[Show abstract][Hide abstract] ABSTRACT: Age-related decreases in neural function result in part from alterations in synapses. To identify molecular defects that lead to such changes, we focused on the outer retina, in which synapses are markedly altered in old rodents and humans. We found that the serine/threonine kinase LKB1 and one of its substrates, AMPK, regulate this process. In old mice, synaptic remodeling was accompanied by specific decreases in the levels of total LKB1 and active (phosphorylated) AMPK. In the absence of either kinase, young adult mice developed retinal defects similar to those that occurred in old wild-type animals. LKB1 and AMPK function in rod photoreceptors where their loss leads to aberrant axonal retraction, the extension of postsynaptic dendrites and the formation of ectopic synapses. Conversely, increasing AMPK activity genetically or pharmacologically attenuates and may reverse age-related synaptic alterations. Together, these results identify molecular determinants of age-related synaptic remodeling and suggest strategies for attenuating these changes.
[Show abstract][Hide abstract] ABSTRACT: AMPK is a sensor of cellular energy status and a promising target for drugs aimed at metabolic disorders. We have studied the selectivity and mechanism of a recently described activator, C2, and its cell-permeable prodrug, C13. C2 was a potent allosteric activator of α1-complexes that, like AMP, also protected against Thr172 dephosphorylation. Compared with AMP, C2 caused only partial allosteric activation of α2-complexes and failed to protect them against dephosphorylation. We show that both effects could be fully restored by exchanging part of the linker between the autoinhibitory and C-terminal domains in α2, containing the equivalent region from α1 thought to interact with AMP bound in site 3 of the γ subunit. Consistent with our results in cell-free assays, C13 potently inhibited lipid synthesis in hepatocytes from wild-type and was largely ineffective in AMPK-knockout hepatocytes; its effects were more severely affected by knockout of α1 than of α2, β1, or β2.
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase α1 knockout (prkaa1(-/-)) mice manifest splenomegaly and anemia. The underlying molecular mechanisms, however, remain to be established. In this study, we tested the hypothesis that defective autophagy-dependent mitochondrial clearance in prkaa1(-/-) mice exacerbates oxidative stress, thereby enhancing erythrocyte destruction. The levels of ULK1 phosphorylation, autophagical flux, mitochondrial contents, and reactive oxygen species (ROS) were examined in human erythroleukemia cell line, K562 cells, as well as prkaa1(-/-) mouse embryonic fibroblasts and erythrocytes. Deletion of Prkaa1 resulted in the inhibition of ULK1 phosphorylation at Ser555, prevented the formation of ULK1 and BECN1- PtdIns3K complexes, and reduced autophagy capacity. The suppression of autophagy was associated with enhanced damaged mitochondrial accumulation and ROS production. Compared with wild-type (WT) mice, prkaa1(-/-) mice exhibited a shortened erythrocyte life span, hemolytic destruction of erythrocytes, splenomegaly, and anemia, all of which were alleviated by the administration of either rapamycin to activate autophagy or Mito-tempol, a mitochondria-targeted antioxidant, to scavenge mitochondrial ROS. Furthermore, transplantation of WT bone marrow into prkaa1(-/-) mice restored mitochondrial removal, reduced intracellular ROS levels, and normalized hematologic parameters and spleen size. Conversely, transplantation of prkaa1 (-/-) bone marrow into WT mice recapitulated the prkaa1(-/-) mouse phenotypes. We conclude that PRKAA1-dependent autophagy-mediated clearance of damaged mitochondria is required for erythrocyte maturation and homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-6 (IL-6) is a major cytokine controlling body weight and metabolism, but because many types of cells can synthesize and respond to IL-6 considerable uncertainty still exists about the mechanisms underlying IL-6 effects. Therefore, the aim of this study was to analyze the effects of tissue-specific deletion of IL-6 by using a fatty acid binding protein (aP2) promoter-Cre inducible system (aP2-Cre-ERT2).
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) functions as a signaling hub to balance energy supply with demand. Phosphorylation of activation loop Thr172 has been considered as an essential step in AMPK activation. In this issue of Chemistry & Biology, Scott and colleagues show that the small molecule direct AMPK activator, A-769662, bypasses this phosphorylation event and acts synergistically with AMP on naive AMPK.
[Show abstract][Hide abstract] ABSTRACT: Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodelling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodelling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodelling, as indicated by increased LV diastolic dimension 30days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.
Journal of Molecular and Cellular Cardiology 05/2014; · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is an attractive therapeutic drug target for treating metabolic disorders. We studied effects of an AMPK activator developed by Merck (ex229 from patent application WO2010036613), comparing chemical activation with contraction in intact incubated skeletal muscles. We also compared effects of ex229 with those of the Abbott A769662 compound and AICA riboside (5-aminoimidazole-4-carboxamide riboside). In rat epitrochlearis muscle, ex229 dose-dependently increased AMPK activity of α1-, α2-, β1- and β2-containing complexes with significant increases in AMPK activity seen at a concentration of 50 μM. At a concentration of 100 μM, AMPK activation was similar to that observed after contraction and importantly led to an ~2-fold increase in glucose uptake. In AMPK α1-/α2-subunit catalytic subunit double knockout myotubes incubated with ex229, the increases in glucose uptake and ACC phosphorylation seen in control cells were completely abolished, suggesting that the effects of the compound were AMPK-dependent. When muscle glycogen levels were reduced by ~50% after starvation, ex229-induced AMPK activation and glucose uptake were amplified in a wortmannin-independent manner. In L6 myotubes incubated with ex229, fatty acid oxidation was increased. Furthermore, in mouse EDL and soleus muscles, ex229 increased both AMPK activity and glucose uptake at least 2-fold. In summary, ex229 efficiently activated skeletal muscle AMPK and elicited metabolic effects in muscle appropriate for treating type2 diabetes by stimulating glucose uptake and increasing fatty acid oxidation.
[Show abstract][Hide abstract] ABSTRACT: AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that plays a central role in skeletal muscle metabolism. We used skeletal muscle-specific AMPKα1α2 double-knockout (mdKO) mice to provide direct genetic evidence of the physiological importance of AMPK in regulating muscle exercise capacity, mitochondrial function, and contraction-stimulated glucose uptake. Exercise performance was significantly reduced in the mdKO mice, with a reduction in maximal force production and fatigue resistance. An increase in the proportion of myofibers with centralized nuclei was noted, as well as an elevated expression of interleukin 6 (IL-6) mRNA, possibly consistent with mild skeletal muscle injury. Notably, we found that AMPKα1 and AMPKα2 isoforms are dispensable for contraction-induced skeletal muscle glucose transport, except for male soleus muscle. However, the lack of skeletal muscle AMPK diminished maximal ADP-stimulated mitochondrial respiration, showing an impairment at complex I. This effect was not accompanied by changes in mitochondrial number, indicating that AMPK regulates muscle metabolic adaptation through the regulation of muscle mitochondrial oxidative capacity and mitochondrial substrate utilization but not baseline mitochondrial muscle content. Together, these results demonstrate that skeletal muscle AMPK has an unexpected role in the regulation of mitochondrial oxidative phosphorylation that contributes to the energy demands of the exercising muscle.-Lantier, L., Fentz, J., Mounier, R., Leclerc, J., Treebak, J. T., Pehmøller, C., Sanz, N., Sakakibara, I., Saint-Amand, E., Rimbaud, S., Maire, P., Marette, A., Ventura-Clapier, R., Ferry, A., Wojtaszewski, J. F. P., Foretz, M., Viollet, B. AMPK controls exercise endurance, mitochondrial oxidative capacity, and skeletal muscle integrity.
[Show abstract][Hide abstract] ABSTRACT: Background
Platelet activation requires sweeping morphological changes, supported by contraction and remodelling of platelet actin cytoskeleton. In various other cell types, AMP-activated protein kinase (AMPK) controls the phosphorylation state of cytoskeletal targets.Objective
We hypothesized that AMPK is activated during platelet aggregation and contributes to the control of cytoskeletal targets.ResultsWe found that AMPK-α1 was mainly activated by thrombin and not by other platelet agonists in purified human platelets. Thrombin activated AMPK-α1 ex vivo via a Ca2+/calmodulin-dependent kinase kinase β CAMKKβ-dependent pathway. Pharmacological inhibition of CAMKKβ blocked thrombin-induced platelet aggregation and counteracted thrombin-induced phosphorylation of several cytoskeletal proteins, namely, regulatory myosin light chains (MLC), cofilin and vasodilator-stimulated phosphoprotein (VASP), three key elements involved in actin cytoskeleton contraction and polymerization. Platelets isolated from mice lacking AMPK-α1 exhibited reduced aggregation in response to thrombin, associated with a defect in MLC, cofilin and VASP phosphorylation and actin polymerization. More importantly, we show for the first time that AMPK pathway was activated in platelets of patients undergoing major cardiac surgery, in a heparin-sensitive manner.ConclusionAMPK-α1 is activated by thrombin in human platelets. It controls phosphorylation of key cytoskeletal targets and actin cytoskeleton remodelling during platelet aggregation.This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 03/2014; · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The multifunctional AMPK-activated protein kinase (AMPK) is an evolutionarily conserved energy sensor that plays an important role in cell proliferation, growth, and survival. It remains unclear whether AMPK functions as a tumor suppressor or a contextual oncogene. This is because although on one hand active AMPK inhibits mammalian target of rapamycin (mTOR) and lipogenesis-two crucial arms of cancer growth-AMPK also ensures viability by metabolic reprogramming in cancer cells. AMPK activation by two indirect AMPK agonists AICAR and metformin (now in over 50 clinical trials on cancer) has been correlated with reduced cancer cell proliferation and viability. Surprisingly, we found that compared with normal tissue, AMPK is constitutively activated in both human and mouse gliomas. Therefore, we questioned whether the antiproliferative actions of AICAR and metformin are AMPK independent. Both AMPK agonists inhibited proliferation, but through unique AMPK-independent mechanisms and both reduced tumor growth in vivo independent of AMPK. Importantly, A769662, a direct AMPK activator, had no effect on proliferation, uncoupling high AMPK activity from inhibition of proliferation. Metformin directly inhibited mTOR by enhancing PRAS40's association with RAPTOR, whereas AICAR blocked the cell cycle through proteasomal degradation of the G2M phosphatase cdc25c. Together, our results suggest that although AICAR and metformin are potent AMPK-independent antiproliferative agents, physiological AMPK activation in glioma may be a response mechanism to metabolic stress and anticancer agents.
Proceedings of the National Academy of Sciences 01/2014; 111(4):E435-44. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Activation of AMP-activated protein kinase (AMPK)-α2 protects the heart against pressure overload-induced heart failure in mice. Although metformin is a known activator of AMPK, it is unclear whether its cardioprotection acts independently of an AMPKα2-dependent pathway. Because the role of AMPKα1 stimulation on remodeling of failing hearts is poorly defined, we first studied the effects of disruption of both the AMPKα1 and AMPKα2 genes on the response to transverse aortic constriction-induced left ventricular (LV) hypertrophy and dysfunction in mice. AMPKα2 gene knockout significantly exacerbated the degree of transverse aortic constriction-induced LV hypertrophy and dysfunction, whereas AMPKα1 gene knockout had no effect on the degree of transverse aortic constriction-induced LV hypertrophy and dysfunction. Administration of metformin was equally effective in attenuating transverse aortic constriction-induced LV remodeling in both wild-type and AMPKα2 knockout mice, as evidenced by reduced LV and lung weights, a preserved LV ejection fraction, and reduced phosphorylation of mammalian target of rapamycin (p-mTOR(Ser2448)) and its downstream target p-p70S6K(Thr389). These data support the notion that activation of AMPKα1 plays a negligible role in protecting the heart against the adverse effects of chronic pressure overload, and that metformin protects against adverse remodeling through a pathway that seems independent of AMPKα2.
[Show abstract][Hide abstract] ABSTRACT: Metabolic stress, as well as several antidiabetic agents, increase hepatic nucleotide monophosphate levels (NMP), activate AMP-activated protein kinase (AMPK), and suppress glucose production. We tested the necessity of hepatic AMPK for the in vivo effects of an acute elevation in NMP on metabolism. AICAR (8mg·kg(-1)·min(-1))-euglycemic clamps were performed to elicit an increase in NMP in wild-type (α1α2(lox/lox)) and liver-specific AMPK-knockout mice (α1α2(lox/lox)+Albcre) in the presence of fixed glucose. Glucose kinetics were equivalent in 5hr fasted (Basal) α1α2(lox/lox) and α1α2(lox/lox)+Albcre mice. AMPK was not required for AICAR-mediated suppression of glucose production and increased glucose clearance. These results demonstrate that AMPK is unnecessary for normal basal glucose kinetics and AICAR-mediated inhibition of glucose production. Moreover, plasma fatty acids and triglycerides also decreased independently of hepatic AMPK during AICAR administration. Although the glucoregulatory effects of AICAR were shown to be independent of AMPK, these studies provide in vivo support for the AMPK-energy sensor paradigm. AICAR reduced hepatic energy charge (EC) by ~20% in α1α2(lox/lox) which was exacerbated by ~2-fold in α1α2(lox/lox)+Albcre. This corresponded to a ~6-fold rise in AMP/ATP in α1α2(lox/lox)+Albcre. Consistent with the effects on adenine nucleotides, maximal mitochondrial respiration was ~30% lower in α1α2(lox/lox)+Albcre than α1α2(lox/lox) livers. Mitochondrial oxidative phosphorylation efficiency was reduced by 25%. In summary, these results demonstrate that the NMP capacity to inhibit glucose production in vivo is independent of liver AMPK. In contrast, AMPK promotes mitochondrial function and protects against a more precipitous fall in ATP during AICAR administration.
Journal of Biological Chemistry 01/2014; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cardiac fibroblasts (CF) are crucial in left ventricular (LV) healing and remodelling after myocardial infarction (MI). They are typically activated into myofibroblasts that express alpha-smooth muscle actin (α-SMA) microfilaments and contribute to the formation of contractile and mature collagen scars that minimize the adverse dilatation of infarcted areas. CF predominantly express the α1 catalytic subunit of AMP-activated protein kinase (AMPKα1), while AMPKα2 is the major catalytic isoform in cardiomyocytes. AMPKα2 is known to protect the heart by preserving the energy charge of cardiac myocytes during injury, but whether AMPKα1 interferes with maladaptative heart responses remains unexplored. In this study, we investigated the role of AMPKα1 in modulating LV dilatation and CF fibrosis during post-MI remodelling. AMPKα1 knockout (KO) and wild type (WT) mice were subjected to permanent ligation of the left anterior descending coronary artery. The absence of AMPKα1 was associated with increased CF proliferation in infarcted areas, while expression of the myodifferentiation marker α-SMA was decreased. Faulty maturation of myofibroblasts might derive from severe down-regulation of the non-canonical transforming growth factor-beta1/p38 mitogen-activated protein kinase pathway in KO infarcts. In addition, lysyl oxidase (LOX) protein expression was dramatically reduced in the scar of KO hearts. Although infarct size was similar in AMPK-KO and WT hearts subjected to MI, these changes resulted in compromised scar contractility, defective scar collagen maturation, and exacerbated adverse remodelling, as indicated by increased LV diastolic dimension 30 days after MI. Our data genetically demonstrate the centrality of AMPKα1 in post-MI scar formation and highlight the specificity of this catalytic isoform in cardiac fibroblast/myofibroblast biology.
Journal of Molecular and Cellular Cardiology 01/2014; · 5.15 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK.
PLoS ONE 01/2014; 9(9):e106710. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aims
SIRT1 and AMP-activated protein kinase (AMPK) share common activators, actions and target molecules. Previous studies have suggested that a putative SIRT1-AMPK regulatory network could act as the prime initial sensor for calorie restriction-induced adaptations in skeletal muscle - the major site of insulin-stimulated glucose disposal. Our study aimed to investigate whether a feedback loop exists between AMPK and SIRT1 in skeletal muscle and how this may be involved glucose tolerance.
To investigate this we used skeletal muscle specific AMPKα1/2 knockout mice (AMPKα1/2 −/−) fed ad libitum (AL) or a 30% calorie restricted (CR) diet and L6 rat myoblasts incubated with SIRT1 inhibitor (EX527).
CR-AMPKα1/2−/− displayed impaired glucose tolerance (*p < 0.05), in association with down-regulated SIRT1 and PGC-1α expression (< 300% vs. CR-WT, ± ± p < 0.01). Moreover, AMPK activity was decreased following SIRT1 inhibition in L6 cells (~ 0.5-fold vs. control, *p < 0.05).
This study demonstrates that skeletal muscle-specific AMPK deficiency impairs the beneficial effects of CR on glucose tolerance, and that these effects may be dependent on reduced SIRT1 levels.
[Show abstract][Hide abstract] ABSTRACT: LKB1 is a master kinase that regulates metabolism and growth through adenosine monophosphate-activated protein kinase (AMPK) and 12 other closely related kinases. Liver-specific ablation of LKB1 causes increased glucose production in hepatocytes in vitro and hyperglycaemia in fasting mice in vivo. Here we report that the salt-inducible kinases (SIK1, 2 and 3), members of the AMPK-related kinase family, play a key role as gluconeogenic suppressors downstream of LKB1 in the liver. The selective SIK inhibitor HG-9-91-01 promotes dephosphorylation of transcriptional co-activators CRTC2/3 resulting in enhanced gluconeogenic gene expression and glucose production in hepatocytes, an effect that is abolished when an HG-9-91-01-insensitive mutant SIK is introduced or LKB1 is ablated. Although SIK2 was proposed as a key regulator of insulin-mediated suppression of gluconeogenesis, we provide genetic evidence that liver-specific ablation of SIK2 alone has no effect on gluconeogenesis and insulin does not modulate SIK2 phosphorylation or activity. Collectively, we demonstrate that the LKB1-SIK pathway functions as a key gluconeogenic gatekeeper in the liver.
[Show abstract][Hide abstract] ABSTRACT: Metformin is currently the drug of first choice for the treatment of type 2 diabetes. However, although prescribed since the end of the 1950s, the mechanism of action of metformin remains as yet incompletely understood but recent work has unveiled novel and surprising properties. Epidemiological reports have suggested that metformin protects against heart failure and has antitumor properties independent of its anti-hyperglycemic effect. Here, we review the proposed mechanisms for metformin action in diabetes, cardiovacular diseases and cancer.
Medecine sciences: M/S 01/2014; 30(1):82-92. · 0.56 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Phagocytosis of apoptotic cells by myeloid cells has been implicated in the maintenance of immune homeostasis. In this study, we found that T cell immunoglobulin- and mucin domain-containing molecule-4 (TIM-4) repressed tumor-specific immunity triggered by chemotherapy-induced tumor cell death. TIM-4 was found to be highly expressed on tumor-associated myeloid cells such as macrophages (TAMs) and dendritic cells (TADCs) and danger-associated molecular patterns (DAMPs) released from chemotherapy-damaged tumor cells induced TIM-4 on tumor-associated myeloid cells recruited from bone marrow-derived precursors. TIM-4 directly interacted with AMPKα1 and activated autophagy-mediated degradation of ingested tumors, leading to reduced antigen presentation and impaired CTL responses. Consistently, blockade of the TIM-4-AMPKα1-autophagy pathway augmented the antitumor effect of chemotherapeutics by enhancing tumor-specific CTL responses. Our finding provides insight into the immune tolerance mediated by phagocytosis of dying cells, and targeting of the TIM-4-AMPKα1 interaction constitutes a unique strategy for augmenting antitumor immunity and improving cancer chemotherapy.
[Show abstract][Hide abstract] ABSTRACT: The purpose of this study was to determine whether AMPK influences the survival of primary cultures of mouse proximal tubular (MPT) cells subjected to metabolic stress. Previous studies, using an immortalized MPT cell line, suggest that AMPK is activated during metabolic stress, and ameliorates stress-induced apoptosis of these cells.
Primary MPT cells were cultured from AMPK knockout (KO) mice lacking either the alpha1 or the alpha2 isoform of the catalytic domain of AMPK. MPT cells were subjected to ATP depletion using antimycin A.
Surprisingly, there was no difference in the amount of death induced by metabolic stress of MPT cells from either type of AMPK KO mice compared to its WT control. Moreover, inhibition of the activity of the alpha1 isoform in primary MPT cells from alpha2-/- mice (pharmacologically, via compound C) or inhibition of the alpha2 isoform in primary MPT cells from alpha1-/- mice (molecularly, via knockdown) both decreased cell viability equivalently in response to metabolic stress. The explanation for this unexpected result appears to be an adaptive increase in expression of the non-deleted alphaisoform. As a consequence, total As a consequence-domain expression (i.e. alpha1 + alpha2), is comparable in kidney cortex and in cultured MPT cells derived from either type of KO mouse versus its WT control. Importantly, each alphaisoform appears able to compensate fully for the absence of the other, with respect to both the phosphorylation of downstream targets of AMPK and the amelioration of stress-induced cell death.
These findings not only confirm the importance of AMPK as a pro-survival kinase in MPT cells during metabolic stress, but also show, for the first time, that each of the two alpha-isoforms can substitute for the other in MPT cells from AMPK KO mice with regard to amelioration of stress-induced loss of cell viability.