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...

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....

Isolated rat hearts were studied by 31 P NMR and 13 C NMR. Hyperpolarized [1-13 C]pyruvate was supplied to control normoxic hearts and production of [1-13 C]lactate, [1-13 C]alanine, 13 CO 2 and H 13 CO 3 ؊ was monitored with 1-s temporal resolution. Hearts were also subjected to 10 min of global ischemia followed by reperfusion. Developed pressure, heart rate, oxygen consumption, [ Myocardial oxygen consumption is sensitive to the ratio of fatty acid versus carbohydrate metabolism (1-3). Fatty acid utilization increases oxygen consumption. This is considered insignificant to physiology in the normoxic heart, but in the setting of ischemia or ischemia-reperfusion, increased metabolism of fatty acids impairs contractility and recovery (1,2). Diverse metabolic (4,5) and pharmacologic (6 -8) interventions share a common featureincreased oxidation of carbohydrates relative to fatty acids improves outcome after myocardial ischemia. Although these benefits have been demonstrated repeatedly, the mechanism is poorly understood and the clinical utility of increased carbohydrate oxidation remains controversial (9).Glucose, pyruvate, and lactate are important substrates for oxidation by the heart. The product of lactate and glucose metabolism, pyruvate, is decarboxylated by pyruvate dehydrogenase (PDH) to produce acetyl-CoA for subsequent oxidation in the citric acid cycle. The other major substrates for energy production, fatty acids and ketones, are metabolized through ␤ oxidation and bypass PDH for generation of acetyl-CoA. Because of the importance of PDH in cardiac metabolism, the classic radiotracer method for assessing PDH flux, 14 CO 2 release from [1-14 C]pyruvate (10 -13), has been extensively developed and widely accepted. Applications, however, are limited in vivo because of radiation containment requirements and the difficulty of collecting blood from the vessels draining the ischemic region. Consequently, there is considerable interest in direct metabolic mapping using hyperpolarized [1-13 C]pyruvate (7,14). 13 C MR images of [1-13 C]lactate, [1-13 C]alanine and H 13 CO 3 Ϫ ([ 13 C]bicarbonate) were relatively homogeneous in the normal myocardium, but reduced signal from [ 13 C]bicarbonate compared with the normal myocardium was observed 2 hr after transient ischemia (15). In the presence of [1-13 C]pyruvate, the appearance of H 13 CO 3 Ϫ in heart tissue is due exclusively to PDH flux (16). When carbohydrates are the only source of acetyl-CoA for oxidation in the TCA cycle, the rate of production of H 13 CO 3 Ϫ is proportional to citric acid cycle flux. However, the heart can derive much of its acetyl-CoA from long chain fatty acids or ketones. It would be expected that reduced bicarbonate signal may be due to reduced flux through the TCA cycle, a switch to oxidation of fats or ketones, or a combination of these two factors.In the present study, oxidation of hyperpolarized [1-13 C]pyruvate was examined in a model widely used for evaluation of tracer kinetics in ischemia and reperfusion, the isolated rat heart...