Metabolism of glucose and glutamine in lymphocytes from graves' hyperthyroid patients: influence of methimazole treatment

Several studies have shown that thyroid hormones are able to influence selected immune responses such as cell mediated immunity, differentiation of B lymphocytes and the activity of NK cells. These hormones can also regulate the metabolism of glucose and glutamine in rat macrophages and their effects seem to occur mainly through the Krebs cycle. Alterations in the hexokinase, citrate synthase, glucose-6-phosphate dehydrogenase and glutaminase activities in lymphocytes from patients with Graves' disease, either untreated or on methimazole (MMI) therapy were investigated. Experiments were also done in vitro to determine the activities of these enzymes in normal lymphocytes cultured for 24 h in the presence of MMI, T₃ and T₄ using concentrations close to the physiological. Changes in the conversion of [U-¹⁴C]-glucose and [U-¹⁴C]-glutamine to ¹⁴CO₂ as caused by the addition of MMI, T₃ or T₄ to the culture medium were also evaluated. The results indicate that high levels of thyroid hormones might stimulate the metabolism of glucose and glutamine for a short period of time but, if the stimulus is maintained, the utilization of glutamine by lymphocytes is then suppressed. Moreover, MMI does affect lymphocyte metabolism but the significance of this finding for its immunosuppressive effect remains to be examined.     

Metabolic Compartmentation in Cortical Synaptosomes: Influence of Glucose and Preferential Incorporation of Endogenous Glutamate into GABA

Metabolism of glutamine was determined under a variety of conditions to study compartmentation in cortical synaptosomes. The combined intracellular and extracellular amounts of [U-¹³C]GABA, [U-¹³C]glutamate and [U-¹³C]glutamine were the same in synaptosomes incubated with [U-¹³C]glutamine in the presence and absence of glucose. However, the concentration of these amino acids was decreased in the latter group, demonstrating the requirement for glucose to maintain the size of neurotransmitter pools. In hypoglycemic synaptosomes more [U-¹³C]glutamine was converted to [U-¹³C]aspartate, and less glutamate was re-synthesized from the tricarboxylic acid (TCA) cycle, suggesting use of the partial TCA cycle from -ketoglutarate to oxaloacetate for energy. Compartmentation was studied in synaptosomes incubated with glucose plus labeled and unlabeled glutamine and glutamate. Incubation with [U-¹³C]glutamine plus unlabeled glutamate gave rise to [U-¹³C]GABA but not labeled aspartate; however, incubation with [U-¹³C]glutamate plus unlabeled glutamine gave rise to [U-¹³C]aspartate, but not labeled GABA. Thus the endogenous glutamate formed via glutaminase in synaptic terminals is preferentially used for GABA synthesis, and is metabolized differently than glutamate taken up from the extracellular milieu.     

Glial Metabolism of Valine

The three essential amino acids, valine, leucine and isoleucine, constitute the group of branched-chain amino acids (BCAAs). BCAAs are rapidly taken up into the brain parenchyma, where they serve several distinct functions including that as fuel material in brain energy metabolism. As one function of astrocytes is considered the production of fuel molecules that support the energy metabolism of adjacent neural cells in brain. Astroglia-rich primary cultures (APC) were shown to rapidly dispose of the BCAAs, including valine, contained in the culture medium. While the metabolisms of leucine and isoleucine by APC have already been studied in detail, some aspects of valine metabolism remained to be determined. Therefore, in the present study an NMR analysis was performed to identify the ¹³C-labelled metabolites that are generated by APC during catabolism of [U-¹³C]valine and that are subsequently released into the incubation medium. The results presented show that APC (1) are potently disposing of the valine contained in the incubation medium; (2) are capable of degrading valine to the tricarboxylic acid (TCA) cycle member succinyl-CoA; and (3) release into the extracellular milieu valine catabolites and compounds generated from them such as [U-¹³C]2-oxoisovalerate, [U-¹³C]3-hydroxyisobutyrate, [U-¹³C]2-methylmalonate, [U-¹³C]isobutyrate, and [U-¹³C]propionate as well as several TCA cycle-dependent metabolites including lactate. This article is dedicated to Dr. George DeVries.     

Glucose and lactate utilization by the amygdala of male and female rats

Gonadal hormones appear to modulate brain energy metabolism, and morphological and functional sexual differences are found in the amygdaloid complex (AC) of rats. Our aim was to study the CO₂ production and lipid synthesis, measured by the rate of L-[U-¹⁴C]lactate or D-[U-¹⁴C]glucose utilization (in pmol.hr^–1.mg^–1), by AC slices in vitro of male and female rats. Lactate was more used than glucose as energy substrate (p < 0.01) but no sex-related difference was observed in glucose or lactate oxidation to CO₂ (p > 0.05) or on lipid synthesis obtained from both substrates (p > 0.05). In addition, there was no effect of the estrous cycle on lactate oxidation to CO₂ by the AC of females (p > 0.05). Based on the present data, it appears that the endogenous normal levels of gonadal hormones are not able to promote sex-related differences in the in vitro glucose or lactate utilization by the AC of rats.     

Amino acids in ram testicular fluid and semen and their metabolism by spermatozoa

1. The testis of the ram secretes considerable amounts of amino acids (200μmoles/day) into the fluid collected from the efferent ducts. The principal amino acid in this testicular fluid is glutamate, which is present in concentrations about eight times those in testicular lymph or in blood from the internal spermatic vein. 2. The concentration of glutamate in seminal plasma from the tail of the epididymis is about ten times that in testicular fluid, and, though glutamate is the major amino acid in ejaculated seminal plasma, its concentration is less than in epididymal plasma. 3. After the intravenous infusion of [U-¹⁴C]glucose, labelled glutamate was found in the testicular fluid. Radioactivity was also detected in alanine, glycine, serine plus glutamine and aspartate. Alanine had the highest specific activity, about 50% of the specific activity of blood glucose. 4. When [U-¹⁴C]glutamate was infused, the specific activity of glutamate in testicular fluid was only about 2% that in the blood plasma. 5. Testicular and ejaculated ram spermatozoa oxidized both [U-¹⁴C]glutamate and [U-¹⁴C]leucine to a small extent, but neither substrate altered the respiration from endogenous levels. 6. No radioactivity was detected in testicular spermatozoal protein after incubation with [U-¹⁴C]glutamate or [U-¹⁴C]leucine. Small amounts of radioactivity were detected in protein from ejaculated ram spermatozoa after incubation with [U-¹⁴C]glutamate. 7. The carbon of [U-¹⁴C]glucose was incorporated into amino acids by testicular spermatozoa; most of the radioactivity occurred in glutamate.     

Metabolic Characterization of Acutely Isolated Hippocampal and Cerebral Cortical Slices Using [U-<Superscript>13</Superscript>C]Glucose and [1,2-<Superscript>13</Superscript>C]Acetate as Substrates

Brain slice preparations from rats, mice and guinea pigs have served as important tools for studies of neurotransmission and metabolism. While hippocampal slices routinely have been used for electrophysiology studies, metabolic processes have mostly been studied in cerebral cortical slices. Few comparative characterization studies exist for acute hippocampal and cerebral cortical slices, hence, the aim of the current study was to characterize and compare glucose and acetate metabolism in these slice preparations in a newly established incubation design. Cerebral cortical and hippocampal slices prepared from 16 to 18-week-old mice were incubated for 15–90 min with unlabeled glucose in combination with [U-¹³C]glucose or [1,2-¹³C]acetate. Our newly developed incubation apparatus allows accurate control of temperature and is designed to avoid evaporation of the incubation medium. Subsequent to incubation, slices were extracted and extracts analyzed for ¹³C-labeling (%) and total amino acid contents (µmol/mg protein) using gas chromatography–mass spectrometry and high performance liquid chromatography, respectively. Release of lactate from the slices was quantified by analysis of the incubation media. Based on the measured ¹³C-labeling (%), total amino acid contents and relative activity of metabolic enzymes/pathways, we conclude that the slice preparations in the current incubation apparatus exhibited a high degree of metabolic integrity. Comparison of ¹³C-labeling observed with [U-¹³C]glucose in slices from cerebral cortex and hippocampus revealed no significant regional differences regarding glycolytic or total TCA cycle activities. On the contrary, results from the incubations with [1,2-¹³C]acetate suggest a higher capacity of the astrocytic TCA cycle in hippocampus compared to cerebral cortex. Finally, we propose a new approach for assessing compartmentation of metabolite pools between astrocytes and neurons using ¹³C-labeling (%) data obtained from mass spectrometry. Based on this approach we suggest that cellular metabolic compartmentation in hippocampus and cerebral cortex is very similar.     

Metabolism of [1,6-13C]Glucose and [U-13C]Glutamine and Depolarization Induced GABA Release in Superfused Mouse Cerebral Cortical Mini-slices

Mouse cerebral cortical mini-slices were used in a superfusion system to monitor depolarization-induced (55 mM K⁺) release of preloaded [2,3-³H]GABA and to investigate the biosynthesis of glutamate, GABA and aspartate during physiological and depolarizing (55 mM K⁺) conditions from either [1,6-¹³C]glucose or [U-¹³C]glutamine. Depolarization-induced GABA release could be reduced (50%) by the GABA transport inhibitor tiagabine (25 μM) or by replacing Ca²⁺ with Co²⁺. In the presence of both tiagabine and Co²⁺ (1 mM), release was abolished completely. The release observed in the presence of 25 μM tiagabine thus represents vesicular release. Superfusion in the presence of [1,6-¹³C]glucose led to considerable labeling in the three amino acids, the labeling in glutamate and aspartate being increased after depolarization. This condition had no effect on GABA labeling. For all three amino acids, the distribution of label in the different carbon atoms revealed on increased tricarboxylic acid (TCA) activity during depolarization. When [U-¹³C]glutamine was used as substrate, labeling in glutamate was higher than that in GABA and aspartate and the fraction of glutamate and aspartate being synthesized by participation of the TCA cycle was increased by depolarization, an effect not seen for GABA. However, GABA synthesis reflected TCA cycle involvement to a much higher extent than for glutamate and aspartate. The results show that this preparation of brain tissue with intact cellular networks is well suited to study metabolism and release of neurotransmitter amino acids under conditions mimicking neural activity. Special issue article in honor of Dr. Ricardo Tapia.     

Integrated 13C-metabolic flux analysis of 14 parallel labeling experiments in Escherichia coli

The use of parallel labeling experiments for ¹³C metabolic flux analysis (¹³C-MFA) has emerged in recent years as the new gold standard in fluxomics. The methodology has been termed COMPLETE-MFA, short for complementary parallel labeling experiments technique for metabolic flux analysis. In this contribution, we have tested the limits of COMPLETE-MFA by demonstrating integrated analysis of 14 parallel labeling experiments with Escherichia coli. An effort on such a massive scale has never been attempted before. In addition to several widely used isotopic tracers such as [1,2-¹³C]glucose and mixtures of [1-¹³C]glucose and [U-¹³C]glucose, four novel tracers were applied in this study: [2,3-¹³C]glucose, [4,5,6-¹³C]glucose, [2,3,4,5,6-¹³C]glucose and a mixture of [1-¹³C]glucose and [4,5,6-¹³C]glucose. This allowed us for the first time to compare the performance of a large number of isotopic tracers. Overall, there was no single best tracer for the entire E. coli metabolic network model. Tracers that produced well-resolved fluxes in the upper part of metabolism (glycolysis and pentose phosphate pathways) showed poor performance for fluxes in the lower part of metabolism (TCA cycle and anaplerotic reactions), and vice versa. The best tracer for upper metabolism was 80% [1-¹³C]glucose+20% [U-¹³C]glucose, while [4,5,6-¹³C]glucose and [5-¹³C]glucose both produced optimal flux resolution in the lower part of metabolism. COMPLETE-MFA improved both flux precision and flux observability, i.e. more independent fluxes were resolved with smaller confidence intervals, especially exchange fluxes. Overall, this study demonstrates that COMPLETE-MFA is a powerful approach for improving flux measurements and that this methodology should be considered in future studies that require very high flux resolution.     

Pyruvate kinase and alanine synthesis in skeletal muscle

L-Phenylalanine is an allosteric inhibitor of M₁-type pyruvate kinase. Accordingly, the effects were studied of 20 mM phenylalanine on the metabolism of 5 mM [U-¹⁴C]glucose and 3 mM L-[U-¹⁴C]glutamate by isolated hemidiaphragms from starved rats. Phenylalanine inhibited lactate and¹⁴CO₂ production from both substrates and stimulated alanine release. It is concluded that pyruvate kinase may have a dual role in intermediary metabolism in skeletal muscle: the enzyme is a component of the lower glycolytic pathway and is implicated in a pathway of amino acid oxidation and alanine synthesis.     

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.     

GLUCOSE AND AMINO ACID METABOLISM IN SOME INVERTEBRATE NERVOUS SYSTEMS

(1) The in vitro metabolism of [U-¹⁴C]glucose and [U-¹⁴C]glutamate was compared in snail, octopus and locust ganglia, and in rat cerebral cortex. (2) The metabolic patterns are quantitatively similar. The major labelled metabolites formed from glucose or glutamate by rat cortex and the invertebrate systems were CO₂, aspartate, glutamate, glutamine and alanine. γ-Aminobutyric acid (GABA) was formed in substantial amounts only by locust and rat. (3) A much larger proportion of labelled glucose and glutamate was converted to alanine by the invertebrates compared with rat cortex, although ¹⁴CO₂ production was lower. (4) The effect of glucose in reducing aspartate formation and stimulating glutamine formation from [U-¹⁴C]glutamate in mammalian cortex was observed in the locust but not in the molluscs. (5) Labelled citric acid cycle intermediates were formed in substantial quantities from glucose and glutamate only by snail and locust.     

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.     

Effects of thyroid states on the Cori cycle,glucose-alanine cycle,and futile cycling of glucose metabolism in rats

The effect of thyroid status on glucose recycling was measured in intact rats by comparing the fates of differently labeled [³H]- and [¹⁴C]glucose. Glucose recycling at the level of three-carbon compounds (i.e., Cori and glucose-alanine cycles) was measured by comparing the rates of turnover of [6-³H]- and [6-¹⁴C]glucose in the same animal. The rate of recycling increased (33–110%) in hyperthyroid rats and decreased (22–30%) in hypothyroid (thyroidectomized) rats. The relative importance of the Cori and glucose-alanine cycles was measured by analyzing the labeled glycolytic intermediates after the injection of labeled glucose; and by measuring the rate of glucose production from the infused labeled lactate and alanine. The results showed that the rate of the Cori cycle is much greater than the glucose-alanine cycle in rats. Substrate cycling at the level of glucokinase-glucose-6-phosphatase was measured by comparing the rates of turnover of [2-³H]- and [6-³H]glucose; and phosphofructokinase-fructose bisphosphatase was measured by comparing the rates of turnover of [3-³H]- and [6-³H]glucose. These cycles were also affected by thyroid states of the animals. The rate of the phosphofructokinase-fructose bisphosphatase cycle increased threefold in hyperthyroid rats and decreased by about half in hypothyroid rats. The glucokinase-glucose-6-phosphatase substrate cycle occurred at the rate of nearly 2 μmol/min/100 g body wt in the hyperthyroid, fasted rats; it was not detectable in hypo- or euthyroid rats. The contribution of the energy released by these cycles to thyroid thermogenesis was discussed. Effects of thyroid states on glucose metabolism in perfused muscles were also studied. There is an apparent shift in the source of energy for oxidation in the hyperthyroid rat. The ratio of lactate production to glucose uptake was significantly elevated in the hyperthyroid rats. This change predisposes for increased glucose recycling in hyperthyroid rats to avoid lactate accumulation and acidosis.     

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.     

Viral effects on metabolism: changes in glucose and glutamine utilization during human cytomegalovirus infection

Human cytomegalovirus (HCMV) infection causes dramatic alterations of intermediary metabolism, similar to those found in tumor cells. In infected cells, glucose carbon is not completely broken down by the tricarboxylic acid (TCA) cycle for energy; instead, it is used biosynthetically. This process requires increased glucose uptake, increased glycolysis and the diversion of glucose carbon, in the form of citrate, from the TCA cycle for use in HCMV-induced fatty acid biosynthesis. The diversion of citrate from the TCA cycle (cataplerosis) requires induction of enzymes to promote glutaminolysis, the conversion of glutamine to α-ketoglutarate to maintain the TCA cycle (anaplerosis) and ATP production. Such changes could result in heretofore uncharacterized pathogenesis, potentially implicating HCMV as a subtle cofactor in many maladies, including oncogenesis. Recognition of the effects of HCMV, and other viruses, on host cell metabolism will provide new understanding of viral pathogenesis and novel avenues for antiviral therapy.     

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.     

Insulin treatment can abolish changes in glucose and glutamine metabolism of lymphocytes and macrophages caused by the implantation of the walker 256 tumour

Activation of lymphocytes and macrophages by the implantation of tumour cells (10⁷ cells per rat) into the left flank of rats increased the conversion of glucose to lactate and of glutamine to glutamate and aspartate and the decarboxylation of [U-¹⁴C]-glucose and [U-¹⁴C]-glutamine in incubated cells. In addition, the amount of GLUT1 was increased in macrophages. The effect of insulin treatment on glucose and glutamine metabolism of lymphocytes and macrophages activated by Walker 256 tumour implantation was also examined. For this purpose, insulin was injected subcutaneously (4 U/100 g b.w. daily) after the fourth day of tumour implantation and the rats were killed 10 days afterwards. Insulin treatment fully reverted the changes due to tumour implantation in the metabolism of glucose and glutamine in lymphocytes and of glucose in macrophages.     

The involvement of sucrose,glucose and other metabolites in the synthesis of triterpenes and dopa in the laticifers of Euphorbia lathyris

A quantitative triterpene analysis was made of latex stem tissue of Euphorbia lathyris. Young plants seedlings of E. lathyris were incubated with various labelled precursors. Incorporation into triterpenes was obtained from [2-¹⁴C]mevalonic acid, [1-¹⁴C]acetate, [3-¹⁴C]pyruvate, [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose, [U-¹⁴C]glyoxylate, [2,3-¹⁴C]succinic acid, [1-¹⁴C]glycerol [U-¹⁴C]serine. Both sugars tyrosine appeared to be effective precursors in DOPA synthesis inside the laticifers. Exogenously supplied mevalonic acid was only involved in triterpene synthesis outside the laticifers. GC-RC of triterpenes synthesized from [U-¹⁴C]glucose revealed the origin of these compounds in the latex. The labelled triterpenes obtained after incorporation of the other mentioned labelled precursors were only partly synthesized in the laticifers. For quantitative data on latex triterpene synthesis seedlings were incubated with [U-¹⁴C]sucrose, [U-¹⁴C]glucose, [U-¹⁴C]xylose [1-¹⁴C]acetate in the presence of increasing amounts of unlabelled substrate. From the amount of ¹⁴C incorporated into the triterpenes the amount of substrate directly involved in triterpene synthesis was calculated, as was the absolute triterpene yield. Sucrose showed the highest triterpene yield, equivalent to the daily increase of the triterpene content of growing seedlings. The possible significance of the other precursors in triterpene synthesis in the laticifers is discussed.     

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.