Mitochondrial Oxidative Phosphorylation Deficiency Inhibits The Differentiation Of Brown Adipocytes And Related Mechanism

Objective:With the enrichment of material culture and the introduction of western diet,the surge of obese crowd in China has caused many diseases such as obesity,type 2 diabetes and related metabolic syndrome.Adipose tissue is not only the main energy storage organ,but also is an important endocrine organ.Brown adipose tissue(BAT)is distributed in the neck,the clavicle and interscapular region of the back of the adult and can be activated by a longterm cold stimulation.Brown adipocytes express uncoupling protein 1(UCP1)which regulates the whole body energy metabolism through non-shivering thermogenesis(NST).Furthermore,aboundant mitochondria which play a key role in the metabolism and status of heat production exist in brown adipocytes.Recently,more and more evidence shows that the mitochondrial oxidative phosphorylation system(OXPHOS)is crucial to brown adipose retrograde regulation,but the knowledge of mechanism is limited and controversial.Rotenone(ROT)reduces cellular respiration by inhibiting mitochondrial electron transfer respiratory chain complex I.Herein,we confirmed that OXPHOS defects can inhibit brown adipocyte differentiation by establishing a pharmacological model of OXPHOS defect through rotenone everyday treatment during brown adipocyte differentiation.The differentiation of brown adipocytes is a complex process which is controlled by multi factors.Therefore,we detected transcription factors expression such as C/EBP? and PPAR? to explore the mechanism of the phenomenon that OXPHOS defects inhibit brown adipogenesis.OXPHOS defect leads to a decrease in celluar ATP content and an increase in reactive oxygen species(ROS)or cytoplasmic calcium.However,the function of these signal molecules in the process that brown adipogenesis is retarted by OXPHOS defects remains to be studied.We also studied whether enhancing glycolysis to supplement ATP or blocking ROS or calcium can restore the differentiation status of brown adipocytes through pyruvate,antioxidants or calcium chelating agents combined with rotenone treatment.In order to explore the key mechanism,we explored the changes of glucose metabolism related signals in ROT group which is compared by the effect of Etomoxir(ETX)on the differentiation of brown adipocytes after inhibiting fatty acid oxidation.Methods:1.The phenotype and gene expression during brown adipocyte differentiationBrown adipocyte differentiation was induced by inductive agents and lipid droplets accumulation was observed during the adipogentic process on day 0,2,4,6,and 8.Lipogenesis factor,brown fat specific marker,mitochondrial and peroxisomal genetic expression were identified to confirm the successful differentiation of brown adipocyte for the following experiments via using real-time fluorescence quantitative detection instrument(Q-PCR).2.The establishment of pharmacological model of mitochondrial OXPHOS defects and its effect on brown adipocyte differentiation100 n M ROT was added daily from preadipocyte to mature brown adipocyte to establish the pharmacological model of mitochondrial OXPHOS defects and the inhibition of respiration was measured via Seahorse.The change of lipid droplets was detected during the inhibition of brown adipogenesis in ROT treatment on day 0,3 and 7 by Oil red staining.3.The effect of mitochondrial OXPHOS defects on gene expression during brown adipogenesisThe m RNA of brown adipocyte was extracted on day 0,3 and 7 of DMSO control group and ROT treatment group.The expression of lipogenesis factor Ap2,brown fat specific marker such as Ucp1,Cidea,Prdm16 and mitochondrial gene like Cox4i1,Cycs,Atpsyn? and peroxisomal gene including Acox1,Pmp70,Pex13,Pex16,Gnpat,Cat were detected via Q-PCR.4.The effect of mitochondrial OXPHOS defects on transcription factor during brown adipogenesisBrown adipocyte differentiation is regulated by multiple complex transcription factors.The m RNA of brown adipocyte was extracted on day 0,3 and 7 of DMSO control group and ROT treatment group.The expression of transcription factors such as Ppar?,Pgc1?,C/Ebp?,C/Ebp?,Pref1,Gata2 and other transcription factors were detected by Q-PCR.5.The recovery impact of increasing in ATP level or decreasing in ROS level on the retardation of brown adipocyte differentiation regulated by mitochondrial OXPHOS defectsMitochondrial OXPHOS defects can reduce ATP production and increase ROS release.ROT was co-treated with pyruvate enhancing glycolysis for ATP supplement,L-Apartic acid providing more nutrients or antioxidant reducing ROS production to examine the change of lipid droplets and expression of thermogenic gene during brown adipogenesis via Q-PCR.6.The recovery impact of decreasing Ca2+ level on the retardation of brown adipocyte differentiation regulated by mitochondrial OXPHOS defectsMitochondrial OXPHOS defects can lead to a rise in cytoplasmic calcium concentration.ROT was co-treated with calcium chelating agent to examine the change of lipid droplets and expression of thermogenic gene during brown adipogenesis via Q-PCR.7.The effect of lactate on the differentiation of brown adipocytesMitochondrial OXPHOS defects can increase extracellular acidification and lactic acid production.20 m M lactate was added daily from preadipocyte to mature brown adipocyte to examine the change of lipid droplets and the expression of thermogentic gene Ucp1 during the differentiation of brown adipocytes in order to understand the role of lactate.8.The effect of fatty acid oxidation inhibition on brown adipogenesisETX can inhibit the oxidation of fatty acid.10 ?M ETX was added daily from preadipocyte to mature brown adipocyte to examine the expression of lipogenesis factor,brown fat specific marker,mitochondrial and peroxisomal gene and transcription factors to study the effect of fatty acid oxidation inhibition on the differentiation of brown adipocytes.9.The changes of signal transduction pathway on retardation of brown adipogenesis regulated by mitochondrial OXPHOS defectsBrown adipocyte differentiation requires sequential activation of AMPK/m TOR signaling to affect the transcription factor expression.The protein of brown adipocyte was extracted on day 0,3 and 7 of DMSO control group and ROT or ETX treatment group.WB showed that the changes of m TOR,ERK,AKT,p38 and GLUT 1/4 signal transduction pathways to investigate the mechanism of retardation of brown adipocyte caused by mitochondrial OXPHOS defects.Results:1.The successful phenotype and gene expression during brown adipocyte differentiation: Light microscopy showed the lipid accumulation during brown adipocyte differentiation and Q-PCR showed an increase in the expression of adipogenic genes,brown specific genes,mitochondrial and peroxisomal genes increased with the increasing differentiation days,indicating that brown adipocytes were successfully differentiated for subsequent research.2.The establishment of pharmacological model of mitochondrial OXPHOS defects and its effect on brown adipocyte differentiation: Seahorse showed that 100 n M ROT inhibits 50% respiration of preadipocytes indicating the successful establishment of a pharmacological model of mitochondrial OXPHOS deficiency.Oil red staining indicated that lipid accumulation in brown adipocytes was reduced in ROT treatment group compared with DMSO control group3.The effect of mitochondrial OXPHOS defects on gene expression during brown adipogenesis: Compared with DMSO control group,the expression of brown specific genes such as Ucp1,Cidea,Prdm16,mitochondrial genes like Cox4i1,Cycs,Atpsyn?,peroxisomal genes including Acox1,Pmp70,Pex13,Pex16,Gnpat and Cat all decreased on day 3 and 7.4.The effect of mitochondrial OXPHOS defects on transcription factor during brown adipogenesis: The expression of Ppar?,Pgc1?,C/Ebp? and C/Ebp? decreased on day 3 after ROT treatment compared with DMSO control group,while the expression of Gata 2,Fgf21 and Cox2 increased without significant change in Pref1.5.The recovery impact of increasing ATP level or decreasing ROS level on the retardation of brown adipocyte differentiation regulated by mitochondrial OXPHOS defects: Compared with ROT treatment group,pyruvate and ROT co-treated group could increase ATP level but could not restore lipid droplet accumulation and expression of brown specific genes such as Ucp1,Cidea and Prdm16.6.The recovery impact of decreasing Ca2+ level on the retardation of brown adipocyte differentiation regulated by mitochondrial OXPHOS defects: Compared with ROT treatment group,calcium chelator and ROT co-treated group could not restore lipid droplet accumulation and expression of brown apecific genes such as Ucp1,Cidea and Prdm16.7.The effect of lactate on the differentiation of brown adipocytes: Compared with control group,Q-PCR showed that Ucp1 expression decreased by 10 % on day 3 in lactate treatment group.8.The effect of fatty acid oxidation inhibition on the differentiation of brown adipocytes: Oil red O staining showed that the accumulation of lipid droplets was retarded by ETX treatment but the change of content is indifferent.Compared with control group,ETX treatment could reduce brown specific gene expression such as Ucp1,Cidea and mitochondrial genes expression like Cox4i1,Cycs,Atpsyn?.9.The changes of signal transduction pathway on retardation of brown adipogenesis regulated by mitochondrial OXPHOS defects: WB showed that compared with DMSO and ETX group,ROT treatment could activate AMPK temporarily and suppress m TOR,p38 and ERK phosphorylation as well as GLUT1 and GLUT4,and total protein acetylation was increased on day 3 but no significant difference occurred on day 7.Conclusion:1.Mitochondria OXPHOS defects that the daily treatment of ROT from pre-adipocyte to mature brown adipocyte continuously inhibits cellular respiration,which results in the retardation of brown adipogenesis and a decrease in the expression of brown specific,mitochondrial and peroxisomal genes.2.Increasing ATP content,supplementing nutrition and reducing ROS or Ca2+ level could not alleviate the inhibition of mitochondrial OXPHOS defects on brown adipocyte differentiation.3.The inhibition of fatty acid oxidation delayed the differentiation of brown adipocytes,but the mechanism was different from the mitochondrial OXPHOS defects.4.Mitochondrial OXPHOS defects could decrease GLUT1 and GLUT4 expression to activate AMPK temporarily and suppress m TOR and ERK phosphorylation,then decrease the expression of transcription factors such as Ppar? and C/Ebp? which further reduce thermogentic,mitochondrial and peroxisomal genes.