On‐line glucose and lactate monitoring in rat striatum: effect of malonate and correlation with histological damage

Microdialysis of glucose, lactate and glycerol was performed to monitor brain insults and to predict brain injury in a rat model using the mitochondrial toxin malonate (5–100 mm ). Striatal dialysates were analyzed off‐line using a CMA 600 microdialysis analyzer or on‐line using flow‐injection analysis and biosensors for glucose and lactate. Histological damage was evaluated using stereological principles. Lactate (baseline ca. 1 mm ) was dose‐dependently increased, reaching a maximum of five‐ to six‐fold increase, whereas glucose (baseline 1–2 mm ) was decreased (>50%) by malonate >20 mm . These changes were reversible upon perfusion with normal Ringer's. Transient increases in glycerol (four‐ to eight‐fold) were only observed in some rats, and were not dose‐dependent. Histological damage was related to the perfused malonate concentration, but was not significantly correlated with lactate or glycerol changes.     

Partitioning of CO2 Production Between Glucose and Lactate in Excised Sympathetic Ganglia, with Implications for Brain

Abstract: Chains of lumbar sympathetic ganglia from 15-day-old chicken embryos were incubated for 4 h at 36°C in a bicarbonate-buffered salt solution equilibrated with 5% CO₂-95% O₂. Glucose (1–10 m M ), lactate (1–10 m M ), [U-¹⁴C]glucose, [1-¹⁴C]glucose, [6-¹⁴C]glucose, and [U-¹⁴C]lactate were added as needed. ¹⁴CO₂ output was measured continuously by counting the radioactivity in gas that had passed through the incubation chamber. Lactate reduced the output of CO₂ from [U-¹⁴C]glucose, and glucose reduced that from [U-¹⁴C]lactate. When using uniformly labeled substrates in the presence of 5.5 m M glucose, the output of CO₂ from lactate exceeded that from glucose when the lactate concentration was >2 m M . The combined outputs at each concentration tested were greater than those from either substrate alone. The ¹⁴CO₂ output from [1-¹⁴C]glucose always exceeded that from [6-¹⁴C]glucose, indicating activity of the hexose monophosphate shunt. Lactate reduced both of these outputs, with the maximum difference between them during incubation remaining constant as the lactate concentration was increased, suggesting that lactate may not affect the shunt. Modeling revealed many details of lactate metabolism as a function of its concentration. Addition of a blood-brain barrier to the model suggested that lactate can be a significant metabolite for brain during hyperlactemia, especially at the high levels reached physiologically during exercise.     

Acute Depression of Energy Metabolism after Microdialysis Probe Implantation is Distinct from Ischemia-Induced Changes in Mouse Brain

The current study used measurements of metabolites and markers of membrane integrity to determine the most suitable time point for microdialysis experiments following probe implantation. Leakage of Evans blue and sodium fluorescein indicated increased BBB permeability only immediately (15 min), but not 1.5 and 24 h following probe implantation. Acute implantation decreased glucose and lactate levels relative to the levels after 24 h (to 13–37% and 25–60%, respectively). No change in extracellular levels of glutamate or glycerol was seen. In comparison to acute probe implantation, the pattern of damage under brain ischemia (middle cerebral artery occlusion) differed: While glucose levels dropped, lactate levels rose after ischemia, and glutamate (tenfold) and glycerol (eightfold) increased sharply. In conclusion, acute implantation of a microdialysis probe causes transient depression of the energy metabolites, glucose and lactate, likely due to injury-induced hypermetabolism. However, no massive tissue damage or severe ischemic conditions around the probe occur.     

Gender-specific response to interstitial angiotensin II in human white adipose tissue.

Angiotensin II is synthesized locally in various tissues. However, the role of interstitial angiotensin II in the regulation of regional metabolism and tissue perfusion has not as yet been clearly defined. We characterized the effect of interstially applied angiotensin II in abdominal subcutaneous adipose tissue of young, normal-weight, healthy men (n = 8) and women (n = 6) using the microdialysis technique. Adipose tissue was perfused with 0.01, 0.1, and 1 micro M angiotensin II. Dialysate concentrations of ethanol, glycerol, glucose, and lactate were measured to assess changes in blood flow (ethanol dilution technique), lipolysis, and glycolysis, respectively. Baseline ethanol ratio and dialysate lactate were both significantly higher, whereas dialysate glucose was significantly lower in men vs. women. In men, ethanol ratio and dialysate glucose, lactate and glycerol did not change significantly during perfusion with angiotensin II. In women, however, angiotensin II induced a significant increase in ethanol ratio and dialysate lactate and a decrease in dialysate glucose close to values found for men and this response was almost maximal at the lowest angiotensin II concentration used. Dialysate glycerol did not change significantly. We conclude that baseline blood flow and glucose supply and metabolism is significantly higher in women than in men. In men, interstitial Ang II has only a minimal effect on adipose tissue blood flow and metabolism. In women, however, a high physiological concentration of interstitial angiotensin II can reduce blood flow down to values found in men. This is associated with an impaired glucose supply and metabolism. Additionally, Ang II inhibits lipolysis.     

Inhibition of Brain Glycolysis by Aluminum

Abstract: Aluminum inhibited both the cytosolic and mitochondrial hexokinase activities in rat brain. The IC₅₀ values were between 4 and 9 μ M . Aluminum was effective at mildly acidic (pH 6.8) or slightly alkaline (pH 7.2–7.5) pH, in the presence of a physiological level of magnesium (0.5 m M ). However, saturating (8 m M ) magnesium antagonized the effect of aluminum on both forms of hexokinase activity. Other enzymes examined were considerably less sensitive to inhibition by aluminum. The IC₅₀ of aluminum for phosphofructokinase was 1.8 m M and for lactate dehydrogenase 0.4 m M . At 10–600 μ M , aluminum actually stimulated pyruvate kinase. Aluminum also inhibited lactate production by rat brain extracts: this effect was much more marked with glucose as substrate than with glucose-6-phosphate. However, the IC₅₀ for inhibiting lactate production using glucose as substrate was 280 μ M , higher than that required to inhibit hexokinase. This concentration of aluminum is comparable to those reportedly found in the brains of patients who had died with dialysis dementia and in the brains of some of the patients who had died with Alzheimer disease. Inhibition of carbohydrate utilization may be one of the mechanisms by which aluminum can act as a neurotoxin.     

Effects of a Single Therapeutic Dose of Glycerol on Cerebral Metabolism in the Brains of Young Mice: Possible Increase in Brain Glucose Transport and Glucose Utilization

Abstract: This is a study of the effects of a single “therapeutic” dose of glycerol [2 g(22 mmol)/kg i.p.] on brain carbohydrate and energy metabolism in normal nursing weanling mice. Findings were correlated with brain water and electrolyte content and with metabolite changes in plasma, red blood cells, and liver. Plasma glycerol levels peaked at 21 mM 7.5 min after injection and returned to the control value, 0.16 mM, by 2 h. Plasma Na⁺ concentration decreased and plasma protein increased for as long as 2 h after injection. Although red blood cells were freely permeable to glycerol, there was no evidence for glycerol metabolism in these cells. Glycerol levels in liver paralleled those in plasma. Glycerol injection increased liver glucose concentration 23% and doubled hepatic glycerol-1-phosphate levels. Liver ATP levels were reduced 24% after glycerol injection. Brain water concentration was significantly reduced from 7.5 min to 30 min after glycerol injection; brain Na⁺ and K⁺ levels were unchanged. There was no evidence for glycerol entry into brain (the amount detected in brain tissue could be explained by the glycerol content in the blood of the brain). While plasma glucose increased 33%, brain glucose increased 87%. Concomitantly there were statistically significant increases in fructose-1,6-diphosphate, lactate, α-ketoglutarate, and malate levels. The disproportionately high brain glucose value suggests increased transport of glucose from the blood to the brain. Increases in fructose-1,6-diphosphate, lactate, α-ketoglutarate, and malate are compatible with an increased metabolic flux in the glycolytic pathway and Krebs citric acid cycle. As has been previously shown for urea and/or mannitol, these changes may result from the effects of the hyperosmolar glycerol solution on the blood-brain barrier and on cerebral glucose utilization. The sustained lowering of plasma Na⁺ concentration after a single “therapeutic” glycerol injection suggests a need for monitoring plasma Na⁺ levels in the clinical situation. Possible lowering of hepatic ATP levels by the use of glycerol in humans is another concern.     

Peroxisome proliferator-activated receptor alpha (PPARalpha) influences substrate utilization for hepatic glucose production

The hypoglycemia seen in the fasting PPARalpha null mouse is thought to be due to impaired liver fatty acid beta-oxidation. The etiology of hypoglycemia in the PPARalpha null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPARalpha null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-(13)C(6)]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-(13)C(3)]lactate or [2-(13)C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPARalpha null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPARalpha null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-(13)C(3)]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPARalpha null mice. In contrast, when [2-(13)C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPARalpha null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPARalpha null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPARalpha null mice than in controls. In conclusion, PPARalpha receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.     

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.     

Endocrine and metabolic responses to stress in a laboratory population of the tropical damselfish Acanthochromis polyacanthus

The effects of confinement and exercise on the stress response of the spiny damselfish Acanthochromis polyacanthus were investigated in a laboratory stock of fish. Cultured spiny damselfish had basal plasma cortisol values (<16 ng ml⁻¹) similar to those found in wild fish, and basal plasma glucose and lactate levels that were similar to those found in other teleosts. Plasma cortisol concentrations increased in response to stress with a latency period of 5–10 min. Removal of the stressor resulted in partial recovery of cortisol levels by 24 h. Plasma glucose levels increased in response to stress in all experiments with significant increases occurring within 15 min of the imposition of stress. Elevations in plasma glucose concentrations were not initially reflected in changes in liver or muscle glycogen content, with significant reductions in liver glycogen concentrations only occurring in response to extended periods of stress. In contrast to many temperate species, plasma lactate concentrations did not consistently increase in response to stress, suggesting that the stress response in spiny damselfish is not strongly characterized by anaerobiosis.     

Acetate transport and utilization in the rat brain

Acetate, a glial-specific substrate, is an attractive alternative to glucose for the study of neuronal-glial interactions. The present study investigates the kinetics of acetate uptake and utilization in the rat brain in vivo during infusion of [2-¹³C]acetate using NMR spectroscopy. When plasma acetate concentration was increased, the rate of brain acetate utilization (CMR_ace) increased progressively and reached close to saturation for plasma acetate concentration > 2–3 mM, whereas brain acetate concentration continued to increase. The Michaelis–Menten constant for brain acetate utilization (      = 0.01 ± 0.14 mM) was much smaller than for acetate transport through the blood–brain barrier (BBB) (      = 4.18 ± 0.83 mM). The maximum transport capacity of acetate through the BBB (      = 0.96 ± 0.18 μmol/g/min) was nearly twofold higher than the maximum rate of brain acetate utilization (      = 0.50 ± 0.08 μmol/g/min). We conclude that, under our experimental conditions, brain acetate utilization is saturated when plasma acetate concentrations increase above 2–3 mM. At such high plasma acetate concentration, the rate-limiting step for glial acetate metabolism is not the BBB, but occurs after entry of acetate into the brain.     

Glycerol Production from Glucose and Fructose by 3T3-L1 Cells: A Mechanism of Adipocyte Defense from Excess Substrate

Cultured adipocytes (3T3-L1) produce large amounts of 3C fragments; largely lactate, depending on medium glucose levels. Increased glycolysis has been observed also in vivo in different sites of rat white adipose tissue. We investigated whether fructose can substitute glucose as source of lactate, and, especially whether the glycerol released to the medium was of lipolytic or glycolytic origin. Fructose conversion to lactate and glycerol was lower than that of glucose. The fast exhaustion of medium glucose was unrelated to significant changes in lipid storage. Fructose inhibited to a higher degree than glucose the expression of lipogenic enzymes. When both hexoses were present, the effects of fructose on gene expression prevailed over those of glucose. Adipocytes expressed fructokinase, but not aldolase b. Substantive release of glycerol accompanied lactate when fructose was the substrate. The mass of cell triacylglycerol (and its lack of change) could not justify the comparatively higher amount of glycerol released. Consequently, most of this glycerol should be derived from the glycolytic pathway, since its lipolytic origin could not be (quantitatively) sustained. Proportionally (with respect to lactate plus glycerol), more glycerol was produced from fructose than from glucose, which suggests that part of fructose was catabolized by the alternate (hepatic) fructose pathway. Earlier described adipose glycerophophatase activity may help explain the glycolytic origin of most of the glycerol. However, no gene is known for this enzyme in mammals, which suggests that this function may be carried out by one of the known phosphatases in the tissue. Break up of glycerol-3P to yield glycerol, may be a limiting factor for the synthesis of triacylglycerols through control of glycerol-3P availability. A phosphatase pathway such as that described may have a potential regulatory function, and explain the production of glycerol by adipocytes in the absence of lipolytic stimulation.     

Effect of eland density and foraging on Combretum apiculatum physiognomy in a semi-arid savannah

We studied the impacts of eland density on the physiognomy of Combretum apiculatum . We divided the study area into two zones: high eland density zone (≤8 km from watering point) and low eland density zone (≥8 km from watering point). Eland density was determined in each zone using walking transects. Combretum apiculatum height and diameter and levels of eland damage were measured in each zone. Eland density was 1.92 and 0.86 km⁻² for high and low-density zones respectively. We recorded 239 C. apiculatum trees, 138 in low-density zone and 101 in high-density zone. Mean C. apiculatum height was 2.88 ± 0.67 m and 5.05 ± 1.04 m for high and low eland density zones respectively. High eland density prevented the recruitment of C. apiculatum from the 2.6–5.5 m height class to the >5.6 m height class. Although extensive tree damage occurred at <2.5 m height stratum, C. apiculatum showed resilience as recruitment into the 2.6–5.5 m height class continued. We concluded that high eland density prevents recruitment of C. apiculatum to higher height classes while at the same time causing extensive tree damage at lower height strata.     

Analysis of glucose and lactate in hippocampal dialysates of rats during the operant conditioned reflex using microdialysis

Changes of extracellular glucose and lactate in hippocampus for freely moving rats during the operant conditioned reflex were examined simultaneously. Samples of the dialysate were assayed for both glucose and lactate using in vivo microdialysis and a microbore flow injection analysis-immobilized enzyme reactor-electrochemical detection (FIA-IMER-ECD) system. Microdialysis samplings were conducted in a Skinner box where lights were delivered as conditioned stimuli (CS) paired with foot shocks as unconditioned stimuli (US). In the treatment group the concentration of glucose and lactate showed no fluctuations during the whole process. However, in the control group in which the rats were exposed to many foot shocks, lactate levels decreased by 19% below baseline during the behavioral session and glucose showed a delayed decrease (by 18%). Compared with glucose, lactate can immediately indicate the dynamic changes in brain.     

Lactate uptake contributes to the NAD(P)H biphasic response and tissue oxygen response during synaptic stimulation in area CA1 of rat hippocampal slices

Synaptic train stimulation (10 Hz × 25 s) in hippocampal slices results in a biphasic response of NAD(P)H fluorescence indicating a transient oxidation followed by a prolonged reduction. The response is accompanied by a transient tissue PO₂ decrease indicating enhanced oxygen utilization. The activation of mitochondrial metabolism and/or glycolysis may contribute to the secondary NAD(P)H peak. We investigated whether extracellular lactate uptake via monocarboxylate transporters (MCTs) contributes to the generation of the NAD(P)H response during neuronal activation. We measured the effect of lactate uptake inhibition [using the MCT inhibitor α-cyano-4-hydroxycinnamate (4-CIN)] on the NAD(P)H biphasic response, tissue PO₂ response, and field excitatory post-synaptic potential in hippocampal slices during synaptic stimulation in area CA1 (stratum radiatum). The application of 4-CIN (150–250 μmol/L) significantly decreased the reduction phase of the NAD(P)H response. When slices were supplemented with 20 mmol/L lactate in 150–250 μmol/L 4-CIN, the secondary NAD(P)H peak was restored; whereas 20 mmol/L pyruvate supplementation did not produce a recovery. Similarly, the tissue PO₂ response was decreased by MCT inhibition; 20 mmol/L lactate restored this response to control levels at all 4-CIN concentrations. These results indicate that lactate uptake via MCTs contributes significantly to energy metabolism in brain tissue and to the generation of the delayed NAD(P)H peak after synaptic stimulation.     

Isoprostanes, a novel markers of oxidative stress in schizophrenic disorders

It is a well known fact that 3H-panthenol (PL) has a high bioavailability, so we studied its biotransformation and its protective action against lipoperoxide activation in homogenates and mitochondrial-synaptosomal fraction (11 000 g) of rat brain. The lipoperoxidation was initialized by Fe²⁺-ascorbate complex (Fe²⁺-Asc). In experiments in vivo , after 30 min, we demonstrated accumulation of intermediate products of CoA biosynthesis – pantothenic acid (PA), phospho-PA, and phosphopantetheine – in postmitochondrial fraction of brain, by using a HPLC technique. Addition of the PL (10 m m ) to brain hemispheres homogenates or mitochondrial-synaptosomal fraction caused a remarkable reduction of malondialdehyde production. However, 30 min preincubation with the PL, but not with PA, was ineffective. The data obtained may be a reason for a high neuroprotective activity of PL in curing brain diseases with vessel or alcohol-induced damages.     

Formation of glycerol from glucose in rat brain and cultured brain cells. Augmentation with kainate or ischemia

An increase in the concentration of glycerol in the ischemic brain is assumed to reflect degradation of phospholipids of plasma membranes. However, glycerol could, theoretically, be formed from glucose, which after glycolytic conversion to dihydroxyacetone phosphate, could be converted to glycerol-3-phosphate and hence to glycerol. We show here that (13)C-labeled glycerol accumulate in incubation media of cultured cerebellar granule cells and astrocytes incubated with [(13)C]glucose, 3 mmol/L, demonstrating the formation of glycerol from glucose. Co-incubation of cerebellar granule cells with kainate, 50 micromol/L, led to increased glucose metabolism and increased accumulation of [(13)C]glycerol. Accumulation of [(13)C]glycerol and its precursor, [(13)C]glycerol-3-phosphate, was evident in brain, but not in serum, of kainate-treated rats that received [U-(13)C]glucose, 5 micromol/g bodyweight, intravenously and survived for 5 min. Global ischemia induced by decapitation also caused accumulation of [(13)C]glycerol and [(13)C]glycerol-3-phosphate. These results show that glycerol can be formed from glucose in brain; they also demonstrate the existence of a cerebral glycerol-3-phosphatase activity. Ischemia-induced increases in brain glycerol may, in part, reflect an altered metabolism of glucose, in which glycerol formation, like lactate formation, acts as a redox sink.     

Effects of a high-fat diet and voluntary wheel running on gluconeogenesis and lipolysis in rats.

The purpose of the present study was to determine the effects of diet composition and exercise on glycerol and glucose appearance rate (Ra) and on nonglycerol gluconeogenesis (Gneo) in vivo. Male Wistar rats were fed a high-starch diet (St, 68% of energy as cornstarch, 12% corn oil) for a 2-wk baseline period and then were randomly assigned to one of four experimental groups: St (n = 7), high-fat (HF; 35% cornstarch, 45% corn oil; n = 8), St with free access to exercise wheels (StEx; n = 7), and HF with free access to exercise wheels (HFEx; n = 7). After 8 wk, glucose Ra when using [3-3H]glucose, glycerol Ra when using [2H5]glycerol (estimate of whole body lipolysis), and [3-13C]alanine incorporation into glucose (estimate of alanine Gneo) were determined. Body weight and fat pad mass were significantly (P < 0.05) decreased in exercise vs. sedentary animals only. The average amount of exercise was not significantly different between StEx (3,212 +/- 659 m/day) and HFEx (3,581 +/- 765 m/day). The ratio of glucose to alanine enrichment and absolute glycerol Ra (micromol/min) were higher (P < 0.05) in HF and HFEx compared with St and StEx rats. In separate experiments, the ratio of 3H in C-2 to C-6 of glucose from 3H2O (estimate of Gneo from pyruvate) was also higher (P < 0.05) in HF (n = 5) and HFEx (n = 5), compared with St (n = 5) and StEx (n = 5) rats. Voluntary wheel running did not significantly increase estimated alanine or pyruvate Gneo or absolute glycerol Ra. Voluntary wheel running increased (P < 0.05) glycerol Ra when normalized to fat pad mass. These data suggest that a high-fat diet can increase in vivo Gneo from precursors that pass through pyruvate. They also suggest that changes in the absolute rate of glycerol Ra may contribute to the high-fat diet-induced increase in Gneo.     

Limits to inducer exclusion: inhibition of the bacterial phosphotransferase system by glycerol kinase

The uptake of methyl α- D -glucopyranoside by the phosphoenolpyruvate-dependent phosphotransferase system of Salmonella typhimurium could be inhibited by prior incubation of the cells with glycerol. Inhibition was only observed for glycerol preincubation times longer than 45 s and required the preinduction of both the glucose and the glycerol-catabolizing systems. Larger extents of inhibition by glycerol correlated with higher intracellular levels of glycerol kinase when the glp regulon had been induced to different extents. Preincubation with lactate did not inhibit methyl α- D -glucopyranoside uptake significantly, although both lactate and glycerol were oxidized by the cells. The cellular free-energy state of the cells (intracellular [ATP]/[ADP] ratio) was virtually identical for lactate and glycerol preincubation, suggesting that the inhibition of phosphotransferase-mediated uptake was not a metabolic effect. In vitro , phosphotransferase activity was inhibited to a maximal extent of 32% upon titrating cell-free extracts with high concentrations of commercial glycerol kinase. The results show that uptake systems that have hitherto been regarded merely as targets of the phosphotransferase system component IIA^Glc also have the capacity themselves to retroinhibit the phosphotransferase system flux, presumably by sequestration of the available IIA^Glc, provided that these systems are induced to appropriate levels.     

ALANINE METABOLISM IN RAT CORTEX IN VITRO

Abstract— (1) The metabolism of [U-¹⁴C]alanine was followed in slices of rat cerebral cortex and its interaction with glucose, pyruvate and the metabolic inhibitors fluoracetate and malonate was studied.
(2) Alanine did not stimulate respiration above endogenous levels or affect the rate of oxygen uptake with glucose or pyruvate as cosubstrate. Radioactivity found in CO₂ from labelled alanine was only 6 per cent of that from labelled pyruvate. Lactate was not formed from alanine.
(3) After 2 h incubation with [U-¹⁴C]alanine the specific activities of glutamate, glutamine and GABA were 20–30 per cent that of alanine. All these specific activities except glutamate were lowered by addition of glucose, but with pyruvate as cosubstrate the specific activity of glutamate was increased by 87 per cent above the level with alanine alone.
(4) The effect of alanine as cosubstrate with [U-¹⁴C]pyruvate was to reduce the specific activity of GABA and of glutamine, but not glutamate or lactate; thus there was not an equal dilution of all the metabolites of pyruvate.
(5) Fluoracetate diminished respiration and the production of CO₂ from [U-¹⁴C]-alanine only slightly; the addition of malonate as well practically abolished both. Fluoracetate lowered incorporation from alanine into all the amino acids, and radioactivity could not be detected in glutamine at all; addition of malonate lowered the specific activity of glutamate to 25 per cent but increased that into aspartate, GABA and glutamine.
(6) The interpretation of these data in terms of known pathways of alanine metabolism is discussed.