Effects Of Gene Silencing Of SOCS-3 On InsR MRNA Expression In Hypothalamus Of Obese Rats Fed With High-fat Diet

Background:Children obesity has shown an upward trend step by step. Obesity is a significant health problem and is associated with increased risks of cardiovascular disease, hypertension, type 2 diabetes mellitus, chronic inflammation and gallstone.Experiments have found that the level of leptin and insulin were high in obese people suggesting obese exist leptin resistance and insulin resistance. Insulin resistance is a state in which a given concentration of insulin produces a less-than-expected biological effect. In particular, it is characterized by a decrease in the ability of insulin to stimulate the use of glucose by muscles and adipose tissue and to suppress hepatic glucose production and output. Insulin and leptin have the same signal transduction pathway that Janus kinase(JAK)/signal transducers and activator of transcription (STAT) pathway. Suppressors of cytokine signaling 3(SOCS-3) is the major negative factor of JAK/STAT pathway.Many cytokines'signaling were regulated by SOCS-3.A large number of basic research results show that serum insulin level were increased, the expression of insulin receptor were decreased and the expression of SOCS-3 gene were increased in the animal models of obesity. The up-regulation of SOCS-3 may be involved in the insulin resistance. As a new technology of suppress gene expression, RNA interference has been widely used for mammalian gene function and disease therapeutic research. Thus,it is possible to increased the expression of InsR and become an effective treatment for insulin resistance in obesity ultimately through down-regulation of SOCS-3 expression by RNAi.Objectives: To imitate the feature of children obesity, 5-week-old (equal to juvenile stage) Spraque-Dawley (SD) rats were selected as rodent in the experiment.Using RNAi of SOCS-3,we observed body weight,serum insulin,glucose and C-peptide concentration as well as levels of InsR mRNA in hypothalamus of SD rats fed with high-fat diet in order to study that the expression of InsR mRNA may be increased and insulin resistance in obesity can also be alleviated.Methods: In pre-experiment , total RNA were extracted from hypothalamus of rats;serum insulin and C-peptide were measured with radioimmunity;the concen- tration of serum glucose were measured by automatic biochemistry analyzer. Samples were selected in two groups randomly.(1)In the RNAi+ high-fat diet group(n=10), designated as RNAi+ HFD, the SOCS-3 lenti were stereotaxically injected into the hypo- thalamus of rats which then fed with high-fat diet containing 20% lard;(2)In the control lenti + high-fat diet group(n=8),designated as Con+HFD,the control lenti were stereotaxically injected into the hypothalamus of rats which then fed with high-fat diet containing 20% lard;The expression of InsR mRNA in the hypothalamus was detected with semi-quantitative RT-PCR (usingβ-actin as an internal standard) and fluorescent quantitative PCR (using GAPDH as an internal standard) respectively.Results:1. The body weight in RNAi+HFD group was decreased evidently compared with Con+HFD group. And there were significant differences between two groups at the end of 8 weeks (p<0.01).2. The concentrations of serum insulin, glucose and C-peptide in rats of RNAi+HFD group were all decreased compared with that of Con+HFD group, and there were significant differences between two groups(p<0.05).3.The expression (relative gray value)of InsR mRNA with semi-quantitative RT-PCR in RNAi+HFD group were elevated obviously compared with that in Con+HFD group, and there were significant differences between two groups (p<0.01).4.The expression (relative value) of InsR mRNA with fluorescent quantitative PCR in RNAi+HFD group were elevated compared with that in Con+HFD group, and there were significant differences between two groups (p<0.05).Conclusions:1.The concentrations of serum insulin, glucose and C-peptide were all decreased through down-regulation of SOCS-3 expression by RNAi, suggesting that down-regulation of SOCS-3 expression can effectively improve glycometabolic disorder and insulin resistance.2.The expression of InsR mRNA were increased through down- regulation of SOCS-3 expression by RNAi,suggesting that down-regulation of SOCS-3 expression may be a promising therapeutic approach for insulin resistance in obesity.