RESUMO
Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H+ and phosphate recycling.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Adenina/metabolismo , Adenosina/metabolismo , Adenosina Desaminase/metabolismo , Cromatografia Líquida/métodos , Células HEK293 , Células Hep G2 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Espectrometria de Massas/métodos , Enzimas Multifuncionais/genética , Fosforilação , Proteínas/genética , Nucleotídeos de Purina/metabolismo , Purinas/metabolismoRESUMO
The regulation of membrane lipid composition is critical for cellular homeostasis. Cells are particularly sensitive to phospholipid saturation, with increased saturation causing membrane rigidification and lipotoxicity. How mammalian cells sense membrane lipid composition and reverse fatty acid (FA)-induced membrane rigidification is poorly understood. Here we systematically identify proteins that differ between mammalian cells fed saturated versus unsaturated FAs. The most differentially expressed proteins were two ER-resident polytopic membrane proteins: the E3 ubiquitin ligase RNF145 and the lipid hydrolase ADIPOR2. In unsaturated lipid membranes, RNF145 is stable, promoting its lipid-sensitive interaction, ubiquitination and degradation of ADIPOR2. When membranes become enriched in saturated FAs, RNF145 is rapidly auto-ubiquitinated and degraded, stabilising ADIPOR2, whose hydrolase activity restores lipid homeostasis and prevents lipotoxicity. We therefore identify RNF145 as a FA-responsive ubiquitin ligase which, together with ADIPOR2, defines an autoregulatory pathway that controls cellular membrane lipid homeostasis and prevents acute lipotoxic stress.
Assuntos
Hidrolases , Fluidez de Membrana , Animais , Ácidos Graxos/metabolismo , Hidrolases/metabolismo , Mamíferos , Proteínas de Membrana/metabolismo , Fosfolipídeos , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Mutations in VHL, which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222CâA, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598CâT) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).
Assuntos
Mutação em Linhagem Germinativa , Transtornos do Crescimento/genética , Hipoglicemia/genética , Fator 1 Induzível por Hipóxia/deficiência , Mitocôndrias/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Expressão Gênica , Crescimento/genética , Humanos , Masculino , Metaboloma/genética , Metaboloma/fisiologia , Síndrome , Adulto JovemRESUMO
Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.
Assuntos
DNA-Citosina Metilases/metabolismo , Metiltransferases/metabolismo , Animais , Linhagem Celular , Citosina/metabolismo , Metilação de DNA/fisiologia , DNA-Citosina Metilases/fisiologia , Humanos , Camundongos , Estresse Oxidativo/fisiologia , Biossíntese de Proteínas/fisiologia , RNA/metabolismo , RNA de Transferência/metabolismoRESUMO
Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (ob/ob), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.
Assuntos
Adipócitos , Tecido Adiposo Branco , Tecido Adiposo/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Metabolismo dos Lipídeos , Lipidômica , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/metabolismoRESUMO
The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A (PPARA) gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature.
Assuntos
Adaptação Fisiológica , Altitude , Etnicidade , Hipóxia/metabolismo , Adaptação Fisiológica/genética , Adulto , Pressão Atmosférica , Ciclo do Ácido Cítrico , Metabolismo Energético , Etnicidade/genética , Ácidos Graxos/metabolismo , Feminino , Frequência do Gene , Glucose/metabolismo , Glicólise , Humanos , Hipóxia/genética , Hipóxia/fisiopatologia , Masculino , Mitocôndrias Musculares/metabolismo , Músculo Esquelético/metabolismo , Nepal , Óxido Nítrico/sangue , Fosforilação Oxidativa , Estresse Oxidativo , Consumo de Oxigênio , PPAR alfa/genética , PPAR alfa/metabolismo , Polimorfismo de Nucleotídeo Único , Tibet/etnologiaRESUMO
BACKGROUND: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)-2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature. METHODS: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l-arginine/nitric oxide pathway. RESULTS: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l-arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function. CONCLUSIONS: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.
Assuntos
Anti-Inflamatórios não Esteroides/efeitos adversos , Arginina/análogos & derivados , Doenças Cardiovasculares/etiologia , Ciclo-Oxigenase 2/metabolismo , Animais , Anti-Inflamatórios não Esteroides/sangue , Anti-Inflamatórios não Esteroides/farmacologia , Anti-Inflamatórios não Esteroides/uso terapêutico , Arginina/sangue , Pressão Sanguínea/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Celecoxib/farmacologia , Creatinina/sangue , Ciclo-Oxigenase 1/metabolismo , Ciclo-Oxigenase 2/química , Ciclo-Oxigenase 2/genética , Inibidores de Ciclo-Oxigenase 2/farmacologia , Humanos , Rim/metabolismo , Metaboloma/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/tratamento farmacológico , Osteoartrite/patologia , Efeito PlaceboRESUMO
INTRODUCTION: Relative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. 13C labelled substrates have become a key tool for studying substrate use-yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle. OBJECTIVES: We aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the 13C labelled proportion of acetyl-CoA entering the Krebs cycle. METHODS: A model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with 13C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture). RESULTS: The contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-13C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively. CONCLUSION: These results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.
Assuntos
Mitocôndrias/metabolismo , Miocárdio/metabolismo , Acetilcoenzima A/análise , Animais , Isótopos de Carbono , Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , Coração/fisiologia , Masculino , Espectrometria de Massas/métodos , Modelos Biológicos , Oxirredução , Ratos , Ratos WistarRESUMO
BACKGROUND: The recent pandemic of obesity and the metabolic syndrome (MetS) has led to the realisation that new drug targets are needed to either reduce obesity or the subsequent pathophysiological consequences associated with excess weight gain. Certain nuclear hormone receptors (NRs) play a pivotal role in lipid and carbohydrate metabolism and have been highlighted as potential treatments for obesity. This realisation started a search for NR agonists in order to understand and successfully treat MetS and associated conditions such as insulin resistance, dyslipidaemia, hypertension, hypertriglyceridemia, obesity and cardiovascular disease. The most studied NRs for treating metabolic diseases are the peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-γ, and PPAR-δ. However, prolonged PPAR treatment in animal models has led to adverse side effects including increased risk of a number of cancers, but how these receptors change metabolism long term in terms of pathology, despite many beneficial effects shorter term, is not fully understood. In the current study, changes in male Sprague Dawley rat liver caused by dietary treatment with a PPAR-pan (PPAR-α, -γ, and -δ) agonist were profiled by classical toxicology (clinical chemistry) and high throughput metabolomics and lipidomics approaches using mass spectrometry. RESULTS: In order to integrate an extensive set of nine different multivariate metabolic and lipidomics datasets with classical toxicological parameters we developed a hypotheses free, data driven machine learning approach. From the data analysis, we examined how the nine datasets were able to model dose and clinical chemistry results, with the different datasets having very different information content. CONCLUSIONS: We found lipidomics (Direct Infusion-Mass Spectrometry) data the most predictive for different dose responses. In addition, associations with the metabolic and lipidomic data with aspartate amino transaminase (AST), a hepatic leakage enzyme to assess organ damage, and albumin, indicative of altered liver synthetic function, were established. Furthermore, by establishing correlations and network connections between eicosanoids, phospholipids and triacylglycerols, we provide evidence that these lipids function as a key link between inflammatory processes and intermediary metabolism.
Assuntos
Aprendizado de Máquina , Metabolômica , Animais , Análise Química do Sangue , Bases de Dados Factuais , Humanos , Metabolismo dos Lipídeos/genética , Fígado/metabolismo , Masculino , Síndrome Metabólica/genética , Síndrome Metabólica/metabolismo , Síndrome Metabólica/patologia , Obesidade/genética , Obesidade/metabolismo , Obesidade/patologia , PPAR alfa/agonistas , PPAR alfa/metabolismo , PPAR gama/agonistas , PPAR gama/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Metabolismo Energético/genética , Metabolismo dos Lipídeos/genética , Miocárdio/metabolismo , Consumo de Oxigênio/genética , Trifosfato de Adenosina/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular/genética , Linhagem Celular , Regulação da Expressão Gênica no Desenvolvimento , Coração/embriologia , Coração/fisiologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Potencial da Membrana Mitocondrial/genética , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Isquemia Miocárdica/genética , Isquemia Miocárdica/metabolismo , Miócitos Cardíacos/metabolismo , Oxigênio/metabolismo , Ativação TranscricionalRESUMO
BACKGROUND: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of ß-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms. RESULTS: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARß/δ- and PPARα-dependent mechanism. Enhanced PPARß/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation. CONCLUSIONS: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.
Assuntos
GMP Cíclico/metabolismo , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Nitratos/metabolismo , Óxido Nítrico/metabolismo , Receptores Ativados por Proliferador de Peroxissomo/metabolismo , Ração Animal/análise , Animais , Dieta , Relação Dose-Resposta a Droga , Masculino , Biogênese de Organelas , Oxirredução , Ratos , Ratos WistarRESUMO
The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography-mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.
Assuntos
Envelhecimento/metabolismo , Dieta Hiperlipídica , Mitocôndrias Cardíacas/fisiologia , Animais , Biomarcadores/metabolismo , Masculino , Camundongos , Estresse OxidativoRESUMO
The role of C17:0 and C15:0 in human health has recently been reinforced following a number of important biological and nutritional observations. Historically, odd chain saturated fatty acids (OCS-FAs) were used as internal standards in GC-MS methods of total fatty acids and LC-MS methods of intact lipids, as it was thought their concentrations were insignificant in humans. However, it has been thought that increased consumption of dairy products has an association with an increase in blood plasma OCS-FAs. However, there is currently no direct evidence but rather a casual association through epidemiology studies. Furthermore, a number of studies on cardiometabolic diseases have shown that plasma concentrations of OCS-FAs are associated with lower disease risk, although the mechanism responsible for this is debated. One possible mechanism for the endogenous production of OCS-FAs is α-oxidation, involving the activation, then hydroxylation of the α-carbon, followed by the removal of the terminal carboxyl group. Differentiation human adipocytes showed a distinct increase in the concentration of OCS-FAs, which was possibly caused through α-oxidation. Further evidence for an endogenous pathway, is in human plasma, where the ratio of C15:0 to C17:0 is approximately 1:2 which is contradictory to the expected levels of C15:0 to C17:0 roughly 2:1 as detected in dairy fat. We review the literature on the dietary consumption of OCS-FAs and their potential endogenous metabolism.
Assuntos
Ácidos Graxos/metabolismo , Animais , Biomarcadores/sangue , Doença das Coronárias/sangue , Diabetes Mellitus Tipo 2/sangue , Humanos , Metabolismo dos Lipídeos , Fatores de RiscoRESUMO
Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l(-1) NaCl (as control) or 0.7 mmol l(-1) NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.
Assuntos
Arginina/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Nitratos/farmacologia , Animais , Arginase/genética , Arginase/metabolismo , Dieta , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/fisiologia , Fator 1 Induzível por Hipóxia/genética , Fator 1 Induzível por Hipóxia/metabolismo , Masculino , Nitratos/administração & dosagem , Nitritos/química , Nitritos/metabolismo , Estresse Oxidativo , Oxigênio , Ratos , Ratos WistarRESUMO
The diabetic heart is characterised by functional, morphological and metabolic alterations predisposing it to contractile failure. Chronic sympathetic activation is a feature of the pathogenesis of heart failure, however the type 1 diabetic heart shows desensitisation to ß-adrenergic stimulation. Here, we sought to understand the impact of repeated isoprenaline-mediated ß-stimulation upon cardiac mitochondrial respiratory capacity and substrate metabolism in the 90% pancreatectomy (Px) rat model of type 1 diabetes. We hypothesised these hearts would be relatively protected against the metabolic impact of stress-induced cardiomyopathy. We found that individually both Px and isoprenaline suppressed cardiac mitochondrial respiration, but that this was preserved in Px rats receiving isoprenaline. Px and isoprenaline had contrasting effects on cardiac substrate metabolism, with increased reliance upon cardiac fatty acid oxidation capacity and altered ketone metabolism in the hearts of Px rats, but enhanced capacity for glucose uptake and metabolism in isoprenaline-treated rats. Moreover, Px rats were protected against isoprenaline-induced mortality, whilst isoprenaline elevated cGMP and protected myocardial energetic status in Px rat hearts. Our work suggests that adrenergic stimulation may be protective in the type 1 diabetic heart, and underlines the importance of studying pathological features in combination when modeling complex disease in rodents.
Assuntos
Agonistas Adrenérgicos beta , Isoproterenol , Animais , Agonistas Adrenérgicos beta/farmacologia , Ratos , Masculino , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/efeitos dos fármacos , Miocárdio/metabolismo , Miocárdio/patologia , Diabetes Mellitus Tipo 1/metabolismo , Glucose/metabolismo , Modelos Animais de Doenças , Coração/efeitos dos fármacosRESUMO
AIMS: Potential loss-of-function variants of ATP13A3, the gene encoding a P5B-type transport ATPase of undefined function, were recently identified in patients with pulmonary arterial hypertension (PAH). ATP13A3 is implicated in polyamine transport but its function has not been fully elucidated. In this study, we sought to determine the biological function of ATP13A3 in vascular endothelial cells (ECs) and how PAH-associated variants may contribute to disease pathogenesis. METHODS AND RESULTS: We studied the impact of ATP13A3 deficiency and overexpression in EC models [human pulmonary ECs, blood outgrowth ECs (BOECs), and human microvascular EC 1], including a PAH patient-derived BOEC line harbouring an ATP13A3 variant (LK726X). We also generated mice harbouring an Atp13a3 variant analogous to a human disease-associated variant to establish whether these mice develop PAH. ATP13A3 localized to the recycling endosomes of human ECs. Knockdown of ATP13A3 in ECs generally reduced the basal polyamine content and altered the expression of enzymes involved in polyamine metabolism. Conversely, overexpression of wild-type ATP13A3 increased polyamine uptake. Functionally, loss of ATP13A3 was associated with reduced EC proliferation, increased apoptosis in serum starvation, and increased monolayer permeability to thrombin. The assessment of five PAH-associated missense ATP13A3 variants (L675V, M850I, V855M, R858H, and L956P) confirmed loss-of-function phenotypes represented by impaired polyamine transport and dysregulated EC function. Furthermore, mice carrying a heterozygous germline Atp13a3 frameshift variant representing a human variant spontaneously developed a PAH phenotype, with increased pulmonary pressures, right ventricular remodelling, and muscularization of pulmonary vessels. CONCLUSION: We identify ATP13A3 as a polyamine transporter controlling polyamine homeostasis in ECs, a deficiency of which leads to EC dysfunction and predisposes to PAH. This suggests a need for targeted therapies to alleviate the imbalances in polyamine homeostasis and EC dysfunction in PAH.
Assuntos
Células Endoteliais , Poliaminas , Animais , Humanos , Poliaminas/metabolismo , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/enzimologia , Proliferação de Células , Artéria Pulmonar/metabolismo , Artéria Pulmonar/fisiopatologia , ATPases Translocadoras de Prótons/metabolismo , ATPases Translocadoras de Prótons/genética , Hipertensão Arterial Pulmonar/metabolismo , Hipertensão Arterial Pulmonar/genética , Hipertensão Arterial Pulmonar/fisiopatologia , Hipertensão Arterial Pulmonar/enzimologia , Hipertensão Arterial Pulmonar/patologia , Apoptose , Hipertensão Pulmonar/metabolismo , Hipertensão Pulmonar/genética , Hipertensão Pulmonar/fisiopatologia , Hipertensão Pulmonar/patologia , Endossomos/metabolismo , Transporte Biológico , Modelos Animais de Doenças , Células Cultivadas , Fenótipo , Camundongos Endogâmicos C57BL , CamundongosRESUMO
One potential approach for treating obesity is to increase energy expenditure in brown and white adipose tissue. Here we aimed to achieve this outcome by targeting mitochondrial uncoupler compounds selectively to adipose tissue, thus avoiding side effects from uncoupling in other tissues. Selective drug accumulation in adipose tissue has been observed with many lipophilic compounds and dyes. Hence, we explored the feasibility of conjugating uncoupler compounds with a lipophilic C8-hydrocarbon chain via an ether bond. We found that substituting the trifluoromethoxy group in the uncoupler FCCP with a C8-hydrocarbon chain resulted in potent uncoupling activity. Nonetheless, the compound did not elicit therapeutic effects in mice, likely as a consequence of metabolic instability resulting from rapid ether bond cleavage. A lipophilic analog of the uncoupler compound 2,6-dinitrophenol, in which a C8-hydrocarbon chain was conjugated via an ether bond in the para-position (2,6-dinitro-4-(octyloxy)phenol), exhibited increased uncoupling activity compared to the parent compound. However, in vivo pharmacokinetics studies suggested that 2,6-dinitro-4-(octyloxy)phenol was also metabolically unstable. In conclusion, conjugation of a hydrophobic hydrocarbon chain to uncoupler compounds resulted in sustained or improved uncoupling activity. However, an ether bond linkage led to metabolic instability, indicating the need to conjugate lipophilic groups via other chemical bonds.
Assuntos
Tecido Adiposo Marrom , Tecido Adiposo , Camundongos , Animais , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo/metabolismo , Obesidade/metabolismo , Metabolismo Energético , Tecido Adiposo Branco/metabolismo , Éteres , Fenóis/farmacologia , Proteína Desacopladora 1/metabolismoRESUMO
Metabolomics has refreshed interest in metabolism across biology and medicine, particularly in the areas of functional genomics and biomarker discovery. In this review we will discuss the experimental techniques and challenges involved in metabolomic profiling and how these technologies have been applied to cardiovascular disease. Open profiling and targeted approaches to metabolomics are compared, focusing on high resolution NMR spectroscopy and mass spectrometry, as well as discussing how to analyse the large amounts of data generated using multivariate statistics. Finally, the current literature on metabolomic profiling in human cardiovascular disease is reviewed to illustrate the diversity of approaches, and discuss some of the key metabolites and pathways found to be perturbed in plasma, urine and tissue from patients with these diseases. This includes studies of coronary artery disease, myocardial infarction, and ischemic heart disease. These studies demonstrate the potential of the technology for biomarker discovery and elucidating metabolic mechanisms associated with given pathologies, but also in some cases provide a warning of the pitfalls of poor study design. This article is part of a Special Issue entitled "Focus on Cardiac Metabolism".
Assuntos
Cardiopatias/metabolismo , Metaboloma , Metabolômica , Biomarcadores/metabolismo , Humanos , Metabolômica/métodosRESUMO
Non-genotoxic carcinogens (NGCs) promote tumor growth by altering gene expression, which ultimately leads to cancer without directly causing a change in DNA sequence. As a result NGCs are not detected in mutagenesis assays. While there are proposed biomarkers of carcinogenic potential, the definitive identification of non-genotoxic carcinogens still rests with the rat and mouse long-term bioassay. Such assays are expensive and time-consuming and require a large number of animals, and their relevance to human health risk assessments is debatable. Metabolomics and lipidomics in combination with pathology and clinical chemistry were used to profile perturbations produced by 10 compounds that represented a range of rat non-genotoxic hepatocarcinogens (NGC), non-genotoxic non-hepatocarcinogens (non-NGC), and a genotoxic hepatocarcinogen. Each compound was administered at its maximum tolerated dose level for 7, 28, and 91 days to male Fisher 344 rats. Changes in liver metabolite concentration differentiated the treated groups across different time points. The most significant differences were driven by pharmacological mode of action, specifically by the peroxisome proliferator activated receptor alpha (PPAR-α) agonists. Despite these dominant effects, good predictions could be made when differentiating NGCs from non-NGCs. Predictive ability measured by leave one out cross validation was 87% and 77% after 28 days of dosing for NGCs and non-NGCs, respectively. Among the discriminatory metabolites we identified free fatty acids, phospholipids, and triacylglycerols, as well as precursors of eicosanoid and the products of reactive oxygen species linked to processes of inflammation, proliferation, and oxidative stress. Thus, metabolic profiling is able to identify changes due to the pharmacological mode of action of xenobiotics and contribute to early screening for non-genotoxic potential.