Gluconeogenesis in the kidney cortex. Effects of d-malate and amino-oxyacetate

1. Rat kidney-cortex slices incubated with d-malate alone formed very little glucose. d-Malate, however, augmented gluconeogenesis from l-lactate and inhibited gluconeogenesis from pyruvate and l-malate. 2. d-Malate had little effect on the rate of the tricarboxylic acid cycle with or without other substrates added. 3. d-Malate inhibited the activity of the l-malate dehydrogenase in a high-speed-supernatant fraction from kidney cortex. 4. It was concluded that d-malate inhibited either the operation of the cytoplasmic l-malate dehydrogenase or malate outflow from the mitochondria in the intact kidney-cortex cell. This supports the hypothesis of Lardy, Paetkau & Walter (1965) and Krebs, Gascoyne & Notton (1967) on the role of malate as carrier for carbon and reducing equivalents in gluconeogenesis. 5. Gluconeogenesis from l-lactate in kidney-cortex slices was strongly inhibited by a low concentration (0.1mm) of amino-oxyacetate, whereas glucose formation from pyruvate, malate, aspartate and several other compounds was only slightly affected. 6. High concentrations of l-aspartate largely reversed the inhibition of gluconeogenesis from l-lactate caused by amino-oxyacetate. 7. Amino-oxyacetate inhibited strongly the glutamate-oxaloacetate transaminase in the 30000g supernatant fraction of a kidney-cortex homogenate. The presence of l-aspartate decreased the inhibition of the transaminase by amino-oxyacetate. 8. Detritiation of l-[2-(3)H]aspartate was inhibited by 90% during an incubation of kidney-cortex slices with l-lactate and amino-oxyacetate. 9. Low concentrations (10mum) of artificial electron acceptors such as Methylene Blue and phenazine methosulphate abolished most of the inhibition of gluconeogenesis from l-lactate by amino-oxyacetate. This is interpreted as an activation of net malate outflow from the mitochondria by-passing the inhibited transfer of oxaloacetate. 10. These findings support the concept that transamination to aspartate is involved in the transfer of oxaloacetate from mitochondria to cytosol required in gluconeogenesis from l-lactate.     

Glucose metabolism in the newborn rat. Temporal studies in vivo

1. The concentrations of plasma d-glucose, l-lactate, free fatty acids and ketone bodies and of liver glycogen were measured in caesarian-delivered newborn rats at time-intervals up to 4h after delivery. Glucose and lactate concentrations decreased markedly during the first hours after delivery, but there was a delay of 60-90min before significant glycogen mobilization occurred. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75min after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into caesarian-delivered rats at 0, 1 and 2h after delivery. Calculations revealed that there was an appreciable rate of glucose formation at all ages studied, but immediately after delivery this was exceeded by the rate of glucose utilization. Around 2h post partum the rate of glucose utilization decreased dramatically and this coincided with a reversal of the immediately postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose and liver glycogen was measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into rats immediately after delivery. The logarithm of the specific radioactivity of plasma l-[U-(14)C]lactate decreased linearly with time for at least 60min after injection and the calculated rate of lactate utilization exceeded the rate of lactate formation. 4. (14)C incorporation into plasma d-glucose was maximal from 30-60min after injection of l-[U-(14)C]lactate and the amount incorporated at 60min was 23% of that present in plasma lactate. Although (14)C was also incorporated into liver glycogen the amount was always less than 3% of that present in plasma glucose. 5. The results are discussed in relationship to the adaptation of the newly born rat to the extra-uterine environment and the possible involvement of gluconeogenesis at this time before feeding is established.     

Gluconeogenesis from lactate in the developing rat. Studies in vivo

1. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose, liver glycogen and skeletal-muscle glycogen were measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into 2-, 10- and 30-day-old rats. 2. Between 15 and 60min after the injection of the l-[U-(14)C]lactate, the specific radioactivity of plasma lactate decreased with a half-life of 20-33min in animals at all three ages. 3. At all times after injection examined, the specific radioactivity of plasma glucose of the 2- and 10-day-old rats was at least fourfold greater than that of the 30-day-old rats. 4. Although (14)C was incorporated into liver glycogen the amount incorporated was always less than 5% of that present in plasma glucose. 5. The results are discussed with reference to the factors that may influence the rate of incorporation of (14)C into plasma glucose, and it is concluded that the rate of gluconeogenesis in the 2- and 10-day-old suckling rat is at least twice that of the weaned 30-day-old animal.     

Observations on the biosynthesis of phytoterpenoid quinone and chromanol nuclei

1. p-Hydroxy[U-(14)C]benzoic acid, except for loss of the carboxyl group, is effectively incorporated into the nucleus of ubiquinone and an unidentified prenylphenol by maize roots, maize shoots, french-bean leaves, french-bean cotyledons and Ochromonas danica. Plastoquinone, alpha-tocopherol, gamma-tocopherol and alpha-tocopherolquinone are all unlabelled from this substrate. The high radioactivity of the prenylphenol and its behaviour in a pulse-labelling experiment with maize shoots suggested that it may be a ubiquinone precursor. 2. Members of the 2-polyprenylphenol and 6-methoxy-2-polyprenylphenol series, compounds that are known ubiquinone precursors in Rhodospirillum rubrum, could not be detected in maize tissues, but possibly they may occur as their glycosides. 3. [G-(14)C]Shikimic acid is incorporated into the nuclei of phylloquinone, plastoquinone, alpha-tocopherolquinone, gamma-tocopherol, alpha-tocopherol and ubiquinone in maize shoots, showing that in plant tissues the nuclei of these compounds arise via the shikimic acid pathway of aromatic biosynthesis. 4. l-[U-(14)C]Phenylalanine and l-[U-(14)C]tyrosine are incorporated into plastoquinone, gamma-tocopherol, alpha-tocopherolquinone and ubiquinone. alpha-Tocopherol, which is absent from shoots incubated with l-[U-(14)C]tyrosine, is also labelled from l-[U-(14)C]phenylalanine. Degradation studies showed that there is little (14)C radioactivity in the terpenoid portions of the molecules and from this it is concluded that the aromatic portions of these amino acids are giving rise to the quinone and chromanol nuclei. 5. It is proposed that in maize the nucleus of ubiquinone can be formed from either phenylalanine or tyrosine by a pathway involving p-coumaric acid and p-hydroxybenzoic acid. Plastoquinone, tocopherols and tocopherolquinones are formed from tyrosine by some pathway in which the aromatic ring and C-3 of the side chain of this amino acid gives rise to the nucleus and one methyl substituent respectively of these compounds.     

Glucose metabolism in the newborn rat. Hormonal effects in vivo

1. The concentrations of liver glycogen and plasma d-glucose were measured in caesarian-delivered newborn rats at time-intervals up to 3h after delivery after treatment of the neonatal rats with glucagon, dibutyryl cyclic AMP, cortisol or cortisol+dibutyryl cyclic AMP. Glycogenolysis was promoted by glucagon or dibutyryl cyclic AMP in the third hour after birth but not at earlier times. Cortisol and dibutyryl cyclic AMP together (but neither agent alone) promoted glycogenolysis in the second hour after birth, but no hormone combination was effective in the first postnatal hour. 2. The specific radioactivity of plasma d-glucose was measured as a function of time for up to 75 min after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into newborn rats at delivery and after treatment with glucagon or actinomycin D. Glucagon-mediated hyperglycaemia at this time was due to an increased rate of glucose formation and a decreased rate of glucose utilization. Actinomycin D prevented glucose formation and accelerated the rate of postnatal hypoglycaemia. 3. The specific radioactivity of plasma l-lactate and the incorporation of (14)C into plasma d-glucose was measured as a function of time after the intraperitoneal injection of l-[U-(14)C]lactate into glucagon- or actinomycin D-treated rats immediately after delivery. The calculated rates of lactate formation were unchanged by either treatment, but lactate utilization was stimulated by glucagon administration. Glucagon stimulated and actinomycin D diminished (14)C incorporation into plasma d-glucose. 4. The factors involved in the initiation of glycogenolysis and gluconeogenesis in the rat immediately after birth are discussed.     

Metabolism of l-Threonic Acid in Rumex x acutus L. and Pelargonium crispum (L.) L'Hér

l-Threonic acid is a natural constituent in leaves of Pelargonium crispum (L.) L'Hér (lemon geranium) and Rumex x acutus L. (sorrel). In both species, l-[(14)C]threonate is formed after feeding l-[U-(14)C]ascorbic acid to detached leaves. R. acutus leaves labeled with l-[4-(3)H]- or l-[6-(3)H]ascorbic acid produce l-[(3)H]threonate, in the first case internally labeled and in the second case confined to the hydroxymethyl group. These results are consistent with the formation of l-threonate from carbons three through six of l-ascorbic acid. Detached leaves of P. crispum oxidize l-[U-(14)C] threonate to l-[(14)C]tartrate whereas leaves of R. acutus produce negligible tartrate and the bulk of the (14)C appears in (14)CO(2), [(14)C]sucrose, and other products of carbohydrate metabolism. R. acutus leaves that are labeled with l-[U-(14)C]threonate release (14)CO(2) at linear rate until a limiting value of 25% of the total [U-(14)C]threonate is metabolized. A small quantity of [(14)C]glycerate is also produced which suggests a process involving decarboxylation of l-[U-(14)C]threonate.     

Biosynthesis of carnitine and 4-N-trimethylaminobutyrate from lysine

The conversion of l-[U-(14)C]lysine into carnitine was demonstrated in normal, choline-deficient and lysine-deficient rats. In other experiments in vivo radioactivity from l-[4,5-(3)H]lysine and dl-[6-(14)C]lysine was incorporated into carnitine; however, radioactivity from dl-[1-(14)C]lysine and dl-[2-(14)C]lysine was not incorporated. Administered l-[Me-(14)C]methionine labelled only the 4-N-methyl groups whereas lysine did not label these groups. Therefore lysine must be incorporated into the main carbon chain of carnitine. The methylation of lysine by a methionine source to form 6-N-trimethyl-lysine is postulated as an intermediate step in the biosynthesis of carnitine. Radioactive 4-N-trimethylaminobutyrate (butyrobetaine) was recovered from the urine of lysine-deficient rats injected with [U-(14)C]lysine. This lysine-derived label was incorporated only into the butyrate carbon chain. The specific radioactivity of the trimethylaminobutyrate was 12 times that of carnitine isolated from the urine or carcasses of the same animals. These data further support the idea that the last step in the formation of carnitine from lysine was the hydroxylation of trimethylaminobutyric acid, and are consistent with the following sequence: lysine+methionine --> 6-N-trimethyl-lysine --> --> 4-N-trimethylaminobutyrate --> carnitine.     

A study of the nature of the immediate precursor of the extracellular α-amylase of Bacillus amyloliquefaciens. A reappraisal

1. A defined medium was devised for use in washed-cell experiments with post-exponential-phase cultures of Bacillus amyloliquefaciens. The medium allowed alpha-amylase to be secreted, bacterial concentration to increase and l-[U-(14)C]valine to be incorporated into protein at a linear rate, which was the same as in a post-exponential-phase culture, for up to 6h. 2. Determination of the specific radioactivity of l-[U-(14)C]valine in the medium, the intracellular amino acid pool, the cellular protein and the isolated alpha-amylase, after a 3h incubation of washed cells in the defined medium, showed that at least 76% of the alpha-amylase secreted was synthesized de novo. 3. By isolating the alpha-amylase formed during a 6h incubation in the presence of l-[U-(14)C]valine it was shown that the specific radioactivity of the N-terminal valine, within the limits of experimental error, was the same as that of the total valine residues from the complete alpha-amylase molecule. 4. A consideration of these results in relation to the whole literature on the subject strongly supports the idea that there is no reason to suppose that extracellular alpha-amylase is formed from a high-molecular-weight precursor in B. amyloliquefaciens and closely related organisms with identical characteristics of exoenzyme secretion.     

The metabolism of glucose 6-phosphate by mammalian cerebral cortex in vitro

1. The metabolism of glucose 6-phosphate in rat cerebral-cortex slices in vitro was compared with that of glucose. It was found that a glucose 6-phosphate concentration of 25mm was required to achieve maximal oxygen uptake rates and ATP concentrations, whereas only 2mm-glucose was required. 2. When 25mm-[U-(14)C]glucose 6-phosphate was used as substrate, the pattern of labelling of metabolites was found to be quantitatively and qualitatively similar to the pattern found with 10mm-[U-(14)C]glucose, except that incorporation into [(14)C]lactate was decreased, and significant amounts of [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate were formed. 3. Unlabelled glucose (10mm) caused a tenfold decrease in the incorporation of 25mm-[U-(14)C]glucose 6-phosphate into all metabolites except [(14)C]glucose and [(14)C]mannose phosphate and [(14)C]fructose phosphate. In contrast, unlabelled glucose 6-phosphate (25mm) had no effect on the metabolism of 10mm-[U-(14)C]glucose other than to increase markedly the incorporation into, and amount of, [(14)C]lactate, the specific radioactivity of this compound remaining approximately the same. 4. The effect of glucose 6-phosphate in increasing lactate formation from glucose was found to occur also with a number of other phosphate esters and with inorganic phosphate. Further investigation indicated that the effect was probably due to binding of medium calcium by the phosphate moiety, thereby de-inhibiting glucose uptake. 5. Incubations carried out in a high-phosphate high-potassium medium gave a pattern of metabolism similar to that found when slices were subjected to depolarizing conditions. Tris-buffered medium gave similar results to bicarbonate-buffered saline, except that it allowed much less lactate formation from glucose. 6. Part of the glucose formed from glucose 6-phosphate was extracellular and was produced at a rate of 12mumol/h per g of tissue in Krebs tris medium when glycolysis was blocked. The amount formed was much less when 25mm-P(i) or 26mm-HCO(3) (-) was present, the latter being in the absence of tris. 7. Glucose 6-phosphate also gave rise to an intracellular glucose pool, whereas no intracellular glucose was detectable when glucose was the substrate.     

Glucose metabolism in the developing rat. Studies in vivo

1. The specific radioactivity of plasma d-glucose and the incorporation of (14)C into plasma l-lactate, liver glycogen and skeletal-muscle glycogen was measured as a function of time after the intraperitoneal injection of d-[6-(14)C]glucose and d-[6-(3)H]glucose into newborn, 2-, 10- and 30-day-old rats. 2. The log of the specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the 2-, 10- and 30-day-old rats decreased linearly with time for at least 60min after injection of labelled glucose. The specific radioactivity of both plasma d-[6-(14)C]- and d-[6-(3)H]-glucose of the newborn rat remained constant for at least 75min after injection. 3. The glucose turnover rate of the 30-day-old rat was significantly greater than (approximately twice) that of the 2- and 10-day-old rats. The relative size of both the glucose pool and the glucose space decreased with age. Less than 10% of the glucose utilized in the 2-, 10- and 30-day-old rats was recycled via the Cori cycle. 4. The results are discussed in relationship to the availability of dietary glucose and other factors that may influence glucose metabolism in the developing rat.     

Modulation of branched-chain amino acid oxidation in rat hemidiaphragms in vitro by glucose and ketone bodies

Branched-chain amino acid metabolism in hemidiaphragms from 40 h-starved rats is influenced by the provision of glucose as co-substrate. Glucose inhibits 14CO2 production from [l-14C]valine and [U-14C]valine but stimulates 14CO2 production from [l-14C]leucine, [U-14C]leucine and [U-14C]isoleucine. In the presence of glucose, ketone bodies inhibit alanine release and 14CO2 production from [l-14C]valine, [l-14C]leucine and [U-14C]isoleucine, but inhibition is not observed in the absence of glucose as cosubstrate. Glucose-dependent inhibition by ketone bodies of branched-chain amino acid oxidation via inhibition of the branched-chain 2-oxo acid dehydrogenase complex or branched-chain amino acid aminotransferase may account in part for the reported hypoalanaemic action of ketone bodies in vivo.     

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

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

Biosynthesis of intestinal mucins. Effect of puromycin on mucoprotein biosynthesis by sheep colonic mucosal tissue

1. Surviving sheep colonic mucosal tissue incorporated l-[U-(14)C]threonine when incubated in Krebs medium III at 37 degrees in an atmosphere of oxygen, into a well-characterized mucoprotein fraction, isolated by papain digestion of the incubated scrapings. 2. Acidic hydrolysis and chromatography of the labelled mucoprotein showed that threonine was the only constituent to become labelled. In the presence of puromycin the incorporation of l-[U-(14)C]threonine was considerably diminished (6.7 and 18.5% of control in duplicate experiments). Furthermore, puromycin also decreased incorporation of radioactivity from d-[U-(14)C]-glucose (48.0 and 31.6% of control) and (35)SO(4) (2-) (21.2 and 23.6% of control) into the mucoprotein fraction. 3. In a puromycin-inhibited system, with d-[U-(14)C]-glucose, where the overall specific radioactivity of the mucoprotein was 48% of control, the labelling of the individual monosaccharide constituents (as% of control) was: N-acetylneuraminic acid, 44%; N-glycollylneuraminic acid, 61%; hexosamines, 46%; fucose, 68%; galactose, 34%.     

Competition among oxidizable substrates in brains of young and adult rats. Dissociated cells.

The rates of conversion of D-(-)-3-hydroxy[3-14C]butyrate, [3-14C]acetoacetate, [6-14C]glucose and [U-14C]glutamine into 14CO2 were measured in the presence and absence of alternative oxidizable substrates in intact dissociated cells from the brains of young and adult rats. When unlabelled glutamine was added to [6-14C]glucose or unlabelled glucose was added to [U-14C]glutamine, the rate of 14CO2 production was decreased in both young and adult rats. The rate of oxidation of 3-hydroxy[3-14C]butyrate was also decreased by the addition of unlabelled glutamine in both age groups, but in the reverse situation, i.e. unlabelled 3-hydroxybutyrate added to [U-14C]glutamine, only the brain cells from young rats were affected. No significant effects were seen when glutamine and acetoacetate were combined. The addition of either of the two ketone bodies to [6-14C]glucose markedly lowered the rate of 14CO2 production in young rats, but in the adult only 3-hydroxybutyrate was effective and the magnitude of decrease in the rate of [6-14C]glucose oxidation was much lower than in young animals. Unlabelled glucose decreased the rate of [3-14C]acetoacetate oxidation to a minor extent in brain cells from both age groups; when added to 3-hydroxy[3-14C]butyrate, glucose had no effect in young rats and greatly enhanced 14CO2 production in adult brain cells. Many of these patterns of substrate interaction in dissociated brain cells differ from those in whole homogenates; they may be a function of the plasma membranes and the role of a carrier-mediated transport system or a reflection of a difference in the population of cell types or subcellular organelles in these two preparations.     

The synthesis of phosphatidylcholine by adult rat lung alveolar type II epithelial cells in primary culture

1. The formation of phosphatidylcholine from radioactive precursors was studied in adult rat lung alveolar type II epithelial cells in primary culture. 2. The incorporation of [Me-14C]choline into total lipids and phosphatidylcholine was stimulated by addition of palmitate, whereas the incorporation of [U-14C]glucose into phosphatidylcholine and disaturated phosphatidylcholine was stimulated by addition of choline. Addition of glucose decreased the absolute rate of incorporation of [1(3)-3H]glycerol into total lipids, phosphatidylcholine and disaturated phosphatidylcholine, decreased the percentage [1(3)-3H]glycerol recovered in phosphatidylcholine, but increased the percentage phosphatidylcholine label in the disaturated species. 3. At saturating substrate concentrations, the percentages of phosphatidylcholine radioactivity found in disaturated phosphatidylcholine after incubation with [1-(14)C]acetate (in the presence of glucose) [1-(14)C]palmitate (in the presence of glucose), [Me-14C]choline (in the presence of glucose and palmitate) and [U-14C]glucose (in the presence of choline and palmitate) were 78, 75, 74 and 90%, respectively. 4. Fatty acids stimulated the incorporation of [U-14C]glucose into the glycerol moiety of phosphatidylcholine. The degree of unsaturation of the added fatty acids was reflected in the distribution of [U-14C]glucose label among the different molecular species of phosphatidylcholine. It is suggested that the glucose concentration in the blood as related to the amount of available fatty acids and their degree of unsaturation may be factors governing the synthesis of surfactant lipids.     

Contribution of propionate to glucose synthesis in sheep

1. The production rate of propionate in the rumen and the entry rate of glucose into the body pool of glucose in sheep were measured by isotope-dilution methods. Propionate production rates were measured by using a continuous infusion of specifically labelled [(14)C]propionate. Glucose entry rates were estimated by using either a primed infusion or a continuous infusion of [U-(14)C]glucose. 2. The specific radioactivity of plasma glucose was constant between 4 and 9hr. after the commencement of intravenous infusion of [U-(14)C]glucose and between 1 and 3hr. when a primed infusion was used. 3. Infusion of [(14)C]propionate intraruminally resulted in a fairly constant specific radioactivity of rumen propionate between about 4 and 9hr. and of plasma glucose between 6 and 9hr. after the commencement of the infusion. Comparison of the mean specific radioactivities of glucose and propionate during these periods allowed estimates to be made of the contribution of propionate to glucose synthesis. 4. Comparisons of the specific radioactivities of plasma glucose and rumen propionate during intraruminal infusions of one of [1-(14)C]-, [2-(14)C]-, [3-(14)C]- and [U-(14)C]-propionate indicated considerable exchange of C-1 of propionate on conversion into glucose. The incorporation of C-2 and C-3 of propionate into glucose and lactate indicated that 54% of both the glucose and lactate synthesized arose from propionate carbon. 5. No differences were found for glucose entry rates measured either by a primed infusion or by a continuous infusion. The mean entry rate (+/-s.e.m.) of glucose estimated by using a continuous infusion into sheep was 0.33+/-0.03 (4) m-mole/min. and by using a primed infusion was 0.32+/-0.01 (4) m-mole/min. The mean propionate production rate was 1.24+/-0.03 (8) m-moles/min. The conversion of propionate into glucose was 0.36 m-mole/min., indicating that 32% of the propionate produced in the rumen is used for glucose synthesis. 6. It was indicated that a considerable amount of the propionate converted into glucose was first converted into lactate.     

Cerebral dicarboxylate transport and metabolism studied with isotopically labelled fumarate,malate and malonate

Transport and metabolism of dicarboxylates may be important in the glial-neuronal metabolic interplay. Further, exogenous dicarboxylates have been suggested as cerebral energy substrates. After intrastriatal injection of [(14) C]fumarate or [(14) C]malate, glutamine attained a specific activity 4.1 and 2.6 times higher than that of glutamate, respectively, indicating predominantly glial uptake of these four-carbon dicarboxylates. In contrast, the three-carbon dicarboxylate [(14) C]malonate gave a specific activity in glutamate which was approximately five times higher than that of glutamine, indicating neuronal uptake of malonate. Therefore, neurones and glia take up different types of dicarboxylates, probably by different transport mechanisms. Labelling of alanine from [(14) C]fumarate and [(14) C]malate demonstrated extensive malate decarboxylation, presumably in glia. Intravenous injection of 75 micromol [U-(13) C]fumarate rapidly led to high concentrations of [U-(13) C]fumarate and [U-(13) C]malate in serum, but neither substrate labelled cerebral metabolites as determined by (13) C NMR spectroscopy. Only after conversion of [U-(13) C]fumarate into serum glucose was there (13) C-labelling of cerebral metabolites, and only at <10% of that obtained with 75 micromol [3-(13) C]lactate or [2-(13) C]acetate. These findings suggest a very low transport capacity for four-carbon dicarboxylates across the blood-brain barrier and rule out a role for exogenous fumarate as a cerebral energy substrate.     

The role of the pseudo-disaccharide neamine as an intermediate in the biosynthesis of neomycin.

By using wild-type and deoxystreptamine-negative mutants of Streptomyces fradiae grown in media containing [6(-3)H]glucose or [U-14C]glucose, and by subsequent hydrolysis of the labelled neomycin produced, neamines labelled with 3H in both rings I and II, but with 14C in ring I only, were prepared. A mixture of these two forms of neamine was converted by deoxystreptamine-negative Streptomyces rimosus forma paromomycinus into neomycin (not paromomycin) with a 30% yield. The3H: 14C ratio in this neomycin was the same as the measured in neamine produced by hydrolysis of the neomycin, and in unused neamine reisolated from the incubation medium. The 3H:14C ratio in the neomycin was not affected by the presence of unlabelled deoxystreptamine during the incubation. The radioactivity in the neomycin was associated with rings I and II only. It is concluded that the added neamine is incorporated into antibiotic intact, without initial hydrolysis, and that the probable first step in the subunit assembly of neomycin is the formation of neamine.     

Niacin Biosynthesis in Seedlings of Zea mays

Evidence obtained from incubation of corn (Zea mays cv. Golden Bantam) seedlings in dl-[benzene ring-U-(14)C]tryptophan, l-[5-(3)H]tryptophan, l-[U-(14)C]aspartate and [U-(14)C]glycerol indicates that niacin is synthesized in these plants via oxidative degradation of tryptophan. Aspartate and glycerol do not appear to be precursors of niacin in corn seedlings.     

Lactate metabolism in the perfused rat hindlimb.

A preparation of isolated rat hindleg was perfused with a medium consisting of bicarbonate buffer containing Ficoll and fluorocarbon, containing glucose and/or lactate. The leg was electrically prestimulated to deplete partially muscle glycogen. The glucose was labelled uniformly with 14C and with 3H in positions 2, 5 or 6, and lactate uniformly with 14C and with 3H in positions 2 or 3. Glucose carbon was predominantly recovered in glycogen, and to a lesser extent in lactate. The 3H/14C ration in glycogen from [5-3H,U-14C]- and [6-3H,U-14C]-glucose was the same as in glucose. Nearly all the utilized 3H from [2-3H]glucose was recovered as water. Insulin increased glucose uptake and glycogen synthesis 3-fold. When the muscle was perfused with a medium containing 10 mM-glucose and 2 mM-lactate, there was little change in lactate concentration. 14C from lactate was incorporated into glycogen. There was a marked exponential decrease in lactate specific radioactivity, much greater with [3H]- than with [14C]-lactate. The 'apparent turnover' of [U-14C]lactate was 0.28 mumol/min per g of muscle, and those of [2-3H]- and [3-3H]-lactate were both about 0.7 mumol/min per g. With 10 mM-lactate as sole substrate, there was a net uptake of lactate, at a rate of about 0.15 mumol/min per g, and the apparent turnover of [U-14C]lactate was 0.3 mumol/min per g. The apparent turnover of [3H]lactate was 3-5 times greater. When glycogen synthesis was low (no prestimulation, no insulin), the incorporation of lactate carbon into glycogen exceeded that from glucose, but at high rates of glycogen deposition the incorporation of lactate carbon was much less than that of glucose. Lactate incorporation into glycogen was similar in fast-twitch white and fast-twitch red muscle, but was very low in slow-twitch red fibres. We find that (a) pyruvate in muscle is incorporated into glycogen without randomization of carbon, and synthesis is not inhibited by mercaptopicolinate or cycloserine; (b) there is extensive lactate turnover in the absence of net lactate uptake, and there is a large dilution of 14C-labelled lactate from endogenous supply; (c) there is extensive detritiation of [2-3H]- and [3-3H]-lactate in excess of 14C utilization.