The Effect Of Insulin Receptor Substrates1and2Knockdown On Porcine Hepatic Glucolipid Metabolism

According to the recent study of epidemiology, type2diabetes is a complex and hetergeneous disorder presently affecting more than100million people worldwide and causing serious socio-economic problems. The pathogenesis of type2diabetes is characterized by two major features:peripheral insulin resistance and impaired insulin secretion from pancreatic (3cells. Gene knockout technology allows to generate homozgous null mutant mice and to examine the effects of a total lack of a particular gene product for more than20years. The most important genes related to type2diabetes are the components in insulin signalling. Insulin binds to the insulin receptor(IR), which is a tyrosine kinase that undergoes autophosphorylation and catalyses the phosphrylation of cellular proteins, especially the members of the proteins between IR and a complex network of intracellular signalling to regulate cell growth differentiation, glucose metabolism, glycogen/protein synthesis and gene expression.There are many limitations to mouse disease models in the genetics and phenotype. Moreover, the reactions of mouse to high glucose and high fat food have differences with human. Pigs have been used as models of type2diabetes because of their phenotypic similarities to human including: cardiovascular anatomy and function, metabolism, lipoprotein profile, size, tendency to obesity, and omnivorous habits. The establishment and application of stem cells are very important for human health. At the same time, the establishment of stem cell means that various complicated transgenic operation can be applied to livestock animal especially pig.The goal of this study is to knockdown both IRS1and IRS2in porcine liver cells, and check its effects on gene expression related to glucolipid metabolism. We cloned porcine IRS1gene by Overlap PCR and found1RS1highly exprssed in liver, and then screened the most effective interference shRNA fragments of IRS1by Real-time PCR in porcine liver cells. Furthermore, we cloned the partial3-untranslated region sequence of IRS2by3’RACE, and obtained the most effective inteference shRNA fragments of IRS2. Then, interference vector pGenesil-shIRSl/shIRS2was constructed, with simultaneously knockdown of IRS1and IRS2in efficiency of78%and64%. Finally, the expression of glucolipid metabolism related genes were detected in the porcine liver cells with both IRS1and IRS2knockdown, and significant increase of gluconeogenic enzymes PEPCK and F-1,6-BP and decrease of Gck, which may lead to an increasing level of blood glucose, were observed. The knockdown also led to the upregulation of lipogenic enzymes SREBP-1, Abcg8and CYP7al, which may cause a disorder of cholesterol metabolism.Taken together, our results demonstrate IRS1and IRS2have important roles in the regulation of hepatic metabolism,with laying a solid foundation for producing the T2DM model pigs.