Novel ketone diet enhances physical and cognitive performance.

Ketone bodies are the most energy-efficient fuel and yield more ATP per mole of substrate than pyruvate and increase the free energy released from ATP hydrolysis. Elevation of circulating ketones via high-fat, low-carbohydrate diets has been used for the treatment of drug-refractory epilepsy and for neurodegenerative diseases, such as Parkinson's disease. Ketones may also be beneficial for muscle and brain in times of stress, such as endurance exercise. The challenge has been to raise circulating ketone levels by using a palatable diet without altering lipid levels. We found that blood ketone levels can be increased and cholesterol and triglycerides decreased by feeding rats a novel ketone ester diet: chow that is supplemented with (R)-3-hydroxybutyl (R)-3-hydroxybutyrate as 30% of calories. For 5 d, rats on the ketone diet ran 32% further on a treadmill than did control rats that ate an isocaloric diet that was supplemented with either corn starch or palm oil (P < 0.05). Ketone-fed rats completed an 8-arm radial maze test 38% faster than did those on the other diets, making more correct decisions before making a mistake (P < 0.05). Isolated, perfused hearts from rats that were fed the ketone diet had greater free energy available from ATP hydrolysis during increased work than did hearts from rats on the other diets as shown by using [31P]-NMR spectroscopy. The novel ketone diet, therefore, improved physical performance and cognitive function in rats, and its energy-sparing properties suggest that it may help to treat a range of human conditions with metabolic abnormalities.-Murray, A. J., Knight, N. S., Cole, M. A., Cochlin, L. E., Carter, E., Tchabanenko, K., Pichulik, T., Gulston, M. K., Atherton, H. J., Schroeder, M. A., Deacon, R. M. J., Kashiwaya, Y., King, M. T., Pawlosky, R., Rawlins, J. N. P., Tyler, D. J., Griffin, J. L., Robertson, J., Veech, R. L., Clarke, K. Novel ketone diet enhances physical and cognitive performance.

Impaired in vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy.

BACKGROUND: Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities. METHODS AND RESULTS: Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates. CONCLUSIONS: The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment.

Differential translocation of the fatty acid transporter, FAT/CD36, and the glucose transporter, GLUT4, coordinates changes in cardiac substrate metabolism during ischemia and reperfusion.

BACKGROUND: Fatty acid and glucose transporters translocate between the sarcolemma and intracellular compartments to regulate substrate metabolism acutely. We hypothesised that during ischemia fatty acid translocase (FAT/CD36) would translocate away from the sarcolemma to limit fatty acid uptake when fatty acid oxidation is inhibited. METHODS AND RESULTS: Wistar rat hearts were perfused during preischemia, low-flow ischemia, and reperfusion, using (3)H-substrates for measurement of metabolic rates, followed by metabolomic analysis and subcellular fractionation. During ischemia, there was a 32% decrease in sarcolemmal FAT/CD36 accompanied by a 95% decrease in fatty acid oxidation rates, with no change in intramyocardial lipids. Concomitantly, the sarcolemmal content of the glucose transporter, GLUT4, increased by 90% during ischemia, associated with an 86% increase in glycolytic rates, 45% decrease in glycogen content, and a 3-fold increase in phosphorylated AMP-activated protein kinase. Following reperfusion, decreased sarcolemmal FAT/CD36 persisted, but fatty acid oxidation rates returned to preischemic levels, resulting in a 35% decrease in myocardial triglyceride content. Elevated sarcolemmal GLUT4 persisted during reperfusion; in contrast, glycolytic rates decreased to 30% of preischemic rates, accompanied by a 5-fold increase in intracellular citrate levels and restoration of glycogen content. CONCLUSIONS: During ischemia, FAT/CD36 moved away from the sarcolemma as GLUT4 moved toward the sarcolemma, associated with a shift from fatty acid oxidation to glycolysis, while intramyocardial lipid accumulation was prevented. This relocation was maintained during reperfusion, which was associated with replenishing glycogen stores as a priority, occurring at the expense of glycolysis and mediated by an increase in citrate levels.

Adaptive changes of the Insig1/SREBP1/SCD1 set point help adipose tissue to cope with increased storage demands of obesity.

The epidemic of obesity imposes unprecedented challenges on human adipose tissue (WAT) storage capacity that may benefit from adaptive mechanisms to maintain adipocyte functionality. Here, we demonstrate that changes in the regulatory feedback set point control of Insig1/SREBP1 represent an adaptive response that preserves WAT lipid homeostasis in obese and insulin-resistant states. In our experiments, we show that Insig1 mRNA expression decreases in WAT from mice with obesity-associated insulin resistance and from morbidly obese humans and in in vitro models of adipocyte insulin resistance. Insig1 downregulation is part of an adaptive response that promotes the maintenance of SREBP1 maturation and facilitates lipogenesis and availability of appropriate levels of fatty acid unsaturation, partially compensating the antilipogenic effect associated with insulin resistance. We describe for the first time the existence of this adaptive mechanism in WAT, which involves Insig1/SREBP1 and preserves the degree of lipid unsaturation under conditions of obesity-induced insulin resistance. These adaptive mechanisms contribute to maintain lipid desaturation through preferential SCD1 regulation and facilitate fat storage in WAT, despite on-going metabolic stress.

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 ß-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.

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-(13)C]pyruvate with magnetic resonance spectroscopy to determine whether carnitine acetyltransferase facilitates carbohydrate oxidation in the heart. METHODS AND RESULTS: Ex vivo, following hyperpolarized [2-(13)C]pyruvate infusion, the [1-(13)C]acetylcarnitine resonance was saturated with a radiofrequency pulse, and the effect of this saturation on [1-(13)C]citrate and [5-(13)C]glutamate was observed. In vivo, [2-(13)C]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-(13)C]acetylcarnitine saturation reduced the [1-(13)C]citrate and [5-(13)C]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-(13)C]acetylcarnitine production by 35% and increased the overall acetylcarnitine pool size by 33%. Dobutamine decreased the rate of [1-(13)C]acetylcarnitine production by 37% and decreased the acetylcarnitine pool size by 40%. CONCLUSIONS: Hyperpolarized (13)C 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.

A Metadata description of the data in "A metabolomic comparison of urinary changes in type 2 diabetes in mouse, rat, and human.".

BACKGROUND: Metabolomics is a rapidly developing functional genomic tool that has a wide range of applications in diverse fields in biology and medicine. However, unlike transcriptomics and proteomics there is currently no central repository for the depositing of data despite efforts by the Metabolomics Standard Initiative (MSI) to develop a standardised description of a metabolomic experiment. FINDINGS: In this manuscript we describe how the MSI description has been applied to a published dataset involving the identification of cross-species metabolic biomarkers associated with type II diabetes. The study describes sample collection of urine from mice, rats and human volunteers, and the subsequent acquisition of data by high resolution 1H NMR spectroscopy. The metadata is described to demonstrate how the MSI descriptions could be applied in a manuscript and the spectra have also been made available for the mouse and rat studies to allow others to process the data. CONCLUSIONS: The intention of this manuscript is to stimulate discussion as to whether the MSI description is sufficient to describe the metadata associated with metabolomic experiments and encourage others to make their data available to other researchers.

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 x kg(-1) x 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-(13)C]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.

Determining the in vivo regulation of cardiac pyruvate dehydrogenase based on label flux from hyperpolarised [1-13C]pyruvate.

Pyruvate dehydrogenase (PDH) is a key regulator of cardiac substrate selection and is regulated by both pyruvate dehydrogenase kinase (PDK)-mediated phosphorylation and feedback inhibition. The extent to which chronic upregulation of PDK protein levels, acutely increased PDK activity and acute feedback inhibition limit PDH flux remains unclear because existing in vitro assessment methods inherently disrupt the regulation of the enzyme complex. We have demonstrated previously that hyperpolarised (13)C-labelled metabolic tracers coupled with MRS can monitor flux through PDH in vivo. The aim of this study was to determine the relative contributions of acute and chronic changes in PDK and PDH activities to in vivo myocardial PDH flux. We examined both fed and fasted rats with either hyperpolarised [1-(13)C]pyruvate alone or hyperpolarised [1-(13)C]pyruvate co-infused with malate [to modulate mitochondrial nicotinamide adenine dinucleotide (NADH/NAD(+)) and acetyl-coenzyme A (acetyl-CoA)/CoA ratios, which alter both PDH activity and flux]. To confirm the metabolic fate of infused malate, we performed in vitro (1)H NMR spectroscopy on cardiac tissue extracts. We observed that, in fed rats, where PDH activity was high, the presence of malate increased PDH flux by 27%, whereas, in the fasted state, malate infusion had no effect on PDH flux. These observations suggest that pyruvate oxidation is limited by feedback inhibition from acetyl-CoA only when PDH activity is high. Therefore, in the case of PDH, and potentially other enzymes, hyperpolarised (13)C MRI can be used to assess noninvasively enzymatic regulation.

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-(13)C]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 (PDH(a)). 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 PDH(a), 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 ß-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.

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).

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 (pH(i)) in the myocardium. The aim of this study was to evaluate the potential of hyperpolarized [1-(13)C]pyruvate, coupled with (13)C magnetic resonance spectroscopy (MRS), to measure pH(i) in the healthy and diseased heart. METHODS AND RESULTS: Hyperpolarized [1-(13)C]pyruvate was infused into isolated rat hearts before and immediately after ischaemia, and the formation of (13)CO(2) and H(13)CO(3)(-) was monitored using (13)C MRS. The HCO(3)(-)/CO(2) ratio was used in the Henderson-Hasselbalch equation to estimate pH(i). We tested the validity of this approach by comparing (13)C-based pH(i) measurements with (31)P MRS measurements of pH(i). There was good agreement between the pH(i) measured using (13)C and (31)P MRS in control hearts, being 7.12 +/- 0.10 and 7.07 +/- 0.02, respectively. In reperfused hearts, (13)C and (31)P measurements of pH(i) also agreed, although (13)C equilibration limited observation of myocardial recovery from acidosis. In hearts pre-treated with the carbonic anhydrase (CA) inhibitor, 6-ethoxyzolamide, the (13)C measurement underestimated the (31)P-measured pH(i) by 0.80 pH units. Mathematical modelling predicted that the validity of measuring pH(i) from the H(13)CO(3)(-)/(13)CO(2) ratio depended on CA activity, and may give an incorrect measure of pH(i) under conditions in which CA was inhibited, such as in acidosis. Hyperpolarized [1-(13)C]pyruvate was also infused into healthy living rats, where in vivo pH(i) from the H(13)CO(3)(-)/(13)CO(2) ratio was measured to be 7.20 +/- 0.03. CONCLUSION: Metabolically generated (13)CO(2) and H(13)CO(3)(-) can be used as a marker of cardiac pH(i) in vivo, provided that CA activity is at normal levels.

Adenosine monophosphate-activated protein kinase activation, substrate transporter translocation, and metabolism in the contracting hyperthyroid rat heart.

Thyroid hormones can modify cardiac metabolism via multiple molecular mechanisms, yet their integrated effect on overall substrate metabolism is poorly understood. Here we determined the effect of hyperthyroidism on substrate metabolism in the isolated, perfused, contracting rat heart. Male Wistar rats were injected for 7 d with T(3) (0.2 mg/kg x d ip). Plasma free fatty acids increased by 97%, heart weights increased by 33%, and cardiac rate pressure product, an indicator of contractile function, increased by 33% in hyperthyroid rats. Insulin-stimulated glycolytic rates and lactate efflux rates were increased by 33% in hyperthyroid rat hearts, mediated by an increased insulin-stimulated translocation of the glucose transporter GLUT4 to the sarcolemma. This was accompanied by a 70% increase in phosphorylated AMP-activated protein kinase (AMPK) and a 100% increase in phosphorylated acetyl CoA carboxylase, confirming downstream signaling from AMPK. Fatty acid oxidation rates increased in direct proportion to the increased heart weight and rate pressure product in the hyperthyroid heart, mediated by synchronized changes in mitochondrial enzymes and respiration. Protein levels of the fatty acid transporter, fatty acid translocase (FAT/CD36), were reduced by 24% but were accompanied by a 19% increase in the sarcolemmal content of fatty acid transport protein 1 (FATP1). Thus, the relationship between fatty acid metabolism, cardiac mass, and contractile function was maintained in the hyperthyroid heart, associated with a sarcolemmal reorganization of fatty acid transporters. The combined effects of T(3)-induced AMPK activation and insulin stimulation were associated with increased sarcolemmal GLUT4 localization and glycolytic flux in the hyperthyroid heart.

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

Regulation between the fed and fasted states in mammals is partially controlled by peroxisome proliferator-activated receptor-alpha (PPAR-alpha). Expression of the receptor is high in the liver, heart and skeletal muscle, but decreases with age. A combined (1)H 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-alpha-null mice and wild-type controls during ageing between 3 and 13 months. For the PPAR-alpha-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-alpha 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-alpha-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.

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-(13)C]Pyruvate 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 (13)C-labeled glutamate was delayed compared with that of other metabolites, indicating that Krebs cycle flux can be measured directly. The production of (13)C-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-(13)C]pyruvate.

The effect of hyperpolarized tracer concentration on myocardial uptake and metabolism.

Hyperpolarized (13)C-labeled substrates directly provide a source of magnetic resonance (MR) signal to observe the substrates' real-time uptake and enzymatic conversion. The aim of this study was to optimize the concentration of hyperpolarized [1-(13)C]pyruvate infused as a metabolic tracer, by observing the mitochondrial conversion of pyruvate to H(13)CO(3)(-) in heart tissue. Hyperpolarized pyruvate was infused into rats at concentrations between 20 mM and 80 mM and the relationships between [1-(13)C]lactate, [1-(13)C]alanine, and H(13)CO(3)(-) production and the infused pyruvate concentration were investigated. H(13)CO(3)(-) production reached saturation above 40 mM infused pyruvate concentration, indicating that hyperpolarized MR experiments performed at this concentration maximize the H(13)CO(3)(-) signal with minimal alterations to in vivo substrate composition. Additionally, the linear dependence of alanine production on pyruvate concentration confirmed that hyperpolarized MR methods in the heart reveal enzyme activity, rather than cellular uptake. H(13)CO(3)(-) production demonstrated evidence of sigmoidal enzyme kinetics, a reflection of the allosteric nature of the pyruvate dehydrogenase (PDH) enzyme complex. This protocol could be useful to optimize the infused concentration of other hyperpolarized metabolites in different organs, to ensure adequate MR signal with minimum metabolic perturbation.

A combined metabolomic and proteomic investigation of the effects of a failure to express dystrophin in the mouse heart.

Muscle degeneration in the heart of 1-9 month-old mdx mice (a model for Duchenne muscular dystrophy) has been monitored using metabolomic and proteomic approaches. In both data sets, a pronounced aging trend was detected in control and mdx mice, and this trend was separate from the disease process. In addition, the characteristic increase in taurine associated with dystrophic tissue is correlated with proteins associated with oxidative phosphorylation and mitochondrial metabolism.

A comparative metabolomic study of NHR-49 in Caenorhabditis elegans and PPAR-alpha 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-alpha null mouse. The metabolomic approach demonstrated a number of similarities including the regulation of lipid synthesis, beta-oxidation of fatty acids and changes in glycolysis/gluconeogenesis.

A combined 1H-NMR spectroscopy- and mass spectrometry-based metabolomic study of the PPAR-alpha null mutant mouse defines profound systemic changes in metabolism linked to the metabolic syndrome.

The mobilization of triacylglycerides from storage in adipocytes to the liver is a vital response to the fasting state in mammalian metabolism. This is accompanied by a rapid translational activation of genes encoding mitochondrial, microsomal, and peroxisomal beta-oxidation in the liver, in part under the regulation of peroxisome proliferator-activated receptor-alpha (PPAR-alpha). A failure to express PPAR-alpha results in profound metabolic perturbations in muscle tissue as well as the liver. These changes represent a number of deficits that accompany diabetes, dyslipidemia, and the metabolic syndrome. In this study, the metabolic role of PPAR-alpha has been investigated in heart, skeletal muscle, liver, and adipose tissue of PPAR-alpha null mice at 1 mo of age using metabolomics. To maximize the coverage of the metabolome in these tissues, (1)H-NMR spectroscopy, magic angle spinning (1)H-NMR spectroscopy, gas chromatography-mass spectrometry, and liquid chromatography-mass spectrometry were used to examine metabolites in aqueous tissue extracts and intact tissue. The data were analyzed by the multivariate approaches of principal components analysis and partial least squares. Across all tissues, there was a profound decrease in glucose and a number of amino acids, including glutamine and alanine, and an increase in lactate, demonstrating that a failure to express PPAR-alpha results in perturbations in glycolysis, the citric acid cycle, and gluconeogenesis. Furthermore, despite PPAR-alpha being weakly expressed in adipose tissue, a profound metabolic perturbation was detected in this tissue.