title: Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD.
abstract: The pathogenic mechanisms underlying the progression of non-alcoholic fatty liver disease (NAFLD) are not fully understood. In this study, we aimed to assess the relationship between endoplasmic reticulum (ER) stress and autophagy in human and mouse hepatocytes during NAFLD. ER stress and autophagy markers were analyzed in livers from patients with biopsy-proven non-alcoholic steatosis (NAS) or non-alcoholic steatohepatitis (NASH) compared with livers from subjects with histologically normal liver, in livers from mice fed with chow diet (CHD) compared with mice fed with high fat diet (HFD) or methionine-choline-deficient (MCD) diet and in primary and Huh7 human hepatocytes loaded with palmitic acid (PA). In NASH patients, significant increases in hepatic messenger RNA levels of markers of ER stress (activating transcription factor 4 (ATF4), glucose-regulated protein 78 (GRP78) and C/EBP homologous protein (CHOP)) and autophagy (BCN1) were found compared with NAS patients. Likewise, protein levels of GRP78, CHOP and p62/SQSTM1 (p62) autophagic substrate were significantly elevated in NASH compared with NAS patients. In livers from mice fed with HFD or MCD, ER stress-mediated signaling was parallel to the blockade of the autophagic flux assessed by increases in p62, microtubule-associated protein 2 light chain 3 (LC3-II)/LC3-I ratio and accumulation of autophagosomes compared with CHD fed mice. In Huh7 hepatic cells, treatment with PA for 8 h triggered activation of both unfolding protein response and the autophagic flux. Conversely, prolonged treatment with PA (24 h) induced ER stress and cell death together with a blockade of the autophagic flux. Under these conditions, cotreatment with rapamycin or CHOP silencing ameliorated these effects and decreased apoptosis. Our results demonstrated that the autophagic flux is impaired in the liver from both NAFLD patients and murine models of NAFLD, as well as in lipid-overloaded human hepatocytes, and it could be due to elevated ER stress leading to apoptosis. Consequently, therapies aimed to restore the autophagic flux might attenuate or prevent the progression of NAFLD.
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Barcelona, Spain [2] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain.
authors.forename: A
authors.initials: A
authors.lastname: González-Rodríguez
authors.affiliation: 1] Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, CA, 92093, USA [2] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Mayoral
authors.affiliation: Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Agra
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Barcelona, Spain [2] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain.
authors.forename: M P
authors.initials: MP
authors.lastname: Valdecantos
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Barcelona, Spain [2] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain.
authors.forename: V
authors.initials: V
authors.lastname: Pardo
authors.affiliation: Liver Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa, Madrid, Spain.
authors.forename: M E
authors.initials: ME
authors.lastname: Miquilena-Colina
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain [2] Liver Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa, Madrid, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Vargas-Castrillón
authors.affiliation: Gastroenterology Unit, Hospital del Tajo, Aranjuez, Madrid, Spain.
authors.forename: O
authors.initials: O
authors.lastname: Lo Iacono
authors.affiliation: National Institute for Infectious Diseases IRCCS 'L Spallanzani', Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Corazzari
authors.affiliation: National Institute for Infectious Diseases IRCCS 'L Spallanzani', Rome, Italy.
authors.forename: G M
authors.initials: GM
authors.lastname: Fimia
authors.affiliation: 1] National Institute for Infectious Diseases IRCCS 'L Spallanzani', Rome, Italy [2] Department of Biology, University of Rome 'Tor Vergata', Rome, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Piacentini
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain [2] Oncology Surgery, Cell Therapy and Transplant Organs, Institute of Biomedicine of Seville (IBiS)/Virgen del Rocio Universitary Hospital/CSIC/University of Seville, Seville, Spain.
authors.forename: J
authors.initials: J
authors.lastname: Muntané
authors.affiliation: 1] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain [2] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Boscá
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain [2] Liver Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa, Madrid, Spain.
authors.forename: C
authors.initials: C
authors.lastname: García-Monzón
authors.affiliation: 1] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain [2] Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII, Barcelona, Spain.
authors.forename: P
authors.initials: P
authors.lastname: Martín-Sanz
authors.affiliation: 1] Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Barcelona, Spain [2] Instituto de Investigaciones Biomédicas 'Alberto Sols' (CSIC/UAM), Madrid, Spain.
authors.forename: Á M
authors.initials: ÁM
authors.lastname: Valverde
mesh_terms: D000818:Animals; D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D003710:Demography; D059305:Diet, High-Fat; D000091342:Endoplasmic Reticulum Chaperone BiP; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects; D005247:Feeding Behavior; D005260:Female; D020868:Gene Silencing / Q000187:drug effects; D022781:Hepatocytes / Q000187:drug effects / Q000473:pathology; D006801:Humans; D008099:Liver / Q000473:pathology; D008297:Male; D008810:Mice, Inbred C57BL; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008875:Middle Aged; D065626:Non-alcoholic Fatty Liver Disease / Q000473:pathology*; D019308:Palmitic Acid / Q000494:pharmacology; D010588:Phagosomes / Q000187:drug effects / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D051742:Transcription Factor CHOP / Q000378:metabolism
chemical_list: D000091342:Endoplasmic Reticulum Chaperone BiP; C000717247:HSPA5 protein, human; C000717248:Hspa5 protein, mouse; D008869:Microtubule-Associated Proteins; D051742:Transcription Factor CHOP; D019308:Palmitic Acid; D020123:Sirolimus
references.citation: Gastroenterology. 2011 Jun;140(7):1895-908
references.pmid: 21530520
references.citation: Autophagy. 2012 Apr;8(4):445-544
references.pmid: 22966490
references.citation: J Biol Chem. 2009 Sep 25;284(39):26591-602
references.pmid: 19638343
references.citation: Am J Gastroenterol. 1999 Sep;94(9):2467-74
references.pmid: 10484010
references.citation: J Biol Chem. 2012 Apr 27;287(18):14364-76
references.pmid: 22408252
references.citation: Apoptosis. 2014 Jan;19(1):210-23
references.pmid: 24114361
references.citation: Autophagy. 2011 Nov;7(11):1397-9
references.pmid: 21921693
references.citation: Curr Opin Gastroenterol. 2010 May;26(3):202-8
references.pmid: 20168226
references.citation: Biol Open. 2013 Aug 27;2(10):1084-90
references.pmid: 24167719
references.citation: Diabetologia. 1985 Jul;28(7):412-9
references.pmid: 3899825
references.citation: J Biol Chem. 2009 Nov 6;284(45):31484-92
references.pmid: 19758991
references.citation: J Cell Sci. 2004 Jun 1;117(Pt 13):2805-12
references.pmid: 15169837
references.citation: Gastroenterology. 2008 Feb;134(2):568-76
references.pmid: 18082745
references.citation: Science. 2004 Oct 15;306(5695):457-61
references.pmid: 15486293
references.citation: Hepatology. 2011 Nov;54(5):1600-9
references.pmid: 21748768
references.citation: J Hepatol. 2011 Jan;54(1):142-52
references.pmid: 20888662
references.citation: Clin Gastroenterol Hepatol. 2011 Jun;9(6):524-530.e1; quiz e60
references.pmid: 21440669
references.citation: Int J Cancer. 2012 Aug 1;131(3):548-57
references.pmid: 21858812
references.citation: Histol Histopathol. 2002;17(3):897-908
references.pmid: 12168801
references.citation: J Lipid Res. 1995 Feb;36(2):229-40
references.pmid: 7751810
references.citation: Cell. 2010 Mar 19;140(6):900-17
references.pmid: 20303879
references.citation: Proc Natl Acad Sci U S A. 2003 Mar 18;100(6):3077-82
references.pmid: 12629214
references.citation: J Biol Chem. 2010 Dec 31;285(53):42115-29
references.pmid: 20971848
references.citation: Gastroenterology. 2001 Apr;120(5):1183-92
references.pmid: 11266382
references.citation: Hepatology. 2005 Jun;41(6):1313-21
references.pmid: 15915461
references.citation: Clin Med (Lond). 2011 Apr;11(2):176-8
references.pmid: 21526706
references.citation: Cell Death Differ. 2012 Feb;19(2):310-20
references.pmid: 21779001
references.citation: Hepatology. 2010 Aug;52(2):774-88
references.pmid: 20683968
references.citation: Curr Opin Cell Biol. 2010 Apr;22(2):157-68
references.pmid: 20006481
references.citation: Nature. 2000 Nov 23;408(6811):488-92
references.pmid: 11100732
references.citation: N Engl J Med. 2006 Nov 30;355(22):2297-307
references.pmid: 17135584
references.citation: J Pharmacol Exp Ther. 2011 Nov;339(2):487-98
references.pmid: 21856859
references.citation: Methods Mol Biol. 2006;320:283-93
references.pmid: 16719400
references.citation: Am J Physiol Cell Physiol. 2010 May;298(5):C973-8
references.pmid: 20089934
references.citation: FEBS Lett. 2010 Apr 2;584(7):1374-8
references.pmid: 20153326
references.citation: Toxicol Sci. 2014 Jan;137(1):26-35
references.pmid: 24097666
references.citation: Nat Cell Biol. 2010 Sep;12(9):823-30
references.pmid: 20811354
references.citation: Nature. 2009 Apr 30;458(7242):1131-5
references.pmid: 19339967
references.citation: J Hepatol. 2013 May;58(5):993-9
references.pmid: 23339953
references.citation: Dev Cell. 2008 Dec;15(6):829-40
references.pmid: 19081072
references.citation: Hepatology. 2012 Jan;55(1):222-32
references.pmid: 21932416
references.citation: Gastroenterology. 2010 Nov;139(5):1740-52
references.pmid: 20659474
references.citation: Trends Cell Biol. 2000 Dec;10(12):524-30
references.pmid: 11121744
references.citation: J Biol Chem. 2006 Apr 28;281(17):12093-101
references.pmid: 16505490
references.citation: J Cell Biol. 2005 Nov 21;171(4):603-14
references.pmid: 16286508
references.citation: Gastroenterology. 2010 Nov;139(5):1730-9, 1739.e1
references.pmid: 20682321
references.citation: Cell Metab. 2010 Jun 9;11(6):467-78
references.pmid: 20519119
references.citation: Semin Liver Dis. 2008 Nov;28(4):360-9
references.pmid: 18956292
references.citation: Autophagy. 2008 Jul;4(5):600-6
references.pmid: 18497563
references.citation: Endocrinology. 2005 Mar;146(3):1473-81
references.pmid: 15604215