In vivo detection of intermediate metabolic products of [1-(13) C]ethanol in the brain using (13) C MRS

In this study, in vivo ¹³C MRS was used to investigate the labeling of brain metabolites after intravenous administration of [1‐¹³C]ethanol. After [1‐¹³C]ethanol had been administered systemically to rats, ¹³C labels were detected in glutamate, glutamine and aspartate in the carboxylic and amide carbon spectral region. ¹³C‐labeled bicarbonate HCO (161.0 ppm) was also detected. Saturating acetaldehyde C1 at 207.0 ppm was found to have no effect on the ethanol C1 (57.7 ppm) signal intensity after extensive signal averaging, providing direct in vivo evidence that direct metabolism of alcohol by brain tissue is minimal. To compare the labeling of brain metabolites by ethanol with labeling by glucose, in vivo time course data were acquired during intravenous co‐infusion of [1‐¹³C]ethanol and [¹³C₆]‐D ‐glucose. In contrast with labeling by [¹³C₆]‐D ‐glucose, which produced doublets of carboxylic/amide carbons with a J coupling constant of 51 Hz, the simultaneously detected glutamate and glutamine singlets were labeled by [1‐¹³C]ethanol. As ¹³C labels originating from ethanol enter the brain after being converted into [1‐¹³C]acetate in the liver, and the direct metabolism of ethanol by brain tissue is negligible, it is suggested that orally or intragastrically administered ¹³C‐labeled ethanol may be used to study brain metabolism and glutamatergic neurotransmission in investigations involving alcohol administration. In vivo ¹³C MRS of rat brain following intragastric administration of ¹³C‐labeled ethanol is demonstrated. Published in 2011 by John Wiley & Sons, Ltd.     

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.     

In vivo single-shot, proton-localized 13C MRS of rhesus monkey brain

A single-shot, proton-localized, polarization transfer (13)C spectroscopic method was proposed and implemented on a 4.7 T scanner for studying rhesus monkey brains. The polarization transfer sequence was mostly adiabatic, minimizing signal loss due to B(1) inhomogeneity. RF pulses in polarization transfer were also used for voxel selection of protons with gradient fields. The transferred (13)C magnetization was refocused by additional refocusing adiabatic pulses. With the intravenous infusion of D-[1-(13)C]glucose solution, (13)C NMR spectra from a 30 mL voxel were acquired for the resonances of C1 of glucose, C2,3,4 of glutamate and glutamine. The time-resolved turnover of glutamate, glutamine and aspartate from intravenously infused D-[1-(13)C]glucose at a temporal resolution of 12 min was demonstrated with excellent spectral resolution and signal-to-noise ratio. Typically, the half-height linewidth of the decoupled (13)C peaks was approximately 4 Hz. Data obtained with infusion of sodium [2-(13)C]acetate using the proposed polarization transfer method and data from the carboxylic carbon region using non-localized acquisition are also presented.     

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.     

Estimating gluconeogenesis by NMR isotopomer distribution analysis of [13C]bicarbonate and [1-13C]lactate

The gluconeogenic contribution to glucose production in livers isolated from rats fasted for 24 h was determined by 13C-NMR isotopomer distribution analysis of secreted glucose enriched from 99% [13C]bicarbonate (n = 4) and 99% [1-13C]lactate (n = 4). Experiments with 3% 2H2O were also performed, allowing the gluconeogenic contribution to be measured by the relative 2H enrichments at positions 5 and 2 of glucose. From 13C-NMR analyses, the contribution of gluconeogenesis to glucose output was estimated to be 93 +/- 3% for [13C]bicarbonate perfusion and 91 +/- 3% for [1-13C]lactate perfusion, in good agreement with the 2H-NMR analysis of the gluconeogenic contribution to glucose production (100 +/- 1% and 99 +/- 1%, respectively) and consistent with the expected negligible contribution from glycogenolysis. These results indicate that 13C-NMR analysis of glucose 13C-isotopomer distribution from either [13C]bicarbonate or [1-13C]lactate precursor provides realistic estimates of the gluconeogenic contribution to hepatic glucose output.     

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.     

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.     

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.     

Influence of altered O2 tension on substrate metabolism in perfused rat lung.

Effects of hypoxia (1.5 h) on glucose and palmitate metabolism were investigated in perfused lungs from normal rats and rats exposed for 24 h to hypobaric conditions (simulated altitude of 24,000 ft). Hypoxic lungs were ventilated with 5% O2-5% CO2 and control lungs with 21% O2-5% CO2. Blood gases and pH remained stable during the 1.5-h perfusion period. Exposure of normal rat lungs to 1.5 h of in vitro hypoxia (blood Po2=34 mmHg) significantly increased lactate production and mean arterial pulmonary pressure, but did not alter glucose uptake, pyruvate levels, and oxidation of either [U-14C]glucose or [1-14C]palmitate to CO2. Incorporation of labeled glucose and palmitate into lung lipids was also unaltered. In contrast to normal lungs, prior exposure to hypoxia for 24 h and subsequent perfusion under hypoxic conditions significantly stimulated glucose uptake (74% increase), markedly increased glucose incorporation into lung lipids, and increased oxidation of glucose to CO2. Lactate/pyruvate ratios also showed a significant 38% increase. Lung glycogen was unchanged following 24 h hypoxia. These data indicate that adaptive changes occur in metabolic processes within the lung during acute changes in O2 tension.     

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.     

Atrial natriuretic peptide during and after maximal and submaximal exercise under normoxic and hypoxic conditions

Summary The present study was designed to investigate the influence of exercise intensity and duration as well as of inspiratory oxygen content on plasma atrial natriuretic peptide concentration ([ANP]) and furthermore to compare ANP with the effect on aldosterone concentration ([Aldo]). Ten untrained male subjects performed a maximal exercise test (ME) on a cycle ergometer and a submaximal test of 60-min duration at 60% of maximal performance (SE) under normoxia (N) and normobaric hypoxia (H) (partial pressure of oxygen: 12.3 kPa). Five subjects were exposed to hypoxia at rest for 90 min. The [ANP] was mostly affected by exercise intensity (5 min after ME-N, +298.1%, SEM 39.1%) and less by exercise duration (at the end of SE-N: +229.5%, SEM 33.2%). Hypoxia had no effect at rest and reduced the exercise response (ME-H, +184.3%, SEM 27.2%; SE-H, +172.4%, SEM 15.7%). In contrast to ANP, the Aldo response was affected more by duration at submaximal level (+290.1%, SEM 34.0%) than by short maximal exercise (+235.7%, SEM 22.2%). Exposure to hypoxia rapidly decreased [Aldo] (–28.5%, SEM 3.7% after 30 min, P<0.01), but did not influence the exercise effects (ME-H, +206.2%, SEM 26.4%; SE-H, +321.6%, SEM 51.6%). The [ANP] increase was faster than that of [Aldo] during the maximal tests and there was no difference during submaximal exercise. Changes in plasma volume (PV), sodium concentration, and osmolality (Osm) were most pronounced during maximal exercise (for ME-N: PV –13.1%, SD 3.6%, sodium +6.2 mmol·1^–1, SD 2.7, Osm +18.4 mosmol·kg H₂O^–1, SD 6.5). Regression analysis showed high correlations between changes in [ANP] and in Osm during and after maximal exercise and between changes in [ANP] and heart rate for submaximal exercise. It is concluded that besides other mechanisms increased Osm might be involved in the exercise-dependent increase of plasma [ANP].     

In a medium containing glucose, lactate carbon is incorporated by gonococci predominantly into fatty acids and glucose carbon incorporation is increased: implications regarding lactate stimulation of metabolism

The reason for stimulation by lactate of metabolism of gonococci growing in a medium containing glucose, which enhances pathogenicity by increasing growth rate, lipopolysaccharide (LPS) synthesis and protein formation, has been investigated. Tricine dodecylpolyacrylamide gel electrophoresis (SDS-PAGE) and thin layer chromatography (TLC) on homogenates of gonococci grown in this medium with [14C]lactate showed that lactate carbon was preferentially incorporated into lipid and LPS. Nuclear magnetic resonance (NMR) spectroscopy on lipid extracted from gonococci grown in the glucose containing medium with [13C]lactate showed that lactate carbon was incorporated into fatty acid moieties and not into ethanolamine or glycerol moieties. In contrast, NMR on lipid from gonococci grown with [13C]glucose indicated glucose carbon in both moieties. When unlabelled lactate was added, lipid synthesis from [l3C]glucose was stimulated and small amounts of different fatty acids were formed. The NMR data shows that gluconeogenesis from lactate carbon does not occur in the presence of glucose, suggesting that lactate is used solely for rapid production, via pyruvate, of acetyl CoA, the precursor not only for fatty acid synthesis but also for the constituents and products of the citric acid cycle, including ATP. The rapid formation of a high level of acetyl CoA is the probable reason for the stimulation of metabolism and oxygen uptake by lactate. 14C label on LPS was detected in its fatty acids. Most proteins that stained with silver in tricine SDS-PAGE were not significantly labelled by [14C]lactate in the glucose-containing medium. Two of three appreciably labelled proteins were identified by N-terminal sequencing as GroEL and porin 1B, and one of the two less labelled proteins was similar to peroxiredoxin type proteins. There were no signs of specific induction of these proteins by lactate and their labelling was consistent with fatty acids in attached lipid.     

13C NMR spectroscopic analysis of phospholipid metabolism in adrenal chromaffin cells

Phosphatidylcholine (PC) metabolism in bovine adrenal chromaffin cells has been studied in vivo with 100 MHz 13C NMR spectroscopy. The incorporation of 13[CH3]choline was examined in cells embedded in 1% agarose strands and perfused at 37 degrees C. The pattern of 13C NMR spectroscopy. The incorporation of [13CH3]choline incorporation into PC of suspension cultures of chromaffin cells. The chemical shifts of labelled carbons in [13CH3]choline before and after their incorporation into PC are identical (ca 56.4 ppm). Incorporated [13CH3]choline can be released from the cells by phospholipase D which reduces the cellular PC signal by greater than 85%, indicating that a major fraction of the labelled PC is on the external membrane surface. This application of NMR spectroscopy provides a basis for direct measurement of the in vivo metabolism of PC in functioning adrenal chromaffin cells.     

Metabolic response to carbon monoxide by isolated rat lungs.

Glucose metabolism was studied in isolated rat lungs ventilated with 95% O2.5% CO2 (control), 95% N2: 5% CO2 (hypoxia), and 95% CO:5% CO2 (carbon monoxide) and perfused for 100-120 min with Krebs-Ringer-bicarbonate buffer, pH 7.4, containing [U-14C] and [3-3H]glucose. The production of 14C-labeled lactate plus pyruvate (L + P) and of 14CO2 represented 48% and 22% respectively, of the total [14C]glucose utilization. The lactate-to-pyruvate ratio (L/P) was 8.7. Tritium was recovered predominantly as 3H2O in the perfusate. Wth carbon monoxide ventilation, L + P production was increased by 357% with an L/P of 52.9, and 14CO2 production was markedly decreased. A 56% decrease in lung ATP content was associated with decreased incorporation of 14C into fatty acids. Compared with CO, changes with N2 ventilation were less marked, indicating that ventilation with CO is a more effective method with which to study inhibtion of oxidative metabolism. The lung exhibits a Pasteur effectbintain ATP content or its supply for synthetic activity.     

Effect of hypoxia on arterial and venous blood levels of oxygen,carbon dioxide,hydrogen ions and lactate during incremental forearm exercise

Summary The purpose of the present study was to investigate whether, in humans, hypoxia results in an elevated lactate production from exercising skeletal muscle. Under conditions of both hypoxia [inspired oxygen fraction (F_IO₂): 11.10%] and normoxia (F_IO₂: 20.94%), incremental exercise of a forearm was performed. The exercise intensity was increased every minute by 1.6 kg·m·min^–1 until exhaustion. During the incremental exercise the partial pressure of oxygen (PO₂) and carbon dioxide (PCO₂), oxygen saturation (SO₂), pH and lactate concentration [HLa] of five subjects, were measured repeatedly in blood from the brachial artery and deep veins from muscles in the forearm of both the active and inactive sides. The hypoxia (arterial SO₂ approximately 70%) resulted in (1) the difference in [HLa] in venous blood from active muscle (values during exercise — resting value) often being more than twice that for normoxia, (2) a significantly greater difference in venous-arterial (v-a) [HLa] for the exercising muscle compared to normoxia, and (3) a difference in v-a [HLa] for non-exercising muscle that was slightly negative during normoxia and more so with hypoxia. These studies suggest that lower O₂ availability to the exercising muscle results in increased lactate production.     

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)     

NMR studies on energy metabolism of immobilized primary neurons and astrocytes during hypoxia, ischemia and hypoglycemia

Changes in high-energy phosphate metabolites (ATP and phosphocreatine) were monitored, in real time, by 31P-nuclear magnetic resonance in primary cell cultures of neurons and astrocytes during periods of hypoxia, ischemia and hypoglycemia, and also during the recovery periods following the re-establishment of standard conditions. Cells were immobilized in basement membrane gel threads and perfused with oxygen-depleted medium (oxygen concentration below 30 microM), to create hypoxic conditions, or with aerobic medium (oxygen concentration approximately 460 microM) containing different concentrations of glucose (hypoglycemia). Ischemic conditions were imposed by stopping perfusion for different periods of time (15 min to 2 h). The experimental set-up enabled the acquisition of 31P-spectra with high signal-to-noise ratio within 10-20 min for both cell types. The effect of hypoxia on glucose metabolism was assessed by 13C-NMR using [1-13C]glucose as substrate. The levels of ATP and PCr in astrocytes were unaffected during hypoxia (up to 2 h), but decreased notably under ischemia. In neurons, hypoxic periods caused a sharp drop of the ATP and PCr levels, and considerable damage to the capacity of neurons to replenish the ATP and PCr pools upon returning to normoxic conditions. However, neurons were remarkably less sensitive to ischemic conditions, the ATP and PCr pools being restored quickly, even after 2 h under challenging conditions. The data show that neurons were more resistant to ischemia than astrocytes, and suggest that the capacity to sustain the pools of ATP and PCr was part of the neuronal protective strategy.     

Ketone bodies effectively compete with glucose for neuronal acetyl‐CoA generation in rat hippocampal slices

Ketone bodies can be used for cerebral energy generation in situ, when their availability is increased as during fasting or ingestion of a ketogenic diet. However, it is not known how effectively ketone bodies compete with glucose, lactate, and pyruvate for energy generation in the brain parenchyma. Hence, the contributions of exogenous 5.0 mM [1‐¹³C]glucose and 1.0 mM [2‐¹³C]lactate + 0.1 mM pyruvate (combined [2‐¹³C]lactate + [2‐¹³C]pyruvate) to acetyl‐CoA production were measured both without and with 5.0 mM [U‐¹³C]3‐hydroxybutyrate in superfused rat hippocampal slices by ¹³C NMR non‐steady‐state isotopomer analysis of tissue glutamate and GABA. Without [U‐¹³C]3‐hydroxybutyrate, glucose, combined lactate + pyruvate, and unlabeled endogenous sources contributed (mean ± SEM) 70 ± 7%, 10 ± 2%, and 20 ± 8% of acetyl‐CoA, respectively. With [U‐¹³C]3‐hydroxybutyrate, glucose contributions significantly fell from 70 ± 7% to 21 ± 3% (p < 0.0001), combined lactate + pyruvate and endogenous contributions were unchanged, and [U‐¹³C]3‐hydroxybutyrate became the major acetyl‐CoA contributor (68 ± 3%) – about three‐times higher than glucose. A direct analysis of the GABA carbon 2 multiplet revealed that [U‐¹³C]3‐hydroxybutyrate contributed approximately the same acetyl‐CoA fraction as glucose, indicating that it was less avidly oxidized by GABAergic than glutamatergic neurons. The appearance of superfusate lactate derived from glycolysis of [1‐¹³C]glucose did not decrease significantly in the presence of 3‐hydroxybutyrate, hence total glycolytic flux (Krebs cycle inflow + exogenous lactate formation) was attenuated by 3‐hydroxybutyrate. This indicates that, under these conditions, 3‐hydroxybutyrate inhibited glycolytic flux upstream of pyruvate kinase. Copyright © 2015 John Wiley & Sons, Ltd.     

Kinetics of lactate and glucose metabolism in the pregnant ewe and conceptus

Two monotocous and two ditocous ewes were infused at 124-130 days of gestation with a mixture of L-[U-14C]lactate and D-[6-3H]glucose via a maternal or umbilical catheter, separate days being used for each infusion. Plateaux-specific radioactivities of plasma lactate and glucose were used to derive a four-pool model describing the fluxes between the lactate and glucose pools of the infused conceptus and mother. The average turnover rate of lactate was 23.3 and 13.2 mg carbon min-1 in the conceptus and 32.0 and 44.0 mg carbon min-1 in the mother for monotocous and ditocous ewes, respectively. Glycolysis rates within both conceptus and mother in all sheep were high, accounting for about 80 and 60% of the respective rates of lactate turnover. The synthesis of glucose from lactate accounted for 13 and 31% of the glucose turnover in the mother in monotocous and ditocous ewes, respectively, but was insignificant in the conceptus. Glycolysis within the conceptus used only glucose which had entered and mixed with the conceptus glucose pool; there was no direct transfer of carbon from the maternal glucose pool to the conceptus lactate pool. This finding is an important validation for the use of tracer methods to determine glucose use within the whole conceptus rather than within fetal corporeal tissues alone.     

Carbon dioxide abolishes the reverse Pasteur effect in Leishmania major promastigotes

The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.