title: Inhibition of DNA-dependent protein kinase reduced palmitate and oleate-induced lipid accumulation in HepG2 cells.
abstract: OBJECTIVE
  The aim of this study is to investigate the involvement of DNA-dependent protein kinase (DNA-PK) in palmitate and oleate-induced lipid accumulation in hepatocytes.
  
  
  METHODS
  We treated HepG2 with free fatty acids (FFA) (0.33 mM palmitate and 0.66 mM oleate) mixture to induce lipid accumulation. Cellular lipid was determined by Nile Red staining followed by flow cytometry detection as well as phase contrast and fluorescence microscope examination. Cell viability was detected by MTT assay. Apoptosis was detected by DAPI staining. Lipogenic gene expression was examined by real-time PCR at mRNA level and Western blotting at protein level. Promoter transcriptional activity was measured by dual luciferase assay.
  
  
  RESULTS
  FFA treatment neither affected HepG2 cells viability nor induced DNA fragmentation, while induced cellular lipid accumulation was associated by the upregulation of sterol regulatory element-binding protein-1 (SREBP1) and fatty acid synthase (FAS) at both mRNA and protein levels. Interestingly, we also found that both the protein phosphatase 2A (PP2A) protein expression and DNA-PK activity were increased in these cells. Inhibition of PP2A by okadaic acid, knockdown of DNA-PK by siRNA or inhibition of DNA-PK by specific DNA-PK inhibitors curtailed the FFA-induced upregulations of the SREBP1 mRNA expression and the nuclear active SREBP1 protein expression, and reduced FFA-induced upregulation of FAS promoter transcriptional activity and lipid accumulation.
  
  
  CONCLUSIONS
  This is the first time suggesting that inhibition of DNA-PK reduced FFA-induced lipid accumulation in hepatocytes. This finding might help us better understand non-alcoholic steatohepatitis pathogenesis.
authors.affiliation: Center for Cancer and Inflammation Research, School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong, Hong Kong, China.
authors.forename: Hiu Yee
authors.initials: HY
authors.lastname: Kwan
authors.forename: Wang Fun
authors.initials: WF
authors.lastname: Fong
authors.forename: Zhijun
authors.initials: Z
authors.lastname: Yang
authors.forename: Zhi-Ling
authors.initials: ZL
authors.lastname: Yu
authors.forename: Wen-Luan Wendy
authors.initials: WL
authors.lastname: Hsiao
mesh_terms: D017209:Apoptosis / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D053938:DNA Fragmentation / Q000187:drug effects; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D064429:Fatty Acid Synthases / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000187:drug effects* / Q000473:pathology; D006801:Humans; D050356:Lipid Metabolism / Q000187:drug effects; D019301:Oleic Acid / Q000009:adverse effects*; D019308:Palmitic Acid / Q000009:adverse effects*; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D054648:Protein Phosphatase 2 / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D051780:Sterol Regulatory Element Binding Protein 1 / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
chemical_list: D012333:RNA, Messenger; D034741:RNA, Small Interfering; C497387:SREBF1 protein, human; D051780:Sterol Regulatory Element Binding Protein 1; D019301:Oleic Acid; D019308:Palmitic Acid; D064429:Fatty Acid Synthases; D051747:DNA-Activated Protein Kinase; D054648:Protein Phosphatase 2