Cerebral metabolism of [1,2-13C2]acetate as detected by in vivo and in vitro 13C NMR

The metabolism of [1,2-13C2]acetate in rat brain was studied by in vivo and in vitro 13C NMR spectroscopy, in particular by taking advantage of the homonuclear 13C-13C spin coupling patterns. Well nourished rats were infused with [1,2-13C2]acetate or [1-13C]acetate in the jugular vein, and the in situ kinetics of 13C labeling during the infusion period was followed by 13C NMR techniques. The in vivo 13C NMR spectra showed signals from (i) the C-1 carbon of [1,2-13C2] acetate or [1-13C]acetate, (ii) 13CO3H-, and (iii) the natural abundance 13C carbons of sufficiently mobile fatty acids. Methanol/HCl/perchloric acid extracts of the brains were prepared and were further analyzed by high resolution 13C NMR. The homonuclear 13C-13C spin coupling patterns after infusion of [1,2-13C2]acetate showed very different isotopomer populations in glutamate, glutamine, and gamma-aminobutyric acid. Analyzing the relative proportions of these isotopomers revealed (i) two different glutamate compartments in the rat brain characterized by the presence and absence, respectively, of glutamine synthase activity, (ii) two different tricarboxylic acid cycles, one preferentially metabolizing [(1,2-13C2]acetate, the other mainly using unlabeled acetyl-coenzyme A, (iii) a hitherto unknown cerebral pyruvate recycling system associated with the tricarboxylic acid cycle, metabolizing primarily unlabeled acetyl-coenzyme A, and (iv) a predominant production of gamma-aminobutyric acid in the glutamate compartment lacking glutamine synthase.     

Glycogen metabolism as detected by in vivo and in vitro 13C-NMR spectroscopy using [1,2-13C2]glucose as substrate

The metabolism of glucose to glycogen in the liver of fasted and well-fed rats was investigated with 13C nuclear magnetic resonance spectroscopy using [1,2-(13)C2]glucose as the main substrate. The unique spectroscopic feature of this molecule is the 13C-13C homonuclear coupling leading to characteristic doublets for the C-1 and C-2 resonances of glucose and its breakdown products as long as the two 13C nuclei remain bonded together. The doublet resonances of [1,2-(13)C2]glucose thus provide an ideal marker to follow the fate of this exogenous substrate through the metabolic pathways. [1,2-(13)C2]Glucose was injected intraperitoneally into anesthetized rats and the in vivo 13C-NMR measurements of the intact animals revealed the transformation of the injected glucose into liver glycogen. Glycogen was extracted from the liver and high resolution 13C-NMR spectra were obtained before and after hydrolysis of glycogen. Intact [1,2-13C2]glucose molecules give rise to doublet resonances, natural abundance [13C]glucose molecules produce singlet resonances. From an analysis of the doublet-to-singlet intensities the following conclusions were derived. (i) In fasted rats virtually 100% of the glycosyl units in glycogen were 13C-NMR visible. In contrast, the 13C-NMR visibility of glycogen decreased to 30-40% in well-fed rats. (ii) In fed rats a minimum of 67 +/- 7% of the exogenous [1,2-(13)C2]glucose was incorporated into the liver glycogen via the direct pathway. No contribution of the indirect pathway could be detected. (iii) In fasted rats externally supplied glucose appeared to be consumed in different metabolic processes and less [1,2-(13)C2]glucose was found to be incorporated into glycogen (13 +/- 1%). However, the observation of [5,6-(13)C2]glucose in liver glycogen provided evidence for the operation of the so-called indirect pathway of glycogen synthesis. The activity of the indirect pathway was at least 9% but not more than 30% of the direct pathway. (vi) The pentose phosphate pathway was of little significance for glucose but became detectable upon injection of [1-(13)C]ribose.     

13C NMR for the assessment of human brain glucose metabolism in vivo

Proton-decoupled 13C NMR spectra of the human head were obtained during hyperglycemic glucose clamping using intravenous infusions of [1-13C]glucose in normal volunteers. In addition to 13C signals of mobile lipids, a variety of new metabolite resonances could be resolved for the first time in the human brain. At an enrichment level of 20% [1-13C]glucose, the signals of alpha- and beta-glucose at 92.7 and 96.6 ppm, respectively, could be detected in the human brain after only an infusion period of 15 min. The spatial localization of the different regions of interest was confirmed by 13C NMR spectroscopic imaging with a time resolution of 9 min. Increasing the enrichment level to 99% [1-13C]glucose not only improved the time resolution but allowed the detection of metabolic breakdown products of [1-13C]glucose. The time course of 13C label incorporation into the C2, C3, and C4 resonances of glutamate/glutamine and into lactate could be recorded in the human brain. These results suggest the possibility of obtaining time-resolved, spatially selective, and chemically specific information on the human body.     

In vivo 13C NMR study of glucose and cellobiose metabolism by four cellulolytic strains of the genus Fibrobacter

The metabolism of glucose and cellobiose, products of cellulose hydrolysis, was investigated in four cellulolytic strains of the genus Fibrobacter: Fibrobacter succinogenes S85, 095, HM2 and Fibrobacter intestinalis NR9. In vivo 13C nuclear magnetic resonance was used to quantify the relative contribution of glucose and cellobiose to metabolite production, glycogen storage and cellodextrins synthesis in these four strains. The same features were found in all four strains of the genus Fibrobacter metabolizing simultaneously glucose and cellobiose: i) differential metabolism of glucose and cellobiose; glucose seems preferentially used for glycogen storage and energy production, while part of cellobiose seems to be diverted from glycolysis, ii) synthesis of cellodextrins, mainly from cellobiose not entering into glycolysis, iii) accumulation of glucose 6-phosphate, iv) simultaneous presence of cellobiose phosphorylase and cellobiase activities.Although genetically diverse, the Fibrobacter genus appears to possess a marked homogeneity in its carbon metabolism.     

Effects of hormone and glucose administration on hepatic glucose and glycogen metabolism in vivo. A 13C NMR study

Effects of peripheral venous injection of glucagon and insulin on [1-13C]glucose incorporation into hepatic glycogen of rats were studied by 13C NMR in vivo. Each animal was given a continuous somatostatin infusion and a 100-mg intravenous injection of [1-13C] glucose in NMR experiments or unlabeled glucose in parallel experiments for determination of serum glucose. Insulin administration caused serum glucose to fall below basal levels and accelerated the loss of hepatic [1-13C]glucose; these effects were counteracted by the addition of glucagon. Glucagon administration alone did not affect serum glucose or hepatic [1-13C] glucose but caused the loss of [1-13C]glucose from glycogen and inhibited [1-13C]glucose incorporation into glycogen. Insulin did not alter [1-13C]glucose incorporation into glycogen when given alone or in combination with glucagon. The data are consistent with a model in which liver glycogen synthesis increases linearly with hepatic glucose concentration above a threshold glucose concentration. Insulin did not alter the rate constant or the threshold for synthesis.     

Formaldehyde metabolism by Escherichia coli. Detection by in vivo 13C NMR spectroscopy of S-(hydroxymethyl)glutathione as a transient intracellular intermediate

In vivo 13C NMR has been used to detect the transient formation of S-(hydroxymethyl)glutathione (GSCH2OH) from glutathione and [13C]formaldehyde in Escherichia coli. Two-dimensional 1H-13C shift correlation was used to locate the chemical shift of the formaldehyde-derived protons of the adduct. The adduct GSCH2OH is formed by chemical reaction in the first few minutes after cells are challenged with formaldehyde and remains within the cell until consumed by metabolism.     

Carbon flux through citric acid cycle pathways in perfused heart by 13C NMR spectroscopy

Mathematical models of the TCA cycle derived previously for 14C tracer studies have been extended to 13C NMR to measure the 13C fractional enrichment of [2-13C]acetyl-CoA entering the cycle and the relative activities of the oxidative versus anaplerotic pathways. The analysis is based upon the steady-state enrichment of 13C into the glutamate carbons. Hearts perfused with [2-13C]acetate show low but significant activity of the anaplerotic pathways. Activation of two different anaplerotic pathways is demonstrated by addition of unlabeled propionate or pyruvate to hearts perfused with [2-13C]acetate. In each case, the amount of [2-13C]acetate being oxidized and the relative carbon flux through anaplerotic versus oxidative pathways are evaluated.     

In vivo 13C NMR studies of compartmentalized cerebral carbohydrate metabolism

Localized 13C nuclear magnetic resonance (NMR) spectroscopy provides a unique window for studying cerebral carbohydrate metabolism through, e.g. the completely non-invasive measurement of cerebral glucose and glycogen metabolism. In addition, label incorporation into amino acid neurotransmitters such as glutamate (Glu), GABA and aspartate can be measured providing information on Krebs cycle flux and oxidative metabolism. Given the compartmentation of key enzymes such as pyruvate carboxylase and glutamine synthetase, the detection of label incorporation into glutamine indicated that neuronal and glial metabolism can be measured in vivo. The purpose of this paper is to provide a critical overview of these recent advances into measuring compartmentation of brain energy metabolism using localized in vivo 13C NMR spectroscopy. The studies reviewed herein showed that anaplerosis is significant in brain, as is oxidative ATP generation in glia and the rate of glial glutamine synthesis attributed to the replenishment of the neuronal Glu pool and that brain glycogen metabolism is slow under resting conditions. This new modality promises to provide a new investigative tool to study aspects of normal and diseased brain hitherto unaccessible, such as the interplay between glutamatergic action, glucose and glycogen metabolism during brain activation, and the derangements thereof in patients with hepatic encephalopathy, neurodegenerative diseases and diabetes.     

Glucose metabolism in resting cells ofCandida albicans as studied by13C NMR

The metabolism of glucose in resting cells ofCandida albicans was studied by¹³C NMR spectrometry using¹³C-labeled glucose. Under aeration, the formation of ethanol, glycerol, arabitol, mannitol, and trehalose was observed. The addition of inhibitors of the respiratory chain or the omission of aeration resulted in a total loss of formation of those polyols and trehalose, with ethanol being the only detectable product. Thus, respiration seems to favor the production of polyols including glycerol, as well as that of trehalose. With regard to glycerol, this finding is in contrast with the previous observation inSaccharomyces cerevisiae that oxygenation represses its production.     

Compartmentation of TCA cycle metabolism in cultured neocortical neurons revealed by 13C MR spectroscopy

Cultured neocortical neurons were incubated in medium containing [U-13C]glucose (0.5 mM) and in some cases unlabeled glutamine (0.5 mM). Subsequently the cells were "superfused" for investigation of the effect of depolarization by 55 mM K+. Cell extracts were analyzed by 13C magnetic resonance spectroscopy and gas chromatography/mass spectrometry to determine incorporation of 13C in glutamate, GABA, aspartate and fumarate. The importance of the tricarboxylic acid (TCA) cycle for conversion of the carbon skeleton of glutamine to GABA was evident from the effect of glutamine on the labeling pattern of GABA and glutamate. Moreover, analysis of the labeling patterns of glutamate in particular indicated a depolarization induced increased oxidative metabolism. This effect was only observed in glutamate and not in neurotransmitter GABA. Based on this a hypothesis of mitochondrial compartmentation may be proposed in which mitochondria associated with neurotransmitter synthesis are distinct from those aimed at energy production and influenced by depolarization. The hypothesis of mitochondrial compartmentation was further supported by the finding that the total percent labeling of fumarate and aspartate differed significantly from each other. This can only be explained by the existence of multiple TCA cycles with different turnover rates.     

13C NMR relaxation times of hepatic glycogen in vitro and in vivo

The field dependence of relaxation times of the C-1 carbon of glycogen was studied in vitro by natural-abundance 13C NMR. T1 is strongly field dependent, while T2 does not change significantly with magnetic field. T1 and T2 were also measured for rat hepatic glycogen enriched with [1-13C]glucose in vivo at 4.7 T, and similar relaxation times were observed as those obtained in vitro at the same field. The in vitro values of T1 were 65 +/- 5 ms at 2.1 T, 142 +/- 10 ms at 4.7 T, and 300 +/- 10 ms at 8.4 T, while T2 values were 6.7 +/- 1 ms at 2.1 T, 9.4 +/- 1 ms at 4.7 T, and 9.5 +/- 1 ms at 8.4 T. Calculations based on the rigid-rotor nearest-neighbor model give qualitatively good agreement with the T1 field dependence with a best-fit correlation time of 6.4 X 10(-9) s, which is significantly smaller than tau M, the estimated overall correlation time for the glycogen molecule (ca. 10(-5) s). A more accurate fit of T1 data using a modified Lipari and Szabo approach indicates that internal fast motions dominate the T1 relaxation in glycogen. On the other hand, the T2 relaxation is dominated by the overall correlation time tau M while the internal motions are almost but not completely unrestricted.     

In vitro metabolism of [2-13C]-ethanol by 1H NMR spectroscopy using 13C decoupling with the reverse dept polarization-transfer pulse sequence

The metabolism of [2-13C]-ethanol by alcohol dehydrogenase purified from Drosophila melanogaster has been observed by proton nuclear magnetic resonance spectroscopy (NMR). The reverse-DEPT pulse sequence, with composite pulse 13C decoupling to simplify and increase the signal-to-noise of spectra, has been used to eliminate the strong water signal while still observing the proton signals of metabolites of interest. Using these techniques the rates of synthesis of acetaldehyde, its diol and acetate from [2-13C] ethanol by alcohol dehydrogenase were measured simultaneously.     

Glutamatergic and GABAergic energy metabolism measured in the rat brain by 13C NMR spectroscopy at 14.1 T

Energy metabolism supports both inhibitory and excitatory neurotransmission processes. This study investigated the specific contribution of astrocytic metabolism to γ‐aminobutyric acid (GABA) synthesis and inhibitory GABAergic neurotransmission that remained to be ilucidated in vivo. Therefore, we measured ¹³C incorporation into brain metabolites by dynamic ¹³C nuclear magnetic resonance spectroscopy at 14.1 T in rats under α‐chloralose anaesthesia during infusion of [1,6‐¹³C]glucose. The enhanced sensitivity at 14.1 T allowed to quantify incorporation of ¹³C into the three aliphatic carbons of GABA non‐invasively. Metabolic fluxes were determined with a mathematical model of brain metabolism comprising glial, glutamatergic and GABAergic compartments. GABA synthesis rate was 0.11 ± 0.01 μmol/g/min. GABA‐glutamine cycle was 0.053 ± 0.003 μmol/g/min and accounted for 22 ± 1% of total neurotransmitter cycling between neurons and glia. Cerebral glucose oxidation was 0.47 ± 0.02 μmol/g/min, of which 35 ± 1% and 7 ± 1% was diverted to the glutamatergic and GABAergic tricarboxylic acid cycles, respectively. The remaining fraction of glucose oxidation was in glia, where 12 ± 1% of the TCA cycle flux was dedicated to oxidation of GABA. 16 ± 2% of glutamine synthesis was provided to GABAergic neurons. We conclude that substantial metabolic activity occurs in GABAergic neurons and that glial metabolism supports both glutamatergic and GABAergic neurons in the living rat brain.

     

Locust flight metabolism studied in vivo by 31P NMR spectroscopy

Flight metabolism of locusts has been extensively studied, but biochemical and physiological methods have led to conflicting results. For this reason the non-invasive and non-destructive method of ³¹P NMR spectroscopy was used to study migratory locusts, Locusta migratoria, at rest and during flight.

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.     

Decreased TCA cycle rate in the rat brain after acute 3-NP treatment measured by in vivo 1H-[13C] NMR spectroscopy

Inhibition of succinate dehydrogenase (SDH) by the mitochondrial toxin 3-nitropropionic acid (3-NP) has gained acceptance as an animal model of Huntington's disease. In this study 13C NMR spectroscopy was used to measure the tricarboxylic acid (TCA) cycle rate in the rat brain after 3-NP treatment. The time course of both glutamate C4 and C3 13C labelling was monitored in vivo during an infusion of [1-13C]glucose. Data were fitted by a mathematical model to yield the TCA cycle rate (Vtca) and the exchange rate between alpha-ketoglutarate and glutamate (Vx). 3-NP treatment induced a 18% decrease in Vtca from 0.71 +/- 0.02 micro mol/g/min in the control group to 0.58 +/- 0.02 micro mol/g/min in the 3-NP group (p < 0.001). Vx increased from 0.88 +/- 0.08 micro mol/g/min in the control group to 1.33 +/- 0.24 micro mol/g/min in the 3-NP group (p < 0.07). Fitting the C4 glutamate time course alone under the assumption that Vx is much higher than Vtca yielded Vtca=0.43 micro mol/g/min in both groups. These results suggest that both Vtca and Vx are altered during 3-NP treatment, and that both glutamate C4 and C3 labelling time courses are necessary to obtain a reliable measurement of Vtca.     

Flux control in the rat gastrocnemius glycogen synthesis pathway by in vivo 13C/31P NMR spectroscopy

To determine the relative contributions of glucose transport/hexokinase, glycogen synthase (GSase), and glycolysis to the control of insulin-stimulated muscle glycogen synthesis, we combined 13C and 31P NMR to quantitate the glycogen synthesis rate and glucose 6-phosphate (G-6-P) levels in rat (Sprague-Dawley) gastrocnemius muscle during hyperinsulinemia at euglycemic (E) and hyperglycemic (H) glucose concentrations under thiopental anesthesia. Flux control was calculated using metabolic control analysis. The combined control coefficient of glucose transport/hexokinase (GT/Hk) for glycogen synthesis was 1.1 +/- 0.03 (direct measure) and 1.14-1.16 (calculated for a range of glycolytic fluxes), whereas the control coefficient for GSase was much lower (0.011-0.448). We also observed that the increase in in vivo [G-6-P] from E to H (0.22 +/- 0.03 to 0.40 +/- 0.03 mM) effects a supralinear increase in the in vitro velocity of GSase, from 14.6 to 26.1 mU. kg(-1). min(-1) (1.8-fold). All measurements suggest that the majority of the flux control of muscle glycogen synthesis is at the GT/Hk step.     

In vivo effects of adenosine A1 receptor agonist and antagonist on neuronal and astrocytic intermediary metabolism studied with ex vivo 13C NMR spectroscopy

Adenosine is a neuromodulator, and it has been suggested that cerebral acetate metabolism induces adenosine formation. In the present study the effects that acetate has on cerebral intermediary metabolism, compared with those of glucose, were studied using the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA) and antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). Fasted rats received an intravenous injection of CCPA, DPCPX, or vehicle. Fifteen minutes later either [1,2-13C]acetate or [1-13C]glucose was given intraperitoneally; after another 30 min the rats were decapitated. Cortical extracts were analyzed with 13C NMR spectroscopy and HPLC analysis. DPCPX affected neuronal and astrocytic metabolism. De novo synthesis of GABA from neuronal and astrocytic precursors was significantly reduced. De novo syntheses of glutamate and aspartate were at control levels, but their degradation was significantly elevated. In glutamine the anaplerotic activity and the amount of label in the position representing the second turn in the tricarboxylic acid cycle were significantly increased, suggesting elevated metabolic activity in astrocytes. CCPA did not influence GABA, aspartate, or glutamine synthesis. In glutamate the contribution from the astrocytic anaplerotic pathway was significantly decreased. In the present study the findings in the [1,2-13C]acetate and [1-13C]glucose control, CCPA, and DPCPX groups were complementary, and no adenosine A1 agonist effects arising from cerebral acetate metabolism were detected.     

Glycerol metabolism in a freeze-tolerant arctic insect: an in vivo13C NMR study

Summary Freeze-tolerance in larvae ofGynaephora groenlandica is enhanced by the accumulation of glycerol in the winter. Since summer larvae remain freeze-tolerant despite the lack of glycerol, we investigated glycerol metabolism as a function of acclimation and body temperature using non-invasive¹³C NMR spectroscopy. Major constituents of hemolymph isolated from cold- and warm-acclimated larvae were identified with the aid of standard NMR spectra and confirmed by TLC and GLC. Spectra obtained on live, warm-acclimated larvae showed the presence of lipids, glycogen, glucose, trehalose and amino acids. Similar spectra of cold-acclimated or previously frozen larvae showed the additional presence of glycerol. In vitro time-lapse¹³C spectra ofd-[1-¹³C]glucose added separately to hemolymph or extracted fat body tissue showed that glycerol is synthesized from glucose in the fat body tissue and distributed to the peripheral tissue via hemolymph. In vivo time-lapse¹³C spectra of cold- and warm-acclimated larvae were obtained after injection withd-[1-¹³C]glucose to monitor the production of labeled metabolic intermediates and end-products. [¹³C]Glycerol was produced between –30°C and 30°C but accumulated only below 5°C. Above 5°C glycerol was degraded and the¹³C label incorporated mainly into glycogen. The mechanism underlying temperature control of glycerol biosynthesis and degradation may provide a clue to the role of glycerol in enhancing freeze-tolerance in these insects.