Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK->ERK1/2 pathway

5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEKERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease. 5'-AMP-activated protein kinase; nuclear transport     

Stimulation of glucose transport by AMP-activated protein kinase via activation of p38 mitogen-activated protein kinase

Activation of AMP-activated protein kinase (AMPK) has been recently demonstrated to be associated with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)-stimulated glucose transport mediated by both GLUT1 and GLUT4 transporters. However, signaling events upstream and downstream of AMPK are unknown. Here we report that 1) p38 mitogen-activated protein kinase (MAPK) and mitogen-activated protein kinase kinase 3 (MKK3) were activated by AICAR in Clone 9 cells, which express only the GLUT1 transporters, and 2) activation of p38 was required for AICAR-stimulated glucose transport since treatment of the cells with p38 inhibitor SB203580 or overexpression of dominant negative p38 mutant inhibited glucose transport. Moreover, we found that overexpression of the constitutively active form of AMPK mutant also resulted in a significant activation of p38, and inhibition of p38 activity by SB203580 did not affect AICAR-stimulated activation of AMPK. These findings demonstrate that AICAR-stimulated activation of p38 is indeed mediated by AMPK, and the p38 MAPK cascade is downstream of AMPK in the signaling pathway of AICAR-stimulated glucose transport in Clone 9 cells.     

AMP-activated protein kinase (AMPK) signaling in endothelial cells is essential for angiogenesis in response to hypoxic stress

AMP-activated protein kinase (AMPK) is a stress-activated protein kinase that is regulated by hypoxia and other cellular stresses that result in diminished cellular ATP levels. Here, we investigated whether AMPK signaling in endothelial cells has a role in regulating angiogenesis. Hypoxia induced the activating phosphorylation of AMPK in human umbilical vein endothelial cells (HUVECs), and AMPK activation was required for the maintenance of pro-angiogenic Akt signaling under these conditions. Suppression of AMPK signaling inhibited both HUVEC migration to VEGF and in vitro differentiation into tube-like structures in hypoxic, but not normoxic cultures. Dominant-negative AMPK also inhibited in vivo angiogenesis in Matrigel plugs that were implanted subcutaneously in mice. These data identify AMPK signaling as a new regulator of angiogenesis that is specifically required for endothelial cell migration and differentiation under conditions of hypoxia. As such, endothelial AMPK signaling may be a critical determinant of blood vessel recruitment to tissues that are subjected to ischemic stress.     

AMP-activated protein kinase--the key role in metabolic regulation

AMP-activated protein kinase (AMPK) is the central component of a protein kinase cascade that acts as an energy sensor maintaining the energy balance at the cellular as well as at the whole body level. Within the healthy cell, metabolic stress leading to an increase in AMP concentration results in AMPK activation. Once activated, AMPK "switches off" many anabolic pathways e.g. fatty acid and protein synthesis while "switches on" catabolic pathways such as fatty acid oxidation or glycolysis which serve to restore intracellular ATP level. Adipocyte derived hormones leptin and adiponectin activate AMPK in peripheral tissues increasing energy expenditure. AMPK also regulates food intake due to response to hormonal and nutrient signals in hypothalamus. Antidiabetic drugs that mimic the action of insulin activate the AMPK signaling pathways. Further studies are needed to clarify the importance of the AMPK activation for therapeutic effects of this drugs.     

Energy-sensing and signaling by AMP-activated protein kinase in skeletal muscle.

AMP-activated protein kinase (AMPK) is emerging as an important energy-sensing/signaling system in skeletal muscle. This kinase is activated allosterically by 5'-AMP and inhibited allosterically by creatine phosphate. Phosphorylation of AMPK by an upstream kinase, AMPK kinase (also activated allosterically by 5'-AMP), results in activation. It is activated in both rat and human muscle in response to muscle contraction, the extent of activation depending on work rate and muscle glycogen concentration. AMPK can also be activated chemically in resting muscle with 5-aminoimidazole-4-carboxamide-riboside, which enters the muscle and is phosphorylated to form ZMP, a nucleotide that mimics the effect of 5'-AMP. Once activated, AMPK is hypothesized to phosphorylate proteins involved in triggering fatty acid oxidation and glucose uptake. Evidence is also accumulating for a role of AMPK in inducing some of the adaptations to endurance training, including the increase in muscle GLUT-4, hexokinase, uncoupling protein 3, and some of the mitochondrial oxidative enzymes. It thus appears that AMPK has the capability of monitoring intramuscular energy charge and then acutely stimulating fat oxidation and glucose uptake to counteract the increased rates of ATP utilization during muscle contraction. In addition, this system may have the capability of enhancing capacity for ATP production when the muscle is exposed to endurance training.     

Inositol polyphosphate multikinase: an emerging player for the central action of AMP-activated protein kinase

AMP-activated protein kinase (AMPK) is an essential enzyme indispensable for energy sensing and metabolic homeostasis at both the cellular and whole-body levels. Phosphorylation of AMPK, a key step for its activation, is known to be regulated by upstream kinases such as liver kinase B1 (LKB1) and calmodulin-dependent protein kinase kinase-beta (CaMKKβ). Recent evidence shows that inositol polyphosphate multikinase (IPMK), which possesses both inositol phosphate kinase and lipid inositol kinase activities, can physiologically regulate AMPK signaling in cultured cells and in the arcuate nucleus. IPMK-mediated regulation of AMPK occurs through the dynamic protein interactions of IPMK with AMPK in response to glucose availability. Here we review and discuss a novel role for the hypothalamic IPMK signaling in the control of AMPK and central energy homeostasis.     

Regulation of Rac1 by Simvastatin in Endothelial Cells: DIFFERENTIAL ROLES OF AMP-ACTIVATED PROTEIN KINASE AND CALMODULIN-DEPENDENT KINASE KINASE-��*

These studies explore the connections between simvastatin, Rac1, and AMP-activated protein kinase (AMPK) pathways in cultured vascular endothelial cells and in arterial preparations isolated from statin-treated mice. In addition to their prominent effects on lipoprotein metabolism, statins can regulate the small GTPase Rac1, and may also affect the phosphorylation of the ubiquitous AMPK. We explored pathways of statin-modulated Rac1 and AMPK activation both in arterial preparations from statin-treated mice as well as in cultured endothelial cells. We treated adult mice with simvastatin daily for 2 weeks and then harvested and analyzed arterial preparations. Simvastatin treatment of mice led to a significant increase in AMPK and LKB1 phosphorylation and to a decrease in protein kinase A activity relative to control animals, associated with a marked increase in Rac1 activation. Exposure of bovine aortic endothelial cells to simvastatin for 24 h strikingly increased GTP-bound Rac1 and led to increased phosphorylation of AMPK as well as the AMPK kinase LKB1. These responses to simvastatin were blocked by mevalonate or geranylgeranyl pyrophosphate but not by farnesyl pyrophosphate. Small interfering RNA (siRNA)-mediated knockdown of AMPK abrogated simvastatin-induced Rac1 activation and LKB1 phosphorylation. Importantly, siRNA-mediated knockdown of the key AMPK kinase, calcium/calmodulin-dependent protein kinase kinase β, completely blocked simvastatin-induced endothelial cell migration and also abrogated statin-promoted phosphorylation of AMPK and LKB1, as did pharmacological inhibition with the specific calcium/calmodulin-dependent protein kinase β inhibitor STO-609. Moreover, siRNA-mediated knockdown of Rac1 completely blocked simvastatin-induced LKB1 phosphorylation, but without affecting simvastatin-induced AMPK phosphorylation. These findings establish a key role for simvastatin in activation of a novel Rac1-dependent signaling pathway in the vascular wall.HMG-CoA² reductase inhibitors, commonly known as statins, are widely prescribed for the prevention and treatment of hypercholesterolemia and cardiovascular diseases (1, 2). The salutary clinical effects of these drugs derive in part from their effects on the levels of serum lipoproteins, yet other statin responses appear to be mediated by alterations in vascular function involving the endothelial isoform of nitric-oxide synthase (3) and related signaling pathways. Inhibition of HMG-CoA reductase suppresses the cellular levels of its enzymatic product mevalonate, thereby attenuating formation both of cholesterol as well as the synthesis of distinct isoprenoid compounds such as farnesyl pyrophosphate (Fpp) and geranylgeranyl pyrophosphate (GGpp). Many key signaling proteins are covalently modified by these isoprenoids, which are the products of a metabolic pathway that diverges from the pathway that leads to cholesterol synthesis downstream of HMG-CoA reductase. These isoprenoid compounds can provide lipophilic anchors that facilitate membrane targeting and modulate protein-protein interactions of many key signaling proteins. One such iso-prenylated signaling protein is the GTP-binding cytoskeletonassociated protein Rac1, a member of the Rho GTPase small G protein family that undergoes geranylgeranylation at its C terminus. Statins also affect post-translational modification of another small GTPase, RhoA, that, like Rac1, is a geranylgeranylated protein that is an important determinant of vascular signaling (4–8). Rac1 has particularly important roles in vascular endothelial cells, where this cytoskeleton regulatory protein modulates activity of the endothelial isoform of nitric-oxide synthase (eNOS), a key determinant of vascular homeostasis (9). Rac1 activation in endothelial cells is influenced by the AMP-activated protein kinase (AMPK) (6), which itself is phosphorylated by the protein kinase LKB1 and by the calcium-calmodulin-dependent protein kinase β (CaMKKβ) (see review (10)). In recent years, numerous reports have described effects of statins on variety of these signaling proteins in different experimental systems (11–14).Statins have been shown to promote the phosphorylation of AMPK (13), a heterotrimeric enzyme involved in the modulation of cellular energy pathways that has also been implicated in eNOS regulation (3, 15–17). AMPK was originally discovered and characterized as a cellular “energy sensor” that can be activated by increases in the intracellular AMP:ATP ratio (18). However, in recent years, it has become clear that AMPK is also regulated through AMP-independent pathways involving enzyme phosphorylation on threonine 172 of the enzyme''s α subunit, leading to marked enzyme activation (19). Protein kinases that phosphorylate AMPK include the tumor suppressor LKB1 and the calcium/calmodulin-dependent kinase CaMKKβ. LKB1 itself is a phosphoprotein. The pathways that regulate LKB1 are incompletely understood, and a variety of upstream protein kinases have been implicated in LKB1 regulation (see review (20)). CaMKKβ is principally regulated by calcium binding, but this kinase may also be phosphorylated by the cAMP-dependent protein kinase PKA (21, 22). Another substrate for PKA in vascular cells is the actin-binding phosphoprotein VASP (23, 24); the phosphorylation state of VASP at its PKA site can serve as a surrogate marker for the activity of cAMP-dependent signaling pathways in the vascular wall (25). CaMKKβ has been shown to be involved in AMPK regulation in endothelial cells in response to receptor tyrosine kinase activation and via G protein-coupled receptor pathways (6). Activated AMPK directly phosphorylates eNOS, and this kinase thereby appears be an important determinant of NO-dependent signaling in endothelial cells. However, much remains to be learned about the molecular mechanisms whereby statins enhance AMPK activation.In cultured cells, statins have been shown to inhibit the geranylgeranylation of Rac1, associated with an increase in Rac1 GTP binding and activation (26). The activation of Rac1 is a key step in eNOS activation: siRNA-mediated Rac1 “knockdown” in endothelial cells markedly suppresses receptor signaling to eNOS (5, 7). siRNA-mediated AMPK knockdown suppresses Rac1 activation, again leading to the attenuation of receptor-dependent activation of eNOS (6). The relationships among these various statin-modulated signaling pathways are incompletely characterized. The present studies identify CaMKKβ and LKB1 as critical determinants of simvastatin-dependent activation of AMPK- and Rac1-modulated signaling and reveal that Rac1 in turn regulates LKB1 phosphorylation.     

AMPK induces MUC5B expression via p38 MAPK in NCI-H292 airway epithelial cells

Adenosine monophosphate-activated protein kinase (AMPK) is a well-known serine/threonine kinase that has been implicated in modulation of glucose and fatty acid metabolism. Recent reports have also implicated AMPK in modulation of mucin secretion. In this study, the effects and signaling pathways of AMPK on MUC5B expression were investigated in human NCI-H292 airway epithelial cells. Metformin, as an activator of AMPK, induced MUC5B expression in a dose-dependent manner. Compound C, as an inhibitor of AMPK, inhibited metformin-induced MUC5B expression in a dose-dependent manner. Metformin significantly activated phosphorylation of AMPK; compound C inhibited metformin-activated phosphorylation of AMPK. Without treatment with metformin, there was no difference in MUC5B mRNA expression between Ad-dnAMPK transfected and wild-type adenovirus transfected NCI-H292 cells. However, after treatment with metformin, MUC5B mRNA expression was increased in wild-type adenovirus transfected NCI-H292 cells; MUC5B mRNA expression was significantly decreased in Ad-dnAMPK transfected NCI-H292 cells. Metformin activated phosphorylation of p38 mitogen-activated protein kinase (MAPK); compound C inhibited metformin-activated phosphorylation of p38 MAPK. SB203580, as an inhibitor of p38 MAPK, significantly inhibited metformin-induced MUC5B mRNA expression, while U0126, as an inhibitor of ERK1/2 MAPK, had no effect. In addition, knockdown of p38 MAPK by p38 MAPK siRNA significantly blocked metformin-induced MUC5B mRNA expression. In conclusion, results of this study show that AMPK induces MUC5B expression through the p38 MAPK signaling pathway in airway epithelial cells.     

Opinion: alternative views of AMP-activated protein kinase

Genes most closely related to adenosine monophosphate (AMP)-activated protein kinase, including SAD kinases and Par-1 regulate cell polarity, although AMP-activated protein kinase (AMPK) modulates cellular energy status. LKB1 (Par-4) is required for normal activation of AMPK in the liver and also regulates cell polarity. AMPK is proposed to inhibit energy consuming activity while initiating energy producing activity during energy limitation. Demonstration that metformin, a common drug for Type 2 diabetes, requires LKB1 for full therapeutic benefit has increased interest in AMPK signaling. Despite the potential importance of AMPK signaling for diabetes, metabolic syndrome and even cancer, the developmental processes regulated by AMPK in genetically mutant animals require further elucidation. Mouse conditional null mutants for AMPK activity will allow genetic elucidation of AMPK function in vivo. This perspective focuses on sequence and structural moieties of AMPK and genetic analysis of AMPK mutations. Interestingly, the predicted protein structure of the carboxy-terminus of AMPKα resembles the carboxy-terminal KA-1 domain of MARK3, a Par-1 orthologue.     

AMPK activation attenuates S6K1, 4E-BP1, and eEF2 signaling responses to high-frequency electrically stimulated skeletal muscle contractions.

Regulation of protein translation through Akt and the downstream mammalian target of rapamycin (mTOR) pathway is an important component of the cellular response to hypertrophic stimuli. It has been proposed that 5'-AMP-activated protein kinase (AMPK) activation during muscle contraction may limit the hypertrophic response to resistance-type exercise by inhibiting translational signaling. However, experimental manipulation of AMPK activity during such a stimulus has not been attempted. Therefore, we investigated whether AMPK activation can attenuate the downstream signaling response of the Akt/mTOR pathway to electrically stimulated lengthening muscle contractions. Extensor digitorum longus muscles (n = 8/group) were subjected to a 22-min bout of lengthening contractions by high-frequency sciatic nerve electrical stimulation (STIM) in young adult (8 mo) Fischer 344 x Brown Norway male rats. Forty minutes before electrical stimulation, rats were subcutaneously injected with saline or 5-aminoimidazole-4-carboxamide-1-4-ribofuranoside (AICAR; 1 mg/g body wt), an AMPK activator. Stimulated and contralateral resting muscles were removed at 0, 20, and 40 min post-STIM, and AMPK, acetyl CoA carboxylase (ACC), Akt, eukaryotic initiation factor 4E-binding protein (4E-BP1), 70-kDa ribosomal protein S6 kinase (S6K1), and eukaryotic elongation factor 2 (eEF2) phosphorylations were assessed by Western blot. AICAR treatment increased (P < or = 0.05) post-STIM AMPK (Thr172) and ACC phosphorylation (Ser79/221), inhibited post-STIM S6K1 (Thr389) and 4E-BP1 (gel shift) phosphorylation, and elevated post-STIM eEF2 phosphorylation (Thr56). These findings suggest that translational signaling downstream of Akt/mTOR can be inhibited after lengthening contractions when preceded by AMPK activation and that energetic stress may be antagonistic to the hypertrophic translational signaling response to loaded muscle contractions.     

Grape seed proanthocyanidin extract promotes skeletal muscle fiber type transformation via AMPK signaling pathway

Transformation of skeletal muscle fiber type from fast twitch to slow twitch has significances for sustained contractile and stretchable events, energy homeostasis and antifatigue ability. However, the regulation of skeletal muscle fiber type transformation through nutritional intervention is still not fully spelled out. Grape seed proanthocyanidin extract (GSPE) has been widely reported to play a broader role in many aspects of diseases with its various pharmacological and health-promoting effects. In this study, we found that GSPE significantly improved the fatigue resistance in mice. GSPE up-regulated slow myosin heavy chain (MyHC) and down-regulated fast MyHC, accompanied by increases in activities of succinic dehydrogenase and malate dehydrogenase and by decreased lactate dehydrogenase activity in muscle of mice and in C2C12 myotubes. The AMP-activated protein kinase (AMPK) signaling can be activated by GSPE. Several upstream and downstream factors of AMPK signaling such as liver kinase B1, nuclear respiratory factor 1, calcium calmodulin-dependent protein kinase kinase β, sirtuin1 and peroxisome proliferator activated receptor-γ coactivator-1α (PGC-1α) were also up-regulated by GSPE. Specific inhibition of AMPK signaling by AMPK inhibitor compound C or by AMPKα1 siRNA significantly abolished the GSPE-induced the activation of AMPK and the increase of PGC-1α, and attenuated the GSPE-induced increase of slow MyHC and decrease of fast MyHC in C2C12 myotubes. Taken together, we revealed that GSPE promotes skeletal muscle fiber type transformation from fast twitch to slow twitch through AMPK signaling pathway, and this GSPE-induced fiber type transformation may contribute to increased fatigue resistance.     

Regulation of ERK1/2 by ouabain and Na-K-ATPase-dependent energy utilization and AMPK activation in parotid acinar cells

We previously found that the phosphorylation of ERK1/2 by submaximal concentrations of the muscarinic receptor ligand carbachol was potentiated in rat parotid acinar cells exposed to ouabain, a cardiac glycoside that inhibits the Na-K-ATPase. We now report that this signaling phenomenon involves the prevention of negative regulation of extracellular signal-regulated kinase-1/2 (ERK1/2) that is normally mediated by AMP-activated protein kinase (AMPK). Carbachol increases the turnover of the ATP-consuming Na-K-ATPase, reducing intracellular ATP and promoting the phosphorylation/activation of the energy sensor AMPK. Ouabain blocks the reduction in ATP and subsequent AMPK phosphorylation, which is regulated by the AMP-to-ATP ratio. The ouabain-promoted enhancement of ERK1/2 phosphorylation was not reproduced in Par-C10 cells, an immortalized rat parotid cell line that did not respond to carbachol with an ATP reduction and that employs an upstream AMPK kinase (Ca(2+)/calmodulin-dependent protein kinase kinase, CaMKK) different from that (LKB1) in native cells. In native parotid cells, inhibitory effects of AMPK on ERK1/2 signaling were examined by activating AMPK with 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), which is converted to an AMP mimetic but does not alter parotid ATP levels. AICAR-treated cells display increases in AMPK phosphorylation and a reduced phosphorylation of ERK1/2 subsequent to activation of muscarinic and P2X(7) receptors, which promote increases in Na-K-ATPase turnover, but not upon epidermal growth factor receptor activation. These results suggest that carbachol-initiated AMPK activation can produce a negative feedback on ERK1/2 signaling in response to submaximal muscarinic receptor activation and that increases in fluid secretion can modulate receptor-initiated signaling events indirectly by producing ion transport-dependent decreases in ATP.     

Rottlerin activates AMPK possibly through LKB1 in vascular cells and tissues

AMP-activated protein kinase (AMPK) is a cellular energy sensor involved in multiple cell signaling pathways that has become an attractive therapeutic target for vascular diseases. It is not clear whether rottlerin, an inhibitor of protein kinase Cδ, activates AMPK in vascular cells and tissues. In the present study, we have examined the effect of rottlerin on AMPK in vascular smooth muscle cells (VSMCs) and isolated rabbit aorta. Rottlerin reduced cellular ATP and activated AMPK in VSMCs and rabbit aorta; however, inhibition of PKCδ by three different methods did not activate AMPK. Both VSMCs and rabbit aorta expressed the upstream AMPK kinase LKB1 protein, and rottlerin-induced AMPK activation was decreased in VSMCs by overexpression of dominant-negative LKB1, suggesting that LKB1 is involved in the upstream regulation of AMPK stimulated by rottlerin. These data suggest for the first time that LKB1 mediates rottlerin-induced activation of AMPK in vascular cells and tissues.     

Activation of AMP-Activated Protein Kinase Alleviates Homocysteine-Mediated Neurotoxicity in SH-SY5Y Cells

Mammalian AMP-activated protein kinase (AMPK) acts as a metabolite-sensing protein kinase in multiple tissues. Recent studies have shown that AMPK activation also regulates intracellular signaling pathways involved in cellular survival and apoptosis. Previously, we have reported that AMPK activation alleviates the endoplasmic reticulum (ER) stress-mediated neurotoxicity and tau hyperphosphorylation caused by palmitate. Therefore, we investigated whether AMPK activation alleviates ER stress-mediated neurotoxicity in SH-SY5Y human neuroblastoma cells incubated with homocysteine. Regulation of AMPK activity by isoflavone was also determined to investigate the underlying mechanism of its neuroprotective effect. Treatment of SH-SY5Y human neuroblastoma cells with N ¹-(β-D-ribofuranosyl)-5-aminoimidazole-4-carboxamide (AICAR), a pharmacological activator of AMPK, significantly protected cells against cytotoxicity imposed by tunicamycin and homocysteine. Homocysteine significantly suppressed AMPK activation, which was alleviated by AICAR. We observed a significant inhibition of the unfolded protein response by AICAR in cells incubated with homocysteine, suggesting a protective role of AMPK activation against ER stress-mediated neurotoxicity. AICAR also significantly reduced tau hyperphosphorylation by inactivating glycogen synthase kinase-3β and c-Jun N-terminal kinase in cells incubated with homocysteine. Furthermore, treatment of cells with soy isoflavone, genistein and daidzein significantly activated AMPK, which was repressed by tunicamycin and homocysteine. Therefore, our results suggest that AMPK activation by isoflavone as well as AICAR alleviates homocysteine-mediated neurotoxicity in SH-SY5Y cells.     

Regulation of AMP-activated protein kinase by multisite phosphorylation in response to agents that elevate cellular cAMP

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 alpha subunit phosphorylation sites, viz. alpha-Thr(172) and alpha1-Ser(485)/alpha2-Ser(491), in the clonal beta-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 alpha-Thr(172) via inhibition of calcium/calmodulin-dependent protein kinase kinase-alpha and -beta, but not LKB1. These agents also enhanced phosphorylation of alpha-Ser(485/491) by the cAMP-dependent protein kinase. AMPK alpha-Ser(485/491) phosphorylation was necessary but not sufficient for inhibition of AMPK activity in response to forskolin/isobutylmethylxanthine. We show that AMPK alpha-Ser(485/491) 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.     

Curcumin stimulates glucose uptake through AMPK‐p38 MAPK pathways in L6 myotube cells

Curcumin has been shown to exert a variety of beneficial human health effects. However, mechanisms by which curcumin acts are poorly understood. In this study, we report that curcumin activated AMP‐activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. In addition, curcumin activated the mitogen‐activated protein kinase kinase (MEK)3/6‐p38 mitogen‐activated protein kinase (MAPK) signaling pathways in the downstream of the AMPK cascade. Moreover, inhibition of either AMPK or p38 MAPK resulted in blockage of curcumin‐induced glucose uptake. Furthermore, the administration of curcumin to mice increased AMPK phosphorylation in the skeletal muscles. Taken together, these results indicate that the beneficial health effect of curcumin can be explained by its ability to activate AMPK‐p38 MAPK pathways in skeletal muscles. J. Cell. Physiol. 223:771–778, 2010. © 2010 Wiley‐Liss, Inc.     

Localization of AMP kinase is regulated by stress, cell density, and signaling through the MEK-->ERK1/2 pathway

5'-AMP-activated protein kinase (AMPK) serves as an energy sensor and is at the center of control for a large number of metabolic reactions, thereby playing a crucial role in Type 2 diabetes and other human diseases. AMPK is present in the nucleus and cytoplasm; however, the mechanisms that regulate the intracellular localization of AMPK are poorly understood. We have now identified several factors that control the distribution of AMPK. Environmental stress regulates the intracellular localization of AMPK, and upon recovery from heat shock or oxidant exposure AMPK accumulates in the nuclei. We show that under normal growth conditions AMPK shuttles between the nucleus and the cytoplasm, a process that depends on the nuclear exporter Crm1. However, nucleocytoplasmic shuttling does not take place in high-density cell cultures, for which AMPK is confined to the cytoplasm. Furthermore, we demonstrate that signaling through the mitogen-activated protein kinase kinase (MEK)-->extracellular signal-regulated kinase 1/2 (ERK1/2) cascade plays a crucial role in controlling the proper localization of AMPK. As such, pharmacological inhibitors that interfere with this pathway alter AMPK distribution under nonstress conditions. Taken together, our studies identify novel links between the physiological state of the cell, the activation of MEK-->ERK1/2 signaling, and the nucleocytoplasmic distribution of AMPK. This sets the stage to develop new strategies to regulate the intracellular localization of AMPK and thereby the modification of targets that are relevant to human disease.     

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.