These data show that a calorie-restricted KD enhances brain metabolism. We propose an anticonvulsant mechanism of the KD involving mitochondrial biogenesis leading to enhanced alternative energy stores.

No abstract

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-(13)C]acetate and [1-(13)C]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 gamma-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-(13)C]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.

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.

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. © 1997 Wiley‐Liss, Inc.

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 uptake 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, gamma-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.

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.

Huntington's disease (HD) is caused by a CAG repeat expansion in the HD gene, but how this mutation causes neuronal dysfunction and degeneration is unclear. Inhibition of glutamate uptake, which could cause excessive stimulation of glutamate receptors, has been found in animals carrying very long CAG repeats in the HD gene. In seven HD patients with moderate CAG expansions (40-52), repeat expansion and HD grade at autopsy were strongly correlated (r=0.88, p=0.0002). Uptake of [(3)H]glutamate was reduced by 43% in prefrontal cortex, but the level of synaptic (synaptophysin, AMPA receptors) and astrocytic markers (GFAP, glutamate transporter EAAT1) were unchanged. Glutamate uptake correlated inversely with CAG repeat expansion (r= -0.82, p=0.015). The reducing agent dithiothreitol improved glutamate uptake in controls, but not in HD brains, suggesting irreversible oxidation of glutamate transporters in HD. We conclude that impairment of glutamate uptake may contribute to neuronal dysfunction and degeneration in HD.