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.     

Triglyceride metabolism in 3T3-L1 cells. An in vivo 13C NMR study.

13C nuclear magnetic resonance spectroscopy has been used to study triglyceride metabolism in 3T3-L1 cells incubated with [1-13/14C] acetate, myristate, palmitate, stearate, or oleate. Labeled cells embedded in agarose filaments were perfused in a specially fitted NMR tube within the spectrometer magnet. Incubation of 3T3-L1 cells with a specific fatty acid enriched the cellular triglycerides with that fatty acid; the NMR signal observed in the carbonyl region of the cell spectrum was due in large part to that fatty acid. NMR data demonstrated that cellular enzymes preferentially esterified saturated fatty acids at the glyceride sn-1,3 position and unsaturated fatty acids at the sn-2 position. cellular triglyceride hydrolysis by hormone-sensitive lipase was monitored by measuring the decrease in the integrated intensities of resonances arising from fatty acyl carbonyls esterified at glycerol carbons sn-1,3 and sn-2. Under basal conditions, the time courses were first-order, and the average rates were 0.14% of signal/min at both carbonyl positions. Under isoproterenol stimulated conditions, these rates were still first-order and increased 6.4-fold at the sn-1,3 position and 2.4-fold at the sn-2 position. The observation that the hydrolysis time courses were first-order suggested that only a small amount of cellular triglyceride was available to hormone-sensitive lipase, supporting the view that lipolytic enzymes operate at lipid surfaces where only small amounts of neutral lipid may be soluble. Attempts to correlate the measured rates with the rates of hydrolysis at the sn-1,3 and sn-2 positions were hindered by the fact that the chemical shifts of the carbonyl carbons of the diglyceride hydrolysis product did not overlie those of the triglyceride. Analysis of hydrolysis kinetics revealed that hormone-sensitive lipase exhibited little preference for a particular esterified fatty acid under basal conditions; however, under stimulated conditions, the enzyme exhibited a preference for certain triglyceride species.     

Cerebral glucose metabolism and the glutamine cycle as detected by in vivo and in vitro 13C NMR spectroscopy

We review briefly 13C NMR studies of cerebral glucose metabolism with an emphasis on the roles of glial energetics and the glutamine cycle. Mathematical modeling analysis of in vivo 13C turnover experiments from the C4 carbons of glutamate and glutamine are consistent with: (i) the glutamine cycle being the major cerebral metabolic route supporting glutamatergic neurotransmission, (ii) glial glutamine synthesis being stoichiometrically coupled to glycolytic ATP production, (iii) glutamine serving as the main precursor of neurotransmitter glutamate and (iv) glutamatergic neurotransmission being supported by lactate oxidation in the neurons in a process accounting for 60-80% of the energy derived from glucose catabolism. However, more recent experimental approaches using inhibitors of the glial tricarboxylic acid (TCA) cycle (trifluoroacetic acid, TFA) or of glutamine synthase (methionine sulfoximine, MSO) reveal that a considerable portion of the energy required to support glutamine synthesis is derived from the oxidative metabolism of glucose in the astroglia and that a significant amount of the neurotransmitter glutamate is produced from neuronal glucose or lactate rather than from glial glutamine. Moreover, a redox switch has been proposed that allows the neurons to use either glucose or lactate as substrates for oxidation, depending on the relative availability of these fuels under resting or activation conditions, respectively. Together, these results suggest that the coupling mechanisms between neuronal and glial metabolism are more complex than initially envisioned.     

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.

     

Carbon 13 NMR study of glycogen metabolism in the baboon liver in vivo.

In vivo glycogen metabolism was investigated at 2 Tesla by 13C NMR in the baboon liver. Two concentric surface coils were used for 13C observation and proton decoupling, respectively. Spectra were acquired in 2 to 10 minutes with a 60 ms repetition time. After 3 hours of glucose infusion in the 48 hr fasted animal, 3 g of 99%-enriched [1-13C]glucose were injected. The distribution of the label on C-1 and also C-2, C-5 and C-6 of glycogen indicated 65% and 35% contributions of the direct and indirect pathways to glycogen synthesis from glucose, respectively. The results show that hepatic metabolic pathways and rates can be followed in vivo in large animals by 13C NMR at 2 Tesla.     

Group I and II metabotropic glutamate receptors alter brain cortical metabolic and glutamate/glutamine cycle activity: a 13C NMR spectroscopy and metabolomic study

Metabotropic glutamate receptors (mGluR) modulate neuronal function. Here, we tested the effect on metabolism of a range of Group I and II mGluR ligands in Guinea pig brain cortical tissue slices, applying 13C NMR spectroscopy and metabolomic analysis using multivariate statistics. The effects of Group I agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (RS)-2-chloro-5-hydroxyphenylglycine (CHPG) depended upon concentration and were mostly stimulatory, increasing both net metabolic flux through the Krebs cycle and glutamate/glutamine cycle activity. Only the higher (50 microm) concentrations of CHPG had the opposite effect. The Group I antagonist (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), consistent with its neuroprotective role, caused significant decreases in metabolism. With principal components analysis of the metabolic profiles generated by these ligands, the effects could be separated by two principal components. Agonists at Group II mGluR [(2S,2'R,3'R)-2-(2',3'-dicarboxycyclopropyl)glycine (DCG IV) and 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (APDC)] generally stimulated metabolism, including glutamate/glutamine cycling, although this varied with concentration. The antagonist (2S)-alpha-ethylglutamic acid (EGLU) stimulated astrocyte metabolism with minimal impact on glutamate/glutamine cycling. (RS)-1-Aminophosphoindan-1-carboxylic acid (APICA) decreased metabolism at 5 microm but had a stimulatory effect at 50 microm. All ligand effects were separated from control and from each other using two principal components. The ramifications of these findings are discussed.     

Metabolic characterization of neurological diseases by proton localized NMR spectroscopy of the human brain.

Proton localized Magnetic Resonance Spectroscopy (MRS) of the brain allows the non invasive detection of intracellular cerebral metabolites. Localized MRS has been performed using short stimulated-echo times in various neurological diseases including stroke, multiple sclerosis, and AIDS-related leukoencephalopathies. Principal component analysis (PCA) was used to determine the critical parameters defining the metabolic profile of normal and diseased brain. PCA clearly differentiates the demyelinating processes from ischaemic lesions and leukoencephalopathies. Localized MRS of the brain appears growingly as a tool of choice to discriminate, quantitate and assess cerebral metabolic damage in patients with neurological disorders.     

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.     

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.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Application of 13C and 31P NMR to the study of hepatic metabolism

Alternate scan 13C and 31P NMR has been used to follow the metabolism of 13C-labeled substrates, in the presence and absence of insulin, in isolated perfused liver from fasted rats. Because both 31P and 13C NMR spectra are recorded almost simultaneously with this method, both phosphate metabolites and 13C-labeled metabolites are measured, noninvasively and repetitively, to give an immediate, broad survey of the hepatic response to a variety of stimuli. During the metabolism of [2-13C]pyruvate, [1,2-13C]ethanol, and NH4+, 13C-labeled glycogen increases synchronously with, and at the same rate as, the synthesis of 13C-labeled glucose; thus, glycogenesis was essentially a gluconeogenic process under our conditions and was unaltered by the presence of insulin. From the position of the 13C-labeled citrate peak observed in liver, the measurement of KD for the citrate-magnesium complex under our conditions, and the expression relating these quantities to the concentration of free Mg2+, the intracellular level of free Mg2+ is estimated to be 0.46 +/- 0.05 mM. Later administration of glucagon led to a rapid decrease in glycogen and citrate and a 44% increase in glycero-3-phosphocholine (GPC); increase in GPC is consistent with stimulation of liver phospholipase activity by glucagon. Simultaneous administration of two different 13C-labeled substrates, or one doubly labeled substrate, introduced multiplet structure arising from spin-spin interaction between labeled adjacent carbons into the peaks of several key metabolites. The 13C NMR intensity distributions within the several multiplets are used, within the context of a first-order model for fluxes into the Krebs cycle, to estimate relative fluxes under the conditions of the experiment.     

Methylamine metabolism in Hansenula polymorpha: an in vivo 13C and 31P nuclear magnetic resonance study.

Methylamine uptake, oxidation, and assimilation were studied in Hansenula polymorpha, a methylotrophic yeast. The constitutive ammonia transport system was shown to be effective at accumulating methylamine within cells cultured with methylamine or ammonia as a nitrogen source. [13C]methylamine oxidation rates were measured in vivo in methylamine-adapted cells by 13C nuclear magnetic resonance and were found to be lower than its uptake rate into the cells. The 13C label of methylamine was found exclusively in trehalose and glycerol, and [13C]formaldehyde was also extensively assimilated, indicating the presence of an assimilation pathway for the methylamine carbon. In vivo 31P nuclear magnetic resonance analysis showed major differences in the endogenous polyphosphate levels and mean chain length during adaptation of the cells from ammonia to methylamine, indicating that methylamine accumulated in the vacuole in the same manner as basic amino acids and purines. [13C]glucose metabolism was drastically altered during adaptation of the cells from ammonia to methylamine as a nitrogen source. The total rate of glucose utilization and the rate of ethanol production fell. Direct trehalose synthesis from glucose increased, indicating a switch from carbon utilization for growth to that for storage. The rate of methylamine oxidation was sufficient to support a much higher flow of carbon into central biosynthetic pathways. These results suggest that this reduction in biosynthetic carbon flow, rather than nitrogen availability, was the main factor responsible for reducing the growth rate of the yeast when ammonia was replaced by methylamine as the nitrogen source.     

Magnetic resonance spectroscopy of cancer metabolism and response to therapy

Magnetic resonance spectroscopy allows noninvasive in vivo measurements of biochemical information from living systems, ranging from cultured cells through experimental animals to humans. Studies of biopsies or extracts offer deeper insights by detecting more metabolites and resolving metabolites that cannot be distinguished in vivo. The pharmacokinetics of certain drugs, especially fluorinated drugs, can be directly measured in vivo. This review briefly describes these methods and their applications to cancer metabolism, including glycolysis, hypoxia, bioenergetics, tumor pH, and tumor responses to radiotherapy and chemotherapy.     

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.     

In vivo quantification of neuro‐glial metabolism and glial glutamate concentration using 1H‐[13C] MRS at 14.1T

Astrocytes have recently become a major center of interest in neurochemistry with the discoveries on their major role in brain energy metabolism. An interesting way to probe this glial contribution is given by in vivo ¹³C NMR spectroscopy coupled with the infusion labeled glial‐specific substrate, such as acetate. In this study, we infused alpha‐chloralose anesthetized rats with [2‐¹³C]acetate and followed the dynamics of the fractional enrichment (FE) in the positions C4 and C3 of glutamate and glutamine with high sensitivity, using ¹H‐[¹³C] magnetic resonance spectroscopy (MRS) at 14.1T. Applying a two‐compartment mathematical model to the measured time courses yielded a glial tricarboxylic acid (TCA) cycle rate (V_g) of 0.27 ± 0.02 μmol/g/min and a glutamatergic neurotransmission rate (V_NT) of 0.15 ± 0.01 μmol/g/min. Glial oxidative ATP metabolism thus accounts for 38% of total oxidative metabolism measured by NMR. Pyruvate carboxylase (V_PC) was 0.09 ± 0.01 μmol/g/min, corresponding to 37% of the glial glutamine synthesis rate. The glial and neuronal transmitochondrial fluxes (V_x^g and V_xⁿ) were of the same order of magnitude as the respective TCA cycle fluxes. In addition, we estimated a glial glutamate pool size of 0.6 ± 0.1 μmol/g. The effect of spectral data quality on the fluxes estimates was analyzed by Monte Carlo simulations.

     

Pyruvate metabolism in Halobacterium salinarium studied by intracellular 13C nuclear magnetic resonance spectroscopy.

13C nuclear magnetic resonance spectroscopy was used to study the metabolism of [2-13C]pyruvate in intact cells of Halobacterium salinarium. The spectra of these cells show that pyruvate is reduced to lactic acid and transaminated to alanine. The intensity of C-2 lactate is higher under anaerobic conditions than under aerobic conditions. When cells are grown in the absence of glucose, the level of C-2 lactate intensity is lower. In extracts of these cells, the level of NADH-dependent lactate dehydrogenase activity is lower than that of cells grown in the presence of glucose. A C-5 glutamate resonance suggests the entry of pyruvate into the tricarboxylic acid cycle through acetyl-coenzyme A. In addition, the label is also observed at C-3 and C-4 of glutamate, signifying a pyruvate carboxylase-type reaction and scrambling of label at the fumarate-succinate stage plus malic enzyme operation, respectively. Citrate synthase and malic enzyme activity appear to be controlled by the growth conditions of H. salinarium.     

Basic principles of metabolic modeling of NMR (13)C isotopic turnover to determine rates of brain metabolism in vivo

Metabolic modeling is a necessary part of the analysis of isotopic labeling data that is being obtained in the brain and other organs. Here are explained the basic principles of metabolic modeling of isotopic labeling studies, particularly with regard to (13)C isotopic measurements performed in vivo. The basic elements needed to simulate isotopic flows are described, and how to combine them to perform modeling analyses is explained. Procedures to introduce and evaluate model constraints and simplifications are discussed. The basic principle of isotopomer analysis is explained, as are mechanics of least-squares fitting of simulations to data. Closely related to the fitting is the effect of data scatter, which is discussed in the context of the non-normal distributions of uncertainty that are often seen with (13)C labeling measurements in vivo. This article is meant to provide a general background for investigators to begin to apply metabolic modeling analysis to (13)C isotopic labeling studies performed in vivo.     

In vitro study of astrocytic tumour metabolism by proton magnetic resonance spectroscopy

In vivo magnetic resonance spectroscopy (MRS) studies of glial brain tumours reported that higher grade of astrocytoma is associated with increased level of choline-containing compounds (Cho) and decreased levels of N-acetylaspartate (NAA) and creatine and phosphocreatine (Cr). In this work, we studied the metabolism of glioma tumours by in vitro proton magnetic resonance spectroscopy (1H-MRS). 1H-MR spectra were recorded in vitro from perchloric acid extracts of astrocytoma (WHO II) and glioblastoma multiforme (WHO IV) samples. We observed differences between astrocytoma and glioblastoma multiforme in the levels of Cho, alanine, lactate, NAA, and glutamate/glutamine. In astrocytoma samples, we found higher MR signal of NAA and lower signal of Cho and alanine. MR spectra of glioblastoma samples reported significantly higher levels of lactate and glutamate/glutamine. In contrast, levels of Cr were the same in both tumour types. We also determined NAA/Cr and Cho/Cr ratios in the tumour samples. The NAA/Cr ratio was higher in astrocytomas than in glioblastomas multiforme. Conversely, the Cho/Cr ratio was higher in glioblastoma multiforme. The results indicate that MRS is a promising method for distinguishing pathologies in human brain and for pre-surgical grading of brain tumours.     

A 13C NMR study on fluxes into the TCA cycle of neuronal and glial tumor cell lines and primary cells.

Two tumor cell lines (C6 glioma and N1E-115 neuroblastoma), primary glia and primary neurons (from rat) were incubated with 2-13C-pyruvate and 3-13C-pyruvate in culture dishes. 13C NMR spectra of the cell extracts were used to determine the ratio of pyruvate carboxylase to pyruvate dehydrogenase activity. Pyruvate carboxylase activity was found higher in primary glia cells than in neurons. Glial cells synthesized more amino acids, ie, their TCA cycle was used to a larger extent for biosynthesis than is the case of neurons, where it is preferentially used for the energy metabolism.