Mechanisms Underlying Effects Of Enoyl Coenzyme A Hydratase 1(ECH1) On Browning Reaction Of White Adipose Tissue In Diet-induced Obesity

Part Ⅰ Expression levels of enoyl coenzyme A hydratase 1 in different models and its effect on high fat diet induced obesity and metabolic disordersObjective: In this part,the relationship of expression levels of ECH1 in inguinal white adipose tissues(iWAT)with energy expenditure was analyzed,and the effect of ECH1 on high fat diet(HFD)induced obesity was explored.Methods: Firstly,the mRNA levels of ECH1 in various tissues from C57BL/6J mice were measured by q-PCR.Then its expression levels in iWAT of HFD or cold treated mice,as well as lean or obese patients,were detected and analyzed.Subsequently,obese mice induced by HFD with ECH1 overexpression or not was built and metabolic parameters during HFD fed were monitored.After 8 weeks of HFD,hepatic and serum lipids,glucose,and insulin levels were detected and analyzed.Results: ECH1 in iWAT was downregulated in HFD mice,while upregulated by cold treatment.Accordantly,the mRNA levels of ECH1 were lower in iWAT of obese patients than that of lean individuals,and it was positively correlated with UCP1 mRNA levels and negatively correlated with BMI.In the HFD fed mice,the overexpression of ECH1 in iWAT ameliorated HFD-induced obesity,as well as accompanied insulin resistance,NAFLD,and dyslipidemia.Conclusions: The ECH1 expression levels in iWAT were closely related to energy metabolism,and it played a benefit role in ameliorating HFD induced obesity and accompanied metabolic disorders.Part Ⅱ Role of ECH1 in energy metabolism and browning of white adipose tissueObjective: To determine the effect of ECH1 overexpression in iWAT on energy metabolism and browning of white adipose tissue.Methods: After 8 weeks of HFD fed,the comprehensive lab animal monitoring system(CLAMS)was used to monitor the energy metabolism of mice in part Ⅰ,of which the rectal temperature at room temperature or cold environment was also detected.After 48 h cold exposure,the iWAT of HFD mice were collected and the browning markers were examined by q-PCR,western blot and immunohistochemistry.In the next cohort study,SCD-fed mice were overexpressed or knocked down ECH1 in unilateral iWAT and exposed to 6℃ for 48 h.Then browning markers in iWAT were detected.Results: The CLAMS data showed that the oxygen consumption rate(VO2),carbon dioxide production rate(VCO2)and heat production were higher in mice with ECH1 overexpression when comparing to that of control mice.At the same time,the mice with ECH1 overexpression showed better maintenance of body temperature,more abundant beige adipocytes in iWAT,and increased ratio of small adipocytes in both iWAT and e WAT.In iWAT of SCD-fed mice,ECH1 overexpression also promoted browning of white adipose tissue and inhibited the mTOR signaling pathway and vice versa.Conclusions: The research presented here confirmed that ECH1 in iWAT was involved in adipose browning,promoted the thermogenic program and the energy expenditure,thus ameliorated HFD-induced obesity and accompanied metabolic disorders.Part Ⅲ Role of ECH1 on browning of adipocyte and the potential mechanismObjective: In this part,we focused on the effect of ECH1 on browning of adipocyte,and whether mTOR signaling pathway was involved in the regulation of ECH1 on adipose browning was further verified.Methods: The precursor adipocyte-derived mature adipocytes were infected with adenovirus mediating overexpression or knockdown of ECH1,then browning and lipolysis were detected by western blot,q-PCR and Oil Red O staining after CL316,243 treatment or not.Next,we treated adipocyte of which ECH1 has been knocked down with rapamycin,the specific inhibitor of mTOR signaling pathway,and analyzed the effect of rapamycin on adipocyte browning as stated above.Results: Adenovirus-mediated ECH1 overexpression promoted browning of adipocyte at both basal or CL316,243 treatment when comparing to that of ad-GFP control.Consistently,knockdown of ECH1 inhibited the response of adipocyte to CL316,243 or 8-Br-cAMP,and rapamycin treatment abated this inhibition.Conclusions: These observations demonstrated that ECH1 directly acted on adipocytes and promoted thermogenesis in a cell-autonomous manner at least partially via the mTOR signaling pathway downstream of the β3-adrenergic receptor.