are expressed as mean ± SEM. (PF-08882961 is a small molecule agonist for human GLP1R).