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.     

LABELLING OF BRAIN PROTEINS AT EARLY PERIODS AFTER SUBCUTANEOUS INJECTION OF A MIXTURE OF [U-14C]GLUCOSE AND [3H]GLUTAMATE

To obtain evidence of the site of conversion of [U-¹⁴C]glucose into glutamate and related amino acids of the brain, a mixture of [U-¹⁴C]glucose and [³H]glutamate was injected subcutaneously into rats. [³H]Glutamate gave rise to several ³H-labelled amino acids in rat liver and blood; only ³H-labelled glutamate, glutamine or γ-aminobutyrate were found in the brain. The specific radioactivity of [³H]glutamine in the brain was higher than that of [³H]glutamate indicating the entry of [³H]glutamate mainly in the ‘small glutamate compartment’. The ¹⁴C-labelling pattern of amino acids in the brain and liver after injection of [U-¹⁴C]glucose was similar to that previously reported (Gaitonde et al., 1965). The specific radioactivity of [¹⁴C]glutamine in the blood and liver after injection of both precursors was greater than that of glutamate between 10 and 60 min after the injection of the precursors. The extent of labelling of alanine and aspartate was greater than that of other amino acids in the blood after injection of [U-¹⁴C]glucose. There was no labelling of brain protein with [³H]glutamate during the 10 min period, but significant label was found at 30 and 60 min. The highest relative incorporation of [¹⁴C]glutamate and [¹⁴C]aspartate in rat brain protein was observed at 5 min after the injection of [U-¹⁴C]glucose. The results have been discussed in the context of transport of glutamine synthesized in the brain and the site of metabolism of [U-¹⁴C]glucose in the 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.     

Albumin synthesis by the perfused rat liver. A comparison of methods with special reference to the effect of dietary protein deprivation

1. Isolated perfused rat livers were used to study synthesis of albumin after donors had been fed on normal or protein-free diets. 2. Methods of determining the liver's ability to produce albumin included incorporation of [¹⁴C]carbonate, [³H]lysine and [¹⁴C]arginine, as well as a direct method based on a heterologous perfusing system of rat erythrocytes and rabbit plasma. 3. Livers from protein-deprived rats were found to form extremely little urea and not to incorporate ¹⁴CO₂ into [¹⁴C]urea, but they were capable of producing [¹⁴C]urea from [¹⁴C]arginine and of incorporating the latter and [³H]lysine into albumin. 4. By immunological means these lives were found to synthesize less albumin than normal, but their ability was only slightly impaired when related to body weight or liver weight. 5. These findings are consistent with a block in urea-cycle enzymes with relative integrity of arginase activity and of amino acid activation.     

METABOLISM OF THE ASPARTYL MOIETY OF N-ACETYL-l-ASPARTIC ACID IN THE RAT BRAIN

The metabolism of N-acetyl-l -aspartic acid (NAA) was studied in rat brain. [Aspartyl-U-¹⁴C]NAA was metabolized predominantly by deacylation. Studies of NAA biosynthesis from l -[U-¹⁴C]aspartic acid have confirmed previous reports that NAA turns over slowly in rat brain. However, intracerebrally-injected N-acetyl-l -[U-¹⁴C]asparticacid was rapidly metabolized. Exogenous NAA appears to be taken up rapidly into a small, metabolically-active pool. This pool serves as substrate for a tricarboxylic acid cycle associated with the production of glutamate for the biosynthesis of glutamine. The bulk of the NAA content in brain appears to be relatively inactive metabolically.     

Distinct patterns of entry of two non-metabolizable amino acids into brain and other organs of infant guinea pigs

Abstract— Entry of [3-¹⁴C] α-aminoisobutyric acid (AIB) and [1-¹⁴C] 1-aminocyclopentanecarboxylic acid (cycloleucine) into the brain and other organs of the infant guinea pig has been investigated in vivo. The entry of [¹⁴C]AIB into brain was markedly restricted in comparison to its entry into other organs. The mean distribution ratio (¹⁴C in tissue water/¹⁴C in plasma water) achieved in brain at 45 min after administration of a pulse of [¹⁴C]AIB was 0.3. All other organs studied concentrated [¹⁴C]AIB from the blood stream, with the greatest uptake occurring in liver and kidney, in which distribution ratios reached values of 5–10. In contrast to AIB, [¹⁴C]cycloleucine entered the brain at a rate approximately the same as that into other organs. Distribution ratios for [¹⁴C]cycloleucine ranged between 0.5 and 2.0 for all organs. During the first few days of postnatal life, there was a sharp increase of concentrative uptake of [¹⁴C]AIB into liver and kidney. The entry of [¹⁴C]AIB into brain remained unchanged during this period. There was a small (35 percent) decrease in the rate of entry of [¹⁴C]cycloleucine into brain during the first 3 days of postnatal life. Since [¹⁴C]AIB is known to be concentrated from the surrounding medium by brain slices in vitro, we concluded that the locus of restriction of the entry of [¹⁴C]AIB into the brain in vivo is at the blood-brain barrier. We hypothesize that this property of the barrier is important in preventing concentrative uptake of pharmacologically active and potentially harmful amino acids by brain tissue.     

Regional distribution study of brain perfusion and metabolic agents in normal and tumour bearing rat brains using autoradiographic technique

Regional distribution of brain perfusion imaging agents, [¹³¹I]N,N,N′-trimethyl-N′-[2-hydroxy-3-methyl-5-iodobenzyl]1,3 propanediamine (HIPDM) and [¹³¹I]N-isopropyl-p-iodoamphetamine (IMP), was compared with the distribution of patterns of [¹⁴C]l-methionine and [¹⁴C]d-glucose in normal and tumour bearing rat brains using autoradiographic technique. There was higher concentration of the radiopharmaceutical in grey than white matter in normal rat brain. Autoradiographs of brain tumour sections showed very low uptake of [¹³¹I]HIPDM and [¹³¹I]IMP as compared to normal brain tissue. There was moderate concentration of [¹⁴C]d-glucose and avid uptake of [¹⁴C]l-methionine in tumours. Autoradiographic study is useful for evaluating distribution patterns of radiopharmaceuticals.     

Some neurochemical aspects of fluorocitrate intoxication

Abstract— Some metabolic and biochemical effects of fluorocitrate were studied in vivo in rat brain and cat spinal cord. During the preconvulsant and convulsant phases of fluorocitrate poisoning the contents of free glutamate, glutamine and aspartate declined progressively, while that of alanine increased. Incorporation of ¹⁴C from [U-¹⁴C]glucose into these amino acids also decreased, although somewhat more gradually. GABA exhibited a biphasic change, its content rising after an initial decrease while its relative specific activity rose initially and subsequently diminished. Incorporation of ¹⁴C from [U-¹⁴C]glucose and [U-¹⁴C]lysine into neural protein declined sharply. The citric acid content rose markedly in rat brain and cat spinal cord. In rat brain the glycogen content declined but ATP and ammonia contents were unchanged. The significance of these results with respect to energy metabolism and the possible mechanism of the convulsions during fluorocitrate poisoning is discussed.     

Riboflavin Homeostasis in the Central Nervous System

Abstract: The mechanisms by which riboflavin, which is not synthesized in mammals, enters and leaves brain, CSF, and choroid plexus were investigated by injecting [¹⁴C]riboflavin intravenously or intraventricularly. Tracer amounts of [¹⁴C]riboflavin with or without FMN were infused intravenously at a constant rate into normal, starved, or probenecid-pretreated rabbits. At 3 h, [¹⁴C]riboflavin readily entered choroid plexus and brain, and, to a much lesser extent, CSF. Over 85% of the [¹⁴C]riboflavin in brain and choroid plexus was present as [¹⁴C]FMN and [¹⁴C]FAD. The addition of 0.2 mmol/kg FMN to the infusate markedly depressed the relative entry of [¹⁴C]riboflavin into brain, choroid plexus, and, less so, CSF, whereas starvation increased the relative entry of [¹⁴C]riboflavin into brain and choroid plexus. After intraventricular injection (2 h), most of the [¹⁴C]riboflavin was extremely rapidly cleared from CSF into blood. Some of the [¹⁴C]riboflavin entered brain, where over 85% of the ¹⁴C was present as [¹⁴C]FMN plus [¹⁴C]FAD. The addition of 1.2³μmol FAD (which was rapidly hydrolyzed to riboflavin) to the injectate decreased the clearance of [¹⁴C]riboflavin from CSF and the phosphorylation of [¹⁴C]riboflavin in brain. Probenecid in the injectate also decreased the clearance of [¹⁴C]riboflavin from CSF. These results show that the control of entry and exit of riboflavin is the mechanism, at least in part, by which total riboflavin levels in brain cells and CSF are regulated. Penetration of riboflavin through the blood-brain barrier, saturable efflux of riboflavin from CSF, and saturable entry of riboflavin into brain cells are three distinct parts of the homeostatic system for total riboflavin in the central nervous system.     

Pyrimidine biosynthesis and its regulation in the developing rat brain.

—Measurements of the incorporation of [¹⁴C]NaHCO₃ into orotic acid, uridine nucleotides and RNA in tissue minces establish the occurrence of the complete orotate pathway for the de novo biosynthesis of pyrimidines in rat brain. Selective inhibition of the incorporation of various radiolabelled precursors into orotic acid by uridine demonstrates the operation of a feedback control mechanism in brain minces and indicates carbamoylphosphate synthetase to be the site of inhibition; purine nucleosides were similarly found to inhibit the de novo biosynthesis of pyrimidines. The activity of the orotate pathway, as assessed by the rate of incorporation of [¹⁴C]NaHCO₃ into orotic acid, was found to be very high in fetal brain and to decline rapidly with neurological development; the mature rat brain exhibits less than 1% of the activity of the fetal brain at 18 days of gestation. Comparative studies on the ability of minces of the brain and several extraneural tissues to utilize [¹⁴C]NaHCO₃ and [¹⁴C]aspartate as precursors of orotic acid lead us to speculate that variations in the ability of tissues to synthesize orotic acid de novo are determined by similar variations in their ability to synthesize carbamoylphosphate.     

Stimulatory effect of gamma-aminobutyric acid upon animo acid incorporation into protein by a ribosomal system from immature rat brain

Abstract—

• 1 GABAstimulated the incorporation of L-[U-¹⁴C]leucine, primarily into the particulate protein of a ribosomal system from immature rat brain, but not from immature rat liver.
• 2 The GABA effect required the presence of Na⁺ and occurred at GABA concentrations which are thought to be physiological (1–5 mM).
• 3 Of all other amino acids tested at tissue extract concentrations in the system, only glycine had a similar effect. No analogues of GABA tested had a significant stimulatory effect upon leucine incorporation into protein, with the exception of homocarnosine which was mildly stimulatory.
• 4 The effect of GABA upon the incorporation of L-[U-¹⁴C]leucine was examined in the presence of added amino acid substrates, both individually and as mixtures. Also, the incorporation of L-[U-¹⁴C]leucine was compared with incorporation of L-[U-¹⁴C]Iysine and L-[U-¹⁴C]phenylalanine. The results are discussed in terms of GABA interaction with activating, transfer and transport mechanisms of other amino acids, inhibition of proteinase activity, and the possibility that GABA is stimulating the synthesis or turnover of specific proteins in the brain ribosomal system.
• 5 The results illustrate the fact that studies of ‘protein synthesis’ in immature rat brain ribosomes, as measured by amino acid incorporation, will yield answers which depend heavily upon substrate conditions and upon the labelled amino acid used as the marker for protein synthesis or turnover.

     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

Biopterin

The active synthesis of [¹⁴C]7,8-dihydrobiopterin (BH₂) from intraventricularly administered U-[¹⁴C]GTP was demonstrated in rat brain. The identity of [¹⁴C]BH₂ isolated from brain was confirmed by mass fragmentography. Evidence is presented that [¹⁴C]BH₂ in brain was not synthesized in the peripheral organs. The rate of cerebral synthesis of [¹⁴C]BH₂ from [¹⁴C]GTP was maximal at 2 h; it was 0.53 nmol/g per h, which is consistent with the estimated turnover rate of cerebral BH₂ (0.43 nmol/g per h). Intraventricularly injected 2,4-diamino-6-hydroxypyrimidine (DAOPyr) and 6-thioguanosine were effective inhibitors of the synthesis. U-[¹⁴C]dGTP and 8-[¹⁴C]GTP, when given intraventricularly, did not yield [¹⁴C]BH₂. Simultaneous intraventricular injection of U-[¹⁴C]GTP and DAOPyr resulted in the accumulation of a compound with properties identical to a formamidopyrimidine derivative isolated from the nonenzymatic hydrolysis of GTP. The data from preliminary experiments demonstrated the synthesis of [¹⁴C]BH₂ from U-[¹⁴C]GTP incubated with 12,000g supernatants of rat brain homogenates.     

NIACIN AND NIACINAMIDE TRANSPORT IN THE CENTRAL NERVOUS SYSTEM. IN VIVO STUDIES

Abstract– The concentration ol niacinamide in plasma and CSF was 0.5 and 0.7 μm respectively. The, mechanisms by which niacin and niacinamide, which are not synthesized in brain, enter brain, CSF and choroid plexus were investigated by injecting [¹⁴C]niacin or [¹⁴C]niacinamide intravenously and intraventricularly. [¹⁴C]Niacin or [¹⁴C]niacinamide, with or without unlabeled niacin or niacinamide, were infused intravenously at a constant rate into conscious rabbits. At 3 h, [¹⁴C]niacinamide, but not [¹⁴C]niacin, readily entered CSF, choroid plexus and brain. The addition of 4.1 mmol/kg niacinamide to the infusate markedly depressed the relative entry of [¹⁴C]niacinamide into choroid plexus and brain but not into CSF. After intraventricular injection, [¹⁴C]niacin was rapidly cleared from CSF and readily entered brain and choroid plexus. The addition of unlabeled niacin to the intraventricular injectate decreased the clearance of [¹⁴C]niacin from CSF and the entry of [¹⁴C]niacin into choroid plexus and brain. Unlike niacin, carrier niacinamide (82 μmol) in the injectate did not depress the extremely rapid clearance of intraventricularly injected [¹⁴C]niacinamide from CSF but did decrease the entry of [¹⁴C]niacinamide into brain. These results show that the control of entry and exit of niacinamide and niacin is the mechanism, at least in part, by which total niacin and NAD levels in brain cells are regulated. In the case of niacinamide which readily passes between CSF and plasma, the regulation of entry of niacinamide into brain cells by a high affinity accumulation system is an integral part of the homeostatic system. In the case of niacin, penetration into CSF and the extracellular space of brain from plasma as well as regulation of entry into brain cells by a saturable accumulation system are two distinct parts of the homeostatic system. In vivo, niacin that enters the central nervous system is converted to the principal plasma vitamer, niacinamide, in its free or bound forms such as NAD.     

A SOLUBLE PREPARATION FROM RAT BRAIN THAT INCORPORATES INTO ITS OWN PROTEINS [14C]ARGININE BY A RIBONUCLEASE-SENSITIVE SYSTEM AND [14C]TYROSINE BY A RIBONUCLEASE-INSENSITIVE SYSTEM

Abstract— A 100,000 g supernatant fraction from rat brain that was passed through a column of Sephadex G-25-40 was able, after addition of some factors, to incorporate [^I4C]arginine (apparent K_m= 5 μM) and [¹⁴C]tyrosine (apparent K_m= 20 μM) into its own proteins. The factors required for the incorporation of [¹⁴C]arginine were: ATP (optimal concentration = 0-25-2 μM) and Mg²⁺ (optimal concentration 5 mM). For the incorporation of [^I4C]tyrosine the required factors were: ATP (apparent K_m= 0-75 μM), Mg²⁺ (optimalconcentration 8-16 mM) and K⁺ (apparent K_m= 16 mM). Addition of 19 amino acids did not enhance these incorporations. Optimal pHs were: for [¹⁴C]arginine and [¹⁴C]tyrosine, respectively, 7-4 and 7-0 in phosphate buffer and 7–9 and 7-3-8-1 in tris-HCl buffer. Pancreatic ribonuclease abolished the incorporation of [¹⁴C]arginine but had practically no effect in the incorporation of [¹⁴C]tyrosine. Furthermore, [¹⁴C]arginyl-tRNA was a more effective donor of arginyl groups than [¹⁴C]arginine, whereas [¹⁴C]tyrosyl-tRNA was considerably less effective than [¹⁴C]tyrosine. The incorporations of [¹⁴C]arginine and [¹⁴C]tyrosine into brain proteins were from 25- to 2000-fold higher than for any other amino acid tested (12 in total). In brain [¹⁴C]arginine incorporation was higher than in liver and thyroid but somewhat lower than in kidney. In comparison to brain, the incorporation of [¹⁴C]tyrosine was negligible in liver, thyroid or kidney. Kinetic studies showed that the macromolecular factor in the brain preparation was complex. The protein nature of the products was inferred from their insolubilities in hot TCA and from the action of pronase that rendered them soluble. [¹⁴C]Arginine was bound so that its a-amino group remained free. Maximal incorporation of [¹⁴C]tyrosine in brain of 30-day-old rats was about one-third of that in the 5-day-old rat. The changes with postnatal age in the incorporation of [¹⁴C]arginine were not statistically significant.     

d-β-HYDROXYBUTYRATE: A MAJOR PRECURSOR OF AMINO ACIDS IN DEVELOPING RAT BRAIN

Abstract— D-β-hydroxybutyrate (β-OHB) was compared to glucose as a precursor for brain amino acids during rat development. In the first study [3-¹⁴C]β-OHB or [2-¹⁴C]glucose was injected subcu-taneously (01 μCi/g body wt) into suckling rats shortly after birth and at 6. 11, 13, 15 and 21 days of age. Blood and brain tissue were obtained 20 min later after decapitation. The specific activity of the labelled precursor in the blood and in the brain tissue was essentially the same for each respective age suggesting that the labelled precursor had equilibrated between the blood and brain pools before decapitation. [3-¹⁴C]β-OHB rapidly labelled brain amino acids at all ages whereas [2-¹⁴C]glucose did not prior to 15 days of age. These observations are consistent with a maturational delay in the flux of metabolites through glycolysis and into the tricarboxylic acid cycle. Brain glutamate, glutamine, asparate and GABA were more heavily labelled by [3-¹⁴C]β-OHB from birth-15 days of age whereas brain alanine was more heavily labelled by [2-¹⁴C]glucose at all ages of development. The relative specific activity of brain glutamine/glutamate was less than one at all ages for both labelled precursors suggesting that β-OHB and glucose are entering the‘large’glutamate compartment throughout development. In a second study, 6 and 15 day old rats were decapitated at 5 min intervals after injection of the labelled precursors to evaluate the flux of the [¹⁴C]label into brain metabolites. At 6 days of age, most of the brain acid soluble radioactivity was recovered in the glucose fraction of the [2-^,4C]glucose injected rats with 72, 74, 65 and 63% after 5, 10, 15 and 20 min. In contrast, the 6 day old rats injected with [3-¹⁴C]β-OHB accumulated much of the brain acid soluble radioactivity in the amino acid fraction with 22, 47, 57 and 54% after 5, 10, 15 and 20 min. At 15 days of age the transfer of the [¹⁴C]label from [2-¹⁴C]glucose into the brain amino acid fraction was more rapid with 29, 40, 45, 61 and 73% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. There was almost quantitative transfer of [¹⁴C]label into the brain amino acids of the 15-day-old [3-¹⁴C]β-OHB injected rats with 66, 89, 89, 89 and 90% of the brain acid soluble radioactivity recovered in the amino acid fraction after 5, 10, 15, 20 and 30 min. The calculated half life for /?-OHB at 6 days was 19 8 min and at 15 days was 12-2 min. Surprisingly, the relative specific activity of brain GABA/glutamate was lower at 15 days of age in the [3-¹⁴C]β-OHB injected rats compared to the [2-¹⁴C]glucose injected rats despite a heavier labelling of brain glutamate in the [3-¹⁴C]β-OHB injected group. We interpreted these data to mean that β-OHB is a less effective precursor for the brain glutamate ‘subcompartment’ which is involved in the synthesis of GABA.     

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.     

METABOLIC COMPARTMENTATION IN THE BRAIN: METABOLISM OF A TRICARBOXYLIC ACID CYCLE INTERMEDIATE, [1,4-14C] SUCCINATE, AFTER INTRACEREBRAL ADMINISTRATION

Abstract— The metabolism of a tricarboxylic acid cycle (cycle) intermediate, [1.4-'¹⁴C]succinate, was studied in the brain at 2 20 min after intracerebral injection. The oxidation of [¹⁴C]succinate was rapid, as shown by the incorporation of ¹⁴C into cycle amino acids which accounted for about 30 per cent and 70 per cent of the tissue -“Cat 2 and 10 min respectively. During the whole experimental period the specific radioactivity of glutamine was about three times higher than that of glutamate. Thus exogenous [¹⁴C]succinate elicited signs of metabolic compartmentation similar to those seen after the administration of short chain fatty acids or amino acids. A computer programme, based on data obtained previously on the metabolic compartmentation of acetate and of glucose in the brain, was used to simulate the kinetics of labelling of cycle amino acids after an input of [1.4-¹⁴C]succinate. The correspondence of the simulated data with the experimental results was good in the first 10 min after injection, although the deviations were significant at later time points. Incorporation of ¹⁴C into GABA was very low (< 1 per cent of the amino acid -¹⁴C) after the injection of [1.4-¹⁴C]succinate. Further, labelled GABA formation was not detected in the decapitated rat brain labelled in vivo with [1.4-¹⁴C]succinate 2 min beforehand. Since the oxidation of [l,4-¹⁴C]succinate via the cycle yields unlabellcd GABA. whereas the reversal of the reactions in the GABA bypath may introduce ¹⁴C from succinate into the GABA pool, the results indicate that this reversal is negligible even under the most favourable conditions, i.e. post mortem when both the NADH/NAD⁺ ratios and [¹⁴C]succinate concentrations arc high. The observations are therefore consistent with the view that glutamate is the predominant and probably the only source of GABA carbon in the brain both in vivo and post mortem.     

Cerebral lipid metabolism in experimental hyperphenylalaninaemia: incorporation of 14C-labelled glucose into total lipids

—1. Effects of the administration of phenylalanine to rats on incorporation in vivo or in vitro of [U-¹⁴C]glucose into cerebral lipids were studied during the first 5–10 days of postnatal development. In addition, the effects of added phenylalanine and its deaminated metabolites on incorporation of [U-¹⁴C]glucose by homogenates into lipids of developing rat brain were investigated. Hyperphenylalaninaemia reduced incorporation both in vivo and in vitro of [U-¹⁴C]glucose into cerebral lipids. 2. Phenylalanine or tyrosine added in vitro at concentrations equivalent to those in the brain of the hyperphenylalaninaemic rat (0-1 μmole/ml incubation medium) did not inhibit incorporation of [U-¹⁴C)glucose into lipids, although at much higher concentrations of phenylalanine (36 μumoles/ml incubation medium) slight inhibition (10 per cent) of incorporation of [U-¹⁴C]glucose into lipids was observed. 3. In contrast, the deaminated metabolites in general exerted greater inhibitory effects at lower concentrations. Phenyllactic acid, in comparison to phenylpyruvic and phenyl-acetic acid, was the most potent inhibitor of the incorporation in vitro of [U-¹⁴C]glucose into cerebral lipids. These results indicated that these metabolites of phenylalanine were the more potent inhibitors of cerebral lipid metabolism in immature animals.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.