ABSTRACT
Objective To explore the role of free fatty acids(FFA) on expression of miR-335 in human matured adipocytes.Methods In order to induce differentiation,confluent human pre-adipocytes (day 0) were subsequently cultured in serum-free PAM containing 50 nmol/L insulin,100 nmol/L dexamethasone,0.5 mmol/L 3-isobutyl1-methylxanthine,and 100 μmol/L rosiglitazone.Then human matured adipocytes (day 16) were treated with 1 mmol/L FFA cocktail composed of lauric acid,myristic acid,linoleic acid,oleic acid,and arachidonic acids for 4,8 and 24 hours.Meanwhile,untreated cells were collected as control group.Total RNA from these adipocytes were extracted and the levels of miR-335 expression were evaluated by real-time PCR.Results The expression of miR-335 at 4,8 and 24 hours showed no statistical significance when compared to 0 hour in untreated matured adipocytes (all P ＞ 0.05).The relative expression of miR-335 after the intervention of FFA in human matured adipocytes were 9.03 ± 0.31,9.85 ±2.41 and 11.23 ± 0.62,respectively at 4,8 and 24 hours when used with snRU6 for normalization,and there was statistical signi-ficance compared with 0 hour in control group (all P ＜ 0.05).The levels of miR-335 were 4.73 ± 0.60,5.38 ± 1.25 and 4.57 ±0.52 at the same time point when used with miR-103 for normalization,and there was statistical significance compared with 0 hour in control group (all P ＜ 0.05).Conclusions FFA exert a positive effect on the miR-335 expression in human matured adipocytes,which provide the basis for the further study about the role of miR-335 in human adipocytes.
ABSTRACT
Objective To observe the effect of uncoupler of mitochondrial oxidative phosphorylation——Cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) on mitochondrial function and insulin sensitivity in mature adipocytes.Me-thods 3T3-L1 pre-adipocytes were differentiated into mature adipocytes and then induced and maintained in medium that contained the chemical uncoupler 7.5 μmol/L FCCP.Glucose uptake was determined in the adipocytes by measu-ring 2-deoxy-D-[3H] glucose uptake.Western blot was used to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4) and measure the phosphorylation and total protein contents of insulin signaling proteins such as insulin receptor substrate(IRS)-1,Akt.The mitochondrial morphology was performed by transmission electron microscope.The mitochondrial DNA (mtDNA) copy number was evaluated by real time PCR.Luciferase-based luminescence assay was used to determine cellular ATP production.The mitochondrial membrane potential(△Ψm) and reactive oxygen species(ROS) were detected by flow cytometry.Results (1) Exposure of mature adipocytes to FCCP basal glucose uptake was similar to mature adipocytes without FCCP(t =-0.07,P ＞ 0.05) ; however,the insulin-stimulated glucose uptake was significantly decreased in FCCP group (t =5.87,P ＜ 0.01).(2)FCCP decreased insulin-stimulated GLUT4 translocation to the plasmalemma and inhibition of insulin-induced phosphorylation of IRS-1 and Akt in 3T3-L1 adipocytes.(3)The size of mitochondria in FCCP-treated adipocytes was smaller than that in 3T3-L1 adipocytes without FCCP,and the morphology was condensed and abnormal.(4) mtDNA copy number in FCCP-treated adipocytes was significantly lower than that in adipocytes without FCCP(t =-1.73,P ＜ 0.001).(5) Exposure of 3T3-L1 adipocytes to FCCP significantly decreased △Ψm (t =4.83,P ＜ 0.01) and total cellular ATP production compared with cells without FCCP (t =6.08,P ＜ 0.0001),as well as the increased intracellular ROS levels (t =-6.82,P ＜ 0.01).Conclusions FCCP may impair mitochondrial morphology and mitochondrion dysfunction,and inhibited the activation of insulin-induced phosphorylation of IRS-1 and Akt in 3T3-L1 adipocytes,which suggests that FCCP-induced insulin resistance may be correlated with the FCCP induced generation of ROS in 3T3-L1 adipocytes.