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      Impaired autophagic flux is associated with increased endoplasmic reticulum stress during the development of NAFLD

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          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.

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          Most cited references 25

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          Guidelines for the use and interpretation of assays for monitoring autophagy.

          In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process vs. those that measure flux through the autophagy pathway (i.e., the complete process); thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from stimuli that result in increased autophagic activity, defined as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (in most higher eukaryotes and some protists such as Dictyostelium) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the field understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field.
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            A ubiquitin-like system mediates protein lipidation.

            Autophagy is a dynamic membrane phenomenon for bulk protein degradation in the lysosome/vacuole. Apg8/Aut7 is an essential factor for autophagy in yeast. We previously found that the carboxy-terminal arginine of nascent Apg8 is removed by Apg4/Aut2 protease, leaving a glycine residue at the C terminus. Apg8 is then converted to a form (Apg8-X) that is tightly bound to the membrane. Here we report a new mode of protein lipidation. Apg8 is covalently conjugated to phosphatidylethanolamine through an amide bond between the C-terminal glycine and the amino group of phosphatidylethanolamine. This lipidation is mediated by a ubiquitination-like system. Apg8 is a ubiquitin-like protein that is activated by an E1 protein, Apg7 (refs 7, 8), and is transferred subsequently to the E2 enzymes Apg3/Aut1 (ref. 9). Apg7 activates two different ubiquitin-like proteins, Apg12 (ref. 10) and Apg8, and assigns them to specific E2 enzymes, Apg10 (ref. 11) and Apg3, respectively. These reactions are necessary for the formation of Apg8-phosphatidylethanolamine. This lipidation has an essential role in membrane dynamics during autophagy.
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              Physiological significance of selective degradation of p62 by autophagy.

              Autophagy is a highly conserved bulk protein degradation pathway responsible for the turnover of long-lived proteins, disposal of damaged organelles, and clearance of aggregate-prone proteins. Thus, inactivation of autophagy results in cytoplasmic protein inclusions, which are composed of misfolded proteins and excess accumulation of deformed organelles, leading to liver injury, diabetes, myopathy, and neurodegeneration. Although autophagy has been considered non-selective, growing lines of evidence indicate the selectivity of autophagy in sorting vacuolar enzymes and in the removal of aggregate-prone proteins, unwanted organelles and microbes. Such selectivity by autophagy enables diverse cellular regulations, similar to the ubiquitin-proteasome pathway. In this review, we introduce the selective turnover of the ubiquitin- and LC3-binding protein 'p62' through autophagy and discuss its physiological significance. Copyright 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
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                Author and article information

                Journal
                Cell Death Dis
                Cell Death Dis
                Cell Death & Disease
                Nature Publishing Group
                2041-4889
                April 2014
                17 April 2014
                1 April 2014
                : 5
                : 4
                : e1179
                Affiliations
                [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
                [3 ]Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego , La Jolla, CA, 92093, USA
                [4 ]Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), ISCIII , Barcelona, Spain
                [5 ]Liver Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa , Madrid, Spain
                [6 ]Gastroenterology Unit, Hospital del Tajo, Aranjuez , Madrid, Spain
                [7 ]National Institute for Infectious Diseases IRCCS ‘L Spallanzani' , Rome, Italy
                [8 ]Department of Biology, University of Rome ‘Tor Vergata' , Rome, Italy
                [9 ]Oncology Surgery, Cell Therapy and Transplant Organs, Institute of Biomedicine of Seville (IBiS)/Virgen del Rocio Universitary Hospital/CSIC/University of Seville , Seville, Spain
                Author notes
                [* ]Liver Research Unit, Hospital Universitario Santa Cristina, Instituto de Investigación Sanitaria Princesa , Calle del Maestro Amadeo Vives, 2, 28009 Madrid, Spain. Tel: +34 91 5574402; Fax: +34 91 5574400; E-mail: cgmonzon@ 123456salud.madrid.org
                [* ]Instituto de Investigaciones Biomédicas ‘Alberto Sols', Consejo Superior de Investigaciones Científicas , C/Arturo Duperier 4, 28029 Madrid, Spain. Tel: +34 91 4972746; Fax: +34 91 5854401; E-mail: pmartins@ 123456iib.uam.es (PM-S) or Tel: +34 915854497; Fax: +34 915854401; E-mail: avalverde@ 123456iib.uam.es (ÁMV)
                [10]

                These authors contributed equally to this work.

                [11]

                These authors share senior authorship.

                Article
                cddis2014162
                10.1038/cddis.2014.162
                4001315
                24743734
                Copyright © 2014 Macmillan Publishers Limited

                Cell Death and Disease is an open-access journal published by Nature Publishing Group. This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-nd/3.0/

                Categories
                Original Article

                Cell biology

                nafld, nash, autophagy, er stress, apoptosis, hepatocytes

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