Integration of [U-13C]glucose and 2H2O for quantification of hepatic glucose production and gluconeogenesis

Glucose metabolism in five healthy subjects fasted for 16 h was measured with a combination of [U-13C]glucose and 2H2O tracers. Phenylbutyric acid was also provided to sample hepatic glutamine for the presence of 13C-isotopomers derived from the incorporation of [U-13C]glucose products into the hepatic Krebs cycle. Glucose production (GP) was quantified by 13C NMR analysis of the monoacetone derivative of plasma glucose following a primed infusion of [U-13C]glucose and provided reasonable estimates (1.90 +/- 0.19 mg/kg/min with a range of 1.60-2.15 mg/kg/min). The same derivative yielded measurements of plasma glucose 2H-enrichment from 2H2O by 2H NMR from which the contribution of glycogenolytic and gluconeogenic fluxes to GP was obtained (0.87 +/- 0.14 and 1.03 +/- 0.10 mg/kg/min, respectively). Hepatic glutamine 13C-isotopomers representing multiply-enriched oxaloacetate and [U-13C]acetyl-CoA were identified as multiplets in the 13C NMR signals of the glutamine moiety of urinary phenylacetylglutamine, demonstrating entry of the [U-13C]glucose tracer into both oxidative and anaplerotic pathways of the hepatic Krebs cycle. These isotopomers contributed 0.1-0.2% excess enrichment to carbons 2 and 3 and approximately 0.05% to carbon 4 of glutamine.     

Carbohydrate metabolism of the rat C6 glioma. An in vivo 13C and in vitro 1H magnetic resonance spectroscopy study

Surface coil 13C nuclear magnetic resonance (NMR) spectroscopy was used to investigate the in vivo carbohydrate metabolism of rat C6 gliomas during and after infusion with [1-13C] glucose. In vivo 1H-decoupled 13C NMR spectra of the glioma following infusion with [1-13C]glucose revealed the direct production of [3-13C]lactic acid, [1-13C]glycogen, and [4-13C], [3-13C], and [2-13C]glutamate/glutamine. Lactate levels of in vivo gliomas increased and reached steady state levels during [1-13C]glucose infusion, and decreased following termination of infusion. Complementary in vitro studies using supernatant media collected from C6 glioma cells incubated with media containing [1-13C] or [6-13C]glucose and glutamine were examined by 1H NMR spectroscopy. The [3-(13C/12C)]lactate ratios obtained from 1H spectra of supernatant media containing [1-13C]glucose revealed the percentage of glucose metabolized through the hexose monophosphate shunt to be 10.01 +/- 0.85% (n = 3), while similar measurements of media containing [6-13C]glucose and glutamine showed that glutaminolysis contributed 9.0 +/- 1.0% of total lactate production under these conditions. Enzymatic analysis of media determined lactate production to be 139 +/- 9 nmol per 10(6) cells per h (n = 4). These measurements demonstrate the ability of NMR to monitor brain tumor carbohydrate metabolism both in vitro and in vivo.     

Metabolism of D-[1,2-13C]glucose and alpha-D-[1,2-13C]glucose pentaacetate in tumoral pancreatic islet cells

Tumoral pancreatic islet cells of the RINm5F line were incubated, in groups of 25x106 cells each, for 120 min at 37 degrees C in media (5. 0 ml) containing either alpha-D-[1,2-13C]glucose pentaacetate (1.7 mM) or both D-[1,2-13C]glucose (1.7 mM) and acetate (8.5 mM). In both cases, the amounts of 13C-enriched metabolites (D-glucose, L-lactate and acetate) and non-enriched metabolites (acetate) recovered in the incubation medium after incubation were close to the initial amount of esterified or non-esterified D-[1, 2-13C]glucose and acetate, respectively. The 13C-enriched metabolites corresponded mainly to double-labelled D-[1, 2-13C]glucose, L-[2,3-13C]lactate and [1,2-13C]acetate. The output of L-[2,3-13C]lactate and [1,2-13C]acetate was about 3-4 times lower in the cells exposed to alpha-D-[1,2-13C]glucose pentaacetate than in those incubated with unesterified D-[1,2-13C]glucose. These findings indicate that, despite extensive hydrolysis of alpha-D-[1, 2-13C]glucose pentaacetate in the RINm5F cells, the hexose moiety of the ester is less efficiently metabolized than unesterified D-[1, 2-13C]glucose tested at the same molar concentration (1.7 mM) in the presence of 8.5 mM acetate. Thus, a higher utilization of the hexose moiety of alpha-D-glucose pentaacetate than that of unesterified D-glucose, as previously documented in isolated pancreatic islets, represents a far-from-universal situation.     

Determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma by gas chromatography-negative chemical ionization mass spectrometry

We describe a reliable method for the simultaneous determination of isotopic enrichments of [1-13C]homocysteine, [1-13C]methionine and [2H3-methyl-1-13C]methionine in human plasma. Accurate [1-13C]homocysteine calibration standards were prepared by chemical conversion via thiolactonisation of [1-13C]methionine standards. Based upon anion-exchange chromatography, (di)acetyl-3,5-bis-trifluoromethylbenzyl derivatives, preparation of accurate calibration curves and gas chromatography-negative chemical ionization mass spectrometry, isotopic enrichments in human plasma could be determined with TTR (%) <+/-0.2% (N=3) for [1-13C]homocysteine (enrichment range 0-8%), [1-13C]methionine (enrichment range 0-3%) and [2H3-methyl-1-13C]methionine (enrichment range 0-12%). The method was applied in a [2H3-methyl-1-13C]methionine tracer infusion study in a biological model.     

Metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C]succinate) in hepatocytes from Goto-Kakizaki rats

The metabolism of [1,3-(13)C]glycerol-1,2,3-tris(methylsuccinate) and glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate) was examined in hepatocytes prepared from hereditarily diabetic Goto-Kakizaki rats. Over 120 min incubation in the presence of one of the two (13)C-labelled esters (2.5 mM), the output of (13)C-enriched glucose averaged 57.1 +/- 18.5 and 54.1 +/- 22.7 nmol per 10(6) cells, when expressed as [1,3-(13)C]glycerol and [2,3-(13)C] succinate equivalent, respectively. In the case of [1,3-(13)C]glycerol-1,2,3-tris(methyl-succinate), the molecules of glucose were symmetrically labelled. In the case of glycerol-1,2,3-tris(methyl[2,3-(13)C] succinate), however, both the single-labelled and double-labelled isotopomers of glucose contained more (13)C atoms in their C(6)-C(5)-C(4) than C(1)-C(2)-C(3) moiety. These findings indicate that glycerol-1,2,3-tris(methylsuccinate), recently proposed as a novel insulinotropic tool for the treatment of non-insulin-dependent diabetes mellitus, is efficiently metabolized in hepatocytes from diabetic rats, the high rate of gluconeogenesis coinciding with channelling of D-glyceraldehyde-3-phosphate between glyceraldehyde-3-phosphate dehydrogenase and phosphofructoaldolase.     

31P and 13C NMR studies of isolated perfused hematopoietic cells from leukemic mice

The myeloproliferative leukemic virus (MPLV) induces within 2-3 weeks a massive infiltration of the adult mouse liver by hematopoietic leukemic cells. Since the metabolism of the infiltrated organ might be correlated with an interaction of two cell populations, it was decided to study the isolated hematopoietic cells separately. The metabolism of these cells embedded in an agarose gel and perfused with labeled substrates was investigated using 31P and 13C NMR. Using [1-13C]glucose as precursor, sequential 13C NMR spectra showed that the hematopoietic cells were able to store glucose as [1-13C]glycogen and to metabolize it through the glycolytic pathway to give [3-13C]lactate as sole end-product. The liver neoglucogenic substrates: [2-13C]pyruvate and [3-13C]alanine are not metabolized by these cells. This suggests that the tricarboxylic acid cycle was not efficient. To investigate further the glycolytic properties of the cells, 10 mM sodium azide was added to the medium containing [1-13C]glucose. When compared to the aerobic conditions, a 40% decrease of nucleotides (0.10 vs 0.17 mumole NTP/10(9) cells), a degradation of [1-13C]glycogen and an increase of ca 35% of the glycolytic rate were observed. The analysis of 13C NMR spectra of the perfusates at the end of the perfusion indicates a total conversion of [1-13C]glucose into [3-13C]lactate and [3-13C]pyruvate under anaerobic conditions. These results permit a better understanding of the metabolism of the perfused leukemic livers which are extensively infiltrated by these hematopoietic cells.     

Effect of perfusate glucose concentration on rat lung glycolysis.

We investigated the relationship between perfusate concentration of glucose and its utilization and lactate production derived from exogenous glucose and from metabolism of endogenous substrates. Isolated rat lungs were ventilated with 5% CO2 in air and perfused for 100 min with Krebs-Ringer bicarbonate buffer containing 3% bovine serum albumin, 10(-2) U/ml insulin, [U-14C]glucose and [5-3H]glucose. Glucose utilization, total lactate production, [14C]lactate production, and 3H2O production were measured. The apparent Km and Vmax for glucose utilization were 3.4 mM and 72.5 mumol/g dry wt per h, respectively. Lactate production from endogenous substrates, calculated as the difference between total and [14C]lactate, was 37.6 +/- 2.2 mumol/g dry wt (n = 36); it was unaffected by perfusate glucose concentration and by omission of insulin, but increased threefold with anoxia. Lactate production from 1.5 mM glucose was significantly less (P less than 0.02) with insulin omitted. Glycogen content was unchanged during perfusion without glucose. These results suggest that: 1) protein catabolism contributes to lung lactate production; 2) glucose utilization by lung is not maximal at resting physiological glucose concentrations; and 3) insulin is required at low glucose concentrations for maximal glycolytic rates.     

NMR study on [1-13C]glucose metabolism in the rat brain during hypoxia

The metabolism of [1-13C]glucose in the rat brain during hypoxia was investigated by 13C NMR spectroscopy. Male Wistar rats, weighing 100-120g, were anesthetized with ketamine (50 approximately 75 mg/kg i.p.) and ventilated mechanically with a mixture of 30% oxygen, 69.5% nitrogen and 0.5% halothane. [1-13C]glucose (250 mg/kg) was infused twice, at 10 minute intervals, through the femoral vein. For the control group (n = 4), the oxygen concentration of the inspiratory gas was maintained at 30% by vol throughout the experiments. For the hypoxia group (n = 6), the oxygen concentration in the inspiratory gas was reduced to 6-7% (93-94% nitrogen) and maintained for 30 min following [1-13C]glucose infusion. 13C NMR spectra were measured by a gated proton-decoupling method without a nuclear Overhauser effect. The [1-13C]glucose infusion gave apparent signals of the C1 carbon in the alpha- and beta-anomers of [1-13C]glucose at 92.7 and 96.7 ppm, respectively. Signals of the C2, C3 and C4 carbon atoms in glutamate and/or glutamine (glx) also appeared at 55, 27 and 34 ppm, respectively. The intensity of glx-C2 and glx-C3 signals increased later than that of glx-C4. The time lag between the different glx signals may reflect the turnover rate of the TCA cycle. Under the hypoxic condition, the signal of C3 carbon in lactate appeared at 21 ppm and increased. The alpha-glucose signal diminished during hypoxia, whereas the beta-glucose signal kept its intensity. The difference in changes of the signal intensity between alpha- and beta-glucose suggests that alpha-glucose is consumed more than beta-glucose in the hypoxic brain.     

Assessing the influence of isoflurane anesthesia on cardiac metabolism using hyperpolarized [1‐13C]pyruvate

Isoflurane is a frequently used anesthetic in small‐animal dissolution dynamic nuclear polarization‐magnetic resonance imaging (DNP‐MRI) studies. Although the literature suggests interactions with mitochondrial metabolism, the influence of the compound on cardiac metabolism has not been assessed systematically to date. In the present study, the impact of low versus high isoflurane concentration was examined in a crossover experiment in healthy rats. The results revealed that cardiac metabolism is modulated by isoflurane concentration, showing increased [1‐¹³C]lactate and reduced [¹³C]bicarbonate production during high isoflurane relative to low isoflurane dose [average differences: +16% [1‐¹³C]lactate/total myocardial carbon, –22% [¹³C]bicarbonate/total myocardial carbon; +51% [1‐¹³C]lactate/[¹³C]bicarbonate]. These findings emphasize that reproducible anesthesia is important when studying cardiac metabolism. As the depth of anesthesia is difficult to control in an experimental animal setting, careful study design is required to exclude confounding factors.     

Enhancing the [13C]bicarbonate signal in cardiac hyperpolarized [1‐13C]pyruvate MRS studies by infusion of glucose,insulin and potassium

A change in myocardial metabolism is a known effect of several diseases. MRS with hyperpolarized ¹³C‐labelled pyruvate is a technique capable of detecting changes in myocardial pyruvate metabolism, and has proven to be useful for the evaluation of myocardial ischaemia in vivo. However, during fasting, the myocardial glucose oxidation is low and the fatty acid oxidation (β‐oxidation) is high, which complicates the interpretation of pyruvate metabolism with the technique. The aim of this study was to investigate whether the infusion of glucose, insulin and potassium (GIK) could increase the myocardial glucose oxidation in the citric acid cycle, reflected as an increase in the [¹³C]bicarbonate signal in cardiac hyperpolarized [1‐¹³C]pyruvate MRS measurements in fasted rats. Two groups of rats were infused with two different doses of GIK and investigated by MRS after injection of hyperpolarized [1‐¹³C]pyruvate. No [¹³C]bicarbonate signal could be detected in the fasted state. However, a significant increase in the [¹³C]bicarbonate signal was observed by the infusion of a high dose of GIK. This study demonstrates that a high [¹³C]bicarbonate signal can be achieved by GIK infusion in fasted rats. The increased [¹³C]bicarbonate signal indicates an increased flux of pyruvate through the pyruvate dehydrogenase enzyme complex and an increase in myocardial glucose oxidation through the citric acid cycle. Copyright © 2013 John Wiley & Sons, Ltd.     

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

Glioblastomas and brain metastases demonstrate avid uptake of 2‐[¹⁸F]fluoro‐2‐deoxyglucose by positron emission tomography and display perturbations of intracellular metabolite pools by ¹H MRS. These observations suggest that metabolic reprogramming contributes to brain tumor growth in vivo. The Warburg effect, excess metabolism of glucose to lactate in the presence of oxygen, is a hallmark of cancer cells in culture. 2‐[¹⁸F]Fluoro‐2‐deoxyglucose‐positive tumors are assumed to metabolize glucose in a similar manner, with high rates of lactate formation relative to mitochondrial glucose oxidation, but few studies have specifically examined the metabolic fates of glucose in vivo. In particular, the capacity of human brain cancers to oxidize glucose in the tricarboxylic acid cycle is unknown. Here, we studied the metabolism of human brain tumors in situ. [U‐¹³ C]Glucose (uniformly labeled glucose, i.e. d ‐glucose labeled with ¹³ C in all six carbons) was infused during surgical resection, and tumor samples were subsequently subjected to ¹³C NMR spectroscopy. The analysis of tumor metabolites revealed lactate production, as expected. We also determined that pyruvate dehydrogenase, turnover of the tricarboxylic acid cycle, anaplerosis and de novo glutamine and glycine synthesis contributed significantly to the ultimate disposition of glucose carbon. Surprisingly, less than 50% of the acetyl‐coenzyme A pool was derived from blood‐borne glucose, suggesting that additional substrates contribute to tumor bioenergetics. This study illustrates a convenient approach that capitalizes on the high information content of ¹³C NMR spectroscopy and enables the analysis of intermediary metabolism in diverse cancers growing in their native microenvironment. Copyright © 2012 John Wiley & Sons, Ltd.     

Metabolism of D-[3-3H]glucose, D-[5-3H]glucose, D-[U-14C]glucose, D-[1-14C]glucose and D-[6-14C]glucose in pancreatic islets in an animal model of type-2 diabetes

This study aims at exploring specific aspects of D-glucose metabolism, so far not yet investigated, in pancreatic islets from adult control rats and animals (STZ rats) injected with streptozotocin during the neonatal period. The latter animals, which represent a current model of type-2 diabetes, displayed a lower body weight, higher plasma D-glucose concentration and lower insulinogenic index than control rats. The protein, DNA and insulin content were all also lower in islets prepared from STZ, rather than control rats. In the presence of 10.0 mM D-glucose, the paired ratio between D-[U-14C]glucose oxidation and D-[5-3H]glucose utilization was also decreased in the islets from STZ rats. No significant difference between control and STZ rats was observed, however, in terms of the ratios between D-[3-3H]glucose and D-[5-3H]glucose utilization, between the generation of radioactive lactate from 14C-labelled D-glucose and tritiated D-glucose utilization and between D-[1-14C]glucose and D-[6-14C]glucose oxidation. These findings reinforce the view that the previously documented preferential impairment of the oxidative modality of glycolysis in islets from STZ rats contrasts with the absence of any major anomaly in other variables of D-glucose catabolism.     

Determination of extracellular and intracellular enrichments of [1-(13)C]-alpha-ketoisovalerate using enzymatically converted [1-(13)C]-valine standardization curves and gas chromatography-mass spectrometry.

We report a validated method for the determination of extra- and intracellular [1-(13)C]-alpha-ketoisovalerate ([1-(13)C]-KIV) enrichments by gas chromatography-mass spectrometry. Standardization curves were prepared by enzymatic oxidation of [1-(13)C]-valine enriched standards of known composition. Slopes of [1-(13)C]-valine standardization curves (mean+/-SD: 0.99+/-0.02, n=5) and [1-(13)C]-KIV standardization curves (mean+/-SD: 0.98+/-0.01, n=7) were not significantly different. The method was applied for the determination of [1-(13)C]-KIV enrichments in plasma and tissues during [1-(13)C]-valine infusion in a piglet. [1-(13)C]-KIV enrichment could be determined+/-0.1 MPE (C.V. 1%), and extracellular [1-(13)C]-KIV enrichment was a reliable estimate of intracellular (skeletal muscle, bone growth plate) [1-(13)C]-KIV enrichment.     

Metabolism of hyperpolarized [1‐13C]pyruvate through alternate pathways in rat liver

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.     

In vivo investigation of cardiac metabolism in the rat using MRS of hyperpolarized [1‐13C] and [2‐13C]pyruvate

Hyperpolarized ¹³C MRS allows the in vivo assessment of pyruvate dehydrogenase complex (PDC) flux, which converts pyruvate to acetyl‐coenzyme A (acetyl‐CoA). [1‐¹³C]pyruvate has been used to measure changes in cardiac PDC flux, with demonstrated increase in ¹³C‐bicarbonate production after dichloroacetate (DCA) administration. With [1‐¹³C]pyruvate, the ¹³C label is released as ¹³CO₂/¹³C‐bicarbonate, and, hence, does not allow us to follow the fate of acetyl‐CoA. Pyruvate labeled in the C₂ position has been used to track the ¹³C label into the TCA (tricarboxylic acid) cycle and measure [5‐¹³C]glutamate as well as study changes in [1‐¹³C]acetylcarnitine with DCA and dobutamine. This work investigates changes in the metabolic fate of acetyl‐CoA in response to metabolic interventions of DCA‐induced increased PDC flux in the fed and fasted state, and increased cardiac workload with dobutamine in vivo in rat heart at two different pyruvate doses. DCA led to a modest increase in the ¹³C labeling of [5‐¹³C]glutamate, and a considerable increase in [1‐¹³C]acetylcarnitine and [1,3‐¹³C]acetoacetate peaks. Dobutamine resulted in an increased labeling of [2‐¹³C]lactate, [2‐¹³C]alanine and [5‐¹³C]glutamate. The change in glutamate with dobutamine was observed using a high pyruvate dose but not with a low dose. The relative changes in the different metabolic products provide information about the relationship between PDC‐mediated oxidation of pyruvate and its subsequent incorporation into the TCA cycle compared with other metabolic pathways. Using a high dose of pyruvate may provide an improved ability to observe changes in glutamate. Copyright © 2013 John Wiley & Sons, Ltd.     

An NMR study on the effect of glucose availability on carbohydrate metabolism in Dipetalonema viteae and Brugia pahangi

Adult Brugia pahangi and Dipetalonema viteae utilise a percentage of absorbed glucose (ca. 15%) in the formation of the disaccharide trehalose [8]. This paper reports an investigation, employing 13C-NMR techniques, of the utilisation of trehalose by these nematodes and also the effect of glucose availability on metabolic product composition. The metabolism of [1-13C]trehalose in D. viteae differed dramatically from that of [1-13C]glucose under normal experimental conditions. A succinate/lactate ratio of 0.73 was obtained from the metabolism of [1-13C]trehalose compared with 0.05 from [1-13C]glucose at an initial concentration of ca. 5 mM. Similar, but less consistent, results were obtained from B. pahangi adults. Macrofilariae of D. viteae were fed variable, low levels of glucose at hourly intervals for 8 h, and a significant relationship (P less than 0.001) between the glucose addition rate and the ratio of succinate to lactate production was obtained. The lower the amount of glucose added each hour, the higher was the observed succinate to lactate ratio. The percentage yield of succinate increased greatly as the amount of added glucose was diminished. Parallel experiments performed on B. pahangi macrofilariae indicated that B. pahangi did not increase their succinate output so greatly with reduced glucose availability. It is clear that in the absence of available external glucose, B. pahangi and D. viteae draw on their internal trehalose reserves as a source of carbohydrate for energy generation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Estimation of glucose turnover and recycling in rabbits using various [3H, 14C]glucose labels.

The glucose replacement rate, percent carbon recycling, mean glucose transit time, and the glucose mass were determined in fasted unanesthetized rabbits after administration of [2-3H,U-14C]-, [3-3H,U-14C]-, [5-3H,U-14C]- or [6-3H,U-14C]glucose using the procedures of Katz et al. (10). The glucose replacement rates and carbon recycling determined with [2-3H,U-14C] and [5-3H,U-14C]glucose are equivalent and greater than those obtained with [3-3H,U-14C]- and [6-3H,U-14C]glucose. Although the means of the glucose replacement rates and percent carbon recycling obtained using [3-3H,U-14C]- and [6-3H,U-14C]glucose are similar, greater variation resulted using the former tracer. Comparisons of detritiation rates and percent carbon recycling using [2-3H,U-14C]- and [6-3H,U-14C]glucose suggest that about 10% of tritium is lost from carbon 2 via futile cycling at the glucose 6-phosphate level. Similarly, comparisons of [5-3H,U-14C]- and [6-3H,U-14C]glucose metabolism suggest that about 10% of tritium lost from carbon 5 occurs via futile cycling at the fructose diphosphate level and/or via the transaldolase reaction. Our results indicate that [6-3H,U-14C]glucose is the more suitable tracer for determining the glucose replacement rate and carbon recycling in vivo.     

Hepatic gluconeogenesis and Krebs cycle fluxes in a CCl4 model of acute liver failure

Acute liver failure was induced in rats by CCl4 administration and its effects on the hepatic Krebs cycle and gluconeogenic fluxes were evaluated in situ by 13C NMR isotopomer analysis of hepatic glucose following infusion of [U-13C]propionate. In fed animals, CCl4 injury caused a significant increase in relative gluconeogenic flux from 0.80+/-0.10 to 1.34 +/-0.24 times the flux through citrate synthase (p<0.01). In 24-h fasted animals, CCl4-injury also significantly increased relative gluconeogenic flux from 1.36+/-0.16 to 1.80+/-0.22 times the flux through citrate synthase (p<0.01). Recycling of PEP via pyruvate and oxaloacetate was extensive under all conditions and was not significantly altered by CCl4 injury. CCl4 injury significantly reduced hepatic glucose output by 26% (42.8+/-7.3 vs 58.1+/-2.4 micromol/kg/min, p=0.005), which was attributed to a 26% decrease in absolute gluconeogenic flux from PEP (85.6+/-14.6 vs 116+/-4.8 micromol/kg/min, p<0.01). These changes were accompanied by a 47% reduction in absolute citrate synthase flux (90.6+/-8.0 to 47.6+/-8.0 micromol/kg/min, p<0.005), indicating that oxidative Krebs cycle flux was more susceptible to CCl4 injury. The reduction in absolute fluxes indicate a significant loss of hepatic metabolic capacity, while the significant increases in relative gluconeogenic fluxes suggest a reorganization of metabolic activity towards preserving hepatic glucose output.     

Imaging porcine cardiac substrate selection modulations by glucose,insulin and potassium intervention: A hyperpolarized [1‐13C]pyruvate study

Cardiac metabolism has received considerable attention in terms of both diagnostics and prognostics, as well as a novel target for treatment. As human trials involving hyperpolarized magnetic resonance in the heart are imminent, we sought to evaluate the general feasibility of detection of an imposed shift in metabolic substrate utilization during metabolic modulation with glucose–insulin–potassium (GIK) infusion, and thus the limitations associated with this strategy, in a large animal model resembling human physiology. Four [1‐¹³C]pyruvate injections did not alter the blood pressure or ejection fraction over 180 min. Hyperpolarized [1‐¹³C]pyruvate conversion showed a generally high reproducibility, with intraclass correlation coefficients between the baseline measurements at 0 and 30 min as follows: lactate to pyruvate, 0.85; alanine to pyruvate, 1.00; bicarbonate to pyruvate, 0.83. This study demonstrates that hyperpolarized [1‐¹³C]pyruvate imaging is a feasible technique for cardiac studies and shows a generally high reproducibility in fasted large animals. GIK infusion increases the metabolic conversion of pyruvate to its metabolic derivatives lactate, alanine and bicarbonate, but with increased variability.     

Assessing the pentose phosphate pathway using [2, 3‐13C2]glucose

The pentose phosphate pathway (PPP) is essential for reductive biosynthesis, antioxidant processes and nucleotide production. Common tracers such as [1,2‐¹³C₂]glucose rely on detection of ¹³C in lactate and require assumptions to correct natural ¹³C abundance. Here, we introduce a novel and specific tracer of the PPP, [2,3‐¹³C₂]glucose. ¹³C NMR analysis of the resulting isotopomers is informative because [1,2‐¹³C₂]lactate arises from glycolysis and [2,3‐¹³C₂]lactate arises exclusively through the PPP. A correction for natural abundance is unnecessary. In rats receiving [2,3‐¹³C₂]glucose, the PPP was more active in the fed versus fasted state in the liver and the heart, consistent with increased expression of key enzymes in the PPP. Both the PPP and glycolysis were substantially increased in hepatoma compared with liver. In summary, [2,3‐¹³C₂]glucose and ¹³C NMR simplify assessment of the PPP.