Endoplasmic reticulum-mitochondria calcium signaling in hepatic metabolic diseases

    Endoplasmic reticulum-mitochondria calcium signalling in hepatic metabolic diseases Jennifer Rieusset PII: DOI: Reference:

S0167-4889(17)30003-4 doi:10.1016/j.bbamcr.2017.01.001 BBAMCR 18026

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BBA - Molecular Cell Research

Received date: Revised date: Accepted date:

7 October 2016 21 December 2016 2 January 2017

Please cite this article as: Jennifer Rieusset, Endoplasmic reticulum-mitochondria calcium signalling in hepatic metabolic diseases, BBA - Molecular Cell Research (2017), doi:10.1016/j.bbamcr.2017.01.001

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ACCEPTED MANUSCRIPT Endoplasmic reticulum-mitochondria calcium signalling in hepatic metabolic diseases

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Jennifer Rieusset1

INSERM UMR-1060, CarMeN Laboratory, Lyon 1 University, INRA U1397, F-69921

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Oullins.

Corresponding author:

RIEUSSET, J.

Address:

UMR INSERM U1060

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Faculté de médecine Lyon-Sud

165 chemin du grand Revoyet, BP12,

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69921 Oullins cedex, France 33 (0)4 26 23 59 20

Fax:

33 (0)4 26 23 59 16

E-mail:

[email protected]

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Phone number:

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Abbreviations: ATP: adenosine triphosphate; Ca2+: calcium; CamK: calmodulin-dependent kinase; ChREBP: carbohydrate response element binding protein; CypD : cylophilin D ;

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DGAT2 : diacylglycerol acyltransferase 2 ; ER: endoplasmic reticulum; FOXO1: Forkhead box O1; G6P: glucose 6-phosphate; G6Pase: glucose 6-phoaphatase; GCK: glucokinase;

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GLUT2: glucose transporter, isoform 2; GRP75: glucose-regulated protein 75; GS: glycogen synthase; IP3: inositol triphosphate; IP3R: inositol trisphosphate receptor; IRS: insulin receptor substrate; JNK: c-Jun N-terminal kinase; Mfn2 : mitofusin 2 ; NADH: reduced nicotinamide adenine nucleotide; NAFLD: non-alcoholic fatty liver disease; NASH: nonalcoholic steatohepatitis; MAM: mitochondria-associated membranes; MAVS: mitochondrial antiviral-signaling protein; MEF: mouse embryonic fibroblasts; mTORC1/2: mammalian target of rapamycin complex 1 or 2; OXPHOS: oxidative phosphorylation; PACS2: phosphofurin acidic cluster sorting protein 2 ; PEP: phosphoenolpyruvate; PEPCK: phosphoenolpyruvate carboxykinase; PK: pyruvate kinase; PKA: protein kinase A; PKR: double-strand RNA-dependent protein kinase; PML : promyelocytic leukaemia ; POMC : proopiomelanocortine, PP : pentose phosphate ; PP2A : protein phosphatase 2A ; R5P: ribulose 5-phosphate; T2DM: type 2 diabetes mellitus; TCA: tricarboxylic acid; TG: triglyceride; UPR: unfolded protein response; VDAC: voltage-dependent anion channel;

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ACCEPTED MANUSCRIPT Abstract: The liver plays a central role in glucose homeostasis, and both metabolic inflexibility and insulin resistance predispose to the development of hepatic metabolic diseases.

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Mitochondria and endoplasmic reticulum (ER), which play a key role in the control of hepatic metabolism, also interact at contact points defined as mitochondria-associated membranes

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(MAM), in order to exchange metabolites and calcium (Ca2+) and regulate cellular homeostasis and signaling. Here, we overview the role of the liver in the control of glucose homeostasis, mainly focusing on the independent involvement of mitochondria, ER and Ca2+ signaling in both healthy and pathological contexts. Then we focus on recent data highlighting

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MAM as important hubs for hormone and nutrient signalling in the liver, thus adapting mitochondria physiology and cellular metabolism to energy availability. Lastly, we discuss

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how chronic ER-mitochondria miscommunication could participate to hepatic metabolic

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diseases, pointing MAM interface as a potential therapeutic target for metabolic disorders.

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Key words: organelle communication, mitochondria-associated membranes (MAM), calcium

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signaling, liver, insulin resistance, type 2 diabetes mellitus, NAFLD

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ACCEPTED MANUSCRIPT 1. Introduction The whole organism is continually exposed to nutritional variations (fed and fasted states) and to changes in the physical activity (high or low activity). To face to these changes, the

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organism has to adapt its metabolism (anabolic or catabolic pathways) in order to maintain energy homeostasis. Particularly, the maintenance of systemic glucose homeostasis is crucial

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in mammalians because some tissues, especially brain and red blood cells, rely on glucose as the sole energy source. Consequently, several mechanisms have evolved to closely monitor glucose and maintain it in a narrow physiological range, through an intricate regulatory and

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counter-regulatory neuro-hormonal system (Hers 1990). At the tissue level, several organs are involved in the control of glucose homeostasis, and their metabolism (glucose intake, storage, mobilization or breakdown) are orchestrated mainly via the secretion of hormones, like

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insulin and glucagon. Among them, the liver plays a major role in the control of glucose homeostasis as it has the capacity to both consume/store and produce glucose during feeding/fasting cycles. In addition to the hormonal control of glucose homeostasis,

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hepatocytes possess multiple glucose-sensing systems that interact to modulate biochemical pathways in order to accommodate to glucose availability (Oosterveer and Schoonjans 2014).

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Among them, several enzymes and transcription factors enable the liver to respond to dynamic changes in glucose availability. However, they are not the only intrahepatic players

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for the maintenance of glucose homeostasis. Intracellular organelles, such as mitochondria and endoplasmic reticulum (ER), recently emerged as key nutrient sensors (Gao et al. 2014,

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Mandl et al. 2009) and regulators of insulin signalling pathways (Murrow and Hoehn 2010, Salvadó et al. 2015), thus actively participating to the control of glucose homeostasis. Deregulated glucose homeostasis mechanisms underlie the physiopathology of type 2 diabetes mellitus (T2DM). Particularly, increased hepatic gluconeogenesis participates to hyperglycemia of type 2 diabetic patients, whereas lipid accumulation within hepatocytes characterizes non-alcoholic fatty liver diseases (NAFLD). Both T2DM and NAFLD are the most common liver metabolic diseases in Western societies, and their prevalence is rising dramatically. These two metabolic diseases are intimately linked with complex and bidirectional relationships; however hepatic insulin resistance is the common manifestation linking both pathologies (García-Ruiz et al. 2013, Valenti et al. 2016). Mitochondrial dysfunction and ER stress have been extensively and independently associated with both hepatic insulin resistance and lipid accumulation, thus playing a key role in metabolic diseases (for review see Martin et al. 2014, Wang et al. 2015, Salvadó et al. 2015). Recently,

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ACCEPTED MANUSCRIPT the interest of scientific community for their functional coupling, through physical interactions at sites defined as mitochondria-associated membranes (MAM) (Giorgi et al. 2015, López-Crisosto et al. 2015), has changed our vision on the role of both organelles in

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hepatic metabolic diseases. It has become clearer that structural and functional ERmitochondria interactions could play a role in hormonal and nutrient signaling of the liver in

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order to maintain glucose homeostasis, and that their miscommunication could be involved in hepatic metabolic diseases, at least in mouse models.

In this review, we will briefly overview the basic physiological mechanisms of hepatic glucose metabolism and their alterations during hepatic metabolic diseases. Particularly, we

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will focus on the role of mitochondria and ER in the metabolic adaptations of the liver, including the novel role of ER-mitochondria communication in both glucose and insulin

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signaling. Lastly, the role of ER-mitochondria miscommunication in the development of hepatic metabolic diseases will be discussed.

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2. Role of the liver in the control of glucose homeostasis The liver plays a key role in the control of glucose homeostasis as it has the capacity to

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both consume and produce glucose depending on the requirement (Figure 1).

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2.1. Hepatic glucose oxidation and storage In the postprandial state, glucose metabolism starts with the intestinal absorption of dietary sugar. For that, glucose crosses the apical membrane of the enterocyte using the

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sodium-glucose co-transporter. Then, it moves through the cytoplasm and exits the enterocyte via the basolateral membrane using the glucose transporter GLUT2 to access blood circulation. The liver has a unique position in glucose utilization since it has the first access to ingested glucose via the hepatic portal vein, and then it is exposed to higher glucose concentrations than peripheral tissues (Moore et al. 2012). After a meal, the liver uses around 30% of ingested glucose in order to either produce energy or to store it into glycogen (Cherrington 1999). Glucose is taken by the hepatocytes through GLUT2, which has a higher Km (17 mM) than other glucose transporters of the same family. This low affinity of GLUT2 allows efficient transport of glucose across the plasma membrane only when glycaemia is high (e.g. following feeding) (Thorens 2015). Once taken by the hepatocytes, glucose is phosphorylated to glucose-6-phosphate (G6P) by the liver glucokinase (GCK), the ratelimiting enzyme for hepatic glucose utilization. In contrast to other hexokinases, GCK has a significantly lower affinity and is only active when glucose levels are relatively high 4

ACCEPTED MANUSCRIPT (Cardenas et al. 1998). Furthermore, GCK activity is not inhibited by its product G6P allowing postprandial glycogen storage within hepatocytes (Storer and Cornish-bowden 1997). In addition, GCK is tightly regulated at the transcriptional level as well as by its

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subcellular localisation. Indeed, short-term regulation of GCK is controlled by its interaction with the GCK regulatory protein, which inhibits and sequesters GCK into the nucleus of

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hepatocytes and releases it into the cytoplasm in response to an increase in glucose concentrations (Choi et al. 2013). Therefore, both GLUT2 and GCK function as glucose sensors as they are activated only during glucose abundance and they shunt glucose into glycolysis (for energy production) or glycogen synthesis (for storage).

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Briefly, glycolysis is a ten-step process metabolizing glucose into pyruvate with a net gain of two adenosine triphosphate (ATP) and two reduced nicotinamide adenine dinucleotide

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(NADH) molecules per glucose molecule (Lodish et al. 2000). In addition to GCK, glycolysis is regulated by the phosphofructokinase (PFK) enzyme which transfers a phosphate group from ATP to fructose-6-phosphate producing fructose-1,6-biphosphate, and by the pyruvate-

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kinase (PK), the last step of glycolysis (Berg et al. 2002). In aerobic conditions, pyruvate is further decarboxylated to acetyl-CoA by the pyruvate dehydrogenase and then processed in

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the tricarboxylic acid (TCA) cycle into mitochondria. Finally, reduced coenzymes are reoxidised by the mitochondrial respiratory chain that transfer electron to oxygen to pump

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proton into the inter-membrane space of mitochondria. The pentose-phosphate pathway is an alternative way for degradation of G6P in hepatocytes. In a first oxidative phase, G6P is oxidised into ribose-5-phosphate (R5P) producing 2 NADPH. R5P is a precursor for

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nucleotide synthesis whereas NADPH is a co-substrate for de novo lipogenesis and cholesterol synthesis. Then, excess of R5P undergoes a series of inter-conversions to produce fructose 6-phosphate and glyceraldehyde 3-phosphate, two intermediates of glycolysis pathway (Berg et al. 2002). Glycogen synthesis allows to the liver to store glucose excess. It is catalysed by the glycogen synthase (GS) after conversion of G6P to UDP-glucose. GS synthetizes the glycogen polymer which is further branched by a branching enzyme (Lodish et al. 2000).

2.2. Hepatic glycogenolysis and gluconeogenesis In the fasting state, the liver maintains glycemia through glycogen breakdown, and following prolonged fasting through gluconeogenesis (Cherrington 1999, Rothman et al. 1991). Glycogenolysis is catalysed by glycogen phosphorylase, which cleaves glucose from glycogen polymer and produces glucose-1-phosphate. Glucose 1-phosphate is then converted 5

ACCEPTED MANUSCRIPT into G6P by the phosphoglucomutase, and the G6P is converted to glucose by the glucose-6phosphatase (G6Pase), which can be liberated into the circulation through GLUT2. However, glycogen is a limited source of glucose since the hepatic stocks of glucose rapidly decrease

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while glucose needs remain during fasting. Therefore, hepatic gluconeogenesis allows to the liver to produce glucose from non-carbohydrate precursors such as lactate, amino acids and

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glycerol, during prolonged fasting. It is partly a reverse glycolysis except for 3 irreversible reactions of glycolysis that are bypassed by gluconeogenesis-specific enzymes and constitutes limiting and regulating steps of the process: the phosphoenolpyruvate carboxykinase (PEPCK), the fructose-1,6-bisphosphatase and the G6Pase. PEPCK catalyses the conversion

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of oxaloacetate to phosphoenolpyruvate (PEP), while G6Pase catalyses the final step of

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gluconeogenesis, the production of free glucose from G6P.

3. Hepatic metabolic adaptations during nutritional transitions Blood glucose concentrations fluctuate during the feeding and fasting cycles and one of

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the primary function of the liver is to maintain blood glycemia within a physiological range. For that, both extra-hepatic and intra-hepatic factors coordinate biochemical pathways in

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order to maintain glucose homeostasis.

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3.1. Hormonal regulation of hepatic metabolism The hormonal regulation by insulin and glucagon of multiple metabolic pathways allows to the liver to store or produce glucose as necessary (Klover and Mooney 2004). Both

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transcriptional regulation of rate limiting enzymes and modulation of enzyme activity through phosphorylation and allosteric regulation are involved (Lin and Accili 2011). In the fasting state, the effects of glucagon avoid hypoglycemia by stimulating gluconeogenesis and glycogenolysis leading to hepatic glucose release (Ramnanan et al. 2011). Briefly, glucagon increases intracellular cAMP levels, which further activates the protein kinase A (PKA), thus regulating both glycogenolysis and gluconeogenesis. At the transcriptional level, glucagonmediated activation of PKA further activates the cAMP-response element binding protein thus regulating transcription of gluconeogenic genes. At the postprandial state, insulin prevents hyperglycemia, by inhibiting both hepatic gluconeogenesis and glycogenolysis and stimulating hepatic glycogen synthesis. For that, insulin signalling through the IR/IRS/PI3K/Akt pathway mainly decreases the transcription of gluconeogenic enzymes via the inactivation of Forkhead box O1 (FOXO1) (Dong et al. 2008), and activates GS thus increasing glycogenesis. 6

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3.2. Intracellular glucose sensing systems of the liver In addition to this hormonal component, hepatocytes possess multiple glucose-sensing

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systems, mainly enzymes and transcription factors, that regulate biochemical pathways in order to adjust metabolism to glucose availability (Oosterveer and Schoonjans 2014). Among

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them, the glucose transporter GLUT2 and the GCK enzyme are major components of the hepatic glucose sensing system as described above. However, recent data found that the hepatic loss of GLUT2 did not result in major alterations of hepatic metabolism during nutritional transitions (Seyer et al. 2013), suggesting that compensative mechanisms probably

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occur. On the contrary, GCK plays a key role in postprandial glucose-sensing in the liver as it remains active over a wide range of glucose concentrations allowing hepatocytes to efficiently

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trap glucose in response to glycemic fluctuations. Importantly, loss of hepatic GCK in mice alters hepatic glucose metabolism (Dentin et al. 2004), whereas overexpression of GCK, but not hexokinase I, induces glycolysis and glycogen storage in hepatocytes (O’Doherty et al.

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1996, Seoane et al. 1996). Downstream G6P, glucose metabolites then act as signalling molecules to acutely regulate the activity of several enzymes involved in glucose

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consumption and storage. Finally, the regulation by glucose of the expression of several key enzymes allow longer-term adaptations to glucose availability. The carbohydrate response

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element binding protein (ChREBP) is a key transcription factor regulating the expression of several glycolytic, neoglucogenic and lipogenic genes (Jeong et al. 2011). It was shown that hepatic ChREBP activation by glucose requires GCK-dependent glucose metabolism (Dentin

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et al. 2004). However, it is actually unclear whether it is G6P (Kabashima et al. 2003) or X5P (Li MV et al. 2010, Dentin et al. 2012) which is responsible of ChREBP activation, promoting its nuclear translocation and transcriptional activity. Lastly, post-translational modifications, such as O-linked b-N-acetylglucosaminylation (Hart et al. 2011) and acetylation (Yang et al. 2011), also participates to glucose-sensing in the liver.

4. Hepatic alterations associated with metabolic diseases Metabolic diseases such as obesity, T2DM, and metabolic syndrome are associated with altered hepatic metabolism, participating to hyperglycemia, hyperinsulinemia and hypertriglyceridemia. Particularly, the hepatic hallmark of the metabolic syndrome is known as NAFLD, which are strongly associated with obesity, insulin resistance and T2DM. The spectrum of NAFLD ranges from simple fatty liver with benign prognosis, to a potentially progressive form, non-alcoholic steatohepatitis (NASH), which may lead to liver fibrosis, 7

ACCEPTED MANUSCRIPT cirrhosis, and hepatocellular carcinomas, resulting in increased mortality (Cohen et al. 2011). Hepatic insulin resistance is a common characteristics associated with hepatic metabolic

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diseases.

4.1. Hepatic insulin resistance

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Insulin resistance is defined as the failure of cells to respond normally to insulin’s glucose-lowering effects, resulting in hyperglycemia. In liver, insulin resistance leads to greater glycogenolysis and gluconeogenesis, leading to increase hepatic glucose release. At the molecular levels, increased serine phosphorylation of insulin receptor substrate 1 and 2

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(IRS1/2) proteins participate to downstream alterations of insulin signaling and to increased hepatic glucose release (Aguirre et al. 2002, Sharfi et al. 2008). Different metabolic stresses in

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liver are involved in the activation of several serine/threonine kinases responsible for serine phosphorylation of IRS proteins. Among them, hepatic lipid accumulation plays a key role in alterations of hepatic insulin sensitivity, and particularly DAG-mediated activation of PKCε

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impairs hepatic insulin signaling (Perry et al. 2014). Furthermore, mitochondrial dysfunction, oxidative stress, ER stress and inflammation are also involved in hepatic insulin resistance,

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through mainly the activation of mammalian target of rapamycin complex 1 (mTORC1), cjun N-terminal kinase (JNK), IkB kinase, protein kinase C and the double stranded RNA-

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activated protein kinase (PKR). This molecular aspect of insulin resistance has been extensively reviewed and will not be covered in detail (for review, see Samuel et al. 2016). Since insulin signaling normally induces de novo lipogenesis in the liver, this pathway

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should be impaired in insulin resistance states. However, increased lipogenesis is preserved in insulin resistant liver, driving lipid accumulation. This paradox was called “selective insulin resistance”, in which the insulin signalling pathways mediating glucose metabolism are impaired while those stimulating lipid metabolism are preserved (Otero et al. 2014). It was proposed that mTORC1 is an essential component in the insulin-regulated pathway for hepatic lipogenesis but not gluconeogenesis, and could participate to the paradox of selective insulin resistance in diabetic liver (Li S et al. 2010).

4.2. Aberrant hepatic glucose sensing In liver metabolic diseases, hepatic glucose sensors are chronically activated as the liver is more frequently exposed to hyperglycemic episodes (Thorens 2008). Therefore, GCK is constitutively active and GCK flux is increased in response to elevated glucose concentrations in obese and diabetic mice (Bandsma et al. 2004, Yen et al. 1981). In addition, 8

ACCEPTED MANUSCRIPT the expression and nuclear localization of ChREBP, another important hepatic glucose sensor, are significantly increased in the liver of the obese mice (Dentin et al. 2006). In agreement, the liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in

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ob/ob mice (Dentin et al. 2006). Aberrant glucose sensing in metabolic diseases results in triglyceride accumulation and excessive glucose production in the liver (Dentin et al. 2008,

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Dentin et al. 2006), favouring liver steatosis and hyperglycemia.

4.3. Hepatic steatosis

Hepatic steatosis results from several alterations of lipid metabolism including: i)

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increased lipolysis of peripheral fat stored in adipose tissue, ii) increased de novo fatty acid synthesis (lipogenesis), iii) impaired beta-oxidation in hepatocytes, and iv) reduced

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triglyceride (TG) export from the liver (Postic and Girard 2008). Studies in humans and in rodents have demonstrated that the mechanisms leading to the excessive accumulation lipids in liver are mainly linked to increased delivery of fatty acids from adipose tissue to the liver

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and to increased de novo lipid synthesis in the liver itself, while lipid disposal via β-oxidation and very low density lipoprotein export are only moderately affected (Lewis et al. 2002).

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Whereas a clear correlation between fatty liver and insulin resistance exists, it is actually unknown who is the culprit of the other (Farese et al. 2012). Indeed, recent studies suggested

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that the accumulation of intrahepatic lipids precedes the state of insulin resistance, whereas others have shown that hepatic TG themselves are not toxic and may in fact protect the liver from lipotoxicity by buffering the accumulation of fatty acids, pointing that hepatic steatosis

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is not necessarily associated with insulin resistance (Sun and Lazar 2013).

5. Respective role of mitochondria, ER and calcium signaling in hepatic metabolic homeostasis Cellular homeostasis depends on compartmentalization of metabolic process within specific subcellular organelles allowing spatial separation of metabolic processes. In agreement, both the ER and mitochondria execute specific functions in hepatic metabolism. In this section, we primarily focus on the independent role of mitochondria, ER and calcium homeostasis in the control of hepatic metabolism, with a heavier focus on glucose metabolism, as well as the implication of organelle and calcium (Ca2+) signal dysregulations in hepatic metabolic diseases (Figure 2).

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ACCEPTED MANUSCRIPT 5.1. Role of mitochondria, ER and calcium homeostasis in hepatic glucose metabolism Both mitochondria and ER are two important metabolic organelles, playing a key role

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in the control of both the catalytic and anabolic pathways of hepatic glucose metabolism. Therefore, it is not surprising that both organelles represent around more than the half of the

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volume of the hepatocytes. In addition, both organelles play an important role in the maintenance on intracellular Ca2+ homeostasis which also participate to the control of hepatic glucose metabolism.

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Importance of mitochondria in hepatic glucose metabolism: Mitochondria are catabolic organelle involved in the oxidation of all substrates (glucose, lipids and proteins). Concerning

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glucose, mitochondria allow more efficient energy production through the final oxidation of glucose. Indeed, when glucose is converted to pyruvate by glycolysis, only a very small fraction of the total free energy potentially available from the glucose is released. In

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mitochondria, the metabolism of glucose is completed: pyruvate is imported into the mitochondrion and oxidized by O2 to CO2 and H2O. This allows 15 times more ATP to be

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made than that produced by glycolysis alone. The TCA cycle into mitochondria plays a key role in the control of glucose metabolism as it provides reduced coenzymes processed by the

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respiratory chain and as it controls acetyl-CoA and oxaloacetate levels with a direct impact on glucose fluxes. Finally, mitochondria are also the first subcellular compartment where gluconeogenesis occurs through the conversion of oxaloacetate into PEP by mitochondrial

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PEPCK (Berg et al. 2002).

Importance of ER in hepatic glucose metabolism: The ER is an anabolic organelle playing a key role in protein biosynthesis and secretion, in intermediary lipid metabolism and more complex lipid formation, in Ca2+ homeostasis, and in detoxification of the liver. By all these functions, the ER may impact glucose homeostasis. Nevertheless, the ER also participates more directly in glucose metabolism, as evidenced by the presence of G6Pase at the ER membrane. Indeed, G6Pase functions as a multicomponent system, comprising the enzyme protein with an intraluminal active site and transporters for the entry of G6P and for the exit of phosphate and glucose (Arion et al. 1980). Importance of Ca2+ homeostasis in hepatic glucose metabolism: Both mitochondria and ER are major Ca2+ homeostasis regulators, controlling therefore Ca2+-dependent signaling in the 10

ACCEPTED MANUSCRIPT cytoplasm. Therefore, it is not surprising that Ca2+-dependent mechanisms are involved in the control of glucose homeostasis in the liver (Bartlett et al. 2014). In particular, intracellular Ca2+ signaling controls both hepatic glucose production and glycogenolysis. Indeed,

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Glucagon increases intracellular Ca2+ directly or via cAMP/PKA-mediated phosphorylation of inositol trisphosphate receptor (IP3R), leading to its activation and to Ca2+ release by the

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ER. Cathecholamines also activate phospholipase C, leading to the hydrolysis of phosphoinositides and producing two important second messengers, inositol triphosphate (IP3) and diacylglycerol. Then, IP3 binds to IP3R and also stimulates Ca2+ release from the ER. The subsequent increase in cytosolic Ca2+ then induces either a direct modulation of

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enzyme activity in the gluconeogenesis program (e.g. pyruvate carboxylase or phosphoenolpyruvate carboxykinase) or modulate the expression of key gluconeogenesis

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genes. For that, cytosolic Ca2+ sensors such as calmodulin-dependent kinases (CamK) and calcineurin activate nuclear transcription factors like FoxO1, cAMP response element-binding protein, nuclear factor of activated T cell and c-AMP responsive element binding protein

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regulated transcription coactivator 2, regulating neoglucogenic genes (Ozcan L et al. 2012). In addition to neoglucogenesis, cytosolic Ca2+ regulates glycogenolysis through the stimulation of the phosphorylase kinase and activation of the glycogen phosphorylase (Amaya and

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Nathanson 2013). Furthermore, mitochondrial Ca2+ flux is also a critical regulator of hepatic metabolism as Ca2+ plays a key role in the regulation of mitochondrial oxidative metabolism

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(Griffiths and Rutter 2009). Indeed, mitochondrial Ca2+ increases the activity of three Ca2+ sensitive dehydrogenases of the TCA cycle (pyruvate dehydrogenase phosphatase, isocitrate

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dehydrogenase and oxoglutarate dehydrogenase) (Denton 2009), increasing respiration and ATP synthesis. Moreover, ATP synthesis is also stimulated by the direct action of Ca2+ on the the mitochondrial ATPase (Territo et al. 2000).

5.2. Role of mitochondria and ER in nutrient sensing Recently, both mitochondria and ER have emerged as key regulators of hepatic metabolism during nutritional transitions, and both organelles are now recognized as nutrient sensors. We recently reviewed the role of both organelles in nutrient sensing and the mechanisms will therefore not be detailed here (Theurey and Rieusset 2016).

Mitochondria in hepatic nutrient sensing: Mitochondria have the capacity to switch from glucose oxidation to lipid oxidation according to physiological and nutritional circumstances, pointing a key role of this organelle in metabolic flexibility. Interestingly, several aspects of 11

ACCEPTED MANUSCRIPT mitochondria physiology participate to the adaptation of hepatic metabolism to nutrient availability, such as changes in mitochondrial dynamics (i.e. fasting is associated with mitochondria fusion, whereas in feeding state mitochondria are fragmented), post-

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through control of both mitochondriogenesis and mitophagy).

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transcriptional modifications (i.e. acetylation) and modulation of mitochondria quantity (i.e.

ER in hepatic nutrient sensing: In the presence of misfolded proteins, ER initiates the unfolded protein response (UPR) through the actions of canonical sensors, protein kinase RNA (PKR)-like ER kinase (PERK), inositol-requiring enzyme-1 (IRE1), and activating

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transcription factor-6 (ATF6) in order to restore ER homeostasis (Walter and Ron 2011). Collectively, these three pathways act to suppress protein synthesis, facilitate protein

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degradation, and to increase the availability of protein chaperones. If all 3 pathways fail, the ER activates apoptotic pathways. Several nutrient-related signals can activate the UPR, such as long-chain saturated fatty acids, hypoglycemia or hyperglycemia, in order to restore

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homeostasis and promote cell survival in response to ER stress. Furthermore, Glycosylation is an essential ER luminal modification for proper stability, folding, translocation, and function

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of many proteins (Kaufman 1999). An increase in the hexosamine biosynthetic pathway, which plays a key role in glycosylation, induced a PERK-dependent ER stress and attenuated

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ApoB100 synthesis (Qiu et al. 2011, Sage et al. 2010). Lastly, the UPR also provides an essential mechanism to sense nutrients and to promote cell differentiation (Schröder 2008).

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5.3. Organelle dysfuntion and calcium homeostasis disruption in hepatic metabolic diseases

Numerous studies have shown that both mitochondrial dysfunction and ER stress are classically associated with hepatic insulin resistance and fatty liver. Concerning mitochondria, ultrastructural changes, reduction of mitochondrial DNA, reduced oxidative phosphorylation (OXPHOS) activity and altered -oxidation have been associated with hepatic insulin resistance and steotosis. Therefore, impaired hepatic mitochondrial function is thought to contribute to altered glucose homeostasis during obesity and T2DM (Rieusset 2015, Koliaki and Roden 2013) and to lipid accumulation in NAFLD (Begriche et al. 2013). Particularly, mitochondrial oxidative capacity seems important for hepatic insulin sensitivity as inhibition of mitochondrial -oxidation mediated by the loss of long-chain acyl-CoA deshydrogenase induces hepatic insulin resistance (Zhang et al. 2007), whereas improving liver mitochondrial lipid oxidation in obese mice increases insulin sensitivity independently of hepatic steatosis 12

ACCEPTED MANUSCRIPT (Monsénégo et al. 2012). Similarly, impaired mitochondria (Caldwell et al. 1999) and reduced mitochondrial chain respiratory activity (Pérez-Carreras et al. 2003) were reported in patients with NASH. However, conflicting results exist concerning the causative role of mitochondria

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dysfunction in obesity-associated hepatic insulin resistance and fatty liver. Some studies described an increase of OXPHOS activity in liver of obese mice (Sunny et al. 2011,

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Takamura et al. 2008), and others reported that OXPHOS deficiency may prevent the development of insulin resistance (Pospisilik et al. 2007).

Concerning the ER, its implication in hepatic insulin resistance and fatty liver diseases is more clear. Indeed, several ER stress markers are increased in the liver of diverse models

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of obese and diabetic mice (Ozcan et al. 2004), whereas reducing ER stress using chemical chaperones improves hepatic insulin sensitivity and lipid accumulation in both obese mice

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(Ozcan et al. 2006) and subjects (Kars et al. 2010). Furthermore, activation of the UPR was also found in liver of animal models of NAFLD (Rinella et al. 2011) and in NAFLD patients (Puri et al. 2008). It was shown that the activation of the UPR mainly alter hepatic

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metabolism through: i) the activation of stress kinases that interfere with insulin signalling, such as JNK or PKR, ii) the activation of transcription factors that modulate the expression of

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gluconeogenic and lipogenic enzymes, iii) the clivage of sterol regulatory element-binding protein 1c promoting fat accumulation in hepatocytes (for review, see Salvadó et al. 2015). Disrupted Ca2+ homeostasis also plays a key role in hepatic metabolic diseases.

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Indeed, an increase in intracellular Ca2+ levels and Ca2+-dependent signaling has been found in primary hepatocytes of obese and diabetic mice (Ozcan et al. 2012) and insulin sensitivity

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was improved by the chelation of intracellular Ca2+ in high fat-fed rats (Jang et al. 2002), suggesting that altered Ca2+ homeostasis could also participate to the aetiology of hepatic insulin resistance. In agreement, activation of CamK by increased intracellular Ca2+ levels in obesity mediates suppression of hepatic insulin signaling (Ozcan et al. 2013), whereas the improvement of Ca2+ homeostasis in the liver of obese mice by overexpressing the sarco/ER Ca2+ ATPase pump improved hepatic insulin sensitivity (Fu et al. 2011). Furthermore, both ER Ca²⁺ homoeostasis (Fu et al. 2011) and store-operated Ca²⁺ entry (Wilson et al. 2015) is altered in steatotic hepatocytes, suggesting that these alterations could also participate to NAFLD.

6. Role of ER-mitochondria coupling in hepatic metabolism Importantly ER and mitochondria are not independent organelles within cell, they physically interact at sites known as mitochondria-associated membranes (MAM), in order to 13

ACCEPTED MANUSCRIPT exchange metabolites and signaling molecules, and reciprocally regulate their functions. The liver, particularly enriched in both organelles, has intense ER-mitochondria communication, and studies in liver have increased our knowledge on the nature and function of MAM.

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Particularly, MAM from liver can be easily isolated by subcellular fractionation (Wieckowski et al. 2009) and have been subjected to proteomic analysis (Sala-Vila et al. 2016), increasing

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our knowledge on MAM components. The physical interaction between both organelles does not involve membrane fusion, but is mediated through protein bridges (Csordas et al. 2006). This is illustrated by the interaction between the voltage-dependent anion channel (VDAC) at the outer mitochondrial membrane and the IP3R at the ER through the molecular chaperone

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glucose-regulated protein 75 (Grp75), allowing Ca2+ transfer from the ER to mitochondria (Szabadkai et al. 2006). In addition, MAM are enriched in several phospholipid enzymes

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(Vance 2014). Nevertheless, the list of MAM actors with widely varied functions has increased dramatically in the last five years, revealing new roles of MAM in several cellular signaling pathways (van Vliet et al. 2014). Therefore, miscommunication between both

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organelles starts to emerge in several diseases, particularly in hepatic metabolic diseases. In agreement, recent evidences indicate that MAM could be a hub of hepatic insulin signaling

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and nutrient sensing (Figure 3), and that insulin resistant and fatty liver is associated with

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chronic disruption of MAM, at least in mouse models (Figure 4).

6.1. Is there a regulation of ER-mitochondria coupling by hormones targeting the liver?

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As hepatic metabolism is regulated by Ca2+-mobilizing hormones, it makes sense to ask about a potential regulation of ER-mitochondria interactions by these hormones. Surprisingly, there are very few studies that have studied the hormonal regulation of ERmitochondria physical interactions. Nevertheless, it is well known that several hormones, such as vasopressin and glucagon (Assimacopoulos-jeannet et al. 1986, Robb-Gaspers et al. 1998a), as well as catecholamines, such as norepinephrine (Knerr et al. 1979), clearly increased cytosolic and mitochondrial Ca2+ through IP3R in hepatocytes, in order to regulate oxidative metabolism. Particularly, the changes in cellular Ca2+ concentrations have been correlated with the

regulation of intra-mitochondrial NAD(P)H levels, pyruvate

dehydrogenase activity and the magnitude of the mitochondrial proton motive force (RobbGaspers et al. 1998b), in order to coordinate cellular energy production to energy demand. Therefore, ER-mitochondria coupling might be acutely regulated by these hormones, in order to favour intra-mitochondrial Ca2+ import. Nevertheless, further works are required to clearly 14

ACCEPTED MANUSCRIPT demonstrate a regulation of hepatic ER-mitochondria interactions by Ca2+-mobilizing hormones. Conversely, insulin opposes to the effects of glucagon and epinephrine (Somogyi et al. 1992) and activates Akt that has been shown to phosphorylate and inhibit IP3R,

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attenuating cytosolic Ca2+ levels (Khan et al/ 2006, Marchi et al. 2012). Whether insulin regulates ER-mitochondria interactions in the liver is actually unclear and requires further

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investigations. Alternativity, insulin signaling could be regulated by ER-mitochondria coupling, as discussed below.

6.2. Role of ER-mitochondria coupling in hepatic insulin signaling

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The first argument in favour of a role of MAM in insulin signaling is that some proteins of this cellular pathway are located at the MAM interface. For example, the protein

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kinase Akt is present at the ER-mitochondria interface in both different cellular models and mouse liver (Betz et al. 2013, Giorgi et al. 2010, Tubbs et al. 2014), where it phosphorylates and inhibits IP3R, thus reducing Ca2+ release and preventing apoptosis (Giorgi et al. 2010).

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Insulin stimulation increases Akt phosphorylation at MAM interface in HuH7 cells and mouse liver (Tubbs et al. 2014), whereas it is actually unclear whether Akt is phosphorylated directly

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at MAM or localized at MAM after its phosphorylation into cytoplasm. It is interesting to note that several MAM resident proteins, such as IP3R, phosphofurin acidic cluster sorting

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protein 2 (PACS2), and the VDAC-interacting protein hexokinase 2 are Akt substrates (Simmen et al. 2005; Khan et al. 2006; Szado et al. 2008; Aslan et al. 2009), confirming the important signaling role of this kinase at MAM interface. Furthermore, the activity of Akt is

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regulated by the protein phosphatase 2 (PP2A) at MAM interface in mouse embryonic fibroblasts (MEF), thus impacting IP3R-mediatred Ca2+ release (Gorgi et al. 2010). In addition, mammalian target of rapamycin complex 2 (mTORC2) is also present at MAM interface in mouse liver and its amount at this subcellular domain increases in response to growth factors or insulin stimulation (Betz et al. 2013). mTORC2 at MAM controls Akt and its targets to ultimately control MAM integrity, Ca2+ release, and mitochondrial physiology (Betz et al. 2013). Recently, a fraction of glycogen synthase kinase-3β (GSK3β), another protein of insulin signaling pathway, was localized to MAMs in mouse heart, and was shown to interact with and activate IP3R in both adult cardiomyocytes and H9c2 cells, thus regulating organelle Ca2+ exchange (Gomez et al. 2016). Lastly, the tumor suppressor PTEN is also enriched in MAM in HEK-293 cells, where it sensitizes cells to apoptosis by dephosphorylating IP3R and restoring ER Ca2+ release (Bononi et al. 2013). Nevertheless, whereas MAM appear as an important hub for mTORC2-Akt signaling, the involvement of 15

ACCEPTED MANUSCRIPT ER-mitochondria coupling in the control of insulin action was unknown. Recently, our laboratory demonstrated that MAM integrity is required for insulin signaling in the liver, at least in mouse hepatocytes and HuH7 cells (Tubbs et al. 2014). Indeed, experimental

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disruption of MAM alters insulin signaling and action in both HuH7 cells and mouse liver, whereas overexpression of MAM proteins enhances it. In agreement, mitochondrial Ca2+

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uptake is critical for effective insulin signaling in both skeletal myocytes (del campo et al. 2014) and cardiomyocytes (Gutierrez et al. 2014). Alternatively, invalidation of key proteins of insulin signaling, such as Akt or mTORC2 (Betz et al. 2013) in MEF, or disruption of insulin signaling by palmitate treatment in HuH7 cells (Tubbs et al. 2014) disrupts MAM

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integrity. This suggests that the impact of MAM integrity on insulin signaling could be reciprocal. Nevertheless, the molecular mechanism underlying this relationship is actually

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unknown.

6.3. Role of ER-mitochondria coupling in hepatic metabolic flexibility

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MAM are highly dynamic structures that can influence mitochondria bioenergetics (Cardenas et al. 2010), suggesting that MAM regulation could allow adaptation of

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mitochondria physiology to nutrient availability. In agreement, ER-mitochondria contacts were found to double when nutrients become limiting in mouse liver (Sood et al. 2014).

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Recently, our group further investigated the role of MAM in hepatic metabolic flexibility and we confirmed that ER-mitochondria interactions are regulated by nutritional transition in mouse liver (Theurey et al. 2016). We found that the fasted to fed transition is associated with

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a reduction of organelle interactions in the liver of mice, and importantly we revealed glucose levels as the main nutritional regulator of hepatic MAM integrity at postprandial state. At the molecular level, we demonstrated that high glucose levels disrupt ER-mitochondria interactions and Ca2+ exchange through the activation of the pentose phosphate (PP)-PP2A pathway in HuH7 cells. Indeed, silencing of the catalytic subunit of PP2A and/or inhibition of its activity using okadaic acid prevents glucose-induced reduction of MAM integrity and function. The importance of PP2A in the control of MAM is supported by the presence of PP2A at MAM interface, where it regulates Akt phosphorylation and Ca2+ release by IP3R in MEF (Giorgi et al. 2010). As both Akt and mTORC2 are activated at MAM interface following insulin stimulation or refeeding, we propose that PP2A should be delocalized from MAM following feeding or glucose stimulation, thus increasing the phosphorylation of some target protein(s)) at MAM interface. Further works are required to identify the key target

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ACCEPTED MANUSCRIPT proteins of PP2A at MAM interface regulating ER-mitochondria interactions and function during nutritional transition. Importantly, we found that the glucose-sensing by MAM is crucial for the regulation

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of mitochondrial dynamics and function in the liver. Indeed, glucose-mediated reduction of MAM is associated with mitochondria fission and impaired respiration in both HuH7 cells

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and mouse liver (Theurey et al. 2016). Blocking MAM reduction, either by overexpression of the MAM protein Grp75 or by genetic or pharmacological inhibition of the PP-PP2A pathway, counteracted glucose-induced mitochondrial alterations in HuH7 cells. Lastly, experimental disruption of MAM integrity mimicked glucose effects on mitochondria

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dynamics and function. Altogether, these data point MAM as a new glucose-sensing system in order to adapt cellular bioenergetics to glucose availability.

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The important role of MAM during nutritional transition is supported by the recent observation that MAM regulate the formation of autophagosome during autophagy. Indeed, autophagy is a lysosomal degradation pathway which is induced by the lack of nutrients in

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order to recycle intracellular components and provide energy. Interestingly, disruption of MAM inhibited starvation-induced autophagy by blocking phosphatidylserine transfer

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between organelles in a normal rat kidney cell line (Hailey et al. 2010). In agreement, two essential autophagy proteins, ATG14L and ATG5, were shown to relocalize at MAM after

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starvation, and disruption of MAM prevented autophagosome formation in mammalian cells (Hamasaki et al. 2013). Furthermore, the promyelocytic leukaemia (PML) protein was recently shown to inhibit autophagy at MAM interface in MEF, thus supporting the role of

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ER-mitochondria contact sites in the regulation of autophagy (Missiroli et al. 2016). Therefore, both the interactions between organelles and the recruitment of starvation-induced proteins at MAM are induced by fasting.

6.4. Potential role of ER-mitochondria coupling in hepatic lipid storage Whereas MAM are recognized as hot-spots for lipid metabolism (Voelker 2005), it is actually unknown whether ER-mitochondria miscommunication could be involved in the development of NAFLD. However, several experimental evidences suggest that MAM may be involved in the control of hepatic lipid storage/secretion. First of all, the previous observation that MAM structure and function are regulated by nutritional state in mouse liver suggest that MAM may contribute to the adaptive fuel partitioning during nutritional transition. Indeed, in fasted state, the liver preferentially uses free fatty acids from adipose tissue stores as substrates, and oxidizes them into mitochondria. ER and mitochondria are 17

ACCEPTED MANUSCRIPT then tightly coupled to increase mitochondria oxidative metabolism. At fed state, the excess of glucose is stored into glycogen and triglycerides; therefore, the reduction of MAM integrity and function by glucose should contribute to reduce mitochondrial lipid oxidation and to

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favour lipogenesis. Whereas it could point a potential role of MAM in the control of lipid storage, further studies are needed to determine precisely the role of MAM in the induction of

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hepatic lipogenesis. Secondly, MAM are enriched in several lipid biosynthetic enzymes, including phosphatidylethanolamine N-methyltransferase and phosphatidylserine synthases, glycerol 3-phosphate acyltransferas 1 and acyl-CoA synthase 4.

In addition, the

diacylglycerol acyltransferase 2 (DGAT2), an enzyme that catalyses the final step of

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triacylglycerol synthesis, and the microsomal triacylglycerol transfer protein, which is required for the assembly/secretion of apolipoprotein B-containing lipoproteins, are also

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present in the MAM (for review see Vance 2014). Importantly, ER-mitochondria miscommunication was associated with fatty liver in ob/ob mice, even one study found a disruption of MAM (Tubbs et al. 2014) whereas another study found an enhanced ER-

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mitochondria coupling (Arruda et al. 2014). Importantly disruption of ER-mitochondria communication in the liver of mice by either the total invalidation of cyclophilin D (CypD)

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(Rieusset et al. 2016, Tubbs et al. 2014) or the hepatic invalidation of mitofusin 2 (Mfn2) (Sebastian et al. 2012) induced hepatic lipid accumulation. However, all these mice models

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are insulin resistant, therefore it is difficult to determine whether lipid accumulation is the result of MAM disruption, or insulin resistance, or both. Recently, calveolin-1 was identified at MAM interface in mouse liver, and was shown to control the cholesterol content of this

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subcellular domain (Sala-Vila et al. 2016), highlighting the role of MAM in lipid metabolism. Lastly, MAM have been shown to be crucial in the autophagic process (Hamasaki et al. 2013) as discussed above, and impaired autophagy is associated with hepatic steatosis (Lavallard and Gual 2014), whereas suppression of autophagy leads to hepatic ER stress and insulin resistance in mouse models (Yang et al. 2010). Finally, de novo lipogenesis in the liver is controlled by the UPR signaling, and recent evidences suggest an interesting relationship between MAM and UPR in both mammalian cells and mouse liver. Indeed, loss of several MAM proteins, such as PACS2 (Simmen et al. 2005), SigR1 (Hayashi and Su 2007), Mfn2 (Sebastian et al. 2012) and CypD (Rieusset et al. 2016) induced an increase of UPR signalling, whereas some ER stress sensors, as PERK (Verfaillie et al. 2012) and Grp78 (Hayashi and Su 2007), were found at MAM interface. Furthermore, moderate and acute ER stress increased ER-mitochondria interactions in order to boost mitochondria bioenergetics, whereas chronic ER stress disturbed mitochondria function in Hela cells (Bravo et al. 2011). 18

ACCEPTED MANUSCRIPT Therefore, ER-mitochondria contact sites could modulate hepatic lipid accumulation through its action on UPR signaling.

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7. ER-mitochondria miscommunication as a central mechanism of hepatic

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metabolic diseases

7.1. MAM disruption and hepatic insulin resistance

As described above, MAM are at the crossroad of several important hormonal and nutrient-regulated signaling pathways in the liver, suggesting that miscommunication between

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ER and mitochondria could be involved in hepatic metabolic diseases (Figure 4). This idea was supported by data showing a strong interplay between mitochondria and ER dysfunction in the context of hepatic insulin resistance. Indeed, experimental mitochondrial dysfunction

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induced ER stress through an elevation of cytosolic free Ca2+, and lead subsequently to aberrant insulin signaling and increase hepatic gluconeogenesis in human liver sk-HepI cells

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(Lim et al. 2009). Furthermore, liver-specific ablation of the mitochondrial Mfn2 in mice also induces ER stress, insulin resistance and impaired glucose tolerance (Sebastian et al. 2012),

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while hepatic overexpression of Mfn2 using adenoviral approaches protected mice from dietinduced IR (Gan et al. 2013). Similarly, loss of the mitochondrial CypD induced ER stress

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and alterations of insulin signaling in mouse liver (Rieusset et al. 2016). Therefore, we recently investigated the potential involvement of MAM disruption in hepatic insulin resistance. We found that MAM integrity is altered in palmitate-induced

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insulin resistant HuH7 cells, as well as in liver of different models of diabetic mice (ob/ob and high-fat/high-sucrose diet). Treatment of diabetic mice with antidiabetic drugs (rosiglitazone, metformin) improved insulin sensitivity and restored organelle communication. In addition, disruption of MAM integrity by genetic or pharmacological inhibition of CypD induced hepatic insulin resistance in mice and disrupted insulin signaling in human primary hepatocytes. Lastly, the rescue of MAM integrity in primary hepatocytes of diabetic mice by adenoviral overexpression of CypD improved insulin action. Therefore, our data demonstrate for the first time a key role of disrupted MAM in hepatic insulin resistance (Tubbs et al. 2014). We further propose that disruption Ca2+ exchange between both organelles links MAM alterations to hepatic insulin resistance, at least in CypD-KO mice (Rieusset et al. 2016). In agreement, loss of mTORC2 in MEF disrupted MAM integrity (Betz et al. 2013), whereas liver-specific loss of rictor, a subunit of mTORC2, showed constitutive gluconeogenesis, and impaired glycolysis and lipogenesis in mouse liver (Hagiwara et al. 2012). Interestingly, ER19

ACCEPTED MANUSCRIPT mitochondria contacts are also reduced in pro-opiomelanocortine (POMC) neurons of high-fat diet mice (Schneeberger et al. 2013). Moreover, loss of Mfn2 in POMC neurons in the hypothalamus resulted in reduction of ER-mitochondria interaction, ER stress-induced leptin

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resistance, hyperphagia, reduced energy expenditure and obesity (Schneeberger et al. 2013). A link between disrupted MAM and insulin resistance was also found in adipose tissue of

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mice deficient in cisd2 (also known as WFS2), an iron-sulfur protein localized at ER and MAM interface (Chen et al. 2009, Wang et al. 2014). Altogether these data highlighted a strong relationship between ER-mitochondria miscommunication and insulin resistance in several mouse tissues, as well as in human hepatocytes, even it is still unclear who is the

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culprit of the other. However, another group recently reported that MAM content is increased in the liver of obese mice, leading to mitochondrial Ca2+ overload and mitochondrial

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dysfunction (Arruda et al. 2014). The discrepancy between studies is actually unclear but could be related to differences in metabolic status of mice, environmental conditions, or experimental analysis. Anyway, reduced or excessive ER-mitochondria contacts, likely

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depending on the timing of the adaptive response upon a metabolic challenge, could represent a new and important mechanism contributing to hepatic mitochondrial dysfunction and

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insulin resistance. Future studies in which MAM will be dynamically studied should be

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required to clarify this element.

7.2. MAM disruption and aberrant nutrient sensing Moreover, chronic disruption of MAM is associated with impaired glucose-sensing by

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MAM in the liver of insulin resistant mice (Theurey et al. 2016). Indeed, we found that the regulation of MAM by glucose levels are loss in liver of ob/ob and CypD-KO mice, both characterized by chronic disruption of MAM integrity, mitochondrial fission and altered mitochondrial respiration. Therefore, chronic disruption of MAM may participate to both hepatic metabolic inflexibility and mitochondrial dysfunction associated with insulin resistance. Furthermore, as ER-mitochondria interactions are controlled by PP2A (Theurey et al. 2016) and as hyperactivation of PP2A is associated with insulin resistance (Kowluru and Matti 2012), increased PP2A activity could participate to disruption of MAM in liver of insulin-resistant mice. Future studies are required to understand the molecular mechanisms of MAM disruption in the context of hepatic metabolic diseases.

7.3. Role of MAM disruption in other liver diseases

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ACCEPTED MANUSCRIPT MAM may also contribute to other liver diseases such as hepatitis C infection and hepatocarcinoma, because of their role in antiviral response and in inflammasome activation. Indeed, it was shown that several proteins involved in immune response to DNA viruses, such

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as the mitochondrial antiviral-signaling protein (MAVS) (Horner et al. 2011) or the stimulator of interferon genes (Ishikawa et al. 2009) are localized to MAM in mammalian cells. During

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viral infection, retinoic acid-inducible gene 1 is recruited at MAM in order to bind MAVS and initiate a signaling cascade leading to the up-regulation of type 1 interferon and other proinflammatory cytokines. Interestingly, the hepatitis C virus NS3/4A protease, involved in the cleavage of MAVS to inhibit a strong antiviral response, was shown to target MAM in HuH7

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cells (Horner et al. 2011), suggesting that MAM participate to the regulation of innate immune signalling. In addition, inflammation at different stages in the development of

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NAFLD involve activation of the inflammasome (Xiao and Tipoe 2016), a multimolecular complex acting as a platform for the activation of signaling pathway leading to the release of pro-inflammatory cytokines such as IL-1 and IL-18 (Schroder and Tschopp 2010).

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Interestingly, it was shown that during the activation of the inflammasome, important proteins of this process, such as NLRP3 and ASC proteins, co-localized to the MAM fractions in a

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human THP1 macrophage cell line (Zhou et al. 2011), suggesting a potential role of MAM in the development and progression of NAFLD. Consequently, the involvement of MAM in

dysregulation.

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inflammation and antiviral immunity may participate and exacerbate hepatic metabolic

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8. The ER-mitochondria axis as a therapeutic target to fight hepatic metabolic diseases

As both organelle function and communication play an important role in the control of glucose homeostasis, their targeting could represent an efficient way to improve hepatic metabolic diseases. In agreement, pharmacological approaches alleviating ER and mitochondrial stresses were shown to improve hepatic insulin signaling. Among pharmacological approaches targeting mitochondrial stress, different molecules with antioxidant and/or anti-inflammatory actions, such as carnitine, Coenzyme Q, a-lipoic acid, N-acteylcysteine and mitoQ have shown beneficial effects on hepatic metabolism in both mice and humans (Chang et al. 2015). Several drugs targeting ER stress have been shown to improve hepatic insulin sensitivity in rodents and humans. For example, chemical chaperones such as tauroursodeoxycholate and

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ACCEPTED MANUSCRIPT 4-phenylbutyrate enhanced hepatic insulin sensitivity and glucose homeostasis in HFD mice (Ozcan et al. 2006) and in obese patients (Kars et al. 2010). Targeting MAM to improve metabolic disease is an attractive strategy. However, we need

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before to better characterize the intracellular triggers that regulate ER-mitochondria interactions. Actually, very few data are available on the physiological regulations of MAM

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within cells. As discussed above, insulin (Tubbs et al. 2014), growth factors (Betz et al. 2013) and glucose levels (Theurey et al. 2016) are able to modulate ER-mitochondria interactions. Therefore, identifying their mechanisms of action could reveal new intracellular targets to modulate MAM integrity and function. However, as no proteins are exclusively expressed at

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MAM, their modulation may have unspecific effects. Modulating proteins specifically at MAM interface could be an interesting alternative; however how to perform is still unclear. It

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was proposed that a mitochondrial targeting sequence found in DGAT2 was required for its targeting to MAM (Stone et al. 2009), whereas a functional lipid transfer domain was required for the localization of oxysterol-binding protein-related proteins at MAM interface (Galmes et

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al. 2016). However, it is not known whether it is a general mechanism for other MAM proteins. In addition, it was recently suggested that palmitoylation of cysteine residues in

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some ER proteins, such as thioredoxin and calnexin, was required for their enrichment at MAM in mammalian cells (Lynes et al. 2012). While searching the mechanisms underlying

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glucose-mediated regulation of MAM integrity in HuH7 cells, we found that PP2A is required for this effect (Theurey et al. 2016), suggesting that phosphorylation status of some MAM proteins could be important for the crosstalk between both organelles. However, the nature of

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these proteins is unknown at this stage, even if candidate targeted proteins of PP2A are present at MAM interface, such as Akt, IP3R and Drp1. Therefore, further studies are required to better understand the role of phosphorylation of MAM protein in the control of MAM integrity, and to identity PP2A target proteins at MAM interface.

9. Conclusion Until now, metabolic diseases have been studied by analysing individually ER and mitochondria, pinpointing mitochondria dysfunction or ER stress as the causal element for pathogenesis. Communication between both organelles start to emerge as a central mechanism for organelle and Ca2+ homeostasis and miscommunication between ER and mitochondria could be a primary cause for organelle dysfunction and altered signaling pathways leading to perturbations in hepatic glucose production, lipogenesis and inflammation, at least in mouse models. These hepatic alterations subsequently impact 22

ACCEPTED MANUSCRIPT systemic metabolism and global glucose homeostasis. Therefore, targeting ER-mitochondria contacts appear as an attractive strategy to improve more efficiently metabolic diseases. However, we are far from being able to offer a pharmacological molecule that modulate ER-

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mitochondria interactions in order to improve glucose homeostasis. But before, further studies are required to better understand the functional components of MAM, how they are

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dynamically regulated, and how their alterations contribute to metabolic diseases. In particular, the role of MAM in the control of insulin signaling in skeletal muscle, as well as their role in the control of insulin secretion by beta cells, are required to develop a comprehensive understanding of the function of MAM in metabolic tissues. Importantly, it

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still remains unclear whether preclinical observations in mice models are relevant in human diseases and importantly whether miscommunication between ER and mitochondria is a cause

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or a consequence of metabolic diseases. A complex challenge to answer to these questions is that MAM are dynamic structures, likely influenced by cell type, cellular context and physiological conditions. Moreover, the approaches to quantify physical and functional ER-

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mitochondria interactions are multiple and combining several of them seems required to avoid wrong interpretations. Particularly, caution should be brought to all genetic strategies that

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modulate ER-mitochondria interactions either to access their importance (e.g. adenoviral overexpression of MAM proteins) or to follow their function (e.g. adenoviral overexpression

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of organelle-targeted probes), as it could have by itself consequences on ER homeostasis, and subsequently disrupt organelle communication. Lastly, as no proteins are specific of MAM, their genetic modulation may have unspecific effects and complicate the interpretation of such

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experiments. Actually, the best strategy to modulate ER-mitochondria interactions, independently of genetic manipulations of endogenous proteins, is to use synthetic linkers. Indeed, Csordas et al. has initially developed a construct that encodes a monomeric red fluorescent protein fused to a OMM targeting sequence at the N terminus and to an ER sequence at the C terminus, in order to tighten the physical coupling between ER and mitochondria (Csordas et al. 2006). Furthermore, drug-inducible inter-organellar linkers that can accommodate different fluorescent proteins have also been developed, in order to deeper analyse the ER-mitochondria coupling in live cells (Csordas et al. 2010). Lastly, these linkers have been sub-cloned by others groups either in lentiviral expression vectors or in adenovirus and successfully used to reinforce ER-mitochondria coupling in both mammalian cells (Bao et al. 2016) and in liver (Arruda et al. 2014). Consequently, the scientific community has to keep all this in mind and try to resolve the above-mentioned important questions in order to improve our knowledge on MAM in health and diseases. 23

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Acknowledgments: I thank Geert Bultynck and the European Calcium Society (ECS) for the

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invitation to the 14th international meeting of the ECS.

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Funding: This work is supported by INSERM. This paper discusses results funded by the National Research Agency (ANR-09-JCJC-0116 to J.R.), the « Fondation pour la recherche

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médicale » and by Servier Laboratories.

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ACCEPTED MANUSCRIPT Figure Legends Figure 1: Control of glucose metabolism in the liver. In the liver, glucose is transported by GLUT2 and converted in G6P by the GCK. Then G6P is either oxidized through glycolysis or

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uses as a substrate for glycogen synthesis or for the pentose phosphate pathway. These pathways are mainly regulated by insulin signaling. In excess of glucose, insulin also

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stimulated de novo lipogenesis. At fasting state, the liver produces glucose through both glycogenolysis and gluconeogenesis, and oxidizes fatty acids coming from lipolysis of

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adipose tissue. These pathways are mainly regulated by glucagon.

Figure 2: Place of ER and mitochondria in the control of hepatic metabolism. Mitochondria play a key role in the final oxidation of substrates (glucose, fatty acids and

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amino acids), producing reduced coenzymes (NADH and FADH2) through the TCA cycle, which are further oxidized through aerobic respiration, thus generating ATP synthesis. ER is involved in the first step of gluconeogenesis due to the presence of G6Pase in ER membrane.

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Both organelles are involved in the control of Ca2+ homeostasis, which plays an important

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role in the control of hepatic glucose production.

Figure 3: Connections between MAM and hepatic metabolism. ER-mitochondria contact

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sites play a key role in insulin signaling, in glucose-sensing and subsequent metabolic adaptations, in autophagy, in immune signaling and in Ca2+ homeostasis, thus impacting

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whole-body glucose homeostasis. Key proteins at MAM interface are coloured in the same colour that the downstream signaling pathway that they regulate.

Figure 4: Role of ER-mitochondria miscommunication in hepatic metabolic diseases. ER-mitochondria miscommunication has been associated with hepatic insulin resistance, metabolic inflexibility, defective autophagy and the activation of both immune signaling and inflammasome.

Therefore,

ER-mitochondria

miscommunication

participates

to

the

physiopathology of type 2 diabetes mellitus (T2DM) and non-alcoholic fatty liver diseases (NAFLD), and could be involved in the development of hepatitis C infection (HCV) and hepato-carcinogenesis.

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ACCEPTED MANUSCRIPT Highlights 

The liver is important for whole-body glucose homeostasis in both healthy and

Not only ER and mitochondria function, but their structural and functional interactions, control hepatic metabolism

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pathological contexts

ER-mitochondria contact sites are important hubs of hormone and nutrient signalling, thus regulating hepatic metabolic homeostasis

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ER-mitochondria miscommunication is involved in hepatic metabolic diseases

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

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