Benoit Viollet
email: <benoit.viollet@inserm.fr

Bruno Guigas

Jocelyne Leclerc

Sophie Hébrard

Louise Lantier

Rémi Mounier

Fabrizio Andreelli

Marc Foretz

R Mi Mounier

AMP-activated protein kinase in the regulation of hepatic energy metabolism: from

Keywords: AMP-Activated Protein Kinases, chemistry, genetics, metabolism, Aminoimidazole Carboxamide, analogs & derivatives, metabolism, Animals, Dyslipidemias, drug therapy, metabolism, physiopathology, Energy Metabolism, physiology, Fatty Liver, drug therapy, metabolism, physiopathology, Gluconeogenesis, physiology, Glucose, metabolism, Homeostasis, Humans, Hypoglycemic Agents, metabolism, Lipid Metabolism, Liver, cytology, enzymology, Liver Cirrhosis, drug therapy, metabolism, physiopathology, Mitochondria, metabolism, Protein Conformation, Protein Subunits, chemistry, genetics, metabolism, Ribonucleotides, metabolism MP-activated protein kinase, energy metabolism, hepatic glucose production, fatty liver, liver, therapeutic, type 2 diabetes

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Introduction

The ability to store nutrients is vital to survival for all organisms in periods of acute or prolonged shortage of nutrient supply. In mammals, the liver is a major organ responsible for maintaining both short-term and long-term whole-body energy needs by its capacity to induce a number of changes in terms of stored and released energy in the body under widely changing physiological conditions.

Alterations in energy intake induce a number of metabolic and hormonal changes in the body. The liver functions as a mediator between dietary, as well as endogenous, sources of energy and the extrahepatic organs that continuously need energy, primarily the brain.

Metabolically, the liver is the most versatile organ of the whole body with a metabolism cycling daily between the fed and fasted states. In the fed (anabolic) state, digestion yields simple sugars that are converted to pyruvate (via glycolysis), which is either oxidized to provide energy or channelled into pathways for synthesis of fatty acids (via lipogenesis) for storage. Excess glucose is also stored as glycogen (the total hepatic glucose store is only sufficient for a few hours use), providing a short-term source of carbohydrates for emergency use. The coordinated regulation of these processes allows for efficient utilization of dietary carbohydrate. Hepatic fuel metabolism changes considerably under the different nutritional situations. In the fasted (catabolic) state, the liver becomes a glucose producer (breakdown of stored hepatic glycogen and synthesis of glucose from lactate, amino acids, glycerol, and pyruvate), lipogenesis is slowed down, and fatty acid oxidation and ketogenesis are activated to supply energy and metabolic fuels for extrahepatic organs. Because the liver is the metabolic factory of the body, alterations in liver function clearly affect whole-body metabolism and energy homeostasis and underlie the development of metabolic diseases, including type 2 diabetes and the metabolic syndrome. Hence, understanding the mechanisms regulating hepatic energy metabolism is a prerequisite for developing new pharmacological strategies aimed at treating these metabolic diseases.

Over the last few years, a growing body of evidence indicates that AMP-activated protein kinase (AMPK), a serine threonine kinase comprising a catalytic subunit and regulatory and subunits, represents a point of convergence of regulatory signals monitoring

α β γ
systemic and cellular energy status. AMPK functions as a fuel sensor in most tissues and organs, including liver, skeletal muscle, heart, hypothalamus and adipose tissue, where it inhibits anabolic pathways and stimulates catabolic pathways in response to limited energy availability, thus simultaneously sparing limited energy resources and acquiring extra energy. AMPK acts first by directly affecting enzymes activities, involved in carbohydrate, lipid and protein biosynthesis, and second by longer-term transcriptional control of key players of these metabolic pathways. AMPK emerges as a major regulator of glucose and lipid metabolism and represents an attractive target for therapeutic intervention in the treatment of hepatic disorders. Important progress has recently been made in the comprehension of the pathophysiological role of AMPK in hepatic energy metabolism and this review provides a general overview of AMPK regulation, its activators and its functions in the liver.

Structure and regulation of AMPK in the liver

AMPK exists as a heterotrimeric complex consisting of a catalytic subunit and two regulatory subunits and involved in

α β γ
heterotrimer formation and ligand sensing. The conventional serine/threonine kinase activity of AMPK is supported by the subunit which α is characterized by the presence of a threonine residue within the activation loop of the kinase domain. The C-terminal region of subunit α is required for the association with the regulatory and subunits. The subunit contains a C-terminal region acting as scaffold binding

β γ β α
and subunits and a central region that allowed AMPK complex to bind glycogen but this has been recently disputed in the liver ( γ

Parker et

). The subunit contains four tandem repeats known as cystathionine -synthase (CBS) motifs which bind AMP or ATP al., 2007 γ β molecules in a competitive manner. Recent structural studies revealed that two sites on the subunit bind either AMP or Mg.ATP, a third γ site contains a tightly bound AMP that does not exchange ( ). Isoforms of all three subunits encoding by different genes [START_REF] Xiao | Structural basis for AMP binding to mammalian AMP-activated protein kinase[END_REF] α 1, 2, 1, 2, 1, 2, 3) have been identified in mammals that allow for the generation of 12 different heterotrimeric combinations. These the various hepatic AMPK complexes combination have been reported. Nevertheless, it should be noted that AMPK complexes distribution can be regulated intracellularly with AMPK 2-containing complexes present in both nucleus and cytoplasm raising the α possibility of direct regulation of gene transcription ( ) and with AMPK 1-containing complexes localized in the cytoplasm [START_REF] Salt | AMP-activated protein kinase: greater AMP dependence, and preferential nuclear localization, of complexes containing the alpha2 isoform[END_REF] α but also at the plasma membrane ( , ). Changes in the subcellular localization of AMPK 2 in [START_REF] Evans | Does AMP-activated protein kinase couple inhibition of mitochondrial oxidative phosphorylation by hypoxia to calcium signaling in O2-sensing cells?[END_REF][START_REF] Hallows | Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells[END_REF] α response to specific stimuli appear to be conserved from yeast to mammals via a mechanism involving the interaction with the regulatory β subunits ( , ). In mammalian muscle cells, AMPK 2 bound to the 2 subunit translocates to the Suzuki et al., 2007Vincent et al., 2001 α β nucleus in a manner dependent on a nuclear localization signal that is present in AMPK 2 but not in AMPK 1 subunit. AMPK 2 bound to α α α the 1 subunit is anchored into the cytoplasm at the outer mitochondrial membrane through the myristoylation of 1 subunit. These data ). It has become evident in the last years that LKB1 plays a [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF][START_REF] Shaw | The tumor suppressor LKB1 kinase directly activates AMP-activated kinase and regulates apoptosis in response to energy stress[END_REF][START_REF] Woods | LKB1 is the upstream kinase in the AMP-activated protein kinase cascade[END_REF] crucial role in activating AMPK to control glucose and lipid metabolism in the liver ( , ). It has been [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF][START_REF] Imai | LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells[END_REF] suggested that LKB1 may be constitutively active ( , ) but recent studies indicates that cytosolic [START_REF] Lizcano | LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1[END_REF][START_REF] Sakamoto | Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR[END_REF] localization and activity of LKB1 can be governed by LKB1 acetylation status in the liver (

). CaMKK is viewed as an [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization and activity of LKB1; possible role in AMP-activated protein kinase activation[END_REF] β alternate upstream kinase that could also phosphorylate Thr-172 and activate AMPK in intact cells by an AMP-independent manner in response to increased intracellular Ca concentrations ( , , ). However, CaMKK 2 + [START_REF] Hawley | Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade[END_REF][START_REF] Hurley | The Ca2 /calmodulin-dependent protein kinase kinases are AMP-activated protein + kinase kinases[END_REF][START_REF] Woods | C 2 /calmodulin-dependent protein kinase kinase-beta acts α + upstream of AMP-activated protein kinase in mammalian cells[END_REF] is highly expressed in neural tissue and the role for Ca -mediated AMPK activation in the liver remains to be investigated. The third

β 2 +
potential upstream kinase is TAK1, which activates the homologue of AMPK, the SNF1 complex, when expressed in the S. cerevisiae yeast (

), and could also phosphorylate Thr-172 and activate AMPK in mouse embryonic fibroblasts ( Momcilovic et al., 2006 Xie et al., ) but its role as an upstream kinase remains controversial ( ). In addition to phosphorylation, AMPK is 2006 [START_REF] Goransson | Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase[END_REF] allosterically activated by AMP which binds to the regulatory subunit. Binding of AMP to AMPK induces a conformational change in γ the kinase domain that protects AMPK from dephosphorylation of Thr-172 ( ), probably catalysed by a form of protein [START_REF] Riek | Structural properties of AMP-activated protein kinase: dimerization, molecular shape, and changes upon ligand binding[END_REF] phosphatase-2C (

). The combination of the allosteric and phosphorylation effects causes >1000-fold increase in kinase Sanders et al., 2007 activity ( ) allowing to respond to small changes in cellular energy status in a highly sensitive manner. [START_REF] Suter | Dissecting the role of 5 -AMP for allosteric stimulation, activation, and deactivation of 12 15[END_REF]

Activators of AMPK in the liver

AMPK is activated in response to a variety of metabolic stresses that typically, but not exclusively, change the cellular AMP:ATP ratio, either by increasing ATP consumption (activation of biosynthetic pathways) or reducing ATP production following hypoxia, glucose deprivation and inhibition of mitochondrial oxidative phosphorylation with metabolic poisons (arsenite, oligomycin, dinitrophenol, azide and antimycin A) (

). AMPK plays a central role in the metabolic adaptation to acute and chronic nutritional Towler and Hardie, 2007 stresses. For instance, AMPK is activated in the liver by the metabolic challenges imposed by either a 24-h fast ( , [START_REF] Munday | The short-term regulation of hepatic acetyl-CoA carboxylase during starvation and re-feeding in the rat[END_REF] ) and dietary energy restriction (

). However, in other studies, fasting and caloric restriction did not Witters et al., 1994[START_REF] Jiang | Dietary energy restriction modulates the activity of AMP-activated protein kinase, Akt, and mammalian target of rapamycin inmammary carcinomas, mammary gland, and liver[END_REF] activate liver AMPK ( , ). Interestingly, it has been noted that the metabolic demands during muscular [START_REF] Gonzalez | Metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not include changes in AMPK activity[END_REF]To et al., 2007 work results in a decrease in energy status of the liver (

). Indeed, increased AMPK activation has been demonstrated [START_REF] Camacho | Energy state of the liver during short-term and exhaustive exercise in C57BL/6J mice[END_REF] following short-term exercise in rat liver ( , ). Long-term exercise also induced significant Carlson andWinder, 1999 Park et al., 2002 increase in AMPK phosphorylation as well as AMPK 1-and 2-subunit mRNA levels in the liver, suggesting a role for hepatic AMPK in α α

long-term exercise-induced hepatic adaptations ( ). Takekoshi et al., 2006 In the liver, the transition from the fasted to the fed state is also associated with physiological changes in energy dynamics. The reversal of the metabolic response to starvation includes alterations in enzyme phosphorylation states and changes in the concentration of key regulatory molecules. It has been reported that AMPK coordinates the changes in the activity and/or expression of a number of enzymes of lipid metabolism during refeeding ( , , , ). [START_REF] Munday | The short-term regulation of hepatic acetyl-CoA carboxylase during starvation and re-feeding in the rat[END_REF][START_REF] Gonzalez | Metabolic adaptations to fasting and chronic caloric restriction in heart, muscle, and liver do not include changes in AMPK activity[END_REF][START_REF] Assifi | AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats[END_REF][START_REF] Dentin | Polyunsaturated fatty acids suppress glycolytic and lipogenic genes through the inhibition of ChREBP nuclear protein translocation[END_REF] Refeeding causes a 40 decrease in the activity of AMPK 1 within 1 h, with additional decrease in both AMPK 1 and AMPK 2

% α α α
activities occurring between 1 and 24 h ( ). It is noteworthy that LKB1 activity could also be modulated during the [START_REF] Assifi | AMP-activated protein kinase and coordination of hepatic fatty acid metabolism of starved/carbohydrate-refed rats[END_REF] starvation-refeeding transition in association with its acetylation and intracellular localization (

). The modification of [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization and activity of LKB1; possible role in AMP-activated protein kinase activation[END_REF] AMPK activity during the starved-fed transition is compatible with earlier studies that linked these changes to increase in plasma insulin [ reported to decrease AMPK activity in isolated hepatocytes ( ) , and decrease in glucagon showed to activate Witters and Kemp, 1992

] [ hepatic AMPK (
) , possibly by a protein kinase A-induced phosphorylation and activation of LKB1 [START_REF] Sim | The low activity of acetyl-CoA carboxylase in basal and glucagon-stimulated hepatocytes is due to phosphorylation by the AMP-activated protein kinase and not cyclic AMP-dependent protein kinase[END_REF] Kimball et al.,

). In addition, changes in circulating levels of various hormones and adipokines during refeeding, may directly affect AMPK activity 2004 in the hepatocytes and could also contribute to the fasted-to-fed transition from catabolism to anabolism in the liver. It has been reported that hepatic AMPK can be regulated by ghrelin ( , ), endocannabinoids ( ), [START_REF] Barazzoni | Ghrelin regulates mitochondrial-lipid metabolism gene expression and tissue fat distribution in liver and skeletal muscle[END_REF][START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF][START_REF] Kola | Cannabinoids and ghrelin have both central and peripheral metabolic and cardiac effects via AMP-activated protein kinase[END_REF] glucocorticoids ( ), resistin ( ) and adiponectin ( ) ( ). [START_REF] Christ-Crain | AMP-activated protein kinase mediates glucocorticoid-induced metabolic changes: a novel mechanism in Cushing s syndrome[END_REF][START_REF] Banerjee | Regulation of fasted blood glucose by resistin[END_REF][START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] Table 1 As well as responding to metabolic stresses, hepatic AMPK activity is modulated by various pharmacological and natural drugs including AICAR (5-aminoimidazole-4-carboxamide-1--D-ribofuranoside) ( ), compound A-769662 (

[ ] β
Corton et al., 1995 Cool et al., ), polyphenols ( ) and two major classes of existing antidiabetic drugs biguanides (metformin and phenformin) ( 2006 [START_REF] Zang | Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice[END_REF] ) and thiazolidinediones (TZDs) ( ). [START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF][START_REF] Saha | Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo[END_REF] AICAR is a cell-permeable nucleoside which could be metabolically converted to 5-aminoimidazole-4-carboxamide ribotide (AICA ribotide or ZMP) by adenosine kinase. ZMP shares some structural similarities with 5 -AMP and can mimic all of the allosteric effects of 5 ). However, this view has been recently challenged, showing that treatment of [START_REF] Corton | 5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?[END_REF] hepatocytes with AICAR concentrations above 200 M depleted intracellular ATP levels ( , ).

μ [START_REF] Guigas | -Aminoimidazole-4-Carboxamide-1-beta[END_REF][START_REF] Mukhtar | Inhibition of glucokinase translocation by AMP-activated protein kinase is associated with phosphorylation of both GKRP and 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase[END_REF] Importantly, AMPK-independent effects of AICAR in the control of hepatic glucose uptake ( ), phosphatidylcholine [START_REF] Guigas | -Aminoimidazole-4-Carboxamide-1-beta[END_REF] synthesis (

) and autophagic proteolysis ( ) probably associated with its effect on ATP depletion where [START_REF] Jacobs | Inhibition of hepatic phosphatidylcholine synthesis by 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside is independent of AMP-activated protein kinase activation[END_REF][START_REF] Meley | AMP-activated protein kinase and the regulation of autophagic proteolysis[END_REF] also recently reported. Detrimental effect of AICAR is likely to be due to an AMPK-independent inhibition of mitochondrial oxidative phosphorylation induced by a concomitant effect of ZMP on the mitochondrial respiratory chain complex 1 and a drop of adenine nucleotides and inorganic phosphate following its phosphorylation ( ). Furthermore, we established that ZTP [START_REF] Guigas | AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside[END_REF] accumulation induced uncoupling of mitochondrial oxidative phosphorylation, an effect that could worsen the change in cellular energetic by decreasing the yield of ATP synthesis (

). In addition, it should be noted another important caveat in the use of [START_REF] Guigas | AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside[END_REF] AICAR because ZMP accumulation does affect certain other AMP-regulated enzymes such as glucokinase and fructose 1,6-bisphosphatase causing inhibition of glycolysis and gluconeogenesis in hepatocytes ( , ). Vincent et al., 1992Vincent et al., 1991 Therefore, like all pharmacological approaches, results of experiments using AICAR must be interpreted with caution and it remains to be clearly determined whether all AICAR effects described are mediated by AMPK. In addition, it has been demonstrated that uptake of AICAR into cells, mediated by the adenosine transport system, is blocked by a number of protein kinase inhibitors, thus preventing ZMP accumulation and subsequent AMPK activation ( , ). Fryer et al., 2002 Guigas, unpublished results Recently, a new class of AMPK activators have been identified after the screening of a chemical library using partially purified AMPK from rat liver. The non nucleoside thienopyridone A-592017 emerged from the initial screen, and after optimization the more potent one, A-769662, was developed. Specificity of this new compound has been tested on a panel of 76 protein kinases and the majority of kinases were not significantly affected at 10 M ( ) suggesting that A-769662 is a new specific activator of AMPK. Unlike μ [START_REF] Goransson | Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase[END_REF] other AMPK activators, A-769662 directly activates native AMPK complex purified from rat liver in cell-free essays by mimicking both effects of AMP on allosteric activation and inhibition of dephosphorylation of AMPK complex ( , Goransson et al., 2007 Sanders et al., ). However, A-769662 and AMP binding sites on AMPK complex are unlikely to be identical. Firstly, in the presence of saturating 2007 AMP concentration, A-769662 stimulated further AMPK activity ( , ). Second, A-769662 [START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF][START_REF] Goransson | Mechanism of action of A-769662, a valuable tool for activation of AMP-activated protein kinase[END_REF] allosterically activates AMPK complexes harboring a mutation in the 1 subunit that abolishes allosteric activation by AMP ( γ

Sanders et

). Interestingly, an AMPK complex lacking the glycogen binding domain of the subunit or containing a mutation of Ser-108 to al., 2007 β alanine (an autophosphorylation site within the glycogen binding domain of the 1 subunit) completely abolished the allosteric effect of β A-769662, while only partially reducing AMP activation (

). It has been reported that AMPK activation by A-769662 [START_REF] Sanders | Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade[END_REF] was independent of the upstream kinase utilized ( , ). Importantly, neither change in adenine Goransson et al., 2007 Sanders et ,  ). Some of the beneficial metabolic actions of in vitro in vivo [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Collins | Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5′ -AMP-activated protein kinase[END_REF][START_REF] Zang | Polyphenols stimulate AMP-activated protein kinase, lower lipids, and inhibit accelerated atherosclerosis in diabetic LDL receptor-deficient mice[END_REF] polyphenols are mediated by their ability to activate sirtuin 1 (SIRT1), a mammalian ortholog of Sir2 (silent information regulator 2), an NAD-dependent deacetylase that acts as a master metabolic sensor of NAD and modulates cellular metabolism and life span ( ). Indeed, activation of SIRT1 deacetylates LKB1, which [START_REF] Lan | SIRT1 modulation of the acetylation status, cytosolic localization and activity of LKB1; possible role in AMP-activated protein kinase activation[END_REF] in turn increases its cytoplasmic localization, its association with the LKB1 activator STRAD and its ability to activate AMPK.

Nevertheless, it should be noted that AMPK activation by polyphenols may involve distinct regulators. It has been shown that the EGCG-induced increase in AMPK phosphorylation is mediated by CaMKK activation through production of ROS (

). [START_REF] Collins | Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5′ -AMP-activated protein kinase[END_REF] Furthermore, the SIRT1 regulation of LKB1/AMPK signaling appears to be tissue-specific as resveratrol-stimulated AMPK activation in neurons was independent of SIRT1 (

). [START_REF] Dasgupta | Resveratrol stimulates AMP kinase activity in neurons[END_REF] The molecular pathway of AMPK activation by the antidiabetic drug metformin has been unclear ( ) but the direct [START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF] inhibition of the respiratory chain complex 1 by the biguanide constitutes the more convincing molecular mechanism, thus, leading to reduced cellular ATP and increased AMP ( , , ). This is supported by the fact El-Mir et al., 2000[START_REF] Brunmair | Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?[END_REF][START_REF] Owen | Evidence that metformin exerts its anti-diabetic effects through inhibition of complex 1 of the mitochondrial respiratory chain[END_REF] that metformin treatment in primary hepatocytes resulted in a decrease in ATP content (

). It should be noted that [START_REF] Guigas | -Aminoimidazole-4-Carboxamide-1-beta[END_REF] metformin could also exert AMPK-independent effects in the liver, probably due to its effect on cellular ATP levels (

). [START_REF] Guigas | -Aminoimidazole-4-Carboxamide-1-beta[END_REF] It has been reported that TZDs acutely activate AMPK by a mechanism independent of PPAR -regulated gene transcription, which γ appears to be associated with change in cellular energy state and could potentially increase AMPK activity ( , [START_REF] Brunmair | Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?[END_REF][START_REF] Saha | Pioglitazone treatment activates AMP-activated protein kinase in rat liver and adipose tissue in vivo[END_REF] Regulation of hepatic lipid metabolism AMPK phosphorylates and inactivates a number of metabolic enzymes involved in lipid metabolism in order to maintain liver energy status. AMPK coordinates the changes in the hepatic lipid metabolism and, so, regulates the partitioning of fatty acids between oxidative and biosynthetic pathways. 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) and acetyl CoA carboxylase (ACC), key enzymes in cholesterol and fatty acid synthesis respectively, were the first enzymes shown to be phosphorylated and inactivated by AMPK ( , ). Although the action of AMPK is achieved by rapid and direct [START_REF] Corton | Role of the AMP-activated protein kinase in the cellular stress response[END_REF][START_REF] Henin | Inhibition of fatty acid and cholesterol synthesis by stimulation of AMP-activated protein kinase[END_REF] phosphorylation of these enzymes, long-term effects have also been clearly demonstrated on gene expression. AMPK activation by AICAR or by the use of adenovirus-mediated overexpression of a constitutively active form of the 2 catalytic subunit (AMPK 2-CA)

α α
inhibits glycolytic and lipogenic gene expression ( , , , , [START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF][START_REF] Leclerc | Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase[END_REF]Woods et al., 2000[START_REF] Foretz | AMP-activated protein kinase inhibits the glucose-activated expression of fatty acid synthase gene in rat hepatocytes[END_REF], preserving glucose for ATP-producing pathways rather than for lipid synthesis. Of note, AMPK activation reduces [START_REF] Leclerc | The 5 -AMP-activated protein kinase inhibits the transcriptional stimulation by glucose in liver cells, acting through the glucose response ′ complex[END_REF] expression of sterol regulatory element-binding protein-1c (SREBP1c) ( , ) and carbohydrate response [START_REF] Zhou | Role of AMP-activated protein kinase in mechanism of metformin action[END_REF][START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF] element binding protein (ChREBP) ( , ), transcription factors playing a key role in the - [START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF][START_REF] Kawaguchi | Mechanism for fatty acid sparing effect on glucose-induced transcription: regulation of " " carbohydrate-responsive element-binding protein by AMP-activated protein kinase[END_REF] transcriptional regulation of lipogenic and glycolytic genes by insulin and glucose, respectively. In addition, it has been also reported that AMPK directly phosphorylates ChREBP and modulates its DNA binding activity ( ). Polyunsaturated fatty acids Kawaguchi et al., 2002 (PUFAs) are known to repress glycolytic and lipogenic gene expression and raised the question about a role of AMPK in mediating the effect of PUFAs on gene transcription. To address this point, we investigated the effect of PUFAs in mice lacking AMPK 1/ 2 catalytic α α subunits in the liver (AMPK 1 2 / ). In the absence of hepatic AMPK, PUFAs continue to inhibit glycolytic and lipogenic gene α α LS -expression indicating the existence of AMPK-independent mechanism(s) ( ). Viollet et al., 2006 In the cholesterol synthesis pathway, AMPK blocks the conversion of HMG-CoA to mevalonate. One could expect detrimental effect on cholesterol homeostasis when AMPK activity is altered. In total and liver-specific AMPK 2 KO mice, plasma levels for total and HDL α cholesterol are not statistically different compared to controls but have a tendency to be higher ( , [START_REF] Andreelli | Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin[END_REF]Viollet et al., 2003 ). This suggested that remaining 1 subunit activity in AMPK 2 KO mice is sufficient to control hepatic cholesterol synthesis and that α α

HMG-CoA reductase is a target for both catalytic isoforms of AMPK.

ACC is an important rate-controlling enzyme for the synthesis of malonyl-CoA, which is both a critical precursor for biosynthesis of ). Thus, these results indicate that AMPK regulates cellular lipid metabolism in large part through stimulation of fatty acid oxidation. However, recent studies showed that AMPK also stimulates a previously unrecognized pathway that involves constitutive exocytosis of lipoproteins ( ). This alternative pathway appears to be quantitatively [START_REF] Puljak | Evidence for AMPK-dependent regulation of exocytosis of lipoproteins in a model liver cell line[END_REF] important for intracellular lipid homeostasis and may function in parallel with fatty acid oxidation to regulate intracellular lipid content.

Regulation of hepatic mitochondrial biogenesis and function

As reported recently, AMPK has been also involved in the control of mitochondrial biogenesis in the liver, a role already evidenced in skeletal muscles (

). ). Interestingly, both mitochondrial respiration and basal level of ATP were also [START_REF] Guigas | AMP-activated protein kinase-independent inhibition of hepatic mitochondrial oxidative phosphorylation by AICA riboside[END_REF] Figure 1 significantly lower in hepatocytes isolated from AMPK 1 2 / mice compared with control mice ( ). This result α α LS -- [START_REF] Guigas | -Aminoimidazole-4-Carboxamide-1-beta[END_REF] emphasizes the importance of AMPK in the regulation of cellular energy homeostasis via the control of adaptive mitochondrial function. Thus, diminished AMPK activity may be an important contributing factor in the reduced mitochondrial function and dysregulated intracellular lipid metabolism associated with hepatic insulin resistance. The exact mechanisms by which deletion of AMPK in the liver affects PGC-1 expression and mitochondrial biogenesis remain to be elucidated but could be due to a lack of protein-protein interaction, α as suggested in skeletal muscle ( ). [START_REF] Jager | AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha[END_REF]

Regulation of hepatic glucose production

Recent results from various animal models confirm the physiological importance of hepatic AMPK for whole-body glucose homeostasis. It has been first shown that systemic infusion of AICAR in normal and insulin-resistant obese rats leads to the inhibition of hepatic glucose production (HGP) ( ). Then, several studies reported that AICAR treatment improve blood glucose [START_REF] Bergeron | Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats[END_REF] levels and glucose tolerance in animal models of type 2 diabetes. However, the relative roles played by skeletal muscle and liver in mediating the hypoglycemic effect of AICAR remain unclear. In muscle-specific AMPK dominant-negative mice, AICAR has a weaker hypoglycemic effect than in control animals ( ), suggesting that this compound lowers glycemia at least partly by increasing [START_REF] Mu | A role for AMP-activated protein kinase in contraction-and hypoxia-regulated glucose transport in skeletal muscle[END_REF] muscle glucose uptake. We demonstrated that AMPK 1 2 / mice display an impaired hypoglycemic response to AICAR, indicating α α LS -that the liver as well participate to the hypoglycemic effect of AICAR ( ). It has been also reported that short-term hepatic Figure 2 expression of AMPK-CA leads to mild hypoglycemia in normal mice ( , ) and abolishes [START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF]Viana et al., 2006 hyperglycemia in diabetic and STZ-induced diabetic mice ( ). The hypoglycemic effect of AMPK activation is ob/ob [START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF] consistent with the abolition of HGP, as suggested by the down-regulation of gluconeogenic gene expression (e.g., phosphoenolpyruvate carboxykinase PEPCK and glucose-6-phosphatase G6Pase ) and inhibition of glucose production in hepatic cells expressing AMPK-CA

[ ] [ ]
or treated with AICAR ( , , ). Using transcriptional profiling of hepatocyte cell [START_REF] Lochhead | 5-aminoimidazole-4-carboxamide riboside mimics the effects of insulin on the expression of the 2 key gluconeogenic genes PEPCK and glucose-6-phosphatase[END_REF][START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF]Viana et al., 2006 lines treated with AICAR, the dual specificity phosphatase Dusp4 and the immediate early transcription factor Egr1 have been identified as transcriptional targets of AMPK that are necessary for its ability to fully repress glucose production (

). Egr1 and [START_REF] Berasi | Inhibition of gluconeogenesis through transcriptional activation of EGR1 and DUSP4 by AMP-activated kinase[END_REF] Dusp4 act sequentially to mediate the inhibitory effect of AMPK on hepatic gluconeogenesis: increase in Egr1 protein is accompanied with increased binding to the Dusp4 promoter leading to Dusp4 expression and inhibition of PEPCK and G6Pase gene transcription (

). [START_REF] Berasi | Inhibition of gluconeogenesis through transcriptional activation of EGR1 and DUSP4 by AMP-activated kinase[END_REF] The importance of AMPK in the control of glucose output by the liver was recently highlighted by the potent effects of circulating adipocyte-derived hormones on whole-body glucose metabolism. A physiological link has been established between circulating resistin levels and hepatic AMPK activity in the maintenance of blood glucose (

). Low blood glucose levels and reduced [START_REF] Banerjee | Regulation of fasted blood glucose by resistin[END_REF] HGP in mice lacking resistin are likely related, at least in part, to activation of AMPK and decreased expression of gluconeogenic enzymes in the liver. Interestingly, adiponectin signaling opposes that of resistin in the control of glucose homeostasis and activation of AMPK is though to underlie the ability of this adipocyte hormone to reduce HGP (

). According to this result, adiponectin [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] failed to regulate HGP in liver-specific AMPK 2 KO mice (

). AdipoR1 and AdipoR2 serve as adiponectin receptors α α [START_REF] Yamauchi | Targeted disruption of AdipoR1 and AdipoR2 causes abrogation of adiponectin binding and metabolic actions[END_REF][START_REF] Bjursell | Opposing effects of adiponectin receptors 1 and 2 on energy metabolism[END_REF] Recently, the transcriptional co-activator transducer of regulated CREB activity 2 (TORC2) has been identified as a pivotal component of the gluconeogenic program ( ). TORC2 mediates CREB-dependent transcription of PGC1 and its subsequent [START_REF] Koo | The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism[END_REF] α gluconeogenic targets PEPCK and G6Pase. TORC2 is regulated by multiple signaling pathways in response to changes in glucagon and insulin levels or intracellular energy status. Phosphorylation on Ser171 confers binding of the protein 14-3-3 and sequestration of TORC2 out of the nucleus, resulting in inhibition of gluconeogenic gene expression. The kinase responsible for phosphorylating Ser171 of TORC2 was initially identified as salt-inducible kinase 2 (SIK2), an AMPK-related protein kinase (

). Recently, activation of [START_REF] Screaton | The CREB coactivator TORC2 functions as a calcium-and cAMP-sensitive coincidence detector[END_REF] AMPK was also found to phosphorylate TORC2 and regulate cytoplasmic translocation of TORC2 in primary hepatocyte cultures (Koo et ). Interestingly, in the absence of LKB1 in the liver, the expression of some of the key gluconeogenic genes are enhanced and the al., 2005 antidiabetic drug metformin no longer reduced blood glucose levels, demonstrating that hepatic LKB1/AMPK axis is required to maintain blood glucose levels (

). LKB1 phosphorylates and activates a number of kinases including AMPK and SIK family Shaw et al., 2005 members ( ), suggesting that genetic deletion of LKB1 in liver will result in the loss of multiple metabolic checkpoints [START_REF] Lizcano | LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1[END_REF] in the regulation of TORC2. In LKB1-deficient liver, AMPK was almost completely inactive, TORC2 was predominantly nuclear and fasting blood glucose levels were highly increased concomitantly with PEPCK and G6Pase gene expression (

). The [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF] reduction of TORC2 protein levels by injection of adenoviruses bearing small hairpin RNA for TORC2 in mice lacking LKB1 in the liver resulted in decreased PGC-1 protein levels accompanied by a significant decrease in fasting blood glucose levels, suggesting that TORC2 α is a critical downstream target of LKB1-dependent kinases in the control of gluconeogenesis (

). In addition, a recent [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF] study has shown that metformin regulates hepatic gluconeogenesis through AMPK-dependent regulation of the orphan nuclear receptor small heterodimer partner (SHP) (

). AMPK activation caused upregulation of SHP gene expression and this in turn, [START_REF] Kim | Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP[END_REF] inhibits PEPCK and G6Pase gene expression. It has been proposed that metformin could regulate the expression of hepatic gluconeogenic genes via both a short-term pathways, involving the protein stabilization or phosphorylation of AMPK-targeted transcription factors and coactivators, such as TORC2 ( ), HNF4 ( , ) and FoxO1 ( ) and a [START_REF] Shaw | The kinase LKB1 mediates glucose homeostasis in liver and therapeutic effects of metformin[END_REF] α [START_REF] Leclerc | Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase[END_REF][START_REF] Hong | AMP-activated protein kinase regulates HNF4alpha transcriptional activity by inhibiting dimer formation and decreasing protein stability[END_REF][START_REF] Barthel | Regulation of the forkhead transcription factor FKHR (FOXO1a) by glucose starvation and AICAR, an activator of AMP-activated protein kinase[END_REF] long-term effect exerted via the induction of SHP gene expression ( ) (

). Lastly, it should be noted that AMPK has [START_REF] Kim | Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP[END_REF] Figure 3 been also involved in the repression of gluconeogenic genes in response to ECGC treatment via a LKB1-independent pathway relying on CaMKK activation through production of ROS (

). [START_REF] Collins | Epigallocatechin-3-gallate (EGCG), a green tea polyphenol, suppresses hepatic gluconeogenesis through 5′ -AMP-activated protein kinase[END_REF]

Management of glucose homeostasis and dyslipidemia

The growing evidence that AMPK regulates the coordination of anabolic and catabolic metabolic processes represents an attractive therapeutic target for intervention in many conditions of disordered energy balance, including obesity, type 2 diabetes, and the metabolic syndrome (

). Support for this idea came from treatment with AICAR of various animal models of insulin Winder and Hardie, 1999 in vivo resistance, causing improvement of metabolic disturbances, at least partly by lowering blood glucose levels and increasing insulin sensitivity ( , , , , , ). [START_REF] Bergeron | Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats[END_REF][START_REF] Buhl | Long-term AICAR administration reduces metabolic disturbances and lowers blood pressure in rats displaying features of the insulin resistance syndrome[END_REF][START_REF] Iglesias | AICAR administration causes an apparent enhancement of muscle and liver insulin action in insulin-resistant high-fat-fed rats[END_REF][START_REF] Song | 5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice[END_REF][START_REF] Pold | Long-term AICAR administration and exercise prevents diabetes in ZDF rats[END_REF][START_REF] Fiedler | 5-aminoimidazole-4-carboxy-amide-1-beta-D-ribofuranoside treatment ameliorates hyperglycaemia and hyperinsulinaemia but not dyslipidaemia in KKAy-CETP mice[END_REF] Chronic treatment with AICAR was also shown to reduce plasma TG levels and adiposity in obese animals ( , Bergeron et al., 2001 Buhl et , ). Furthermore, similar beneficial metabolic effects have been also obtained by direct AMPK activation in the al., [START_REF] Angulo | Nonalcoholic fatty liver disease[END_REF][START_REF] Song | 5-Aminoimidazole-4-carboxamide ribonucleoside treatment improves glucose homeostasis in insulin-resistant diabetic (ob/ob) mice[END_REF] liver by overexpression of AMPK-CA or by the use of the AMPK activator A-769662, leading to decreased plasma glucose, plasma TG levels and adiposity in diabetic, obese and high fat fed mice ( , , ). These results [START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF][START_REF] Foretz | Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver[END_REF][START_REF] Yang | Chronic activation of AMP-activated protein kinase-alpha1 in liver leads to decreased adiposity in mice[END_REF] suggest that direct targeting of the liver is crucial for improving glucose and lipid metabolism, thereby preventing and treating type 2 diabetes. Accordingly, it appears that the major effect of intravenous AICAR infusion in type 2 diabetic patients was on the liver with inhibition of hepatic glucose output and decreased blood glucose levels (

). However, as liver samples were not collected [START_REF] Boon | Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients[END_REF] in this study, one can only speculate on the role of hepatic AMPK in this therapeutic effect of AICAR. Indeed, inhibition of hepatic glucose output could have resulted from AMPK-independent effects, such as inhibition of fructose-1,6-bisphosphatase by ZMP accumulation in the liver ( ) and see above Furthermore, AICAR administration could also stimulate hepatic fatty acid [ Vincent et al., 1991 ] oxidation and/or inhibit whole body lipolysis, thereby reducing plasma non-esterified fatty acids (NEFA) concentration [START_REF] Boon | Intravenous AICAR administration reduces hepatic glucose output and inhibits whole body lipolysis in type 2 diabetic patients[END_REF]. Unexpectedly, the effect of AICAR infusion on skeletal muscle glucose uptake was not evident and no changes in AMPK activity were detected in skeletal muscle. Similarly, in another study performed in healthy humans, AMPK activity was not modified in skeletal muscle after AICAR infusion (

),reinforcing the role of the liver in the action of AICAR. [START_REF] Cuthbertson | 5-aminoimidazole-4-carboxamide 1-beta-D-ribofuranoside acutely stimulates skeletal muscle 2-deoxyglucose uptake in healthy men[END_REF] Several reports indicate that metformin and TZDs can reduce risk for type 2 diabetes in people with glucose intolerance (Knowler et ). Interestingly, these two drugs have been reported to activate AMPK which is now considered as a new target for the al., 2002 management of insulin-resistant state. Consistent with these data, AMPK now appears to be activated by a multitude of natural products such as polyphenols and traditional Chinese medicine able to reduce blood glucose levels in obese and diabetic animal models (

). table 1 Pathophysiology and management of type 2 diabetes are complex. Increased concentrations of NEFA and inflammatory cytokines (e.g., tumor necrosis factor TNF and interleukin 6 IL-6 ) released by expanded visceral adipose tissue are crucial mechanisms involved in

α [ α] [ ]
the alteration of insulin signaling cascade ( ). Adiponectin is another important key of the pathophysiology of type 2 [START_REF] Stumvoll | Type 2 diabetes: principles of pathogenesis and therapy[END_REF] diabetes. Adiponectin has favourable effects on insulin resistance, hepatic steatosis and inflammation (

). It is now [START_REF] Kadowaki | Adiponectin and adiponectin receptors in insulin resistance, diabetes, and the metabolic syndrome[END_REF] well established that circulating levels of adiponectin are decreased in individuals with obesity and insulin resistance, suggesting that its deficiency may have a causal role in the etiopathogenesis of these diseases and their consequences. Thus, adiponectin replacement or restoration of endogenous secretion in humans may represent a promising approach to prevent and/or treat obesity, insulin resistance and the metabolic syndrome. The chronic effects of adiponectin on insulin resistance were investigated by generating adiponectin in vivo transgenic mice. Globular adiponectin transgenic mice showed partial amelioration of insulin resistance and diabetes ( ob/ob Yamauchi et

) and full length adiponectin showed suppression of insulin-mediated endogenous glucose production ( ). al., 2003[START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] Interestingly, it has been shown that metabolic and insulin-sensitizing effects of TZDs are in part mediated through increase in adiponectin availability. Indeed, TZDs metabolic effects are abolished in lipoatrophic mice suggesting that adipose tissue is an essential key of TZDs action ( ). In addition, TZDs can markedly enhance the expression and secretion of adiponectin and [START_REF] Chao | Adipose tissue is required for the antidiabetic, but not for the hypolipidemic, effect of thiazolidinediones[END_REF] in vitro in vivo through the activation of its promoter and also by blocking the suppressive effect of TNF on the production of adiponectin. Even if the α intracellular adiponectin signaling is not yet completely described, it is clearly demonstrated that adiponectin activates hepatic AMPK (

). In consequence, TZDs probably activate AMPK by an indirect mechanism, through increase of adiponectin levels. [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF] This is suggested by the reduction of AMPK activation by rosiglitazone in adiponectin KO mice ( ). A possible [START_REF] Nawrocki | Mice lacking adiponectin show decreased hepatic insulin sensitivity and reduced responsiveness to peroxisome proliferator-activated receptor gamma agonists[END_REF] similar mechanism of action could concern AICAR (but not metformin) which increase the expression of adiponectin in human adipose tissue as TZDs (

). [START_REF] Lihn | AICAR stimulates adiponectin and inhibits cytokines in adipose tissue[END_REF]

Management of fatty liver and hepatic fibrosis

Nonalcoholic fatty liver disease (NAFLD), the most common chronic liver disease in western countries, is considered as the hepatic manifestation of the metabolic syndrome (

). NAFLD is highly associated with obesity and insulin resistance and represent a [START_REF] Angulo | Nonalcoholic fatty liver disease[END_REF] broad spectrum of liver abnormalities ranging from simple hepatic steatosis (accumulation of TG inside hepatocytes) to a more severe form, nonalcoholic steatohepatitis (NASH), which is associated with hepatocyte damage, chronic inflammation, and fibrosis, and may progress to cirrhosis and liver failure. Studies in humans and various animal models have suggested that efforts to enhance insulin sensitivity might improve fatty liver disease, a situation frequently observed in patients with metabolic syndrome. The efficacy of metformin as a treatment for fatty liver disease has been confirmed in obese, mice, which develop hyperinsulinemia, insulin ob/ob resistance and fatty livers (

). Recent studies suggest that activation of AMPK accounts for the lipid-lowering effect of [START_REF] Lin | Metformin reverses fatty liver disease in obese, leptin-deficient mice[END_REF] metformin in cultured hepatocytes (

). Similarly, adiponectin restores insulin sensitivity and decreases hepatic steatosis by [START_REF] Zang | AMP-activated protein kinase is required for the lipid-lowering effect of metformin in insulin-resistant human HepG2 cells[END_REF] lowering TG content in the liver of obese mice ( , ). The action of adiponectin is linked to an [START_REF] Xu | The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice[END_REF][START_REF] Yamauchi | The fat-derived hormone adiponectin reverses insulin resistance associated with both lipoatrophy and obesity[END_REF] activation of hepatic AMPK, ultimately leading to decreased fatty acid biosynthesis and increased mitochondrial fatty acid oxidation (

). The role of AMPK has been confirmed by the decrease in liver TG content in lean and obese rodents during [START_REF] Yamauchi | Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase[END_REF]

AICAR infusion (

) and treatment with direct AMPK activator A-769662 ( ). In addition, it has been [START_REF] Bergeron | Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats[END_REF][START_REF] Cool | Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome[END_REF] recently demonstrated that resveratrol improves insulin sensitivity and protects against lipid accumulation in the liver of diabetic and high-fat fed animals concomitantly with activation of hepatic AMPK ( , ). These effects have been [START_REF] Baur | Resveratrol improves health and survival of mice on a high-calorie diet[END_REF][START_REF] Shang | Resveratrol improves non-alcoholic fatty liver disease by activating AMP-activated protein kinase[END_REF] correlated to increased mitochondrial number and SIRT1-mediated PCG-1 deacetylation, and decreased expression of lipogenic genes in α the liver. Similarly, the beneficial effect of betaine (trimethylglycine), a naturally occurring metabolite of choline, on high-sucrose diet-induced hepatic steatosis in mice is associated with increased activation of hepatic AMPK (

). Promising therapeutic [START_REF] Song | Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis[END_REF] effects of betaine supplementation on human NAFLD have been reported in a pilot clinical studies ( ) but the use [START_REF] Abdelmalek | Betaine, a promising new agent for patients with nonalcoholic steatohepatitis: results of a pilot study[END_REF] of betaine has been also described earlier in the treatment of alcoholic fatty liver disease (AFLD) (

). Although, the [START_REF] Barak | The effect of betaine in reversing alcoholic steatosis[END_REF] underlying causes of NAFLD and AFLD are clearly different, there are similarities in the disturbances of hepatic metabolism. This is supported by reports showing that treatement with adiponectin alleviated alcoholic and non-alcoholic fatty liver disease in mice, partly due to enhanced hepatic fatty acid oxidation and decreased fatty acid synthesis (

). Interestingly, chronic ethanol ingestion [START_REF] Xu | The fat-derived hormone adiponectin alleviates alcoholic and nonalcoholic fatty liver diseases in mice[END_REF] causes the impairment of AMPK-mediated regulation of fatty acid metabolism and may have an important role in the development of alcoholic fatty liver ( , ). Activation of AMPK by AICAR or metformin largely blocked the [START_REF] You | The role of AMP-activated protein kinase in the action of ethanol in the liver[END_REF][START_REF] Garcia-Villafranca | Ethanol consumption impairs regulation of fatty acid metabolism by decreasing the activity of AMP-activated protein kinase in rat liver[END_REF] ability of ethanol to increase levels of SREBP1c protein and expression of SREBP1c-regulated lipogenic enzymes and also appears to protect the liver from fatty changes associated with chronic alcohol use ( , ). Very recently, treatment [START_REF] You | The role of AMP-activated protein kinase in the action of ethanol in the liver[END_REF]Tomita et al., 2005 with resveratrol has been also shown to prevent the development of alcoholic liver steatosis through the SIRT1-AMPK signaling system associated with increased circulating adiponectin levels and enhanced expression of hepatic AdipoR1 and R2 receptors [START_REF] Ajmo | Resveratrol Alleviates Alcoholic Fatty Liver in Mice[END_REF].

It is now established that hepatic stellate cells (HSCs) play a crucial role in the fibrotic response during the progression of NASH ( ). Stimuli such as liver injury activate and transdifferentiate HSCs from vitamin A-storing [START_REF] Bataller | Liver fibrosis[END_REF]Brenner, 2005 Friedman, 2004 pericytes to myofibroblast-like cells. Once activated, human HSCs become proliferative, proinflammatory and profibrogenic through increased responsiveness to several soluble mediators (

). Despite the clear role of insulin resistance in the progression of [START_REF] Friedman | Mechanisms of disease: Mechanisms of hepatic fibrosis and therapeutic implications[END_REF] fibrosis, the molecular mechanisms involved in these conditions are still unclear. Adiponectin levels which have been directly correlated with insulin sensitivity are closely and inversely associated with degree of hepatic steatosis, necroinflammation, and fibrosis in NAFLD (

). Recent studies have demonstrated that in rat HSCs, adiponectin inhibits proliferation, migration, and expression of Targher et al., 2006 fibrogenic genes, and it may induce apoptosis of activated cells (

). Furthermore, administration of adiponectin [START_REF] Ding | The roles of leptin and adiponectin: a novel paradigm in adipocytokine regulation of liver fibrosis and stellate cell biology[END_REF] in vivo prevents proliferation of activated HSCs and reduces the development of fibrosis and liver damage during experimental steatohepatitis (Xu , ). In addition to adiponectin, AICAR and metformin significantly inhibited proliferation and migration of et al., 2003[START_REF] Kamada | Enhanced carbon tetrachloride-induced liver fibrosis in mice lacking adiponectin[END_REF] human HSCs in a dose-dependent manner (

). The beneficial effect of these compounds is linked to suppression of [START_REF] Caligiuri | Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells[END_REF] platelet-derived growth factor (PDGF) expression in HSCs and subsequent inhibition of type I procollagen secretion [START_REF] Caligiuri | Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells[END_REF]. Activation of AMPK by adiponectin plays a pivotal role in this molecular pathway since the dose-dependent [START_REF] Adachi | High molecular weight adiponectin inhibits proliferation of hepatic stellate cells via activation of adenosine monophosphate-activated protein kinase[END_REF] PDGF suppression is abrogated in the presence of dominant-negative AMPK (

) or by the knock-down of [START_REF] Adachi | High molecular weight adiponectin inhibits proliferation of hepatic stellate cells via activation of adenosine monophosphate-activated protein kinase[END_REF]

AMPK (

). Additional mechanisms, such as short-term inhibition of PDGF-mediated phosphorylation of ribosomal S6 [START_REF] Caligiuri | Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells[END_REF] kinase (p70S6K) and 4E binding protein-1 (4EBP1) (downstream effectors of the mammalian target of rapamycin complex 1 (mTORC1) pathway) by AICAR have been demonstrated (

). AICAR and metformin could also inhibit HSCs proliferation via [START_REF] Caligiuri | Adenosine monophosphate-activated protein kinase modulates the activated phenotype of hepatic stellate cells[END_REF] suppression of ROS production and subsequent inhibition of AKT pathway (

). Taken together, data from recent Adachi and Brenner, 2008 studies provide evidence that AMPK and adiponectin inhibit HSCs proliferation and hepatic fibrosis via multiple molecular mechanisms and suggest that use of drugs activating hepatic AMPK may have an additional rationale in their antifibrogenic properties.

Conclusion

Hepatic metabolism is highly sensitive to the variations in nutritional and hormonal signals reflecting changes in whole body energy demands. The AMPK system plays a major role in the regulation of hepatic glucose and lipid metabolism through its acute effect on energy metabolism and long-term effects on gene expression pattern in the liver. Activation of hepatic AMPK causes a switch from anabolic to catabolic state and entails metabolic consequences that are beneficial for diabetic patients, such as inhibition of HGP and restoration of blood glucose levels. AMPK is a key player in regulating energy balance at both cellular and whole-body levels, placing it as an ideal therapeutic target for the treatment of altered energy metabolism that occur in conditions like insulin resistance, type 2 diabetes and the metabolic syndrome. A large number of AMPK activators have been employed with promising results in animal models with obesity or type 2 diabetes and also in preliminary studies with diabetic patients. These encouraging results have provided the rationale for the development of new pharmacological but also nutritional AMPK activators. However, the widespread cellular functions of AMPK make its selective targeting in therapeutics a difficult one, with simultaneous advantageous and deleterious consequences being possible.

One of the major caveats in the use of AMPK activators is their possible role in the regulation of food intake. Stimulation of AMPK expressed in specific nuclei of the hypothalamus has been shown to increase food intake (

). The ideal AMPK [START_REF] Minokoshi | AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus[END_REF] activators will be administrated by the oral route, will activate AMPK at low concentration and be effective in specific target tissue, such as the liver but not the hypothalamus, and will also have minimal off-target and/or side effects. Finally, it is now becoming apparent that distinct expression patterns of AMPK isoforms exist between rodent and human liver. This finding will have significant implications for the understanding of the physiological role of AMPK complexes across species and for the design of new therapeutic agents targeting human AMPK complexes. The hypoglycemic effect of AICAR is dependent partially on hepatic AMPK AMPK 1 2 / mice deleted for both AMPK catalytic subunit in the liver are resistant to hypoglycemic effect of AICAR. AICAR tolerance α α LS -test was performed on overnight fasted AMPK 1 2 / mice injected intraperitonealy with AICAR at 0.12 g/kg and 0.25 g/kg and tail α α LS -blood was collected at 0, 20, 40, 60 and 80 min for determination of glucose concentration using a glucometer. p<0.005, p<0.001,

AMPK controls gluconeogenic gene expression by promoting TORC2 phosphorylation In response to fasting, glucagon promotes dephosphorylation of TORC2 on Ser171 through the PKA-dependent inhibition of SIK, leading (A) to its nuclear translocation and association with CREB transcription factor, driving the expression of the PGC1 co-activator. Expression of α the coactivator PGC-1 drives the transcription of key gluconeogenic enzymes such as PEPCK and G6Pase in association with the α transcription factor HNF4a and the forkhead family activator FoxO1.

Phosphorylation of TORC2 (Ser171) is controlled by insulin (B) through AKT2-dependent activation of SIK2 and by AMPK. Phosphorylated TORC2 is sequestered in the cytoplasm via a phosphorylation-dependent interaction with 14-3-3 proteins and degraded by 26S proteasome to inhibit gluconeogenic program. The antidiabetic drug metformin inhibits gluconeogenic gene expression through the LKB1/AMPK/TORC2 axis. TORC2 represents a central point in the control of hepatic gluconeogenesis that senses both hormone and energy status. AMPK activation also caused upregulation of SHP gene expression and this in turn, inhibits PEPCK and G6Pase gene expression.

  α β β γ γ γ heterotrimeric AMPK complexes show relative tissue specificity. AMPK 1 and 2-containing complexes account each for about half of α α total AMPK activity in rodent liver ( ) but AMPK 1-containing complexes are predominant in human hepatocytes (B. Cheung et al., 2000 αGuigas, unpublished results). Expression of the 1 and 1 regulatory subunits predominates in rodent liver (). To the β γ[START_REF] Cheung | Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding[END_REF] best of our knowledge, no selective association between catalytic 1 and 2 and regulatory and subunits or differences in the activity of

  of AMPK complexes may elicit distinct metabolic effects in tissues and cells depending on the expression of the different -and -subunit isoforms and illustrate the complexity of the molecular mechanisms by which energy metabolism can be α β regulated by AMPK. Regulation of AMPK activity involves both direct allosteric activation and reversible phosphorylation. Activation of AMPK requires phosphorylation on Thr-172 within the catalytic subunit and three upstream kinases have been identified corresponding to the tumor suppressor LKB1 kinase, CaMKK (C 2 /calmodulin-dependent protein kinase kinase ) and possibly TAK1 (TGF -activated kinase-1, a β α + β β member of the mitogen-activated protein kinase kinase family). LKB1 is mainly involved in Thr-172 phosphorylation following change in AMP:ATP ratio ( , ,

+

  Baur et al., ). It has been demonstrated that SIRT1 activation by resveratrol functions as an upstream regulator in the LKB1/AMPK signaling 2006 axis (). The ability of resveratrol to stimulate AMPK was mimicked by overexpression of SIRT1 and abolished by[START_REF] Hou | SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase[END_REF] knockdown or pharmacological inhibition of SIRT1. Furthermore, AMPK activation by resveratrol-activated SIRT1 is mediated by the upstream kinase, LKB1, but not CaMKK . The mechanism by which SIRT1 activation by polyphenols leads to LKB1-dependent AMPK β activation relies on the direct deacetylation of LKB1 by SIRT1 (

  fatty acids and a potent inhibitor of mitochondrial fatty acid oxidation via the allosteric regulation of carnitine palmitoyltransferase-1 (CPT-1) which catalyzes the entry of long-chain fatty acyl-CoA into mitochondria. Inhibition of ACC by AMPK leads to a fall in malonyl-CoA content and a subsequent decrease in fatty acid synthesis concomitantly with an increase in -oxidation ( , β[START_REF] Brusq | Inhibition of lipid synthesis through activation of AMP kinase: an additional mechanism for the hypolipidemic effects of berberine[END_REF] ). Exposure of hepatocytes to AICAR also produces a strong stimulation of long-chain fatty acid oxidation as a resultVelasco et al., 1997 of an increase in the rate of ketogenesis and an inhibition of triacylglycerol synthesis via phosphorylation of sn-glycerol-3-phosphate acyltransferase (GPAT) (). In addition, overexpression of AMPK 2-CA in the liver increases plasma ketone bodies[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF] α levels, a surrogate marker for hepatic -oxidation (). Interestingly, AMPK-induced ACC phosphorylation is impaired in β Foretz et al., 2005 hepatocytes deleted of both catalytic subunit, contributing to increase intracellular malonyl CoA levels and triglyceride (TG) accumulation in the liver (B. Viollet, M. Foretz, unpublished results

  First evidences were brought by treatment with resveratrol, a polyphenol constituent of Reznick and Shulman, 2006 red wine, which increases mitochondrial number in liver in association with AMPK activation ( ). Second, AMPK 1 2 Baur et al., 2006 α α LS / mice have reduced mitochondrial biogenesis as suggested by decreased transcript and protein expression of key mitochondrial -constituents such as peroxisome proliferator-activated receptor-coactivator-1 (PGC-1 ), cytochrome c oxidase I (COX I), COX IV and

α

  [START_REF] Andreelli | Liver adenosine monophosphate-activated kinase-alpha2 catalytic subunit is a key target for the control of hepatic glucose production by adiponectin and leptin but not insulin[END_REF] and functional differences in adiponectin-signaling pathways in the liver have been recently demonstrated with AdipoR1 being tightly linked to activation of AMPK pathways whereas AdipoR2 being more associated with the activation of peroxisome proliferator-activated receptor-(PPAR-) pathways ( , ).

Figure 1

 1 Figure 1 Alterations in mitochondrial biogenesis in hepatocytes from AMPK 1 2 / mice α α LS --Hepatocytes from wild-type (black bars) or AMPK 1 2 / mice (open bars) were isolated and AMPK 1 and 2 activities, oxygen (A) α α LS --α α consumption rate ( O ), intracellular ATP concentrations, cytochrome c (Cyt. C) and cytochrome oxidase (COX IV) contents, and citrate J 2 synthase activity (CS) were measured. In separate experiments, RNA was extracted from freeze-clamped livers of wild-type (black bars) (B) and AMPK 1 2 / (open bars) mice starved for 24h and quantitative real-time PCR was performed on cDNA. The mRNA contents for the α α LS -indicated gene were normalized for -actin and expressed relative to that in wild-type mice. The results of all experiments indicating that β

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

[START_REF] Muoio | AMP-activated kinase reciprocally regulates triacylglycerol synthesis and fatty acid oxidation in liver and muscle: evidence that sn-glycerol-3-phosphate acyltransferase is a novel target[END_REF]

Ackowledgements:

The authors were supported by the EXGENESIS Integrated Project (LSHM- CT-2004-005272) funded by the European Commission, Programme Nationale de Recherche sur le Diab te, Agence Nationale de la Recherche, Association de Langue Fran aise pour l Etude du Diab