Activation of cardiac AMP-activated protein kinase by LKB1 expression or chemical hypoxia is blunted by increased Akt activity

AMP-activated protein kinase (AMPK) plays a major role in the regulation of cardiac energy substrate utilization and can be negatively regulated by Akt activation in the heart. It has recently been shown that Akt directly phosphorylates AMPKalpha(1)/alpha(2) on Ser(485/491) in vitro and prevents the AMPK kinase (AMPKK) LKB1 from phosphorylating AMPKalpha at its primary activation site, Thr(172) (S Horman, D Vertommen, R Heath, D Neumann, V Mouton, A Woods, U Schlattner, T Wallimann, D Carling, L Hue, and MH Rider. J Biol Chem 281: 5335-5340, 2006). To determine whether this is also the case in the cardiac myocyte, neonatal rat cardiac myocytes (NRCM) were infected with a recombinant adenovirus expressing a constitutively active mutant of Akt1 (myrAkt1) and then with or without adenoviruses expressing the active LKB1 complex. Expression of myrAkt1 blunted LKB1-induced phosphorylation of AMPKalpha at Thr(172), which resulted in a dramatic decrease in phosphorylation of AMPK's target, acetyl CoA-carboxylase. This decrease in AMPK activity was associated with prior Akt1-dependent phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491). To investigate whether Akt1 activation was also able to prevent other AMPKKs from phosphorylating AMPKalpha, we subjected NRCM to chemical hypoxia and noted a marked increase in phosphorylation of AMPKalpha at Thr(172), despite no change in LKB1 activity. NRCM expressing myrAkt1 demonstrated increased phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491) and a complete inhibition of chemical hypoxia-induced phosphorylation of AMPKalpha at Thr(172). Taken together, our data show that activation of Akt1 is able to prevent activation of cardiac AMPK by LKB1 and at least one other AMPKK, likely by prior phosphorylation of AMPKalpha(1)/alpha(2) at Ser(485/491).     

Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival

The serine/threonine kinase Akt (also known as protein kinase B) is activated in response to various stimuli by a mechanism involving phosphoinositide 3-kinase (PI3-K). Akt provides a survival signal that protects cells from apoptosis induced by growth factor withdrawal, but its function in other forms of stress is less clear. Here we investigated the role of PI3-K/Akt during the cellular response to oxidant injury. H(2)O(2) treatment elevated Akt activity in multiple cell types in a time- (5-30 min) and dose (400 microM-2 mm)-dependent manner. Expression of a dominant negative mutant of p85 (regulatory component of PI3-K) and treatment with inhibitors of PI3-K (wortmannin and LY294002) prevented H(2)O(2)-induced Akt activation. Akt activation by H(2)O(2) also depended on epidermal growth factor receptor (EGFR) signaling; H(2)O(2) treatment led to EGFR phosphorylation, and inhibition of EGFR activation prevented Akt activation by H(2)O(2). As H(2)O(2) causes apoptosis of HeLa cells, we investigated whether alterations of PI3-K/Akt signaling would affect this response. Wortmannin and LY294002 treatment significantly enhanced H(2)O(2)-induced apoptosis, whereas expression of exogenous myristoylated Akt (an activated form) inhibited cell death. Constitutive expression of v-Akt likewise enhanced survival of H(2)O(2)-treated NIH3T3 cells. These results suggest that H(2)O(2) activates Akt via an EGFR/PI3-K-dependent pathway and that elevated Akt activity confers protection against oxidative stress-induced apoptosis.     

AMP-activated protein kinase is involved in COX-2 expression in response to ultrasound in cultured osteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and in clinical studies. Cyclooxygenase-2 (COX-2) is a crucial mediator in mechanically induced bone formation. AMP-activated protein kinase (AMPK) has reported to sense and regulate the cellular energy status in various cell types. Here we found that US-mediated COX-2 expression was attenuated by LKB1 and AMPKalpha1 small interference RNA (siRNA) in human osteoblasts. Pretreatment of osteoblasts with AMPK inhibitor (araA and compound C), p38 inhibitor (SB203580), NF-kappaB inhibitor (PDTC), IkappaB protease inhibitor (TPCK) and NF-kappaB inhibitor peptide also inhibited the potentiating action of US. US increased the kinase activity and phosphorylation of LKB1, AMPK and p38. Stimulation of osteoblasts with US activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 phosphorylation at Ser(276), p65 and p50 translocation from the cytosol to the nucleus, and kappaB-luciferase activity. US-mediated an increase of IKKalpha/beta activity, kappaB-luciferase activity and p65 and p50 binding to the NF-kappaB element was inhibited by araA, SB203580 and LKB1 siRNA. Our results suggest that US increased COX-2 expression in osteoblasts via the LKB1/AMPKalpha1/p38/IKKalphabeta and NF-kappaB signaling pathway.     

Effect of ethanol on hydrogen peroxide-induced AMPK phosphorylation

AMP-activated protein kinase (AMPK) responds to oxidative stress. Previous work has shown that ethanol treatment of cultured hepatoma cells and of mice inhibited the activity of AMPK and reduced the amount of AMPK protein. Ethanol generates oxidative stress in the liver. Since AMPK is activated by reactive oxygen species, it seems paradoxical that ethanol would inhibit AMPK in the hepatoma cells. In an attempt to understand the mechanism whereby ethanol inhibits AMPK, we studied the effect of ethanol on AMPK activation by exogenous hydrogen peroxide. The effects of ethanol, hydrogen peroxide, and inhibitors of protein phosphatase 2A (PP2A) [either okadaic acid or PP2A small interference RNA (siRNA)] on AMPK phosphorylation and activity were examined in rat hepatoma cells (H4IIEC3) and HeLa cells. In H4IIEC3 cells, hydrogen peroxide (H(2)O(2), 1 mM) transiently increased the level of phospho-AMPK to 1.5-fold over control (P < 0.05). Similar findings were observed in HeLa cells, which do not express the upstream AMPK kinase, LKB1. H(2)O(2) markedly increased the phosphorylation of LKB1 in H4IIEC3 cells. Ethanol significantly inhibited the phosphorylation of PKC-zeta, LKB1, and AMPK caused by exposure to H(2)O(2). This inhibitory effect of ethanol required its metabolism. More importantly, the inhibitory effects of ethanol on H(2)O(2)-induced AMPK phosphorylation were attenuated by the presence of the PP2A inhibitor, okadaic acid, or PP2A siRNA. The inhibitory effect of ethanol on AMPK phosphorylation is exerted through the inhibition of PKC-zeta and LKB1 phosphorylation and the activation of PP2A.     

AMP-Activated protein kinase is activated by the stimulations of G(q)-coupled receptors

The AMP-activated protein kinase (AMPK) functions as a metabolic sensor that monitors cellular AMP and ATP levels. Platelet-activating factor (PAF) activates endogeneous AMPKalpha1 in Chinese hamster ovary cells expressing the PAF receptor coupled with both G(i) and G(q), but its activity was not inhibited after treatment with islet-activating protein. Norepinephrine and bradykinin also activated AMPKalpha1 in cells expressing the G(q)-coupled alpha(1b)-adrenergic receptor and bradykinin receptor, respectively. Stimulations of the G(i)-coupled alpha(2A)-adrenergic receptor, fMet-Leu-Phe receptor, prostaglandin EP3alpha receptor, and G(s)-coupled beta(2)-adrenergic receptor did not activate AMPKalpha1. AMPKalpha1 thus is activated specifically by stimulation of G(q)-coupled receptors. G(q)-coupled receptors transmit the signal for GLUT4 translocation and glucose uptake through an insulin-independent pathway. However, direct activation of AMPKalpha1 with treatment of 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside did not trigger GLUT4 translocation nor stimulate glucose uptake in our cells. Thus, activation of AMPKalpha1 via G(q) is not sufficient to trigger GLUT4 translocation or stimulate glucose uptake.     

NCS-1 inhibits insulin-stimulated GLUT4 translocation in 3T3L1 adipocytes through a phosphatidylinositol 4-kinase-dependent pathway

Expression of NCS-1 (neuronal calcium sensor-1, also termed frequenin) in 3T3L1 adipocytes strongly inhibited insulin-stimulated translocation of GLUT4 and insulin-responsive aminopeptidase. The effect of NCS-1 was specific for GLUT4 and the insulin-responsive aminopeptidase translocation as there was no effect on the trafficking of the cation-independent mannose 6-phosphate receptor or the GLUT1 glucose transporter isoform. Moreover, NCS-1 showed partial colocalization with GLUT4-EGFP in the perinuclear region. The inhibitory action of NCS-1 was independent of calcium sequestration since neither treatment with ionomycin nor endothelin-1, both of which elevated the intracellular calcium concentration, restored insulin-stimulated GLUT4 translocation. Furthermore, NCS-1 did not alter the insulin-stimulated protein kinase B (PKB/Akt) phosphorylation or the recruitment of Cbl to the plasma membrane. In contrast, expression of the NCS-1 effector phosphatidylinositol 4-kinase (PI 4-kinase) inhibited insulin-stimulated GLUT4 translocation, whereas co-transfection with an inactive PI 4-kinase mutant prevented the NCS-1-induced inhibition. These data demonstrate that PI 4-kinase functions to negatively regulate GLUT4 translocation through its interaction with NCS-1.     

Toll-like receptor 4 engagement inhibits adenosine 5'-monophosphate-activated protein kinase activation through a high mobility group box 1 protein-dependent mechanism

Despite the potent antiinflammatory effects of pharmacologically induced adenosine 5'-monophosphate kinase (AMPK) activation on Toll-like receptor 4 (TLR4)-induced cellular activation, there is little evidence that AMPK is activated during inflammatory conditions. In the present studies, we examined mechanisms by which TLR4 engagement may affect the ability of AMPK to become activated in neutrophils and macrophages under in vitro conditions and in the lungs during lipopolysaccharide (LPS)-induced acute lung injury. We found that incubation of neutrophils or macrophages with LPS diminished the ability of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) or hydrogen peroxide (H(2)O(2)) to activate AMPK. Although ratios of AMP to adenosine 5'-triphosphate (ATP) were increased in LPS-treated neutrophils and in the lungs of LPS exposed mice, a condition that should result in AMPK activation, no activation of AMPK was found. Immunocytochemistry and Western blot analysis revealed that nuclear to cytosolic translocation of the proinflammatory mediator high mobility group box 1 protein (HMGB1) correlated with inhibition of AMPK activation in LPS-stimulated macrophages. Moreover, while induced overexpression of HMGB1 resulted in inhibition of AMPK activation, Small interfering RNA (siRNA)-induced knockdown of HMGB1 was associated with enhanced activation of AMPK in macrophages incubated with AICAR. Increased interaction between liver kinase B1 (LKB1), an upstream activator of AMPK, and HMGB1 was found in LPS-stimulated macrophages and in the lungs of mice exposed to LPS. These results suggest that nuclear to cytoplasmic translocation of HMGB1 in TLR4-activated cells potentiates inflammatory responses by binding to LKB1, thereby inhibiting the antiinflammatory effects of AMPK activation.     

Differential effects of phosphatidylinositol 3-kinase inhibition on intracellular signals regulating GLUT4 translocation and glucose transport

Phosphatidylinositol (PI) 3-kinase is required for insulin-stimulated translocation of GLUT4 to the surface of muscle and fat cells. Recent evidence suggests that the full stimulation of glucose uptake by insulin also requires activation of GLUT4, possibly via a p38 mitogen-activated protein kinase (p38 MAPK)-dependent pathway. Here we used L6 myotubes expressing Myc-tagged GLUT4 to examine at what level the signals regulating GLUT4 translocation and activation bifurcate. We compared the sensitivity of each process, as well as of signals leading to GLUT4 translocation (Akt and atypical protein kinase C) to PI 3-kinase inhibition. Wortmannin inhibited insulin-stimulated glucose uptake with an IC(50) of 3 nm. In contrast, GLUT4myc appearance at the cell surface was less sensitive to inhibition (IC(50) = 43 nm). This dissociation between insulin-stimulated glucose uptake and GLUT4myc translocation was not observed with LY294002 (IC(50) = 8 and 10 microm, respectively). The sensitivity of insulin-stimulated activation of PKC zeta/lambda, Akt1, Akt2, and Akt3 to wortmannin (IC(50) = 24, 30, 35, and 60 nm, respectively) correlated closely with inhibition of GLUT4 translocation. In contrast, insulin-dependent p38 MAPK phosphorylation was efficiently reduced in cells pretreated with wortmannin, with an IC(50) of 7 nm. Insulin-dependent p38 alpha and p38 beta MAPK activities were also markedly reduced by wortmannin (IC(50) = 6 and 2 nm, respectively). LY294002 or transient expression of a dominant inhibitory PI 3-kinase construct (Delta p85), however, did not affect p38 MAPK phosphorylation. These results uncover a striking correlation between PI 3-kinase, Akt, PKC zeta/lambda, and GLUT4 translocation on one hand and their segregation from glucose uptake and p38 MAPK activation on the other, based on their wortmannin sensitivity. We propose that a distinct, high affinity target of wortmannin, other than PI 3-kinase, may be necessary for activation of p38 MAPK and GLUT4 in response to insulin.     

Inhibition of GLUT4 translocation by Tbc1d1, a Rab GTPase-activating protein abundant in skeletal muscle, is partially relieved by AMP-activated protein kinase activation

Insulin increases glucose transport by stimulating the trafficking of intracellular GLUT4 to the cell surface, a process known as GLUT4 translocation. A key protein in signaling this process is AS160, a Rab GTPase-activating protein (GAP) whose activity appears to be suppressed by Akt phosphorylation. Tbc1d1 is a Rab GAP with a sequence highly similar to that of AS160 and with the same Rab specificity as that of AS160. The role of Tbc1d1 in regulating GLUT4 trafficking has been unclear. Our previous study showed that overexpressed Tbc1d1 inhibited insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes, even though insulin caused phosphorylation on its single canonical Akt motif. In the present study, we show in 3T3-L1 adipocytes that Tbc1d1 is only 1/20 as abundant as AS160, that knockdown of Tbc1d1 has no effect on insulin-stimulated GLUT4 translocation, and that overexpressed Tbc1d1 also inhibits GLUT4 translocation elicited by activated Akt expression. These results indicate that endogenous Tbc1d1 does not participate in insulin-regulated GLUT4 translocation in adipocytes and suggest that the GAP activity of Tbc1d1 is not suppressed by Akt phosphorylation. In addition, we discovered that Tbc1d1 is much more highly expressed in skeletal muscle than fat and that the AMP-activated protein kinase (AMPK) activator 5'-aminoimidazole-4-carboxamide ribonucleoside partially reversed the inhibition of insulin-stimulated GLUT4 translocation by overexpressed Tbc1d1 in 3T3-L1 adipocytes. 5'-Aminoimidazole-4-carboxamide ribonucleoside activation of the kinase AMPK is known to cause GLUT4 translocation in muscle. The above findings strongly suggest that Tbc1d1 is a component in the signal transduction pathway leading to AMPK-stimulated GLUT4 translocation in muscle.     

Muscle cell depolarization induces a gain in surface GLUT4 via reduced endocytosis independently of AMPK

Contracting skeletal muscle increases glucose uptake to sustain energy demand. This is achieved through a gain in GLUT4 at the membrane, but the traffic mechanisms and regulatory signals involved are unknown. Muscle contraction is elicited by membrane depolarization followed by a rise in cytosolic Ca2+ and actomyosin activation, drawing on ATP stores. It is unknown whether one or more of these events triggers the rise in surface GLUT4. Here, we investigate the effect of membrane depolarization on GLUT4 cycling using GLUT4myc-expressing L6 myotubes devoid of sarcomeres and thus unable to contract. K+-induced membrane depolarization elevated surface GLUT4myc, and this effect was additive to that of insulin, was not prevented by inhibiting phosphatidylinositol 3-kinase (PI3K) or actin polymerization, and did not involve Akt activation. Instead, depolarization elevated cytosolic Ca2+, and the surface GLUT4myc elevation was prevented by dantrolene (an inhibitor of Ca2+ release from sarcoplasmic reticulum) and by extracellular Ca2+ chelation. Ca2+-calmodulin-dependent protein kinase-II (CaMKII) was not phosphorylated after 10 min of K+ depolarization, and the CaMK inhibitor KN62 did not prevent the gain in surface GLUT4myc. Interestingly, although 5'-AMP-activated protein kinase (AMPK) was phosphorylated upon depolarization, lowering AMPKalpha via siRNA did not alter the surface GLUT4myc gain. Conversely, the latter response was abolished by the PKC inhibitors bisindolylmaleimide I and calphostin C. Unlike insulin, K+ depolarization caused only a small increase in GLUT4myc exocytosis and a major reduction in its endocytosis. We propose that K+ depolarization reduces GLUT4 internalization through signals and mechanisms distinct from those engaged by insulin. Such a pathway(s) is largely independent of PI3K, Akt, AMPK, and CaMKII but may involve PKC.     

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.     

Hydrogen peroxide activates endothelial nitric-oxide synthase through coordinated phosphorylation and dephosphorylation via a phosphoinositide 3-kinase-dependent signaling pathway.

Endothelial nitric-oxide synthase (eNOS) is an important component of vascular homeostasis. During vascular disease, endothelial cells are exposed to excess reactive oxygen species that can alter cellular phenotype by inducing various signaling pathways. In the current study, we examined the implications of H(2)O(2)-induced signaling for eNOS phosphorylation status and activity in porcine aortic endothelial cells. We found that H(2)O(2) treatment enhanced eNOS activity and NO bioactivity as determined by the conversion of l-[(3)H]arginine to l-[(3)H]citrulline and cellular cGMP content. Concomitant with eNOS activation, H(2)O(2) also activated Akt, increased eNOS phosphorylation at Ser-1177, and decreased eNOS phosphorylation at Thr-495. H(2)O(2)-induced promotion of eNOS activity and modulation of the eNOS phosphorylation status at Ser-1177 and Thr-495 were significantly attenuated by selective inhibitors of Src kinase, the ErbB receptor family, and phosphoinositide 3-kinase (PI 3-K). We found that Akt activation, eNOS Ser-1177 phosphorylation, and eNOS activation by H(2)O(2) were calcium-dependent, whereas eNOS dephosphorylation at Thr-495 was not, suggesting a branch point in the signaling cascade downstream from PI 3-K. Consistent with this, overexpression of a dominant negative isoform of Akt inhibited H(2)O(2)-induced phosphorylation of eNOS at Ser-1177 but not dephosphorylation of eNOS at Thr-495. Together, these data indicate that H(2)O(2) promotes calcium-dependent eNOS activity through a coordinated change in the phosphorylation status of the enzyme mediated by Src- and ErbB receptor-dependent PI 3-K activation. In turn, PI 3-K mediates eNOS Ser-1177 phosphorylation via a calcium- and Akt-dependent pathway, whereas eNOS Thr-495 dephosphorylation does not involve calcium or Akt. This response may represent an attempt by endothelial cells to maintain NO bioactivity under conditions of enhanced oxidative stress.     

Identification of protein kinase D as a novel contraction-activated kinase linked to GLUT4-mediated glucose uptake, independent of AMPK

Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.     

Akt2 regulates Rac1 activity in the insulin-dependent signaling pathway leading to GLUT4 translocation to the plasma membrane in skeletal muscle cells

The small GTPase Rac1 plays a pivotal role in insulin-stimulated glucose uptake in skeletal muscle, which is mediated by GLUT4 translocation to the plasma membrane. However, regulatory mechanisms for Rac1 and its role in the signaling pathway composed of phosphoinositide 3-kinase and the serine/threonine kinase Akt remain obscure. Here, we investigate the role of Akt in the regulation of Rac1 in myocytes. Insulin-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt inhibitor IV. Insulin-induced Rac1 activation, on the other hand, was completely inhibited by this inhibitor. Constitutively activated phosphoinositide 3-kinase induced Rac1 activation and GLUT4 translocation. This GLUT4 translocation was almost completely suppressed by Rac1 knockdown. Furthermore, constitutively activated phosphoinositide 3-kinase-induced, but not constitutively activated Rac1-induced, GLUT4 translocation was suppressed by Akt2 knockdown. Finally, insulin-induced Rac1 activation was indeed inhibited by Akt2 knockdown. Together, these results reveal a novel regulatory mechanism involving Akt2 for insulin-dependent Rac1 activation.     

Regulation of H(2)O(2)-induced necrosis by PKC and AMP-activated kinase signaling in primary cultured hepatocytes

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H(2)O(2) is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H(2)O(2) treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H(2)O(2) treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H(2)O(2) (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H(2)O(2)-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H(2)O(2) even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H(2)O(2). Furthermore, AMPK activators protected against H(2)O(2)-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H(2)O(2). Knockdown of PKC-alpha using antisense oligonucleotides also slightly protected (~22%) against H(2)O(2). Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H(2)O(2)-induced necrosis, suggesting that a signaling "program" is important in mediating H(2)O(2)-induced necrosis in primary hepatocytes.     

Deficiency of LKB1 in skeletal muscle prevents AMPK activation and glucose uptake during contraction

Recent studies indicate that the LKB1 tumour suppressor protein kinase is the major "upstream" activator of the energy sensor AMP-activated protein kinase (AMPK). We have used mice in which LKB1 is expressed at only approximately 10% of the normal levels in muscle and most other tissues, or that lack LKB1 entirely in skeletal muscle. Muscle expressing only 10% of the normal level of LKB1 had significantly reduced phosphorylation and activation of AMPKalpha2. In LKB1-lacking muscle, the basal activity of the AMPKalpha2 isoform was greatly reduced and was not increased by the AMP-mimetic agent, 5-aminoimidazole-4-carboxamide riboside (AICAR), by the antidiabetic drug phenformin, or by muscle contraction. Moreover, phosphorylation of acetyl CoA carboxylase-2, a downstream target of AMPK, was profoundly reduced. Glucose uptake stimulated by AICAR or muscle contraction, but not by insulin, was inhibited in the absence of LKB1. Contraction increased the AMP:ATP ratio to a greater extent in LKB1-deficient muscles than in LKB1-expressing muscles. These studies establish the importance of LKB1 in regulating AMPK activity and cellular energy levels in response to contraction and phenformin.     

Palmitic acid acutely stimulates glucose uptake via activation of Akt and ERK1/2 in skeletal muscle cells

Chronic exposure to saturated fatty acids can cause insulin resistance. However, the acute effects of fatty acids are not clear and need to be elucidated because plasma fatty acid concentrations fluctuate postprandially. Here, we present the acute effects of palmitate (PA) on skeletal muscle cells and their underlying molecular mechanisms. Immuno-fluorescence results showed that PA rapidly induced GLUT4 translocation and stimulated glucose uptake in rat skeletal muscle cell line L6. Phosphorylation of AMP-activated protein kinase (AMPK), Akt, and extracellular signal-related kinase1/2 (ERK1/2) was enhanced by PA in a time-dependent manner. Cell surface-bound PA was sufficient to stimulate Akt phosphorylation. The inhibitors of PI3 kinase (PI3K), AMPK, Akt, and ERK1/2 could decrease PA-induced glucose uptake, and PI3K inhibitor decreased AMPK, Akt, and ERK1/2 phosphorylation. Weakening AMPK activity reduced phosphorylation of Akt but not ERK1/2, and Akt inhibitor could not affect ERK1/2 activation either. Meanwhile, ERK1/2 inhibitors had no effect on Akt phosphorylation. Taken together, our data suggest that PA-mediated glucose uptake in skeletal muscle cells may be stimulated by the binding of PA to cell surface and followed by PI3K/AMPK/Akt and PI3K/ERK1/2 pathways independently.     

Possible involvement of the alpha1 isoform of 5'AMP-activated protein kinase in oxidative stress-stimulated glucose transport in skeletal muscle

Recent studies have suggested that 5'AMP-activated protein kinase (AMPK) is activated in response to metabolic stresses, such as contraction, hypoxia, and the inhibition of oxidative phosphorylation, which leads to insulin-independent glucose transport in skeletal muscle. In the present study, we hypothesized that acute oxidative stress increases the rate of glucose transport via an AMPK-mediated mechanism. When rat epitrochlearis muscles were isolated and incubated in vitro in Krebs buffer containing the oxidative agent H(2)O(2), AMPKalpha1 activity increased in a time- and dose-dependent manner, whereas AMPKalpha2 activity remained unchanged. The activation of AMPKalpha1 was associated with phosphorylation of AMPK Thr(172), suggesting that an upstream kinase is involved in the activation process. H(2)O(2)-induced AMPKalpha1 activation was blocked in the presence of the antioxidant N-acetyl-l-cysteine (NAC), and H(2)O(2) significantly increased the ratio of oxidized glutathione to glutathione (GSSG/GSH) concentrations, a sensitive marker of oxidative stress. H(2)O(2) did not cause an increase in the conventional parameters of AMPK activation, such as AMP and AMP/ATP. H(2)O(2) increased 3-O-methyl-d-glucose transport, and this increase was partially, but significantly, blocked in the presence of NAC. Results were similar when the muscles were incubated in a superoxide-generating system using hypoxanthine and xanthine oxidase. Taken together, our data suggest that acute oxidative stress activates AMPKalpha1 in skeletal muscle via an AMP-independent mechanism and leads to an increase in the rate of glucose transport, at least in part, via an AMPKalpha1-mediated mechanism.     

5-Aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside inhibits cancer cell proliferation in vitro and in vivo via AMP-activated protein kinase

5-Aminoimidazole-4-carboxamide-1-beta-4-ribofuranoside (AICAR) is widely used as an AMP-kinase activator, which regulates energy homeostasis and response to metabolic stress. Here, we investigated the effect of AICAR, an AMPK activator, on proliferation of various cancer cells and observed that proliferation of all the examined cell lines was significantly inhibited by AICAR treatment due to arrest in S-phase accompanied with increased expression of p21, p27, and p53 proteins and inhibition of PI3K-Akt pathway. Inhibition in in vitro growth of cancer cells was mirrored in vivo with increased expression of p21, p27, and p53 and attenuation of Akt phosphorylation. Anti-proliferative effect of AICAR is mediated through activated AMP-activated protein kinase (AMPK) as iodotubericidin and dominant-negative AMPK expression vector reversed the AICAR-mediated growth arrest. Moreover, constitutive active AMPK arrested the cells in S-phase by inducing the expression of p21, p27, and p53 proteins and inhibiting Akt phosphorylation, suggesting the involvement of AMPK. AICAR inhibited proliferation in both LKB and LKB knock-out mouse embryo fibroblasts to similar extent and arrested cells at S-phase when transfected with dominant negative expression vector of LKB. Altogether, these results indicate that AICAR can be utilized as a therapeutic drug to inhibit cancer, and AMPK can be a potential target for treatment of various cancers independent of the functional tumor suppressor gene, LKB.