Effect Of Mitofusin2on Fatty Acid Metabolism In High Fat Diet Induced Insulin Resistance Rat Muscle

Insulin Resistance （IR） means insensitivity of peripheral tissues （skeletalmuscle, adipose tissue, or liver） to insulin, characterized by peripheral tissuedysfunction to glucose uptake or utilization. A recent study suggested that theactive lipid metabolites such as LCACoAs, diacylglycerols and ceramides arethe culprits leading to insulin resistance.Fatty acid is the main energy source for human and mammals.Intramuscular lipids accumulate when the uptake of fatty acids exceeds therate of β-oxidation. Fatty acid translocase CD36plays an important role in theuptake of longchain fatty acids （LCFAs） in skeletal muscle. Carnitinepalmitoyltransferase1（CPT-1） is the rate-limiting enzyme that controls thetransfer of cytosolic LCACoAs into mitochondria for oxidation.Mitochondrial dysfunction has been proposed as a possible mechanism ofskeletal muscle insulin resistance and lipid accumulation. Mitofusin2（MFN2）plays an important role in mitochondrial function. It is proved that MFN2overexpression can improve insulin resistance. However, the mechanism remainsunclear. In the current study, we established insulin-resistant rat model byhigh-fat-feeding and used MFN2-expressing adenovirus to investigate themechanism by which MFN2improves skeletal muscle lipid intermediatesaccumulation and ameliorates insulin resistance.Objective: To investigate the effects of mitofusion-2（MFN2） on theaccumulation of lipid intermediates in high-fat feeding rats skeletal muscle,lipid metabolism and the possible underlying mechanisms.Methods: Male Sprague-Dawley （SD） rats were divided randomly intotwo groups of64: normal control （8N） group, high-fat diet （8F） group,8Ngroup were fed with normal diets,8F with high-fat diets for8weeks. Insulinsensitivity in each group was evaluated by glucose infusion rate （GIR） of hyperinsulinemic euglycemic clamp technique under constant state. And then8rats in each group were killed randomly by abdominal aorta bleeding. Bloodglucose（BG）, triglyceride （TG）, total cholesterol（TC） and plasmainsulin（INS） were determined at the end of treatment. The changes ofmitochondrial morphology were observed by transmission electronmicroscope （TEM）. The lipid intermediates in muscle, including long chainfatty acid CoAs （LCACoAs）, diacylglycerols （DAG） and ceramides （CEA）,were assayed by ELISA kit. The skeletal muscle triglyceride was assayed byGPO-PAP method. The expression of MFN2, carnitine palmitoyltransferase I（CPT1） and fatty acid translocase CD36were detected by real-time PCR andWestern blot. After8weeks, high-fat diet （8F） group were divided randomlyinto five groups: high-fat diet control （Control） group, empty Ad adenovirus（Ad） group, low Ad-MFN2group （Ad-MFN210⁸） group, medium Ad-MFN2（Ad-MFN210⁹） group, high Ad-MFN2(Ad-MFN210¹⁰) group. All rats wererespectively infected with control or MFN2expression adenovirus once aweek for3weeks. Hyperinsulinemic clamp studies were performed toreevaluate insulin sensitivity. Blood glucose（BG）, triglyceride（TG）,totalcholesterol（TC） and plasma insulin（INS） were determined at the end oftreatment. The changes of mitochondrial morphology were observed bytransmission electron microscope （TEM）. The lipid intermediates in muscle:long chain fatty acid CoAs （LCACoAs）, diacylglycerols （DAG） andceramides （CEA） were assayed by ELISA kit. The skeletal muscle triglyceridewas assayed by GPO-PAP method. The expression levels of MFN2, carnitinepalmitoyltransferase I （CPT1） and fatty acid translocase CD36were detectedby real-time PCR and Western blot.Results：1The change after8weeks high-fat feeding:（1） the establishment ofinsulin resistance models: at the end of8weeks high-fat feeding, bloodglucose （BG）（7.2±0.6mmol/Lvs5.7±0.7mmol/L） and plasma insulin （INS）（40.6±3.9mU/L vs28.7±3.3mU/L） were significantly higher in HF groupcompared with NC group （P<0.01）; GIR was significantly lower in HF group compared with NC group (17.7±2.3mg/kg/min vs29.4±2.4mg/kg/min,（P<0.01）.（2） the change in blood lipids: at the end of8weeks high-fat-feeding,triglyceride （TG0.35±0.04mmol/Lvs0.22±0.02mmol/L） and totalcholesterol（TC,1.51±0.07mmol/L vs1.13±0.06mmol/L） were significantlyhigher in HF group compared with NC group （P<0.01）.（3） The change ofmitochondria morphology： at the end of8weeks high-fat-feeding, themitochondrial of HF group was less in quantities and smaller in volumn thannormal control （NC） group.（4） the accumulation of muscle lipid intermediates:at the end of8weeks high-fat-feeding, the levels of lipid intermediates,including TG, LCCoAs, CER and DAG in skeletal muscle of high-fat dietsrats were significantly increased （P＜0.01）.（5） Change in expression of CPT1and CD36: at the end of8weeks high-fat-feeding, the expression of CD36was increased markedly （P<0.01） compared with NC group, meanwhile, theexpression of CPT1in HF group was decreased significantly （P<0.01）.2The change after MFN2over expression:（1） the change of insulinresistance: after MFN2over expression, the expression of MFN2in MFN2over expression group was increased markedly （P<0.01） compared with Adgroup. GIR was increased markedly after Ad-MFN2infection with differentdoses compared with Ad group （P<0.01）. The levels of blood glucose （BG）and plasma insulin （INS） in MFN2over-expression group were significantlylower than Ad group （P<0.01）.（2） the change of blood lipids: after MFN2over-expression, there were no significantly difference between Ad groupand MFN2over-expression group in triglyceride（TG） and total cholesterol（TC）（P＞0.05）.（3） The change of mitochondria morphology：compared withthose from Ad rats, mitochondria from muscle in MFN2over-expressiongroup have a more clearly defined internal membrane structure, includingwider crista.（4） The accumulation of muscle lipid intermediates: after MFN2over expression, there were no significant changes in the accumulation of TG（P＞0.05）. However, the accumulation of lipid intermediates, includingLCCoAs, CER and DAG were markedly reduced （P＜0.01）.（5） Change inexpression of CPT1and CD36: after MFN2over expression, the expression of CD36was not significantly changed, whereas both mRNA and protein levelsof CPT1were up-regulated markedly （P＜0.01） after MFN2over-expression.Conclusions:1High fat feeding induce insulin resistance in rats represented withelevated blood glucose, increased serum insulin, and decreased GIR; whileMFN2over-expression improved insulin sensitivity.2After high fat feeding, the mitochondria of high fat group were less inquantities and smaller in volumn. MFN2over-expression group have a moreclearly defined internal membrane structure, including wider crista.3High fat feeding lead to an increase in blood triglyceride, and totalcholesterol, as well as accumulation of cytosolic lipid intermediates in musclein rats; MFN2over-expression reduced lipid intermediates in muscle withoutsignificant effect on blood triglyceride or total cholesterol.4High fat feeding lead to up-regulation of CD36expression anddown-regulation of CPT1expression in skeletal muscle in rats; MFN2over-expression increased CPT1expression in muscle whereas have nosignificant effect on CD36expression.