5′‐AMP‐activated protein kinase (AMPK) has been suggested to play a key role in the regulation of metabolism in skeletal muscle. AMPK is activated in treadmill‐exercised and electrically stimulated rodent muscles. Whether AMPK is activated during exercise in humans is unknown. We investigated the degree of activation and deactivation of α‐isoforms of AMPK during and after exercise. Healthy human subjects performed bicycle exercise on two separate occasions at either a low (∼50% maximum rate of O2 uptake (V̇O2,max) for 90 min) or a high (∼75% V̇O2,max for 60 min) intensity. Biopsies from the vastus lateralis muscle were obtained before and immediately after exercise, and after 3 h of recovery. We observed a 3‐ to 4‐fold activation of the α2‐AMPK isoform immediately after high intensity exercise, whereas no activation was observed after low intensity exercise. The activation of α2‐AMPK was totally reversed 3 h after exercise. In contrast, α1‐AMPK was not activated during either of the two exercise trials. The in vitro AMP dependency of α2‐AMPK was significantly greater than that of α1‐AMPK (∼3‐ vs.∼2‐fold). We conclude that in humans activation of α2‐AMPK during exercise is dependent upon exercise intensity. The stable activation of α2‐AMPK, presumably due to the activation of an upstream AMPK kinase, is compatible with a role for this kinase complex in the regulation of skeletal muscle metabolism during exercise, whereas the lack of stable α1‐AMPK activation makes this kinase complex a less likely candidate.

Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 p m o l / l ) -e u g l y c e m i c clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t 1 / 2 ] = 11 vs. 3 4 min) and glycogen synthase activity (t 1 / 2 = 8 vs. 17 min), and the magnitude of increase was two-to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase ( I RTK) activity (t 1 / 2 = 50 min), serine phosphorylation of Akt (t 1 / 2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t 1 / 2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t 1 / 2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase a c t i v i t y, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulinsignaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (1). In human skeletal muscle, the effects of exercise per se on muscle glucose transport are relatively short-lived (2-4 h), whereas the enhanced sensitivity for glucose transport activation by insulin has been observed >48 h after an exercise bout in human subjects (3-5). In rat skeletal muscle, it has been demonstrated that there is a marked increase in insulin sensitivity for both glucose transport and glycogen synthase activation after exercise (2,6). These changes facilitate glycogen resynthesis, and they may be the mechanism by which muscle glycogen storage is increased above pre-exercise values, known as "supercompensation" (7,8). Whether prior exercise also increases the sensitivity for glycogen synthase activation by insulin in human skeletal muscle is unknown.We have previously hypothesized that an upregulation of insulin signaling is involved in the increased insulin sensitivity after exercise (9). However, if humans are subjected to physiological hyperinsulinemia or if rat muscles are incubated in the presence of insulin 3-4 h after exercise, insulin receptor tyrosine kinase (IRTK) a c t i v i t y, insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation, and phosphatidylinositol (PI) 3-kinase activity are not enhanced in skeletal muscle (9,10). This suggests that exercise may modulate insulin signaling further downstream or affect processes directly involved in glucose transporter translocation and activation.Signaling involving D-3 phosphorylated inositol lipids, generated by the action of PI 3-kinases, has been suggested to lead to the metabolic effects of insulin, including the activation of glucose transport and glycogen sy...

The 5AMP-activated protein kinase (AMPK) is a potential antidiabetic drug target. Here we show that the pharmacological activation of AMPK by 5-aminoimidazole-1-␤-4-carboxamide ribofuranoside (AICAR) leads to inactivation of glycogen synthase (GS) and phosphorylation of GS at Ser 7 (site 2). In muscle of mice with targeted deletion of the ␣2-AMPK gene, phosphorylation of GS site 2 was decreased under basal conditions and unchanged by AICAR treatment. In contrast, in ␣1-AMPK knockout mice, the response to AICAR was normal. Fuel surplus (glucose loading) decreased AMPK activation by AICAR, but the phosphorylation of the downstream targets acetyl-CoA carboxylase-␤ and GS was normal. Fractionation studies suggest that this suppression of AMPK activation was not a direct consequence of AMPK association with membranes or glycogen, because AMPK was phosphorylated to a greater extent in response to AICAR in the membrane/glycogen fraction than in the cytosolic fraction. Thus, the downstream action of AMPK in response to AICAR was unaffected by glucose loading, whereas the action of the kinase upstream of AMPK, as judged by AMPK phosphorylation, was decreased. The fact that ␣2-AMPK is a GS kinase that inactivates GS while simultaneously activating glucose transport suggests that a balanced view on the suitability for AMPK as an antidiabetic drug target should be taken. Diabetes 53:3074 -3081, 2004 T he 5ЈAMP-activated protein kinase (AMPK) system is a sensor of cellular energy status that adjusts the supply of ATP to the demand for the nucleotide (1). Activation of ␣2-AMPK stimulates muscle glucose transport (2,3). Once glucose has been taken up and converted to glucose-6-phosphate (G6P), it can be stored as glycogen or metabolized by glycolysis to generate ATP. It has been reported that AMPK phosphorylates muscle glycogen synthase (GS) in cell-free assays at site 2 (Ser 7) (4). Thus, AMPK activation may under some conditions decrease the potential for glycogen synthesis. Recently, we and others showed that GS activity decreases in response to acute 5-aminoimidazole-1-␤-4-carboxamide ribofuranoside (AICAR) treatment of muscle-like cells in culture (5), isolated and perfused skeletal muscle (6 -8), and fast twitch, but not slow twitch, muscle in vivo (7). AICAR treatment leads to decreased gel mobility of GS in perfused muscle, which together with the decreased activity, is reversed by protein phosphatase treatment (6). These observations indicate that regulation of GS activity by AICAR involves phosphorylation of GS. Although it has been suggested that AICAR-induced GS deactivation is mediated by AMPK due to the negative correlation between ␣2-AMPK and GS activity (6), these data do not prove a causal relation. Thus, by studying muscle from ␣-AMPK knockout (KO) mice in the present study, we aimed to verify that AMPK is a muscle GS kinase in vivo.AMPK activity decreases when muscle is exposed to fuel surplus. For example, glucose loading and glycogen accumulation suppress muscle AMPK phosphorylation/ activation at basal co...

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In type 2 diabetes, insulin activation of muscle glycogen synthase (GS) is impaired. This defect plays a major role for the development of insulin resistance and hyperglycemia. In animal muscle, insulin activates GS by reducing phosphorylation at both NH 2 -and COOH-terminal sites, but the mechanism involved in human muscle and the defect in type 2 diabetes remain unclear. We studied the effect of insulin at physiological concentrations on glucose metabolism, insulin signaling and phosphorylation of GS in skeletal muscle from type 2 diabetic and well-matched control subjects during euglycemic-hyperinsulinemic clamps. Analysis using phospho-specific antibodies revealed that insulin decreases phosphorylation of sites 3a ؉ 3b in human muscle, and this was accompanied by activation of Akt and inhibition of glycogen synthase kinase-3␣. In type 2 diabetic subjects these effects of insulin were fully intact. Despite that, insulin-mediated glucose disposal and storage were reduced and activation of GS was virtually absent in type 2 diabetic subjects. Insulin did not decrease phosphorylation of sites 2 ؉ 2a in healthy human muscle, whereas in diabetic muscle insulin infusion in fact caused a marked increase in the phosphorylation of sites 2 ؉ 2a. This phosphorylation abnormality likely caused the impaired GS activation and glucose storage, thereby contributing to skeletal muscle insulin resistance, and may therefore play a pathophysiological role in type 2 diabetes.

We investigated the effects of caffeine ingestion on skeletal muscle glucose uptake, glycogen synthase (GS) activity, and insulin signaling intermediates during a 100-min euglycemic-hyperinsulinemic (100 U/ml) clamp. On two occasions, seven men performed 1-h one-legged knee extensor exercise at 3 h before the clamp. Caffeine (5 mg/kg) or placebo was administered in a randomized, double-blind fashion 1 h before the clamp. During the clamp, whole-body glucose disposal was reduced (P < 0.05) in caffeine (37.5 ؎ 3.1 mol ⅐ min ؊1 ⅐ kg ؊1 ) vs. placebo (54.1 ؎ 2.9 mol ⅐ min ؊1 ⅐ kg ؊1 ). In accordance, the total area under the curve over 100 min (AUC 0 -100 min ) for insulin-stimulated glucose uptake in caffeine was reduced (P < 0.05) by ϳ50% in rested and exercised muscle. Caffeine also reduced (P < 0.05) GS activity before and during insulin infusion in both legs. Exercise increased insulin sensitivity of leg glucose uptake in both caffeine and placebo. Insulin increased insulin receptor tyrosine kinase (IRTK), insulin receptor substrate 1-associated phosphatidylinositol (PI) 3-kinase activities, and Ser 473 phosphorylation of protein kinase B (PKB)/Akt significantly but similarly in rested and exercised legs. Furthermore, insulin significantly decreased glycogen synthase kinase-3␣ (GSK-3␣) activity equally in both legs. Caffeine did not alter insulin signaling in either leg. Plasma epinephrine and muscle cAMP concentrations were increased in caffeine. We conclude that 1) caffeine impairs insulin-stimulated glucose uptake and GS activity in rested and exercised human skeletal muscle; 2) caffeine-induced impairment of insulin-stimulated muscle glucose uptake and downregulation of GS activity are not accompanied by alterations in IRTK, PI 3-kinase, PKB/Akt, or GSK-3␣ but may be associated with increases in epinephrine and intramuscular cAMP concentrations; and 3) exercise reduces the detrimental effects of caffeine on insulin action in muscle. Diabetes 51:583-590, 2002

We investigated the possible regulatory role of glycogen in insulin-stimulated glucose transport and insulin signaling in skeletal muscle. Rats were preconditioned to obtain low (LG), normal, or high (HG) muscle glycogen content, and perfused isolated hindlimbs were exposed to 0, 100, or 10,000 microU/ml insulin. In the fast-twitch white gastrocnemius, insulin-stimulated glucose transport was significantly higher in LG compared with HG. This difference was less pronounced in the mixed-fiber red gastrocnemius and was absent in the slow-twitch soleus. In the white gastrocnemius, insulin activation of insulin receptor tyrosine kinase and phosphoinositide 3-kinase was unaffected by glycogen levels, whereas protein kinase B activity was significantly higher in LG compared with HG. In additional incubation experiments on fast-twitch epitrochlearis muscles, insulin-stimulated cell surface GLUT-4 content was significantly higher in LG compared with HG. The data indicate that, in fast-twitch muscle, the effect of insulin on glucose transport and cell surface GLUT-4 content is modulated by glycogen content, which does not involve initial but possibly more downstream signaling events.

A-769662 activates AMPK ␤1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle.