The Effect Of Oxidative Stress And Intervention Of Pioglitazone On The Expression Of SOCS-3in3T3-L1Adipocytes

Obesity is a worldwide epidemic disease. The incidence of obesity in develpoing countries increasd gradually in several decades,accompanied with the great economic achievement and altered lifestyle.The developing countries was becoming the new harder-hit area of obesity.Obesity is not only simple disease but the important risk factor of diabetes,athrosclerosis diseases or tumor. Besides of the function of energy storage,fat tissue take the important role in mechanism of insulin resistance, and has more endocrine function by releasing some cytokines to regulate insulin sensitivity in insulin target organs.SOCS-3(suppressor of cytokine signaling-3)is the principal member of SOCS family.In recent researches,SOCS-3was found take part in the mechanism of leptin resistance,the signal induction of insulin and insulin resistance.The enhanced oxidative stress was known as the mediator of insulin resistance,but the mechanism was not clearly illustrated.There was little research refered to the effect of oxidative stress on SOCS-3in adipocytes. Thiazolidinediones(TZDs), agonist of PPAR-γ,which was represented by pioglitazone were widely used as antidiabetic drugs, has the multiple effects by PPAR-γ activation to ameliorate the insulin sensitivity. Whether the intervention of pioglitazone has effect on SOCS-3of adipocytes under oxidative situation was not mentioned.Our study investigated the mature3T3-L1adipocytes,which was given the stimulation of H2O2. The imitation of oxidative stress was attained by increased intracellular level of ROS.We detected the increased intracellular ROS by flow cytometry, the altered expression of SOCS-3gene and protein was measured by real time PCR or Western blot. The another part of study focus on the investigation of effect of antioxidant (a-lipoic acid)and PPAR-Y agonist (pioglitazone) on SOCS-3.Our study aimed to understand more about mechanism of insulin resistance and find new therapeutic attempt.Material and Methods1.Murine3T3-L1preadipocytes were cultured and were treated with a differentiation mixture containing insulin, dexamethasone and isobuthylmetylxantine. Mature adipocytes were confirmed by Oil Red O staining and detection of adiponectin mRNA expression(which is the marker gene of mature adipocytes).2.We utilized two ways to mimic the different conditions of oxidative stress for3T3-L1adipocytes.Acute oxidative stress:Direct exposure to0.5mM H2O2for just15min Chronic oxidative stress:Addition to100mU/ml glucose oxidase (GO) into culture containing DMEM for4hours,which resulted in a relatively lower H2O2concentration in the medium.3. Treatment:Control:3T3-L1adipocytes without H2O2treatmentH:3T3-L1adipocytes exposure to0.5mM H2O2for15minH1:3T3-L1adipocytes exposure to0.5mM H2O2for5minH2:3T3-L1adipocytes exposure to0.5mM H2O2for10minGO:100mU/ml glucose oxidase (GO) in3T3-L1adipocytes medium for4hoursGO+LA:100mU/ml glucose oxidase (GO) and2.5mmol/Lα-lipoic acid in3T3-L1adipocytes medium for4hoursGO+PGZ:10μmol/L Pioglitazone pre-treatment and100mU/ml glucose oxidase (GO) treatment for4hours.4.Experimental methodsThe increased level of intracellular ROS (Reactive Oxygen Species) was detected by flow cytometry. The expression of SOCS-3mRNA and protein were measured by real-time PCR and western blot. A series of adipokines including adiponectin,resistin,tumor necrosis factor-mRNA was determined by PCR. The concentration of adiponectin in medium was measured by ELISA.Results1①Mature3T3-L1adipocytes were confirmed by detection of adiponectin mRNA and Oil red staining on the10th day after induction of differentiation. Lipid droplets were accumulated in mature adipocyte. Adiponectin mRNA increased gradually with the progression of differentiation.②Exposure to H2O2(0.5mM) for5,10min resulted in dramatically increased intracellular ROS detected by FACS (P<0.05).After4hours GO treatment or H2O2treatment for15min, the level of intracellular ROS was significantly increased.(P<0.05), the treatment of α-lipoic acid attenuated the increased level of ROS in3T3-L1adipocytes treated by GO(P<0.05).③The expression of SOCS-3was up-regulated after the treatment of0.5mM H2O2, the change was significant after10min (P<0.05). The SOCS-3expression in those cells treated by GO was also significantly up-regulated, SOCS-3mRNA expression increased4to6folds compared with control.(P<0.05) In cells treated by GO and α-lipoic acid the expression of SOCS-3mRNA was decreased approximately50%(P<0.05) compared with that of cells treated by GO.④The level of adiponectin mRNA decreased in cells treated by either H2O2or GO(P<0.05). The levels of TNF-α and resistin mRNA increased (P<0.05). The concentration of secretory adiponectin in the culture medium of3T3-L1adipoytes were decreased significantly in cells by GO treatment (P<0.05). In cells treated by GO and α-lipoic acid, the above change were attenuated significantly(P<0.05).2①In3T3-L1adipocytes which was pretreated by10μ mol/L pioglitazone, the level of intracellular ROS was increased significangtly compared with control after4-hour GO treatment(P<0.05). Although the difference was not significant(P>0.05), the level of intracellular ROS was a little lower than that of those cells untreated by pioglitazone.②The level of SOCS-3mRNA and protein were significantly lower in cells pretreated by pioglitazone(P<0.05) compared with which in3T3-L1adipocytes treated by GO.③The level of adiponectin mRNA was significantly higher in cells pretreated by pioglitazone(P<0.05) compared with which in3T3-L1adipocytes treated by GO.Conclusions1.The increased level of intracellular ROS could induce the up-regulation of SOCS-3in mature3T3-L1adipocytes,accompanied with the modulation of adipokines,above changes were hypothesized to have a role in the molecular mechanisms of resulting insulin resistance in fat tissue.The antioxidant, a-lipoic acid could attenuated the effect of ROS,which implied the antioxidant therapy could have the potential value of prevention or treatment on IR by partly inhibiting SOCS-3.2. PPPAR γ agonist pioglitazone could partly inhibit the up-regulation of SOCS-3in3T3-L1adipocytes under oxidative stress and this effect perhaps was not totally dependent on the lowered intracellular ROS.We speculated pioglitazone take effect on ameliorating insulin resistance not only by PPPAR γ activation but inhibiting SOCS-3through some unknown mechanism. TZD maybe have more beneficial biological effect besides of insulin sensitilizer.