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PubMed1994

Effect of taurine, L-glutamine and L-histidine addition in an amino acid glucose solution on the cellular bioavailability of zinc.

Radioactive zinc was used to study the effect of a binary parenteral nutrient solution, composed of amino acids and glucose, on zinc uptake by fibroblasts. The influence of addition of taurine, L-glutamine and of the increase in L-histidine content of the admixture was assessed. The pure mixture was highly toxic for cells and so it was diluted 1/5 in tyrode buffer with 2% albumin. As compared with cells incubated in the buffer containing albumin, zinc absorption was significantly higher (P < 0.05) in the presence of the amino acids of the mixture. Amino acids thus increased bioavailability by displacing zinc bound to albumin. When the histidine concentration in the nutrient medium (4.2 mM) was doubled, inhibition was noted after 30 min of incubation and zinc uptake thereafter remained comparable to that in histidine-free medium. The addition of glutamine (4.2 mM), usually not present in binary mixtures, resulted in significant differences as compared with glutamine-free control medium. Taurine (0.8 mM), led to a constant increase in zinc uptake by fibroblasts as compared with that obtained with taurine-free mixture. However, ultrafiltration showed that taurine was not able to displace zinc from albumin.

Biological Availability↗
PubMed2005

Endothelial Na+-D-glucose cotransporter: no role in insulin-mediated glucose uptake.

A recent report indicates that the Na+-D-glucose cotransporter SGLT1 is present in capillaries of skeletal muscle and is required for insulin-mediated glucose uptake in myocytes. This result is based on the complete inhibition of insulin-mediated muscle glucose uptake by phlorizin, an inhibitor of SGLT1. Using the pump-perfused rat hind limb, we measured glucose uptake, lactate efflux, and radioactive 2-deoxyglucose uptake into individual muscles with saline (control), phlorizin, insulin, and insulin plus phlorizin, as well as with saline and insulin using normal and low Na+ perfusion buffer. Insulin-mediated glucose uptake was not inhibited after correction for phlorizin interference in the glucose assay. Lactate efflux and 2-deoxyglucose uptake by individual muscles were unaffected by phlorizin. Low Na+ buffer did not affect insulin-mediated glucose uptake, lactate efflux, or 2-deoxyglucose uptake. We conclude that endothelial SGLT1 exerts no barrier for glucose delivery to myocytes.

Animals↗
PubMed1980

Comparative utilization in vivo of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose and [1-C14] palmitate in the rat.

Female rats were injected i.v. with comparable trace amounts of [U-14C] glycerol, [2-3H] glycerol, [U-14C] glucose, or [1-14C] palmitate, and killed 30 min afterwards. The radioactivity remaining in plasma at that time was maximal in animals receiving [U-14C] glucose while the appearance of radioactive lipids was higher in the [U-14C] glycerol animals than in other groups receiving hydrosoluble substrates. The carcass, more than the liver, was the tissue where the greatest proportion of radioactivity was recovered, while the greatest percentage of radioactivity appeared in the liver in the form of lipids. The values of total radioactivity found in different tissues were very similar when using either labelled glucose or glycerol but the amount recovered as lipids was much greater in the latter. The maximal proportion of radioactive lipids appeared in the fatty-acid form in the liver, carcass, and lumbar fat pads when using [U-14C] glycerol as a hydrosoluble substrate, and the highest lipidic fraction appeared in adipose tissue as labelled, esterified fatty acids. In the spleen, heart, and kidney, most of the lipidic radioactivity from any of the hydrosoluble substrates appeared as glyceride glycerol. The highest proportion of radioactivity from [1-14C] palmitate appeared in the esterified fatty acid in adipose tissue, being followed in decreasing proportion by the heart, carcass, liver, kidney, and spleen. Thus at least in part, both labelled glucose and glycerol are used throughout different routes for their conversion in vivo to lipids. A certain proportion of glycerol is directly utilized by adipose tissue. The fatty acids esterification ability differs among the tissues and does not correspond directly with the reported activities of glycerokinase, suggesting that the alpha-glycerophosphate for esterification comes mainly from glucose and not from glycerol.

Animals↗
PubMed1978

Utilization of pyruvate, alanine and glutamate by isolated fat cells and their effects on glycerol metabolism.

To study the metabolic interactions of different substrates in adipose tissue in vitro, isolated fat cells from fed rats were incubated in medium containing either (U-14C)-pyruvate, L-(U-14C)-alanine, L-(U-14C)-glutamate or (1(-14)C)-glycerol, and supplemented or not with 5 mM glucose or with non-radioactive pyruvate, alanine or glutamate (2 or 10 mM). The utilization of pyruvate for CO2 or fatty acid formation was greater than that of alanine and glutamate, both in the absence and presence of glucose. Glucose enhanced the formation of fatty acids from all the labelled substrates, decreased the synthesis of glyceride glycerol from pyruvate and glycerol, and enhanced it from alanine and glutamate. Pyruvate and glutamate enhanced the utilization of glycerol by the adipocytes and these effects were significantly reduced in the presence of glucose. Thus, the metabolic fate of the glycerol taken up by the cells varied according to the nature of the available substrates.

Adipose Tissue↗
PubMed1984

Nonenzymatic glucosylation of lysyl and hydroxylysyl residues in type I and type II collagens.

Nonenzymatic glucosylation of type I and type II collagens was examined by incubating collagen substrates with D-glucose in vitro. In one set of experiments, unlabeled collagen was incubated with [14C]-glucose and the incorporation of [14C]-radioactivity into protein was determined by TCA precipitation. The incorporation was dependent on the concentration of glucose and the time of incubation. The glucosylated product was also examined by SDS-polyacrylamide slab gel electrophoresis. The results indicated that both alpha 1(I)- and alpha 2(I)-chains of type I collagen were glucosylated and the glucosylation occurred both with native and denatured collagen as substrate. In further studies [3H]-lysine-labeled collagens were glucosylated, the products reduced by NaBH4, and the [3H]-lysine-derived residues were separated by amino acid analyzer. After a 144 h incubation in vitro, 18.9% of [3H]-lysyl residues and 36.5% of [3H]-hydroxylysyl residues in type I collagen were substituted with glucose. In contrast, 47.9% of [3H]-lysyl residues and 68.1% of [3H]-hydroxylysyl residues in type II collagen were glucosylated after 144 h incubation. Based on quantitative amino acid analyses of the substrates, these values represent 27.6 lysine plus hydroxylysine residues substituted per triple-helical type I collagen molecule and 65.3 residues per triple-helical type II collagen molecule. Thus, type I and type II collagens display differential susceptibilities to nonenzymatic glucosylation. Finally, [3H]-proline-labeled type I collagen was glucosylated to varying extents, and the glucosylated products were used as substrates for human polymorphonuclear leukocyte collagenase. No difference in susceptibility to this collagenase was noted, irrespective of the extent of glucosylation.

Amino Acids↗
PubMed1990

Subcutaneous injection with an insulin pen. Insulin absorption and plasma concentrations of glucose and free insulin.

In 6 type 1 diabetic patients treated with a basal-prandial injection regimen with a pen, plasma concentrations of free insulin and glucose were measured over a period of 24 h. We obtained an insulin peak 1 h after injection of regular insulin and lower levels at night. Between peaks, basal levels of free insulin were not reached. Glucose concentrations fluctuated only slightly. In 10 healthy volunteers, the absorption rate of insulin was studied after subcutaneous injection of 125I-Actrapid using a conventional technique and with a pen. During an 8-h period residual radioactivity and plasma insulin and glucose were measured. No significant differences were found, except for the insulin concentration 60 min after injection (higher after pen injection, P less than 0.05). The variability of the parameters was greater following pen injection (P less than 0.02). A basal-prandial injection regimen can thus produce stable glucose levels but only with considerable hyperinsulinaemia. Injection with an insulin pen causes a modest increase in absorption rate and plasma concentrations of insulin.

Absorption↗
PubMed1983

Glycosylation of yeast aspartyl-tRNA synthetase. Affinity labelling by glucose and glucose 6-phosphate.

Several lines of evidence establish that the crystallizable aspartyl-tRNA synthetase from Baker's yeast contains some covalently bound glucose: (i) a positive staining of the enzyme was obtained after polyacrylamide gel electrophoresis followed by the concanavalin A-peroxidase test which is specific for glucose and mannose containing proteins; (ii) thin-layer chromatography and gas-liquid chromatography revealed the presence of glucose in enzyme hydrolysates; (iii) immunoaffinoelectrophoresis in agarose gels containing concanavalin A and antibodies raised against aspartyl-tRNA synthetase showed that the enzyme was able to precipitate entirely in the lectin. Finally incubation of the enzyme with [14C]glucose or [14C]glucose 6-phosphate led to the incorporation of radioactivity into trichloroacetic acid-precipitable protein. Indeed immunoprecipitation of [14C]glucose-labelled aspartyl-tRNA synthetase with specific antibodies using the rocket method followed by autoradiography gave a radioactive peak. This last result also demonstrates the possibility of in vitro glycosylation of yeast aspartyl-tRNA synthetase.

Affinity Labels↗
PubMed1977

Preferential uptake of D-glucose by plasma membranes isolated from human adipose tissue.

A sensitive method for the measurement of the stereospecific uptake of D-glucose by plasma membranes isolated from human adipose tissue has been developed. The method is based on the difference in uptake of L-[14C]glucose and D-[3H]glucose as measured by the retention of radioactivity by the membrane preparation collected on Millipore filters. This D-glucose-uptake activity was reversible and did not involve any chemical alteration of the sugar. All uptake activity was lost upon boiling the membrane preparation for 5-10 min. All of the hydroxyl groups of D-glucose appear to be involved in a concerted fashion in the uptake reaction. The D-glucose-uptake activity was shown to be closely associated with glucose transport in adipose cells, since it exhibited the following properties characteristic of this carrier-mediated transport system. (a) The uptake was specific for the D-isomer of glucose. (b) Saturation of D-glucose uptake occurred with increasing concentrations of D-glucose, (c) The uptake activity was inhibited by N-ethylamleimide and phloretin, two reagents previously reported to inhibit D-glucose transport. We conclude that plasma membranes isolated from human adipose tissue retain the glucose transport activity of the intact cells and can be used in subsequent attempts at the isolation and characterization of this transport system.

Adipose Tissue↗
PubMed1976

Rotation induced increases of glucose uptake in rat vestibular nuclei and vestibulocerebellum.

A new technique for the autoradiographic measurement of regional cerebral glucose consumption using tracer amounts of radioactive 2-deoxyglucose (2-DG) was employed to study the effects of vestibular inputs on local cerebral glucose metabolism. Rats rotated in the vertical plane showed localized increases of glucose utilization in the vestibular nuclei and several areas of the cerebellum: flocculus, nodulus, ventral uvula, and accessory paraflocculus. The changes in the cerebellum occurred both in the neuronal perikarya-rich granular layer and the neuropil-rich molecular layer. Differential changes resulting from rotation occurred within the granular layer of the nodulus, where at least 7 separate longitudinal zones of differing glucose consumption were seen. This type of longitudinal organization has been described previously in other areas of the vermis with other techniques.

Animals↗
PubMed1979

Quantitative evaluation of the in vitro biosynthesis of gastric mucus glycoproteins. Standardization of the methodology.

For the evaluation of a normal or pathological function of gastric mucosa, a reproducible method for the estimation of the biosynthesis of mucus glycoproteins appears to be necessary. Rat gastric mucosal scrapings incorporate in vitro several labeled compounds such as 14C-U-D-glucose, 35SO4, 14C-I-L-fucose and L-G-3H-proline in glycoproteins similar to those synthetized in vivo by native mucosa. The aim of our present work is to describe in detail the procedure we use and to show its reproducibility in 9 separate experiments. Secreted glycoproteins (fraction II) and intracellular glycoproteins (fraction III) obtained during a 4 hours in vitro incubation of rat gastric mucosal scrapings at 37 degrees C in standardized conditions were separately studied. The protein and hexose contents, total incorporated radioactivity and specific radioactivity (cpm/mg protein) were determined in these two fractions. The protein content of fraction II from 2 rat gastric mucosal scrapings incubated in 5 ml was 2 mg +/- 0.4 SEM and its hexose was 38.7 mg per 100 mg protein +/- 3.9. Fraction III contained 6.3 mg protein +/- 1.3 and 15.7 mg hexose per 100 mg protein +/- 3.5. About 27% of the total radioactivity incorporated (from 14C-glucose) was in fraction II (+/- 2.6 SEM) and 73% (+/- 2.6 SEM) in the cell bound fraction III. The distribution of total radioactivity was quite similar to that of total proteins : about 25% proteins were in fraction II and 75% in fraction III with a SEM of about 5 to 10% of the average value. If the biosynthetic activity is expressed as specific radioactivity, i.e. the ratio of incorporated radioactivity to mg proteins, the average for both fraction II and fraction III was about 10,000 cpm/mg protein and the standard error of the mean values are +/- 5.2% of the mean for in vitro secreted mucus glycoproteins (fraction II) and 9.1% in the case of intracellular glycoproteins (fraction III). These values can be considered as a satisfactory index of reproducibility of the method of mucosal scrapings if performed as described above.

Animals↗
PubMed2002

Immobilized-biomembrane affinity chromatography for binding studies of membrane proteins.

Analyses of specific interactions between solutes and a membrane protein can serve to characterize the protein. Frontal affinity chromatography of an interactant on a column containing the membrane protein immobilized in a lipid environment is a simple and robust approach for series of experiments with particular protein molecules. Regression analysis of the retention volumes at a series of interactant concentrations shows the affinity of the protein for the interactant and the amount of active binding sites. The higher the affinity, the fewer sites are required to give sufficient retention. Competition experiments provide the affinities of even weakly binding solutes and the non-specific retention of the primary interactant. Hummel and Dreyer size-exclusion chromatography allows complementary analyses of non-immobilized membrane materials. Analyses of the human facilitative glucose transporter GLUT1 by use of the inhibitor cytochalasin B (radioactively labeled) and the competitive substrate D-glucose (non-labeled) showed that GLUT1 interconverted between two states, exhibiting one or two cytochalasin B-binding sites per two GLUTI monomers, dependent on the membrane composition and environment. Similar analyses of a nucleoside transporter, a photosynthetic reaction center, nicotinic acetylcholine receptors and a P-glycoprotein, alternative techniques, and immobilized-liposome chromatographic approaches are presented briefly.

Chromatography, Affinity↗
PubMed1987

Biochemical events during stapling of the proximal tibial epiphyseal plate in pigs.

Epiphyseal stapling is considered a safe, effective method of correcting angular deformities of the knee in growing children. A study of the underlying biochemical events would contribute to a better understanding of the control of longitudinal growth rate in epiphyseal growth plates and of the molecular pathology of exaggerated physiologic bowleg or knock-knee deformities. Blount stainless staples were applied to the medial side of the proximal tibia epiphyseal growth plate of domestic pigs. The resulting biochemical changes were investigated by incubation of the isolated growth plate cartilage under organ culture conditions. On the fourth to the eighth day after stapling, a significant decrease of [14C]glucose uptake and lactate formation and a significantly lower specific radioactivity of the isolated glycosaminoglycans were observed in relation to the controls. During the second to 12th week, the incorporation of [3H]thymidine and the specific radioactivity of the glycosaminoglycans following incubation with [14C]glucose showed statistically significant decreases. The glycosaminoglycan content of the cartilage was not altered, but the chondroitin sulfate/keratan sulfate ratio was lower in the stapled than in the nonstapled areas. In this period, glucose uptake and lactate production were found to be normalized.

Animals↗
PubMed1992

Distribution of radioactivity of 14C-amino acids added to the medium in cells and metabolites in cultures of rumen fungi.

A mixture of L-(U-14C) amino acids was added to cultures of 11 strains of rumen anaerobic fungi belonging to Neocallimastix frontalis, Neocallimastix joyonii, Sphaeromonas communis and Piromonas communis. Fungi were grown in a complex medium with glucose for 4 days. The radioactivity was found in cellular protein (27.7-65.3% of the total radioactivity recovered), lactate (16.9-41.8%), volatile fatty acids (7.4-25.7%) and ethanol (4.6-10.5%). A small amount of radioactivity was recovered in lipids (0.2-1.8%) and CO2 (0.3-1.0%). The results suggest that the assimilation of amino acids by growing fungal cells was quantitatively comparable with their dissimilation to metabolites.

Amino Acids↗
PubMed2004

In vitro glycation of human immunoglobulin G.

BACKGROUND: Glucose can covalently bind to the proteins by nonenzymatic process often termed glycation. Glycation of IgG is of special interest due to its possible influence on the functionality of immunoglobulins and overall immunocompetence. METHODS: The glycation of IgG was studied using radioactive D-[U-14C]-glucose. RESULTS: The kinetics, concentration/temperature dependence and distribution of glycation binding sites of human IgG were studied under various conditions. (a) In average, 0.7 glucose molecules were found to be bound per IgG after 90 days of incubation at 37 degrees C with normal serum IgG and glucose concentrations, and 3.1 molecules after incubation with glucose in the concentration characteristic for diabetics. (b) Incubation of the same solutions for 270 days at 4-8 degrees C resulted in binding of 0.3 and 0.8 glucose molecules per IgG, respectively. (c) After 90 days at 37 degrees C in the presence of 0.56 mol/l of glucose, IgG was glycated with averagely 47 glucose molecules per IgG and with 9 after 270 days at 4-8 degrees C. (d) At very high glucose concentration (1.67 mol/l) in concentrated IgG solution (87 g/l), the molar ratio glucose/IgG reached 41 after incubation at 60 degrees C for 10 h. CONCLUSIONS: Glycation of IgG with glucose nearly resembled the first order reaction kinetics. No preferential glycation sites were found as bound glucose was equally distributed throughout F(ab)2 and Fc fragments as well as on the light and heavy immunoglobulin chains.

Drug Stability↗
PubMed2005

Energizing cell-free protein synthesis with glucose metabolism.

In traditional cell-free protein synthesis reactions, the energy source (typically phosphoenolpyruvate (PEP) or creatine phosphate) is the most expensive substrate. However, for most biotechnology applications glucose is the preferred commercial substrate. Previous attempts to use glucose in cell-free protein synthesis reactions have been unsuccessful. We have now developed a cell-free protein synthesis reaction where PEP is replaced by either glucose or glucose-6-phosphate (G6P) as the energy source, thus allowing these reactions to compete more effectively with in vivo protein production technologies. We demonstrate high protein yields in a simple batch-format reaction through pH control and alleviation of phosphate limitation. G6P reactions can produce high protein levels ( approximately 700 microg/mL of chloramphenical acetyl transferase (CAT)) when pH is stabilized through replacement of the HEPES buffer with Bis-Tris. Protein synthesis with glucose as an energy source is also possible, and CAT yields of approximately 550 mug/mL are seen when both 10 mM phosphate is added to alleviate phosphate limitations and the Bis-Tris buffer concentration is increased to stabilize pH. By following radioactivity from [U-(14)C]-glucose, we find that glucose is primarily metabolized to the anaerobic products, acetate and lactate. The ability to use glucose as an energy source in cell-free reactions is important not only for inexpensive ATP generation during protein synthesis, but also as an example of how complex biological systems can be understood and exploited through cell-free biology.

Cell Culture Techniques↗
PubMed1984

Gastric emptying, glucose tolerance and associated hormonal changes in heroin addiction.

The gastric emptying rate of 8 heroin-dependent males was measured by means of a radioactive isotope method. A simultaneous oral glucose tolerance test was carried out. The glucose response showed a low, flat curve with a delayed peak, and increased secretion of insulin and growth hormone occurred. As the gastric emptying rate proved to be normal the observed metabolic abnormalities are not due to the effect of heroin on the gastro-intestinal tract, but must be attributed to some other effect of chronic heroin administration.

Adult↗
PubMed1977

Utilization of long-chain free fatty acids and glucose by human leukemic blast cells.

We have studied the utilization of free fatty acid and glucose by human leukemic blast cells. Palmitate was both incorporated into complex cellular lipids, primarily phospholipids and triglycerides, and oxidized to CO2. The predominant phospholipid synthesized was phosphatidylcholine. Only a small proportion of the incoming fatty acid was modified structurally before incorporation into lipid esters. After incubation with [1-14 C]palmitate, 91% of the radioactivity recovered in cell lipids remained in fatty acids containing 16 carbon atoms. Studies with labeled glucose revealed little de novo synthesis of fatty acid, and the majority of the radioactivity from glucose was located in the water-soluble fraction after saponification of the esters. We conclude that the free fatty acids contained in the extracellular fluid provide much of the fatty acid for required cellular lipid synthesis in human leukemic blast cells. Since there is little elongation of incoming palmitate before incorporation into cellular lipids, it may be possible to alter the fatty acid composition of membrane phospholipids by changing the proportion of the various free fatty acids available to the leukemic cells.

Carbon Dioxide↗