Different Effects Of N-3 And N-6 PUFA On Adiponectin Expression And The Underlying Mechanism Mediated By CDK5/PPARγ

Background and ObjectiveAdiponectin has been considered as a valuable target for the prevention and treatment of obesity and its related diseases.Effect of PUFA on adiponectin has attracted a wide attention.Although existing evidences suggest that n-3 and n-6 PUFA may have distinct impacts on adiponectin,there is limited study designed specifically to explore it.The present study therefore aimed to observe and compare the individual effects of n-3 and n-6 PUFA on adiponectin under normal and inflammatory conditions using 3T3-L1 adipocytes.Meanwhile,whether n-3 and n-6 PUFA regulated adiponectin through PPARγ and CDK5-mediated PPARγphosphorylation were tested to find a possible explanation for their distinct actions.Results of the study will provide a new scientific evidence and new perspective for the rational intake of dietary PUFA as well as the prevention and treatment of obesity and its related diseases.MethodsAfter becoming 100%confluent,3T3-L1 preadipocytes were induced to differentiate by dexamethasone,insulin and IBMX.Cells that more than 80%became mature adipocytes could be used for the subsequent experiments.Under normal condition,3T3-L1 adipocytes were firstly treated with 100μmol/L n-3(EPA,DHA,α-ALA)and n-6 PUFA(LA,AA)for 24h respectively.Secondly,3T3-L1 adipocytes were treated with n-3 and n-6 PUFA in the presence or absence of GW9662 for 24h.Thirdly,3T3-L1 adipocytes were pre-treated with EPA,α-ALA and LA for 24h,followed by a co-incubation with roscovitine for another 2h.Finally,3T3-L1 adipocytes were pre-treated with DHA and AA for 24h,followed by a co-incubation with TNF-α for another 2h.In terms of inflammatory condition,3T3-L1 adipocytes were firstly treated with 100,200 and 300μmol/L PA to establish inflammatory model.Secondly,3T3-L1 adipocytes were treated with PA in isolation or in conjunction with rosiglitazone for 24h.Thirdly,3T3-L1 adipocytes were pre-treated with PA for 24h,followed by a co-incubation with roscovitine for another 2h.Finally,3T3-L1 adipocytes were co-treated with PA and 100μmol/L n-3 or n-6 PUFA for 24h.Adiponectin,PPARγ,p-PPARγ,CDK5,p-CDK5 IL-6,MCP-1,TNF-α,interaction between CDK5 and PPARγ were measured using real-time RT-PCR,Western blot,ELISA and Co-IP,respectively.ResultsUnder normal condition,n-3PUFA increased adiponectin and PPARγ expression,among which DHA worked best.LA,as a kind of n-6PUFA,only led to transcriptional increases in adiponectin and PPARγ,while AA had no effects on any of them.The co-incubation of PUFA and GW9662 resulted in a reduction in PPARy as well as adiponectin.EPA,α-ALA and LA stimulated PPARγ phosphorylation,but DHA and AA inhibited it.Although the addition of roscovitine blocked EPA-,α-ALA-and LA-induced increases in PPARγ phosphorylation,the corresponding increases in adiponectin were slight.The addition of TNF-α blocked DHA-and AA-induced decreases in PPARγ phosphorylation,which eventually reduced adiponectin expression.After PUFA treatments,changes in CDK5 activity and interaction between CDK5 and PPARγ were similar to PPARy phosphorylation:EPA,α-ALA and LA stimulated them but DHA and AA inhibited them.Under inflammatory condition,200μmol/L PA appeared to be more potent than 100 and 300μmol/L in enhancing IL-6 and MCP-1.In the process of establishing inflammatory model,PA decreased both adiponectin and PPARγ.The addition of rosiglitazone inhibited PA-induced decrease in PPARγ,with a concurrent increment in adiponectin.Meanwhile,PA stimulated CDK5 activity,interaction between CDK5 and PPARγ and PPARγ phosphorylation.The addition of roscovitine led to an increase in adiponectin by blocking PA-induced PPARγ phosphorylation.Except for AA,n-3PUFA and LA were found to be capable of improving PA-induced decreases in adiponectin and PPARγ.Only EPA and DHA attenuated PA-induced stimulations of CDK5 activity,interaction between CDK5 and PPARγ and PPARγphosphorylation.Conclusion1.In 3T3-L1 adipocytes,n-3PUFA increased adiponectin expression whereas n-6PUFA had no obvious effects.N-3PUFA regulated adiponectin through both PPARγ and CDK5/p-PPARγ pathways,while n-6PUFA mainly affected CDK5/p-PPARγ.PPARγ expression compared with CDK5/p-PPARγ led to a greater impact on adiponectin expression,the different effects of n-3 and n-6 PUFA on adiponectin were hence attributed to their distinct regulations of PPARγ expression.2.In the process of establishing inflammatory model,PA inhibited adiponectin expression via PPARγ and CDK5/p-PPARγ.Under inflammatory condition,n-3PUFA was more effectively than n-6PUFA in improving adiponectin expression.EPA and DHA regulated adiponectin by PPARγ and CDK5/p-PPARγ,while α-ALA and LA regulated adiponectin only by PPARγ expression.Different effects of n-3 and n-6 PUFA on adiponectin were considered to be associated with their distinct impacts on PPARy expression as well as CDK5/p-PPARγ.3.n-3PUFA except for DHA stimulated adiponectin expression in different way under normal and inflammatory conditions.As a kind of n-6PUFA,LA only increased adiponectin in the inflammatory state by modulating PPARγ expression.n-3PUFA appeared to be more effective than n-6PUFA in the prevention and treatment of obesity and its related diseases.