Two Metabolic Fuels,Glucose and Lactate,Differentially Modulate Exocytotic Glutamate Release from Cultured Astrocytes

Neurochemical Research - Astrocytes have a prominent role in metabolic homeostasis of the brain and can signal to adjacent neurons by releasing glutamate via a process of regulated exocytosis....     

Effects of Potassium and Glutamine on Metabolism of Glucose in Astrocytes

The metabolic effects of extracellular glutamine (2.5 mM) or high potassium (25 mM) on glucose metabolism were studied in cultured cerebellar astrocytes. High potassium caused an increased glycolytic flux and an increase in glutamine release. Exposure to glutamine increased glycolytic flux and alanine formation, indicating that glutamine uptake is an energy requiring process. The effects of glutamine and high potassium on glycolytic flux were additive. Formation of metabolites from [1-¹³C]glucose and [2-¹³C]acetate confirmed the effects of glutamine and high potassium on glycolytic metabolism. In the presence of extracellular glutamine, analysis of the ¹³C labeling patterns of citrate and glutamine indicated a decrease in the cycling ratio and/or pyruvate carboxylation and glutamine synthesis from [1-¹³C]glucose did occur, but was decreased. Exposure to high potassium led to extracellular accumulation of acetate, presumably through non-enzymatic decarboxylation of pyruvate.     

Carbon Balance Studies of Glucose Metabolism in Rat Cerebral Cortical Synaptosomes

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.     

Cellular Location of Glutamine Synthetase and Lactate Dehydrogenase in Oligodendrocyte-Enriched Cultures from Rat Brain

Glial cells were isolated from 1-week-old rat brain and cultured in a serum-free medium supplemented with the hormones insulin, hydrocortisone, and triiodothyronine. After 1 week in culture the cell population consisted mainly of galactocerebroside-positive cells (GC+; oligodendrocytes), the remainder of the cells being positive for glial fibrillary acidic protein (GFAP+; astrocytes). Oligodendrocytes were selectively removed from the cultures by complement-mediated cytolysis. The activities of glutamine synthetase and of various marker enzymes were measured in the nonlysed cells remaining after complement treatment of the cultures and in the culture medium containing proteins of the lysed cells. We found that the cellular activity of glutamine synthetase decreased in parallel with the lysis of GC+ cells and that the activity of glutamine synthetase in the supernatant increased. The activity of glycerol-3-phosphate dehydrogenase, a marker enzyme for oligodendrocytes, was no longer detectable in complement-treated cultures and the activity of glutamine synthetase was markedly lowered, whereas the activity of lactate dehydrogenase was as high as in untreated cultures. The location of glutamine synthetase both in oligodendrocytes and in astrocytes was confirmed by double-label immunocytochemistry with antisera against glutamine synthetase, GC, and GFAP. We conclude that in this culture system glutamine synthetase is expressed in both types of glial cells and that the activity of lactate dehydrogenase is at least one order of magnitude higher in astrocytes than in oligodendrocytes.     

Glucose,Lactate and Glutamine but not Glutamate Support Depolarization-Induced Increased Respiration in Isolated Nerve Terminals

Synaptosomes prepared from various aged and gene modified experimental animals constitute a valuable model system to study pre-synaptic mechanisms. Synaptosomes were isolated from whole brain and the XFe96 extracellular flux analyzer (Seahorse Bioscience) was used to study mitochondrial respiration and glycolytic rate in presence of different substrates. Mitochondrial function was tested by sequentially exposure of the synaptosomes to the ATP synthase inhibitor, oligomycin, the uncoupler FCCP (carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone) and the electron transport chain inhibitors rotenone and antimycin A. The synaptosomes exhibited intense respiratory activity using glucose as substrate. The FCCP-dependent respiration was significantly higher with 10 mM glucose compared to 1 mM glucose. Synaptosomes also readily used pyruvate as substrate, which elevated basal respiration, activity-dependent respiration induced by veratridine and the respiratory response to uncoupling compared to that obtained with glucose as substrate. Also lactate was used as substrate by synaptosomes but in contrast to pyruvate, mitochondrial lactate mediated respiration was comparable to respiration using glucose as substrate. Synaptosomal respiration using glutamate and glutamine as substrates was significantly higher compared to basal respiration, whereas oligomycin-dependent and FCCP-induced respiration was lower compared to the responses obtained in the presence of glucose as substrate. We provide evidence that synaptosomes are able to use besides glucose and pyruvate also the substrates lactate, glutamate and glutamine to support their basal respiration. Veratridine was found to increase respiration supported by glucose, pyruvate, lactate and glutamine and FCCP was found to increase respiration supported by glucose, pyruvate and lactate. This was not the case when glutamate was the only energy substrate.     

Effect of Aging on the Metabolism of Pyruvate and 3-Hydroxybutyrate in Nonsynaptic and Synaptic Mitochondria from Rat Brain

Abstract: Age-dependent changes in the oxidative metabolism in nonsynaptic and synaptic mitochondria from brains of 3, 12, and 24-month-old rats were investigated. When pyruvate and malate were used in conjunction as substrates, a significant reduction in State 3 respiration was observed in both mitochondrial populations from 12-and 24-month-old rats compared with 3-month-old animals. A similar age-dependent reduction in the oxidation of [1-¹¹C]pyruvate was also observed in nonsynaptic and synaptic mitochondria from senescent rats. Pyruvate dehydrogenase complex activity (both active and total) was, however, not decreased in the two mitochondrial populations from brains of 3, 12, and 24-month-old rats. When DL-3-hydroxybutyrate plus malate were used as substrates, a decrease in State 3 respiration was observed only in synaptic mitochondria from 24-month-old rats compared with 3- month-old animals. Similarly, an age-dependent reduction in the oxidation of 3-hydroxy[3-¹¹C]butyrate was also observed only in synaptic mitochondria from 12-and 24-month-old rats. However, a significant reduction in the activities of ketone body-metabolizing enzymes, namely, 3-hydroxybutyrate dehydrogenase, 3-ketoacid CoA transferase, and acetoacetyl-CoA thiolase was observed in both mitochondrlal populations from 12- and 24-month-old rats compared with 3 month-old animals. These findings show that specific alterations in oxidative metabolism occur in nonsynaptic and synaptic mitochondria from aging rats. The data also suggest that in addition to alterations in enzyme activities, permeability of anions (e.g. pyruvate) across the inner mitochondrial membrane may be altered in nonsynaptic and synaptic mitochondria from senescent animals.     

Energy Metabolism in Rat Brain: Inhibition of Pyruvate Decarboxylation by 3-Hydroxybutyrate in Neonatal Mitochondria

Abstract: The effect of 3-hydroxybutyrate on pyruvate decarboxylation by neonatal rat brain mitochondria and synaptosomes was investigated. The rate of [1 -¹⁴C]pyruvate decarboxylation (1 mm final concentration) by brain synaptosomes derived from 8-day-old rats was inhibited by 10% in the presence of 2 mm -d ,l -3-hydroxybutyrate and by more than 20% in the presence of 20 mm -d ,l -3-hydroxybutyrate. The presence of 2 mm -l ,d -3-hydroxybutyrate did not affect the rate of [1-¹⁴T]pyruvate decarboxylation (1 mm final concentration) by brain mitochondria; however, at a concentration of 20 mm -d ,l -3-hydroxybutyrate, a marked inhibition was seen in preparations from both 8-day-old (35% inhibition) and 21-day-old (24% inhibition) but not in those from adult rats. Although the presence of 100 mm -K⁺ in the incubation medium stimulated the rate of pyruvate decarboxylation by approximately 50% compared with the rate in the presence of 1 mm -K⁺, the presence of 20 mm -d ,l -3-hydroxybutyrate still caused a marked inhibition in both media (1 and 100 mm -K⁺). The presence of 20 mm -d ,l -3-hydroxybutyrate during the incubation caused an approximately 20% decrease in the level of the active form of the pyruvate dehydrogenase complex in brain mitochondria from 8-day-old rats. The concentrations of ATP, ADP, NAD⁺, NADH, acetyl CoA, and CoA were measured in brain mitochondria from 8-day-old rats incubated in the presence of 1 mm -pyruvate alone or 1 mm -pyruvate plus 20 mm -d ,l -3-hydroxybutyrate. Neither the ATP/ADP nor the NADH/NAD⁺ ratio showed significant changes. The acetyl CoA/CoA ratio was significantly increased by more than twofold in the presence of 3-hydroxybutyrate. The possible mechanisms and physiological significance of 3-hydroxybutyrate inhibition of pyruvate decarboxylation in neonatal rat brain mitochondria are discussed.     

Characteristics of Glucose Transport in Neuronal Cells and Astrocytes from Rat Brain in Primary Culture

Glucose transport systems in cultured neuronal cells and astrocytes of rats were characterized by measuring the uptake of 2-deoxy-D-[3H]glucose ([3H]2-DG) into the cells. Various sugars inhibited 2-DG uptake by neuronal cells and astrocytes similarly, a finding indicating that the substrate specificities of the transporters in the two types of cells were almost the same. However, the Km values for 2-DG of neuronal cells and astrocytes were 1.7 and 0.36 mM, respectively. The uptake of 2-DG was strongly inhibited by cytochalasin B. Nucleosides, such as adenosine, inosine, and uridine, inhibited 2-DG uptake competitively in both neuronal cells and astrocytes. The uptake by both types of cells were also inhibited by forskolin, but not by cyclic AMP, an observation suggesting that forskolin bound directly to the transporters to cause inhibition. Its inhibition was competitive in astrocytes and noncompetitive in neuronal cells. Astrocytes contained a glucose transporter with a subunit molecular weight of 45K, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after photoaffinity labeling using [3H]cytochalasin B as a probe.     

Compartmentation of Lactate Originating from Glycogen and Glucose in Cultured Astrocytes

Brain glycogen metabolism was investigated by employing isofagomine, an inhibitor of glycogen phosphorylase. Cultured cerebellar and neocortical astrocytes were incubated in medium containing [U-¹³C]glucose in the absence or presence of isofagomine and the amounts and percent labeling of intra- and extracellular metabolites were determined by mass spectrometry (MS). The percent labeling in glycogen was markedly decreased in the presence of isofagomine. Surprisingly, the percent labeling of intracellular lactate was also decreased demonstrating the importance of glycogen turnover. The decrease was limited to the percent labeling in the intracellular pool of lactate, which was considerably lower compared to that observed in the medium in which it was close to 100%. These findings indicate compartmentation of lactate derived from glycogenolysis and that derived from glycolysis. Inhibiting glycogen degradation had no effect on the percent labeling in citrate. However, the percent labeling of extracellular glutamine was slightly decreased in neocortical astrocytes exposed to isofagomine, indicating an importance of glycogen turnover in the synthesis of releasable glutamine. In conclusion, the results demonstrate that glycogen in cultured astrocytes is continuously synthesized and degraded. Moreover, it was found that lactate originating from glycogen is compartmentalized from that derived from glucose, which lends further support to a compartmentalized metabolism in astrocytes. Special issue dedicated to Dr. Bernd Hamprecht.     

Effect of pH on Glutamine Content Derived from Exogenous Glutamate in Astrocytes

Abstract: A shift in pH from 7.4 to 7.8 in the incubation solution caused a 3.4-fold increase in the free glutamine content of mouse cerebral astrocytes that were incubated with glutamate (100 μ M ) and ammonium (100 μ M ). This large and reversible steady-state increase in glutamine content was accompanied by smaller transient increases in the following: (a) net formation of glutamine; (b) clearance of glutamate from the incubation solution; and (c) glutamate content. The content of glutamine was reduced markedly by omission of either glutamate or ammonium from the incubation solution, or by inhibition of glutamine synthetase activity with methionine sulfoximine. The rate at which glutamine was exported from the astrocytes was unaffected by the pH change. The effects of pH on the concentration of free ammonia or on glutamate uptake do not appear to mediate the increase in glutamine content. Uptake of exogenous glutamine was little affected by the pH change. Therefore, possible mediation of the effect by an increase in intracellular pH must be considered. The response to altered pH described here may provide a cellular basis for the increased level of brain glutamine observed in hyperammonemia.     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的：应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法：选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果：中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）,乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义（P〈0．05）,葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义（P〈0．05）;重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值（P〈0．05）,乳酸值亦明显高于中度昏迷组测量值（P〈O．05）,葡萄糖测量值两组术前测量值差异无统计学意义（P〉0．05）,自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论：结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

脑内谷氨酸、乳酸以及葡萄糖对中重型脑外伤患者病情的评价意义

目的:应用微透析技术对于中重型脑外伤患者进行持续脑内谷氨酸、乳酸以及葡萄糖,分析结果以评价以上因素与患者病情的关系。方法:选择我院2006年3月-2009年11月颅脑外科和ICU收治的急性颅脑损伤患者32例,根据GCS分为重度昏迷组和中度昏迷组,均行急诊手术治疗,并在手术直视下置入微透析探针,置入后第4天拔除,定时收集透析液约10μl,于术前以及术后第1、2、3、4天收取标本并立即送检,分别检测患者标本中的谷氨酸、乳酸和葡萄糖含量,并结合患者预后进行分析。结果:中度昏迷组乳酸与谷氨酸值在手术后呈进行性下降,与术前比较,术后第2、3、4天差异有统计学意义(P<0.05),乳酸值的变化与谷氨酸变化趋势相近,与术前比较,在术后第3、4天差异有统计学意义(P<0.05),葡萄糖值与术前比较,术后第2、3、4天差异有统计学意义(P<0.05);重度昏迷组谷氨酸、乳酸和葡萄糖与术前比较,三者均在第4天出现有统计学意义的变化。重度昏迷组谷氨酸测量值在各个观察点均高于中度昏迷组测量值(P<0.05),乳酸值亦明显高于中度昏迷组测量值(P<0.05),葡萄糖测量值两组术前测量值差异无统计学意义(P>0.05),自术后第1天始,中度昏迷组各个时间点测量值明显高于重度昏迷组。结论:结合患者的GCS评分,应用微透析技术实时监测患者脑内谷氨酸、乳酸以及葡萄糖的含量变化,能很好的把握患者的病情,有效指导临床治疗。     

Effects of Bilobalide on Hypoxia/Hypoglycemia-Stimulated Glutamate Efflux from Rat Cortical Brain Slices

The aim of this study was to investigate the effects of bilobalide, the postulated active constituent of Ginkgo biloba, on the release of glutamate elicited by hypoxia/hypoglycemia. Cortical slices were prepared from rat brain and perfused with normal artificial cerebrospinal fluid (aCSF) or aCSF made hypoxic by gassing with nitrogen, and hypoglycemic by removal of glucose. The perfusate was assayed for glutamate by HPLC. After 30 minutes, perfusion with hypoxic/hypoglycemic aCSF glutamate levels in the perfusate were increased approximately 5-fold. Bilobalide at 1, 10, and 100 M, when perfused together with hypoxic/hypoglycemic aCSF, significantly reduced the release of glutamate. This study suggests that the reported neuroprotective properties of bilobalide may, in part, be mediated through its ability to reduce glutamate efflux, thus leading to a decrease in the excitotoxic effects of this neurotransmitter.     

Valproate Increases Glutaminase and Decreases Glutamine Synthetase Activities in Primary Cultures of Rat Brain Astrocytes

Abstract: It has been proposed that hyperammonemia may be associated with valproate therapy. As astrocytes are the primary site of ammonia detoxification in brain, the effects of valproate on glutamate and glutamine metabolism in astrocytes were studied. It is well established that, because of compartmentation of glutamine synthetase, astrocytes are the site of synthesis of glutamine from glutamate and ammonia. The reverse reaction is catalyzed by the ubiquitous enzyme glutaminase, which is present in both neurons and astrocytes. In astrocytes exposed to 1.2 mM valproate, glutaminase activity increased 80% by day 2 and remained elevated at day 4; glutamine synthetase activity was decreased 30%. Direct addition of valproate to assay tubes with enzyme extracts from untreated astrocytes had significant effects only at concentrations of 10 and 20 mM, When astrocytes were exposed for 4 days to 0.3, 0.6, or 1.2 mM valproate and subsequently incubated with l -[U-¹⁴C]glutamate, label incorporation into [¹⁴C]glutamine was decreased by 11, 25, and 48%, respectively, and is consistent with a reduction in glutamine synthetase activity. Label incorporation from l -[U-¹⁴C]glutamate into [¹⁴C]aspartate also decreased with increasing concentrations of valproate. Following a 4-day exposure to 0.6 mM valproate, the glutamine levels increased 40% and the glutamate levels 100%. These effects were not directly proportional to valproate concentration, because exposure to 1.2 mM valproate resulted in a 15% decrease in glutamine levels and a 25% increase in glutamate levels compared with control cultures. Intracellular aspartate was inversely proportional to all concentrations of extracellular valproate, decreasing 60% with exposure to 1.2 mM valproate. These results indicate that valproate increases glutaminase activity, decreases glutamine synthetase activity, and alters Krebs-cycle activity in astrocytes, suggesting a possible mechanism for hyperammonemia in brain during valproate therapy.     

Oxygen Dependence of Glucose and Acetylcholine Metabolism in Slices and Synaptosomes from Rat Brain

Abstract: Previous studies have shown that a reduction in the O₂ tension of the blood from 120 torr to 57 torr (hypoxic hypoxia) decreases brain acetylcholine (ACh) synthesis. To determine if this decrease is due to a direct impairment of ACh metabolism or to an indirect effect mediated by other neurotransmitter systems, we studied ACh formation in rat brain slices and synaptosomes. At O₂ tensions ranging from 760 to less than 1 torr, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were determined. In slices, the first decreases were observed in the rate of ¹⁴CO₂ production and [¹⁴C]ACh synthesis at an O₂ tension of 152 torr. The ATP level started to decline at 53–38 torr, and a reduction in the ATP/ADP ratio was first found at and below 19 torr. Lactate formation was maximally stimulated at 38–19 torr. Synaptosomes responded differently than brain slices to reduced O₂ tensions. In synaptosomes, ¹⁴CO₂ production and [¹⁴C]ACh synthesis from [U-¹⁴C]glucose, the levels of lactate and ATP, and the ATP/ADP ratio were unaltered if a minimum O₂ tension of 19 torr was maintained. Despite the difference in sensitivities to decreases in O₂ levels, there is a curvilinear relationship between [U-¹⁴C]glucose decarboxylation and [¹⁴C]ACh synthesis at various O₂ tensions for both tissue preparations with a high coefficient of determination (R²= 0.970). The difference in the metabolic sensitivity of slices and synaptosomes to a reduced O₂ level may be explained by the greater distance O₂ must diffuse in slices. The results are discussed in comparison with hypoxia in vivo.     

Interrelationships of Leucine and Glutamate Metabolism in Cultured Astrocytes

Abstract: The aim was to study the extent to which leu-cine furnishes α-NH₂ groups for glutamate synthesis via branched-chain amino acid aminotransferase. The transfer of N from leucine to glutamate was determined by incubating astrocytes in a medium containing [¹⁵N]leucine and 15 unlabeled amino acids; isotopic abundance was measured with gas chromatography-mass spectrometry. The ratio of labeling in both [¹⁵N]glutamate/[¹⁵N]leucine and [2-¹⁵N]glutamine/[¹⁵N]leucine suggested that at least one-fifth of all glutamate N had been derived from leucine nitrogen. At the same time, enrichment in [¹⁵N]leucine declined, reflecting dilution of the ¹⁶N label by the unlabeled amino acids that were in the medium. Isotopic abundance in [¹⁶N]-isoleucine increased very quickly, suggesting the rapidity of transamination between these amino acids. The appearance of ¹⁵N in valine was more gradual. Measurement of branched-chain amino acid transaminase showed that the reaction from leucine to glutamate was approximately six times more active than from glutamate to leucine (8.72 vs. 1.46 nmol/min/mg of protein). However, when the medium was supplemented with α-ketoisocaproate (1 mM), the ketoacid of leucine, the reaction readily ran in the “reverse” direction and intraastrocytic [glutamate] was reduced by ～50% in only 5 min. Extracellular concentrations of α-ketoisocaproate as low as 0.05 mM significantly lowered intracellular [glutamate]. The relative efficiency of branched-chain amino acid transamination was studied by incubating astrocytes with 15 unlabeled amino acids (0.1 mM each) and [¹⁵N]glutamate. After 45 min, the most highly labeled amino acid was [¹⁵N]alanine, which was closely followed by [¹⁵N]leucine and [¹⁵N]isoleucine. Relatively little ¹⁵N was detected in any other amino acids, except for [¹⁵N]serine. The transamination of leucine was ～17 times greater than the rate of [1-¹⁴C]leucine oxidation. These data indicate that leucine is a major source of glutamate nitrogen. Conversely, reamination of a-ketoisocaproate, the ketoacid of leucine, affords a mechanism for the temporary “buffering” of intracellular glutamate.     

High Sensitivity of Glutamate Uptake to Extracellular Free Arachidonic Acid Levels in Rat Cortical Synaptosomes and Astrocytes

By using both synaptosomes and cultured astrocytes from rat cerebral cortex, we have investigated the inhibitory action of arachidonic acid on the high-affinity glutamate uptake systems, focusing on the possible physiological significance of this mechanism. Application of arachidonic acid (1-100 microM) to either preparation leads to fast (within 30 s) and largely reversible reduction in the uptake rate. When either melittin (0.2-1 microgram/ml), a phospholipase A2 activator, or thimerosal (50-200 microM), which inhibits fatty acid reacylation in phospholipids, is applied to astrocytes, both an enhancement in extracellular free arachidonate and a reduction in glutamate uptake are seen. The two effects display similar dose dependency and time course. In particular, 10% uptake inhibition correlates with 30% elevation in free arachidonate, whereas inhibition greater than or equal to 60% is paralleled by threefold stimulation of arachidonate release. In the presence of albumin (1-10 mg/ml), a free fatty acid-binding protein, inhibition by either melittin, thimerosal, or arachidonic acid is prevented and an enhancement of glutamate uptake above the control levels is observed. Our data show that neuronal and glial glutamate transport systems are highly sensitive to changes in extracellular free arachidonate levels and suggest that uptake inhibition may be a relevant mechanism in the action of arachidonic acid at glutamatergic synapses.     

Metabolism of Lactate in the Rat Brain During the Early Neonatal Period

The metabolism of lactate in isolated cells from early neonatal rat brain has been studied. In these circumstances, lactate was mainly oxidized to CO2, although a significant portion was incorporated into lipids (78% sterols, 4% phosphatidylcholine, 2% phosphatidylethanolamine, and 1% phosphatidylserine). The rate of lactate incorporation into CO2 and lipids was higher than those found for glucose and 3-hydroxybutyrate. Lactate strongly inhibited glucose oxidation through the pyruvate dehydrogenase-catalyzed reaction and the tricarboxylic acid cycle while scarcely affecting glucose utilization by the pentose phosphate pathway. Lipogenesis from glucose was strongly inhibited by lactate without relevant changes in the rate of glycerol phosphate synthesis. These results suggest that lactate inhibits glucose utilization at the level of the pyruvate dehydrogenase-catalyzed reaction, which may be a mechanism to spare glucose for glycerol and NADPH synthesis. The effect of 3-hydroxybutyrate inhibiting lactate utilization only at high concentrations of 3-hydroxybutyrate suggests that before ketogenesis becomes active, lactate may be the major fuel for the neonatal brain. (-)-Hydroxycitrate and aminooxyacetate markedly inhibited lipogenesis from lactate, suggesting that the transfer of lactate carbons through the mitochondrial membrane is accomplished by the translocation of both citrate and N-acetylaspartate.