Metabolism of [U-13 C]glucose in human brain tumors in vivo.

Glioblastomas and brain metastases demonstrate avid uptake of 2-[(18) F]fluoro-2-deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by (1) H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2-[(18) F]Fluoro-2-deoxyglucose-positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U-(13) C]Glucose (uniformly labeled glucose, i.e. d-glucose labeled with (13) C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to (13) C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl-coenzyme A pool was derived from blood-borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of (13) C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment.

Hyperpolarized 13C allows a direct measure of flux through a single enzyme-catalyzed step by NMR.

(13)C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing (13)C nuclei now offers the potential to measure metabolic fluxes through select enzyme-catalyzed steps with substantially improved sensitivity. Here, we investigated the metabolism of hyperpolarized [1-(13)C(1)]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved (13)CO(2), the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of approximately 1 s along with H(13)CO(3)(-), the hydrated form of (13)CO(2) generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-(13)C(1)]pyruvate, production of (13)CO(2) and H(13)CO(3)(-) was suppressed by approximately 90%, whereas the signal from [1-(13)C(1)]lactate was enhanced. In separate experiments, it was shown that O(2) consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of (13)CO(2) and H(13)CO(3)(-) from [1-(13)C(1)]pyruvate does not reflect production of CO(2) in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

Effects of insulin and cytosolic redox state on glucose production pathways in the isolated perfused mouse liver measured by integrated 2H and 13C NMR.

A great deal is known about hepatic glucose production and its response to a variety of factors such as redox state, substrate supply and hormonal control, but the effects of these parameters on the flux through biochemical pathways which integrate to control glucose production are less clear. A combination of 13C and [2H]water tracers and NMR isotopomer analysis were used to investigate metabolic fluxes in response to altered cytosolic redox state and insulin. In livers isolated from fed mice and perfused with a mixture of substrates including lactate/pyruvate (10:1, w/w), hepatic glucose production had substantial contributions from glycogen, PEP (phosphoenolpyruvate) and glycerol. Inversion of the lactate/pyruvate ratio (1:10, w/w) resulted in a surprising decrease in the contribution from glycogen and an increase in that from PEP to glucose production. A change in the lactate/pyruvate ratio from 10:1 to 1:10 also stimulated flux through the tricarboxylic acid cycle (2-fold), while leaving oxygen consumption and overall glucose output unchanged. When lactate and pyruvate were eliminated from the perfusion medium, both gluconeogenesis and tricarboxylic-acid-cycle flux were dramatically lower. Insulin lowered glucose production by inhibiting glycogenolysis at both low and high doses, but only at high levels of insulin did gluconeogenesis or tricarboxylic-acid-cycle flux tend towards lower values (P<0.1). Our data demonstrate that, in the isolated mouse liver, substrate availability and cellular redox state have a dramatic impact on liver metabolism in both the tricarboxylic acid cycle and gluconeogenesis. The tight correlation of these two pathways under multiple conditions suggest that interventions which increase or decrease hepatic tricarboxylic-acid-cycle flux will have a concomitant effect on gluconeogenesis and vice versa.

Tricarboxylic acid cycle inhibition by Li+ in the human neuroblastoma SH-SY5Y cell line: a 13C NMR isotopomer analysis.

Li+ effects on glucose metabolism and on the competitive metabolism of glucose and lactate were investigated in the human neuroblastoma SH-SY5Y cell line using 13C NMR spectroscopy. The metabolic model proposed for glucose and lactate metabolism in these cells, based on tcaCALC best fitting solutions, for both control and Li+ conditions, was consistent with: (i) a single pyruvate pool; (ii) anaplerotic flux from endogenous unlabelled substrates; (iii) no cycling between pyruvate and oxaloacetate. Li+ was shown to induce a 38 and 53% decrease, for 1 and 15 mM Li+, respectively, in the rate of glucose conversion into pyruvate, when [U-13C]glucose was present, while no effects on lactate production were observed. Pyruvate oxidation by the tricarboxylic acid cycle and citrate synthase flux were shown to be significantly reduced by 64 and 84% in the presence of 1 and 15 mM Li+, respectively, suggesting a direct inhibitory effect of Li+ on tricarboxylic acid cycle flux. This work also showed that when both glucose and lactate are present as energetic substrates, SH-SY5Y cells preferentially consumed exogenous lactate over glucose, as 62% of the acetyl-CoA was derived from [3-13C]lactate while only 26% was derived from [U-13C]glucose. Li+ did not significantly affect the relative utilisation of these two substrates by the cells or the residual contribution of unlabelled endogenous sources for the acetyl-CoA pool.

Modeling of brain metabolism and pyruvate compartmentation using (13)C NMR in vivo: caution required.

Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,6-(13)C2]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ. Furthermore, the models included the capacity to analyze multiplets arising from (13)C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-(13)C2]glucose metabolism. Four analyses were used, two comparing the effect of different pyruvate enrichment in glia and neurons, and two for determining the effect of multiplets present in the data. When fit independently, the enrichment in glial pyruvate was less than in neurons. In the absence of multiplets, fit quality and parameter values were typical of those in the literature, whereas the multiplet curves were not modeled well. This prompted the use of robust statistical analysis (the Kolmogorov-Smirnov test of goodness of fit) to determine whether individual curves were modeled appropriately. At least 50% of the curves in each experiment were considered poorly fit. It was concluded that the model does not include all metabolic features required to analyze the data.

High-resolution detection of ¹³C multiplets from the conscious mouse brain by ex vivo NMR spectroscopy.

Glucose readily supplies the brain with the majority of carbon needed to sustain neurotransmitter production and utilization. The rate of brain glucose metabolism can be computed using (13)C nuclear magnetic resonance (NMR) spectroscopy by detecting changes in (13)C contents of products generated by cerebral metabolism. As previously observed, scalar coupling between adjacent (13)C carbons (multiplets) can provide additional information to (13)C contents for the computation of metabolic rates. Most NMR studies have been conducted in large animals (often under anesthesia) because the mass of the target organ is a limiting factor for NMR. Yet, despite the challengingly small size of the mouse brain, NMR studies are highly desirable because the mouse constitutes a common animal model for human neurological disorders. We have developed a method for the ex vivo resolution of NMR multiplets arising from the brain of an awake mouse after the infusion of [1,6-(13)C(2)]glucose. NMR spectra obtained by this method display favorable signal-to-noise ratios. With this infusion protocol, the (13)C multiplets of glutamate, glutamine, GABA and aspartate achieved steady state after 150 min. The method enables the accurate resolution of multiplets over time in the awake mouse brain. We anticipate that this method can be broadly applicable to compute brain fluxes in normal and transgenic mouse models of neurological disorders.

Myc controls transcriptional regulation of cardiac metabolism and mitochondrial biogenesis in response to pathological stress in mice.

In the adult heart, regulation of fatty acid oxidation and mitochondrial genes is controlled by the PPARgamma coactivator-1 (PGC-1) family of transcriptional coactivators. However, in response to pathological stressors such as hemodynamic load or ischemia, cardiac myocytes downregulate PGC-1 activity and fatty acid oxidation genes in preference for glucose metabolism pathways. Interestingly, despite the reduced PGC-1 activity, these pathological stressors are associated with mitochondrial biogenesis, at least initially. The transcription factors that regulate these changes in the setting of reduced PGC-1 are unknown, but Myc can regulate glucose metabolism and mitochondrial biogenesis during cell proliferation and tumorigenesis in cancer cells. Here we have demonstrated that Myc activation in the myocardium of adult mice increases glucose uptake and utilization, downregulates fatty acid oxidation by reducing PGC-1alpha levels, and induces mitochondrial biogenesis. Inactivation of Myc in the adult myocardium attenuated hypertrophic growth and decreased the expression of glycolytic and mitochondrial biogenesis genes in response to hemodynamic load. Surprisingly, the Myc-orchestrated metabolic alterations were associated with preserved cardiac function and improved recovery from ischemia. Our data suggest that Myc directly regulates glucose metabolism and mitochondrial biogenesis in cardiac myocytes and is an important regulator of energy metabolism in the heart in response to pathologic stress.

Effect of murine strain on metabolic pathways of glucose production after brief or prolonged fasting.

Background strain is known to influence the way a genetic manipulation affects mouse phenotypes. Despite data that demonstrate variations in the primary phenotype of basic inbred strains of mice, there is limited data available about specific metabolic fluxes in vivo that may be responsible for the differences in strain phenotypes. In this study, a simple stable isotope tracer/NMR spectroscopic protocol has been used to compare metabolic fluxes in ICR, FVB/N (FVB), C57BL/6J (B6), and 129S1/SvImJ (129) mouse strains. After a short-term fast in these mice, there were no detectable differences in the pathway fluxes that contribute to glucose synthesis. However, after a 24-h fast, B6 mice retain some residual glycogenolysis compared with other strains. FVB mice also had a 30% higher in vivo phosphoenolpyruvate carboxykinase flux and total glucose production from the level of the TCA cycle compared with B6 and 129 strains, while total body glucose production in the 129 strain was approximately 30% lower than in either FVB or B6 mice. These data indicate that there are inherent differences in several pathways involving glucose metabolism of inbred strains of mice that may contribute to a phenotype after genetic manipulation in these animals. The techniques used here are amenable to use as a secondary or tertiary tool for studying mouse models with disruptions of intermediary metabolism.

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o).

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility. Hearts were frozen at various times after exposure to these substrates, were extracted, and were then analyzed by (13)C NMR spectroscopy. The time-dependent multiplets observed in the (13)C NMR resonances of glutamate in all hearts and in malate and aspartate in hearts perfused with high-pyruvate/low-lactate concentrations were analyzed using a kinetic model of the tricarboxylic acid (TCA) cycle. The analysis showed that TCA cycle flux (V(TCA)) and exchange flux (V(X)) that involved cycle intermediates were both sensitive to cell redox and altered Ca(2+) concentration, and the ratio of these fluxes (V(X)/V(TCA)) varied >10-fold.

13C isotopomer analysis of glutamate by tandem mass spectrometry.

Tandem mass spectrometry allows a compound to be isolated from the rest of the sample and dissociated into smaller fragments. We show here that fragmentation of glutamate mass isotopomers yields additional mass spectral data that significantly improve the analysis of metabolic fluxes compared to full-scan mass spectrometry. In order to validate the technique, tandem and full-scan mass spectrometry were used along with (13)C NMR to analyze glutamate from rat hearts perfused with three substrate mixtures (5 mM glucose plus 5 mM [2-(13)C]acetate, 5 mM [1-(13)C]glucose plus 5 U/L insulin, and 5 mM glucose plus 1 mM [3-(13)C]pyruvate). Analysis by tandem mass spectrometry showed that the enriched substrate contributed 98 +/- 2, 53 +/- 2, and 84 +/- 7%, respectively, of acetyl-coenzyme A while the rate of anaplerotic substrate entry was 7 +/- 3, 25 +/- 8, and 16 +/- 8%. Similar results were obtained with (13)C NMR data, while values from full-scan data had higher error. We believe that this is the first use of tandem mass spectrometry to determine pathway flux using (13)C-enriched substrates. Although analysis of the citric acid cycle by NMR is simpler (and more intuitive), tandem mass spectrometry has the potential to combine high sensitivity with the high information yield previously available only by NMR.

Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase.

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent. Upon an extended 24-h fast, livers that lack PEPCK exhibit both 2-fold lower glucose production and oxygen consumption, compared with the controls, with all glucose production being derived only from glycerol. The mitochondrial reduction-oxidation (red-ox) state, as indicated by the NADH/NAD+ ratio, is 5-fold higher, and hepatic TCA cycle intermediate concentrations are dramatically increased in the PEPCK null livers. Consistent with this, flux through the TCA cycle and pyruvate cycling pathways is 10- and 40-fold lower, respectively. Disruption of hepatic cataplerosis due to loss of PEPCK leads to the accumulation of TCA cycle intermediates and a nearly complete blockage of gluconeogenesis from amino acids and lactate (an energy demanding process) but intact gluconeogenesis from glycerol (which contributes to net NADH production). Inhibition of the TCA cycle and fatty acid oxidation due to increased TCA cycle intermediate concentrations and reduced mitochondrial red-ox state lead to the development of steatosis.