The stimulus–secretion coupling of amino acid-induced insulin release. Insulinotropic action of l-alanine

Available information on the fate and insulinotropic action of l-alanine in isolated pancreatic islets is restricted to data collected in obese hyperglycemic mice. Recent data, however, collected mostly in tumoral islet cells of either the RINm5F line or BRIN-BD11 line, have drawn attention to the possible role of Na⁺ co-transport in the insulinotropic action of l-alanine. In the present study conducted in islets prepared from normal adult rats, l-alanine was found (i) to inhibit pyruvate kinase in islet homogenates, (ii) not to affect the oxidation of endogenous fatty acids in islets prelabelled with [U-¹⁴C]palmitate, (iii) to stimulate ⁴⁵Ca uptake in islets deprived of any other exogenous nutrient, and (iv) to augment insulin release evoked by either 2-ketoisocaproate or l-leucine, whilst failing to significantly affect glucose-induced insulin secretion. The oxidation of l-[U-¹⁴C]alanine was unaffected by d-glucose, but inhibited by l-leucine. Inversely, l-alanine decreased the oxidation of d-[U-¹⁴C]glucose, but failed to affect l-[U-¹⁴C]leucine oxidation. It is concluded that the occurrence of a positive insulinotropic action of l-alanine is restricted to selected experimental conditions, the secretory data being compatible with the view that stimulation of insulin secretion by the tested nutrient(s) reflects, as a rule, their capacity to augment ATP generation in the islet B cells. However, the possible role of Na⁺ co-transport in the secretory response to l-alanine cannot be ignored.     

d-Glucose Metabolism in BRIN-BD11 Islet Cells

A novel insulin-secreting cell line, BRIN-BD11, was recently established following electrofusion of RINm5F cells with NEDH rat pancreatic islet cells. In the present study,d-glucose metabolism was compared in BRIN-BD11 and RINm5F cells. The concentration dependency ofd-[5-³H]glucose utilization displayed a comparable pattern in the two cell lines, but the absolute values were lower in BRIN-BD11 than RINm5F cells. Except in the case ofd-[1-¹⁴C]glucose, the ratio between¹⁴C labeledd-glucose oxidation andd-[5-³H]glucose utilization was higher, however, in BRIN-BD11 than RINm5F cells. Moreover, BRIN-BD11 cells were less affected than RINm5F cells by a rise ind-glucose concentration, in terms of the inhibitory action of the hexose upon oxidative variables, such as oxidative glycolysis, pyruvate decarboxylation, and oxidation of glucose-derived acetyl residues in the Krebs cycle. The total energy yield fromd-glucose catabolism appeared similar, however, in BRIN-BD11 and RINm5F cells. These findings extend the knowledge that BRIN-BD11 cells display an improved metabolic and secretory behavior, when considering the difference otherwise found between normal and tumoral islet cells.     

D-glucose metabolism in normal dispersed islet cells and tumoral INS-1 cells

The metabolism of D-glucose was characterized in both normal dispersed rat islet cells and the 2-mercaptoethanol-dependent insulin-secreting cells of the INS-1 line. The normal and tumoral islet cells differed from one another by the relative magnitude, concentration dependency and hierarchy of the increase in the production of ³HOH from D-[5-³H]glucose and ¹⁴C-labelled CO₂, acidic metabolites and amino acids from D-[U-¹⁴C]glucose at increasing concentrations of the hexose. For instance, whilst the paired ratio between D-[U-¹⁴C]glucose oxidation and D-[5-³H]glucose utilization augmented in a typical sigmoidal manner in normal islet cells exposed to increasing concentrations of D-glucose, it progressively decreased under the same experimental conditions in INS-1 cells. Nevertheless, the absolute values and concentration-response relationship for the increase in ATP generation rate attributable to the catabolism of D-glucose were virtually identical in normal and tumoral cells. These findings indicate that the analogy in the secretory response to D-glucose of normal and INS-1 islet cells, although coinciding with a comparable response to the hexose in terms of ATP generation, contrasts with a vastly different pattern of D-glucose metabolism in these two cell types.     

Fructose utilization and altered cytochrome P-450 in cultured hepatocytes from adult rats

Cultured adult rat hepatocytes incubated in media containing fructose exhibit increased levels of cytochrome P-450, relative to cells incubated with equimolar glucose, and the effect of fructose is proportional to its concentration between 2 and 10 mM. For investigating the mechanism of the effect of fructose on cytochrome P-450 in cultured cells, [U-¹⁴C]fructose or [U-¹⁴C]-glucose were added to the incubation medium, and their uptake and utilization were compared. While the uptake kinetics of the two hexoses were similar, the rate of phosphorylation of fructose was more than 10-fold that of glucose. Similarly, the appearance of fructose carbon in metabolic pools, as well as its conversion to CO₂ and cellular glycerolipid, was increased. The latter finding suggested that fructose might alter cytochrome P-450 by stimulating glycerolipid synthesis, since the stability of the cytochrome is lipid-dependent. However, the changes in glycerolipid formation failed to parallel changes in the level of cytochrome P-450 in fructose-treated cells. Moreover, the relative distribution of ¹⁴C into specific lipids was similar for both hexoses, suggesting that an increased carbon flux in cells incubated with fructose did not directly impose a qualitative change in cellular lipid synthesis. We conclude that the fructose-mediated alteration of cytochrome P-450 in cultured rat hepatocytes reflects a process other than increased incorporation of fructose carbon into metabolic pools.     

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.     

Ontogenetic Study of the Effects of Energetic Nutrients on Amino Acid Metabolism of Rat Cerebral Cortex

We studied the effect of various energetic nutrients on metabolism of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in cerebral cortex of rats at different ages. At gestational age, glucose and lactate stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine and from l-[U-¹⁴C]leucine, respectively; glucose, -OH-butyrate and lactate stimulated lipid synthesis from l-[U-¹⁴C]leucine. At 10 days of age, glucose, mannose, and fructose stimulated protein synthesis, and glucose and mannose stimulated oxidation to CO₂ as well as lipid synthesis from l-[U-¹⁴C]leucine. In adult rats, glucose, mannose, and fructose stimulated protein synthesis from l-[U-¹⁴C]leucine and [1–¹⁴C]glycine; glutamine also markedly decreased the oxidation of l-[U-¹⁴C]leucine and [1–¹⁴C]glycine in 10–day-old and adult rats.     

Characterization of particulate D-apiosyl-and D-xylosyltransferase from Lemna minor.

A particulate enzyme preparation capable of catalyzing the transfer of d-[U-¹⁴C]apiose and d-[U-¹⁴C]xylose from uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (UDP[U-¹⁴C]Api) and uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate) (UDP[U-¹⁴C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-¹⁴C]apiose or d-[U-¹⁴C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a K_m for UDP[U-¹⁴C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-¹⁴C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-^l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min.     

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.     

INTERACTION OF LEUCINE, GLUCOSE, AND KETONE METABOLISM IN RAT BRAIN IN VITRO

Brain cortex slices from fed, 48 h and 120 h fasted rats were incubated and ¹⁴CO₂ was measured from (a) [U-¹⁴C]glucose (5 mm ) either alone or in the presence of l -lcucine (0.1 or 1 mm ), and (b) [U-¹⁴C]leucine or [l-¹⁴C]leucine at 0.1 or 1 mm with or without glucose (5 mm ). In other experiments, sodium dl -3-hydroxybutyrate (3-OHB) or acetoacetate (AcAc) at 1 or 5 mm were added in the above incubation mixture. The rate of conversion of [U¹⁴C]glucose to CO₂ was decreased 20% by leucine at 1 mm and 30–50% by 3-OHB at 1 or 5 mm but not by leucine at 0.1 mm . The effects of 3-OHB and of leucine (1 mm ) were not additive. The effects of leucine were similar in the fed and fasted rats. The rate of conversion of [U-¹⁴C]leucine or [l-^,4C]leucine to ¹⁴CO₂ at 0.1 mm and 1.0 mm was increased by glucose (35%) in the fed or fasted rats. Ketone bodies in the absence of glucose had no effect on leucine oxidation. However, the stimulatory effect of glucose on the rate of conversion of leucine to CO₂ was inhibited by 3-OHB at 5 mm . These results suggest that (a) leucine in increased concentrations (1 mm ) may reduce glucose oxidation by brain cortex while itself becoming an oxidative fuel for brain, and (b) leucine oxidation by brain may be influenced by the prevailing glucose and ketone concentrations.     

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.     

The inhibition of oxalate biosynthesis in isolated perfused rat liver by DL-phenyllactate and n-heptanoate

Glycolate oxidase was isolated and partially purified from human and rat liver. The enzyme preparation readily catalyzed the oxidation of glycolate, glyoxylate, lactate, hydroxyisocaproate and α-hydroxybutyrate. The oxidation of glycolate and glyoxylate by glycolate oxidase was completely inhibited by 0.02 m dl-phenyllactate or n-heptanoate. The oxidation of glyoxylate by lactic dehydrogenase or xanthine oxidase was not inhibited by 0.067 m dl-phenyllactate or n-heptanoate. The conversion of [U-¹⁴C] glyoxylate to [¹⁴C] oxalate by isolated perfused rat liver was completely inhibited by dl-phenyllactate and n-heptanoate confirming the major contribution of glycolate oxidase in oxalate synthesis. Since the inhibition of oxalate was 100%, lactic dehydrogenase and xanthine oxidase do not contribute to oxalate biosynthesis in isolated perfused rat liver. dl-Phenyllactate also inhibited [¹⁴C] oxalate synthesis from [1-¹⁴C] glycolate, [U-¹⁴C] ethylene glycol, [U-¹⁴C] glycine, [3-¹⁴C] serine, and [U-¹⁴C] ethanolamine in isolated perfused rat liver. Oxalate synthesis from ethylene glycol was inhibited by dl-phenyllactate in the intact male rat confirming the role of glycolate oxidase in oxalate synthesis in vivo and indicating the feasibility of regulating oxalate metabolism in primary hyperoxaluria, ethylene glycol poisoning, and kidney stone formation by enzyme inhibitors.     

Resistance to alloxan of tumoral insulin-producing cells

Rat pancreatic islets and insulin-producing cells of the RINm5F line were incubated for 5 min at 7 or 23 degrees C in media containing 3H2O and either L-[1-14C]glucose or [2-14C]alloxan. In the islets the intracellular distribution space of [2-14C]alloxan represented, at 7 and 23 degrees C respectively, 11.4 +/- 1.0 and 25.5 +/- 2.3% of the intracellular 3H2O space. In the RINm5F cells, the distribution space of [2-14C]alloxan failed to be affected by the ambient temperature and represented, after correction for extracellular contamination, no more than 5.2 +/- 0.5% of the intracellular 3H2O space. Preincubation for 30 min at 7 degrees C in the presence of alloxan (10 mM) failed to affect subsequent D-[U-14C]glucose oxidation in the tumoral cells, whilst causing a 70% inhibition of glucose oxidation in the islets. It is proposed that RINm5F cells are resistant to the cytotoxic action of alloxan, this being attributable, in part at least, to poor uptake of the diabetogenic agent.     

Characterization of [14C]apiogalacturonans synthesized in a cell-free system from Lemna minor.

Radioactive polysaccharide was synthesized when uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (containing some uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate)) was incubated with a particulate enzyme preparation from Lemna minor. Characterization experiments established that the product: (i) was insoluble in methanol and water, (ii) contained d-[U-¹⁴C]apiose (75%) and d-[U-¹⁴C]xylose (25%), and (iii) was soluble in 1% ammonium oxalate. The material solubilized by ammonium oxalate (solubilized product): (i) was separated into five fractions by column chromatography with diethylaminoethyl-Sephadex (DEAE-Sephadex), (ii) contained [U-¹⁴C]apiobiose side chains that were removed by hydrolysis at pH 4, and (iii) was degraded by fungal pectinase. Both d-[U-¹⁴C]apiose residues of the [U-¹⁴C]apiobiose side chains were synthesized in vivo since radioactivity was distributed equally between the two residues. The presence of uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) during synthesis of radioactive polysaccharide resulted in: (i) an increase in the incorporation of radioactive d-[U-¹⁴C]apiose into solubilized product, (ii) an increase in the ratio of d-[U-¹⁴C]apiose to d-[U-¹⁴C]xylose present in solubilized product, (iii) an increase in the amount of [U-¹⁴C]apiobiose plus d-[U-¹⁴C]apiose released from the solubilized product by hydrolysis at pH 4, and (iv) a tighter binding of the solubilized product to DEAE-Sephadex. These results show that apiogalacturonans similar to or the same as those synthesized by the intact plant were synthesized in the particulate enzyme preparation isolated from L. minor. [¹⁴C]Apiogalacturonans completely free of d-[U-^l4C]xylose were not isolated. The [¹⁴C]apiogalacturonan with the least d-[U-¹⁴C]xylose still had 4.8% of its radioactivity present in d-[U-¹⁴C]xylose. The possibility remains that d-xylose is a normal constituent of the apiogalacturonans of the cell wall of L. minor.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Studies in lipogenesis in vivo: Fatty acid and cholesterol synthesis during starvation and re-feeding

1. Lipogenesis in vivo has been studied in mice given a 250mg. meal of [U-¹⁴C]glucose (2·5μc) or given an intraperitoneal injection of 25μg. of [U-¹⁴C]glucose (2·0μc). 2. The ability to convert a [U-¹⁴C]glucose meal into fatty acid was not significantly depressed by 6–7hr. of starvation. In contrast, incorporation of ¹⁴C into fatty acid in the liver after the intraperitoneal dose of [¹⁴C]glucose was depressed by 80% and by more than 90% by 1 and 2hr. of starvation respectively. Carcass fatty acid synthesis from the [U-¹⁴C]glucose meal was not depressed by 12hr. of starvation, whereas from the tracer dose of [U-¹⁴C]glucose the depression in incorporation was 80% after 6hr. of starvation. 3. Re-feeding for 3 days, after 3 days' starvation, raised fatty acid synthesis and cholesterol synthesis in the liver fivefold and tenfold respectively above the levels in non-starved control mice. These increases were associated with an increased amount of both fatty acid and cholesterol in the liver. 4. After 18hr. of starvation incorporation of a [U-¹⁴C]glucose meal into carcass and liver glycogen were both increased threefold.     

Channeling of alpha-D-glucose 6-phosphate in tumoral islet cells exposed to D-galactose

In human erythrocytes, in which the fractional turnover rate of glucose 6-phosphate is rather low, menadione increases to almost the same relative extent the oxidation of D-[U-14C]glucose and D-[U-14C]galactose. However, in pancreatic tumoral islet cells (RINm5F line), in which the fractional turnover rate of glucose 6-phosphate is considerably higher, menadione increases the oxidation of D-[1-14C]glucose but not that of D-[1-14C]galactose. These results suggest that alpha-D-glucose 6-phosphate generated from exogenous D-galactose is channeled preferentially into the glycolytic rather than pentose phosphate pathway. Such was no more the case, however, when the RINm5F cells were exposed simultaneously to both D-glucose and D-galactose.     

Metabolite pools during starch synthesis and carbohydrate oxidation in amyloplasts isolated from wheat endosperm

Starch synthesis and CO₂ evolution were determined after incubating intact and lysed wheat (Triticum aestivum L. cv. Axona) endosperm amyloplasts with ¹⁴C-labelled hexose-phosphates. Amyloplasts converted [U-¹⁴C]glucose 1-phosphate (Glc1P) but not [U-¹⁴C]glucose 6-phosphate (Glc6P) into starch in the presence of ATP. When the oxidative pentose-phosphate pathway (OPPP) was stimulated, both [U-¹⁴C]Glc1P and [U-¹⁴C]Glc6P were metabolized to CO₂, but Glc6P was the better precursor for the OPPP, and Glc1P-mediated starch synthesis was reduced by 75%. In order to understand the basis for the partitioning of carbon between the two potentially competing metabolic pathways, metabolite pools were measured in purified amyloplasts under conditions which promote both starch synthesis and carbohydrate oxidation via the OPPP. Amyloplasts incubated with Glc1P or Glc6P alone showed little or no interconversion of these hexose-phosphates inside the organelle. When amyloplasts were synthesizing starch, the stromal concentrations of Glc1P and ADP-glucose were high. By contrast, when flux through the OPPP was highest, Glc1P and ADP-glucose inside the organelle were undetectable, and there was an increase in metabolites involved in carbohydrate oxidation. Measurements of the plastidial hexose-monophosphate pool during starch synthesis and carbohydrate oxidation indicate that the phosphoglucose isomerase reaction is at equilibrium whereas the reaction catalysed by phosphoglucomutase is significantly displaced from equilibrium. Received: 29 March 1997 / Accepted: 5 June 1997     

Metabolism of 5-thio-D-glucopyranose and 6-thio-D-fructopyranose in rats.

5-Thio-α-d-[U-¹⁴C]glucopyranose and 6-thio-β-d-[U-¹⁴C]fructopyranose were administered orally and intravenously to rats. On intravenous administration of 5-thio-d-[U-¹⁴C]glucopyranose, 1% was oxidized to [¹⁴C]carbon dioxide, 93% was excreted in the urine, and 1.6% was retained in the carcass. On oral administration of 5-thio-d-[U-¹⁴C]glucopyranose, 1% was exhaled as [¹⁴C]carbon dioxide, 90% was excreted in feces and urine, and 4% was retained in the carcass after 72 h. On intravenous administration of 6-thio-β-d-[U-¹⁴C]fructopyranose, 56% was exhaled as [¹⁴C]carbon dioxide, 23% was excreted in the urine, and 7.5% was retained in the carcass; after oral administration, 35% was oxidized to [¹⁴C]carbon dioxide, 50% was excreted in feces and urine, and 6% was retained in the animal after 72 h.On intravenous administration of 5-thio-d-glucose to fasted male rats, blood d-glucose levels increased at lower doses than on oral administration. A dose of 50 mg/kg raised blood d-glucose to 226 mg/100 ml within 2.5 h after intravenous but only to 173 mg/100 ml within 2 h after oral administration from basal level of 70–90 mg/100 ml. Blood d-glucose concentration returned to normal levels within 9 h in both cases. 6-Thio-d-fructopyranose showed no diabetogenic action. The LD₅₀ of 6-thio-d-fructopyranose was 11,200 mg/kg when tested in mice.     

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.     

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.