AMPK上游激酶——肿瘤抑制因子LKB1

AMPK是哺乳动物细胞中高度保守的蛋白质,是细胞的“代谢感受器”。AMPK的活化需要上游激酶(AMPKK)对AMPKα亚基活化环上Thr172进行磷酸化来完成。最近的研究发现,肿瘤抑制因子LKB1可以磷酸化Thr172进而激活AMPK,因此认为它是AMPKK家族的成员。     

Inhibition of Connexin 26 by the AMP-Activated Protein Kinase

Connexins provide intercellular connections that allow passage of ions and small organic molecules. They clamp the cell membrane potential and cellular ion composition to that of neighboring cells. The cell membrane potential and ion composition of an energy-depleted cell could thus be maintained despite its compromised Na⁺/K⁺ activity. By the same token, however, the breakdown of ion gradients in that cell imposes an additional challenge to the neighboring cells, which may jeopardize their survival. Thus, timely closure of connexins may be critically important for the survival of those cells. Energy depletion stimulates the AMP-activated protein kinase (AMPK), a serine/threonine kinase that senses energy depletion and stimulates several cellular mechanisms to enhance energy production and to limit energy utilization. The present study explored whether AMPK regulates connexin 26. To this end, cRNA encoding connexin 26 was injected into Xenopus oocytes with and without additional injection of wild-type AMPK (α1β1γ1), of the constitutively active ^γR70QAMPK (α1β1γ1[R70Q]) or of the inactive mutant ^αK45RAMPK (α1[K45R]β1γ1). Connexin 26 activity was determined in dual-electrode voltage-clamp experiments. Moreover, connexin 26 abundance was determined in the oocyte cell membrane by chemiluminescence and confocal microscopy. As a result, connexin 26-mediated current and connexin 26 protein abundance were significantly decreased by coexpression of ^γR70QAMPK and, to a lower extent, by wild-type AMPK but not by ^αK45RAMPK. In conclusion, AMPK is a potent regulator of connexin 26.     

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.     

Activation of AMP-Activated Protein Kinase by Adenine Alleviates TNF-Alpha-Induced Inflammation in Human Umbilical Vein Endothelial Cells

The AMP-activated protein kinase (AMPK) signaling system plays a key role in cellular stress by repressing the inflammatory responses induced by the nuclear factor-kappa B (NF-κB) system. Previous studies suggest that the anti-inflammatory role of AMPK involves activation by adenine, but the mechanism that allows adenine to produce these effects has not yet been elucidated. In human umbilical vein endothelial cells (HUVECs), adenine was observed to induce the phosphorylation of AMPK in both a time- and dose-dependent manner as well as its downstream target acetyl Co-A carboxylase (ACC). Adenine also attenuated NF-κB targeting of gene expression in a dose-dependent manner and decreased monocyte adhesion to HUVECs following tumor necrosis factor (TNF-α) treatment. The short hairpin RNA (shRNA) against AMPK α1 in HUVECs attenuated the adenine-induced inhibition of NF-κB activation in response to TNF-α, thereby suggesting that the anti-inflammatory role of adenine is mediated by AMPK. Following the knockdown of adenosyl phosphoribosyl transferase (APRT) in HUVECs, adenine supplementation failed to induce the phosphorylation of AMPK and ACC. Similarly, the expression of a shRNA against APRT nullified the anti-inflammatory effects of adenine in HUVECs. These results suggested that the role of adenine as an AMPK activator is related to catabolism by APRT, which increases the cellular AMP levels to activate AMPK.     

A Cell-Autonomous Molecular Cascade Initiated by AMP-Activated Protein Kinase Represses Steroidogenesis

Steroid hormones regulate essential physiological processes, and inadequate levels are associated with various pathological conditions. In testosterone-producing Leydig cells, steroidogenesis is strongly stimulated by luteinizing hormone (LH) via its receptor leading to increased cyclic AMP (cAMP) production and expression of the steroidogenic acute regulatory (STAR) protein, which is essential for the initiation of steroidogenesis. Steroidogenesis then passively decreases with the degradation of cAMP into AMP by phosphodiesterases. In this study, we show that AMP-activated protein kinase (AMPK) is activated following cAMP-to-AMP breakdown in MA-10 and MLTC-1 Leydig cells. Activated AMPK then actively inhibits cAMP-induced steroidogenesis by repressing the expression of key regulators of steroidogenesis, including Star and Nr4a1. Similar results were obtained in Y-1 adrenal cells and in the constitutively steroidogenic R2C cells. We have also determined that maximum AMPK activation following stimulation of steroidogenesis in MA-10 Leydig cells occurs when steroid hormone production has reached a plateau. Our data identify AMPK as a molecular rheostat that actively represses steroid hormone biosynthesis to preserve cellular energy homeostasis and prevent excess steroid production.     

Translocation of Protein Kinase C in Anterior Pituitary Tumor Cells

Previous studies have shown that phorbol esters and lithium each stimulate the secretion of adrenocorticotropic hormone (ACTH) by the anterior pituitary tumor cell line AtT20/D16-16. Pretreatment with either lithium or phorbol ester desensitizes the cells to subsequent stimulation by phorbol ester. An early consequence of phorbol ester action in other systems is the translocation of protein kinase C from cytosol to membranes. We have assayed protein kinase C activity in cytosol and membranes of AtT20 cells after treatment with phorbol dibutyrate, lithium, or other agents that stimulate secretion of ACTH in these cells. Phorbol dibutyrate clearly induced translocation of protein kinase C, but lithium treatment did not cause translocation itself, nor did pretreatment with lithium affect the translocation induced by phorbol dibutyrate. These results are consistent with a role for translocation of protein kinase C in the stimulatory and desensitizing effects of phorbol esters but fail to implicate translocation in the actions of lithium on AtT20 cells.     

Raloxifene Induces Autophagy-Dependent Cell Death in Breast Cancer Cells via the Activation of AMP-Activated Protein Kinase

Raloxifene is a selective estrogen receptor modulator (SERM) that binds to the estrogen receptor (ER), and exhibits potent anti-tumor and autophagy-inducing effects in breast cancer cells. However, the mechanism of raloxifene-induced cell death and autophagy is not well-established. So, we analyzed mechanism underlying death and autophagy induced by raloxifene in MCF-7 breast cancer cells.Treatment with raloxifene significantly induced death in MCF-7 cells. Raloxifene accumulated GFP-LC3 puncta and increased the level of autophagic marker proteins, such as LC3-II, BECN1, and ATG12-ATG5 conjugates, indicating activated autophagy. Raloxifene also increased autophagic flux indicators, the cleavage of GFP from GFP-LC3 and only red fluorescence-positive puncta in mRFP-GFP-LC3-expressing cells. An autophagy inhibitor, 3-methyladenine (3-MA), suppressed the level of LC3-II and blocked the formation of GFP-LC3 puncta. Moreover, siRNA targeting BECN1 markedly reversed cell death and the level of LC3-II increased by raloxifene. Besides, raloxifene-induced cell death was not related to cleavage of caspases-7, -9, and PARP. These results indicate that raloxifene activates autophagy-dependent cell death but not apoptosis. Interestingly, raloxifene decreased the level of intracellular adenosine triphosphate (ATP) and activated the AMPK/ULK1 pathway. However it was not suppressed the AKT/mTOR pathway. Addition of ATP decreased the phosphorylation of AMPK as well as the accumulation of LC3-II, finally attenuating raloxifene-induced cell death.Our current study demonstrates that raloxifene induces autophagy via the activation of AMPK by sensing decreases in ATP, and that the overactivation of autophagy promotes cell death and thereby mediates the anti-cancer effects of raloxifene in breast cancer cells.     

The Opposing Actions of Target of Rapamycin and AMP-Activated Protein Kinase in Cell Growth Control

Cell growth is a highly regulated, plastic process. Its control involves balancing positive regulation of anabolic processes with negative regulation of catabolic processes. Although target of rapamycin (TOR) is a major promoter of growth in response to nutrients and growth factors, AMP-activated protein kinase (AMPK) suppresses anabolic processes in response to energy stress. Both TOR and AMPK are conserved throughout eukaryotic evolution. Here, we review the fundamentally important roles of these two kinases in the regulation of cell growth with particular emphasis on their mutually antagonistic signaling.An efficient homeostatic response to maintain cellular energy despite a noncontinuous supply of nutrients is crucial for the survival of organisms. Cells have, therefore, evolved a host of molecular pathways to sense both intra- and extracellular nutrients and thereby quickly adapt their metabolism to changing conditions. The target of rapamycin (TOR) and AMP-activated protein kinase (AMPK) signaling pathways control growth and metabolism in a complementary manner with TOR promoting anabolic processes under nutrient- and energy-rich conditions, whereas AMPK promotes a catabolic response when cells are low on nutrients and energy. Both pathways are highly conserved from yeast to human. This review summarizes the cross talk between TOR and AMPK in different organisms.     

AICAR Enhances the Phagocytic Ability of Macrophages towards Apoptotic Cells through P38 Mitogen Activated Protein Kinase Activation Independent of AMP-Activated Protein Kinase

Recent studies have suggested that 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) increases macrophage phagocytosis through adenosine monophosphate-activated protein kinase (AMPK). However, little information is available on the effects of AICAR on the clearance of apoptotic cells by macrophages, known as efferocytosis, which is essential in maintaining tissue homeostasis and resolving inflammation. AICAR increased p38 MAPK activation and the phagocytosis of apoptotic cells by macrophages, which were inhibited by the p38 MAPK inhibitor, SB203580, the TGF-beta-activated kinase 1 (TAK1) inhibitor, (5Z)-7-oxozeaenol, and siRNA-mediated knock-down of p38α. AICAR increased phosphorylation of Akt, but the inhibition of PI3K/Akt activity using {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 did not affect the AICAR-induced changes in efferocytosis in macrophages. {"type":"entrez-protein","attrs":{"text":"CGS15943","term_id":"875345334","term_text":"CGS15943"}}CGS15943, a non-selective adenosine receptor antagonist, did not affect AICAR-induced changes in efferocytosis, but dipyridamole, an adenosine transporter inhibitor, diminished the AICAR-mediated increases in efferocytosis. AICAR-induced p38 MAPK phosphorylation was not inhibited by the AMPK inhibitor, compound C, or siRNA-mediated knock-down of AMPKα1. Inhibition of AMPK using compound C or 5’-iodotubercidin did not completely block AICAR-mediated increases in efferocytosis. Furthermore, AICAR also increased the removal of apoptotic neutrophils or thymocytes in mouse lungs. These results reveal a novel mechanism by which AICAR increases macrophage-mediated phagocytosis of apoptotic cells and suggest that AICAR may be used to treat efferocytosis-related inflammatory conditions.     

Berberine Promotes Glucose Consumption Independently of AMP-Activated Protein Kinase Activation

Berberine is a plant alkaloid with anti-diabetic action. Activation of AMP-activated protein kinase (AMPK) pathway has been proposed as mechanism for berberine’s action. This study aimed to examine whether AMPK activation was necessary for berberine’s glucose-lowering effect. We found that in HepG2 hepatocytes and C2C12 myotubes, berberine significantly increased glucose consumption and lactate release in a dose-dependent manner. AMPK and acetyl coenzyme A synthetase (ACC) phosphorylation were stimulated by 20 µmol/L berberine. Nevertheless, berberine was still effective on stimulating glucose utilization and lactate production, when the AMPK activation was blocked by (1) inhibition of AMPK activity by Compound C, (2) suppression of AMPKα expression by siRNA, and (3) blockade of AMPK pathway by adenoviruses containing dominant-negative forms of AMPKα1/α2. To test the effect of berberine on oxygen consumption, extracellular flux analysis was performed in Seahorse XF24 analyzer. The activity of respiratory chain complex I was almost fully blocked in C2C12 myotubes by berberine. Metformin, as a positive control, showed similar effects as berberine. These results suggest that berberine and metformin promote glucose metabolism by stimulating glycolysis, which probably results from inhibition of mitochondrial respiratory chain complex I, independent of AMPK activation.     

Activation of AMP-Activated Protein Kinase Contributes to Doxorubicin-Induced Cell Death and Apoptosis in Cultured Myocardial H9c2 Cells

Despite its potent antitumor effect, clinical use of Doxorubicin is limited because of serious side effects including myocardial toxicity. Understanding the cellular mechanism involved in this process in a better manner is beneficial for optimizing Doxorubicin treatment. In the current study, the authors focus on the AMP-activated protein kinase (AMPK) in the said process. In this study, the authors discovered for the first time that Doxorubicin induces AMPK activation in cultured rat embryonic ventricular myocardial H9c2 cells. Reactive oxygen species (ROS)-dependent LKB1 activation serves as the upstream signal for AMPK activation by Doxorubicin. Evidence in support of the activation of AMPK contributing to Doxorubicin-induced H9c2 cell death/apoptosis—probably by modulating multiple downstream signal targets, including regulating JNK, p53, and inhibiting mTORC1—is provided in this article.     

Activation of AMP-Activated Protein Kinase Revealed by Hydrogen/Deuterium Exchange Mass Spectrometry

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Resveratrol Inhibits the Epithelial Sodium Channel via Phopshoinositides and AMP-Activated Protein Kinase in Kidney Collecting Duct Cells

Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCD_c14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCD_c14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P₃ reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol''s known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4–24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP₃, and a sustained late (4–24 h) effect secondary to activation of AMPK.     

AMP-Activated Protein Kinase Induces p53 by Phosphorylating MDMX and Inhibiting Its Activity

AMP-activated protein kinase (AMPK) has been shown to activate p53 in response to metabolic stress. However, the underlying mechanisms remain unclear. Here we show that metabolic stresses induce AMPK-mediated phosphorylation of human MDMX on Ser342 in vitro and in cells, leading to enhanced association between MDMX and 14-3-3. This markedly inhibits p53 ubiquitylation and significantly stabilizes and activates p53. By striking contrast, no phosphorylation of MDM2 by AMPK was noted. AMPK-mediated MDMX phosphorylation, MDMX–14-3-3 binding, and p53 activation were drastically reduced in mouse embryo fibroblasts harboring endogenous MDMX with S341A (mouse homologue of human serine 342), S367A, and S402A (mouse homologue of human serine 403) mutations. Moreover, deficiency of AMPK prevented MDMX–14-3-3 interaction and p53 activation. The activation of p53 through AMPK-mediated MDMX phosphorylation and inactivation was further confirmed by using cell and animal model systems with two AMPK activators, metformin and salicylate (the active form of aspirin). Together, the results unveil a mechanism by which metabolic stresses activate AMPK, which, in turn, phosphorylates and inactivates MDMX, resulting in p53 stabilization and activation.     

Energy-Dependent Modulation of Glucagon-Like Signaling in Drosophila via the AMP-Activated Protein Kinase

Adipokinetic hormone (AKH) is the equivalent of mammalian glucagon, as it is the primary insect hormone that causes energy mobilization. In Drosophila, current knowledge of the mechanisms regulating AKH signaling is limited. Here, we report that AMP-activated protein kinase (AMPK) is critical for normal AKH secretion during periods of metabolic challenges. Reduction of AMPK in AKH cells causes a suite of behavioral and physiological phenotypes resembling AKH cell ablations. Specifically, reduced AMPK function increases life span during starvation and delays starvation-induced hyperactivity. Neither AKH cell survival nor gene expression is significantly impacted by reduced AMPK function. AKH immunolabeling was significantly higher in animals with reduced AMPK function; this result is paralleled by genetic inhibition of synaptic release, suggesting that AMPK promotes AKH secretion. We observed reduced secretion in AKH cells bearing AMPK mutations employing a specific secretion reporter, confirming that AMPK functions in AKH secretion. Live-cell imaging of wild-type AKH neuroendocrine cells shows heightened excitability under reduced sugar levels, and this response was delayed and reduced in AMPK-deficient backgrounds. Furthermore, AMPK activation in AKH cells increases intracellular calcium levels in constant high sugar levels, suggesting that the underlying mechanism of AMPK action is modification of ionic currents. These results demonstrate that AMPK signaling is a critical feature that regulates AKH secretion, and, ultimately, metabolic homeostasis. The significance of these findings is that AMPK is important in the regulation of glucagon signaling, suggesting that the organization of metabolic networks is highly conserved and that AMPK plays a prominent role in these networks.     

The AMP-Activated Protein Kinase Is Involved in the Regulation of Ketone Body Production by Astrocytes

The possible role of the AMP-activated protein kinase (AMPK), a highly conserved stress-activated kinase, in the regulation of ketone body production by astrocytes was studied. AMPK activity in rat cortical astrocytes was three times higher than in rat cortical neurons. AMPK in astrocytes was shown to be functionally active. Thus, incubation of astrocytes with 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), a cell-permeable activator of AMPK, stimulated both ketogenesis from palmitate and carnitine palmitoyltransferase I. This was concomitant to a decrease of intracellular malonyl-CoA levels and an inhibition of acetyl-CoA carboxylase/fatty acid synthesis and 3-hydroxy-3-methylglutaryl-CoA reductase/cholesterol synthesis. Moreover, in microdialysis experiments AICAR was shown to stimulate brain ketogenesis markedly. The effect of chemical hypoxia on AMPK and the ketogenic pathway was studied subsequently. Incubation of astrocytes with azide led to a remarkable drop of fatty acid beta-oxidation. However, activation of AMPK during hypoxia compensated the depression of beta-oxidation, thereby sustaining ketone body production. This effect seemed to rely on the cascade hypoxia --> increase of the AMP/ATP ratio --> AMPK stimulation --> acetyl-CoA carboxylase inhibition --> decrease of malonyl-CoA concentration --> carnitine palmitoyltransferase I deinhibition --> enhanced ketogenesis. Furthermore, incubation of neurons with azide blunted lactate oxidation, but not 3-hydroxybutyrate oxidation. Results show that (a) AMPK plays an active role in the regulation of ketone body production by astrocytes, and (b) ketone bodies produced by astrocytes during hypoxia might be a substrate for neuronal oxidative metabolism.     

AMP-Activated Protein Kinase Is Required for the Macropinocytic Internalization of Ebolavirus

Identification of host factors that are needed for Zaire Ebolavirus (EBOV) entry provides insights into the mechanism(s) of filovirus uptake, and these factors may serve as potential antiviral targets. In order to identify novel host genes and pathways involved in EBOV entry, gene array findings in the National Cancer Institute''s NCI-60 panel of human tumor cell lines were correlated with permissivity for EBOV glycoprotein (GP)-mediated entry. We found that the gene encoding the γ2 subunit of AMP-activated protein kinase (AMPK) strongly correlated with EBOV transduction in the tumor panel. The AMPK inhibitor compound C inhibited infectious EBOV replication in Vero cells and diminished EBOV GP-dependent, but not Lassa fever virus GPC-dependent, entry into a variety of cell lines in a dose-dependent manner. Compound C also prevented EBOV GP-mediated infection of primary human macrophages, a major target of filoviral replication in vivo. Consistent with a role for AMPK in filovirus entry, time-of-addition studies demonstrated that compound C abrogated infection when it was added at early time points but became progressively less effective when added later. Compound C prevented EBOV pseudovirion internalization at 37°C as cell-bound particles remained susceptible to trypsin digestion in the presence of the inhibitor but not in its absence. Mouse embryonic fibroblasts lacking the AMPKα1 and AMPKα2 catalytic subunits were significantly less permissive to EBOV GP-mediated infection than their wild-type counterparts, likely due to decreased macropinocytic uptake. In total, these findings implicate AMPK in macropinocytic events needed for EBOV GP-dependent entry and identify a novel cellular target for new filoviral antivirals.     

BMS309403 Stimulates Glucose Uptake in Myotubes through Activation of AMP-Activated Protein Kinase

BMS309403 is a biphenyl azole inhibitor against fatty acid binding protein 4 (FABP4) and regarded as a lead compound for effective treatment of obesity related cardio-metabolic diseases. Here we discovered an off-target activity of BMS309403 in that it stimulates glucose uptake in C2C12 myotubes in a temporal and dose dependent manner via activation of AMP-activated protein kinase (AMPK) signaling pathway but independent of FABPs. Further analysis indicated that BMS309403 activates AMPK through increasing the ratio of intracellular AMP:ATP while decreasing mitochondrial membrane potential. These findings provide mechanistic insights on the action of BMS309403.