Unsaturated fatty acids induce non‐canonical autophagy
2015; Springer Nature; Volume: 34; Issue: 8 Linguagem: Inglês
10.15252/embj.201489363
ISSN1460-2075
AutoresMireia Niso‐Santano, Shoaib Ahmad Malik, Federico Pietrocola, José Manuel Bravo‐San Pedro, Guillermo Mariño, Valentina Cianfanelli, Amena BenYounès, Rodrigo Troncoso, Maria Markaki, Valentina Sica, Valentina Izzo, Kariman Chaba, Chantal Bauvy, Nicolas Dupont, Oliver Kepp, Patrick Rockenfeller, Heimo Wolinski, Frank Madeo, Sergio Lavandero, Patrice Codogno, Francis Harper, Gérard Pierron, Nektarios Tavernarakis, Francesco Cecconi, Maria Chiara Maiuri, Lorenzo Galluzzi, Guido Kroemer,
Tópico(s)RNA modifications and cancer
ResumoArticle16 January 2015free access Unsaturated fatty acids induce non-canonical autophagy Mireia Niso-Santano Mireia Niso-Santano Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Government College University, Faisalabad, Pakistan Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Guillermo Mariño Guillermo Mariño Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Valentina Cianfanelli Valentina Cianfanelli Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Amena Ben-Younès Amena Ben-Younès Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Rodrigo Troncoso Rodrigo Troncoso Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Search for more papers by this author Valentina Sica Valentina Sica Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author Valentina Izzo Valentina Izzo Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Kariman Chaba Kariman Chaba Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Chantal Bauvy Chantal Bauvy Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Oliver Kepp Oliver Kepp Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Search for more papers by this author Patrick Rockenfeller Patrick Rockenfeller Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Heimo Wolinski Heimo Wolinski Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Sergio Lavandero Sergio Lavandero Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Francis Harper Francis Harper Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece Search for more papers by this author Francesco Cecconi Francesco Cecconi Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Search for more papers by this author Mireia Niso-Santano Mireia Niso-Santano Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Shoaib Ahmad Malik Shoaib Ahmad Malik Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Government College University, Faisalabad, Pakistan Search for more papers by this author Federico Pietrocola Federico Pietrocola Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author José Manuel Bravo-San Pedro José Manuel Bravo-San Pedro Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Guillermo Mariño Guillermo Mariño Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Valentina Cianfanelli Valentina Cianfanelli Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Search for more papers by this author Amena Ben-Younès Amena Ben-Younès Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Rodrigo Troncoso Rodrigo Troncoso Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Search for more papers by this author Maria Markaki Maria Markaki Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Search for more papers by this author Valentina Sica Valentina Sica Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France Search for more papers by this author Valentina Izzo Valentina Izzo Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Kariman Chaba Kariman Chaba Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Chantal Bauvy Chantal Bauvy Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Nicolas Dupont Nicolas Dupont Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Oliver Kepp Oliver Kepp Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Search for more papers by this author Patrick Rockenfeller Patrick Rockenfeller Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Heimo Wolinski Heimo Wolinski Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Frank Madeo Frank Madeo Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria BioTechMed Graz, Graz, Austria Search for more papers by this author Sergio Lavandero Sergio Lavandero Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA Search for more papers by this author Patrice Codogno Patrice Codogno Université Paris Descartes, Sorbonne Paris Cité, Paris, France INSERM, U1151, Paris, France Institut Necker Enfants-Malades, Paris, France Search for more papers by this author Francis Harper Francis Harper Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Gérard Pierron Gérard Pierron Gustave Roussy Comprehensive Cancer Center, Villejuif, France CNRS, UMR8122, Villejuif, France Search for more papers by this author Nektarios Tavernarakis Nektarios Tavernarakis orcid.org/0000-0002-5253-1466 Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece Search for more papers by this author Francesco Cecconi Francesco Cecconi Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy Search for more papers by this author Maria Chiara Maiuri Maria Chiara Maiuri Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Search for more papers by this author Lorenzo Galluzzi Lorenzo Galluzzi Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France Gustave Roussy Comprehensive Cancer Center, Villejuif, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Search for more papers by this author Guido Kroemer Corresponding Author Guido Kroemer Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France INSERM, U1138, Paris, France Université Paris Descartes, Sorbonne Paris Cité, Paris, France Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France Search for more papers by this author Author Information Mireia Niso-Santano1,2,3,‡, Shoaib Ahmad Malik1,2,3,4,‡, Federico Pietrocola1,2,3,5, José Manuel Bravo-San Pedro1,2,3, Guillermo Mariño1,2,3, Valentina Cianfanelli6,7, Amena Ben-Younès1,2,3, Rodrigo Troncoso8,9,10, Maria Markaki11, Valentina Sica1,2,3,5, Valentina Izzo1,2,3, Kariman Chaba1,12, Chantal Bauvy12,13,14, Nicolas Dupont12,13,14, Oliver Kepp1,3,15, Patrick Rockenfeller16,17, Heimo Wolinski16,17, Frank Madeo16,17, Sergio Lavandero8,9,10,18, Patrice Codogno12,13,14, Francis Harper2,19, Gérard Pierron2,19, Nektarios Tavernarakis11,20, Francesco Cecconi6,7,21, Maria Chiara Maiuri1,2,3, Lorenzo Galluzzi1,2,3,12,‡ and Guido Kroemer 1,3,12,15,22,‡ 1Equipe 11 labellisée par la Ligue contre le Cancer, Centre de Recherche des Cordeliers, Paris, France 2Gustave Roussy Comprehensive Cancer Center, Villejuif, France 3INSERM, U1138, Paris, France 4Government College University, Faisalabad, Pakistan 5Université Paris Sud/Paris 11, Le Kremlin Bicêtre, France 6Department of Biology, University of Rome ‘Tor Vergata’, Rome, Italy 7Unit of Cell Stress and Survival, Danish Cancer Society Research Center, Copenhagen, Denmark 8Advanced Center for Chronic Disease (ACCDiS), Faculty of Chemical & Pharmaceutical Sciences/Faculty of Medicine, University of Chile, Santiago, Chile 9Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile 10Faculty of Medicine, Institute of Nutrition and Food Technology, University of Chile, Santiago, Chile 11Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, Heraklion, Greece 12Université Paris Descartes, Sorbonne Paris Cité, Paris, France 13INSERM, U1151, Paris, France 14Institut Necker Enfants-Malades, Paris, France 15Cell Biology & Metabolomics Platforms, Gustave Roussy Comprehensive Cancer Center, Villejuif, France 16Institute of Molecular Biosciences, NAWI Graz, University of Graz, Graz, Austria 17BioTechMed Graz, Graz, Austria 18Department of Internal Medicine (Cardiology Division), University of Texas Southwestern Medical Center, Dallas, TX, USA 19CNRS, UMR8122, Villejuif, France 20Department of Basic Sciences, Faculty of Medicine, University of Crete, Heraklion, Greece 21Laboratory of Molecular Neuroembryology, IRCCS Fondazione Santa Lucia, Rome, Italy 22Pôle de Biologie, Hôpital Européen Georges Pompidou, AP-HP, Paris, France ‡MN-S and SAM equally contributed to this paper ‡LG and GK share co-senior authorship *Corresponding author. Tel: +33 1 4211 6046; Fax: +33 1 4211 6047; E-mail: [email protected] The EMBO Journal (2015)34:1025-1041https://doi.org/10.15252/embj.201489363 See also: VA Bankaitis (April 2015) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract To obtain mechanistic insights into the cross talk between lipolysis and autophagy, two key metabolic responses to starvation, we screened the autophagy-inducing potential of a panel of fatty acids in human cancer cells. Both saturated and unsaturated fatty acids such as palmitate and oleate, respectively, triggered autophagy, but the underlying molecular mechanisms differed. Oleate, but not palmitate, stimulated an autophagic response that required an intact Golgi apparatus. Conversely, autophagy triggered by palmitate, but not oleate, required AMPK, PKR and JNK1 and involved the activation of the BECN1/PIK3C3 lipid kinase complex. Accordingly, the downregulation of BECN1 and PIK3C3 abolished palmitate-induced, but not oleate-induced, autophagy in human cancer cells. Moreover, Becn1+/− mice as well as yeast cells and nematodes lacking the ortholog of human BECN1 mounted an autophagic response to oleate, but not palmitate. Thus, unsaturated fatty acids induce a non-canonical, phylogenetically conserved, autophagic response that in mammalian cells relies on the Golgi apparatus. Synopsis A systematic screen in cancer cells reveals that unsaturated and saturated fatty acids induce autophagy via distinct pathways, with unsaturated fatty acids acting in a Golgi-dependent but Beclin-1-independent manner. Saturated and unsaturated fatty acids promote autophagy, in vitro and in vivo, via different molecular mechanisms. The saturated fatty acid palmitate stimulates canonical, BECN1- and PIK3C3-dependent autophagic responses that involve JNK1, PKR and AMPK. The unsaturated fatty acid oleate promotes a non-canonical BECN1-independent autophagic response that requires an intact Golgi apparatus. Oleate-induced non-canonical autophagy is conserved in human cells, mice, yeast and nematodes. Introduction Macroautophagy (here referred to as “autophagy”) relies on the sequestration of cytoplasmic structures in double-membraned vesicles that are commonly known as autophagosomes. Upon closure, autophagosomes fuse with lysosomes to generate autolysosomes, resulting in the degradation of the autophagosomal cargo and inner membrane by acidic hydrolases. The nucleation of autophagosomes is often initiated by unc-51-like autophagy-activating kinase 1 (ULK1) and generally requires phosphatidylinositol 3-kinase, catalytic subunit type 3 (PIK3C3, also known as VPS34). PIK3C3 encodes the catalytic subunit of a class III phosphatidylinositol-3 kinase that operates within a large macromolecular complex involving beclin 1, autophagy related (BECN1), ATG14 and phosphoinositide-3-kinase, regulatory subunit 4 (PIK3R4, also known as VPS15) (Weidberg et al, 2011). The elongation of autophagosomal membranes relies on two conjugation reactions, that is, the conjugation of ATG12 to ATG5 and that of mammalian orthologs of yeast Atg8 to phosphatidylethanolamine. In this setting, microtubule-associated protein 1 light chain 3 (MAP1LC3, best known as LC3) and GABA(A) receptor-associated protein (GABARAP) family members are conjugated to phosphatidylethanolamine in process that resembles ubiquitination. Such a cascade of reactions involves ATG7 (an E1-like enzyme) and ATG3 (an E2-like enzyme), eventually yielding phosphatidylethanolamine-conjugated and autophagosome-associated LC3 and GABARAP proteins (Rubinsztein et al, 2012). Thus, the formation of autophagosomes can be quantified by following the redistribution of LC3B (the most studied member of the LC3/Atg8 family) to cytoplasmic puncta or by assessing its lipidation (which increases its electrophoretic mobility). In addition, the so-called autophagic flux (i.e. the actual ability of autophagosomes to degrade intracellular components) can be monitored by measuring the turnover of long-lived proteins or by determining the degradation of specific autophagic substrates such as sequestosome 1 (SQSTM1, best known as p62) (Bjorkoy et al, 2009). Of note, autophagic responses are negatively regulated by mechanistic target of rapamycin (MTOR) complex I (MTORCI), a key hub for the control of cell survival, growth and proliferation (Laplante & Sabatini, 2012). Autophagy plays a major role in the differentiation and function of adipocytes (Singh et al, 2009b; Zhang et al, 2009), in the mobilization of lipid droplets within hepatocytes (a process that has been dubbed “lipophagy”) (Singh et al, 2009a), as well as in the oxidation of fatty acids (FAs) by cancer cells (Guo et al, 2013), underscoring its significant impact on lipid metabolism. Conversely, several saturated FAs (SFAs) and unsaturated FAs (UFAs) appear to modulate autophagy (Brenner et al, 2013). Moreover, neutral lipid droplets have recently been shown to contribute to autophagic responses by providing substrates for the formation of autophagosomes (Dupont et al, 2014a). The UFAs oleate (C18:1) and linoleate (C18:2) stimulate the autophagic flux in both mammary epithelial cells (Pauloin et al, 2010) and hepatocytes (Mei et al, 2011). Along similar lines, polyunsaturated FAs including di-homo-γ-linoleic acid (C20:3), arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) induce autophagy in multiple cell types (Fukui et al, 2013; O'Rourke et al, 2013). Conversely, the autophagy-modulatory potential of the SFA palmitate (C16:0) exhibits a significant degree of variation. Indeed, palmitate has been shown to inhibit (Las et al, 2011; Kim et al, 2013) as well as to activate (Khan et al, 2012; Martino et al, 2012) autophagy in pancreatic islet and vascular endothelial cells. Moreover, palmitate reportedly limits autophagy in hepatocytes (Mei et al, 2011), yet promotes it in mouse embryonic fibroblasts (Tan et al, 2012). At least in some cell types, the autophagic response to palmitate involves protein kinase C (PKC) (Tan et al, 2012), eukaryotic translation initiation factor 2, subunit 1α, 35 kDa (EIF2S1, best known as eIF2α) and its substrate eIF2α kinase 2 (EIF2AK2, best known as PKR), and mitogen-activated protein kinase 8 (MAPK8, best known as JNK1) (Shen et al, 2012). In addition, palmitate has been shown to stimulate or inhibit AMP-activated protein kinase (AMPK), an upstream regulator of autophagy impinging on MTORCI signaling, depending on experimental variables including cell type, concentration and exposure time (Fediuc et al, 2006; Sun et al, 2008). Although these discrepancies have not been investigated in detail, they may reflect the ability of palmitate to modulate autophagy in a manner that changes with time, initially promoting it and then inhibiting it. In support of this notion, feeding mice with a high-fat diet for a prolonged period has been shown to promote alterations in intracellular lipids that are accompanied by defects in the fusion between autophagosomes and lysosomes (Koga et al, 2010). Driven by the consideration that distinct FAs influence the composition of cellular membranes and affect long-term health in a different manner (Wijendran & Hayes, 2004; Cascio et al, 2012), we performed a systematic analysis of FAs for their capacity to induce autophagy in short-term experiments. Here, we report the unexpected finding that SFAs and UFAs promote autophagy by activating different molecular mechanisms. Thus, while SFAs stimulate canonical autophagy, UFAs trigger a BECN1- and PIK3C3-independent autophagic response that is accompanied by the redistribution of LC3 to Golgi-associated vesicles. Results Palmitate and oleate induce autophagy in vitro and in vivo To identify FAs that trigger autophagy, we exposed human osteosarcoma U2OS cells that stably express a green fluorescent protein (GFP)-LC3 chimera (Shen et al, 2011) to a panel of 26 FAs differing in the length of the carbon chain as well as in saturation status, followed by assessment of GFP-LC3+ puncta by automated fluorescence microscopy. SFAs with 15–18 carbon atoms such as pentadecanoic, hexadecanoic, heptadecanoic and octadecanoic acid (stearate), but neither smaller nor larger SFAs, promoted the formation of GFP-LC3+ dots in U2OS cells (Fig 1A). Similarly, several UFAs with 14–20 carbon atoms, including myristoleic, palmitoleic, oleic, linoleic and arachidonic acid, efficiently triggered the aggregation of GFP-LC3 in cytoplasmic dots (Fig 1A). Based on solubility considerations and natural abundance in human cells (as well as in commonly employed oils, i.e. palm and olive oil), we chose to focus our study on one UFA, that is, oleic acid (oleate) and one SFA, that is, hexadecanoic acid (palmitate). Figure 1. Induction of autophagy by palmitate and oleate A. Screening for autophagy-inducing fatty acids. U2OS cells expressing GFP-LC3 were cultured in control conditions (Co) or treated with the indicated saturated (in black) or unsaturated (in blue) fatty acids for 4 h, followed by the quantification of the number of cytoplasmic GFP-LC3+ dots per cell by automated fluorescence microscopy. Data are means ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01 versus untreated cells). B–D. Autophagic flux induced by palmitate, stearate and oleate. Wild-type (WT, C) and GFP-LC3-expressing (B) U2OS cells as well as HeLa cells (D) were maintained in control conditions, exposed to nutrient-free (NF) conditions or treated with 500 μM oleate (OL), 500 μM stearate (ST) or 500 μM palmitate (PA), alone or in combination with 10 μg/ml E-64d and 10 μg/ml pepstatin (Pep) for 4 (D) or 6 (B, C) h. Thereafter, the number of cytoplasmic GFP-LC3+ dots per cell (B), LC3 lipidation and p62 degradation (C) or the degradation of labeled long-lived proteins (D) was quantified. In (B) and (D), data are means ± SEM (B) or normalized means ± SD (D) of at least three independent experiments (B), or four replicate assessments from one representative (D) experiment(s) out of three performed (*P < 0.05, **P < 0.01 versus untreated cells; ##P < 0.01 versus cells treated with PA or OL only; n.s., not significant versus cells treated with E-64d plus Pep). In (C), β-actin levels were monitored to ensure equal loading of lanes, and densitometry was employed to quantify the abundance of lipidated LC3 (LC3-II) and p62 (both normalized to β-actin levels). E, F. Involvement of ATG5 and ATG7 in fatty acid-induced autophagy. WT (F) or GFP-LC3-expressing (E) U2OS cells were transfected with a control siRNA (siUNR) or with siRNAs targeting ATG5 (siATG5) or ATG7 (siATG7) for 48 h and either maintained in control conditions or treated with 500 μM PA or 500 μM OL. Six hours later, cells were processed for the quantification of the number of cytoplasmic GFP-LC3+ dots per cell by automated fluorescence microscopy (E) or for the assessment of LC3 lipidation and p62 degradation by immunoblotting (F). In (E), data are means ± SEM of at least three independent experiments (*P < 0.05, **P < 0.01 versus untreated siUNR-transfected cells; #P < 0.05 versus siUNR-transfected cells treated with PA or OL only). In (F), β-actin levels were monitored to ensure equal loading of lanes. G. Induction of autophagy by fatty acids in mice. C57BL/6 mice were injected i.p. with vehicle only, 100 mg/kg PA or 100 mg/kg OL. One or two hours later, animals were euthanatized and LC3 lipidation, p62 degradation and AMPK phosphorylation were assessed by immunoblotting in the indicated tissues. GAPDH levels were monitored to ensure equal loading of lanes. Densitometry was employed to quantify the abundance of lipidated LC3 (LC3-II) and p62 (both normalized to GAPDH levels) and AMPK phosphorylation (normalized to total AMPK levels). Results are means ± SD of three mice (*P < 0.05, **P < 0.01, ***P < 0.001, versus vehicle-treated mice). Download figure Download PowerPoint The increase in GFP-LC3+ puncta, LC3 lipidation and p62 degradation induced by palmitate and oleate was exacerbated by the addition of E-64d and pepstatin (Fig 1B and C), two well-known inhibitors of lysosomal proteases. This indicates that palmitate and oleate do not stimulate the mere accumulation of LC3
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