PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Effects of Exercise on AMPK Signaling and Downstream Components to PI3K in Rat with Type 2 Diabetes

Exercise can increase skeletal muscle sensitivity to insulin, improve insulin resistance and regulate glucose homeostasis in rat models of type 2 diabetes. However, the potential mechanism remains poorly understood. In this study, we established a male Sprague–Dawley rat model of type 2 diabetes, with insulin resistance and β cell dysfunction, which was induced by a high-fat diet and low-dose streptozotocin to replicate the pathogenesis and metabolic characteristics of type 2 diabetes in humans. We also investigated the possible mechanism by which chronic and acute exercise improves metabolism, and the phosphorylation and expression of components of AMP-activated protein kinase (AMPK) and downstream components of phosphatidylinositol 3-kinase (PI3K) signaling pathways in the soleus. As a result, blood glucose, triglyceride, total cholesterol, and free fatty acid were significantly increased, whereas insulin level progressively declined in diabetic rats. Interestingly, chronic and acute exercise reduced blood glucose, increased phosphorylation and expression of AMPKα1/2 and the isoforms AMPKα1 and AMPKα2, and decreased phosphorylation and expression of AMPK substrate, acetyl CoA carboxylase (ACC). Chronic exercise upregulated phosphorylation and expression of AMPK upstream kinase, LKB1. But acute exercise only increased LKB1 expression. In particular, exercise reversed the changes in protein kinase C (PKC)ζ/λ phosphorylation, and PKCζ phosphorylation and expression. Additionally, exercise also increased protein kinase B (PKB)/Akt1, Akt2 and GLUT4 expression, but AS160 protein expression was unchanged. Chronic exercise elevated Akt (Thr³⁰⁸) and (Ser⁴⁷³) and AS160 phosphorylation. Finally, we found that exercise increased peroxisome proliferator-activated receptor-γ coactivator 1 (PGC1) mRNA expression in the soleus of diabetic rats. These results indicate that both chronic and acute exercise influence the phosphorylation and expression of components of the AMPK and downstream to PIK3 (aPKC, Akt), and improve GLUT4 trafficking in skeletal muscle. These data help explain the mechanism how exercise regulates glucose homeostasis in diabetic rats.     

AMPK activation by AICAR inhibits myogenic differentiation and myostatin expression in Cattle

AMP-activated protein kinase (AMPK) regulates metabolism in skeletal muscle, and myostatin (MSTN) negatively regulates skeletal muscle development and growth. In the present study, AMPK activation and the relationship between AMPK and MSTN during myogenic differentiation were investigated in cultures derived from bovine skeletal muscle. Myoblasts capable of forming myotubes were obtained from bovine skeletal muscle and treated with AICAR to activate AMPK, resulting in suppressed myotube formation. AICAR treatment significantly reduced the expression of MSTN mRNA during myogenic differentiation. Combined treatment with AICAR and MSTN suppressed myotube formation to a greater extent than AICAR alone. SB431542, an inhibitor of MSTN signaling, promoted myotube formation during myogenic differentiation. However, simultaneous treatment with AICAR blocked this effect of SB431542. Therefore, AMPK activation inhibits myogenic differentiation but may suppress MSTN expression to balance muscle development.     

Discovery of TBC1D1 as an insulin-, AICAR-, and contraction-stimulated signaling nexus in mouse skeletal muscle

The Akt substrate of 160 kDa (AS160) is phosphorylated on Akt substrate (PAS) motifs in response to insulin and contraction in skeletal muscle, regulating glucose uptake. Here we discovered a dissociation between AS160 protein expression and apparent AS160 PAS phosphorylation among soleus, tibialis anterior, and extensor digitorum longus muscles. Immunodepletion of AS160 in tibialis anterior muscle lysates resulted in minimal depletion of the PAS band at 160 kDa, suggesting the presence of an additional PAS immunoreactive protein. By immunoprecipitation and mass spectrometry, we identified this protein as the AS160 paralog TBC1D1, an obesity candidate gene regulating GLUT4 translocation in adipocytes. TBC1D1 expression was severalfold higher in skeletal muscles compared with all other tissues and was the dominant protein detected by the anti-PAS antibody at 160 kDa in tibialis anterior and extensor digitorum longus but not soleus muscles. In vivo stimulation by insulin, contraction, and the AMP-activated protein kinase (AMPK) activator AICAR increased TBC1D1 PAS phosphorylation. Using mass spectrometry on TBC1D1 from mouse skeletal muscle, we identified several novel phosphorylation sites on TBC1D1 and found the majority were consensus or near consensus sites for AMPK. Semiquantitative analysis of spectra suggested that AICAR caused greater overall phosphorylation of TBC1D1 sites compared with insulin. Purified Akt and AMPK phosphorylated TBC1D1 in vitro, and AMPK, but not Akt, reduced TBC1D1 electrophoretic mobility. TBC1D1 is a major PAS immunoreactive protein in skeletal muscle that is phosphorylated in vivo by insulin, AICAR, and contraction. Both Akt and AMPK phosphorylate TBC1D1, but AMPK may be the more robust regulator.     

Inhibition of AMPK expression in skeletal muscle by systemic inflammation in COPD rats

Background

Chronic obstructive pulmonary disease (COPD) is a disease characterized by airflow limitation and inflammation. Meanwhile, COPD also is associated with metabolic disorders, such as skeletal muscle weakness. Strikingly, activation of AMP-activated protein kinase (AMPK) exerts critical roles in energy metabolism. However, it remains unclear whether and how the expression levels of AMPK are affected in the COPD model rats which may lead to the dysfunction of the skeletal muscle in these rats.

Methods

Here we developed a rat model of COPD, and we investigated the morphological changes of peripheral skeletal muscle and measured the levels of tumor necrosis factor -α (TNF-α) and AMPK in skeletal muscle by using approaches that include immunohistochemistry and polymerase chain reaction (PCR).

Results

We found that the expression levels of both AMPK mRNA and protein in skeletal muscles were significantly reduced in the COPD model rats, in comparison to those from the control rats, the COPD model rats that received treatments with AICAR and resveratrol, whereas the expression levels of TNF-α were elevated in COPD rats.

Conclusion

Such findings indicate that AMPK may serve as a target for therapeutic intervention in the treatment of muscle weakness in COPD patients.     

Activation of Wnt/β-Catenin Signaling Increases Insulin Sensitivity through a Reciprocal Regulation of Wnt10b and SREBP-1c in Skeletal Muscle Cells

Background

Intramyocellular lipid accumulation is strongly related to insulin resistance in humans, and we have shown that high glucose concentration induced de novo lipogenesis and insulin resistance in murin muscle cells. Alterations in Wnt signaling impact the balance between myogenic and adipogenic programs in myoblasts, partly due to the decrease of Wnt10b protein. As recent studies point towards a role for Wnt signaling in the pathogenesis of type 2 diabetes, we hypothesized that activation of Wnt signaling could play a crucial role in muscle insulin sensitivity.

Methodology/Principal Findings

Here we demonstrate that SREBP-1c and Wnt10b display inverse expression patterns during muscle ontogenesis and regeneration, as well as during satellite cells differentiation. The Wnt/β-catenin pathway was reactivated in contracting myotubes using siRNA mediated SREBP-1 knockdown, Wnt10b over-expression or inhibition of GSK-3β, whereas Wnt signaling was inhibited in myoblasts through silencing of Wnt10b. SREBP-1 knockdown was sufficient to induce Wnt10b protein expression in contracting myotubes and to activate the Wnt/β-catenin pathway. Conversely, silencing Wnt10b in myoblasts induced SREBP-1c protein expression, suggesting a reciprocal regulation. Stimulation of the Wnt/β-catenin pathway i) drastically decreased SREBP-1c protein and intramyocellular lipid deposition in myotubes; ii) increased basal glucose transport in both insulin-sensitive and insulin-resistant myotubes through a differential activation of Akt and AMPK pathways; iii) restored insulin sensitivity in insulin-resistant myotubes.

Conclusions/Significance

We conclude that activation of Wnt/β-catenin signaling in skeletal muscle cells improved insulin sensitivity by i) decreasing intramyocellular lipid deposition through downregulation of SREBP-1c; ii) increasing insulin effects through a differential activation of the Akt/PKB and AMPK pathways; iii) inhibiting the MAPK pathway. A crosstalk between these pathways and Wnt/β-catenin signaling in skeletal muscle opens the exciting possibility that organ-selective modulation of Wnt signaling might become an attractive therapeutic target in regenerative medicine and to treat obese and diabetic populations.     

Whole Body Deletion of AMP-activated Protein Kinase β2 Reduces Muscle AMPK Activity and Exercise Capacity

AMP-activated protein kinase (AMPK) β subunits (β1 and β2) provide scaffolds for binding α and γ subunits and contain a carbohydrate-binding module important for regulating enzyme activity. We generated C57Bl/6 mice with germline deletion of AMPK β2 (β2 KO) and examined AMPK expression and activity, exercise capacity, metabolic control during muscle contractions, aminoimidazole carboxamide ribonucleotide (AICAR) sensitivity, and susceptibility to obesity-induced insulin resistance. We find that β2 KO mice are viable and breed normally. β2 KO mice had a reduction in skeletal muscle AMPK α1 and α2 expression despite up-regulation of the β1 isoform. Heart AMPK α2 expression was also reduced but this did not affect resting AMPK α1 or α2 activities. AMPK α1 and α2 activities were not changed in liver, fat, or hypothalamus. AICAR-stimulated glucose uptake but not fatty acid oxidation was impaired in β2 KO mice. During treadmill running β2 KO mice had reduced maximal and endurance exercise capacity, which was associated with lower muscle and heart AMPK activity and reduced levels of muscle and liver glycogen. Reductions in exercise capacity of β2 KO mice were not due to lower muscle mitochondrial content or defects in contraction-stimulated glucose uptake or fatty acid oxidation. When challenged with a high-fat diet β2 KO mice gained more weight and were more susceptible to the development of hyperinsulinemia and glucose intolerance. In summary these data show that deletion of AMPK β2 reduces AMPK activity in skeletal muscle resulting in impaired exercise capacity and the worsening of diet-induced obesity and glucose intolerance.     

5'-AMP-activated protein kinase phosphorylates IRS-1 on Ser-789 in mouse C2C12 myotubes in response to 5-aminoimidazole-4-carboxamide riboside

Exercise is known to increase insulin sensitivity and is an effective form of treatment for the hyperglycemia observed in type 2 diabetes. Activation of 5'-AMP-activated protein kinase (AMPK) by 5-aminoimidazole-4-carboxamide riboside (AICAR), exercise, or electrically stimulated contraction leads to increased glucose transport in skeletal muscle. Here we report the first evidence of a direct interaction between AMPK and the most upstream component of the insulin-signaling cascade, insulin receptor substrate-1 (IRS-1). We find that AMPK rapidly phosphorylates IRS-1 on Ser-789 in cell-free assays as well as in mouse C2C12 myotubes incubated with AICAR. In the C2C12 myotubes activation of AMPK by AICAR matched the phosphorylation of IRS-1 on Ser-789. This phosphorylation correlates with a 65% increase in insulin-stimulated IRS-1-associated phosphatidylinositol 3-kinase activity in C2C12 myotubes preincubated with AICAR. The binding of phosphatidylinositol 3-kinase to IRS-1 was not affected by AICAR. These results demonstrate the existence of an interaction between AMPK and early insulin signaling that could be of importance to our understanding of the potentiating effects of exercise on insulin signaling.     

Role of adenosine 5'-monophosphate-activated protein kinase subunits in skeletal muscle mammalian target of rapamycin signaling

AMP-activated protein kinase (AMPK) is an important energy-sensing protein in skeletal muscle. Mammalian target of rapamycin (mTOR) mediates translation initiation and protein synthesis through ribosomal S6 kinase 1 (S6K1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). AMPK activation reduces muscle protein synthesis by down-regulating mTOR signaling, whereas insulin mediates mTOR signaling via Akt activation. We hypothesized that AMPK-mediated inhibitory effects on mTOR signaling depend on catalytic alpha2 and regulatory gamma3 subunits. Extensor digitorum longus muscle from AMPK alpha2 knockout (KO), AMPK gamma3 KO, and respective wild-type (WT) littermates (C57BL/6) were incubated in the presence of 5-aminoimidazole-4-carboxamide-1-beta-d-ribonucleoside (AICAR), insulin, or AICAR plus insulin. Phosphorylation of AMPK, Akt, and mTOR-associated signaling proteins were assessed. Insulin increased Akt Ser473 phosphorylation (P < 0.01), irrespective of genotype or presence of AICAR. AICAR increased phosphorylation of AMPK Thr172 (P < 0.01) in WT but not KO mice. Insulin stimulation increased phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46) (P < 0.01) in WT, AMPK alpha2 KO, and AMPK gamma3 KO mice. However, in WT mice, preincubation with AICAR completely inhibited insulin-induced phosphorylation of mTOR targets, suggesting mTOR signaling is blocked by prior AMPK activation. The AICAR-induced inhibition was partly rescued in extensor digitorum longus muscle from either alpha2 or gamma3 AMPK KO mice, indicating functional alpha2 and gamma3 subunits of AMPK are required for the reduction in mTOR signaling. AICAR alone was without effect on basal phosphorylation of S6K1 (Thr389), ribosomal protein S6 (Ser235/236), and 4E-BP1 (Thr37/46). In conclusion, functional alpha2 and gamma3 AMPK subunits are required for AICAR-induced inhibitory effects on mTOR signaling.     

Glycogen synthase kinase-3β is required for the induction of skeletal muscle atrophy

Skeletal muscle atrophy commonly occurs in acute and chronic disease. The expression of the muscle-specific E3 ligases atrogin-1 (MAFbx) and muscle RING finger 1 (MuRF1) is induced by atrophy stimuli such as glucocorticoids or absence of IGF-I/insulin and subsequent Akt signaling. We investigated whether glycogen synthase kinase-3β (GSK-3β), a downstream molecule in IGF-I/Akt signaling, is required for basal and atrophy stimulus-induced expression of atrogin-1 and MuRF1, and myofibrillar protein loss in C(2)C(12) skeletal myotubes. Abrogation of basal IGF-I signaling, using LY294002, resulted in a prominent induction of atrogin-1 and MuRF1 mRNA and was accompanied by a loss of myosin heavy chain fast (MyHC-f) and myosin light chains 1 (MyLC-1) and -3 (MyLC-3). The synthetic glucocorticoid dexamethasone (Dex) also induced the expression of both atrogenes and likewise resulted in the loss of myosin protein abundance. Genetic ablation of GSK-3β using small interfering RNA resulted in specific sparing of MyHC-f, MyLC-1, and MyLC-3 protein levels after Dex treatment or impaired IGF-I/Akt signaling. Interestingly, loss of endogenous GSK-3β suppressed both basal and atrophy stimulus-induced atrogin-1 and MuRF1 expression, whereas pharmacological GSK-3β inhibition, using CHIR99021 or LiCl, only reduced atrogin-1 mRNA levels in response to LY294002 or Dex. In conclusion, our data reveal that myotube atrophy and myofibrillar protein loss are GSK-3β dependent, and demonstrate for the first time that basal and atrophy stimulus-induced atrogin-1 mRNA expression requires GSK-3β enzymatic activity, whereas MuRF1 expression depends solely on the physical presence of GSK-3β.     

Activation of AMP-activated protein kinase stimulates Na+,K+-ATPase activity in skeletal muscle cells

Contraction stimulates Na(+),K(+)-ATPase and AMP-activated protein kinase (AMPK) activity in skeletal muscle. Whether AMPK activation affects Na(+),K(+)-ATPase activity in skeletal muscle remains to be determined. Short term stimulation of rat L6 myotubes with the AMPK activator 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR), activates AMPK and promotes translocation of the Na(+),K(+)-ATPase α(1)-subunit to the plasma membrane and increases Na(+),K(+)-ATPase activity as assessed by ouabain-sensitive (86)Rb(+) uptake. Cyanide-induced artificial anoxia, as well as a direct AMPK activator (A-769662) also increase AMPK phosphorylation and Na(+),K(+)-ATPase activity. Thus, different stimuli that target AMPK concomitantly increase Na(+),K(+)-ATPase activity. The effect of AICAR on Na(+),K(+)-ATPase in L6 myotubes was attenuated by Compound C, an AMPK inhibitor, as well as siRNA-mediated AMPK silencing. The effects of AICAR on Na(+),K(+)-ATPase were completely abolished in cultured primary mouse muscle cells lacking AMPK α-subunits. AMPK stimulation leads to Na(+),K(+)-ATPase α(1)-subunit dephosphorylation at Ser(18), which may prevent endocytosis of the sodium pump. AICAR stimulation leads to methylation and dephosphorylation of the catalytic subunit of the protein phosphatase (PP) 2A in L6 myotubes. Moreover, AICAR-triggered dephosphorylation of the Na(+),K(+)-ATPase was prevented in L6 myotubes deficient in PP2A-specific protein phosphatase methylesterase-1 (PME-1), indicating a role for the PP2A·PME-1 complex in AMPK-mediated regulation of Na(+),K(+)-ATPase. Thus contrary to the common paradigm, we report AMPK-dependent activation of an energy-consuming ion pumping process. This activation may be a potential mechanism by which exercise and metabolic stress activate the sodium pump in skeletal muscle.     

AMP-activated protein kinase plays an important evolutionary conserved role in the regulation of glucose metabolism in fish skeletal muscle cells

AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.     

推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制*

目的:探讨推拿对失神经骨骼肌萎缩大鼠的治疗作用及其机制。方法:48只雄性SD大鼠随机分为模型组(n=24)和推拿组(n=24),通过切断右侧胫神经制备腓肠肌萎缩大鼠模型。术后第2日开始给推拿组大鼠手术侧腓肠肌给予手法干预,模型组不予干预。两组分别在0 d、7 d、14 d、21 d四个时间点各处死6只大鼠,取大鼠双侧腓肠肌,称重后计算各组大鼠腓肠肌湿重比;HE染色测定肌纤维截面积和直径,实时荧光定量PCR检测腓肠肌中miR-23a、Akt、MuRF1、MAFbx基因相对表达量。结果:与0 d比较,模型组和推拿组大鼠腓肠肌湿重比、肌纤维截面积和直径呈现进行性下降的趋势,其中7 d、14 d、21 d推拿组腓肠肌湿重比、肌纤维截面积和直径均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组MuRF1、MAFbx、Akt mRNA表达均呈现先升后降的趋势,其中7 d、21 d推拿组MuRF1 mRNA表达均显著低于模型组(P＜0.05,P＜0.01),7 d、14 d、21 d推拿组MAFbx mRNA表达均显著低于模型组(P＜0.01,P＜0.05,P＜0.01),7 d、14 d、21 d推拿组Akt mRNA表达均显著高于模型组(P＜0.05,P＜0.01);与0 d比较,模型组和推拿组21 d时miR-23a mRNA表达升高,推拿组miR-23a mRNA表达显著高于模型组(P＜0.05)。结论:推拿能延缓失神经骨骼肌的萎缩,其机制可能与上调miR-23a、Akt基因的表达,下调 MuRF1、MAFbx基因的表达,使蛋白降解速度受到抑制,从而减轻骨骼肌蛋白的降解程度有关。     

Muscle contractions, AICAR, and insulin cause phosphorylation of an AMPK-related kinase

We hypothesized that AMP-activated protein kinase-related kinase 5 (ARK5)/novel kinase family 1 (NUAK1), an AMP-activated protein kinase (AMPK)-related kinase that has been found to be stimulated by protein kinase B (Akt), would be expressed in rat skeletal muscle and activated by electrically elicited contractions, 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), or insulin. We verified expression of ARK5 in muscle through RT-PCR and Western blot. Cross-reactivity of ARK5 immunoprecipitates with antibodies against phospho-AMPK was increased by approximately 30% by muscle contractions and approximately 60% by incubation of muscle with AICAR. AMPK was not detected in the ARK5 immunoprecipitates. Despite the apparent increase in phosphorylation of ARK5 at a site essential to its activation, neither contractions nor AICAR increased ARK5 activity. For muscles from animals injected with saline or insulin, we probed nonimmunoprecipitated samples in sequence for phosphotyrosine (P-Tyr), ARK5, and phosphorylated substrates of Akt (P-AS) and found that the ARK5 band could be precisely superimposed on phosphoprotein bands from the P-Tyr and P-AS blots. In the band corresponding to ARK5, insulin increased P-Tyr content by approximately 45% and cross-reactivity with the antibody against P-AS by approximately threefold. We also detected ARK5 in phosphotyrosine immunoprecipitates. Our data suggest that increased phosphorylation of ARK5 by muscle contractions or exposure to AICAR is insufficient to activate ARK5 in skeletal muscle, suggesting that some other modification (e.g., phosphorylation on tyrosine or by Akt) may be necessary to its activity in muscle.