Effects Of N-3、n-6PUFAs On Adiponectin And It’s PPARγ-dependent Mechanism In3T3-L1Adipocytes

Background:In recent years there has been a marked rise in over-nutrition, obesity, and obesity-associated diseases such as type2diabetes and cardiovascular disease (CVD). An increase in the prevalence of such conditions has led to much research aimed at establishing the underlying causes. Adipose tissue can not only store energy, but also has the function of endocrine. Adipokines secreted from adipose tissue are thought to loss weight, adjust glucolipid metabolism, regulate fertility function, improve insulin resistance, anti-inflammatory, anti-atherosclerosis, antitumor and so on. Polyunsa--turated acids, including n-3PUFAs, such as Docosahexaenoic acid (DHA), Eicosapentaenoic Acid (EPA) and n-6PUFAs such as Linoleic acid (LA), Arachidonic acid (AA), paly an important role in regulating glucolipid metabolism and the differentiation of adipocytes, but the mechanism of PUFAs adjusting adiponectin expression has not entirely clear.Objective:To study the effects of n-3、n-6polyunsaturated fatty acids (PUFAs) on adiponectin and PPARγ mRNA,as well as to research it’s mechanism in3T3-L1adipocytes to provide a new thought and theory basis of the prevention and cure of metabolic syndrome、type2diabetes and obesity related disease. Method:3T3-L1adipocytes were incubated with25μmol/L、50μmoLT、100μmol/L200μmol/L、400μmol/L DHA、EPA、LA or with Bull Serum Albumin (BSA) alone (control) for24h. Adipocytes were also incubated for24h with DHA plus GW-9662, a PPARy antagonist, or with PUFAs alone. The mRNA expression of adiponectin and PPARy was assessed by real-time fluorescence quantitative PCR. The secreted adiponectin protein was analysed by western blotting. PUFAs can be attacked easily by harmful substances such as free radicals and happened to be lipid peroxidation. To ensure whether the high concentration PUFAs cutting adiponectin expression and secretion is relevant to lipid peroxidation,24h cell cultures were collected and examined the concentration of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxide shift enzyme (GSH).The data were shown with the mean±tandard deviation (X±s) by SPSS13.0software.The differences between sample mean and overall mean were compared by one-sample T test. 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 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. The paper shows the statistical variance result, F and P value in all of the groups. 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.Result:(1) The effects of n-3PUFAs on adiponectin and PPARy mRNA:Effects of the different concentrations of DHA on adiponectin and PPAR y mRNA expression have a statistical significance(F=59.944, P<0.001; F=41.051, P<0.001). Both50μmol/L and100μmol/L DHA increased the expression of adiponectin and PPARy mRNA compared with the control, the highest was in group100μmol/L (P<0.001). With the increasing of the concentration of adiponectin and PPAR y mRNA expression was lower, especially in group400μmol/L. Pearson correlation analysis was r=0.775,P<0.001; Effects of the different concentrations of EPA on adiponectin and PPAR y mRNA expression have a statistical significance (F=140.564,P<0.001; F=27.601, P<0.001). When the EPA concentration was25μ mol/L, adiponectin and PPAR y mRNA express reached the highest level (P<0.001),when the EPA concentration was in50μmol/L to100μ mol/L, adiponectin and PPAR y mRNA increased, adiponectin and PPAR y mRNA expression gradually reduced with the further concentrations increasing. When the EPA concentration was400μ mol/L, adiponectin and PPAR y mRNA expression was the lowest, Pearson correlation analysis was r=0.678, P=0.001.(2) The effects of n-6PUFAs on adiponectin and PPAR y mRNA expression: Effects of the different concentrations of LA on adiponectin and PPAR y mRNA expression have a statistical significance(F=47.752, P<0.001; F=76.943, P<0.001). Compared with the control, when the LA concentration were50μmol/L、100μmol/L, adiponectin and PPAR y mRNA expression increased (P=0.060; P=0.276), the highest express quantity in50μmol/L group. Adiponectin and PPAR y mRNA expression gradually reduced with the further concentrations increasing. When LA concentration was400μmol/L, adiponectin and PPAR y mRNA expression caught the bottom (P<0.001), Pearson correlation analysis was r=0.972, P=0.003.(3) Effects of antagonist (GW9662) on adiponectin and PPARy mRNA:Effects of diferent n-3、n-6PUFAs with PPAR y antagonist GW9662on adiponectin and PPARy mRNA expression have a statistical significance (F=29.033, P=0.002; F=130.858, P<0.001). Compared with the control,100μmol/L DHA, EPA, LA plused with100μmol/L GW9662respectively, adiponectin and PPAR y mRNA decreased significatively.(4) Effects of n-3、n-6PUFAs on adiponectin secretion:compared with the control,100μmol/L of DHA、EPA and LA enhanced adiponectin secretion by109.56%(P=0.030),221.89%(P=0.020) and110.62%(P=0.008) respectively; when adipocytes were incubated with GW9662, adiponectin secretion was reduced by94.83%,83.04%, and88.46%respectively (P<0.001).(5) Effects of DHA、EPA、LA on lipid peroxidation:MDA content was the lowest in the group100μmol/L and gradually enhanced with increased concentration (P=0.018; P=0.011; P=0.004);The high concentration PUFAs increased SOD vitality and GSH level to a greater extent.Conclusion:(1) Our results demonstrate that n-3、n-6PUFAs affects the expression of adiponectin mRNA in3T3-L1adipocytes concentration-dependently.When DHA, EPA, LA are in the concentration of100μmol/L,25μmol/L,50μmol/L respect--tively,adiponectin mRNA expression reaches the peak with statistical significance. N-3PUFAs can promote adiponectin expression and secretion more than n-6PUFAs in physiological concentrations(100μmol/L). N-3, n-6PUFAs can significantly reduce adiponectin expression in more than two times of physiological concentrations.(2) As the same as PPAR y agonists Pioglitazone, n-3,n-6PUFAs can also improve adiponectin expression and secretion levels in physiological concentrations, but PPAR y antagonists GW9662can significantly block the PUFAs promoting function of adiponectin expression and secretion, so that PUFAs may regulate adiponectin expression through PPAR y in3T3-L1adipocytes. (3) High levels of PUFAs can reduce adiponectin expression that may be relative to lipid peroxidation.Innovation points:Interest in obesity and the metabolic syndrome has prompted renewed research into the physiology of adipose tissue. Adiponectin is the potential molecular target to the prevention and control of the current obesity, diabetes and metabolic syndrome. The present studys consider that the change of dietary structure and lifestyle are linked to obesity, related insulin resistance and diabetes. High fat diet and fatty acid composition may be one of the important factors. The domestic and foreign researchs have few reports about the effects of fatty acid on adiponectin expression. This experiment, in vitro cell culture experiment, focused on polyunsaturated fatty acids on adiponectin express, and further discussed the molecular mechanism of polyunsaturated fatty acids adjust adiponectin expression which affect obesity and insulin resistance.