Effects Of N-6/n-3PUFAs Composition On Adiponectin PPARγ In3T3-L1Adipocytes And Its Modulation

BackgroundAdiponectin is a newly discovered cytokine secreted by fat cells, and paid close attention to the role in the regulation of glucose metabolism and energy balance, improve insulin sensitivity and resistance to atherosclerosis by people. More and more evidence shows, n-6and n-3polyunsaturated fatty acids (PUAFs) have different role in the regulation of lipids, inflammation, and other chronic diseases. Peroxisome proliferator-activated receptor (PPARy), as fatty acid receptors, plays an important role in the regulation of fat cell differentiation, and the regulation of glucose and lipid metabolism.the mechanism of PUFAs adjusting adiponectin expression has not clear.PurposeTo study the effects of n-3and n-6PUFAs on the gene and protein expression of adiponectin and the gene expression of PPARy in3T3-L1adipocytes, and observe the possible relationship between adiponectin and peroxisome proliferator activated receptor. It is aim to seek the possible fatty acid or concentration that can up regulating the expression of adiponectin, and the mechanism in3T3-L1adipocytes. It will have great theory value and practical importance to provide the therapeutics or prevention on obesity related disease with original scientific proofs.Methods1. After induced into adipocytes and identified by Oil Red O staining, the3T3-L1 adipocytes were treated for Linoleic acid (LA), Docosahe--xaenoic acid (DHA) at different ratio(1:1,5:1,10:1,20:1and30:1) concentration (25、50、100、200、400μmol/L) for24h, and the gene expression of adiponectin mRNA and PPARy mRNA was determined by real-time PCR(relative quantity).1. And then the3T3-L1adipocytes were treated for Linoleic acid (LA), Docosahe-xaenoic acid (DHA) at concentration (25、50、100、200、400μmol/L) for24h, and the protein expression of adiponectin was determined by western blotting(relative quantity).2. The data were shown with the mean±standard deviation (X±s) by SPSS13.0software. Use One-Way ANOVA analysis on multiple sample mean, before which the variance analysis of homogeneity was made for all inspection, if variance was homogeneous, use the analysis of variance and LSD T-test and dunnett T-test method for two groups of comparisons and more than two; if variance was not homogeneous, use Welch method and Dunnett’s T3method for two groups of comparisons and more than two comparison. Pearson correlation analysis tested if the two variables were correlated. Pearson correlation coefficient r and P were given in statistical results, P<0.05means significantly different.Results1. Effects of n-6/n-3PUFAs composition on expression of adiponectin mRNA and PPARy mRNA1.1Effects of n-6/n-3PUFAs (1:1) on expression of adiponectin mRNA and PPARy mRNAThe mRNA expression of adiponectin in n-6/n-3PUFAs group were increased at25、50、100、200、400μmol/L(118.18%、152.73%、154.55%、56.36%、38.18%),and both with a significant increased compared with the control at the concentration of25、50、100μmol/L(p=0.001;p=0.000;p=0.000).Compared with the control, when the n-6/n-3PUFAs concentration were50μmol/L、100μmol/L,PPARy mRNA expression increased(p=0.008; p=0.045), Adiponectin and PPARy mRNA expression display a significant positive correlation, coefficient r=0.863, P=0.000.1.2Effects of n-6/n-3PUFAs(5:1) on expression of adiponectin mRNA and PPARy mRNAThe mRNA expression of adiponectin in n-6/n-3PUFAs (5:1) group were increased at25、50、100μmol/L,and both with a significant increased at the concentration of25μmol/L(p=0.000). And then display a descent tendency by a dose-dependent manner with the increased of fatty acid concentration, to a significant at400μmol/L (p=0.000).The mRNA expression of PPARy in n-6/n-3PUFAs (5:1) group were increased at25、50μmol/L,and both with a significant increased at the concentration of50μmol/L(p=0.003). And then display a descent tendency by a dose-dependent manner with the increased of fatty acid concentration, to a significant at200(p=0.029)、400μmol/L(p=0.008). Adiponectin and PPARy mRNA expression display a significant positive correlation, coefficient r=0.590, P=0.010.1.3Effects of n-6/n-3PUFAs (10:1) on expression of adiponectin mRNA and PPARy mRNAThe mRNA expression of adiponectin and PPARγ mRNA in n-6/n-3PUFAs (10:1) group were increased at50μmol/L (p=0.019, p>0.05).At the concentration of400μmol/L significant decrease of both adiponectin and PPARy mRNA was observed. Adiponectin and PPARy mRNA expression display a significant positive correlation, coefficient r=0.791, P=0.000.1.4Effects of n-6/n-3PUFAs (20:1) on expression of adiponectin mRNA and PPARy mRNAThe mRNA expression of adiponectin and PPARy in n-6/n-3PUFAs(20:1) group were decreased in a dose-dependent manner, in addition to50μmol/L(P>0.05).And at the concentration of200,400μmol/L to show a significant decreased compared with control(P<0.05). Adiponectin and PPARy mRNA expression display a significant positive correlation, coefficient r=0.926,P=0.000. 1.5Effects of n-6/n-3PUFAs (30:1) on expression of adiponectin mRNA and PPARy mRNAThe mRNA expression of adiponectin and PPARy in n-6/n-3PUFAs(30:1) group were decreased in a dose-dependent manner.And at the concentration of50,100,200,400μmol/L to show a significant decreased compared with control(P<0.05). Adiponectin and PPARy mRNA expression display a significant positive correlation, coefficient r=0.793,P=0.000.2. Effects of n-6/n-3PUFAs composition on expression of adiponectin protein2.1Effects of n-6/n-3PUFAs (1:1) on expression of adiponectin proteinThe protein expression of adiponectin in n-6/n-3PUFAs (1:1) group were increased at25、50、100、200、400μmol/L (24.77%,21.18%,28.50%,5.17%,2.04%),and both with a significant increased compared with the control at the concentration of25(P<0.05)、100μmol/L(P<0.05).2.2Effects of n-6/n-3PUFAs (5:1) on expression of adiponectin proteinThe protein expression of adiponectin in n-6/n-3PUFAs (5:1) group were increased at25、50、100、200μmol/L (15.11%、8.16%、4.88%、2.54%),and both with a significant increased compared with the control at the concentration of25(P<0.05).2.3Effects of n-6/n-3PUFAs (10:1) on expression of adiponectin proteinThe protein expression of adiponectin in n-6/n-3PUFAs (10:1) group were increased at25、50、100μmol/L (3.01%、8.94%、14.32%),and both with a significant increased compared with the control at the concentration of100μmol/L (P<0.05). And then display a descent tendency by a dose-dependent manner with the increased of fatty acid concentration, to a significant at400μmol/L(P<0.05)2.4Effects of n-6/n-3PUFAs (20:1) on expression of adiponectin proteinThe protein expression of adiponectin in n-6/n-3PUFAs (20:1) groups display a descent tendency by a dose-dependent manner with the decreased of fatty acid concentration, to a significant at400μmol/L(P<0.05). 2.5Effects of n-6/n-3PUFAs (30:1) on expression of adiponectin proteinThe protein expression of adiponectin in n-6/n-3PUFAs (30:1) groups display a descent tendency by a dose-dependent manner with the increased of fatty acid concentration, to a significant at51.54%(P=0.000)、52.04%(P=0.000)62.34%(P=0.000)、61.93%(P=0.000),compared with the control.Conclusion1. Different n-6/n-3PUFAs ratios make different impact on gene expression of adiponectin in3T3-L1adipocytes cells.n-6/n-3PUFAs (1:1and5:1) can promote gene expression of adiponectin more than other ratios.the increase in n-6/n-3PUFAs ratios decreases expression of adiponectin,which completely inhibits gene expression of adiponectin and PPARy in n-6/n-3PUFAs(30:1).2. N-6/n-3PUFAs can induce a dose-dependent manner at the gene expression of adiponectin and PPARy mRNA in3T3-L1adipocytes.When n-6/n-3PUFAs are in the concentration of25、50、100μmol/L respectively, gene expression of adiponectin increase;when more than the concentration of200μmol/L, gene expression of adiponectin decreases.N-3PUFAs can promote adiponectin expression more than n-6PUFAs in physiological concentrations.3. The molecular mechanism of adiponectin gene expression might be correlated with a PPARy activation signaling pathway, for there is a positive correlation between adiponectin and PPARγ.n-6/n-3PUFAs indue the expression of adiponectin mRNA in3T3-L1adipocytes, possibly by a mechanism involving PPARγ.4. It is prompting in this study that both PUFAs ratios, dose are the important factor affecting the expression of adiponectin.In the proper range (n-6/n-3PUFAs are1:1、5:1) n-6PUFAs may promote adiponectin gene expression of adiponectin.However,n-3PUFAs plays a mainly role in increasing adiponectin gene expression.And it is possible to educe its beneficial contribution through the very category, construction,dose of n-6/n-3PUFAs.