The Influence Of Down-regulation Of Suppressor Of Cellular Signaling Proteins On Glucose Transport Of Intrauterine Growth Retardation Rats

Objective To primary culture and identify skeletal muscle cell of intrauterine growth restriction (IUGR) rats. To investigate the expression change of insulin receptor substrate-1(IRS-1), suppressors of cytokine signaling-1 (SOCS-1) and suppressors of cytokine signaling-3 (SOCS-3) which is important in insulin signaling pathway in skeletal muscle cell of intrauterine growth restriction (IUGR) rats, in order to explore the effect on the incidence of insulin resistance in adult IUGR.Methods IUGR animal models were established by maternal nutrition restriction. Skeletal muscle cell was primary cultured by tissue dry-up method. Identified skeletal muscle cell by the method of SABC. The levels of SOCS-1, SOCS-3 and IRS-1 in skeletal muscle cells of new-born and 12-week IUGR rats were detected by Real-time PCR and Western blot.Results Birth weight and birth length of IUGR rats were less than the control(P< 0.05). The relative expression levels of SOCS-1, SOCS-3 were higher than those in control group (both P<0.05). The relative expression level of IRS-1 was lower than control group (P<0.05). The amount of IRS-1 mRNA and protein expression were negatively correlated with the mRNA and protein expression of SOCSs.Conclusion Increased expression of SOCS-1,SOCS-3 contributed to the decreased expression of IRS-1, which maybe play an important role in insulin resistance in IUGR rats. SOCS-1 and SOCS-3 might provide new ideas and methods in type 2 diabetes. Objective To construct the recombinant vector containing the suppressors of cytokine signaling-1(SOCS-1) short hairpin RNA(shRNA) and suppressors of cytokine signaling-3(SOCS-3) shRNA. To select most effective of shRNA for follow-up study.Methods Two DNA sequences containing a small hairpin structure were designed and synthesized. The complement form was obtained by annealing and cloning into vector pGPU6/GFP/Neo, and the recombinant plasmid was transformed into strain DH5a. The plasmid identified by restriction enzyme analysis was used for sequencing. Skeletal muscle cells were transfected with plasmid vector. The levels of SOCS-3 skeletal muscle cells were detected by Real-time PCR and Western blot.Results The recombinant plasmid were cloned, and the sequences were obtained. Transfection efficiency was above 80%. The mRNA and protein expression of SOCS-1 and SOCS-3 were lower than the blank control group and the negative control group (both P<0.05), and SOCS-1-shRNAb and SOCS-3-shRNAa were the most effective one.Conclusion The recombinant vector containing SOCS-1-shRNA and SOCS-3-shRNA gene were successfully constructed and could specifically inhibit SOCS-1 and SOCS-3 expression, which will provide a gene therapy protocol for the metabolic syndrome. Objective To evaluate the inhibition effect of short hairpin RNA (shRNA) on the expression of suppressor of cellular signaling-1(SOCS-1) and suppressor of cellular signaling-1(SOCS-3) and insulin-stimulated glucose transport in intrauterine growth retardation (IUGR) skeletal muscle cells in basal and insulin-stimulated state.Methods An IUGR animal model was established by maternal food restriction in Sprague Dawley rats. Skeletal muscle cell was primary cultured by tissue dry-up method. Constructed shRNAs targeting SOCS-1 and SOCS-3 were transfected into 12-week old IUGR skeletal muscle cells. Insulin-stimulated glucose transport was detected via immunofluorescence. Levels of glucose transporter4(GLUT4) protein in the quadriceps femoris were measured by Western blot.Results Recombination plasmids pGPU6/GFP/Neo-SOCS-1-shRNA and pGPU6/ GFP/Neo-SOCS-3-shRNA were successfully constructed and the shRNAs efficiently inhibited the expression of SOCS-1 and SOCS-3 of 12-week old intrauterine growth retardation (IUGR) skeletal muscle cells. Immunofluorescence showed that insulin-stimulated GLUT4 translocation was elevated compared to the blank group in basal and insulin-stimulated (10-11-,10-9,10-7mol/l) states, and the level was proportional to the concentration of insulin. Levels of GLUT4 protein in the quadriceps femoris were increased in 10-7mol/l insulin-stimulated state compared to the basal state in each group. GLUT4 protein level was proportional to phosphorylation of Akt.Conclusion This data provides additional information on the mechanism of insulin resistance associated with IUGR. Down-regulation of SOCS-1 and SOCS-3 ameliorates the capacity of glucose transport, and provides a potential gene therapy approach to managing metabolic syndrome.