Effect Of Mitochondrial Aconitase Aco2 On Lipid Deposition Of 3T3-L1 Cells And Its Mechanism

Mitochondria are necessary for maintaining white adipocyte metabolic homeostasis,TCA cycle produced ATP sustains normal lipid synthesis during the early period of adipogenesis.In the TCA cycle,mitochondrial aconitase(Aco2)catalyzes the conversion of citrate to isocitrate,which produces NADH and FADH2 driving synthesis of ATP through OXPHOS.In this study,we explore the relationship between adipogenesis and mitochondrial energy metabolism through overexpression and deficiency of Aco2.Our work is helpful to further understand the relationship between TCA cycle enzymes and mitochondrial function and lipid metabolism of adipose tissue,providing a potential method for treatment obesity and related metabolic syndrome and providing a theoretical basis for improving meat quality.The main results of this study are as follows:1.Expression patterns of Aco2 in adipose tissue of mice and during adipocyte differentiation in 3T3-L1 cells.Compared to CD mice,m RNA levels of Aco2 was significantly lower in i WAT,e WAT,BAT and livers of HFD mice.However,Aco2 enzyme activity in i WAT showing higher levels in HFD mice than that of CD mice.Levels of Aco2 m RNA and activity in i WAT of mice exposed at 4?significantly higher than those at room temperature.During3T3-L1 cells differentiation,levels of Aco2 m RNA and activity showing stable expressions in the early differentiation period from day 0 to day 4,with increased levels in the late differentiation phase from day 4 to day 8.2.Overexpression of Aco2 promotes adipogenesis in 3T3-L1 cells.Overexpression of Aco2 promote triglyceride synthesis,increased m RNA and protein levels of lipogenesis Acc,Fas and TG synthesis Glut4,Ppar-?,Dgat2 and reduced protein levels of two major adipose tissue lipases ATGL and HSL without affecting cell proliferation.3.Aco2 overexpression enhances mitochondrial ATP production.Aco2 overexpression significantly enhances mitochondrial biogenesis by increasing the m RNA expression of mitochondrially encoded genes,mitochondrial mass,mitochondrial DNA copy number and the m RNA levels of mitochondrial biogenesis regulators in adipocytes.By increasing the activity of citrate synthase(Cs),the ratio of NADPH/NADP~+enhances the rate of mitochondrial TCA cycling.By increasing the protein expression of select subunits of the OXPHOS complexes promoted the ATP level.Thus,proving that overexpression of Aco2enhances mitochondrial biogenesis and ATP synthesis in 3T3-L1 cells.Furthermore,in order to test that increased adipogenesis in Aco2-overexpressing cells was attributed to elevated ATP production.By added 20?m rotenone to Aco2-overexpressing cells to consume the ATP content in the cells,we found the promoting effect of Aco2 overexpressing on adipogenesis was abolished by rotenone treatment,suggesting that Aco2 overexpression mediates adipogenesis through elevation of ATP production4.Deficiency of Aco2 inhibits adipocytes differentiation.CRISPR/Cas9-mediated Aco2deficiency was applied in 3T3-L1 cells,the results show that deficiency of Aco2 inhibit triglyceride synthesis,reduced m RNA and protein levels of lipogenesis and TG synthesis,increased protein levels of two major adipose tissue lipases ATGL and HSL and show lower levels of citrate,isocitrate,fumarate as well as malate without affecting cell proliferation.Further,in order to study the mechanism of Aco2 deficiency inhibiting lipid synthesis,we examined the expression levels of mitochondrial biogenesis,TCA cycle rate and ATP content in Aco2 deficiency and control cells,these results demonstrate that deficiency of Aco2 reduces mitochondrial biogenesis and ATP synthesis in 3T3-L1 cells.5.Supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.Considering the role of Aco2 in the conversion of citrate to isocitrate in the TCA cycle,and we found that the level of isocitrate is reduced in Aco2-deficient cells.Next,we explored whether the supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.By add 5m M of isocitrate to the media of Aco2-deficient cells,lipid accumulation was efficiently restored morphologically and the m RNA and protein levels of genes related to adipogenesis and TG synthesis were also increased.Meanwhile,mitochondrial biogenesis and ATP content were also increased.These results indicate that supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.To further test whether production of ATP was the limiting factor for the stimulation of adipogenesis by isocitrate,20?m of rotenone was added into isocitrate-treated Aco2-deficient cells,the results show depletion of ATP production abolishes the effect of isocitrate,suggesting that ATP is the rate-limiting factor for isocitrate to restore adipogenesis in Aco2-deficient cells.In summary,our results demonstrate an important capacity for adipocyte Aco2 in adipogenesis,overexpression of Aco2 promotes lipid accumulation in 3T3-L1 cells,deficiency of Aco2 inhibit adipogenesis in adipocytes and supplementation of isocitrate restores adipocyte differentiation in Aco2-deficient cells.On the other hand,mechanistically,the role of Aco2 in adipogenesis is ATP-dependent.By ATP is depleted,the promotive role of Aco2 in adipogenesis is abolished.