F. Mark Jeffrey

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

13C NMR is a powerful tool for monitoring metabolic fluxes in vivo. The recent availability of automated dynamic nuclear polarization equipment for hyperpolarizing 13C 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 [1-13C1]pyruvate in a widely used model for physiology and pharmacology, the perfused rat heart. Dissolved 13CO2, the immediate product of the first step of the reaction catalyzed by pyruvate dehydrogenase, was observed with a temporal resolution of ≈1 s along with H13CO3−, the hydrated form of 13CO2 generated catalytically by carbonic anhydrase. In hearts presented with the medium-chain fatty acid octanoate in addition to hyperpolarized [1-13C1]pyruvate, production of 13CO2 and H13CO3− was suppressed by ≈90%, whereas the signal from [1-13C1]lactate was enhanced. In separate experiments, it was shown that O2 consumption and tricarboxylic acid (TCA) cycle flux were unchanged in the presence of added octanoate. Thus, the rate of appearance of 13CO2 and H13CO3− from [1-13C1]pyruvate does not reflect production of CO2 in the TCA cycle but rather reflects flux through pyruvate dehydrogenase exclusively.

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

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[18F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by 1H 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‐[18F]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 13C 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 13C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright

C-NMR: a simple yet comprehensive method for analysis of intermediary metabolism

13C-NMR is a particularly attractive tool for metabolic studies because of its inherent simplicity: all labeled products at sufficient concentration may be identified and analysed in a single spectrum. However, the real power behind the approach presented here is the ability to measure groups of individual 13C-isotopomers (isotope isomers). The information that this provides is superior to conventional tracer techniques, allowing a very detailed description of metabolic events. Several examples are given of the value of this convenient and straightforward analysis for some problems of current interest in intermediary metabolism.

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.

Contribution of various substrates to total citric acid cycle flux and ]anaplerosis as determined by13C isotopomer analysis and O2 consumption in the heart

A simple relationship between parameters derived from a13C NMR isotopomer analysis and O2 consumption is presented that allows measurement of the absolute rate of acetyl-CoA oxidation and anaplerotic flux in tissues oxidizing a mixture of four substrates. The method was first applied in a study of the effects of work state and β-adrenergic stimulation on net acetate oxidation and anaplerosis in the isolated working rat heart. The results demonstrate that the anticipated ratio of 2 between O2 consumption and TCA cycle flux for hearts oxidizing only acetate holds at low workload when anaplerosis is low, but deviates toward a factor of 3 under high workload conditions when anaplerosis is increased. This analysis was also extended to hearts that oxidize a more physiological mixture of substrates including long-chain fatty acids, acetoacetate, lactate, pyruvate, and glucose. We show that the contribution each substrate makes to total TCA cycle flux can be determined by combined13C NMR and O2 consumption measurements. The present study also demonstrates that stimulation of anaplerosis (by addition of propionate) can significantly alter the relative contribution each substrate makes to total TCA cycle flux. We conclude that if13C labeling patterns are selected appropriately, a comprehensive picture of flux through all major metabolic pathways feeding the cycle can be determined in a single experiment even when complex physiological mixtures of substrates are provided.

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.

Use of a single 13C NMR resonance of glutamate for measuring oxygen consumption in tissue

A kinetic model of the citric acid cycle for calculating oxygen consumption from13C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MV˙o 2) was compared with MV˙o 2 predicted by the model with 13C NMR data obtained from rat hearts perfused with glucose and either [2-13C]acetate or [3-13C]pyruvate. The accuracy of MV˙o 2 measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MV˙o 2 determined by set 2(C4D34 only) gave the same degree of accuracy as set 3(complete data); both were superior to set 1(C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between α-ketoglutarate and glutamate, resulting in greater error in estimating MV˙o 2. Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.A kinetic model of the citric acid cycle for calculating oxygen consumption from (13)C nuclear magnetic resonance (NMR) multiplet data has been developed. Measured oxygen consumption (MVO(2)) was compared with MVO(2) predicted by the model with (13)C NMR data obtained from rat hearts perfused with glucose and either [2-(13)C]acetate or [3-(13)C]pyruvate. The accuracy of MVO(2) measured from three subsets of NMR data was compared: glutamate C-4 and C-3 resonance areas; the doublet C4D34 (expressed as a fraction of C-4 area); and C-4 and C-3 areas plus several multiplets of C-2, C-3, and C-4. MVO(2) determined by set 2 (C4D34 only) gave the same degree of accuracy as set 3 (complete data); both were superior to set 1 (C-4 and C-3 areas). Analysis of the latter suffers from the correlation between citric acid cycle flux and exchange between alpha-ketoglutarate and glutamate, resulting in greater error in estimating MVO(2). Analysis of C4D34 is less influenced by correlation between parameters, and this single measurement provides the best opportunity for a noninvasive measurement of oxygen consumption.

NMR indirect detection of glutamate to measure citric acid cycle flux in the isolated perfused mouse heart

13C‐edited proton nuclear magnetic resonance (NMR) spectroscopy was used to follow enrichment of glutamate C3 and C4 with a temporal resolution of ∼20 s in mouse hearts perfused with 13C‐enriched substrates. A fit of the NMR data to a kinetic model of the tricarboxylic acid (TCA) cycle and related exchange reactions yielded TCA cycle (V tca) and exchange (V x) fluxes between α‐ketoglutarate and glutamate. These fluxes were substrate‐dependent and decreased in the order acetate (V tca=14.1 μmol g−1 min−1; V x=26.5 μmol g−1 min−1)>octanoate (V tca=6.0 μmol g−1 min−1; V x=16.1 μmol g−1 min−1)>lactate (V tca=4.2 μmol g−1 min−1; V x=6.3 μmol g−1 min−1).

13C isotopomer analysis of glutamate by heteronuclear multiple quantum coherence-total correlation spectroscopy (HMQC-TOCSY)

13C has become an important tracer isotope for studies of intermediary metabolism. Information about relative flux through pathways is encoded by the distribution of 13C isotopomers in an intermediate pool such as glutamate. This information is commonly decoded either by mass spectrometry or by measuring relative multiplet areas in a 13C NMR spectrum. We demonstrate here that groups of glutamate 13C isotopomers may be quantified by indirect detection of protons in a 2D HMQC‐TOCSY NMR spectrum and that fitting of these data to a metabolic model provides an identical measure of the 13C fractional enrichment of acetyl‐CoA and relative anaplerotic flux to that given by direct 13C NMR analysis. The sensitivity gain provided by HMQC‐TOCSY spectroscopy will allow an extension of 13C isotopomer analysis to tissue samples not amenable to direct 13C detection (∼10 mg soleus muscle) and to tissue metabolites other than glutamate that are typically present at lower concentrations.

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 [1,6-13C2]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 13C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-13C2]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.