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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.

Renal metabolism is essential for kidney functions and energy homeostasis in the body. The TCA cycle is the hub of metabolism, but the metabolic activities of the cycle in the kidney have rarely been investigated. This study is to assess metabolic processes at the level of the TCA cycle in the kidney based on isotopomer distributions in multiple metabolites. Isolated rat kidneys were perfused with media containing common substrates including lactate and alanine for an hour. One group of kidneys received [U‐13C3]lactate instead of natural abundance lactate while the other group received [U‐13C3]alanine instead of natural abundance alanine. Perfused kidneys and effluent were prepared for analysis using NMR spectroscopy. 13C‐labeling patterns in glutamate, fumarate, aspartate and succinate from the kidney extracts showed that pyruvate carboxylase and oxidative metabolism through the TCA cycle were comparably very active, but pyruvate cycling and pyruvate dehydrogenase were relatively less active. Isotopomer analyses with fumarate and malate from effluent, however, indicated that pyruvate carboxylase was much more active than the TCA cycle and other metabolic processes. The reverse equilibrium of oxaloacetate with four‐carbon intermediates of the cycle was nearly complete (92%), based on the ratio of [2,3,4‐13C3]/[1,2,3‐13C3] in aspartate or malate. 13C enrichment in glucose with 13C‐lactate supply was higher than that with 13C‐alanine. Isotopomer analyses with multiple metabolites (i.e., glutamate, fumarate, aspartate, succinate and malate) allowed us to assess relative metabolic processes in the TCA cycle in the kidney supplied with [U‐13C3]lactate. Data from the analytes were generally consistent, indicating highly active pyruvate carboxylase and oxidative metabolism through the TCA cycle. Different 13C‐labeling patterns in analytes from the kidney extracts versus effluent suggested metabolic compartmentalization. [ABSTRACT FROM AUTHOR]

Advanced imaging technologies, large‐scale metabolomics, and the measurement of gene transcripts or enzyme expression all enable investigations of intermediary metabolism in human patients. Complementary information about fluxes in individual metabolic pathways may be obtained by ex vivo 13C NMR of blood or tissue biopsies. Simple molecules such as 13C‐labeled glucose are readily administered to patients prior to surgical biopsies, and 13C‐labeled glycerol is easily administered orally to outpatients. Here, we review recent progress in practical applications of 13C NMR to study cancer biology, the response to oxidative stress, gluconeogenesis, triglyceride synthesis in patients, as well as new insights into compartmentation of metabolism in the cytosol. The technical aspects of obtaining the sample, preparing material for analysis, and acquiring the spectra are relatively simple. This approach enables convenient, valuable, and quantitative insights into intermediary metabolism in patients. [ABSTRACT FROM AUTHOR]

Mitochondrial metabolism provides precursors to build macromolecules in growing cancer cells (1,2). In normally functioning tumour cell mitochondria, oxidative metabolism of glucose- and glutamine-derived carbon produces citrate and acetyl-coenzyme A for lipid synthesis, which is required for tumorigenesis (3). Yet some tumours harbour mutations in the citric acid cycle (CAC) or electron transport chain (ETC) that disable normal oxidative mitochondrial function (4-7), and it is unknown how cells from such tumours generate precursors for macromolecular synthesis. Here we show that tumour cells with defective mitochondria use glutamine-dependent reductive carboxylation rather than oxidative metabolism as the major pathway of citrate formation. This pathway uses mitochondrial and cytosolic isoforms of NADP1/ NADPH-dependent isocitrate dehydrogenase, and subsequent metabolism of glutamine-derived citrate provides both the acetylcoenzyme A for lipid synthesis and the four-carbon intermediates needed to produce the remaining CAC metabolites and related macromolecular precursors. This reductive, glutamine-dependent pathway is the dominant mode of metabolism in rapidly growing malignant cells containing mutations in complex I or complex III of the ETC, in patient-derived renal carcinoma cells with mutations in fumarate hydratase, and in cells with normal mitochondria subjected to acute pharmacological ETC inhibition. Our findings reveal the novel induction of a versatile glutamine-dependent pathway that reverses many of the reactions of the canonical CAC, supports tumour cell growth, and explains how cells generate pools of CAC intermediates in the face of impaired mitochondrial metabolism., We first studied the metabolism of isogenic 143B human osteosarcoma cells that contained or lacked a loss-of-function mutation in ETC complex III (cytochrome b-c1 complex). These cell lines were generated [...]

Obesity is a risk factor for type 2 diabetes in cats. The risk of developing diabetes is severalfold greater for male cats than for females, even after having been neutered early in life. The purpose of this study was to investigate the role of different metabolic pathways in the regulation of endogenous glucose production (EGP) during the fasted state considering these risk factors. A triple tracer protocol using [sup.2][H.sub.2][O.sub.2], [U-[sup.13][C.sub.3]]propionate, and [3,4-[sup.13][C.sub.2]]glucose was applied in overnight-fasted cats (12 lean and 12 obese ; equal sex distribution) fed three different diets. Compared with lean cats, obese cats had higher insulin (P < 0.001) but similar blood glucose concentrations. EGP was lower in obese cats (P < 0.001) due to lower glycogenolysis and gluconeogenesis (GNG P < 0.03). Insulin, body mass index, and girth correlated negatively with EGP (P < 0.003). Female obese cats had ~1.5 times higher fluxes through phosphoenolpyruvate carboxykinase (P < 0.02) and citrate synthase (P < 0.05) than male obese cats. However, GNG was not higher because pyruvate cycling was increased 1.5-fold (P < 0.03). These results support the notion that fasted obese cats have lower hepatic EGP compared with lean cats and are still capable of maintaining fasting euglycemia, despite the well-documented existence of peripheral insulin resistance in obese cats. Our data further suggest that sex-related differences exist in the regulation of hepatic glucose metabolism in obese cats, suggesting that pyruvate cycling acts as a controlling mechanism to modulate EGP. Increased pyruvate cycling could therefore be an important factor in modulating the diabetes risk in female cats. nuclear magnetic resonance spectroscopy; diabetes mellitus; pyruvate cycling; thyroxine; polyunsaturated fatty acids

Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to [sup.2][H.sub.2]O or [sup.13]C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-[sup.13][C.sub.3]]lactate released small amounts of [1,2,3-[sup.13][C.sub.3]]- or [4,5,6-[sup.13][C.sub.3]]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the tricarboxylic acid (TCA) cycle. After exposure of incubated muscle to [sup.2][H.sub.2]O, [U-[sup.13][C.sub.3]]lactate, glucose, and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of [sup.2]H enrichment in medium glucose but 50- to 100-fold greater [sup.2]H enrichment in glucosyl units from glycogen. The [sup.13]C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates, [sup.13]C nuclear magnetic resonance (NMR) spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the TCA cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phosphoenolpyruvate. In vivo, the [sup.13]C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-[sup.13][C.sub.3]]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-[sup.13][C.sub.3]]- and [4,5,6-[sup.13][C.sub.3]]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle. pyruvate kinase; phosphoenolpyruvate carboxykinase; glyconeogenesis; nuclear magnetic resonance; skeletal muscle

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-L-thyronine ([T.sub.3])-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. [sup.2][H.sub.2]O and [U- [sup.13][C.sub.3]]propionate were administered intraperitoneally, and [3,4-[sub.13][C.sub.2]]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. [sup.13]C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both [T.sub.3]-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by [sup.2]H NMR analysis. In [T.sub.3]-treated rats, the hepatic glycogen content and hence the contribution of glycogenolysis to glucose production was essentially zero; in this case, excess glucose production was due to a dramatic increase in gluconeogenesis from TCA cycle intermediates. [sup.13]C NMR analysis also revealed increased phosphoenolpyruvate carboxykinase flux (4x), increased pyruvate cycling flux (4x), and increased TCA flux (5x) in [T.sub.3]-treated animals. ZDF rats had substantial glycogen stores after a 24-h fast, and consequently nearly 50% of plasma glucose originated from glycogenolysis; other fluxes related to the TCA cycle were not different from controls. The differing mechanisms of excess glucose production in these models were easily distinguished by integrated [sup.2]H and [sup.13]C NMR analysis of plasma glucose. gluconeogenesis; glycogenolysis; type 2 diabetes; thyroid hormone; isotopes; nuclear magnetic resonance

After administration of 13C‐labeled glucose, the activity of the pentose phosphate pathway (PPP) is often assessed by the distribution of 13C in lactate. However, in some tissues, such as the well‐oxygenated heart, the concentration of lactate may be too low for convenient analysis by NMR. Here, we examined 13C‐labeled glutamate as an alternative biomarker of the PPP in the heart. Isolated rat hearts were perfused with media containing [2,3‐13C2]glucose and the tissue extracts were analyzed. Metabolism of [2,3‐13C2]glucose yields [1,2‐13C2]pyruvate via glycolysis and [2,3‐13C2]pyruvate via the PPP. Pyruvate is in exchange with lactate or is further metabolized to glutamate through pyruvate dehydrogenase and the TCA cycle. A doublet from [4,5‐13C2]glutamate, indicating flux through the PPP, was readily detected in 13C NMR of heart extracts even when the corresponding doublet from [2,3‐13C2]lactate was minimal. Benfotiamine, known to induce the PPP, caused an increase in production of [4,5‐13C2]glutamate. In rats receiving [2,3‐13C2]glucose, brain extracts showed well‐resolved signals from both [2,3‐13C2]lactate and [4,5‐13C2]glutamate in 13C NMR spectra. Assessment of the PPP in the brain based on glutamate had a strong linear correlation with lactate‐based assessment. In summary, 13C NMR analysis of glutamate enabled detection of the low PPP activity in isolated hearts. This analyte is an alternative to lactate for monitoring the PPP with the use of [2,3‐13C2]glucose. [ABSTRACT FROM AUTHOR]

Stable isotope tracers have been used to gain an understanding of integrative animal and human physiology. More commonly studied organ systems include hepatic glucose metabolism, lipolysis from adipose tissue, and whole body protein metabolism. Recent improvements in isotope methodology have included the use of novel physiologic methods/models and mathematical modeling of data during different physiologic states. Here we review some of the latest advancements in this field and highlight future research needs. First we discuss the use of an oral [U-13C3]-glycerol tracer to determine the relative contribution of glycerol carbons to hepatic glucose production after first cycling through the tricarboxylic acid cycle, entry of glycerol into the pentose phosphate pathway or direct conversion of glycerol into the glucose. Second, we describe an adaptation of the established oral minimal model used to define postprandial glucose dynamics to include glycerol dynamics in an oral glucose tolerance test with a [2H5]-glycerol tracer to determine dynamic changes in lipolysis. Simulation results were optimized when parameters describing glycerol flux were determined with a hybrid approach using both tracer-based calculations and constrained parameter optimization. Both of these methodologies can be used to expand our knowledge of not only human physiology, but also the effects of various nutritional strategies and medications on metabolism. [ABSTRACT FROM AUTHOR]

Drugs and other interventions for high impact hepatic diseases often target biochemical pathways such as gluconeogenesis, lipogenesis, or the metabolic response to oxidative stress. However, traditional liver function tests do not provide quantitative data about these pathways. In this study, we developed a simple method to evaluate these processes by NMR analysis of plasma metabolites. Healthy subjects ingested [U-13C3]glycerol, and blood was drawn at multiple times. Each subject completed three visits under differing nutritional states. High resolution 13C NMR spectra of plasma triacylglycerols and glucose provided new insights into a number of hepatic processes including fatty acid esterification, the pentose phosphate pathway, and gluconeogenesis through the tricarboxylic acid cycle. Fasting stimulated pentose phosphate pathway activity and metabolism of [U-13C3]glycerol in the tricarboxylic acid cycle prior to gluconeogenesis or glyceroneogenesis. Fatty acid esterification was transient in the fasted state but continuous under fed conditions. We conclude that a simple NMR analysis of blood metabolites provides an important biomarker of pentose phosphate pathway activity, triacylglycerol synthesis, and flux through anaplerotic pathways in mitochondria of human liver. [ABSTRACT FROM AUTHOR]

The source of hyperpolarized (HP) [13C]bicarbonate in the liver during metabolism of HP [1-13C]pyruvate is uncertain and likely changes with physiology. Multiple processes including decarboxylation through pyruvate dehydrogenase or pyruvate carboxylase followed by subsequent decarboxylation via phosphoenolpyruvate carboxykinase (gluconeogenesis) could play a role. Here we tested which metabolic fate of pyruvate contributed to the appearance of HP [13C]bicarbonate during metabolism of HP [1-13C]pyruvate by the liver in rats after 21 h of fasting compared to rats with free access to food. The 13C NMR of HP [13C]bicarbonate was observed in the liver of fed rats, but not in fasted rats where pyruvate carboxylation and gluconeogenesis was active. To further explore the relative fluxes through pyruvate carboxylase versus pyruvate dehydrogenase in the liver under typical conditions of hyperpolarization studies, separate parallel experiments were performed with rats given non-hyperpolarized [2,3-13C]pyruvate. 13C NMR analysis of glutamate isolated from the liver of rats revealed that flux from injected pyruvate through pyruvate dehydrogenase was dominant under fed conditions whereas flux through pyruvate carboxylase dominated under fasted conditions. The NMR signal of HP [13C]bicarbonate does not parallel pyruvate carboxylase activity followed by subsequent decarboxylation reaction leading to glucose production. In the liver of healthy well-fed rats, the appearance of HP [13C]bicarbonate exclusively reflects decarboxylation of HP [1-13C]pyruvate via pyruvate dehydrogenase. © 2016 The Authors. NMR in Biomedicine published by John Wiley & Sons Ltd. [ABSTRACT FROM AUTHOR]