Bjørnar Hassel

Mitochondrial biogenesis in the anticonvulsant mechanism of the ketogenic diet

The full anticonvulsant effect of the ketogenic diet (KD) can require weeks to develop in rats, suggesting that altered gene expression is involved. The KD typically is used in pediatric epilepsies, but is effective also in adolescents and adults. Our goal was to use microarray and complementary technologies in adolescent rats to understand its anticonvulsant effect.

Use of fluorocitrate and fluoroacetate in the study of brain metabolism.

Fluoroacetate and its toxic metabolite fluorocitrate cause inhibition of aconitase. In brain tissue, both substances are preferentially taken up by glial cells and leads to inhibition of the glial TCA cycle. It is important to realise, however, that the glia‐specificity of these compounds depends both on the dosage and on the model used. The glia‐inhibitory effect of fluorocitrate as obtained by intracerebral microinjection in vivo is reversible within 24 h. A substantial inhibition of the glial TCA cycle by systemic administration of fluoroacetate requires a lethal dose.  Inhibition of the glial aconitase leads to accumulation of citrate and to a reduction in the formation of glutamine. Whereas the former is likely to be responsible for the main toxic effect of these compounds possibly by chelation of free calcium ions, it is the latter that has received most attention in the study of glial‐neuronal interactions, since glutamine is an important precursor for transmitter glutamate and GABA. GLIA 21: 106–113, 1997.

Trafficking of Amino Acids Between Neurons and Glia In Vivo. Effects of Inhibition of Glial Metabolism by Fluoroacetate

Glial-neuronal interchange of amino acids was studied by 13C nuclear magnetic resonance spectroscopy of brain extracts from fluoroacetate-treated mice that received [1,2-13C]acetate and [1-13C]glucose simultaneously. [13C]Acetate was found to be a specific marker for glial metabolism even with the large doses necessary for nuclear magnetic resonance spectroscopy. Fluoroacetate, 100 mg/kg, blocked the glial, but not the neuronal tricarboxylic acid cycles as seen from the 13C labeling of glutamine, glutamate, and γ-aminobutyric acid. Glutamine, but not citrate, was the only glial metabolite that could account for the transfer of 13C from glia to neurons. Massive glial uptake of transmitter glutamate was indicated by the labeling of glutamine from [1-13C]glucose in fluoroacetate-treated mice. The C-3/C-4 enrichment ratio, which indicates the degree of cycling of label, was higher in glutamine than in glutamate in the presence of fluoroacetate, suggesting that transmitter glutamate (which was converted to glutamine after release) is associated with a tricarboxylic acid cycle that turns more rapidly than the overall cerebral tricarboxylic acid cycle.

Glial-neuronal interactions as studied by cerebral metabolism of [2-13C]acetate and [1-13C]glucose: an ex vivo 13C NMR spectroscopic study.

Abstract: Mice were injected intravenously with [2‐13C]‐acetate or [1‐13C]glucose and killed after 5, 15, or 30 min. Another group of animals was injected three times subcutaneously during 30 min with [2‐13C]acetate to achieve a steady‐state‐like situation. Brain extracts were analyzed by 13C NMR spectroscopy, and the percent enrichment of various carbon positions was calculated for amino acids, lactate, and glucose. Results obtained with [2‐13C]acetate, which is metabolized by glia and not by neurons, showed that glutamine originated from a glial tricarboxylic acid cycle (TCA cycle) that loses 65% of its intermediates per turn of the cycle. This TCA cycle was associated with pyruvate carboxylation, which may replenish virtually all of this loss, as seen from the labeling of glutamine from [1‐13C]glucose. From the C‐3/C‐4 labeling ratios in glutamine and glutamate and from the corresponding C‐3/C‐2 labeling ratio in GABA obtained with [2‐13C]acetate, it was concluded that the carbon skeleton of glutamine to some extent was passed through TCA cycles before glutamate and GABA were formed. Thus, astrocytically derived glutamine is not only a precursor for transmitter amino acids but is also an energy substrate for neurons in vivo. Furthermore, the neuronal TCA cycles may be control points in the synthesis of transmitter amino acids. Injection of [2‐13C]acetate led to a higher 13C enrichment of the C‐2 in glutamate and of the corresponding C‐4 in GABA than in the C‐3 of either compound. This could reflect cleavage of [2‐13C]‐citrate and formation of [3‐13C]oxaloacetate and acetyl‐CoA, i.e., the first step in fatty acid synthesis. [3‐13C]‐Oxaloacetate would, after entry into a TCA cycle, give the observed labeling of glutamate and GABA.

Cerebral Metabolism of Lactate in Vivo: Evidence for Neuronal Pyruvate Carboxylation

The cerebral metabolism of lactate was investigated. Awake mice received [3-13C]lactate or [1-13C]glucose intravenously, and brain and blood extracts were analyzed by 13C nuclear magnetic resonance spectroscopy. The cerebral up-take and metabolism of [3-13C]lactate was 50% that of [1-13C]glucose. [3-13C]Lactate was almost exclusively metabolized by neurons and hardly at all by glia, as revealed by the 13C labeling of glutamate, γ-aminobutyric acid and glutamine. Injection of [3-13C]lactate led to extensive formation of [2-13C]lactate, which was not seen with [1-13C]glucose, nor has it been seen in previous studies with [2-13C]acetate. This formation probably reflected reversible carboxylation of [3-13C]pyruvate to malate and equilibration with fumarate, because inhibition of succinate dehydrogenase with nitropropionic acid did not block it. Of the [3-13C]lactate that reached the brain, 20% underwent this reaction, which probably involved neuronal mitochondrial malic enzyme. The activities of mitochondrial malic enzyme, fumarase, and lactate dehydrogenase were high enough to account for the formation of [2-13C]lactate in neurons. Neuronal pyruvate carboxylation was confirmed by the higher specific activity of glutamate than of glutamine after intrastriatal injection of [1-14C]pyruvate into anesthetized mice. This procedure also demonstrated equilibration of malate, formed through pyruvate carboxylation, with fumarate. The demonstration of neuronal pyruvate carboxylation demands reconsideration of the metabolic interrelationship between neurons and glia.

Selective Inhibition of the Tricarboxylic Acid Cycle of GABAergic Neurons with 3-Nitropropionic Acid In Vivo

Abstract: The effects of 3‐nitropropionic acid (3‐NPA), an inhibitor of succinate dehydrogenase, on cerebral metabolism were investigated in mice by NMR spectroscopy. 3‐NPA, 180 mg/kg, caused a dramatic buildup of succinate. Succinate was labeled 5.5 times better from [1‐13C]glucose than from [2‐13C]acetate, showing a predominantly neuronal accumulation. [1‐13C]Glucose labeled GABA in the C‐2 position only, compatible with inhibition of the tricarboxylic acid (TCA) cycle associated with GABA formation, at the level of succinate dehydrogenase. Aspartate was not labeled by [1‐13C]glucose in 3‐NPA‐intoxicated animals. In contrast, [1‐13C]glucose labeled glutamate in the C‐2, C‐3, and C‐4 positions showing uninhibited cycling of label in the TCA cycle associated with the large, neuronal pool of glutamate. The labeling of glutamine, and hence GABA, from [2‐13C]acetate showed that the TCA cycle of glial cells was unaffected by 3‐NPA and that transfer of glutamine from glia to neurons took place during 3‐NPA intoxication. The high 13C enrichment of the C‐2 position of glutamine from [1‐13C]glucose showed that pyruvate carboxylation was active in glia during 3‐NPA intoxication. These findings suggest that 3‐NPA in the initial phase of intoxication fairly selectively inhibited the TCA cycle of GABAergic neurons; whereas the TCA cycle of glia remained uninhibited as did the TCA cycle associated with the large neuronal pool of glutamate, which includes glutamatergic neurons. This may help explain why the caudoputamen, which is especially rich in GABAergic neurons, selectively undergoes degeneration both in humans and animals intoxicated with 3‐NPA. Further, the present results may be of relevance for the study of basal ganglia disorders such as Huntingtons disease.

Glial Formation of Pyruvate and Lactate from TCA Cycle Intermediates: Implications for the Inactivation of Transmitter Amino Acids?

Abstract: Cerebral formation of lactate via the tricarboxylic acid (TCA) cycle was investigated through the labeling of lactate from [2‐13C]acetate and [1‐13C]glucose as shown by 13C NMR spectroscopy. In fasted mice that had received [2‐13C]acetate intravenously, brain lactate C‐2 and C‐3 were labeled at 5, 15, and 30 min, reflecting formation of pyruvate and hence lactate from TCA cycle intermediates. In contrast, [1‐13C]glucose strongly labeled lactate C‐3, reflecting glycolysis, whereas lactate C‐2 was weakly labeled only at 15 min. These data show that formation of pyruvate, and hence lactate, from TCA cycle intermediates took place predominantly in the acetate‐metabolizing compartment, i.e., glia. The enrichment of total brain lactate from [2‐13C]acetate reached ∼1% in both the C‐2 and the C‐3 position in fasted mice. It was calculated that this could account for 20% of the lactate formed in the glial compartment. In fasted mice, there was no significant difference between the labeling of lactate C‐2 and C‐3 from [2‐13C]acetate, whereas in fed mice, lactate C‐3 was more highly labeled than the C‐2, reflecting adaptive metabolic changes in glia in response to the nutritional state of the animal. It is hypothesized that conversion of TCA cycle intermediates into pyruvate and lactate may be operative in the glial metabolism of extracellular glutamate and GABA in vivo. Given the vasodilating effect of lactate on cerebral vessels, which are ensheathed by astrocytic processes, conversion of glutamate and GABA into lactate could be one mechanism mediating increases in cerebral blood flow during nervous activity.

Neuronal uptake and metabolism of glycerol and the neuronal expression of mitochondrial glycerol-3-phosphate dehydrogenase.

Glycerol is effective in the treatment of brain oedema but it is unclear if this is due solely to osmotic effects of glycerol or whether the brain may metabolize glycerol. We found that intracerebral injection of [14C]glycerol in rat gave a higher specific activity of glutamate than of glutamine, indicating neuronal metabolism of glycerol. Interestingly, the specific activity of GABA became higher than that of glutamate. NMR spectroscopy of brains of mice given 150 µmol [U‐13C]glycerol (0.5 m i.v.) confirmed this predominant labelling of GABA, indicating avid glycerol metabolism in GABAergic neurones. Uptake of [14C]glycerol into cultured cerebellar granule cells was inhibited by Hg2+, suggesting uptake through aquaporins, whereas Hg2+ stimulated glycerol uptake into cultured astrocytes. The neuronal metabolism of glycerol, which was confirmed in experiments with purified synaptosomes and cultured cerebellar granule cells, suggested neuronal expression of glycerol kinase and some isoform of glycerol‐3‐phosphate dehydrogenase. Histochemically, we demonstrated mitochondrial glycerol‐3‐phosphate dehydrogenase in neurones, whereas cytosolic glycerol‐3‐phosphate dehydrogenase was three to four times more active in white matter than in grey matter, reflecting its selective expression in oligodendroglia. The localization of mitochondrial and cytosolic glycerol‐3‐phosphate dehydrogenases in different cell types implies that the glycerol‐3‐phosphate shuttle is of little importance in the brain.

Up-regulation of hippocampal glutamate transport during chronic treatment with sodium valproate

Excessive glutamatergic neurotransmission has been implicated in some neurodegenerative disorders. It would be of value to know whether glutamate transport, which terminates the glutamate signal, can be up‐regulated pharmacologically. Here we show that chronic treatment of rats with the anti‐epileptic drug sodium valproate (200 mg or 400 mg/kg bodyweight, twice per day for 90 days) leads to a dose‐dependent increase in hippocampal glutamate uptake capacity as measured by uptake of [3H]glutamate into proteoliposomes. The level of glutamate transporters EAAT1 and EAAT2 in hippocampus also increased dose‐dependently. No effect of sodium valproate on glutamate transport was seen in frontal or parietal cortices or in cerebellum. The hippocampal levels of glial fibrillary acidic protein and glutamine synthetase were unaffected by valproate treatment, whereas the levels of synapsin I and phosphate‐activated glutaminase were reduced by valproate treatment, suggesting that the increase in glutamate transporters was not caused by astrocytosis or increased synaptogenesis. A direct effect of sodium valproate on the glutamate transporters could be excluded. The results show that hippocampal glutamate transport is an accessible target for pharmacological intervention and that sodium valproate may have a role in the treatment of excitotoxic states in the hippocampus.

Quantification of the GABA Shunt and the Importance of the GABA Shunt Versus the 2-Oxoglutarate Dehydrogenase Pathway in GABAergic Neurons

Abstract: We investigated the activity of the cerebral GABA shunt relative to the overall cerebral tricarboxylic acid (TCA) cycle and the importance of the GABA shunt versus 2‐oxoglutarate dehydrogenase for the conversion of 2‐oxoglutarate into succinate in GABAergic neurons. Awake mice were dosed with [1‐13C]glucose, and brain extracts were analyzed by 13C NMR spectroscopy. The percent enrichments of GABA C‐2 and glutamate C‐4 were the same: 5.0 ± 1.6 and 5.1 ± 0.2%, respectively (mean ± SD). This, together with previous data, indicates that the flux through the GABA shunt relative to the overall cerebral TCA cycle flux equals the GABA/glutamate pool size ratio, which in the mouse is 17%. It has previously been shown that under the experimental conditions used in this study, the 13C labeling of aspartate from [1‐13C]glucose specifically reflects the metabolic activity of GABAergic neurons. In the present study, the reduction in the formation of [13C]aspartate during inhibition of the GABA shunt by γ‐vinyl‐GABA indicated that not more than half the flux from 2‐oxoglutarate to succinate in GABAergic neurons goes via the GABA shunt. Therefore, because fluxes through the GABA shunt and 2‐oxoglutarate dehydrogenase in GABAergic neurons are approximately the same, the TCA cycle activity of GABAergic neurons could account for one‐third of the overall cerebral TCA cycle activity in the mouse. Treatment with γ‐vinyl‐GABA, which increased GABA levels dramatically, caused changes in the 13C labeling of glutamate and glutamine, which indicated a reduction in the transfer of glutamate from neurons to glia, implying reduced glutamatergic neurotransmission. In the most severely affected animals these alterations were associated with convulsions.