Pyruvate Carboxylase Flux Research Articles

MCT1 Inhibitor AZD3965 Increases Mitochondrial Metabolism, Facilitating Combination Therapy and Noninvasive Magnetic Resonance Spectroscopy.

Monocarboxylate transporters (MCT) modulate tumor cell metabolism and offer promising therapeutic targets for cancer treatment. Understanding the impact of MCT blockade on tumor cell metabolism may help develop combination strategies or identify pharmacodynamic biomarkers to support the clinical development of MCT inhibitors now in clinical trials. In this study, we assessed the impact of the MCT1 inhibitor AZD3965 on cancer cell metabolism in vitro and in vivo Exposing human lymphoma and colon carcinoma cells to AZD3965 increased MCT4-dependent accumulation of intracellular lactate, inhibiting monocarboxylate influx and efflux. AZD3965 also increased the levels of TCA cycle-related metabolites and 13C-glucose mitochondrial metabolism, enhancing oxidative pyruvate dehydrogenase and anaplerotic pyruvate carboxylase fluxes. Increased mitochondrial metabolism was necessary to maintain cell survival under drug stress. These effects were counteracted by coadministration of the mitochondrial complex I inhibitor metformin and the mitochondrial pyruvate carrier inhibitor UK5099. Improved bioenergetics were confirmed in vivo after dosing with AZD3965 in mouse xenograft models of human lymphoma. Our results reveal new metabolic consequences of MCT1 inhibition that might be exploited for therapeutic and pharmacodynamic purposes. Cancer Res; 77(21); 5913-24. ©2017 AACR.

Non-invasive assessment of hepatic mitochondrial metabolism by positional isotopomer NMR tracer analysis (PINTA)

Hepatic mitochondria play a central role in the regulation of intermediary metabolism and maintenance of normoglycemia, and there is great interest in assessing rates of hepatic mitochondrial citrate synthase flux (VCS) and pyruvate carboxylase flux (VPC) in vivo. Here, we show that a positional isotopomer NMR tracer analysis (PINTA) method can be used to non-invasively assess rates of VCS and VPC fluxes using a combined NMR/gas chromatography-mass spectrometry analysis of plasma following infusion of [3-13C]lactate and glucose tracer. PINTA measures VCS and VPC fluxes over a wide range of physiological conditions with minimal pyruvate cycling and detects increased hepatic VCS following treatment with a liver-targeted mitochondrial uncoupler. Finally, validation studies in humans demonstrate that the VPC/VCS ratio measured by PINTA is similar to that determined by in vivo NMR spectroscopy. This method will provide investigators with a relatively simple tool to non-invasively examine the role of altered hepatic mitochondrial metabolism.

CHO Cells adapted to inorganic phosphate limitation show higher growth and higher pyruvate carboxylase flux in phosphate replete conditions.

Inorganic phosphate (Pi ) is an essential ion involved in diverse cellular processes including metabolism. Changes in cellular metabolism upon long term adaptation to Pi limitation have been reported in E. coli. Given the essential role of Pi , adaptation to Pi limitation may also result in metabolic changes in animal cells. In this study, we have adapted CHO cells producing recombinant IgG to limiting Pi conditions for 75 days. Not surprisingly, adapted cells showed better survival under Pi limitation. Here, we report the finding that such cells also showed better growth characteristics compared to control in batch culture replete with Pi (higher peak density and integral viable cell density), accompanied by a lower specific oxygen uptake rate and cytochrome oxidase activity towards the end of exponential phase. Surprisingly, the adapted cells grew to a lower peak density under glucose limitation. This suggests long term Pi limitation may lead to selection for an altered metabolism with higher dependence on glucose availability for biomass assimilation compared to control. Steady state U-13 C glucose labeling experiments suggest that adapted cells have a higher pyruvate carboxylase flux. Consistent with this observation, supplementation with aspartate abolished the peak density difference whereas supplementation with serine did not abolish the difference. This supports the hypothesis that cell growth in the adapted culture might be higher due to a higher pyruvate carboxylase flux. Decreased fitness under carbon limitation and mutations in the sucABCD operon has been previously reported in E. coli upon long term adaptation to Pi limitation, suggestive of a similarity in cellular response among such diverse species. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:749-758, 2017.

Hepatic Acetyl CoA Links Adipose Tissue Inflammation to Hepatic Insulin Resistance and Type 2 Diabetes

Impaired insulin-mediated suppression of hepatic glucose production (HGP) plays a major role in the pathogenesis of type 2 diabetes (T2D), yet the molecular mechanism by which this occurs remains unknown. Using a novel in vivo metabolomics approach, we show that the major mechanism by which insulin suppresses HGP is through reductions in hepatic acetyl CoA by suppression of lipolysis in white adipose tissue (WAT) leading to reductions in pyruvate carboxylase flux. This mechanism was confirmed in mice and rats with genetic ablation of insulin signaling and mice lacking adipose triglyceride lipase. Insulin's ability to suppress hepatic acetyl CoA, PC activity, and lipolysis was lost in high-fat-fed rats, a phenomenon reversible by IL-6 neutralization and inducible by IL-6 infusion. Taken together, these data identify WAT-derived hepatic acetyl CoA as the main regulator of HGP by insulin and link it to inflammation-induced hepatic insulin resistance associated with obesity and T2D.

In Vivo Hyperpolarized Carbon-13 Magnetic Resonance Spectroscopy Reveals Increased Pyruvate Carboxylase Flux in an Insulin-Resistant Mouse Model

The pathogenesis of type 2 diabetes is characterized by impaired insulin action and increased hepatic glucose production (HGP). Despite the importance of hepatic metabolic aberrations in diabetes development, there is currently no molecular probe that allows measurement of hepatic gluconeogenic pathways in vivo and in a noninvasive manner. In this study, we used hyperpolarized carbon 13 ((13)C)-labeled pyruvate magnetic resonance spectroscopy (MRS) to determine changes in hepatic gluconeogenesis in a high-fat diet (HFD)-induced mouse model of type 2 diabetes. Compared with mice on chow diet, HFD-fed mice displayed higher levels of oxaloacetate, aspartate, and malate, along with increased (13)C label exchange rates between hyperpolarized [1-(13) C]pyruvate and its downstream metabolites, [1-(13)C]malate and [1-(13)C]aspartate. Biochemical assays using liver extract revealed up-regulated malate dehydrogenase activity, but not aspartate transaminase activity, in HFD-fed mice. Moreover, the (13) C label exchange rate between [1-(13)C]pyruvate and [1-(13)C]aspartate (k(pyr->asp)) exhibited apparent correlation with gluconeogenic pyruvate carboxylase (PC) activity in hepatocytes. Finally, up-regulated HGP by glucagon stimulation was detected by an increase in aspartate signal and k(pyr->asp), whereas HFD mice treated with metformin for 2 weeks displayed lower production of aspartate and malate, as well as reduced k(pyr->asp) and (13)C-label exchange rate between pyruvate and malate, consistent with down-regulated gluconeogenesis. Taken together, we demonstrate that increased PC flux is an important pathway responsible for increased HGP in diabetes development, and that pharmacologically induced metabolic changes specific to the liver can be detected in vivo with a hyperpolarized (13)C-biomolecular probe. Hyperpolarized (13)C MRS and the determination of metabolite exchange rates may allow longitudinal monitoring of liver function in disease development.

Rational design of 13C-labeling experiments for metabolic flux analysis in mammalian cells

Background13C-Metabolic flux analysis (13C-MFA) is a standard technique to probe cellular metabolism and elucidate in vivo metabolic fluxes. 13C-Tracer selection is an important step in conducting 13C-MFA, however, current methods are restricted to trial-and-error approaches, which commonly focus on an arbitrary subset of the tracer design space. To systematically probe the complete tracer design space, especially for complex systems such as mammalian cells, there is a pressing need for new rational approaches to identify optimal tracers.ResultsRecently, we introduced a new framework for optimal 13C-tracer design based on elementary metabolite units (EMU) decomposition, in which a measured metabolite is decomposed into a linear combination of so-called EMU basis vectors. In this contribution, we applied the EMU method to a realistic network model of mammalian metabolism with lactate as the measured metabolite. The method was used to select optimal tracers for two free fluxes in the system, the oxidative pentose phosphate pathway (oxPPP) flux and anaplerosis by pyruvate carboxylase (PC). Our approach was based on sensitivity analysis of EMU basis vector coefficients with respect to free fluxes. Through efficient grouping of coefficient sensitivities, simple tracer selection rules were derived for high-resolution quantification of the fluxes in the mammalian network model. The approach resulted in a significant reduction of the number of possible tracers and the feasible tracers were evaluated using numerical simulations. Two optimal, novel tracers were identified that have not been previously considered for 13C-MFA of mammalian cells, specifically [2,3,4,5,6-13C]glucose for elucidating oxPPP flux and [3,4-13C]glucose for elucidating PC flux. We demonstrate that 13C-glutamine tracers perform poorly in this system in comparison to the optimal glucose tracers.ConclusionsIn this work, we have demonstrated that optimal tracer design does not need to be a pure simulation-based trial-and-error process; rather, rational insights into tracer design can be gained through the application of the EMU basis vector methodology. Using this approach, rational labeling rules can be established a priori to guide the selection of optimal 13C-tracers for high-resolution flux elucidation in complex metabolic network models.

Metabolism as means for hypoxia adaptation: metabolic profiling and flux balance analysis

BackgroundCellular hypoxia is a component of many diseases, but mechanisms of global hypoxic adaptation and resistance are not completely understood. Previously, a population of Drosophila flies was experimentally selected over several generations to survive a chronically hypoxic environment. NMR-based metabolomics, combined with flux-balance simulations of genome-scale metabolic networks, can generate specific hypotheses for global reaction fluxes within the cell. We applied these techniques to compare metabolic activity during acute hypoxia in muscle tissue of adapted versus "naïve" control flies.ResultsMetabolic profiles were gathered for adapted and control flies after exposure to acute hypoxia using 1H NMR spectroscopy. Principal Component Analysis suggested that the adapted flies are tuned to survive a specific oxygen level. Adapted flies better tolerate acute hypoxic stress, and we explored the mechanisms of this tolerance using a flux-balance model of central metabolism. In the model, adapted flies produced more ATP per glucose and created fewer protons than control flies, had lower pyruvate carboxylase flux, and had greater usage of Complex I over Complex II.ConclusionWe suggest a network-level hypothesis of metabolic regulation in hypoxia-adapted flies, in which lower baseline rates of biosynthesis in adapted flies draws less anaplerotic flux, resulting in lower rates of glycolysis, less acidosis, and more efficient use of substrate during acute hypoxic stress. In addition we suggest new specific hypothesis, which were found to be consistent with existing data.

Partial resistance to peroxisome proliferator-activated receptor-alpha agonists in ZDF rats is associated with defective hepatic mitochondrial metabolism.

OBJECTIVE—Fluxes through mitochondrial pathways are defective in insulin-resistant skeletal muscle, but it is unclear whether similar mitochondrial defects play a role in the liver during insulin resistance and/or diabetes. The purpose of this study is to determine whether abnormal mitochondrial metabolism plays a role in the dysregulation of both hepatic fat and glucose metabolism during diabetes.RESEARCH DESIGN AND METHODS—Mitochondrial fluxes were measured using 2H/13C tracers and nuclear magnetic resonance spectroscopy in ZDF rats during early and advanced diabetes. To determine whether defects in hepatic fat oxidation can be corrected by peroxisome proliferator–activated receptor (PPAR-)-α activation, rats were treated with WY14,643 for 3 weeks before tracer administration.RESULTS—Hepatic mitochondrial fat oxidation in the diabetic liver was impaired twofold secondary to decreased ketogenesis, but tricarboxylic acid (TCA) cycle activity and pyruvate carboxylase flux were normal in newly diabetic rats and elevated in older rats. Treatment of diabetic rats with a PPAR–α agonist induced hepatic fat oxidation via ketogenesis and hepatic TCA cycle activity but failed to lower fasting glycemia or endogenous glucose production. In fact, PPAR-α agonism overstimulated mitochondrial TCA cycle flux and induced pyruvate carboxylase flux and gluconeogenesis in lean rats.CONCLUSIONS—The impairment of certain mitochondrial fluxes, but preservation or induction of others, suggests a complex defect in mitochondrial metabolism in the diabetic liver. These data indicate an important codependence between hepatic fat oxidation and gluconeogenesis in the normal and diabetic state and potentially explain the sometimes equivocal effect of PPAR-α agonists on glycemia.

Carbohydrate-response Element-binding Protein Deletion Alters Substrate Utilization Producing an Energy-deficient Liver

Livers from mice lacking the carbohydrate-responsive element-binding protein (ChREBP) were compared with wild type (WT) mice to determine the effect of this transcription factor on hepatic energy metabolism. The pyruvate dehydrogenase complex was considerably more active in ChREBP(-/-) mice because of diminished pyruvate dehydrogenase kinase activity. Greater pyruvate dehydrogenase complex activity caused a stimulation of lactate and pyruvate oxidation, and it significantly impaired fatty acid oxidation in perfused livers from ChREBP(-/-) mice. This shift in mitochondrial substrate utilization led to a 3-fold reduction of the free cytosolic [NAD(+)]/[NADH] ratio, a 1.7-fold increase in the free mitochondrial [NAD(+)]/[NADH] ratio, and a 2-fold decrease in the free cytosolic [ATP]/[ADP][P(i)] ratio in the ChREBP(-/-) liver compared with control. Hepatic pyruvate carboxylase flux was impaired with ChREBP deletion secondary to decreased fatty acid oxidation, increased pyruvate oxidation, and limited pyruvate availability because of reduced activity of liver pyruvate kinase and malic enzyme, which replenish pyruvate via glycolysis and pyruvate cycling. Overall, the shift from fat utilization to pyruvate and lactate utilization resulted in a decrease in the energy of ATP hydrolysis and a hypo-energetic state in the livers of ChREBP(-/-) mice.

Stirred vessel cultures of rat brain cells aggregates: Characterization of major metabolic pathways and cell population dynamics

We report a study on neural metabolism of long-term three-dimensional cultures of rat embryonic brain cells in stirred vessels. Our experimental setup was optimized to keep viable aggregate cultures with neuronal maintenance for up to 44 days. Results show that aggregate size and shape could be hydrodynamically controlled depending on the impeller design, avoiding necrotic centers or significant losses in cell viability. Aggregates were composed mainly of neurons until day 16, whereas an effective growth of the glial population was observed after day 21. Cell metabolic status was evaluated by quantification of several metabolites in the culture medium; amino acid metabolism was used as a marker of metabolic interrelationships between neural cell types. Furthermore, (13)C-NMR spectroscopy was used on day 31 to explore specific metabolic pathways: incubation with [1-(13)C]glucose for 45 hr produced an increase in label incorporation in extracellular alanine, lactate, and glutamine, reflecting mainly astrocytic metabolism. The contribution of anaplerotic vs. oxidative pathways for glutamine synthesis was determined: a 92% reduction in the pyruvate carboxylase flux during the first 41 hr of incubation suggested a decrease in the need for replacing tricarboxylic acid cycle intermediates. We believe that our data corroborate the aggregating cultures as an attractive system to analyze brain cell metabolism being a valuable tool to model metabolic fluxes for in vitro brain diseases.

Cytosolic and Mitochondrial Malic Enzyme Isoforms Differentially Control Insulin Secretion

In islet beta-cells and INS-1 cells both the high activity of malic enzyme and the correlation of insulin secretion rates with pyruvate carboxylase (PC) flux suggest that a pyruvate-malate cycle is functionally relevant to insulin secretion. Expression of the malic enzyme isoforms in INS-1 cells and rat islets was measured, and small interfering RNA was used to selectively reduce isoform mRNA expression in INS-1 cells to evaluate its impact on insulin secretion. The cytosolic NADP(+)-specific isoform (ME1) was the most abundant, with the mitochondrial isoforms NAD(+)-preferred (ME2) expressed at approximately 50%, and the NADP(+)-specific (ME3) at approximately 10% compared with ME1. Selective reduction (89 +/- 2%) of cytosolic ME1 mRNA expression and enzyme activity significantly reduced glucose (15 mM:41 +/- 6%, p < 0.01) and amino acid (4 mM glutamine +/- 10 mM leucine: 39 +/- 6%, p < 0.01)-stimulated insulin secretion. Selective small interfering RNA reduction (51 +/- 6%) of mitochondrial ME2 mRNA expression did not impact glucose-induced insulin secretion, but decreased amino acid-stimulated insulin secretion by 25 +/- 4% (p < 0.01). Modeling of the metabolism of [U-(13)C]glucose by its isotopic distribution in glutamate indicates a second pool of pyruvate distinct from glycolytically derived pyruvate in INS-1 cells. ME1 knockdown decreased flux of both pools of pyruvate through PC. In contrast, ME2 knockdown affected only PC flux of the pyruvate derived from glutamate metabolism. These results suggest a physiological basis for two metabolically and functionally distinct pyruvate cycles. The cycling of pyruvate by ME1 generates cytosolic NADPH, whereas mitochondrial ME2 responds to elevated amino acids and serves to supply sufficient pyruvate for increased Krebs cycle flux when glucose is limiting.

Measurements of the anaplerotic rate in the human cerebral cortex using 13C magnetic resonance spectroscopy and [1‐13C] and [2‐13C] glucose

Recent studies in rodent and human cerebral cortex have shown that glutamate-glutamine neurotransmitter cycling is rapid and the major pathway of neuronal glutamate repletion. The rate of the cycle remains controversial in humans, because glutamine may come either from cycling or from anaplerosis via glial pyruvate carboxylase. Most studies have determined cycling from isotopic labeling of glutamine and glutamate using a [1-(13)C]glucose tracer, which provides label through neuronal and glial pyruvate dehydrogenase or via glial pyruvate carboxylase. To measure the anaplerotic contribution, we measured (13)C incorporation into glutamate and glutamine in the occipital-parietal region of awake humans while infusing [2-(13)C]glucose, which labels the C2 and C3 positions of glutamine and glutamate exclusively via pyruvate carboxylase. Relative to [1-(13)C]glucose, [2-(13)C]glucose provided little label to C2 and C3 glutamine and glutamate. Metabolic modeling of the labeling data indicated that pyruvate carboxylase accounts for 6 +/- 4% of the rate of glutamine synthesis, or 0.02 micromol/g/min. Comparison with estimates of human brain glutamine efflux suggests that the majority of the pyruvate carboxylase flux is used for replacing glutamate lost due to glial oxidation and therefore can be considered to support neurotransmitter trafficking. These results are consistent with observations made with arterial-venous differences and radiotracer methods.

Cerebral pyruvate carboxylase flux is unaltered during bicuculline‐seizures

AbstractThe original article of this erratum was published in J Neurosci Res 2005; 79: 128–138

Cerebral pyruvate carboxylase flux is unaltered during bicuculline‐seizures

Glutamine synthesis in the astroglia reflects the sum of neurotransmitter cycling (glutamate and gamma-aminobutyric acid [GABA]) and de novo synthesis (anaplerosis), the latter catalyzed by pyruvate carboxylase. Previous studies have shown that the glutamate plus GABA cycling flux is correlated strongly with neuronal activity; however, the relationship between pyruvate carboxylase flux and neuronal activity is not known. In this study, pyruvate carboxylase flux was assessed during intravenous infusion of [2-(13)C]glucose using localized (1)H-[(13)C] NMR spectroscopy at 7 Tesla in vivo in halothane-anesthetized and ventilated adult Wistar rats during 85 min of bicuculline-induced seizures (1 mg/kg, intravenously) and in nontreated controls. During seizures, concentrations of lactate, alanine, glutamine, GABA, and succinate increased whereas glutamate and aspartate decreased such that the decrease in glutamate plus aspartate equaled the increase in glutamine plus GABA. Pyruvate carboxylase flux was assessed by the sum of [2-(13)C] and [3-(13)C] of glutamine and glutamate (Glx(2+3)) labeling during [2-(13)C]glucose infusion. During seizures the initial rate of Glx(2+3) synthesis (0.069 +/- 0.013 micromol/g/min) was not significantly different (P = 0.68) from that of the controls (0.059 +/- 0.010 micromol/g/min), indicating that anaplerotic flow through pyruvate carboxylase was unaltered. Intense neuronal activation of seizures did not seem to increase anaplerosis through pyruvate carboxylase, despite the substantial increase in neuronal activity and glutamate/glutamine cycling shown in a previous study (Patel et al., 2004b).

13C NMR Isotopomer Analysis of Anaplerotic Pathways in INS-1 Cells

Anaplerotic flux into the Kreb's cycle is crucial for glucose-stimulated insulin secretion from pancreatic beta-cells. However, the regulation of flux through various anaplerotic pathways in response to combinations of physiologically relevant substrates and its impact on glucose-stimulated insulin secretion is unclear. Because different pathways of anaplerosis generate distinct products, they may differentially modulate the insulin secretory response. To examine this question, we applied 13C-isotopomer analysis to quantify flux through three anaplerotic pathways: 1) pyruvate carboxylase of pyruvate derived from glycolytic sources; 2) pyruvate carboxylase of pyruvate derived from nonglycolytic sources; and 3) glutamate dehydrogenase (GDH). At substimulatory glucose, anaplerotic flux rate in the clonal INS-1 832/13 cells was approximately 40% of Kreb's cycle flux, with similar contributions from each pathway. Increasing glucose to 15 mm stimulated insulin secretion approximately 4-fold, and was associated with a approximately 4-fold increase in anaplerotic flux that could mostly be attributed to an increase in PC flux. In contrast, the addition of glutamine to the perfusion media stimulated GDH flux approximately 6-fold at both glucose concentrations without affecting insulin secretion rates. In conclusion, these data support the hypothesis that a signal generated by anaplerosis from increased pyruvate carboxylase flux is essential for glucose-stimulated insulin secretion in beta-cells and that anaplerosis through GDH does not play a major role in this process.

Metabolic-flux analysis of continuously cultured hybridoma cells using (13)CO(2) mass spectrometry in combination with (13)C-lactate nuclear magnetic resonance spectroscopy and metabolite balancing.

Protein production of mammalian-cell culture is limited due to accumulation of waste products such as lactate, CO(2), and ammonia. In this study, the intracellular fluxes of hybridoma cells are measured to determine the amount by which various metabolic pathways contribute to the secretion of waste products derived from glucose. Continuously cultured hybridoma cells are grown in medium containing either 1-(13)C-, 2-(13)C-, or 6-(13)C-glucose. The uptake and production rates of amino acids, glucose, ammonia, O(2), and CO(2) as well as the cellular composition are measured. In addition, the (13)C distribution of the lactate produced and alanine produced by the hybridomas is determined by (1)H-NMR spectroscopy, and the (13)CO(2)/(12)CO(2) ratio is measured by on-line mass spectrometry. These data are used to calculate the intracellular fluxes of the glycolysis, the pentose phosphate pathway, the TCA cycle, and fluxes involved in amino acid metabolism. It is shown that: (i) approximately 20% of the glucose consumed is channeled through the pentose shunt; (ii) the glycolysis pathway contributes the most to lactate production, and most of the CO(2) is produced by the TCA cycle; (iii) the pyruvate-carboxylase flux is negligibly small; and (iv) the malic-enzyme flux is estimated to be 10% of the glucose uptake rate. Based on these flux data suggestions are made to engineer a more efficient glucose metabolism in mammalian cells.

Localized in vivo 13C-NMR of Glutamate Metabolism in the Human Brain: Initial Results at 4 Tesla

Using optimized administration of ¹³C-labeled glucose, the time course of the specific activity of glucose was measured directly by in vivo ¹³C-NMR in the human brain at 4 Tesla. Subsequent label incorporation was measured at the C2, C3 and C4 positions of both glutamate and the well-resolved C2, C3 and C4 resonances of glutamine and at the C2 and C3 positions of aspartate. GABA was clearly observed for the first time in vivo, suggesting a substantial GABA turnover in the normal human visual cortex. Likewise, lactate C3 labeled with an estimated active pool size on the order of 0.5 mM. A model of cerebral glutamate metabolism is proposed which predicts that glutamatergic action (‘neurotransmission’), pyruvate carboxylase flux, TCA cycle activity, glucose consumption and exchange across the mitochondrial membrane can be assessed simultaneously in the human brain.

Role of pyruvate carboxylase in facilitation of synthesis of glutamate and glutamine in cultured astrocytes.

CO2 fixation was measured in cultured astrocytes isolated from neonatal rat brain to test the hypothesis that the activity of pyruvate carboxylase influences the rate of de novo glutamate and glutamine synthesis in astrocytes. Astrocytes were incubated with 14CO2 and the incorporation of 14C into medium or cell extract products was determined. After chromatographic separation of 14C-labelled products, the fractions of 14C cycled back to pyruvate, incorporated into citric acid cycle intermediates, and converted to the amino acids glutamate and glutamine were determined as a function of increasing pyruvate carboxylase flux. The consequences of increasing pyruvate, bicarbonate, and ammonia were investigated. Increasing extracellular pyruvate from 0 to 5 mM increased pyruvate carboxylase flux as observed by increases in the 14C incorporated into pyruvate and citric acid cycle intermediates, but incorporation into glutamate and glutamine, although relatively high at low pyruvate levels, did not increase as pyruvate carboxylase flux increased. Increasing added bicarbonate from 15 to 25 mM almost doubled CO2 fixation. When 25 mM bicarbonate plus 0.5 mM pyruvate increased pyruvate carboxylase flux to approximately the same extent as 15 mM bicarbonate plus 5 mM pyruvate, the rate of appearance of [14C] glutamate and glutamine was higher with the lower level of pyruvate. The conclusion was drawn that, in addition to stimulating pyruvate carboxylase, added pyruvate (but not added bicarbonate) increases alanine aminotransferase flux in the direction of glutamate utilization, thereby decreasing glutamate as pyruvate + glutamate --> alpha-ketoglutarate + alanine. In contrast to previous in vivo studies, the addition of ammonia (0.1 and 5 mM) had no effect on net 14CO2 fixation, but did alter the distribution of 14C-labelled products by decreasing glutamate and increasing glutamine. Rather unexpectedly, ammonia did not increase the sum of glutamate plus glutamine (mass amounts or 14C incorporation). Low rates of conversion of alpha-[14C]ketoglutarate to [14C]glutamate, even in the presence of excess added ammonia, suggested that reductive amination of alpha-ketoglutarate is inactive under conditions studied in these cultured astrocytes. We conclude that pyruvate carboxylase is required for de novo synthesis of glutamate plus glutamine, but that conversion of alpha-ketoglutarate to glutamate may frequently be the rate-limiting step in this process of glutamate synthesis.

Measurement of Metabolic Fluxes through Pyruvate Kinase, Phosphoenolpyruvate Carboxykinase, Pyruvate Dehydrogenase, and Pyruvate Carboxylase in Hepatocytes of Different Acinar Origin

Isolated hepatocytes were prepared from the periportal and perivenous regions of the liver of 18-h-starved rats. These showed characteristic enzyme patterns and an enhanced rate of ureagenesis in the periportal cells; however, total cellular ATP content was unchanged in the two cell types. Measurements of py- ruvate kinase flux showed no significant difference in the overall rate in the two cell types; however, the flux through phosphoenolpyruvate (PEP) carboxykinase was significantly higher in the periportal cells, such that the percentage of PEP being metabolized by py- ruvate kinase was enhanced in the perivenous cells. The increase in partitioning of PEP through pyruvate kinase could account for only a small percentage of the difference in gluconeogenic flux in the two cell types, suggesting that the rate of provision of PEP was the principal limiting factor for glucose synthesis. The flux through pyruvate dehydrogenase showed no significant metabolic zonation, whereas pyruvate carboxylase flux was enhanced in the periportal zone. The partitioning of pyruvate between pyruvate carboxylase and pyruvate dehydrogenase was increased 2.8-fold in the periportal cells compared to that in the perivenous cells and it is suggested that this, together with possible alterations in phosphoenolpyruvate carboxykinase, is primarily responsible for the different gluconeogenic rates in the two zones of the liver.

Effect of dietary L-carnitine on growth, carcass characteristics, and metabolism of swine (1996)

Thirty six Yorkshire gilts (initially 123 lb BW) were used to investigate the effect of dietary carnitine on growth performance, carcass characteristics, fatty acid oxidation, and enzyme kinetics. Dietary carnitine reduced fat deposition in favor of protein deposition, stimulated fatty acid oxidation, induced the expression of pyruvate carboxylase, increased the capacity of pyruvate carboxylase flux, and decreased the capacity of branch chain keto dehydrogenase.; Swine Day, Manhattan, KS, November 21, 1996