RESUMO
NADPH, a highly compartmentalized electron donor in mammalian cells, plays essential roles in cell metabolism. However, little is known about how cytosolic and mitochondrial NADPH dynamics relate to cancer cell growth rates in response to varying nutrient conditions. To address this issue, we present NADPH composite index analysis, which quantifies the relationship between compartmentalized NADPH dynamics and growth rates using genetically encoded NADPH sensors, automated image analysis pipeline, and correlation analysis. Through this analysis, we demonstrated that compartmentalized NADPH dynamics patterns were cancer cell-type dependent. Specifically, cytosolic and mitochondrial NADPH dynamics of MDA-MB-231 decreased in response to serine deprivation, while those of HCT-116 increased in response to serine or glutamine deprivation. Furthermore, by introducing a fractional contribution parameter, we correlated cytosolic and mitochondrial NADPH dynamics to growth rates. Using this parameter, we identified cancer cell lines whose growth rates were selectively inhibited by targeting cytosolic or mitochondrial NADPH metabolism. Mechanistically, we identified citrate transporter as a key mitochondrial transporter that maintains compartmentalized NADPH dynamics and growth rates. Altogether, our results present a significant advance in quantifying the relationship between compartmentalized NADPH dynamics and cancer cell growth rates, highlighting a potential of targeting compartmentalized NADPH metabolism for selective cancer cell growth inhibitions.
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Many metabolic phenotypes in cancer cells are also characteristic of proliferating nontransformed mammalian cells, and attempts to distinguish between phenotypes resulting from oncogenic perturbation from those associated with increased proliferation are limited. Here, we examined the extent to which metabolic changes corresponding to oncogenic KRAS expression differed from those corresponding to epidermal growth factor (EGF)-driven proliferation in human mammary epithelial cells (HMECs). Removal of EGF from culture medium reduced growth rates and glucose/glutamine consumption in control HMECs despite limited changes in respiration and fatty acid synthesis, while the relative contribution of branched-chain amino acids to the TCA cycle and lipogenesis increased in the near-quiescent conditions. Most metabolic phenotypes measured in HMECs expressing mutant KRAS were similar to those observed in EGF-stimulated control HMECs that were growing at comparable rates. However, glucose and glutamine consumption as well as lactate and glutamate production were lower in KRAS-expressing cells cultured in media without added EGF, and these changes correlated with reduced sensitivity to GLUT1 inhibitor and phenformin treatment. Our results demonstrate the strong dependence of metabolic behavior on growth rate and provide a model to distinguish the metabolic influences of oncogenic mutations and nononcogenic growth.
Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Fator de Crescimento Epidérmico/genética , Transportador de Glucose Tipo 1/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Mama/crescimento & desenvolvimento , Mama/patologia , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Glucose/metabolismo , Transportador de Glucose Tipo 1/antagonistas & inibidores , Ácido Glutâmico/metabolismo , Glutamina/metabolismo , Humanos , Ácido Láctico/metabolismo , Glândulas Mamárias Humanas/crescimento & desenvolvimento , Glândulas Mamárias Humanas/patologia , Células Tumorais CultivadasRESUMO
BACKGROUND: The study of cancer metabolism has been largely dedicated to exploring the hypothesis that oncogenic transformation rewires cellular metabolism to sustain elevated rates of growth and division. Intense examination of tumors and cancer cell lines has confirmed that many cancer-associated metabolic phenotypes allow robust growth and survival; however, little attention has been given to explicitly identifying the biochemical requirements for cell proliferation in a rigorous manner in the context of cancer metabolism. RESULTS: Using a well-studied hybridoma line as a model, we comprehensively and quantitatively enumerate the metabolic requirements for generating new biomass in mammalian cells; this indicated a large biosynthetic requirement for ATP, NADPH, NAD(+), acetyl-CoA, and amino acids. Extension of this approach to serine/glycine and glutamine metabolic pathways suggested lower limits on serine and glycine catabolism to supply one-carbon unit synthesis and significant availability of glutamine-derived carbon for biosynthesis resulting from nitrogen demands alone, respectively. We integrated our biomass composition results into a flux balance analysis model, placing upper bounds on mitochondrial NADH oxidation to simulate metformin treatment; these simulations reproduced several empirically observed metabolic phenotypes, including increased reductive isocitrate dehydrogenase flux. CONCLUSIONS: Our analysis clarifies the differential needs for central carbon metabolism precursors, glutamine-derived nitrogen, and cofactors such as ATP, NADPH, and NAD(+), while also providing justification for various extracellular nutrient uptake behaviors observed in tumors. Collectively, these results demonstrate how stoichiometric considerations alone can successfully predict empirically observed phenotypes and provide insight into biochemical dynamics that underlie responses to metabolic perturbations.
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Cholesterol is a lipid that is critical for steroid hormone production and the integrity of cellular membranes, and, as such, it is essential for cell growth. The epidermal growth factor receptor (EGFR) family member ERBB4, which forms signaling complexes with other EGFR family members, can undergo ligand-induced proteolytic cleavage to release a soluble intracellular domain (ICD) that enters the nucleus to modify transcription. We found that ERBB4 activates sterol regulatory element binding protein-2 (SREBP-2) to enhance low-density lipoprotein (LDL) uptake and cholesterol biosynthesis. Expression of the ERBB4 ICD in mammary epithelial cells or activation of ERBB4 with the ligand neuregulin 1 (NRG1) induced the expression of SREBP target genes involved in cholesterol biosynthesis, including HMGCR and HMGCS1, and lipid uptake, LDLR, which encodes the LDL receptor. Addition of NRG1 increased the abundance of the cleaved, mature form of SREBP-2 through a pathway that was blocked by addition of inhibitors of PI3K (phosphatidylinositol 3-kinase) or dual inhibition of mammalian target of rapamycin complex 1 (mTORC1) and mTORC2, but not by inhibition of AKT or mTORC1. Pharmacological inhibition of the activity of SREBP site 1 protease or of all EGFR family members (with lapatinib), but not EGFR alone (with erlotinib), impaired NRG1-induced expression of cholesterol biosynthesis genes. Collectively, our findings indicated that activation of ERBB4 promotes SREBP-2-regulated cholesterol metabolism. The connections of EGFR and ERBB4 signaling with SREBP-2-regulated cholesterol metabolism are likely to be important in ERBB-regulated developmental processes and may contribute to metabolic remodeling in ERBB-driven cancers.
Assuntos
Colesterol/biossíntese , Lipoproteínas LDL/metabolismo , Neuregulina-1/metabolismo , Receptor ErbB-4/metabolismo , Receptores de LDL/metabolismo , Proteína de Ligação a Elemento Regulador de Esterol 2/metabolismo , Linhagem Celular Tumoral , Colesterol/genética , Feminino , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Lipoproteínas LDL/genética , Alvo Mecanístico do Complexo 1 de Rapamicina , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neuregulina-1/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor ErbB-4/genética , Receptores de LDL/genética , Proteína de Ligação a Elemento Regulador de Esterol 2/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
Cancer and proliferating cells exhibit an increased demand for glutamine-derived carbons to support anabolic processes. In addition, reductive carboxylation of α-ketoglutarate by isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2) was recently shown to be a major source of citrate synthesis from glutamine. The role of NAD(P)H/NAD(P)(+) cofactors in coordinating glucose and glutamine utilization in the tricarboxylic acid (TCA) cycle is not well understood, with the source(s) of NADPH for the reductive carboxylation reaction remaining unexplored. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial enzyme that transfers reducing equivalents from NADH to NADPH. Here, we show that knockdown of NNT inhibits the contribution of glutamine to the TCA cycle and activates glucose catabolism in SkMel5 melanoma cells. The increase in glucose oxidation partially occurred through pyruvate carboxylase and rendered NNT knockdown cells more sensitive to glucose deprivation. Importantly, knocking down NNT inhibits reductive carboxylation in SkMel5 and 786-O renal carcinoma cells. Overexpression of NNT is sufficient to stimulate glutamine oxidation and reductive carboxylation, whereas it inhibits glucose catabolism in the TCA cycle. These observations are supported by an impairment of the NAD(P)H/NAD(P)(+) ratios. Our findings underscore the role of NNT in regulating central carbon metabolism via redox balance, calling for other mechanisms that coordinate substrate preference to maintain a functional TCA cycle.
Assuntos
Ciclo do Ácido Cítrico/fisiologia , Glucose/metabolismo , NADP Trans-Hidrogenase Específica para A ou B/metabolismo , NADP/metabolismo , NAD/metabolismo , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Glucose/genética , Camundongos , Camundongos Nus , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , NAD/genética , NADP/genética , NADP Trans-Hidrogenase Específica para A ou B/genética , OxirreduçãoRESUMO
Acetyl coenzyme A (AcCoA) is the central biosynthetic precursor for fatty-acid synthesis and protein acetylation. In the conventional view of mammalian cell metabolism, AcCoA is primarily generated from glucose-derived pyruvate through the citrate shuttle and ATP citrate lyase in the cytosol. However, proliferating cells that exhibit aerobic glycolysis and those exposed to hypoxia convert glucose to lactate at near-stoichiometric levels, directing glucose carbon away from the tricarboxylic acid cycle and fatty-acid synthesis. Although glutamine is consumed at levels exceeding that required for nitrogen biosynthesis, the regulation and use of glutamine metabolism in hypoxic cells is not well understood. Here we show that human cells use reductive metabolism of α-ketoglutarate to synthesize AcCoA for lipid synthesis. This isocitrate dehydrogenase-1 (IDH1)-dependent pathway is active in most cell lines under normal culture conditions, but cells grown under hypoxia rely almost exclusively on the reductive carboxylation of glutamine-derived α-ketoglutarate for de novo lipogenesis. Furthermore, renal cell lines deficient in the von Hippel-Lindau tumour suppressor protein preferentially use reductive glutamine metabolism for lipid biosynthesis even at normal oxygen levels. These results identify a critical role for oxygen in regulating carbon use to produce AcCoA and support lipid synthesis in mammalian cells.
Assuntos
Hipóxia Celular , Glutamina/metabolismo , Isocitrato Desidrogenase/metabolismo , Lipogênese , Acetilcoenzima A/biossíntese , Acetilcoenzima A/metabolismo , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linfócitos T CD8-Positivos/citologia , Carbono/metabolismo , Carcinoma de Células Renais/metabolismo , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Células Cultivadas , Ciclo do Ácido Cítrico , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Isocitrato Desidrogenase/deficiência , Isocitrato Desidrogenase/genética , Ácidos Cetoglutáricos/metabolismo , Neoplasias Renais/metabolismo , Neoplasias Renais/patologia , Oxirredução , Oxigênio/metabolismo , Ácido Palmítico/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
We developed a simple and accurate method for determining deuterium enrichment of glucose hydrogen atoms by electron impact gas chromatography mass spectrometry (GC/MS). First, we prepared 18 derivatives of glucose and screened over 200 glucose fragments to evaluate the accuracy and precision of mass isotopomer data for each fragment. We identified three glucose derivatives that gave six analytically useful ions: (1) glucose aldonitrile pentapropionate (m/z 173 derived from C4-C5 bond cleavage; m/z 259 from C3-C4 cleavage; m/z 284 from C4-C5 cleavage; and m/z 370 from C5-C6 cleavage); (2) glucose 1,2,5,6-di-isopropylidene propionate (m/z 301, no cleavage of glucose carbon atoms); and (3) glucose methyloxime pentapropionate (m/z 145 from C2-C3 cleavage). Deuterium enrichment at each carbon position of glucose was determined by least-squares regression of mass isotopomer distributions. The validity of the approach was tested using labeled glucose standards and carefully prepared mixtures of standards. Our method determines deuterium enrichment of glucose hydrogen atoms with an accuracy of 0.3 mol %, or better, without the use of any calibration curves or correction factors. The analysis requires only 20 µL of plasma, which makes the method applicable for studying gluconeogenesis using deuterated water in cell culture and animal experiments.
Assuntos
Glucose/análise , Hidrogênio/sangue , Animais , Cromatografia Gasosa-Espectrometria de Massas , Camundongos , Camundongos Endogâmicos C57BL , Estrutura MolecularRESUMO
Palmitate (PA) is known to induce reactive oxygen species (ROS) formation and apoptosis in liver cells, whereas concurrent treatment of oleate (OA) with PA predominately induces steatosis without ROS in liver cells. We previously reported that PA treatment induces the decoupling of glycolysis and tricarboxylic acid cycle (TCA cycle) fluxes, but OA co-treatment restored most metabolic fluxes to their control levels. However, the mechanisms by which metabolites are linked to metabolic fluxes and subsequent lipoapoptotic or steatotic phenotypes remain unclear. To determine the link, we used GC-MS-based polar and non-polar metabolic profiling in lipoapoptosis- or steatosis-developing H4IIEC3 hepatoma cells, to examine the metabolome at different time points after treatment with either PA alone (PA cells) or both PA and OA (PA/OA cells). Metabolic profiles revealed various changes in metabolite levels for TCA cycle intermediates, pentose phosphate pathway (PPP) intermediates, and energy storage metabolites between PA and PA/OA cells. For example, adenosine was markedly increased only in PA cells, whereas gluconate was increased in PA/OA cells. To assess the interaction among these metabolites, the metabolite-to-metabolite correlations were calculated and correlation networks were visualized. These correlation networks demonstrate that a dissociation among PPP metabolites was introduced in PA-treated cells, and this dissociation was restored in PA/OA-treated cells. Thus, our data suggest that abnormal PPP fluxes, in addition to increased adenosine levels, might be related to the decoupling of glycolysis and the resulting lipoapoptotic phenotype.
Assuntos
Apoptose/efeitos dos fármacos , Metaboloma/efeitos dos fármacos , Metabolômica/métodos , Ácido Oleico/farmacologia , Palmitatos/farmacologia , Animais , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Ciclo do Ácido Cítrico/efeitos dos fármacos , Fígado Gorduroso/metabolismo , Fígado Gorduroso/patologia , Cromatografia Gasosa-Espectrometria de Massas , Glicólise/efeitos dos fármacos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Via de Pentose Fosfato/efeitos dos fármacos , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Systems level tools for the quantitative analysis of metabolic networks are required to engineer metabolism for biomedical and industrial applications. While current metabolomics techniques enable high-throughput quantification of metabolites, these methods provide minimal information on the rates and connectivity of metabolic pathways. Here we present a new method, nontargeted tracer fate detection (NTFD), that expands upon the concept of metabolomics to solve the above problems. Through the combined use of stable isotope tracers and chromatography coupled to mass spectrometry, our computational analysis enables the quantitative detection of all measurable metabolites derived from a specific labeled compound. Without a priori knowledge of a reaction network or compound library, NTFD provides information about relative flux magnitudes into each metabolite pool by determining the mass isotopomer distribution for all labeled compounds. This novel method adds a new dimension to the metabolomics tool box and provides a framework for global analysis of metabolic fluxes.
Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Metabolômica/métodos , Algoritmos , Linhagem Celular Tumoral , Glutamina/química , Glutamina/metabolismo , Humanos , Marcação por Isótopo , Redes e Vias MetabólicasRESUMO
To identify metabolic pathways involved in hepatic lipoapoptosis, metabolic flux analysis using [U-(13)C(5)]glutamine as an isotopic tracer was applied to quantify phenotypic changes in H4IIEC3 hepatoma cells treated with either palmitate alone (PA-cells) or both palmitate and oleate in combination (PA/OA-cells). Our results indicate that palmitate inhibited glycolysis and lactate dehydrogenase fluxes while activating citric acid cycle (CAC) flux and glutamine uptake. This decoupling of glycolysis and CAC fluxes occurred during the period following palmitate exposure but preceding the onset of apoptosis. Oleate co-treatment restored most fluxes to their control levels, resulting in steatotic lipid accumulation while preventing apoptosis. In addition, palmitate strongly increased the cytosolic NAD(+)/NADH ratio, whereas oleate co-treatment had the opposite effect on cellular redox. We next examined the influence of amino acids on these free fatty acid-induced phenotypic changes. Increased medium amino acids enhanced reactive oxygen species (ROS) generation and apoptosis in PA-cells but not in PA/OA-cells. Overloading the medium with non-essential amino acids induced apoptosis, but essential amino acid overloading partially ameliorated apoptosis. Glutamate was the most effective single amino acid in promoting ROS. Amino acid overloading also increased cellular palmitoyl-ceramide; however, ceramide synthesis inhibitors had no effect on measurable indicators of apoptosis. Our results indicate that free fatty acid-induced ROS generation and apoptosis are accompanied by the decoupling of glycolysis and CAC fluxes leading to abnormal cytosolic redox states. Amino acids play a modulatory role in these processes via a mechanism that does not involve ceramide accumulation.
Assuntos
Aminoácidos/farmacologia , Apoptose/efeitos dos fármacos , Ácidos Graxos não Esterificados/farmacologia , Aminoácidos/sangue , Aminoácidos/farmacocinética , Animais , Linhagem Celular Tumoral , Ceramidas/metabolismo , Citosol/efeitos dos fármacos , Citosol/metabolismo , Relação Dose-Resposta a Droga , Cromatografia Gasosa-Espectrometria de Massas , Glutamina/sangue , Glutamina/farmacocinética , Glutamina/farmacologia , Glicólise/efeitos dos fármacos , Humanos , L-Lactato Desidrogenase/metabolismo , Fígado/metabolismo , Fígado/patologia , Ácido Oleico/farmacologia , Oxirredução/efeitos dos fármacos , Palmitatos/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismoRESUMO
We developed a LC-MS-MS assay of the (2)H labeling of free glutathione (GSH) and bound glutathione [GSSR; which includes all DTT-reducible forms, primarily glutathione disulfide (GSSG) and mixed disulfides with proteins] and ophthalmate (an index of GSH depletion) labeled from (2)H-enriched body water. In rats whose body water was 2.5% (2)H enriched for up to 31 days, GSH labeling follows a complex pattern because of different rates of labeling of its constitutive amino acids. In rats infused with [(13)C(2),(15)N-glycine]glutathione, the rate of appearance of plasma GSH was 2.1 micromol.min(-1).kg(-1), and the half-life of plasma GSH/GSSR was 6-8 min. In healthy humans whose body fluids were 0.5% (2)H enriched, the (2)H labeling of GSH/GSSR and ophthalmate can be precisely measured after 4 h, with GSH being more rapidly labeled than GSSR. Since plasma GSH/GSSR derives mostly from liver, this technique opens the way to 2) probe noninvasively the labeling pattern and redox status of the liver GSH system in humans and 2) assess the usefulness of ophthalmate as an index of GSH depletion.
Assuntos
Água Corporal/metabolismo , Deutério/farmacocinética , Glutationa/farmacocinética , Oligopeptídeos/farmacocinética , Adulto , Animais , Óxido de Deutério/farmacocinética , Feminino , Glutationa/sangue , Glutationa/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Fragmentos de Peptídeos/análise , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Glutathione (GSH), an intracellular tripeptide that combats oxidative stress, must be continually replaced due to loss through conjugation and destruction. Previous methods, estimating the synthesis of GSH in vivo, used constant infusions of labeled amino acid precursors. We developed a new method based on incorporation of (2)H from orally supplied (2)H(2)O into stable C-H bonds on the tripeptide. The incorporation of (2)H(2)O into GSH was studied in rabbits over a 2-week period. The method estimated N, the maximum number of C-H bonds in GSH that equilibrate with (2)H(2)O as amino acids. GSH was analyzed by liquid chromatography/mass spectrometry after derivatization to yield GSH-N-ethylmaleimide (GSNEM). A model, which simulated the expected abundance at each mass isotopomer for the GSNEM ion at various values for N, was used to find the best fit to the data. The plateau labeling fit best a model with N=6 of a possible 10 C-H bonds. Thus, the amino acid precursors do not completely equilibrate with (2)H(2)O prior to GSH synthesis. Advantages of this new method include replacing costly amino acid infusions with the oral administration of (2)H(2)O and a statistical basis for estimating N.
Assuntos
Óxido de Deutério/metabolismo , Glutationa/biossíntese , Algoritmos , Animais , Cromatografia Líquida , Óxido de Deutério/química , Glutationa/sangue , Glutationa/química , Espectrometria de Massas , CoelhosRESUMO
Previous studies have indicated that the early steps in the isoprenoid/cholesterol biosynthetic pathway occur in peroxisomes. However, the role of peroxisomes in cholesterol biosynthesis has recently been questioned in several reports concluding that three of the peroxisomal cholesterol biosynthetic enzymes, namely mevalonate kinase, phosphomevalonate kinase, and mevalonate diphosphate decarboxylase, do not localize to peroxisomes in human cells even though they contain consensus peroxisomal targeting signals. We re-investigated the subcellular localization of the cholesterol biosynthetic enzymes of the pre-squalene segment in human cells by using new stable isotopic techniques and data computations with isotopomer spectral analysis, in combination with immunofluorescence and cell permeabilization techniques. Our present findings clearly show and confirm previous studies that the pre-squalene segment of the cholesterol biosynthetic pathway is localized to peroxisomes. In addition, our data are consistent with the hypothesis that acetyl-CoA derived from peroxisomal beta-oxidation of very long-chain fatty acids and medium-chain dicarboxylic acids is preferentially channeled to cholesterol synthesis inside the peroxisomes without mixing with the cytosolic acetyl-CoA pool.
Assuntos
Peroxissomos/metabolismo , Esqualeno/metabolismo , Terpenos/metabolismo , Acetil-CoA C-Acetiltransferase/metabolismo , Animais , Células CHO , Isomerases de Ligação Dupla Carbono-Carbono/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colesterol/biossíntese , Cricetinae , Cricetulus , Digitonina/farmacologia , Ácidos Graxos/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Geraniltranstransferase/metabolismo , Hemiterpenos , Humanos , Hidroximetilglutaril-CoA Sintase/metabolismo , Imuno-Histoquímica , Indicadores e Reagentes/farmacologia , Neoplasias Hepáticas/patologia , Peroxissomos/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Fosfotransferases (Aceptor do Grupo Fosfato)/metabolismoRESUMO
Four patients with cerebrotendinous xanthomatosis (CTX) and 2 healthy controls received a constant proximal intraduodenal infusion of 1- 13 C-acetate as a stable-isotope-labeled marker of sterol synthesis. One patient was treated with pravastatin (20 mg twice daily) and another patient with chenodeoxycholic acid (250 mg tid). Every hour, venous blood and duodenal samples were obtained. Stable-isotope enrichment of neutral and polar sterols in serum and bile was assessed by gas chromatography/mass spectrometry. Isotopomer spectral analysis was performed on cholesterol, lathosterol, Delta-8-cholesterol, methylsterol, and lanosterol. Stable-isotope labeling of cholestanol, bile acids, and bile alcohols was analyzed by assessing the change over time of the ratio of M + 3 to M + 0. Eleven hours after marker infusion, we found up to 50% newly synthesized lathosterol in serum and up to 80% in bile, with similar results for other cholesterol precursors. In cholesterol, stable-isotope labeling could be demonstrated in all study subjects with a more prominent labeling in bile than in serum. No stable-isotope labeling was detected in cholestanol. Only minor stable-isotope incorporation was detectable in polar sterols in some subjects. Therapy with pravastatin did not have any effect on fractional or absolute synthesis rates or on the concentrations of cholestanol or cholesterol precursors compared to untreated patients with CTX. In contrast, therapy with chenodeoxycholic acid markedly lowered the concentrations of cholestanol and cholesterol precursors, led to a disappearance of bile alcohols, and reduced absolute synthesis rates of lathosterol. Isotopomer spectral analysis proved to be a powerful method to assess the endogenous synthesis of cholesterol precursors in patients with CTX. Higher fractional synthesis in bile than in serum may be due to the size of the pools in bile vs serum. Cholestanol exhibits no marker uptake and is therefore probably synthesized from preformed cholesterol. Biliary cholesterol secretion in patients with CTX is decreased compared to healthy controls.
Assuntos
Bile/química , Colesterol/biossíntese , Esteróis/análise , Esteróis/sangue , Xantomatose Cerebrotendinosa/metabolismo , Adulto , Ácidos e Sais Biliares/análise , Ácidos e Sais Biliares/metabolismo , Isótopos de Carbono , Ácido Quenodesoxicólico/uso terapêutico , Colestanol/análise , Colestanol/sangue , Colestanol/metabolismo , Colestanóis/análise , Colestanóis/metabolismo , Colesterol/análogos & derivados , Colesterol/análise , Colesterol/sangue , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Marcação por Isótopo , Lanosterol/análise , Lanosterol/sangue , Masculino , Pessoa de Meia-Idade , Pravastatina/uso terapêutico , Xantomatose Cerebrotendinosa/tratamento farmacológicoRESUMO
An important objective in postgenomic biology is to link gene expression to function by developing physiological networks that include data from the genomic and functional levels. Here, we develop a model for the analysis of time-dependent changes in metabolites, fluxes, and gene expression in a hepatic model system. The experimental framework chosen was modulation of extracellular glutamine in confluent cultures of mouse Hepa1-6 cells. The importance of glutamine has been demonstrated previously in mammalian cell culture by precipitating metabolic shifts with glutamine depletion and repletion. Our protocol removed glutamine from the medium for 24 h and returned it for a second 24 h. Flux assays of glycolysis, the tricarboxylic acid (TCA) cycle, and lipogenesis were used at specified intervals. All of these fluxes declined in the absence of glutamine and were restored when glutamine was repleted. Isotopomer spectral analysis identified glucose and glutamine as equal sources of lipogenic carbon. Metabolite measurements of organic acids and amino acids indicated that most metabolites changed in parallel with the fluxes. Experiments with actinomycin D indicated that de novo mRNA synthesis was required for observed flux changes during the depletion/repletion of glutamine. Analysis of gene expression data from DNA microarrays revealed that many more genes were anticorrelated with the glycolytic flux and glutamine level than were correlated with these indicators. In conclusion, this model may be useful as a prototype physiological regulatory network where gene expression profiles are analyzed in concert with changes in cell function.