Search PubMed⌕ Search

Biomedical subjects

J Gliemann

Publications and source records attributed to J Gliemann.

At least 109 records · Page 6Linked to original sources
PubMed1985

The effect of catecholamines and adenosine deaminase on the glucose transport system in rat adipocytes.

2-Deoxyglucose uptake (3 min) and 3-O-methylglucose transport (2 s) was measured in rat adipocytes preincubated with 5 microM epinephrine plus adenosine deaminase as described by Green (Green, A. (1983) FEBS Lett. 152, 261-264). 2-Deoxyglucose uptake was about 95% depressed in insulin-treated, but not in 'basal', cells preincubated with epinephrine plus adenosine deaminase for 60 min in broad agreement with Green's report. However, this depression was caused by a decrease in sugar phosphorylation rather than transport. In similarly incubated cells, transport of 3-O-methylglucose, a sugar analogue not phosphorylated in the adipocytes, was not affected by catecholamine plus adenosine deaminase. However, a decrease in transport of about 60% was observed both in the absence and the presence of insulin when the albumin concentration was high enough and the cell concentration low enough to prevent accumulation of free fatty acids in the medium. In addition, the insulin sensitivity with regard to hexose transport was markedly reduced. Transport was approximately doubled in cells incubated with 5 microM epinephrine in the absence of adenosine deaminase. Thus, epinephrine at a high concentration stimulates hexose transport in the absence of adenosine deaminase (presence of adenosine) whereas it inhibits both basal and insulin-stimulated transport in the presence of adenosine deaminase (absence of adenosine).

3-O-Methylglucose↗
PubMed1985

Uptake and degradation of insulin and alpha 2-macroglobulin-trypsin complex in rat adipocytes. Evidence for different pathways.

The cell association and degradation of insulin and alpha 2-macroglobulin-trypsin complex were measured in rat adipocytes with or without various inhibitors in the attempt to clarify whether the two ligands were taken up by the same or by different pathways. Several inhibitors, and particularly those of membrane traffic, lysosomal function and transglutaminase activity, affected the two ligands differently. Thus, chloroquine (100 microM) reduced both the uptake of alpha 2-macroglobulin X trypsin and its receptor-mediated degradation by about 70%. In contrast, the uptake of insulin was increased 2-3-times and the receptor-mediated degradation was only slightly reduced. Methylamine (10 mM) and ammonium chloride (10 mM) reduced degradation of alpha 2-macroglobulin X trypsin markedly without affecting that of insulin. Leupeptin (100 microM) increased uptake and reduced degradation of alpha 2-macroglobulin X trypsin without affecting insulin. Dansylcadaverine (500 microM) almost abolished uptake and degradation of alpha 2-macroglobulin X trypsin but had little effect on insulin. Moreover, uptake and degradation of alpha 2-macroglobulin X trypsin was much more sensitive than insulin to the action of metabolic inhibitors such as dinitrophenol and cyanide. The results show that the two ligands are taken up by functionally different systems. In addition, they support the hypothesis that lysosomes play a relatively minor role in the receptor-mediated degradation of insulin.

Adipose Tissue↗
PubMed1985

A kinetic analysis of hexose transport in cultured human lymphocytes (IM-9).

3-O-Methyl-D-glucose transport across the plasma membrane of cultured human lymphocytes of the IM-9 line was followed for net entry into sugar-free cells (zero trans entry), net exit into sugar-free medium (zero trans exit) and for equilibration of labelled sugar in cells with the same sugar concentration in the intracellular water as in the medium (equilibrium exchange). The measurements were performed at 37 degrees C (pH 7.4). Equilibrium exchange of 1 mM 3-O-methylglucose (t 1/2 about 7 S) was exponential, suggesting a homogeneous cell suspension. Initial rates of transport showed a Michaelis-Menten dependency on the sugar concentration. The transport system was found to be asymmetric with the following kinetic parameters. Zero trans entry: Km = 2.8 mM, Vmax = 10.7 mM X min-1. Zero trans exit: Km = 9.5 mM, Vmax = 37.9 mM X min-1. Equilibrium exchange: Km = 9.9 mM, Vmax = 44.0 mM X min-1. Finally, the affinity constant for the internal site was measured as approx. 1.2 mM using the infinite cis protocol.

3-O-Methylglucose↗
PubMed1984

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

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

Adipose Tissue↗
PubMed1983

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver.

The cleavage of insulin by the partially purified insulin protease was studied using the four [125I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 greater than A-14 greater than A-19 greater than B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the 'insulin' peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the 'insulin' peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).

Animals↗
PubMed1983

Cell association and degradation of alpha 2-macroglobulin-trypsin complexes in hepatocytes and adipocytes.

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.

Adipose Tissue↗
PubMed1983

The mechanism of receptor-mediated degradation of insulin in isolated rat adipocytes: indirect evidence for a non-lysosomal pathway.

Receptor-bound insulin is substrate for a degradation leading to the release of about half the cell-associated [125I]monoiodoinsulin as [125I]monoiodotyrosine. Classical lysosomal inhibitors of the amine type (cloroquine, methylamine and NH+4) only partly inhibited this receptor-mediated degradation. Leupeptin, which is very effective in other systems, was without any effect in the present system. The degradation could not be reduced by lowering the ATP content of the cells. Sulphydryl reagents strongly inhibited the degradation as has also been shown for the cytosolic insulin-specific protease. Microtubules and microfilaments are probably not involved since inhibitors of the cytoskeleton were without marked effects. It is suggested that in the rat adipocyte only a minor part of the receptor-mediated degradation of insulin takes place via the classical endocytotic lysosomal pathway.

Acyltransferases↗
PubMed1983

Monoiodoinsulin labelled in tyrosine residue 16 or 26 of the B-chain or 19 of the A-chain. II. Characterization of the kinetic binding constants and determination of the biological potency.

The binding affinity to insulin receptors in isolated rat adipocytes at 37 degrees C of the four isomers of [125I]monoiodoinsulin was ranked as B26 greater than B16 = A14 greater than A19. It was demonstrated that the difference in affinity was mainly due to a change in the association rate constant, rather than in the dissociation rate constant. At steady state in the binding process the fraction of cell-associated 125I-activity eluting from a Sephadex G-50 Fine column at a position identical to that of iodoinsulin was greater than 90% and independent of the position of the iodine. It was also shown that the formation of [125I]-monoiodotyrosine as a consequence of receptor-mediated degradation was proportional to the respective binding affinities of the four isomers. The two isomers with binding affinities different from that of [A14-Tyr-125I]monoiodoinsulin (i.e. the B26 and the A19 isomers, respectively) were shown to have biological potencies which corresponded within +/- 8% to the observed changed binding affinities. In cultured human lymphocytes of the IM-9 line the hierarchy of binding affinities at 37 degrees C was B26 greater than B16 greater than A14 greater than A19, and in cultured human colon adenocarcinoma cells of the HT-29 line the binding affinities were ranked in the order B26 greater than B16 greater than A14 greater than or equal to A19 indicating that the functional properties of the insulin receptor vary within cell types and/or species.

Adipose Tissue↗
PubMed1983

(N-methylpyridinium)insulins. Modification at the A19- and B16-tyrosines.

Reaction of 2-iodo-N-methylpyridinium iodide with insulin gave, amongst others, A19- and B16-(N-methylpyridinium)insulin derivatives in which the phenolic groups of the respective tyrosines had been modified. Under carefully controlled conditions the A19 derivative gave small rhombohedral crystals, whereas the B16 derivative failed to crystallize. Both these derivatives had low biological activities.

Amino Acids↗
PubMed1982

[125I]diiodoinsulins. Binding affinities, biologic potencies, and properties of their decay products.

UNLABELLED: Insulin was iodinated with 0.3-0.4 mol 125I/mol insulin using the lactoperoxidase method. About one-third of the radioactivity incorporated into insulin was in diiodoinsulins and about 40% of these molecules contained diiodotyrosine in residue 14 of the A chain. Most of the remaining molecules contained one A14-monoiodotyrosine and one monoiodotyrosine in either position A19, B16, or B26. The binding affinity and biologic potency of this heterogeneous diiodoinsulin preparation was not significantly different from that of A14-[125I]monoiodoinsulin in rat adipocytes, whereas it was slightly reduced in hepatocytes and IM-9 lymphocytes. From the iodine distribution and previous data on the binding affinity of each of the four monoiodoinsulin isomers it was calculated that A14-diiodotyrosine-insulin possesses full binding affinity and biologic potency in adipocytes. Diiodoinsulins isolated from another iodoinsulin preparation (iodate method) contained 58% A19-diiodotyrosine-insulin, and most remaining molecules contained one A19-monoiodotyrosine. The binding affinity of this mixed diiodoinsulin preparation was approximately one-fourth of that of A14-monoiodoinsulin in adipocytes, IM-9 lymphocytes, and hepatocytes. It was calculated that A19-diiodotyrosine-insulin is nearly devoid of binding affinity. The diiodoinsulins (lactoperoxidase method) decayed to iodide (probably from diiodotyrosine-insulin) or to polymers with little specific but a markedly increased nonspecific binding. In addition, the polymers had a marked tendency to adsorb to cellulose acetate filters. CONCLUSIONS: 1. The binding affinities of diiodoinsulins range from very low values to values at least as high as that of insulin depending on the positions of the iodine moieties. 2. The relative binding affinities vary among tissues. 3. Polymeric decay products give high nonspecific binding.

Electrophoresis, Polyacrylamide Gel↗
PubMed1981

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes.

Rat adipocytes were incubated at 37 degrees C with 2-deoxy-D-[1-14C]glucose ([14C]2dGlc) at various concentrations and the intracellular concentrations of [14C]2dGlc and deoxy[14C]glucose phosphate ([14C]2dGlcP) were measured. Using 7 microM extracellular [14C]2dGlc, the intracellular [14C]2dGlc concentration approached the extra-cellular by 5 min insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of 2dGlcP. The rate of accumulation of total radioactivity ([14C]2dGlc plus [14C]2dGlcP) decreased after 20 min using 7 microM extracellular [14C]2dGlc. This change occurred later at 22 degrees C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [14C]2dGlc, but radio-activity disappearing from the cells was largely [14C]2dGlcP. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 microM [14C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM 3-O-methylglucose, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of 2dGlcP and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.

Adipose Tissue↗
PubMed1981

Effect of pH on binding kinetics and biological effect of insulin in rat adipocytes.

The pH dependence of binding of [TyrA14-125I]monoiodoinsulin to isolated rat adipocytes was investigated at 37 degrees C. Although the amount of tracer bound at steady state at pH 7.8 was 6 times that at pH 6.8, the fractional degradation of receptor-bound iodoinsulin remained the same. The decreased binding at acidic pH could be accounted for by a decrease in the affinity. The pH-induced change in affinity was similar for iodoinsulin and insulin. The half-time of dissociation was not changed in the pH range 7.0 to 7.8. The presence of 25 nM insulin in the wash out medium caused a small increase in the dissociation of tracer independent of pH. Kinetic experiments revealed that the decreased affinity at acidic pH was mainly due to a decrease in the association rate constant. The decreased binding at acidic pH was reflected in a shift to the right of the insulin dose-response curve for initial rate of uptake of 3-O-[methyl-14C]glucose and the rate of conversion of glucose to lipids. It is concluded that the degradation of bound iodoinsulin is independent of pH (6.8 to 8.8), that the decreased binding at acidic pH is due to a decreased association rate constant, and that cells in acidic milieu are relatively insulin-resistant.

Adipose Tissue↗
PubMed1981

Termination of insulin-induced hexose transport in adipocytes.

The hexose transport of insulin-pretreated (80 pM) adipocytes remained elevated for at least 45 min when the cells were depleted of ATP by treatment with dinitrophenol. On the other hand, the half-time of deactivation of hexose transport in insulin-pretreated cells was of the same magnitude as that of dissociation of receptor-bound insulin both in the absence and presence of glucose (about 8 min). Thus, a high ATP-level, but not ongoing glucose metabolism appears to be important for termination of the insulin effect shortly after dissociation of insulin from its receptor.

Adenosine Triphosphate↗
PubMed1981

Hexose transport in human adipocytes: factors influencing the response to insulin and kinetics of methylglucose and glucose transport.

Optimal experimental conditions were defined for measuring the initial uptake rate of the non-metabolizable sugar analogue 3-O-methylglucose in non-stimulated and insulin-stimulated human adipocytes. The permeability of the adipocyte plasma membrane for tracer methylglucose (100 mumol/l) was 2.9 X 10(-7) cm X s-1 at 37 degrees C and slightly lower at 20 degrees C. At 37 degrees C and pH 7.4 insulin (5 nmol/l) increased the permeability about twofold (range 1.5 to fivefold) with half maximal effect at about 100 pmol/l). At pH 7.0 the dose response curve for the insulin effect on the uptake rate of methylglucose was shifted about 2.5-fold to the right. The permeability to L-glucose due to simple diffusion was estimated as 3.0 X 10(-10) cm X s-1 suggesting that uptake of methylglucose occurs almost exclusively by facilitated diffusion. The Km for methylglucose equilibrium exchange in insulin stimulated cells was about 4.8 mmol/l. The initial uptake of tracer methylglucose in insulin-stimulated cells was inhibited by unlabelled methylglucose and by D-glucose with inhibition constants of about 3.8 and 7.7 mmol/l respectively. Uptake of tracer 2-deoxyglucose (50 mumol/l) in insulin-stimulated adipocytes was linear from 10 s to 5 min whereas the rate of uptake in the presence of 3 mmol/l of D-glucose was markedly decreased suggesting that deoxyglucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose.

Adipose Tissue↗
PubMed1981

Monoiodoinsulin labelled in tyrosine residue 16 or 26 of the insulin B-chain. Preparation and characterization of some binding properties.

By the combination of polyacrylamide gel electrophoresis and QAE-Sephadex ion exchange chromatography it was possible to isolate the four isomers of porcine [125I]monoiodoinsulin to a purity of more than 97%. The yield of the two B-chain-labelled isomers was increased by iodinating in buffer containing 6M urea. The apparent binding affinity to isolated rat adipocytes was 0.65 for the A19 isomer, 1.0 for the B16 isomer, and 2.0 for the B26 isomer, respectively, relative to the A14 isomer. In contrast, the B26 isomer had only an apparent binding affinity of 1.2 relative to the A14 isomer in isolated hepatocytes.

Adipose Tissue↗