Neuronal cultures in vitro readily oxidized both D-[ 14 C]glucose and L-[14C]lactate to 14 CO2, whereas astroglial cultures oxidized both substrates sparingly and metabolized glucose predominantly to lactate and released it into the medium. [ 14 C]Glucose oxidation to 14 CO2 varied inversely with unlabeled lactate concentration in the medium, particularly in neurons, and increased progressively with decreasing lactate concentration. Adding unlabeled glucose to the medium inhibited [ 14 C]lactate oxidation to 14 CO2 only in astroglia but not in neurons, indicating a kinetic preference in neurons for oxidation of extracellular lactate over intracellular pyruvate͞ lactate produced by glycolysis. Protein kinase-catalyzed phosphorylation inactivates pyruvate dehydrogenase (PDH), which regulates pyruvate entry into the tricarboxylic acid cycle. Dichloroacetate inhibits this kinase, thus enhancing PDH activity. In vitro dichloroacetate stimulated glucose and lactate oxidation to CO2 and reduced lactate release mainly in astroglia, indicating that limitations in glucose and lactate oxidation by astroglia may be due to a greater balance of PDH toward the inactive form. To assess the significance of astroglial export of lactate to neurons in vivo, we attempted to diminish this traffic in rats by administering dichloroacetate (50 mg͞kg) intravenously to stimulate astroglial lactate oxidation and then examined the effects on baseline and functionally activated local cerebral glucose utilization (lCMRglc). Dichloroacetate raised baseline lCMRglc throughout the brain and decreased the percent increases in lCMRglc evoked by functional activation. These studies provide evidence in support of the compartmentalization of glucose metabolism between astroglia and neurons but indicate that the compartmentalization may be neither complete nor entirely obligatory.G lucose is an essential and normally almost exclusive substrate for cerebral energy metabolism (1). As in other tissues, it is metabolized in brain in two sequential pathways, first to pyruvate͞lactate by glycolysis in cytosol, followed by oxidation in mitochondria to CO 2 and H 2 O. It was recently proposed that the glycolytic and oxidative components of glucose and glycogen metabolism are compartmentalized not only between cytosol and mitochondria but also between astroglia and neurons, i.e., glucose and glycogen metabolism in astroglia to lactate, which is then exported to neurons where it is oxidized to provide the ATP needed for neuronal function (2, 3). Arguments in support of this hypothesis are: (i) capillaries in brain are largely enveloped by astroglial processes that present a barrier to the transport of glucose from blood to neurons; (ii) glycogen in brain is confined almost entirely to astrocytes; (iii) astrocytes in culture readily metabolize glucose to lactate and release it into the medium (4, 5); and (iv) glutamate, the most prevalent excitatory neurotransmitter in brain, stimulates aerobic glycolysis in cultured astrocytes (6, 7). Much of the evidence suppo...

ABSTRACT

Studies of local glucose utilization in neural tissues in vivo with the autoradiographic [¹⁴C]deoxyglucose method have demonstrated that energy metabolism increases almost linearly with the degree of functional activation, i.e. spike frequency, in the terminal projection zones of activated pathways. The increased metabolism is found in neuropil and is minimal or undetectable in neuronal cell bodies. Electrical stimulation, increased extracellular [K⁺] ([K⁺]_o), or opening of Na⁺ channels with veratridine stimulates metabolism in neural tissues, and this increase is blocked by ouabain, a specific inhibitor of Na⁺,K⁺-ATPase. Activation of this enzyme to restore ionic gradients across cellular membranes appears to mediate the function-related increase in energy metabolism. The metabolic activation is, therefore, not directly related to the functional activity itself but to processes operating to recover from that activity. The limited spatial resolution of the [¹⁴C]DG method precludes identification of cellular elements in neuropil participating in the metabolic activation, e.g. axonal terminals, dendrites, or astrocytic processes enveloping the synapses. We have, therefore, attempted to simulate in vitro conditions to be expected from functional activation and increased spike activity in vivo, e.g. increased extracellular [K⁺], intracellular [Na⁺], or extracellular neurotransmitter levels, and examined their effects on glucose metabolism in neurons and astroglia in culture. Increased [K⁺]_o stimulated [¹⁴C]DG phosphorylation in neuronal and mixed neuronal-astroglial cultures, but not in astroglial cultures assayed in bicarbonate buffer; it did occasionally stimulate metabolism in astroglia when assayed in HEPES or phosphate buffers, but these effects were variable and inconsistent. Veratridine (75 μM) stimulated [¹⁴C]DG phosphorylation in neurons and astroglia; these stimulations were blocked by 1 mM ouabain or 10 μM tetrodotoxin (TTX), which blocks voltage-dependent Na⁺ channels. The Na⁺ ionophore monensin (10 μM) doubled the rate of metabolism, a stimulation that was only partially blocked by ouabain and unaffected by TTX. L-Glutamate (500 μM) stimulated [¹⁴C]DG phosphorylation in astroglia, but this stimulation was probably secondary to Na⁺ uptake into the cells via a sodium/glutamate co-transporter because it was not blocked by inhibitors of NMDA or non-NMDA receptors but was absent in Na⁺-free medium. These results indicate that astroglia contribute to the increased energy metabolism in neuropil during functional activation by mechanisms that promote Na⁺ entry into the cells.

Changing the medium of primary cell cultures of CNS origin causes severe damage that is mediated via the N-methyl-D-aspartate (NMDA)-type of glutamate receptors and dependent on the presence of glutamine in the medium. Data presented here show that glutamine has two roles in culture damage: glutamine is contaminated with a small amount of glutamate, which is responsible for initiating culture damage, and glutamine is the source of the glutamate that is produced extracellularly in damaged cultures. The NMDA receptor plays a critical role minutes after medium change when the glutamate contaminating the glutamine binds to NMDA receptors; during this time, addition of a low level (10-20 microM) of 2-amino-5-phosphonovaleric acid can block most culture damage and the appearance of extracellular glutamate. A higher level (300 microM) of 2-amino-5-phosphonovaleric acid can protect cultures when added at much later times (30-60 min). Between 3 and 6 h after medium change, the concentration of extracellular glutamate starts to rise and accumulates until the end of the culture period (20 h). Medium removed from cultures at 3 h or later after medium change and incubated alone (i.e., with no cells) also continues to generate glutamate; filtration (0.22 microns pore size) or centrifugation (18,000 g) stops the appearance of this glutamate. 6-Diazo-5-oxo-L-norleucine, an inhibitor of the mitochondrial enzyme glutaminase, blocks the generation of glutamate. Mitochondria or mitochondrial fragments are probably released from the damaged cells and then convert extracellular glutamine to glutamate, resulting in generation of a high extracellular glutamate concentration.