Inhibition of DNA-dependent protein kinase reduced palmitate and oleate-induced lipid accumulation in HepG2 cells
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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.
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.
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.
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.
KeywordsDNA-PK PP2A Palmitate Oleate Lipid Hepatocellular steatosis
This study was supported by grants FRG1/10-11/035, FRG2/10-11/023 and FRG2/11-12/057 from Hong Kong Baptist University.
- 1.Brunt EM (2004) Nonalcoholic steatohepatitis. Semin Liver Dis 24:3–20Google Scholar
- 2.Cohen JC, Horton JD, Hobbs HH (2011) Human fatty liver disease: old questions and new insights. Science 32:1519–1523Google Scholar
- 14.Wang Q, Gao F, Wang T, Flagg T, Deng X (2009) A nonhomologous end-joining pathway is required for protein phosphatase 2A promotion of DNA double-strand break repair. Neoplasia 11:1012–1021Google Scholar
- 21.Donnelly KL, Smith Ci, Schwarzenberg SJ, Jessurun J, Boldt MD, Parks EJ (2005) Sources of fatty acids stored in liver and secreted via lipoproteins in patients with nonalcoholic fatty acid liver disease. J Clin Invest 115:1343–1351Google Scholar
- 23.Nakamuta M, Kohjima M, Morizono S, Kotoh K, Yoshimoto Y, Miyagi I, Enjoji M (2005) Evaluation of fatty acid metabolism-related gene expression in nonalcoholic fatty liver disease. Int J Mol Med 16:631Google Scholar
- 30.Shimizu S, Ugi S, Maegawa H, Eqawa K, Nishio Y, Yoshizaki T, Shi K, Nagai Y, Morino K, Nemoto K, Nakamura T, Bryer-Ash M, Kashiwagi A (2003) Protein-tyrosine phosphatase 1B as new activator for hepatic lipogenesis via sterol regulatory element-binding protein-1 gene expression. J Biol Chem 278:43095–43101CrossRefGoogle Scholar
- 32.Shi K, Ugi S, Shimizu S, Sekine O, Ikede K, Eqawa K, Yoshizaki Y, Nagai Y, Nishio Y, Takada T, Torii R, Kimura H, Kashiwagi A, Maegawa H (2007) Membrane localization of protein-tyrosine phosphatase 1B is essential for its activation of sterol regulatory element-binding protein-1 gene expression. Biochem Biophys Res Commun 363:626–632CrossRefGoogle Scholar