Glioblastomas (GBMs) and brain metastases demonstrate avid uptake of 18fluoro-2-deoxyglucose (FDG) by positron emission tomography (PET) and display perturbations of intracellular metabolite pools by 1H magnetic resonance spectroscopy (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. FDG-positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation compared to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain malignancies to oxidize glucose in the tricarboxylic acid cycle is unknown. Here we studied the metabolism of human brain tumors in situ. [U-13C]glucose was infused during surgical resection, and tumor samples were subsequently subjected to 13C NMR spectroscopy. Analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the TCA 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-CoA 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 13C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse malignancies growing in their native microenvironment.

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 C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13 CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of Ϸ1 s along with H 13 CO 3 ؊ , the carbon dioxide ͉ heart ͉ hyperpolarization ͉ NMR spectroscopy ͉ pyruvate N oninvasive measures of flux through specific enzymecatalyzed reactions remain an important goal in physiology and clinical medicine. Standard radionuclide imaging methods do not provide information about individual reactions because the measured signal represents the weighted sum of the tracer plus the biochemical products produced by tissue. 13 C NMR spectroscopy is much more powerful in this regard because it can easily differentiate between specific 13 C-labeled products of biochemical reactions (1). The sensitivity enhancement gained by hyperpolarization of 13 C nuclei (2) offers the possibility of using noninvasive 13 C NMR spectroscopy and imaging to measure f luxes through individual enzyme-catalyzed reactions.[1-13 C 1 ]Pyruvate, for example, is a substrate that is avidly metabolized in most tissues. The individual metabolic products of this tracer, [1-13 C 1 ]lactate, [1-13 C 1 ]alanine, and H 13 CO 3 Ϫ , can be separately detected in vivo because of the inherent chemical shift dispersion of 13 C NMR (3). Because decarboxylation of [1-13 C 1 ]pyruvate via pyruvate dehydrogenase (PDH) must produce 13 CO 2 , the appearance of H 13 CO 3 Ϫ potentially directly reflects flux through PDH. A substantially reduced H 13 CO 3 Ϫ signal in hearts after coronary occlusion and reflow was previously attributed to an effect of transient ischemia on the tricarboxylic acid (TCA) cycle (3). However, it is known that the heart switches rapidly among a wide variety of substrates to supply acetyl-CoA (4, 5), and because fats and ketones are not metabolized via PDH the rate of production of H 13 CO 3 Ϫ from [1-13 C 1 ]pyruvate in vivo should be sensitive to the availability of other substrates.Noninvasive detection of flux through PDH would unquestionably be of value in understanding potentially high-impact therapies for heart disease. Pharmacological and metabolic interventions that would be expected to increase flux through PDH have been examined since the 1960s with the goal of protecting ischemic myocardium (6-9) or improving function in the failing heart (10, 11). However, because of the complex interconnections between fatty acid and carbohydrate oxidation, it has been difficult to separate effects of metabolism through a specific reaction, PDH, from effects on oxygen consumption, myocardial efficiency, and overa...

13C-nuclear magnetic resonance (NMR) spectroscopy provides a new approach to the analysis of metabolic pathways, because it detects an interaction between adjacent 13C nuclei. Previous models of isotope distribution in the tricarboxylic acid cycle were designed for analysis of radioisotope data and did not consider the information provided by 13C-13C coupling. A mathematical model of the tricarboxylic acid cycle was developed that preserves all isotope isomer (isotopomer) information and yields simple relationships between 13C-NMR spectra of glutamate and metabolic parameters under steady-state conditions. With the use of relative peak areas measured from the spectra of tissues supplied with 13C-enriched substrate(s), the relative fluxes through both oxidative (acetyl-CoA utilization) and nonoxidative (anaplerotic) pathways of the tricarboxylic acid cycle can be determined. Furthermore, with the judicious selection of 13C-labeling patterns in a mixture of substrates, direct substrate competition experiments can be performed. The perchloric acid extracts of Langendorff and working rat hearts oxidizing 13C-enriched fatty acids or carbohydrates are analyzed to illustrate this approach, and the importance of measuring the fractional enrichment of the available substrate is demonstrated. The technique can of course be used with all tissues, not just heart, and is applicable to the analysis of in vivo 13C-NMR spectra.

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 2 H and 13 C 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 reductionoxidation (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. Hepatic phosphoenolpyruvate carboxykinase (PEPCK)1 is a major control point for gluconeogenesis (1). Excess PEPCK expression in mice causes hyperglycemia (2), hyperinsulinemia, and increased glucose turnover (3). Inhibition of PEPCK by pharmaceutical interventions causes hypoglycemia (4) and, as expected, the global ablation of the cytosolic isoform of PEPCK in mice by genetic manipulation results in nonviable offspring (5). Most surprisingly, the liver-specific deletion of cytosolic PEPCK yielded a phenotype that, except during fasting and exercise, was virtually indistinguishable from control mice (5, 6). Even after a 24-h fast, when liver glycogen is depleted and flux through liver PEPCK should be essential to maintain plasma glucose, these animals are euglycemic and glucose turnover is normal. By using NMR spectroscopy and stable isotope tracers, we demonstrated that approximately ϳ60% of whole body glucose production in liver-specific PEPCK knock-out animals is derived from lactate and alanine (6). This suggests that either an alternative route to glucose production that bypasses PEPCK in these livers exists or that the majority of whole body gluconeogenesis is extrahepatic (6). In marked contrast to the minimal impact that the absence of hepatic PEPCK has on systemic glucose kinetics, these mice develop dramatic hepatic steatosis after fasting (6) even though enzymes of the TCA cycle and ␤-oxidation are up-regulated (5) in liver tissue....