Growth factors and nutrients, such as amino acids and glucose, regulate mammalian target of rapamycin complex 1 (mTORC1) signaling and subsequent translational control in a coordinated manner. Brain-derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, activates mTORC1 and induces phosphorylation of its target, p70S6 kinase (p70S6K), at Thr389 in neurons. BDNF also increases mammalian target of rapamycin-dependent novel protein synthesis in neurons. Here, we report that BDNF-induced p70S6K activation is dependent on glucose, but not amino acids, sufficiency in cultured cortical neurons. AMP-activated protein kinase (AMPK) is the molecular background to this specific nutrient dependency. Activation of AMPK, which is induced by glucose deprivation, treatment with pharmacological agents such as 2-Deoxy-d-glucose, metformin, and 5-aminoimidazole-4-carboxamide ribonucleoside or forced expression of a constitutively active AMPK subunit, counteracts BDNF-induced phosphorylation of p70S6K and enhanced protein synthesis in cortical neurons. These results indicate that AMPK inhibits the effects of BDNF on mTORC1-mediated translation in neurons.

Background: Peroxisome proliferator-activated receptor alpha (PPAR alpha) regulates the expression of fatty acid metabolismgenes and is thought to play a role in the regulation of insulin secretion and lipid detoxification. We have examined the mechanism whereby glucose decreases PPAR alpha gene expression in the pancreatic beta-cell.Methods: INS832/13 beta-cell and isolated rat islets were incubated at 3 and 20 mM glucose for 18 h in the absence or presence of adenosine monophosphate (AMP)-activated protein kinase (AMPK) activators and inhibitors, as well as p38 mitogen-activated protein kinase (p38 MAPK) inhibitors. In another set of experiments, INS832/13 were infected with an adenovirus expressing a dominant-negative form of AMPK. PPAR alpha expression levels were measured by reverse transcription polymerase chain reaction and Western blot.Results: Elevated glucose reduced the abundance of the PPAR alpha transcript and protein, and its target genes acyl-coenzyme A (CoA) oxidase (ACO) and uncoupling protein 2 (UCP-2) in INS832/13 beta-cell and isolated rat islets. Glucose reduced AMPK activity, while the AMPK activators 5-amino-4-imidazolecarboxamide riboside and metformin increased PPAR alpha expression and suppressed the action of glucose. By contrast, the AMPK inhibitor compound C mimicked the glucose effect. A dominant negative form of AMPK alpha reduced the PPAR alpha, ACO and UCP-2 transcripts to the same extent as elevated glucose. Pharmacological evidence indicated that glucose-regulated PPAR alpha expression does not involve p38 MAPK, a target of AMPK in several cell types.Conclusions: The results indicate that glucose represses PPAR alpha gene expression via AMPK, but not via p38 MAPK in the beta-cell.

Detailed knowledge of the pathways by which ghrelin and leptin signal to AMPK in hypothalamic neurons and lead to regulation of appetite and glucose homeostasis is central to the development of effective means to combat obesity. Here we identify CaMKK2 as a component of one of these pathways, show that it regulates hypothalamic production of the orexigenic hormone NPY, provide evidence that it functions as an AMPKalpha kinase in the hypothalamus, and demonstrate that it forms a unique signaling complex with AMPKalpha and beta. Acute pharmacologic inhibition of CaMKK2 in wild-type mice, but not CaMKK2 null mice, inhibits appetite and promotes weight loss consistent with decreased NPY and AgRP mRNAs. Moreover, the loss of CaMKK2 protects mice from high-fat diet-induced obesity, insulin resistance, and glucose intolerance. These data underscore the potential of targeting CaMKK2 as a therapeutic intervention.

Hypercapnia (elevated CO(2) levels) occurs as a consequence of poor alveolar ventilation and impairs alveolar fluid reabsorption (AFR) by promoting Na,K-ATPase endocytosis. We studied the mechanisms regulating CO(2)-induced Na,K-ATPase endocytosis in alveolar epithelial cells (AECs) and alveolar epithelial dysfunction in rats. Elevated CO(2) levels caused a rapid activation of AMP-activated protein kinase (AMPK) in AECs, a key regulator of metabolic homeostasis. Activation of AMPK was mediated by a CO(2)-triggered increase in intracellular Ca(2+) concentration and Ca(2+)/calmodulin-dependent kinase kinase-beta (CaMKK-beta). Chelating intracellular Ca(2+) or abrogating CaMKK-beta function by gene silencing or chemical inhibition prevented the CO(2)-induced AMPK activation in AECs. Activation of AMPK or overexpression of constitutively active AMPK was sufficient to activate PKC-zeta and promote Na,K-ATPase endocytosis. Inhibition or downregulation of AMPK via adenoviral delivery of dominant-negative AMPK-alpha(1) prevented CO(2)-induced Na,K-ATPase endocytosis. The hypercapnia effects were independent of intracellular ROS. Exposure of rats to hypercapnia for up to 7 days caused a sustained decrease in AFR. Pretreatment with a beta-adrenergic agonist, isoproterenol, or a cAMP analog ameliorated the hypercapnia-induced impairment of AFR. Accordingly, we provide evidence that elevated CO(2) levels are sensed by AECs and that AMPK mediates CO(2)-induced Na,K-ATPase endocytosis and alveolar epithelial dysfunction, which can be prevented with beta-adrenergic agonists and cAMP.

Pre‐B‐cell enhancing factor 1 (PBEF‐1; also known as visfatin) is a stress‐inducible regulator of intracellular NAD+. Elevated NAD+ levels are associated with increased Sirtuin activity, which can contribute to life span extension in some organisms. Caloric restriction may serve to extend life span via this mechanism. Since AMP‐activated protein kinase (AMPK) serves an important role as a cellular fuel gauge, we investigated the role of AMPK in regulating muscle PBEF‐1 levels. PBEF‐1 was significantly increased (50%) in tibialis anterior muscle from mice expressing an activating mutation of AMPK (γ1R70Q) in skeletal muscle (p<0.01). PBEF‐1 was significantly decreased in the muscles of two models of impaired AMPK activity, muscle‐specific AMPKα2 inactive transgenic mice (13%; P<0.05) and muscle‐specific LKB1 knockout mice (30%; p<0.01). In addition to skeletal muscle, PBEF‐1 was also decreased in cardiac tissue from the AMPKα2 inactive transgenic mice and LKB1 knockouts, and this effect was relatively greater in the heart compared to skeletal muscle. We next investigated the effects of seven weeks of voluntary wheel cage running on PBEF‐1 protein expression in skeletal muscle and heart from rats. Training resulted in 26% increased PBEF‐1 levels in cardiac muscle (p<0.05), but had no effect in triceps muscle despite clearly increased citrate synthase activity in this muscle. In conclusion, AMPK may be an important regulator of the purported anti‐aging protein, PBEF‐1 in skeletal and cardiac muscles. PBEF‐1 levels are significantly elevated after training in cardiac muscle, suggesting a previously unidentified potential aging‐protective role of chronic exercise in the heart.

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic α subunit and two non-catalytic subunits, β and γ, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK α1β1γ1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian αβγ complex shows that β binds α and γ. Here we show that a β1(186–270)γ1 complex can form in the absence of detectable α subunit. Moreover, using alanine mutagenesis we show that β1 Thr-263 and Tyr-267 are required for βγ association but not αβ association.

Regular endurance exercise has profound benefits on overall health, including the prevention of obesity, cardiovascular disease, and diabetes. The objective of this study was to determine whether AMP-activated protein kinase (AMPK) mediates commonly observed adaptive responses to exercise training in skeletal muscle. Six weeks of voluntary wheel running induced a significant (P < 0.05) fiber type IIb to IIa/x shift in triceps muscle of wild-type mice. Despite similar wheel running capacities, this training-induced shift was reduced by ∼40% in transgenic mice expressing a muscle-specific AMPKα2 inactive subunit. Sedentary mice carrying an AMPK-activating mutation (γ1TG) showed a 2.6-fold increase in type IIa/x fibers but no further increase with training. To determine whether AMPK is involved in concomitant metabolic adaptations to training, we measured markers of mitochondria (citrate synthase and succinate dehydrogenase) and glucose uptake capacity (GLUT4 and hexokinase II). Mitochondrial markers increased similarly in wild-type and AMPKα2-inactive mice. Sedentary γ1TG mice showed a ∼25% increase in citrate synthase activity but no further increase with training. GLUT4 protein expression was not different in either line of transgenic mice compared with wild-type mice and tended to increase with training, although this increase was not statistically significant. Training induced a ∼65% increase in hexokinase II protein in wild-type mice but not in AMPKα2-inactive mice. Hexokinase II was significantly elevated in sedentary γ1TG mice, without an additional increase with training. AMPK is not necessary for exercise training-induced increases in mitochondrial markers, but it is essential for fiber type IIb to IIa/x transformation and increases in hexokinase II protein.

Although mutations in the gamma-subunit of AMP-activated protein kinase (AMPK) can result in excessive glycogen accumulation and cardiac hypertrophy, the mechanisms by which this occurs have not been well defined. Because >65% of cardiac AMPK activity is associated with the gamma1-subunit of AMPK, we investigated the effects of expression of an AMPK-activating gamma1-subunit mutant (gamma1 R70Q) on regulatory pathways controlling glycogen accumulation and cardiac hypertrophy in neonatal rat cardiac myocytes. Whereas expression of gamma1 R70Q displayed the expected increase in palmitate oxidation rates, rates of glycolysis were significantly depressed. In addition, glycogen synthase activity was increased in cardiac myocytes expressing gamma1 R70Q, due to both increased expression and decreased phosphorylation of glycogen synthase. The inhibition of glycolysis and increased glycogen synthase activity were correlated with elevated glycogen levels in gamma1 R70Q-expressing myocytes. In association with the reduced phosphorylation of glycogen synthase, glycogen synthase kinase (GSK)-3beta protein and mRNA levels were profoundly decreased in the gamma1 R70Q-expressing myocytes. Consistent with GSK-3beta negatively regulating hypertrophy via inhibition of nuclear factor of activated T cells (NFAT), the dramatic downregulation of GSK-3beta was associated with increased nuclear activity of NFAT. Together, these data provide important new information about the mechanisms by which a mutation in the gamma-subunit of AMPK causes altered AMPK signaling and identify multiple pathways involved in regulating both cardiac myocyte metabolism and growth that may contribute to the development of the gamma mutant-associated cardiomyopathy.

AMP-activated protein kinase (AMPK) is widely recognized as an important regulator of glucose transport in skeletal muscle. The p38 mitogen-activated protein kinase (MAPK) has been proposed to be a component of AMPK-mediated signaling. Here we used several different models of altered AMPK activity to determine whether p38 MAPK is a downstream intermediate of AMPK-mediated signaling in skeletal muscle. First, L6 myoblasts and myotubes were treated with AICAR, an AMPK stimulator. AMPK phosphorylation was significantly increased, but there was no change in p38 MAPK phosphorylation. Similarly, AICAR incubation of isolated rat extensor digitorum longus (EDL) muscles did not increase p38 phosphorylation. Next, we used transgenic mice expressing an inactive form of the AMPKalpha2 catalytic subunit in skeletal muscle (AMPKalpha2i TG mice). AMPKalpha2i TG mice did not exhibit any defect in basal or contraction-induced p38 MAPK phosphorylation. We also used transgenic mice expressing an activating mutation in the AMPKgamma1 subunit (gamma1R70Q TG mice). Despite activated AMPK, basal p38 MAPK phosphorylation was not different between wild type and gamma1R70Q TG mice. In addition, muscle contraction-induced p38 MAPK phosphorylation was significantly blunted in the gamma1R70Q TG mice. In conclusion, increasing AMPK activity by AICAR and AMPKgamma1 mutation does not increase p38 MAPK phosphorylation in skeletal muscle. Furthermore, AMPKalpha2i TG mice lacking contraction-stimulated AMPK activity have normal p38 MAPK phosphorylation. These results suggest that p38 MAPK is not a downstream component of AMPK-mediated signaling in skeletal muscle.

The AMP-activated protein kinase (AMPK) is a central regulator of the energy status of the cell, based on its unique ability to respond directly to fluctuations in the ratio of AMP:ATP. Because glucose and amino acids stimulate insulin release from pancreatic beta-cells by the regulation of metabolic intermediates, AMPK represents an attractive candidate for control of beta-cell function. Here, we show that inhibition of AMPK in beta-cells by high glucose inversely correlates with activation of the mammalian Target of Rapamycin (mTOR) pathway, another cellular sensor for nutritional conditions. Forced activation of AMPK by AICAR, phenformin, or oligomycin significantly blocked phosphorylation of p70S6K, a downstream target of mTOR, in response to the combination of glucose and amino acids. Amino acids also suppressed the activity of AMPK, and this at a minimum required the presence of leucine and glutamine. It is unlikely that the ability of AMPK to sense both glucose and amino acids plays a role in regulation of insulin secretion, as inhibition of AMPK by amino acids did not influence insulin secretion. Moreover, activation of AMPK by AICAR or phenformin did not antagonize glucose-stimulated insulin secretion, and insulin secretion was also unaffected in response to suppression of AMPK activity by expression of a dominant negative AMPK construct (K45R). Taken together, these results suggest that the inhibition of AMPK activity by glucose and amino acids might be an important component of the mechanism for nutrient-stimulated mTOR activity but not insulin secretion in the beta-cell.

AMP-activated protein kinase (AMPK) plays multiple roles in the body's overall metabolic balance and response to exercise, nutritional stress, hormonal stimulation, and the glucose-lowering drugs metformin and rosiglitazone. AMPK consists of a catalytic alpha subunit and two non-catalytic subunits, beta and gamma, each with multiple isoforms that form active 1:1:1 heterotrimers. Here we show that recombinant human AMPK alpha1beta1gamma1 expressed in insect cells is monomeric and displays specific activity and AMP responsiveness similar to rat liver AMPK. The previously determined crystal structure of the core of mammalian alphabetagamma complex shows that beta binds alpha and gamma. Here we show that a beta1(186-270)gamma1 complex can form in the absence of detectable alpha subunit. Moreover, using alanine mutagenesis we show that beta1 Thr-263 and Tyr-267 are required for betagamma association but not alphabeta association.

AMP-activated kinase (AMPK) is a ubiquitous metabolic sensor that inhibits the cystic fibrosis (CF) transmembrane conductance regulator (CFTR). To determine whether CFTR reciprocally regulates AMPK function in airway epithelia and whether such regulation is involved in lung inflammation, AMPK localization, expression, and activity and cellular metabolic profiles were compared as a function of CFTR status in CF and non-CF primary human bronchial epithelial (HBE) cells. As compared with non-CF HBE cells, CF cells had greater and more diffuse AMPK staining and had greater AMPK activity than their morphologically matched non-CF counterparts. The cellular [AMP]/[ATP] ratio was higher in undifferentiated than in differentiated non-CF cells, which correlated with AMPK activity under these conditions. However, this nucleotide ratio did not predict AMPK activity in differentiating CF cells. Inhibiting channel activity in non-CF cells did not affect AMPK activity or metabolic status, but expressing functional CFTR in CF cells reduced AMPK activity without affecting cellular [AMP]/[ATP]. Therefore, lack of functional CFTR expression and not loss of channel activity in CF cells appears to up-regulate AMPK activity in CF HBE cells, presumably through non-metabolic effects on upstream regulatory pathways. Compared with wild-type CFTR-expressing immortalized CF bronchial epithelial (CFBE) cells, DeltaF508-CFTR-expressing CFBE cells had greater AMPK activity and greater secretion of tumor necrosis factor-alpha and the interleukins IL-6 and IL-8. Further pharmacologic AMPK activation inhibited inflammatory mediator secretion in both wild type- and DeltaF508-expressing cells, suggesting that AMPK activation in CF airway cells is an adaptive response that reduces inflammation. We propose that therapies to activate AMPK in the CF airway may be beneficial in reducing excessive airway inflammation, a major cause of CF morbidity.

All proteins assembled by the ribosome must pass through the nascent-peptide exit tunnel. Some nascent peptides can specifically interact with the tunnel and affect the functions of the ribosome. The molecular mechanisms of such interactions and of the ribosome response are currently unknown. However, a recent study has revealed elements of the tunnel that might be involved in sensing the nascent-peptide sequence.

The AMP-activated protein kinase (AMPK) is an important metabolic sensor/effector that coordinates many of the changes in mammalian tissues during variations in energy availability. We have sought to create an in vivo genetic model of chronic AMPK activation, selecting murine skeletal muscle as a representative tissue where AMPK plays important roles. Muscle-selective expression of a mutant noncatalytic gamma1 subunit (R70Qgamma) of AMPK activates AMPK and increases muscle glycogen content. The increase in glycogen content requires the presence of the endogenous AMPK catalytic alpha-subunit, since the offspring of cross-breeding of these mice with mice expressing a dominant negative AMPKalpha subunit have normal glycogen content. In R70Qgamma1-expressing mice, there is a small, but significant, increase in muscle glycogen synthase (GSY) activity associated with an increase in the muscle expression of the liver isoform GSY2. The increase in glycogen content is accompanied, as might be expected, by an increase in exercise capacity. Transgene expression of this mutant AMPKgamma1 subunit may provide a useful model for the chronic activation of AMPK in other tissues to clarify its multiple roles in the regulation of metabolism and other physiological processes.

The AMP-activated protein kinase (AMPK) and cAMP signaling systems are both key regulators of cellular metabolism. In this study, we show that AMPK activity is attenuated in response to cAMP-elevating agents through modulation of at least two of its α subunit phosphorylation sites, viz. α-Thr172 and α1-Ser485/α2-Ser491, in the clonal β-cell line INS-1 as well as in mouse embryonic fibroblasts and COS cells. Forskolin, isobutylmethylxanthine, and the glucose-dependent insulinotropic peptide inhibited AMPK activity and reduced phosphorylation of the activation loop α-Thr172 via inhibition of calcium/calmodulin-dependent protein kinase kinase-α and -β, but not LKB1. These agents also enhanced phosphorylation of α-Ser485/491 by the cAMP-dependent protein kinase. AMPK α-Ser485/491 phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK α-Ser485/491 can be a site for autophosphorylation, which may play a role in limiting AMPK activation in response to energy depletion or other regulators. Thus, our findings not only demonstrate cross-talk between the cAMP/cAMP-dependent protein kinase and AMPK signaling modules, but also describe a novel mechanism by which multisite phosphorylation of AMPK contributes to regulation of its enzyme activity.