Role And Mechanism Of Mitochondrial Trifunctional Protein ?-subunit In Adipocyte Differentiation

ObjectiveMitochondrial trifunctional protein(MTP)is a hetero-multimeric enzyme located in the inner mitochondrial membrane,which consists of four ?-subunits and four ?-subunits and catalyzes last three of the four chain-shortening reactions in fatty acid ?-oxidation(FAO)cycle.Recent studies found that MTP? have been linked with the occurrence and development of obesity and obesity-related disorders.Obesity is essentially by an increase in adipocyte number and adipocyte size,namely adipocyte hypertrophy,which is the result of much triglyceride accumulated in adipocytes;and the increased number of adipocytes is caused by adipocyte differentiation,namely the more formation of mature adipocytes from preadipocytes.For nnow,the role of MTP? in the pathogenesis of obesity has not been evaluated,and the function of MTP? in adipocyte differentiation has not been determined.Therefore,this research has focused on the role of MTP? during adipocyte differentiation and its mechanisms.MethodsTo observe the expression of MTP? during adipocyte differentiation through in vitro adipogenesis models,namely MDI induced adipogenic differentiation of 3T3-L1 preadipocyte;Permanent silencing was performed using MTP?-targeted and control sh RNA lentiviral particles(GV248),then MTP?-targeted sh RNA transfected 3T3-L1 preadipocyte were used to study the function of MTP? during adipocyte differentiation.And next to confirm the function of MTP? by overexpressing MTP? in 3T3-L1 preadipocyte with lentiviral vectors(GV358)containing an MTP? expression cassette.The m RNA and protein expression of MTP? and adipocyte-differentiation related factor,including CCAAT/enhancer binding protein ?(C/EBP?),peroxisome proliferator-activated receptor ?(PPAR?),and fatty acid binding protein 4(FABP4)were detected by real-time fluorescent quantitative PCR(QPCR)and Western blot(WB);Oil Red O staining to detect the adipogenic differentiation of 3T3-L1 preadipocyte.On the basis of above experiment,we try to reveal the mechanism of MTP? in preadipocyte differentiation,on the one hand,through QPCR to observe the expression changes of cell-proliferation-associated genes,including proliferating cell nuclear antigen(PCNA),Cyclin B1,Cyclin E1,and insulin-like growth factor 1(IGF-1),in MTP? knockdown and overexpression 3T3-L1 preadipocytes;on the other hand,we treated MTP?-overexpressing 3T3-L1 preadipocyte with the SIRT1 inhibitor EX-527,and meantime through MDI induced adipogenic differentiation.Based on the in vitro experimental results.To explore whether MTP? regulates the adipogenic differentiation of the preadipocytes in vivo,we adopted an in vivo model of adipocyte differentiation,injection of MTP? overexpressing(ov MTP?)or control(ov CON)3T3-F442 A preadipocytes in the back of 6-8 weeks nude mice,resulted in the formation of de novo fat pads after 5 weeks of feeding.To detect the in vivo adipogenic differentiation of de novo fat pads by using H&E,and to anylysis MTP? and SIRT1 expression of fat pads using immunohistochemistry.ResultsFirst,we found that MTP? expression was upregulated during the differentiation of 3T3-L1 preadipocyte cells into adipocytes,thus we speculated that MTP? may play a role inadipocyte differentiation.In order to verify this deduction,we constucted MTP?-targeted sh RNA transfected 3T3-L1 preadipocyte and MDI induced adipogenic differentiation;QPCR and WB analysis indicated that the knockdown efficiency of MTP? in transfected cells is low during differentiation.Compared with control group,lipid droplet accumulation significantly increased in MTP?-knockdown cells on day 8 of differentiation;and MTP? knockdown induced the increases of PPAR?,C/EBP?,and FABP4 expression during mid-late stage of differentiation.The results have shown that MTP? inhibit differentiation.We next confirmed whether MTP? overexpression might repress adipocyte differentiation in 3T3-L1 cells.MTP? was overexpressed in 3T3-L1 cells by transfecting lentiviral vector containing an MTP? expression cassette.The accumulation of lipid droplets significantly decreased in MTP?-overexpression cells on day 8 of differentiation,when compared with ov CON cells;and PPAR?,C/EBP?,FABP4 also were repressed in MTP? overexpressing cells.Further,we found that MTP? overexpression obviously enhanced SIRT1 expreesion.Previous studies have found that SIRT1 inhibit the adipocyte differentiation of 3T3-L1 preadipocyte and mesenchymal stem cells.Thus,we speculate MTP? inhibit differentiation may partly by promoting SIRT1 expression.To test this speculation,the MTP? overexpression 3T3-L1 preadipocytes were treated with the SIRT1 inhibitor EX-527;the results showed that EX-527 did not affect MTP? expression,but did reduce SIRT1 levels;and the compensation of EX-527 to impairment of MTP? overexpression on 3T3-L1 preadipocytes differentiation.We also found that MTP? overexpression suppressed cell-proliferation-associated genes expression,including PCNA,Cyclin B1,Cyclin E1 and IGF-1.Finally,we adopted an in vivo model of adipocyte differentiation,to study the role and mechanism of MTP? in vivo adipocyte differentiation.H&E staining of the de novo fat pad showed that the control lentiviral-transfected(ov CON)3T3-F442 A cells were normally differentiated into adipocytes,whereas MTP? lentiviral-transfected(ov MTP?)cells were not differentiated into adipocytes.Immunohistochemistry with anti-MTP? and anti-SIRT1 antibody showing that MTP? and SIRT1 expression was strongly positive(stained brown)in the tissues of mice which were injected with 3T3-F442 A cells containing MTP? lentivirals,whereas differentiated adipocytes in the tissue of control mice demonstrated less expression of MTP? and SIRT1.ConclusionMTP? Plays a negative regulation role in adipocyte differentiation in vitro,during which the proliferation of preadipocyte was supressed and the expression of SIRT1 was promoted.And MTP? also blocks adipocyte differentiation through upregulating SIRT1 expression in a physiological in vivo system.