PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

AMP-Activated Protein Kinase α1 but Not α2 Catalytic Subunit Potentiates Myogenin Expression and Myogenesis

The link between AMP-activated protein kinase (AMPK) and myogenesis remains poorly defined. AMPK has two catalytic α subunits, α1 and α2. We postulated that AMPK promotes myogenesis in an isoform-specific manner. Primary myoblasts were prepared from AMPK knockout (KO) mice and AMPK conditional KO mice, and knockout of the α1 but not the α2 subunit resulted in downregulation of myogenin and reduced myogenesis. Myogenin expression and myogenesis were nearly abolished in the absence of both AMPKα1 and AMPKα2, while enhanced AMPK activity promoted myogenesis and myotube formation. The AMPKα1-specific effect on myogenesis was likely due to the dominant expression of α1 in myoblasts. These results were confirmed in C2C12 cells. To further evaluate the necessity of the AMPKα1 subunit for myogenesis in vivo, we prepared both DsRed AMPKα1 knockout myoblasts and enhanced green fluorescent protein (EGFP) wild-type myoblasts, which were cotransplanted into tibialis anterior muscle. A number of green fluorescent muscle fibers were observed, showing the fusion of engrafted wild-type myoblasts with muscle fibers; on the other hand, very few or no red muscle fibers were observed, indicating the absence of myogenic capacity of AMPKα1 knockout myoblasts. In summary, these results indicate that AMPK activity promotes myogenesis through a mechanism mediated by AMPKα1.     

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.     

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

The Ca²⁺ sensor STIM1 is crucial for activation of store-operated Ca²⁺ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca²⁺ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca²⁺ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.     

Sumoylation of AMPKβ2 subunit enhances AMP-activated protein kinase activity

AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. It is a heterotrimer composed of a catalytic α and two regulatory subunits (β and γ). AMPK activity is regulated allosterically by AMP and by the phosphorylation of residue Thr-172 within the catalytic domain of the AMPKα subunit by upstream kinases. We present evidence that the AMPKβ2 subunit may be posttranslationally modified by sumoylation. This process is carried out by the E3-small ubiquitin-like modifier (SUMO) ligase protein inhibitor of activated STAT PIASy, which modifies the AMPKβ2 subunit by the attachment of SUMO2 but not SUMO1 moieties. Of interest, AMPKβ1 is not a substrate for this modification. We also demonstrate that sumoylation of AMPKβ2 enhances the activity of the trimeric α2β2γ1 AMPK complex. In addition, our results indicate that sumoylation is antagonist and competes with the ubiquitination of the AMPKβ2 subunit. This adds a new layer of complexity to the regulation of the activity of the AMPK complex, since conditions that promote ubiquitination result in inactivation, whereas those that promote sumoylation result in the activation of the AMPK complex.     

Identification of a Nuclear Export Signal in the Catalytic Subunit of AMP-activated Protein Kinase

The metabolic regulator AMP-activated protein kinase (AMPK) maintains cellular homeostasis through regulation of proteins involved in energy-producing and -consuming pathways. Although AMPK phosphorylation targets include cytoplasmic and nuclear proteins, the precise mechanisms that regulate AMPK localization, and thus its access to these substrates, are unclear. We identify highly conserved carboxy-terminal hydrophobic amino acids that function as a leptomycin B–sensitive, CRM1-dependent nuclear export sequence (NES) in the AMPK catalytic subunit (AMPKα). When this sequence is modified AMPKα shows increased nuclear localization via a Ran-dependent import pathway. Cytoplasmic localization can be restored by substituting well-defined snurportin-1 or protein kinase A inhibitor (PKIA) CRM1-binding NESs into AMPKα. We demonstrate a functional requirement in vivo for the AMPKα carboxy-terminal NES, as transgenic Drosophila expressing AMPKα lacking this NES fail to rescue lethality of AMPKα null mutant flies and show decreased activation loop phosphorylation under heat-shock stress. Sequestered to the nucleus, this truncated protein shows highly reduced phosphorylation at the key Thr172 activation residue, suggesting that AMPK activation predominantly occurs in the cytoplasm under unstressed conditions. Thus, modulation of CRM1-mediated export of AMPKα via its C-terminal NES provides an additional mechanism for cells to use in the regulation of AMPK activity and localization.     

Quercetin reduces obesity-associated ATM infiltration and inflammation in mice: a mechanism including AMPKα1/SIRT1

Adipose tissue macrophage (ATM) plays a central role in obesity-associated inflammation and insulin resistance. Quercetin, a dietary flavonoid, possesses anti-inflammation and anti-insulin resistance properties. However, it is unclear whether quercetin can alleviate high-fat diet (HFD)-induced ATM infiltration and inflammation in mice. In this study, 5-week-old C57BL/6 mice were fed low-fat diet, HFD, or HFD with 0.l% quercetin for 12 weeks, respectively. Dietary quercetin reduced HFD-induced body weight gain and improved insulin sensitivity and glucose intolerance in mice. Meanwhile, dietary quercetin enhanced glucose transporter 4 translocation and protein kinase B signal in epididymis adipose tissues (EATs), suggesting that it heightened glucose uptake in adipose tissues. Histological and real-time PCR analysis revealed that quercetin attenuated mast cell and macrophage infiltration into EATs in HFD-fed mice. Dietary quercetin also modified the phenotype ratio of M1/M2 macrophages, lowered the levels of proinflammatory cytokines, and enhanced adenosine monophosphate-activated protein kinase (AMPK) α1 phosphorylation and silent information regulator 1 (SIRT1) expression in EATs. Further, using AMPK activator 5-aminoimidazole-4-carboxamide-1-β4-ribofuranoside and inhibitor Compound C, we found that quercetin inhibited polarization and inflammation of mouse bone marrow-derived macrophages through an AMPKα1/SIRT1-mediated mechanism. In conclusion, dietary quercetin might suppress ATM infiltration and inflammation through the AMPKα1/SIRT1 pathway in HFD-fed mice     

Cardiac Fibrosis Alleviated by Exercise Training Is AMPK-Dependent

Regular exercise can protect the heart against external stimuli, but the mechanism is not well understood. We determined the role of adenosine monophosphate-activated protein kinase (AMPK) in regulating swimming exercise-mediated cardiac protection against β-adrenergic receptor overstimulation with isoproterenol (ISO) in mice. Ten-week-old AMPKα2^+/+ and AMPKα2-knockout (AMPKα2^-/-) littermates were subjected to 4 weeks of swimming training (50 min daily, 6 days a week) or housed under sedentary conditions. The mice received daily subcutaneous injection of ISO (5 mg/kg/d), a nonselective β-adrenergic receptor agonist, during the last 2 weeks of swimming training. Swimming training alleviated ISO-induced cardiac fibrosis in AMPKα2^+/+ mice but not AMPKα2^-/- mice. Swimming training activated cardiac AMPK in AMPKα2^+/+ mice. Furthermore, swimming training attenuated ISO-induced production of reactive oxygen species (ROS) and expression of NADPH oxidase and promoted the expression of antioxidant enzymes in AMPKα2^+/+ mice but not AMPKα2^-/- mice. In conclusion, swimming training attenuates ISO-induced cardiac fibrosis by inhibiting the NADPH oxidase–ROS pathway mediated by AMPK activation. Our findings provide a new mechanism for the cardioprotective effects of exercise.     

CaMKKβ-AMPKα2 signaling contributes to mitotic Golgi fragmentation and the G2/M transition in mammalian cells

Before a cell enters mitosis, the Golgi apparatus undergoes extensive fragmentation. This is required for the correct partitioning of the Golgi apparatus into daughter cells, and inhibition of this process leads to cell cycle arrest in G2 phase. AMP-activated protein kinase (AMPK) plays critical roles in regulating growth and reprogramming metabolism. Recent studies have suggested that AMPK promotes mitotic progression and Golgi disassembly, and that this seems independent of the cellular energy status. However, the molecular mechanism underlying these events is not well understood. Here, we show that both treatment with compound C and depletion of AMPKα2 (but not AMPKα1) delays the G2/M transition in synchronized HeLa cells, as evidenced by flow cytometry and mitotic index analysis. Furthermore, knockdown of AMPKα2 specifically delays further fragmentation of isolated Golgi stacks. Interestingly, pAMPKα^Thr172 signals transiently appear in the perinuclear region of late G2/early prophase cells, partially co-localizing with the Golgi matrix protein, GM-130. These Golgi pAMPKα^Thr172 signals were also specifically abolished by AMPKα2 knockdown, indicating specific spatio-temporal activation of AMPKα2 at Golgi complex during late G2/early prophases. We also found that the specific CaMKKβ inhibitor, STO-609, reduces the pAMPKα ^Thr172 signals in the perinuclear region of G2 phase cells and delays mitotic Golgi fragmentation. Taken together, these data suggest that AMPKα2 is the major catalytic subunit of AMPKα which regulates Golgi fragmentation and G2/M transition, and that the CaMKKβ activates AMPKα2 during late G2 phase.     

Induction of Cell-Cell Fusion by Ebola Virus Glycoprotein: Low pH Is Not a Trigger

Ebola virus (EBOV) is a highly pathogenic filovirus that causes hemorrhagic fever in humans and animals. Currently, how EBOV fuses its envelope membrane within an endosomal membrane to cause infection is poorly understood. We successfully measure cell-cell fusion mediated by the EBOV fusion protein, GP, assayed by the transfer of both cytoplasmic and membrane dyes. A small molecule fusion inhibitor, a neutralizing antibody, as well as mutations in EBOV GP known to reduce viral infection, all greatly reduce fusion. By monitoring redistribution of small aqueous dyes between cells and by electrical capacitance measurements, we discovered that EBOV GP-mediated fusion pores do not readily enlarge—a marked difference from the behavior of other viral fusion proteins. EBOV GP must be cleaved by late endosome-resident cathepsins B or L in order to become fusion-competent. Cleavage of cell surface-expressed GP appears to occur in endosomes, as evidenced by the fusion block imposed by cathepsin inhibitors, agents that raise endosomal pH, or an inhibitor of anterograde trafficking. Treating effector cells with a recombinant soluble cathepsin B or thermolysin, which cleaves GP into an active form, increases the extent of fusion, suggesting that a fraction of surface-expressed GP is not cleaved. Whereas the rate of fusion is increased by a brief exposure to acidic pH, fusion does occur at neutral pH. Importantly, the extent of fusion is independent of external pH in experiments in which cathepsin activity is blocked and EBOV GP is cleaved by thermolysin. These results imply that low pH promotes fusion through the well-known pH-dependent activity of cathepsins; fusion induced by cleaved EBOV GP is a process that is fundamentally independent of pH. The cell-cell fusion system has revealed some previously unappreciated features of EBOV entry, which could not be readily elucidated in the context of endosomal entry.     

AMPKα1 Deletion Shortens Erythrocyte Life Span in Mice: ROLE OF OXIDATIVE STRESS*

AMP-activated protein kinase (AMPK) is an energy sensor essential for maintaining cellular energy homeostasis. Here, we report that AMPKα1 is the predominant isoform of AMPK in murine erythrocytes and mice globally deficient in AMPKα1 (AMPKα1^−/−), but not in those lacking AMPKα2, and the mice had markedly enlarged spleens with dramatically increased proportions of Ter119-positive erythroid cells. Blood tests revealed significantly decreased erythrocyte and hemoglobin levels with increased reticulocyte counts and elevated plasma erythropoietin concentrations in AMPKα1^−/− mice. The life span of erythrocytes from AMPKα1^−/− mice was less than that in wild-type littermates, and the levels of reactive oxygen species and oxidized proteins were significantly increased in AMPKα1^−/− erythrocytes. In keeping with the elevated oxidative stress, treatment of AMPKα1^−/− mice with the antioxidant, tempol, resulted in decreased reticulocyte counts and improved erythrocyte survival. Furthermore, the expression of Foxo3 and reactive oxygen species scavenging enzymes was significantly decreased in erythroblasts from AMPKα1^−/− mice. Collectively, these results establish an essential role for AMPKα1 in regulating oxidative stress and life span in erythrocytes.     

Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-Troglitazone-induced Apoptosis in Prostate Cancer Cells Involve AMP-activated Protein Kinase

Prostate cancer (PCa) is one of the most frequently diagnosed cancers in men with limited treatment options for the hormone-resistant forms. Development of novel therapeutic options is critically needed to target advanced forms. Here we demonstrate that combinatorial treatment with the thiazolidinedione troglitazone (TZD) and TNF-related apoptosis-inducing ligand (TRAIL) can induce significant apoptosis in various PCa cells independent of androgen receptor status. Because TZD is known to activate AMP-activated protein kinase (AMPK), we determined whether AMPK is a molecular target mediating this apoptotic cascade by utilizing PCa cell lines stably overexpressing AMPKα1 dominant negative (C4-2-DN) or empty vector (C4-2-EV). Our results indicated a significantly higher degree of apoptosis with TRAIL-TZD combination in C4-2-EV cells compared with C4-2-DN cells. Similarly, results from a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a larger reduction of viability of C4-2-EV cells compared with C4-2-DN cells when treated with TRAIL-TZD, thus suggesting that C4-2-DN cells were more apoptosis-resistant. Additionally, siRNA-mediated knockdown of endogenous AMPKα1 expression showed a reduction of TRAIL-TZD-induced apoptosis, further confirming the participation of AMPK in mediating this apoptosis. Apoptosis induction by this combinatorial treatment was also associated with a cleavage of β-catenin that was inhibited in both C4-2-DN cells and those cells in which AMPKα1 was knocked down. In addition, time course studies showed an increase in pACC^S79 (AMPK target) levels coinciding with the time of apoptosis. These studies indicate the involvement of AMPK in TRAIL-TZD-mediated apoptosis and β-catenin cleavage and suggest the possibility of utilizing AMPK as a therapeutic target in apoptosis-resistant prostate cancer.     

Identification of 5′ AMP-activated Kinase as a Target of Reactive Aldehydes during Chronic Ingestion of High Concentrations of Ethanol

The production of reactive aldehydes including 4-hydroxy-2-nonenal (4-HNE) is a key component of the pathogenesis in a spectrum of chronic inflammatory hepatic diseases including alcoholic liver disease (ALD). One consequence of ALD is increased oxidative stress and altered β-oxidation in hepatocytes. A major regulator of β-oxidation is 5′ AMP protein kinase (AMPK). In an in vitro cellular model, we identified AMPK as a direct target of 4-HNE adduction resulting in inhibition of both H₂O₂ and 5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR)-induced downstream signaling. By employing biotin hydrazide capture, it was confirmed that 4-HNE treatment of cells resulted in carbonylation of AMPKα/β, which was not observed in untreated cells. Using a murine model of alcoholic liver disease, treatment with high concentrations of ethanol resulted in an increase in phosphorylated as well as carbonylated AMPKα. Despite increased AMPK phosphorylation, there was no significant change in phosphorylation of acetyl CoA carboxylase. Mass spectrometry identified Michael addition adducts of 4-HNE on Cys¹³⁰, Cys¹⁷⁴, Cys²²⁷, and Cys³⁰⁴ on recombinant AMPKα and Cys²²⁵ on recombinant AMPKβ. Molecular modeling analysis of identified 4-HNE adducts on AMPKα suggest that inhibition of AMPK occurs by steric hindrance of the active site pocket and by inhibition of hydrogen peroxide induced oxidation. The observed inhibition of AMPK by 4-HNE provides a novel mechanism for altered β-oxidation in ALD, and these data demonstrate for the first time that AMPK is subject to regulation by reactive aldehydes in vivo.     

Activation of AMPK Enhances Neutrophil Chemotaxis and Bacterial Killing

An inability of neutrophils to eliminate invading microorganisms is frequently associated with severe infection and may contribute to the high mortality rates associated with sepsis. In the present studies, we examined whether metformin and other 5′ adenosine monophosphate-activated protein kinase (AMPK) activators affect neutrophil motility, phagocytosis and bacterial killing. We found that activation of AMPK enhanced neutrophil chemotaxis in vitro and in vivo, and also counteracted the inhibition of chemotaxis induced by exposure of neutrophils to lipopolysaccharide (LPS). In contrast, small interfering RNA (siRNA)-mediated knockdown of AMPKα1 or blockade of AMPK activation through treatment of neutrophils with the AMPK inhibitor compound C diminished neutrophil chemotaxis. In addition to their effects on chemotaxis, treatment of neutrophils with metformin or aminoimidazole carboxamide ribonucleotide (AICAR) improved phagocytosis and bacterial killing, including more efficient eradication of bacteria in a mouse model of peritonitis-induced sepsis. Immunocytochemistry showed that, in contrast to LPS, metformin or AICAR induced robust actin polymerization and distinct formation of neutrophil leading edges. Although LPS diminished AMPK phosphorylation, metformin or AICAR was able to partially decrease the effects of LPS/toll-like receptor 4 (TLR4) engagement on downstream signaling events, particularly LPS-induced IκBα degradation. The IκB kinase (IKK) inhibitor PS-1145 diminished IκBα degradation and also prevented LPS-induced inhibition of chemotaxis. These results suggest that AMPK activation with clinically approved agents, such as metformin, may facilitate bacterial eradication in sepsis and other inflammatory conditions associated with inhibition of neutrophil activation and chemotaxis.     

Exposure to Hydrogen Peroxide Induces Oxidation and Activation of AMP-activated Protein Kinase

Although metabolic conditions associated with an increased AMP/ATP ratio are primary factors in the activation of 5′-adenosine monophosphate-activated protein kinase (AMPK), a number of recent studies have shown that increased intracellular levels of reactive oxygen species can stimulate AMPK activity, even without a decrease in cellular levels of ATP. We found that exposure of recombinant AMPKαβγ complex or HEK 293 cells to H₂O₂ was associated with increased kinase activity and also resulted in oxidative modification of AMPK, including S-glutathionylation of the AMPKα and AMPKβ subunits. In experiments using C-terminal truncation mutants of AMPKα (amino acids 1–312), we found that mutation of cysteine 299 to alanine diminished the ability of H₂O₂ to induce kinase activation, and mutation of cysteine 304 to alanine totally abrogated the enhancing effect of H₂O₂ on kinase activity. Similar to the results obtained with H₂O₂-treated HEK 293 cells, activation and S-glutathionylation of the AMPKα subunit were present in the lungs of acatalasemic mice or mice treated with the catalase inhibitor aminotriazole, conditions in which intracellular steady state levels of H₂O₂ are increased. These results demonstrate that physiologically relevant concentrations of H₂O₂ can activate AMPK through oxidative modification of the AMPKα subunit. The present findings also imply that AMPK activation, in addition to being a response to alterations in intracellular metabolic pathways, is directly influenced by cellular redox status.     

The Recruitment of AMP-activated Protein Kinase to Glycogen Is Regulated by Autophosphorylation

The mammalian AMP-activated protein kinase (AMPK) is an obligatory αβγ heterotrimeric complex carrying a carbohydrate-binding module (CBM) in the β-subunit (AMPKβ) capable of attaching AMPK to glycogen. Nonetheless, AMPK localizes at many different cellular compartments, implying the existence of mechanisms that prevent AMPK from glycogen binding. Cell-free carbohydrate binding assays revealed that AMPK autophosphorylation abolished its carbohydrate-binding capacity. X-ray structural data of the CBM displays the central positioning of threonine 148 within the binding pocket. Substitution of Thr-148 for a phospho-mimicking aspartate (T148D) prevents AMPK from binding to carbohydrate. Overexpression of isolated CBM or β1-containing AMPK in cellular models revealed that wild type (WT) localizes to glycogen particles, whereas T148D shows a diffuse pattern. Pharmacological AMPK activation and glycogen degradation by glucose deprivation but not forskolin enhanced cellular Thr-148 phosphorylation. Cellular glycogen content was higher if pharmacological AMPK activation was combined with overexpression of T148D mutant relative to WT AMPK. In summary, these data show that glycogen-binding capacity of AMPKβ is regulated by Thr-148 autophosphorylation with likely implications in the regulation of glycogen turnover. The findings further raise the possibility of regulated carbohydrate-binding function in a wider variety of CBM-containing proteins.     

AMP Activated Protein Kinase Is Indispensable for Myocardial Adaptation to Caloric Restriction in Mice

Caloric restriction (CR) is a robust dietary intervention known to enhance cardiovascular health. AMP activated protein kinase (AMPK) has been suggested to mediate the cardioprotective effects of CR. However, this hypothesis remains to be tested by using definitive loss-of-function animal models. In the present study, we subjected AMPKα2 knockout (KO) mice and their wild type (WT) littermates to a CR regimen that reduces caloric intake by 20%–40% for 4 weeks. CR decreased body weight, heart weight and serum levels of insulin in both WT and KO mice to the same degree, indicating the effectiveness of the CR protocol. CR activated cardiac AMPK signaling in WT mice, but not in AMPKα2 KO mice. Correspondingly, AMPKα2 KO mice had markedly reduced cardiac function during CR as determined by echocardiography and hemodynamic measurements. The compromised cardiac function was associated with increased markers of oxidative stress, endoplasmic reticulum stress and myocyte apoptosis. Mechanistically, CR down-regulated the expression of ATP5g2, a subunit of mitochondrial ATP synthase, and reduced ATP content in AMPKα2 KO hearts, but not in WT hearts. In addition, CR accelerated cardiac autophagic flux in WT mice, but failed to do so in AMPKα2 KO mice. These results demonstrated that without AMPK, CR triggers adverse effects that can lead to cardiac dysfunction, suggesting that AMPK signaling pathway is indispensible for energy homeostasis and myocardial adaptation to CR, a dietary intervention that normally produces beneficial cardiac effects.     

PP2A Mediated AMPK Inhibition Promotes HSP70 Expression in Heat Shock Response

Background

Under stress, AMP-activated protein kinase (AMPK) plays a central role in energy balance, and the heat shock response is a protective mechanism for cell survival. The relationship between AMPK activity and heat shock protein (HSP) expression under stress is unclear.

Methodology/Principal Findings

We found that heat stress induced dephosphorylation of AMPKα subunit (AMPKα) in various cell types from human and rodent. In HepG2 cells, the dephosphorylation of AMPKα under heat stress in turn caused dephosphorylation of acetyl-CoA carboxylase and upregulation of phosphoenolpyruvate carboxykinase, two downstream targets of AMPK, confirming the inhibition of AMPK activity by heat stress. Treatment of HepG2 cells with phosphatase 2A (PP2A) inhibitor okadaic acid or inhibition of PP2A expression by RNA interference efficiently reversed heat stress-induced AMPKα dephosphorylation, suggesting that heat stress inhibited AMPK through activation of PP2A. Heat stress- and other HSP inducer (CdCl₂, celastrol, MG132)-induced HSP70 expression could be inhibited by AICAR, an AMPK specific activator. Inhibition of AMPKα expression by RNA interference reversed the inhibitory effect of AICAR on HSP70 expression under heat stress. These results indicate that AMPK inhibition under stress contribute to HSP70 expression. Mechanistic studies showed that activation of AMPK by AICAR had no effect on heat stress-induced HSF1 nuclear translocation, phosphorylation and binding with heat response element in the promoter region of HSP70 gene, but significantly decreased HSP70 mRNA stability.

Conclusions/Significance

These results demonstrate that during heat shock response, PP2A mediated AMPK inhibition upregulates HSP70 expression at least partially through stabilizing its mRNA, which suggests a novel mechanism for HSP induction under stress.