Research On The Uncoupling Activity Of Liver Mitochondrial Carrier Protein HDMCP And Its Relation With Nonalcoholic Fatty Liver Disease

Backgrond/aims:Nonalcoholic fatty liver disease(NAFLD) represents a common clinicopathologic condition characterized by lipid deposition in hepatocytes of liver parenchyma without alcohol consumption in amounts considered to be harmful to the liver,ranging from simple steatosis to steatohepatitis,fibrosis,cirrhosis and hepatocellular carcinoma. Nowadays,there are accumulating evidences that mitochondrial dysfunction plays a pivotal role in NAFLD.Uncoupling proteins(UCPs) belong to the superfamily of mitochondrial anion-carrier proteins,which are located on the mitochondrial inner membrane and are identified in various tissues.UCPs uncouple the mitochondrial respiration from ATP synthesis by dissipating the transmembrane proton gradient to further influence mitochondrial function and metabolic process.Therefore,UCPs might be a key cluster of proteins involved in mitochondrial dysfunction.Liver is the largest metabolic organ in human body and mitochondrial proton leak accounts for 20-30%of the oxygen consumption of isolated resting hepatocytes.Due to the uncoupling character of UCPs,it is plausible that they might participate in hepatic mitochondrial proton leak and certain dysregulated metabolic pathways.Nevertheless, there are currently no UCPs(UCP 1 to 5) detected in normal hepatocytes. Hepatocellular carcinoma down-regulated mitochondrial carrier protein(HDMCP) was first cloned in the year of 2004 and proved to be exclusively expressed in liver.This protein bears all the hallmark features of the mitochondrial anion-carrier proteins and is significantly down-regulated during the development of hepatocellular carcinoma. However,whether it does have an uncoupling activity needs to be examined by more experimental systems.Therefore,our aim of this study is to explore the uncoupling activity of HDMCP in a yeast expression system and its function in NAFLD.Methods:CDNAs of UCP1,OXO and HDMCP were obtained using routine RT-PCR amplification from mice livers and then sequentially cloned into PMDT₁₈ and pYES2 expression vector.Thereafter,Saccharomyces cerevisae was transformed with purified pYES2 vectors containing UCP1,OXO,HDMCP and empty vector and further grown in SC-ura liquid medium.After approximately 48h growth,mitochondria were isolated from the yeast and the uncoupling activity was measured.NAFLD cell model was established by exposing L02 and HepG2 cells at 80%confluency to HFFA,a mixture of oleate(OA) and palmitate(PA),at the final ratio of 2:1 and concentration of 1mM for serial time spots(24 h,36h,48h,60h and 72h).Nile Red(1 mg/ml in PBS) was used to display the cellular lipid content.Cellular protein content was assessed by the lowry method and TG level was measured using the method of GPO/POD enzymatic reaction. For NAFLD animal model,a total of 24 twelve-week-old SDrats weighing 158g-172g were randomly divided into NAFLD(n=12) and control group(n=12).Control group was given basic diet while NAFLD group was given fat-rich diet.The successful establishment of both two models was confirmed by pathologic(Nile red and H-E staining) and biochemical changes(TG,Tch,fasting glucose,fasting insulin,et al).Routine real time RT-PCR and western blot were used to detect HDMCP mRNA and protein levels.RNA interference was used to knockdown HDMCP level by adding HDMCP-shRNA into cell models using Lipofectamine 2000.Mitochondrial ATP was extracted and measured using a luciferin-luciferase bioluminescent assay.The rate of mitochondrial H₂O₂ production was determined according to the manufacturer's instructions.Each experiment was performed in at least triplicate and data were expressed as means±SE(standard error).Unpaired student's t-test for normal distributed numerical variance and Mann-Whitney for skewed data were executed by SPSS 11.0.Linear correlation was used to explore the association between two variables.The differences were considered statistically significant at p＜0.05.Results:Firstly,we successfully expressed HDMCP in yeast mitochondria and found a significant GDP insensitive uncoupling activity of HDMCP.Secondly,on the basis of successfully established animal and cell NAFLD models,we found a significantly increased expression of HDMCP in both two models,where the increment of HDMCP in cell model was correlated with culture time and steatosis was aggravated when HDMCP level was knocked down by RNA interference.Finally,we found that the effect of HDMCP in steatosis alleviation might function through promoting ATP depletion and decreasing H₂O₂ production,as these two markers were both significantly decreased in L02 and HepG2 cells with high expression of HDMCP.Furthermore, down-regulation of HDMCP could significantly antagonize these changes.Conclusion:This study adds supportive data to the hypothesis that HDMCP might be a long postulated liver specific uncoupling protein and broaden our understanding of the pathogenesis of NAFLD.More importantly,HDMCP might become a novel drug target for its property in alleviating hepatic steatosis.