Effect Of Oleic Acid On Adiponectin In 3T3-L1 Adipocytes And The Underlying Mechanisms Mediated By CDK5/PPAR?

BackgroundAdiponectin is a cytokine secreted by fat cells,which plays an important role in the regulation of glucose and lipid metabolism,improving insulin sensitivity,and anti atherosclerosis.It was found that dietary fatty acids may affect the expression of adiponectin.Oleic acid is a monounsaturated fatty acid,which can regulate glucose and lipid metabolism,improve endothelial function and induce an anti-inflammatory effect.But the effects of oleic acid on adiponectin remain controversial,as well as the mechanism that how oleic acid regulates adiponectin is unclear.Peroxisome proliferator activated receptor ?(PPAR?)is a nuclear receptor and it response element PPRE exists in the adiponectin gene promoter.PPAR? agonist can promote the expression of adiponectin by upregulating PPAR?.Another study found that CDK5 in obese mice induced by high-fat diet were abnormally activated,which can phosphorylate PPAR? in the 273 serine and down-regulate the expression of adiponectin.So whether the regulating effect of oleic acid on adiponectin is mediated by PPAR? or CDK5/PPAR? pathway is waiting for explore and study systematically.ObjectiveIn this study,to comprehensive and systematic discussion on the effect of oleic acid on adiponectin,different concentrations of oleic acid were used on induced differentiation maturely 3T3-L1 adipocytes,and observation the effects of different concentrations oleic acid on adiponectin,PPAR?,CDK5 and other related molecules.Then choose the concentration of oleic acid which can affect adiponectin treated cells respectively,in order to explore the underlying molecular mechanism of different oleic acid concentration effects of adiponectin,so that provide theoretical basis for guiding the prevention and treatment of obesity and related metabolic disease.Methods1 In vitro cultured and induced differentiated maturation of 3T3-L1 fat cells.Choosing different concentrations(25,50,100,200,400?mol/L)oleic acid treated cells 24h.Positive control group are the cells treated Rosiglitazone(PPARy agonist,which can promote the expression of adiponectin)only.Blank control group are cells treated BSA only.Then we detected the expression of adiponectin,PPAR?,CDK5 mRNA and intracellular protein by real-time PCR and Western-blot method.2 Choosing the concentration of oleic acid which can affect adiponectin treat 3T3-L1 mature fat cells 24h,then determining the adiponectin,PPAR?,CDK5 mRNA and protein expression by real-time PCR and Western-blot,and determining the level of PPAR?ser273 phosphorylation.3 Using the appropriate concentration(100?mol/L)of oleic aicd with or without GW9662 which is a PPARy inhibitor treat 3T3-L1 mature fat cells 24h.The inhibitor group is cells treated GW9662 only.Blank control group are cells treated BSA only.Then we detected the adiponectin,PPARy,CDK5 mRNA and protein expression by real-time PCR and Western-blot,and determining the level of PPARyser273 phosphorylation.4 Using the high concentration(400?nol/L)of oleic aicd with or without Roscovitine which is a inhibitor of CDK5 activity treat 3T3-L1 adipocytes 24h.The inhibitor group is cells treated Roscovitine only.Blank control group are cells treated BSA only.Determining the adiponectin,PPARy,CDK5 mRNA and protein expression by real-time PCR and Western-blot,and determining the level of PPAR?ser273 and p-CDK5(15Tyr)phosphorylation.Results1 There are differents effect of different concentrations of oleic acid on adiponectin expression,which showed that low concentrations of oleic acid is promotion and the high concentration is inhibition.In 50,100?mol/L,oleic acid can promote the expression of adiponectin mRNA and protein(P<0.01;P<0.01),and 200?mol/L oleic acid can promote adiponectin mRNA expression(P<0.05).The effect of promotion is the most significant in 100?mol/L.With the increase of concentration,the effect of oleic acid on the promotion of adiponectin has decreased,and oleic acid could inhibit the expression of adiponectin at 400?mol/L(P<0.01;P<0.05).2 The effect of different concentrations of oleic acid on the expression of PPARy is also different:oleic acid can increase PPARy mRNA and protein expression at 50,100?mol/L(P<0.01;P<0.01),but the level of PPAR? ser273 phosphorylation did not change(P>0.05).PPARy protein did not change at the 400?mol/L oleic acid(P>0.05),but the level of PPAR? ser273 phosphorylation had increased(P<0.01).Different concentrations of oleic acid had no significant effect on CDK5 mRNA and protein(,P>0.05;P>0.05).3 The suitable concentration(100?mol/L)of oleic acid upregulated adiponectin and PPARy mRNA and protein expression(P<0.01;P<0.01).Adding PPARy inhibitor which can inhibit PPAR? effectively inhibited the upregulated effect of oleic acid on adiponectin mRNA and protein significantly(P<0.01;P<0.01),and the adiponectin mRNA and protein significantly decreased,even lower than the control group(P<0.01;P<0.05).4 High concentration(400?mol/L)of oleic acid could downregulate adiponectin,and upregulate the level of PPAR?ser273 phosphorylation and p-CDK5(15Tyr)(P<0.01;P<0.05).Using an inhibitor of CDK5 activity could decrease the level of p-CDK5(15Tyr)(P<0.05)and make PPAR?ser273 phosphorylation return to the level of the control group(P>0.05),and increse the adiponectin mRNA and protein to the level of control group(P>0.05;P>0.05).Conclusions1 The regulated effect of oleic acid on adiponectin was different with the concentrations.The expression of adiponectin mRNA and protein could be up-regulated by oleic acid at 50,100,200?mol/L,and be inhibited at 400?mol/L.2 The suitable concentration(100?mol/L)of oleic acid could increase the expression of adiponectin mediated by upregulating PPARy mRNA and protein expression.3 The high concentration(400p,mol/L)of oleic acid could inhibit the expression of adiponectin mediated by increasing the activity of CDK5 and the level of PPAR?ser273 phosphorylation.