Skeletal Muscle AMP-activated Protein Kinase Research Articles

Effect of l-Arginine Infusion on Glucose Disposal during Exercise in Humans

We have previously shown that local infusion of a nitric oxide synthase (NOS) inhibitor attenuates increases in leg glucose uptake during exercise in humans. We have also shown that infusion of the NOS substrate, l-arginine (l-Arg), increases glucose clearance, although the mechanisms involved were not determined. A potential mechanism for NO-mediated glucose disposal is via interactions with NOS and the energy sensor AMP-activated protein kinase (AMPK). The aim of this study was to determine the mechanism(s) by which l-Arg infusion increases glucose disposal during exercise in humans by examining total NOS activity and AMPK signaling. Seven males cycled for 120 min at 64% ± 1% VO(2)peak, during which the [6,6-H]glucose tracer was infused. During the final 60 min of exercise, either saline alone (Control, CON), or saline containing l-Arg HCl (l-Arg, 30 g at 0.5 g·min(-1)) was coinfused in a double-blind, randomized, counterbalanced order. l-Arg increased the glucose rate of disappearance and glucose clearance rate during exercise; however, this was accompanied by a 150% increase in plasma insulin concentration from 65 to 75 min (P < 0.05) that remained significantly elevated until 90 min of exercise. Skeletal muscle AMPK signaling, nNOSμ phosphorylation by AMPK, and total NOS activity increased to a similar extent in the two trials. The increase in glucose disposal after l-Arg infusion during exercise is likely due to the significantly higher plasma insulin concentration.

Men Have Greater Basal Skeletal Muscle Ampk Phosphorylation Than Women

AMP-activated protein kinase (AMPK) plays a central role in the regulation of cellular metabolism. It is activated when the energy charge of the cell is low. Acetyl-coenzyme A carboxylase (ACC) is a downstream target for AMPK wich produces its phosphorylation and inhibition, leading to reduced malonyl-coenzyme A and increased fatty acid transport into the mitochondria. Since women have greater leptin levels and higher capacity to oxide fat during exercise we hypothesized that women will have greater basal skeletal muscle AMPK and ACC phosphorylation compared to men. PURPOSE. To determine if there is an influence of fiber type and gender in skeletal muscle basal phosphorylation of AMPK and ACC. METHODS. Muscle biopsies were obtained in twenty five healthy men and nine women (age: 24.1 ± 3.3, 25.3 ± 4 yrs, and weight: 78.6 ± 8.8, 56.9 ± 6.7 kg, respectively). Protein expression of AMPK and ACC was measured by Western Blot. MHC distribution was determined by SDS-PAGE electrophoresis. RESULTS. Thr172-AMPKα phosphorylation was 2.4-fold higher in men compared to women (2.33 ± 1.4 and 0.99 ± 0.5 a.u., respectively, P < 0.01). However, ACC phosphorylation was similar in both genders (P=0.8). There was a significant positive association between type I MHC and basal Thr172-AMPKα phosphorylation (r=0.54; P<0.01), but only in men. CONCLUSION. Skeletal muscle basal AMPKα phosphorylation is higher in men compared to women, but both sexes have similar basal ACC phosphorylation. This could be in part explained by the lower percentage of type II muscle fibres in women compared to men. Our results suggest that other mechanisms in addition to AMPK regulate the basal ACC phosphorylation state in human skeletal muscle. Granted by ULPGC2009-07, INNOVA2020, proyecto 2010/01RM Cátedra del Real Madrid de la UEM.

Myostatin-deficient mice exhibit reduced insulin resistance through activating the AMP-activated protein kinase signalling pathway

Myostatin-null mice (Mstn(-/-)) have reduced body fat and increased tolerance to glucose. To date the molecular mechanisms through which myostatin regulates body fat content and insulin sensitivity are not known. Therefore, the aim of the current study was to identify signalling pathways through which myostatin regulates insulin sensitivity. Wild-type (WT) mice and Mstn(-/-) mice were fed either a control chow diet or a high fat diet (HFD) for 12 weeks. Glucose tolerance testing and insulin stimulated glucose uptake by M. extensor digitorum longus (EDL) were used as variables to determine insulin sensitivity. Quantitative PCR, Western blotting and enzyme assays were used to monitor AMP-activated protein kinase (AMPK) levels and activity. Mstn(-/-) mice exhibited reduced fat accumulation and peripheral insulin resistance when compared with WT mice, even when they were fed an HFD. Furthermore, treatment with a myostatin antagonist also increased insulin sensitivity during HFD. Consistent with increased insulin sensitivity, we also detected elevated levels of GLUT4, AKT, p-AKT and insulin receptor substrate-1 in Mstn(-/-) muscles. Molecular analysis showed that there is increased expression and activity of AMPK in Mstn(-/-) muscles. Furthermore, we also observed an increase in the AMPK downstream target genes, Sirt1 and Pgc-1α (also known as Ppargc1a), in skeletal muscle of Mstn(-/-) mice. We conclude that myostatin inactivation leads to increased AMPK levels and activity resulting in increased insulin sensitivity of skeletal muscle. We propose that, by regulating AMPK in skeletal muscle and adipose tissues, myostatin plays a major role in regulating insulin signalling.

Anti-Diabetic Effects of aCoptis chinensisContaining New Traditional Chinese Medicine Formula in Type 2 Diabetic Rats

The Chinese formula Tang-Min-Ling (TML), an improved product of the decoction of Dachaihu which has a history of more than 2000 years, has main constituents of Coptis chinensis Franch, Scutellaria baicalensis Georgi, Rheum officinale Baill and Bupleurum chinense DC. A multi-central randomized controlled investigation performed previously by us has showed that TML has positive effects on regulating glycometabolism in type 2 diabetes (T2DM) patients, but the mechanisms remain unclear. Using Otsuka Long-Evans Tokushima Fatty (OLETF) rats as an animal model with rosiglitazone as a positive control, we were able to detect TML's effect on the serum glucose, serum lipid, serum leptin and adiponcetin after oral administration for 12 weeks. We were also able to detect the insulin resistance level by a glucose clamp test and study the mechanisms of TML in improving insulin resistance by detecting skeletal muscle AMP-activated protein kinase (AMPK) and glucose transporter 4 (GLUT4). Results showed that TML significantly reduced the glucose area under a curve of the oral glucose tolerance test, and had a positive effect in regulating serum lipid metabolism. TML treatment also significantly reduced the serum leptin level, but it had no effect on the serum adiponectin level. The AMPK enzymatic activity and GLUT4 expression in Skeletal Muscle were also upregulated in the TML group. The results suggest that the Chinese medicine TML, which contains Coptis chinensis Franch as one of its components, improves glycometabolism and its possible mechanisms may involve in improvement of insulin resistance of OLETF rats.

Obesity impairs skeletal muscle AMPK signaling during exercise: role of AMPKα2 in the regulation of exercise capacity in vivo

ObjectiveSkeletal muscle AMP-activated protein kinase (AMPK)α2 activity is impaired in obese, insulin resistant individuals during exercise. We determined whether this defect contributes to the metabolic dysregulation and reduced exercise capacity observed in the obese state.DesignC57BL/6J wild-type (WT) mice and/or mice expressing a kinase dead AMPKα2 subunit in skeletal muscle (α2-KD) were fed chow or high fat (HF) diets from 3–16 weeks (wks) of age. At 15wks mice performed an exercise stress test to determine exercise capacity. In WT mice, muscle glucose uptake and skeletal muscle AMPKα2 activity was assessed in chronically catheterized mice (carotid artery/jugular vein) at 16wks. In a separate study, HF-fed WT and α2-KD mice performed 5wks of exercise training (from 15–20wks of age) to test whether AMPKα2 is necessary to restore work tolerance.ResultsHF-fed WT mice had reduced exercise tolerance during an exercise stress test, and an attenuation in muscle glucose uptake and AMPKα2 activity during a single bout of exercise (p<0.05 vs. chow). In chow-fed α2-KD mice running speed and time were impaired ~45% and ~55%, respectively (p<0.05 vs. WT chow); HF feeding further reduced running time ~25% (p<0.05 vs. α2-KD chow). In response to 5wks of exercise training, HF-fed WT and α2-KD mice increased maximum running speed ~35% (p<0.05 vs. pre-training) and maintained body weight at pre-training levels, whereas body weight increased in untrained HF WT and α2-KD mice. Exercise training restored running speed to levels seen in healthy, chow-fed mice.ConclusionHF feeding impairs AMPKα2 activity in skeletal muscle during exercise in vivo. While this defect directly contributes to reduced exercise capacity, findings in HF-fed α2-KD mice show that AMPKα2-independent mechanisms are also involved. Importantly, α2-KD mice on a HF-fed diet adapt to regular exercise by increasing exercise tolerance, demonstrating that this adaptation is independent of skeletal muscle AMPKα2 activity.

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.

N‐Acetylcysteine infusion does not affect glucose disposal during prolonged moderate‐intensity exercise in humans

There is evidence that reactive oxygen species (ROS) signalling is required for normal increases in glucose uptake during contraction of isolated mouse skeletal muscle, and that AMP-activated protein kinase (AMPK) is involved. The aim of this study was to determine whether ROS signalling is involved in the regulation of glucose disposal and AMPK activation during moderate-intensity exercise in humans. Nine healthy males completed 80 min of cycle ergometry at 62 +/- 1% of peak oxygen consumption ( V(O(2)peak).A 6,6-(2)H-glucose tracer was infused at rest and during exercise, and in a double-blind randomised cross-over design, N-acetylcysteine (NAC) or saline (CON) was co-infused. NAC was infused at 125 mg kg(1) h(1) for 15 min and then at 25 mg kg(1) h(1) for 20 min before and throughout exercise. NAC infusion elevated plasma NAC and cysteine, and muscle NAC and cysteine concentrations during exercise. Although neither NAC infusion nor exercise significantly affected muscle reduced or oxidised glutathione (GSH or GSSG) concentration (P > 0.05), S-glutathionylation (an indicator of oxidative stress) of a protein band of approximately 270 kDa was increased approximately 3-fold with contraction and this increase was prevented by NAC infusion. Despite this, exercised-induced increases in tracer determined glucose disposal, plasma lactate, plasma non-esterified fatty acids (NEFAs), and decreases in plasma insulin were not affected by NAC infusion. In addition, skeletal muscle AMPKalpha and acetyl-CoA carboxylase-beta (ACCbeta) phosphorylation increased during exercise by approximately 3- and approximately 6-fold (P < 0.05), respectively, and this was not affected by NAC infusion. Unlike findings in mouse muscle ex vivo, NAC does not attenuate skeletal muscle glucose disposal or AMPK activation during moderate-intensity exercise in humans.

Local hindlimb antioxidant infusion does not affect muscle glucose uptake during in situ contractions in rat

There is evidence that reactive oxygen species (ROS) contribute to the regulation of skeletal muscle glucose uptake during highly fatiguing ex vivo contraction conditions via AMP-activated protein kinase (AMPK). In this study we investigated the role of ROS in the regulation of glucose uptake and AMPK signaling during low-moderate intensity in situ hindlimb muscle contractions in rats, which is a more physiological protocol and preparation. Male hooded Wistar rats were anesthetized, and then N-acetylcysteine (NAC) was infused into the epigastric artery (125 mg.kg(-1).h(-1)) of one hindlimb (contracted leg) for 15 min before this leg was electrically stimulated (0.1-ms impulse at 2 Hz and 35 V) to contract at a low-moderate intensity for 15 min. The contralateral leg did not receive stimulation or local NAC infusion (rest leg). NAC infusion increased (P<0.05) plasma cysteine and cystine (by approximately 360- and 1.4-fold, respectively) and muscle cysteine (by 1.5-fold, P=0.001). Although contraction did not significantly alter muscle tyrosine nitration, reduced (GSH) or oxidized glutathione (GSSG) content, S-glutathionylation of protein bands at approximately 250 and 150 kDa was increased (P<0.05) approximately 1.7-fold by contraction, and this increase was prevented by NAC. Contraction increased (P<0.05) skeletal muscle glucose uptake 20-fold, AMPK phosphorylation 6-fold, ACCbeta phosphorylation 10-fold, and p38 MAPK phosphorylation 60-fold, and the muscle fatigued by approximately 30% during contraction and NAC infusion had no significant effect on any of these responses. This was despite NAC preventing increases in S-glutathionylation with contraction. In conclusion, unlike during highly fatiguing ex vivo contractions, local NAC infusion during in situ low-moderate intensity hindlimb contractions in rats, a more physiological preparation, does not attenuate increases in skeletal muscle glucose uptake or AMPK signaling.

Prevention of high-fat diet-induced muscular lipid accumulation in rats by α lipoic acid is not mediated by AMPK activation

Skeletal muscle triglyceride accumulation is associated with insulin resistance in obesity. Recently, it has been suggested that alpha lipoic acid (ALA) improves insulin sensitivity by lowering triglyceride accumulation in nonadipose tissues via activation of skeletal muscle AMP-activated protein kinase (AMPK). We examined whether chronic ALA supplementation prevents muscular lipid accumulation that is associated with high-fat diets via activation of AMPK. In addition, we tested if ALA supplementation was able to improve insulin sensitivity in rats fed low- and high-fat diets (LFD, HFD). Supplementing male Wistar rats with 0.5% ALA for 8 weeks significantly reduced body weight, both on LFD and HFD (-24% LFD+ALA vs. LFD, P < 0.01, and -29% HFD+ALA vs. HFD, P < 0.001). Oil red O lipid staining revealed a 3-fold higher lipid content in skeletal muscle after HFD compared with LFD and ALA-supplemented groups (P < 0.05). ALA improved whole body glucose tolerance ( approximately 20% lower total area under the curve (AUC) in ALA supplemented groups vs. controls, P < 0.05). These effects were not mediated by increased muscular AMPK activation or ALA-induced improvement of muscular insulin sensitivity. To conclude, the prevention of HFD-induced muscular lipid accumulation and the improved whole body glucose tolerance are likely secondary effects due to the anorexic nature of ALA.

Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

TBC1D4 (also known as AS160) regulates glucose transporter 4 (GLUT4) translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of serine (S)/threonine (T) residues by upstream kinases resulting in inactivation of Rab-GTPase-activating protein (Rab-GAP) activity leading to GLUT4 mobilization. The majority of known phosphorylation sites on TBC1D4 lie within the Akt consensus motif and are phosphorylated by insulin stimulation. However, the 5'-AMP-activated protein kinase (AMPK) and other kinases may also phosphorylate TBC1D4, and therefore we hypothesized the presence of additional phosphorylation sites. Mouse skeletal muscles were contracted or stimulated with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), and muscle lysates were subjected to mass spectrometry analyses resulting in identification of novel putative phosphorylation sites on TBC1D4. The surrounding amino acid sequence predicted that S711 would be recognized by AMPK. Using a phosphospecific antibody against S711, we found that AICAR and contraction increased S711 phosphorylation in mouse skeletal muscle, and this increase was abolished in muscle-specific AMPKalpha2 kinase-dead transgenic mice. Exercise in human vastus lateralis muscle also increased TBC1D4 S711 phosphorylation. Recombinant AMPK, but not Akt1, Akt2, or PKCzeta, phosphorylated purified muscle TBC1D4 on S711 in vitro. Interestingly, S711 was also phosphorylated in response to insulin in an Akt2- and rapamycin-independent, but a wortmannin-sensitive, manner, suggesting this site is regulated by one or more additional upstream kinases. Despite increased S711 phosphorylation with AICAR, contraction, and insulin, mutation of S711 to alanine did not alter glucose uptake in response to these stimuli. S711 is a novel TBC1D4 phosphorylation site regulated by AMPK in skeletal muscle.

Skeletal Muscle AMP-activated Protein Kinase Is Essential for the Metabolic Response to Exercise in Vivo

AMP-activated protein kinase (AMPK) has been postulated as a super-metabolic regulator, thought to exert numerous effects on skeletal muscle function, metabolism, and enzymatic signaling. Despite these assertions, little is known regarding the direct role(s) of AMPK in vivo, and results obtained in vitro or in situ are conflicting. Using a chronically catheterized mouse model (carotid artery and jugular vein), we show that AMPK regulates skeletal muscle metabolism in vivo at several levels, with the result that a deficit in AMPK activity markedly impairs exercise tolerance. Compared with wild-type littermates at the same relative exercise capacity, vascular glucose delivery and skeletal muscle glucose uptake were impaired; skeletal muscle ATP degradation was accelerated, and arterial lactate concentrations were increased in mice expressing a kinase-dead AMPKalpha2 subunit (alpha2-KD) in skeletal muscle. Nitric-oxide synthase (NOS) activity was significantly impaired at rest and in response to exercise in alpha2-KD mice; expression of neuronal NOS (NOSmicro) was also reduced. Moreover, complex I and IV activities of the electron transport chain were impaired 32 +/- 8 and 50 +/- 7%, respectively, in skeletal muscle of alpha2-KD mice (p < 0.05 versus wild type), indicative of impaired mitochondrial function. Thus, AMPK regulates neuronal NOSmicro expression, NOS activity, and mitochondrial function in skeletal muscle. In addition, these results clarify the role of AMPK in the control of muscle glucose uptake during exercise. Collectively, these findings demonstrate that AMPK is central to substrate metabolism in vivo, which has important implications for exercise tolerance in health and certain disease states characterized by impaired AMPK activation in skeletal muscle.

Central leptin action improves skeletal muscle AKT, AMPK, and PGC1α activation by hypothalamic PI3K-dependent mechanism

Central leptin action requires PI3K activity to modulate glucose homeostasis and peripheral metabolism. However, the mechanism behind this phenomenon is not clearly understood. We hypothesize that hypothalamic PI3K activity is important for the modulation of the AMP-activated protein kinase (AMPK)/acetyl-CoA carboxylase (ACC) pathway, PGC1α, and AKT in skeletal muscle (SM). To address this issue, we injected leptin into the lateral ventricle of rats. Hypothalamic JAK2 and AKT were activated by intracerebroventricular (ICV) injection of leptin in a time-dependent manner. Central leptin improved tolerance to glucose (GTT), increased PGC1α expression, and AKT, AMPK, ACC and JAK2 phosphorylation in the soleus muscle. Previous ICV administration of either LY294002 or propranolol (IP) blocked these effects. We concluded that the activation of the hypothalamic PI3K pathway is important for leptin-induced AKT phosphorylation, as well as for active catabolic pathway through AMPK and PGC1α in SM. Thus, a defective leptin signalling PI3K pathway in the hypothalamus may contribute to peripheral resistance to insulin associated to diet-induced obesity.

Genetic and metabolic effects on skeletal muscle AMPK in young and older twins

The protein complex AMP-activated protein kinase (AMPK) is believed to play an important role in the regulation of skeletal muscle glucose and lipid metabolism. Defects in the AMPK system might therefore be an important factor in the pathogenesis of type 2 diabetes. We aimed to identify genetic and environmental mechanisms involved in the regulation of AMPK expression and activity and to examine the association between AMPK protein levels and activity on the one hand, and glucose and fat metabolism on the other. We investigated skeletal muscle biopsies from 100 young and 82 older mono- and dizygotic nondiabetic twins excised during the basal and insulin-stimulated states of a physiological hyperinsulinemic-euglycemic clamp. AMPKalpha1, -alpha2, and -gamma3 mRNA expression was investigated using real-time PCR, and Western blotting was employed to measure protein levels. Multiple regression analyses indicated that skeletal muscle AMPK mRNA and protein expression as well as activity were regulated by sex, age, obesity, and aerobic capacity. Comparison of intraclass correlations on AMPK measurements from mono- and dizygotic twins suggested that skeletal muscle AMPK expression was under minor genetic influence. AMPKgamma3 protein expression and activity were negatively related to whole body glucose uptake through the nonoxidative metabolic pathway and positively related to phosphorylation of glycogen synthase. Our results suggest that skeletal muscle AMPK expression is under minor genetic control but regulated by age and sex and associated with obesity and aerobic capacity. Furthermore, our results indicate a role for gamma3-containing AMPK complexes in downregulation of insulin-stimulated nonoxidative glucose metabolism possibly through inhibition of glycogen synthase activity.

A-769662 activates AMPK β1-containing complexes but induces glucose uptake through a PI3-kinase-dependent pathway in mouse skeletal muscle

5'-AMP-activated protein kinase (AMPK) regulates several aspects of metabolism. Recently, A-769662 was shown to activate AMPK in skeletal muscle. However, no biological effects of AMPK activation by A-769662 in this tissue have been reported. We hypothesized that A-769662 would increase glucose uptake in skeletal muscle. We studied incubated soleus and extensor digitorum longus (EDL) muscles from 129S6/sv and C57BL/6 mice. Glucose uptake increased only in soleus from 129S6/sv when concentrations of A-769662 were 500 microM (approximately 15%, P < 0.05) and 1 mM (approximately 60%, P < 0.01). AMPK beta1- but not beta2-containing complexes were dose dependently activated by A-769662 in muscles from both genotypes (approximately 100% at 200 microM and 300-600% at 1 mM). The discrepancy between the A-769662-induced AMPK activation pattern and stimulation of glucose uptake suggested that these effects were unrelated. A-769662 increased phosphorylation of Akt in both muscles from both genotypes, with phosphorylation of T308 being significantly higher in soleus than in EDL in 129S6/sv mice (P < 0.01). In soleus from 129S6/sv mice, insulin receptor substrate 1-associated phosphatidylinositol 3 (PI3)-kinase activity was markedly increased with A-769662, and Akt phosphorylation and glucose uptake were inhibited by wortmannin while phosphorylation of acetyl-CoA carboxylase (S227) was unaffected. Thus, A-769662 activates beta1-containing AMPK complexes in skeletal muscle but induces glucose uptake through a PI3-kinase-dependent pathway. Although development of A-769662 has constituted a step forward in the search for AMPK activators targeting specific AMPK trimers, our data suggest that in intact muscle, A-769662 has off-target effects. This may limit use of A-769662 to study the role of AMPK in skeletal muscle metabolism.

Role of the atypical protein kinase Cζ in regulation of 5′-AMP-activated protein kinase in cardiac and skeletal muscle

During metabolic stress, phosphorylation and activation of 5'-AMP-activated protein kinase (AMPK) becomes a major regulator of cellular energy metabolism in heart and skeletal muscle. Despite this, the upstream regulation of AMPK in both heart and muscle is poorly understood. Recent work has implicated the atypical protein kinase Czeta (PKCzeta) as a regulator of AMPK in endothelial cells via phosphorylation of LKB1, an upstream AMPK kinase (AMPKK). Our goal was to determine the potential role PKCzeta plays in regulating AMPK in cardiac and skeletal muscle. Cultures of H9c2 myocytes (cardiac) and C(2)C(12) myotubes (skeletal muscle) were pretreated with a selective PKCzeta pseudosubstrate peptide inhibitor and treated with various AMPK activating agents to determine whether PKCzeta regulates AMPK. PKCzeta activity was also examined in isolated working rat hearts subjected to ischemia. We show that PKCzeta is not involved in regulating threonine 172 AMPK phosphorylation induced by metformin or phenformin in either cardiac or skeletal muscle cells but is involved in 5-aminoimidazole-4-carboxamine-1-beta-D-ribofuranoside (AICAR)-induced AMPK phosphorylation in cardiac muscle cells. Activation of PKCzeta with high palmitate concentrations is also insufficient to increase AMPK phosphorylation. Furthermore, we show that the ischemia-induced activation of AMPK is not accompanied by increased PKCzeta activity. Finally, we show that PKCzeta may actually be a downstream target of AMPK in skeletal muscle, since adenoviral expression of a dominant-negative mutant of AMPK prevented metformin- and AICAR-induced phosphorylation of PKCzeta. We conclude that PKCzeta plays a very minor role in the regulation of AMPK in cardiac and skeletal muscle and may actually be a downstream target of AMPK in skeletal muscle.

Two-hybrid analysis identifies PSMD11, a non-ATPase subunit of the proteasome, as a novel interaction partner of AMP-activated protein kinase

Mammalian AMP-activated protein kinase (AMPK) is a heterotrimeric serine/threonine protein kinase that acts as a sensor of cellular energy status. It interacts with a great variety of different substrates leading to short-term (i.e. regulation of the activity of different enzymes by direct phosphorylation) and long-term effects (i.e. regulation of transcriptional activity of different transcription factors). In this work, we describe the use of the yeast two-hybrid technology to identify additional proteins that interact with the different subunits of AMPK. We have performed three yeast two-hybrid screenings of a human skeletal muscle cDNA library using three different baits: a constitutively active form of AMPKα2 (LexA-AMPKα2-T172D) co-expressed with AMPKγ1, LexA-AMPKβ2 and LexA-AMPKγ3. Our results identify novel interaction partners of AMPK in human skeletal muscle. We also further characterize the interaction of AMPK with one of these novel interacting proteins, the non-ATPase subunit of the proteasome PSMD11. Our results indicate that AMPK is able to interact physically with this subunit and modify its phosphorylation status, supporting a possible role for AMPK in regulating proteasome function.

Genetic disruption of AMPK signaling abolishes both contraction- and insulin-stimulated TBC1D1 phosphorylation and 14-3-3 binding in mouse skeletal muscle

TBC1D1 is a Rab-GTPase-activating protein (GAP) known to be phosphorylated in response to insulin, growth factors, pharmacological agonists that activate 5'-AMP-activated protein kinase (AMPK), and muscle contraction. Silencing TBC1D1 in L6 muscle cells by siRNA increases insulin-stimulated GLUT4 translocation, and overexpression of TBC1D1 in 3T3-L1 adipocytes with low endogenous TBC1D1 expression inhibits insulin-stimulated GLUT4 translocation, suggesting a role of TBC1D1 in regulating GLUT4 translocation. Aiming to unravel the regulation of TBC1D1 during contraction and the potential role of AMPK in intact skeletal muscle, we used EDL muscles from wild-type (WT) and AMPK kinase dead (KD) mice. We explored the site-specific phosphorylation of TBC1D1 Ser(237) and Thr(596) and their relation to 14-3-3 binding, a proposed mechanism for regulation of GAP function of TBC1D1. We show that muscle contraction increases 14-3-3 binding to TBC1D1 as well as phosphorylation of Ser(237) and Thr(596) in an AMPK-dependent manner. AMPK activation by AICAR induced similar Ser(237) and Thr(596) phosphorylation of, and 14-3-3 binding to, TBC1D1 as muscle contraction. Insulin did not increase Ser(237) phosphorylation or 14-3-3 binding to TBC1D1. However, insulin increased Thr(596) phosphorylation, and intriguingly this response was fully abolished in the AMPK KD mice. Thus, TBC1D1 is differentially regulated in response to insulin and contraction. This study provides genetic evidence to support an important role for AMPK in regulating TBC1D1 in response to both of these physiological stimuli.

Does training fasted make you fast?

exercise training allows one to perform the same absolute exercise bout with less of a challenge to homeostasis. The metabolic adaptations include increases in the activity of tricarboxylic acid (TCA) cycle enzymes such as citrate synthase and the β-oxidation enzyme β-hydroxyacyl-CoA dehydrogenase

AMPK activation is fiber type specific in human skeletal muscle: effects of exercise and short-term exercise training

AMP-activated protein kinase (AMPK) has been extensively studied in whole muscle biopsy samples of humans, yet the fiber type-specific expression and/or activation of AMPK is unknown. We examined basal and exercise AMPK-alpha Thr(172) phosphorylation and AMPK subunit expression (alpha(1), alpha(2), and gamma(3)) in type I, IIa, and IIx fibers of human skeletal muscle before and after 10 days of exercise training. Before training basal AMPK phosphorylation was greatest in type IIa fibers (P < 0.05 vs. type I and IIx), while an acute bout of exercise increased AMPK phosphorylation in all fibers (P < 0.05), with the greatest increase occurring in type IIx fibers. Exercise training significantly increased basal AMPK phosphorylation in all fibers, and the exercise-induced increases were uniformly suppressed compared with pretraining exercise. Expression of AMPK-alpha(1) and -alpha(2) was similar between fibers and was not altered by exercise training. However, AMPK-gamma(3) was differentially expressed in skeletal muscle fibers (type IIx > type IIa > type I), irrespective of training status. Thus skeletal muscle AMPK phosphorylation and AMPK expression are fiber type specific in humans in the basal state, as well as during exercise. Our findings reveal fiber type-specific differences that have been masked in previous studies examining mixed muscle samples.

Brought in by force: AMPK, TBC1D1, and contraction-stimulated glucose transport in skeletal muscle

transient intracellular calcium oscillations were the first signaling mediator described to regulate contraction-stimulated glucose transport in skeletal muscle ([6][1]). Thirty years later, the discoveries, that 5′-AMP-activated protein kinase (AMPK) activator 5-aminoimidazole-4-carboxamide 1-β-