| Mitochondria are at the crossroads of energy metabolism, of whichalterations in structure and function may participate in the pathophysiologicprocedure of insulin resistance and type2diabetes. However, whether themitochondrial defects are the cause, the consequence, or the parallel process ofinsulin resistance remains controversial. In fact, one of the first evidencesconnecting mitochondrial activity to diabetes mellitus was the identification ofa mutation in the mitochondrial DNA that caused a maternal-inherited form ofdiabetes and associated deafness. Nonetheless, data obtained from humansubjects concerning this putative relation indicated that the mitochondrialdefect observed in diabetic muscle might be secondary to the insulin-resistantstate instead of being a causal factor. Indeed, functional defects ofmitochondria especially reduced oxidative and phosphorylation capacities,even secondary to insulin resistance, may aggravate insulin resistance.Mitofusin2(Mfn2) is a mitochondrial membrane protein that participatesin mitochondrial fusion and regulates mitochondrial metabolism inmammalian cells. It is highly expressed in tissues with high energy demands,such as skeletal muscle, heart, and brain. Recent studies indicate that Mfn2seems to play a positive role in maintaining the glucose homeostasis. Inaddition, the aberrant expression of Mfn2may underlie the pathophysiology ofinsulin resistance. Therefore, as it was found in Mfn2/shRNA BALB/c mice,the hepatic glucose production and insulin resistance had a significant increase.Another study also support the view that Mfn2deficiency causesmitochondrial dysfunction, which leads to enhanced ROS production,enhanced JNK activity, and inactivation of IRS1, a key protein in insulinsignaling. In addition, in myoblasts with a limited oxidative capacity, Mfn2gain of function caused an increase in glucose oxidation rate and a parallel increase in mitochondrial membrane potential, which indicated augmentedpyruvate oxidation in mitochondria and enhanced Krebs cycle and oxidativephosphorylation. A positive correlation between the Mfn2expression and theinsulin sensitivity was also detected in nondiabetic and type2diabeticsubjects.Skeletal muscle glucose uptake and metabolism plays a major role in theregulation of whole-body glucose homeostasis in both normal and diabeticsubjects. Glucose uptake is the rate-limiting step in glucose utilization and isdepressed in case of insulin resistance, due to defective translocation ofglucose transporter4(GLUT4) to the cell surface. GLUT4is a glucosetransport protein highly expressed in skeletal muscle and adipose tissue, whichtranslocation was induced by two separate signal transduction pathways:dependent or independent of insulin. Insulin-dependent pathway results inGLUT4translocation via activation of phosphatidylinositol-3kinase (PI-3K)and PKB/Akt, whereas the AMPK signaling pathway provides an alternativeto the glucose uptake pathway in muscle. A defect in GLUT4expression andtranslocation has been reported to be the primary metabolic abnormality indiabetic skeletal muscle, and both signal transduction pathways could elicit atranslocation of GLUT4from internal membranes to plasma membrane.Impaired glucose transport in skeletal muscle leads to impaired whole bodyglucose uptake, and contributes to the pathogenesis of Type2diabetes mellitus.Previous studies have reported that whole-body glucose uptake was found tobe a linear function of GLUT4expression in skeletal muscle. Exerciseincreases GLUT4gene and protein expression, and a binding site for themyocyte enhancer factor2(MEF-2) is required on the GLUT4promoter forthis response. It appears that HDAC5, PGC-1, and p38regulate MEF-2andcould be potential targets for modulating GLUT4expression. In addition,obese insulin-resistant rodents have abnormalities in the LKB1-AMPK-PGC-1pathway in muscle, and these abnormalities may underlie the pathophysiologyof GLUT4expression. Observations suggest that AMPK activation leads toincreased PGC-1α activation and/or expression and thereby a concomitant induction of muscle GLUT4expression. Therefore, the aim of the subsequentresearch was to test the hypothesis that AMPK-PGC-1α-mediated signalingregulates the expression of GLUT4. Recent in vivo studies have shown thatrepeated AICAR treatment induced increases in muscle mitochondrial proteinsas well as GLUT4protein in WT mice but not in PGC-1α-/-mice do suggestthat PGC-1αis required for AMPK-induced GLUT4protein changes.Recently, it has been reported in six morbidly obese women aftermalabsorptive bariatric surgery, that the increase in Mfn2mRNA levels wasassociated with the improvement of whole-body glucose uptake as well aswith the rise in GLUT4expression. However, the direct evidence that Mfn2isinvolved in glucose uptake and insulin sensitivity has not been reported. Onthe basis of previous studies, we have reported a high-fat diet (HFD) causes adecrease in Mfn2and GLUT4expression in skeletal muscle, which mayexplain, at least in part, the development of insulin resistance. Another studyalso have shown that Mfn2and GLUT4expression is dysregulated in skeletalmuscle from obese or type2diabetic subjects, and a positive correlationbetween the Mfn2expression and the insulin sensitivity was also detected.Based on the presence of mitochondrial dysfunction in insulin resistantconditions, we sought to determine the effects of Mfn2in glucose homeostasisin vivo. However, the reason determining the relationship between Mfn2andGLUT4in a direct manner remains unclear. Therefore, the aim of the presentstudy was to test the hypothesis that Mfn2overexpression regulates theexpression and translocation of GLUT4in skeletal muscle. This was addressedby performing a repeated AdMfn2treatment experiment in HFD rats and in L6cells.Part Ⅰ Overexpression of Mfn2improves translocation of GLUT4inskeletal muscle of high-fat diet-fed ratsObjectives: The aim of the present study was to investigate whetherMfn2overexpression can improve insulin sensitivity of high-fat diet (HFD)rats and the possible underlying mechanisms.Methods: Forty male4-week-old Sprague–Dawley rats (80-100g) were obtained from the animal center of Hebei Medical University and maintainedin an optimal environment for1week before experimentation. Animals wererandomly divided into four groups: the negative control group (NC, n=10), thehigh-fat diet group (HF, n=10), the high-fat diet plus adenoviral vectors group(HF+Ado, n=10), and the high-fat diet plus adenoviral vectors encoding Mfn2group (HF+AdMfn2, n=10). The rats were housed in a12-h light-dark cycleand given free access to a rodent standard diet (65.5%carbohydrate,10.3%fat,and24.2%protein) or a HFD (20.1%carbohydrate,59.8%fat, and20.1%protein), starting at5weeks of age, for8weeks. Eight-weeks after the HFDtreatment, rats were subjected to a euglycemic-hyperinsulinemic clamp asdescribed previously to assess insulin sensitivity. Briefly, after two basalsamples were taken, insulin was infused intravenously at a constant rate (0.25units·kg-1·h-1). Blood samples (50μl) were taken every10min to determineblood glucose and adjust the glucose infusion until the glucose infusion ratewas stabilized. After8-weeks HFD treatment, rats in the intervention groups(the HF+Ado and HF+AdMfn2group) received0.1ml Ado or AdMfn2adenoviruses at a dose of1×1010plaque-forming units per ml via tail veininjection for3weeks, and the non-intervention rats (the NC and HF group)were injected with saline buffer only. At the end of the11-week study, the ratswere fasted overnight and then euglycemic-hyperinsulinemic clamp techniquewas applied to evaluate the improvement degree of insulin resistance. Ratswere anesthetized by intraperitoneal injections of pentobarbital sodium (50mg/kg body weight), and blood samples were drawn from the abdominal aortato measure the concentrations of glucose, insulin, and FFA in plasma. Then,the muscle tissues were collected, quickly placed in a liquid nitrogen container,and then stored at80°C for analysis. The experimental protocols wereapproved by the Animal Welfare Committee of the University and done inaccordance with the institutional guidelines for animal research. Mfn2andGLUT4mRNA levels were measured by real-time PCR and normalized withthe internal reference gene GAPDH. The expression of Mfn2, IRβ, PI-3K, Akt,p-Akt, AMPKα, p-AMPKα and GLUT4protein levels were measured by Western blot analysis.Results:1. After a total of11-week-HFD treatment, HF group had higherplasma glucose, insulin, and FFA levels than NC group. We also determinedplasma parameters after Ado or AdMfn2administration in subsets of HFD rats.The parameters did not alter in HF+Ado group compared with HF group, butdecreased in HF+AdMfn2group.2. AdMfn2increases the clamp glucoseinfusion rate. As expected, HFD caused a decrease in the clamp glucoseinfusion rate (GIR) required to maintain euglycemia. AdMfn2administrationincreased GIR by67%，whereas treatment of rats with Ado did not cause anyalteration compared with HF group.3. Mfn2mRNA and protein expression inresponse to HFD and AdMfn2. To study the regulatory profile of Mfn2, weanalyzed its expression under conditions such as exposure to HFD ortreatment with AdMfn2that stimulate whole-body Mfn2expression. Exposureto HFD for11weeks caused downregulation of Mfn2mRNA and proteinlevels in skeletal muscles (51%and44%of the NC Group, respectively,P<0.05), which is in keeping with previous reports. Administration of AdMfn2for3weeks caused a3.4-and3.3-fold stimulation in Mfn2mRNA and proteinexpression in muscle tissues, whereas in HF+Ado group the mRNA andprotein levels were not altered in muscle compared with HF group.4. AdMfn2specifically induces GLUT4mRNA and protein expression in HFD ratsmuscle tissues. Glucose uptake is mediated by GLUT4in muscle tissues. Toevaluate the role of the transporter in AdMfn2-induced insulin sensitivityimprovement, GLUT4expression levels were examined. GLUT4mRNAlevels were measured by real-time PCR and normalized with the internalreference gene GAPDH. Results indicated that, HFD down-regulated theGLUT4mRNA to a significant level (56%of the NC Group, P<0.05) and Adotreatment had no significant effect on the GLUT4mRNA level compared toHF Group. In contrast, after intervention of AdMfn2, the GLUT4mRNA wassignificantly higher in HF+AdMfn2Group compared to HF Group (149%ofthe HF Group, P<0.05). In keeping with previous studies, muscle expressionof GLUT4protein was clearly reduced in HF group (66%of NC Group, P<0.05). After AdMfn2treatment, the total GLUT4protein level wassignificantly increased up to131%of the HF group (P<0.05). On the contrary,there was no difference of the total GLUT4protein level between the HF+AdoGroup and the HF Group (P>0.05). Therefore, administration of AdMfn2for3weeks not only enhanced GLUT4gene expression but also stabilized GLUT4protein expression.5. AdMfn2promotes GLUT4translocation in HFD ratsmuscle tissues. To investigate the effect of AdMfn2on GLUT4translocation,the ratio of plasma membrane (PM) to total GLUT4protein was analyzed. ThePM and total GLUT4protein of muscle tissues was fractionated and subjectedto Western blot analysis. With administration of AdMfn2in HFD rats, the ratiowas significantly increased up to187%of the HF group (P<0.05). However,the ratio was not significantly altered in HF+Ado Group compared with theHF Group (P>0.05). These results demonstrated that AdMfn2inducedGLUT4-redistributed into the PM fraction in skeletal muscle tissues.6.Involvement of Akt and AMPK pathways in AdMfn2-induced glucose uptakeand GLUT4translocation. To elucidate the effect of AdMfn2on the signalingpathways involved in GLUT4translocation, the IR-PI-3K-Akt and AMPKpathways were analyzed by Western blot analysis. Results demonstrated thatAdMfn2had no effect on the phosphorylation of Akt (89%of the HF group,P>0.05) but significantly increased the phosphorylation of AMPKα (276%ofthe HF group, P<0.05). No differences were detected with regard to theexpression of AMPKα (P>0.05). Expression of the IRβ, PI-3K, p-Akt and Aktprotein was significantly decreased in HF Group compared to NCGroup(P<0.05). No significant differences were found in the expression levelsof the IRβ, PI-3K, p-Akt and Akt protein among the three groups (HF Group,HF+Ado Group, and HF+AdMfn2Group; P>0.05). These resultsdemonstrated that the effect of AdMfn2on GLUT4translocation partlycontributing to the increased AMPK activation.Conclusions:1. In parallel with glucose intolerance, HFD increased theplasma insulin and FFA levels in rats, and Mfn2overexpression improvesthese metabolic parameters.2. Mfn2is repressed in skeletal muscle in response to treatment with HFD and induced by administration of AdMfn2.3.HFD decreased GLUT4expression and translocation in skeletal muscleswhich were upregulated after Mfn2overexpression in HFD rats.4. Mfn2overexpression may induce GLUT4expression and translocation via AMPKphosphorylation rather than Akt phosphorylation in skeletal muscles of HFDrats, which was instrumental in improving insulin sensitivity.Part Ⅱ Effect of Mfn2on GLUT4expression in L6cellsObjectives: The aim of the present study was to test the hypothesis thatMfn2regulates the expression of GLUT4via an AMPK-PGC-1α-mediatedsignaling in L6cells.Methods: L6cells were maintained in DMEM supplemented with10%(v/v) FBS,100units/ml penicillin, and100μg/ml streptomycin at37°C with5%CO2. Differentiation was induced by switching to medium containing2%FBS for7days before treatments were begun. After differentiation, L6myotubes were incubated with adenovirus encoding either siMfn2or AdMfn2at a multiplicity of infection100plaque-forming units (pfu) cell-1(100MOI)for24h in DMEM containing0.5%FBS. Empty recombinant adenovirusserved as a control. After24h infection, the analysis was performed. Theexpression of Mfn2, AMPKα, p-AMPKα, PGC-1αand GLUT4protein levelswere measured by Western blot analysis.Results:1. Effect of infection of Ado, siMfn2or AdMfn2on Mfn2protein expression in differentiated L6cells. Exposure to siMfn2for24hcaused downregulation of Mfn2protein levels in L6cells (13.9%of the NCGroup, P<0.05). AdMfn2treatment for24h caused a2.61-fold stimulation inMfn2protein expression in L6cells compared to NC Group, whereas in Adogroup the protein levels were not altered compared with NC group.2. Effectof infection of Ado, siMfn2or AdMfn2on AMPK phosphorylation indifferentiated L6cells. Phosphorylation of AMPKα (p-AMPKα) wasdecreased in L6cells treated with siMfn2for12h (61.1%of the NC Group,P<0.05), and increased by AdMfn2for12h (1.49-fold of the NC Group,P<0.05), whereas without changes between Ado Group and NC Group (P>0.05). There were no differences in the total AMPKα from the four groups(P>0.05).3. Effect of infection of Ado, siMfn2or AdMfn2on PGC-1αprotein expression in differentiated L6cells. PGC-1α protein expression wasdecreased in L6cells treated with siMfn2for12h (32.4%of the NC Group,P<0.05), and increased by AdMfn2for12h (1.95-fold of the NC Group,P<0.05), whereas without change between Ado Group and NC Group(P>0.05).4. Effect of infection of Ado, siMfn2or AdMfn2on GLUT4proteinexpression in differentiated L6cells. GLUT4protein expression wasdecreased in L6cells treated with siMfn2for12h (54.5%of the NC Group,P<0.05), and increased by AdMfn2for12h (1.62-fold of the NC Group,P<0.05), whereas without change between Ado Group and NC Group(P>0.05).Conclusions: Mfn2induces AMPKα phosphorylation, increases PGC-1α protein expression, and upregulates GLUT4protein expression in L6cells.Part Ⅲ AMPK is required for Mfn2-induced expression andtranslocation of GLUT4protein in L6cellsObjectives: The aim of the present study was to test the hypothesis thatMfn2regulates the expression and translocation of GLUT4protein via anAMPK-dependent mechanism in L6cells.Methods: L6cells were maintained in DMEM supplemented with10%(v/v) FBS,100units/ml penicillin, and100μg/ml streptomycin at37°C with5%CO2. Differentiation was induced by switching to medium containing2%FBS for7days before treatments were begun. Differentiated L6myotubeswere treated with or without10μM compound C for30min, and thenincubated with or without adenovirus encoding AdMfn2at a multiplicity ofinfection100plaque-forming units (pfu) cell-1(100MOI) for24h in DMEMcontaining0.5%FBS. After24h infection, the analysis was performed. Theexpression of Mfn2, AMPKα, p-AMPKα, Akt, p-Akt, PGC-1αand GLUT4protein levels were measured by Western blot analysis.Results:1. Effect of AdMfn2without or with compound C （AMPKinhibitor）cotreatment on Mfn2protein expression in differentiated L6cells. Exposure to AdMfn2for24h caused upregulation of Mfn2protein levels in L6cells (3.0-fold of the NC Group, P<0.05), whereas compound C treatmentcaused a slight decrease in Mfn2protein expression compared with NC Group(P<0.05). Cotreatment with AdMfn2and compound C caused upregulation ofMfn2protein expression compared with the NC Group (P<0.05), butprevented the AdMfn2-induced increase of Mfn2protein expression (P<0.05).2. Effect of AdMfn2without or with compound C cotreatment on AMPKphosphorylation in differentiated L6cells. Exposure to AdMfn2for24hcaused upregulation of AMPK phosphorylation compared with NC Group(P<0.05). Compound C without or with AdMfn2cotreatment causeddownregulation of AMPK phosphorylation compared with NC Group(P<0.05), and also decreased compared with AdMfn2Group (P<0.05),whereas without changes between CC Group and AdMfn2+CC Group(P>0.05). There were no differences in the total AMPKα from the four groups(P>0.05).3. Effect of AdMfn2without or with compound C cotreatment onAkt phosphorylation and Akt protein expression in differentiated L6cells. Nosignificant differences were found in the expression levels of the p-Akt andAkt protein among the four groups (NC Group, AdMfn2Group, CC Group,and AdMfn2+CC Group; P>0.05).4. Effect of AdMfn2without or withcompound C cotreatment on GLUT4expression and translocation indifferentiated L6cells. Exposure to AdMfn2for24h caused upregulation ofGLUT4plasma membrane protein expression compared with NC Group(P<0.05). Compound C without or with AdMfn2cotreatment causeddownregulation of GLUT4plasma membrane protein expression comparedwith NC Group (P<0.05), and also decreased compared with AdMfn2Group(P<0.05), whereas without changes between CC Group and AdMfn2+CCGroup (P>0.05). The total GLUT4protein expression also follows the sametrend. There were no differences in the ratio of PM to total GLUT4proteinfrom the four groups (P>0.05).5. Effect of AdMfn2without or withcompound C cotreatment on PGC-1αprotein expression in differentiated L6cells. Exposure to AdMfn2for24h caused upregulation of PGC-1αprotein expression compared with NC Group (P<0.05). Compound C without or withAdMfn2cotreatment caused downregulation of PGC-1αprotein expressioncompared with NC Group (P<0.05), and also decreased compared withAdMfn2Group (P<0.05), whereas without changes between CC Group andAdMfn2+CC Group (P>0.05).Conclusions: Mfn2increases GLUT4expression via anAMPK-dependent mechanism in L6cells, without affecting the translocationof GLUT4. |
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