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1.
EMBO J ; 42(4): e110620, 2023 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-36637036

RESUMO

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine-resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain-of-function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif-1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine-resistant UC cells. Interestingly, IDH2-mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2-mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo-resistant urothelial carcinoma.


Assuntos
Carcinoma de Células de Transição , Neoplasias da Bexiga Urinária , Humanos , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genética , Gencitabina , Glicólise , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Via de Pentose Fosfato , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/genética
2.
J Clin Biochem Nutr ; 70(1): 54-63, 2022 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-35068682

RESUMO

The liver has been thought to protect against oxidative stress through mechanisms involving reduced glutathione (GSH) that consumes high-energy phosphor-nucleotides on its synthesis. However, hepatoprotective mechanisms in acute liver failure (ALF) where the phosphor-nucleotides are decreased in remain to be solved. Liver tissues were collected from patients with ALF and liver cirrhosis (LC) and living donors (HD) who had undergone liver transplantation. Tissues were used for metabolomic analyses to determine metabolites belonging to the central carbon metabolism, and to determine sulfur-containing metabolites. ALF and LC exhibited a significant decline in metabolites of glycolysis and pentose phosphate pathways and high-energy phosphor-nucleotides such as adenosine triphosphate as compared with HD. Conversely, methionine, S-adenosyl-l-methionine, and the ratio of serine to 3-phosphoglycerate were elevated significantly in ALF as compared with LC and HD, suggesting a metabolic boost from glycolysis towards trans-sulfuration. Notably in ALF, the increases in hypotaurine (HTU) + taurine (TU) coincided with decreases in the total amounts of reduced and oxidized glutathione (GSH + 2GSSG). Plasma NH3 levels correlated with the ratio of HTU + TU to GSH + 2GSSG. Increased tissue levels of HTU + TU vs total glutathione appear to serve as a biomarker correlating with hyperammonemia, suggesting putative roles of the HTU-TU pathway in anti-oxidative protective mechanisms.

3.
Clin Chem ; 64(10): 1463-1473, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30021922

RESUMO

BACKGROUND: Although circulating exosomes in blood play crucial roles in cancer development and progression, difficulties in quantifying exosomes hamper their application for reliable clinical testing. By combining the properties of nanobeads with optical disc technology, we have developed a novel device named the ExoCounter to determine the exact number of exosomes in the sera of patients with various types of cancer. METHOD: In this system, individual exosomes were captured in the groove of an optical disc coated with antibodies against exosome surface antigens. The captured exosomes were labeled with antibody-conjugated magnetic nanobeads, and the number of the labeled exosomes was counted with an optical disc drive. RESULTS: We showed that the ExoCounter could detect specific exosomes derived from cells or human serum without any enrichment procedures. The detection sensitivity and linearity with this system were higher than those with conventional detection methods such as ELISA or flow cytometry. In addition to the ubiquitous exosome markers CD9 and CD63, the cancer-related antigens CD147, carcinoembryonic antigen, and human epidermal growth factor receptor 2 (HER2) were also used to quantify cancer cell line-derived exosomes. Furthermore, analyses of a cross-sectional cohort of sera samples revealed that HER2-positive exosomes were significantly increased in patients with breast cancer or ovarian cancer compared with healthy individuals and those with noncancer diseases. CONCLUSIONS: The ExoCounter system exhibits high performance in the direct detection of exosomes in cell culture and human sera. This method may enable reliable analysis of liquid biopsies.


Assuntos
Antígenos de Neoplasias/sangue , Biomarcadores Tumorais/sangue , Exossomos , Dispositivos Lab-On-A-Chip , Procedimentos Analíticos em Microchip/métodos , Neoplasias/sangue , Células A549 , Exossomos/imunologia , Células HCT116 , Células HEK293 , Humanos , Sensibilidade e Especificidade , Coloração e Rotulagem , Tetraspanina 30/imunologia
4.
Proc Natl Acad Sci U S A ; 112(37): 11642-7, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26324945

RESUMO

Loss of prolyl hydroxylase 2 (PHD2) activates the hypoxia-inducible factor-dependent hypoxic response, including anaerobic glycolysis, which causes large amounts of lactate to be released from cells into the circulation. We found that Phd2-null mouse embryonic fibroblasts (MEFs) produced more lactate than wild-type MEFs, as expected, whereas systemic inactivation of PHD2 in mice did not cause hyperlacticacidemia. This unexpected observation led us to hypothesize that the hypoxic response activated in the liver enhances the Cori cycle, a lactate-glucose carbon recycling system between muscle and liver, and thereby decreases circulating lactate. Consistent with this hypothesis, blood lactate levels measured after a treadmill or lactate tolerance test were significantly lower in Phd2-liver-specific knockout (Phd2-LKO) mice than in control mice. An in vivo (13)C-labeled lactate incorporation assay revealed that the livers of Phd2-LKO mice produce significantly more glucose derived from (13)C-labeled lactate than control mice, suggesting that blockade of PHD2 in the liver ameliorates lactic acidosis by activating gluconeogenesis from lactate. Phd2-LKO mice were resistant to lactic acidosis induced by injection of a lethal dose of lactate, displaying a significant elongation of survival. Moreover, oral administration of a PHD inhibitor improved survival in an endotoxin shock mice model. These data suggest that PHD2 is a potentially novel drug target for the treatment of lactic acidosis, which is a serious and often fatal complication observed in some critically ill patients.


Assuntos
Acidose Láctica/metabolismo , Prolina Dioxigenases do Fator Induzível por Hipóxia/metabolismo , Fígado/metabolismo , Oxigênio/metabolismo , Animais , Gasometria , Glicemia/metabolismo , Genótipo , Hepatócitos/metabolismo , Hipóxia/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Lactatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Condicionamento Físico Animal , Sepse/metabolismo
5.
J Neuroinflammation ; 13(1): 99, 2016 May 04.
Artigo em Inglês | MEDLINE | ID: mdl-27143001

RESUMO

BACKGROUND: Toll-like receptor 4 (TLR4) plays a pivotal role in the pathophysiology of stroke-induced inflammation. Both astroglia and microglia express TLR4, and endogenous ligands produced in the ischemic brain induce inflammatory responses. Reactive oxygen species (ROS), nitric oxide (NO), and inflammatory cytokines produced by TLR4 activation play harmful roles in neuronal damage after stroke. Although astroglia exhibit pro-inflammatory responses upon TLR4 stimulation by lipopolysaccharide (LPS), they may also play cytoprotective roles via the activation of the pentose phosphate pathway (PPP), reducing oxidative stress by glutathione peroxidase. We investigated the mechanisms by which astroglia reduce oxidative stress via the activation of PPP, using TLR4 stimulation and hypoxia in concert with microglia. METHODS: In vitro experiments were performed using cells prepared from Sprague-Dawley rats. Coexisting microglia in the astroglial culture were chemically eliminated using L-leucine methyl ester (LME). Cells were exposed to LPS (0.01 µg/mL) or hypoxia (1 % O2) for 12-15 h. PPP activity was measured using [1-(14)C]glucose and [6-(14)C]glucose. ROS and NO production were measured using 2',7'-dichlorodihydrofluorescein diacetate and diaminofluorescein-FM diacetate, respectively. The involvement of nuclear factor-erythroid-2-related factor 2 (Nrf2), a cardinal transcriptional factor under stress conditions that regulates glucose 6-phosphate dehydrogenase, the rate-limiting enzyme of PPP, was evaluated using immunohistochemistry. RESULTS: Cultured astroglia exposed to LPS elicited 20 % increases in PPP flux, and these actions of astroglia appeared to involve Nrf2. However, the chemical depletion of coexisting microglia eliminated both increases in PPP and astroglial nuclear translocation of Nrf2. LPS induced ROS and NO production in the astroglial culture containing microglia but not in the microglia-depleted astroglial culture. LPS enhanced astroglial ROS production after glutathione depletion. U0126, an upstream inhibitor of mitogen-activated protein kinase, eliminated LPS-induced NO production, whereas ROS production was unaffected. U0126 also eliminated LPS-induced PPP activation in astroglial-microglial culture, indicating that microglia-derived NO mediated astroglial PPP activation. Hypoxia induced astroglial PPP activation independent of the microglia-NO pathway. Elimination of ROS and NO production by sulforaphane, a natural Nrf2 activator, confirmed the astroglial protective mechanism. CONCLUSIONS: Astroglia in concert with microglia may play a cytoprotective role for countering oxidative stress in stroke.


Assuntos
Astrócitos/metabolismo , Microglia/metabolismo , Óxido Nítrico/metabolismo , Estresse Oxidativo/fisiologia , Acidente Vascular Cerebral/metabolismo , Animais , Western Blotting , Células Cultivadas , Modelos Animais de Doenças , Imuno-Histoquímica , Técnicas In Vitro , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lipopolissacarídeos/toxicidade , Fator 2 Relacionado a NF-E2/fisiologia , Via de Pentose Fosfato/fisiologia , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Espectrometria de Massas por Ionização por Electrospray
6.
J Mol Cell Cardiol ; 82: 116-24, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25744081

RESUMO

Dichloroacetate (DCA) promotes pyruvate entry into the Krebs cycle by inhibiting pyruvate dehydrogenase (PDH) kinase and thereby maintaining PDH in the active dephosphorylated state. DCA has recently gained attention as a potential metabolic-targeting therapy for heart failure but the molecular basis of the therapeutic effect of DCA in the heart remains a mystery. Once-daily oral administration of DCA alleviates pressure overload-induced left ventricular remodeling. We examined changes in the metabolic fate of pyruvate carbon (derived from glucose) entering the Krebs cycle by metabolic interventions of DCA. (13)C6-glucose pathway tracing analysis revealed that instead of being completely oxidized in the mitochondria for ATP production, DCA-mediated PDH dephosphorylation results in an increased acetyl-CoA pool both in control and pressure-overloaded hearts. DCA induces hyperacetylation of histone H3K9 and H4 in a dose-dependent manner in parallel to the dephosphorylation of PDH in cultured cardiomyocytes. DCA administration increases histone H3K9 acetylation in in vivo mouse heart. Interestingly, DCA-dependent histone acetylation was associated with an up-regulation of 2.3% of genes (545 out of 23,474 examined). Gene ontology analysis revealed that these genes are highly enriched in transcription-related categories. This evidence suggests that sustained activation of PDH by DCA results in an overproduction of acetyl-CoA, which exceeds oxidation in the Krebs cycle and results in histone acetylation. We propose that DCA-mediated PDH activation has the potential to induce epigenetic remodeling in the heart, which, at least in part, forms the molecular basis for the therapeutic effect of DCA in the heart.


Assuntos
Ácido Dicloroacético/farmacologia , Epigênese Genética/efeitos dos fármacos , Complexo Piruvato Desidrogenase/metabolismo , Remodelação Ventricular/efeitos dos fármacos , Remodelação Ventricular/genética , Ácido 3-Hidroxibutírico/metabolismo , Acetilcoenzima A/metabolismo , Acetilação , Animais , Regulação da Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Histonas/metabolismo , Masculino , Metaboloma , Metabolômica/métodos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Miocárdio/metabolismo , Especificidade de Órgãos/genética , Fosforilação , Complexo Piruvato Desidrogenase/farmacologia , Ratos , Transcrição Gênica
7.
Nitric Oxide ; 46: 102-13, 2015 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-25461272

RESUMO

CD44 expressed in cancer cells was shown to stabilize cystine transporter (xCT) that uptakes cystine and excretes glutamate to supply cysteine as a substrate for reduced glutathione (GSH) for survival. While targeting CD44 serves as a potentially therapeutic stratagem to attack cancer growth and chemoresistance, the impact of CD44 targeting in cancer cells on metabolic systems of tumors and host tissues in vivo remains to be fully determined. This study aimed to reveal effects of CD44 silencing on alterations in energy metabolism and sulfur-containing metabolites in vitro and in vivo using capillary electrophoresis-mass spectrometry and quantitative imaging mass spectrometry (Q-IMS), respectively. In an experimental model of xenograft transplantation of human colon cancer HCT116 cells in superimmunodeficient NOG mice, snap-frozen liver tissues containing metastatic tumors were examined by Q-IMS. As reported previously, short hairpin CD44 RNA interference (shCD44) in cancer cells caused significant regression of tumor growth in the host liver. Under these circumstances, the CD44 knockdown suppressed polyamines, GSH and energy charges not only in metastatic tumors but also in the host liver. In culture, HCT116 cells treated with shCD44 decreased total amounts of methionine-pool metabolites including spermidine and spermine, and reactive cysteine persulfides, suggesting roles of these metabolites for cancer growth. Collectively, these results suggest that CD44 expressed in cancer accounts for a key regulator of metabolic interplay between tumor and the host tissue.


Assuntos
Cisteína/metabolismo , Receptores de Hialuronatos/metabolismo , Metaboloma/fisiologia , Neoplasias/metabolismo , Animais , Técnicas de Silenciamento de Genes , Células HCT116 , Humanos , Receptores de Hialuronatos/genética , Fígado/química , Fígado/metabolismo , Masculino , Espectrometria de Massas , Metaboloma/efeitos dos fármacos , Metaboloma/genética , Metabolômica , Camundongos , Neoplasias/genética , Interferência de RNA , RNA Interferente Pequeno/farmacologia , Transplante Heterólogo
8.
Proc Natl Acad Sci U S A ; 109(4): 1293-8, 2012 Jan 24.
Artigo em Inglês | MEDLINE | ID: mdl-22232681

RESUMO

Enhancement of cerebral blood flow by hypoxia is critical for brain function, but signaling systems underlying its regulation have been unclear. We report a pathway mediating hypoxia-induced cerebral vasodilation in studies monitoring vascular disposition in cerebellar slices and in intact mouse brains using two-photon intravital laser scanning microscopy. In this cascade, hypoxia elicits cerebral vasodilation via the coordinate actions of H(2)S formed by cystathionine ß-synthase (CBS) and CO generated by heme oxygenase (HO)-2. Hypoxia diminishes CO generation by HO-2, an oxygen sensor. The constitutive CO physiologically inhibits CBS, and hypoxia leads to increased levels of H(2)S that mediate the vasodilation of precapillary arterioles. Mice with targeted deletion of HO-2 or CBS display impaired vascular responses to hypoxia. Thus, in intact adult brain cerebral cortex of HO-2-null mice, imaging mass spectrometry reveals an impaired ability to maintain ATP levels on hypoxia.


Assuntos
Monóxido de Carbono/metabolismo , Cérebro/irrigação sanguínea , Sulfeto de Hidrogênio/metabolismo , Hipóxia/fisiopatologia , Microcirculação/fisiologia , Fluxo Sanguíneo Regional/fisiologia , Vasodilatação/fisiologia , Ácido 15-Hidroxi-11 alfa,9 alfa-(epoximetano)prosta-5,13-dienoico , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Western Blotting , Cistationina beta-Sintase/metabolismo , Primers do DNA/genética , Heme Oxigenase (Desciclizante)/genética , Heme Oxigenase (Desciclizante)/metabolismo , Imuno-Histoquímica , Espectrometria de Massas , Camundongos , Microscopia Confocal
9.
Am J Physiol Renal Physiol ; 306(12): F1462-76, 2014 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-24761004

RESUMO

The kidney is one of the major loci for the expression of cystathionine ß-synthase (CBS) and cystathionine γ-lyase (CTH). While CBS-deficient (Cbs(-/-)) mice display homocysteinemia/methioninemia and severe growth retardation, and rarely survive beyond the first 4 wk, CTH-deficient (Cth(-/-)) mice show homocysteinemia/cystathioninemia but develop with no apparent abnormality. This study examined renal amino acid reabsorption in those mice. Although both 2-wk-old Cbs(-/-) and Cth(-/-) mice had normal renal architecture, their serum/urinary amino acid profiles largely differed from wild-type mice. The most striking feature was marked accumulation of Met and cystathionine in serum/urine/kidney samples of Cbs(-/-) and Cth(-/-) mice, respectively. Levels of some neutral amino acids (Val, Leu, Ile, and Tyr) that were not elevated in Cbs(-/-) serum were highly elevated in Cbs(-/-) urine, and urinary excretion of other neutral amino acids (except Met) was much higher than expected from their serum levels, demonstrating neutral aminoaciduria in Cbs(-/-) (not Cth(-/-)) mice. Because the bulk of neutral amino acids is absorbed via a B(0)AT1 transporter and Met has the highest substrate affinity for B(0)AT1 than other neutral amino acids, hypermethioninemia may cause hyperexcretion of neutral amino acids.


Assuntos
Aminoácidos Neutros/metabolismo , Cistationina beta-Sintase/deficiência , Homocistinúria/epidemiologia , Homocistinúria/metabolismo , Aminoacidúrias Renais/epidemiologia , Aminoacidúrias Renais/metabolismo , Animais , Comorbidade , Cistationina/metabolismo , Cistationina beta-Sintase/genética , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/deficiência , Cistationina gama-Liase/genética , Modelos Animais de Doenças , Feminino , Hiper-Homocisteinemia/metabolismo , Túbulos Renais Proximais/patologia , Masculino , Metionina/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
10.
Arterioscler Thromb Vasc Biol ; 33(11): 2549-57, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23968980

RESUMO

OBJECTIVE: Fatty acids (FAs) are the major substrate for energy production in the heart. Here, we hypothesize that capillary endothelial fatty acid binding protein 4 (FABP4) and FABP5 play an important role in providing sufficient FAs to the myocardium. APPROACH AND RESULTS: Both FABP4/5 were abundantly expressed in capillary endothelium in the heart and skeletal muscle. The uptake of a FA analogue, 125I-15-(p-iodophenyl)-3-(R,S)-methyl pentadecanoic acid, was significantly reduced in these tissues in double-knockout (DKO) mice for FABP4/5 compared with wild-type mice. In contrast, the uptake of a glucose analogue, 18F-fluorodeoxyglucose, was remarkably increased in DKO mice. The expression of transcripts for the oxidative catabolism of FAs was reduced during fasting, whereas transcripts for the glycolytic pathway were not altered in DKO hearts. Notably, metabolome analysis revealed that phosphocreatine and ADP levels were significantly lower in DKO hearts, whereas ATP content was kept at a normal level. The protein expression levels of the glucose transporter Glut4 and the phosphorylated form of phosphofructokinase-2 were increased in DKO hearts, whereas the phosphorylation of insulin receptor-ß and Akt was comparable between wild-type and DKO hearts during fasting, suggesting that a dramatic increase in glucose usage during fasting is insulin independent and is at least partly attributed to the post-transcriptional and allosteric regulation of key proteins that regulate glucose uptake and glycolysis. CONCLUSIONS: Capillary endothelial FABP4/5 are required for FA transport into FA-consuming tissues that include the heart. These findings identify FABP4/5 as promising targets for controlling the metabolism of energy substrates in FA-consuming organs that have muscle-type continuous capillary.


Assuntos
Metabolismo Energético/fisiologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Ácidos Graxos/metabolismo , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Proteínas de Neoplasias/metabolismo , Difosfato de Adenosina/metabolismo , Animais , Endotélio Vascular/metabolismo , Proteínas de Ligação a Ácido Graxo/genética , Ácidos Graxos/farmacocinética , Fluordesoxiglucose F18/farmacocinética , Iodobenzenos/farmacocinética , Camundongos , Camundongos Knockout , Proteínas de Neoplasias/genética , Fosfocreatina/metabolismo , Fosfofrutoquinase-2/metabolismo
11.
Cancer Res ; 84(7): 1065-1083, 2024 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-38383964

RESUMO

Triple-negative breast cancer (TNBC) chemoresistance hampers the ability to effectively treat patients. Identification of mechanisms driving chemoresistance can lead to strategies to improve treatment. Here, we revealed that protein arginine methyltransferase-1 (PRMT1) simultaneously methylates D-3-phosphoglycerate dehydrogenase (PHGDH), a critical enzyme in serine synthesis, and the glycolytic enzymes PFKFB3 and PKM2 in TNBC cells. 13C metabolic flux analyses showed that PRMT1-dependent methylation of these three enzymes diverts glucose toward intermediates in the serine-synthesizing and serine/glycine cleavage pathways, thereby accelerating the production of methyl donors in TNBC cells. Mechanistically, PRMT1-dependent methylation of PHGDH at R54 or R20 activated its enzymatic activity by stabilizing 3-phosphoglycerate binding and suppressing polyubiquitination. PRMT1-mediated PHGDH methylation drove chemoresistance independently of glutathione synthesis. Rather, activation of the serine synthesis pathway supplied α-ketoglutarate and citrate to increase palmitate levels through activation of fatty acid synthase (FASN). Increased palmitate induced protein S-palmitoylation of PHGDH and FASN to further enhance fatty acid synthesis in a PRMT1-dependent manner. Loss of PRMT1 or pharmacologic inhibition of FASN or protein S-palmitoyltransferase reversed chemoresistance in TNBC. Furthermore, IHC coupled with imaging MS in clinical TNBC specimens substantiated that PRMT1-mediated methylation of PHGDH, PFKFB3, and PKM2 correlates with chemoresistance and that metabolites required for methylation and fatty acid synthesis are enriched in TNBC. Together, these results suggest that enhanced de novo fatty acid synthesis mediated by coordinated protein arginine methylation and protein S-palmitoylation is a therapeutic target for overcoming chemoresistance in TNBC. SIGNIFICANCE: PRMT1 promotes chemoresistance in TNBC by methylating metabolic enzymes PFKFB3, PKM2, and PHGDH to augment de novo fatty acid synthesis, indicating that targeting this axis is a potential treatment strategy.


Assuntos
Fosfoglicerato Desidrogenase , Neoplasias de Mama Triplo Negativas , Humanos , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Resistencia a Medicamentos Antineoplásicos , Serina/metabolismo , Palmitatos , Ácidos Graxos , Linhagem Celular Tumoral , Proteína-Arginina N-Metiltransferases/genética , Proteínas Repressoras
12.
Mol Genet Metab Rep ; 35: 100968, 2023 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-36974075

RESUMO

The pyruvate dehydrogenase complex serves as the main connection between cytosolic glycolysis and the tricarboxylic acid cycle within mitochondria. An infant with pyruvate dehydrogenase complex deficiency was treated with vitamin B1 supplementation and a ketogenic diet. These dietary modifications resolved the renal tubular reabsorption, central apnea, and transfusion-dependent anemia. A concurrent metabolome analysis demonstrated the resolution of the amino aciduria and an increased total amount of substrates in the tricarboxylic acid cycle, reflecting the improved mitochondrial energetics. Glutamate was first detected in the cerebrospinal fluid, accompanied by a clinical improvement, after the ketogenic ratio was increased to 3:1; thus, glutamate levels in cerebrospinal fluid may represent a biomarker for neuronal recovery. Metabolomic analyses of body fluids are useful for monitoring therapeutic effects in infants with inborn errors of carbohydrate metabolism.

13.
Antioxidants (Basel) ; 12(2)2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36829799

RESUMO

Pathological examination of formalin-fixed paraffin-embedded (FFPE) needle-biopsied samples by certified pathologists represents the gold standard for differential diagnosis between ductal carcinoma in situ (DCIS) and invasive breast cancers (IBC), while information of marker metabolites in the samples is lost in the samples. Infrared laser-scanning large-area surface-enhanced Raman spectroscopy (SERS) equipped with gold-nanoparticle-based SERS substrate enables us to visualize metabolites in fresh-frozen needle-biopsied samples with spatial matching between SERS and HE staining images with pathological annotations. DCIS (n = 14) and IBC (n = 32) samples generated many different SERS peaks in finger-print regions of SERS spectra among pathologically annotated lesions including cancer cell nests and the surrounding stroma. The results showed that SERS peaks in IBC stroma exhibit significantly increased polysulfide that coincides with decreased hypotaurine as compared with DCIS, suggesting that alterations of these redox metabolites account for fingerprints of desmoplastic reactions to distinguish IBC from DCIS. Furthermore, the application of supervised machine learning to the stroma-specific multiple SERS signals enables us to support automated differential diagnosis with high accuracy. The results suggest that SERS-derived biochemical fingerprints derived from redox metabolites account for a hallmark of desmoplastic reaction of IBC that is absent in DCIS, and thus, they serve as a useful method for precision diagnosis in breast cancer.

14.
Biochem Biophys Res Commun ; 415(3): 445-9, 2011 Nov 25.
Artigo em Inglês | MEDLINE | ID: mdl-22051049

RESUMO

The liver plays a central role in glucose homeostasis in the whole-body by responding to environmental factors including nutrients, hormones, and oxygen. In conditions of metabolic overload such as diabetes mellitus and obesity, coordinated regulation between oxygen supply and consumption has been reported to be disrupted and subsequently cause tissue hypoxia, although pathological significance of the disease-related hypoxia remains elusive. To investigate the role of tissue hypoxia in the liver on systemic glucose homeostasis, mice lacking HIF-1α gene, a critical component of a master regulator of hypoxic response, in hepatocytes were exposed to high fat/sucrose diet (HFSD). Exposure to HFSD for 5 weeks elicited liver hypoxia with a transient increase in HIF-1α protein expression in the liver of control mice. Glucose disposal was marginally impaired in control mice when challenged oral glucose tolerance test, but such impairment was enhanced in the mutant mice. This alteration was accompanied by a complete inhibition of glucokinase induction with a significant reduction of hepatic glucose uptake. Mice fed HFSD for 20 weeks exhibited fasting hyperglycemia and glucose intolerance, whereas these metabolic phenotypes deteriorated considerably with severe insulin resistance in skeletal muscles and adipose tissues in the mutant mice. These findings suggest that HIF-1 in hepatocytes plays protective roles against the progression of diabetes mellitus.


Assuntos
Dieta Hiperlipídica/efeitos adversos , Glucose/metabolismo , Hepatócitos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Obesidade/metabolismo , Animais , Diabetes Mellitus/metabolismo , Teste de Tolerância a Glucose , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Resistência à Insulina , Camundongos , Camundongos Obesos , Obesidade/etiologia , Sacarose/administração & dosagem , Sacarose/efeitos adversos
15.
Circ Res ; 105(11): 1118-27, 2009 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-19815821

RESUMO

RATIONALE: Aldehyde accumulation is regarded as a pathognomonic feature of oxidative stress-associated cardiovascular disease. OBJECTIVE: We investigated how the heart compensates for the accelerated accumulation of aldehydes. METHODS AND RESULTS: Aldehyde dehydrogenase 2 (ALDH2) has a major role in aldehyde detoxification in the mitochondria, a major source of aldehydes. Transgenic (Tg) mice carrying an Aldh2 gene with a single nucleotide polymorphism (Aldh2*2) were developed. This polymorphism has a dominant-negative effect and the Tg mice exhibited impaired ALDH activity against a broad range of aldehydes. Despite a shift toward the oxidative state in mitochondrial matrices, Aldh2*2 Tg hearts displayed normal left ventricular function by echocardiography and, because of metabolic remodeling, an unexpected tolerance to oxidative stress induced by ischemia/reperfusion injury. Mitochondrial aldehyde stress stimulated eukaryotic translation initiation factor 2alpha phosphorylation. Subsequent translational and transcriptional activation of activating transcription factor-4 promoted the expression of enzymes involved in amino acid biosynthesis and transport, ultimately providing precursor amino acids for glutathione biosynthesis. Intracellular glutathione levels were increased 1.37-fold in Aldh2*2 Tg hearts compared with wild-type controls. Heterozygous knockout of Atf4 blunted the increase in intracellular glutathione levels in Aldh2*2 Tg hearts, thereby attenuating the oxidative stress-resistant phenotype. Furthermore, glycolysis and NADPH generation via the pentose phosphate pathway were activated in Aldh2*2 Tg hearts. (NADPH is required for the recycling of oxidized glutathione.) CONCLUSIONS: The findings of the present study indicate that mitochondrial aldehyde stress in the heart induces metabolic remodeling, leading to activation of the glutathione-redox cycle, which confers resistance against acute oxidative stress induced by ischemia/reperfusion.


Assuntos
Aldeído Desidrogenase/genética , Aldeído Desidrogenase/metabolismo , Aldeídos/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/metabolismo , Estresse Oxidativo/fisiologia , Fator 4 Ativador da Transcrição/metabolismo , Adaptação Fisiológica/fisiologia , Aldeído-Desidrogenase Mitocondrial , Animais , Modelos Animais de Doenças , Ecocardiografia , Ativação Enzimática/fisiologia , Indução Enzimática/fisiologia , Glucose/metabolismo , Glutationa/metabolismo , Metaboloma/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/fisiologia , Traumatismo por Reperfusão Miocárdica/diagnóstico por imagem , Traumatismo por Reperfusão Miocárdica/patologia , Miocárdio/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Via de Pentose Fosfato/fisiologia , Transcrição Gênica/fisiologia
16.
Anal Bioanal Chem ; 400(7): 1853-63, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21301820

RESUMO

Fatty liver is one of the typical manifestations in homocysteinemia/homocystinuria patients and their genetic animal model, mice lacking cystathionine ß-synthase (Cbs(-/-)). The vast majority of Cbs(-/-) die within 4 weeks after birth via yet unknown mechanisms, whereas a small portion survive to adulthood, escaping fatty degeneration of the liver during lactation periods, through regeneration. To investigate the molecular basis of such fatty changes, we analyzed lipid components in fatty livers of 2-week-old Cbs(-/-) and regenerated non-fatty livers of 8-week-old Cbs(-/-) survivors using a chip-based nanoESI (electrospray ionization)-MS system, which allows quantitative detection of triacylglycerol/phospholipid molecular species. Hepatic levels of all major triacylglycerol species were much higher in Cbs(-/-) than in wild-type mice at 2 weeks, although not at 8 weeks. Levels of some phospholipid species were either up- or downregulated in 2-week-old Cbs(-/-); e.g. saturated (16:0 and 18:0) or mono-unsaturated (16:1 and 18:1) fatty acids-containing phosphatidylcholine/phosphatidylethanolamine species were upregulated, while poly-unsaturated fatty acids-containing phosphatidylcholine (18:2-18:2 and 18:2-20:5), phosphatidylethanolamine (18:1-20:4), and phosphatidylinositol (18:0-20:4) were downregulated. Capillary electrophoresis-MS analysis identified high-level accumulation of S-adenosylmethionine and S-adenosylhomocysteine in fatty livers of 2-week-old Cbs(-/-) but much less in non-fatty livers of 8-week-old Cbs(-/-). Although hepatic S-adenosylmethionine/S-adenosylhomocysteine ratios were comparable between 2-week-old Cbs(-/-) and wild-type, global protein arginine methylation was disturbed in fatty livers of Cbs(-/-). Our results suggest that cellular signaling mediated by altered phospholipid contents might be involved in pathogenesis of fatty liver in Cbs(-/-).


Assuntos
Fígado Gorduroso/metabolismo , Homocisteína/sangue , Homocistinúria/metabolismo , Regeneração Hepática , Fosfolipídeos/metabolismo , Triglicerídeos/metabolismo , Animais , Western Blotting , Estudos de Casos e Controles , Modelos Animais de Doenças , Camundongos , Espectrometria de Massas por Ionização por Electrospray
17.
Curr Res Struct Biol ; 3: 192-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34485929

RESUMO

Human cytochromes P45011ß (CYP11B1) and P450aldo (CYP11B2) are monooxygenases that synthesize cortisol through steroid 11ß-hydroxylation and aldosterone through a three-step process comprising 11ß-hydroxylation and two 18-hydroxylations, respectively. CYP11B1 also catalyzes 18-monohydroxylation and 11ß,18-dihydroxylation. To study the molecular basis of such catalytic divergence of the two enzymes, we examined a CYP11B1 mutant (Mt-CYP11B1) with amino acid replacements on the distal surface by determining the catalytic activities and crystal structure in the metyrapone-bound form at 1.4-Å resolution. Mt-CY11B1 retained both 11ß-hydroxylase and 18-hydroxylase activities of the wild type (Wt-CYP11B1) but lacked 11ß,18-dihydroxylase activity. Comparisons of the crystal structure of Mt-CYP11B1 to those of Wt-CYP11B1 and CYP11B2 that were already reported show that the mutation reduced the innermost space putatively surrounding the C3 side of substrate 11-deoxycorticosterone (DOC) bound to Wt-CYP11B1, while the corresponding space in CYP11B2 is enlarged markedly and accessible to bulk water through a channel. Molecular dynamics simulations of their DOC-bound forms supported the above findings and revealed that the enlarged space of CYP11B2 had a hydrogen bonding network involving water molecules that position DOC. Thus, upon positioning 11ß-hydroxysteroid for 18-hydroxylation in their substrate-binding sites, steric hindrance could occur more strongly in Mt-CYP11B1 than in Wt-CYP11B1 but less in CYP11B2. Our investigation employing Mt-CYP11B1 sheds light on the divergence in structure and function between CYP11B1 and CYP11B2 and suggests that CYP11B1 with spatially-restricted substrate-binding site serves as 11ß-hydroxylase, while CYP11B2 with spatially-extended substrate-binding site successively processes additional 18-hydroxylations to produce aldosterone.

18.
Metabolites ; 11(12)2021 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-34940639

RESUMO

Cardiac dysfunction is induced by multifactorial mechanisms in diabetes. Deranged fatty acid (FA) utilization, known as lipotoxicity, has long been postulated as one of the upstream events in the development of diabetic cardiomyopathy. CD36, a transmembrane glycoprotein, plays a major role in FA uptake in the heart. CD36 knockout (CD36KO) hearts exhibit reduced rates of FA transport with marked enhancement of glucose use. In this study, we explore whether reduced FA use by CD36 ablation suppresses the development of streptozotocin (STZ)-induced diabetic cardiomyopathy. We found that cardiac contractile dysfunction had deteriorated 16 weeks after STZ treatment in CD36KO mice. Although accelerated glucose uptake was not reduced in CD36KO-STZ hearts, the total energy supply, estimated by the pool size in the TCA cycle, was significantly reduced. The isotopomer analysis with 13C6-glucose revealed that accelerated glycolysis, estimated by enrichment of 13C2-citrate and 13C2-malate, was markedly suppressed in CD36KO-STZ hearts. Levels of ceramides, which are cardiotoxic lipids, were not elevated in CD36KO-STZ hearts compared to wild-type-STZ ones. Furthermore, increased energy demand by transverse aortic constriction resulted in synergistic exacerbation of contractile dysfunction in CD36KO-STZ mice. These findings suggest that CD36KO-STZ hearts are energetically compromised by reduced FA use and suppressed glycolysis; therefore, the limitation of FA utilization is detrimental to cardiac energetics in this model of diabetic cardiomyopathy.

19.
Cell Rep ; 34(1): 108579, 2021 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-33406421

RESUMO

O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Mitocôndrias/metabolismo , Mitofagia , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Quinases/metabolismo , Acetilglucosamina/metabolismo , Animais , Apoptose , Ciclo Celular , Linhagem Celular , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , N-Acetilglucosaminiltransferases/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico
20.
Redox Biol ; 41: 101926, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33752108

RESUMO

Chemosensitivity to cisplatin derivatives varies among individual patients with intractable malignancies including ovarian cancer, while how to unlock the resistance remain unknown. Ovarian cancer tissues were collected the debulking surgery in discovery- (n = 135) and validation- (n = 47) cohorts, to be analyzed with high-throughput automated immunohistochemistry which identified cystathionine γ-lyase (CSE) as an independent marker distinguishing non-responders from responders to post-operative platinum-based chemotherapy. We aimed to identify CSE-derived metabolites responsible for chemoresistant mechanisms: gold-nanoparticle (AuN)-based surface-enhanced Raman spectroscopy (SERS) was used to enhance electromagnetic fields which enabled to visualize multiple sulfur-containing metabolites through detecting scattering light from Au-S vibration two-dimensionally. Clear cell carcinoma (CCC) who turned out less sensitive to cisplatin than serous adenocarcinoma was classified into two groups by the intensities of SERS intensities at 480 cm-1; patients with greater intensities displayed the shorter overall survival after the debulking surgery. The SERS signals were eliminated by topically applied monobromobimane that breaks sulfane-sulfur bonds of polysulfides to result in formation of sulfodibimane which was detected at 580 cm-1, manifesting the presence of polysulfides in cancer tissues. CCC-derived cancer cell lines in culture were resistant against cisplatin, but treatment with ambroxol, an expectorant degrading polysulfides, renders the cells CDDP-susceptible. Co-administration of ambroxol with cisplatin significantly suppressed growth of cancer xenografts in nude mice. Furthermore, polysulfides, but neither glutathione nor hypotaurine, attenuated cisplatin-induced disturbance of DNA supercoiling. Polysulfide detection by on-tissue SERS thus enables to predict prognosis of cisplatin-based chemotherapy. The current findings suggest polysulfide degradation as a stratagem unlocking cisplatin chemoresistance.


Assuntos
Antineoplásicos , Neoplasias Ovarianas , Animais , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Cisplatino , Resistencia a Medicamentos Antineoplásicos , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Ovarianas/tratamento farmacológico , Análise Espectral Raman , Sulfetos
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