Platelet lipidomics and de novo lipogenesis: impact on health and disease.

PURPOSE OF REVIEW: Lipids play vital roles in platelet structure, signaling, and metabolism. In addition to capturing exogenous lipids, platelets possess the capacity for de novo lipogenesis, regulated by acetyl-coA carboxylase 1 (ACC1). This review aims to cover the critical roles of platelet de novo lipogenesis and lipidome in platelet production, function, and diseases. RECENT FINDINGS: Upon platelet activation, approximately 20% of the platelet lipidome undergoes significant modifications, primarily affecting arachidonic acid-containing species. Multiple studies emphasize the impact of de novo lipogenesis, with ACC1 as key player, on platelet functions. Mouse models suggest the importance of the AMPK-ACC1 axis in regulating platelet membrane arachidonic acid content, associated with TXA2 secretion, and thrombus formation. In human platelets, ACC1 inhibition leads to reduced platelet reactivity. Remodeling of the platelet lipidome, alongside with de novo lipogenesis, is also crucial for platelet biogenesis. Disruptions in the platelet lipidome are observed in various pathological conditions, including cardiovascular and inflammatory diseases, with associations between these alterations and shifts in platelet reactivity highlighted. SUMMARY: The platelet lipidome, partially regulated by ACC-driven de novo lipogenesis, is indispensable for platelet production and function. It is implicated in various pathological conditions involving platelets.

Fibroblasts and platelets: a face-to-face dialogue at the heart of cardiac fibrosis.

Myocardial fibrosis is a feature found in most cardiac diseases and a key element contributing to heart failure and its progression. It has therefore become a subject of particular interest in cardiac research. Mechanisms leading to pathological cardiac remodeling and heart failure are diverse, including effects on cardiac fibroblasts, the main players in cardiac extracellular matrix synthesis, but also on cardiomyocytes, immune cells, endothelial cells, and more recently, platelets. Although transforming growth factor-ß (TGF-ß) is a primary regulator of fibrosis development, the cellular and molecular mechanisms that trigger its activation after cardiac injury remain poorly understood. Different types of anti-TGF-ß drugs have been tested for the treatment of cardiac fibrosis and have been associated with side effects. Therefore, a better understanding of these mechanisms is of great clinical relevance and could allow us to identify new therapeutic targets. Interestingly, it has been shown that platelets infiltrate the myocardium at an early stage after cardiac injury, producing large amounts of cytokines and growth factors. These molecules can directly or indirectly regulate cells involved in the fibrotic response, including cardiac fibroblasts and immune cells. In particular, platelets are known to be a major source of TGF-ß1. In this review, we have provided an overview of the classical cellular effectors involved in the pathogenesis of cardiac fibrosis, focusing on the emergent role of platelets, while discussing opportunities for novel therapeutic interventions.

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.

Enhanced protein acetylation initiates fatty acid-mediated inhibition of cardiac glucose transport.

Fatty acids (FAs) rapidly and efficiently reduce cardiac glucose uptake in the Randle cycle or glucose-FA cycle. This fine-tuned physiological regulation is critical to allow optimal substrate allocation during fasted and fed states. However, the mechanisms involved in the direct FA-mediated control of glucose transport have not been totally elucidated yet. We previously reported that leucine and ketone bodies, other cardiac substrates, impair glucose uptake by increasing global protein acetylation from acetyl-CoA. As FAs generate acetyl-CoA as well, we postulated that protein acetylation is enhanced by FAs and participates in their inhibitory action on cardiac glucose uptake. Here, we demonstrated that both palmitate and oleate promoted a rapid increase in protein acetylation in primary cultured adult rat cardiomyocytes, which correlated with an inhibition of insulin-stimulated glucose uptake. This glucose absorption deficit was caused by an impairment in the translocation of vesicles containing the glucose transporter GLUT4 to the plasma membrane, although insulin signaling remained unaffected. Interestingly, pharmacological inhibition of lysine acetyltransferases (KATs) prevented this increase in protein acetylation and glucose uptake inhibition induced by FAs. Similarly, FA-mediated inhibition of insulin-stimulated glucose uptake could be prevented by KAT inhibitors in perfused hearts. To summarize, enhanced protein acetylation can be considered as an early event in the FA-induced inhibition of glucose transport in the heart, explaining part of the Randle cycle.NEW & NOTEWORTHY Our results show that cardiac metabolic overload by oleate or palmitate leads to increased protein acetylation inhibiting GLUT4 translocation to the plasma membrane and glucose uptake. This observation suggests an additional regulation mechanism in the physiological glucose-FA cycle originally discovered by Randle.

Dual antiplatelet therapy is associated with high α-tubulin acetylation in circulating platelets from coronary artery disease patients.

Platelet inhibition is the main treatment strategy to prevent atherothrombotic complications after acute coronary syndrome or percutaneous coronary intervention. Despite dual antiplatelet therapy (DAPT) combining aspirin and a P2Y12 receptor inhibitor, high on-treatment platelet reactivity (HPR) persists in some patients due to poor response to treatment and is associated with ischemic risk. Tubulin acetylation has been pointed out as a hallmark of stable microtubules responsible for the discoid shape of resting platelets. However, the impact of antiplatelet treatments on this post-translational modification has never been studied. This study investigated whether tubulin acetylation differs according to antiplatelet therapy and on-treatment platelet reactivity. Platelets were isolated from arterial blood samples of 240 patients admitted for coronary angiography, and levels of α-tubulin acetylation on lysine 40 (α-tubulin K40 acetylation) were assessed by western blot. We show that platelet α-tubulin K40 acetylation was significantly increased in DAPT-treated patients. In addition, the proportion of patients with high levels of α-tubulin K40 acetylation was drastically reduced among DAPT-treated patients with HPR. Multivariate logistic regression confirmed that DAPT resulting in adequate platelet inhibition was strongly associated with elevated α-tubulin K40 acetylation. In conclusion, our study highlights the role of elevated platelet α-tubulin K40 acetylation as a marker of platelet inhibition in response to DAPT.Clinical trial registration: https://clinicaltrials.gov - NCT03034148.

What is the context? High on-treatment platelet reactivity due to dual antiplatelet therapy poor response is associated with thrombotic risk.Acetylation of α-tubulin K40 plays a crucial role in regulating platelet shape.High α-tubulin K40 acetylation is a hallmark of stable microtubules.What is new? α-tubulin K40 acetylation is increased in platelets from dual antiplatelet therapy-treated patients.High platelet α-tubulin K40 acetylation is mainly observed in clopidogrel-responsive patients.What is the impact? Elevated acetylated K40 α-tubulin could be used as a readout of adequate platelet inhibition in response to dual antiplatelet therapy.High α-tubulin K40 acetylation could contribute to maintaining the resting morphology of circulating platelets and therefore modify their capacity to be involved in thrombotic events.

Mean platelet volume: a prognostic marker in heart failure with preserved ejection fraction.

Heart failure (HF) with preserved ejection fraction (HFpEF) is associated with high burden of comorbidities known to increase the mean platelet volume (MPV). This parameter has been associated with morbidity and mortality in HF. However, the role of platelets and the prognostic relevance of MPV in HFpEF remain largely unexplored. We aimed to evaluate the clinical usefulness of MPV as a prognostic marker in HFpEF. We prospectively enrolled 228 patients with HFpEF (79 ± 9 years; 66% females) and 38 controls of similar age and gender (78 ± 5 years; 63% females). All subjects underwent two-dimensional echocardiography and MPV measurements. Patients were followed-up for a primary end point of all-cause mortality or first HF hospitalization. The prognostic impact of MPV was determined using Cox proportional hazard models. Mean MPV was significantly higher in HFpEF patients compared with controls (MPV: 10.7 ± 1.1fL vs. 10.1 ± 1.1fL, p = .005). HFpEF patients (n = 56) with MPV >75th percentile (11.3 fL) displayed more commonly a history of ischemic cardiomyopathy. Over a median follow-up of 26 months, 136 HFpEF patients reached the composite endpoint. MPV >75th percentile was a significant predictor of the primary endpoint (HR: 1.70 [1.08; 2.67], p = .023) adjusted for NYHA class, chronic obstructive pulmonary disease, loop diuretics, renal function, and hemoglobin. We demonstrated that MPV was significantly higher in HFpEF patients compared with controls of similar age and gender. Elevated MPV was a strong and independent predictor of poor outcome in HFpEF patients and may be relevant for clinical use.

What is the context? Heart failure with preserved ejection fraction (HFpEF) is associated with several comorbidities known to increase the mean platelet volume (MPV).MPV is a measure of platelet size and a potential marker of platelet reactivity. An increased MPV results from an increased platelet turnover.MPV has been associated with morbidity and mortality from heart failure.No study has previously compared MPV between HFpEF and controls and investigated the prognostic relevance of MPV in HFpEF disease.What is new? In this study, we compared the MPV between HFpEF patients and controls of similar age and gender, prospectively enrolled between 2015 and 2021. We evaluated the prognostic role of elevated MPV in HFpEF patients.Our main results:The MPV was higher in HFpEF patients compared to controls of similar age and gender.HFpEF patients with elevated MPV displayed more commonly a history of ischemic cardiomyopathy.Elevated MPV was a strong and independent predictor of poor outcome in HFpEF patients.What is the impact? MPV may be relevant for clinical use to predict clinical outcome in HFpEF patients.Elevated MPV reflecting platelet activity supports the potential role of platelets in HFpEF's pathophysiology.

Dexamethasone Modulates the Cytokine Response but Not COVID-19-Induced Coagulopathy in Critically Ill.

Severe forms of coronavirus 2019 (COVID-19) disease are caused by an exaggerated systemic inflammatory response and subsequent inflammation-related coagulopathy. Anti-inflammatory treatment with low dose dexamethasone has been shown to reduce mortality in COVID-19 patients requiring oxygen therapy. However, the mechanisms of action of corticosteroids have not been extensively studied in critically ill patients in the context of COVID-19. Plasma biomarkers of inflammatory and immune responses, endothelial and platelet activation, neutrophil extracellular trap formation, and coagulopathy were compared between patients treated or not by systemic dexamethasone for severe forms of COVID-19. Dexamethasone treatment significantly reduced the inflammatory and lymphoid immune response in critical COVID-19 patients but had little effect on the myeloid immune response and no effect on endothelial activation, platelet activation, neutrophil extracellular trap formation, and coagulopathy. The benefits of low dose dexamethasone on outcome in critical COVID-19 can be partially explained by a modulation of the inflammatory response but not by reduction of coagulopathy. Future studies should explore the impact of combining dexamethasone with other immunomodulatory or anticoagulant drugs in severe COVID-19.

α-Tubulin acetylation on lysine 40 controls cardiac glucose uptake.

Our group previously demonstrated that an excess of nutrients, as observed in diabetes, provokes an increase in cardiac protein acetylation responsible for a reduced insulin-stimulated translocation of the glucose transporter GLUT4 to the plasma membrane. The acetylated proteins involved in this event have yet not been identified. α-Tubulin is a promising candidate as a major cytoskeleton component involved, among other things, in the translocation of GLUT4-containing vesicles from their intracellular pools toward the plasma membrane. Moreover, α-tubulin is known to be acetylated, Lys40 (K40) being its best characterized acetylated residue. The present work sought to evaluate the impact of α-tubulin K40 acetylation on cardiac glucose entry, with a particular interest in GLUT4 translocation. First, we observed that a mouse model of high-fat diet-induced obesity presented an increase in cardiac α-tubulin K40 acetylation level. We next showed that treatment of insulin-sensitive primary cultured adult rat cardiomyocytes with tubacin, a specific tubulin acetylation inducer, reduced insulin-stimulated glucose uptake and GLUT4 translocation. Conversely, decreasing α-tubulin K40 acetylation by expressing a nonacetylable dominant form of α-tubulin (mCherry α-tubulin K40A mutant) remarkably intensified insulin-induced glucose transport. Finally, mCherry α-tubulin K40A expression similarly improved glucose transport in insulin-resistant cardiomyocytes or after AMP-activated protein kinase activation. Taken together, our study demonstrates that modulation of α-tubulin K40 acetylation level affects glucose transport in cardiomyocytes, offering new putative therapeutic insights regarding modulation of glucose metabolism in insulin-resistant and diabetic hearts.NEW & NOTEWORTHY Acetylation level of α-tubulin on K40 is increased in the heart of a diet-induced mouse model of type 2 diabetes. Pharmacological stimulation of α-tubulin K40 acetylation lowers insulin-mediated GLUT4 vesicles translocation to the plasma membrane, reducing glucose transport. Expressing a nonacetylable dominant form of α-tubulin boosts glucose uptake in both insulin-sensitive and insulin-resistant cardiomyocytes.

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.

Anti-GPVI (HY101) activates platelets in a multicolor flow cytometry panel.

The platelet transmembrane receptor GPVI can be assessed together with other platelet membrane markers in a whole blood multicolor flow cytometry panel. The advantage of combining multiple antibodies in a single tube is the possibility of distinguishing multiple platelet subgroups. In this short communication, we describe an activation problem encountered with anti-GPVI, clone HY101. Activation of platelets was seen after the addition of anti-GPVI in a flow cytometry panel, highlighted by the expression of the activation markers CD62P, PAC-1, CD63, and CD107a. This was also confirmed by platelet aggregation studies.

A new degree of complexi(n)ty in the regulation of GLUT4 trafficking.

Loss of the insulin-stimulated glucose uptake in muscle is a crucial event participating in the defect of whole-body metabolism in type 2 diabetes. Therefore, identification by Pavarotti et al. (Biochem. J (2021) 478 (2): 407-422) of complexin-2 as an important contributor to glucose transporter 4 (GLUT4) translocation to muscle cell plasma membrane upon insulin stimulation is essential. The present commentary discusses the biological importance of the findings and proposes future challenges and opportunities.

New insight in understanding the contribution of SGLT1 in cardiac glucose uptake: evidence for a truncated form in mice and humans.

Although sodium glucose cotransporter 1 (SGLT1) has been identified as one of the major SGLT isoforms expressed in the heart, its exact role remains elusive. Evidence using phlorizin, the most common inhibitor of SGLTs, has suggested its role in glucose transport. However, phlorizin could also affect classical facilitated diffusion via glucose transporters (GLUTs), bringing into question the relevance of SGLT1 in overall cardiac glucose uptake. Accordingly, we assessed the contribution of SGLT1 in cardiac glucose uptake using the SGLT1 knockout mouse model, which lacks exon 1. Glucose uptake was similar in cardiomyocytes isolated from SGLT1-knockout (Δex1KO) and control littermate (WT) mice either under basal state, insulin, or hyperglycemia. Similarly, in vivo basal and insulin-stimulated cardiac glucose transport measured by micro-PET scan technology did not differ between WT and Δex1KO mice. Micromolar concentrations of phlorizin had no impact on glucose uptake in either isolated WT or Δex1KO-derived cardiomyocytes. However, higher concentrations (1 mM) completely inhibited insulin-stimulated glucose transport without affecting insulin signaling nor GLUT4 translocation independently from cardiomyocyte genotype. Interestingly, we discovered that mouse and human hearts expressed a shorter slc5a1 transcript, leading to SGLT1 protein lacking transmembrane domains and residues involved in glucose and sodium bindings. In conclusion, cardiac SGLT1 does not contribute to overall glucose uptake, probably due to the expression of slc5a1 transcript variant. The inhibitory effect of phlorizin on cardiac glucose uptake is SGLT1-independent and can be explained by GLUT transporter inhibition. These data open new perspectives in understanding the role of SGLT1 in the heart.NEW & NOTEWORTHY Ever since the discovery of its expression in the heart, SGLT1 has been considered as similar as the intestine and a potential contributor to cardiac glucose transport. For the first time, we have demonstrated that a slc5a1 transcript variant is present in the heart that has no significant impact on cardiac glucose handling.

AMPKα1 deletion in myofibroblasts exacerbates post-myocardial infarction fibrosis by a connexin 43 mechanism.

We have previously demonstrated that systemic AMP-activated protein kinase α1 (AMPKα1) invalidation enhanced adverse LV remodelling by increasing fibroblast proliferation, while myodifferentiation and scar maturation were impaired. We thus hypothesised that fibroblastic AMPKα1 was a key signalling element in regulating fibrosis in the infarcted myocardium and an attractive target for therapeutic intervention. The present study investigates the effects of myofibroblast (MF)-specific deletion of AMPKα1 on left ventricular (LV) adaptation following myocardial infarction (MI), and the underlying molecular mechanisms. MF-restricted AMPKα1 conditional knockout (cKO) mice were subjected to permanent ligation of the left anterior descending coronary artery. cKO hearts exhibit exacerbated post-MI adverse LV remodelling and are characterised by exaggerated fibrotic response, compared to wild-type (WT) hearts. Cardiac fibroblast proliferation and MF content significantly increase in cKO infarcted hearts, coincident with a significant reduction of connexin 43 (Cx43) expression in MFs. Mechanistically, AMPKα1 influences Cx43 expression by both a transcriptional and a post-transcriptional mechanism involving miR-125b-5p. Collectively, our data demonstrate that MF-AMPKα1 functions as a master regulator of cardiac fibrosis and remodelling and might constitute a novel potential target for pharmacological anti-fibrotic applications.

Acetyl-CoA Carboxylase Inhibitor CP640.186 Increases Tubulin Acetylation and Impairs Thrombin-Induced Platelet Aggregation.

Acetyl-CoA carboxylase (ACC) is the first enzyme regulating de novo lipid synthesis via the carboxylation of acetyl-CoA into malonyl-CoA. The inhibition of its activity decreases lipogenesis and, in parallel, increases the acetyl-CoA content, which serves as a substrate for protein acetylation. Several findings support a role for acetylation signaling in coordinating signaling systems that drive platelet cytoskeletal changes and aggregation. Therefore, we investigated the impact of ACC inhibition on tubulin acetylation and platelet functions. Human platelets were incubated 2 h with CP640.186, a pharmacological ACC inhibitor, prior to thrombin stimulation. We have herein demonstrated that CP640.186 treatment does not affect overall platelet lipid content, yet it is associated with increased tubulin acetylation levels, both at the basal state and after thrombin stimulation. This resulted in impaired platelet aggregation. Similar results were obtained using human platelets that were pretreated with tubacin, an inhibitor of tubulin deacetylase HDAC6. In addition, both ACC and HDAC6 inhibitions block key platelet cytoskeleton signaling events, including Rac1 GTPase activation and the phosphorylation of its downstream effector, p21-activated kinase 2 (PAK2). However, neither CP640.186 nor tubacin affects thrombin-induced actin cytoskeleton remodeling, while ACC inhibition results in decreased thrombin-induced reactive oxygen species (ROS) production and extracellular signal-regulated kinase (ERK) phosphorylation. We conclude that when using washed human platelets, ACC inhibition limits tubulin deacetylation upon thrombin stimulation, which in turn impairs platelet aggregation. The mechanism involves a downregulation of the Rac1/PAK2 pathway, being independent of actin cytoskeleton.

Glucose transporters in cardiovascular system in health and disease.

Glucose transporters are essential for the heart to sustain its function. Due to its nature as a high energy-consuming organ, the heart needs to catabolize a huge quantity of metabolic substrates. For optimized energy production, the healthy heart constantly switches between various metabolites in accordance with substrate availability and hormonal status. This metabolic flexibility is essential for the maintenance of cardiac function. Glucose is part of the main substrates catabolized by the heart and its use is fine-tuned via complex molecular mechanisms that include the regulation of the glucose transporters GLUTs, mainly GLUT4 and GLUT1. Besides GLUTs, glucose can also be transported by cotransporters of the sodium-glucose cotransporter (SGLT) (SLC5 gene) family, in which SGLT1 and SMIT1 were shown to be expressed in the heart. This SGLT-mediated uptake does not seem to be directly linked to energy production but is rather associated with intracellular signalling triggering important processes such as the production of reactive oxygen species. Glucose transport is markedly affected in cardiac diseases such as cardiac hypertrophy, diabetic cardiomyopathy and heart failure. These alterations are not only fingerprints of these diseases but are involved in their onset and progression. The present review will depict the importance of glucose transport in healthy and diseased heart, as well as proposed therapies targeting glucose transporters.

AMPK-ACC signaling modulates platelet phospholipids and potentiates thrombus formation.

AMP-activated protein kinase (AMPK) α1 is activated in platelets on thrombin or collagen stimulation, and as a consequence, phosphorylates and inhibits acetyl-CoA carboxylase (ACC). Because ACC is crucial for the synthesis of fatty acids, which are essential for platelet activation, we hypothesized that this enzyme plays a central regulatory role in platelet function. To investigate this, we used a double knock-in (DKI) mouse model in which the AMPK phosphorylation sites Ser79 on ACC1 and Ser212 on ACC2 were mutated to prevent AMPK signaling to ACC. Suppression of ACC phosphorylation promoted injury-induced arterial thrombosis in vivo and enhanced thrombus growth ex vivo on collagen-coated surfaces under flow. After collagen stimulation, loss of AMPK-ACC signaling was associated with amplified thromboxane generation and dense granule secretion. ACC DKI platelets had increased arachidonic acid-containing phosphatidylethanolamine plasmalogen lipids. In conclusion, AMPK-ACC signaling is coupled to the control of thrombosis by specifically modulating thromboxane and granule release in response to collagen. It appears to achieve this by increasing platelet phospholipid content required for the generation of arachidonic acid, a key mediator of platelet activation.

α1AMP-Activated Protein Kinase Protects against Lipopolysaccharide-Induced Endothelial Barrier Disruption via Junctional Reinforcement and Activation of the p38 MAPK/HSP27 Pathway.

Vascular hyperpermeability is a determinant factor in the pathophysiology of sepsis. While, AMP-activated protein kinase (AMPK) is known to play a role in maintaining endothelial barrier function in this condition. Therefore, we investigated the underlying molecular mechanisms of this protective effect. α1AMPK expression and/or activity was modulated in human dermal microvascular endothelial cells using either α1AMPK-targeting small interfering RNA or the direct pharmacological AMPK activator 991, prior to lipopolysaccharide (LPS) treatment. Western blotting was used to analyze the expression and/or phosphorylation of proteins that compose cellular junctions (zonula occludens-1 (ZO-1), vascular endothelial cadherin (VE-Cad), connexin 43 (Cx43)) or that regulate actin cytoskeleton (p38 MAPK; heat shock protein 27 (HSP27)). Functional endothelial permeability was assessed by in vitro Transwell assays, and quantification of cellular junctions in the plasma membrane was assessed by immunofluorescence. Actin cytoskeleton remodeling was evaluated through actin fluorescent staining. We consequently demonstrate that α1AMPK deficiency is associated with reduced expression of CX43, ZO-1, and VE-Cad, and that the drastic loss of CX43 is likely responsible for the subsequent decreased expression and localization of ZO-1 and VE-Cad in the plasma membrane. Moreover, α1AMPK activation by 991 protects against LPS-induced endothelial barrier disruption by reinforcing cortical actin cytoskeleton. This is due to a mechanism that involves the phosphorylation of p38 MAPK and HSP27, which is nonetheless independent of the small GTPase Rac1. This results in a drastic decrease of LPS-induced hyperpermeability. We conclude that α1AMPK activators that are suitable for clinical use may provide a specific therapeutic intervention that limits sepsis-induced vascular leakage.

Cardiac myocyte ß3-adrenergic receptors prevent myocardial fibrosis by modulating oxidant stress-dependent paracrine signaling.

Aims: Human and mouse cardiac beta3-adrenergic receptors (beta3AR) exert antipathetic effects to those of beta1-2AR stimulation. We examined their role in modulating myocardial remodelling, particularly fibrosis in response to haemodynamic stress. Methods and results: Mice with cardiac myocyte-specific expression of beta3AR (ADRB3-tg) or tamoxifen-inducible homozygous deletion (c-Adrb3-ko, with loxP-targeted Adrb3) were submitted to transaortic constriction. A superfusion assay was used for proteomic analysis of paracrine mediators between beta3AR-expressing cardiac myocytes and cardiac fibroblasts cultured separately. We show that cardiac beta3AR attenuate myocardial fibrosis in response to haemodynamic stress. Interstitial fibrosis and collagen content were reduced in ADRB3-tg, but augmented in c-Adrb3-ko. ADRB3 and collagen (COL1A1) expression were also inversely related in ventricular biopsies of patients with valve disease. Incubation of cardiac fibroblasts with media conditioned by hypertrophic myocytes induced fibroblast proliferation, myo-differentiation, and collagen production. These effects were abrogated upon ADRB3 expression in myocytes. Comparative shotgun proteomic analysis of the myocyte secretomes revealed a number of factors differentially regulated by beta3AR, among which connective tissue growth factor [CTGF (CCN2)] was prominently reduced. CTGF was similarly reduced in stressed hearts from ADRB3-tg, but increased in hearts from c-Adrb3-ko mice. CTGF expression was mediated by reactive oxygen species production which was reduced by ADRB3 expression in vitro and in vivo. This antioxidant and anti-fibrotic effect involved beta3AR coupling to the neuronal isoform of nitric oxide synthase (nNOS) in cardiac myocytes, as both were abrogated upon nNOS inhibition or Nos1 homozygous deletion. Conclusion: Cardiac beta3AR protect from fibrosis in response to haemodynamic stress by modulating nitric oxide and oxidant stress-dependent paracrine signaling to fibroblasts. Specific agonism at beta3AR may offer a new therapeutic modality to prevent cardiac fibrosis.

O-GlcNAcylation, enemy or ally during cardiac hypertrophy development?

O-linked attachment of the monosaccharide ß-N-acetyl-glucosamine (O-GlcNAcylation) is a post-translational modification occurring on serine and threonine residues, which is evolving as an important mechanism for the regulation of various cellular processes. The present review will, first, provide a general background on the molecular regulation of protein O-GlcNAcylation and will summarize the role of this post-translational modification in various acute cardiac pathologies including ischemia-reperfusion. Then, we will focus on research studies examining protein O-GlcNAcylation in the context of cardiac hypertrophy. A particular emphasis will be laid on the convergent but also divergent actions of O-GlcNAcylation according to the type of hypertrophy investigated, including physiological, pressure overload-induced and diabetes-linked cardiac hypertrophy. In an attempt to distinguish whether O-GlcNAcylation is detrimental or beneficial, this review will present the different O-GlcNAcylated targets involved in hypertrophy development. We will finally argue on potential interest to target O-GlcNAc processes to treat cardiac hypertrophy. This article is part of a Special Issue entitled: The role of post-translational protein modifications on heart and vascular metabolism edited by Jason R.B. Dyck & Jan F.C. Glatz.

Metabolism and acetylation contribute to leucine-mediated inhibition of cardiac glucose uptake.

High plasma leucine levels strongly correlate with type 2 diabetes. Studies of muscle cells have suggested that leucine alters the insulin response for glucose transport by activating an insulin-negative feedback loop driven by the mammalian target of rapamycin/p70 ribosomal S6 kinase (mTOR/p70S6K) pathway. Here, we examined the molecular mechanism involved in leucine's action on cardiac glucose uptake. Leucine was indeed able to curb glucose uptake after insulin stimulation in both cultured cardiomyocytes and perfused hearts. Although leucine activated mTOR/p70S6K, the mTOR inhibitor rapamycin did not prevent leucine's inhibitory action on glucose uptake, ruling out the contribution of the insulin-negative feedback loop. α-Ketoisocaproate, the first metabolite of leucine catabolism, mimicked leucine's effect on glucose uptake. Incubation of cardiomyocytes with [13C]leucine ascertained its metabolism to ketone bodies (KBs), which had a similar negative impact on insulin-stimulated glucose transport. Both leucine and KBs reduced glucose uptake by affecting translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Finally, we found that leucine elevated the global protein acetylation level. Pharmacological inhibition of lysine acetyltransferases counteracted this increase in protein acetylation and prevented leucine's inhibitory action on both glucose uptake and GLUT4 translocation. Taken together, these results indicate that leucine metabolism into KBs contributes to inhibition of cardiac glucose uptake by hampering the translocation of GLUT4-containing vesicles via acetylation. They offer new insights into the establishment of insulin resistance in the heart.NEW & NOTEWORTHY Catabolism of the branched-chain amino acid leucine into ketone bodies efficiently inhibits cardiac glucose uptake through decreased translocation of glucose transporter 4 to the plasma membrane. Leucine increases protein acetylation. Pharmacological inhibition of acetylation reverses leucine's action, suggesting acetylation involvement in this phenomenon.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/leucine-metabolism-inhibits-cardiac-glucose-uptake/.