title: Saturated free fatty acids induce cholangiocyte lipoapoptosis.
abstract: Recent studies have identified a cholestatic variant of nonalcoholic fatty liver disease (NAFLD) with portal inflammation and ductular reaction. Based on reports of biliary damage, as well as increased circulating free fatty acids (FFAs) in NAFLD, we hypothesized the involvement of cholangiocyte lipoapoptosis as a mechanism of cellular injury. Here, we demonstrate that the saturated FFAs palmitate and stearate induced robust and rapid cell death in cholangiocytes. Palmitate and stearate induced cholangiocyte lipoapoptosis in a concentration-dependent manner in multiple cholangiocyte-derived cell lines. The mechanism of lipoapoptosis relied on the activation of caspase 3/7 activity. There was also a significant up-regulation of the proapoptotic BH3-containing protein, PUMA. In addition, palmitate-induced cholangiocyte lipoapoptosis involved a time-dependent increase in the nuclear localization of forkhead family of transcription factor 3 (FoxO3). We show evidence for posttranslational modification of FoxO3, including early (6 hours) deacetylation and dephosphorylation that coincide with localization of FoxO3 in the nuclear compartment. By 16 hours, nuclear FoxO3 is both phosphorylated and acetylated. Knockdown studies confirmed that FoxO3 and its downstream target, PUMA, were critical for palmitate- and stearate-induced cholangiocyte lipoapoptosis. Interestingly, cultured cholangiocyte-derived cells did not accumulate appreciable amounts of neutral lipid upon FFA treatment.
  
  
  CONCLUSIONS
  Our data show that the saturated FFAs palmitate and stearate induced cholangiocyte lipoapoptosis by way of caspase activation, nuclear translocation of FoxO3, and increased proapoptotic PUMA expression. These results suggest that cholangiocyte injury may occur through lipoapoptosis in NAFLD and nonalcoholic steatohepatitis patients.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Nebraska Medical Center, Omaha, NE.
authors.forename: Sathish Kumar
authors.initials: SK
authors.lastname: Natarajan
authors.forename: Sally A
authors.initials: SA
authors.lastname: Ingham
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Mohr
authors.forename: Cody J
authors.initials: CJ
authors.lastname: Wehrkamp
authors.forename: Anuttoma
authors.initials: A
authors.lastname: Ray
authors.forename: Sohini
authors.initials: S
authors.lastname: Roy
authors.forename: Sophie C
authors.initials: SC
authors.lastname: Cazanave
authors.forename: Mary Anne
authors.initials: MA
authors.lastname: Phillippi
authors.forename: Justin L
authors.initials: JL
authors.lastname: Mott
mesh_terms: D017209:Apoptosis; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D001653:Bile Ducts, Intrahepatic / Q000201:enzymology*; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D004789:Enzyme Activation; D005230:Fatty Acids, Nonesterified / Q000009:adverse effects*; D005234:Fatty Liver / Q000209:etiology* / Q000378:metabolism; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors / Q000378:metabolism; D006801:Humans; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D010168:Palmitates / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism
chemical_list: D051017:Apoptosis Regulatory Proteins; C438378:BBC3 protein, human; C499517:FOXO3 protein, human; D005230:Fatty Acids, Nonesterified; D000071316:Forkhead Box Protein O3; D051858:Forkhead Transcription Factors; D010168:Palmitates; D011518:Proto-Oncogene Proteins; D020928:Mitogen-Activated Protein Kinases; D020169:Caspases
references.citation: Hepatology. 2008 Apr;47(4):1178-90
references.pmid: 18081149
references.citation: PLoS One. 2010 Dec 29;5(12):e15726
references.pmid: 21209957
references.citation: Am J Physiol Gastrointest Liver Physiol. 2013 Jun 1;304(11):G1013-24
references.pmid: 23578785
references.citation: PLoS One. 2010 Nov 17;5(11):e14037
references.pmid: 21103345
references.citation: Free Radic Biol Med. 2012 Sep 1;53(5):1181-91
references.pmid: 22796327
references.citation: Hepatology. 2007 Jun;45(6):1366-74
references.pmid: 17476695
references.citation: Biochem Biophys Res Commun. 2005 Oct 14;336(1):357-63
references.pmid: 16139248
references.citation: J Biol Chem. 2009 Sep 25;284(39):26591-602
references.pmid: 19638343
references.citation: Hepatology. 2003 Jun;37(6):1286-92
references.pmid: 12774006
references.citation: Nat Cell Biol. 2008 Feb;10(2):138-48
references.pmid: 18204439
references.citation: Nature. 1993 Mar 4;362(6415):79-83
references.pmid: 7680434
references.citation: Lab Invest. 2011 Jul;91(7):1007-17
references.pmid: 21464819
references.citation: Clin Liver Dis. 2004 Aug;8(3):481-500, vii
references.pmid: 15331059
references.citation: Cell Physiol Biochem. 2013;31(2-3):277-89
references.pmid: 23466956
references.citation: World J Gastroenterol. 2004 Jan;10(1):105-11
references.pmid: 14695779
references.citation: J Biol Chem. 2007 Sep 14;282(37):27141-27154
references.pmid: 17626006
references.citation: Biochim Biophys Acta. 2012 Nov;1822(11):1815-25
references.pmid: 22968147
references.citation: Science. 2004 Mar 26;303(5666):2011-5
references.pmid: 14976264
references.citation: Dig Dis Sci. 2005 Jun;50(6):1130-5
references.pmid: 15986869
references.citation: J Biol Chem. 2011 Nov 11;286(45):39336-48
references.pmid: 21941003
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3077-82
references.pmid: 12629214
references.citation: Diabetologia. 2011 Jul;54(7):1788-98
references.pmid: 21455726
references.citation: Hepatology. 2011 Jul;54(1):122-32
references.pmid: 21465509
references.citation: Cell Signal. 2007 Mar;19(3):519-27
references.pmid: 17113751
references.citation: Hepatology. 2010 Aug;52(2):774-88
references.pmid: 20683968
references.citation: Lab Invest. 2009 Dec;89(12):1374-86
references.pmid: 19823170
references.citation: Diabetes. 2009 Feb;58(2):344-51
references.pmid: 19008341
references.citation: Dig Dis Sci. 2001 Nov;46(11):2347-52
references.pmid: 11713934
references.citation: Nucleic Acids Res. 2011 Dec;39(22):9498-507
references.pmid: 21835778
references.citation: J Hepatol. 2014 Mar;60(3):599-605
references.pmid: 24239776
references.citation: Proc Natl Acad Sci U S A. 2008 Sep 9;105(36):13421-6
references.pmid: 18755897
references.citation: Neoplasia. 2009 Apr;11(4):313-24
references.pmid: 19308286
references.citation: Hepatology. 2012 Aug;56(2):687-97
references.pmid: 22383162
references.citation: Cell Health Cytoskelet. 2012 Feb 1;2012(4):11-27
references.pmid: 22593641
references.citation: J Clin Biochem Nutr. 2010 Jan;46(1):30-5
references.pmid: 20104262
references.citation: J Biol Chem. 2006 Apr 28;281(17):12093-101
references.pmid: 16505490
references.citation: Clin Gastroenterol Hepatol. 2006 Feb;4(2):226-32
references.pmid: 16469684
references.citation: J Biol Chem. 2012 Jan 6;287(2):1545-55
references.pmid: 22128155
references.citation: J Pediatr Gastroenterol Nutr. 2011 Aug;53(2):131-40
references.pmid: 21629127
references.citation: J Clin Pathol. 2011 Jul;64(7):564-70
references.pmid: 21486894