Autophagy, signaling and obesity

Pharmacological Research 66 (2012) 513–525

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Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Invited review

Autophagy, signaling and obesity Vanessa J. Lavallard a,b , Alfred J. Meijer d , Patrice Codogno e , Philippe Gual a,b,c,∗ a

INSERM, U1065, Equipe 8 «Complications hépatiques de l’obésité», Nice, France Université de Nice-Sophia Antipolis, Faculté de Médecine, Nice, France c Centre Hospitalier Universitaire de Nice, Hôpital de l’Archet, Département Digestif, Nice, France d Department of Medical Biochemistry, Academic Medical Center, Amsterdam, The Netherlands e INSERM, U984, University Paris-Sud 11, 92296 Châtenay-Malabry, France b

a r t i c l e

i n f o

Article history: Received 3 September 2012 Accepted 4 September 2012 Keywords: Autophagy Lipophagy Liver Adipose tissue Pancreas NAFLD

a b s t r a c t Autophagy is a cellular pathway crucial for development, differentiation, survival and homeostasis. Autophagy can provide protection against aging and a number of pathologies such as cancer, neurodegeneration, cardiac disease and infection. Recent studies have reported new functions of autophagy in the regulation of cellular processes such as lipid metabolism and insulin sensitivity. Important links between the regulation of autophagy and obesity including food intake, adipose tissue development, ␤ cell function, insulin sensitivity and hepatic steatosis exist. This review will provide insight into the current understanding of autophagy, its regulation, and its role in the complications associated with obesity. © 2012 Elsevier Ltd. All rights reserved.

Contents 1. 2.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulation of autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The main signaling pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.1. The insulin–PI3K–PKB–mTOR pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. The ERK pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. The p38 MAPK pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Short-term regulation of autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. The Beclin-1–PI3K class III network . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Amino acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Energy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Oxidative stress: reactive oxygen species (ROS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Endoplasmic reticulum stress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.6. Acetylation/deacetylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Long-term regulation of autophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.1. FoxO proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2. The transcription factor EB . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Autophagy and obesity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Food intake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Adipose tissue development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Liver complications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.1. Hepatic steatosis and lipophagy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.2. Hepatic insulin resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Pancreas and type 2 diabetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Abbreviations: Atg, autophagy related genes; ER, endoplasmic reticulum; mTOR, mammalian target of rapamycin; PKB, protein kinase B. ∗ Corresponding author at: INSERM U1065, Bâtiment Universitaire ARCHIMED, Team 8 “Hepatic complications in obesity”, 151 Route Saint Antoine de Ginestière, BP 2 3194, 06204 Nice Cedex 03, France. Tel.: +33 489 064 223; fax: +33 489 064 221. E-mail address: [email protected] (P. Gual). 1043-6618/$ – see front matter © 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2012.09.003

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3.5. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disclosures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Financial support . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Autophagy, or cellular self-digestion, is a catabolic process which targets cell constituents (damaged organelles, unfolded proteins, intracellular pathogens) to lysosomes for degradation [1,2]. Under basal conditions, autophagy is involved in the degradation of long-lived proteins whereas the ubiquitin–proteasome pathway, another catabolic process, is responsible for the degradation of short-lived proteins [3,4]. In response to cellular stress such as nutrient deprivation, an increase in autophagic turnover maintains cellular energy homeostasis. Three types of autophagy have been identified: macroautophagy, chaperone-mediated autophagy (CMA) and microautophagy (Fig. 1). Macroautophagy (hereafter referred to as autophagy) involves the formation of a small vesicular sac called the isolation membrane or phagophore. The phagophore encloses a portion of cytosol resulting in the formation of a double-membraned structure termed an autophagosome. The autophagosome then fuses with a lysosome leading to the degradation of the cellular constituents sequestered in the autophagosome. Amino acids and other compounds generated by autophagic degradation of macromolecules are released into the cytoplasm for recycling or for energy production [1]. The origin of the membranes involved in the formation of autophagosomes is not yet known. Recent studies suggest that the lipids for autophagosome formation may be derived from the endoplasmic reticulum (ER) [5–7] but additional membrane sources, such as the plasma membrane, mitochondria and Golgi, cannot be excluded [8]. Microautophagy also involves the sequestration of cellular constituents within lysosomes but in this case through the invagination of the lysosomal membrane. CMA, concerns the sequestration of polypeptides and soluble proteins containing a KFERQ motif in their amino acids sequence. These proteins are bound to a chaperone protein for translocation to the lysosomes where binding to the lysosome-associated membrane protein type 2A (LAMP-2A) receptor leads to protein internalization and degradation [9]. In autophagy, the formation of the phagophore and the autophagosomes requires 18 different Atg proteins (AuTophaGyrelated) initially identified in yeast [10]. The process of autophagosome formation involves three major steps: initiation, nucleation and elongation/enclosure (Fig. 2). The initiation step is controlled by the ULK1–Atg13–FIP200 complex [11,12]. The serine/threonine kinase mammalian target of rapamycin (mTOR), a component of the mTORC1 complex, is the main inhibitor of autophagy. The nucleation step requires the Beclin-1–class III phosphatidylinositol 3-kinase (PI3K) complex that includes Beclin-1, Vps34 (class III PI3K), Vps15, Atg14L (Barkor) and Ambra-1 [13]. The involvement of mTOR and the Beclin-1–class III PI3K complex in the regulation of autophagy are discussed below. Two conjugation systems are involved in the elongation/enclosure step. The first is the conjugation of Atg12–Atg5 mediated by two ligases Atg7 and Atg10. Atg5 also associates with Atg16 to form the Atg12–Atg5–Atg16 complex. The second involves the cleavage of LC3 (Atg8) by Atg4 leading to the soluble form LC3-I, which is then conjugated to phosphatidylethanolamine (PE) via the participation of Atg7 and Atg3. This lipid conjugation forms the autophagic double membrane-associated LC3-II protein allowing the closure of

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autophagic vacuole [14,15]. LC3-II is used as a marker of autophagosomes [16]. Autophagy is a cellular pathway crucial for the maintenance of cellular homeostasis, normal mammalian physiology and could play a protective or deleterious role in a range of disease. Recent studies have reported its role and its regulation in the complications associated with obesity. 2. Regulation of autophagy Autophagy is regulated by multiple signal-transduction pathways. Insulin and glucagon inhibit and enhance autophagy, respectively. Other mechanisms are also involved in the regulation of autophagy. We will describe some of these in this chapter. 2.1. The main signaling pathways 2.1.1. The insulin–PI3K–PKB–mTOR pathway The mTOR signaling pathway [17] is the major pathway regulating autophagy (Fig. 3), as first described by Blommaart et al. [18] for hepatocytes and by Noda and Ohsumi [19] for yeast cells. In response to insulin, mTOR is activated and inhibits autophagy. Insulin binds to its tyrosine kinase receptor, leading to its autophosphorylation, and the recruitment and phosphorylation of its major substrates insulin receptor substrate 1 and 2 (IRS1 and IRS2). Phosphorylated IRSs then recruit mainly class I PI3K. The PIP3 (phosphatidylinositol 3,4,5-triphosphate) produced by PI3K recruits Akt/PKB (protein kinase B), which is activated by PDK1 (phosphoinositide-dependent kinase-1)-mediated phosphorylation. Akt/PKB then phosphorylates the TSC2 protein and prevents TSC1/2 complex formation (tuberous sclerosis complex 1/2). Downstream of the TSC1/2 complex which acts as a GTPase, the Rheb protein, in its GTP-form, binds and activates mTOR, which leads to the inhibition of autophagy [20–22]. For complete activation of mTOR, however, the presence of amino acids is essential [18,23] (see Section 2.2.2). In contrast, the lipid phosphatase PTEN (Phosphatase and tensin homolog) inhibits signaling downstream of Akt/PKB, by hydrolyzing phosphatidylinositol 3,4,5-triphosphate to phosphatidylinositol 4,5-bisphosphate, and prevents mTORinduced inhibition of autophagy (Fig. 3) [24]. mTOR is present in two complexes: mTORC1 and mTORC2 (TORC, target of rapamycin complex). The mTORC1 complex includes mTOR and the proteins raptor, G␤L and the inhibitory protein PRAS40. The active mTORC1 complex interacts and inactivates the protein kinase ULK1 (the homolog of the yeast Atg1) by phosphorylation. Under conditions of starvation or in response to rapamycin, inactivation of mTOR allows the dissociation of the mTORC1 complex, resulting in dephosphorylation and activation of ULK1, phosphorylation of Atg13 and FIP200, and consequently the induction of autophagy [25–27]. The mTORC2 complex includes, in addition to mTOR, the proteins rictor, G␤L, SIN1 and PROTOR. This complex is less rapamycin-sensitive and is involved in Akt/PKB phosphorylation [23]. Glucagon inhibits mTOR signaling, and stimulates autophagy [18], at least in part through a protein kinase A-dependent mechanism [28].

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Fig. 1. The three types of autophagy. Macroautophagy involves the formation of a double-membrane vesicle called autophagosome. This structure sequesters damaged organelles and mis-folded proteins which are degraded by lysosomal enzymes. In chaperone-mediated autophagy, proteins containing a KFERQ motif, bound to a chaperone protein for translocation to the lysosome leading to their internalization and degradation. Microautophagy involves the direct sequestration of cellular components by the lysosome through the invagination of the lysosomal membrane.

2.1.2. The ERK pathway Several studies have reported the potential role of the ERK (extracellular signal-regulated kinases) pathway, which is also activated in response to insulin, in the regulation of autophagy. However, this pathway activates or inhibits autophagic turnover depending on the stimuli and the cell type used. For example, autophagy is stimulated by ERK activity in response: to amino acid depletion [29] and aurintricarboxylic acid administration [30] in the human colorectal cancer cell line HT29, to soya-saponins in human colon adenocarcinoma HCT-15 [31], to dihydrocapsaicin in WI38 lung fibroblasts [32], to cadmium in mesengial MES-13 cells [33,34], to TNF␣ treatment in MCF-7 and L929 cells [35,36] and to daunorubicin in k562 cells [37], but inhibited by the carcinogen lindane in the mouse Sertoli cell line [22]. Activation of autophagy by the ERK pathway has been evaluated with specific autophagic markers such as conversion of LC3-I to LC3-II [32,34,35], induction of Beclin-1 [35] and induction of BNIP3 [38]. In the human colon cancer HT-29, Codogno et al. reported the implication of ERK in the regulation of autophagy through the regulation of a cytoplasmic heterotrimeric Gi3 protein and its regulator the Galpha-interacting protein (GAIP) [39,40]. The Gi3 protein, in the GTP-bound form, inhibits autophagy, and when bound to the GDP, stimulates autophagy. The hydrolysis of GTP for the Gi3 protein is regulated by the GTPase GAIP. ERK1/2 phosphorylate and activate GAIP, which generates the GDP-bound form of Gi3 leading to activation of autophagy. Phosphorylation of GAIP by ERK1/2 is further sensitive to amino acids. Indeed, amino acids inhibit ERK1/2 kinases, leading to a low level of GAIP phosphorylation/activation

and consequently a decrease in autophagic turnover [29]. The antiautophagic action of insulin via Gi3 protein has also been reported in the liver [41]. A recent review of the current literature has indicated that active ERK1/2 is essential in mitophagy, i.e. the specific autophagic elimination of damaged mitochondria, the presence of which would otherwise lead to cell death [42]. The reason for the apparent conflicting effects of ERK1/2 on autophagy may be that, in addition to its mTOR-independent stimulation of autophagy, ERK1/2 can also cause activation of signaling upstream of mTOR [23,43] (Fig. 3) and in this way inhibits autophagy. 2.1.3. The p38 MAPK pathway As reported for the ERK pathway, the negative or positive regulation of autophagy by p38 MAPK is cell type dependent and stimuli specific. In hepatocytes, an increase in cell volume following Na+ -dependent concentrative uptake of amino acids inhibits autophagic proteolysis through activation of p38 MAPK by an integrin-dependent mechanism [44]. Activation of p38␣ with anisomycin, a protein synthesis inhibitor, or UV irradiation leads to inhibition of the autophagic pathway in HEK293 cells. The p38␣ MAPK protein negatively regulates the interaction of mAtg9, a multi-spanning membrane protein that is essential for autophagy, with p38 IP, a novel mAtg9 binding partner. This interaction controls the level of autophagy when induced in response to starvation [45]. Exposure of macrophages to LDL cholesterol inhibits autophagy through activation of p38 MAPK which may contribute to foam cell formation in the development of atherosclerosis [46].

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Fig. 2. The mains steps of autophagosome formation. The initiation step is controlled by the ULK1–Atg13–FIP200 complex, which also contains Atg101. mTOR interacts with, and inactivates ULK1 by phosphorylation. Under starvation conditions, mTOR is inactivated leading to ULK1 activation, phosphorylation of Atg13 and FIP200 and consequently the induction of autophagy. The nucleation step requires the Beclin-1–class III PI3K complex including Beclin-1, class III PI3K, Vps15, Atg14L and Ambra1. The ULK1–Atg13–FIP200 and Beclin-1–class III PI3K complexes recruit two conjugation systems essential for the elongation and enclosure step: the Atg12–Atg5 and the Atg8–PE conjugation systems.

Fig. 3. Short-term regulation of autophagy in response to amino acids, insulin, energy and endoplasmic reticulum (ER) stress. Amino acids inhibit autophagy through class III PI3K and Rag-mediated mTOR activation. ERK1/2 phosphorylates and activates GAIP which promotes the GDP-bound form of Gi3 protein leading to activation of autophagy. Amino acids inhibit ERK1/2 leading to a low level of GAIP phosphorylation/activation and consequently the decrease in autophagic turnover. The activation of the insulin receptor promotes the activation of class I PI3K and Akt/PKB, the inhibition of the TSC1/2 complex, the activation of mTOR by Rheb and consequently the inhibition of autophagy. Upon a decrease in cellular energy, LKB1 kinase activates AMPK which inhibits mTOR through the TSC1/2 complex and Rheb, and also by phosphorylation of raptor in the mTORC1 complex. AMPK also directly activates autophagy by phosphorylating p27(kip1). ER stress induces the release of Ca2+ from ER to cytosol. The increase in intracellular level of Ca2+ stimulates CaMKK␤ kinase which activates AMPK. Autophagy is thus activated by AMPK-mediated inhibition of mTOR.

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On the other hand, accumulation of the mutant glial fibrillary acidic protein (GFAP) in Alexander disease induces autophagy through p38 MAPK activation and mTOR inhibition [47]. 2.2. Short-term regulation of autophagy 2.2.1. The Beclin-1–PI3K class III network Beclin-1, the mammalian homolog of Atg6, binds to class III PI3K and is, like class III PI3K [48], essential for the activation of autophagy [49]. Beclin-1 constitutively interacts with the antiapoptotic members of the Bcl-2 family (Bcl-2, Bcl-XL) through the BH3-domain in Beclin-1. This interaction prevents the association of Beclin-1 with the class III PI3K and thus the activation of autophagy [49–51]. Induction of autophagy requires the dissociation of Beclin-1 from Bcl-2, which is mediated by: (i) Bcl-2 degradation [52], (ii) competition with other BH3-containing proteins such as BAD and BNIP3 [53] and (iii) Bcl-2 phosphorylation by JNK1. In response to amino acid deprivation or ceramides, JNK1, but not JNK2, phosphorylates Bcl-2 on threonine 69, serine 70 and serine 87. This triple phosphorylation induces the dissociation of Bcl-2 from Beclin-1 in promoting activation of autophagy [54,55]. A recent study has also reported that DAPK (death-associated protein kinase) dissociates the Bcl-2/Beclin-1 complex by phosphorylating Beclin-1, leading to induction of autophagy [56]. The Beclin-1–PI3K class III complex also includes the proteins Ambra1, Vmp1, Bif-1 and UVRAG, which stabilize the complex and which may play an important role in the stimulation of autophagy [57–62]. 2.2.2. Amino acids Amino acids inhibit autophagy by stimulation of mTOR signaling. However, the mechanisms involved in this regulation are not yet fully elucidated. Among the various amino acids, leucine, but not valine or isoleucine, is most active in stimulating mTOR and inhibiting autophagy [63,64]. Metabolism of leucine is not required [65]. Any proposed mechanism of amino acid sensing must account for this high specificity. The amino acid sensor is intra-rather than extra-cellular [66,67]. In mammalian cells, the transporters SLC1A5 (solute carrier family 1 member 5), responsible for cellular uptake of glutamine, and SLC7A5/SLC3A2, mediating glutamine–leucine exchange across the plasma membrane, per se, control the intracellular concentration of leucine [68,69]. The inhibition of SLC1A5 leads to the inhibition of mTORC1 and consequently the activation of autophagy [69]. Several mechanisms of amino acid sensing have been proposed in the past and have been reviewed in detail elsewhere [66,67,70,71]. Among them, these are the stimulation of mTOR/mTORC1 by amino acids via the class III PI3K [72,73] and the Rag GTPases [74–76] resulting in inhibition of autophagic turnover (Fig. 3). The involvement of class III PI3K in amino acid signaling has been puzzling because the enzyme is also involved in the initiation of autophagy. Presumably, class III PI3K is part of different protein complexes with different functions [66,67]. It is generally accepted that Rag is essential for the activation of mTOR by amino acids [74,75]. In one of the most recent studies on amino acid sensing it is proposed that the v-ATPase in the lysosomal membrane, in addition to its role in proton pumping, actually functions as the amino acid sensor in mTOR signaling [77]. The v-ATPase responds to an increase in the intra-lysosomal, rather than the cytosolic, amino acid concentration with a conformational change which causes increased binding of Ragulator, a scaffolding protein complex that anchors Rag to the lysosomal surface. This promotes the translocation of the mTORC1 complex to the extra-lysosomal surface, upon which mTOR becomes activated. This is in agreement with studies showing that active mTORC1 is localized at the lysosomal membrane [78,79]. In this mechanism, the activity of the lysosomal proton-assisted amino acid transporter PAT1, responsible for the efflux of amino acids from the

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lysosomes, controls the concentration of amino acids within the lysosomal lumen and thus the extent of mTORC1 activation [77]. If this mechanism is correct, it must be speculated that among the various amino acids, leucine is especially active in inducing the conformation change of the v-ATPase. In another very recent study, it is proposed that leucyl-tRNA synthetase is the actual amino acid sensor in mammalian cells [80], and also in yeast cells [81]. The enzyme directly binds to Rag GTPase in a leucine-dependent manner and functions as a GTPase-activating protein for Rag GTPase to activate mTORC1. In this mechanism, leucylation of the tRNA is not required. It is sufficient that leucine binds to the leucine-binding domain of the leucyl-tRNA synthetase and activates the enzyme, as measured by ATP-[32 P]PPi exchange activity [80]. It is important to note that these two mechanisms of amino acid sensing detect different pools of amino acids: the v-ATPase senses the intralysosomal pool of amino acids while the leucyl-tRNA synthetase senses cytosolic leucine.

2.2.3. Energy The cellular energy status is also an important regulator of autophagy. Low levels of cellular energy (ATP) activate AMPK (5 -AMP-activated protein kinase) and stimulate autophagy [82]. When the ATP/AMP ratio decreases, the LKB1 kinase activates AMPK, which then activates the TSC1/2 complex leading to inhibition of mTOR through Rheb [20] (Fig. 3) and at the same time inhibits raptor in the mTORC1 complex [83]. Suppression of mTORinduced inhibition of autophagy enhances autophagic turnover, which promotes an increase in ATP production via the degradation and recycling of nutrients. The energy sensing LKB1–AMPK pathway also regulates p27(kip1) phosphorylation which mediates the decision to enter autophagy or apoptosis in response to a bio-energetic stress [84]. Finally, AMPK can directly phosphorylate ULK1 on serines 317 and 777 leading to its dissociation from the mTORC1 complex and consequently enhances autophagic turnover in response to glucose privation. Under basal conditions, mTOR phosphorylates ULK1 on serine 757 preventing its activation and its interaction with AMPK [85–87].

2.2.4. Oxidative stress: reactive oxygen species (ROS) The major source of ROS comes from the electron transport chain of mitochondria [88]. When the production of ROS outstrips degradation, excessive ROS levels induce oxidative stress and damage of cellular components including DNA, proteins and lipids. A rise in ROS levels activates autophagy as a protective mechanism. Indeed, upregulation of autophagy tends to reduce the ROS levels and prevents the deleterious effects of elevated ROS levels. Autophagy is initiated by ROS in a manner that is sensitive to antioxidants (also in starvation) [89,90]. It is proposed that the protease Atg4 is the target of ROS [61]. Atg4 induces the cleavage of LC3/Atg8 and the delipidation of LC3-II on the cytosolic surface of autophagosomes, which allows recycling of LC3. Thus the inactivation of Atg4 by ROS promotes LC3 lipidation and induction of autophagy. Further, the localization of Atg4 in a ROS-poor environment during the maturation of autophagosomes to autophagolysosomes leads to Atg4 activation and can still delipidate and recycle LC3 [89]. While Atg4 is an important factor in the regulation of autophagy, ROS-mediated activation of autophagy has also been explained by additional mechanisms: upregulation of Beclin-1 [91] and the activation of the TSC2 by LKB1–AMPK pathway, which in turn alleviates the mTOR-inhibited autophagic response [92]. Upon starvation, activation of autophagy by ROS is clearly a survival response, which promotes the degradation of damaged mitochondria (mitophagy) and of oxidized proteins, and protects from ROS-induced apoptosis [93–95].

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2.2.5. Endoplasmic reticulum stress The endoplasmic reticulum (ER) is an important cellular compartment. It serves as the reservoir of Ca2+ and promotes the folding of newly synthesized proteins. Accumulation of un-folded or misfolded proteins, glucose privation or oxidative stress causes UPR activation (unfolded protein response). UPR promotes protein folding, degradation and maintenance of ER homeostasis [96,97]. In mammals, three UPR sensors control signal-transduction pathways leading to transcription of a large profile of genes including PERK (RNA-dependent protein kinase-like ER kinase), ATF6 (activating transcription factor 6) and IRE1 (inositol-requiring kinase 1). However, sustained activation of UPR induces ER stress. Several studies have reported that ER stress can induce autophagy. The treatment of neuroblastoma cells with ER stress activators induces autophagy, which is dependent on IRE1. In addition, suppression of JNK, a target of IRE1, inhibits induction of autophagy [96]. PolyQ72 aggregate-induced ER stress triggers phosphorylation of eiF2␣, which is mediated by PERK leading to conversion of LC3-I to LC3-II and to increased Atg12 expression [98]. The transcription factors CHOP and ATF4, which are regulated by PERK, increase Atg8 and Atg5 expression, respectively [99]. Other mechanisms have been proposed such as inhibition of the PKB–mTOR pathway [100], activation of the p38 MAPK pathway [101] and an elevated intracellular level of Ca2+ . Indeed, ER stress induces the release of Ca2+ from the ER to the cytosol. An increase in the intracellular level of Ca2+ stimulates CaMKK␤ kinase (calcium-activated calmodulin-dependent kinase kinase-␤), which activates AMPK. Autophagy is thus activated by AMPK-mediated mTOR inhibition [102]. High levels of Ca2+ also promote the phosphorylation/activation of PKC␪ (Ca2+ -dependent protein kinase C theta) leading to the conversion of LC3-I to LC3-II and activation of autophagy in hepatocytes in response to ER stressors [103]. 2.2.6. Acetylation/deacetylation In nutrient-rich conditions some Atg proteins such as Atg5, Atg7, Atg8 and Atg12 are specifically acetylated by the p300 acetyltransferase and during starvation they are de-acetylated by the sirtuin 1 deacetylase (also known as NAD-dependent deacetylase sirtuin-1 and SIRT1). SIRT1 regulates several cellular processes, such as the maintenance of energy homeostasis and cellular survival in response to calorie restriction. In this context, it is important to note that AMPK is also controlled by its acetylation status. The deacetylation of the enzyme promotes its association with LKB1, resulting in AMPK phosphorylation and activation [104]. Suppression of SIRT1 activity in cultured cells and in tissues increases the acetylation level of these Atg proteins and prevents induction of autophagy. In contrast, the overexpression of SIRT1 stimulates the basal level of autophagy [105]. SIRT1 and Atg5 null mice share several similarities including accumulation of damaged organelles and deficiencies in energy metabolism. While the deacetylase activity of SIRT1 regulates autophagy, the precise function of the deacetylated Atg protein in autophagic regulation is not yet elucidated. In addition, how SIRT1 and p300 sense upstream input signals, has not been demonstrated. Perhaps, the level of NAD+ [106] and the insulin/growth factor pathway could control reversible acetylation [97]. 2.3. Long-term regulation of autophagy 2.3.1. FoxO proteins The transcription factors FoxO (class O of forkhead box transcription factors) including FoxO1, FoxO3 and FoxO4 are involved in the regulation of glucose metabolism, the cell cycle and cell death. In response to insulin, the phosphorylation of FoxO proteins by Akt/PKB sequesters FoxO in the cytoplasm and inactivates it [107,108]. When Akt/PKB is inactivated, FoxO proteins translocate

into the nucleus and transactivate their target genes [109]. Recent studies have reported a role of FoxO proteins in induction of autophagy. In skeletal muscle atrophy, Mammucari et al. have shown that FoxO3 induces the transcription of the autophagy genes LC3, Gabarapl1 (Atg8 homolog), Atg4b, Atg12, Vps34 and Bnip3 [110]. In hepatocytes, overexpression of the constitutive nuclear form of FoxO1 prevents the decrease in Vps34, Atg12 and Gabarapl1 gene expression that is induced by insulin [111]. Furthermore, under conditions of cellular stress, FoxO1 is acetylated and interacts with Atg7 to induce autophagy in cancer cells [112]. AMPK can also directly regulate FoxO3 transcriptional activity by phosphorylation [113]. In neurons, JNK regulates FoxO-dependent autophagy. In contrast to other cell types, neuronal JNK negatively regulates autophagy and the upregulation of BNIP3 by FoxO releases Beclin1 from Bcl-XL leading to activation of autophagy in JNK-deficient cells [114]. 2.3.2. The transcription factor EB The transcription factor EB (TFEB) is involved in the control of lysosomal biogenesis. Under normal conditions, TFEB is phosphorylated by ERK on serine 142 leading to its localization in the cytoplasm. Under starvation conditions, TFEB is not phosphorylated, and translocates to the nucleus and transactivates its target genes [115]. TFEB could also be a regulator of autophagy. Overexpression of TFEB in different cell types enhanced the expression of genes encoding proteins involved in the crucial steps of the autophagic pathway: (i) cargo recognition, (ii) to formation of vesicle and (iii) substrate degradation. The cell overexpressing TFEB further displayed more autophagosomes and autophagolysosomes. This has also been validated in vivo [115]. After starvation (16 h), mice displayed downregulation of the ERK pathway, a decrease in TFEB phosphorylation, an increase in level of nuclear TFEB and induction of both autophagic and lysosomal TFEB target genes in the liver [115]. The overexpression of TFEB in GFP-LC3 transgenic mice compared to control mice increases the number of autophagosomes in the liver and increases the upregulation of genes encoding proteins involved in lysosomal function and autophagy. Similar results have been obtained in mice specifically overexpressing TFEB in the liver [115]. A very recent study by Settembre et al. [116] shows that under fed conditions TFEB is phosphorylated by, and colocalizes with, the mTORC1 complex on the lysosomal membrane in an amino acid- and Rag-dependent manner. In starvation, or upon pharmacological inhibition of mTORC1 by administration of rapamycin or the ATP-competitive inhibitor Torin, TFEB is released and translocates to the nucleus. These studies highlight a crucial role for TFEB in the transcriptional regulation of autophagy. 3. Autophagy and obesity The incidence of overweight and obesity is rapidly increasing in many Western countries. Obesity leads to numerous adverse metabolic disorders such as dyslipidemia, hypertension, reduced HDL cholesterol, and glucose intolerance. This cluster of metabolic abnormalities is grouped into the so-call metabolic syndrome that increase the risk of cardiovascular diseases, type 2 diabetes and liver complications (non-alcoholic fatty liver diseases: NAFLD). The spectrum of these hepatic abnormalities extends from isolated steatosis (triglyceride accumulation) to steatohepatitis (steatosis with inflammation) (non-alcoholic steato-hepatitis: NASH), steatofibrosis, which sometimes leads to cirrhosis and hepatocellular carcinoma. NAFLD is one of the three main causes of cirrhosis [117]. Insulin resistance is at the core of the pathophysiology of the metabolic syndrome and type 2 diabetes. Insulin resistance is

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characterized by a decrease in insulin signaling and action. Adipose tissue plays a central role in the control of glucose and lipid metabolism through its ability to control glucose transport, lipid storage and adipokines secretion. In obesity, the excessive gain of adipose tissue, in particular visceral adipose tissue causes its dysfunction, which could participate in the development of insulin resistance and other obesity-linked complications such as NAFLD. Adipose tissue expansion is associated with chronic low grade inflammation with infiltration of the tissue by immune cells (dendritic cells, macrophages, T lymphocytes). Consequently, adipose tissue produces inflammatory cytokines and free fatty acids in excess, and adipokine secretion is perturbed. Inflammatory cytokines and free fatty acids antagonize local insulin signaling in adipocytes, and also in muscles and liver. In liver, combined hyperglycemia and hyperinsulinemia promote de novo lipid synthesis and mitochondrial structural defects within hepatocytes. Moreover, insulin-resistance of adipose tissue leads to an enhanced free fatty acid flux to the liver, per se, in contributing to insulinresistance and steatosis. Oxidative and endoplasmic reticulum stresses play an important role in the alteration of insulin signaling and in the development of the liver complications. Interestingly, it has been recently reported that autophagy regulates food intake, adipose tissue development, hepatic steatosis, insulin-resistance and plays a protective role against lipotoxicity in ␤ cells. We will thus provide insight into the current understanding of the role of autophagy in these different responses.

autophagy in pre-adipocytes reduces accumulation of triglycerides and expression of transcription factors involved in adipocyte differentiation. While mice invalidated for Atg7 in adipose tissue are resistant to high fat diet (HFD)-induced obesity, the amount of brown adipose tissue (BAT) increased. Interestingly, the authors reported conversion of white adipocytes to brown adipocytes. In contrast to white adipose tissue, BAT stores little triglycerides and generates energy from fatty acids in part as heat (thermogenesis), because of the presence of UCP1 (uncoupling protein 1) in their mitochondrial inner membrane. The development of BAT induces weight loss, improves insulin sensitivity and enhances ␤-oxidation [122]. Finally, Atg7−/− mice displayed a decrease in the plasmatic levels of leptin, triglycerides and cholesterol and are resistant to obesity induced by HFD challenge [123]. In line with these studies, recent data show that autophagy in adipocytes of obese man is upregulated [124,125], presumably caused by attenuation of mTOR signaling [124]. These findings indicate that inhibition of autophagy in adipose tissue, or more specifically in adipocytes, may play a protective role against obesity and insulin resistance by reducing white adipose tissue and promoting BAT development. Identification of the molecular mechanisms of induction by autophagy of the transdifferentiation of white adipose tissue to BAT constitutes the next exciting and important challenge.

3.1. Food intake

3.2. Adipose tissue development

An overview of the role of hepatic autophagy and liver complications has been recently published [126,127]. Under physiological conditions, autophagy participates in the basal turnover of lipids by engulfing and degrading lipid droplets. As discussed in Section 2, autophagy is inhibited by the insulin-, amino acid–mTOR signaling pathway via both short-term and long-term mechanisms of regulation. Short-term inhibition can be produced by the mTOR complex. Long-term regulation occurs via the transcription factors FoxO and TFEB, which control the transcription of autophagic genes and are inhibited by insulin-induced activation of Akt/PKB and mTOR, respectively. In obesity, the level of autophagy is decreased in hepatocytes. Several mechanisms may account for this decline. (i) Obesity-induced increase in the calcium-dependent protease calpain 2, which leads to down-regulation of Atg7 and then to defective autophagy. Acute inhibition of calpain is able to restore Atg7 expression [128]. (ii) In obese mice, the autophagy inhibitor mTOR is over-activated in the liver, presumably as a result of an increased amino acid concentration following over-nutrition. The over-activation of mTOR by itself can result in liver and muscle insulin resistance because of phosphorylation, and inhibition of IRS1 by S6K, a downstream target of mTOR [129–131]. (iii) Although controversial, hyperinsulinemia may also contribute to down-regulation of autophagy in obese mice. Indeed, elevated activation of Akt/PKB, a key molecule in the insulin pathway, decreases autophagy in the liver of obese mice [111]. However, destruction of insulin production by ␤ cells by streptozotocin does not increase autophagy in the liver of obese mice [128], in contrast to lean mice [111]. The reasons for these discrepancies are unclear. In obesity, a defect in hepatic autophagy and its associated decrease in the rate of lysosomal degradation contribute to further increasing the ER stress induced by nutrient overload, in an inflammatory milieu [128,132]. Together, a decline in autophagy and increased ER stress lead to insulin resistance [128].

Adipocytes are the cells that primarily compose adipose tissue, specialized in storing energy as fat. During obesity, the size and number of adipocytes increases, resulting in adipose tissue expansion. Interestingly, it has recently been reported that autophagy regulates adipose mass and differentiation [122,123]. Inhibition of

3.3.1. Hepatic steatosis and lipophagy In response to a moderate increase in lipid availability or during nutrient deprivation, hepatic autophagy degrades lipid droplets to provide free fatty acids for ATP production. In contrast, a sustained availability of lipids induced by a long-lasting

Recent reports have suggested that autophagy could regulate food intake. Meng and Cai reported that Atg7 protein expression is reduced in the hypothalamus of mice when exposed to a chronic high-fat diet (HFD). The defect of hypothalamic autophagy by knock down of Atg7 causes an increase in food intake, a weight gain and an impairment of energy expenditure [118]. This correlated with induction of the NF-␬B pathway and changes in inflammation. Knock down of IKK␤ in neurons corrected the glucose intolerance; hyperinsulinemia and hyperleptinemia induced by Atg7 knock down. The decline in hypothalamic autophagy can disrupt the central regulation of the energy balance and of glucose/insulin homeostasis leading to the development of obesity and type 2 diabetes [118]. In contrast, Kaushik et al. reported that starvation increased hypothalamic lipid delivery to the lysosomes via autophagy and promoted the generation of intracellular free fatty acids which stimulate expression of hypothalamic agouti-related protein (AgRP), resulting in increased food intake [119]. Furthermore, suppression of autophagy in AgRP neurons, a neuronal population of the arcuate nucleus in the hypothalamus, reduced food intake, body weight and total fat mass. In follow-up work, Kaushik et al. have studied the role of autophagy in POMC neurons, the other neuronal population of the arcuate nucleus. AgRP and POMC neurons have complementary effects on appetite [120]. Loss of autophagy in POMC neurons promotes adiposity, impairs peripheral lipolysis regulation and glucose tolerance [121]. While these reports are important for the obesity field, additional investigations are necessary to better understand, in which hypothalamic neuron subtype, the regulation of autophagic flux takes place and regulate food intake.

3.3. Liver complications

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Fig. 4. Molecular mechanisms of the impairment of hepatic autophagy in obesity. Short-term inhibition can be produced by the mTOR complex. Long-term regulation could occur via the transcription factors FoxO and TFEB, which control the transcription of autophagic genes and are inhibited by insulin-induced activation of Akt/PKB and mTOR, respectively. In obesity, mTOR is over-activated in the liver, presumably as a result of an increased amino acid concentration following over-nutrition and/or hyper-insulinemia. In obesity, a defect in hepatic autophagy and the associated decrease in the rate of lysosomal degradation contribute to a further increase in the ER stress induced by nutrient overload and insulin resistance. Obesity-induced increase in the calcium-dependent protease calpain leads to down-regulation of Atg7 and then to defective autophagy.

HFD challenge inhibits hepatic autophagic turnover [133]. This ability of autophagy to degrade lipid droplets in hepatocytes has been termed lipophagy. The authors reported for the first time that autophagy regulates lipid metabolism by eliminating triglycerides and by preventing development of steatosis. An inhibition of macroautophagy by genetic knockdown of the autophagy gene atg5, or pharmacological inhibition with 3-methyladenine in cultured hepatocytes challenged with a lipid load from fatty acid supplementation or culture in methionine and cholinedeficient medium, significantly increased cellular triglycerides content. Excessive triglycerides and cholesterol were retained in lipid droplets because of a decreased rate of lipolysis and the resultant reduction in fatty acid ␤-oxidation in cells with an inhibition of macroautophagy. Lipid movement through the autophagic pathway was confirmed by fluorescence microscopy demonstrating co-localization of lipid with autophagosomes and lysosomes, electron microscopic evidence of lipid in autophagic vacuoles and immunogold staining demonstrating an association of the autophagosome-associated protein microtubule-associated LC3 protein with lipid droplets. The LC3 protein directly interacts with lipid droplets before autophagosome formation. Lysosomes merge only with autophagosome-associated lipid droplets. These results have been confirmed [134]. However, the autophagic system does not accommodate to an exogenous lipid load in vitro and in vivo. Oleate challenge in hepatocyte is associated with the absence of the increase in autophagyc flux but with increased triglyceride content and the size and number of lipid droplets. HFD-fed mice (16 weeks) also have an impairment in hepatic autophagic function, as demonstrated by decreased mobilization of lipid into the autophagic compartment [133]. Lipid accumulation alters membrane structure, and a resultant decrease in fusion efficiency between autophagosomes and lysosomes may explain the inhibitory effect of lipid accumulation induced by a HFD on macroautophagy. Starvation induces hepatic autophagy and increases delivery to the liver of free fatty acid from adipose tissue. The liver

of starved mice displayed an increase in the number of lipid droplets, autophagosomes, lysosomes and autophagolysosomes [133]. Hepatocyte-specific Atg7-deficient mice are characterized by hepatomegaly and accumulation of poly-ubiquitinylated proteins, as previously reported by Komatsu et al. [135]. Hepatic triglycerides and the cholesterol content are also increased in these mice, which confirm the crucial role of autophagy in the regulation of lipid storage. 3.3.2. Hepatic insulin resistance In obesity and steatotic liver, autophagy is inhibited in hepatocytes. This inhibition could be mediated by amino acids and/or insulin through Akt/PKB and mTOR signaling pathways (Fig. 4). Obesity is associated with insulin resistance and hyperinsulinemia, which could contribute to the inhibition of hepatic autophagic turnover. In obese mice with hepatic steatosis and insulin resistance, hepatic autophagy is reduced, as reflected by a decrease in LC3-II and an increase in levels of p62, a protein mainly degraded by autophagy [111]. Several mechanisms could be involved. It has been proposed that hyper-insulinemia associated with HFD-induced obesity activates Akt/PKB, which, in turn, phosphorylates and inactivates FoxO1 leading to down-regulation of the expression of Vps34, Atg12 and Gabarapl1 at the mRNA level. Inhibition of Akt/PKB prevents down-regulation of these genes. In contrast, hepatic autophagy is increased, as evaluated by the accumulation of LC3-II and the decrease in p62 protein, in type 1 diabetic mice deficient in insulin [111]. However, destruction of insulin production by ␤ cells by streptozotocin does not increase autophagy in the liver of obese mice [128]. The sustained activation of mTOR due to an elevated amino acid concentration associated with obesity, but also the inhibition of ULK1 [136] could also negatively regulate hepatic autophagy (Fig. 4). Recent studies have suggested that ER stress could be the link between obesity, insulin-resistance and type 2 diabetes [136–138], and inhibition of hepatic ER stress inhibited liver steatosis [139]. The defective hepatic autophagy in obesity could promote ER

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Fig. 5. Potential role of autophagy in obesity. In the arcuate nucleus in the hypothalamus, AgRP and POMC neurons regulate appetite and food intake. In contrast to AgRP neurons, the invalidation of autophagy in POMC neurons increase food intake. Activation of autophagy in adipose tissue is involved in the development of adipose mass and the enhancement in fat accumulation. The inflammation of adipose tissue plays an important role in its insulin resistance and the release of free fatty acids. In obesity, the liver macroautophagy is defective leading to hepatic steatosis (lipophagy), ER stress and insulin resistance. The increase in circulating amino acids levels and hyper-insulinemia could enhance the activation of the mTOR leading to the inhibition of autophagy in hepatocytes. The increase in the hepatic calcium-dependent protease calpain 2 activity with obesity could also lead to impairment of hepatic autophagy via the down-regulation of Atg7. The elevated fatty acids level associated with over-nutrition and the insulin resistance of adipose tissue can directly or via the ROS–JNK pathway enhance the ER stress too. In ␤-cells, activation of autophagy by free fatty acid could preserve the architectural structure and function of pancreatic ␤ cells. Activation of autophagy could also prevent the ␤ cells injury and the development of type 2 diabetes.

stress and causes insulin resistance [136]. Since autophagy is known to eliminate mis/un-folded proteins, impairment in hepatic autophagy could lead to accumulation of mis/un-folded proteins and induction of ER stress. In the liver of obese mice, it has been reported that a decrease in autophagy promotes ER stress leading to insulin resistance [128,140]. Overexpression of Atg7 in the liver of obese mice significantly reduced ER stress, decreased the triglyceride content, improved glucose tolerance and insulin sensitivity [128]. Thus, a vicious cycle takes place, hyperinsulinemia negatively regulates hepatic autophagy in the steatotic liver and the decline in hepatic autophagy enhances ER stress and insulin resistance (Figs. 4 and 5).

3.4. Pancreas and type 2 diabetes Obesity is associated with low grade chronic inflammation and an increase in circulating free fatty acids levels. Free fatty acids are involved in the development of insulin resistance [141,142] and the progressive defect in insulin secretion [142,143]. Upon insulinresistance, pancreatic ␤ cells enhance their insulin secretion (hyper-insulinemia) to compensate for hyperglycemia. However, the progressive diminution of the number of pancreatic ␤ cells, mainly due to apoptosis, and the decrease of their function leads to the development of type 2 diabetes. Autophagy plays a protective role by limiting death of ␤ cells. Upon exposure to a HFD, ␤-cell-specific Atg7-deficient mice displayed a decrease in the ␤ cell number and an impairment of glucose tolerance due to a reduction in insulin secretion. Autophagy is thus crucial in preserving the architectural structure and function of pancreatic ␤ cells in

insulin resistant mice [144–146]. Autophagy is also involved in the turnover of insulin secretory granules within the ␤ cell, thereby regulating insulin secretion [147]. Autophagy may play a pro-survival role but chronic activation of autophagy can cause autophagic cell death (or type 2 cell death), which is distinct from apoptosis. A recent report showed that autophagy might contribute to autophagic cell death in obese mice [148]. Pdx1 is a transcription factor essential for normal pancreatic ␤ cell function and survival. Hemizygous loss of this gene leads to ␤ cell death and diabetes. In MIN6 cells, a decrease in Pdx1 expression induces an increase in autophagy, which precedes cell death. Inhibition of autophagy delays apoptosis in this in vitro model. Double hemizygous Pdx+/−/Beclin-1+/− mice displayed inhibition of autophagy, which correlated with reduced activation of caspase-3 and increased proliferation of ␤ cells compared to Pdx+/− mice on a one-week HFD. This leads to increased insulin secretion and preserved glucose tolerance and ␤ cell mass. However, this protective effect of reduced autophagy wears off after 7 weeks on a HFD [148]. In view of these apparent opposing results, further studies are clearly required for a better understanding of the mechanism by which autophagy regulates ␤ cell survival and function.

3.5. Aging The prevalence of type 2 diabetes and liver complications increases with age and it is well established that autophagy and CMA are impaired in aging [149,150]. This decline is associated with an increase in the lipid content in different organs such as the liver. As seen above, excessive accumulation of lipids further

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alters autophagic turnover and its protective role against accumulation of lipid droplets and insulin resistance. A link between aging, loss of mitochondrial function and ROS production has also been reported [149,151]. Calorie restriction enhances longevity and this may be due, at least in part, to activation of autophagy, in particular lipophagy and mitophagy leading to a decrease in oxidative stress and in the lipid content [66,152,153]. Aging, dysfunctional mitochondria and oxidative stress contribute to the development of type 2 diabetes and liver complications [154,155]. The increase in autophagic turnover could enhance mitochondrial turnover and biogenesis, and, consequently, decrease ROS production. This may contribute to life span extension and prevent or delay the development of complications associated with obesity [156].

Financial support This work was supported by grants from INSERM (France), the University of Nice, the Programme Hospitalier de Recherche Clinique (Centre Hospitalier Universitaire of Nice), and charities (SFD/BMS and AFEF/Schering-Plough to PG). VJL was supported by the Programme Hospitalier de Recherche Clinique (Centre Hospitalier Universitaire of Nice) and the Association pour la Recherche sur le Cancer (France). PG is a recipient of an Interface Grant from the Centre Hospitalier Universitaire of Nice.

Acknowledgment We thank Dr. M.C. Brahimi-Horn for editorial correction.

4. Conclusions Autophagy is a crucial physiological process in providing nutrients to maintain vital cellular functions during fasting, but also to purge the cell of superfluous or damaged organelles, lipids and mis-folded proteins in obesity. Obesity is a complex multi-factorial chronic disease affecting multi-organs and physiological responses. It is clear that modifications in autophagic turnover during obesity mediate protective or deleterious responses depending on the cell/organ. As argued elsewhere [136], it is possible that initially insulin resistance functions as an adaptive mechanism to increase autophagy in order to protect cells against death because, after all, insulin inhibits autophagy, but that sustained stress results in down-regulation of autophagy [157]. This review summarizes the knowledge of the role of autophagy in specific cells, including neurons, adipocytes, hepatocytes and ␤ cells in the context of obesity (Fig. 5). A better understanding of the molecular mechanism of autophagy and its implication in food intake, glucose/lipid homeostasis, diabetes, liver complications but also inflammation, highlight new potential therapeutic targets. Interestingly, some of the drugs that can modulate autophagy are already approved for other human clinical uses. For example, rapamycin, carbamazepine and cisplatin promote autophagy, while the anti-malaria compound chloroquine (which increases the intralysosomal pH) inhibits autophagic turnover. However, the lack of specificity, and the absence of organ or cell selectivity, are the major limitations of these compounds. Lastly, metformin is an activator of AMPK, inhibits mTOR and promotes autophagy, which may contribute to its anti-diabetic effect [158]. Although perhaps trivial, but the safest way to combat insulin resistance and type-2 diabetes is calorie restriction. This acts as a two-sided sword: on the one hand it diminishes the production of ROS by the mitochondrial respiratory chain and on the other hand it stimulates autophagy to promote cell survival. In this context, a fascinating new development is that another strategy used to combat obesity and insulin resistance, i.e. increased energy utilization by exercise, also appears to be a potent inducer of autophagy in skeletal muscle and in the heart which contributes to the improvement of glucose tolerance after high-fat feeding [159]. Finally, production of irisin, a newly identified hormone by muscle after exercise, causes an increase in total body energy expenditure and resistance to obesity-linked insulin resistance by promoting brown fat formation in the absence of changes in food intake [160]. Thus, like calorie restriction, exercise appears to act as another two-sided sword in combating insulin resistance.

Disclosures Authors declare no conflict of interest

References [1] Yorimitsu T, Klionsky DJ. Autophagy: molecular machinery for self-eating. Cell Death and Differentiation 2005;12(Suppl. 2):1542–52. [2] Codogno P, Meijer AJ. Autophagy and signaling: their role in cell survival and cell death. Cell Death and Differentiation 2005;12(Suppl. 2):1509–18. [3] Ciechanover A, Orian A, Schwartz AL. Ubiquitin-mediated proteolysis: biological regulation via destruction. Bioessays 2000;22:442–51. [4] Nedelsky NB, Todd PK, Taylor JP. Autophagy and the ubiquitin–proteasome system: collaborators in neuroprotection. Biochimica et Biophysica Acta 2008;1782:691–9. [5] Axe EL, Walker SA, Manifava M, Chandra P, Roderick HL, Habermann A, et al. Autophagosome formation from membrane compartments enriched in phosphatidylinositol 3-phosphate and dynamically connected to the endoplasmic reticulum. Journal of Cell Biology 2008;182:685–701. [6] Hayashi-Nishino M, Fujita N, Noda T, Yamaguchi A, Yoshimori T, Yamamoto A. A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation. Nature Cell Biology 2009;11:1433–7. [7] Yla-Anttila P, Vihinen H, Jokitalo E, Eskelinen EL. 3D tomography reveals connections between the phagophore and endoplasmic reticulum. Autophagy 2009;5:1180–5. [8] Mari M, Tooze SA, Reggiori F. The puzzling origin of the autophagosomal membrane. F1000 Biol Rep 2011;3:25. [9] Arias E, Cuervo AM. Chaperone-mediated autophagy in protein quality control. Current Opinion in Cell Biology 2011;23:184–9. [10] Nakatogawa H, Suzuki K, Kamada Y, Ohsumi Y. Dynamics and diversity in autophagy mechanisms: lessons from yeast. Nature Reviews Molecular Cell Biology 2009;10:458–67. [11] Chan EY, Longatti A, McKnight NC, Tooze SA. Kinase-inactivated ULK proteins inhibit autophagy via their conserved C-terminal domains using an Atg13independent mechanism. Molecular and Cellular Biology 2009;29:157–71. [12] Chan EY. mTORC1 phosphorylates the ULK1–mAtg13–FIP200 autophagy regulatory complex. Science Signaling 2009;2:pe51. [13] Mizushima N, Komatsu M. Autophagy: renovation of cells and tissues. Cell 2011;147:728–41. [14] Ohsumi Y. Molecular dissection of autophagy: two ubiquitin-like systems. Nature Reviews Molecular Cell Biology 2001;2:211–6. [15] Kabeya Y, Mizushima N, Yamamoto A, Oshitani-Okamoto S, Ohsumi Y, Yoshimori T. LC3, GABARAP and GATE16 localize to autophagosomal membrane depending on form-ii formation. Journal of Cell Science 2004;117:2805–12. [16] Klionsky DJ, Abdalla FC, Abeliovich H, Abraham RT, Acevedo-Arozena A, Adeli K, et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 2012;8:1–100. [17] Wullschleger S, Loewith R, Hall MN. Tor signaling in growth and metabolism. Cell 2006;124:471–84. [18] Blommaart EF, Luiken JJ, Blommaart PJ, van Woerkom GM, Meijer AJ. Phosphorylation of ribosomal protein S6 is inhibitory for autophagy in isolated rat hepatocytes. Journal of Biological Chemistry 1995;270:2320–6. [19] Noda T, Ohsumi Y. Tor, a phosphatidylinositol kinase homologue, controls autophagy in yeast. Journal of Biological Chemistry 1998;273:3963–6. [20] Inoki K, Li Y, Xu T, Guan KL. Rheb GTPase is a direct target of TSC2 GAP activity and regulates mTOR signaling. Genes and Development 2003;17:1829–34. [21] Zhang Y, Gao X, Saucedo LJ, Ru B, Edgar BA, Pan D. Rheb is a direct target of the tuberous sclerosis tumour suppressor proteins. Nature Cell Biology 2003;5:578–81. [22] Long X, Lin Y, Ortiz-Vega S, Yonezawa K, Avruch J. Rheb binds and regulates the mTOR kinase. Current Biology 2005;15:702–13. [23] Zoncu R, Efeyan A, Sabatini DM. mTOR: from growth signal integration to cancer, diabetes and ageing. Nature Reviews Molecular Cell Biology 2011;12:21–35. [24] Arico S, Petiot A, Bauvy C, Dubbelhuis PF, Meijer AJ, Codogno P, et al. The tumor suppressor pten positively regulates macroautophagy by inhibiting the phosphatidylinositol 3-kinase/protein kinase B pathway. Journal of Biological Chemistry 2001;276:35243–6.

V.J. Lavallard et al. / Pharmacological Research 66 (2012) 513–525 [25] Jung CH, Jun CB, Ro SH, Kim YM, Otto NM, Cao J, et al. Ulk–Atg13–FIP200 complexes mediate mTOR signaling to the autophagy machinery. Molecular Biology of the Cell 2009;20:1992–2003. [26] Ganley IG, Lam du H, Wang J, Ding X, Chen S, Jiang X. ULK1.Atg13.FIP200 complex mediates mTOR signaling and is essential for autophagy. Journal of Biological Chemistry 2009;284:12297–305. [27] Hosokawa N, Hara T, Kaizuka T, Kishi C, Takamura A, Miura Y, et al. Nutrient-dependent mTORC1 association with the ULK1–Atg13–FIP200 complex required for autophagy. Molecular Biology of the Cell 2009;20:1981–91. [28] Baum JI, Kimball SR, Jefferson LS. Glucagon acts in a dominant manner to repress insulin-induced mammalian target of rapamycin complex 1 signaling in perfused rat liver. American Journal of Physiology Endocrinology and Metabolism 2009;297:E410–5. [29] Ogier-Denis E, Pattingre S, El Benna J, Codogno P. ERK1/2-dependent phosphorylation of galpha-interacting protein stimulates its gtpase accelerating activity and autophagy in human colon cancer cells. Journal of Biological Chemistry 2000;275:39090–5. [30] Pattingre S, Bauvy C, Codogno P. Amino acids interfere with the ERK1/2dependent control of macroautophagy by controlling the activation of Raf-1 in human colon cancer HT-29 cells. Journal of Biological Chemistry 2003;278:16667–74. [31] Ellington AA, Berhow MA, Singletary KW. Inhibition of Akt signaling and enhanced ERK1/2 activity are involved in induction of macroautophagy by triterpenoid B-group soyasaponins in colon cancer cells. Carcinogenesis 2006;27:298–306. [32] Oh SH, Lim SC. Endoplasmic reticulum stress-mediated autophagy/apoptosis induced by capsaicin (8-methyl-n-vanillyl-6-nonenamide) and dihydrocapsaicin is regulated by the extent of c-Jun NH2-terminal kinase/extracellular signal-regulated kinase activation in WI38 lung epithelial fibroblast cells. Journal of Pharmacology and Experimental Therapeutics 2009;329:112–22. [33] Wang SH, Shih YL, Kuo TC, Ko WC, Shih CM. Cadmium toxicity toward autophagy through ROS-activated GSK-3beta in mesangial cells. Toxicological Sciences 2009;108:124–31. [34] Wang SH, Shih YL, Ko WC, Wei YH, Shih CM. Cadmium-induced autophagy and apoptosis are mediated by a calcium signaling pathway. Cellular and Molecular Life Sciences 2008;65:3640–52. [35] Cheng Y, Qiu F, Tashiro S, Onodera S, Ikejima T. ERK and JNK mediate TNFalpha-induced p53 activation in apoptotic and autophagic L929 cell death. Biochemical and Biophysical Research Communications 2008;376:483–8. [36] Sivaprasad U, Basu A. Inhibition of ERK attenuates autophagy and potentiates tumour necrosis factor-alpha-induced cell death in MCF-7 cells. Journal of Cellular and Molecular Medicine 2008;12:1265–71. [37] Han W, Sun J, Feng L, Wang K, Li D, Pan Q, et al. Autophagy inhibition enhances daunorubicin-induced apoptosis in K562 cells. PLoS ONE 2011;6:e28491. [38] An HJ, Maeng O, Kang KH, Lee JO, Kim YS, Paik SG, et al. Activation of Ras up-regulates pro-apoptotic BNIP3 in nitric oxide-induced cell death. Journal of Biological Chemistry 2006;281:33939–48. [39] Ogier-Denis E, Couvineau A, Maoret JJ, Houri JJ, Bauvy C, De Stefanis D, et al. A heterotrimeric Gi3-protein controls autophagic sequestration in the human colon cancer cell line HT-29. Journal of Biological Chemistry 1995;270:13–6. [40] Ogier-Denis E, Petiot A, Bauvy C, Codogno P. Control of the expression and activity of the Galpha-interacting protein (GAIP) in human intestinal cells. Journal of Biological Chemistry 1997;272:24599–603. [41] Gohla A, Klement K, Piekorz RP, Pexa K, vom Dahl S, Spicher K, et al. An obligatory requirement for the heterotrimeric G protein Gi3 in the antiautophagic action of insulin in the liver. Proceedings of the National Academy of Sciences of the United States of America 2007;104:3003–8. [42] Gusdon AM, Chu CT. To eat or not to eat: neuronal metabolism, mitophagy, and parkinson’s disease. Antioxidants and Redox Signalling 2011;14:1979–87. [43] Winter JN, Jefferson LS, Kimball SR. ERK and Akt signaling pathways function through parallel mechanisms to promote mTORC1 signaling. American Journal of Physiology Cell Physiology 2011;300:C1172–80. [44] Haussinger D, Reinehr R, Schliess F. The hepatocyte integrin system and cell volume sensing. Acta Physiologica (Oxford, England) 2006;187:249–55. [45] Webber JL, Tooze SA. Coordinated regulation of autophagy by p38alpha MAPK through mAtg9 and p38iP. EMBO Journal 2010;29:27–40. [46] Mei S, Gu H, Ward A, Yang X, Guo H, He K, et al. P38 MAPK promotes cholesterol ester accumulation in macrophages through inhibition of macroautophagy. Journal of Biological Chemistry 2012;287:11761–8. [47] Tang G, Yue Z, Talloczy Z, Hagemann T, Cho W, Messing A, et al. Autophagy induced by Alexander disease-mutant GFAP accumulation is regulated by p38/MAPK and mTOR signaling pathways. Human Molecular Genetics 2008;17:1540–55. [48] Petiot A, Ogier-Denis E, Blommaart EF, Meijer AJ, Codogno P. Distinct classes of phosphatidylinositol 3 -kinases are involved in signaling pathways that control macroautophagy in HT-29 cells. Journal of Biological Chemistry 2000;275:992–8. [49] Liang XH, Jackson S, Seaman M, Brown K, Kempkes B, Hibshoosh H, et al. Induction of autophagy and inhibition of tumorigenesis by beclin 1. Nature 1999;402:672–6. [50] Furuya N, Yu J, Byfield M, Pattingre S, Levine B. The evolutionarily conserved domain of beclin 1 is required for Vps34 binding, autophagy and tumor suppressor function. Autophagy 2005;1:46–52. [51] He C, Levine B. The beclin 1 interactome. Current Opinion in Cell Biology 2010;22:140–9.

523

[52] Li B, Hu Q, Wang H, Man N, Ren H, Wen L, et al. Omi/HtrA2 is a positive regulator of autophagy that facilitates the degradation of mutant proteins involved in neurodegenerative diseases. Cell Death and Differentiation 2010;17:1773–84. [53] Maiuri MC, Criollo A, Tasdemir E, Vicencio JM, Tajeddine N, Hickman JA, et al. BH3-only proteins and BH3 mimetics induce autophagy by competitively disrupting the interaction between beclin 1 and Bcl-2/Bcl-x(l). Autophagy 2007;3:374–6. [54] Wei Y, Pattingre S, Sinha S, Bassik M, Levine B. JNK1-mediated phosphorylation of Bcl-2 regulates starvation-induced autophagy. Molecular Cell 2008;30:678–88. [55] Pattingre S, Bauvy C, Carpentier S, Levade T, Levine B, Codogno P. Role of Jnk1-dependent Bcl-2 phosphorylation in ceramide-induced macroautophagy. Journal of Biological Chemistry 2009;284:2719–28. [56] Zalckvar E, Berissi H, Mizrachy L, Idelchuk Y, Koren I, Eisenstein M, et al. Dapkinase-mediated phosphorylation on the BH3 domain of beclin 1 promotes dissociation of beclin 1 from Bcl-xl and induction of autophagy. EMBO Reports 2009;10:285–92. [57] Fimia GM, Stoykova A, Romagnoli A, Giunta L, Di Bartolomeo S, Nardacci R, et al. Ambra1 regulates autophagy and development of the nervous system. Nature 2007;447:1121–5. [58] Ropolo A, Grasso D, Pardo R, Sacchetti ML, Archange C, Lo Re A, et al. The pancreatitis-induced vacuole membrane protein 1 triggers autophagy in mammalian cells. Journal of Biological Chemistry 2007;282:37124–33. [59] Takahashi Y, Coppola D, Matsushita N, Cualing HD, Sun M, Sato Y, et al. Bif-1 interacts with beclin 1 through UVRAG and regulates autophagy and tumorigenesis. Nature Cell Biology 2007;9:1142–51. [60] Liang C, Feng P, Ku B, Dotan I, Canaani D, Oh BH, et al. Autophagic and tumour suppressor activity of a novel beclin1-binding protein UVRAG. Nature Cell Biology 2006;8:688–99. [61] Liang C, Lee JS, Inn KS, Gack MU, Li Q, Roberts EA, et al. Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking. Nature Cell Biology 2008;10:776–87. [62] Itakura E, Kishi C, Inoue K, Mizushima N. Beclin 1 forms two distinct phosphatidylinositol 3-kinase complexes with mammalian Atg14 and UVRAG. Molecular Biology of the Cell 2008;19:5360–72. [63] van Sluijters DA, Dubbelhuis PF, Blommaart EF, Meijer AJ. Amino-aciddependent signal transduction. Biochemical Journal 2000;351(Pt 3):545–50. [64] Blommaart EF, Luiken JJ, Meijer AJ. Autophagic proteolysis: control and specificity. Histochemical Journal 1997;29:365–85. [65] Lynch CJ, Halle B, Fujii H, Vary TC, Wallin R, Damuni Z, et al. Potential role of leucine metabolism in the leucine-signaling pathway involving mTOR. American Journal of Physiology Endocrinology and Metabolism 2003;285: E854–63. [66] Meijer AJ, Codogno P. Autophagy: regulation and role in disease. Critical Reviews in Clinical Laboratory Sciences 2009;46:210–40. [67] Kim J, Guan KL. Amino acid signaling in tor activation. Annual Review of Biochemistry 2011;80:1001–32. [68] Hundal HS, Taylor PM. Amino acid transceptors: gate keepers of nutrient exchange and regulators of nutrient signaling. American Journal of Physiology Endocrinology and Metabolism 2009;296:E603–13. [69] Nicklin P, Bergman P, Zhang B, Triantafellow E, Wang H, Nyfeler B, et al. Bidirectional transport of amino acids regulates mTOR and autophagy. Cell 2009;136:521–34. [70] Avruch J, Long X, Ortiz-Vega S, Rapley J, Papageorgiou A, Dai N. Amino acid regulation of tor complex 1. American Journal of Physiology Endocrinology and Metabolism 2009;296:E592–602. [71] Wiczer BM, Thomas G. Phospholipase d and mTORC1: nutrients are what bring them together. Science Signaling 2012;5:pe13. [72] Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, Gulati P, et al. Amino acids mediate mtor/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proceedings of the National Academy of Sciences of the United States of America 2005;102:14238–43. [73] Byfield MP, Murray JT, Backer JM. hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. Journal of Biological Chemistry 2005;280:33076–82. [74] Kim E, Goraksha-Hicks P, Li L, Neufeld TP, Guan KL. Regulation of TORC1 by Rag GTPases in nutrient response. Nature Cell Biology 2008;10:935–45. [75] Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled L, et al. The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science 2008;320:1496–501. [76] Long X, Ortiz-Vega S, Lin Y, Avruch J. Rheb binding to mammalian target of rapamycin (mTOR) is regulated by amino acid sufficiency. Journal of Biological Chemistry 2005;280:23433–6. [77] Zoncu R, Bar-Peled L, Efeyan A, Wang S, Sancak Y, Sabatini DM. mTORC1 senses lysosomal amino acids through an inside-out mechanism that requires the vacuolar H(+)-ATPase. Science 2011;334:678–83. [78] Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM. Ragulator–Rag complex targets mtorc1 to the lysosomal surface and is necessary for its activation by amino acids. Cell 2010;141:290–303. [79] Korolchuk VI, Saiki S, Lichtenberg M, Siddiqi FH, Roberts EA, Imarisio S, et al. Lysosomal positioning coordinates cellular nutrient responses. Nature Cell Biology 2011;13:453–60. [80] Han JM, Jeong SJ, Park MC, Kim G, Kwon NH, Kim HK, et al. Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway. Cell 2012;149:410–24.

524

V.J. Lavallard et al. / Pharmacological Research 66 (2012) 513–525

[81] Bonfils G, Jaquenoud M, Bontron S, Ostrowicz C, Ungermann C, De Virgilio C. Leucyl-tRNA synthetase controls TORC1 via the ego complex. Molecular Cell 2012;46:105–10. [82] Meley D, Bauvy C, Houben-Weerts JH, Dubbelhuis PF, Helmond MT, Codogno P, et al. AMP-activated protein kinase and the regulation of autophagic proteolysis. Journal of Biological Chemistry 2006;281:34870–9. [83] Gwinn DM, Shackelford DB, Egan DF, Mihaylova MM, Mery A, Vasquez DS, et al. AMPK phosphorylation of raptor mediates a metabolic checkpoint. Molecular Cell 2008;30:214–26. [84] Liang J, Shao SH, Xu ZX, Hennessy B, Ding Z, Larrea M, et al. The energy sensing LKB1–AMPK pathway regulates p27(kip1) phosphorylation mediating the decision to enter autophagy or apoptosis. Nature Cell Biology 2007;9:218–24. [85] Egan D, Kim J, Shaw RJ, Guan KL. The autophagy initiating kinase ULK1 is regulated via opposing phosphorylation by ampk and mtor. Autophagy 2011;7:643–4. [86] Kim J, Kundu M, Viollet B, Guan KL. Ampk and mTOR regulate autophagy through direct phosphorylation of ULK1. Nature Cell Biology 2011;13:132–41. [87] Lee JW, Park S, Takahashi Y, Wang HG. The association of AMPK with ULK1 regulates autophagy. PLoS ONE 2010;5:e15394. [88] Murphy MP. How mitochondria produce reactive oxygen species. Biochemical Journal 2009;417:1–13. [89] Scherz-Shouval R, Shvets E, Fass E, Shorer H, Gil L, Elazar Z. Reactive oxygen species are essential for autophagy and specifically regulate the activity of Atg4. EMBO Journal 2007;26:1749–60. [90] Azad MB, Chen Y, Gibson SB. Regulation of autophagy by reactive oxygen species (ROS): implications for cancer progression and treatment. Antioxidants and Redox Signalling 2009;11:777–90. [91] Chen Y, McMillan-Ward E, Kong J, Israels SJ, Gibson SB. Oxidative stress induces autophagic cell death independent of apoptosis in transformed and cancer cells. Cell Death and Differentiation 2008;15:171–82. [92] Alexander A, Cai SL, Kim J, Nanez A, Sahin M, MacLean KH, et al. ATM signals to TSC2 in the cytoplasm to regulate mTORC1 in response to ROS. Proceedings of the National Academy of Sciences of the United States of America 2010;107:4153–8. [93] Lemasters JJ. Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging. Rejuvenation Research 2005;8:3–5. [94] Xiong Y, Contento AL, Nguyen PQ, Bassham DC. Degradation of oxidized proteins by autophagy during oxidative stress in arabidopsis. Plant Physiology 2007;143:291–9. [95] Yang J, Wu LJ, Tashino S, Onodera S, Ikejima T. Reactive oxygen species and nitric oxide regulate mitochondria-dependent apoptosis and autophagy in evodiamine-treated human cervix carcinoma HeLa cells. Free Radical Research 2008;42:492–504. [96] Ogata M, Hino S, Saito A, Morikawa K, Kondo S, Kanemoto S, et al. Autophagy is activated for cell survival after endoplasmic reticulum stress. Molecular and Cellular Biology 2006;26:9220–31. [97] He C, Klionsky DJ. Regulation mechanisms and signaling pathways of autophagy. Annual Review of Genetics 2009;43:67–93. [98] Kouroku Y, Fujita E, Tanida I, Ueno T, Isoai A, Kumagai H, et al. ER stress (PERK/eiF2alpha phosphorylation) mediates the polyglutamine-induced LC3 conversion, an essential step for autophagy formation. Cell Death and Differentiation 2007;14:230–9. [99] Rouschop KM, van den Beucken T, Dubois L, Niessen H, Bussink J, Savelkouls K, et al. The unfolded protein response protects human tumor cells during hypoxia through regulation of the autophagy genes MAP1LC3B and Atg5. Journal of Clinical Investigation 2010;120:127–41. [100] Qin L, Wang Z, Tao L, Wang Y. ER stress negatively regulates Akt/TSC/mTOR pathway to enhance autophagy. Autophagy 2010;6:239–47. [101] Kim DS, Kim JH, Lee GH, Kim HT, Lim JM, Chae SW, et al. P38 mitogen-activated protein kinase is involved in endoplasmic reticulum stress-induced cell death and autophagy in human gingival fibroblasts. Biological and Pharmaceutical Bulletin 2010;33:545–9. [102] Hoyer-Hansen M, Jaattela M. Connecting endoplasmic reticulum stress to autophagy by unfolded protein response and calcium. Cell Death and Differentiation 2007;14:1576–82. [103] Sakaki K, Wu J, Kaufman RJ. Protein kinase ctheta is required for autophagy in response to stress in the endoplasmic reticulum. Journal of Biological Chemistry 2008;283:15370–80. [104] Lin YY, Kiihl S, Suhail Y, Liu SY, Chou YH, Kuang Z, et al. Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK. Nature 2012;482:251–5. [105] Lee IH, Cao L, Mostoslavsky R, Lombard DB, Liu J, Bruns NE, et al. A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proceedings of the National Academy of Sciences of the United States of America 2008;105:3374–9. [106] Canto C, Auwerx J. Targeting sirtuin 1 to improve metabolism: all you need is nad(+)? Pharmacological Reviews 2012;64:166–87. [107] Biggs 3rd WH, Meisenhelder J, Hunter T, Cavenee WK, Arden KC. Protein kinase B/Akt-mediated phosphorylation promotes nuclear exclusion of the winged helix transcription factor FKHR1. Proceedings of the National Academy of Sciences of the United States of America 1999;96: 7421–6. [108] Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, et al. Akt promotes cell survival by phosphorylating and inhibiting a forkhead transcription factor. Cell 1999;96:857–68.

[109] Salih DA, Brunet A. Foxo transcription factors in the maintenance of cellular homeostasis during aging. Current Opinion in Cell Biology 2008;20: 126–36. [110] Mammucari C, Milan G, Romanello V, Masiero E, Rudolf R, Del Piccolo P, et al. Foxo3 controls autophagy in skeletal muscle in vivo. Cell Metabolism 2007;6:458–71. [111] Liu HY, Han J, Cao SY, Hong T, Zhuo D, Shi J, et al. Hepatic autophagy is suppressed in the presence of insulin resistance and hyperinsulinemia: inhibition of foxo1-dependent expression of key autophagy genes by insulin. Journal of Biological Chemistry 2009;284:31484–92. [112] Zhao Y, Yang J, Liao W, Liu X, Zhang H, Wang S, et al. Cytosolic FoxO1 is essential for the induction of autophagy and tumour suppressor activity. Nature Cell Biology 2010;12:665–75. [113] Greer EL, Oskoui PR, Banko MR, Maniar JM, Gygi MP, Gygi SP, et al. The energy sensor amp-activated protein kinase directly regulates the mammalian FoxO3 transcription factor. Journal of Biological Chemistry 2007;282:30107–19. [114] Xu P, Das M, Reilly J, Davis RJ. JNK regulates FoxO-dependent autophagy in neurons. Genes and Development 2011;25:310–22. [115] Settembre C, Di Malta C, Polito VA, Garcia Arencibia M, Vetrini F, Erdin S, et al. TFEB links autophagy to lysosomal biogenesis. Science 2011;332:1429–33. [116] Settembre C, Zoncu R, Medina DL, Vetrini F, Erdin S, Huynh T, et al. A lysosometo-nucleus signalling mechanism senses and regulates the lysosome via mtor and TFEB. EMBO Journal 2012;31:1095–108. [117] Hardy OT, Czech MP, Corvera S. What causes the insulin resistance underlying obesity? Current Opinion in Endocrinology, Diabetes and Obesity 2012;19:81–7. [118] Meng Q, Cai D. Defective hypothalamic autophagy directs the central pathogenesis of obesity via the ikappab kinase beta (IKKbeta)/NF-kappaB pathway. Journal of Biological Chemistry 2011;286:32324–32. [119] Kaushik S, Rodriguez-Navarro JA, Arias E, Kiffin R, Sahu S, Schwartz GJ, et al. Autophagy in hypothalamic AgRP neurons regulates food intake and energy balance. Cell Metabolism 2011;14:173–83. [120] Rubinsztein DC. Autophagy – alias self-eating – appetite and ageing. EMBO Reports 2012;13:173–4. [121] Kaushik S, Arias E, Kwon H, Lopez NM, Athonvarangkul D, Sahu S, et al. Loss of autophagy in hypothalamic POMC neurons impairs lipolysis. EMBO Reports 2012;13:258–65. [122] Singh R, Xiang Y, Wang Y, Baikati K, Cuervo AM, Luu YK, et al. Autophagy regulates adipose mass and differentiation in mice. Journal of Clinical Investigation 2009;119:3329–39. [123] Zhang Y, Goldman S, Baerga R, Zhao Y, Komatsu M, Jin S. Adipose-specific deletion of autophagy-related gene 7 (Atg7) in mice reveals a role in adipogenesis. Proceedings of the National Academy of Sciences of the United States of America 2009;106:19860–5. [124] Ost A, Svensson K, Ruishalme I, Brannmark C, Franck N, Krook H, et al. Attenuated mtor signaling and enhanced autophagy in adipocytes from obese patients with type 2 diabetes. Molecular Medicine 2010;16:235–46. [125] Kovsan J, Bluher M, Tarnovscki T, Kloting N, Kirshtein B, Madar L, et al. Altered autophagy in human adipose tissues in obesity. Journal of Clinical Endocrinology and Metabolism 2011;96:E268–77. [126] Rautou PE, Mansouri A, Lebrec D, Durand F, Valla D, Moreau R. Autophagy in liver diseases. Journal of Hepatology 2010;53:1123–34. [127] Czaja MJ. Functions of autophagy in hepatic and pancreatic physiology and disease. Gastroenterology 2011;140:1895–908. [128] Yang L, Li P, Fu S, Calay ES, Hotamisligil GS. Defective hepatic autophagy in obesity promotes er stress and causes insulin resistance. Cell Metabolism 2010;11:467–78. [129] Tremblay F, Marette A. Amino acid and insulin signaling via the mTOR/p70 S6 kinase pathway. A negative feedback mechanism leading to insulin resistance in skeletal muscle cells. Journal of Biological Chemistry 2001;276: 38052–60. [130] Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, et al. Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 2004;431:200–5. [131] Tremblay F, Krebs M, Dombrowski L, Brehm A, Bernroider E, Roth E, et al. Overactivation of S6 kinase 1 as a cause of human insulin resistance during increased amino acid availability. Diabetes 2005;54:2674–84. [132] Hotamisligil GS. Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 2010;140:900–17. [133] Singh R, Kaushik S, Wang Y, Xiang Y, Novak I, Komatsu M, et al. Autophagy regulates lipid metabolism. Nature 2009;458:1131–5. [134] Shibata M, Yoshimura K, Furuya N, Koike M, Ueno T, Komatsu M, et al. The MAP1-LC3 conjugation system is involved in lipid droplet formation. Biochemical and Biophysical Research Communications 2009;382:419–23. [135] Komatsu M, Waguri S, Ueno T, Iwata J, Murata S, Tanida I, et al. Impairment of starvation-induced and constitutive autophagy in Atg7-deficient mice. Journal of Cell Biology 2005;169:425–34. [136] Codogno P, Meijer AJ. Autophagy: a potential link between obesity and insulin resistance. Cell Metabolism 2010;11:449–51. [137] Harding HP, Zeng H, Zhang Y, Jungries R, Chung P, Plesken H, et al. Diabetes mellitus and exocrine pancreatic dysfunction in perk−/− mice reveals a role for translational control in secretory cell survival. Molecular Cell 2001;7:1153–63. [138] Kaneto H, Matsuoka TA, Nakatani Y, Kawamori D, Miyatsuka T, Matsuhisa M, et al. Oxidative stress, ER stress, and the JNK pathway in type 2 diabetes. Journal of Molecular Medicine (Berlin, Germany) 2005;83:429–39.

V.J. Lavallard et al. / Pharmacological Research 66 (2012) 513–525 [139] Kammoun HL, Chabanon H, Hainault I, Luquet S, Magnan C, Koike T, et al. GRP78 expression inhibits insulin and ER stress-induced SREBP-1c activation and reduces hepatic steatosis in mice. Journal of Clinical Investigation 2009;119:1201–15. [140] Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, et al. Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes. Science 2004;306:457–61. [141] Hotamisligil GS. The role of TNFalpha and TNF receptors in obesity and insulin resistance. Journal of Internal Medicine 1999;245:621–5. [142] Osborn O, Sears DD, Olefsky JM. Fat-induced inflammation unchecked. Cell Metabolism 2010;12:553–4. [143] Leung N, Sakaue T, Carpentier A, Uffelman K, Giacca A, Lewis GF. Prolonged increase of plasma non-esterified fatty acids fully abolishes the stimulatory effect of 24 hours of moderate hyperglycaemia on insulin sensitivity and pancreatic beta-cell function in obese men. Diabetologia 2004;47: 204–13. [144] Ebato C, Uchida T, Arakawa M, Komatsu M, Ueno T, Komiya K, et al. Autophagy is important in islet homeostasis and compensatory increase of beta cell mass in response to high-fat diet. Cell Metabolism 2008;8: 325–32. [145] Jung HS, Chung KW, Won Kim J, Kim J, Komatsu M, Tanaka K, et al. Loss of autophagy diminishes pancreatic beta cell mass and function with resultant hyperglycemia. Cell Metabolism 2008;8:318–24. [146] Quan W, Hur KY, Lim Y, Oh SH, Lee JC, Kim KH, et al. Autophagy deficiency in beta cells leads to compromised unfolded protein response and progression from obesity to diabetes in mice. Diabetologia 2012;55: 392–403. [147] Marsh BJ, Soden C, Alarcon C, Wicksteed BL, Yaekura K, Costin AJ, et al. Regulated autophagy controls hormone content in secretory-deficient pancreatic endocrine beta-cells. Molecular Endocrinology 2007;21:2255–69. [148] Fujimoto K, Hanson PT, Tran H, Ford EL, Han Z, Johnson JD, et al. Autophagy regulates pancreatic beta cell death in response to Pdx1 deficiency and nutrient deprivation. Journal of Biological Chemistry 2009;284:27664–73.

525

[149] Cuervo AM, Bergamini E, Brunk UT, Droge W, Ffrench M, Terman A. Autophagy and aging: the importance of maintaining “clean” cells. Autophagy 2005;1:131–40. [150] Yen WL, Klionsky DJ. How to live long and prosper: autophagy, mitochondria, and aging. Physiology (Bethesda) 2008;23:248–62. [151] Shigenaga MK, Hagen TM, Ames BN. Oxidative damage and mitochondrial decay in aging. Proceedings of the National Academy of Sciences of the United States of America 1994;91:10771–8. [152] Bergamini E, Cavallini G, Donati A, Gori Z. The role of autophagy in aging: its essential part in the anti-aging mechanism of caloric restriction. Annals of the New York Academy of Sciences 2007;1114:69–78. [153] Miwa S, Lawless C, von Zglinicki T. Mitochondrial turnover in liver is fast in vivo and is accelerated by dietary restriction: application of a simple dynamic model. Aging Cell 2008;7:920–3. [154] Evans JL, Goldfine ID, Maddux BA, Grodsky GM. Oxidative stress and stressactivated signaling pathways: a unifying hypothesis of type 2 diabetes. Endocrine Reviews 2002;23:599–622. [155] Lowell BB, Shulman GI. Mitochondrial dysfunction and type 2 diabetes. Science 2005;307:384–7. [156] Guarente L. Mitochondria – a nexus for aging, calorie restriction, and sirtuins? Cell 2008;132:171–6. [157] Yin JJ, Li YB, Wang Y, Liu GD, Wang J, Zhu XO, et al. The role of autophagy in endoplasmic reticulum stress-induced pancreatic beta cell death. Autophagy 2012;8:158–64. [158] Gonzalez CD, Lee MS, Marchetti P, Pietropaolo M, Towns R, Vaccaro MI, et al. The emerging role of autophagy in the pathophysiology of diabetes mellitus. Autophagy 2011;7:2–11. [159] He C, Bassik MC, Moresi V, Sun K, Wei Y, Zou Z, et al. Exercise-induced Bcl2-regulated autophagy is required for muscle glucose homeostasis. Nature 2012;481:511–5. [160] Bostrom P, Wu J, Jedrychowski MP, Korde A, Ye L, Lo JC, et al. A PGC1-alphadependent myokine that drives brown-fat-like development of white fat and thermogenesis. Nature 2012;481:463–8.