Disruption of GCN2 Pathway Aggravates Vascular and Parenchymal Remodeling during Pulmonary Fibrosis.

Pulmonary fibrosis (PF) and pulmonary hypertension (PH) are chronic diseases of the pulmonary parenchyma and circulation, respectively, which may coexist, but underlying mechanisms remain elusive. Mutations in the GCN2 (general control nonderepressible 2) gene (EIF2AK4 [eukaryotic translation initiation factor 2 alpha kinase 4]) were recently associated with pulmonary veno-occlusive disease. The aim of this study is to explore the involvement of the GCN2/eIF2α (eukaryotic initiation factor 2α) pathway in the development of PH during PF, in both human disease and in a laboratory animal model. Lung tissue from patients with PF with or without PH was collected at the time of lung transplantation, and control tissue was obtained from tumor resection surgery. Experimental lung disease was induced in either male wild-type or EIF2AK4-mutated Sprague-Dawley rats, randomly receiving a single intratracheal instillation of bleomycin or saline. Hemodynamic studies and organ collection were performed 3 weeks after instillation. Only significant results (P < 0.05) are presented. In PF lung tissue, GCN2 protein expression was decreased compared with control tissue. GCN2 expression was reduced in CD31+ endothelial cells. In line with human data, GCN2 protein expression was decreased in the lung of bleomycin rats compared with saline. EIF2AK4-mutated rats treated with bleomycin showed increased parenchymal fibrosis (hydroxyproline concentrations) and vascular remodeling (media wall thickness) as well as increased right ventricular systolic pressure compared with wild-type animals. Our data show that GCN2 is dysregulated in both humans and in an animal model of combined PF and PH. The possibility of a causative implication of GCN2 dysregulation in PF and/or PH development should be further studied.

Cryogenic contrast-enhanced microCT enables nondestructive 3D quantitative histopathology of soft biological tissues.

Biological tissues comprise a spatially complex structure, composition and organization at the microscale, named the microstructure. Given the close structure-function relationships in tissues, structural characterization is essential to fully understand the functioning of healthy and pathological tissues, as well as the impact of possible treatments. Here, we present a nondestructive imaging approach to perform quantitative 3D histo(patho)logy of biological tissues, termed Cryogenic Contrast-Enhanced MicroCT (cryo-CECT). By combining sample staining, using an X-ray contrast-enhancing staining agent, with freezing the sample at the optimal freezing rate, cryo-CECT enables 3D visualization and structural analysis of individual tissue constituents, such as muscle and collagen fibers. We applied cryo-CECT on murine hearts subjected to pressure overload following transverse aortic constriction surgery. Cryo-CECT allowed to analyze, in an unprecedented manner, the orientation and diameter of the individual muscle fibers in the entire heart, as well as the 3D localization of fibrotic regions within the myocardial layers. We foresee further applications of cryo-CECT in the optimization of tissue/food preservation and donor banking, showing that cryo-CECT also has clinical and industrial potential.

At the crossroads of fertility and metabolism: the importance of AMPK-dependent signaling in female infertility associated with hyperandrogenism.

STUDY QUESTION: What biological processes are linked to the signaling of the energy sensor 5'-AMP-activated protein kinase (AMPK) in mouse and human granulosa cells (GCs)? SUMMARY ANSWER: The lack of α1AMPK in GCs impacted cell cycle, adhesion, lipid metabolism and induced a hyperandrogenic response. WHAT IS KNOWN ALREADY: AMPK is expressed in the ovarian follicle, and its activation by pharmacological medications, such as metformin, inhibits the production of steroids. Polycystic ovary syndrome (PCOS) is responsible for infertility in approximately 5-20% of women of childbearing age and possible treatments include reducing body weight, improving lifestyle and the administration of a combination of drugs to improve insulin resistance, such as metformin. STUDY DESIGN, SIZE, DURATION: AMPK signaling was evaluated by analyzing differential gene expression in immortalized human granulosa cells (KGNs) with and without silencing α1AMPK using CRISPR/Cas9. In vivo studies included the use of a α1AMPK knock-out mouse model to evaluate the role of α1AMPK in folliculogenesis and fertility. Expression of α1AMPK was evaluated in primary human granulosa-luteal cells retrieved from women undergoing IVF with and without a lean PCOS phenotype (i.e. BMI: 18-25 kg/m2). PARTICIPANTS/MATERIALS, SETTING, METHODS: α1AMPK was disrupted in KGN cells and a transgenic mouse model. Cell viability, proliferation and metabolism were evaluated. Androgen production was evaluated by analyzing protein levels of relevant enzymes in the steroid pathway by western blots, and steroid levels obtained from in vitro and in vivo models by mass spectrometry. Differential gene expression in human GC was obtained by RNA sequencing. Analysis of in vivo murine folliculogenesis was performed by histology and immunochemistry, including evaluation of the anti-Müllerian hormone (AMH) marker. The α1AMPK gene expression was evaluated by quantitative RT-PCR in primary GCs obtained from women with the lean PCOS phenotype (n = 8) and without PCOS (n = 9). MAIN RESULTS AND THE ROLE OF CHANCE: Silencing of α1AMPK in KGN increased cell proliferation (P < 0.05 versus control, n = 4), promoted the use of fatty acids over glucose, and induced a hyperandrogenic response resulting from upregulation of two of the enzymes involved in steroid production, namely 3ß-hydroxysteroid dehydrogenase (3ßHSD) and P450 side-chain cleavage enzyme (P450scc) (P < 0.05, n = 3). Female mice deficient in α1AMPK had a 30% decrease in their ovulation rate (P < 0.05, n = 7) and litter size, a hyperandrogenic response (P < 0.05, n = 7) with higher levels of 3ßHSD and p450scc levels in the ovaries, and an increase in the population of antral follicles (P < 0.01, n = 10) compared to controls. Primary GCs from lean women with PCOS had lower α1AMPK mRNA expression levels than the control group (P < 0.05, n = 8-9). LARGE SCALE DATA: The FastQ files and metadata were submitted to the European Nucleotide Archive (ENA) at EMBL-EBI under accession number PRJEB46048. LIMITATIONS, REASONS FOR CAUTION: The human KGN is a not fully differentiated, transformed cell line. As such, to confirm the role of AMPK in GC and the PCOS phenotype, this model was compared to two others: an α1AMPK transgenic mouse model and primary differentiated granulosa-lutein cells from non-obese women undergoing IVF (with and without PCOS). A clear limitation is the small number of patients with PCOS utilized in this study and that the collection of human GCs was performed after hormonal stimulation. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal that AMPK is directly involved in steroid production in human GCs. In addition, AMPK signaling was associated with other processes frequently reported as dysfunctional in PCOS models, such as cell adhesion, lipid metabolism and inflammation. Silencing of α1AMPK in KGN promoted folliculogenesis, with increases in AMH. Evaluating the expression of the α1AMPK subunit could be considered as a marker of interest in infertility cases related to hormonal imbalances and metabolic disorders, including PCOS. STUDY FUNDING/COMPETING INTEREST(S): This study was financially supported by the Institut National de la Recherche Agronomique (INRA) and the national programme « FERTiNERGY ¼ funded by the French National Research Agency (ANR). The authors report no intellectual or financial conflicts of interest related to this work. R.K. is identified as personnel of the International Agency for Research on Cancer/World Health Organization. R.K. alone is responsible for the views expressed in this article and she does not necessarily represent the decisions, policy or views of the International Agency for Research on Cancer/World Health Organization. TRIAL REGISTRATION NUMBER: N/A.

AMP-activated protein kinase: A remarkable contributor to preserve a healthy heart against ROS injury.

Heart failure is one of the leading causes of death and disability worldwide. Left ventricle remodeling, fibrosis, and ischemia/reperfusion injury all contribute to the deterioration of cardiac function and predispose to the onset of heart failure. Adenosine monophosphate-activated protein kinase (AMPK) is the universally recognized energy sensor which responds to low ATP levels and restores cellular metabolism. AMPK activation controls numerous cellular processes and, in the heart, it plays a pivotal role in preventing onset and progression of disease. Excessive reactive oxygen species (ROS) generation, known as oxidative stress, can activate AMPK, conferring an additional role of AMPK as a redox-sensor. In this review, we discuss recent insights into the crosstalk between ROS and AMPK. We describe the molecular mechanisms by which ROS activate AMPK and how AMPK signaling can further prevent heart failure progression. Ultimately, we review the potential therapeutic approaches to target AMPK for the treatment of cardiovascular disease and prevention of heart failure.

Myocardial glucotoxicity: Mechanisms and potential therapeutic targets.

Besides coronary artery disease, which remains the main cause of heart failure in patients with diabetes, factors independent of coronary artery disease are involved in the development of heart failure in the onset of what is called diabetic cardiomyopathy. Among them, hyperglycaemia - a hallmark of type 2 diabetes - has both acute and chronic deleterious effects on myocardial function, and clearly participates in the establishment of diabetic cardiomyopathy. In the present review, we summarize the cellular and tissular events that occur in a heart exposed to hyperglycaemia, and depict the complex molecular mechanisms proposed to be involved in glucotoxicity. Finally, from a more translational perspective, different therapeutic strategies targeting hyperglycaemia-mediated molecular mechanisms will be detailed.

Platelet Acetyl-CoA Carboxylase Phosphorylation: A Risk Stratification Marker That Reveals Platelet-Lipid Interplay in Coronary Artery Disease Patients.

Adenosine monophosphate-activated protein kinase (AMPK) acetyl-CoA carboxylase (ACC) signaling is activated in platelets by atherogenic lipids, particularly by oxidized low-density lipoproteins, through a CD36-dependent pathway. More interestingly, increased platelet AMPK-induced ACC phosphorylation is associated with the severity of coronary artery calcification as well as acute coronary events in coronary artery disease patients. Therefore, AMPK-induced ACC phosphorylation is a potential marker for risk stratification in suspected coronary artery disease patients. The inhibition of ACC resulting from its phosphorylation impacts platelet lipid content by down-regulating triglycerides, which in turn may affect platelet function.

Clinical Protocol to Prevent Thrombogenic Effect of Liver-Derived Mesenchymal Cells for Cell-Based Therapies.

The efficacy of mesenchymal stem cell infusion is currently tested in numerous clinical trials. However, therapy-induced thrombotic consequences have been reported in several patients. The aim of this study was to optimize protocols for heterologous human adult liver-derived progenitor cell (HHALPC) infusion, in order to eliminate acute thrombogenesis in liver-based metabolic or acute decompensated cirrhotic (ADC) patients. In rats, thrombotic effects were absent when HHALPCs were infused at low cell dose (5 × 106 cells/kg), or at high cell dose (5 × 107 cells/kg) when combined with anticoagulants. When HHALPCs were exposed to human blood in a whole blood perfusion assay, blocking of the tissue factor (TF) coagulation pathway suppressed fibrin generation and platelet activation. In a Chandler tubing loop model, HHALPCs induced less explosive activation of coagulation with blood from ADC patients, when compared to blood from healthy controls, without alterations in coagulation factor levels other than fibrinogen. These studies confirm a link between TF and thrombogenesis, when TF-expressing cells are exposed to human blood. This phenomenon however, could be controlled using either a low, or a high cell dose combined with anticoagulants. In clinical practice, this points to the suitability of a low HHALPC dose infusion to cirrhotic patients, provided that platelet and fibrinogen levels are monitored.

AMP-Activated Protein Kinase and O-GlcNAcylation, Two Partners Tightly Connected to Regulate Key Cellular Processes.

The AMP-activated protein kinase (AMPK) is an important cellular energy sensor. Its activation under energetic stress is known to activate energy-producing pathways and to inactivate energy-consuming pathways, promoting ATP preservation and cell survival. AMPK has been shown to play protective role in many pathophysiological processes including cardiovascular diseases, diabetes, and cancer. Its action is multi-faceted and comprises short-term regulation of enzymes by direct phosphorylation as well as long-term adaptation via control of transcription factors and cellular events such as autophagy. During the last decade, several studies underline the particular importance of the interaction between AMPK and the post-translational modification called O-GlcNAcylation. O-GlcNAcylation means the O-linked attachment of a single N-acetylglucosamine moiety on serine or threonine residues. O-GlcNAcylation plays a role in multiple physiological cellular processes but is also associated with the development of various diseases. The first goal of the present review is to present the tight molecular relationship between AMPK and enzymes regulating O-GlcNAcylation. We then draw the attention of the reader on the putative importance of this interaction in different pathophysiological events.

Sodium-myoinositol cotransporter-1, SMIT1, mediates the production of reactive oxygen species induced by hyperglycemia in the heart.

Hyperglycemia (HG) stimulates the production of reactive oxygen species in the heart through activation of NADPH oxidase 2 (NOX2). This production is independent of glucose metabolism but requires sodium/glucose cotransporters (SGLT). Seven SGLT isoforms (SGLT1 to 6 and sodium-myoinositol cotransporter-1, SMIT1) are known, although their expression and function in the heart remain elusive. We investigated these 7 isoforms and found that only SGLT1 and SMIT1 were expressed in mouse, rat and human hearts. In cardiomyocytes, galactose (transported through SGLT1) did not activate NOX2. Accordingly, SGLT1 deficiency did not prevent HG-induced NOX2 activation, ruling it out in the cellular response to HG. In contrast, myo-inositol (transported through SMIT1) reproduced the toxic effects of HG. SMIT1 overexpression exacerbated glucotoxicity and sensitized cardiomyocytes to HG, whereas its deletion prevented HG-induced NOX2 activation. In conclusion, our results show that heart SMIT1 senses HG and triggers NOX2 activation. This could participate in the redox signaling in hyperglycemic heart and contribute to the pathophysiology of diabetic cardiomyopathy.

Hypothermic continuous machine perfusion enables preservation of energy charge and functional recovery of heart grafts in an ex vivo model of donation following circulatory death.

OBJECTIVES: Cardiac transplantation using hearts from donors after circulatory death (DCD) is critically limited by the unavoidable warm ischaemia and its related unpredictable graft function. Inasmuch as hypothermic machine perfusion (MP) has been shown to improve heart preservation, we hypothesized that MP could enable the use of DCD hearts for transplantation. METHODS: We recovered 16 pig hearts following anoxia-induced cardiac arrest and cardioplegia. Grafts were randomly assigned to two different groups of 4-h preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems©, Itasca, Il). After preservation, the grafts were reperfused ex vivo using the Langendorff method for 60 min. Energetic charge was quantified at baseline, post-preservation and post-reperfusion by measuring lactate and high-energy phosphate levels. Left ventricular contractility parameters were assessed both in vivo prior to ischaemia and ex vivo during reperfusion. RESULTS: Following preservation, the hearts that were preserved using CS exhibited higher lactate levels (57.1 ± 23.7 vs 21.4 ± 12.2 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate ratio (0.53 ± 0.25 vs 0.11 ± 0.11; P < 0.001) and lower phosphocreatine/creatine ratio (9.7 ± 5.3 vs 25.2 ± 11; P < 0.001) in comparison with the MP hearts. Coronary flow was similar in both groups during reperfusion (107 ± 9 vs 125 ± 9 ml/100 g/min heart; P = ns). Contractility decreased in the CS group, yet remained well preserved in the MP group. CONCLUSION: MP preservation of DCD hearts results in improved preservation of the energy and improved functional recovery of heart grafts compared with CS.

Hypothermic continuous machine perfusion improves metabolic preservation and functional recovery in heart grafts.

The number of heart transplants is decreasing due to organ shortage, yet the donor pool could be enlarged by improving graft preservation. Hypothermic machine perfusion (MP) has been shown to improve kidney, liver, or lung graft preservation. Sixteen pig hearts were recovered following cardioplegia and randomized to two different groups of 4-hour preservation using either static cold storage (CS) or MP (Modified LifePort© System, Organ Recovery Systems, Itasca, Il). The grafts then underwent reperfusion on a Langendorff for 60 min. Energetic metabolism was quantified at baseline, postpreservation, and postreperfusion by measuring lactate and high-energy phosphates. The contractility index (CI) was assessed both in vivo prior to cardioplegia and during reperfusion. Following reperfusion, the hearts preserved using CS exhibited higher lactate levels (56.63 ± 23.57 vs. 11.25 ± 3.92 µmol/g; P < 0.001), increased adenosine monophosphate/adenosine triphosphate (AMP/ATP) ratio (0.4 ± 0.23 vs. 0.04 ± 0.04; P < 0.001), and lower phosphocreatine/creatine (PCr/Cr) ratio (33.5 ± 12.6 vs. 55.3 ± 5.8; P <0.001). Coronary flow was similar in both groups during reperfusion (107 ± 9 vs. 125 + /-9 ml/100 g/min heart; P = ns). CI decreased in the CS group, yet being well-preserved in the MP group. Compared with CS, MP resulted in improved preservation of the energy state and more successful functional recovery of heart graft.

AMPK activation by glucagon-like peptide-1 prevents NADPH oxidase activation induced by hyperglycemia in adult cardiomyocytes.

Exposure of cardiomyocytes to high glucose concentrations (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase (NOX2). NOX2 activation is triggered by enhanced glucose transport through a sodium-glucose cotransporter (SGLT) but not by a stimulation of glucose metabolism. The aim of this work was to identify potential therapeutic approaches to counteract this glucotoxicity. In cultured adult rat cardiomyocytes incubated with 21 mM glucose (HG), AMP-activated protein kinase (AMPK) activation by A769662 or phenformin nearly suppressed ROS production. Interestingly, glucagon-like peptide 1 (GLP-1), a new antidiabetic drug, concomitantly induced AMPK activation and prevented the HG-mediated ROS production (maximal effect at 100 nM). α2-AMPK, the major isoform expressed in cardiomyocytes (but not α1-AMPK), was activated in response to GLP-1. Anti-ROS properties of AMPK activators were not related to changes in glucose uptake or glycolysis. Using in situ proximity ligation assay, we demonstrated that AMPK activation prevented the HG-induced p47phox translocation to caveolae, whatever the AMPK activators used. NOX2 activation by either α-methyl-d-glucopyranoside, a glucose analog transported through SGLT, or angiotensin II was also counteracted by GLP-1. The crucial role of AMPK in limiting HG-mediated NOX2 activation was demonstrated by overexpressing a constitutively active form of α2-AMPK using adenoviral infection. This overexpression prevented NOX2 activation in response to HG, whereas GLP-1 lost its protective action in α2-AMPK-deficient mouse cardiomyocytes. Under HG, the GLP-1/AMPK pathway inhibited PKC-ß2 phosphorylation, a key element mediating p47phox translocation. In conclusion, GLP-1 induces α2-AMPK activation and blocks HG-induced p47phox translocation to the plasma membrane, thereby preventing glucotoxicity.

A-769662 potentiates the effect of other AMP-activated protein kinase activators on cardiac glucose uptake.

AMP-activated protein kinase (AMPK), a key cellular sensor of energy, regulates metabolic homeostasis and plays a protective role in the ischemic or diabetic heart. Stimulation of cardiac glucose uptake contributes to this AMPK-mediated protection. The small-molecule AMPK activator A-769662, which binds and directly activates AMPK, has recently been characterized. A-769662-dependent AMPK activation protects the heart against an ischemia-reperfusion episode but is unable to stimulate skeletal muscle glucose uptake. Here, we tried to reconcile these conflicting findings by investigating the impact of A-769662 on cardiac AMPK signaling and glucose uptake. We showed that A-769662 promoted AMPK activation, resulting in the phosphorylation of several downstream targets, but was incapable of stimulating glucose uptake in cultured cardiomyocytes and the perfused heart. The lack of glucose uptake stimulation can be explained by A-769662's narrow specificity, since it selectively activates cardiac AMPK heterotrimeric complexes containing α2/ß1-subunits, the others being presumably required for this metabolic outcome. However, when combined with classical AMPK activators, such as metformin, phenformin, oligomycin, or hypoxia, which impact AMPK heterotrimers more broadly via elevation of cellular AMP levels, A-769662 induced more profound AMPK phosphorylation and subsequent glucose uptake stimulation. The synergistic effect of A-769662 under such ischemia-mimetic conditions protected cardiomyocytes against ROS production and cell death. In conclusion, despite the fact that A-769662 activates AMPK, it alone does not significantly stimulate glucose uptake. However, strikingly, its ability of potentiating the action on other AMPK activators makes it a potentially useful participant in the protective role of AMPK in the heart.

Role of AMP-activated protein kinase in regulating hypoxic survival and proliferation of mesenchymal stem cells.

AIMS: Mesenchymal stem cells (MSCs) are widely used for cell therapy, particularly for the treatment of ischaemic heart disease. Mechanisms underlying control of their metabolism and proliferation capacity, critical elements for their survival and differentiation, have not been fully characterized. AMP-activated protein kinase (AMPK) is a key regulator known to metabolically protect cardiomyocytes against ischaemic injuries and, more generally, to inhibit cell proliferation. We hypothesized that AMPK plays a role in control of MSC metabolism and proliferation. METHODS AND RESULTS: MSCs isolated from murine bone marrow exclusively expressed the AMPKα1 catalytic subunit. In contrast to cardiomyocytes, a chronic exposure of MSCs to hypoxia failed to induce cell death despite the absence of AMPK activation. This hypoxic tolerance was the consequence of a preference of MSC towards glycolytic metabolism independently of oxygen availability and AMPK signalling. On the other hand, A-769662, a well-characterized AMPK activator, was able to induce a robust and sustained AMPK activation. We showed that A-769662-induced AMPK activation inhibited MSC proliferation. Proliferation was not arrested in MSCs derived from AMPKα1-knockout mice, providing genetic evidence that AMPK is essential for this process. Among AMPK downstream targets proposed to regulate cell proliferation, we showed that neither the p70 ribosomal S6 protein kinase/eukaryotic elongation factor 2-dependent protein synthesis pathway nor p21 was involved, whereas p27 expression was increased by A-769662. Silencing p27 expression partially prevented the A-769662-dependent inhibition of MSC proliferation. CONCLUSION: MSCs resist hypoxia independently of AMPK whereas chronic AMPK activation inhibits MSC proliferation, p27 being involved in this regulation.

Redundant control of migration and adhesion by ERM proteins in vascular smooth muscle cells.

Ezrin, radixin, and moesin possess a very similar structure with a C-terminal actin-binding domain and a N-terminal FERM interacting domain. They are known to be involved in cytoskeleton organization in several cell types but their function in vascular smooth muscle cells (VSMC) is still unknown. The aim of this study was to investigate the role of ERM proteins in cell migration induced by PDGF, a growth factor involved in pathophysiological processes like angiogenesis or atherosclerosis. We used primary cultured VSMC obtained from rat aorta, which express the three ERM proteins. Simultaneous depletion of the three ERM proteins with specific siRNAs abolished the effects of PDGF on cell architecture and migration and markedly increased cell adhesion and focal adhesion size, while these parameters were only slightly affected by depletion of ezrin, radixin or moesin alone. Rac1 activation, cell proliferation, and Ca(2+) signal in response to PDGF were unaffected by ERM depletion. These results indicate that ERM proteins exert a redundant control on PDGF-induced VSMC migration by regulating focal adhesion turn-over and cell adhesion to substrate.

Connection between cardiac vascular permeability, myocardial edema, and inflammation during sepsis: role of the α1AMP-activated protein kinase isoform.

OBJECTIVE: As adenosine monophosphate (AMP)-activated protein kinase both controls cytoskeleton organization in endothelial cells and exerts anti-inflammatory effects, we here postulated that it could influence vascular permeability and inflammation, thereby counteracting cardiac wall edema during sepsis. DESIGN: Controlled animal study. SETTINGS: University research laboratory. SUBJECTS: C57BL/6J, α1AMPK, and α1AMPK mice. INTERVENTION: Sepsis was triggered in vivo using a sublethal injection of lipopolysaccharide (O55B5, 10 mg/kg), inducing systolic left ventricular dysfunction. Left ventricular function, edema, vascular permeability, and inflammation were assessed in vivo in both wild-type mice (α1AMPK) and α1AMP-activated protein kinase-deficient mice (α1AMPK). The 5-aminoimidazole-4-carboxamide riboside served to study the impact of AMP-activated protein kinase activation on vascular permeability in vivo. The integrity of endothelial cell monolayers was also examined in vitro after lipopolysaccharide challenge in the presence of aminoimidazole-4-carboxamide riboside and/or after α1AMP-activated protein kinase silencing. MEASUREMENTS AND MAIN RESULTS: α1AMP-activated protein kinase deficiency dramatically impaired tolerance to lipopolysaccharide challenge. Indeed, α1AMPK exhibited heightened cardiac vascular permeability after lipopolysaccharide challenge compared with α1AMPK. Consequently, an increase in left ventricular mass corresponding to exaggerated wall edema occurred in α1AMPK, without any further decrease in systolic function. Mechanistically, the lipopolysaccharide-induced α1AMPK cardiac phenotype could not be attributed to major changes in the systemic inflammatory response but was due to an increased disruption of interendothelial tight junctions. Accordingly, AMP-activated protein kinase activation by aminoimidazole-4-carboxamide riboside counteracted lipopolysaccharide-induced hyperpermeability in wild-type mice in vivo as well as in endothelial cells in vitro. This effect was associated with a potent protection of zonula occludens-1 linear border pattern in endothelial cells. CONCLUSIONS: Our results demonstrate for the first time the involvement of a signaling pathway in the control of left ventricular wall edema during sepsis. AMP-activated protein kinase exerts a protective action through the preservation of interendothelial tight junctions. Interestingly, exaggerated left ventricular wall edema was not coupled with aggravated systolic dysfunction. However, it could contribute to diastolic dysfunction in patients with sepsis.

NADPH oxidase activation by hyperglycaemia in cardiomyocytes is independent of glucose metabolism but requires SGLT1.

AIMS: Exposure to high glucose (HG) stimulates reactive oxygen species (ROS) production by NADPH oxidase in cardiomyocytes, but the underlying mechanism remains elusive. In this study, we have dissected the link between glucose transport and metabolism and NADPH oxidase activation under hyperglycaemic conditions. METHODS AND RESULTS: Primary cultures of adult rat cardiomyocytes were exposed to HG concentration (HG, 21 mM) and compared with the normal glucose level (LG, 5 mM). HG exposure activated Rac1GTP and induced p47phox translocation to the plasma membrane, resulting in NADPH oxidase (NOX2) activation, increased ROS production, insulin resistance, and eventually cell death. Comparison of the level of O-linked N-acetylglucosamine (O-GlcNAc) residues in LG- and HG-treated cells did not reveal any significant difference. Inhibition of the pentose phosphate pathway (PPP) by 6-aminonicotinamide counteracted ROS production in response to HG but did not prevent Rac-1 upregulation and p47phox translocation leading to NOX2 activation. Modulation of glucose uptake barely affected oxidative stress and toxicity induced by HG. More interestingly, non-metabolizable glucose analogues (i.e. 3-O-methyl-D-glucopyranoside and α-methyl-D-glucopyranoside) reproduced the toxic effect of HG. Inhibition of the sodium/glucose cotransporter SGLT1 by phlorizin counteracted HG-induced NOX2 activation and ROS production. CONCLUSION: Increased glucose metabolism by itself does not trigger NADPH oxidase activation, although PPP is required to provide NOX2 with NADPH and to produce ROS. NOX2 activation results from glucose transport through SGLT1, suggesting that an extracellular metabolic signal transduces into an intracellular ionic signal.

Inhibition of the mTOR/p70S6K pathway is not involved in the insulin-sensitizing effect of AMPK on cardiac glucose uptake.

The AMP-activated protein kinase (AMPK) is known to increase cardiac insulin sensitivity on glucose uptake. AMPK also inhibits the mammalian target of rapamycin (mTOR)/p70 ribosomal S6 kinase (p70S6K) pathway. Once activated by insulin, mTOR/p70S6K phosphorylates insulin receptor substrate-1 (IRS-1) on serine residues, resulting in its inhibition and reduction of insulin signaling. AMPK was postulated to act on insulin by inhibiting this mTOR/p70S6K-mediated negative feedback loop. We tested this hypothesis in cardiomyocytes. The stimulation of glucose uptake by AMPK activators and insulin correlated with AMPK and protein kinase B (PKB/Akt) activation, respectively. Both treatments induced the phosphorylation of Akt substrate 160 (AS160) known to control glucose uptake. Together, insulin and AMPK activators acted synergistically to induce PKB/Akt overactivation, AS160 overphosphorylation, and glucose uptake overstimulation. This correlated with p70S6K inhibition and with a decrease in serine phosphorylation of IRS-1, indicating the inhibition of the negative feedback loop. We used the mTOR inhibitor rapamycin to confirm these results. Mimicking AMPK activators in the presence of insulin, rapamycin inhibited p70S6K and reduced IRS-1 phosphorylation on serine, resulting in the overphosphorylation of PKB/Akt and AS160. However, rapamycin did not enhance the insulin-induced stimulation of glucose uptake. In conclusion, although the insulin-sensitizing effect of AMPK on PKB/Akt is explained by the inhibition of the insulin-induced negative feedback loop, its effect on glucose uptake is independent of this mechanism. This disconnection revealed that the PKB/Akt/AS160 pathway does not seem to be the rate-limiting step in the control of glucose uptake under insulin treatment.

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells.

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca(2+)-dependent AMPK activation via calmodulin-dependent protein kinase kinase-beta(CaMKKbeta), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKbeta inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase.

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.