Different Effects Of DHA And EPA On Insulin Resistance In 3T3-L1 Adipocytes

ObjectivesThe prevalence of noncommunicable diseases(NCDs)has steadily increased in recent decades,placing an enormous burden on already struggling health system globally.Improving insulin resistance(IR)exerts a critical effect on the early prevention and treatment of NCDs.Docosahexaenoic acid(DHA)and Eicosapentaenoic acid(EPA)has attracted great attention due to the property of anti-IR.However,DHA and EPA appear to be different in regulating IR in 3T3-L1 adipocytes which is not fully clarified.Based on that,we seek to probe into the dose-response relationship and difference between DHA and EPA in regulating adipocytes under IR status.Results of the present study will provide scientific evidence and novel perspective for the rational supplementation of DHA and EPA targeting ameliorating IR.MethodsAfter achieving confluence,3T3-L1 preadipocytes were induced to differentiate into mature adipocytes for further experiments.To induce IR in adipocytes,cells were treated with TNF-? for 48h at the concentration of 5,10,15 and 20ng/mL respectively.Insulin-stimulated glucose uptake of adipocytes was measured using GOD-POD method.Simultaneously,key molecules of insulin signaling pathway were detected by Real-time PCR and Western blot.Finally,an optimal concentration of TNF-? was selected to induce IR in the further study.Firstly,3T3-L1 adipocytes were treated with 10ng/mL TNF-? for 24h,after which DHA or EPA at the concentration of 25,50,100,200 and 400?mol/L in combination with lOng/mL TNF-? were exposed to cells respectively for another 24h.BSA treatment served as the control.MTT method was utilized to verify the cell viability.Insulin-stimulated glucose uptake,intracellular TG as well as extracellular MDA were detected by corresponding commercial kits.To explore the effects of different concentrations of DHA and EPA on lipid synthesis and lipolysis,the mRNA expression of SREBP-lc,ACC,FAS,HSL and ATGL were detected by Real-time PCR.To test the effects on adipokines,adiponectin and leptin were determined by Real-time PCR and ELISA.IRS1/PI3K/Akt/GLUT4 signaling pathway was detected by Real-time PCR and Western blot to reveal the effects of DHA and EPA on IR.Further,GPR120 and PPARy,main regulators of adipocytes,were detected by Real-time PCR and Western blot to unveil the underlying mechanism.ResultsIn the current study,reduced insulin-stimulated glucose uptake and impaired insulin signaling pathway were observed to confirm the induction of IR in adipocytes by 10ng/mL TNF-?.DHA and EPA at various concentrations showed no significant repression on cell viability,and therefore,all concentrations were used in the further study.DHA and EPA at the concentration of 400?mol/L significantly increased extracellular MDA.Inhibition of insulin-stimulated glucose uptake induced by TNF-? was significantly ameliorated by 100,200?mol/L DHA and EPA without significance difference compared with control,in which DHA exerted more potent effect than EPA at the concentration of 200?mol/L.Increment of intracellular TG was observed by DHA and EPA treatments.DHA was more conducive to stimulate gene expressions related to lipid synthesis than EPA.DHA and EPA significantly reduced the expression of HSL mRNA except 400?mol/L EPA.DHA at the concentration of 200 and 400?mol/L significantly increased adiponectin mRNA expression and secretion,and the effect was stronger than EPA.With the exception of 25?mol/L DHA and EPA,all treatments reduced the expression and secretion of leptin.DHA and EPA were able to improve IR by regulating the IRS1/PI3K/Akt/GLUT4 signaling pathway.DHA and EPA could regulate PPARy expression.DHA at the concentration of 100 and 200?mol/L significantly enhanced mRNA and protein expressions of GPR120,while EPA showed no significant effect on GPR120 protein expression.Conclusions1.DHA and EPA at the concentration of 100 and 200?mol/L showed the most significant effect on improving the ability of glucose uptake.At the concentration of 200?mol/L,the effect of DHA was more potent than that of EPA.2.The differences of DHA and EPA in regulating IR adipocytes were as follows:?DHA exerted more potent effect in promoting lipid synthesis than EPA.?At the concentration of 200 and 400?mol/L,DHA increased adiponectin expression and secretion better than EPA.?DHA and EPA could improve IR by regulating IRS1/PI3K/Akt/GLUT4 signaling pathway and DHA showed more potent effect than EPA.?DHA might improve adipocyte IR through GPR120/PPARy signaling pathway.EPA could regulate PPARy expression,but exerted no significant effect on GPR120 protein expression.