Establishment Of Transgenic Pig Model Of Type 2 Diabetes Mellitus With GIPR Function

The concept that certain factors produced by the intestinal mucosa in response to nutrient ingestion are capable of stimulating the release of substances from the endocrine pancreas and thereby reducing blood glucose glucose levels was first introduced in the early1900s. It mainly includes glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1(GLP-1).Incretin induced a much greater increase in plasma insulin levels in oral glucose administration when compared with the same amount of glucose given intravenously. Its effect has been estimated to account for50-70%of total postprandial insulin secretion. GIP and GLP-1exert their effects by binding to their specific receptors,the GIP receptor (GIPR) and the GLP-1receptor (GLP-1R) and increase the level of intracellular cAMP in pancreatic β cells. In addition to their insulinotropic effects, GIP and GLP-1have been shown to preserve pancreatic β cell mass by inhibiting apoptosis of β cells and enhancing their proliferation.GIP is a42-amino acid peptide produced by enteroendocrine K-cells which reaching a peak just15-30min after meal ingestion and is hydrolysed rapidly by the proteolytic enzyme dipeptidyl-peptidase4(DPP Ⅳ) into a truncated and inactive product. Impaired GIP/GIPR axis is considered the pathogenesis of diabetes. In the present study we constructed the diabetic model by impairing the GIP/GIPR axis.Pigs have the similar organ size and physiology to the human and resemble suitable diabetic model. We generated a plasmid over expressing a dominant-negative GIPR which combine with the ligands but with null intrinsic activity. The GIPRdn transgenic pigs were generated by somotic cell nuclear transfer and the diabetic phenotype was analysised. We performed the first domeatic GIPRdn transgenic pig which resembled the T2DM model for further study of diabetes research and antidiabetic drug screening. PartⅠPlasmid construction of over expressing a dominant-negative GIPR (GIPRdn)Objective We generated a plasmid over expressing a dominant-negative GIPR.Methods The cDNA of human GIP receptor was mutated in the region coding for the third intracellular loop by introducing a point (mutation at amino acid position340(Ala-Glu) and a deletion of8amino acids at position319-326which is essential for signal transduction. The plasmid consists of the rat ins2promotor (RIP Ⅱ) and the cDNA of a reformed human GIPRdn.Results The plasmid was composed of hGIPRdn and Pnmu102by recombination, whereafter was verified by sequence.Conclusion The plasmid over expressing a dominant-negative GIPR was successfully constructed.Part ⅡGeneration of over expressing a dominant-negative GIPR (GIPRdn) transgenic pigObjective We generated over expressing a dominant-negative GIPR (GIPRdn) transgenic pigs. Methods The fibroblasts of male pig were transfected with lineared GIPRdn plasmid by lipofectamine2000and were screened by G418.Blastocysts developed from the transfected fibroblasts by cloning were transferred to the horn of uterus of recipient pigs. Piglets were genotyped by PCR.. Expression of GIPRdn mRNA in the pancrease tissure was determined by RT-PCR.Results Transfected fibroblasts were identified by PCR. Seven over expressing a dominant-negative GIPR (GIPRdn) transgenic pigs was generated by somatic cell nuclear transfer.Conclusion Over expressing a dominant-negative GIPR (GIPRdn) transgenic pigs were born healthly.Part ⅢAnalysis the phenotype of over expressing a dominant-negative GIPR (GIPRdn) transgenic pigObjective We observed the devolpment of glucose metabolism and evaluated the insulin secretion.Methods Oral glucose tolerance test was performed in2M/5M wild type and transgnic pigs. Pancreas weight was measured, immunohistochemistry of insulin and Ki67in pancreas were performed.Results The area under the insulin curve(AUC insulin) during the experimental period increased about2.3times in2-month-old GIPRdn transgenic pigs versus the controls (P=0.02). The glucose level and insulin content have no significant difference between5-month-old GIPRdn transgenic pigs and controls. However, the area under the insulin curve in5-month-old GIPRdn transgenic pigs declined about44% compared to2-month-old transgenic pigs, while the insulin secretion profile is similar in2-month-old and5-month-old controls. Weight of pancreas in5-month-old GIPRdn transgenic pigs decreased about44%compaired with age matched controls. transgenic pigs show insulin resistance and a defective β3cell development, implying the importance of GIP signaling for postnatal islet development and potential induction of insulin resistance.Conclusion Over expressing a dominant-negative GIPR (GIPRdn) transgenic pig exhibited the impaired glucose tolerance and disturbed pancreatic development.