Glucose Regulation Related Genes And Its Function

Differential display was applied to clone genes relative to blood-glucose regulation as candidate for diabetes mellitus. We build autonomous model of blood glucose regulation of SD rat through jugular vein right atrium intubation and glucose stimulation. After blood glucose stimulation, the skeletal muscle was separated and applied to mRNA differential display.181 differentially expressed fragments were got from RT-PCR products with 3 single-base anchored primers in combination with 10 random primers. After slot blotting, we got 56 ESTs with up-regulation expression and 18 ESTs with down-regulation expression. After Northern blotting, we confirmed EST-89, EST-62, EST-119 are true positive fragments with up-regulation expression; EST-17, EST-76, EST-124 are true positive with down-regulation expression. A cDNA library of rat skeletal muscle was screened with these 6 ESTs and we got two full-length cDNA, named as Fang-1(2.3Kb)and Fang-2(0.9Kb), GenBank Accession No. 399873 and 399874, respectively.With blast software we found Fang-1 has homolog (GenBank Accession NO. AK001644) in human, whose function keeps unclear. After high concentration of glucose stimulation, compared with control, Fang-1 and Fang-2 were up and down-regulated expressed, respectively.In order to determine whether Fang-1 can effect glucose metabolism of cells, full-length Fang-1 was cloned into expression vector pCDNA3.1 and transfected into CBRH7919. After RT-PCR and Northern blotting, we found over-expression of Fang-1 can up-regulate the expression of IRS1.One fragment of Fang-1 was cloned into pGEX-4T3 and expressed in E. ColiBL21. The protein was purified with affinity chromatography and used to immunize rabbit as antigen to prepare polyantibody. The full length cDNA...