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PubMed1987

Increase in the glucosylated form of erythrocyte Cu-Zn-superoxide dismutase in diabetes and close association of the nonenzymatic glucosylation with the enzyme activity.

Human erythrocytes contain glucosylated and nonglucosylated Cu-Zn-superoxide dismutases which can be separated by boronate affinity chromatography. The percentage of the glucosylated form is significantly increased in the erythrocytes of patients with diabetes as compared to normal erythrocytes. The nonglucosylated form of Cu-Zn-superoxide dismutase, which was washed through the boronate column, was glucosylated in vitro upon exposure to radioactive or non-radioactive D-glucose. Incorporation of D-glucose into the protein was observed, and with the increase in glucosylation, the enzymatic activity decreased, indicating that the glucosylation of the enzyme led to a low active form. This is the first demonstration that superoxide dismutase is glucosylated in erythrocytes and that the glucosylation leads to the inactivation of the enzyme.

Blood Glucose↗

PubMed1984

Selective metabolism of L-serine by Moniliformis (Acanthocephala) in vitro.

In a series of in vitro experiments, adult male and female Moniliformis dubius were incubated at pH 6.88 and 37 degrees C for 3 h in a 2.5 mM solution of 18 amino acids. Fifteen of these were absorbed slightly from the medium, but L-serine was almost completely absorbed while the concentrations of glycine and alanine in the medium increased during the course of the incubation. By using L-[U-14C]serine, it was found that labelled ethanol and CO2 were the main end-products of metabolism excreted into the medium, with smaller amounts of labelled alanine, lactate and acetate. Small amounts of cystathionine with high specific radioactivity were found in extracts of the worms at the end of incubation, together with other radioactive metabolites including glucose, ethanol, lactate, succinate, malate, serine, glycine and alanine. Ammonia was found to be an excretory product of the amino acid metabolism of M. dubius. Possible metabolic pathways and suggestions for the significance of serine metabolism in this parasite are discussed.

Acanthocephala↗

PubMed1979

Membrane glycopeptides from virus-transformed hamster fibroblasts and the normal counterpart.

Comparisons of membrane glycopeptides from baby hamster kidney fibroblasts (BHK21/C13) and a clone transformed by Rous sarcoma virus (C13/B4) were made by using cells metabolically labeled with radioactive D-glucose and L-fucose. Most of the glycopeptides were metabolically labeled with both the general and the specific glycoprotein precursors. The glycopeptides obtained from the cell surface by controlled trypsinization were representative of the surface membrane as shown by comparing them with those of purified membrane preparations. The trypsin-removable glycopeptides from both cell types were further processed and examined by successive chromatography on Sephadex G-50 and DEAE-cellulose. The chromatographic distribution patterns showed that each cell type had glycopeptides of similar characteristics, although the proportions of the glycopeptides differed dramatically between the two cell types. After transformation there was an increase in the larger, more highly charged glycopeptides. This was verified by the increased sialic acid content in these glycopeptides. Some of the glycopeptides were homogeneous after the size and charge separations, since a variety of procedures did not separate them further. The apparent homogeneity and reasonably few species obtained may be due to the methods of isolation, with the procedures selecting particular glycopeptides from the external portion of the membrane. These results corroborate the concept and show for the first time that virus transformation is accompanied by an increase in certain species of glycopeptides rather than de novo synthesis.

Animals↗

PubMed1969

The development of gluconeogenesis in rat liver: experiments in vivo.

1. The injection of substrate amounts of lactate into newborn rats produced an increase in the concentration of phosphoenolpyruvate in liver. Similar experiments with foetal rats showed no increase in phosphoenolpyruvate concentration although pyruvate formation was observed. 2. The administration of pyruvate to foetal rats was also without effect on the hepatic phosphoenolpyruvate concentration, although a 20-fold increase in this was observed when pyruvate was injected into newborn animals. 3. Analogous experiments with aspartate produced qualitatively similar differences between foetal and newborn rats. 4. When [(14)C]-lactate, -pyruvate or -aspartate was injected into foetal or newborn rats incorporation of radioactivity into liver glucose was observed only in the newborn animals. 5. Lactate/pyruvate ratios of 213 in foetal liver and 13.5 in the livers of newborn rats indicated a relatively reduced environment in the cytosol of foetal liver. This difference in redox state was illustrated experimentally by a greater conversion of pyruvate into lactate and an increased formation of malate in foetal liver. 6. Although both the substrate-loading and tracer experiments indicated a block in gluconeogenesis in foetal liver at the stage of conversion of oxaloacetate into phosphoenolpyruvate, gluconeogenesis was also hindered by a highly reduced environment.

Animals↗

PubMed1985

Autoradiographic method to screen for soil microorganisms which accumulate zinc.

An autoradiographic method was developed to screen for and isolate soil microorganisms which accumulate zinc (Zn). Diluted soil samples (Rubicon fine sand, Entic Haplorthods [pH 5.9]) were plated on soil extract-glucose agar containing radioactive 65Zn. After 7 days of incubation, individual colonies which accumulated sufficient 65Zn could be detected by autoradiography. These colonies were isolated and confirmed as Zn accumulators in pure culture by using the autoradiographic plate technique. Most Zn accumulators were filamentous fungi, identified as Penicillium janthinellum, Aspergillus fumigatus, and Paecilomyces sp. Isolates of Penicillium janthinellum were the most common Zn accumulators. The most abundant Zn-accumulating bacteria were Bacillus spp. The validity of the autoradiographic plate technique to differentiate soil microbes which accumulate Zn was examined independently by energy dispersive X-ray analysis in a scanning electron microscope. This method confirmed that fungal isolates which gave positive autoradiographic responses in the plate assay bioaccumulated more Zn in their biomass than fungal isolates from the same soil sample which gave negative autoradiographic responses. Thus, this technique can be applied to specifically screen for and isolate microbes from the environment which bioaccumulate Zn.

Aspergillus fumigatus↗

PubMed1980

Leucogenenol a thymothyroid hormone.

Thymectomy and, to a lesser extent, thyroidectomy cause a significant decrease in the serum leucogenenol levels of rats. Daily injections of neonatally thymectomized mice with leucogenenol causes the mice to maintain a normal level of neutrophils and lymphocytes in their circulation. The thymus and thyroid are the only tissues in rats and mice that afford radioactive leucogenenol when incubated in a medium containing radioactive D-glucose. It is concluded that the thymus and possibly the thyroid are responsible for the normal biosynthesis of leucogenenol in animals.

Adrenal Glands↗

PubMed1984

Adrenalectomy reverses insulin resistance in muscle from obese (ob/ob) mice.

Glucose transport and insulin resistance in the skeletal muscle of the genetically obese (ob/ob) mouse has been studied in animals at 6-7 wk of age using an in situ perfused hindquarter preparation. Rates of glucose uptake and the uptake of radioactively labeled 2-deoxy-D-glucose were lower in muscles from intact ob/ob mice than in those from their lean littermates. Following adrenalectomy the basal rate of glucose or 2-deoxy-D-glucose uptake in the ob/ob mice was restored to normal. The uptake of 2-deoxyglucose was tested at two insulin concentrations. In lean mice, adrenalectomy had no effect on 2-deoxyglucose uptake. Adrenalectomy, however, restored the responsiveness to insulin in the ob/ob mouse to normal. The activity ratio of muscle glycogen synthase was elevated by insulin but the effect was greater in the lean animals with or without adrenal glands than in the obese mouse. Adrenalectomy in the obese mouse did not correct this defect.

Adrenal Glands↗

PubMed1985

Measures of de novo synthesis of milk components from propionate in lactating goats.

Possible direct contributions of propionate to de novo synthesis of milk components by the mammary gland of lactating goats fed a concentrate-roughage diet have been studied in vivo by primed constant infusion of [1-carbon-14]propionate into the right mammary artery. Specific radioactivities of milk galactose, fatty acids, and protein were higher in the infused than in the uninfused half of the mammary gland, suggesting de novo synthesis of these compounds in the udder. Specific radioactivities of milk glucose in both udder halves were identical, ruling out any possibility of mammary gland-derived glucose from propionate of blood plasma under the experimental conditions. Of milk galactose, .8% was derived from propionate of blood plasma, and of milk glucose, 98% was derived from glucose of blood plasma. After intraruminal infusion of unlabeled propionic acid at 11 g/h, concentration of propionate in blood plasma was doubled, its contribution to milk galactose was increased to 1.5%, and proportions of milk odd-numbered fatty acids were increased. Propionate was incorporated largely into milk odd-numbered fatty acids (9:0, 11:0, 13:0, 15:0, 17:0). We conclude that small amounts of propionate can be incorporated into principal components of milk in the mammary gland of lactating goats.

Animals↗

PubMed1977

Metabolim of blood-borne lactate in rat brain in vivo.

L-Lactate uniformly labelled with 14C was administered to rats as a single intravenous injection. In experiments concerning the determination of lactate flux into total forebrain, the tissue was obtained by a freeze-clamping technique; in experiments concerning the determination of lactate flux in discrete brain areas the tissue was coagulated by microwave irradiation of the head. In the acid extract of brain tissue the contents and radioactivities of lactate, glucose and cycle amino acids were measured. The following results were obtained: 1. Labelled lactate in plasma equilibrates rapidly with lactate in a small pool in brain tissue. This accessible pool comprises one quarter of total brain lactate. Its size varies in brain regions being largest in cortex and smallest in pons and medulla. 2. Approximately one half of plasma-borne lactate in brain is used metabolically as is seen from the incorporation of lactate carbon in amino acids. The regional activity of lactate metabolism correlates with the local size of the accessible lactate pool. 3. The rates of lactate flux in total forebrain were approximated, the invasion of lactate from plasma to brain tissue to 0.4 mumol, the metabolisms of plasma-borne lactate to 0.2 mumol per g brain tissue min-1. 4. The labelling pattern of cycle amino acids is intermediate between the pattern from [14C]-glucose and [14c]-bicarbonate as precursors. This gives evidence for the influx of part of lactate into the "small" compartment of the citrate cycle along the CO2 fixation route with a predominant accumulation of lactate carbon in aspartate and glutamine.

Animals↗

PubMed1984

Nonenzymatic glycosylation of bovine retinal microvessel basement membranes in vitro. Kinetic analysis and inhibition by aspirin.

Incubation of intact bovine retinal microvessels or isolated retinal microvessel basement membranes (RVBM) with radioactive D-glucose or L-glucose, followed by basement membrane collagenous protein purification, resulted in the isolation of nonenzymatically glycosylated RVBM collagens. Type IV collagen was identified in the RVBM by selective salt fractionation, SDS-polyacrylamide gel electrophoresis, amino acid analysis, and immunoprecipitation with specific antibody. Kinetic analysis of the condensation of glucose with RVBM was carried out by labeling retinal microvessel basement membranes with D-[2-3 H]-glucose and D-[6-14 C]-glucose. The rate constant for aldimine product formation, k1, was 1.95 +/- 0.24 (SD) X 10(-4) mM-1 h-1, and the rate constant for the reversed reaction, k-1, was 5.9 +/- 1.0 X 10(-2) h-1. Based on a rate constant for the Amadori rearrangement, k2, of 8.8 +/- 1.0 X 10(-3) h-1, which was the rate-determining step, the half life of this reaction was 80 +/- 9 h. These data may be useful in estimating the glycosylation of retinal microvessel basement membranes in vivo. The nonenzymatic glycosylation of retinal microvessel basement membrane proteins was progressively inhibited by increasing concentrations (0.1 to 2.0 mM) of aspirin.

Animals↗

PubMed1989

Freeze-mount microautoradiographic study in the mouse hippocampus after intravenous injection of tritiated 2-deoxyglucose and glucose.

Differences in the uptake of tracers from radioactive 2-deoxyglucose ([1,2-3H] and [2,6-3H]), and glucose ([1-3H], [3-3H]) into hippocampal regions were investigated by freeze-mount microautoradiography after 45 min for 2-deoxyglucose, and after 15 and 45 min for glucose. Silver grains were assessed quantitatively by an image analyser. (1) The radioactivity (silver grains/mm2) in the stratum lacunosum-moleculare of Ammon's horn from 2-deoxyglucose autoradiograms was significantly higher than that in other hippocampal regions (P less than 0.01), while lowest in the hilus fascia dentata (P less than 0.01). (2) Autoradiograms of [1-3H]glucose and 15 min of [3-3H]glucose showed the radioactivity in the dentate molecular layer to be significantly higher than that in other regions, excepting the stratum lacunosummoleculare (P less than 0.05). (3) The 2-deoxyglucose and 45-min glucose autoradiograms showed intensely labeled perikarya of pyramidal cells in the CA3a sector. (4) Radioactivity in the dentate granular layer from the 45-min autoradiogram of [3-3H]glucose was significantly higher than that in the molecular layer (P less than 0.05). The results imply that the metabolic fate of glucose, i.e. whether it is mainly used for energy production or amino acid synthesis, depends on each structure of the hippocampus.

Animals↗

PubMed1982

Evidence that glucose "marks" beta cells resulting in preferential release of newly synthesized insulin.

Studies of isolated islets labeled with radioactive leucine show that glucose at a critical time "marks" islets in such a way as to cause preferential release of newly synthesized insulin. The preferential release of insulin from marked islets is relatively independent of subsequent secretagogues or rates of insulin secretion. Previous kinetic studies have indicated that the critical time at which marking occurs is after proinsulin biosynthesis but before the secretory event. Thus, secretory cells may regulate the diversion of newly synthesized material for immediate release as it is approaching or transiting the Golgi apparatus.

1-Methyl-3-isobutylxanthine↗

PubMedDate not supplied

[Degree of leucine utilization in the provision of substrate for lipid synthesis in animal tissues in vivo].

Radioactivity of lipids synthesized in the encephalon, liver kidneys, lungs, pancreas, stomach mucosa, duodenum, heart, skeletal muscle, fat tissue and rat skin 2h after intraperitoneal administration of [U-14C] leucin is 0.38-19.3% of protein radioactivity, 31.2-615.3% of radioactivity of lipids synthesized in the mentioned organs and tissues of rats whom [U-14C] glucose was administered. CO2 radioactivity in rats released with the expired air in 1.5% of medium radioactivity of proteins in the studied organs and tissues, 19.5% of radioactivity of 14CO2 released with the expired air in rats who were administered [U-14C] glucose.

Animals↗

PubMed1983

The metabolic fate of exogenous sorbitol in the rat.

Dietary sorbitol is rapidly converted to fructose and other carbohydrates in the liver, but its metabolic fate has not been studied rigorously. Twenty-four rats were given 20.4 muCi [14C]sorbitol with 100 mg of sorbitol, and groups of six were killed at 1, 3, 6, and 24 hours after sorbitol administration. Rats were also fed 6.9 muCi [14C]sorbitol for 7 or 14 days. Serum, liver, and lens were analyzed for 14C-labeled sorbitol, fructose, and glucose by using high-performance liquid chromatography. Negligible radioactivity (1.1%) was found in the gastrointestinal content at 24 hours indicating virtually complete absorption. Most of the radioactivity was recovered in the glucose fraction in serum, liver and lens. Glucose and fructose concentrations showed some decline by day 14 compared with day 7 in serum and liver. However, in the lens, sorbitol showed a peak value at the end of the 14th day (37.5 +/- 9.9 micrograms/pair). These findings suggest that: 1) after oral administration, sorbitol is completely absorbed, and 2) that there is a finite accumulation of sorbitol and fructose in the lens in 14 days. Although the radioactive label indicated the exogenous origin of these carbohydrates, it is not certain whether the sorbitol is converted to glucose before entering and accumulating in the lens.

Administration, Oral↗

PubMed1978

Glucose inhibition of 45Ca efflux from pancreatic islets.

Pancreatic islets were microdissected from ob/ob mice, loaded for 2 h with 45Ca and perfused with calcium-deficient medium. Irrespective of the glucose and calcium concentrations in the loading medium, increased glucose in the perfusion medium resulted in reduced amounts of radioactivity in the perfusate. A glucose inhibition of 45Ca washout was also evident when the specific radioactivity of the islets approached that of the labeling medium, indicating that the effect was not simply due to isotopic dilution. The depression of 45Ca washout diminished after culture of the islets in a serum-free medium and it was absent in islets taken from mice homozygous for the gene diabetes. The glucose effect became less pronounced when 50 micron D-600, an inhibitor of the calcium inward transport, was added to the calcium-deficient perfusion medium and abolished in the presence of 20 mM Ca-EGTA. The inhibition of the 45Ca washout observed is not necessarily due to a direct glucose interaction with the outward calcium transport but may also result from stimulation of the uptake and intracellular trapping of the cation.

Animals↗

PubMed1970

Studies on the incorporation of (U-14C)glucose and (35S)sulphate into the acid glycosaminoglycans of neonatal rat skin.

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.

Animals↗

PubMed1990

Phosphoglucoisomerase-catalyzed interconversion of hexose phosphates: a model for the interconversion of D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate.

Based on experimental data, a model is proposed for the interconversion of either unlabelled hexose phosphates or D-[2-3H]glucose 6-phosphate and D-[1-3H]fructose 6-phosphate in the reaction catalyzed by phosphoglucoisomerase. This model takes into account the known differences in maximal velocity and affinity for each substrate, the intramolecular transfer of tritium between C1 and C2, and the isotopic discrimination between unlabelled and tritiated esters. This model reveals that, in a close system characterized by the progressive detritiation of hexose phosphates, the concentration ratio of D-glucose 6-phosphate to D-fructose 6-phosphate is much higher with the tritiated than unlabelled esters, a paradoxical increase in the specific radioactivity of D-glucose 6-phosphate above its initial value being even observed during the initial period of exposure of D-[2-3H]glucose 6-phosphate to phosphoglucoisomerase. The extension of this model to an open system may be essential for the correct interpretation of radioactive data collected in intact cells exposed to D-[2-3H]glucose.

Fructosephosphates↗

PubMed1987

Lipid metabolism in various regions of squid giant nerve fiber.

The purpose of this investigation was to compare the incorporation of radioactivity from various precursors into lipids of different regions of squid giant nerve fiber systems including axoplasm, axon sheath, giant fiber lobes which contain stellate ganglion cell bodies, and the remaining ganglion including giant synapses. To identify the labeled lipids, stellate ganglia including giant fiber lobes and the remaining tissue were first incubated separately with [14C]glucose, [32P]phosphate, [14C]serine, [14C]acetate and [3H]myristate. The radioactivity from glucose, after conversion to glycerol and fatty acids, was incorporated into most lipids, including triacylglycerol, free fatty acids, cardiolipin, phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, phosphatidylinositol, phosphatidylserine, sphingomyelin and ceramide 2-aminoethylphosphanate [corrected]. The radioactivity from serine was largely incorporated into phosphatidylserine and, to a lesser extent, into other phospholipids, mainly as the base component. The sphingoid bases of ceramide and sphingomyelin were also significantly labeled. Saturated and monounsaturated and, to a lesser extent, polyunsaturated fatty acids of these lipids were synthesized from acetate, glucose and myristate. Among the major lipids, cholesterol was not labeled by any of the radioactive compounds used. Ganglion residues incorporated the most radioactivity in total lipids from either [14C]glucose or [14C]serine, followed by giant fiber lobes and then sheath. Axoplasm incorporated the least. Among various lipids, phosphatidylethanolamine with shorter saturated fatty acids and phosphatidylglycerol contained the most radioactivity from glucose in all regions. Axoplasm was characterized by a higher proportion of glucose radioactivity in ceramide, sphingomyelin and phosphatidylglycerol. Axoplasm and sheath contained a higher proportion of serine radioactivity than did the other two regions in ceramide. Essentially no radioactivity from [14C]galactose was incorporated in any region.

Acetates↗