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.     

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.     

Aging Decreases Oxidative Metabolism and the Release and Synthesis of Acetylcholine

Acetylcholine (ACh) synthesis in vivo is known to decrease during the aging process (senescence). To elucidate the molecular mechanism(s) of this age-related decline, we studied brain slices from 3-, 10-, and 30-month-old mice of two strains (C57B1 and Balb/c). In low K⁺ media, oxidative metabolism as measured by ¹⁴CO₂ production decreased with aging from 100% (3 months) to 85% (10 months) or 71% (30 months) whether [U^?14C]glucose, [3,4-¹⁴C]glucose, or [l-¹⁴C]pyruvate was the substrate. In the aged brain (3 months) the increase in ¹⁴CO₂ production with K⁺ stimulation was about twofold higher than in the young brain (3 months). Thus, in high K⁺ media, only slight decreases (<10%) in oxidative metabolism occurred with aging. Changes in ACh synthesis paralleled the decreases in ¹⁴CO₂ production. Synthesis of [¹⁴C]ACh from [U-¹⁴C]glucose in low K⁺ media declined from 100% (3 months) to 85% (10 months) or 66% (30 months), while in high K⁺ media only slight decreases (<10.5%) occurred with aging. The Ca²⁺-dependent, K⁺-stimulated release of [¹⁴C]ACh declined from 100% (3 months) to 58% (10 months) or 25% (30 months). Only the decrease in the release of ACh declined to the same extent as the reduced in vivo synthesis of ACh with aging. The results suggest that decreases in oxidative metabolism, ACh synthesis, and in the release of ACh contribute to a reduction in cholinergic function in the senescent brain.     

Effect of 2-deoxy-d-Glucose on Aminoacids Metabolism in Rats’ Cerebral Cortex Slices

We studied the effect of different concentrations of 2-deoxy-d-glucose on the l-[U-¹⁴C]leucine, l-[1-¹⁴C]leucine and [1-¹⁴C]glycine metabolism in slices of cerebral cortex of 10-day-old rats. 2-deoxy-d-glucose since 0.5 mM concentration has inhibited significantly the protein synthesis from l-[U-¹⁴C]leucine and from [1-¹⁴C]glycine in relation to the medium containing only Krebs Ringer bicarbonate. Potassium 8.0 mM in incubation medium did not stimulate the protein synthesis compared to the medium containing 2.7 mM, and at 50 mM diminishes more than 2.5 times the protein synthesis compared to the other concentration. Only at the concentration of 5.0 mM, 2-deoxy-d-glucose inhibited the CO₂ production and lipid synthesis from l-[U-¹⁴C] leucine. This compound did not inhibit either CO₂ production, or lipid synthesis from [1-¹⁴C]glycine. Lactate at 10 mM and glucose 5.0 mM did not revert the inhibitory effect of 2-deoxy-d-glucose on the protein synthesis from l-[U-¹⁴C]leucine. 2-deoxy-d-glucose at 2.0 mM did not show any effect either on CO₂ production, or on lipid synthesis from l-[U-¹⁴C]lactate 10 mM and glucose 5.0 mM.     

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.     

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.     

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.     

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.     

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 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.     

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.     

Selective damage in striatum and hippocampus with in vitro anoxia

An in vitro model of anoxia-induced brain damage was utilized to help elucidate the biochemical basis of cell damage due to reduced oxygen availability. Previous studies suggest that anoxia-induced damage may vary presynaptically, post-synaptically or in the cell body. Thus, the consequences of an anoxic treatment incubation were examined with hippocampal slices, which contain cholinergic nerve terminals but not cell bodies, and with slices from whole striatum or its subregions, which contain both cholinergic cell bodies and nerve terminals. Slices were preincubated with either oxygen or nitrogen (treatment incubation) and the persistent effects of this treatment on [¹⁴C]acetylcholine and¹⁴CO₂ production from [U-¹⁴C]glucose were assessed in a subsequent incubation under optimal conditions (test incubation). An anoxic treatment incubation reduced the subsequent test incubation production of CO₂ about 40% in the hippocampus and striatum, The anoxic treatment incubation diminished ACh production by 46% in the striatum, but only minimally affected that in the hippocampus. Anoxic treatment incubations of synaptosomes did not alter test-incubation ACh synthesis or CO₂ production. Omission of calcium from the anoxic treatment incubation increased striatal ACh synthesis by 88% and CO₂ production in both regions. These results suggest that anoxia produces persistent changes in postsynaptic processes or cell bodies (in this model cholinergic ones) that differ from those in nerve terminals and that calcium is important in the production of these deficits.     

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.     

The effect of glucose on the control of carbohydrate metabolism in ripening bananas

The effect of exogenous glucose on the major fluxes of carbohydrate metabolism in cores of climacteric fruit of banana (Musa cavendishii Lamb ex Paxton) was determined with the intention of using the effects in the application of top-down metabolic control analysis. Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21 °C. Cores were removed from climacteric fruit and incubated in 100 or 200 mM glucose for 4 or 6 h. The rates of starch breakdown, sucrose and fructose accumulation and CO₂ production were measured. The steady-state contents of hexose monophosphates, adenylates and pyruvate were determined. In addition, the detailed distribution of label was determined after supply of the following: [U-¹⁴C]-, [1-¹⁴C]-, [3,4¹⁴C]and [6-¹⁴C]glucose, and [U-¹⁴C]glycerol. The data were used to estimate the major fluxes of carbohydrate metabolism. Supply of exogenous glucose led to increases in the size of the hexose-monophosphate pools. There was a small stimulation of the rate of sugar synthesis and a major increase in the rate of starch synthesis. Starch breakdown was inhibited. Respiration responded to the demand for ATP by sugar synthesis. The effect of glucose on fluxes and metabolite pools is discussed in relation to our understanding of the control and regulation of carbohydrate metabolism in ripening fruit.Abbreviations Glc6P glucose-6-phosphate - Glc1P glucose-1-phosphate - Fru6P fructose-6-phosphate - AEC adenylate energy charge We thank Geest Foods Group, Great Dunmow, Essex, UK for giving us the bananas. SAH thanks the managers of the Broodbank Fund for a fellowship.     

Oxidation of Glucose,Ribose, Alanine,and Glutamate by Leishmania braziliensis panamensis1

The metabolism of [1-¹⁴C]- and [6-¹⁴C]glucose, [1-¹⁴]ribose, [1-¹⁴C]- and [U-¹⁴C]alanine, and [1-¹⁴C]- and [5-¹⁴C]glutamate by the promastigotes of Leishmania braziliensis panamensis was investigated in cells resuspended in Hanks' balanced salt solution supplemented with ribose, alanine, or glutamate. The ratio of ¹⁴CO₂ produced from [1-¹⁴C]glucose to that from [6-¹⁴C]glucose ranged from about two to six, indicating appreciable carbon flow through the pentose phosphate pathway. A functional pentose phosphate pathway was further demonstrated by the production of ¹⁴CO₂ from [1-¹⁴C]ribose although the rate of ribose oxidation was much lower than the rate of glucose oxidation. The rate of ¹⁴CO₂ production from [1-¹⁴C]glucose was almost linear with time of incubation, whereas that of [6-¹⁴C]glucose accelerated, consistent with an increasing rate of flux through the Embden-Meyerhof pathway during incubation. Increasing the assay temperature from 26°C to 34°C had no appreciable effect on the rates or time courses of oxidation of either [1-¹⁴C]- or [6-¹⁴C]glucose or of [1-¹⁴C]ribose. Both alanine and glutamate were oxidized by L. b. panamensis, and at rates comparable to or appreciably greater than the rate of oxidation of glucose. The ratios of ¹⁴CO₂ produced from [1-¹⁴C]- to [U-¹⁴C]alanine and from [1-¹⁴C]- to [5^-14C]glutamate indicated that these compounds were metabolized via a functioning tricarboxylic acid cycle and that most of the label that entered the tricarboxylic acid cycle was oxidized to carbon dioxide. Heating the cultures for 6 or 12 h at 34°C, which converts the promastigotes into an ellipsoidally shaped intermediate form, decreased the rates of oxidation of glucose, alanine, and glutamate. The oxidation of glutamate decreased by about 50% and 70% after a 6-h or 12-h heat treatment, respectively. Returning the heated cultures to 26°C initiated a reversion to the promastigote form and recovery of the rate of glucose oxidation, but glutamate oxidation did not return to control levels by 19 h at 26°C.     

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.     

Activity of the pentose phosphate pathway during lignification

Summary We did this work to see if there is a correlation between lignin synthesis and the activity of the pentose phosphate pathway. Excision of the third internode of the stem of Coleus blumei Benth. followed by incubation on sucrose and indoleacetic acid led to extensive formation of tracheids. During this lignification we determined the activities of glucose-6-phosphate dehydrogenase and fructose-1,6-diphosphate aldolase, and the extent to which [1-¹⁴C]-,[3,4-¹⁴C]-, and [6-¹⁴C]glucose labelled CO₂ and the major cellular components. The results indicate that the pentose phosphate pathway was active during lignification, and that the activity of this pathway relative to glycolysis increased at the onset of lignification. Explants of storage tissue of Helianthus tuberosus L. were cultured under conditions which caused extensive lignification. ¹⁴CO₂ production from [1-¹⁴C]-, [3,4-¹⁴C]-, and [6-¹⁴C]glucose indicated activity of the pentose phosphate pathway during tracheid formation. We suggest that lignification is accompanied by appreciable activity of the pentose phosphate pathway and that this could provide the reducing power for lignin synthesis.Abbreviations NADP nicotinamide-adenine dinucleotide phosphate - IAA indoleacetic acid     

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.     

Fluxes of carbohydrate metabolism in ripening bananas

The major fluxes of carbohydrate metabolism were estimated during starch breakdown by ripening bananas (Musa cavendishii Lamb ex Paxton). Hands of bananas, untreated with ethylene, were allowed to ripen in the dark at 21° C. Production of CO₂ and the contents of starch, sucrose, glucose and fructose of intact fruit were determined for a period of 10 d that included the climacteric. The detailed distribution of label was determined after supplying the following to cores of pulp from climacteric fruit: [U-¹⁴C]-, [1-¹⁴C]-, [3,4-¹⁴C]-and [6-¹⁴C]glucose, [U-¹⁴C]glycerol, ¹⁴CO₂. The data obtained were used to estimate the following fluxes, values given as mol hexose · (g FW)^–1 · h^–1 in parenthesis: starch to hexose monophosphates (5.9) and vice versa (0.4); hexose monophosphates to sucrose (7.7); sucrose to hexose (4.7); hexose to hexose monophosphate (3.8); glycolysis (0.5–1.6); triose phosphate to hexose monophosphates (0.14); oxidative pentose-phosphate pathway (0.48); CO₂ fixation in the dark (0.005). These estimates are related to our understanding of carbohydrate metabolism during ripening.We both thank Mr Richard Trethewey for his constructive criticism: S.A.H. thanks the Managers of the Broodbank Fund for a fellowship.     

(14C) acetylcholine synthesis by cortex slices of rat brain

Abstract—

• 1 A procedure has been developed to measure ACh synthesis from [¹⁴C]-precursors. As little as 10^?9 moles of ACh were detected as the result of de nova synthesis. Following incubation of cortex slices of rat brain with eserine and a tagged metabolite, ACh carrier was added to the incubation medium and to an extract from the slices. ACh was purified by chromatography on Amberlite CG-50, precipitation and recrystallization of ACh chloroaurate.
• 2 [U^?14C]glucose and [2^?14C]pyruvate formed similar amounts of [¹⁴C]ACh. Hydrolysis of ACh with subsequent chromatography of the resultant acetic acid demonstrated that all of the label was located in the acetyl moiety. [¹⁴C]acetate did not serve as a precursor of the acetyl group of ACh. Equivalent incorporation of carbons 1 and 6 of glucose into ACh indicated that glucose metabolism to ACh occurred via the Embden-Meyerhof pathway.
• 3 The amount of ACh detected by bioassay after incubation of cortex slices with [U^?14C]glucose was approximately the same as that calculated as labelled ACh; this demonstrates that all of the acetyl groups of ACh formed during incubation were derived from glucose.
• 4 [¹⁴C]choline, either methyl or chain labelled, formed [¹⁴C]ACh while labelled ethanolamine, serine and methionine did not. Synthesis from labelled choline did not occur in the absence of glucose.
• 5 When both [U^?14C]glucose and [¹⁴C]choline were incubated with brain slices, the acetyl and choline moieties of ACh were equally labelled; this demonstrates that the entire molecule was formed from added precursors. Slices supported a high rate of ACh synthesis without addition of choline. The addition of 10^?4m -hemicholinium-3 inhibited ACh formation by more than 90 per cent from either [U-¹⁴C]glucose or [Me-¹⁴C]choline.
• 6 Study of the time course of ACh synthesis from glucose demonstrated a rapid formation of [¹⁴C]ACh within the slices which reached a maximum during the first hour of incubation. [¹⁴C]ACh in the incubation medium accumulated at a linear rate for 3 hr. Replacement of a portion of the sodium chloride of the incubation medium by potassium chloride to a final concentration of 31 mm -KCI markedly increased the formation of [¹⁴C]ACh found in the incubation medium. Decreased amounts of [¹⁴C]ACh were extracted from the slices by homogenization or by subsequent heating at pH 4 in the high potassium ion medium.