Glial uptake of neurotransmitter glutamate from the extracellular fluid studied in vivo by microdialysis and (13)C NMR

Glial uptake of neurotransmitter glutamate (GLU) from the extracellular fluid was studied in vivo in rat brain by (13)C NMR and microdialysis combined with gas-chromatography/mass-spectrometry. Brain GLU C5 was (13)C enriched by intravenous [2,5-(13)C]glucose infusion, followed by [(12)C]glucose infusion to chase (13)C from the small glial GLU pool. This leaves [5-(13)C]GLU mainly in the large neuronal metabolic pool and the vesicular neurotransmitter pool. During the chase, the (13)C enrichment of whole-brain GLU C5 was significantly lower than that of extracellular GLU (GLU(ECF)) derived from exocytosis of vesicular GLU. Glial uptake of neurotransmitter [5-(13)C]GLU(ECF) was monitored in vivo through the formation of [5-(13)C,(15)N]GLN during (15)NH(4)Ac infusion. From the rate of [5-(13)C,(15)N]GLN synthesis (1.7 +/- 0.03 micromol/g/h), the mean (13)C enrichment of extracellular GLU (0.304 +/- 0.011) and the (15)N enrichment of precursor NH(3) (0.87 +/- 0.014), the rate of synthesis of GLN (V'(GLN)), derived from neurotransmitter GLU(ECF), was determined to be 6.4 +/- 0.44 micromol/g/h. Comparison with V(GLN) measured previously by an independent method showed that the neurotransmitter provides 80-90% of the substrate GLU pool for GLN synthesis. Hence, under our experimental conditions, the rate of 6.4 +/- 0.44 micromol/g/h also represents a reasonable estimate for the rate of glial uptake of GLU(ECF), a process that is crucial for protecting the brain from GLU excitotoxicity.     

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.     

In vivo (13)C NMR measurement of neurotransmitter glutamate cycling, anaplerosis and TCA cycle flux in rat brain during

The aims of this study were twofold: (i) to determine quantitatively the contribution of glutamate/glutamine cycling to total astrocyte/neuron substrate trafficking for the replenishment of neurotransmitter glutamate; and (ii) to determine the relative contributions of anaplerotic flux and glutamate/glutamine cycling to total glutamine synthesis. In this work in vivo and in vitro (13)C NMR spectroscopy were used, with a [2-(13)C]glucose or [5-(13)C]glucose infusion, to determine the rates of glutamate/glutamine cycling, de novo glutamine synthesis via anaplerosis, and the neuronal and astrocytic tricarboxylic acid cycles in the rat cerebral cortex. The rate of glutamate/glutamine cycling measured in this study is compared with that determined from re-analysis of (13)C NMR data acquired during a [1-(13)C]glucose infusion. The excellent agreement between these rates supports the hypothesis that glutamate/glutamine cycling is a major metabolic flux ( approximately 0.20 micromol/min/g) in the cerebral cortex of anesthetized rats and the predominant pathway of astrocyte/neuron trafficking of neurotransmitter glutamate precursors. Under normoammonemic conditions anaplerosis was found to comprise 19-26% of the total glutamine synthesis, whilst this fraction increased significantly during hyperammonemia ( approximately 32%). These findings indicate that anaplerotic glutamine synthesis is coupled to nitrogen removal from the brain (ammonia detoxification) under hyperammonemic conditions.     

A radiometric assay for aspartoacylase activity in cultured oligodendrocytes

Recent studies have shown that aspartoacylase (ASPA), the defective enzyme in Canavan disease, is detectable in the brain only in the oligodendrocytes. Studying the regulation of ASPA is central to the understanding the pathogenesis of Canavan disease and to the development of therapeutic strategies. Toward this goal, we have developed a sensitive method for the assay of ASPA in cultured oligodendrocytes. The method involves: (a) chemical synthesis of [14C]N-acetylaspartate (NAA) from L-[14C]Asp; (b) use of [14C]NAA as substrate in the assay; and (c) separation and quantitation of the product L-[14C]Asp using a TLC system. This method can detect as low as 10pmol of product and has been optimized for cultured oligodendrocytes. Thus, this method promises to be a valuable tool for understanding the biochemical mechanisms involved in the cell-specific expression and regulation of ASPA in oligodendrocytes.     

Recovery of (13)CO2 during rest and exercise after [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO3 infusions

For estimating the oxidation rates (Rox) of glucose and other substrates by use of (13)C-labeled tracers, we obtained correction factors to account for label dilution in endogenous bicarbonate pools and TCA cycle exchange reactions. Fractional recoveries of (13)C label in respiratory gases were determined during 225 min of rest and 90 min of leg cycle ergometry at 45 and 65% peak oxygen uptake (VO(2 peak)) after continuous infusions of [1-(13)C]acetate, [2-(13)C]acetate, or NaH(13)CO(3). In parallel trials, [6,6-(2)H]glucose and [1-(13)C]glucose were given. Experiments were conducted after an overnight fast with exercise commencing 12 h after the last meal. During the transition from rest to exercise, CO(2) production increased (P < 0.05) in an intensity-dependent manner. Significant differences were observed in the fractional recoveries of (13)C label as (13)CO(2) at rest (NaH(13)CO(3), 77.5 +/- 2.8%; [1-(13)C]acetate, 49.8 +/- 2.4%; [2-(13)C]acetate, 26.1 +/- 1.4%). During exercise, fractional recoveries of (13)C label from [1-(13)C]acetate, [2-(13)C]acetate, and NaH(13)CO(3) were increased compared with rest. Magnitudes of label recoveries during both exercise intensities were tracer specific (NaH(13)CO(3), 93%; [1-(13)C]acetate, 80%; [2-(13)C]acetate, 65%). Use of an acetate-derived correction factor for estimating glucose oxidation resulted in Rox values in excess (P < 0.05) of glucose rate of disappearance during hard exercise. We conclude that, after an overnight fast: 1) recovery of (13)C label as (13)CO(2) from [(13)C]acetate is decreased compared with bicarbonate; 2) the position of (13)C acetate label affects carbon dilution estimations; 3) recovery of (13)C label increases in the transition from rest to exercise in an isotope-dependent manner; and 4) application of an acetate correction factor in glucose oxidation measurements results in oxidation rates in excess of glucose disappearance during exercise at 65% of VO(2 peak). Therefore, bicarbonate, not acetate, correction factors are advocated for estimating glucose oxidation from carbon tracers in exercising men.     

Blood glycerol is an important precursor for intramuscular triacylglycerol synthesis.

The utilization of blood glycerol and glucose as precursors for intramuscular triglyceride synthesis was examined in rats using an intravenous infusion of [2-(14)C]glycerol and [6-(3)H]glucose or [6-(14)C]glucose. In 24-h fasted rats, more glycerol than glucose was incorporated into intramuscular triglyceride glycerol in soleus (69 +/- 23 versus 4 +/- 1 nmol/micromol triglyceride/h, respectively, p = 0.02 glycerol versus glucose) and in gastrocnemius (25 +/- 5 versus 9 +/- 2 nmol/micromol triglyceride/h, respectively, p = 0.02). Blood glucose was utilized more than blood glycerol for triglyceride glycerol synthesis in quadriceps. In fed rats, the blood glycerol incorporation rates (4 +/- 2, 8 +/- 3, and 9 +/- 3 nmol/micromol triglyceride/h) were similar (p > 0.3) to those of glucose (5 +/- 2, 8 +/- 2, and 5 +/- 2 nmol/micromol triglyceride/h for quadriceps, gastrocnemius, and soleus muscle, respectively). Glucose incorporation into intramuscular triglycerides was less with [6-(3)H]glucose than with [6-(14)C]glucose, suggesting an indirect pathway for glucose carbon entry into muscle triglyceride. The isotopic equilibrium between plasma and intramuscular free glycerol ([U-(13)C]glycerol) was complete in quadriceps and gastrocnemius, but not soleus, within 2 h after beginning the tracer infusion. We conclude that blood glycerol is a direct and important precursor for muscle triglyceride synthesis in rats, confirming the presence of functionally important amounts of glycerol kinase in skeletal muscle.     

Hepatic glycogen synthesis from duodenal glucose and alanine. An in situ 13C NMR study

An in situ and in vivo surface coil 13C NMR study was performed to study hepatic glycogen synthesis from [3-13C]alanine and [1-13C]glucose administered by intraduodenal infusion in 18-h fasted male Sprague-Dawley rats. Combined, equimolar amounts of alanine and glucose were given. Hepatic appearance and disappearance of substrate and concurrent glycogen synthesis was followed over 150 min, with 5-min time resolution. Active glycogen synthesis from glucose via the direct (glucose----glycogen) and indirect (glucose----lactate----glycogen) pathways and from alanine via gluconeogenesis was observed. The indirect pathway of glycogen synthesis from [1-13C]glucose accounted for 30% (+/- 6 S.E.) of total glycogen formed from labeled glucose. This estimate does not take into account dilution of label in the hepatic oxaloacetate pool and is, therefore, somewhat uncertain. Hepatic levels of [3-13C]alanine achieved were significantly lower than levels of [1-13C]glucose in the liver, and the period of active glycogen synthesis from [3-13C]alanine was longer than from glucose. However, the overall pseudo-first-order rate constant during the period of active glycogen synthesis from [3-13C]alanine (0.075 min-1 +/- 0.026 S.E.) was almost 3 times that from [1-13C]glucose via the direct pathway (0.025 min-1 +/- 0.005 S.E.). The most likely reason for the small rate constant governing direct glycogen formation from duodenally administered glucose compared to that from duodenally administered alanine is a low level of glucose phosphorylating capacity in the liver.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

13C/31P NMR assessment of mitochondrial energy coupling in skeletal muscle of awake fed and fasted rats. Relationship with uncoupling protein 3 expression

To examine the relationship between mitochondrial energy coupling in skeletal muscle and change in uncoupling protein 3 (UCP3) expression during the transition from the fed to fasted state, we used a novel noninvasive (31)P/(13)C NMR spectroscopic approach to measure the degree of mitochondrial energy coupling in the hind limb muscles of awake rats before and after a 48-h fast. Compared with fed levels, UCP3 mRNA and protein levels in the gastrocnemius increased 1.7- (p < 0.01) and 2.9-fold (p < 0.001), respectively, following a 48-h fast. Tricarboxylic acid cycle flux measured using (13)C NMR as an index of mitochondrial substrate oxidation was 212 +/- 23 and 173 +/- 25 nmol/g/min (p not significant) in the fed and 48-h fasted groups, respectively. Unidirectional ATP synthesis flux measured using (31)P NMR was 79 +/- 15 and 57 +/- 9 nmol/g/s (p not significant) in the fed and 48-h fasted groups, respectively. Mitochondrial energy coupling as expressed by the ratio of ATP synthesis to tricarboxylic acid cycle flux was not different between the fed and fasted states. To test the hypothesis that UCP3 may be involved in the translocation of long chain free fatty acids (FFA) into the mitochondrial matrix under conditions of elevated FFA availability, [U-(13)C]palmitate/albumin was administered in a separate group of rats with (+) or without (-) etomoxir (an inhibitor of carnitine palmitoyltransferase I). The ratio of glutamate enrichment ((+) etomoxir/(-) etomoxir) in the hind limb muscles was the same between groups, indicating that UCP3 does not appear to function as a translocator for long chain FFA in skeletal muscle following a 48-h fast. In summary, these data demonstrate that despite a 2-3-fold increase in UCP3 mRNA and protein expression in skeletal muscle during the transition from the fed to fasted state, mitochondrial energy coupling does not change. Furthermore, UCP3 does not appear to have a major role in FFA translocation into the mitochondria. The physiological role of UCP3 following a 48-h fast in skeletal muscle remains to be elucidated.     

Influx and incorporation into protein of L-phenylalanine in the perfused rat pancreas: effects of amino acid deprivation and carbachol.

The rate of protein synthesis in the isolated perfused rat pancreas was measured from the rate of incorporation of L-[3H]phenylalanine into total protein, and was compared with the transport of this amino acid into the epithelium. Unidirectional (15 s) and net (15-30 min) uptake of L-[3H]phenylalanine was measured relative to D-[14C]mannitol (extracellular marker) using a cell loading technique. The fractional rate of protein synthesis in the pancreas was also measured in vivo using a flooding dose technique and found to be 118 +/- 10% day-1 (corresponding to an absolute rate of incorporation of L-Phe into protein of 36.1 +/- 3 nmol min-1 g-1) in overnight fasted rats. Compared with the in vivo rate, the perfused pancreas exhibited a markedly lower rate of protein synthesis which increased significantly when amino acids were added to the perfusate (15.6 +/- 1.9 vs. 22.5 +/- 0.9% day-1 or 4.7 +/- 0.6 vs. 6.9 +/- 0.3 nmol L-Phe min-1 g-1). Carbachol (3 x 10(-7) M) stimulated protein synthesis provided amino acids were also supplied in the perfusate. Protein synthesis rates measured under all conditions in vivo and in vitro were at least an order of magnitude lower than the unidirectional influx (121 +/- 14 nmol min-1 g-1) of L-phenylalanine into the pancreatic epithelium. These results demonstrate that amino acid transport across the basolateral membrane of the epithelium is not rate-limiting for pancreatic protein synthesis.     

Synthesis and biodistribution of [(11)C]R116301, a promising PET ligand for central NK(1) receptors

N1-(2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(11)C]R116301) was prepared and evaluated as a potential positron emission tomography (PET) ligand for investigation of central neurokinin(1) (NK(1)) receptors. 1-Bromo-3,5-di(trifluoromethyl)benzene was converted in three steps into 3,5-di(trifluoromethyl)[carbonyl-(11)C]benzoyl chloride, which was reacted with N1-(2,6-dimethylphenyl)-2-{4-[(2R,4S)-2-benzylhexahydro-4-pyridinyl]piperazino}acetamide providing [(11)C]R116301 in 45-57% decay-corrected radiochemical yield. The total synthesis time, from end of bombardment (EOB) to the formulated product, was 35 min. Specific activity (SA) was 82-172 GBq/micromol (n=10) at the end of synthesis. N1-([4-(3)H]-2,6-Dimethylphenyl)-2-(4-{(2R,4S)-2-benzyl-1-[3,5-di(trifluoromethyl)benzoyl]hexahydro-4-pyridinyl}piperazino)acetamide ([(3)H]R116301) was also synthesized (SA: 467 GBq/mmol). The B(max) for [(3)H]R116301 measured in vitro on Chinese hamster ovary cell membranes stably transfected with the human NK(1) receptor was 19.10+/-1.02 pmol/mg protein with an apparent dissociation constant of 0.08+/-0.01 nM. Ex vivo, in vivo and in vitro autoradiography studies with [(3)H]R116301 in gerbils demonstrated a preferential accumulation of the radioactivity in the striatum, olfactory tubercule, olfactory bulb and locus coeruleus. In vivo, the biodistribution of [(11)C]R116301 in gerbils revealed that the highest initial uptake is in the lung, followed by the liver and kidney. In the brain, maximum accumulation was found in the olfactory tubercules (1.10+/-0.08 injected dose (ID)/g 20 min post injection (p.i.)) and the nucleus accumbens (1.00+/-0.12ID/g 10 min p.i.). Tissue/cerebellum concentration ratios for striatum and nucleus accumbens increased with time due to rapid uptake followed by a slow wash out (1.29 and 1.64, respectively, 30 min p.i.). A tissue to cerebellum ratio of 1.33 and 1.62 was also observed for olfactory bulb and olfactory tubercules, respectively (20 min p.i.). In summary, [(11)C]R116301 appears to be a promising radioligand suitable for the visualization of NK(1) receptors in vivo using PET.     

Elucidation of the role of fructose 2,6-bisphosphate in the regulation of glucose fluxes in mice using in vivo (13)C NMR measurements of hepatic carbohydrate metabolism.

Fructose 2,6-bisphosphate (Fru-2,6-P2) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru-2,6-P2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru-2,6-P2 levels were elevated in ADM mice with adenovirus-mediated overexpression of a double mutant bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (n = 6), in comparison to normal control mice (control, n = 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13C NMR including 3D localization in conjunction with [1-13C]glucose infusion. In addition to glycogen C1, the C6 and C2-C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver. The rate of label incorporation into glycogen C1 was not different between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 +/- 0.5 micro mol.g-1.h-1, which was higher than that of the control group of 3.7 +/- 0.5 micro mol.g-1.h-1 (P < 0.02). The rates of net glycogen synthesis, determined by the glycogen C2-C5 signal changes, were twofold higher in the ADM group (P = 0.04). The results provide direct in vivo evidence that the effects of elevated Fru-2,6-P2 levels in the liver include increased glycogen storage through indirect synthesis of glycogen. These observations provide a key to understanding the mechanisms by which elevated hepatic Fru-2,6-P2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.     

In vivo effects of uncoupling protein-3 gene disruption on mitochondrial energy metabolism

To clarify the role of uncoupling protein-3 (UCP3) in skeletal muscle, we used NMR and isotopic labeling experiments to evaluate the effect of UCP3 knockout (UCP3KO) in mice on the regulation of energy metabolism in vivo. Whole body energy expenditure was determined from the turnover of doubly labeled body water. Coupling of mitochondrial oxidative phosphorylation in skeletal muscle was evaluated from measurements of rates of ATP synthesis (using (31)P NMR magnetization transfer experiments) and tricarboxylic acid (TCA) cycle flux (calculated from the time course of (13)C enrichment in C-4 and C-2 of glutamate during an infusion of [2-(13)C]acetate). At the whole body level, we observed no change in energy expenditure. However, at the cellular level, skeletal muscle UCP3KO increased the rate of ATP synthesis from P(i) more than 4-fold under fasting conditions (wild type, 2.2 +/- 0.6 versus knockout, 9.1 +/- 1.4 micromol/g of muscle/min, p < 0.001) with no change in TCA cycle flux rate (wild type, 0.74 +/- 0.04 versus knockout, 0.71 +/- 0.03 micromol/g of muscle/min). The increased efficiency of ATP production may account for the significant (p < 0.05) increase in the ratio of ATP to ADP in the muscle of UCP3KO mice (5.9 +/- 0.3) compared with controls (4.5 +/- 0.4). The data presented here provide the first evidence of uncoupling activity by UCP3 in skeletal muscle in vivo.     

NMR measurements of in vivo myocardial glycogen metabolism

Using 13C and 1H NMR we measured the rate of glycogen synthesis (0.23 +/- 0.10 mumol/min gram wet weight tissue (gww) in rat heart in vivo during an intravenous infusion of D-[1-13C]glucose and insulin. Glycogen was observed within 10 min of starting and increased linearly throughout a 50-min infusion. This compared closely with the average activity of glycogen synthase I (0.22 +/- 0.03 mumol/min gww) measured at physiologic concentrations of UDP-glucose (92 microM) and glucose-6-phosphate (110 microM). When unlabeled glycogen replaced D-[1-13C]glucose in the infusate after 50 min the D-[1-13C]glycogen signal remained stable for another 60 min, indicating that no turnover of the newly synthesized glycogen had occurred. Despite this phosphorylase a activity in heart extracts from rats given a 1 h glucose and insulin infusion (3.8 +/- 2.4 mumol/min gww) greatly exceeded the total synthase activity and if active in vivo should promote glycogenolysis. We conclude that during glucose and insulin infusion in the rat: (a) the absolute rate of myocardial glycogen synthesis can be measured in vivo by NMR; (b) glycogen synthase I can account for the observed rates of heart glycogen synthesis; (c) there is no futile cycling of glucose in and out of heart glycogen; and (d) the activity of phosphorylase a measured in tissue extracts is not reflected in vivo. These studies raise the question whether significant regulation of phosphorylase a activity in vivo is mediated by factors in addition to its phosphorylation state.     

Regulation of myocardial [(13)C]glucose metabolism in conscious rats

Administration of supplemental glucose and/or insulin is postulated to improve the outcome from myocardial ischemia by increasing the heart's relative utilization of glucose as an energy substrate. To examine the degree to which circulating glucose and insulin levels actually influence myocardial substrate preference in vivo, we infused conscious, chronically catheterized rats with D-[1-(13)C]glucose and compared steady-state (13)C enrichment of plasma glucose with that of myocardial glycolytic ([3-(13)C]alanine) and oxidative ([4-(13)C]glutamate) intermediary metabolites. In fasting rats, [3-(13)C]alanine-to-[1-(13)C]glucose and [4-(13)C]glutamate-to-[3-(13)C]alanine ratios averaged 0.16 +/- 0.12 and 0.14 +/- 0.03, respectively, indicating that circulating glucose contributed 32% of myocardial glycolytic flux, whereas subsequent flux through pyruvate dehydrogenase contributed 14% of total tricarboxylic acid (TCA) cycle activity. Raising plasma glucose to 11 mmol/l, or insulin to 500 pmol/l, increased these contributions equivalently. At supraphysiological (>6,500 pmol/l) insulin levels, the plasma glucose contribution to glycolysis increased further, and addition of hyperglycemia made it the sole glycolytic substrate, yet [4-(13)C]glutamate-to-[3-(13)C]alanine ratios remained /=40% of myocardial TCA cycle flux.     

Dynamic or inert metabolism? Turnover of N‐acetyl aspartate and glutathione from d‐[1‐13C]glucose in the rat brain in vivo

The rate of (13)C-label incorporation into both aspartyl (NAA C3) and acetyl (NAA C6) groups of N-acetyl aspartate (NAA) was simultaneously measured in the rat brain in vivo for up to 19 h of [1-(13)C]glucose infusion (n = 8). Label incorporation was detected in NAA C6 approximately 1.5 h earlier than in NAA C3 because of the delayed labeling of the precursor of NAA C3, aspartate, compared to that of NAA C6, glucose. The time courses of NAA were fitted using a mathematical model assuming synthesis of NAA in one kinetic compartment with the respective precursor pools of aspartate and acetyl coenzyme A (acetyl-CoA). The turnover rates of NAA C6 and C3 were 0.7 +/- 0.1 and 0.6 +/- 0.1 micromol/(g h) with the time constants 14 +/- 2 and 13 +/- 2 h, respectively, with an estimated pool size of 8 micromol/g. The results suggest that complete label turnover of NAA from glucose occurs in approximately 70 h. Several hours after starting the glucose infusion, label incorporation into glutathione (GSH) was also detected. The turnover rate of GSH was 0.06 +/- 0.02 micromol/(g h) with a time constant of 13 +/- 2 h. The estimated pool size of GSH was 0.8 micromol/g, comparable to the cortical glutathione concentration. We conclude that NAA and GSH are completely turned over and that the metabolism is extremely slow (< 0.05% of the glucose metabolic rate).     

Short-term metabolic fate of 13N-labeled glutamate, alanine, and glutamine(amide) in rat liver

Tracer quantities (in 0.2 ml) of 13N-labeled glutamate, alanine, or glutamine(amide) were administered rapidly (less than or equal to 2 s) via the portal vein of anesthetized adult male rats. Liver content of tracer at 5 s was 57 +/- 6 (n = 6), 24 +/- 1 (n = 3), and 69 +/- 7 (n = 3)% of the injected dose, respectively. Portal-hepatic vein differences for the corresponding amino acids were 17 +/- 6, 26 +/- 8, and 19 +/- 9% (n = 4), respectively, suggesting some export of glutamate and glutamine, but not of alanine, to the hepatic vein. Following L-[13N]glutamate administration, label rapidly appeared in liver alanine and aspartate (within seconds). The data emphasize the rapidity of nitrogen exchange via linked transaminases. By 30 s following administration of either L-[13N]glutamate or L-[13N]alanine, label in liver glutamate was comparable; yet, by 1 min greater than or equal to 9 times as much label was present in liver glutamine(amine) following L-[13N]glutamate administration than following L-[13N]alanine administration. Conversely, label in liver urea at 1 min was more pronounced in the latter case despite: (a) comparable total pool sizes of glutamate and alanine in liver; and (b) label incorporation from alanine into urea must occur via prior transfer of alanine nitrogen to glutamate. The data provide evidence for zonal differences in uptake of alanine and glutamate from the portal vein in vivo. The rate of turnover of L-[amide-13N]glutamine was considerably slower than that of L-[13N]alanine or of L-[13N]glutamate, presumably due in part to the higher concentration of glutamine in that organ. Nevertheless, it was possible to show that despite occasional suggestions to the contrary, glutamine(amide) is a source of urea nitrogen in vivo. The present findings continue to emphasize the rapidity of nitrogen exchange reactions in vivo.     

Intraneuronal N-acetylaspartate supplies acetyl groups for myelin lipid synthesis: evidence for myelin-associated aspartoacylase

Despite its growing use as a radiological indicator of neuronal viability, the biological function of N-acetylaspartate (NAA) has remained elusive. This is due in part to its unusual metabolic compartmentalization wherein the synthetic enzyme occurs in neuronal mitochondria whereas the principal metabolizing enzyme, N-acetyl-L-aspartate amidohydrolase (aspartoacylase), is located primarily in white matter elements. This study demonstrates that within white matter, aspartoacylase is an integral component of the myelin sheath where it is ideally situated to produce acetyl groups for synthesis of myelin lipids. That it functions in this manner is suggested by the fact that myelin lipids of the rat optic system are well labeled following intraocular injection of [14C-acetyl]NAA. This is attributed to uptake of radiolabeled NAA by retinal ganglion cells followed by axonal transport and transaxonal transfer of NAA into myelin, a membrane previously shown to contain many lipid synthesizing enzymes. This study identifies a group of myelin lipids that are so labeled by neuronal [14C]NAA, and demonstrates a different labeling pattern from that produced by neuronal [14C]acetate. High performance liquid chromatographic analysis of the deproteinated soluble materials from the optic system following intraocular injection of [14C]NAA revealed only the latter substance and no radiolabeled acetate, suggesting little or no hydrolysis of NAA within mature neurons of the optic system. These results suggest a rationale for the unusual compartmentalization of NAA metabolism and point to NAA as a neuronal constituent that is essential for the formation and/or maintenance of myelin. The relevance of these findings to Canavan disease is discussed.     

Noninvasive Measurements of [1-13C] Glycogen Concentrations and Metabolism in Rat Brain In Vivo

Using a specific 13C NMR localization method, 13C label incorporation into the glycogen C1 resonance was measured while infusing [1-(13)C]glucose in intact rats. The maximal concentration of [1-(13)C]glycogen was 5.1 +/- 0.6 micromol g(-1) (mean +/- SE, n = 8). During the first 60 min of acute hyperglycemia, the rate of 13C label incorporation (synthase flux) was 2.3 +/- 0.7 micromol g(-1) h(-1) (mean +/- SE, n = 9 rats), which was higher (p < 0.01) than the rate of 0.49 +/- 0.14 micromol g(-1) h(-1) measured > or = 2 h later. To assess whether the incorporation of 13C label was due to turnover or net synthesis, the infusion was continued in seven rats with unlabeled glucose. The rate of 13C label decline (phosphorylase flux) was lower (0.33 +/- 0.10 micromol g(-1) h(-1)) than the initial rate of label incorporation (p < 0.01) and appeared to be independent of the duration of the preceding infusion of [1-(13)C]glucose (p > 0.05 for correlation). The results implied that net glycogen synthesis of approximately 3 micromol g(-1) had occurred, similar to previous reports. When infusing unlabeled glucose before [1-(13)C]glucose in three studies, the rate of glycogen C1 accumulation was 0.46 +/- 0.08 micromol g(-1) h(-1). The results suggest that steady-state glycogen turnover rates during hyperglycemia are approximately 1% of glucose consumption.     

MRL/lpr mice have alterations in brain metabolism as shown with [1H-13C] NMR spectroscopy

Cerebral glucose metabolism and cerebral blood flow are altered in patients with lupus who have neuropsychiatric manifestations. However, the dynamics of changes in glucose metabolism remain unclear. The present study was undertaken using 1H and 13C nuclear magnetic resonance (NMR) spectroscopy to determine the rates of incorporation of glucose into amino acids and lactate via cell-specific pathways in mice with lupus. In the well-established MRL/lpr lupus mouse model, 24-week-old mice had a significant increase of 30-80% (P<0.001) in total brain glutamine, glutamate and lactate concentrations, while alanine, aspartate, N-acetyl aspartate (NAA) and gamma-aminobutyric acid (GABA) remained unchanged as compared to the congenic MRL+/+control mice. Although succinate concentration was increased in lupus brain, it did not reach statistical significance. Furthermore, 13C isotopomer analysis showed a selective increase of de novo synthesis of lactate from [1-(13)C] glucose through glycolysis resulting in 1.5-fold increased fractional 13C enrichments in lactate in MRL/lpr mice. [4-(13)C] Glutamate, which is synthesized mainly by the neuronal pyruvate dehydogenase, was selectively increased, while [2-(13)C] and [3-(13)C] GABA synthesis were decreased by 25% compared to controls. In accordance with the total concentrations, aspartate synthesis remained unaltered in brains of lupus mice, while alanine synthesis was elevated, indicating increased utilization of alanine. Creatine was unchanged in MRL/lpr mice as compared to controls. An interesting finding was a significant increase (158%, P<0.005) in choline concentration in MRL/lpr mice while the myo-inositol concentration remained the same in both groups. Furthermore a significant increase in total brain water content was observed, indicative of possible edema. In conclusion, the cumulative effect of increased brain lactate synthesis, altered glucose metabolism and intracellular glutamine accumulation could be an important mechanism causing brain pathology in SLE. The alteration in metabolites could alter downstream pathways and cause neurological dysfunction. Future NMR spectroscopic studies using stable isotopes and real-time measurements of metabolic rates, along with levels of metabolites in plasma and cerebrospinal fluid, could be valuable in the elucidation of the cerebral metabolic consequences of systemic lupus erythematosis (SLE) in humans.