We investigated the importance of the two catalytic ␣-isoforms of the 5-AMP-activated protein kinase (AMPK) in 5-aminoimidazole-4-carboxamide-1-␤-4-ribofuranoside (AICAR) and contraction-induced glucose uptake in skeletal muscle. Incubated soleus and EDL muscle from whole-body ␣ 2 -or ␣ 1 -AMPK knockout (KO) and wild type (WT) mice were incubated with 2.0 mM AICAR or electrically stimulated to contraction. Both AICAR and contraction increased 2DG uptake in WT muscles. KO of ␣ 2 , but not ␣ 1 , abolished AICAR-induced glucose uptake, whereas neither KO affected contraction-induced glucose uptake. AICAR and contraction increased ␣ 2 -and ␣ 1 -AMPK activity in wild type (WT) muscles. During AICAR stimulation, the remaining AMPK activity in KO muscles increased to the same level as in WT. During contraction, the remaining AMPK activity in ␣ 2 -KO muscles was elevated by 100% probably explained by a 2-3-fold increase in ␣ 1 -protein. In ␣ 1 -KO muscles, ␣ 2 -AMPK activity increased to similar levels as in WT. Both interventions increased total AMPK activity, as expressed by AMPK-P and ACC␤-P, in WT muscles. During AICAR stimulation, this was dramatically reduced in ␣ 2 -KO but not in ␣1-KO, whereas during contraction, both measurements were essentially similar to WT in both KO-muscles. The results show that ␣ 2 -AMPK is the main donor of basal and AICAR-stimulated AMPK activity and is responsible for AICAR-induced glucose uptake. In contrast, during contraction, the two ␣-isoforms seem to substitute for each other in terms of activity, which may explain the normal glucose uptake despite the lack of either ␣ 2 -or ␣ 1 -AMPK. Alternatively, neither ␣-isoform of AMPK is involved in contractioninduced muscle glucose uptake.The 5Ј-AMP-activated protein kinase (AMPK) 1 is a multisubstrate serine/threonine protein kinase that is ubiquitously expressed and functions as an intracellular fuel sensor activated by depletion of high energy phosphor compounds (1, 2). Activation of AMPK initiates a complex series of signaling events, causing an increase in uptake and oxidation of substrates for ATP synthesis concurrent with decreasing ATP consuming biosynthetic processes such as protein (3, 4), lipid (1), and glycogen synthesis (5, 6).Both human and rodent studies have shown that AMPK in skeletal muscle is activated during exercise in vivo (7-10) and during contraction in vitro (11-15) probably by several coinciding mechanisms. These involve decreased ATP/AMP and PCr/Cr ratios (16, 17), decreased pH (16), and reduction of muscle glycogen content (6, 15) and substrate delivery (18 -20). Therefore, it is tempting to ascribe a role for AMPK in muscle metabolism in response to exercise, and in particular investigators have hypothesized a role for AMPK in contraction-stimulated glucose uptake (11,13,21).AMPK may also be activated by treatment with the adenosine analogue 5-aminoimidazole-4-carboxamide-1-␤-4-ribofuranoside (AICAR) in rat, mouse, and human skeletal muscle in vitro (6,11,21,22) and in vivo in conscious rats (23). AICAR is...

5 AMP-activated protein kinase (AMPK) is a key regulator of cellular metabolism and is regulated in muscle during exercise. We have previously established that only three of 12 possible AMPK α/β/γ-heterotrimers are present in human skeletal muscle. Previous studies describe discrepancies between total AMPK activity and regulation of its target acetyl-CoA-carboxylase (ACC)β. Also, exercise training decreases expression of the regulatory γ3 AMPK subunit and attenuates α2 AMPK activity during exercise. We hypothesize that these observations reflect a differential regulation of the AMPK heterotrimers. We provide evidence here that only the α2/β2/γ3 subunit is phosphorylated and activated during high-intensity exercise in vivo. The activity associated with the remaining two AMPK heterotrimers, α1/β2/γ1 and α2/β2/γ1, is either unchanged (20 min, 80% maximal oxygen uptake (V O 2 ,peak )) or decreased (30 or 120 s sprint-exercise). The differential activity of the heterotrimers leads to a total α-AMPK activity, that is decreased (30 s trial), unchanged (120 s trial) and increased (20 min trial). AMPK activity associated with the α2/β2/γ3 heterotrimer was strongly correlated to γ3-associated α-Thr-172 AMPK phosphorylation (r 2 = 0.84, P < 0.001) and to ACCβ Ser-221 phosphorylation (r 2 = 0.65, P < 0.001). These data single out the α2/β2/γ3 heterotrimer as an important actor in exercise-regulated AMPK signalling in human skeletal muscle, probably mediating phosphorylation of ACCβ.

We tested the hypothesis that 5'AMP-activated protein kinase (AMPK) plays an important role in regulating the acute, exercise-induced activation of metabolic genes in skeletal muscle, which were dissected from whole-body alpha2- and alpha1-AMPK knockout (KO) and wild-type (WT) mice at rest, after treadmill running (90 min), and in recovery. Running increased alpha1-AMPK kinase activity, phosphorylation (P) of AMPK, and acetyl-CoA carboxylase (ACC)beta in alpha2-WT and alpha2-KO muscles and increased alpha2-AMPK kinase activity in alpha2-WT. In alpha2-KO muscles, AMPK-P and ACCbeta-P were markedly lower compared with alpha2-WT. However, in alpha1-WT and alpha1-KO muscles, AMPK-P and ACCbeta-P levels were identical at rest and increased similarly during exercise in the two genotypes. The alpha2-KO decreased peroxisome-proliferator-activated receptor gamma coactivator (PGC)-1alpha, uncoupling protein-3 (UCP3), and hexokinase II (HKII) transcription at rest but did not affect exercise-induced transcription. Exercise increased the mRNA content of PGC-1alpha, Forkhead box class O (FOXO)1, HKII, and pyruvate dehydrogenase kinase 4 (PDK4) similarly in alpha2-WT and alpha2-KO mice, whereas glucose transporter GLUT 4, carnitine palmitoyltransferase 1 (CPTI), lipoprotein lipase, and UCP3 mRNA were unchanged by exercise in both genotypes. CPTI mRNA was lower in alpha2-KO muscles than in alpha2-WT muscles at all time-points. In alpha1-WT and alpha1-KO muscles, running increased the mRNA content of PGC-1alpha and FOXO1 similarly. The alpha2-KO was associated with lower muscle adenosine 5'-triphosphate content, and the inosine monophosphate content increased substantially at the end of exercise only in alpha2-KO muscles. In addition, subcutaneous injection of 5-aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) increased the mRNA content of PGC-1alpha, HKII, FOXO1, PDK4, and UCP3, and alpha2-KO abolished the AICAR-induced increases in PGC-1alpha and HKII mRNA. In conclusion, KO of the alpha2- but not the alpha1-AMPK isoform markedly diminished AMPK activation during running. Nevertheless, exercise-induced activation of the investigated genes in mouse skeletal muscle was not impaired in alpha1- or alpha2-AMPK KO muscles. Although it cannot be ruled out that activation of the remaining alpha-isoform is sufficient to increase gene activation during exercise, the present data do not support an essential role of AMPK in regulating exercise-induced gene activation in skeletal muscle.

An acute bout of exercise increases glucose uptake in skeletal muscle by an insulin-independent mechanism. In the period after exercise, insulin sensitivity to increased glucose uptake is enhanced. The molecular mechanisms underpinning this phenomenon are poorly understood but appear to involve an increased cell surface abundance of GLUT4. While increased proximal insulin signaling does not seem to mediate this effect, elevated phosphorylation of TBC1D4, a downstream target of both insulin (Akt) and exercise (AMPK) signaling, appears to play a role. The main purpose of this study was to determine whether AMPK activation increases skeletal muscle insulin sensitivity. We found that prior AICAR stimulation of wild-type mouse muscle increases insulin sensitivity to stimulate glucose uptake. However, this was not observed in mice with reduced or ablated AMPK activity in skeletal muscle. Furthermore, prior AICAR stimulation enhanced insulin-stimulated phosphorylation of TBC1D4 at Thr 649 and Ser 711 in wild-type muscle only. These phosphorylation events were positively correlated with glucose uptake. Our results provide evidence to support that AMPK activation is sufficient to increase skeletal muscle insulin sensitivity. Moreover, TBC1D4 phosphorylation may facilitate the effect of prior AMPK activation to enhance glucose uptake in response to insulin.The effect of insulin on skeletal muscle glucose uptake is increased in the period after a single bout of exercise. This phenomenon is observed in muscle from both humans and rodents (1-6) and may persist for up to 48 h after exercise, depending on carbohydrate availability (7-9). Improved muscle insulin sensitivity postexercise is mediated by one or several local contraction-induced mechanisms (10) involving both enhanced transport and intracellular processing of glucose. This period is characterized by increased GLUT4 protein abundance at the plasma membrane and enhanced glycogen synthase activity (11,12). These changes occur independent of global protein synthesis (13), including both total GLUT4 and glycogen synthase protein content (4,11), and are independent of changes in proximal insulin signaling, including Akt activation (3,4,(13)(14)(15)(16)(17).AMPK is a heterotrimeric complex consisting of catalytic (a1/a2) and regulatory subunits (b1/b2 and g1/g2/g3). Of the 12 heterotrimeric combinations, only 3 and 5 combinations have been found in the skeletal muscle of human and mouse, respectively (18,19). AMPK is activated in response to various stimuli that increase cellular energy stress (e.g., metformin, hypoxia, hyperosmolarity, muscle contraction, and exercise) (20). With energy stress, intracellular concentrations of AMP and ADP accumulate. This activates AMPK allosterically and decreases the ability of upstream phosphatases to dephosphorylate Thr 172 , which further increases AMPK phosphorylation and activity (21). Like exercise, AICAR increases AMPK activity in skeletal muscle (22), which partly mimics the metabolic changes observed during muscle contraction (...

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...