Targeted Deletion Of Grb10 Gene In The Pancreas Improves Beta-Cell Function

Objectives:To generate pancreas-specific Grb10 knockout mice and test the phenotype.Methods:Grb10flox/- mice have been generated and bred for 6 generation, and then heterozygote mice have been bred with heterozygote mice to get homozygote mice. Breed Grb10flox/flox mice with PDX-Cre+/-mice then get pancreas-specific Grb10 knockout mice (pGrb10 KO mice) and Grb10flox/- mice (WT control mice). DNA has been digested from tails for Sounthern blot, and DNA has been digested from different tissues for PCR to confirm in DNA level that Grb10 has been deleted only in pancreas. Western Blot and Immunohistochemistry have been used to detect whether Grb10 protein has been removed yet.Results:Southern Blot shows that in pGrb10 KO mice the gene target sites locate right. PCR shows that deletion happens only in pancreas, no any effect on other tissues. According to Western Blot, Grb10 is highest expressed in pancreas, but completely deleted in pGrb10 KO mice. Immunohistochemistry of pancreas shows that Grb10 expresses in all of the endocrine cells and exocrine cells, conparablly higher expresses in islets.Conclusion:pGrb10 KO mice have been successfully generated. Grb10 has been completely deleted in pancreas in pGrb10 KO mice, without effect on any other tissues.Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance after deleting Grb10 in pancreas.Methods:PGrb10 KO mice group and WT mice group(n=8-13) have been set to measure body weight, food intake(from 4 weeks old to 22 weeks, once per week), fat percentage(22 weeks old), organ weight. Feed with normal chow for 18 weeks, Glucose Tolerance Test(GTT) will be carried out to study the insulin secretion after glucose stimulation and periphery tissues'insulin sensitivity; Insulin Tolerance Test(ITT) will be carried out to study periphery tissues'insulin sensitivity. Fasting plasma insulin level and beta cell mass weight will also be tested. Pancreatic beta cell proliferation rate will be tested by BrdU labeling.Results:pGrb10 KO mice have no difference with WT mice in Body weight, food intake, fat percentage, different organs (excluding pancreas), and periphery tisuue insulin sensitivity. pGrb10 KO mice have bigger pancreas, lower glucose level but pGrb10 KO mice have similar insulin level and beta cell mass weight. pGrb10 KO mice have higher proliferation rate than WT mice.Conclusion:Compared with WT control mice, pGrb10 KO mice have bigger pancreas, lower Glucose level, and much higher pancreatic beta cell proliferation rate.Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance in High Fat Diet induced type 2 diabetes mice model after deleting Grb10 in pancreas.Methods:When mice are 4 weeks old, feed them with 60%HFD for 18 weeks. pGrb10 KO micegroup and WT mice group(n≥10) have been set up to measure body weight, food intake(from 4 weeks old to 22 weeks, once per week), fat percentage(22 weeks old). Glucose Tolerance Test(GTT) will be carried out to study the insulin secretion after glucose stimulation and periphery tissues'insulin sensitivity; Insulin Tolerance Test(ITT) will be carried out to study periphery tissues'insulin sensitivity. Fasting plasma insulin level and beta cell mass weight will also be tested. Islet isolation will be done to study the detail mechanism of Grb10 effect in beta cells. After digested with Collagenase P, the pancreas tissue will be dispersed into plate and then islets will be hand-picked and culture in 12-well plate. After stimulated with IGF-1 and insulin, cells will be collected to test whether p-AKT and downstream have been changed in pGrb10 KO mice. TEM will be used to test insulin granule in pancreatic beta cell in both WT and pGrb10 KO mice.Results:Under high fat diet, PGrb10 KO micehave no difference with WT mice in Body weight, food intake, fat percentage, and periphery tissue insulin sensitivity. pGrb10 KO micehave significantly more beta cells, much lower glucose level, higher insulin level and much more insulin granule in pancreatic beta cells. In pGrb10 KO mice, p-Akt, p-Foxo and p-Erk have been upregulated.Conclusion:In type 2 diabetes model, compared with WT control mice, pGrb10 KO mice have lower Glucose level, higher insulin lever, much more beta cell mass and insulin granules in pancreatic beta cells. Insulin signaling has been upregulated in pGrb10 KO mice.Objectives:Study the effect on insulin releasing from the beta cell, islet mass size and glucose tolerance in STZ-induced type 1 diabetes mice model after deleting Grb10 in pancreas.Methods:3-months-old normal chow mice have been chosen in pGrb10 KO mice and WT control mice(n=8-14). The male mice have been injected STZ 75mg/kgBW and females have been injected 80mg/kgBW for 5 days, and then test blood glucose every 3 days at the same time point.21 days later, the mice have been sacrificed, and the pancreas weight, islets weight and blood insulin will be tested. Similar age of mice will be chosen and inject with STZ for 2days,48 hours later, mice will be sacrificed and the pancreas will be taken, fixed in 10%formalin, bedded in wax, and cut sections. In Situ Cell Death Detection Kit (Roche) will be used to detect beta cell apoptosis.Results:Compared with Wt control mice, pGrb10 KO mice have significant low glucose level much less weight losing, significant more beta cells and much higher plasma insulin level. TUNEL assay shows that pGrb10 KO mice have lower apoptosis rate than the WT mice.Conclusion:In type 1 diabetes model, Compared with WT control mice, pGrb10 KO micehave higher ability to conquer apoptosis.