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.     

Development of Na+-stimulated glucose oxidation in synaptosomes

Abstract— d -[¹⁴C]Glucose was oxidized to ¹⁴CO₂ by synaptosomes prepared from adult rat brain. Added Na⁺ stimulated glucose oxidation by 179%, but K⁺ and choline were without effect. Li⁺ stimulated glucose oxidation by 64%. Ouabain largely prevented the stimulatory effect of Na⁺ on glucose oxidation but had no effect in the absence of Na⁺. 2-Deoxy-d -glucose competitively inhibited glucose oxidation differently at two different ranges of deoxyglucose and glucose concentrations; the K_i was 0.54 and 16 mm , respectively. In the presence or absence of Na⁺ 2,4-DNP-stimulated glucose oxidation by 370% while iodoacetate inhibited glucose oxidation by 87–95%. There was a striking increase in Na⁺-stimulated glucose oxidation with development but glucose oxidation in the absence of Na⁺ did not change dramatically with age. Taken together the data suggest synaptosomes exhibit coupled respiration which can be modulated by Na⁺. In addition, the appearance of Na⁺-stimulated glucose oxidation with maturation probably is linked to the development of Na⁺-K⁺-ATPase acitivity in the synaptosomal membrane.     

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.     

Amino Acid Metabolism in Rat Hippocampus During the Period of Brain Growth Spurt

We studied protein synthesis, lipid synthesis and CO₂ production by oxidation of glycine, alanine and leucine by slices of rat hippocampus during the period of brain growth spurt. The metabolism of the three amino acids decreased with the age of the animals, A major reduction was observed in protein synthesis, which was 4 times higher at 7 days of age than at 21 days of age for all amino acids studied. Glycine oxidation to CO₂ was twice as high as alanine oxidation and ten times higher than leucine oxidation. The major pathway of leucine utilization was incorporation into proteins. Glycine was the amino acid that had the highest metabolic rate.     

Glycine,Serine, and Leucine Metabolism in Different Regions of Rat Central Nervous System

We have investigated the glycine, serine and leucine metabolism in slices of various rat brain regions of 14-day-old or adult rats, using [1-¹⁴C]glycine, [2-¹⁴C]glycine, L-[3-¹⁴C]serine and L-[U-¹⁴C]leucine. We showed that the [1-¹⁴C]glycine oxidation to CO₂ in all regions studied occurs almost exclusively through its cleavage system (GCS) in brains of both 14-day-old and adults rats. In 14-day-old rats, the highest oxidation of [1-¹⁴C]glycine was in cerebellum and the lowest in medulla oblongata. In these animals, the L-[U-¹⁴C]leucine oxidation was lower than the [1-¹⁴C]glycine oxidation, except in medulla oblongata where both oxidations were the same. Serine was the amino acid that showed lowest oxidation to CO₂ in all structure studied. In adult rats brains, the highest oxidation of [1-¹⁴C]glycine was in cerebral cortex and the lowest in medulla oblongata. We have not seen difference in the lipid synthesis from both glycine labeled, neither in 14-day-old rats nor in adult ones, indicating that the lipids formed from glycine were not neutral. Lipid synthesis from serine was significantly high than lipid synthesis and from all other amino acids studied in all studied structures. Protein synthesis from L-[U-¹⁴C]leucine was significantly higher than that from glycine in all regions and ages studied.     

Role of liver-cell potassium ions in secretion of serum albumin and lipoproteins

1. The incorporation of l-[1-¹⁴C]leucine into the proteins of liver slices and into the serum albumin and lipoproteins transported by these slices was investigated. 2. Transport rates were found to be dependent on the K⁺ content of the slices. 3. The effect of K⁺ on transport of serum albumin and of serum lipoprotein can be separated from any effect on synthesis by altering K⁺ concentrations after inhibition of protein synthesis by cycloheximide or puromycin. 4. The effect of low K⁺ concentrations is reversible. 5. There is linear relationship between the K⁺ content of the slices and the transport of protein. A simple method is described for maintaining various steady concentrations of K⁺ in the liver slices. 6. K⁺ may be replaced by Rb⁺. Cs⁺ is partly effective, but NH₄⁺ and Li⁺ are no more effective than Na⁺. 7. We found evidence that K⁺ content rather than the flux rates of K⁺ or Na⁺ is important in this effect. 8. These results are probably important in ethionine and carbon tetrachloride poisoning in the rat, and may be significant in liver transplantation.     

THE HEXOSE MONOPHOSPHATE PATHWAY IN ETHYLNITROSOUREA INDUCED TUMORS OF THE NERVOUS SYSTEM

Respiration studies in vitro, in which tissue slices were incubated with [1-¹⁴C]glucose or [6-¹⁴C]glucose and ¹⁴CO₂ collected, resulted in C-1/C-6 ¹⁴CO₂ ratios that were higher in slices of tumor and newborn brain than in slices of adult brain. In adult brain, the C-1/C-6 ¹⁴CO₂ ratio averaged close to unity. In slices of tumor and newborn brain however, the mean C-1/C-6 ratio was greater than three. Addition of phenazine methosulfate (PMS) increased conversion of [1-¹⁴C]glucose to ¹⁴CO₂ in slices of normal adult brain 5-fold, and in slices of newborn brain and tumor, approx 12-fold. Injection of animals with 6-aminonicotinamide (6-AN) decreased conversion of [1-¹⁴C]glucose in slices of normal brain 30% but decreased conversion in tumor slices by 80%. Evidence supports the presence of an active hexose monophosphate pathway (HMP) in tumors of the nervous system regulated in part by available NADP⁺ levels. Inhibition by 6-AN was more effective in tumors than in normal adult brain.     

Effect of experimental colchicine encephalopathy on brain protein synthesis and tubulin metabolism

Colchicine blocks axoplasmic flow and produces neurofibrillary degeneration. Brain slices from mice injected intracerebrally with colchicine incorporated more [¹⁴C]leucine into protein and had a decreased uptake of [¹⁴C]leucine into the perchloric acid-soluble pool than did their controls. Brain RNA content was decreased and free leucine increased by colchicine-induced encephalopathy. The specific activities of proteins from subcellular fractions of colchicine-injected brain were increased in the nuclear fraction, the 100,000-g supernatant, and its vinblastine-precipitable tubulin. The ratio of the specific activity of the crude mitochondrial fraction to that of the total homogenate was decreased, as would consistent with impaired movement of newly labeled protein into synaptosomes. Colchicine-injected brain extracts contained one or more cytosol fractions that stimulated ribosomal incorporation of [¹⁴C]leucine into protein in a cell-free system. Colchicine-binding-activity measurements indicated loss of soluble and particulate tubulin in colchicine-injected brains; the decrease of soluble tubulin was verified by its selective precipitation with vinblastine. Colchicine encephalopathy did not affect the rate of spontaneous breakdown of in vitro colchicine binding activity. Similarities of colchicine encephalopathy to the neuron's response to axonal damage suggest that colchicine-induced increase in protein synthesis may, in part, reflect a neuronal response to blockage of neuroplasmic transport.     

THE MECHANISM OF ACTION OF β-BUNGAROTOXIN

—β-Bungarotoxin, a presynaptically-acting polypeptide neurotoxin, caused an efflux from synaptosomes of previously accumulated γ-aminobutyric acid and 2-deoxy-d -glucose. The toxin-induced efflux of γ-aminobutyric acid occurred by a Na⁺ -dependent process while that of 2-deoxyglucose was Na⁺ -independent. These effects were also produced by treating synaptosomes with low molecular weight compounds, including fatty acids, that inhibit oxidative phosphorylation. After incubation with β-bungarotoxin, synaptosomes exhibited increased production of ¹⁴CO₂ from [U-¹⁴C]glucose and decreased ATP levels. β-Bungarotoxin treatment of various subcellular membrane fractions caused the production of a factor that uncoupled oxidative phosphorylation when added to mitochondria. Mitochondria from toxin-treated brain tissue exhibited a limitation in the maximal rate of substrate utilization. We conclude that β-bungarotoxin acts by inhibiting oxidative phosphorylation in the mitochondria of nerve terminals. This inhibition accounts for the observed β-bungarotoxin effects on synaptosomes and at neuromuscular junctions. We suggest that the effects on energy metabolism result from a phospholipase A activity found to be associated with the toxin.     

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.

     

Thyroid hormone inhibition of synaptosome amino acid uptake and protein synthesis.

Abstract— 3,3′,5-Triiodothyronine (T₃) inhibited L-[¹⁴C]leucine uptake into synaptosomes. Inhibition was competitive with a K_i of 3.1 × 10^?5m . Hofstee plot revealed an inverted hyperbolic curve suggestive of a two carrier or carrier plus diffusion mediated system for amino acid uptake. Both the carrier mediated and diffusional components were inhibited by thyroid analogues. l -Thyroxine and analogues inhibited the incorporation of l -[¹⁴C] leucine into cerebral synaptosome protein. At 50 μm , the triiodo-compounds were more inhibitory than tetraiodo->3,5-triiodo-l -thyronine >3,3′,5-triiodothyropro-pionic> l -thyroxine >3,5-diiodo-l -tyrosine. Thyroid analogue inhibition was not seen in liver or brain mitochondrial protein synthesis. 3,3′,5-Triiodothyronine had no effect on respiratory control or 2,4-DNP stimulated synaptosome respiration supported by malate plus pyruvate. Ouabain did not inhibit [¹⁴C]leucine uptake into adult synaptosomes. There was synergistic inhibition of synaptosome protein synthesis by thyroid analogues in the presence of 0.2 mm -ouabain. 3,3′,5-Triiodothyronine had no effect on synaptosome fraction ATPase or Na-K ATPase. Addition of T₃ induced further inhibition of synaptosome protein synthesis in the presence of either chloramphenicol (100μm ) or cycloheximide (50μg/ml). [¹⁴C]Glycine uptake and incorporation into synaptosome protein was inhibited by 3,3′,5-triiodothyronine. There was no inhibition of [¹⁴C]proline uptake or incorporation. The above evidence and kinetic data strongly favor a selective competitive block in amino acid transport at the synaptosome membrane leading to a decreased rate of protein synthesis.     

Preincubation stimulates fatty acid synthesis by liver slices and isolated hypatocytes from fasted and diabetic rats.

We have observed that preincubation of 48 hour-fasted or alloxan diabetic rat liver slices, with no exogenous energy supply, for 3 hours resulted in an increased rate of incorporation of [1-¹⁴C] acetate into fatty acids and cholesterol during the following 2 hours. This preincubation effect was enhanced by the presence of glucose (25mM) in or prevented by the addition of dibutyryl cyclic adenosine 3′,5′ monophosphate (10^?4M) to the preincubation medium. Preincubation of normal rat liver slices did not change their rate of incorporation of [1-¹⁴C] acetate into fatty acids or cholesterol. The rate of ¹⁴CO₂ synthesized by normal, fasted or diabetic liver slices was little affected by preincubation. The preincubation effect, i.e. enhanced fatty acid synthesis was also observed in suspensions of hepatocytes from fasted and diabetic rats, preincubated for 2 hours, followed by a 1 hour incubation with either [1-¹⁴C] acetate or [³H] H₂O as precursor. We conclude from these data that there is concurrent and coordinated short- and long-term regulation of fatty acid biosynthesis in fasted and diabetic rat livers. Further, we suggest that the release of inhibition by preincubation of these tissues provides a useful tool for studying the coordinated control     

Role and Metabolism of Free Leucine in Skeletal Muscle in Protein Sparing Action of Dietary Carbohydrate and Fat

Feeding rats with either a carbohydrate meal or a fat meal to the previously fasted rats caused significant decrease in urinary output of urea and total nitrogen. The content of free leucine in skeletal muscle decreased in the rats fed either a carbohydrate meal or a fat meal. Feeding of either a carbohydrate meal or a fat meal stimulated incoiporation of l-leucine-1–¹⁴C into protein fraction of skeletal muscle and reduced its oxidation to ¹⁴CO₂.

These results suggest that the metabolism of leucine is under nutritional regulation and that the decrease in content of free leucine in skeletal muscle might be caused by enhanced reutilization of leucine into protein by the feeding of a carbohydrate meal or a fat meal. The role of free leucine in skeletal muscle as a regulator of protein turnover in the tissue are discussed in relation to the metabolism of this branched chain amino acid.     

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.     

Effects of exogenous phospholipases on brain membrane phospholipid perturbation, (Na+ + K+)-ATPase activity and cellular swelling of brain slices

Rat brain membranes were incubated with bee venom phospholipase A₂ (PLA₂) or phospholipase C (PLC) from Clostridium perfringens. PLA₂ caused a significant increase in free polyunsaturated fatty acids concomitant with membrane phospholipid degradation as monitored by HPLC and by gas chromatography. Equal concentrations of PLC had a much lesser effect than PLA₂. Divergent and differential effects were shown on deacylation and incorporation of [³H]arachidonic acid in membrane phospholipids. The incorporation of [³H]arachidonic acid into various phospholipids was greatly reduced by PLA₂ (0.018 units/ml) whereas PLC at identical concentration was not effective. PLA₂ inhibited (Na⁺ + K⁺)-ATPase but was not effective on p-nitrophenyl-phosphatase activity whereas PLC stimulated both enzymes. PLA₂ induced swelling of cortical brain slices whereas PLC was not effective. Thus, the severity of the perturbation of membrane integrity, and the inhibition of (Na⁺ + K⁺)-ATPase in brain membranes may play an important role in cellular swelling of brain slices induced by PLA₂.     

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.     

Acetylcholine Synthesis and CO2 Production from Variously Labeled Glucose in Rat Brain Slices and Synaptosomes

Abstract: The molecular basis of the close linkage between oxidative metabolism and acetylcholine (ACh) synthesis is still unclear. We studied this problem in slices and synaptosomes by measurement of ACh synthesis from [U-¹⁴C]glucose, and ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose, an index of glucose decarboxylation by the pyruvate dehydrogenase complex (PDH) and the enzymes of the Krebs cycle, respectively. We examined both under conditions that either inhibited (low O₂ or antimycin) or stimulated (2,4- dinitrophenol [DNP] or 35 mm -K⁺) ¹⁴CO₂ production from [2-¹⁴C]- or [3,4-¹⁴C]glucose. Incorporation of [U-¹⁴C]glucose into ACh was reduced under low O₂ and by antimycin or DNP (by 51-93%) and stimulated by 35 mm -K⁺ (by 30-60%). Under all of these conditions, ACh synthesis and the decarboxylation of [3,4-¹⁴C]- and [2-¹⁴C]glucose were linearly related (r= 0.741 and 0.579, respectively). The difference in the rate of ¹⁴CO₂ production from [3,4-¹⁴C]- and [2-¹⁴C]glucose was used as a measure of the amount of glucose that was not oxidatively decarboxylated (efflux). We found that efflux was reduced (low 0₂ and antimycin), unchanged (DNP in slices), or increased (DNP in synaptosomes and K⁺ stimulation in slices) compared with control values under 100% O₂. ACh synthesis and efflux were more closely related (r= 0.860) than ACh synthesis and ¹⁴CO₂ production from variously labeled glucoses.     

Incorporation of l-[C]leucine into egg proteins by liver slices from cod

1. Liver slices from cod (Gadus morhua L.) were incubated with l-[¹⁴C]leucine and the incorporation of label into total protein, precipitated with trichloroacetic acid, and into egg proteins, precipitated with an antibody after addition of carrier egg proteins, was measured. 2. Liver slices from immature male or female cod, and from male fish with developing testes, did not incorporate significant amounts of l-[¹⁴C]leucine into egg proteins, whereas with slices from female cod with developing ovaries the rate of incorporation into egg proteins was 8% of the rate of incorporation into total protein. 3. Liver slices from immature male or female fish that had received an intramuscular injection of oestradiol benzoate (1mg/kg) 5–8 days previously incorporated l-[¹⁴C]leucine into egg proteins at about 26% of the rate of incorporation into total protein. 4. Incorporation into total protein and into egg proteins was inhibited by puromycin, and 1.2 and 0.13μg of puromycin/mg of tissue protein, respectively, gave 50% inhibition.     

Effect of methylmalonate on ketone body metabolism in developing rat brain

The oxidation of 3-hydroxy[3-¹⁴C]butyrate to CO₂ and its incorporation into cerebral lipids by cortex slices from one-week old rats were markedly inhibited by methylmalonate. However, methylmalonate had no effect on the metabolism of labelled aceto- acetate, glucose and acetate by brain slices. Addition of propionate in the incubation medium reduced cerebral lipogenesis from labelled 3-hydroxybutyrate and acetate. Acute methylmalonic acidemia induced in one-week old pups by injecting 3% methylmalonate solution caused a reduction in the incorporation of labelled 3-hydroxybutyrate into cerebral lipids. However, acute methylmalonic acidemia had no effect on cerebral lipogensis $\text{in vivo}$ from labelled acetate. These findings show (i) the conversion of 3-hydroxybutyrate to acetoacetate by 3-hydroxybutyrate dehydrogenase in the brain is inhibited by methylmalonate, and (ii) an inhibition of cerebral lipid synthesis by propionate, which also accumulates in patients with methylmalonic aciduria.     

Alternative pathways of glucose utilization in brain. Changes in the pattern of glucose utilization in brain during development and the effect of phenazine methosulfate on the integration of metabolic routes.

The activities of alternative pathways of glucose metabolism in developing rat brain were evaluated by measurement of the yields of ¹⁴CO₂ from glucose labeled with ¹⁴C on carbons 1, 2, 3 + 4, 6 and uniformly labeled glucose, from the detritiation of [2-³H]glucose and from the incorporation of ¹⁴C from specifically labeled glucose into lipids by brain slices from cerebral hemispheres and cerebellum. The glycolytic route and tricarboxylic acid cycle (¹⁴CO₂ yield from carbons 3, 4, and 6 of glucose) increased during development. The flux through the glutamate-γ-aminobutyric route (¹⁴CO₂ yield from carbon 2-carbon 6 of glucose) also showed an increase with development. In contrast, the proportion of glucose metabolized via the pentose phosphate pathway was markedly decreased as development progressed. The artificial electron acceptor, phenazine methosulfate, was used as a probe to investigate the effect of alterations in the redox state of $\text{NADP}{}^{\mathrm{+}}\text{NADPH}$ couple on a number of NADP-linked systems in developing brain. Phenazine methosulfate produced a massive (20- to 50-fold) stimulation of the pentose phosphate pathway, in contrast, the incorporation of glucose carbon into fatty acids and flux through the glutamate-γ-aminobutyrate shunt were sharply decreased. The effects of phenazine methosulfate on the incorporation of glucose into glyceride glycerol, on the flux of glucose through the pyruvate dehydrogenase reaction and tricarboxylic acid cycle, all processes linked to the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ couple, appeared to be minimal in the brain at the stages of development studied, i.e., 1, 5, 10, 20 days, and in the adult rat. The significance of the massive reserve potential of the pentose phosphate pathway in the developing brain is discussed.