AMP-activated Protein Kinase Catalytic Subunit Research Articles

Myeloid-Restricted AMPKα1 Promotes Host Immunity and Protects against IL-12/23p40-Dependent Lung Injury during Hookworm Infection.

How the metabolic demand of parasitism affects immune-mediated resistance is poorly understood. Immunity against parasitic helminths requires M2 cells and IL-13, secreted by CD4(+) Th2 and group 2 innate lymphoid cells (ILC2), but whether certain metabolic enzymes control disease outcome has not been addressed. This study demonstrates that AMP-activated protein kinase (AMPK), a key driver of cellular energy, regulates type 2 immunity and restricts lung injury following hookworm infection. Mice with a selective deficiency in the AMPK catalytic α1 subunit in alveolar macrophages and conventional dendritic cells produced less IL-13 and CCL17 and had impaired expansion of ILC2 in damaged lung tissue compared with wild-type controls. Defective type 2 responses were marked by increased intestinal worm burdens, exacerbated lung injury, and increased production of IL-12/23p40, which, when neutralized, restored IL-13 production and improved lung recovery. Taken together, these data indicate that defective AMPK activity in myeloid cells negatively impacts type 2 responses through increased IL-12/23p40 production. These data support an emerging concept that myeloid cells and ILC2 can coordinately regulate tissue damage at mucosal sites through mechanisms dependent on metabolic enzyme function.

Risk prediction for early-onset gastric carcinoma: a case-control study of polygenic gastric cancer in Han Chinese with hereditary background.

Recent genomewide studies have identified several germline variations associated with gastric cancer. The aim of the present study was to identify, in a Chinese Han population, the individual and combined effects of those single nucleotide polymorphisms (SNPs) that increase the risk of early-onset gastric cancer. We conducted a case-control study comprising 116 patients with gastric cancer as well as 102 sex- and age-matched controls and confirmed that the SNPs MUC1 (mucin 1) rs9841504 and ZBTB20 (zinc finger and BTB domain containing 20) rs4072037 were associated with an increased gastric cancer risk. Of the 116 patients diagnosed with cancer, 65 had at least 1 direct lineal relative with carcinoma of the digestive system or breast/ovarian cancer. These 65 had another 4 SNPs associated with gastric cancer susceptibility: PSCA (prostate stem cell antigen) rs2294008, PLCE1 (phospholipase C epsilon 1) rs2274223, PTGER4/PRKAA1 (prostaglandin E receptor 4/protein kinase AMP-activated catalytic subunit alpha 1) rs13361707, and TYMS (thymidylate synthetase) rs2790. However, each of these low-penetrance susceptibility polymorphisms alone is not considered influential enough to predict the absolute risk of early-onset gastric cancer. Thus we decided to study different combinations of polygenes as they affected for our population. Those subjects with both the risk alleles MUC1 rs9841504 and ZBTB20 rs4072037 had a greater than 3-fold increased risk of gastric cancer. Also those with a hereditary background including the risk alleles PLCE1 rs2274223 and PTGER4/PRKAA1 rs13361707 were 3 times more susceptible to cardia cancer than those without. These findings show that the study of combined polymorphisms, instead of single low-penetrance variations in susceptibility, may lead to a high-risk classification for a specific population.

AMPK antagonizes hepatic glucagon-stimulated cyclic AMP signalling via phosphorylation-induced activation of cyclic nucleotide phosphodiesterase 4B.

Biguanides such as metformin have previously been shown to antagonize hepatic glucagon-stimulated cyclic AMP (cAMP) signalling independently of AMP-activated protein kinase (AMPK) via direct inhibition of adenylate cyclase by AMP. Here we show that incubation of hepatocytes with the small-molecule AMPK activator 991 decreases glucagon-stimulated cAMP accumulation, cAMP-dependent protein kinase (PKA) activity and downstream PKA target phosphorylation. Moreover, incubation of hepatocytes with 991 increases the Vmax of cyclic nucleotide phosphodiesterase 4B (PDE4B) without affecting intracellular adenine nucleotide concentrations. The effects of 991 to decrease glucagon-stimulated cAMP concentrations and activate PDE4B are lost in hepatocytes deleted for both catalytic subunits of AMPK. PDE4B is phosphorylated by AMPK at three sites, and by site-directed mutagenesis, Ser304 phosphorylation is important for activation. In conclusion, we provide a new mechanism by which AMPK antagonizes hepatic glucagon signalling via phosphorylation-induced PDE4B activation.

Specific deletion of AMP-activated protein kinase (α1AMPK) in mouse Sertoli cells modifies germ cell quality

The AMP-activated protein kinase (AMPK) is an important regulator of cellular energy homeostasis which plays a role in fertility. Complete disruption of the AMPK catalytic subunit α1 gene (α1AMPK KO) in male mice results in a decrease in litter size which is associated with the production of altered sperm morphology and motility. Because of the importance of Sertoli cells in the formation of germ cells, we have chosen to selectively disrupt α1AMPK only in the Sertoli cells in mice (Sc-α1AMPK-KO mice). Specific deletion of the α1AMPK gene in Sertoli cells resulted in a 25% reduction in male fertility associated with abnormal spermatozoa with a thin head. No clear alterations in testis morphology or modification in the number of Sertoli cells in vivo were observed, but a dysregulation in energy metabolism in Sertoli cells occurred. We have reported an increase in lactate production, in lipid droplets, and a reduction in ATP production in Sc-α1AMPK-KO Sertoli cells. These perturbations were associated with lower expression of mitochondrial markers (cytochrome c and PGC1-α). In addition another metabolic sensor, the deacetylase SIRT1, had a reduction in expression which is correlated with a decline in deacetylase activity. Finally, expression and localization of junctions forming the blood-testis barrier between Sertoli cells themselves and with germ cells were deregulated in Sc-α1AMPK-KO. In conclusion, these results suggest that dysregulation of the energy sensing machinery exclusively through disruption of α1AMPK in Sertoli cells translates to a reduction in the quality of germ cells and fertility.

Cigarette smoke exposure triggers the autophagic cascade via activation of the AMPK pathway in mice.

We previously demonstrated that cigarette smoke (CS) exposure decreases primordial follicle counts and induces autophagy in ovarian granulosa cells in preference to apoptosis. Therefore, the objective of this study was to investigate molecular targets underlying smoke-induced activation of the reparative autophagy pathway in the ovary. Briefly, ovarian homogenates were prepared from adult female mice exposed to mainstream CS twice daily for 8 wk, using a whole-body exposure system. A gene array revealed that CS exposure induced a greater than 2-fold significant increase in the expression of proautophagic genes Cdkn1b, Map1lc3a, Bad, and Sqstm1/p62. A significant increase in Prkaa2, Pik3c3, and Maplc31b expression, as well as a significant decrease in Akt1 and Mtor expression, was detected by quantitative PCR. The 5'-AMP-activated protein kinase catalytic subunit (AMPK) alpha1 + alpha2 and ATG7 protein expression was significantly increased, whereas AKT1, mTOR, CDKN1B/p27, and CXCR4 proteins were significantly decreased in CS exposed versus control ovaries. Up-regulation of AMPK alpha1 + alpha2, a known initiator of autophagic signaling, and ATG7 further suggests activation of the autophagy cascade. Two prosurvival factors, AKT and mTOR, were decreased in expression, an outcome that favors induction of the autophagy pathway, whereas decreased levels of CDKN1B is suggestive of cell cycle dysregulation. In summary, our data suggest that CS exposure induces ovarian follicle loss through induction of the autophagic cascade via the AMPK pathway together with inhibition of antiautophagic markers AKT and mTOR. We further postulate that toxicant-induced dysregulation of reparative autophagy is a novel pathway central to impaired follicle development and subfertility.

Pharmacological and Morphological Evidence of AMPK-Mediated Energy Sensing in the Lower Brain Stem Ependymocytes to Control Reproduction in Female Rodents.

Ependymocytes are one of the energy-sensing cells that regulate animal reproduction through their responsiveness to changes in extracellular glucose levels and the expression of pancreatic-type glucokinase and glucose transporter 2, which play a critical role in sensing blood glucose levels in pancreatic β-cells. Molecular mechanisms underlying glucose sensing in the ependymocytes remain poorly understood. The AMP-activated protein kinase (AMPK), a serine/threonine kinase highly conserved in all eukaryotic cells, has been suggested to be an intracellular fuel gauge that detects cellular energy status. The present study aims to clarify the role AMPK of the lower brainstem ependymocytes has in sensing glucose levels to regulate reproductive functions. First, we will show that administration of 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, into the 4th ventricle suppressed pulsatile LH release in female rats. Second, we will demonstrate the presence of AMPK catalytic subunit immunoreactivities in the rat lower brainstem ependymocytes. Third, transgenic mice were generated to visualize the ependymocytes with Venus, a green fluorescent protein, expressed under the control of the mouse vimentin promoter for further in vitro study. The Venus-labeled ependymocytes taken from the lower brainstem of transgenic mice revealed that AMPK activation by 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside, an AMPK activator, increased in vitro intracellular calcium concentrations. Taken together, malnutrition-induced AMPK activation of ependymocytes of the lower brainstem might be involved in suppression of GnRH/LH release and then gonadal activities.

AMP‐activated Protein Kinase is Necessary for Cardiorespiratory Adjustments during Hypoxia

The AMP‐activated protein kinase (AMPK) has been proposed to underpin appropriate cardiorespiratory adjustments during hypoxia (Evans et al., 2006), and has been identified as a mediator of hypoxic pulmonary vasoconstriction (HPV) (Evans et al., 2005). Our objective was to assess by non‐invasive Echo Doppler ultrasound, the effect(s) on HPV of deleting LKB1, CAMKK‐β or the AMPKα1 & α2 catalytic subunits. Our preliminary investigations on wild type and transgenic mice suggest that the LKB1‐AMPK signalling pathway, but not CAMKK‐β, are required for HPV. Moreover, deletion of AMPK catalytic subunit(s) blocked hypoxia‐induced inhibition of voltage‐gated potassium currents (Kv) in acutely isolated pulmonary arterial smooth muscle cells. Therefore, AMPK underpins the regulation of pulmonary vascular responses to hypoxia at the molecular, cellular and system level.Evans et al. (2005). J. Biol Chem., 280, 41505‐41511.Evans (2006). J. Physiol., 574, 113‐123.

Inhibition of AMP-activated protein kinase signaling alleviates impairments in hippocampal synaptic plasticity induced by amyloid β.

The AMP-activated protein kinase (AMPK) is a Ser/Thr kinase that is activated in response to low-energy states to coordinate multiple signaling pathways to maintain cellular energy homeostasis. Dysregulation of AMPK signaling has been observed in Alzheimer's disease (AD), which is associated with abnormal neuronal energy metabolism. In the current study we tested the hypothesis that aberrant AMPK signaling underlies AD-associated synaptic plasticity impairments by using pharmacological and genetic approaches. We found that amyloid β (Aβ)-induced inhibition of long-term potentiation (LTP) and enhancement of long-term depression were corrected by the AMPK inhibitor compound C (CC). Similarly, LTP impairments in APP/PS1 transgenic mice that model AD were improved by CC treatment. In addition, Aβ-induced LTP failure was prevented in mice with genetic deletion of the AMPK α2-subunit, the predominant AMPK catalytic subunit in the brain. Furthermore, we found that eukaryotic elongation factor 2 (eEF2) and its kinase eEF2K are key downstream effectors that mediate the detrimental effects of hyperactive AMPK in AD pathophysiology. Our findings describe a previously unrecognized role of aberrant AMPK signaling in AD-related synaptic pathophysiology and reveal a potential therapeutic target for AD.

Protein expression profiling during wallerian degeneration after rat sciatic nerve injury

Wallerian degeneration (WD) is an important area of research in modern neuroscience. Many protein expressions are regulated by differentially expressed genes in WD, but the precise mechanisms are elusive. In this study, we profiled differentially expressed proteins in WD after rat sciatic nerve injury using an antibody array. Functional analysis positively identified cell proliferation, regulation of cell proliferation, and immune system processes. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed molecular networks related mainly to cytokine-cytokine receptor interaction, the mitogen-activated proteinkinase (MAPK) signaling pathway, apoptosis, the toll-like receptor (TLR) signaling pathway, and the Janus kinase (Jak) - signal transducer and activator of transcription (STAT) signaling pathway. Interactions between these differential proteins were well established and regulated by the key factors transforming growth factor beta 1 (TGF-β1), toll-like receptor 4 (TLR4), Fas ligand (FasL), and 5'-AMP-activated protein kinase catalytic subunit alpha-1 (PRKAA1). These results provide information related to functional analysis of differentially expressed genes during WD.

Loss of a neural AMP-activated kinase mimics the effects of elevated serotonin on fat, movement, and hormonal secretions.

AMP-activated protein kinase (AMPK) is an evolutionarily conserved master regulator of metabolism and a therapeutic target in type 2 diabetes. As an energy sensor, AMPK activity is responsive to both metabolic inputs, for instance the ratio of AMP to ATP, and numerous hormonal cues. As in mammals, each of two genes, aak-1 and aak-2, encode for the catalytic subunit of AMPK in C. elegans. Here we show that in C. elegans loss of aak-2 mimics the effects of elevated serotonin signaling on fat reduction, slowed movement, and promoting exit from dauer arrest. Reconstitution of aak-2 in only the nervous system restored wild type fat levels and movement rate to aak-2 mutants and reconstitution in only the ASI neurons was sufficient to significantly restore dauer maintenance to the mutant animals. As in elevated serotonin signaling, inactivation of AAK-2 in the ASI neurons caused enhanced secretion of dense core vesicles from these neurons. The ASI neurons are the site of production of the DAF-7 TGF-β ligand and the DAF-28 insulin, both of which are secreted by dense core vesicles and play critical roles in whether animals stay in dauer or undergo reproductive development. These findings show that elevated levels of serotonin promote enhanced secretions of systemic regulators of pro-growth and differentiation pathways through inactivation of AAK-2. As such, AMPK is not only a recipient of hormonal signals but can also be an upstream regulator. Our data suggest that some of the physiological phenotypes previously attributed to peripheral AAK-2 activity on metabolic targets may instead be due to the role of this kinase in neural serotonin signaling.

Ablation of cereblon attenuates myocardial ischemia–reperfusion injury

Cereblon (CRBN) was originally identified as a target protein for a mild type of mental retardation in humans. However, recent studies showed that CRBN acts as a negative regulator of AMP-activated protein kinase (AMPK) by binding directly to the AMPK catalytic subunit. Because AMPK is implicated in myocardial ischemia–reperfusion (I–R) injury, we reasoned that CRBN might play a role in the pathology of myocardial I–R through regulation of AMPK activity. To test this hypothesis, wild-type (WT) and crbn knockout (KO) mice were subjected to I–R (complete ligation of the coronary artery for 30min followed by 24h of reperfusion). We found significantly smaller infarct sizes and less fibrosis in the hearts of KO mice than in those of WT mice. Apoptosis was also significantly reduced in the KO mice compared with that in WT mice, as shown by the reduced numbers of TUNEL-positive cells. In parallel, AMPK activity remained at normal levels in KO mice undergoing I–R, whereas it was significantly reduced in WT mice under the same conditions. In rat neonatal cardiomyocytes, overexpression of CRBN significantly reduced AMPK activity, as demonstrated by reductions in both phosphorylation levels of AMPK and the expression of its downstream target genes. Collectively, these data demonstrate that CRBN plays an important role in myocardial I–R injury through modulation of AMPK activity.

The effects of age and muscle contraction on AMPK activity and heterotrimer composition

Sarcopenia is characterized by increased skeletal muscle atrophy due in part to alterations in muscle metabolism. AMP-activated protein kinase (AMPK) is a master regulator of skeletal muscle metabolic pathways which regulate many cellular processes that are disrupted in old-age. Functional AMPK is a heterotrimer composed of α, β and γ subunits, and each subunit can be represented in the heterotrimer by one of two (α1/α2, β1/β2) or three (γ1/γ2/γ3) isoforms. Altered isoform composition affects AMPK localization and function. Previous work has shown that overall AMPK activation with endurance-type exercise is blunted in old vs. young skeletal muscle. However, details regarding the activation of the specific isoforms of AMPK, as well as the heterotrimeric composition of AMPK in old skeletal muscle, are unknown. Our purpose here, therefore, was to determine the effect of old-age on 1) the activation of the α1 and α2 catalytic subunits of AMPK in skeletal muscle by a continuous contraction bout, and 2) the heterotrimeric composition of skeletal muscle AMPK. We studied gastrocnemius (GAST) and tibialis anterior (TA) muscles from young adult (YA; 8months old) and old (O; 30months old) male Fischer344×Brown Norway F1 hybrid rats after an in situ bout of endurance-type contractions produced via electrical stimulation of the sciatic nerve (STIM). AMPKα phosphorylation and AMPKα1 and α2 activities were unaffected by age at rest. However, AMPKα phosphorylation and AMPKα2 protein content and activity were lower in O vs. YA after STIM. Conversely, AMPKα1 content was greater in O vs. YA muscle, and α1 activity increased with STIM in O but not YA muscles. AMPKγ3 overall concentration and its association with AMPKα1 and α2 were lower in O vs. YA GAST. We conclude that activation of AMPKα1 is enhanced, while activation of α2 is suppressed immediately after repeated skeletal muscle contractions in O vs. YA skeletal muscle. These changes are associated with changes in the AMPK heterotrimer composition. Given the known roles of AMPK α1, α2 and γ3, this may contribute to sarcopenia and associated muscle metabolic dysfunction.

0358: Differential AMP-activated protein kinase (AMPK) activation in platelets, in response to various agonists. Comparison between human and murine platelets

AMPK is activated during platelet aggregation and controls the phosphorylation state of key cytoskeletal targets. CaMKKβ is responsible for thrombin-induced AMPK activation. Substantial differences have been observed between human and murine platelets. This study aims to bring out the differences in AMPK signalling pathway between human and murine platelets. Only α1 catalytic subunit of AMPK is present in human platelets. Thrombin induces a dramatic AMPKα1 activation and subsequent acetyl-CoA carboxylase (ACC) phosphorylation, while thromboxane A2 (U46619) and collagen exert a marginal effect on both enzymes. ADP has no effect at all. These differences cannot be entirely related to a lower calcium production in response to U46619, collagen and ADP. Indeed, U46619 increases calcium with a similar extent than thrombin while it does not modify ACC phosphorylation. PAR1 but not PAR4-activating peptide reproduces the effect of thrombin on ACC. In addition, inhibition of CaMKKβ/AMPKα1 activation blunts platelet aggregation in response to thrombin and does not affect the response upon stimulation by the other agonists. Murine platelets express AMPKα1 and AMPKα2. AMPK can be activated either by thrombin or U46619 or collagen but not by ADP. The absence of AMPKα1 blocks ACC phosphorylation induced by the three agonists. It mainly inhibits the thrombin-induced platelet aggregation but also significantly reduces aggregation in response to U46619 and collagen. The absence of AMPKα2 does not affect the increase in ACC phosphorylation, whatever the agonist used. AMPKα1 is the sole catalytic subunit of AMPK expressed in human platelets and is activated by thrombin through PAR1. Murine platelets contain both catalytic subunits although α1 plays a predominant role. In mice, thrombin is not the predominant AMPK activator probably because of the difference in the type of PAR expression. Signalling events of mouse platelets can diverge from those of human platelets.

Isolation of AMP-activated protein kinase (AMPK) alleles required for neuronal maintenance in Drosophila melanogaster

SummaryThe maintenance of energetic homeostasis in the face of limited available nutrients is a complex problem faced by all organisms. One important mechanism to maintain energetic homeostasis involves the activation of the energy sensor AMP-activated protein kinase (AMPK). AMPK is a cell-autonomous energy sensor that is highly sensitive to and regulated by the ATP to ADP and ATP to AMP ratios. However, the genetic analysis of AMPK signaling in vertebrates has been complicated by the existence of multiple redundant AMPK subunits. Here, we describe the identification of mutations in the single Drosophila melanogaster AMPK catalytic subunit (AMPKα) and their implications for neural maintenance and integrity. This article provides a citation replacement for previously published ampkα alleles, transgenes and neuronal phenotypes, which remain accurate; however, they were used in a previously published study that has subsequently been retracted (Mirouse et al., 2013).

SUMOylation regulates the SNF1 protein kinase

The AMP-activated protein kinase (AMPK) is a major stress sensor of mammalian cells. AMPK's homolog in the yeast Saccharomyces cerevisiae, the SNF1 protein kinase, is a central regulator of carbon metabolism that inhibits the Snf3/Rgt2-Rgt1 glucose sensing pathway and activates genes involved in respiration. We present evidence that glucose induces modification of the Snf1 catalytic subunt of SNF1 with the small ubiquitin-like modifier protein SUMO, catalyzed by the SUMO (E3) ligase Mms21. Our results suggest that SUMOylation of Snf1 inhibits its function in two ways: by interaction of SUMO attached to lysine 549 with a SUMO-interacting sequence motif located near the active site of Snf1, and by targeting Snf1 for destruction via the Slx5-Slx8 (SUMO-directed) ubiquitin ligase. These findings reveal another way SNF1 function is regulated in response to carbon source.

AMPKα1 controls hepatocyte proliferation independently of energy balance by regulating Cyclin A2 expression

AMP-activated protein kinase (AMPK) is an evolutionarily conserved sensor of cellular energy status that contributes to restoration of energy homeostasis by slowing down ATP-consuming pathways and activating ATP-producing pathways. Unexpectedly, in different systems, AMPK is also required for proper cell division. In the current study, we evaluated the potential effect of the AMPK catalytic subunit, AMPKα1, on hepatocyte proliferation. Hepatocyte proliferation was determined in AMPKα1 knockout and wild-type mice in vivo after two thirds partial hepatectomy, and in vitro in primary hepatocyte cultures. The activities of metabolic and cell cycle-related signaling pathways were measured. After partial hepatectomy, hepatocytes proliferated rapidly, correlating with increased AMPK phosphorylation. Deletion of AMPKα1 delayed liver regeneration by impacting on G1/S transition phase. The proliferative defect of AMPKα1-deficient hepatocytes was cell autonomous, and independent of energy balance. The priming phase, lipid droplet accumulation, protein anabolic responses and growth factor activation after partial hepatectomy occurred normally in the absence of AMPKα1 activity. By contrast, mRNA and protein expression of cyclin A2, a key driver of S phase progression, were compromised in the absence of AMPK activity. Importantly, AMPKα1 controlled cyclin A2 transcription mainly through the ATF/CREB element. Our study highlights a novel role for AMPKα1 as a positive regulator of hepatocyte division occurring independently of energy balance.

Insulin Resistance due to Nutrient Excess: Mechanisms of AMPK Downregulation

Excesses of glucose and the branched chain amino acids lead to insulin resistance in skeletal muscle by activating mTOR/p70S6 kinase (p70S6K). In a recent study we demonstrated that glucose and leucine may produce these effects by downregulating AMP‐activated protein kinase (AMPK) which, when activated, diminishes mTOR/p70S6 K signaling. How these excesses of glucose and leucine decrease AMPK activity is unclear, since they do not alter the cell's energy state (AMP/ATP or CrP). We explored whether the decrease in AMPK activity might be due to decreases in the activity of SIRT1, a histone protein deacetylase that can activate AMPK or by changes in the activity of an unidentified kinase or phosphatase that results in the phosphorylation of Ser485/491 on the catalytic subunit of AMPK leading to its inhibition. Incubation of extensor digitorum longus muscle, with high glucose (25 mM) or 5 mM glucose and leucine (100 μM) persistently diminished AMPK activity within 30 min an effect that lasted for 2 h. Likewise both high glucose and leucine decreased the activity of SIRT1as reflected by increases in the deacetylation of histone H3 and a decrease in the activity of the rate‐limiting enzyme for SIRT1 activation, nicotinamide phosphoribosyltransferase (NAMPT). In addition, SIRT1 abundance was decreased. These changes only become evident after 2 h, however. In contrast, excess glucose and leucine caused increased phosphorylation of AMPK at Ser485/491 after 1 h. In studies with intact rats, we found that prolonged (3–8 h) hyperglycemia, produced by a glucose infusion, downregulates AMPK Thr172 phosphorylation and causes insulin resistance as early as 3 h. However, SIRT1, AMPK (Ser485/491) and NAMPT activity only decreased between 5–8 h. The results indicate that both leucine and high glucose downregulate AMPK by at least two mechanisms. This could account for sustained decreases in AMPK activity; however, temporally they do not account for the initial decrease in AMPK activity caused by these fuels.

Genetic Deletion of Catalytic Subunits of AMP-activated Protein Kinase Increases Osteoclasts and Reduces Bone Mass in Young Adult Mice

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis and a potential therapeutic target for the intervention of cancer and metabolic disorders. However, the role of AMPK in bone homeostasis remains incompletely understood. Here we assessed the skeletal phenotype of mice lacking catalytic subunits of AMPK and found that mice lacking AMPKα1 (Prkaa1(-/-)) or AMPKα2 (Prkaa2(-/-)) had reduced bone mass compared with the WT mice, although the reduction was less in Prkaa2(-/-) mice than in Prkaa1(-/-) mice. Static and dynamic bone histomorphometric analyses revealed that Prkaa1(-/-) mice had an elevated rate of bone remodeling because of increases in bone formation and resorption, whereas AMPKα2 KO-induced bone mass reduction was largely attributable to elevated bone resorption. In agreement with our in vivo results, AMPKα deficiency was associated with increased osteoclastogenesis in vitro. Moreover, we found that AMPKα1 inhibited the receptor activator of nuclear factor κB (RANK) signaling, providing an explanation for AMPK-mediated inhibition of osteoclastogenesis. Therefore, our findings further underscore the importance of AMPK in bone homeostasis, in particular osteoclastogenesis, in young adult mammals.

Differential regulation of eEF2 and p70S6K by AMPKalpha2 in heart

Eukaryotic elongation factor 2 (eEF-2) and mammalian target of rapamycin (mTOR)–p70 ribosomal protein S6 kinase (p70S6K) signaling pathways control protein synthesis and are inhibited during myocardial ischemia. Intracellular acidosis and AMP-activated protein kinase (AMPK) activation, both occurring during ischemia, have been proposed to participate in this inhibition. We evaluated the contribution of AMPKα2, the main cardiac AMPK catalytic subunit isoform, in eEF2 and mTOR–p70S6K regulation using AMPKα2 KO mice. Hearts were perfused ex vivo with or without insulin, and then submitted or not to ischemia. Insulin pre-incubation was necessary to activate mTOR–p70S6K and evaluate their subsequent inhibition by ischemia. Ischemia decreased insulin-induced mTOR–p70S6K phosphorylation in WT and AMPKα2 KO mice to a similar extent. This AMPKα2-independent p70S6K inhibition correlated well with the inhibition of PKB/Akt, located upstream of mTOR–p70S6K and can be mimicked in cardiomyocytes by decreasing pH. By contrast, ischemia-induced inhibitory phosphorylation of eEF-2 was drastically reduced in AMPKα2 KO mice. Interestingly, AMPKα2 also played a role under normoxia. Its deletion increased the insulin-induced p70S6K stimulation. This p70S6K over-stimulation was associated with a decrease in inhibitory phosphorylation of Raptor, an mTOR partner identified as an AMPK target. In conclusion, AMPKα2 controls cardiac p70S6K under normoxia and regulates eEF-2 but not the mTOR–p70S6K pathway during ischemia. This challenges the accepted notion that mTOR–p70S6K is inhibited by myocardial ischemia mainly via an AMPK-dependent mechanism.

Dephosphorylation of endogenous GABAB receptor R2 subunit and AMPK α subunits which were measured by in vitro method using transfer membrane

Protein phosphorylation can be regulated by changes in kinase activity, phosphatase activity, or both. GABAB receptor R2 subunit (GABABR2) is phosphorylated at S783 by 5′-AMP-activated-protein kinase (AMPK), and this phosphorylation modulates GABAB receptor desensitization. Since the GABAB receptor is an integral membrane protein, solubilizing GABABR2 is difficult. To circumvent this problem and to identify specific phosphatases that dephosphorylate S783, we employed an in vitro assay based on dephosphorylation of proteins on PVDF membranes by purified phosphatases. Our method allowed us to demonstrate that S783 in GABABR2 is directly dephosphorylated by PP2A (but not by PP1, PP2B nor PP2C) in a dose-dependent and okadaic acid-sensitive manner. We also show that the level of phosphorylation of the catalytic subunit of AMPK at T172 is reduced by PP1, PP2A and PP2C. Our data indicate that PP2A dephosphorylates GABABR2(S783) less efficiently than AMPK(T172), and that additional phosphatases might be involved in S783 dephosphorylation.