Helen J. Atherton

Systems level studies of mammalian metabolomes: the roles of mass spectrometry and nuclear magnetic resonance spectroscopy

The study of biological systems in a holistic manner (systems biology) is increasingly being viewed as a necessity to provide qualitative and quantitative descriptions of the emergent properties of the complete system. Systems biology performs studies focussed on the complex interactions of system components; emphasising the whole system rather than the individual parts. Many perturbations to mammalian systems (diet, disease, drugs) are multi-factorial and the study of small parts of the system is insufficient to understand the complete phenotypic changes induced. Metabolomics is one functional level tool being employed to investigate the complex interactions of metabolites with other metabolites (metabolism) but also the regulatory role metabolites provide through interaction with genes, transcripts and proteins (e.g. allosteric regulation). Technological developments are the driving force behind advances in scientific knowledge. Recent advances in the two analytical platforms of mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy have driven forward the discipline of metabolomics. In this critical review, an introduction to metabolites, metabolomes, metabolomics and the role of MS and NMR spectroscopy will be provided. The applications of metabolomics in mammalian systems biology for the study of the health-disease continuum, drug efficacy and toxicity and dietary effects on mammalian health will be reviewed. The current limitations and future goals of metabolomics in systems biology will also be discussed (374 references).

Real-time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy

The Krebs cycle plays a fundamental role in cardiac energy production and is often implicated in the energetic imbalance characteristic of heart disease. In this study, we measured Krebs cycle flux in real time in perfused rat hearts using hyperpolarized magnetic resonance spectroscopy (MRS). [2‐13C]Pyru‐ vate was hyperpolarized and infused into isolated perfused hearts in both healthy and postischemic metabolic states. We followed the enzymatic conversion of pyruvate to lactate, acetylcarnitine, citrate, and glutamate with 1 s temporal resolution. The appearance of 13C‐labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of 13C‐ labeled citrate and glutamate was decreased postischemia, as opposed to lactate, which was significantly elevated. These results showed that the control and fluxes of the Krebs cycle in heart disease can be studied using hyperpolarized [2‐13C]pyruvate.— Schroeder, M. A., Atherton, H. J., Ball, D. R., Cole, M. A., Heather, L. C., Griffin, J. L., Clarke, K., Radda, G. K., Tyler, D. J. Real‐time assessment of Krebs cycle metabolism using hyperpolarized 13C magnetic resonance spectroscopy. FASEBJ. 23, 2529–2538 (2009)

Metabolomics as a tool for cardiac research

Metabolomics represents a paradigm shift in metabolic research, away from approaches that focus on a limited number of enzymatic reactions or single pathways, to approaches that attempt to capture the complexity of metabolic networks. Additionally, the high-throughput nature of metabolomics makes it ideal to perform biomarker screens for diseases or follow drug efficacy. In this Review, we explore the role of metabolomics in gaining mechanistic insight into cardiac disease processes, and in the search for novel biomarkers. High-resolution NMR spectroscopy and mass spectrometry are both highly discriminatory for a range of pathological processes affecting the heart, including cardiac ischemia, myocardial infarction, and heart failure. We also discuss the position of metabolomics in the range of functional-genomic approaches, being complementary to proteomic and transcriptomic studies, and having subdivisions such as lipidomics (the study of intact lipid species). In addition to techniques that monitor changes in the total sizes of pools of metabolites in the heart and biofluids, the role of stable-isotope methods for monitoring fluxes through pathways is examined. The use of these novel functional-genomic tools to study metabolism provides a unique insight into cardiac disease progression.

Measuring intracellular pH in the heart using hyperpolarized carbon dioxide and bicarbonate: a 13C and 31P magnetic resonance spectroscopy study.

Aims Technological limitations have restricted in vivo assessment of intracellular pH (pHi) in the myocardium. The aim of this study was to evaluate the potential of hyperpolarized [1-13C]pyruvate, coupled with 13C magnetic resonance spectroscopy (MRS), to measure pHi in the healthy and diseased heart. Methods and results Hyperpolarized [1-13C]pyruvate was infused into isolated rat hearts before and immediately after ischaemia, and the formation of 13CO2 and H13CO3− was monitored using 13C MRS. The HCO3−/CO2 ratio was used in the Henderson–Hasselbalch equation to estimate pHi. We tested the validity of this approach by comparing 13C-based pHi measurements with 31P MRS measurements of pHi. There was good agreement between the pHi measured using 13C and 31P MRS in control hearts, being 7.12 ± 0.10 and 7.07 ± 0.02, respectively. In reperfused hearts, 13C and 31P measurements of pHi also agreed, although 13C equilibration limited observation of myocardial recovery from acidosis. In hearts pre-treated with the carbonic anhydrase (CA) inhibitor, 6-ethoxyzolamide, the 13C measurement underestimated the 31P-measured pHi by 0.80 pH units. Mathematical modelling predicted that the validity of measuring pHi from the H13CO3−/13CO2 ratio depended on CA activity, and may give an incorrect measure of pHi under conditions in which CA was inhibited, such as in acidosis. Hyperpolarized [1-13C]pyruvate was also infused into healthy living rats, where in vivo pHi from the H13CO3−/13CO2 ratio was measured to be 7.20 ± 0.03. Conclusion Metabolically generated 13CO2 and H13CO3− can be used as a marker of cardiac pHi in vivo, provided that CA activity is at normal levels.

Role of Pyruvate Dehydrogenase Inhibition in the Development of Hypertrophy in the Hyperthyroid Rat Heart A Combined Magnetic Resonance Imaging and Hyperpolarized Magnetic Resonance Spectroscopy Study

Background— Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Methods and Results— Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg · kg−1 · d−1). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022±0.0002 versus 0.0055±0.0005 second−1; P=0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-13C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100±20 versus 200±30 mg; P=0.04) despite no change in the increase observed in cardiac output. Conclusions— This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand.Background Hyperthyroidism increases heart rate, contractility and cardiac output, as well as metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate utilisation. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase (PDK), thereby inhibiting glucose oxidation via pyruvate dehydrogenase (PDH). Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy (MRS) to investigate the rate and regulation of in vivo pyruvate dehydrogenase (PDH) flux in the hyperthyroid heart, and to establish whether modulation of flux through PDH would alter cardiac hypertrophy.Background— Hyperthyroidism increases heart rate, contractility, cardiac output, and metabolic rate. It is also accompanied by alterations in the regulation of cardiac substrate use. Specifically, hyperthyroidism increases the ex vivo activity of pyruvate dehydrogenase kinase, thereby inhibiting glucose oxidation via pyruvate dehydrogenase. Cardiac hypertrophy is another effect of hyperthyroidism, with an increase in the abundance of mitochondria. Although the hypertrophy is initially beneficial, it can eventually lead to heart failure. The aim of this study was to use hyperpolarized magnetic resonance spectroscopy to investigate the rate and regulation of in vivo pyruvate dehydrogenase flux in the hyperthyroid heart and to establish whether modulation of flux through pyruvate dehydrogenase would alter cardiac hypertrophy. Methods and Results— Hyperthyroidism was induced in 18 male Wistar rats with 7 daily intraperitoneal injections of freshly prepared triiodothyronine (0.2 mg · kg−1 · d−1). In vivo pyruvate dehydrogenase flux, assessed with hyperpolarized magnetic resonance spectroscopy, was reduced by 59% in hyperthyroid animals (0.0022±0.0002 versus 0.0055±0.0005 second−1; P =0.0003), and this reduction was completely reversed by both short- and long-term delivery of dichloroacetic acid, a pyruvate dehydrogenase kinase inhibitor. Hyperpolarized [2-13C]pyruvate was also used to evaluate Krebs cycle metabolism and demonstrated a unique marker of anaplerosis, the level of which was significantly increased in the hyperthyroid heart. Cine magnetic resonance imaging showed that long-term dichloroacetic acid treatment significantly reduced the hypertrophy observed in hyperthyroid animals (100±20 versus 200±30 mg; P =0.04) despite no change in the increase observed in cardiac output. Conclusions— This work has demonstrated that inhibition of glucose oxidation in the hyperthyroid heart in vivo is mediated by pyruvate dehydrogenase kinase. Relieving this inhibition can increase the metabolic flexibility of the hyperthyroid heart and reduce the level of hypertrophy that develops while maintaining the increased cardiac output required to meet the higher systemic metabolic demand. # Clinical Perspective {#article-title-52}

Validation of the in vivo assessment of pyruvate dehydrogenase activity using hyperpolarised 13C MRS.

Many diseases of the heart are characterised by changes in substrate utilisation, which is regulated in part by the activity of the enzyme pyruvate dehydrogenase (PDH). Consequently, there is much interest in the in vivo evaluation of PDH activity in a range of physiological and pathological states to obtain information on the metabolic mechanisms of cardiac diseases. Hyperpolarised [1‐13C]pyruvate, detected using MRS, is a novel technique for the noninvasive evaluation of PDH flux. PDH flux has been assumed to directly reflect in vivo PDH activity, although to date this assumption remains unproven. Control animals and animals undergoing interventions known to modulate PDH activity, namely high fat feeding and dichloroacetate infusion, were used to investigate the relationship between in vivo hyperpolarised MRS measurements of PDH flux and ex vivo measurements of PDH enzyme activity (PDHa). Further, the plasma concentrations of pyruvate and other important metabolites were evaluated following pyruvate infusion to assess the metabolic consequences of pyruvate infusion during hyperpolarised MRS experiments. Hyperpolarised MRS measurements of PDH flux correlated significantly with ex vivo measurements of PDHa, confirming that PDH activity influences directly the in vivo flux of hyperpolarised pyruvate through cardiac PDH. The maximum plasma concentration of pyruvate reached during hyperpolarised MRS experiments was approximately 250 µM, equivalent to physiological pyruvate concentrations reached during exercise or with dietary interventions. The concentrations of other metabolites, including lactate, glucose and β‐hydroxybutyrate, did not vary during the 60 s following pyruvate infusion. Hence, during the 60‐s data acquisition period, metabolism was minimally affected by pyruvate infusion. Copyright

Metabolomics of the interaction between PPAR-α and age in the PPAR-α-null mouse

Regulation between the fed and fasted states in mammals is partially controlled by peroxisome proliferator‐activated receptor‐α (PPAR‐α). Expression of the receptor is high in the liver, heart and skeletal muscle, but decreases with age. A combined 1H nuclear magnetic resonance (NMR) spectroscopy and gas chromatography‐mass spectrometry metabolomic approach has been used to examine metabolism in the liver, heart, skeletal muscle and adipose tissue in PPAR‐α‐null mice and wild‐type controls during ageing between 3 and 13 months. For the PPAR‐α‐null mouse, multivariate statistics highlighted hepatic steatosis, reductions in the concentrations of glucose and glycogen in both the liver and muscle tissue, and profound changes in lipid metabolism in each tissue, reflecting known expression targets of the PPAR‐α receptor. Hepatic glycogen and glucose also decreased with age for both genotypes. These findings indicate the development of age‐related hepatic steatosis in the PPAR‐α‐null mouse, with the normal metabolic changes associated with ageing exacerbating changes associated with genotype. Furthermore, the combined metabolomic and multivariate statistics approach provides a robust method for examining the interaction between age and genotype.

The Cycling of Acetyl-Coenzyme A Through Acetylcarnitine Buffers Cardiac Substrate Supply A Hyperpolarized 13C Magnetic Resonance Study

Background— Carnitine acetyltransferase catalyzes the reversible conversion of acetyl-coenzyme A (CoA) into acetylcarnitine. The aim of this study was to use the metabolic tracer hyperpolarized [2-13C]pyruvate with magnetic resonance spectroscopy to determine whether carnitine acetyltransferase facilitates carbohydrate oxidation in the heart. Methods and Results— Ex vivo, following hyperpolarized [2-13C]pyruvate infusion, the [1-13C]acetylcarnitine resonance was saturated with a radiofrequency pulse, and the effect of this saturation on [1-13C]citrate and [5-13C]glutamate was observed. In vivo, [2-13C]pyruvate was infused into 3 groups of fed male Wistar rats: (1) controls, (2) rats in which dichloroacetate enhanced pyruvate dehydrogenase flux, and (3) rats in which dobutamine elevated cardiac workload. In the perfused heart, [1-13C]acetylcarnitine saturation reduced the [1-13C]citrate and [5-13C]glutamate resonances by 63% and 51%, respectively, indicating a rapid exchange between pyruvate-derived acetyl-CoA and the acetylcarnitine pool. In vivo, dichloroacetate increased the rate of [1-13C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1-13C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%. Conclusions— Hyperpolarized 13C magnetic resonance spectroscopy has revealed that acetylcarnitine provides a route of disposal for excess acetyl-CoA and a means to replenish acetyl-CoA when cardiac workload is increased. Cycling of acetyl-CoA through acetylcarnitine appears key to matching instantaneous acetyl-CoA supply with metabolic demand, thereby helping to balance myocardial substrate supply and contractile function.

In vivo alterations in cardiac metabolism and function in the spontaneously hypertensive rat heart.

AIMS The aim of this work was to use hyperpolarized carbon-13 ((13)C) magnetic resonance (MR) spectroscopy and cine MR imaging (MRI) to assess in vivo cardiac metabolism and function in the 15-week-old spontaneously hypertensive rat (SHR) heart. At this time point, the SHR displays hypertension and concentric hypertrophy. One of the cellular adaptations to hypertrophy is a reduction in β-oxidation, and it has previously been shown that in response to hypertrophy the SHR heart switches to a glycolytic/glucose-oxidative phenotype. METHODS AND RESULTS Cine-MRI (magnetic resonance imaging) was used to assess cardiac function and degree of cardiac hypertrophy. Wistar rats were used as controls. SHRs displayed functional changes in stroke volume, heart rate, and late peak-diastolic filling alongside significant hypertrophy (a 56% increase in left ventricular mass). Using hyperpolarized [1-(13)C] and [2-(13)C]pyruvate, an 85% increase in (13)C label flux through pyruvate dehydrogenase (PDH) was seen in the SHR heart and (13)C label incorporation into citrate, acetylcarnitine, and glutamate pools was elevated in proportion to the increase in PDH flux. These findings were confirmed using biochemical analysis of PDH activity and protein expression of PDH regulatory enzymes. CONCLUSIONS Functional and structural alterations in the SHR heart are consistent with the hypertrophied phenotype. Our in vivo work indicates a preference for glucose metabolism in the SHR heart, a move away from predominantly fatty acid oxidative metabolism. Interestingly, (13)C label flux into lactate was unchanged, indicating no switch to an anaerobic glycolytic phenotype, but rather an increased reliance on glucose oxidation in the SHR heart.

A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-α in the mouse

Proton Nuclear Magnetic Resonance spectroscopy and Gas Chromatography Mass Spectrometry based metabolomics has been used in conjunction with multivariate statistics to examine the metabolic changes in Caenorhabditis elegans following the deletion of nuclear hormone receptor‐49 (nhr‐49). Deletion of the receptor produced profound changes in fatty acid metabolism, in particular an increase in the ratio of unsaturated to saturated fatty acids, a decrease in the concentration of glucose and increases in lactate and alanine. Given the proposed functional similarity between nhr‐49 and the mammalian peroxisome proliferator‐activated receptors (PPARs) these changes were compared with the metabolome of the PPAR‐α null mouse. The metabolomic approach demonstrated a number of similarities including the regulation of lipid synthesis, β‐oxidation of fatty acids and changes in glycolysis/gluconeogenesis.