Diabetes Publish Ahead of Print, published online January 31, 2011 ORIGINAL ARTICLE Molecular Mechanism by Which AMP-Activated Protein Kinase Activation Promotes Glycogen Accumulation in Muscle Roger W. Hunter,1 Jonas T. Treebak,2 Jørgen F.P. Wojtaszewski,2 and Kei Sakamoto1 OBJECTIVE—During energy stress, AMP-activated protein energy-consuming conditions such as during contraction kinase (AMPK) promotes glucose transport and glycolysis for and also energy-depleting processes such as hypoxia, ATP production, while it is thought to inhibit anabolic glycogen which leads to an increase in fatty acid oxidation, glucose synthesis by suppressing the activity of glycogen synthase (GS) uptake, and inhibition of protein synthesis (1,5). The most to maintain the energy balance in muscle. Paradoxically, chronic well established function of AMPK activation in muscle is activation of AMPK causes an increase in glycogen accumulation to stimulate glucose transport by promoting the redis- in skeletal and cardiac muscles, which in some cases is tribution of GLUT4 from intracellular compartments to the associated with cardiac dysfunction. The aim of this study was cell surface (5–7). to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation. The resulting increase in glucose transport and phos- phorylation of glucose by hexokinase II leads to an in- RESEARCH DESIGN AND METHODS—We recently gener- crease in the intracellular level of glucose-6-phosphate ated knock-in mice in which wild-type muscle GS was replaced (G6P) (8,9). G6P can be used for the synthesis of glycogen by a mutant (Arg582Ala) that could not be activated by glucose-6- or metabolized in the glycolytic pathway to generate ATP. phosphate (G6P), but possessed full catalytic activity and could During glycogen synthesis, G6P is converted to uridine still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead diphosphate (UDP) glucose, and the glucosyl moiety from (KD) AMPK were incubated with glucose tracers and the AMPK- UDP glucose is used to elongate a growing glycogen chain activating compound 5-aminoimidazole-4-carboxamide ribonu- through a-1,4-glycosidic bonds by the action of glycogen cleoside (AICAR) ex vivo. GS activity and glucose uptake and synthase (GS) (10,11). There are two GS isoforms in utilization (glycolysis and glycogen synthesis) were assessed. mammals encoded by separate genes. GYS1, encoding the muscle isoform, is expressed in muscle and many other RESULTS—Even though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompa- organs, including kidney, heart, and brain, whereas GYS2, nied by increases in glucose transport and intracellular [G6P]. encoding the liver GS isoform, is expressed exclusively in These effects of AICAR required the catalytic activity of AMPK. the liver (11). GS activity of both isoforms is regulated by Strikingly, AICAR-induced glycogen synthesis was completely G6P, an allosteric activator, and by covalent phosphory- abolished in G6P-insensitive GS knock-in mice, although AICAR- lation, which inhibits enzyme activity (10). stimulated AMPK activation, glucose transport, and total glucose Carling and Hardie (12) reported that AMPK phosphor- utilization were normal. ylates muscle GS at site 2 (Ser8 [amino acid numbering CONCLUSIONS—We provide genetic evidence that AMPK starts from the initiator methionine residue] in human, activation promotes muscle glycogen accumulation by allosteric mouse, and rat), a known inhibitory site of the enzyme, in activation of GS through an increase in glucose uptake and cell-free assays. Recent work has shown in intact skeletal subsequent rise in cellular [G6P]. muscle tissue that acute stimulation of AMPK by a phar- macologic activator, 5-aminoimidazole-4-carboxamide ri- bonucleoside (AICAR), promotes phosphorylation of GS at site 2 (13), resulting in a decrease in enzymatic activity MPK is a major regulator of cellular and whole- (13–15). From these findings, it was anticipated that acti- body energy homeostasis that coordinates vation of AMPK would reduce muscle glycogen levels. metabolic pathways to balance nutrient supply However, in apparent conflict with this anticipation, long- Awith energy demand (1–4). In response to cel- term/chronic activation of AMPK increases glycogen stor- lular stress, AMPK inhibits anabolic pathways and stim- age in skeletal (16,17) and cardiac (18) muscles. Some ulates catabolic pathways to restore cellular energy have speculated that AMPK-mediated increases in glucose charge. In skeletal muscle, AMPK is activated under transport and the subsequent elevation of intracellular [G6P] are able to allosterically stimulate GS and thus gly- cogen synthesis by overriding the inhibitory phosphoryla- From the 1MRC Protein Phosphorylation Unit, College of Life Sciences, Uni- tion of GS in muscles (8,9). versity of Dundee, Dundee, U.K.; and the 2Molecular Physiology Group, This hypothesis, however, has not been directly tested, Department of Exercise and Sport Sciences, University of Copenhagen, Copenhagen, Denmark. mainly because there are currently no experimental or Corresponding author: Roger W. Hunter, [email protected]. assay systems to assess G6P-mediated regulation of GS in Received 13 August 2010 and accepted 24 December 2010. vivo. GS activity is routinely assayed in vitro using cell/ DOI: 10.2337/db10-1148 tissue extracts in which the rate of incorporation of UDP- This article contains Supplementary Data online at http://diabetes. 14 diabetesjournals.org/lookup/suppl/doi:10.2337/db10-1148/-/DC1. [ C]glucose into glycogen is measured in the absence or Ó 2011 by the American Diabetes Association. Readers may use this article as presence of G6P (19). GS activity in the presence of sat- long as the work is properly cited, the use is educational and not for profit, urating concentrations of G6P is independent of the state and the work is not altered. See http://creativecommons.org/licenses/by -nc-nd/3.0/ for details. of phosphorylation, and the activity ratio in the absence of diabetes.diabetesjournals.org DIABETES 1 Copyright American Diabetes Association, Inc., 2011 AMPK IN CONTROL OF MUSCLE GLYCOGEN STORAGE G6P relative to that in the presence of G6P is used as an liquid nitrogen and [14C]glycogen extracted by ethanol precipitation from KOH index of GS activity. However, it has been virtually im- digests as described (26). Measurement of glycolysis. The rate of glycolysis was determined by the possible to prove that G6P activates GS in vivo or to assess 3 fi detritiation of [5- H]glucose as described (22). Briefly, muscles were incubated its physiologic signi cance because G6P binds non- in 2 mL KRB containing 5.5 mmol/L glucose and 0.5 mCi/mmol [5-3H]glucose covalently to GS and therefore dissociates from it when for 40 min at 37°C. Muscles were frozen in liquid nitrogen, and the rate of muscle tissue is homogenized in a protein extraction [5-3H]glucose incorporation into glycogen was determined as described for D- 14 3 buffer. [U- C]glucose. [ H]H2O was isolated from conditioned KRB by borate com- We have recently identified a key residue, Arg582, which plex ion exchange chromatography and measured by scintillation counting. is located in a highly basic segment comprising a putative Preparation of muscle lysates. Muscle lysates were prepared as described (26). Homogenates were clarified at 3,000g for 10 min at 4°C, and protein G6P-binding pocket at the C-terminus of GS (20,21). Sub- concentration was estimated using Bradford reagent and bovine serum albu- stitution of Arg582 to Ala (R582A) caused a complete loss min (BSA) as standard. Lysates were frozen in liquid nitrogen and stored at 2 of allosteric activation of GS by G6P without affecting 80°C. phosphorylation-dependent enzymatic activity and robustly Immunoblotting. Muscle extracts (20–30 mg) were denatured in SDS sample reduced insulin-mediated glycogen synthesis in skeletal buffer, separated by SDS-PAGE, and transferred to polyvinylidene fluoride muscle (22). To investigate the physiologic involvement of membrane. Membranes were blocked for 1 h in 20 mmol/L Tris-HCl (pH 7.5), 137 mmol/L NaCl, and 0.1% (v/v) Tween-20 (TBST) containing 5% (w/v) allosteric activation of GS in regulating muscle glycogen skimmed milk. Membranes were incubated in primary antibody prepared in metabolism in vivo, a knock-in mouse expressing a G6P- TBST containing 5% (w/v) BSA overnight at 4°C. Detection was performed R582A/R582A insensitive GS mutant (GS mouse) has recently using horseradish peroxidase-conjugated secondary antibodies and enhanced been generated (22). Using this mouse model, we demon- chemiluminescence reagent. strate here that acute activation of AMPK promotes muscle Assay of glycogen synthase and phosphorylase. Muscle homogenates (25 mg) were assayed for glycogen synthase and phosphorylase activity (reverse glycogen synthesis through allosteric activation of GS 14 14 direction) by measuring the incorporation of UDP-[U C]glucose and [U- C] through increasing glucose uptake and the subsequent rise glucose-1-phosphate respectively into glycogen, as described (22). Results are in intracellular [G6P]. expressed as the activity ratio in the absence and presence of 10 mmol/L G6P (glycogen synthase) or 2 mmol/L AMP (phosphorylase). AMPK activity assay. AMPK was immunoprecipitated from 30 mg lysate with RESEARCH DESIGN AND METHODS antibodies against the a1 and a2 subunits and assayed for phosphotransferase 14 Materials. D-[U- C]glucose-1-phosphate was purchased from Hartmann An- activity toward AMARA peptide (AMARAASAAALARRR) using [g-32P]ATP, as alytic GmbH (Brunswick, Germany). All other radiochemicals were from previously described (28). Perkin Elmer (Buckinghamshire, U.K.). Human insulin (Actrapid) was from Assay of muscle glycogen. Frozen muscles were digested in 100 mL of 1 mol/L Novo-Nordisk (Bagsværd, Denmark). AICAR was from Toronto Research KOH for 20 min at 80°C. The pH was adjusted to 4.8 with 50 mL of 4 mol/L Chemicals (Ontario, Canada).