RESUMEN
Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U-13 C3 ]lactate instead of natural abundance lactate while the other group received [U-13 C3 ]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13 C-labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four-carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4-13 C3 ]/[1,2,3-13 C3 ] in aspartate or malate. 13 C enrichment in glucose with 13 C-lactate supply was higher than that with 13 C-alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U-13 C3 ]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13 C-labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization.
Asunto(s)
Ciclo del Ácido Cítrico , Malatos , Ratas , Animales , Malatos/metabolismo , Piruvato Carboxilasa/metabolismo , Ácido Aspártico/metabolismo , Glucosa/metabolismo , Ácido Glutámico/metabolismo , Ácido Pirúvico/metabolismo , Ácido Láctico , Succinatos , Alanina/metabolismo , Isótopos de Carbono/metabolismoRESUMEN
Advanced imaging technologies, large-scale metabolomics, and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13 C NMR of blood or tissue biopsies. Simple molecules such as 13 C-labeled glucose are readily administered to patients prior to surgical biopsies, and 13 C-labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13 C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable, and quantitative insights into intermediary metabolism in patients.
Asunto(s)
Imagen por Resonancia Magnética , Metabolómica , Humanos , Isótopos de Carbono/química , Espectroscopía de Resonancia Magnética/métodos , Metabolómica/métodos , Redes y Vías MetabólicasRESUMEN
After administration of 13 C-labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of 13 C in lactate. However, in some tissues, such as the well-oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined 13 C-labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3-13 C2 ]glucose and the tissue extracts were analyzed. Metabolism of [2,3-13 C2 ]glucose yields [1,2-13 C2 ]pyruvate via glycolysis and [2,3-13 C2 ]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5-13 C2 ]glutamate, indicating flux through the PPP, was readily detected in 13 C NMR of heart extracts even when the corresponding doublet from [2,3-13 C2 ]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5-13 C2 ]glutamate. In rats receiving [2,3-13 C2 ]glucose, brain extracts showed well-resolved signals from both [2,3-13 C2 ]lactate and [4,5-13 C2 ]glutamate in 13 C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate-based assessment. In summary, 13 C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3-13 C2 ]glucose.
Asunto(s)
Espectroscopía de Resonancia Magnética con Carbono-13 , Ácido Glutámico/metabolismo , Miocardio/metabolismo , Vía de Pentosa Fosfato , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Glutamina/metabolismo , Ácido Láctico/metabolismo , Masculino , Metaboloma , Ratas Sprague-Dawley , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Hepatic energy metabolism is a key element in many metabolic diseases. Hepatic anaplerosis provides carbons for gluconeogenesis (GNG) and triglyceride (TG) synthesis. We aimed to optimize a protocol that measures hepatic anaplerotic contribution for GNG, TG synthesis, and hepatic pentose phosphate pathway (PPP) activity using a single dose of oral [U-13C3]glycerol paired with an oral sugar tolerance test (OSTT) in a population with significant insulin resistance. The OSTT (75 g glucose + 25 g fructose) was administered to eight obese adolescents with polycystic ovarian syndrome (PCOS) followed by ingestion of [U-13C3]glycerol at t = 180 or t = 210 min. 13C-labeling patterns of serum glucose and TG-glycerol were determined by nuclear magnetic resonance. 13C enrichment in plasma TG-glycerol was detectable and stable from 240 to 390 min with the [U-13C3]glycerol drink at t = 180 min(3.65 ± 2.3 to 4.47 ± 1.4%; P > 0.4), but the enrichment was undetectable at 240 min with the glycerol drink at t = 210 min. The relative contribution from anaplerosis was determined at the end of the OSTT [18.5 ±3.4% (t = 180 min) vs. 16.0 ± 3.5% (t = 210 min); P = 0.27]. [U-13C3]glycerol was incorporated into GNG 390 min after the OSTT with an enrichment of 7.5-12.5%. Glucose derived from TCA cycle activity was 0.3-1%, and the PPP activity was 2.8-4.7%. In conclusion, it is possible to obtain relative measurements of hepatic anaplerotic contribution to both GNG and TG esterification following an OSTT in a highly insulin-resistant population using a minimally invasive technique. Tracer administration should be timed to allow enough de novo TG esterification and endogenous glucose release after the sugar drink.
Asunto(s)
Gluconeogénesis/fisiología , Hígado/metabolismo , Obesidad Infantil , Síndrome del Ovario Poliquístico , Triglicéridos/biosíntesis , Adolescente , Glucemia , Isótopos de Carbono , Femenino , Glucosa/metabolismo , Glicerol/metabolismo , Humanos , Resistencia a la Insulina , Lipogénesis , Adulto JovenRESUMEN
The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2-13 C2 ]glucose rely on detection of 13 C in lactate and require assumptions to correct natural 13 C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3-13 C2 ]glucose. 13 C NMR analysis of the resulting isotopomers is informative because [1,2-13 C2 ]lactate arises from glycolysis and [2,3-13 C2 ]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3-13 C2 ]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3-13 C2 ]glucose and 13 C NMR simplify assessment of the PPP.
Asunto(s)
Isótopos de Carbono/metabolismo , Glucosa/metabolismo , Vía de Pentosa Fosfato , Animales , Encéfalo/enzimología , Espectroscopía de Resonancia Magnética con Carbono-13 , Carcinoma Hepatocelular/metabolismo , Glucólisis , Hígado/metabolismo , Masculino , Miocardio/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Sprague-Dawley , Procesamiento de Señales Asistido por ComputadorRESUMEN
It is a challenge to assess metabolic dysregulation in fatty liver of human patients prior to clinical manifestations. Here, we recruited obese, but otherwise healthy, subjects to examine biochemical processes in the liver with simple triglyceride accumulation using stable isotopes and NMR analysis of metabolic products in blood. Intrahepatic triglycerides were measured using 1H magnetic resonance spectroscopy, and volunteers received 2H2O and [U-13C3]glycerol orally, followed by a series of blood draws. NMR analysis of plasma triglycerides and glucose provided detailed information about metabolic pathways in patients with simple hepatic steatosis. Compared with subjects with low hepatic fat, patients with hepatic steatosis were characterized by the following: lower 13C enrichments in the glycerol backbones of triglycerides (i.e., TG-[13C]glycerol), higher [U-13C3]glycerol metabolism through the tricarboxylic acid (TCA) cycle, delayed gluconeogenesis from [U-13C3]glycerol, and less flexibility in adjusting supporting fluxes of glucose production upon an oral load of glycerol. In summary, simple hepatic steatosis was associated with enhanced [U-13C3]glycerol metabolism through pathways that intersect the TCA cycle and delayed gluconeogenesis from glycerol.
Asunto(s)
Hígado Graso/metabolismo , Gluconeogénesis , Glicerol/metabolismo , Lipogénesis , Adulto , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
The pentose phosphate pathway (PPP) is widely assumed to play a key role in both reductive biosynthesis and protection from oxidative stress because it is the major source of NADPH. However, little is known about the activity of the PPP in fatty liver, which is characterized by both oxidative stress and lipogenesis. This study was designed to test whether the PPP is active in parallel with lipogenesis and antioxidant processes in the fatty liver of whole animals. Eight- and 16-wk-old obese Zucker diabetic fatty rats and their lean littermates received [U-13C3]glycerol, and 13C labeling patterns of glucose and triglycerides were analyzed for the assessment of hepatic PPP activity and the potentially related processes simultaneously. Oxidative stress, antioxidant activity, and NADPH-producing enzymes in the liver were further examined. Both PPP activity and lipogenesis increased in the fatty liver of young obese Zucker rats but decreased together in older obese Zucker rats. As expected, lipid peroxidation measured by malondialdehyde increased in the fatty liver of obese Zucker rats at both ages. However, evidence for antioxidant processes such as [glutathione] or activities of glutathione reductase, glutathione peroxidase, and catalase was not altered. Hepatic PPP activity paralleled lipogenesis but was dissociated from biomarkers of oxidative stress or antioxidant processes. In summary, NADPH from the PPP was presumably consumed for reductive biosynthesis rather than antioxidant defense in the fatty liver.
Asunto(s)
Antioxidantes/metabolismo , Lipogénesis/fisiología , Hígado/metabolismo , Vía de Pentosa Fosfato/fisiología , Animales , Catalasa/metabolismo , Hígado Graso/complicaciones , Hígado Graso/metabolismo , Hígado Graso/patología , Glutatión/metabolismo , Masculino , Malondialdehído/metabolismo , Obesidad/complicaciones , Obesidad/metabolismo , Obesidad/patología , Estrés Oxidativo/fisiología , Ratas , Ratas Zucker , Triglicéridos/metabolismoRESUMEN
Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-(13)C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution (13)C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-(13)C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver.
Asunto(s)
Ciclo del Ácido Cítrico/efectos de los fármacos , Ácidos Grasos/sangre , Glicerol/administración & dosificación , Hígado/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Vía de Pentosa Fosfato/efectos de los fármacos , Administración Oral , Adulto , Biomarcadores/sangre , Isótopos de Carbono/administración & dosificación , Isótopos de Carbono/farmacocinética , Esterificación/efectos de los fármacos , Esterificación/fisiología , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells. In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis. Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function, and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP(+)/NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetyl-coenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism.
Asunto(s)
Mitocondrias/metabolismo , Mitocondrias/patología , Neoplasias/metabolismo , Neoplasias/patología , Acetilcoenzima A/metabolismo , Animales , Carcinoma de Células Renales/genética , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/patología , Hipoxia de la Célula , Línea Celular Tumoral , Ácido Cítrico/metabolismo , Transporte de Electrón , Complejo I de Transporte de Electrón/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Fumarato Hidratasa/genética , Fumarato Hidratasa/metabolismo , Glucosa/metabolismo , Glutamina/metabolismo , Humanos , Isocitrato Deshidrogenasa/metabolismo , Neoplasias Renales/genética , Neoplasias Renales/metabolismo , Neoplasias Renales/patología , Ratones , NADP/metabolismoRESUMEN
Phosphoenolpyruvate (PEP) generated from pyruvate is required for de novo synthesis of glycerol and glycogen in skeletal muscle. One possible pathway involves synthesis of PEP from the citric acid cycle intermediates via PEP carboxykinase, whereas another could involve reversal of pyruvate kinase (PK). Earlier studies have reported that reverse flux through PK can contribute carbon precursors for glycogen synthesis in muscle, but the physiological importance of this pathway remains uncertain especially in the setting of high plasma glucose. In addition, although PEP is a common intermediate for both glyconeogenesis and glyceroneogenesis, the importance of reverse PK in de novo glycerol synthesis has not been examined. Here we studied the contribution of reverse PK to synthesis of glycogen and the glycerol moiety of acylglycerols in skeletal muscle of animals with high plasma glucose. Rats received a single intraperitoneal bolus of glucose, glycerol, and lactate under a fed or fasted state. Only one of the three substrates was (13)C-labeled in each experiment. After 3 h of normal awake activity, the animals were sacrificed, and the contribution from each substrate to glycogen and the glycerol moiety of acylglycerols was evaluated. The fraction of (13)C labeling in glycogen and the glycerol moiety exceeded the possible contribution from either plasma glucose or muscle oxaloacetate. The reverse PK served as a common route for both glyconeogenesis and glyceroneogenesis in the skeletal muscle of rats with high plasma glucose. The activity of pyruvate carboxylase was low in muscle, and no PEP carboxykinase activity was detected.
Asunto(s)
Glucemia/metabolismo , Gluconeogénesis/efectos de los fármacos , Glicerol/metabolismo , Ácido Láctico/farmacología , Músculo Esquelético/metabolismo , Piruvato Quinasa/metabolismo , Animales , Masculino , Fosfoenolpiruvato/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
The source of hyperpolarized (HP) [(13)C]bicarbonate in the liver during metabolism of HP [1-(13)C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [(13)C]bicarbonate during metabolism of HP [1-(13)C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The (13)C NMR of HP [(13)C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-(13)C]pyruvate. (13)C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [(13)C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [(13)C]bicarbonate exclusively reflects decarboxylation of HP [1-(13)C]pyruvate via pyruvate dehydrogenase.
Asunto(s)
Hígado/metabolismo , Espectroscopía de Resonancia Magnética/métodos , Ácido Pirúvico/metabolismo , Alanina/metabolismo , Animales , Bicarbonatos/metabolismo , Isótopos de Carbono , Espectroscopía de Resonancia Magnética con Carbono-13 , Ciclo del Ácido Cítrico , Gluconeogénesis , Ácido Láctico/metabolismo , Fosfoenolpiruvato Carboxiquinasa (ATP) , Espectroscopía de Protones por Resonancia Magnética , Piruvato Carboxilasa/metabolismo , Complejo Piruvato Deshidrogenasa/metabolismo , Ratas Sprague-DawleyRESUMEN
After exposure to [U-(13)C3]glycerol, the liver produces primarily [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other (13)C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex (13)C-labeling patterns in exported glucose. Here, we investigated (13)C labeling in plasma glucose in rats given [U-(13)C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a (13)C-(13)C bond within the trioses contributing to glucose, the appearance of [1,2-(13)C2]-, [2,3-(13)C2]-, [5,6-(13)C2]-, and [4,5-(13)C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-(13)C2]glucose/[2,3-(13)C2]glucose was significantly greater than [5,6-(13)C2]glucose/[4,5-(13)C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by (13)C distribution in blood glucose after [U-(13)C3]glycerol administration.
Asunto(s)
Gluconeogénesis , Glicerol/metabolismo , Hígado/metabolismo , Vía de Pentosa Fosfato , Animales , Glucemia/metabolismo , Isótopos de Carbono , Lactatos/metabolismo , Espectroscopía de Resonancia Magnética , Masculino , Ratas Sprague-Dawley , Factores de Tiempo , Transaldolasa/metabolismo , Triosa-Fosfato Isomerasa/metabolismoRESUMEN
Metabolic reprogramming facilitates cancer cell growth, so quantitative metabolic flux measurements could produce useful biomarkers. However, current methods to analyze flux in vivo provide either a steady-state overview of relative activities (infusion of (13)C and analysis of extracted metabolites) or a dynamic view of a few reactions (hyperpolarized (13)C spectroscopy). Moreover, although hyperpolarization has successfully quantified pyruvate-lactate exchanges, its ability to assess mitochondrial pyruvate metabolism is unproven in cancer. Here, we combined (13)C hyperpolarization and isotopomer analysis to quantify multiple fates of pyruvate simultaneously. Two cancer cell lines with divergent pyruvate metabolism were incubated with thermally polarized [3-(13)C]pyruvate for several hours, then briefly exposed to hyperpolarized [1-(13)C]pyruvate during acquisition of NMR spectra using selective excitation to maximize detection of H[(13)C]O3(-) and [1-(13)C]lactate. Metabolites were then extracted and subjected to isotopomer analysis to determine relative rates of pathways involving [3-(13)C]pyruvate. Quantitation of hyperpolarized H[(13)C]O3(-) provided a single definitive metabolic rate, which was then used to convert relative rates derived from isotopomer analysis into quantitative fluxes. This revealed that H[(13)C]O3(-) appearance reflects activity of pyruvate dehydrogenase rather than pyruvate carboxylation followed by subsequent decarboxylation reactions. Glucose substantially altered [1-(13)C]pyruvate metabolism, enhancing exchanges with [1-(13)C]lactate and suppressing H[(13)C]O3(-) formation. Furthermore, inhibiting Akt, an oncogenic kinase that stimulates glycolysis, reversed these effects, indicating that metabolism of pyruvate by both LDH and pyruvate dehydrogenase is subject to the acute effects of oncogenic signaling on glycolysis. The data suggest that combining (13)C isotopomer analyses and dynamic hyperpolarized (13)C spectroscopy may enable quantitative flux measurements in living tumors.
Asunto(s)
Glucosa/metabolismo , Glucólisis , Espectroscopía de Resonancia Magnética , Neoplasias/diagnóstico por imagen , Neoplasias/metabolismo , Ácido Pirúvico/metabolismo , Isótopos de Carbono/farmacocinética , Isótopos de Carbono/farmacología , Línea Celular Tumoral , Humanos , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/metabolismo , RadiografíaRESUMEN
Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-(13)C(3)]glycerol, and (2)H(2)O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, (13)C from [U-(13)C(3)]glycerol appeared in glycogen in carbon positions 4-6 of glucosyl units but none in positions 1-3. The extent of [4,5,6-(13)C(3)]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H(2)O(2). Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked (13)C asymmetry in glycogen can only be attributed to conversion of [U-(13)C(3)]glycerol to [U-(13)C(3)]dihydroxyacetone phosphate and [U-(13)C(3)]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this (13)C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.
Asunto(s)
Glicerol/farmacología , Miocardio/enzimología , Transaldolasa/metabolismo , Animales , Isótopos de Carbono , Glucógeno/metabolismo , Técnicas In Vitro , Masculino , Redes y Vías Metabólicas/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
During hepatic lipogenesis, the glycerol backbone of acylglycerols originates from one of three sources: glucose, glycerol, or substrates passing through the citric acid cycle via glyceroneogenesis. The relative contribution of each substrate source to glycerol in rat liver acylglycerols was determined using (13)C-enriched substrates and NMR. Animals received a fixed mixture of glucose, glycerol, and lactate; one group received [U-(13)C6]glucose, another received [U-(13)C3]glycerol, and the third received [U-(13)C3]lactate. After 3 h, the livers were harvested to extract fats, and the glycerol moiety from hydrolyzed acylglycerols was analyzed by (13)C NMR. In either fed or fasted animals, glucose and glycerol provided the majority of the glycerol backbone carbons, whereas the contribution of lactate was small. In fed animals, glucose contributed >50% of the total newly synthesized glycerol backbone, and 35% of this contribution occurred after glucose had passed through the citric acid cycle. By comparison, the glycerol contribution was ~40%, and of this, 17% of the exogenous glycerol passed first through the cycle. In fasted animals, exogenous glycerol became the major contributor to acylglycerols. The contribution from exogenous lactate did increase in fasted animals, but its overall contribution remained small. The contributions of glucose and glycerol that had passed through the citric acid cycle first increased in fasted animals from 35 to 71% for glucose and from 17 to 24% for glycerol. Thus, a substantial fraction from both substrate sources passed through the cycle prior to incorporation into the glycerol moiety of acylglycerols in the liver.
Asunto(s)
Ciclo del Ácido Cítrico , Glucosa/metabolismo , Glicéridos/metabolismo , Glicerol/metabolismo , Ácido Láctico/metabolismo , Hígado/metabolismo , Animales , Ayuno , Espectroscopía de Resonancia Magnética , Masculino , Músculo Esquelético/metabolismo , Ácido Pirúvico/metabolismo , Ratas , Ratas Sprague-DawleyRESUMEN
Tumor cells require a constant supply of macromolecular precursors, and interrupting this supply has been proposed as a therapeutic strategy in cancer. Precursors for lipids, nucleic acids, and proteins are generated in the tricarboxylic acid (TCA) cycle and removed from the mitochondria to participate in biosynthetic reactions. Refilling the pool of precursor molecules (anaplerosis) is therefore crucial to maintain cell growth. Many tumor cells use glutamine to feed anaplerosis. Here we studied how "glutamine-addicted" cells react to interruptions of glutamine metabolism. Silencing of glutaminase (GLS), which catalyzes the first step in glutamine-dependent anaplerosis, suppressed but did not eliminate the growth of glioblastoma cells in culture and in vivo. Profiling metabolic fluxes in GLS-suppressed cells revealed induction of a compensatory anaplerotic mechanism catalyzed by pyruvate carboxylase (PC), allowing the cells to use glucose-derived pyruvate rather than glutamine for anaplerosis. Although PC was dispensable when glutamine was available, forcing cells to adapt to low-glutamine conditions rendered them absolutely dependent on PC for growth. Furthermore, in other cell lines, measuring PC activity in nutrient-replete conditions predicted dependence on specific anaplerotic enzymes. Cells with high PC activity were resistant to GLS silencing and did not require glutamine for survival or growth, but displayed suppressed growth when PC was silenced. Thus, PC-mediated, glucose-dependent anaplerosis allows cells to achieve glutamine independence. Induction of PC during chronic suppression of glutamine metabolism may prove to be a mechanism of resistance to therapies targeting glutaminolysis.
Asunto(s)
Proliferación Celular , Glioblastoma/metabolismo , Glutamina/metabolismo , Piruvato Carboxilasa/fisiología , Línea Celular Tumoral , Ciclo del Ácido Cítrico , Glioblastoma/patología , Glutaminasa/antagonistas & inhibidores , Glutamina/deficiencia , Humanos , Piruvato Carboxilasa/metabolismo , Ácido Pirúvico/metabolismoRESUMEN
OBJECTIVE: Polycystic ovary syndrome (PCOS) is characterized by hyperandrogenism, insulin resistance, and hepatic steatosis (HS). Because dietary essential amino acid (EAA) supplementation has been shown to decrease HS in various populations, this study's objective was to determine whether supplementation would decrease HS in PCOS. METHODS: A randomized, double-blind, crossover, placebo-controlled trial was conducted in 21 adolescents with PCOS (BMI 37.3 ± 6.5 kg/m2, age 15.6 ± 1.3 years). Liver fat, very low-density lipoprotein (VLDL) lipogenesis, and triacylglycerol (TG) metabolism were measured following each 28-day phase of placebo or EAA. RESULTS: Compared to placebo, EAA was associated with no difference in body weight (p = 0.673). Two markers of liver health improved: HS was lower (-0.8% absolute, -7.5% relative reduction, p = 0.013), as was plasma aspartate aminotransferase (AST) (-8%, p = 0.004). Plasma TG (-9%, p = 0.015) and VLDL-TG (-21%, p = 0.031) were reduced as well. VLDL-TG palmitate derived from lipogenesis was not different between the phases, nor was insulin sensitivity (p > 0.400 for both). Surprisingly, during the EAA phase, participants reported consuming fewer carbohydrates (p = 0.038) and total sugars (p = 0.046). CONCLUSIONS: Similar to studies in older adults, short-term EAA supplementation in adolescents resulted in significantly lower liver fat, AST, and plasma lipids and thus may prove to be an effective treatment in this population. Additional research is needed to elucidate the mechanisms for these effects.
Asunto(s)
Hígado Graso , Hiperandrogenismo , Resistencia a la Insulina , Síndrome del Ovario Poliquístico , Adolescente , Femenino , Humanos , Hiperandrogenismo/complicaciones , Insulina , Lipoproteínas VLDL , Obesidad/complicaciones , Síndrome del Ovario Poliquístico/tratamiento farmacológico , Síndrome del Ovario Poliquístico/complicacionesRESUMEN
BACKGROUND: Glycerol is a substrate for gluconeogenesis and fatty acid esterification in the liver, processes which are upregulated in obesity and may contribute to excess fat accumulation. Glycine and glutamate, in addition to cysteine, are components of glutathione, the major antioxidant in the liver. In principle, glycerol could be incorporated into glutathione via the TCA cycle or 3-phosphoglycerate, but it is unknown whether glycerol contributes to hepatic de novo glutathione biosynthesis. METHODS: Glycerol metabolism to hepatic metabolic products including glutathione was examined in the liver from adolescents undergoing bariatric surgery. Participants received oral [U-13C3]glycerol (50 mg/kg) prior to surgery and liver tissue (0.2-0.7g) was obtained during surgery. Glutathione, amino acids, and other water-soluble metabolites were extracted from the liver tissue and isotopomers were quantified with nuclear magnetic resonance spectroscopy. RESULTS: Data were collected from 8 participants (2 male, 6 female; age 17.1 years [range 14-19]; BMI 47.4 kg/m2 [range 41.3-63.3]). The concentrations of free glutamate, cysteine, and glycine were similar among participants, and so were the fractions of 13C-labeled glutamate and glycine derived from [U-13C3]glycerol. The signals from all component amino acids of glutathione - glutamate, cysteine and glycine - were strong and analyzed to obtain the relative concentrations of the antioxidant in the liver. The signals from glutathione containing [13C2]glycine or [13C2]glutamate derived from the [U-13C3]glycerol drink were readily detected, and 13C-labelling patterns in the moieties were consistent with the patterns in corresponding free amino acids from the de novo glutathione synthesis pathway. The newly synthesized glutathione with [U-13C3]glycerol trended to be lower in obese adolescents with liver pathology. CONCLUSIONS: This is the first report of glycerol incorporation into glutathione through glycine or glutamate metabolism in human liver. This could represent a compensatory mechanism to increase glutathione in the setting of excess glycerol delivery to the liver.
Asunto(s)
Hígado , Humanos , Hígado/metabolismo , Glutatión/metabolismo , Glicerol/metabolismo , Masculino , Femenino , Adolescente , Adulto Joven , Espectroscopía de Resonancia MagnéticaRESUMEN
Stable isotopes are powerful tools to assess metabolism. 13C labeling is detected using nuclear magnetic resonance (NMR) spectroscopy or mass spectrometry (MS). MS has excellent sensitivity but generally cannot discriminate among different 13C positions (isotopomers), whereas NMR is less sensitive but reports some isotopomers. Here, we develop an MS method that reports all 16 aspartate and 32 glutamate isotopomers while requiring less than 1% of the sample used for NMR. This method discriminates between pathways that result in the same number of 13C labels in aspartate and glutamate, providing enhanced specificity over conventional MS. We demonstrate regional metabolic heterogeneity within human tumors, document the impact of fumarate hydratase (FH) deficiency in human renal cancers, and investigate the contributions of tricarboxylic acid (TCA) cycle turnover and CO2 recycling to isotope labeling in vivo. This method can accompany NMR or standard MS to provide outstanding sensitivity in isotope-labeling experiments, particularly in vivo.
Asunto(s)
Ácido Aspártico , Ácido Glutámico , Humanos , Isótopos de Carbono , Ciclo del Ácido Cítrico , Espectrometría de MasasRESUMEN
BACKGROUND: Empagliflozin, a sodium glucose cotransporter 2 inhibitor, is a medication to treat type 2 diabetes. The effect of empagliflozin in persons without diabetes has received less attention. Here we conducted a randomized, double-blind placebo-controlled clinical trial to examine the effect of empagliflozin on plasma triglycerides in obese non-diabetic adults. METHODS: Participants (n = 35; BMI ≥ 30 kg/m2) underwent body composition assessments using MRI, and were randomly assigned to either placebo or empagliflozin (10 mg/d) for three months. At the baseline and post-treatment visit, after an overnight fast, blood was drawn for biochemical analysis. Participants received [U-13C3]glycerol orally followed by multiple blood draws over 3 h to examine glycerol incorporation into triglycerides using NMR spectroscopy. RESULTS: The changes in blood triglyceride concentration with empagliflozin therapy related to the mass of baseline visceral adipose tissue (VAT; r = 0.53, p = 0.04). Empagliflozin slightly lowered triglycerides in obese subjects with low VAT, but increased triglycerides in the subjects with high VAT. Consistently, empagliflozin effectively suppressed triglyceride synthesis following [U-13C3]glycerol administration in the subjects with low VAT (p < 0.05), but not in the subjects with high VAT. The subjects with high VAT lost body weight after three months of empagliflozin treatment. In all subjects, about 20% of the triglyceride backbone originated from mitochondrial metabolism of glycerol. CONCLUSIONS: The effect of empagliflozin on triglycerides in obese adults differed depending on VAT. Empagliflozin suppressed triglyceride synthesis in the subjects with low VAT, but tended to increase triglycerides in those with high VAT.