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Thematic Review Section: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases| Volume 53, ISSUE 12, P2490-2514, December 01, 2012

AMP-activated protein kinase: an emerging drug target to regulate imbalances in lipid and carbohydrate metabolism to treat cardio-metabolic diseases

Thematic Review Series: New Lipid and Lipoprotein Targets for the Treatment of Cardiometabolic Diseases
Open AccessPublished:July 13, 2012DOI:https://doi.org/10.1194/jlr.R025882
      The adenosine monophosphate-activated protein kinase (AMPK) is a metabolic sensor of energy metabolism at the cellular as well as whole-body level. It is activated by low energy status that triggers a switch from ATP-consuming anabolic pathways to ATP-producing catabolic pathways. AMPK is involved in a wide range of biological activities that normalizes lipid, glucose, and energy imbalances. These pathways are dysregulated in patients with metabolic syndrome (MetS), which represents a clustering of major cardiovascular risk factors including diabetes, lipid abnormalities, and energy imbalances. Clearly, there is an unmet medical need to find a molecule to treat alarming number of patients with MetS. AMPK, with multifaceted activities in various tissues, has emerged as an attractive drug target to manage lipid and glucose abnormalities and maintain energy homeostasis. A number of AMPK activators have been tested in preclinical models, but many of them have yet to reach to the clinic. This review focuses on the structure-function and role of AMPK in lipid, carbohydrate, and energy metabolism. The mode of action of AMPK activators, mechanism of anti-inflammatory activities, and preclinical and clinical findings as well as future prospects of AMPK as a drug target in treating cardio-metabolic disease are discussed.

      Abbreviations:

      ACC
      acetyl CoA carboxylase
      ACE
      angiotensin-converting enzyme
      AICAR
      5-aminoimidazole-4-carboxamide riboside
      AIS
      autoinhibitory sequence
      AMPK
      5′ adenosine monophosphate-activated protein kinase
      ATGL
      adipocyte-triglyceride lipase
      CaMKKβ
      Ca2+ /calmodulin-dependent kinase β
      CBS
      cystathionone-β-synthase
      CDC42
      cell division control protein 42 homolog
      ChREBP
      carbohydrate response element binding protein 1
      CNS
      central nervous system
      CPT1
      carnitine palmitoyltransferase 1
      CRTC2
      CREB-regulated transcription coactivator-2
      CTRP9
      C1q/TNF-related protein 9
      CXCR4
      C-X-C chemokine receptor type 4
      DDI
      drug-drug interaction
      DGAT2
      diacylglycerol acyltransferase 2
      DIO
      diet-induced obese
      EGCG
      epigallocathechin-3-gallate
      eNOS
      endothelial nitric oxide synthase
      FOXO
      forkhead box
      GBD
      glycogen-binding domain
      GPAT
      glycerol phosphate acyl transferase
      GS
      glycogen synthase
      HDAC
      class IIA histone deacetylases
      HF
      heart failure
      HKII
      hexokinase II
      HMGR
      3-hydroxy-3-methylglutaryl-CoA reductase
      HNF-4α
      hepatic nuclear factor-4α
      hsCRP
      high-sensitivity C-reactive protein
      HSL
      hormone-sensitive lipase
      HUVEC
      human umbilical vein endothelial cell
      ICAM-1
      intercellular adhesion molecule-1
      IL
      interleukin
      LCFA
      long chain fatty acid
      LKB1
      liver kinase B1
      LPS
      lipopolysaccharide
      MetS
      metabolic syndrome
      MGAT
      monoacylglycerol acyltransferase
      MIF
      macrophage inhibitory factor
      mTOR
      mammalian target of rapamycin
      NAFLD
      nonalcoholic fatty liver disease
      NF-kB
      nuclear factor kappa B
      OCT1
      Organic cation transporter 1
      PEPCK
      phosphoenolpyruvate carboxylase
      PGC1-α
      PPAR-γ coactivator-1α
      SCD1
      steroyl CoA desaturase 1
      SDF1α
      stromal cell-derived factor 1 α
      SERCA
      sarcoplasmic/endoplasmic reticulum Ca2+ ATPase
      SIRT1
      sirtuin (silent mating type information regulation 2 homolog) 1
      SREBP1
      sterol regulatory element binding protein 1
      STRAD
      ste20 related adapter
      TBC1D4
      TBC1 domain family member 4
      TGF-β
      transforming growth factor-β
      TNF-α
      tumor necrosis factor-α
      TORC1
      target-of-rapamycin complex-1
      TSC2
      tuberous sclerosis complex 2
      UCP1
      uncoupling protein 1
      UPR
      unfolded protein response
      VCAM-1
      vascular cell adhesion molecule-1
      WAT
      white adipose tissue
      The pathophysiology of diabetes and obesity is a very complex process involving many pathways. Deregulation of these pathways gives rise to metabolic abnormalities termed as metabolic syndrome (MetS) (
      • Grundy S.M.
      Metabolic complications of obesity.
      ), characterized by a clustering of major risk factors for developing cardiovascular disease. Obesity, representing derangement in energy balances, is tightly linked with the development of type-2 diabetes through its ability to engender insulin resistance, leading to glucose intolerance and development of type-2 diabetes and dyslipidemia. Thus, insulin resistance is associated with a wide array of the pathophysiological sequelae, including hyperlipidemia, hypertension, and atherosclerosis (
      • Grundy S.M.
      Metabolic complications of obesity.
      ,
      • Han S.
      • Liang C.P.
      • Westerterp M.
      • Senokuchi T.
      • Welch C.L.
      • Wang Q.
      • Matsumoto M.
      • Accili D.
      • Tall A.R.
      Hepatic insulin signaling regulates VLDL secretion and atherogenesis in mice.
      ). Because the number of patients with MetS is growing at an alarming rate worldwide and 25% of the developed world's population have pathological conditions of MetS, there is global unmet medical need to find a molecule for monotherapy. Currently, patients with MetS are on 3–5 concomitant medications to treat conditions of hyperglycemia, hypertension, dyslipidemia, and elevated levels of pro-inflammatory proteins. This requirement for concomitant treatment is associated with poor compliance and risk of drug-drug interactions (DDI) (
      • Boyd S.T.
      • Scott D.M.
      • Pick A.M.
      Pill burden in low-income patients with metabolic syndrome and diabetes.
      ), leading to discontinuing therapy.
      5′ adenosine monophosphate-activated protein kinase (AMPK) is an enzyme that controls key players of metabolic pathways, thus emerging as a major regulator of glucose and lipid metabolism through multiple beneficial roles in the target tissues, liver, adipose, and muscle (
      • Srivastava R.A.
      • Srivastava N.
      Search for obesity drugs: targeting central and peripheral pathways.
      ). AMPK is a phylogenetically conserved serine/threonine kinase that mediates cellular energy homeostasis (
      • Hardie D.G.
      The AMP-activated protein kinase pathway—new players upstream and downstream.
      ,
      • Cheung P.C.
      • Salt I.P.
      • Davies S.P.
      • Hardie D.G.
      • Carling D.
      Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding.
      ) through the enzymatic activity stimulated by phosphorylation of threonine-172 (
      • Birnbaum M.J.
      Activating AMP-activated protein kinase without AMP.
      ,
      • Hardie D.G.
      Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status.
      ). AMPK was discovered as an enzyme whose activity catalyzes the phosphorylation and subsequent inhibition of acetyl-CoA carboxylase (ACC) and 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase, HMGR) (
      • Beg Z.H.
      • Allmann D.W.
      • Gibson D.M.
      Modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with cAMP and wth protein fractions of rat liver cytosol.
      ,
      • Carlson C.A.
      • Kim K.H.
      Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation.
      ). These activities attributable to AMPK together with regulation of insulin signaling make it an attractive target for the management of hepatic metabolic disorders and insulin resistance (
      • Viollet B.
      • Foretz M.
      • Guigas B.
      • Horman S.
      • Dentin R.
      • Bertrand L.
      • Hue L.
      • Andreelli F.
      Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders.
      ,
      • Towler M.C.
      • Hardie D.G.
      AMP-activated protein kinase in metabolic control and insulin signaling.
      ). The hypothalamic functions involved in the regulation of satiety may impact obesity and diabetes (
      • Kola B.
      Role of AMP-activated protein kinase in the control of appetite.
      ). AMPK activation is associated with the stimulation of hepatic fatty acid oxidation; inhibition of cholesterol synthesis, lipogenesis, and triglyceride synthesis (
      • Hardie D.G.
      AMP-activated protein kinase: a master switch in glucose and lipid metabolism.
      ); inhibition of adipocyte lipolysis and lipogenesis (
      • Daval M.
      • Foufelle F.
      • Ferre P.
      Functions of AMP-activated protein kinase in adipose tissue.
      ); stimulation of skeletal muscle fatty acid oxidation and muscle glucose uptake (
      • Hardie D.G.
      • Pan D.A.
      Regulation of fatty acid synthesis and oxidation by the AMP-activated protein kinase.
      ); and modulation of insulin secretion by pancreatic β-cells (
      • Düfer M.
      • Noack K.
      • Krippeit-Drews P.
      • Drews G.
      Activation of the AMP-activated protein kinase enhances glucose-stimulated insulin secretion in mouse beta-cells.
      ). All these attributes of AMPK show promise and offer opportunity to develop a selective AMPK activator to treat lipid and glucose abnormalities. This review highlights the multifaceted actions of AMPK and its role in the liver and other tissues, with special emphasis on lipid metabolism, inflammation, and mode of action.

      AMPK STRUCTURE AND REGULATION

      Mammalian AMPK exists as a heterotrimeric complex consisting of three subunits that occur as multiple isoforms. The catalytic α-subunits (α1/α2) and the regulatory β- (β1/β2) and γ-subunits (γ1/γ2/γ3) are encoded by seven different genes. The catalytic α subunit contains a highly conserved Ser/Thr kinase domain near the N-terminus in the activation loop (
      • Hawley S.A.
      • Davison M.
      • Woods A.
      • Davies S.P.
      • Beri R.K.
      • Carling D.
      • Hardie D.G.
      Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.
      ). Phosphorylation of Thr-172 within this loop is critical for enzyme activity (
      • Hawley S.A.
      • Davison M.
      • Woods A.
      • Davies S.P.
      • Beri R.K.
      • Carling D.
      • Hardie D.G.
      Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.
      ). In addition to regulation through phosphorylation, AMPKα-subunit activity has been shown to be self-regulated by a region identified C-terminal to the kinase domain (
      • Crute B.E.
      • Seefeld K.
      • Gamble J.
      • Kemp B.E.
      • Witters L.A.
      Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase.
      ,
      • Pang T.
      • Xiong B.
      • Li J.Y.
      • Qiu B.Y.
      • Jin G.Z.
      • Shen J.K.
      • Li J.
      Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits.
      ). This autoinhibitory sequence (AIS) is similar to ubiquitin-associated domains, which are highly conserved sequences within the AMPK subfamily of kinases. Studies performed using AIS deletion constructs show a greater than 10-fold increase in kinase activity as compared with constructs containing the AIS (
      • Crute B.E.
      • Seefeld K.
      • Gamble J.
      • Kemp B.E.
      • Witters L.A.
      Functional domains of the alpha1 catalytic subunit of the AMP-activated protein kinase.
      ,
      • Pang T.
      • Xiong B.
      • Li J.Y.
      • Qiu B.Y.
      • Jin G.Z.
      • Shen J.K.
      • Li J.
      Conserved alpha-helix acts as autoinhibitory sequence in AMP-activated protein kinase alpha subunits.
      ).
      The AMPKβ-subunits do not display catalytic activity, but they appear to be critical for AMPK αβγ complex assembly and glycogen sensing in the cell (
      • Bendayan M.
      • Londono I.
      • Kemp B.E.
      • Hardie G.D.
      • Ruderman N.
      • Prentki M.
      Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy.
      • Polekhina G.
      • Gupta A.
      • Michell B.J.
      • van Denderen B.
      • Murthy S.
      • Feil S.C.
      • Jennings I.G.
      • Campbell D.J.
      • Witters L.A.
      • Parker M.W.
      • et al.
      AMPK beta subunit targets metabolic stress sensing to glycogen.
      ). Domains located near the C terminus of the β-subunit have been shown to interact with regions on the α- and γ-subunits, which suggests that the β-subunit, in part, may function as a scaffold to support AMPK heterotrimeric complex assembly. The β-subunits also contain glycogen-binding domains (GBD) that mediate AMPK's association with glycogen, which could facilitate colocalization of AMPK with its substrate glycogen synthase (GS). This might allow the cell to coordinate the control of glycogen synthesis with glycogen levels and energy availability (
      • Bendayan M.
      • Londono I.
      • Kemp B.E.
      • Hardie G.D.
      • Ruderman N.
      • Prentki M.
      Association of AMP-activated protein kinase subunits with glycogen particles as revealed in situ by immunoelectron microscopy.
      • Polekhina G.
      • Gupta A.
      • Michell B.J.
      • van Denderen B.
      • Murthy S.
      • Feil S.C.
      • Jennings I.G.
      • Campbell D.J.
      • Witters L.A.
      • Parker M.W.
      • et al.
      AMPK beta subunit targets metabolic stress sensing to glycogen.
      ). Additionally, a potential role for β-subunit myristoylation has been suggested to be important for appropriate AMPK cell membrane localization and activation (
      • Oakhill J.S.
      • Chen Z.P.
      • Scott J.W.
      • Steel R.
      • Castelli L.A.
      • Ling N.
      • Macaulay S.L.
      • Kemp B.E.
      beta-Subunit myristoylation is the gatekeeper for initiating metabolic stress sensing by AMP-activated protein kinase (AMPK).
      ).
      The AMPKγ-subunits regulate enzyme activity through sensing relative intracellular ATP, ADP, and AMP concentrations. This is accomplished through complex interactions of adenine nucleotide with four repeated cystathionone-β-synthase (CBS) motifs occurring in pairs called Bateman domains (
      • Cheung P.C.
      • Salt I.P.
      • Davies S.P.
      • Hardie D.G.
      • Carling D.
      Characterization of AMP-activated protein kinase gamma-subunit isoforms and their role in AMP binding.
      ,
      • Kemp B.E.
      Bateman domains and adenosine derivatives form a binding contract.
      ,
      • Bateman A.
      The structure of a domain common to archaebacteria and the homocystinuria disease protein.
      ). It is now thought that the CBS motifs arrange in a manner that results in the formation of four adenine binding sites. Site 4 has a very high affinity for AMP and does not readily exchange for ATP or ADP, whereas sites 1 and 3 bind AMP, ADP, and ATP competitively (
      • Kemp B.E.
      • Oakhill J.S.
      • Scott J.W.
      AMPK structure and regulation from three angles.
      ). Site 2 seems to be largely unoccupied, and its role in regulating AMPK activity has not been fully elucidated (
      • Morrison A.
      • Yan X.
      • Tong C.
      • Li J.
      Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice.
      ,
      • Bonen A.
      • Han X.X.
      • Habets D.D.
      • Febbraio M.
      • Glatz J.F.
      • Luiken J.J.
      A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulin- and AICAR-stimulated fatty acid metabolism.
      ). Initially, AMPK activation was thought to be mediated solely by AMP binding; however, recent work has shown that both AMP and ADP binding result in conformational changes which activate AMPK in two ways: i) promoting Thr-172 phosphorylation by upstream kinases, and ii) antagonizing its dephosphorylation by protein phosphatase(s) (
      • Davies S.P.
      • Helps N.R.
      • Cohen P.T.
      • Hardie D.G.
      5′-AMP inhibits dephosphorylation, as well as promoting phosphorylation, of the AMP-activated protein kinase. Studies using bacterially expressed human protein phosphatase-2C alpha and native bovine protein phosphatase-2AC.
      ,
      • Sanders M.J.
      • Grondin P.O.
      • Hegarty B.D.
      • Snowden M.A.
      • Carling D.
      Investigating the mechanism for AMP activation of the AMP-activated protein kinase cascade.
      ). Conversely, only AMP has been shown to directly increase phosphorylated AMPK (Thr-172) activity through an allosteric mechanism. Kinetic enzyme assays have shown that allosteric activation by AMP results in a greater than 10-fold increase in activity, while the activation resulting from Thr-172 phosphorylation of the α-subunit is greater than 100-fold. In combination, these two activation mechanisms yield a greater than 1000-fold increase in activity (
      • Suter M.
      • Riek U.
      • Tuerk R.
      • Schlattner U.
      • Wallimann T.
      • Neumann D.
      Dissecting the role of 5′-AMP for allosteric stimulation, activation, and deactivation of AMP-activated protein kinase.
      ). In cells, intracellular adenine nucleotide ratios are very tightly regulated, and AMPK activation appears to be sensitive to small changes in these ratios. Although many ATP consuming processes produce ADP, adenylate kinase maintains AMP and ADP concentrations close to equilibrium in most cell types. Therefore, an increase in the ADP:ATP ratio is associated with a concomitant increase in AMP concentration, and the intracellular AMP:ATP ratio also increases. Given that both AMP and ADP activate AMPK through increasing phosphorylation and that ADP concentrations usually significantly exceed AMP concentrations (
      • Chen Z.P.
      • Stephens T.J.
      • Murthy S.
      • Canny B.J.
      • Hargreaves M.
      • Witters L.A.
      • Kemp B.E.
      • McConell G.K.
      Effect of exercise intensity on skeletal muscle AMPK signaling in humans.
      • Veech R.L.
      • Lawson J.W.
      • Cornell N.W.
      • Krebs H.A.
      Cytosolic phosphorylation potential.
      ), it is possible ADP may be the physiologically relevant adenine nucleotide activator of AMPK through its ability to maintain AMPK in its phosphorylated state (
      • Jin X.
      • Townley R.
      • Shapiro L.
      Structural insight into AMPK regulation: ADP comes into play.
      • Carling D.
      • Mayer F.V.
      • Sanders M.J.
      • Gamblin S.J.
      AMP-activated protein kinase: nature's energy sensor.
      ).

      AMPK phosphorylation/activation

      Multiple AMPK kinases have been shown to mediate AMPKα Thr-172 phosphorylation in vitro; however, only two have proven to be physiologically relevant in vivo. The major AMPK kinase is the ubiquitously expressed tumor suppressor liver kinase B1 (LKB1) (
      • Woods A.
      • Johnstone S.R.
      • Dickerson K.
      • Leiper F.C.
      • Fryer L.G.
      • Neumann D.
      • Schlattner U.
      • Wallimann T.
      • Carlson M.
      • Carling D.
      LKB1 is the upstream kinase in the AMP-activated protein kinase cascade.
      ,
      • Brajenovic M.
      • Joberty G.
      • Kuster B.
      • Bouwmeester T.
      • Drewes G.
      Comprehensive proteomic analysis of human Par protein complexes reveals an interconnected protein network.
      ). Although LKB1 activity is not dependent on phosphorylation of its activation loop, significant activity requires binding of the scaffold protein MO25 (
      • Boudeau J.
      • Baas A.F.
      • Deak M.
      • Morrice N.A.
      • Kieloch A.
      • Schutkowski M.
      • Prescott A.R.
      • Clevers H.C.
      • Alessi D.R.
      MO25alpha/beta interact with STRADalpha/beta enhancing their ability to bind, activate and localize LKB1 in the cytoplasm.
      ) and stabilization by Ste20-related adaptor (STRAD) protein binding (
      • Baas A.F.
      • Boudeau J.
      • Sapkota G.P.
      • Smit L.
      • Medema R.
      • Morrice N.A.
      • Alessi D.R.
      • Clevers H.C.
      Activation of the tumour suppressor kinase LKB1 by the STE20-like pseudokinase STRAD.
      ). Together, these three proteins form a constitutively active trimeric LKB1-STRAD-MO25 complex that has been shown to mediate AMPK Thr-172 phosphorylation in multiple mammalian systems. Quick to follow the discovery of LKB1 as an AMPK kinase was the identification of Ca2+/calmodulin-dependent kinase β (CaMKKβ), which phosphorylates Thr-172 in response to increased cytosolic Ca2+ concentrations independent of changes in adenine nucleotides (
      • Hawley S.A.
      • Pan D.A.
      • Mustard K.J.
      • Ross L.
      • Bain J.
      • Edelman A.M.
      • Frenguelli B.G.
      • Hardie D.G.
      Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase.
      • Hawley S.A.
      • Selbert M.A.
      • Goldstein E.G.
      • Edelman A.M.
      • Carling D.
      • Hardie D.G.
      5′-AMP activates the AMP-activated protein kinase cascade, and Ca2+/calmodulin activates the calmodulin-dependent protein kinase I cascade, via three independent mechanisms.
      ). It is thought that this mechanism may couple ATP-requiring processes, which often signal through increases in cytosolic Ca2+, with AMPK-mediated restoration of energy charge before a significant decrease in ATP levels occur (
      • Hardie D.G.
      Sensing of energy and nutrients by AMP-activated protein kinase.
      ). The physiological significance of another AMPK kinase, TAK1, remains to be established (
      • Momcilovic M.
      • Hong S.P.
      • Carlson M.
      Mammalian TAK1 activates Snf1 protein kinase in yeast and phosphorylates AMP-activated protein kinase in vitro.
      ).

      DOWNSTREAM METABOLIC TARGETS OF AMPK

      AMPK maintains energy homeostasis by activating catabolic pathways (ATP producing) and inhibiting anabolic pathways (ATP consuming). Downstream effects of AMPK activation can be tissue-dependent and impact, directly or indirectly, a broad range of cellular processes, including lipid and glucose metabolism, energy expenditure, immune response, and cell growth and polarity.
      Key lipid metabolic enzymes in the liver that are substrates for AMPK include ACC1 (
      • Munday M.R.
      • Campbell D.G.
      • Carling D.
      • Hardie D.G.
      Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase.
      ), and HMGR (
      • Clarke P.R.
      • Hardie D.G.
      Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.
      ), which are the rate-limiting enzymes of fatty acid and sterol synthesis, respectively. Glycerol phosphate acyl transferase (GPAT), a key enzyme in triglyceride and phospholipid synthesis, has also been shown to be susceptible to AMPK-mediated inhibitory phosphorylation (
      • Muoio D.M.
      • Seefeld K.
      • Witters L.A.
      • Coleman R.A.
      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.
      ). Additionally, AMPK activation in the liver was shown to directly increase fatty acid uptake through promoting FAT/CD36 translocation to the plasma membrane (
      • Bonen A.
      • Han X.X.
      • Habets D.D.
      • Febbraio M.
      • Glatz J.F.
      • Luiken J.J.
      A null mutation in skeletal muscle FAT/CD36 reveals its essential role in insulin- and AICAR-stimulated fatty acid metabolism.
      ) and to enhance fatty acid oxidation through the inhibitory phosphorylation of ACC2, the enzyme responsible for producing the CPT1 inhibitor malonyl-CoA (
      • Merrill G.F.
      • Kurth E.J.
      • Hardie D.G.
      • Winder W.W.
      AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle.
      ). In adipose tissue, AMPK phosphorylates lipases including hormone-sensitive lipase (HSL) (
      • Watt M.J.
      • Holmes A.G.
      • Pinnamaneni S.K.
      • Garnham A.P.
      • Steinberg G.R.
      • Kemp B.E.
      • Febbraio M.A.
      Regulation of HSL serine phosphorylation in skeletal muscle and adipose tissue.
      ) and adipocyte-triglyceride lipase (ATGL) (
      • Ahmadian M.
      • Abbott M.J.
      • Tang T.
      • Hudak C.S.
      • Kim Y.
      • Bruss M.
      • Hellerstein M.K.
      • Lee H.Y.
      • Samuel V.T.
      • Shulman G.I.
      • et al.
      Desnutrin/ATGL is regulated by AMPK and is required for a brown adipose phenotype.
      ). AMPK also coordinates long-term adaptation of lipid metabolism by downregulating mRNA levels of the transcriptional factor sterol regulatory element binding protein 1 (SREBP1) (
      • Morrison A.
      • Yan X.
      • Tong C.
      • Li J.
      Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice.
      ,
      • Zhou G.
      • Myers R.
      • Li Y.
      • Chen Y.
      • Shen X.
      • Fenyk-Melody J.
      • Wu M.
      • Ventre J.
      • Doebber T.
      • Fujii N.
      • et al.
      Role of AMP-activated protein kinase in mechanism of metformin action.
      ) and protein levels of hepatic nuclear factor-4α (HNF-4α) (
      • Leclerc I.
      • Lenzner C.
      • Gourdon L.
      • Vaulont S.
      • Kahn A.
      • Viollet B.
      Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase.
      ), which reduces lipogenic genes including PK, ACC1, and FAS. AMPK also regulates at the intersection of lipid and carbohydrate metabolism by phosphorylating and reducing DNA binding of the glucose-sensitive ChREBP (
      • Kawaguchi T.
      • Osatomi K.
      • Yamashita H.
      • Kabashima T.
      • Uyeda K.
      Mechanism for fatty acid “sparing” effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
      ), resulting in effects on hepatic lipogenic gene targets, which significantly overlap with effects observed on SREBP1 (Fig. 1).
      Figure thumbnail gr1
      Fig. 1Current model for AMPK activation. Constitutively active LKB1-STRAD-MO25 complex continuously phosphorylates AMPK, which in the absence of low energy signals is rapidly dephosphorylated by protein phosphatases. In times of energy stress, when the rate of ATP consumption exceeds the rate of production, the ADP:ATP ratio increases. Through the action of adenylate kinase, increases in ADP concentrations are associated with concomitant increases in AMP concentrations, and the intracellular AMP:ATP ratio also increases. This energy stress favors binding of ADP/AMP over ATP to the γ-subunit, leading to a conformational change that increases LKB1-STRAD-MO25-dependent phosphorylation and decreases susceptibility to protein phosphatase-dependent dephosphorylation. The combined effect of increased AMPKα phosphorylation and AMP allosteric activation can yield > 1,000-fold increase in activity. Activated AMPK then mediates its control on metabolism by phosphorylation of many downstream targets, resulting in restored energy homeostasis.
      AMPK regulates carbohydrate metabolism by increasing GLUT 4-dependent glucose uptake through i) phosphorylation of the Rab-GTPase-activating proteins AS160/TBC1D1 (
      • Sakamoto K.
      • Holman G.D.
      Emerging role for AS160/TBC1D4 and TBC1D1 in the regulation of GLUT4 traffic.
      ) and ii) increasing the glycolysis-stimulator fructose-2,6-bisphosphate concentrations through phosphorylation of specific 6-phosphofructose-2-kinase isoforms (
      • Marsin A.S.
      • Bouzin C.
      • Bertrand L.
      • Hue L.
      The stimulation of glycolysis by hypoxia in activated monocytes is mediated by AMP-activated protein kinase and inducible 6-phosphofructo-2-kinase.
      ). Additionally, AMPK inhibits glycogen synthase via phosphorylation (
      • J⊘rgensen S.B.
      • Nielsen J.N.
      • Birk J.B.
      • Olsen G.S.
      • Viollet B.
      • Andreelli F.
      • Schjerling P.
      • Vaulont S.
      • Hardie D.G.
      • Hansen B.F.
      • et al.
      The alpha2–5′AMP-activated protein kinase is a site 2 glycogen synthase kinase in skeletal muscle and is responsive to glucose loading.
      ). AMPK activation in the liver also results in the downregulation of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase (PEPCK) through multiple mechanisms, including phosphorylation of CREB-regulated transcription coactivator-2 (CRTC2) (
      • Koo S.H.
      • Flechner L.
      • Qi L.
      • Zhang X.
      • Screaton R.A.
      • Jeffries S.
      • Hedrick S.
      • Xu W.
      • Boussouar F.
      • Brindle P.
      • et al.
      The CREB coactivator TORC2 is a key regulator of fasting glucose metabolism.
      ) and class IIA histone deacetylases (HDAC-4, 5, and 7), which results in decreases in HDAC3 nuclear recruitment and FOXO activation (
      • Mihaylova M.M.
      • Vasquez D.S.
      • Ravnskjaer K.
      • Denechaud P.D.
      • Yu R.T.
      • Alvarez J.G.
      • Downes M.
      • Evans R.M.
      • Montminy M.
      • Shaw R.J.
      Class IIa histone deacetylases are hormone-activated regulators of FOXO and mammalian glucose homeostasis.
      ).
      Independent of effects on lipid and carbohydrate metabolism, AMPK also coordinates energy homeostasis by increasing cellular catabolic capacity through enhancing mitochondrial function and serving as a metabolic checkpoint for cell growth. Mitochondrial function is enhanced through upregulation of the transcriptional coactivator PPAR-γ coactivator-1α (PGC-1α) (
      • Zong H.
      • Ren J.M.
      • Young L.H.
      • Pypaert M.
      • Mu J.
      • Birnbaum M.J.
      • Shulman G.I.
      AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation.
      ), which promotes the expression of nuclear-encoded genes and mitochondrial biogenesis. It has recently been shown that AMPK also promotes mitophagy, a process that recycles damaged or surplus mitochondria (see Ref.
      • Hardie D.G.
      AMPK and autophagy get connected.
      for review). AMPK serves as a metabolic checkpoint inhibiting energy-demanding cellular functions, such as cell growth, migration, and immune response, when energy availability is limited. This has been shown to be mediated either by inhibition of the protein synthesis activator target-of-rapamycin complex-1 (TORC1) through the direct phosphorylation of its Raptor subunit (
      • Gwinn D.M.
      • Shackelford D.B.
      • Egan D.F.
      • Mihaylova M.M.
      • Mery A.
      • Vasquez D.S.
      • Turk B.E.
      • Shaw R.J.
      AMPK phosphorylation of raptor mediates a metabolic checkpoint.
      ) and its regulator tumor suppressor tuberous sclerosis complex 2 (TSC2) (
      • Inoki K.
      • Zhu T.
      • Guan K.L.
      TSC2 mediates cellular energy response to control cell growth and survival.
      ) or by indirect inhibition of NF-κB signaling.

      ROLE OF AMPK IN HEPATIC LIPID AND GLUCOSE METABOLISM

      Hepatic lipid and glucose dysregulation in metabolic syndrome

      Liver is a key organ involved in the lipid and glucose metabolism. It is the major site for storage and release of carbohydrates as well as for the synthesis of fatty acid; thus playing a key role in the control of whole-body energy metabolism (
      • Viollet B.
      • Foretz M.
      • Guigas B.
      • Horman S.
      • Dentin R.
      • Bertrand L.
      • Hue L.
      • Andreelli F.
      Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders.
      ). During over- and under-nutritional states, AMPK coordinates activity of enzymes in lipid and glucose metabolism (
      • Suchankova G.
      • Tekle M.
      • Saha A.K.
      • Ruderman N.B.
      • Clarke S.D.
      • Gettys T.W.
      Dietary polyunsaturated fatty acids enhance hepatic AMP-activated protein kinase activity in rats.
      ), and it regulates the partitioning of fatty acids between oxidative and biosynthetic pathways. Derangement in lipid and glucose metabolism in the liver, characterized by enhanced production and impaired catabolism, contributes to insulin resistance and dyslipidemia/hyperlipidemia, leading to diabetes and liver disease (
      • Reaven G.M.
      Pathophysiology of insulin resistance in human disease.
      ) and setting the stage of MetS (
      • Watanabe S.
      • Yaginuma R.
      • Ikejima K.
      • Miyazaki A.
      Liver diseases and metabolic syndrome.
      ) and associated cardiovascular complications (
      • Han S.
      • Liang C.P.
      • Westerterp M.
      • Senokuchi T.
      • Welch C.L.
      • Wang Q.
      • Matsumoto M.
      • Accili D.
      • Tall A.R.
      Hepatic insulin signaling regulates VLDL secretion and atherogenesis in mice.
      ). The pathophysiological conditions leading to the abnormal metabolic pathways and eventually diabetes and obesity are often caused by the hepatic elevation of the enzymes synthesizing lipid and glucose (
      • Muoio D.M.
      • Newgard C.B.
      Obesity-related derangements in metabolic regulation.
      ). Hyperlipidemia in MetS appears to occur as a result of overproduction of VLDL and glucose by the liver through lipogenic and gluconeogenic pathways (
      • Han S.
      • Liang C.P.
      • Westerterp M.
      • Senokuchi T.
      • Welch C.L.
      • Wang Q.
      • Matsumoto M.
      • Accili D.
      • Tall A.R.
      Hepatic insulin signaling regulates VLDL secretion and atherogenesis in mice.
      ,
      • Boizard M.
      • Le Liepvre X.
      • Lemarchand P.
      • Foufelle F.
      • Ferre P.
      • Dugail I.
      Obesity-related overexpression of fatty-acid synthase gene in adipose tissue involves sterol regulatory element-binding protein transcription factors.
      ).
      The role of liver in insulin resistance and lipoprotein overproduction has been demonstrated in animal disease models of excess energy intake (
      • Srivastava R.A.
      • Jahagirdar R.
      • Azhar S.
      • Sharma S.
      • Bisgaier C.L.
      Peroxisome proliferator-activated receptor-alpha selective ligand reduces adiposity, improves insulin sensitivity and inhibits atherosclerosis in LDL receptor-deficient mice.
      ,
      • Srivastava R.A.
      • He S.
      Anti-hyperlipidemic and insulin sensitizing activities of fenofibrate reduces aortic lipid deposition in hyperlipidemic Golden Syrian hamster.
      ) and in genetic animal models of diabetes, obesity, and hyperlipidemia (
      • Godbole V.Y.
      • Grundleger M.L.
      • Thenen S.W.
      Early development of lipogenesis in genetically obese (ob/ob) mice.
      ), in which the production of lipids and glucose by liver is elevated and clearance is impaired (
      • Han S.
      • Liang C.P.
      • Westerterp M.
      • Senokuchi T.
      • Welch C.L.
      • Wang Q.
      • Matsumoto M.
      • Accili D.
      • Tall A.R.
      Hepatic insulin signaling regulates VLDL secretion and atherogenesis in mice.
      ,
      • Srivastava R.A.
      • Srivastava N.
      Search for obesity drugs: targeting central and peripheral pathways.
      ). Therefore, liver not only plays an important role in dyslipidemia but also in the development of insulin resistance (
      • Viollet B.
      • Foretz M.
      • Guigas B.
      • Horman S.
      • Dentin R.
      • Bertrand L.
      • Hue L.
      • Andreelli F.
      Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders.
      ) through imbalances in energy status. AMPK plays an important role in balancing this liver-centric dysregulation by functioning as an energy sensor and augmenting fatty acid oxidation and inhibiting the biosynthesis of cholesterol, triglycerides, and glucose.

      Hepatic AMPK in glucose metabolism

      While AMPK-mediated effects are observed in multiple tissues, liver appears to be the major organ responsible for lipids and glucose production. AMPK-mediated balancing of lipid and glucose production in the liver is a key step in maintaining hepatic metabolism. This function of AMPK in the liver is brought about by modulating a number of genes, both at the enzyme phosphorylation level as well as transcriptional level (
      • Munday M.R.
      • Campbell D.G.
      • Carling D.
      • Hardie D.G.
      Identification by amino acid sequencing of three major regulatory phosphorylation sites on rat acetyl-CoA carboxylase.
      ,
      • Clarke P.R.
      • Hardie D.G.
      Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.
      ,
      • Zhou G.
      • Myers R.
      • Li Y.
      • Chen Y.
      • Shen X.
      • Fenyk-Melody J.
      • Wu M.
      • Ventre J.
      • Doebber T.
      • Fujii N.
      • et al.
      Role of AMP-activated protein kinase in mechanism of metformin action.
      ,
      • Leclerc I.
      • Lenzner C.
      • Gourdon L.
      • Vaulont S.
      • Kahn A.
      • Viollet B.
      Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase.
      ). For instance, phosphorylation of ChREBP by AMPK mediates inhibition of glucose-induced gene transcription (
      • Kawaguchi T.
      • Osatomi K.
      • Yamashita H.
      • Kabashima T.
      • Uyeda K.
      Mechanism for fatty acid “sparing” effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
      ). Over-function of SREBP1, a lipogenic transcription factor, is associated with the increased prevalence of dyslipidemia in type-2 diabetes (
      • Kaplan M.L.
      • Leveille G.A.
      Development of lipogenesis and insulin sensitivity in tissues of the ob/ob mouse.
      ). A key contributing factor in type-2 diabetes is the failure of insulin to suppress gluconeogenesis and hepatic glucose production. AMPK prevents overproduction through downregulation of the SREBP1 and inhibition of lipogenic and gluconeogenic enzymes as evidenced by the liver-specific AMPKα2 knockout mice that develop hyperglycemia and glucose intolerance as a result of increased hepatic glucose production (
      • Andreelli F.
      • Foretz M.
      • Knauf C.
      • Cani P.D.
      • Perrin C.
      • Iglesias M.A.
      • Pillot B.
      • Bado A.
      • Tronche F.
      • Mithieux G.
      • et al.
      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.
      ). Conversely, the stimulation of AMPK in wild-type mice dramatically reduces hepatic glucose output (
      • Foretz M.
      • Ancellin N.
      • Andreelli F.
      • Saintillan Y.
      • Grondin P.
      • Kahn A.
      • Thorens B.
      • Vaulont S.
      • Viollet B.
      Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.
      ).
      Results from animal models confirm the physiological importance of hepatic AMPK for whole-body glucose homeostasis. For instance, systemic infusion of an AMPK activator, 5-aminoimidazole-4-carboxamide riboside (AICAR), in normal and insulin-resistant obese rats decreased hepatic glucose production (
      • Bergeron R.
      • Previs S.F.
      • Cline G.W.
      • Perret P.
      • Russell 3rd, R.R.
      • Young L.H.
      • Shulman G.I.
      Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats.
      ), further corroborated by blood glucose lowering in mouse models of diabetes following liver-specific short-term activation of AMPK using adenovirus-mediated expression of a constitutively active form of AMPKα2 (
      • Foretz M.
      • Ancellin N.
      • Andreelli F.
      • Saintillan Y.
      • Grondin P.
      • Kahn A.
      • Thorens B.
      • Vaulont S.
      • Viollet B.
      Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.
      ). Additionally, liver-specific deletion of AMPKα2 caused mild hyperglycemia and glucose intolerance as a result of enhanced gluconeogensis (
      • Andreelli F.
      • Foretz M.
      • Knauf C.
      • Cani P.D.
      • Perrin C.
      • Iglesias M.A.
      • Pillot B.
      • Bado A.
      • Tronche F.
      • Mithieux G.
      • et al.
      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.
      ). These results demonstrate that hepatic AMPKα2 is essential to inhibit gluconeogenesis and maintain blood glucose levels in the physiological range. AMPK appears to be acting through LKB1, because lack of hepatic LKB1 abolished AMPK activity and resulted in increased levels of blood glucose (
      • Sakamoto K.
      • Goransson O.
      • Hardie D.G.
      • Alessi D.R.
      Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR.
      ,
      • Brunmair B.
      • Staniek K.
      • Gras F.
      • Scharf N.
      • Althaym A.
      • Clara R.
      • Roden M.
      • Gnaiger E.
      • Nohl H.
      • Waldhausl W.
      • et al.
      Thiazolidinediones, like metformin, inhibit respiratory complex I: a common mechanism contributing to their antidiabetic actions?.
      ). Furthermore, treatment with metformin, an AMPK activator, failed to reduce blood glucose in mice lacking LKB1, again suggesting the key role of LKB1 in the hepatic AMPK activation (
      • Sakamoto K.
      • Goransson O.
      • Hardie D.G.
      • Alessi D.R.
      Activity of LKB1 and AMPK-related kinases in skeletal muscle: effects of contraction, phenformin, and AICAR.
      ). Additionally, liver-specific deletion of AMPK α subunits (α1 and α2) in mice showed lack of efficacy following AICAR treatment, indicating crucial role of hepatic AMPK in the AICAR-mediated control of blood glucose levels (
      • J⊘rgensen S.B.
      • Viollet B.
      • Andreelli F.
      • Frosig C.
      • Birk J.B.
      • Schjerling P.
      • Vaulont S.
      • Richter E.A.
      • Wojtaszewski J.F.
      Knockout of the alpha2 but not alpha1 5′-AMP-activated protein kinase isoform abolishes 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranosidebut not contraction-induced glucose uptake in skeletal muscle.
      ). The suppression of gluconeogenesis by AMPK results from the inhibition of the transcription of PEPCK, the key regulatory gluconeogenic enzyme (
      • Shirai T.
      • Inoue E.
      • Ishimi Y.
      • Yamauchi J.
      AICAR response element binding protein (AREBP), a key modulator of hepatic glucose production regulated by AMPK in vivo.
      ). In addition, AMPK attenuates the synthesis of glycogen in hepatocytes by deactivation of glycogen synthase (
      • Bultot L.
      • Guigas B.
      • Von Wilamowitz-Moellendorff A.
      • Maisin L.
      • Vertommen D.
      • Hussain N.
      • Beullens M.
      • Guinovart J.J.
      • Foretz M.
      • Viollet B.
      • et al.
      AMP-activated protein kinase phosphorylates and inactivates liver glycogen synthase.
      ). Thus, AMPK downregulates expression of enzymes that are centrally involved in fatty acid synthesis and gluconeogenesis by inhibiting the transcription factors SREBP-1c (
      • Zhou G.
      • Myers R.
      • Li Y.
      • Chen Y.
      • Shen X.
      • Fenyk-Melody J.
      • Wu M.
      • Ventre J.
      • Doebber T.
      • Fujii N.
      • et al.
      Role of AMP-activated protein kinase in mechanism of metformin action.
      ), ChREBP (
      • Kawaguchi T.
      • Osatomi K.
      • Yamashita H.
      • Kabashima T.
      • Uyeda K.
      Mechanism for fatty acid “sparing” effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
      ), and HNF-4α (
      • Leclerc I.
      • Lenzner C.
      • Gourdon L.
      • Vaulont S.
      • Kahn A.
      • Viollet B.
      Hepatocyte nuclear factor-4alpha involved in type 1 maturity-onset diabetes of the young is a novel target of AMP-activated protein kinase.
      ), and by attenuating the activity of transcriptional coactivators (
      • Zong H.
      • Ren J.M.
      • Young L.H.
      • Pypaert M.
      • Mu J.
      • Birnbaum M.J.
      • Shulman G.I.
      AMP kinase is required for mitochondrial biogenesis in skeletal muscle in response to chronic energy deprivation.
      ,
      • Gwinn D.M.
      • Shackelford D.B.
      • Egan D.F.
      • Mihaylova M.M.
      • Mery A.
      • Vasquez D.S.
      • Turk B.E.
      • Shaw R.J.
      AMPK phosphorylation of raptor mediates a metabolic checkpoint.
      ), which inhibits the transcription of gluconeogenic enzymes. Thus, in the liver, AMPK normalizes glucose metabolism in disease states.

      Hepatic AMPK in lipid metabolism

      Lipid overproduction in the MetS is caused by elevated activity of enzymes of fatty acid and cholesterol synthesis, and these enzymes are controlled by AMPK (
      • Beg Z.H.
      • Allmann D.W.
      • Gibson D.M.
      Modulation of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity with cAMP and wth protein fractions of rat liver cytosol.
      ,
      • Carlson C.A.
      • Kim K.H.
      Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation.
      ,
      • Carlson C.L.
      • Winder W.W.
      Liver AMP-activated protein kinase and acetyl-CoA carboxylase during and after exercise.
      ). Leptin-deficient ob/ob mice provide an animal model of type-2 diabetes, exhibiting hyperglycemia, hyperinsulinemia, and obesity as a result of a high level of hepatic lipogenesis linked with increased expression of lipogenic and glycolytic genes (
      • Godbole V.Y.
      • Grundleger M.L.
      • Thenen S.W.
      Early development of lipogenesis in genetically obese (ob/ob) mice.
      ). Overexpression of constitutively active (CA)-AMPKα2 in the liver of ob/ob mice normalizes the expression pattern of these genes. Importantly, AMPK activation reduces expression of SREBP1c (
      • Zhou G.
      • Myers R.
      • Li Y.
      • Chen Y.
      • Shen X.
      • Fenyk-Melody J.
      • Wu M.
      • Ventre J.
      • Doebber T.
      • Fujii N.
      • et al.
      Role of AMP-activated protein kinase in mechanism of metformin action.
      ) and ChREBP (
      • Kawaguchi T.
      • Osatomi K.
      • Yamashita H.
      • Kabashima T.
      • Uyeda K.
      Mechanism for fatty acid “sparing” effect on glucose-induced transcription: regulation of carbohydrate-responsive element-binding protein by AMP-activated protein kinase.
      ) transcription factors, which play a key role in the transcriptional regulation of lipogenic and glycolytic genes, respectively. The role of ChREBP in hepatic lipogenesis was confirmed by improved hepatic steatosis and insulin resistance in ob/ob mice with liver-specific inhibition of ChREBP (
      • Dentin R.
      • Benhamed F.
      • Hainault I.
      • Fauveau V.
      • Foufelle F.
      • Dyck J.R.
      • Girard J.
      • Postic C.
      Liver-specific inhibition of ChREBP improves hepatic steatosis and insulin resistance in ob/ob mice.
      ) and activation of AMPK. This was further corroborated by the liver-specific AMPKα2 deletion in mice showing enhanced hepatic lipogenesis and increased plasma triglyceride levels and hepatic glucose production (
      • Andreelli F.
      • Foretz M.
      • Knauf C.
      • Cani P.D.
      • Perrin C.
      • Iglesias M.A.
      • Pillot B.
      • Bado A.
      • Tronche F.
      • Mithieux G.
      • et al.
      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.
      ). Conversely, overexpression of AMPKα2 in hepatocytes decreases plasma triglyceride levels (
      • Foretz M.
      • Ancellin N.
      • Andreelli F.
      • Saintillan Y.
      • Grondin P.
      • Kahn A.
      • Thorens B.
      • Vaulont S.
      • Viollet B.
      Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.
      ).
      Phosphorylation of ACC1 at Ser-79 and ACC2 at Ser-218 by AMPK leads to inhibition of ACC activity and decreased malonyl-CoA content, leading to reduced fatty acid biosynthesis and increased CPT1, the rate-limiting step in the import and oxidation of fatty acids in mitochondria (
      • Merrill G.F.
      • Kurth E.J.
      • Hardie D.G.
      • Winder W.W.
      AICA riboside increases AMP-activated protein kinase, fatty acid oxidation, and glucose uptake in rat muscle.
      ). Thus, a reduction in malonyl-CoA concentration and a subsequent increase in β-oxidation results in decreased triglyceride synthesis (Fig. 2). ACC2 knockout mice show increased fatty acid oxidation in muscle, heart, and liver, and they are lean and have reduced fat content despite eating 20–30% more food than the WT mice (
      • Choi C.S.
      • Savage D.B.
      • Abu-Elheiga L.
      • Liu Z.X.
      • Kim S.
      • Kulkarni A.
      • Distefano A.
      • Hwang Y.J.
      • Reznick R.M.
      • Codella R.
      • et al.
      Continuous fat oxidation in acetyl-CoA carboxylase 2 knockout mice increases total energy expenditure, reduces fat mass, and improves insulin sensitivity.
      ). The liver triglycerides were also reduced substantially in these mice. Further evidence of AMPK-mediated triglyceride lowering was obtained by infusion of AICAR in lean and obese rodents (
      • Bergeron R.
      • Previs S.F.
      • Cline G.W.
      • Perret P.
      • Russell 3rd, R.R.
      • Young L.H.
      • Shulman G.I.
      Effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside infusion on in vivo glucose and lipid metabolism in lean and obese Zucker rats.
      ). These results are consistent with ex vivo findings demonstrating the AICAR-induced inhibition of mitochondrial GPAT activity and subsequent inhibition of triacylglycerol synthesis (
      • Ruderman N.
      • Prentki M.
      AMP kinase and malonyl-CoA: targets for therapy of the metabolic syndrome.
      ). Further evidences supporting the role of AMPK in triglycerides lowering came from two separate studies: i) overexpression of AMPKα2 in the liver decreased plasma triglyceride and increased plasma ketone bodies, a surrogate marker for hepatic β-oxidation (
      • Foretz M.
      • Ancellin N.
      • Andreelli F.
      • Saintillan Y.
      • Grondin P.
      • Kahn A.
      • Thorens B.
      • Vaulont S.
      • Viollet B.
      Short-term overexpression of a constitutively active form of AMP-activated protein kinase in the liver leads to mild hypoglycemia and fatty liver.
      ); and ii) liver-specific AMPKα2 deletion increased plasma triglyceride levels and reduced plasma ketone bodies (
      • Andreelli F.
      • Foretz M.
      • Knauf C.
      • Cani P.D.
      • Perrin C.
      • Iglesias M.A.
      • Pillot B.
      • Bado A.
      • Tronche F.
      • Mithieux G.
      • et al.
      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.
      ). These observations further emphasize the critical role for AMPKα2 subunit in balancing between hepatic lipogenesis and β-oxidation. Thus, diminished AMPK activity may be an important contributing factor in the reduced mitochondrial function and deregulated intracellular lipid metabolism associated with hepatic insulin resistance.
      Figure thumbnail gr2
      Fig. 2Hepatic regulation of lipid and carbohydrate metabolism. Nutritional, hormonal, and pharmacological stimuli modulate AMPK activation, resulting in inhibition of FA and cholesterol synthesis and stimulation of FA oxidation. ACC inhibition lowers malonyl CoA, which in turn inhibits lipid synthesis and activates CPT1. Hepatic activation of AMPK also reduces glucose output. Both ACC and SCD1 are key enzymes in controlling liver lipids.
      Another player in the AMPK activation pathway is SIRT1. In human hepatoma cells, AMPK activation through SIRT1 decreased glucose induced FAS and triglyceride accumulation (
      • Imai K.
      • Inukai K.
      • Ikegami Y.
      • Awata T.
      • Katayama S.
      LKB1, an upstream AMPK kinase, regulates glucose and lipid metabolism in cultured liver and muscle cells.
      ). Overexpression of SIRT1 increased, while shRNA silencing of SIRT1 dramatically decreased LKB1, AMPK, and ACC phosphorylation, and glucose stimulated triglyceride accumulation (
      • Hou X.
      • Xu S.
      • Maitland-Toolan K.A.
      • Sato K.
      • Jiang B.
      • Ido Y.
      • Lan F.
      • Walsh K.
      • Wierzbicki M.
      • Verbeuren T.J.
      • et al.
      SIRT1 regulates hepatocyte lipid metabolism through activating AMP-activated protein kinase.
      ), suggesting an important role of AMPK-SIRT1 axis in hepatic lipid metabolism. Decreased phosphorylation of SIRT1 and LKB1 in mice fed high-fat diet, and subsequent phosphorylation of AMPK and ACC further support this concept. This study suggests a regulatory role for SIRT1 in high-fat diet-induced regulation of AMPK-dependent effects (
      • Yoneda M.
      • Guo Y.
      • Ono H.
      • Nakatsu Y.
      • Zhang J.
      • Cui X.
      • Iwashita M.
      • Kumamoto S.
      • Tsuchiya Y.
      • Sakoda H.
      • et al.
      Decreased SIRT1 expression and LKB1 phosphorylation occur with long-term high-fat diet feeding, in addition to AMPK phosphorylation impairment in the early phase.
      ). Furthermore, liver-specific SIRT1 knockout mice developed hepatic steatosis, possibly through AMPK-mediated pathway (
      • Purushotham A.
      • Schug T.T.
      • Xu Q.
      • Surapureddi S.
      • Guo X.
      • Li X.
      Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation.
      ). Similarly, liver-specific deletion of LKB1 in mice dramatically reduced the phosphorylation of AMPK, leading to decreased glucose clearance, increased TORC2, and increased expression of gluconeogenic enzymes PEPCK and G6Pase, suggesting the importance of AMPK-LKB1-SIRT1 axis in regulating hepatic lipid metabolism (
      • Fulco M.
      • Sartorelli V.
      Comparing and contrasting the roles of AMPK and SIRT1 in metabolic tissues.
      ). Additionally, expression of lipogenic genes, such as Fas, Acc, Srebp1c, and ChREBP, were elevated in liver-specific LKB1-deficient mice. Taken together, these data support the role of SIRT1, LKB1, and AMPK in suppressing gluconeogenic and lipogenic pathways in the acute regulation of energy balance in the liver.

      AMPK and hepatic steatosis

      Obesity is a risk factor for developing chronic nonalcoholic fatty liver disease (NAFLD), which is caused by the overexpression of the lipogenic genes srebp1c, acc1, and diacylglycerol acyltransferase 2 (dgat2) (
      • Srivastava R.A.
      • He S.
      Anti-hyperlipidemic and insulin sensitizing activities of fenofibrate reduces aortic lipid deposition in hyperlipidemic Golden Syrian hamster.
      ). Transgenic mice overexpressing srebp1 show massive fatty liver and increased accumulation of cholesteryl ester and triglycerides (
      • Shimano H.
      • Horton J.D.
      • Hammer R.E.
      • Shimomura I.
      • Brown M.S.
      • Goldstein J.L.
      Overproduction of cholesterol and fatty acids causes massive liver enlargement in transgenic mice expressing truncated SREBP-1a.
      ). AMPK activation reduces the lipogenic enzymes and transcription factors, including SREBP1, and increases mitochondrial oxidation of fatty acids in the liver. AMPK activator S17834, a synthetic polyphenol, regulates SREBP1 at the posttranscriptional level by phosphorylating SREBP1c at Ser-372, leading to inhibition of SREBP activity and attenuating hepatic steatosis and atherosclerosis in diet-induced insulin-resistance mice (
      • Li Y.
      • Xu S.
      • Mihaylova M.M.
      • Zheng B.
      • Hou X.
      • Jiang B.
      • Park O.
      • Luo Z.
      • Lefai E.
      • Shyy J.Y.
      • et al.
      AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.
      ). AMPK is also involved in the mitochondrial biogenesis in the liver. For instance, AMPK activator resveratrol increases mitochondrial number in liver in association with AMPK activation, and mouse liver lacking AMPK shows reduced mitochondrial biogenesis (
      • Um J.H.
      • Park S.J.
      • Kang H.
      • Yang S.
      • Foretz M.
      • McBurney M.W.
      • Kim M.K.
      • Viollet B.
      • Chung J.H.
      AMP-activated protein kinase-deficient mice are resistant to the metabolic effects of resveratrol.
      ). Thus, AMPK-mediated improvement in mitochondrial function together with inhibition of hepatic fatty acid synthesis and increase in fatty acid oxidation contributes to improvements in hepatic steatosis.
      High sucrose-fed rats develop NAFLD concomitant with decreases in AMPK activity in the liver (
      • Song Z.
      • Deaciuc I.
      • Zhou Z.
      • Song M.
      • Chen T.
      • Hill D.
      • McClain C.J.
      Involvement of AMP-activated protein kinase in beneficial effects of betaine on high-sucrose diet-induced hepatic steatosis.
      ). Activation of AMPK in the liver leads to reduced lipid synthesis and increased fatty acid oxidation (Fig. 2). Transgenic mice expressing constitutively active (CA)-AMPK-α1 in the liver exhibited resistance to weight gain and accumulation of liver lipids on high-fat diet (
      • Yang J.
      • Maika S.
      • Craddock L.
      • King J.A.
      • Liu Z.M.
      Chronic activation of AMP-activated protein kinase-alpha1 in liver leads to decreased adiposity in mice.
      ). Similar to ACC2−/− mice, steroyl CoA desaturase 1-deficient (SCD1−/−) mice on high-fat diet shows reduced body weight, body fat mass, hepatic lipids, and increased oxygen consumption. These mice also showed increased expression of genes involved in fatty acid oxidation and improved insulin sensitivity (
      • Ntambi J.M.
      • Miyazaki M.
      • Stoehr J.P.
      • Lan H.
      • Kendziorski C.M.
      • Yandell B.S.
      • Song Y.
      • Cohen P.
      • Friedman J.M.
      • Attie A.D.
      Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity.
      ). This effect is thought to occur through increased AMPK phosphorylation (52%) and activity (40%), leading to increased ACC phosphorylation (62%) and CPT1 activity (63%) (
      • Dobrzyn P.
      • Dobrzyn A.
      • Miyazaki M.
      • Cohen P.
      • Asilmaz E.
      • Hardie D.G.
      • Friedman J.M.
      • Ntambi J.M.
      Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver.
      ). Absence of SCD1 in ob/ob mice ameliorated the severe obesity observed in the ob/ob mice (
      • Dobrzyn P.
      • Dobrzyn A.
      • Miyazaki M.
      • Cohen P.
      • Asilmaz E.
      • Hardie D.G.
      • Friedman J.M.
      • Ntambi J.M.
      Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver.
      ), suggesting the inhibition of ACC as one mechanism accounting for increased fatty acid oxidation in the livers of SCD1-deficient mice, as phosphorylation and activity of AMPK and ACC increased in SCD1−/− mice (
      • Ntambi J.M.
      • Miyazaki M.
      • Stoehr J.P.
      • Lan H.
      • Kendziorski C.M.
      • Yandell B.S.
      • Song Y.
      • Cohen P.
      • Friedman J.M.
      • Attie A.D.
      Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity.
      ), resulting in decreased levels of malonyl-CoA and activation of CPT1, leading to increased palmitate oxidation. Leptin-deficient ob/ob mice with SCD1 mutations were significantly less obese than ob/ob controls and had reduced triglyceride storage in liver (
      • Dobrzyn P.
      • Dobrzyn A.
      • Miyazaki M.
      • Cohen P.
      • Asilmaz E.
      • Hardie D.G.
      • Friedman J.M.
      • Ntambi J.M.
      Stearoyl-CoA desaturase 1 deficiency increases fatty acid oxidation by activating AMP-activated protein kinase in liver.
      ). SCD1 is therefore a component of the novel metabolic response to hepatic lipid accumulation. These results suggest that the inhibition of SCD1 leads to the activation of AMPK and downstream effects. Thus, SCD1 deficiency appears to be involved in AMPK activation and hepatic lipid metabolism.
      More importantly, the antidiabetic drug metformin activates AMPK and markedly reduces hepatic lipid content in ob/ob mice (
      • Kim Y.D.
      • Park K.G.
      • Lee Y.S.
      • Park Y.Y.
      • Kim D.K.
      • Nedumaran B.
      • Jang W.G.
      • Cho W.J.
      • Ha J.
      • Lee I.K.
      • et al.
      Metformin inhibits hepatic gluconeogenesis through AMP-activated protein kinase-dependent regulation of the orphan nuclear receptor SHP.
      ). The metabolic improvements of adiponectin are linked to the activation of AMPK, resulting in decreased liver lipids (
      • Yamauchi T.
      • Kamon J.
      • Minokoshi Y.
      • Ito Y.
      • Waki H.
      • Uchida S.
      • Yamashita S.
      • Noda M.
      • Kita S.
      • Ueki K.
      • et al.
      Adiponectin stimulates glucose utilization and fatty-acid oxidation by activating AMP-activated protein kinase.
      ). AMPK activators AICAR and thienopyridine reduce hepatic fat content in rodents through AMPK activation (
      • Cool B.
      • Zinker B.
      • Chiou W.
      • Kifle L.
      • Cao N.
      • Perham M.
      • Dickinson R.
      • Adler A.
      • Gagne G.
      • Iyengar R.
      • et al.
      Identification and characterization of a small molecule AMPK activator that treats key components of type 2 diabetes and the metabolic syndrome.
      ). Another protein, fetuin A, produced exclusively from the liver has been suggested to regulate fat-derived hormone adiponectin. Fetuin A inhibits insulin receptor kinase and induces insulin resistance (
      • Rauth G.
      • Poschke O.
      • Fink E.
      • Eulitz M.
      • Tippmer S.
      • Kellerer M.
      • Haring H.U.
      • Nawratil P.
      • Haasemann M.
      • Jahnen-Dechent W.
      • et al.
      The nucleotide and partial amino acid sequences of rat fetuin. Identity with the natural tyrosine kinase inhibitor of the rat insulin receptor.
      ), and fetuin A-deficient mice show improved insulin resistance (
      • Mathews S.T.
      • Rakhade S.
      • Zhou X.
      • Parker G.C.
      • Coscina D.V.
      • Grunberger G.
      Fetuin-null mice are protected against obesity and insulin resistance associated with aging.
      ). Conversely, wild-type mice treated with fetuin A develop insulin resistance, consistent with association of fetuin A and insulin resistance in humans (
      • Stefan N.
      • Hennige A.M.
      • Staiger H.
      • Machann J.
      • Schick F.
      • Krober S.M.
      • Machicao F.
      • Fritsche A.
      • Haring H.U.
      Alpha2-Heremans-Schmid glycoprotein/fetuin-A is associated with insulin resistance and fat accumulation in the liver in humans.
      ). This effect of fetuin A is thought to be linked to adiponectin, as individuals with NAFLD have lower adiponectin (
      • Musso G.
      • Gambino R.
      • Durazzo M.
      • Biroli G.
      • Carello M.
      • Faga E.
      • Pacini G.
      • De Michieli F.
      • Rabbione L.
      • Premoli A.
      • et al.
      Adipokines in NASH: postprandial lipid metabolism as a link between adiponectin and liver disease.
      ). These studies suggest a cross-talk between adipose tissue and liver involving AMPK, fetuin A, and adiponectin. In humans with nonalcoholic steatotic hepatitis, metformin administration improved liver function tests and decreased liver size (
      • Sofer E.
      • Boaz M.
      • Matas Z.
      • Mashavi M.
      • Shargorodsky M.
      Treatment with insulin sensitizer metformin improves arterial properties, metabolic parameters, and liver function in patients with nonalcoholic fatty liver disease: a randomized, placebo-controlled trial.
      ), suggesting a key role of AMPK in improving hepatic steatosis, consistent with its mechanism of action. Thus, hepatic steatosis presents a pathological condition of deregulated lipogenesis in the liver in the insulin resistance and diabetic individuals, leading to increased hepatic triglycerides. AMPK activation improves hepatic steatosis directly by inhibition of lipogenesis and indirectly by lowering malonyl-CoA, leading to increased CPT1 and fatty acid oxidation.

      AMPK ROLE IN WHOLE-BODY ENERGY BALANCE

      AMPK in white adipose

      White adipose tissue (WAT) is a primary depot of lipid storage. In lean healthy subjects, this compartment maintains a reserve of energy substrate for utilization during energy depleting states. However, excess nutrients by diets high in fat and carbohydrate can lead to excessive adipose storage of triglycerides, resulting in obesity. During ATP-consuming states with increased lipolysis, such as fasting, exercise, and hypoxia, AMPK is activated in WAT to generate substrate for ATP production (
      • Daval M.
      • Foufelle F.
      • Ferre P.
      Functions of AMP-activated protein kinase in adipose tissue.
      ,
      • Park H.
      • Kaushik V.K.
      • Constant S.
      • Prentki M.
      • Przybytkowski E.
      • Ruderman N.B.
      • Saha A.K.
      Coordinate regulation of malonyl-CoA decarboxylase, sn-glycerol-3-phosphate acyltransferase, and acetyl-CoA carboxylase by AMP-activated protein kinase in rat tissues in response to exercise.
      • Gauthier M.S.
      • Miyoshi H.
      • Souza S.C.
      • Cacicedo J.M.
      • Saha A.K.
      • Greenberg A.S.
      • Ruderman N.B.
      AMP-activated protein kinase is activated as a consequence of lipolysis in the adipocyte: potential mechanism and physiological relevance.
      ). AMPK activation suppresses the lipogenic pathway in WAT. Additionally, the downstream reduction of malonyl-CoA levels (a repressor of CPT1) leads to increases in CPT1 and uncoupling protein 1 (UCP1)-dependent fatty acid oxidation (
      • Daval M.
      • Foufelle F.
      • Ferre P.
      Functions of AMP-activated protein kinase in adipose tissue.
      ,
      • Viollet B.
      • Athea Y.
      • Mounier R.
      • Guigas B.
      • Zarrinpashneh E.
      • Horman S.
      • Lantier L.
      • Hebrard S.
      • Devin-Leclerc J.
      • Beauloye C.
      • et al.
      AMPK: lessons from transgenic and knockout animals.
      ). Mitochondrial β-oxidation restores the AMP:ATP ratio. Adipokines, leptin, and adiponectin, which are secreted by the adipose, promote positive feedback by activating AMPK, thereby enhancing oxidation of fatty acids (
      • Orci L.
      • Cook W.S.
      • Ravazzola M.
      • Wang M.Y.
      • Park B.H.
      • Montesano R.
      • Unger R.H.
      Rapid transformation of white adipocytes into fat-oxidizing machines.
      ,
      • Yin W.
      • Mu J.
      • Birnbaum M.J.
      Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis In 3T3–L1 adipocytes.
      ).
      In terms of β-adrenergic effects on AMPK and triglyceride lipolysis, some studies in primary rat adipocytes and F442a adipocytes demonstrate inhibition of lipolysis with AMPK activators, such as AICAR (
      • Corton J.M.
      • Gillespie J.G.
      • Hawley S.A.
      • Hardie D.G.
      5-aminoimidazole-4-carboxamide ribonucleoside. A specific method for activating AMP-activated protein kinase in intact cells?.
      • Sullivan J.E.
      • Brocklehurst K.J.
      • Marley A.E.
      • Carey F.
      • Carling D.
      • Beri R.K.
      Inhibition of lipolysis and lipogenesis in isolated rat adipocytes with AICAR, a cell-permeable activator of AMP-activated protein kinase.
      ), via inactivation of HSL (
      • Djouder N.
      • Tuerk R.D.
      • Suter M.
      • Salvioni P.
      • Thali R.F.
      • Scholz R.
      • Vaahtomeri K.
      • Auchli Y.
      • Rechsteiner H.
      • Brunisholz R.A.
      • et al.
      PKA phosphorylates and inactivates AMPKalpha to promote efficient lipolysis.
      ). Yet other researchers have described phosphorylation of HSL by AMPK, leading to lipolysis and resulting in reduced plasma triglycerides and fatty acids (
      • Yin W.
      • Mu J.
      • Birnbaum M.J.
      Role of AMP-activated protein kinase in cyclic AMP-dependent lipolysis In 3T3–L1 adipocytes.
      ,
      • Su C.L.
      • Sztalryd C.
      • Contreras J.A.
      • Holm C.
      • Kimmel A.R.
      • Londos C.
      Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes.
      ). Furthermore, AMPKa2−/− mice fed a high-fat diet showed increased white adipose tissue mass associated with increases in lipid content, supporting a role of AMPK facilitated lipolysis (
      • Viollet B.
      • Athea Y.
      • Mounier R.
      • Guigas B.
      • Zarrinpashneh E.
      • Horman S.
      • Lantier L.
      • Hebrard S.
      • Devin-Leclerc J.
      • Beauloye C.
      • et al.
      AMPK: lessons from transgenic and knockout animals.
      ,
      • Villena J.A.
      • Viollet B.
      • Andreelli F.
      • Kahn A.
      • Vaulont S.
      • Sul H.S.
      Induced adiposity and adipocyte hypertrophy in mice lacking the AMP-activated protein kinase-alpha2 subunit.
      ). The role of AMPK on lipolysis is complex and requires further research to elucidate clinical relevance.
      AMPK is also involved in the phenotypic changes associated with mitochondrial uncoupling. Mice with hyperactivated brown adipose thermogenesis (
      • Klaus S.
      • Keipert S.
      • Rossmeisl M.
      • Kopecky J.
      Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase.
      ) as well as mice having ectopic expression of UCP1 in white adipose (
      • Matejkova O.
      • Mustard K.J.
      • Sponarova J.
      • Flachs P.
      • Rossmeisl M.
      • Miksik I.
      • Thomason-Hughes M.
      • Grahame Hardie D.
      • Kopecky J.
      Possible involvement of AMP-activated protein kinase in obesity resistance induced by respiratory uncoupling in white fat.
      ,
      • Rossmeisl M.
      • Flachs P.
      • Brauner P.
      • Sponarova J.
      • Matejkova O.
      • Prazak T.
      • Ruzickova J.
      • Bardova K.
      • Kuda O.
      • Kopecky J.
      Role of energy charge and AMP-activated protein kinase in adipocytes in the control of body fat stores.
      ) have increased AMPK. These animals exhibit associated decreases in lipogenesis and lipolysis and increases in fatty acid oxidation and glycolysis, resulting in decreased body weight. Similarly, 3T3-L1 adipocytes overexpressing UCP1 have decreased triglyceride content, accompanied by decreased lipogenesis and increased glycolysis (
      • Si Y.
      • Shi H.
      • Lee K.
      Metabolic flux analysis of mitochondrial uncoupling in 3T3–L1 adipocytes.
      ).
      In addition to the direct effects of AMPK in adipocytes, adipose tissue mediates a critical role in whole-body energy balance through secretion of adipokines leptin and adiponectin (
      • Friedman J.
      Fat in all the wrong places.
      ). Leptin, when administered to normal rats via adenovirus transfer, results in whole-body fat loss associated with lipid depletion of adipocytes, as well as increased circulating nonesterified fatty acids and ketones derived from increased fatty acid oxidation (
      • Orci L.
      • Cook W.S.
      • Ravazzola M.
      • Wang M.Y.
      • Park B.H.
      • Montesano R.
      • Unger R.H.
      Rapid transformation of white adipocytes into fat-oxidizing machines.
      ).
      Mouse AMPK activity is localized to epididymal fat rather than subcutaneous fat (
      • Sponarova J.
      • Mustard K.J.
      • Horakova O.
      • Flachs P.
      • Rossmeisl M.
      • Brauner P.
      • Bardova K.
      • Thomason-Hughes M.
      • Braunerova R.
      • Janovska P.
      • et al.
      Involvement of AMP-activated protein kinase in fat depot-specific metabolic changes during starvation.
      ). AMPK has lower expression and is less active in visceral fat than subcutaneous fat in morbidly obese humans (
      • Gauthier M.S.
      • O'Brien E.L.
      • Bigornia S.
      • Mott M.
      • Cacicedo J.M.
      • Xu X.J.
      • Gokce N.
      • Apovian C.
      • Ruderman N.
      Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans.
      ,
      • Martínez-Agustin O.
      • Hernandez-Morante J.J.
      • Martinez-Plata E.
      • Sanchez de Medina F.
      • Garaulet M.
      Differences in AMPK expression between subcutaneous and visceral adipose tissue in morbid obesity.
      ). WAT biopsies from obese insulin-resistant subjects and body-weight-matched obese insulin-sensitive subjects revealed an association of lower AMPK activity in WAT from insulin-resistant subjects (
      • Gauthier M.S.
      • O'Brien E.L.
      • Bigornia S.
      • Mott M.
      • Cacicedo J.M.
      • Xu X.J.
      • Gokce N.
      • Apovian C.
      • Ruderman N.
      Decreased AMP-activated protein kinase activity is associated with increased inflammation in visceral adipose tissue and with whole-body insulin resistance in morbidly obese humans.
      ). Adiponectin expression and lipolysis rates are highly correlated to AMPK expression in subcutaneous fat (
      • Martínez-Agustin O.
      • Hernandez-Morante J.J.
      • Martinez-Plata E.
      • Sanchez de Medina F.
      • Garaulet M.
      Differences in AMPK expression between subcutaneous and visceral adipose tissue in morbid obesity.
      ). Subjects with low acylation-stimulating protein (ASP) and triglycerides have associated increases in adipose AMPK, UCP1, and CPT1 as well as decreased ACC (
      • MacLaren R.E.
      • Cui W.
      • Lu H.
      • Simard S.
      • Cianflone K.
      Association of adipocyte genes with ASP expression: a microarray analysis of subcutaneous and omental adipose tissue in morbidly obese subjects.
      ). Finally, a recent study involving type-2 diabetic subjects receiving oral doses of either metformin or sulfonylurea monotherapy demonstrated enhanced AMPK and ACC phosphorylation with metformin treatment (
      • Boyle J.G.
      • Logan P.J.
      • Jones G.C.
      • Small M.
      • Sattar N.
      • Connell J.M.
      • Cleland S.J.
      • Salt I.P.
      AMP-activated protein kinase is activated in adipose tissue of individuals with type 2 diabetes treated with metformin: a randomised glycaemia-controlled crossover study.
      ). However, there was no associated improvement in BMI, blood pressure, circulating lipids, or glucose outcomes compared with the sulfonylurea group. Additional studies in larger populations of obese type-2 diabetics are needed to fully understand the therapeutic efficacy of AMPK activation in obesity.

      AMPK in the hypothalamus

      AMPK phosphorylation in metabolically active tissues such as adipose and muscle yields a lean phenotype with decreased adipose stores. However, tissue-specific AMPK activation in the hypothalamus may increase appetite and food consumption. AMPK is activated during fasting, yet suppressed during refeeding (
      • Minokoshi Y.
      • Alquier T.
      • Furukawa N.
      • Kim Y.B.
      • Lee A.
      • Xue B.
      • Mu J.
      • Foufelle F.
      • Ferre P.
      • Birnbaum M.J.
      • et al.
      AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus.
      ). During fasting, AMPK-mediated phosphorylation of ACC (inhibition) reduces malonyl-CoA levels in the hypothalamus and increases food consumption (
      • López M.
      • Lage R.
      • Saha A.K.
      • Perez-Tilve D.
      • Vazquez M.J.
      • Varela L.
      • Sangiao-Alvarellos S.
      • Tovar S.
      • Raghay K.
      • Rodriguez-Cuenca S.
      • et al.
      Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
      ). Subsequently, CPT1 activity is stimulated resulting in increased mitochondrial fatty acid oxidation (
      • Kahn B.B.
      • Alquier T.
      • Carling D.
      • Hardie D.G.
      AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism.
      ). Fasting induces phosphorylation of ACC and AMPK in rats, and it suppresses FAS mRNA expression in the ventromedial nuclei subsequently, yielding decreased malonyl-CoA in the hypothalamus (
      • López M.
      • Lage R.
      • Saha A.K.
      • Perez-Tilve D.
      • Vazquez M.J.
      • Varela L.
      • Sangiao-Alvarellos S.
      • Tovar S.
      • Raghay K.
      • Rodriguez-Cuenca S.
      • et al.
      Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
      ). Expression of active AMPK in the hypothalamus of mice increases food consumption, while hypothalamus of AMPK dominant negative mice results in a hypophagic phenotype (
      • Minokoshi Y.
      • Alquier T.
      • Furukawa N.
      • Kim Y.B.
      • Lee A.
      • Xue B.
      • Mu J.
      • Foufelle F.
      • Ferre P.
      • Birnbaum M.J.
      • et al.
      AMP-kinase regulates food intake by responding to hormonal and nutrient signals in the hypothalamus.
      ). Treatment of mice with AMPK inhibitor, compound C, decreases food consumption and body weight, and treatment with AICAR, an activator of AMPK increases feeding behavior (
      • Ronnett G.V.
      • Aja S.
      AMP-activated protein kinase in the brain.
      ,
      • Xue B.
      • Kahn B.B.
      AMPK integrates nutrient and hormonal signals to regulate food intake and energy balance through effects in the hypothalamus and peripheral tissues.
      ), suggesting a direct role of AMPK in feeding behavior.
      The role of hypothalamic AMPK on control of feeding behavior is promoted by the hormones leptin and ghrelin. Leptin, secreted by the WAT in response to feeding, has been shown to inhibit AMPK activity in the hypothalamus, resulting in satiety of appetite (
      • Lim C.T.
      • Kola B.
      • Korbonits M.
      AMPK as a mediator of hormonal signalling.
      ). Other groups have demonstrated that centrally administered ghrelin can increase feeding behavior via AMPK activation, independent of leptin (
      • López M.
      • Lage R.
      • Saha A.K.
      • Perez-Tilve D.
      • Vazquez M.J.
      • Varela L.
      • Sangiao-Alvarellos S.
      • Tovar S.
      • Raghay K.
      • Rodriguez-Cuenca S.
      • et al.
      Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
      ). Ghrelin, secreted by the stomach, stimulates signaling in neuropeptide Y (NPY) neurons and the cannabinoid pathway via mechanisms involving AMPK activation (
      • Kola B.
      • Farkas I.
      • Christ-Crain M.
      • Wittmann G.
      • Lolli F.
      • Amin F.
      • Harvey-White J.
      • Liposits Z.
      • Kunos G.
      • Grossman A.B.
      • et al.
      The orexigenic effect of ghrelin is mediated through central activation of the endogenous cannabinoid system.
      ). Intracerebroventricular (ICV) administration of ghrelin in rats increases phosphorylation of both AMPK and ACC causing increased food consumption, an effect prevented by coadministration with the AMPK inhibitor compound C (
      • López M.
      • Lage R.
      • Saha A.K.
      • Perez-Tilve D.
      • Vazquez M.J.
      • Varela L.
      • Sangiao-Alvarellos S.
      • Tovar S.
      • Raghay K.
      • Rodriguez-Cuenca S.
      • et al.
      Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
      ). These findings suggest that the orexigenic activity of ghrelin may be mediated directly via AMPK activation. The net result of AMPK activation in the hypothalamus is increased appetite (
      • Hardie D.G.
      Sensing of energy and nutrients by AMP-activated protein kinase.
      ,
      • López M.
      • Lage R.
      • Saha A.K.
      • Perez-Tilve D.
      • Vazquez M.J.
      • Varela L.
      • Sangiao-Alvarellos S.
      • Tovar S.
      • Raghay K.
      • Rodriguez-Cuenca S.
      • et al.
      Hypothalamic fatty acid metabolism mediates the orexigenic action of ghrelin.
      ,
      • Ahima R.S.
      • Qi Y.
      • Singhal N.S.
      Adipokines that link obesity and diabetes to the hypothalamus.
      ,
      • Lage R.
      • Dieguez C.
      • Vidal-Puig A.
      • Lopez M.
      AMPK: a metabolic gauge regulating whole-body energy homeostasis.
      ). Based on these findings, it is possible that AMPK activators capable of crossing the blood-brain barrier may increase feeding behavior. Conversely, therapeutic AMPK activators with exposure limited to tissues such as liver, muscle, or adipose may yield beneficial reductions in peripheral lipid stores without affecting food consumption. Thus, tissue-selective AMPK activators provide an attractive therapeutic approach to reduce adipocity, body weight, circulating lipids, hepatic steatosis, or insulin resistance without affecting appetite.

      AMPK in skeletal muscle

      AMPK is activated by the increase in AMP:ATP ratio associated with ATP consumption during exercise, muscle contraction, and hypoxia (
      • Klaus S.
      • Keipert S.
      • Rossmeisl M.
      • Kopecky J.
      Augmenting energy expenditure by mitochondrial uncoupling: a role of AMP-activated protein kinase.
      ,
      • Hardie D.G.
      AMPK: a key regulator of energy balance in the single cell and the whole organism.
      ,
      • Zhang B.B.
      • Zhou G.
      • Li C.
      AMPK: an emerging drug target for diabetes and the metabolic syndrome.
      ). During stress conditions in which ATP levels are reduced, AMPK increases catabolic processes, such as glycolysis and fatty acid oxidation, and represses anabolic processes, including glycogen, protein, and lipid synthesis (
      • Hardie D.G.
      AMPK: a key regulator of energy balance in the single cell and the whole organism.
      ,
      • Zhang B.B.
      • Zhou G.
      • Li C.
      AMPK: an emerging drug target for diabetes and the metabolic syndrome.
      ). By the ACC/malonyl-CoA/CPT1 pathway, AMPK enhances fatty acid oxidation in muscle, depleting stored triacylglycerides and diacylglycerides contributing to insulin resistance (
      • Lowell B.B.
      • Shulman G.I.
      Mitochondrial dysfunction and type 2 diabetes.
      ). AMPK restores the AMP:ATP ratio through ATP production mediated by increased fatty acid uptake and lipid oxidation (
      • Viollet B.
      • Athea Y.
      • Mounier R.
      • Guigas B.
      • Zarrinpashneh E.
      • Horman S.
      • Lantier L.
      • Hebrard S.
      • Devin-Leclerc J.
      • Beauloye C.
      • et al.
      AMPK: lessons from transgenic and knockout animals.
      ,
      • Hardie D.G.
      AMPK: a key regulator of energy balance in the single cell and the whole organism.
      ,
      • Zhang B.B.
      • Zhou G.
      • Li C.
      AMPK: an emerging drug target for diabetes and the metabolic syndrome.
      ,
      • Cantó C.
      • Jiang L.Q.
      • Deshmukh A.S.
      • Mataki C.
      • Coste A.
      • Lagouge M.
      • Zierath J.R.
      • Auwerx J.
      Interdependence of AMPK and SIRT1 for metabolic adaptation to fasting and exercise in skeletal muscle.
      ,
      • McGee S.L.
      • Hargreaves M.
      AMPK-mediated regulation of transcription in skeletal muscle.
      ). Leptin secreted from adipose contributes to increased lipid catabolism and energy expenditure in resting muscle (
      • Kus V.
      • Prazak T.
      • Brauner P.
      • Hensler M.
      • Kuda O.
      • Flachs P.
      • Janovska P.
      • Medrikova D.
      • Rossmeisl M.
      • Jilkova Z.
      • et al.
      Induction of muscle thermogenesis by high-fat diet in mice: association with obesity-resistance.
      ,
      • Minokoshi Y.
      • Kim Y.B.
      • Peroni O.D.
      • Fryer L.G.
      • Muller C.
      • Carling D.
      • Kahn B.B.
      Leptin stimulates fatty-acid oxidation by activating AMP-activated protein kinase.
      ).
      Similar to liver, AMPK also mediates changes in cellular glucose homeostasis in muscle cells. In response to muscle contraction, AMPK activation phosphorylates and stimulates 14-3-3 binding to TBC1D1, as well as TBC1D4 (AS160), thereby inhibiting this Rab-GTPase-activating protein repressor of GLUT4 translocation, and resulting in GLUT4 translocation to the plasma membrane of myocytes to facilitate glucose uptake (
      • Karagounis L.G.
      • Hawley J.A.
      The 5′ adenosine monophosphate-activated protein kinase: regulating the ebb and flow of cellular energetics.
      ,
      • Pehm⊘ller C.
      • Treebak J.T.
      • Birk J.B.
      • Chen S.
      • Mackintosh C.
      • Hardie D.G.
      • Richter E.A.
      • Wojtaszewski J.F.
      Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14–3-3 binding in mouse skeletal muscle.
      ).
      Acute effects of ATP depletion stimulate AMPK phosphorylation; however, chronic responses include AMPK-derived expression including increased mitochondrial hexokinase II (HKII) involved in glucose transport (
      • Holmes B.F.
      • Kurth-Kraczek E.J.
      • Winder W.W.
      Chronic activation of 5′-AMP-activated protein kinase increases GLUT-4, hexokinase, and glycogen in muscle.
      ), PGC-1α involved in mitochondrial biogenesis, and CPT1 involved in fatty acid oxidation via α1- and α2-AMPK subunits (
      • J⊘rgensen S.B.
      • Wojtaszewski J.F.
      • Viollet B.
      • Andreelli F.
      • Birk J.B.
      • Hellsten Y.
      • Schjerling P.
      • Vaulont S.
      • Neufer P.D.
      • Richter E.A.
      • et al.
      Effects of alpha-AMPK knockout on exercise-induced gene activation in mouse skeletal muscle.
      ). Increased HKII expression is associated with the translocation of GLUT4 in fast-twitch muscle fibers but not slow-twitch (
      • Wright D.C.
      • Geiger P.C.
      • Holloszy J.O.
      • Han D.H.
      Contraction- and hypoxia-stimulated glucose transport is mediated by a Ca2+-dependent mechanism in slow-twitch rat soleus muscle.
      ). Furthermore, AICAR stimulates glucose uptake in mouse muscle via AMPK γ3 subunit expressed strictly in glycolytic skeletal muscle (
      • Barnes B.R.
      • Marklund S.
      • Steiler T.L.
      • Walter M.
      • Hjalm G.
      • Amarger V.
      • Mahlapuu M.
      • Leng Y.
      • Johansson C.
      • Galuska D.
      • et al.
      The 5′-AMP-activated protein kinase gamma3 isoform has a key role in carbohydrate and lipid metabolism in glycolytic skeletal muscle.
      ). HKII also mediates glucose phosphorylation to glucose-6-phosphate, a process that is diminished in insulin resistant subjects as a result of impaired glucose tolerance (
      • Lehto M.
      • Huang X.
      • Davis E.M.
      • Le Beau M.M.
      • Laurila E.
      • Eriksson K.F.
      • Bell G.I.
      • Groop L.
      Human hexokinase II gene: exon-intron organization, mutation screening in NIDDM, and its relationship to muscle hexokinase activity.
      ). These findings suggest AMPK may play a role in the regulation of fuel balance from triacylglycerol and diacylglycerol stores (increased fat oxidation) to glycogen stores, thereby increasing the glycolytic potential of skeletal muscle and potentially reducing insulin resistance.
      During endurance exercise, AMPK mediates a transition from fast-twitch to the oxidative slow-twitch muscle fiber phenotype (
      • McGee S.L.
      • Hargreaves M.
      AMPK-mediated regulation of transcription in skeletal muscle.
      ) by directly phosphorylating PGC-1α (
      • Viollet B.
      • Athea Y.
      • Mounier R.
      • Guigas B.
      • Zarrinpashneh E.
      • Horman S.
      • Lantier L.
      • Hebrard S.
      • Devin-Leclerc J.
      • Beauloye C.
      • et al.
      AMPK: lessons from transgenic and knockout animals.
      ,
      • Jäger S.
      • Handschin C.
      • St-Pierre J.
      • Spiegelman B.M.
      AMP-activated protein kinase (AMPK) action in skeletal muscle via direct phosphorylation of PGC-1alpha.
      ). AICAR treatment of rats inactivates GS via AMPK phosphorylation (
      • Wojtaszewski J.F.
      • Jorgensen S.B.
      • Hellsten Y.
      • Hardie D.G.
      • Richter E.A.
      Glycogen-dependent effects of 5-aminoimidazole-4-carboxamide (AICA)-riboside on AMP-activated protein kinase and glycogen synthase activities in rat skeletal muscle.
      ). Because glycogen surplus results in reduced glucose uptake, the inhibition of GS should favorably increase glucose tolerance in muscle.
      The role of AMPK activity has been assessed in muscle of obese and type-2 diabetic human subjects in several contexts. While one study reported that obese diabetics had an exercise time- and intensity- associated reduction in AMPK activity and AS160 phosphorylation, type-2 diabetics had lower basal PGC-1α gene expression (
      • Sriwijitkamol A.
      • Coletta D.K.
      • Wajcberg E.
      • Balbontin G.B.
      • Reyna S.M.
      • Barrientes J.
      • Eagan P.A.
      • Jenkinson C.P.
      • Cersosimo E.
      • DeFronzo R.A.
      • et al.
      Effect of acute exercise on AMPK signaling in skeletal muscle of subjects with type 2 diabetes: a time-course and dose-response study.
      ). Other research groups have reported no differences in AMPK activities or fatty acid oxidation in a similar setting (
      • Steinberg G.R.
      • Smith A.C.
      • Van Denderen B.J.
      • Chen Z.
      • Murthy S.
      • Campbell D.J.
      • Heigenhauser G.J.
      • Dyck D.J.
      • Kemp B.E.
      AMP-activated protein kinase is not down-regulated in human skeletal muscle of obese females.
      ). Reduced basal glycerol release, muscle biopsy HSL protein levels, HSL phosphorylation, and increased skeletal muscle triglyceride have been observed in obese subjects (
      • Jocken J.W.
      • Roepstorff C.
      • Goossens G.H.
      • van der Baan P.
      • van Baak M.
      • Saris W.H.
      • Kiens B.
      • Blaak E.E.
      Hormone-sensitive lipase serine phosphorylation and glycerol exchange across skeletal muscle in lean and obese subjects: effect of beta-adrenergic stimulation.
      ). Finally, a recent study explored the effect of short- and long-term fasting on AMPK activity in healthy human subjects and reported elevated lipid and glycogen content in muscle and suppression of AS160 phosphorylation, with no change in AMPK phosphorylation (
      • Vendelbo M.H.
      • Clasen B.F.
      • Treebak J.T.
      • Moller L.
      • Krusenstjerna-Hafstrom T.
      • Madsen M.
      • Nielsen T.S.
      • Stodkilde-Jorgensen H.
      • Pedersen S.B.
      • Jorgensen J.O.
      • et al.
      Insulin resistance after a 72 hour fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle.
      ). Thus, the mixed data in human subjects warrants a more careful, appropriately designed and adequately powered study.

      ROLE OF AMPK IN CARDIAC FUNCTION

      Although the expression of AMPK in the normal functioning heart is very low compared with skeletal muscle, the role of AMPK signaling in heart has been suggested in physiological (
      • Musi N.
      • Hirshman M.F.
      • Arad M.
      • Xing Y.
      • Fujii N.
      • Pomerleau J.
      • Ahmad F.
      • Berul C.I.
      • Seidman J.G.
      • Tian R.
      • et al.
      Functional role of AMP-activated protein kinase in the heart during exercise.
      ) and pathological conditions like hypertrophy (
      • Tian R.
      • Musi N.
      • D'Agostino J.
      • Hirshman M.F.
      • Goodyear L.J.
      Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.
      ,
      • Li H.L.
      • Yin R.
      • Chen D.
      • Liu D.
      • Wang D.
      • Yang Q.
      • Dong Y.G.
      Long-term activation of adenosine monophosphate-activated protein kinase attenuates pressure-overload-induced cardiac hypertrophy.
      ) and ischemia (
      • Li J.
      • Coven D.L.
      • Miller E.J.
      • Hu X.
      • Young M.E.
      • Carling D.
      • Sinusas A.J.
      • Young L.H.
      Activation of AMPK alpha- and gamma-isoform complexes in the intact ischemic rat heart.
      ). One of the primary functions of AMPK activation is to induce glucose uptake, which has been observed in hypertrophied hearts of animal models (
      • Zhang J.
      • Duncker D.J.
      • Ya X.
      • Zhang Y.
      • Pavek T.
      • Wei H.
      • Merkle H.
      • Ugurbil K.
      • From A.H.
      • Bache R.J.
      Effect of left ventricular hypertrophy secondary to chronic pressure overload on transmural myocardial 2-deoxyglucose uptake. A 31P NMR spectroscopic study.
      ) and humans (
      • Nuutila P.
      • Maki M.
      • Laine H.
      • Knuuti M.J.
      • Ruotsalainen U.
      • Luotolahti M.
      • Haaparanta M.
      • Solin O.
      • Jula A.
      • Koivisto V.A.
      • et al.
      Insulin action on heart and skeletal muscle glucose uptake in essential hypertension.
      ), suggesting a functional role of AMPK in cardiac hypertrophy (
      • Tian R.
      • Musi N.
      • D'Agostino J.
      • Hirshman M.F.
      • Goodyear L.J.
      Increased adenosine monophosphate-activated protein kinase activity in rat hearts with pressure-overload hypertrophy.
      ,
      • Arad M.
      • Benson D.W.
      • Perez-Atayde A.R.
      • McKenna W.J.
      • Sparks E.A.
      • Kanter R.J.
      • McGarry K.
      • Seidman J.G.
      • Seidman C.E.
      Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy.
      ) and ischemia (
      • Kim A.S.
      • Miller E.J.
      • Wright T.M.
      • Li J.
      • Qi D.
      • Atsina K.
      • Zaha V.
      • Sakamoto K.
      • Young L.H.
      A small molecule AMPK activator protects the heart against ischemia-reperfusion injury.
      ,
      • Russell 3rd, R.R.
      • Li J.
      • Coven D.L.
      • Pypaert M.
      • Zechner C.
      • Palmeri M.
      • Giordano F.J.
      • Mu J.
      • Birnbaum M.J.
      • Young L.H.
      AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury.
      ). Further evidence of AMPK in cardiac function comes from AMPK mutation that leads to myocardial metabolic storage disease with coexistence of hypertrophy and defects in conduction system (
      • Arad M.
      • Benson D.W.
      • Perez-Atayde A.R.
      • McKenna W.J.
      • Sparks E.A.
      • Kanter R.J.
      • McGarry K.
      • Seidman J.G.
      • Seidman C.E.
      Constitutively active AMP kinase mutations cause glycogen storage disease mimicking hypertrophic cardiomyopathy.
      ). Using a muscle-specific α2 kinase dead (KD) transgenic mouse model, Russell 3rd et al. (
      • Russell 3rd, R.R.
      • Li J.
      • Coven D.L.
      • Pypaert M.
      • Zechner C.
      • Palmeri M.
      • Giordano F.J.
      • Mu J.
      • Birnbaum M.J.
      • Young L.H.
      AMP-activated protein kinase mediates ischemic glucose uptake and prevents postischemic cardiac dysfunction, apoptosis, and injury.
      ) demonstrated that AMPK in heart is involved in glucose uptake during low flow ischemia and reperfusion, thus playing a cardioprotective role by reducing ischemia and reperfusion associated damage. Indeed, infusion of an AMPK activator, AICAR, in ischemia-induced rats caused myocardial AMPK activation and GLUT-4 translocation (
      • Russell 3rd, R.R.
      • Bergeron R.
      • Shulman G.I.
      • Young L.H.
      Translocation of myocardial GLUT-4 and increased glucose uptake through activation of AMPK by AICAR.
      ). Thus, AMPK-mediated glucose metabolism is suggested to have protective effects in cardiac hypertrophy (
      • Kolwicz Jr, S.C.
      • Tian R.
      Glucose metabolism and cardiac hypertrophy.
      ).
      The protective role of AMPK in failing heart has been extensively studied (reviewed in Ref.
      • Beauloye C.
      • Bertrand L.
      • Horman S.
      • Hue L.
      AMPK activation, a preventive therapeutic target in the transition from cardiac injury to heart failure.
      ). While a vast amount of literature exists on the role of AMPK on heart function, here we focus on two known and widely studied AMPK activators, AICAR and metformin, in relation to cardiac function. Acute therapy of metformin imparts cardioprotection against myocardial infarction both in diabetic and nondiabetic mice, suggesting direct action of AMPK activator on the ventricular muscle (
      • Calvert J.W.
      • Gundewar S.
      • Jha S.
      • Greer J.J.
      • Bestermann W.H.
      • Tian R.
      • Lefer D.J.
      Acute metformin therapy confers cardioprotection against myocardial infarction via AMPK-eNOS-mediated signaling.
      ). Long-term activation of AMPK by AICAR in pressure overload-induced cardiac hypertrophy rats showed attenuation of cardiac hypertrophy and improved cardiac function (
      • Li H.L.
      • Yin R.
      • Chen D.
      • Liu D.
      • Wang D.
      • Yang Q.
      • Dong Y.G.
      Long-term activation of adenosine monophosphate-activated protein kinase attenuates pressure-overload-induced cardiac hypertrophy.
      ). To directly address the cardioprotective effect of AMPK, Nagata et al. (
      • Nagata D.
      • Kiyosue A.
      • Takahashi M.
      • Satonaka H.
      • Tanaka K.
      • Sata M.
      • Nagano T.
      • Nagai R.
      • Hirata Y.
      A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell.
      ) produced a constitutively active mutant of AMPK by replacing Thr-172 with Asp, and they demonstrated that overexpression of this construct in HUVECs inhibited anoxia-induced apoptosis, suggesting AMPK's role in protecting against ischemic cardiac injury. As the major effect of metformin is in glucose metabolism, it is possible that the cardioprotective effect of metformin occurs primarily via enhanced glucose metabolism in the heart. Glucose-independent long-term effect of metformin was investigated in nondiabetic rat model of post-MI heart failure (
      • Yin M.
      • van der Horst I.C.
      • van Melle J.P.
      • Qian C.
      • van Gilst W.H.
      • Sillje H.H.
      • de Boer R.A.
      Metformin improves cardiac function in a nondiabetic rat model of post-MI heart failure.
      ). In this animal model, metformin showed attenuation of the development of heart failure after myocardial infarction, suggesting glucose-independent effect of AMPK activator on cardiac function. Altered substrate metabolism is associated with advanced heart failure (HF), and metformin is known to affect substrate metabolism leading to improved outcome in diabetic HF. Kazdova et al. (
      • Benes J.
      • Kazdova L.
      • Drahota Z.
      • Houstek J.
      • Medrikova D.
      • Kopecky J.
      • Kovarova N.
      • Vrbacky M.
      • Sedmera D.
      • Strnad H.
      • et al.
      Effect of metformin therapy on cardiac function and survival in a volume-overload model of heart failure in rats.
      ) examined the effect of metformin on the improvement of cardiac function in nondiabetic volume-overload HF rat model and observed normalization of NEFA and increased palmitate oxidation but no effect on mitochondrial respiration, cardiac morphology, and mortality, suggesting that the use of metformin is safe in diabetic HF.
      Obesity is associated with insulin resistance and glucose intolerance, which is linked to increased cardiac injury. A fat-derived plasma protein, C1q/TNF-related protein 9 (CTRP9), has shown beneficial effect on glucose metabolism (
      • Wong G.W.
      • Krawczyk S.A.
      • Kitidis-Mitrokostas C.
      • Ge G.
      • Spooner E.
      • Hug C.
      • Gimeno R.
      • Lodish H.F.
      Identification and characterization of CTRP9, a novel secreted glycoprotein, from adipose tissue that reduces serum glucose in mice and forms heterotrimers with adiponectin.
      ) and vascular function (
      • Zheng Q.
      • Yuan Y.
      • Yi W.
      • Lau W.B.
      • Wang Y.
      • Wang X.
      • Sun Y.
      • Lopez B.L.
      • Christopher T.A.
      • Peterson J.M.
      • et al.
      C1q/TNF-related proteins, a family of novel adipokines, induce vascular relaxation through the adiponectin receptor-1/AMPK/eNOS/nitric oxide signaling pathway.
      ). Decreased levels of CTRP9 have been observed in mouse model of ischemia-reperfusion injury and in DIO mice (
      • Kambara T.
      • Ohashi K.
      • Shibata R.
      • Ogura Y.
      • Maruyama S.
      • Enomoto T.
      • Uemura Y.
      • Shimizu Y.
      • Yuasa D.
      • Matsuo K.
      • et al.
      CTRP9 protein protects against myocardial injury following ischemia-reperfusion through AMP-activated protein kinase (AMPK)-dependent mechanism.
      ). Delivery of adenovirus expressing CTRP9 attenuated infarct size after ischemia reperfusion, and this effect was found to be mediated via AMPK signaling. In another study, activation of AMPK by metformin in mouse model of HF showed improvement in left ventricular function, attenuation of cardiac hypertrophy, and survival (
      • Gundewar S.
      • Calvert J.W.
      • Jha S.
      • Toedt-Pingel I.
      • Ji S.Y.
      • Nunez D.
      • Ramachandran A.
      • Anaya-Cisneros M.
      • Tian R.
      • Lefer D.J.
      Activation of AMP-activated protein kinase by metformin improves left ventricular function and survival in heart failure.
      ). These cardioprotective effects were associated with phosphorylation of AMPK and eNOS and increases in PGC1-α. Furthermore, metformin-mediated improvements in cardiac function were found to be dampened in AMPK-deficient mice, suggesting the important role of AMPK in reducing cardiac damage. In a canine pacing-induced HF model (
      • Sasaki H.
      • Asanuma H.
      • Fujita M.
      • Takahama H.
      • Wakeno M.
      • Ito S.
      • Ogai A.
      • Asakura M.
      • Kim J.
      • Minamino T.
      • et al.
      Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase.
      ), AMPK activators AICAR and metformin attenuated oxidative stress-induced cardiomyocyte death and improved cardiac function via AMPK-mediated mechanism, as the beneficial effects of cardioprotection was blunted by Compound C, an inhibitor of AMPK (
      • Gundewar S.
      • Calvert J.W.
      • Jha S.
      • Toedt-Pingel I.
      • Ji S.Y.
      • Nunez D.
      • Ramachandran A.
      • Anaya-Cisneros M.
      • Tian R.
      • Lefer D.J.
      Activation of AMP-activated protein kinase by metformin improves left ventricular function and survival in heart failure.
      ,
      • Sasaki H.
      • Asanuma H.
      • Fujita M.
      • Takahama H.
      • Wakeno M.
      • Ito S.
      • Ogai A.
      • Asakura M.
      • Kim J.
      • Minamino T.
      • et al.
      Metformin prevents progression of heart failure in dogs: role of AMP-activated protein kinase.
      ). Together, these findings suggest a cardioprotective role of AMPK activators in established animal models of cardiac injury and dysfunction.

      ROLE OF AMPK IN IMMUNE RESPONSE ASSOCIATED WITH METABOLIC DISORDERS

      Anti-inflammatory effects of AMPK signaling have been demonstrated in multiple in vivo models of inflammatory diseases with AMPK activators AICAR and metformin. AICAR-mediated activation of AMPK was shown to largely reduce inflammation and minimize organ damage in models of acute lung injury, acute and chronic colitis, and autoimmune encephalomyelitis (
      • Bai A.
      • Ma A.G.
      • Yong M.
      • Weiss C.R.
      • Ma Y.
      • Guan Q.
      • Bernstein C.N.
      • Peng Z.
      AMPK agonist downregulates innate and adaptive immune responses in TNBS-induced murine acute and relapsing colitis.
      • Zhao X.
      • Zmijewski J.W.
      • Lorne E.
      • Liu G.
      • Park Y.J.
      • Tsuruta Y.
      • Abraham E.
      Activation of AMPK attenuates neutrophil proinflammatory activity and decreases the severity of acute lung injury.
      ).
      In patients with type-2 diabetes or mild metabolic syndrome, treatment with metformin reduces plasma levels of high-sensitivity C-reactive protein (hsCRP) as well as levels of IL-6, ICAM-1, MIF, and TNF-α (
      • Akbar D.H.
      Effect of metformin and sulfonylurea on C-reactive protein level in well-controlled type 2 diabetics with metabolic syndrome.
      ,
      • Fidan E.
      • Onder Ersoz H.
      • Yilmaz M.
      • Yilmaz H.
      • Kocak M.
      • Karahan C.
      • Erem C.
      The effects of rosiglitazone and metformin on inflammation and endothelial dysfunction in patients with type 2 diabetes mellitus.
      ). Interestingly, while metformin affects hsCRP levels in much greater extent than insulin secretagoge glibenclamide (
      • Akbar D.H.
      Effect of metformin and sulfonylurea on C-reactive protein level in well-controlled type 2 diabetics with metabolic syndrome.
      ,
      • Ashcroft F.M.
      ATP-sensitive potassium channelopathies: focus on insulin secretion.
      ), both metformin and PPAR-γ agonist rosiglitazone, which also promotes AMPK activity (
      • Morrison A.
      • Yan X.
      • Tong C.
      • Li J.
      Acute rosiglitazone treatment is cardioprotective against ischemia-reperfusion injury by modulating AMPK, Akt, and JNK signaling in nondiabetic mice.
      ), were similarly effective in controlling inflammatory markers in addition to metabolic parameters (
      • Fidan E.
      • Onder Ersoz H.
      • Yilmaz M.
      • Yilmaz H.
      • Kocak M.
      • Karahan C.
      • Erem C.
      The effects of rosiglitazone and metformin on inflammation and endothelial dysfunction in patients with type 2 diabetes mellitus.
      ).
      The most common attributes of chronic inflammation associated with obesity, type-2 diabetes, and atherosclerosis include i) phenotypic changes in circulating and resident monocyte-macrophages; ii) leukocyte migration, lipid accumulation, and foam cell formation in subendothelial space of the diseased arteries; iii) activation of NF-kB signaling; iv) unfolded protein response (UPR); and v) local and systemic oxidative stress (
      • Olefsky J.M.
      • Glass C.K.
      Macrophages, inflammation, and insulin resistance.
      ,
      • Schenk S.
      • Saberi M.
      • Olefsky J.M.
      Insulin sensitivity: modulation by nutrients and inflammation.
      ) (Fig. 3).
      Figure thumbnail gr3
      Fig. 3Downstream anti-inflammatory effects of AMPK signaling. Activation of AMPK reduces vascular inflammation via inhibiting NF-κB signaling pathway, leading to decreases in cytokine production, lipid peroxidation, and leukocyte homing. On the other hand, AMP activation improves vascular function by influencing eNOS and acting on the SIRT1-PGC1α-FoxO axis.
      Polarization of resident macrophages toward pro-inflammatory M1 or mixed M1/M2 phenotypes is a hallmark of low-grade inflammation associated with metabolic disorders (
      • Olefsky J.M.
      • Glass C.K.
      Macrophages, inflammation, and insulin resistance.
      ). Similar to other tissues, AMPK is also believed to act as a “master switch” of macrophage functional polarization, as it can be rapidly activated or inactivated upon exposure to anti-inflammatory (IL-10, TGF-β) or pro-inflammatory (LPS) stimuli, respectively (
      • Sag D.
      • Carling D.
      • Stout R.D.
      • Suttles J.
      Adenosine 5′-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype.
      ). In murine macrophages, constitutively active AMPKα1 significantly lowered production of LPS-induced TNF-α and IL-6, whereas IL-10 secretion remained substantially upregulated coincidently with modulation of IkB-B, Akt/GSKβ, mTOR, and CREB (
      • Sag D.
      • Carling D.
      • Stout R.D.
      • Suttles J.
      Adenosine 5′-monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype.
      ), known downstream mediators of AMPK signaling (
      • Mihaylova M.M.
      • Shaw R.J.
      The AMPK signalling pathway coordinates cell growth, autophagy and metabolism.
      ). Interestingly, AMPK/angiotensin-converting enzyme (ACE) axis also has been recently shown to support macrophage M2 polarization. Although controversial, this study demonstrates that unidentified lipid mediators secreted by adipocytes from nonobese individuals stimulate AMPK phosphorylation and ACE expression and promote macrophage phenotypic transition toward anti-inflammatory state (
      • Kohlstedt K.
      • Trouvain C.
      • Namgaladze D.
      • Fleming I.
      Adipocyte-derived lipids increase angiotensin-converting enzyme (ACE) expression and modulate macrophage phenotype.
      ).
      In the subendothelial space of the large and small arteries, differentiation of monocyte-derived macrophages into lipid-laden foam cells is considered to be a crucial event for the development and progression of atherosclerotic lesion (
      • Rocha V.Z.
      • Libby P.
      Obesity, inflammation, and atherosclerosis.
      ). While uncontrolled uptake of modified LDL by macrophage scavenger receptors is largely responsible for this process, cholesterol efflux mediated by the ATP-binding cassette transporters ABCA1 and ABCG1 is recognized as a critical mechanism to balance tissue cholesterol homeostasis (
      • Yvan-Charvet L.
      • Wang N.
      • Tall A.R.
      Role of HDL, ABCA1, and ABCG1 transporters in cholesterol efflux and immune responses.
      ). AMPK activation attenuates oxLDL-induced lipid accumulation in murine macrophages by upregulation of ABCG1 expression and promoting cholesterol efflux from lipid-laden cells (
      • Li D.
      • Wang D.
      • Wang Y.
      • Ling W.
      • Feng X.
      • Xia M.
      Adenosine monophosphate-activated protein kinase induces cholesterol efflux from macrophage-derived foam cells and alleviates atherosclerosis in apolipoprotein E-deficient mice.
      ). Furthermore, infusion of AICAR in vivo augments ABCG1 expression levels and markedly reduces atherosclerosis in ApoE−/− mice (
      • Li D.
      • Wang D.
      • Wang Y.
      • Ling W.
      • Feng X.
      • Xia M.
      Adenosine monophosphate-activated protein kinase induces cholesterol efflux from macrophage-derived foam cells and alleviates atherosclerosis in apolipoprotein E-deficient mice.
      ).
      Migration of circulating monocytes into vascular intima, followed by proliferation of monocyte/macrophage lineage within atherosclerotic lesion, represents a key feature of human atheroma as well as plaque instability and rupture (
      • Rocha V.Z.
      • Libby P.
      Obesity, inflammation, and atherosclerosis.
      ,
      • Rekhter M.D.
      • Gordon D.
      Active proliferation of different cell types, including lymphocytes, in human atherosclerotic plaques.
      ,
      • Koenig W.
      • Khuseyinova N.
      Biomarkers of atherosclerotic plaque instability and rupture.
      ). Consistent with the ability of AMPK to inhibit growth and proliferation of numerous mesenchymal cell types (
      • Hardie D.G.
      AMP-activated protein kinase: an energy sensor that regulates all aspects of cell function.
      ), AMPK activation also suppresses oxLDL-induced macrophage proliferation by directly causing cell cycle arrest independent of p38 MAPK/Akt signaling (
      • Ishii N.
      • Matsumura T.
      • Kinoshita H.
      • Motoshima H.
      • Kojima K.
      • Tsutsumi A.
      • Kawasaki S.
      • Yano M.
      • Senokuchi T.
      • Asano T.
      • et al.
      Activation of AMP-activated protein kinase suppresses oxidized low-density lipoprotein-induced macrophage proliferation.
      ). Migration and homing of leukocytes into inflammatory sites is an energy consuming function that is highly dependent on availability of energy substrates (
      • Carlier M.F.
      • Pantaloni D.
      Control of actin assembly dynamics in cell motility.
      ) and, therefore, can potentially lead to a shortfall in energy supply (
      • Kanellis J.
      • Kandane R.K.
      • Etemadmoghadam D.
      • Fraser S.A.
      • Mount P.F.
      • Levidiotis V.
      • Kemp B.E.
      • Power D.A.
      Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells.
      ). AMPK activation in monocyte-like cell line rapidly reduced random cell migration as well as chemotaxis toward SDF1α gradient independent of CXCR4 receptor binding (
      • Kanellis J.
      • Kandane R.K.
      • Etemadmoghadam D.
      • Fraser S.A.
      • Mount P.F.
      • Levidiotis V.
      • Kemp B.E.
      • Power D.A.
      Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells.
      ) or subcellular localization of the Rho GTPases, RhoA, and cdc42, emphasizing the role of AMPK in control of cell movement via change in energy dynamics at the site of inflammation (
      • Kanellis J.
      • Kandane R.K.
      • Etemadmoghadam D.
      • Fraser S.A.
      • Mount P.F.
      • Levidiotis V.
      • Kemp B.E.
      • Power D.A.
      Activators of the energy sensing kinase AMPK inhibit random cell movement and chemotaxis in U937 cells.
      ).
      Energy deficiency, hypoxia, and a highly aggressive oxidative environment at the area of inflammation trigger significant endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in multiple resident cell types tangled with variety of human pathologies, including diabetes and obesity (
      • Ozcan U.
      • Cao Q.
      • Yilmaz E.
      • Lee A.H.
      • Iwakoshi N.N.
      • Ozdelen E.
      • Tuncman G.
      • Gorgun C.
      • Glimcher L.H.
      • Hotamisligil G.S.
      Endoplasmic reticulum stress links obesity, insulin action, and type 2 diabetes.
      ,
      • Erbay E.
      • Babaev V.R.
      • Mayers J.R.
      • Makowski L.
      • Charles K.N.
      • Snitow M.E.
      • Fazio S.
      • Wiest M.M.
      • Watkins S.M.
      • Linton M.F.
      • et al.
      Reducing endoplasmic reticulum stress through a macrophage lipid chaperone alleviates atherosclerosis.
      ). Circulating oxidized and glycated LDLs (HOG-LDL) damage and induce apoptosis in vascular endothelial cells (EC), further promoting progression of atherosclerosis as well as other vascular complications of metabolic disorders (
      • Napoli C.
      Oxidation of LDL, atherogenesis, and apoptosis.
      ,
      • Artwohl M.
      • Graier W.F.
      • Roden M.
      • Bischof M.
      • Freudenthaler A.
      • Waldhausl W.
      • Baumgartner-Parzer S.M.
      Diabetic LDL triggers apoptosis in vascular endothelial cells.
      ). HOG-LDL-mediated impairment of vascular endothelium has been linked to increased ER stress via oxidation and inhibition of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) (
      • Dong Y.
      • Zhang M.
      • Liang B.
      • Xie Z.
      • Zhao Z.
      • Asfa S.
      • Choi H.C.
      • Zou M.H.
      Reduction of AMP-activated protein kinase alpha2 increases endoplasmic reticulum stress and atherosclerosis in vivo.
      ). AMPKα2-deficient mice revealed higher expression of ER stress markers, increased levels of intracellular Ca2+, and significantly reduced SERCA activity in aortic EC, strongly supporting protective role for AMPKα2 subunit in maintaining SERCA function (
      • Dong Y.
      • Zhang M.
      • Wang S.
      • Liang B.
      • Zhao Z.
      • Liu C.
      • Wu M.
      • Choi H.C.
      • Lyons T.J.
      • Zou M.H.
      Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.
      ). Consistently, deletion of AMPKα2 subunit in ApoE−/− or LDLR−/− mice results in escalation of vascular inflammation, oxidative stress, and largely increased atherosclerotic burden due to impaired endothelium-dependent vasorelaxation and SERCA activity (
      • Dong Y.
      • Zhang M.
      • Liang B.
      • Xie Z.
      • Zhao Z.
      • Asfa S.
      • Choi H.C.
      • Zou M.H.
      Reduction of AMP-activated protein kinase alpha2 increases endoplasmic reticulum stress and atherosclerosis in vivo.
      ,
      • Dong Y.
      • Zhang M.
      • Wang S.
      • Liang B.
      • Zhao Z.
      • Liu C.
      • Wu M.
      • Choi H.C.
      • Lyons T.J.
      • Zou M.H.
      Activation of AMP-activated protein kinase inhibits oxidized LDL-triggered endoplasmic reticulum stress in vivo.
      ).
      Activation of the NADPH oxidase system is aligned with metabolic disorders and believed to function as a primary mechanism of endothelial dysfunction and chronic vascular spasm (
      • Fernández-Sánchez A.
      • Madrigal-Santillan E.
      • Bautista M.
      • Esquivel-Soto J.
      • Morales-Gonzalez A.
      • Esquivel-Chirino C.
      • Durante-Montiel I.
      • Sanchez-Rivera G.
      • Valadez-Vega C.
      • Morales-Gonzalez J.A.
      Inflammation, oxidative stress, and obesity.
      ). Reduced availability of nitric oxide (NO) coupled with generation of multiple highly reactive oxidant species (ROS) modulates the activity of diverse intracellular signaling molecules via “redox signaling” (
      • Cave A.C.
      • Brewer A.C.
      • Narayanapanicker A.
      • Ray R.
      • Grieve D.J.
      • Walker S.
      • Shah A.M.
      NADPH oxidases in cardiovascular health and disease.
      ). Endothelial NO synthase (eNOS) is a direct AMPK target (
      • Towler M.C.
      • Hardie D.G.
      AMP-activated protein kinase in metabolic control and insulin signaling.
      ) linked to immune response and vascular inflammation. Several studies implicate AMPK activation as an effective pathway in improving a disturbed redox balance (
      • Towler M.C.
      • Hardie D.G.
      AMP-activated protein kinase in metabolic control and insulin signaling.
      ,
      • Cave A.C.
      • Brewer A.C.
      • Narayanapanicker A.
      • Ray R.
      • Grieve D.J.
      • Walker S.
      • Shah A.M.
      NADPH oxidases in cardiovascular health and disease.
      ) through restoration of vascular function via NO-dependent vasorelaxation and inhibition of leukocyte adhesion (
      • Ewart M.A.
      • Kohlhaas C.F.
      • Salt I.P.
      Inhibition of tumor necrosis factor alpha-stimulated monocyte adhesion to human aortic endothelial cells by AMP-activated protein kinase.
      ,
      • Wang S.
      • Xu J.
      • Song P.
      • Viollet B.
      • Zou M.H.
      In vivo activation of AMP-activated protein kinase attenuates diabetes-enhanced degradation of GTP cyclohydrolase I.
      ). Metformin and AICAR treatments result in increased superoxide dismutase mRNA expression and inhibition of hyperglycemia-induced intracellular and mitochondrial ROS production (
      • Kukidome D.
      • Nishikawa T.
      • Sonoda K.
      • Imoto K.
      • Fujisawa K.
      • Yano M.
      • Motoshima H.
      • Taguchi T.
      • Matsumura T.
      • Araki E.
      Activation of AMP-activated protein kinase reduces hyperglycemia-induced mitochondrial reactive oxygen species production and promotes mitochondrial biogenesis in human umbilical vein endothelial cells.
      ). AMPK upregulates UCP-2 expression in cultured EC and in aorta from diabetic mice, resulting in decreased superoxide and 3-nitrotyrosine production and increased NO bioactivity (
      • Xie Z.
      • Zhang J.
      • Wu J.
      • Viollet B.
      • Zou M.H.
      Upregulation of mitochondrial uncoupling protein-2 by the AMP-activated protein kinase in endothelial cells attenuates oxidative stress in diabetes.
      ). Interestingly, recent data suggest that antioxidant effect of rosiglitazone is strictly dependent on its ability to activate AMPK and is not mediated by PPAR-γ (
      • Ceolotto G.
      • Gallo A.
      • Papparella I.
      • Franco L.
      • Murphy E.
      • Iori E.
      • Pagnin E.
      • Fadini G.P.
      • Albiero M.
      • Semplicini A.
      • et al.
      Rosiglitazone reduces glucose-induced oxidative stress mediated by NAD(P)H oxidase via AMPK-dependent mechanism.
      ).
      There is now evidence that in human neutrophils, AMPK inhibits NADPH oxidase and suppresses superoxide production, suggesting that activation of AMPK could be an important mechanism for regulating an immune response when energy supply is limited (
      • Alba G.
      • El Bekay R.
      • Alvarez-Maqueda M.
      • Chacon P.
      • Vega A.
      • Monteseirin J.
      • Santa Maria C.
      • Pintado E.
      • Bedoya F.J.
      • Bartrons R.
      • et al.
      Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils.
      ).
      Mechanistically, AMPK directly phosphorylates and controls activity of numerous key metabolic regulators and transcription factors predominantly involved in control of glucose and lipid metabolism (
      • Towler M.C.
      • Hardie D.G.
      AMP-activated protein kinase in metabolic control and insulin signaling.
      ). Multiple studies demonstrated that AMPK activation downregulates NF-kB system, a prime mechanism for low-grade chronic inflammation associated with obesity, type-2 diabetes, and atherosclerosis (
      • Olefsky J.M.
      • Glass C.K.
      Macrophages, inflammation, and insulin resistance.
      ,
      • Schenk S.
      • Saberi M.
      • Olefsky J.M.
      Insulin sensitivity: modulation by nutrients and inflammation.
      ,
      • Rocha V.Z.
      • Libby P.
      Obesity, inflammation, and atherosclerosis.
      ). However, inhibitory effects of AMPK on NF-kB signaling are likely to be indirect and governed by downstream mediators, such as SIRT1 and PGC1α (
      • Salminen A.
      • Hyttinen J.M.
      • Kaarniranta K.
      AMP-activated protein kinase inhibits NF-κB signaling and inflammation: impact on healthspan and lifespan.
      ). Exposure of macrophages to pro-inflammatory stimuli results in decreased sirtuin levels associated with increased activation of RelA/p65 subunit of NF-kB and increased pro-inflammatory cytokine release (
      • Yang S.R.
      • Wright J.
      • Bauter M.
      • Seweryniak K.
      • Kode A.
      • Rahman I.
      Sirtuin regulates cigarette smoke-induced proinflammatory mediator release via RelA/p65 NF-kappaB in macrophages in vitro and in rat lungs in vivo: implications for chronic inflammation and aging.
      ). Constitutively active AMPKα1 mimics the effect of SIRT1 on deacetylating NF-kB and, as a result, significantly inhibits LPS- and free fatty acid (FFA)-induced TNF-α expression and restores macrophage anti-inflammatory phenotype (
      • Yang Z.
      • Kahn B.B.
      • Shi H.
      • Xue B.Z.
      Macrophage alpha1 AMP-activated protein kinase (alpha1AMPK) antagonizes fatty acid-induced inflammation through SIRT1.
      ). Consistently, myeloid-specific deletion of SIRT1 gene results in increased accumulation of activated macrophages in liver and adipose tissue of mice challenged with a high-fat diet and promotes development of systemic insulin resistance and metabolic imbalance (
      • Schug T.T.
      • Xu Q.
      • Gao H.
      • Peres-da-Silva A.
      • Draper D.W.
      • Fessler M.B.
      • Purushotham A.
      • Li X.
      Myeloid deletion of SIRT1 induces inflammatory signaling in response to environmental stress.
      ).
      The p65 subunit of NF-kB constitutively bound to PGC-1α and NF-kB activation increases physical interaction between p65 and PGC1α, resulting in reduction in PGC1α expression and subsequent dysregulation of glucose oxidation (
      • Alvarez-Guardia D.
      • Palomer X.
      • Coll T.
      • Davidson M.M.
      • Chan T.O.
      • Feldman A.M.
      • Laguna J.C.
      • Vazquez-Carrera M.
      The p65 subunit of NF-kappaB binds to PGC-1alpha, linking inflammation and metabolic disturbances in cardiac cells.
      ). Adenovirus-mediated overexpression of the PGC1α gene in human aortic smooth muscle cells (SMC) and EC alleviates intracellular and mitochondrial ROS production and NF-kB activity, as well as MCP-1 and VCAM-1 expression, supporting the possibility that signaling molecules stimulating PGC-1α in the vasculature, such as AMPK, will exert beneficial effects on vascular inflammation and development of atherosclerosis (
      • Kim H.J.
      • Park K.G.
      • Yoo E.K.
      • Kim Y.H.
      • Kim Y.N.
      • Kim H.S.
      • Kim H.T.
      • Park J.Y.
      • Lee K.U.
      • Jang W.G.
      • et al.
      Effects of PGC-1alpha on TNF-alpha-induced MCP-1 and VCAM-1 expression and NF-kappaB activation in human aortic smooth muscle and endothelial cells.
      ). Thus, anti-inflammatory consequences of AMPK activation strongly support a rational to develop novel pharmacological interventions designed to stimulate AMPK activity and potentially provide additional clinical benefits for patients with metabolic disorders by reducing local and systemic inflammation linked to vascular complications of the disease.

      PHARMACOLOGICAL ACTIVATORS OF AMPK

      The effects on glucose and lipid metabolism described in the previous sections suggest that AMPK activation may be a potential mechanism in the effective treatment of type-2 diabetes and the MetS (
      • Winder W.W.
      • Hardie D.G.
      AMP-activated protein kinase, a metabolic master switch: possible roles in type 2 diabetes.
      ) and perhaps even obesity. Numerous new compounds have been reported as a result of searching novel AMPK activators by rational drug design, screening of chemical libraries, and testing of plant extracts. While a wide variety of agents activate AMPK, in many cases, the mechanisms remain poorly understood. Some agents directly interact with one or more of the AMPK protein subunits, while others influence AMPK activation indirectly through a number of players in the metabolic and signaling pathways (Fig. 4). The indirect activators of AMPK include pharmacological agents, nutritional staging, hormones, natural bioactives, and long chain fatty acids. The direct activators are selective small molecules.
      Figure thumbnail gr4
      Fig. 4As shown, many activators, such as resveratrol, berberine, and TZDs, inhibit mitochondrial ATP production and thus activate AMPK indirectly by increasing the cellular AMP:ATP ratio. Resveratrol also acts via SIRT1-mediated mechanism to influence LKB1 and, eventually, phosphorylation of AMPK. The biguanide drugs metformin and phenformin require the transporter OCT1 to enter cells but also work through modulation of AMP:ATP ratio. EGCG and α-lipoic acid activate AMPK via CaMKKβ. AICAR is taken up by cells and converted to ZMP, an AMP mimetic that binds to the AMPKγ subunit. A-769662, a small molecule, mimics the effect of AMP but binds to a different site, probably involving the β subunit. Activation of AMPK by PT1 occurs by relieving inhibition by the auto-inhibitory domain on the α subunit. Adipokines, leptin, and adiponectin activate AMPK by influencing α subunit indirectly.
      In the context of how indirect and direct AMPK activators induce AMPK activation, it is important to mention here the two upstream kinases in mammals, the LKB1 (
      • Hawley S.A.
      • Boudeau J.
      • Reid J.L.
      • Mustard K.J.
      • Udd L.
      • Makela T.P.
      • Alessi D.R.
      • Hardie D.G.
      Complexes between the LKB1 tumor suppressor, STRAD alpha/beta and MO25 alpha/beta are upstream kinases in the AMP-activated protein kinase cascade.
      ) and the CaMKK-β (
      • Hawley S.A.
      • Pan D.A.
      • Mustard K.J.
      • Ross L.
      • Bain J.
      • Edelman A.M.
      • Frenguelli B.G.
      • Hardie D.G.
      Calmodulin-dependent protein kinase kinase-beta is an alternative upstream kinase for AMP-activated protein kinase.