Study On The Role Of Free Fatty Acids In Metabolic Syndrome And Intervention Mechanism Of Metformin

BackgroudOxidative stress induced by free fatty acids plays a critical role in the pathogenesis of endothelial dysfunction and atherosclerosis in patients with metabolic syndrome. Reducing oxidative stress in these patients may prevent the development of cardiovascular complications. In addition to glycemic control, metformin has been reported to directly protect the cardiovascular system. In this study, we investigated whether metformin could reduce intracellular reactive oxygen species (ROS) levels induced by palmitic acid (PA) in human aortic endothelial cells and how this happened.Methods1. Cell culturePrimary human aortic endothelial cells were cultured at 37℃in EGM-2 media. The cells were transfected with Trx, AMPK or FOXO3 small interfering RNAs (siRNAs) or treated with metformin or PA at various concentrations and for the time periods indicated.2. siRNA-induced gene silencing Trx, AMPK and FOXO3 gene expression were silenced with Trx siRNA, AMPK siRNA and FOXO 3 siRNA respectively. Transfection of HAECs with siRNAs was carried out using LipofectaminTM 2000 according to the manufacturer's instructions. Transfected cells were then treated with PA and metformin at the designated concentrations for the indicated time periods.3. Intracellular ROS level detectionIntracellular ROS levels were determined using the oxidant-sensitive fluorogenic probe CM-H2DCFDA. Fluorescence was detected by a fluorescent microscope. Fuorescent intensity was measured and normalized to cell number. ROS levels were compared with the no-treatment control and expressed as the percentage of the control.4. Western Blot AnalysisTreated cells were collected and lysed. Protein concentration was measured by the Bradford method. Fifteen micrograms of protein sample per lane were subjected to SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidine fluoride membranes. The membranes were blocked by 5% nonfat powdered milk, incubated with the primary antibody, and then with the secondary HRP-labeled antibody. Bands were visualized with Enhanced Chemiluminescence. The Quantity One imaging program was used to quantify the densitometry of protein bands. The relative protein levels were normalized to P-actin and expressed as the percentage of the non-treatment control. Quantitative RT-PCRTotal RNA from treated cells was extracted with Trizol. The mRNAs were reverse-transcribed into cDNA with the iScript cDNA synthesis kit. Quantitiative RT-PCR was performed using the iCycler iQ RT-PCR detection system. Primers were designed through Beacon Designer 2.0 software. The mRNA levels were acquired from the value of the threshold cycle (Ct) of Trx normalized against the Ct ofβ-actin. The relative levels of mRNA were compared and expressed as the percentage of the control. The data shown was representative of three separate experiments.Results1. Metformin suppressed ROS production induced by PA in endothelial cells.We found the PA-induced increase of intracellular ROS levels was reduced by metformin in a dose-dependent manner. This result indicates that metformin is capable of reducing intracellular ROS levels.2. Metformin upregulated the antioxidant Trx.We found that metformin alone slightly increased Trx expression. In the presence of PA, metformin greatly increased Trx expression in a dose-dependent manner. Moreover, we found metformin induced Trx expression at mRNA level.3. Trx mediated metformin induced reduction of ROSSilencing Trx with siRNA prevented metformin-induced reduction in ROS levels in the absence or the presence of PA, indicating that Trx is involved in metformin-mediated reduction in ROS levels in both basal and PA-induced stress states.4. Activation of the AMPK pathway mediates the metformin induced upregulation of Trx.Metformin induced AMPK phosphorylation in a dose-dependent manner, indicating that activation of this pathway by metformin. Knockdown of AMPK by its specific siRNA inhibited the basal and metformin-induced upregulation of Trx protein indicating that AMPK pathway is involved in basal as well as in metformin-induced Trx regulation.5. FOXO 3 is required for metformin-induced upregulation of Trx.Silencing FOXO3 with siRNA significantly inhibited expression of the basal Trx and metformin-induced Trx protein and mRNA, further suggesting that the FOXO3 transcription factor is required for the metformin-induced upregulation of Trx.Conclusions1. FFA induced the over production of ROS in HAECs,and metformin inhibited this production.2. Metformin reduced ROS level through increasing the expression of antioxidation agent thioredoxin.3. Metformin increased the expression of thioredoxin through the activation of AMPK-FOXO3 pathway. BackgroudVisceral obesity is associated with a higher risk of cardiovascular disease which is the most common cause to mortality in the modern society. In humans the storage of fatty acids as triglyceride (TG) is the major energy depot and lipid resources for cellular function. Different people store fat differently, resulting in different types of obesity by positive energy balance over a prolonged time. Previous studies have shown that women most accumulate more subcutaneous fat and more lower body fat, whereas men are prone to store fat in visceral (omental and mesenteric) depots. DGAT catalyses the final step of TG synthesis and is seen as the regulating and rate limiting factor in the TG synthetic pathway.We hypothesized that the differences in fat distribution may be due to different activities of the enzyme Acyl-CoA:diacylglycerol acyltransferase (DGAT) in different fat tissues. Different DGAT activity in omental and subcutaneous adipocytes could therefore explain the differences in fat storage between men and women.MethodsWe obtained omental and abdominal subcutaneous adipose tissue-samples from seven non obese women, five non obese men (BMI<30 kg/m2), fourteen obese women and nine obese men (BMI>30 kg/m2) undergoing elective surgery. Free fatty acid uptake and fat cell size were measured immediately after surgery. Two different methods of DGAT activity measurement were compared:using either the cytosolic fraction or microsomal fraction from adipocytes. Then the DGAT activity in defferent fat depots was examined in men and women, as well as FFA uptake and fat cell size. DGAT activity was determined by measuring the incorporation of [14C]palmitoyl-CoA into triglyceride of the cytosolic fraction and was calculated in nmol/min/mg lipid. Fat distribution was assessed by measuring the circumferences of abdomen and thigh one day after surgery. Using clinically obtained computed tomography (CT) images we also calculated the visceral fat mass and abdominal subcutaneous fat mass of our subjects with the Slice-O-Matic software programs.ResutsDGAT activity was significantly higher in omental adipose tissue than in abdominal subcutaneous adipose tissue in non obese patients, but there was no significant difference in DGAT activity between omental and abdominal subcutaneous fat in obese subjects. No differences were found between men and women, neither in omental nor in subcutaneous fat DGAT activity. There was a significant relationship between free fatty acid (FFA) uptake and DGAT activity in omental adipose tissue in the whole group. No other significant associations were observed between FFA uptake and DGAT activity. Cell sizing showed a significant difference in cell diameter between non obese women and obese men. A significant difference in DGAT activity and subcutaneous fat cell size was found between non obese patients and obese patients.ConclusionOur results indicate that differences in DGAT activity are related to weight. There was no difference between DGAT activity in men and women. Therefore differences in the fat deposits between men and women are not likely to be due to differences in the DGAT activity. Further research is necessary to determine the role of DGAT activity on fat metabolism in humans.