Effects Of Different Fatty Acids On Expression Of Adiponectin In 3T3-L1 Adipocytes And The Relationships Between Adiponectin,PPARγ And Cdk5

BackgroundAdiponectin is a special cytokine which is secreted by adipose tissue. It plays a very important role in the processes of cardiovascular diseases, weight control, and glucose and lipid metabolism. Increasing evidences have shown that dietary factors espically fatty acids can influence the expression of adiponectin. Aprevious study found that promoter reigon of adiponectin gene contained peroxisome proliferator activated receptor gamma (PPARy) reponse element. As a result, PPARy agonist can enhance insulin sensitivity by stimulating the expression of adiponectin. Besides, cyclin dependent kinase 5(Cdk5) activated by high-fat diet was proved to inhibit the expression of adiponectin through regulation of PPARy phosphorylation in mice. To date, definite effects of fatty acids on adiponectin and the underlying mechanism are still unclear, which need to be futher studied.ObjectiveTo explore changes in adiponectin expression in 3T3-L1 adipocytes incubated with different types of fatty acids with different doses (25,50,100,200 and 400μmol/L), and then seek the fatty acidsand concentrations that can obviously increase adiponectin. Finally, analyze the relationships between adiponectin, PPARy and Cdk5. Results of the present study will provide a great theory value and practical importance to the prevention and treatment of obesity and its related diseases.Methods1. After differentiation of 3T3-L1 pre-adipocytes, the mature 3T3-L1 adipocytes were incubated with n-3 polyunsaturated fatty acids [eicosapntemacnioc acid(EPA), docosahexaenoic acid(DHA) and a-Linolenic acid(a-ALA)], n-6 polyunsaturated fatty acids [arachidonic acid (AA) and linoleic acid (LA)], monosaturated fatty acidoleic acid (OA) and saturated fatty acid [palmitic acid(PA)]with different concentrations (25,50,100,200 and 400μmol/L) for 24h.Blank control, negative control (10mol/L of GW9662) and positive control (10mol/L of rosiglitazone) were set up.The relative expression of adiponectin,PPARyand Cdk5 mRNA were determined by real-time PCR.2.3T3-L1 adipocytes were respectively incubated with 100μmol/L or 400μmol/L of EPA, DHA, a-ALA, AA, LA, OA and PA, repectively.The intracellular protein expression of adiponectin, PPARy and Cdk5 were determined by Western-blot.3.3T3-L1 adipocytes were respectively incubated with 100μmol/L of EPA, DHA, a-ALA, AA, LA, OA and PA. The medium protein expression of adiponectin was determined by Western-blot.4. All data were expressed as mean±standard deviation (X±SD) by SPSS 19.0 software. One-Way ANOVA analysis was used on multiple samples, followed by variance analysis of homogeneity.If variances were homogeneous, LSD T-test was usedfor comparisons of two groups, other wise, Dunnett’s T3 was used. Pearson correlation analysis tested whether two variables were correlated. P< 0.05 means significant difference.Results1. Effects of different n-3 PUFAs on expression of adiponectin, PPARy and Cdk5 mRNA(1)Effects of DHA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, compared with control, the expressions of adiponectinand PPARy mRNA significantly increased (P<0.01) in 25,50,100 and 200μ/L group, among which 100μmol/L worked best.400 mol/L DHA decreased the expression of adiponectin and PPARy mRNA obviously (P<0.01). The expression of Cdk5 mRNA increased (P<0.05) in 100μmol/L group but decreased (P<0.05) in 400μmol/L group.(2)Effects of EPA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, compared with the control, the expression of adiponectin, PPARymRNA significantly increased in 25,50,100 and 200μmol/L group. The highest adiponectin and PPARymRNA expression wereobserved in 200μmol/L group (P<0.05).The expression of Cdk5 mRNA increased (P<0.05) in 50,100 and 200μmol/L groups. In 400μmol/L group, the adiponectin mRNA expression significantly decreased (P<0.05), but the PPARy and Cdk5 mRNA expressions had no significant changes (P> 0.05).(3) Effects of a-ALA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, the expression of adiponectin mRNA increased markedlyin 50 and 100μmol/L group (P<0.05), and 100μmol/L was more effective. In 25 and 50μmol/Lgroup, the expression of PPARy mRNA markedly increased (P<0.05), but it decreased significantly in 400μmol/L group (P<0.05). a-ALA had on effect of Cdk5 mRNA.2. Effects of different n-6 PUFAs on expression of adiponectin, PPARy and Cdk5 mRNA(1) Effects of AA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, adiponectin gene expression gradually reduced along with increase of concentrations. Compared with the control, the mRNA expression of adiponectin increased (P<0.05) in 25μmol/L group, but deceased (.P<0.05) in 400μmol/L group. Both the mRNA expression of PPAR y and Cdk5 increased significantly in 50μmol/L group. As the increase of the concentration, PPARγ and Cdk5 mRNA expression decreased, and both of them were the lowest in 400μmol/L group (P<0.05).(2) Effects of LA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, compared with the control, the expression of adiponectin mRNA increased obviously (P<0.05) in 50 and 100μmol/L group. The expression of PPARy mRNA increased significantly (P<0.05) in 200μmol/L group. But both of adiponectin and PPARγ mRNA expression decreased in 400μmol/L group. Cdk5 mRNA increased in 400μmol/L group.3.Effects of OA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, compared with the control, the expression of adiponectin and PPARy mRNA increased in 25,50 and 100μmol/L group, among which 100μmol/L had the best effects. Both of adiponectin and PPARy mRNA expression decreased (P<0.05) in 400μmol/L group. The expression of Cdk5 mRNA decreased obviously (P<0.05) in high dose groups.4.Effects of PA with different doses on expression of adiponectin, PPARy and Cdk5 mRNAAfter incubation, although the mRNA expression of adiponectin and PPARy decreased in a dose-dependent manner, a significant increased (P<0.05) were observed in 25μmol/L group. Compared with the control, the mRNA expressions of adiponectin and PPARy were the lowest at the concentration of 400μmol/L. The expression of Cdk5 mRNA decreased in 400μmol/L group.5. Effects of different fatty acids at the concentration of 100 and 400μmol/L on the intracellular protein expression of adiponectin, PPARy and Cdk5(1) Effects of different fatty acids on the intracellular protein expression of adiponectin, PPARy and Cdk5 at the concentration of 100μmol/L3T3-L1 adipocytes were incubated with EPA, DHA, a-ALA, LA, AA, OA and PA respectively at concentration of 100μmol/L for 24h. Compared with control, EPA, DHA, ALA and OA significantly increased (P<0.05) the intracellular protein levels of adiponectin, while AA, LA and PA had no effect on it. The intracellular protein levels of adiponectin were significant different between n-3 PUFAs, AA, LA and PA group.Compared with control, the expression of PPARy intracellular protein levels increased only in the group of DHA, ALA and LA, and it was significant different between n-3 PUFAs and n-6 PUFAs.The intracellular protein levels of Cdk5 had no significant changes in all groups.(2) Effects of different fatty acids on the intracellular protein expression of adiponectin, PPARy and Cdk5 at the concentration of 400μmol/L3T3-L1 adipocytes were incubated with EPA, DHA, a-ALA, LA, AA, OA and PA respectively at concentration of 400μmol/L for 24h. The intracellular protein levels of adiponectin significantly reduced (P<0.01) in all the groups except for control.Compared with the control, the intracellular protein levels of PPARysignificantly reduced (P<0.01) in all the groups except for control, which was similar with adiponectin intracellular expression.400μmol/L of different fatty acids made no changes in the intracellular protein levles of Cdk5(P>0.05).6.Effects of different fatty acids at the concentration of 100μmol/L on the expression of adiponectin protein in the medium3T3-L1 adipocytes were incubated with EPA, DHA, a-ALA, LA, AA, OA and PA respectively at concentration of 100μmol/L for 24h. The adiponectin protein levels in the medium increased markedly (P<0.05) in group EPA, DHA and ALA. The group of AA, LA and OA made no changes in the expression of adiponectin protein in the medium. The secretion of adiponectin in PA group decreased without statistical significance (P>0.05).The expressions of adiponectin protein in the medium were significant different between n-3PUFAs groups, AA, LA, OA and PA (P<0.05).7.Relationships between adiponectin, PPARy and Cdk53T3-L1 adipocytes were incubated with EPA, DHA, a-ALA, LA, AA, OA and PA at concentration of 100 or 400μmol/L respectively. Resultes of correlation analysis indicated that adiponectin mRNA positively related to PPARy mRNA (P<0.05), while Cdk5 did not relate to either adiponectin mRNA or PPARy mRNA. In terms of intracellular protein, adiponectin positivitly related to PPARy (P<0.01),but Cdk5 negatively related to adiponectin (P<0.05) and PPARy (P<0.05)Conclusions1.Compared with other doses,100μmol/L is more effective in stimulating expression of adiponectin, while 400μmol/L inhibits it.2.Compared with n-6PUFAs, n-3PUFAs and OA increase the expression of adiponectin more significantly in 25μmol/L,50μmol/L and 100μmol/L group. But PA only increases the expression of adiponectin in 25μmol/L group.3.In all 100μmol/L groups, DHA increases the expression of adiponectin more effectively than other n-3PUFAs like EPA and a-ALA.4. AA, which belongs to n-6PUFAs, increases the expression of adiponectin only in low concentration (25μmol/L). But LA, another n-6PUFA, can increase the expression of adiponectin in 100μmol/L5. Both mRNA and intracellular protein of adiponectin are positively relate d to PPARγ in 3T3-L1 adipocytes incubated with 100 or 400 μmol/L of fatty acids, while Cdk5 mRNA dose not relate to either adiponectin mRNA or PPA Rγ mRNA, Cdk5 intracellular protein nagetively relates to adiponectin and PP ARγ intracellular protein.