Effect Of Mfn2Expression On Glucose And Lipid Metabolism In Liver Of High-Fat Diet Induced Insulin Resistant Rats And Mechanism

The incidence of type2diabetes(T2DM) has increased dramatically inrecent years. It is estimated that more than300million individuals worldwidewill be at high risk of developing T2DM by the year2025. Insulin resistanceplays a primary role in the pathogenesis of T2DM, and becomes a majorpublic health issue. Metabolic efficiency of glucose and lipid has decreasedduring insulin resistance. Mitochondria are core organelles that have a centralrole in the energy metabolism, and its role in metabolic diseases can not beignored. Studies have shown that mitochondrial dysfunction is closely relatedto the development of insulin resistance and type2diabetes, andmitochondrial damage may be one of the mechanisms of insulin resistance.Mitochondrial dysfunction characterized by reduced oxidative capacity andATP generation can lead to lipid deposition and induce insulin resistance.The maintenance of mitochondrial morphology play a pivotal rolein energy control. Mitochondria is a dynamic organelle, maintaining abalance by continuous fusion and division. Mitofusin (Mfn) promotesmitochondrial fusion, and regulates the biosynthesis and maintenance of themitochondrial network structure. Mitofusin2(Mfn2) is a mitochondrial fusionprotein, mainly expressed in mammalian cells. Mitofusin2(Mfn2) is amitochondrial fusion protein, mainly expressed in mammalian cells. Researchin vitro and in vivo experimental models suggest that the physiological orpathological conditions can lead to changes in Mfn2expression, for example,diabetes, obesity and insulin resistance down-regulate expression of Mfn2,exercise and weight loss up-regulate expression of Mfn2. Data sugguest thatdown-regulation of Mfn2expression lead to low mitochondrial membranepotential and reduced oxygen consumption and glucose oxidation, while up-regulation of Mfn2expression lead to high mitochondrial membranepotential and enhanced glucose oxidation. In addition, previous studies haveshown that high-fat diet down-regulated Mfn2expression.Increasing evidence indicates that expression of Mfn2is related to insulinresistance. However, the specific mechanism remains unclear. To enhance ourunderstanding to these questions, we established a high-fat diet inducedinsulin resistant rat model, at the same time HepG2cells were incubated withpalmitate to establish a insulin resistant HepG2cell model, infected insulinresistant rats with Mfn2expression adenovirus, the effect of Mfn2over-expression on the glucose and lipid metabolism in rats liver wasevaluated. Then, the specific molecular mechanism at the animal and cellularlevels through which Mfn2ameliorates insulin resistance was exploredrespectively.The paper contains four parts as below:Part â… : High-fat diet induces insulin resistance and its effect on theexpression of Mfn2Objective: To establish an animal model of insulin resistance induced byhigh-fat diet and to determine the effect of high-fat diet on expression ofMfn2.Methods: Male SD rats, weight about60-80g, were randomly dividedinto2groups: normal control (ND, n=12) and high fat diet group (HFD, n=72).Rats were fed with a regular low fatty acids diet contained10.3%fat,24.2%protein, and65.5%carbohydrate (kcal) or high-fat diet which consists of59.8%fat,20.1%protein, and20.1%carbohydrate (kcal). Rats in every grouphad free access to water and chow. The environment was controlled in termsof light (12:12-h light-dark cycle starting at6:00AM) and humidity. Insulinresistance was evaluated by glucose infusion rate (GIR) of hyperinsulinemiceuglycemic clamp technique at the end of8weeks(six rats in each group).The blood samples were obtained from the abdominal aorta. Fasting bloodglucose (FBG) levels were measured by Accu-chek Active Meter(ACCU-CHEK Active; Roche). Insulin (INS) and free fatty acids (FFA) levels were analyzed by using a Rat insulin or FFA ELISA kit (Crystal Chem.Inc). The liver tissue samples of rats were taken immediately and kept at-70℃after quick frozen in liquid nitrogen. The expression of Mfn2wasdetected by quantitative Real time PCR and Western blot. The morphologicalchanges in the ultrastructure of their hepatic cells were investigated by meansof electron microscopy assay.Results:1The body weight between two groups had no significant difference (P>0.05). Compared with ND group, GIR in HFD group was significantlydecreased (P<0.01); while the levels of BG, TG, TC, INS and FFA in HFDgroup were significantly higher than these in the ND group (P <0.05).2Expression of Mfn2mRNA and protein were significantly decreased inHFD group (P<0.01), compared with ND group.3Rats liver tissue morphology by histological HE staining: normal liver cellswere regularly aligned, liver lobular architecture was intact, and only a smallnumber of fibers were formed in blood vessel walls. There was diffusesteatosis, especially surrounding central veins, significant cell swelling,massive lipid droplet vacuole and inflammation cellular infiltration in lobulaof liver in HFD group. Electron microscopy assay revealed that the shape andsize of mitochondria in the hepatocytes of the control group were normal andhad few lipid droplets. In contrast, many mitochondria from rats in thehigh-fat diet group were enlarged and swollen, and showed morphologicalchanges, including large lipid droplet, and mitochondrial inner-outermenbrance meromixis. The cristae on the inner membrane of mitochondriawere disappeared or unclear.Conclusions:1High-fat diet-fed rats showed high concentrations of glucose,hyperlipidemia, hyperinsulinemia and GIR was lower. High-fat diet inducedinsulin resistance in rats.2High-fat diet cause hepatic steatosis and ultrastructure damage inhepatocellular mitochondria. 3Expression of Mfn2in the liver tissues of High-fat diet-fed rats wasdown-regulated, implicating that the development of insulin resistance may berelated to low expression of Mfn2.Part â…¡: Effect of Mfn2over-expression on the activity ofglucoskinase and phosphoenolpy ruvate carboxykinase in the liver ofinsulin resistant ratsObjective: To investigate the effects of Mfn2on the insulin sensitivityand the activity of Glucoskinase (GK) and Phosphoenolpyruvatecarboxykinase (PEPCK) in liver of rats fed with high-fat diets.Methods: After8weeks, rats fed high-fat diets were randomly dividedinto5groups: high-fat diet control group infused with PBS buffer (Con, n=12),high fat diet group infected with empty control adenovirus (Ad, n=12),high-fat diet group were respectively infected with different amount ofAd-Mfn2(108,109or1010v.p/kg body weight) once a week, for3weeks. Afterintervention with adenovirus for3weeks, insulin resistance was evaluated byGIR of hyperinsulinemic euglycemic clamp technique. The blood sampleswere obtained from the abdominal aorta. The liver tissue samples of rats weretaken immediately and kept at-70℃after quick frozen in liquid nitrogen.FBG, TG, TC, INS and FFA were tested by methods as part one. The activityof GK and PEPCK were detected by enzyme-coupled colorimetric assay. Thelevel of hepatic glycogen was detected by using a glycogen assay kit. Theexpression levels of Mfn2were detected by Real time PCR and Western blot.Results:1The expression of Mfn2of rats infected with different amount of Ad-Mfn2(10⁸,10⁹or10¹⁰v.p/kg body weight) for3weeks, were increased dramatically(P<0.01).2After intervention with adenovirus for3weeks, the body weight had nosignificant difference among groups (P>0.05). FBG, INS of rats infected withdifferent amount of Ad-Mfn2(10⁸,10⁹or10¹⁰v.p/kg body weight) for3weeks were decreased (P<0.05), while GIR was significantly increased(P<0.01). 3At the end of8weeks, concentration of hepatic glycogen in rats fed withhigh-fat diet was significantly decreased (P<0.05), compared with the NDgroup. After intervention with adenovirus for3weeks, concentrations ofhepatic glycogen in rats infected with Ad-Mfn2was increased with theincreasing of dose. Compared with the rats fed with high-fat diet, the glycogencontents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) wereincreased (P<0.05).4The activity of GK was increased (P<0.01)and PEPCK decreased in theliver of rats fed high-fat diet for8weeks (P<0.01). After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the activty of GKwas increased (P<0.01), while the activity of PEPCK was decreased (P<0.01).Compared with rats high-fat diet-fed,the activity of GK and PEPCK of liver inrats infected with Ad-Mfn2(109or1010v.p/kg body weight),there weresignificant difference (P<0.05or P<0.01).5All of the sections in the Con and Ad group exhibited diffuse hepaticsteatosis under a light microscope. Hepatic steatosis was most obvious aroundthe portal area (mostly microvesicular and macrovesicular mixed steatosis)and was accompanied by inflammatory cell infiltration. The liver HE stainingof rat infected with Ad-Mfn2shows less cell volume and fat dropletaccumulation. Electron microscope revealed that mitochondria from rats in theCon and Ad group were enlarged and swollen, and showed morphologicalchanges, including larger lipid droplet, and mitochondrial membrane isunclear, even had vacuolar degeneration. The cristae on the inner membraneof mitochondria were almost disappeared.Conclusions:1High-fat diets can lead to abnormal glucose metabolism, accompanied bylower concentration of hepatic glycogen,lower activity of GK and higher ofPEPCK.2Over-expression of Mfn2improves the glucose metabolism.The activity ofGK was increased, the activity of PEPCK was decreased, and theconcentration of hepatic glycogen was higher. It also alleviates hepatic steatosis and ultrastructure damage in hepatocellular mitochondria.Over-expression of Mfn2ameliorated high-fat diet induced insulin resistance,and maybe related to the increasing activity of GK,decreasing activity ofPEPCK,and alleviating the demage of the liver cell and mitochondria.Part â…¢: Effect of Mfn2on expression of ACCα and CPT-Ⅰα in liverof insulin resistant ratsObjective: To investigate the effects of Mfn2on the expression of AcetylCoA carboxylase α (ACCα) and Carnitine palmitoyltransferase-Ⅰα (CPT-Ⅰα)in liver of rats fed with high-fat diets.Methods: Animal grouping and treatment was the same as the partâ… and partâ…¡. Hepatic triglyceride (TG) content was detected with a triglyceridedetermination kit. The expression levels of ACCα and CPT-Ⅰα were detectedby Real time PCR and Western blot.Results:1After intervention with adenovirus for3weeks, the plasma TG, TC and FFAlevels in liver of rats infected with Ad-Mfn2(108,109or1010v.p/kg bodyweight) for3weeks were decreased (P<0.05), compared with Ad group.2At the end of8weeks, hepatic TG content of rats fed with high-fat diet wassignificantly increased (P<0.05), compared with the ND group. Afterintervention with adenovirus for3weeks, hepatic TG contents of rats infectedwith Ad-Mfn2was increased with the increasing dose of Ad-Mfn2, Comparedwith Ad, hepatic TG contents in rats infected with Ad-Mfn2(109or1010v.p/kg body weight) were decreased (P<0.05or P<0.01).3The mRNA expression of ACCα was increased and CPT-Ⅰα was decreased(P<0.05). Phosphorylation of ACCα protein was decreased (P<0.05), butCPT-Ⅰα protein was decreased at the end of8weeks. After intervention withadenovirus for3weeks, with increasing dose of Ad-Mfn2, the mRNAexpression of ACCα was decreased (P<0.01) and CPT-Ⅰα was increased(P<0.05). Phosphorylation of ACCα protein was increased (P<0.01), butCPT-â…  α protein expression was increased, compared with Ad group(P<0.05). Conclusions:1High-fat diet can lead to abnormal lipid metabolism, accompanied by highhyperlipidemia, high FFA levels, and Lipid deposition. The activity of ACCαwas increased and expression of CPT-Ⅰα was down-regulated in rats fed withhigh-fat diet.2Over-expression of Mfn2improved the lipid metabolism. Mfn2amelioratedinsulin resistance, probably by decreasing the activity of ACCα and heptic TGcontents, while enhancing expression of CPT-Ⅰα..Part â…£: Mechanism of over-expression Mfn2ameliorating insulinresistanceObjective: To explore the mechanism of Mfn2over-expressionameliorating insulin resistance by testing the expression of relative moleculesof the insulin signaling pathway.Methods: The model of IR was established with HepG2cells cultured athigh concentrations of palmitate (PA,0.25mmol/L) for24h. The glucosecontent was measured by glucose assay kit. Insulin resistant HepG2cells weredivided into4groups: HepG2cells cultured with PA (Con) group, HepG2cells cultured with PA infected with empty control adenovirus (Ad) group,HepG2cells cultured with PA infected with Ad-Mfn2(50or100pfu/cell), andcultured for24h. The expression of INSR, IRS2, PI3K, AKT2and GLUT2were detected by Real time PCR and Western blot.Results:1The glucose contents of PA group were significantly higher than those incontrol (P<0.01).2The expression of Mfn2of insulin resistant HepG2cells was decreasedsignificantly (P<0.01). The expression of INSR, IRS2and GLUT2weredown-regulated in HepG2cells cultured with PA (P<0.01). There were nochanges in PI3K and AKT2expression, but their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group. 3After infected with Ad-Mfn2for24h, the glucose contents of HepG2cellscultured with PA infected with Ad-Mfn250and100pfu/cell were lower thanAd group (P<0.01)。4The expression of Mfn2in HepG2cells cultured with PA infected withAd-Mfn250or100pfu/cell were increased, compared with Ad group.Over-expression of Mfn2were confirmed (P<0.01). Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, and phosphorylationlevels of PI3K and AKT2were increased (P<0.01).5At the end of8weeks, the expression of INSR, IRS2and GLUT2weredown-regulated markedly by high-fat diets at8weeks (P<0.01). There wereno changes in PI3K and AKT2expression, their phosphorylation levelsdecreased significantly (P<0.01), compared with Ad group.6After intervention with adenovirus for3weeks, Over-expression of Mfn2up-regulated the expression of INSR, IRS2and GLUT2, phosphorylationlevels of PI3K-P85and AKT2increased (P<0.01).Conclusions:1High-fat diets inhibited the expression of insulin signaling pathway. Theexpressions of INSR, IRS2and GLUT2were down-regulated by high-fat dietand protein phosphorylation levels of PI3K and AKT2were decreased.2High concentrations of palmitate could induce HepG2cells hepatic insulinresistance, and down-regulate the expression of INSR, IRS2and GLUT2andprotein phosphorylation levels of PI3K and AKT2.3Over-expression of Mfn2improved insulin resistance of rats or HepG2cells,and up-regulated the expression of INSR, IRS2and GLUT2. Proteinphosphorylation levels of PI3K and AKT2were decreased. Our data suggestthat Mfn2might ameliorate insulin resistance by enhancing the expression ofinsulin signaling pathway that are import in glucose and lipid metablism.