Free Fatty Acids Induce JNK-dependent Hepatocyte Lipoapoptosis

Abstract

Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway. Elevated serum free fatty acids (FFAs) and hepatocyte lipoapoptosis are features of non-alcoholic fatty liver disease. However, the mechanism by which FFAs mediate lipoapoptosis is unclear. Because JNK activation is pivotal in both the metabolic syndrome accompanying non-alcoholic fatty liver disease and cellular apoptosis, we examined the role of JNK activation in FFA-induced lipoapoptosis. Multiple hepatocyte cell lines and primary mouse hepatocytes were treated in culture with monounsaturated fatty acids and saturated fatty acids. Despite equal cellular steatosis, apoptosis and JNK activation were greater during exposure to saturated versus monounsaturated FFAs. Inhibition of JNK, pharmacologically as well as genetically, reduced saturated FFA-mediated hepatocyte lipoapoptosis. Cell death was caspase-dependent and associated with mitochondrial membrane depolarization and cytochrome c release indicating activation of the mitochondrial pathway of apoptosis. JNK-dependent lipoapoptosis was associated with activation of Bax, a known mediator of mitochondrial dysfunction. As JNK can activate Bim, a BH3 domain-only protein capable of binding to and activating Bax, its role in lipoapoptosis was also examined. Small interfering RNA-targeted knock-down of Bim attenuated both Bax activation and cell death. Collectively the data indicate that saturated FFAs induce JNK-dependent hepatocyte lipoapoptosis by activating the proapoptotic Bcl-2 proteins Bim and Bax, which trigger the mitochondrial apoptotic pathway. Non-alcoholic fatty liver disease (NAFLD) 2The abbreviations used are: NAFLD, non-alcoholic fatty liver disease; FFA, free fatty acid; JNK, c-Jun NH2-terminal kinase; NASH, non-alcoholic steatohepatitis; BH3, Bcl-2 homology domain 3; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TMRM, tetramethylrhodamine methyl ester; Δψ, mitochondrial membrane potential; MOPS, 4-morpholinepropanesulfonic acid; MAPK, mitogen-activated protein kinase; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; ELISA, enzyme-linked immunosorbent assay; si, small interfering; CYP, cytochrome P450; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. 2The abbreviations used are: NAFLD, non-alcoholic fatty liver disease; FFA, free fatty acid; JNK, c-Jun NH2-terminal kinase; NASH, non-alcoholic steatohepatitis; BH3, Bcl-2 homology domain 3; DAPI, 4′,6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; TMRM, tetramethylrhodamine methyl ester; Δψ, mitochondrial membrane potential; MOPS, 4-morpholinepropanesulfonic acid; MAPK, mitogen-activated protein kinase; Z, benzyloxycarbonyl; fmk, fluoromethyl ketone; ELISA, enzyme-linked immunosorbent assay; si, small interfering; CYP, cytochrome P450; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand. is associated with obesity and affects a third of the population of the United States. Because a subset of patients with NAFLD progresses to non-alcoholic steatohepatitis (NASH) and end stage liver disease (1Matteoni C.A. Younossi Z.M. Gramlich T. Boparai N. Liu Y.C. McCullough A.J. Gastroenterology. 1999; 116: 1413-1419Abstract Full Text Full Text PDF PubMed Scopus (2773) Google Scholar), NAFLD has emerged as a substantial public health concern. This syndrome is associated with hepatocyte steatosis and elevated serum free fatty acids (2Feldstein A.E. Canbay A. Guicciardi M.E. Higuchi H. Bronk S.F. Gores G.J. J. Hepatol. 2003; 39: 978-983Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). However, the cellular mechanisms linking elevated serum free fatty acids and hepatocyte steatosis to tissue injury remain obscure. Liver cell apoptosis is a prominent feature of NASH and correlates with disease severity (3Feldstein A.E. Canbay A. Angulo P. Taniai M. Burgart L.J. Lindor K.D. Gores G.J. Gastroenterology. 2003; 125: 437-443Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar). The toxicity of lipids, or lipotoxicity, and specifically lipid-induced apoptosis, or lipoapoptosis, is a potential mechanism relating apoptosis to NASH. In support of this concept, FFAs have been reported to cause cellular steatosis and enhance expression of the apoptosis effectors tumor necrosis factor-α and Fas (2Feldstein A.E. Canbay A. Guicciardi M.E. Higuchi H. Bronk S.F. Gores G.J. J. Hepatol. 2003; 39: 978-983Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 4Feldstein A.E. Werneburg N.W. Canbay A. Guicciardi M.E. Bronk S.F. Rydzewski R. Burgart L.J. Gores G.J. Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (663) Google Scholar). However, direct evidence for hepatocyte lipoapoptosis by individual FFAs is lacking as is the potential mechanism by which they may engage the cellular apoptotic machinery. c-Jun NH2-terminal kinase (JNK) is activated by various stress signals and interestingly also in obesity (5Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2614) Google Scholar, 6Liu W. Schoenkerman A. Lowe Jr., W.L. Am. J. Physiol. 2000; 279: E782-E790Crossref PubMed Google Scholar). The JNK pathway contributes to stress-induced apoptosis in several cell types, including pancreatic β-cells, neurons, and hepatocytes (7Ammendrup A. Maillard A. Nielsen K. Aabenhus Andersen N. Serup P. Dragsbaek Madsen O. Mandrup-Poulsen T. Bonny C. Diabetes. 2000; 49: 1468-1476Crossref PubMed Scopus (182) Google Scholar, 8Becker E.B. Howell J. Kodama Y. Barker P.A. Bonni A. J. Neurosci. 2004; 24: 8762-8770Crossref PubMed Scopus (111) Google Scholar, 9Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (138) Google Scholar). Interactions between FFAs and JNK signaling may, therefore, be a potential link between obesity and lipoapoptosis. JNK has three isoforms of which only two are expressed in the liver, JNK1 and JNK2 (10Davis R.J. Cell. 2000; 103: 239-252Abstract Full Text Full Text PDF PubMed Scopus (3612) Google Scholar). Sustained JNK2 and JNK3 activation are cytotoxic in neurons (11Keramaris E. Vanderluit J.L. Bahadori M. Mousavi K. Davis R.J. Flavell R. Slack R.S. Park D.S. J. Biol. Chem. 2005; 280: 1132-1141Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar), whereas JNK1 potentiates hepatocyte apoptosis by the toxic bile acid deoxycholate (9Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (138) Google Scholar). Thus, JNK isoform cytotoxicity appears to be cell type- and stimulus-specific. JNK may induce cell injury through both transcriptional and non-transcriptional mechanisms. Growth factor withdrawal-, UV light-, and oxidative stress-associated JNK-mediated apoptosis requires activation of the transcription factor c-Jun/AP-1 (12Hochedlinger K. Wagner E.F. Sabapathy K. Oncogene. 2002; 21: 2441-2445Crossref PubMed Scopus (91) Google Scholar, 13Bruckner S.R. Tammariello S.P. Kuan C.Y. Flavell R.A. Rakic P. Estus S. J. Neurochem. 2001; 78: 298-303Crossref PubMed Scopus (68) Google Scholar, 14Wang Y. Schattenberg J.M. Rigoli R.M. Storz P. Czaja M.J. J. Biol. Chem. 2004; 279: 31089-31097Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 15Czaja M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Crossref PubMed Scopus (120) Google Scholar). In contrast, JNK may also induce apoptosis in a transcription-independent process by activating proapoptotic members of the Bcl-2 family, including Bim and Bax, or inactivating Bcl-2 and Bcl-xL, antiapoptotic members of this family (16Aoki H. Kang P.M. Hampe J. Yoshimura K. Noma T. Matsuzaki M. Izumo S. J. Biol. Chem. 2002; 277: 10244-10250Abstract Full Text Full Text PDF PubMed Scopus (294) Google Scholar, 17Deng X. Xiao L. Lang W. Gao F. Ruvolo P. May Jr., W.S. J. Biol. Chem. 2001; 276: 23681-23688Abstract Full Text Full Text PDF PubMed Scopus (252) Google Scholar, 18Kharbanda S. Saxena S. Yoshida K. Pandey P. Kaneki M. Wang Q. Cheng K. Chen Y.N. Campbell A. Sudha T. Yuan Z.M. Narula J. Weichselbaum R. Nalin C. Kufe D. J. Biol. Chem. 2000; 275: 322-327Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 19Okuno S. Saito A. Hayashi T. Chan P.H. J. Neurosci. 2004; 24: 7879-7887Crossref PubMed Scopus (220) Google Scholar, 20Zu K. Hawthorn L. Ip C. Mol. Cancer Ther. 2005; 4: 43-50Crossref PubMed Scopus (20) Google Scholar). Activation of the proapoptotic proteins or inhibition of their antiapoptotic counterparts triggers mitochondrial dysfunction and cell death. The dual objectives of this study were to determine whether individual free fatty acids induce hepatocyte apoptosis and whether the observed lipoapoptosis is JNK-dependent. We induced steatosis in cultured hepatocytes and demonstrated apoptotic cell death with free fatty acid treatment. Sustained JNK activation was a prominent feature of this lipoapoptosis, and inhibition of JNK abrogated hepatocyte lipotoxicity. Cells—Mouse hepatocytes were isolated from C57/Bl6 wild type, JNK1-/-, JNK2-/- (Jackson Laboratories, Bar Harbor, ME), and cathepsin B-/- mice (21Guicciardi M.E. Deussing J. Miyoshi H. Bronk S.F. Svingen P.A. Peters C. Kaufmann S.H. Gores G.J. J. Clin. Investig. 2000; 106: 1127-1137Crossref PubMed Scopus (627) Google Scholar) by collagenase perfusion (22Faubion W.A. Guicciardi M.E. Miyoshi H. Bronk S.F. Roberts P.J. Svingen P.A. Kaufmann S.H. Gores G.J. J. Clin. Investig. 1999; 103: 137-145Crossref PubMed Scopus (465) Google Scholar). After Percoll purification hepatocytes were cultured in Dulbecco's modified Eagle's medium containing high glucose (25 mm), 100,000 units/liter penicillin, 100 mg/liter streptomycin, and 10% fetal bovine serum. HepG2, a well differentiated human hepatoblastoma cell line frequently used for studies of lipid metabolism (23Dashti N. G. J. Full Text PDF PubMed Google Scholar), a human cell and a cell were also cultured in Dulbecco's modified Eagle's medium containing and 10% fetal bovine serum. acid acid acid and acid were from acid and acid were in a of or acid and in were in to and The of the was in Dulbecco's modified Eagle's medium containing bovine serum was used in The of fatty acids used from to by fatty were with for lipid was with for P. J. Cell Biol. PubMed Scopus Google Scholar). were by with was lipid was expressed as in cellular cell of with and were used to apoptotic were in with the of and were were by and by in After with phosphate-buffered were by in was with in the of J. F. PubMed Scopus Google Scholar). data were a and of were cultured was a the of A. Bronk S.F. Roberts P.J. R. Gores G.J. Hepatology. 1999; PubMed Scopus Google Scholar). The mitochondrial membrane potential in of the which mitochondrial of mitochondrial membrane is tetramethylrhodamine methyl also a of mitochondrial is by in However, as is of in were treated with free fatty acid for to with for in the were with for After the was This in mitochondrial depolarization and of of from in was for and treated and expressed as a of this in correlates with of was and were a and by were with in was with in PBS, in PBS, and in for Bax, cytochrome and were mouse Bax and mouse c were and Laboratories, was used as were by with Cell and mitochondrial were from to with free fatty acids. cell were the medium was and the were with The were in of containing and to and for cell were for to cultured were by in MOPS, and to After 100 and in the the was for to The was for to from The from this is the and the is the mitochondrial The mitochondrial was in MOPS, 100 by and in 10% and a After for with the mitochondrial was for to The protein of was by the The and cell were to as was by and in with was used to binding JNK, MAPK, and kinase Cell kinase cytochrome c Bax, and cytochrome c Bim and Bim were used to were a and were by and the to the of to were in was the to the this of a acid to its its was and of and JNK and by and was a were in were with by of with and in containing well was with to JNK or was and with the was a Cell was by JNK and was to cell Bim by was used to Bim expression in and human Bim was and a was by of lipid to the was used as a was by protein with of with in were with lipid in a of of After in for of medium was were for apoptosis, for Bax activation by or by as was from cultured and were used to The was as The for Bim were and the for human cytochrome were and the for were and were used as The was in a and a The of in the was was the The between and was was used to the in The was expressed to the for The and were to a of the and were from DAPI, and acid were from was from protein kinase and and JNK were from was from data are expressed as the between were cell culture of cellular steatosis by with various FFAs in medium containing bovine serum were with acid and acid both monounsaturated fatty and acid and acid both saturated fatty for lipid were by equal of saturated and fatty cellular steatosis, expressed as a of cellular was steatosis was in in in in and in Thus, the individual FFAs cellular expression is associated with human NASH P. M. C. Hepatology. PubMed Scopus Google Scholar). expression in was In contrast, free fatty acid in expression in primary hepatocytes or Thus, cellular steatosis, a feature of human NASH, and features of human NASH in a FFAs cytotoxicity was and and In treated for with acid toxicity was and acid toxicity was whereas only acid and acid toxicity was observed and were observed in and primary mouse hepatocytes Thus, saturated fatty and were toxic monounsaturated fatty and in primary hepatocytes and cell lines examined. of apoptosis was by and both of apoptosis M. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). In cell demonstrated death in this was to with acid and with acid for acid also to activation with the of apoptosis, was greater in and acid as with The attenuated cell death by FFAs examined in a The also reduced cell death by in with acid or acid data data that FFAs induce caspase-dependent apoptosis. FFA-mediated the of dysfunction with of and release of cytochrome c the are prominent features of the mitochondrial pathway of apoptosis. was of the with acid and of the with acid for with fatty cytochrome c was also from the as demonstrated by a from to in of of and of and of cytochrome c the by acid and acid was by of data that saturated FFAs mediate apoptosis by the mitochondrial pathway of apoptosis. dysfunction in hepatocytes may be death or stress mitochondrial dysfunction by of the Bcl-2 family protein to to as C. Ther. 2001; PubMed Scopus Google Scholar). However, in whereas to was in inhibition of with and also acid or acid toxicity has also been reported to trigger mitochondrial dysfunction in a is a mediator of this pathway (21Guicciardi M.E. Deussing J. Miyoshi H. Bronk S.F. Svingen P.A. Peters C. Kaufmann S.H. Gores G.J. J. Clin. Investig. 2000; 106: 1127-1137Crossref PubMed Scopus (627) Google Scholar). However, and acid induced and apoptosis in cathepsin B-/- hepatocytes to that observed in wild hepatocytes and apoptosis with and acid Thus, free fatty apoptosis is associated with and is cathepsin data that of the mitochondrial pathway of apoptosis death and c-Jun NH2-terminal JNK a feature of the metabolic is associated with NAFLD and has been in apoptosis, we whether JNK activation is for the lipoapoptosis observed in hepatocytes and and In primary hepatocytes acid to a in JNK whereas acid JNK during acid to a in and by both and in in was observed by as as with acid This in JNK was and as by was and with and with acid acid the to only and in and In primary mouse hepatocytes was and with and with acid acid the only to a in of and and mitogen-activated protein of the mitogen-activated protein kinase family, was acid In contrast, was exposure to Thus, both JNK and were activated by proapoptotic free fatty acids. whether JNK or contributes to lipoapoptosis in studies were small JNK reduced acid toxicity by acid toxicity by and acid toxicity by this a JNK was also used W. A. S. Y. S. A. 2001; PubMed Scopus Google Scholar). This also reduced acid toxicity by and acid and acid toxicity by determine the role of the two JNK isoforms expressed by hepatocytes in of lipoapoptosis, hepatocytes were isolated from mice in JNK2 and JNK1 and cell death was reduced in JNK2-/- mice In contrast, hepatocytes from mice were to acid or acid cytotoxicity the role of in this apoptotic pathway was the and that reduced or lipoapoptosis Collectively that FFA-mediated lipoapoptosis in and in the JNK2 isoform this JNK the of by a proapoptotic BH3 family can be activated by JNK Mol. 2003; PubMed Scopus (120) Google Scholar). Bax a which is specifically by the R.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). to Bax activation as demonstrated by for of with JNK and and Bax activation Bax activation to mitochondrial and release of cytochrome cytochrome c release was by and by the JNK in inhibition of the mitochondrial pathway of apoptosis. data Bax activation and mitochondrial of JNK activation in lipoapoptosis. proteins of the Bcl-2 family, as Bax, are known to be activated by Bcl-2 family the known proteins only Bim and and activate Bax T. L. C. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). As activation was observed we Bim in FFA-mediated lipoapoptosis. We examined Bim expression in free fatty three known and only the two were in with its toxicity acid to and in cellular Bim that acid to a in cellular whereas acid to a The in Bim protein was also associated with in Bim as by acid to a in Bim expression in and a in in was observed in The observed in Bim is a of cell death as of well apoptosis. Thus, JNK-dependent lipoapoptosis is associated with Bim Bim is of acid or a role in lipoapoptosis was by Bim in and in Bim expression was lipoapoptosis was reduced by acid toxicity was was reduced by Bim expression Bax the of mitochondrial in this was In with Bim Bax activation was reduced by Collectively data that JNK lipoapoptosis Bax The of this study to the mechanism of FFA-induced hepatocyte lipoapoptosis. The data that saturated FFAs are cytotoxic monounsaturated cytotoxicity caspase-dependent apoptosis, and JNK triggers the mitochondrial pathway of apoptosis by Bax data the cellular mechanism of hepatocyte lipoapoptosis, a to human NASH. In the FFAs induced apoptosis in FFAs were toxic monounsaturated FFAs a of cellular data are with in cell types, as pancreatic neurons, and Am. J. Physiol. 2000; 279: PubMed Google Scholar, S. T. S. Y. K. M. H. N. S. M. Mol. 2002; PubMed Google Scholar), saturated acid and also greater cytotoxicity monounsaturated acid and are in to FFAs potentiates 2004; PubMed Scopus Google Scholar). The mechanisms of may well be from of NAFLD the that both are by indicating that FFAs have toxic that the in serum liver tissue may which with NAFLD liver disease. of lipid metabolic as of death and direct activation of cellular proapoptotic are mechanisms by which lipoapoptosis is to (2Feldstein A.E. Canbay A. Guicciardi M.E. Higuchi H. Bronk S.F. Gores G.J. J. Hepatol. 2003; 39: 978-983Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, A.E. Canbay A. Angulo P. Taniai M. Burgart L.J. Lindor K.D. Gores G.J. Gastroenterology. 2003; 125: 437-443Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar, 4Feldstein A.E. Werneburg N.W. Canbay A. Guicciardi M.E. Bronk S.F. Rydzewski R. Burgart L.J. Gores G.J. Hepatology. 2004; 40: 185-194Crossref PubMed Scopus (663) Google Scholar, P. A. M. M. D. L. S. A. 2000; PubMed Scopus Google Scholar, 2002; PubMed Scopus Google Scholar). However, of and FFAs engage the cell death machinery. The data this The demonstrated that FFA-mediated lipoapoptosis by a caspase-dependent mechanism by activating the mitochondrial pathway of apoptosis. was and lipoapoptosis was attenuated by The activation with mitochondrial dysfunction and was by the death pathway with a or by the pathway by of cathepsin Collectively data indicate that cytotoxic FFAs engage the mitochondrial cell death pathway. FFAs are known to several cellular signaling including the A. M. A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, X. A. D. M.J. M. J. Mol. 2004; PubMed Scopus Google Scholar). In the hepatocyte cell lines acid activated both and JNK with the proapoptotic of JNK and the of M.J. Liu H. Wang Y. Hepatology. 2003; 37: 1405-1413Crossref PubMed Scopus (120) Google Scholar), only inhibition of JNK abrogated FFA-induced lipoapoptosis. data link the known of JNK to with the lipoapoptosis observed in this In this JNK activation by FFAs may only to signaling also to cellular JNK to a pivotal role in liver to JNK activation are also by In neurons, JNK3 apoptosis S.R. Estus S. J. Neurosci. 2002; PubMed Scopus Google Scholar) and cell T. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). in JNK1 and JNK2 have proapoptotic or in a and (5Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2614) Google Scholar, 9Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (138) Google Scholar, D. A.J. T. Davis R.J. M. Flavell R.A. Full Text Full Text PDF PubMed Scopus Google Scholar, K. T. Karin M. Wagner E.F. J. 2001; PubMed Scopus Google Scholar). In JNK1 and (5Hirosumi J. Tuncman G. Chang L. Gorgun C.Z. Uysal K.T. Maeda K. Karin M. Hotamisligil G.S. Nature. 2002; 420: 333-336Crossref PubMed Scopus (2614) Google Scholar) and also bile hepatocyte apoptosis (9Qiao L. Han S.I. Fang Y. Park J.S. Gupta S. Gilfor D. Amorino G. Valerie K. Sealy L. Engelhardt J.F. Grant S. Hylemon P.B. Dent P. Mol. Cell. Biol. 2003; 23: 3052-3066Crossref PubMed Scopus (138) Google Scholar). data a role for the JNK2 isoform in FFA-mediated lipoapoptosis as JNK2-/- were to cell death. Thus, in cytotoxicity by JNK isoforms is stimulus-specific. release of cytochrome c is by the proapoptotic members of the Bcl-2 family, Bax and Bax is for apoptosis cellular L. J. Park 2000; PubMed Scopus Google Scholar, J.L. Jr., Full Text Full Text PDF PubMed Scopus Google Scholar). in hepatocytes Bax and bile mitochondrial and apoptosis M. Higuchi H. S. T. S. Saito H. S. H. Am. J. Physiol. 2004; PubMed Scopus Google Scholar, S.H. 2003; PubMed Scopus Google Scholar). with the role of Bax in hepatocyte apoptosis, acid to Bax activation and cytochrome c inhibition of JNK both Bax activation and to of lipoapoptosis. BH3 domain proteins are the of cellular stress and are for the mitochondrial pathway of apoptosis. only Bim and have been to activate Bax T. L. C. Mol. Cell. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar). As was activated in we FFA-induced JNK activation was to the mitochondrial pathway of apoptosis by Bim we observed and in cellular Bim protein of with acid and of apoptosis in are with data from JNK-dependent Bim as well as with activation of apoptosis has been reported E.B. Howell J. Kodama Y. Barker P.A. Bonni A. J. Neurosci. 2004; 24: 8762-8770Crossref PubMed Scopus (111) Google Scholar, 19Okuno S. Saito A. Hayashi T. Chan P.H. J. Neurosci. 2004; 24: 7879-7887Crossref PubMed Scopus (220) Google Scholar, 20Zu K. Hawthorn L. Ip C. Mol. Cancer Ther. 2005; 4: 43-50Crossref PubMed Scopus (20) Google Scholar, C.A. Jr., J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Google Scholar). Thus, the data support a JNK hepatocyte lipoapoptosis a mitochondrial pathway of cell death. lipoapoptosis is a public health the obesity and the between apoptosis and liver disease in this syndrome (3Feldstein A.E. Canbay A. Angulo P. Taniai M. Burgart L.J. Lindor K.D. Gores G.J. Gastroenterology. 2003; 125: 437-443Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar). cellular is to this syndrome as toxic FFAs are to to hepatocyte lipoapoptosis in data indicate that FFAs cause JNK activation to of the mitochondrial proapoptotic with Bax JNK as a potential to the of JNK H. Y. T. D. M. Y. H. Y. M. 2004; PubMed Scopus Google Scholar).