The Effect Of Mfn2in Palmitate-induced Oxidative Stress And Insulin Resistance In Skeletal Muscle Cells

In recent years, the global prevalence of type2diabetes (T2DM) hasincreased dramatically and it has become the major concern of the publichealth problems.The important pathogenesis of type2diabetes is insulinresistance (IR). Mitochondria play a central role in energy metabolism.Studies have shown that mitochondrial dysfunction closely related to theoccurrence of insulin resistance. Mitochondrial dysfunction includes reducedability of mitochondrial oxidative phosphorylation, ATP synthesis, lipiddeposition, increased oxidative stress level, etc.Mitofusin2(Mfn2) is a kind of protein which set in outer membrane ofmitochondria. Mfn2not only balance the fission and fussion of mitochondrial,it also regulates the metabolism of mitochondria and improves insulinsensitivity. Diabetes, obesity and exercise could alter the expression level ofMfn2. Our previous data suggested that high fat diet induced insulin resistance,it related to lower expression of Mfn2in skeletal muscle tissue.Objective: To investigate whether Mfn2leads to insulin resistance byregulating oxidative stress in rat skeletal muscle cells.Methods：Rat L6myoblasts were cultured and induced in monolayers tothe stage of myotubes. The cells were then randomly divided into two groups:normal control (NC) group and palmitic acid (PA) treatment group, as a modelof insulin resistance. The normal control (NC) group was randomly dividedinto four subgroups: normal control (NC) group, normal empty-adenovirus(NAd0) group, Mfn2overexpression(Mfn2) group, knock-down of Mfn2expression (siMfn2) group; palmitic acid (PA) group was randomly dividedinto three subgroups: palmitic acid (PA) group, PA cells infected withempty-adenovirus (PAd0) group, Mfn2overexpression of PA cells (PMfn2)group.The expression of Mfn2mRNA and protein was detected to evaluate whether adenovirs transfection was success. Glucose uptake was performedusing the glucose oxidase-peroxidase method to evaluate insulin sensitivity.Mitochondrial membrane potential was measured using JC-1fluorescentprobe. DHE fluorescent probe was used to test cells reactive oxygen species(ROS). Total superoxide dismutase (T-SOD), catalase (CAT) and glutathioneperoxidase (GSH-Px) activity and the content malondialdehyde (MDA) levelswere measured with commercial enzyme assay kits. The phosphorylation of c-Jun amino terminal kinase (JNK), nuclear factor-κ B (the nf-kappa B) weredeteced using western-blot. Data were expressed as mean±SE. Significance ofdifferences among groups was determined using Student’s t-test or ANOVA.Results:1. The change after palmitate treatment: compared with NC, the insulinsensitivity of PA group decreased, Both Mfn2mRNA and protein level,mitochondrial membrane potential, T-SOD, CAT and GSH-Px activityreduced significantly, ROS, MDA and phosphorylation of JNK, NF κ Bincreased significantly.2. The changes after modulating Mfn2expression level in normal cells:NAd0group have no significant change compared with NC group. Mfn2mRNA and protein expression levels were significantly higher in Mfn2groupcompared with NC group (P<0.01). Meanwhile, skeletal muscle cells in Mfn2group had significantly higer glucose uptake rate, mitochondrial membranepotential, ROS, T-SOD, CAT activity,phosphorylation of JNK and NF κ B,however, the MDA content decreased significantly. Mfn2mRNA and proteinexpression levels in siMfn2group were reduced significantly (P<0.01),glucose uptake rate, mitochondrial membrane potential level, T-SOD andCAT activity were significantly decreased, while ROS, MDA, activation ofJNK and NFκB compared with NC group.3. The change after Mfn2overexpression in PA cells. All resuls had nostatistical differences between PA group and PAd0group. Mfn2expressionwas increased in PMfn2group compared with PA group (P<0.01). In skeletalmuscle cells, the glucose uptake rate, mitochondrial membrane potential, T-SOD and CAT activity were significantly increased, ROS, MDA, activationof JNK and NF κ B reduced significantly, oxidative stress level improvementobvioursly.Conclusion:1. Mfn2expression in the palmitate-cultured cells was significantly loweras results of oxidative stress state, reduced glucose uptake and declined insulinsensitivity.2. Mfn2over-expression led to increase glucose utilization in skeletalmuscle cells and had no obvious effect to oxidative stress level; Knock-downMfn2expression led to mitochondrial dysfunction, elevated oxidative stresslevel and insulin resistance.3. Mfn2could improve mitochondrial function, reduce oxidative stress inpalmitate-cultured skeletal muscle cells thereby ameliorate the insulinresistance.