Reversal of metabolic deficits by lipoic acid in a triple transgenic mouse model of Alzheimer's disease: a 13C NMR study

Alzheimer''s disease is an age-related neurodegenerative disease characterized by deterioration of cognition and loss of memory. Several clinical studies have shown Alzheimer''s disease to be associated with disturbances in glucose metabolism and the subsequent tricarboxylic acid (TCA) cycle-related metabolites like glutamate (Glu), glutamine (Gln), and N-acetylaspartate (NAA). These metabolites have been viewed as biomarkers by (a) assisting early diagnosis of Alzheimer''s disease and (b) evaluating the efficacy of a treatment regimen. In this study, 13-month-old triple transgenic mice (a mouse model of Alzheimer''s disease (3xTg-AD)) were given intravenous infusion of [1-¹³C]glucose followed by an ex vivo ¹³C NMR to determine the concentrations of ¹³C-labeled isotopomers of Glu, Gln, aspartate (Asp), GABA, myo-inositol, and NAA. Total (¹²C+¹³C) Glu, Gln, and Asp were quantified by high-performance liquid chromatography to calculate enrichment. Furthermore, we examined the effects of lipoic acid in modulating these metabolites, based on its previously established insulin mimetic effects. Total ¹³C labeling and percent enrichment decreased by ∼50% in the 3xTg-AD mice. This hypometabolism was partially or completely restored by lipoic acid feeding. The ability of lipoic acid to restore glucose metabolism and subsequent TCA cycle-related metabolites further substantiates its role in overcoming the hypometabolic state inherent in early stages of Alzheimer''s disease.     

Effects of ammonia on the anaplerotic pathway and amino acid metabolism in the brain: an ex vivo C NMR spectroscopic study of rats after administering [2-C] glucose with or without ammonium acetate

The ¹³C-label incorporation into glutamate, glutamine, aspartate and γ-aminobutyric acid (GABA) from [2-¹³C] glucose was measured by ¹³C nuclear magnetic resonance (NMR) spectroscopy to directly examine the effects of ammonia on the activity of pyruvate carboxylase (i.e., the anaplerotic pathway) and the amino acid metabolism in the rat brain in vivo. Rats were sacrificed by exposure to microwaves at 7.5, 15, 30, and 60 min after an i.v. injection of [2-¹³C] glucose with or without ammonium acetate. After the injection of ammonium acetate, the brain contents of glutamate, aspartate and GABA had decreased, however, the percentage of ¹³C enrichment of C3 of glutamine, glutamate and GABA, and C2 and C3 of aspartate had increased. The ¹³C entered the TCA cycle via pyruvate carboxylase from [2-¹³C] glucose, labeling the C2 or C3 positions of aspartate, the C2 or C3 positions of glutamate and glutamine, and the C3 or C4 positions of GABA first and second turns of the tricarboxylic acid (TCA) cycle. The C4/C3 labeling ratio in GABA was lower than the analogous ratio in glutamate (C2/C3) and higher than that of glutamine (C2/C3). The order of these ratios (glutamate>GABA>glutamine) was not altered by the injection of ammonium acetate. These findings directly indicate that ammonia increases the anaplerotic pathway and that the ¹³C-skeletons entered glial glutamine through the anaplerotic pathway flow from glia to neuron. A fraction of the glutamine is used in the direct synthesis of GABA via glutamate, whereas the remaining fraction of glutamine passed through the neuronal TCA cycle before synthesizing GABA.     

Brain mitochondrial metabolic dysfunction and glutamate level reduction in the pilocarpine model of temporal lobe epilepsy in mice

Although certain metabolic characteristics such as interictal glucose hypometabolism are well established for temporal lobe epilepsy (TLE), its pathogenesis still remains unclear. Here, we performed a comprehensive study of brain metabolism in a mouse model of TLE, induced by pilocarpine–status epilepticus (SE). To investigate glucose metabolism, we injected mice 3.5–4 weeks after SE with [1,2-¹³C]glucose before microwave fixation of the head. Using ¹H and ¹³C nuclear magnetic resonance spectroscopy, gas chromatography—mass spectrometry and high-pressure liquid chromatography, we quantified metabolites and ¹³C labeling in extracts of cortex and hippocampal formation (HF). Hippocampal levels of glutamate, glutathione and alanine were decreased in pilocarpine–SE mice compared with controls. Moreover, the contents of N-acetyl aspartate, succinate and reduced nicotinamide adenine dinucleotide (phosphate) NAD(P)H were decreased in HF indicating impairment of mitochondrial function. In addition, the reduction in ¹³C enrichment of hippocampal citrate and malate suggests decreased tricarboxylic acid (TCA) cycle turnover in this region. In cortex, we found reduced ¹³C labeling of glutamate, glutamine and aspartate via the pyruvate carboxylation and pyruvate dehydrogenation pathways, suggesting slower turnover of these amino acids and/or the TCA cycle. In conclusion, mitochondrial metabolic dysfunction and altered amino-acid metabolism is found in both cortex and HF in this epilepsy model.     

Knockout of GAD65 has major impact on synaptic GABA synthesized from astrocyte-derived glutamine

γ-Aminobutyric acid (GABA) synthesis from glutamate is catalyzed by glutamate decarboxylase (GAD) of which two isoforms, GAD65 and GAD67, have been identified. The GAD65 has repeatedly been shown to be important during intensified synaptic activity. To specifically elucidate the significance of GAD65 for maintenance of the highly compartmentalized intracellular and intercellular GABA homeostasis, GAD65 knockout and corresponding wild-type mice were injected with [1-¹³C]glucose and the astrocyte-specific substrate [1,2-¹³C]acetate. Synthesis of GABA from glutamine in the GABAergic synapses was further investigated in GAD65 knockout and wild-type mice using [1,2-¹³C]acetate and in some cases γ-vinylGABA (GVG, Vigabatrin), an inhibitor of GABA degradation. A detailed metabolic mapping was obtained by nuclear magnetic resonance (NMR) spectroscopic analysis of tissue extracts of cerebral cortex and hippocampus. The GABA content in both brain regions was reduced by ∼20%. Moreover, it was revealed that GAD65 is crucial for maintenance of biosynthesis of synaptic GABA particularly by direct synthesis from astrocytic glutamine via glutamate. The GAD67 was found to be important for synthesis of GABA from glutamine both via direct synthesis and via a pathway involving mitochondrial metabolism. Furthermore, a severe neuronal hypometabolism, involving glycolysis and tricarboxylic acid (TCA) cycle activity, was observed in cerebral cortex of GAD65 knockout mice.     

Reduced astrocytic contribution to the turnover of glutamate,glutamine, and GABA characterizes the latent phase in the kainate model of temporal lobe epilepsy

The occurrence of spontaneous seizures in mesial temporal lobe epilepsy (MTLE) is preceded by a latent phase that provides a time window for identifying and treating patients at risk. However, a reliable biomarker of epileptogenesis has not been established and the underlying processes remain unclear. Growing evidence suggests that astrocytes contribute to an imbalance between excitation and inhibition in epilepsy. Here, astrocytic and neuronal neurotransmitter metabolism was analyzed in the latent phase of the kainate model of MTLE in an attempt to identify epileptogenic processes and potential biomarkers. Fourteen days after status epilepticus, [1-¹³C]glucose and [1,2-¹³C]acetate were injected and the hippocampal formation, entorhinal/piriform cortex, and neocortex were analyzed by ¹H and ¹³C magnetic resonance spectroscopy. The ¹³C enrichment in glutamate, glutamine, and γ-aminobutyric acid (GABA) from [1-¹³C]glucose was decreased in all areas. Decreased GABA content was specific for the hippocampal formation, together with a pronounced decrease in astrocyte-derived [1,2-¹³C]GABA and a decreased transfer of glutamine for the synthesis of GABA. Accumulation of branched-chain amino acids combined with decreased [4,5-¹³C]glutamate in hippocampal formation could signify decreased transamination via branched-chain aminotransferase in astrocytes. The results point to astrocytes as major players in the epileptogenic process, and ¹³C enrichment of glutamate and GABA as potential biomarkers.     

Altered brain mitochondrial metabolism in healthy aging as assessed by in vivo magnetic resonance spectroscopy

A decline in brain function is a characteristic feature of healthy aging; however, little is known about the biologic basis of this phenomenon. To determine whether there are alterations in brain mitochondrial metabolism associated with healthy aging, we combined ¹³C/¹H magnetic resonance spectroscopy with infusions of [1-¹³C]glucose and [2-¹³C]acetate to quantitatively characterize rates of neuronal and astroglial tricarboxylic acid cycles, as well as neuroglial glutamate–glutamine cycling, in healthy elderly and young volunteers. Compared with young subjects, neuronal mitochondrial metabolism and glutamate–glutamine cycle flux was ∼30% lower in elderly subjects. The reduction in individual subjects correlated strongly with reductions in N-acetylaspartate and glutamate concentrations consistent with chronic reductions in brain mitochondrial function. In elderly subjects infused with [2-¹³C]acetate labeling of glutamine, C4 and C3 differed from that of the young subjects, indicating age-related changes in glial mitochondrial metabolism. Taken together, these studies show that healthy aging is associated with reduced neuronal mitochondrial metabolism and altered glial mitochondrial metabolism, which may in part be responsible for declines in brain function.     

A subconvulsive dose of kainate selectively compromises astrocytic metabolism in the mouse brain in vivo

Despite the well-established use of kainate as a model for seizure activity and temporal lobe epilepsy, most studies have been performed at doses giving rise to general limbic seizures and have mainly focused on neuronal function. Little is known about the effect of lower doses of kainate on cerebral metabolism and particularly that associated with astrocytes. We investigated astrocytic and neuronal metabolism in the cerebral cortex of adult mice after treatment with saline (controls), a subconvulsive or a mildly convulsive dose of kainate. A combination of [1,2-¹³C]acetate and [1-¹³C]glucose was injected and subsequent nuclear magnetic resonance spectroscopy of cortical extracts was employed to distinctively map astrocytic and neuronal metabolism. The subconvulsive dose of kainate led to an instantaneous increase in the cortical lactate content, a subsequent reduction in the amount of [4,5-¹³C]glutamine and an increase in the calculated astrocytic TCA cycle activity. In contrast, the convulsive dose led to decrements in the cortical content and ¹³C labeling of glutamate, glutamine, GABA, and aspartate. Evidence is provided that astrocytic metabolism is affected by a subconvulsive dose of kainate, whereas a higher dose is required to affect neuronal metabolism. The cerebral glycogen content was dose-dependently reduced by kainate supporting a role for glycogen during seizure activity.     

Rapid adaptation of rat brain and liver metabolism to a ketogenic diet: an integrated study using 1H- and 13C-NMR spectroscopy

The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-¹³C]glucose (Glc) or [2,4-¹³C₂]β-hydroxybutyrate (β-HB). Specific enrichments of amino acids (AAs) measured by ¹H- and ¹³C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-¹³C]Glc infusion whereas they were higher in KD rats after [2,4-¹³C₂]β-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.     

Cerebral dynamics and metabolism of hyperpolarized [1-13C]pyruvate using time-resolved MR spectroscopic imaging

Dynamic hyperpolarized [1-¹³C]pyruvate metabolic imaging in the normal anesthetized rat brain is demonstrated on a clinical 3-T magnetic resonance imaging scanner. A 12-second bolus injection of hyperpolarized [1-¹³C]pyruvate is imaged at a 3-second temporal resolution. The observed dynamics are evaluated with regard to cerebral blood volume (CBV), flow, transport, and metabolic exchange with the cerebral lactate pool. A model for brain [1-¹³C]lactate, based on blood–brain transport kinetics, CBV, and the observed pyruvate dynamics is described.     

Altered 13C glucose metabolism in the cortico–striato–thalamo–cortical loop in the MK-801 rat model of schizophrenia

Using a modified MK-801 (dizocilpine) N-methyl--aspartic acid (NMDA) receptor hypofunction model for schizophrenia, we analyzed glycolysis, as well as glutamatergic, GABAergic, and monoaminergic neurotransmitter synthesis and degradation. Rats received an injection of MK-801 daily for 6 days and on day 6, they also received an injection of [1-¹³C]glucose. Extracts of frontal cortex (FCX), parietal and temporal cortex (PTCX), thalamus, striatum, nucleus accumbens (NAc), and hippocampus were analyzed using ¹³C nuclear magnetic resonance spectroscopy, high-performance liquid chromatography, and gas chromatography–mass spectrometry. A pronounced reduction in glycolysis was found only in PTCX, in which ¹³C labeling of glucose, lactate, and alanine was decreased. ¹³C enrichment in lactate, however, was reduced in all areas investigated. The largest reductions in glutamate labeling were detected in FCX and PTCX, whereas in hippocampus, striatum, and Nac, ¹³C labeling of glutamate was only slightly but significantly reduced. The thalamus was the only region with unaffected glutamate labeling. γ-Aminobutyric acid (GABA) labeling was reduced in all areas, but most significantly in FCX. Glutamine and aspartate labeling was unchanged. Mitochondrial metabolites were also affected. Fumarate labeling was reduced in FCX and thalamus, whereas malate labeling was reduced in FCX, PTCX, striatum, and NAc. Dopamine turnover was decreased in FCX and thalamus, whereas that of serotonin was unchanged in all regions. In conclusion, neurotransmitter metabolism in the cortico–striato–thalamo–cortical loop is severely impaired in the MK-801 (dizocilpine) NMDA receptor hypofunction animal model for schizophrenia.     

Imaging cerebral 2-ketoisocaproate metabolism with hyperpolarized (13)C magnetic resonance spectroscopic imaging

The branched chain amino acid transaminase (BCAT) has an important role in nitrogen shuttling and glutamate metabolism in the brain. The purpose of this study was to describe the cerebral distribution and metabolism of hyperpolarized 2-keto[1-¹³C]isocaproate (KIC) in the normal rat using magnetic resonance modalities. Hyperpolarized KIC is metabolized to [1-¹³C]leucine (leucine) by BCAT. The results show that KIC and its metabolic product, leucine, are present at imageable quantities 20 seconds after end of KIC administration throughout the brain. Further, significantly higher metabolism was observed in hippocampal regions compared with the muscle tissue. In conclusion, the cerebral metabolism of hyperpolarized KIC is imaged and hyperpolarized KIC may be a promising substrate for evaluation of cerebral BCAT activity in conjunction with neurodegenerative disease.     

NMR spectroscopy study of the effect of 3-nitropropionic acid on glutamate metabolism in cultured astrocytes

3-Nitropropionic acid (3-NPA) is a selective and irreversible inhibitor of succinate dehydrogenase. The effect of this compound on the metabolism of [U-¹³C]glutamate was studied in astrocytes using ¹³C nuclear magnetic resonance spectroscopy. The appearance of [1,2,3-¹³C]glutamate in cell extracts and [1,2,3-¹³C]glutamine and [U-¹³C]lactate in cell media demonstrated the metabolism of labeled glutamate via the tricarboxylic acid cycle. Such labeling was observed in the control situation and also in cells treated with 3 mM 3-NPA. In the cells treated with 3 mM 3-NPA, however, the labeling was significantly reduced, and with 10 mM 3-NPA no such labeling was observed. Labeled aspartate was observed in untreated cells only. Labeled succinate was not detectable under control conditions, but increased dose dependently in the presence of 3-NPA. Glutamate uptake and conversion of [U-¹³C]glutamate to U-¹³C]glutamine was largely unaffected by 3-NPA, and ATP content was unchanged. In a previous study using cerebellar neurons, tricarboxylic acid cycle metabolism was blocked with 3 mM 3-NPA. The present results show that astrocyte metabolism is more adaptable to blockade of the tricarboxylic acid cycle by 3-NPA than neuronal metabolism. J. Neurosci. Res. 47:650–654, 1997. © 1997 Wiley-Liss, Inc.     

The entry of [1-C]glucose into biochemical pathways reveals a complex compartmentation and metabolite trafficking between glia and neurons: a study by C-NMR spectroscopy

Glial–neuronal interactions were investigated in rats injected intraperitoneally with [1-¹³C]glucose and killed after 15, 30, 45, or 60 min. Brain extracts were analyzed by ¹³C-NMR spectroscopy and the fractional ¹³C-enrichment at individual carbon positions was measured for amino acids, lactate, and N-acetyl-aspartate. [1-¹³C]Glucose was shown to be metabolized by both neurons and glia, with the anaplerotic pathway through pyruvate carboxylase (PC) accounting for 10% of total cerebral glucose metabolism. The PC-mediated pathway accounted for 39% of the glutamine synthesis, and for 8, 6, 14% of glutamate, GABA, and aspartate synthesis, respectively. These results reflect a compartmentation of the cerebral amino acids synthesis within glial and neuronal cells. The appearance of the ¹³C-label in C5 of glutamate and glutamine, C1 of GABA and C2 of lactate, is suggestive of pyruvate, formation from TCA cycle intermediates and provides evidence of metabolite trafficking between astrocytes and neurons.     

Brain slices from glutaminase-deficient mice metabolize less glutamine: a cellular metabolomic study with carbon 13 NMR

In the brain, glutaminase is considered to have a key role in the provision of glutamate, a major excitatory neurotransmitter. Brain slices obtained from wild-type (control) and glutaminase-deficient (GLS1+/−) mice were incubated without glucose and with 5 or 1 mmol/L [3-¹³C]glutamine as substrate. At the end of the incubation, substrate removal and product formation were measured by both enzymatic and carbon 13 nuclear magnetic resonance (¹³C-NMR) techniques. Slices from GLS1+/− mice consumed less [3-¹³C]glutamine and accumulated less [3-¹³C]glutamate. They also produced less ¹³CO₂ but accumulated amounts of ¹³C-aspartate and ¹³C-gamma-aminobutyric acid (GABA) that were similar to those found with brain slices from control mice. The newly formed glutamine observed in slices from control mice remained unchanged in slices from GLS1+/− mice. As expected, flux through glutaminase in slices from GLS1+/− mice was found diminished. Fluxes through all enzymes of the tricarboxylic acid cycle were also reduced in brain slices from GLS1+/− mice except through malate dehydrogenase with 5 mmol/L [3-¹³C]glutamine. The latter diminutions are consistent with the decreases in the production of ¹³CO₂ also observed in the slices from these mice. It is concluded that the genetic approach used in this study confirms the key role of glutaminase for the provision of glutamate.     

Detection of reduced GABA synthesis following inhibition of GABA transaminase using in vivo magnetic resonance signal of [C]GABA C1

Previous in vivo magnetic resonance spectroscopy (MRS) studies of gamma-aminobutyric acid (GABA) synthesis have relied on ¹³C label incorporation into GABA C2 from [1-¹³C] or [1,6-¹³C₂]glucose. In this study, the [¹³C]GABA C1 signal at 182.3 ppm in the carboxylic/amide spectral region of localized in vivo ¹³C spectra was detected. GABA-transaminase of rat brain was inhibited by administration of gabaculine after pre-labeling of GABA C1 and its metabolic precursors with exogenous [2,5-¹³C₂]glucose. A subsequent isotope chase experiment was performed by infusing unlabeled glucose, which revealed a markedly slow change in the labeling of GABA C1 accompanying the blockade of the GABA shunt. This slow labeling of GABA at elevated GABA concentration was attributed to the relatively small intercompartmental GABA-glutamine cycling flux that constitutes the main route of ¹³C label loss during the isotope chase. Because this study showed that using low RF power broadband stochastic proton decoupling is feasible at very high field strength, it has important implications for the development of carboxylic/amide ¹³C MRS methods to study brain metabolism and neurotransmission in human subjects at high magnetic fields.     

[U-13C]glutamate metabolism in astrocytes during hypoglycemia and hypoxia

The ability of cultured astrocytes to metabolize [U-¹³C]glutamate in the absence of glucose was investigated by utilizing ¹³C nuclear magnetic resonance spectroscopy to identify ¹³C-labeled metabolites. Control cultures (3 mM glucose), hypoglycemic cultures (glucose-deprived), severe hypoglycemic cultures (glucose-deprived, 0.5 mM iodoacetate as an inhibitor of glycolysis), hypoglycemic/hypoxic cultures, and cultures deprived of all additional substrates were incubated for 2 hr in medium containing 0.5 mM glutamate (50% [U-¹³C]glutamate). Glucose deprivation alone had little effect on removal of glutamate from the culture medium, but the presence of iodoacetate or incubating cultures in a low-oxygen atmosphere decreased glutamate clearance. Only the withdrawal of all substrates other than glutamate decreased glutamine synthesis. Metabolism of glutamate through the tricarboxylic acid (TCA) cycle was evident by the appearance of [1,2,3-¹³C]glutamate and [U-¹³C]aspartate in cell extracts and [U-¹³C]lactate in cell media. Lactate derived from TCA cycle intermediates was significantly reduced after glucose deprivation and even more so after severe hypoglycemia. Release of glutamate from astrocytes was observed under all incubation conditions. [U-¹³C]Aspartate was not detected in control media but was released from glucose-deprived cells when oxygen was available. Increased release was observed in the presence of iodoacetate. After withdrawal of all substrates other than glutamate, [U-¹³C]aspartate was the only metabolite observed intracellularly, whereas aspartate, glutamine, and 5-oxoproline were detected in the incubation medium. The present results indicate that glutamate-to-aspartate conversion is preferentially utilized by astrocytes when oxygen is available but glycolysis is impaired. J. Neurosci. Res. 51:636–645, 1998. © 1998 Wiley-Liss, Inc.     

Microdialysis with radiometric monitoring of -[β-11C]DOPA to assess dopaminergic metabolism: effect of inhibitors of -amino acid decarboxylase,monoamine oxidase,and catechol-O-methyltransferase on rat striatal dialysate

The catecholamine, dopamine (DA), is synthesized from 3,4-dihydroxy--phenylalanine (-DOPA) by aromatic -amino acid decarboxylase (AADC). Dopamine metabolism is regulated by monoamine oxidase (MAO) and catechol-O-methyltransferase (COMT). To measure dopaminergic metabolism, we used microdialysis with radiometric detection to monitor -[β-¹¹C]DOPA metabolites in the extracellular space of the rat striatum. We also evaluated the effects of AADC, MAO, and COMT inhibitors on metabolite profiles. The major early species measured after administration of -[β-¹¹C]DOPA were [¹¹C]3,4-dihydroxyphenylacetic acid ([¹¹C]DOPAC) and [¹¹C]homovanillic acid ([¹¹C]HVA) in a 1:1 ratio, which shifted toward [¹¹C]HVA with time. An AADC inhibitor increased the uptake of -[β-¹¹C]DOPA and -3-O-methyl-[¹¹C]DOPA and delayed the accumulation of [¹¹C]DOPAC and [¹¹C]HVA. The MAO and COMT inhibitors increased the production of [¹¹C]3-methoxytyramine and [¹¹C]DOPAC, respectively. These results reflect the -DOPA metabolic pathway, suggesting that this method may be useful for assessing dopaminergic metabolism.     

A two-compartment mathematical model of neuroglial metabolism using [1-(11)C] acetate

The purpose of this study was to develop a two-compartment metabolic model of brain metabolism to assess oxidative metabolism from [1-¹¹C] acetate radiotracer experiments, using an approach previously applied in ¹³C magnetic resonance spectroscopy (MRS), and compared with an one-tissue compartment model previously used in brain [1-¹¹C] acetate studies. Compared with ¹³C MRS studies, ¹¹C radiotracer measurements provide a single uptake curve representing the sum of all labeled metabolites, without chemical differentiation, but with higher temporal resolution. The reliability of the adjusted metabolic fluxes was analyzed with Monte-Carlo simulations using synthetic ¹¹C uptake curves, based on a typical arterial input function and previously published values of the neuroglial fluxes V_tca^g, V_x, V_nt, and V_tcaⁿ measured in dynamic ¹³C MRS experiments. Assuming V_x^g=10 × V_tca^g and V_xⁿ=V_tcaⁿ, it was possible to assess the composite glial tricarboxylic acid (TCA) cycle flux V_gt^g (V_gt^g=V_x^g × V_tca^g/(V_x^g+V_tca^g)) and the neurotransmission flux V_nt from ¹¹C tissue-activity curves obtained within 30 minutes in the rat cortex with a beta-probe after a bolus infusion of [1-¹¹C] acetate (n=9), resulting in V_gt^g=0.136±0.042 and V_nt=0.170±0.103 μmol/g per minute (mean±s.d. of the group), in good agreement with ¹³C MRS measurements.     

Brain glycogen content and metabolism in subjects with type 1 diabetes and hypoglycemia unawareness

Supercompensated brain glycogen may contribute to the development of hypoglycemia unawareness in patients with type 1 diabetes by providing energy for the brain during periods of hypoglycemia. Our goal was to determine if brain glycogen content is elevated in patients with type 1 diabetes and hypoglycemia unawareness. We used in vivo ¹³C nuclear magnetic resonance spectroscopy in conjunction with [1-¹³C]glucose administration in five patients with type 1 diabetes and hypoglycemia unawareness and five age-, gender-, and body mass index-matched healthy volunteers to measure brain glycogen content and metabolism. Glucose and insulin were administered intravenously over ∼51 hours at a rate titrated to maintain a blood glucose concentration of 7 mmol/L. ¹³C-glycogen levels in the occipital lobe were measured at ∼5, 8, 13, 23, 32, 37, and 50 hours, during label wash-in and wash-out. Newly synthesized glycogen levels were higher in controls than in patients (P<0.0001) for matched average blood glucose and insulin levels, which may be due to higher brain glycogen content or faster turnover in controls. Metabolic modeling indicated lower brain glycogen content in patients than in controls (P=0.07), implying that glycogen supercompensation does not contribute to the development of hypoglycemia unawareness in humans with type 1 diabetes.     

Modeling of brain metabolism and pyruvate compartmentation using 13C NMR in vivo: caution required

Two variants of a widely used two-compartment model were prepared for fitting the time course of [1,6-¹³C₂]glucose metabolism in rat brain. Features common to most models were included, but in one model the enrichment of the substrates entering the glia and neuronal citric acid cycles was allowed to differ. Furthermore, the models included the capacity to analyze multiplets arising from ¹³C spin-spin coupling, known to improve parameter estimates in heart. Data analyzed were from a literature report providing time courses of [1,6-¹³C₂]glucose metabolism. Four analyses were used, two comparing the effect of different pyruvate enrichment in glia and neurons, and two for determining the effect of multiplets present in the data. When fit independently, the enrichment in glial pyruvate was less than in neurons. In the absence of multiplets, fit quality and parameter values were typical of those in the literature, whereas the multiplet curves were not modeled well. This prompted the use of robust statistical analysis (the Kolmogorov–Smirnov test of goodness of fit) to determine whether individual curves were modeled appropriately. At least 50% of the curves in each experiment were considered poorly fit. It was concluded that the model does not include all metabolic features required to analyze the data.