Inhibiting FoxO1 Effects On Free Fatty Acid-induced Insulin Resistance And Steatosis Through SREBP-1 Pathway

Background & ObjectiveT2DM is a serious public health topic and in high incidence in the world.CDS found that the incidence of diabetes in china has made up 9.7 percent in the latest survey. The namble of diabetes patients is almost a million, and china has become a global is the fastest growing regions in diabetes in the global range.IR is a important character in the mechanism of T2DM and is also an important feature to ir is not only to t2dm of the occurrence of an important reason is also the risk factor of hypertension, stein-Leventhal syndrome and atherosclerosis.In recently, IR become a hot topic in the development of the study of the pathogenesis about T2DM.NAFLD is the most common liver damage in Type 2 diabetes as a complication, the mechanism is recognized by the insulin resistance and oxidative stress, Lipid Peroxidation, Inflammatory factors relaxing, therefore, to improve the insulin resistance and oxidative stress theoretically could protect the NAFLD. there are two ways in the IR of NAFLD:continuing lipid accumulate and metabolic disturbance in liver could induce the insulin resistance of liver and in various tissues; the unbalanced between two adipokine could induce the insulin resistance. a model of fat rich diet feeding and liver cell induced by FFA in vitro could the insulin resistance and Endoplasmic reticulum stress. Forkhead transcription factor O1 (FoxO1) is a member of Fox protein family,. The distribution of FoxO1 in insulin-responsive tissues is broad such as hypothalamus, muscle tissue, adipose tissue and liver. And it is a key regulator of intracellular energy metabolism.Liver is a major tissue to keep the level of blood glucose in fasting and FoxO1 is the key transcription factor. The character of metabolic disturbance in the insulin resistant is hyperlipermia when fasting or postprandial.Sterol regulatory element-binding proteins 1c (SREBP-lc) is a regulator of lipid gene and transcription factor lipogenesis in liver, and it also takes a important action in NAFLD. The molecules mechanism between NAFLD and the insulin resistance has not clearly define.In RXR/LXR model, SREBP-lc take a negative action by FoxO1.Materials & Methods1. HepG-2 cells were induced to a status of IR and steatosis by being exposed to 5×10-4mol/L FFA for 24 hours.2. The experiment is based on 4 groups:HepG-2 cell group cultured with normal medium, palmitic acid group, blank plasmid group, and siFoxO1 group. The transfection was carried on when 80%-90% of the cells have been fused and according to the Lipofectamine2000 prospectus.3. FoxOl siRNA vector carried a red fluorescence, so the transfection efficiency could estimate by observing the expression of red fluorescence. The expression of red fluorescence was observed under fluorescence inverted microscope and the The expression levels of FoxOl in different groups were detected by RT-PCR4. Then glucose in medium was measured by GOD-POD assay, and the glucose consumption was was counted. MTT method was used to detect the proliferation of HepG2 cells.5. Steatosis of HepG2 cells was observed by Oil Red O staining.6. The mRNA and protein expression of SREBP-1c were determined by RT-PCR and Western blot ResultsAfter transfected of siFoxO1 plasmids using Lipofectamine 2000, the mRNA expression of FoxO1 was obviously reduced by 51.2%, but the glucose consumption was obviously increased (P<0.01). However, transfection of siFoxO1 can decrease cellular lipid accumulation. The SREBP-lc mRNA and the SREBP-lc protein expression were significantly decreased after transfected of siFoxO1 plasmids (P<0.01). There was no remarkable difference between palmitic acid group, and blank plasmid group.Conclusions1. In the insulin resistant and Steatosis HepG-2 cells, the expression of FoxO1 mRNA and lipid accumulation increased; High level of FFA could affect normal insulin signal transduction, disorder of lipid metabolic and cause IR and Lipid accumulation.2. Inhibiting the expression of FoxO1 could improve FFA-induced IR and steatosis by down-regulating SREBP-1c expression.