Molecular mechanism by which AMP-activated protein kinase activation promotes glycogen accumulation in muscle

OBJECTIVE

During energy stress, AMP-activated protein kinase (AMPK) promotes glucose transport and glycolysis for ATP production, while it is thought to inhibit anabolic glycogen synthesis by suppressing the activity of glycogen synthase (GS) to maintain the energy balance in muscle. Paradoxically, chronic activation of AMPK causes an increase in glycogen accumulation in skeletal and cardiac muscles, which in some cases is associated with cardiac dysfunction. The aim of this study was to elucidate the molecular mechanism by which AMPK activation promotes muscle glycogen accumulation.

RESEARCH DESIGN AND METHODS

We recently generated knock-in mice in which wild-type muscle GS was replaced by a mutant (Arg582Ala) that could not be activated by glucose-6-phosphate (G6P), but possessed full catalytic activity and could still be activated normally by dephosphorylation. Muscles from GS knock-in or transgenic mice overexpressing a kinase dead (KD) AMPK were incubated with glucose tracers and the AMPK-activating compound 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) ex vivo. GS activity and glucose uptake and utilization (glycolysis and glycogen synthesis) were assessed.

RESULTS

Even though AICAR caused a modest inactivation of GS, it stimulated muscle glycogen synthesis that was accompanied by increases in glucose transport and intracellular [G6P]. These effects of AICAR required the catalytic activity of AMPK. Strikingly, AICAR-induced glycogen synthesis was completely abolished in G6P-insensitive GS knock-in mice, although AICAR-stimulated AMPK activation, glucose transport, and total glucose utilization were normal.

CONCLUSIONS

We provide genetic evidence that AMPK activation promotes muscle glycogen accumulation by allosteric activation of GS through an increase in glucose uptake and subsequent rise in cellular [G6P].AMPK is a major regulator of cellular and whole-body energy homeostasis that coordinates metabolic pathways to balance nutrient supply with energy demand (1–4). In response to cellular stress, AMPK inhibits anabolic pathways and stimulates catabolic pathways to restore cellular energy charge. In skeletal muscle, AMPK is activated under energy-consuming conditions such as during contraction and also energy-depleting processes such as hypoxia, which leads to an increase in fatty acid oxidation, glucose uptake, and inhibition of protein synthesis (1,5). The most well established function of AMPK activation in muscle is to stimulate glucose transport by promoting the redistribution of GLUT4 from intracellular compartments to the cell surface (5–7).The resulting increase in glucose transport and phosphorylation of glucose by hexokinase II leads to an increase in the intracellular level of glucose-6-phosphate (G6P) (8,9). G6P can be used for the synthesis of glycogen or metabolized in the glycolytic pathway to generate ATP. During glycogen synthesis, G6P is converted to uridine diphosphate (UDP) glucose, and the glucosyl moiety from UDP glucose is used to elongate a growing glycogen chain through α-1,4-glycosidic bonds by the action of glycogen synthase (GS) (10,11). There are two GS isoforms in mammals encoded by separate genes. GYS1, encoding the muscle isoform, is expressed in muscle and many other organs, including kidney, heart, and brain, whereas GYS2, encoding the liver GS isoform, is expressed exclusively in the liver (11). GS activity of both isoforms is regulated by G6P, an allosteric activator, and by covalent phosphorylation, which inhibits enzyme activity (10).Carling and Hardie (12) reported that AMPK phosphorylates muscle GS at site 2 (Ser8 [amino acid numbering starts from the initiator methionine residue] in human, mouse, and rat), a known inhibitory site of the enzyme, in cell-free assays. Recent work has shown in intact skeletal muscle tissue that acute stimulation of AMPK by a pharmacologic activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), promotes phosphorylation of GS at site 2 (13), resulting in a decrease in enzymatic activity (13–15). From these findings, it was anticipated that activation of AMPK would reduce muscle glycogen levels. However, in apparent conflict with this anticipation, long-term/chronic activation of AMPK increases glycogen storage in skeletal (16,17) and cardiac (18) muscles. Some have speculated that AMPK-mediated increases in glucose transport and the subsequent elevation of intracellular [G6P] are able to allosterically stimulate GS and thus glycogen synthesis by overriding the inhibitory phosphorylation of GS in muscles (8,9).This hypothesis, however, has not been directly tested, mainly because there are currently no experimental or assay systems to assess G6P-mediated regulation of GS in vivo. GS activity is routinely assayed in vitro using cell/tissue extracts in which the rate of incorporation of UDP-[¹⁴C]glucose into glycogen is measured in the absence or presence of G6P (19). GS activity in the presence of saturating concentrations of G6P is independent of the state of phosphorylation, and the activity ratio in the absence of G6P relative to that in the presence of G6P is used as an index of GS activity. However, it has been virtually impossible to prove that G6P activates GS in vivo or to assess its physiologic significance because G6P binds noncovalently to GS and therefore dissociates from it when muscle tissue is homogenized in a protein extraction buffer.We have recently identified a key residue, Arg582, which is located in a highly basic segment comprising a putative G6P-binding pocket at the C-terminus of GS (20,21). Substitution of Arg582 to Ala (R582A) caused a complete loss of allosteric activation of GS by G6P without affecting phosphorylation-dependent enzymatic activity and robustly reduced insulin-mediated glycogen synthesis in skeletal muscle (22). To investigate the physiologic involvement of allosteric activation of GS in regulating muscle glycogen metabolism in vivo, a knock-in mouse expressing a G6P-insensitive GS mutant (GS^R582A/R582A mouse) has recently been generated (22). Using this mouse model, we demonstrate here that acute activation of AMPK promotes muscle glycogen synthesis through allosteric activation of GS through increasing glucose uptake and the subsequent rise in intracellular [G6P].