DEVELOPMENT OF LIPOGENESIS IN RAT BRAIN CORTEX: THE DIFFERENTIAL INCORPORATION OF GLUCOSE AND ACETATE INTO BRAIN LIPIDS IN VITRO

—The oxidation to CO₂ and the incorporation of [U-¹⁴C]glucose and [U-¹⁴C]acetate into lipids by cortex slices from rat brain during the postnatal period were investigated. The oxidation of [U-¹⁴C]glucose was low in 2-day-old rat brain, and increased by about two-fold during the 2nd and 3rd postnatal weeks. The oxidation of [U-¹⁴C]acetate was increased markedly in the second postnatal week, but decreased to rates observed in 2-day-old rat brain at the time of weaning. Both labeled substrates were readily incorporated into non-saponifiable lipids and fatty acids by brain slices from 2-day-old rat. Their rates of incorporation and the days on which maximum rates occurred were different, however, maximum incorporation of [U-¹⁴C]glucose and [U-¹⁴]acetate into lipid fractions being observed on about the 7th and 12th postanatal days, respectively. The metabolic compartmentation in the utilization of these substrates for lipogenesis is suggested. The activities of glucose-6-phosphate dehydrogenase, cytosolic NADP-malate dehydrogenase, cytosolic NADP-isocitrate dehydrogenase, ATP-citrate lyase and acetyl CoA carboxylase were measured in rat brain during the postnatal period. All enzymes followed somewhat different courses of development; the activity of acetyl CoA carboxylase was, however, the lowest among other key enzymes in the biosynthetic pathway, and its developmental pattern paralleled closely the fatty acid synthesis from [U-¹⁴C]glucose. It is suggested that acetyl CoA carboxylase is a rate-limiting step in the synthesis de novo of fatty acids in developing rat brain.     

GLUCOSE AND AMINO ACID METABOLISM IN OCTOPUS OPTIC AND VERTICAL LOBES IN VITRO

(1) The metabolism of glucose and amino acids in vitro was compared in the rat cerebral cortex and the optic and vertical lobes of the octopus brain. (2) Specific activities and pool sizes of the five amino acids, glutamate, aspartate, glutamine, alanine and γ-aminobutyric acid (GABA), were determined in octopus and rat brain slices after 2 hr incubation with 10 mm -[U-¹⁴C]glucose, 10 mm -L-[U-¹⁴C]glutamate, and 10mm -^L-[U-¹⁴C]glutamate with added 10 mM-glucose. Amino acid pool sizes were similar in rat and octopus brain, with the exception of alanine, which was higher in the octopus. Generally specific activities were from four- to 20-fold higher in rat brain. With [U-¹⁴C]glucose as substrate, specific activities of GABA and glutamate were highest in rat; those of alanine and glutamine highest in octopus brain. With ^L-[U-¹⁴C]glutamate the specific activities of GABA and aspartate were highest in rat, that of aspartate highest and GABA lowest in octopus. The addition of glucose to ^L-[U-¹⁴C]glutamate as substrate had little effect on the specific activities of any of the amino acids. (3) The uptake of some amino acids was determined by incubation with [U-¹⁴C]amino acids for 2 hr, and ¹⁴CO₂ formation was also measured. The amount of label taken up by octopus was uniformly 20-25 per cent of that found for rat brain. The amount of ¹⁴CO₂, however, differed according to the amino acid. Four times as much ¹⁴CO₂ was generated from alanine by octopus optic lobe and twice as much by the vertical lobe than rat cortex, but from glutamate, only 24 per cent in the optic and 15 per cent in the vertical lobe. No ¹⁴CO₂ was generated from [U-¹⁴C]GABA in the octopus, by contrast with the rat. (4) Activity of some of the enzymes involved in amino acid metabolism was determined in homogenates of rat cortex and octopus optic and vertical lobes, with and without activation by Triton X-100. Enzymic activities in the octopus, with the exception of alanine aminotransferase, were lower than in the rat, and glutamate decarboxylase could not be detected in octopus brain, in the absence of detergent.     

Glucose handling by hepatocytes from obese Zucker rats

Hepatocytes isolated from obese Zucker rats showed a significantly higher rate of both [U-¹⁴C]glucose and [U-¹⁴C]lactate incorporation into [¹⁴C]lipid than those from their lean counterparts. This was associated with a marked increase in the lipogenic rate measured by the incorporation of³H₂O into the cell esterified fatty acids. Although there were no changes in the incorporation of the tracer into either [¹⁴C]glycogen or¹⁴CO₂, the [¹⁴C] total uptake was significantly higher in the obese animals. The high rate of [¹⁴C]lipid synthesis from glucose was observed both at 15 and 30 mM substrate concentrations and was linked to an enhanced uptake of the tracer into the cell as measured using the decarboxilation of [1-¹⁴C]glucose in the presence of phenazine methosulphate. The presence of insulin in the incubation medium had no effect on the uptake of glucose by the liver cells. However, the large uptake of glucose by the hepatocytes from the obese animals was not related to an enhanced rate of transport as measured using 3-O-methyl[U-¹⁴C]glucose. The activity of glucose-6-phosphate dehydrogenase together with a higher [1-¹⁴C]glucose/[U-¹⁴C]glucose descarboxylation ratio indicate a predominant very active pentose phosphate pathway which may be responsible for the enhanced glucose uptake observed in the hepatocytes from the obese animals.     

Intermediary Metabolism of Organic Matter in the Sediments of a Eutrophic Lake

The rates, products, and controls of the metabolism of fermentation intermediates in the sediments of a eutrophic lake were examined. ¹⁴C-fatty acids were directly injected into sediment subcores for turnover rate measurements. The highest rates of acetate turnover were in surface sediments (0- to 2-cm depth). Methane was the dominant product of acetate metabolism at all depths. Simultaneous measurements of acetate, propionate, and lactate turnover in surface sediments gave turnover rates of 159, 20, and 3 μM/h, respectively. [2-¹⁴C]propionate and [U-¹⁴C]lactate were metabolized to [¹⁴C]acetate, ¹⁴CO₂, and ¹⁴CH₄. [¹⁴C]formate was completely converted to ¹⁴CO₂ in less than 1 min. Inhibition of methanogenesis with chloroform resulted in an immediate accumulation of volatile fatty acids and hydrogen. Hydrogen inhibited the metabolism of C₃-C₅ volatile fatty acids. The rates of fatty acid production were estimated from the rates of fatty acid accumulation in the presence of chloroform or hydrogen. The mean molar rates of production were acetate, 82%; propionate, 13%; butyrates, 2%; and valerates, 3%. A working model for carbon and electron flow is presented which illustrates that fermentation and methanogenesis are the predominate steps in carbon flow and that there is a close interaction between fermentative bacteria, acetogenic hydrogen-producing bacteria, and methanogens.     

Substrate utilization for energy production and lipid synthesis in oligodendrocyte-enriched cultures prepared from rat brain

The metabolism of oligodendrocytes has been studied using cultures of oligodendrocyte-enriched glial cells isolated from cerebra of 5–8-day old rats. Cultures containing 60–80% oligodendrocytes were incubated for 16h with [3-¹⁴C]acetoacetate, d-[3-¹⁴C]3-hydroxybutyrate, [U-¹⁴C]glucose, l-[U-¹⁴C]glutamine and [1-¹⁴C]pyruvate or [2-¹⁴C]pyruvate in the presence or absence of other oxidizable substrates. Labelled CO₂ was collected as an index of oxidative metabolism and the incorporation of label into total lipids, fatty acids and cholesterol was used as an index of the de novo synthesis of lipids. Glucose, acetoacetate, D-3-hydroxybutyrate, pyruvate and l-lactate were measured to determine substrate utilization and product formation under various conditions. Our results indicate that glucose is rapidly converted to lactate and is a relatively poor substrate for oxidative metabolism and lipid synthesis. Ketone bodies were used as an energy source and as precursors for the synthesis of fatty acids and cholesterol. Preferential incorporation of acetoacetate into cholesterol was not observed. Exogenous pyruvate was incorporated into both the glycerol skeleton of complex lipids and into cholesterol and fatty acids. l-Glutamine appeared to be an important substrate for the energy metabolism of these cells.     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

Carbohydrate oxidation by leucoplasts from suspension cultures of soybean (Glycine max L.)

The aim of this work was to discover how leucoplasts from suspension cultures of soybean (Glycine max L.) oxidize hexose monophosphates. Leucoplasts were isolated from protoplast lysates on a continuous gradient of Nycodenz with a yield of 28% and an intactness of 80%. Incubation of the leucoplasts with ¹⁴C-labelled substrates led to ¹⁴CO₂ production, that was dependent upon leucoplast intactness, from [U-¹⁴C]glucose 6-phosphate, [U-¹⁴C]glucose 1-phosphate, [U-¹⁴C] fructose 6-phosphate and [U-¹⁴C]glucose+ATP, but not from [U-¹⁴C]fructose-1,6-bisphosphate or [U-¹⁴C]triose phosphate. The yield from [U-¹⁴C]glucose 6-phosphate was at least four times greater than that from any of the other substrates. When [1-¹⁴C]-, [2-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose 6-phosphate were supplied to leucoplasts significant ¹⁴CO₂ production that was dependent upon leucoplast intactness was found only for [1-¹⁴C]glucose 6-phosphate. It is argued that soybean cell leucoplasts oxidize glucose 6-phosphate via the oxidative pentose phosphate pathway with very little recycling, and that in these plastids glycolysis to acetyl CoA is negligible.S.A.C. thanks the Science and Engineering Research Council for a research studentship.     

Inhibition of glucose utilization for acetylcholine synthesis by cerebrospinal fluid.

Abstract— Replacement of bicarbonate-Locke incubation medium with feline CSF reduced [¹⁴C]ACh formation from [U-¹⁴C]glucose by rat brain mince approx 30%. CSF was obtained from a cannula leading to the cisterna magna of freely moving cats. The component of CSF responsible for inhibition was characterized as a dialyzable heat-stable organic anion. Choline acetyltransferase activity was not altered by CSF. [¹⁴C]ACh synthesis and ¹⁴CO₂ production from [U-¹⁴C]glucose but not from [2-¹⁴C]-pyruvate were inhibited by CSF, suggesting inhibition in the metabolism of glucose to pyruvate. The anionic fraction of human CSF was as potent as that from feline CSF in inhibiting ¹⁴CO₂ production from [U-¹⁴C]glucose. Brain hexokinase was inhibited by the anionic fraction of feline CSF. The inhibition was non-competitive with respect to glucose and uncompetitive with respect to ATP. It is suggested that inhibition of hexokinase by CSF was responsible at least in part for the inhibition of glucose metabolism which resulted in decreased [¹⁴C]ACh synthesis and ¹⁴CO₂ production.     

Lactate Metabolism and Its Effects on Glucose Metabolism in an Excised Neural Tissue

Abstract: Chains of lumbar sympathetic ganglia, excised from 15-day-old chicken embryos, were incubated for 4 h at 36°C in a bicarbonate-buffered physiological salt solution containing 5.5 mM glucose and equilibrated with 5% CO₂–95% O₂. [U-¹⁴C]Glucose and [U-¹⁴C]lactate were used as tracers to measure the products of glucose and lactate metabolism, respectively, including CO₂, lactate, and constituents of the tissue. When 5 mM lactate was added to bathing solution containing 5.5 mM glucose, lactate carbon displaced 50–70% of the glucose carbon otherwise used for CO₂ production and provided about three times as much carbon for CO₂ as did glucose. The lactate addition increased the total carbon incorporated into CO₂ and into constituents of the tissue above those observed with glucose alone and also increased the lactate released to the bathing solution from [U-¹⁴C]-glucose. The latter increase was evidently due to an interference with reuptake of the lactate released from the ganglion cells, not to an increase in the cellular release itself. When the volume of bathing solution was increased 10-fold relative to that of the tissue, the average output of CO₂ from [U-¹⁴C]glucose during a 4-h incubation was decreased by 50% when 5 mM lactate was present but was not affected significantly in the absence of added lactate. It is concluded that the effect of changing volume in the presence of lactate was due to the effects of lactate on glucose metabolism described above and resulted from a lower average lactate concentration in the smaller volume than in the larger one, due to metabolic depletion of the added lactate. Consumable substrates other than lactate, such as glutamine and certain amino acids, also affected glucose metabolism.     

Energy metabolism in hypoxic astrocytes: Protective mechanism of fructose-1,6-bisphosphate

The protective effects of fructose-1,6-biphosphate (FBP) during hypoxia/ischemia are thought to result from uptake and utilization of FBP as a substrate for glycolysis or from stimulation of glucose metabolism. To test these hypotheses, we measumed CO₂ and lactate production from [6-¹⁴C]glucose, [1-¹⁴C]glucose, and [U-¹⁴C]FBP in normoxic and hypoxic cultured astrocytes with and without FBP present. FBP had little effect on CO₂ production by glycolysis, but increased CO₂ production by the pentose phosphate pathway. Labeled FBP produced very small amounts of CO₂. Lactate production from [1-, and 6-¹⁴C]glucose increased similarly during hypoxic hypoxia; the increase was independent of added FBP. Labeled lactate from [U-¹⁴C]FBP was minimal. We conclude that exogenous FBP is not used by astrocytes as a substrate for glycolysis and that FBP alters glucose metabolism.     

Metabolism of [4-14C]levulinic acid by etiolated and greening leaves of Hordeum vulgare

When etiolated barley (Hordeum vulgare L. var. Larker) shoots are incubated with [4-¹⁴C]levulinic acid, ¹⁴CO₂ is evolved, and amino and organic acids are labelled. Respiratory inhibitors and short-chain fatty acids, similar in size to levulinic acid, reduce the production of ¹⁴CO₂ from [4-¹⁴C]levulinic acid, while δ-aminolevulinic acid treatment or illuminating the tissue increase ¹⁴CO₂ evolution. The contribution of levulinic acid metabolism to α-aminolevulinic acid biosynthesis is no greater than that of a general cellular metabolite. The data suggest that fatty acid oxidation and the citric acid cycle are involved in levulinic acid metabolism.     

The fate of labelled glucose molecules in the rat adipocyte: Dependence on glucose concentration

Isolated rat adipocytes were incubated with 15 nM [3-³H]glucose or 100 nM [U-¹⁴C]glucose with or without insulin and in the absence or presence of unlabelled glucose. Following a 2 h incubation with 15 nM [3-³H]glucose, about two thirds of the cell-associated ³H-labelled metabolic products were hydrophilic largely anionic intermediates and about one third was lipids. The equivalent values were 40 and 60%, respectively, when using 100 nM [U-¹⁴C]glucose. The only ¹⁴C-labelled metabolite escaping to the incubation medium was ¹⁴CO₂, which accounted for about 15% of the rate of metabolism. Therefore, the rate of incorporation of 100 nM [U-¹⁴C]glucose into the cell-associated metabolites was quite a good measure of its net influx rate. The conversion of the two tracers to the sum of the metabolic products in cells treated with a maximally stimulating insulin concentration remained constant with glucose concentrations up to about 100 μM and then decreased progressively. The incorporation of radioactivity into the different metabolites varied markedly over the glucose concentration range 0–100 μM, presumably due to the saturation of different metabolic pools at different glucose concentrations. This variation was much less in cells not stimulated with insulin. Consequently, the maximal effect of insulin on the incorporation of the tracers into a given metabolite (e.g., labelled lipids) varied over the entire glucose concentration range. In addition, the apparent sensitivity (ED₅₀) with respect to the incorporation into a given metabolite was also dependent on the glucose concentration.     

METABOLISM OF HEXOSES IN RAT CEREBRAL CORTEX SLICES

Abstract—

• 1 The metabolism of two ¹⁴C-labelled hexoses and one hexose analogue, viz. mannose, fructose and glucosamine, has been compared with that of glucose for slices of rat cerebral cortex incubated in vitro.
• 2 The metabolism of [U-¹⁴C]mannose was essentially identical to that of glucose; oxygen consumption and CO₃ production were similar and maximal at a substrate concentration of 2·75 mM. Incorporation of label into lactate, aspartate, glutamate and GABA was similar for the two substrates at 5·5 mM substrate concentration.
• 3 With [U-¹⁴C]fructose, maximal oxygen consumption and CO₃ production were obtained at a substrate concentration of 11 mM. At 5·5 mM, incorporation into lactate was 5 per cent, into glutamate and GABA 30 per cent, into alanine 63 per cent and into aspartate 152 per cent of that from glucose. Increasing substrate concentration to 27·5 mm was without effect on incorporation into amino acids from glucose and raised incorporation from fructose into glutamate, GABA and alanine to a level similar to that found with glucose; at the higher substrate concentration aspartate incorporation from fructose was 200 per cent and lactate 42 per cent of that with glucose. Unlabelled fructose was without effect on incorporation of radioactivity from [3-¹⁴C]pyruvate into CO₂ or amino acids; it increased incorporation into lactate by 36 per cent. Unlabelled glucose diminished incorporation into CO₂ from [U-¹⁴C]fructose to 35 per cent; incorporation into lactate was stimulated 178 per cent at 5·5 mM fructose; at 27·5 mM it was diminished to 75 per cent.
• 4 By comparison with [1-¹⁴C]glucose, incorporation of radioactivity from [1-¹⁴C]-glucosamine into lactate, CO₂, alanine, GABA and glutamine was very low; incorporation into aspartate was similar to glucose. Thus the metabolism of glucosamine resembled that of fructose. Glucosamine-1-phosphate, glucosamine-6-phosphate, and an unidentified metabolite, all accumulated.

     

INHIBITION OF ACETYLCHOLINE SYNTHESIS AND OF CARBOHYDRATE UTILIZATION BY MAPLE-SYRUP-URINE DISEASE METABOLITES

The branched-chain 2-oxo acids which accumulate in maple-syrup-urine disease inhibited the production of acetylcholine and of lipids, proteins, nucleic acids and of CO₂. in sliced adult rat brains incubated with [U-¹⁴C] glucose. Inhibition of the biosynthetic reactions was proportional to the inhibition of CO₂ production, even though the flux of radioactivity into the biosynthetic products was less than 2% of that to CO². The oxo acids reduced the production of ¹⁴CO₂, from [U-¹⁴C] glucose and from [2-¹⁴C]pyruvic acid more than from [1-¹⁴C]pyruvic acid in sliced brains. They inhibited the solubilized oxoglutarate dehydrogenase complex more than they did the solubilized pyruvate dehydrogenase complex. Valine and isoleucine, which also accumulate in maple-syrup-urine disease, inhibited pyruvate kinase from rat brain allosterically. Quantitative comparison of the effects of the disease metabolites on cell-free systems with their effects on fluxes in intact cells indicated that the inhibition of oxoglutarate dehydrogenase appeared to be functionally significant. The residual activities of the other enzymes studied were adequate to support the normal flux of carbohydrates. The oxo acids were effective at concentrations within the range reported to occur in patients with maple-syrup-urine disease. The effects on biosyntheses including that of acetylcholine would be expected to impair brain development and function and could be important in the development of brain disease in the patients. In contrast to the results with metabolites from maple-syrup-urine disease, metabolites which accumulate in phenylketonuria (phenylalanine and 2-oxo-3-phenylpropionic acid) did not inhibit carbohydrate utilization or the biosynthetic reactions studied, under the conditions of these experiments.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Further studies on the metabolism of phospholipids inStreptomyces griseus: Turnover of phospholipid moieties

Phospholipid turnover inStreptomyces griseus was studied by pulse-chase techniques using 1-[¹⁴C]sodium acetate and [U-¹⁴C]glucose. Different phospholipids and their individual moieties were found to have different turnover rates. The moieties of inositol-containing phospholipids exhibited the fastest turnover rates among the major phospholipids, while only fatty acyl moieties of phosphatidylethanolamine showed rapid turnover. Cardiolipin did not show any significant turnover with both precursors.     

Biosynthesis and metabolism of steryl glycosides in Sinapis alba seedlings

With ¹⁴CO₂, d-glucose-[U-¹⁴C] and dl-mevalonate-[4R-4-³H₁] used as precursors, a study was made of the labelling dynamics of the steryl glucosides (SG) and steryl acylglucosides (ASG) in Sinapis alba seedlings. The radioactivity of the sterol and sugar moieties, as well as of the fatty acid moieties in the case of ASG, was analysed separately. The course of incorporation of ¹⁴C from ¹⁴ CO₂ and glucose-[U-¹⁴C] into the sugar part of SG and ASG indicated that about $\text{2}\text{3}$ of the whole pool of the newly synthesized sterol glycosides of both types underwent rapid deglucosylation. Likewise, fatty acids in the ASG pool were rapidly exchanged. The present results point to a high metabolic activity of the sterol glycoside derivatives in plant cells.     

Diurnal change of tartrate dissimilation during the ripening of grapes

l(+)-tartrate-[U-¹⁴C] or sucrose-[U-¹⁴C] was fed into grape berries and ¹⁴CO₂ evolution was determined. ¹⁴CO₂ evolution front l(+)-tartrate-[U-¹⁴C] was slightly higher in mature than immature berries, and that from sucrose-[U-¹⁴C] was higher in immature than mature ones. ¹⁴CO₂ evolution from l(+)-tartrate-[U-¹⁴C] was irregular throughout the day until 2 or 3 weeks after flowering. This stage shifted to regular ¹⁴CO₂ evolution until 6 or 7 weeks after flowering, and the mode of ¹⁴CO₂ evolution showed diurnal variation; higher in the day than at night. Then the stage without variation of ¹⁴CO₂ evolution followed 10 weeks after flowering. These observations indicate that tartrate is not biochemically inert in grape berries, while the amount of ¹⁴CO₂ evolution from sucrose-[U-¹⁴C] was higher at night than in the day through the whole ripening process, except in the early stage.     

THE METABOLISM OF LEUCINE BY DEVELOPING RAT BRAIN: EFFECT OF LEUCINE AND 2-OXO-4-METHYLVALERATE ON LIPID SYNTHESIS FROM GLUCOSE AND KETONE BODIES

Abstract— The oxidation of l -[U-¹⁴C]leucine and l -[l-¹⁴C]leucine at varying concentrations from 0.1 to 5mM to CO₂ and the incorporation into cerebral lipids and proteins by brain slices from 1-week old rats were markedly stimulated by glucose. Although the addition of S mM-dl -3-hydroxybutyrate had no effect on the metabolism of [U-¹⁴C]leucine by brain slices from suckling rats, the stimulatory effects of glucose on the metabolism of l -[U-¹⁴C]leucine were markedly reduced in the presence of dl -3-hydroxybutyrate. The stimulatory effect of glucose on leucine oxidation was, however, not observed in adult rat brain. Furthermore, the incorporation of leucine-carbon into cerebral lipids and proteins was also very low in the adult brain. The incorporation of l -[U-¹⁴C]leucine into cerebral lipids by cortex slices was higher during the first 2 postnatal weeks, which then declined to the adult level. During this time span, the oxidation of l -[U-¹⁴C]leucine to CO₂ remained relatively unchanged. The incorporation in vivo of D-3-hydroxy[3-¹⁴C]butyrate into cerebral lipids was markedly decreased by acute hyperleucinemia induced by injecting leucine into 9-day old rats. In in vitro experiments, 5 mM-leucine had no effect on the oxidation of [U-¹⁴C]glucose to CO₂ or its incorporation into lipids by brain slices from 1-week old rats. However, 5 mM-leucine inhibited the oxidation of d -3-hydroxy-[3-¹⁴C]butyrate, [3-¹⁴C]acetoacetate and [1-¹⁴C]acetate to CO₂ by brain slices, but their incorporation into cerebral lipids was not affected by leucine. In contrast 2-oxo-4-methylvalerate, a deaminated metabolite of leucine, markedly inhibited both the oxidation to CO₂ and the incorporation into lipids of labelled glucose, ketone bodies and acetate by cortex slices from 1-week old rats. These findings suggest that the reduction in the incorporation in vivo of d -3-hydroxy[3-¹⁴C]butyrate into cerebral lipids in rats injected with leucine is most likely caused by 2-oxo-4-methylvalerate formed from leucine. Since the concentrations of leucine and 2-oxo-4-methylvalerate in plasma of untreated patients with maple-syrup urine disease are markedly elevated, our findings are compatible with the possibility that an alteration in the metabolism of glucose and ketone bodies in the brain may contribute to the pathophysiology of this disease.     

INTERACTION OF LEUCINE, GLUCOSE, AND KETONE METABOLISM IN RAT BRAIN IN VITRO

Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and ¹⁴CO₂ was measured from (a) [U-¹⁴C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-¹⁴C]leucine or [l-¹⁴C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U¹⁴C]glucose to CO₂ was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-¹⁴C]leucine or [l-^,4C]leucine to ¹⁴CO₂ at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO₂ was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations.