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PubMed1986

Solubilization and separation of the human erythrocyte D-glucose transporter covalently and noncovalently photoaffinity-labeled with [3H]cytochalasin B.

The D-glucose transporter in the human erythrocyte membranes was photoaffinity-labeled with [3H]cytochalasin B and solubilized with n-octyl beta-D-glucopyranoside (octyl glucoside). [3H]Cytochalasin B-bound proteins were further isolated by using Sephadex G-50 chromatography. The amount of [3H]cytochalasin B associated with the membrane proteins was approximately 10% of the total radioactivity in the octyl glucoside extract. The solubilized photoaffinity-labeled D-glucose transporter was isolated and found to consist of two major peaks by DEAE-Sephacel chromatography. The radioactivity of peak II was considerably greater than that of peak I. The incorporation of [3H]cytochalasin B into both peaks was blocked by the presence of D-glucose during photolysis. With preparative NaDod-SO4/polyacrylamide gel electrophoresis, the radioactivity of peak I could be released, but that of peak II remained with the D-glucose transporter. These results indicate that [3H]cytochalasin B was covalently bound to the D-glucose transporter only in peak II and that peak II could be generated by the photoaffinity labeling of peak I. However, the D-glucose transport activity was associated only with peak I. These findings suggest that the anionic domain of the D-glucose transporter becomes exposed because of conformational changes of the protein as a result of covalent binding with [3H]cytochalasin B by photoaffinity labeling.

Affinity Labels↗

PubMed1982

Carbon balance studies of glucose metabolism in rat cerebral cortical synaptosomes.

Synaptosomes were isolated from rat cerebral cortex and incubated with [U-14C]-, [1-14C]- or [6-14C]glucose. Glucose utilization and the metabolic partitioning of glucose carbon in products were determined by isotopic methods. From the data obtained a carbon balance was constructed, showing lactate to be the main product of glucose metabolism, followed by CO2, amino acids and pyruvate. Measuring the release of 14CO2 from glucose labelled in three different positions allowed the construction of a flow diagram of glucose carbon atoms in synaptosomes, which provides information about the contribution of the various pathways of glucose metabolism. Some 2% of glucose utilized was calculated to be degraded via the pentose phosphate pathway. Addition of chlorpromazine, imipramine or haloperidol at concentrations of 10(-5) M reduced glucose utilisation by 30% without changing the distribution pattern of radioactivity in the various products.

Amino Acids↗

PubMed1988

Gluconeogenesis from glycine and serine in fasted normal and diabetic rats.

1. Non-anaesthetized normal and diabetic rats were fasted for 1 day, and [U-14C]glycine, or [U-14C]serine, or [U-14C]- plus [3-3H]-glucose was injected intra-arterially. The rates of synthesis de novo/irreversible disposal for glycine, serine and glucose, as well as the contribution of carbon atoms by the amino acids to plasma glucose, were calculated from the integrals of the specific-radioactivity-versus-time curves in plasma. 2. The concentrations of both glycine and serine in blood plasma were lower in diabetic than in fasted normal animals. 3. The rates of synthesis de novo/irreversible disposal of both amino acids tended to be lower in diabetic animals, but the decrease was statistically significant only for serine (14.3 compared with 10.5 mumol/min per kg). 4. Of the carbon atoms of plasma glucose, 2.9% arose from glycine in both fasted normal and diabetic rats, whereas 4.46% of glucose carbon originated from serine in fasted normal and 6.77% in diabetic rats. 5. As judged by their specific radioactivities, plasma serine and glycine exchange carbon atoms rapidly and extensively. 6. It was concluded that the turnover of glycine remains essentially unchanged, whereas that of serine is decreased in diabetic as compared with fasted normal rats. The plasma concentration of both amino acids was lower in diabetic rats. Both glycine and serine are glucogenic. In diabetic rats the contribution of carbon atoms from glycine to glucose increases in direct proportion to the increased glucose turnover, whereas the contribution by serine becomes also proportionally higher.

Animals↗

PubMed1978

A protein-glucan intermediate during paramylon synthesis.

A sodium deoxycholate extract containing glucosyltransferase activity was obtained from a particulate preparation from Euglena gracilis. It transferred glucose from UDP-[14C]glucose into material that was precipitated by trichloroacetic acid. This material released beta-(1 leads to 3)-glucan oligosaccharides into solution on incubation with weak acid, weak alkali and beta-(1 leads to 3)-glucosidase. The products of the incubation of the deoxycholate extract with UDP-[14C]glucose were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioactive bands were obtained that had the properties of beta-(1 leads to 3)-glucan covalently linked to protein by a bond labile to weak acid. High-molecular-weight material containing a beta-(1 leads to 3)-glucan was also shown to be present by gel filtration. The bond linking glucan to aglycone is possibly a pyrophosphate linkage. It is proposed that in Euglena gracilis beta-(1 leads to 3)-glucan (paramylon) is synthesized on a protein primer.

Animals↗

PubMed1995

The effects of oophorectomy and female sex steroids on glucose kinetics in the rat.

In order to clarify the roles played by female sex steroids on glucose metabolism, basal glucose kinetics were studied in control sham operated (C), oophorectomised (O), 17-beta-oestradiol treated oophorectomised (1.5 micrograms/day) (E) and progesterone treated oophorectomised (1.5 mg/day) (P) female rats. Hormone (or vehicle only) delivery was via osmotic pumps which were inserted at the time of oophorectomy (or sham operation) 7 days prior to metabolic testing. In fasted anaesthetised rats, hepatic glucose production (HGP), plasma glucose metabolic clearance rate (MCR) and glucose uptake indices (GUI) of selected peripheral tissues were measured using radioactive tracer methodology. Following surgery, the O rats significantly gained and the E rats significantly lost weight compared to the C rats. Plasma insulin and glucose were not different between groups. HGP and MCR were increased by 24-29% (P < 0.005) and 19-28% (P < 0.001), respectively, in the O compared to the C, E and P rats. The GUI of brown adipose tissue was significantly reduced in the P compared to the C rats (3 +/- 1 vs 25 +/- 10 micromol/100 g/min). In conclusion, female sex steroid hormones significantly influence rat body weight, hepatic glucose metabolism and the metabolism of brown adipose tissue.

Animals↗

PubMed1992

An inhibitor of sweet taste response modulates glucose-induced phosphoinositide breakdown in rat pancreatic islets.

We studied the effect of a specific-competitive inhibitor of the sucrose taste response, p-nitrophenyl-D-glucopyranoside (PNP-Glu) on insulin release and phosphoinositide metabolism in rat pancreatic islets. The alpha-anomer, but not the beta-anomer, of PNP-Glu at a concentration of 5 mM inhibited insulin release induced by 10 mM glucose. Islets were labeled by exposure for 2 h to 10 uCi of myo-[2-3H] inositol solution supplemented with 2.8 mM glucose. Forty islets were then incubated in the presence of 10 mM LiCl, 1 mM inositol and 10 mM glucose with or without the anomers of PNP-Glu. [3H] radioactivity in the incubation medium remained significantly greater in the presence of the alpha-anomer of PNP-Glu than in the presence of glucose alone after 5- and 20-min incubation. The inositol monophosphate levels in the islets incubated with glucose alone were increased more than in the islets with alpha-anomer. The beta-anomer of PNP-Glu did not change either glucose-induced insulin release or phosphoinositide breakdown. A patch-clamp study revealed that neither anomer affected the glucose-dependent ATP-sensitive K(+)-channels. These results indicate that the anomeric preference for glucose in insulin release in the pancreatic islets is closely associated with phosphoinositide breakdown.

Animals↗

PubMed1980

Calcium movements in relation to glucose-stimulated insulin secretion.

The influence of glucose on the beta-cell handling of Ca2+ was studied in pancreatic islets isolated from ob/ob mice. Glucose had both stimulatory and inhibitory effects on the washout of radioactivity from islets preloaded with 45Ca. Although the phenomenon of stimulation may be essentially associated with an increased turnover of 45Ca incorporated in response to glucose, the inhibitory effect might merely reflect a lowering of Ca2+ in the cytoplasm following from its accumulation into secretory granules and mitochondria. It is suggested that the Ca2+ uptake by the granules is mediated by an ATP-dependent proton gradient and that these organelles serve as a regulator of the cytoplasmic Ca2+ involved in stimulus-secretion coupling. Alterations in the beta-cell handling of Ca2+ may not only explain the role of glucose as an initiator of insulin release, but also the potentiation by cAMP of the action of glucose. The latter effect can tentatively be ascribed to an increase of cytoplasmic Ca2+ following cAMP-induced inhibition of the net Ca2+ uptake by mitochondria.

1-Methyl-3-isobutylxanthine↗

PubMed1964

DISTRIBUTION OF RADIOACTIVITY IN AUTOLYZED CELL WALL OF BACILLUS CEREUS DURING SPHEROPLAST FORMATION.

Kronish, Donald P. (Warner-Lambert Research Institute, Morris Plains, N.J.), Raam R. Mohan, and Benjamin S. Schwartz. Distribution of radioactivity in autolyzed cell wall of Bacillus cereus during spheroplast formation. J. Bacteriol. 87:581-587. 1964.-Spheroplasts of Bacillus cereus strain T were produced from cells grown in the presence of uniformly labeled C(14)-glucose. At regular intervals during spheroplast formation, enzymatically degraded cell wall was isolated by a new procedure. Radioactivity of solubilized cell wall in cell-free material increased from 2.5 to 42% of the total incorporated label during spheroplast formation. The rate of cell-wall degradation as measured by increase in radioactivity was biphasic with relative slopes of 2.0 and 5.0. During autolytic depolymerization of B. cereus cell wall, two major components were solubilized at different rates. Chemical fractionation revealed these to be a peptide and a mucopeptide. The possibility of two enzymes being involved in spheroplast formation and cell-wall degradation is discussed.

Amino Acids↗

PubMed2006

Effect of high glucose concentration on corneal collagen biosynthesis.

The effect of high glucose concentration (3 g/l) on bovine corneal total protein and collagen biosynthesis was studied, using 3H-proline incorporation in explant cultures with protein and collagen determinations. The high glucose concentration increased the incorporation of 3H-proline in total corneal proteins as well as in collagens. The specific radioactivity of stromal collagens was strongly increased in these conditions. Mannitol was used to control the osmotic effect of the high glucose concentration, both at 1 and 3 g/l concentrations. Mannitol did not increase the incorporation of 3H-proline in total proteins or collagens, but on the contrary decreased it. The high glucose concentration decreased the excretion of neosynthesized proteins and collagens in the culture medium, but did not affect the total protein or collagen content of the corneas. The strong increase in the specific radioactivity of corneal collagens in the presence of 3 g/l glucose suggests an increased turnover of collagens in diabetic corneas. The increased biosynthesis of collagens together with their decreased elimination in the extracellular compartment can create the conditions for the formation and accumulation of advanced glycation endproducts by the Maillard reaction. This can induce and stimulate the liquefaction of the vitreous body leading to sight-threatening disorders such as diabetic retinopathy, retinal detachment, glaucoma, cataract formation and age-related macular degeneration.

Animals↗

PubMed1998

Effect of oral glucose loading on the biodistribution of BMIPP in normal volunteers.

UNLABELLED: We have evaluated whether myocardial uptake of the fatty acid analog 123I-15-(p-iodophenyl)-3-R,S-methyl pentadecanoic acid (BMIPP) is dependent on the dietary state. METHODS: We compared the biodistribution of 150 MBq of 123I-BMIPP in six healthy volunteers in two states: after at least 12 hr of fasting and after oral glucose loading (75 g) 60 min before tracer administration, followed by a meal enriched in carbohydrates and protein. Planar and tomographic acquisitions were performed over a 4-hr time period after tracer injection; data were corrected for radioactive decay and injected dose. Radioactivity was measured in blood samples drawn at several points. RESULTS: Significant increases of glycemia and insulinemia and a significant drop in plasma nonesterified acids were documented after glucose loading. Half-time values for plasma radioactivity were significantly shorter in the glucose-loaded state than in the fasted state (4.3 +/- 1.4 min compared to 6.3 +/- 1.3 min, p < 0.05). Activity in the heart and liver tended to be higher in the glucose-loaded state than in the fasted state. SPECT images at 0.5 hr after tracer injection demonstrated that the myocardial wall-to-cavity ratio was higher after glucose than in the fasted state (2.53 +/- 0.59 compared to 2.11 +/- 0.21, p = 0.15). Washout from the liver between 1 and 4 hr after injection increased from 18.6% +/- 4.4% in the fasted study to 24.1% +/- 2.4% after glucose (p = 0.04). Washout from the myocardium between 0.5 and 3.5 hr after injection increased from 13.1% +/- 8.8% in the fasted study to 24.0% +/- 3.7% after glucose (p = 0.05). CONCLUSION: These results indicate that fasting before BMIPP scintigraphy is not mandatory to obtain adequate SPECT images. At the tire when SPECT is usually performed, glucose loading may provide improved ratios between myocardial and blood pool activity.

Administration, Oral↗

PubMedDate not supplied

Phosphorylation of Escherichia coli enolase.

In vivo labeling of Escherichia coli JA200 pLC 11-8 resulted in 32P incorporation into enolase as demonstrated by immunoaffinity chromatography and electrophoresis followed by autoradiography. Complete acid hydrolysis, followed by thin layer chromatography was employed for determination of the phosphoamino acid residue. Comparison with phosphoamino acid standards resulted in the identification of a labeled residue corresponding to phosphoserine. In vitro labeling of cell extracts from glucose and acetate grown cells resulted in differential labeling of enolase. When specific radioactivities of in vivo labeled enolase were compared, 7 times more label was incorporated at late log phase in glucose grown cells than in late log acetate grown cells. At stationary phase, only 2.5 times more label was incorporated into glucose compared to acetate. When 32P-labeled enolase from glucose grown cells was subjected to treatment with potato acid phosphatase, dephosphorylation of the enzyme could be observed. Monitoring enzyme activity during the acid phosphatase treatment revealed a 70% decrease for the forward enzyme reaction, and a 3-fold increase, followed by a gradual decrease to almost zero, for the reverse enzyme reaction. Complete reversal of the changes in activity was possible by adding an aliquot of partially purified enolase kinase plus ATP.

Acid Phosphatase↗

PubMed1975

Regulation of cell wall synthesis in Avena stem segments by gibberellic Acid.

Gibberellic acid induces (a) increased elongation of Avena sativa stem segments, (b) increased formation of cell wall material, measured on the basis of dry weight, and (c) increased incorporation of (14)C-glucose into all fractions of the cell wall material. This increased incorporation of radioactivity correlates well with increased formation of cell wall material and shows a time-course pattern similar to the time course of the elongation response. Approximately one hour after the application of gibberellic acid, the rates both of growth and of incorporation of radioactivity accelerate to about 2-fold over the control rate. Gibberellic acid does not stimulate the incorporation of labeled glucose into the cell wall material simply by increasing the rate of uptake of glucose by internodal cells. The stimulation of the incorporation of (14)C-glucose into cell wall material, which reflects the stimulation of cell wall synthesis, seems to be an important and relatively early effect of gibberellic acid in this system and probably contributes significantly to the elongation response elicited by the hormone.

Journal Article↗

PubMed1979

In vitro lipid metabolism in the rat pancreas. II. Effects of secretagogues on fatty acid metabolism, net lipolysis and ATP levels.

1. The concentration of carbamylcholine, bombesin, pancreozymin, pentagastrin and secretin evoking a similar 4--5-fold maximal increase in amylase secretion from rat pancreatic fragments were 3.10(-6), 10(-7), 10(-8), 3.10(-6), and 3.10(-6) M, respectively. The maximal concentration of vasoactive intestinal peptide tested (3.10(-6) M) increased amylase secretion by 250%. The six secretagogues could be separated into two groups according to their effects on lipid metabolism and ATP levels. 2. When used at their optimal concentrations, carbamylcholine, bombesin, pancreozymin, and pentagastrin lowered pancreatic ATP levels by 18-26% and increased net release of free fatty acids by 68-105%. 3. The effects of 3.10(-6) M carbamylcholine and 10(-8) M pancreozymin on the metabolism of 3H2O, D-[U-14C]glucose and [1-14C]acetate were similar; the incorporation of radioactivity in the fatty acid moiety of glycerolipids decreased by 20--50% whereas the incorporation of 3H from 3H2O and of 14C from [U-14C]glucose increased by 20--35% in the glycerol moiety. In addition, the oxidation of [U-14C]glucose, [1-14C]acetate and [1-14C]palmitate to 14CO2 increased by 15--32% while the esterification of [1-14C]palmitate, [1-14C]-linoleate, and [1-14C]arachidonate was inhibited by 14--23%. The spectrum of fatty acids labeled with [1-14C]acetate indicated an inhibition of the malonic acid pathway whereas the elongation of polyenoic fatty acids was unaltered.

Acetyl Coenzyme A↗

PubMed1978

Formation and function of N-acetyloglucosamine-linked phosphoryl- and pyrophosphorylundecaprenols in membranes from Bacillus cereus.

Membranes from Bacillus cereus AHU 1356 incorporated radioactivity from UDP-N-acetyl[14C]glucosamine into three alkaline-stable and acid-labile lipids which were extracted into chloroform:methanol (2:1) and separated from each other by thin layer chromatography on silica gel plates. The major labeled lipid (Lipid 1) and a minor one (Lipid 2) were identified as N-actetylglucosaminyl phosphorylundecaprenol from several analytical criteria involving mass spectral data and from reversal of their formation by UDP. These two lipids appear to differ in geometry of their polyprenol moieties. The third labeled lipid (Lipid 3) was identified as N-acetylglucosaminyl pyrophosphorylundecaprenol. Antibiotic 24010, a tunicamycin-like antibiotic, at 1 microgram/ml was found to inhibit almost completely the formation of Lipid 3, whereas it inhibited the formation of Lipid 1 much more weakly and rather enhanced the formation of Lipid 2. Radioactivity was also incorporated into a polymer from UDP-GlcNAc and from Lipid 3. UDP-N-acetylmannosamine, UDP-N-acetylgalactosamine, and UDP-glucose supported the incorporation. Antibiotic 24010 strongly inhibited the incorporation of radioactivity from UDP-GlcNAc into polymer, whereas it did not affect the incorporation from Lipid 3. Thus, it is concluded that N-acetylglucosaminyl pyrophosphorylundecaprenol serves as a precursor in the synthesis of a polymer presumed as the cell wall polysaccharide of this bacterial strain.

Acetylglucosamine↗

PubMed1981

A 2-phase liquid scintillation assay for glycolipid synthetases.

Glycolipid synthetases can be assayed conveniently by incubating the lipid substrate with the radiosugar-labeled nucleotide in a small plastic scintillation vial. At the end of the incubation period, water and perchloric acid are added, then n-butanol, then a toluene-based scintillation cocktail. The radioactive lipid partitions into the scintillation fluid, leaving excess sugar nucleotide in the aqueous phase. Only a small fraction of the total radioactivity in the aqueous layer is detectable. This method is illustrated for ceramide:UDP-glucose glucosyltransferase. The approach should be applicable to other lipid synthetases that can be assayed with radioactive hydrophilic substrate.

Animals↗

PubMed1985

[Neutral glycosphingolipids of Fabry's disease lymphoblastoid lines established by Epstein-Barr virus transformation].

Human lymphoid cell lines established by Epstein-Barr viral transformation of peripheral B-lymphocytes from normal subjects and from Fabry patients, were investigated for their ability to biosynthesize neutral glycosphingolipids from [14C]galactose and [14C]glucose as precursors. Galactose was taken up in the presence of high concentrations of glucose and selectively utilised by the cells in the synthesis of galactosphingolipids. The pattern of neutral glycosphingolipids labelled from [14C]galactose was slightly modified with time of labelling in either lymphoid cell line: the first labelled glycosphingolipid was lactosylceramide (LacCer) in the normal line and globotetraosylceramide (GbOse4Cer) in the Fabry line. After labelling for 96 h, a steady state was reached and the percentage of every type of labelled glycosphingolipid was stable in each cell line; however, differences in the neutral sphingolipid composition appeared between the various cell lines. When using radiolabelled glucose as precursor, the major part of the radioactivity was incorporated into neutral lipids and phospholipids; neutral sphingolipids were much less labelled than when using galactose. Catabolism of endogeneous labelled glycosphingolipids (synthesized by the cells during the 'pulse') was studied after cultivating the cells without radiolabelled precursors ('chase'). In the cells from normal subjects, all the neutral glycosphingolipids were slowly degraded (half-life time around 15-25 days for LacCer and GbOse3Cer). In contrast, in a lymphoid line from a Fabry patient, no appreciable degradation of GbOse3Cer occurred during 30 days. This block in the catabolism of GbOse3Cer is in good agreement with the previously reported deficiency of alpha-galactosidase A activity in this Fabry lymphoid cell line [Salvayre, R. et al. (1981) Biochim. Biophys. Acta 659, 445-456] and demonstrates that alpha-galactosidase B does not hydrolyze GbOse3Cer in the living cell (in contrast to the situation in vitro).

Adult↗

PubMed1998

Glucose metabolism in vivo in crossbred Holstein cattle feeding on different types of roughage during late pregnancy and early lactation.

An experiment was carried out to study the glucose kinetics of crossbred Holstein cattle feeding on either hay or 5% urea treated rice straw during late pregnancy (21 days prepartum) and early lactation (30 days postpartum). In all 16 pregnant heifers (23-25 months of age) were selected for the experiments, including eight animals of two breed types, Holstein Friesian x Red Sindhi (50:50 = 50% HF) and Holstein Friesian x Red Sindhi (87.5:12.5 = 87.5% HF). They were divided into four groups of four animals each. Animals from the same breed type in each group were fed with either rice straw treated with 5% urea or pangola hay (Digitaria decumbens) as the source of roughage throughout the experiments. The glucose turnover rate in both types of crossbred Holstein cattle was determined using a continuous infusion of [U-14C] and 3-[3H]glucose during late pregnancy and early lactation. Total glucose entry and utilization rates increased significantly during lactation for all groups. Recycling of [C]glucose was, approximately 20% in both crossbred cattle fed either hay or urea treated rice straw and was unaffected by the stage of late pregnancy or early lactation. Comparing 50 and 87.5% HF animals, arterial plasma glucose concentrations were slightly higher during pregnant periods but significantly higher in lactating periods in 50% HF animals. The ratio of specific radioactivity of arterial blood bicarbonate relative to that of arterial blood [14C]glucose in the lactating period, significantly decreased in 50% HF animals fed either urea treated rice straw or hay. An increase in udder blood flow during early lactation was significantly higher in 87.5% HF animals than in 50% HF animals. The uptake, arteriovenous differences and extraction ratio for glucose across the udder, significantly increased in the lactating period for all crossbred animals. Glucose uptake by the udder of 87.5% HF animals accounted for 65% of the total glucose turnover rate compared to a value of 46% in the lactating 50% HF animals. It can be concluded that both crossbred cattle fed either urea treated rice straw or hay exhibit the same body glucose turnover rate. The 87.5% HF animal has the genetic potential for a high milk yield and has high body and udder glucose metabolisms compared with 50% HF animals.

Animal Feed↗

PubMed1997

Evidence for channeling of intermediates in the oxidative pentose phosphate pathway by soybean and pea nodule extracts, yeast extracts, and purified yeast enzymes.

Evidence is presented that intermediates of the oxidative pentose phosphate pathway (OPPP) are channeled from one pathway enzyme to the next. CO2 produced from [1-14C]glucose in the presence of unlabelled pathway intermediates contained much more radioactivity than predicted by a model in which pathway-produced intermediates are in equilibrium with identical molecules in the bulk phase. This was the case whether glucose 6-phosphate (Glc6P), 6-phosphogluconolactone, or 6-phosphogluconate was added. Assumptions involved in calculating the amount of 14CO2 predicted for free mixing of 14C-labelled and unlabelled intermediates are discussed, together with the following results. (a) 14CO2 production by pea nodules in the presence of 3 mM 6-phosphogluconate was higher than in its absence. (b) Apparent channeling of intermediates was much higher for purified yeast enzymes than for yeast extract. (c) 6-Phosphogluconate and 6-phosphogluconolactone were channeled between yeast Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase despite the absence of 6-phosphogluconolactonase in the purified yeast enzyme mixture. (d) When purified yeast hexokinase was physically separated from Glc6P dehydrogenase and 6-phosphogluconate dehydrogenase by a dialysis membrane, there was no apparent channeling. (e) Poly(ethylene glycol), high salt and detergents had little effect on apparent channeling of OPPP intermediates, which is consistent with a stable complex of enzymes. On the other hand, density gradient centrifugation experiments suggested a more transient interaction between the enzymes. Taken together, the results support channeling of OPPP pathway intermediates.

Carbon Radioisotopes↗