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Ectopic fat accumulation in non-adipose tissues is closely related to diabetes-related myocardial dysfunction. Nevertheless, the complete picture of the lipid metabolites involved in the metabolic-related myocardial alterations is not fully characterized. The aim of this study was to characterize the specific lipid profile in hearts in an animal model of obesity/insulin resistance induced by a high-fat diet (HFD). The cardiac lipidome profiles were assessed via liquid chromatography-mass spectrometry (LC-MS)/MS-MS and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging in hearts from C57BL/6J mice fed with an HFD or standard-diet (STD) for 12 weeks. Targeted lipidome analysis identified a total of 63 lipids (i.e., 48 triacylglycerols (TG), 5 diacylglycerols (DG), 1 sphingomyelin (SM), 3 phosphatidylcholines (PC), 1 DihydroPC, and 5 carnitines) modified in hearts from HFD-fed mice compared to animals fed with STD. Whereas most of the TG were up-regulated in hearts from animals fed with an HFD, most of the carnitines were down-regulated, thereby suggesting a reduction in the mitochondrial ß-oxidation. Roughly 30% of the identified metabolites were oxidated, pointing to an increase in lipid peroxidation. Cardiac lipidome was associated with a specific biochemical profile and a specific liver TG pattern. Overall, our study reveals a specific cardiac lipid fingerprint associated with metabolic alterations induced by HFD.
Assuntos
Resistência à Insulina , Camundongos , Animais , Lipidômica , Modelos Animais de Doenças , Dieta Hiperlipídica , Camundongos Endogâmicos C57BL , Fígado/metabolismo , Lipídeos/análise , Metabolismo dos LipídeosRESUMO
Isotopic-labeling experiments have been valuable to monitor the flux of metabolic reactions in biological systems, which is crucial to understand homeostatic alterations with disease. Experimental determination of metabolic fluxes can be inferred from a characteristic rearrangement of stable isotope tracers (e.g., 13C or 15N) that can be detected by mass spectrometry (MS). Metabolites measured are generally members of well-known metabolic pathways, and most of them can be detected using both gas chromatography (GC)-MS and liquid chromatography (LC)-MS. In here, we show that GC methods coupled to chemical ionization (CI) MS have a clear advantage over alternative methodologies due to GC's superior chromatography separation efficiency and the fact that CI is a soft ionization technique that yields identifiable protonated molecular ion peaks. We tested diverse GC-CI-MS setups, including methane and isobutane reagent gases, triple quadrupole (QqQ) MS in SIM mode, or selected ion clusters using optimized narrow windows (â¼10 Da) in scan mode, and standard full scan methods using high resolution GC-(q)TOF and GC-Orbitrap systems. Isobutane as a reagent gas in combination with both low-resolution (LR) and high-resolution (HR) MS showed the best performance, enabling precise detection of isotopologues in most metabolic intermediates of central carbon metabolism. Finally, with the aim of overcoming manual operations, we developed an R-based tool called isoSCAN that automatically quantifies all isotopologues of intermediate metabolites of glycolysis, TCA cycle, amino acids, pentose phosphate pathway, and urea cycle, from LRMS and HRMS data.
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Butanos/metabolismo , Metabolômica , Butanos/análise , Cromatografia Gasosa-Espectrometria de Massas , Gases/análise , Gases/metabolismo , Marcação por IsótopoRESUMO
Pharmaceuticals from veterinary treatments may enter terrestrial food webs when medicated livestock are available to wildlife in supplementary feeding stations aimed at the conservation of endangered scavengers. Here, we hypothesized that the exposure risk to livestock fluoroquinolones, as indicators of pharmaceutical burden in food, is related to the variable reliance of scavengers on domestic versus wild animal carcasses. Since the misuse of broad-spectrum antibiotics is a major predisposing factor for opportunistic mycoses, we evaluated disease signs potentially associated with diet-dependent drug exposure in nestlings of two threatened vultures. A greater occurrence (100%, n=14) and concentration of fluoroquinolones (mean±SD=73.0±27.5µgL-1, range=33.2-132.7), mostly enrofloxacin, were found in Cinereous vultures, Aegypius monachus, due to their greater dependence on livestock carcasses than Egyptian vultures, Neophron percnopterus (fluoroquinolones occurrence: 44%, n=16, concentration: 37.9±16.6µgL-1, range=11.5-55.9), which rely much more on carcasses of wild animals (42% of remains vs. 23% in the cinereous vulture). The chaotic, chronic and pulsed ingestion of these drugs throughout nestling development is proposed as one of the most plausible explanations for the high occurrence and intensity of oral Candida-like lesions in nestling vultures. The high occurrence of fluoroquinolone residues and disease hindered the probing of a cause-effect relationship between both factors in individual vultures. This relationship could be evaluated through a population-based approach by sampling vultures not exposed to these drugs. The high dependence of vultures on domestic animals today compared to past decades and the growing intensification of livestock farming, imply an expected increase in the impact of pharmaceuticals on scavenger populations. This requires further evaluation due to potential consequences in biodiversity conservation and environmental health. We encourage the prioritization of efforts to promote the use of less medicated free-ranging livestock carcasses left in the countryside, rather than stabled stocks made available in vulture restaurants. Additionally, attention should be paid to the population recovery of wild species that dominated scavenger diets in the past.
Assuntos
Antibacterianos/efeitos adversos , Doenças das Aves/microbiologia , Gado , Micoses/veterinária , Infecções Oportunistas/veterinária , Aves Predatórias/microbiologia , Drogas Veterinárias/efeitos adversos , Animais , Antibacterianos/sangue , Conservação dos Recursos Naturais , Dieta , Resíduos de Drogas , Egito , Espécies em Perigo de Extinção , Enrofloxacina , Fluoroquinolonas/efeitos adversos , Fluoroquinolonas/sangue , Aves Predatórias/sangue , Drogas Veterinárias/sangueRESUMO
The high standards required for food safety make it necessary to trace unambiguously raw or cooked food products coming from medicated animals. Nevertheless, considering the lability of ß-lactams and their degradation, the detection of the presence of antibiotics in meat either raw or submitted to a cooking process is not easily affordable. To achieve this goal, an evaluation of the effect of common domestic cooking procedures, such as boiling and grilling, on the fate of phenoxymethylpenicillin (PENV) residues was performed. Finally, in this work, the penilloic acid from PENV (MET02) and the corresponding penicilloic acid (PENV-HYDRO) are suggested as biomarkers. These compounds present the highest relative abundances 5 days after the treatment was stopped (5PT) and show enough thermal stability to be considered suitable biomarker candidates for the pharmacological treatment instead of the parent compound. Nevertheless, the peaks corresponding to MET02 are significantly more intense than those for PENV-HYDRO, which makes preferential the use of MET02 to perform the control of samples.
Assuntos
Antibacterianos , Biomarcadores , Galinhas , Culinária , Contaminação de Alimentos , Carne , Animais , Contaminação de Alimentos/análise , Carne/análise , Biomarcadores/análise , Antibacterianos/análise , Espectrometria de Massas por Ionização por Electrospray/métodos , Cromatografia Líquida de Alta Pressão/métodos , Resíduos de Drogas/análise , Temperatura AltaRESUMO
AIMS: To uncover novel candidate metabolomic and lipidomic biomarkers in newly-diagnosed type 1 diabetes (T1DM) after achieving optimal glucose control. METHODS: Comprehensive lipidomic and metabolomic analysis was performed in serum of 12 adults with T1DM at onset and after achieving optimal glycemic control (HbA1c < 7 %) (after 2-6 months). RESULTS: After intensive therapy, subjects (mean age 25.2 years, 58.3 % men) showed decreases in blood glucose (p < 0.001), HbA1c [11.5 % (9.2-13.4) to 6.2 % (5.2 - 6.7); p < 0.001] and changes in 51 identified lipids. Among these changes, we found that triglycerides (TG) containing medium chain fatty acids (TG45:0, TG47:1), sphingomyelins (SM) (SM(d18:2/20:0), SM42:4)), and phosphatidylcholines (PC) (PC(O-26:2), PC(O-30:0), PC(O-32:0), PC(O-42:6), PC(O-44:5), PC(O-38:3), PC(O-33:0), PC(O-46:8), PC(O-44:6), PC(O-40:3), PC(O-42:4), PC(O-46:7), PC(O-46:6), PC(O-44:5), PC(O-42:3), PC(O-44:4)) decreased; whereas PC(35:1), PC(37:1) and TG containing longer chain fatty acids (TG(52:1), TG(55:7), TG(51:2), TG(53:3), TG52:2), TG(53:2), TG(57:3), TG(61:3), TG(61:2) increased. Further, dihydro O-acylceramide (18:1/18:0/16:0), diacylglycerophosphoethanolamine (PE(34:1)), diacylglycerophosphoinositol (PI(38:6), and dihydrosphingomyelins (dihydroSM(36:0), dihydroSM(40:0), dihydroSM(41:0), dihydroSM(42:0)) increased. Uric acid, mannitol, and mannitol-1-acetate levels also increased. CONCLUSIONS: Our data uncovered potential favorable changes in the metabolism of glycerophospholipids, glycerolipids, and sphingolipids in new-onset T1DM after achieving optimal glycemic control. Further research on their potential role in developing diabetes-related complications is needed.
Assuntos
Diabetes Mellitus Tipo 1 , Masculino , Adulto , Humanos , Feminino , Diabetes Mellitus Tipo 1/tratamento farmacológico , Lipidômica , Controle Glicêmico , Hemoglobinas Glicadas , Triglicerídeos , Fosfatidilcolinas , Ácidos GraxosRESUMO
Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.
Assuntos
Plasticidade Celular , Reprogramação Celular , Animais , Camundongos , Vitamina B 12 , Cicatrização , VitaminasRESUMO
In this work, metabolomic profile changes in milk from cows affected by mastitis and treated with enrofloxacin (ENR) have been studied using LC-HRMS techniques. Principal component analysis was applied to the obtained results, and the interest was focused on changes affecting compounds without a structural relationship to ENR. Most of the compounds, whose concentrations were modified as a result of the pharmacological treatment and/or the pathological status, were related to amino acids and peptides. Compounds that may become possible biomarkers for either disease or treatment have been detected. Additionally, the alterations caused by thermal processes, such as those applied to milk before consumption, on the identified metabolites have also been considered.
Assuntos
Mastite Bovina , Leite , Animais , Bovinos , Enrofloxacina/análise , Enrofloxacina/metabolismo , Enrofloxacina/uso terapêutico , Feminino , Fluoroquinolonas/análise , Mastite Bovina/metabolismo , Leite/química , TemperaturaRESUMO
High circulating concentrations of the gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) are significantly associated with the risk of obesity and type 2 diabetes (T2D). We aimed at evaluating the impact of glycemic control and bariatric surgery on circulating concentrations of TMAO and its microbiota-dependent intermediate, γ-butyrobetaine (γBB), in newly diagnosed T2D patients and morbidly obese subjects following a within-subject design. Based on HbA1c concentrations, T2D patients achieved glycemic control. However, the plasma TMAO and γBB concentrations were significantly increased, without changes in estimated glomerular filtration rate. Bariatric surgery was very effective in reducing weight in obese subjects. Nevertheless, the surgery reduced plasma γBB concentrations without affecting TMAO concentrations and the estimated glomerular filtration rate. Considering these results, an additional experiment was carried out in male C57BL/6J mice fed a Western-type diet for twelve weeks. Neither diet-induced obesity nor insulin resistance were associated with circulating TMAO and γBB concentrations in these genetically defined mice strains. Our findings do not support that glycemic control or bariatric surgery improve the circulating concentrations of TMAO in newly diagnosed T2D and morbidly obese patients.
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Adenylosuccinate lyase (ADSL) functions in de novo purine synthesis (DNPS) and the purine nucleotide cycle. ADSL deficiency (ADSLD) causes numerous neurodevelopmental pathologies, including microcephaly and autism spectrum disorder. ADSLD patients have normal serum purine nucleotide levels but exhibit accumulation of dephosphorylated ADSL substrates, S-Ado, and SAICAr, the latter being implicated in neurotoxic effects through unknown mechanisms. We examined the phenotypic effects of ADSL depletion in human cells and their relation to phenotypic outcomes. Using specific interventions to compensate for reduced purine levels or modulate SAICAr accumulation, we found that diminished AMP levels resulted in increased DNA damage signaling and cell cycle delays, while primary ciliogenesis was impaired specifically by loss of ADSL or administration of SAICAr. ADSL-deficient chicken and zebrafish embryos displayed impaired neurogenesis and microcephaly. Neuroprogenitor attrition in zebrafish embryos was rescued by pharmacological inhibition of DNPS, but not increased nucleotide concentration. Zebrafish also displayed phenotypes commonly linked to ciliopathies. Our results suggest that both reduced purine levels and impaired DNPS contribute to neurodevelopmental pathology in ADSLD and that defective ciliogenesis may influence the ADSLD phenotypic spectrum.
Assuntos
Adenilossuccinato Liase/deficiência , Adenilossuccinato Liase/metabolismo , Transtorno Autístico/metabolismo , Neurogênese , Erros Inatos do Metabolismo da Purina-Pirimidina/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Animais , Transtorno do Espectro Autista/metabolismo , Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Galinhas/metabolismo , Ciliopatias/metabolismo , Dano ao DNA , Humanos , Microcefalia/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Fenótipo , Fosfoproteínas/metabolismo , Purinas/metabolismo , Ribonucleotídeos/metabolismo , Peixe-Zebra/metabolismoRESUMO
Mitochondria constantly undergo fusion and fission events, referred as mitochondrial dynamics, which determine mitochondrial architecture and bioenergetics. Cultured cell studies demonstrate that mitochondrial dynamics are acutely regulated by phosphorylation of the mitochondrial fission orchestrator dynamin-related protein 1 (Drp1) at S579 or S600. However, the physiological impact and crosstalk of these phosphorylation sites is poorly understood. Here, we describe the functional interrelation between S579 and S600 phosphorylation sites in vivo and their role on mitochondrial remodeling. Mice carrying a homozygous Drp1 S600A knockin (Drp1 KI) mutation display larger mitochondria and enhanced lipid oxidation and respiratory capacities, granting improved glucose tolerance and thermogenic response upon high-fat feeding. Housing mice at thermoneutrality blunts these differences, suggesting a role for the brown adipose tissue in the protection of Drp1 KI mice against metabolic damage. Overall, we demonstrate crosstalk between Drp1 phosphorylation sites and provide evidence that their modulation could be used in the treatment and prevention of metabolic diseases.
Assuntos
Tecido Adiposo Marrom/metabolismo , Dinaminas/metabolismo , Metabolismo dos Lipídeos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Animais , Dinaminas/genética , Camundongos , Camundongos Knockout , Mitocôndrias/genética , Mutação , Oxirredução , FosforilaçãoRESUMO
Impaired HDL-mediated macrophage cholesterol efflux and higher circulating concentrations of trimethylamine N-oxide (TMAO) levels are independent risk factors for cardiovascular mortality. The TMAO precursors, γ-butyrobetaine (γBB) and Trimethyllysine (TML), have also been recently associated with cardiovascular death, but their interactions with HDL-mediated cholesterol efflux remain unclear. We aimed to determine the associations between APOB depleted plasma-mediated macrophage cholesterol efflux and plasma TMAO, γBB, and TML concentrations and explore their association with two-year follow-up mortality in patients with acute ST-elevation myocardial infarction (STEMI) and unstable angina (UA). Baseline and ATP-binding cassette transporter ABCA1 and ABCG1 (ABCA1/G1)-mediated macrophage cholesterol efflux to APOB-depleted plasma was decreased in patients with STEMI, and the latter was further impaired in those who died during follow-up. Moreover, the circulating concentrations of TMAO, γBB, and TML were higher in the deceased STEMI patients when compared with the STEMI survivors or UA patients. However, after statistical adjustment, only ABCA1/G1-mediated macrophage cholesterol efflux remained significantly associated with mortality. Furthermore, neither the TMAO, γBB, nor TML levels altered the HDL-mediated macrophage cholesterol efflux in vitro. We conclude that impaired ABCA1/G1-mediated macrophage cholesterol efflux is independently associated with mortality at follow-up in STEMI patients.
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For millions of years, endogenous retroelements have remained transcriptionally silent within mammalian genomes by epigenetic mechanisms. Modern anticancer therapies targeting the epigenetic machinery awaken retroelement expression, inducing antiviral responses that eliminate tumors through mechanisms not completely understood. Here, we find that massive binding of epigenetically activated retroelements by RIG-I and MDA5 viral sensors promotes ATP hydrolysis and depletes intracellular energy, driving tumor killing independently of immune signaling. Energy depletion boosts compensatory ATP production by switching glycolysis to mitochondrial oxidative phosphorylation, thereby reversing the Warburg effect. However, hyperfunctional succinate dehydrogenase in mitochondrial electron transport chain generates excessive oxidative stress that unleashes RIP1-mediated necroptosis. To maintain ATP generation, hyperactive mitochondrial membrane blocks intrinsic apoptosis by increasing BCL2 dependency. Accordingly, drugs targeting BCL2 family proteins and epigenetic inhibitors yield synergistic responses in multiple cancer types. Thus, epigenetic therapy kills cancer cells by rewiring mitochondrial metabolism upon retroelement activation, which primes mitochondria to apoptosis by BH3-mimetics. SIGNIFICANCE: The state of viral mimicry induced by epigenetic therapies in cancer cells remodels mitochondrial metabolism and drives caspase-independent tumor cell death, which sensitizes to BCL2 inhibitor drugs. This novel mechanism underlies clinical efficacy of hypomethylating agents and venetoclax in acute myeloid leukemia, suggesting similar combination therapies for other incurable cancers.This article is highlighted in the In This Issue feature, p. 995.
Assuntos
Antineoplásicos/farmacologia , Epigênese Genética/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , HumanosRESUMO
Inhibition of mTOR is the standard of care for lymphangioleiomyomatosis (LAM). However, this therapy has variable tolerability and some patients show progressive decline of lung function despite treatment. LAM diagnosis and monitoring can also be challenging due to the heterogeneity of symptoms and insufficiency of non-invasive tests. Here, we propose monoamine-derived biomarkers that provide preclinical evidence for novel therapeutic approaches. The major histamine-derived metabolite methylimidazoleacetic acid (MIAA) is relatively more abundant in LAM plasma, and MIAA values are independent of VEGF-D. Higher levels of histamine are associated with poorer lung function and greater disease burden. Molecular and cellular analyses, and metabolic profiling confirmed active histamine signaling and metabolism. LAM tumorigenesis is reduced using approved drugs targeting monoamine oxidases A/B (clorgyline and rasagiline) or histamine H1 receptor (loratadine), and loratadine synergizes with rapamycin. Depletion of Maoa or Hrh1 expression, and administration of an L-histidine analog, or a low L-histidine diet, also reduce LAM tumorigenesis. These findings extend our knowledge of LAM biology and suggest possible ways of improving disease management.
Assuntos
Neoplasias Pulmonares , Linfangioleiomiomatose , Biomarcadores , Histamina , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Linfangioleiomiomatose/tratamento farmacológico , Transdução de SinaisRESUMO
BACKGROUND: The bicuspid aortic valve (BAV) is the most common cardiac congenital disease and is associated with an increased risk of developing ascending aorta dilation; which can have fatal consequences. Currently; no established risk biomarkers exist to facilitate the diagnosis and prognosis of BAV. METHODS: Using an untargeted metabolomic approach; we identified the levels of metabolites in plasma samples and compared them depending on the bicuspid or tricuspid morphology of the aortic valve. Including those patients with ascending aortic dilation and/or aortic stenosis (n = 212), we analyzed the role possibly played by alpha-Tocopherol in BAV disease; considering its association with the pathophysiological characteristics of BAV and biomarkers related to inflammation, oxidative stress and endothelial damage, as well as characteristics related to alpha-Tocopherol functionality and metabolism. RESULTS: We found that BAV patients; especially those with ascending aortic dilation; presented lower antioxidant capacity; as determined by decreased plasma levels of alpha-Tocopherol; paraoxonase 1 and high-density lipoprotein (HDL), as well as increased levels of C-reactive protein (CRP; a biomarker of inflammation) and endothelial microparticles (EMPs; an endothelial damage biomarker). By applying random forest analyses; we evaluated the significant screening capacity of alpha-Tocopherol; CRP and EMPs to classify patients depending on the morphology of the aortic valve. DISCUSSION: Our findings support the role of decreased antioxidant capacity; increased inflammation and endothelial damage in the pathogenesis of BAV and the progression of aortic dilation. Moreover; determining the plasma levels of alpha-Tocopherol; CRP and EMPs could improve BAV diagnosis in large populations.
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An imbalance between hepatic fatty acid uptake and removal results in ectopic fat accumulation, which leads to non-alcoholic fatty liver disease (NAFLD). The amount and type of accumulated triglycerides seem to play roles in NAFLD progression; however, a complete understanding of how triglycerides contribute to NAFLD evolution is lacking. Our aim was to evaluate triglyceride accumulation in NAFLD in a murine model and its associations with molecular mechanisms involved in liver damage and adipose tissue-liver cross talk by employing lipidomic and molecular imaging techniques. C57BL/6J mice fed a high-fat diet (HFD) for 12 weeks were used as a NAFLD model. Standard-diet (STD)-fed animals were used as controls. Standard liver pathology was assessed using conventional techniques. The liver lipidome was analyzed by liquid chromatography-mass spectrometry (LC-MS) and laser desorption/ionization-mass spectrometry (LDI-MS) tissue imaging. Liver triglycerides were identified by MS/MS. The transcriptome of genes involved in intracellular lipid metabolism and inflammation was assessed by RT-PCR. Plasma leptin, resistin, adiponectin, and FABP4 levels were determined using commercial kits. HFD-fed mice displayed increased liver lipid content. LC-MS analyses identified 14 triglyceride types that were upregulated in livers from HFD-fed animals. Among these 14 types, 10 were identified in liver cross sections by LDI-MS tissue imaging. The accumulation of these triglycerides was associated with the upregulation of lipogenesis and inflammatory genes and the downregulation of ß-oxidation genes. Interestingly, the levels of plasma FABP4, but not of other adipokines, were positively associated with 8 of these triglycerides in HFD-fed mice but not in STD-fed mice. Our findings suggest a putative role of FABP4 in the liver-adipose tissue cross talk in NAFLD.
Assuntos
Fígado/química , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/sangue , Hepatopatia Gordurosa não Alcoólica/genética , Adipocinas/sangue , Adiponectina/metabolismo , Tecido Adiposo/metabolismo , Animais , Cromatografia Líquida , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Leptina/metabolismo , Metabolismo dos Lipídeos/genética , Lipidômica/métodos , Masculino , Camundongos Endogâmicos C57BL , Imagem Molecular , Hepatopatia Gordurosa não Alcoólica/induzido quimicamente , Resistina/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Triglicerídeos/metabolismoRESUMO
We report the in vivo regulation of Inosine-5´-monophosphate dehydrogenase 1 (IMPDH1) in the retina. IMPDH1 catalyzes the rate-limiting step in the de novo synthesis of guanine nucleotides, impacting the cellular pools of GMP, GDP and GTP. Guanine nucleotide homeostasis is central to photoreceptor cells, where cGMP is the signal transducing molecule in the light response. Mutations in IMPDH1 lead to inherited blindness. We unveil a light-dependent phosphorylation of retinal IMPDH1 at Thr159/Ser160 in the Bateman domain that desensitizes the enzyme to allosteric inhibition by GDP/GTP. When exposed to bright light, living mice increase the rate of GTP and ATP synthesis in their retinas; concomitant with IMPDH1 aggregate formation at the outer segment layer. Inhibiting IMPDH activity in living mice delays rod mass recovery. We unveil a novel mechanism of regulation of IMPDH1 in vivo, important for understanding GTP homeostasis in the retina and the pathogenesis of adRP10 IMPDH1 mutations.
Assuntos
Guanosina Trifosfato/biossíntese , IMP Desidrogenase/genética , Luz , Processamento de Proteína Pós-Traducional , Retina/metabolismo , Retina/efeitos da radiação , Trifosfato de Adenosina/biossíntese , Animais , Fenômenos Bioquímicos , Regulação da Expressão Gênica , Homeostase , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Fosforilação , Estimulação Luminosa , Células Fotorreceptoras/fisiologiaRESUMO
Mitochondrial metabolism and the generation of reactive oxygen species (ROS) contribute to the acquisition of DNA mutations and genomic instability in cancer. How genomic instability influences the metabolic capacity of cancer cells is nevertheless poorly understood. Here, we show that homologous recombination-defective (HRD) cancers rely on oxidative metabolism to supply NAD+ and ATP for poly(ADP-ribose) polymerase (PARP)-dependent DNA repair mechanisms. Studies in breast and ovarian cancer HRD models depict a metabolic shift that includes enhanced expression of the oxidative phosphorylation (OXPHOS) pathway and its key components and a decline in the glycolytic Warburg phenotype. Hence, HRD cells are more sensitive to metformin and NAD+ concentration changes. On the other hand, shifting from an OXPHOS to a highly glycolytic metabolism interferes with the sensitivity to PARP inhibitors (PARPi) in these HRD cells. This feature is associated with a weak response to PARP inhibition in patient-derived xenografts, emerging as a new mechanism to determine PARPi sensitivity. This study shows a mechanistic link between two major cancer hallmarks, which in turn suggests novel possibilities for specifically treating HRD cancers with OXPHOS inhibitors.
Assuntos
Neoplasias Ovarianas , Inibidores de Poli(ADP-Ribose) Polimerases , Carcinoma Epitelial do Ovário , Feminino , Recombinação Homóloga , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Estresse Oxidativo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologiaRESUMO
Multinucleated giant cells (MGCs) are implicated in many diseases including schistosomiasis, sarcoidosis and arthritis. MGC generation is energy intensive to enforce membrane fusion and cytoplasmic expansion. Using receptor activator of nuclear factor kappa-Β ligand (RANKL) induced osteoclastogenesis to model MGC formation, here we report RANKL cellular programming requires extracellular arginine. Systemic arginine restriction improves outcome in multiple murine arthritis models and its removal induces preosteoclast metabolic quiescence, associated with impaired tricarboxylic acid (TCA) cycle function and metabolite induction. Effects of arginine deprivation on osteoclastogenesis are independent of mTORC1 activity or global transcriptional and translational inhibition. Arginine scarcity also dampens generation of IL-4 induced MGCs. Strikingly, in extracellular arginine absence, both cell types display flexibility as their formation can be restored with select arginine precursors. These data establish how environmental amino acids control the metabolic fate of polykaryons and suggest metabolic ways to manipulate MGC-associated pathologies and bone remodelling.
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Arginina/metabolismo , Células Gigantes/imunologia , Animais , Artrite/genética , Artrite/metabolismo , Artrite/fisiopatologia , Remodelação Óssea , Ciclo do Ácido Cítrico , Feminino , Células Gigantes/citologia , Humanos , Interleucina-4/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Osteoclastos/metabolismo , Osteogênese , Ligante RANK/genética , Ligante RANK/metabolismoRESUMO
CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.
Assuntos
Aminoácidos/metabolismo , Ciclo Celular , Cadeia Pesada da Proteína-1 Reguladora de Fusão/metabolismo , Nucleotídeos/metabolismo , Aminoácidos Aromáticos/metabolismo , Aminoácidos de Cadeia Ramificada/metabolismo , Divisão Celular , Dano ao DNA , Reparo do DNA , Cadeia Pesada da Proteína-1 Reguladora de Fusão/fisiologia , Perfilação da Expressão Gênica , Técnicas de Inativação de Genes , Humanos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Estresse OxidativoRESUMO
The endoplasmic reticulum (ER) of cancer cells needs to adapt to the enhanced proteotoxic stress associated with the accumulation of unfolded, misfolded and transformation-associated proteins. One way by which tumors thrive in the context of ER stress is by promoting ER-Associated Degradation (ERAD), although the mechanisms are poorly understood. Here, we show that the Small p97/VCP Interacting Protein (SVIP), an endogenous inhibitor of ERAD, undergoes DNA hypermethylation-associated silencing in tumorigenesis to achieve this goal. SVIP exhibits tumor suppressor features and its recovery is associated with increased ER stress and growth inhibition. Proteomic and metabolomic analyses show that cancer cells with epigenetic loss of SVIP are depleted in mitochondrial enzymes and oxidative respiration activity. This phenotype is reverted upon SVIP restoration. The dependence of SVIP hypermethylated cancer cells on aerobic glycolysis and glucose was also associated with sensitivity to an inhibitor of the glucose transporter GLUT1. This could be relevant to the management of tumors carrying SVIP epigenetic loss, because these occur in high-risk patients who manifest poor clinical outcomes. Overall, our study provides insights into how epigenetics helps deal with ER stress and how SVIP epigenetic loss in cancer may be amenable to therapies that target glucose transporters.