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Biomedical subjects

J Gliemann

Publications and source records attributed to J Gliemann.

At least 127 records · Page 7Linked to original sources
PubMed1981

NA1, NA1, NA1-trimethylinsulin--an insulin analogue with a quaternary amino group at the A1 terminus.

By utilizing the differing reactivity of the amino groups in aqueous organic solvents, des-GlyA1-NB1,N epsilon B29-(Msc)2-insulin was prepared. Its reaction with the phenyl ester of N,N,N-trimethylglycine in the presence of N-hydroxysuccinimide afforded the crystalline NA1,NA1,NA1-trimethylinsulin analogue. In the fat cell assay this analogue has an activity of 49% and, in the mouse convulsion assay, it is 70%.

Adipose Tissue↗
PubMed1981

Tyrosine A14[125I]monoiodoinsulin: Preparation, Biologic Properties, and long-term stability.

125I-insulin was prepared by reacting 17.4 nmol porcine insulin (100 micrograms) with 5 mCi 125I (about 2.4 nmol) using the lactoperoxidase method. The reaction product was subjected to gel electrophoresis and the band containing A14 [125I]monoiodoinsulin was eluted. This preparation showed a specific activity of about 1.5 Ci/mumol as evaluated by radioimmunoassay and bioassay, i.e., about 75% of the theoretical maximum. The content of radioactive derivatives other than A14 monoiodoinsulin was less than 2%. The binding affinity of tracer A14 monoiodoinsulin to adipocytes, hepatocytes, and cultured human lymphocytes was twice as high as that of A19 monoiodoinsulin. Binding to antibodies was examined to 10 guinea pig anti-insulin sera. Three sera did not distinguish between the two tracers, whereas seven exhibited higher binding of the A14 tracer. A detailed analysis of one of the discriminating sera showed that the average affinity constant was about 2.5 times lower for the A19 tracer than for the A14 tracer. The A14 monoiodoinsulin tracer is remarkably stable. After 200 days the specific activity had declined to about half of its original value which is consistent with the hypothesis that the physical decay of [125I]monoiodoinsulin (T 1/2 equals 60 days) extinguishes the activity of the molecule without causing major damage of other molecules. By this time 96% of the radioactivity migrated with insulin when subjected to gel filtration on Sephadex G-50, 4% was in the void volume, and nothing in the total column volume or later. Binding to receptors was indistinguishable from that obtained at time zero. It is concluded that Tyr A14[125I]monoiodoinsulin represents an advance in biologic work as compared with previous tracers for insulin.

Adipose Tissue↗
PubMed1981

Insulin receptor binding and receptor-mediated insulin degradation in human adipocytes.

UNLABELLED: 125I-insulin binding and receptor-mediated insulin degradation were studied in isolated human fat cells from subcutaneous tissue. A high albumin concentration during cell isolation and incubation protected the fragile human adipocyte from lysis. Binding of tracer was pH dependent with an optimum between 7.4 and 7.6. At 37 degrees C steady state was reached by 45 min and maintained for at least 2 h. The binding of labelled insulin in the presence of 10 mumol/l unlabelled insulin was only 1-4% of the total insulin binding. The half-maximal displacement of tracer iodoinsulin (10 pmol/l) by unlabelled insulin occurred at 0.25 nmol/l. Kinetic studies of the dissociation of labelled iodoinsulin from fat cells showed a slight acceleration in the presence of a high concentration of unlabelled insulin in the washout buffer as compared to a buffer containing no insulin. At steady state binding about 95% of the cell-associated radioactivity was extracted as iodoinsulin as judged by gel filtration. The remaining 5% co-eluted with iodotyrosine. During 60 min about 90% of the cell-associated radioactivity dissociated as iodoinsulin and the rest as iodotyrosine. CONCLUSIONS: 1) A high albumin content of buffers prevents traumatization of the human adipocyte; 2) under these conditions steady state binding of insulin is readily measured at 37 degrees C; 3) the use of a washing procedure makes the non-specific binding negligible; 4) the human adipocyte insulin receptor has a very high affinity; 5) receptor-mediated insulin degradation is minimal.

Adipose Tissue↗
PubMed1981

Glucose transport in isolated rat adipose cells.

Several different approaches to the measurement of hexose transport rates in isolated adipocytes from lean and obese rats have been utilized in the last six years. The uptake as a function of time of the non-metabolizable sugar 3-0-methylglucose is so fast that until recently no measurements of the 'true' transport rates (ie initial velocity of influx) could be made. Other nonmetabolizable sugars such as L-arabinose are taken up more slowly, due to their low affinities and their influxes at early times are measureable. Alternatively, the uptake with time of 2-deoxyglucose, an analogue of glucose which is transported and phosphorylated, but not further metabolized, has been used extensively as a measure of transport rate. The data from different studies comparing the transport rates in adipocytes from lean and obese rats are at variance. The present discussion is an attempt to explain why the results of such studies have not always agreed. In addition, as a result of our recent studies comparing 2-deoxyglucose and 3-0-methylglucose transport, we introduce a new model of hexose transport in adipocytes.

3-O-Methylglucose↗
PubMed1980

Glucose-induced acceleration of deoxyglucose transport in rat adipocytes. Evidence for a second barrier to sugar entry.

The transport of trace (a concentration much less than Km for transport) 2-deoxyglucose (deoxyglucose) and 3-O-methylglucose (methylglucose) into rat adipocytes was measured using 1- to 2-s pulses at 37 degrees C in the absence of glucose and in the presence of glucose with and without pre-exposure to glucose. Pre-exposure of insulin-stimulated adipocytes to 10 mM glucose for 30 min resulted in an acceleration of trace deoxyglucose transport above that seen in the absence of glucose. In contrast, when glucose was added together with deoxyglucose, an inhibition of transport was observed. There was no effect up to 5-min pre-exposure to 10 mM glucose, but acceleration appeared thereafter and reached a steady state by 30 min, i.e. a 3-fold higher transport rate than that seen when deoxyglucose and glucose were added together. Glucose (3.5 mM) appears to be a threshold concentration for this effect. A smaller effect was seen in the absence of insulin (1.5-fold) using 10 mM glucose and in the presence of insulin using 10 mM mannose (2.7-fold). No effect was seen using 10 mM galactose, fructose, pyruvate-lactate, deoxyglucose, and methylglucose. There was no effect of glucose pre-exposure on insulin-stimulated methylglucose transport. We propose that entry occurs across two independent barriers in series separated by an aqueous pore, that transport across the second barrier is more rate limiting for deoxyglucose than for methylglucose, and than metabolites of glucose decrease the resistance of the second barrier to deoxyglucose transport.

Adipose Tissue↗
PubMed1980

Insulin receptors of cultured human lymphocytes (IM-9). Lack of receptor-mediated degradation.

The binding of insulin to cultured human lymphocytes of the IM-9 line was investigated at near-physiological conditions, i.e. 37 degrees C, pH 7.4, using a monoiodinated insulin exclusively labeled in tyrosyl residue 14 of the A chain. Although the apparent dissociation constant did not differ much from that found in rat adipocytes, the rate constants were about 1 order of magnitude greater. The rate of dissociation of 60 pM of labeled insulin was accelerated more in the lymphocytes than in adipocytes by the addition of 50 nM insulin to the wash-out medium. In rat adipocytes insulin is degraded both by receptor-independent and receptor-mediated processes (Gliemann, J., and Sonne, O. (1978) J. Biol. Chem. 253, 7857-7863). Lymphocytes possessed a receptor-independent insulin-degrading system with the same properties as that of adipocytes. However, receptor-mediated degradation was absent and insulin binding is therefore a true reversible process. The following is concluded: 1) The binding sites of the receptors in IM-9 lymphocytes are different from those of the insulin-responsive adipocytes and hepatocytes. 2) The lymphocytes lack the enzyme responsible for the receptor-mediated degradation or a coupling factor between the receptor and the enzyme.

Adipose Tissue↗
PubMed1980

Insulin binding and hexose transport in rat adipocytes. Relation to cell size.

Insulin binding, initial velocity of [14C]methylglucose transport, uptake of [14C]deoxyglucose and conversion of [U-14C]glucose to CO2, glyceride-glycerol and fatty acids were measured at 37 degrees C in adipocytes from rats of different weights (135-450 g) and therefore with different mean cell volumes (53-389 pl). Insulin binding per cell increased with increasing cell size and binding was 2.3 times higher in the largest cells than in the smallest cells with tracer alone. The difference was largely accounted for by an increase in the apparent affinity. Influx of methylglucose per cell increased with increasing cell size in the absence of insulin and remained constant as a function of cell size in its presence. The effect of insulin ranged from 11 fold in small cells to 3.5 fold in large cells. The rat of conversion of [U-14C]glucose to CO2 and lipids was about half of the rate of methylglucose transport under all conditions. In contrast, the uptake of deoxyglucose in insulin-stimulated cells decreased markedly with increasing cell size. Increasing cell size caused a small decrease in sensitivity which could be explained by a smaller amont of insulin bound per unit surface area. The results show that increasing cell size/animal weight causes changes in insulin binding which may explain changes in sensitivity. In addition, the hexose transport system is modified in a way which is not explained by changes in insulin binding. Finally, changes in deoxyglucose uptake with cell size do not parallel changes in methylglucose transport.

Adipose Tissue↗
PubMed1980

Rate-limiting steps of 2-deoxyglucose uptake in rat adipocytes.

2-Deoxy[1-14C]glucose uptake in rat adipocytes was measured as a function of time in the absence and presence of unlabelled glucose or 2-deoxyglucose. Uptake of tracer alone was linear from 2s to 6 min. At 37 degrees C the rate of uptake in insulin-stimulated cells decreased markedly after a few seconds in the preence of glucose (0.5-10 mM) and after 0.5-2 min in the presence of deoxyglucose (2-10 mM). Similar data were obtained at 22 degrees C. With 10 mM glucose (37 degrees C, 30 s) approx. 80% of the intracellular radioactivity was non-phosphorylated deoxyglucose and with 10 mM deoxyglucose approx. 40% was non-phosphorylated. The results show that deoxy[14C]glucose uptake after a few minutes is mainly limited by hexokinase in the presence of glucose and at least partially in the presence of deoxyglucose. The data suggest caution in using deoxyglucose uptake as a measure of transport, especially in complex kinetic studies. In addition, the initial velocity of tracer * 3-O-methylglucose was found to be approx. 2-fold higher than that of tracer deoxyglucose even though both sugars inhibited the initial velocity of labelled methylglucose half-maximally at a concentration of 5 mM. These data suggest a fundamental difference between deoxyglucose and methylglucose transport.

Adipose Tissue↗
PubMed1980

Hagfish insulin: the discrepancy between binding affinity and biologic activity.

Hagfish insulin exhibits a binding affinity of about 25% of that of pig insulin in rat adipocytes and IM-9 lymphocytes, even though its relative biologic potency is only about 5% in adipocytes. The dissociation rate constant of hagfish insulin is about half that of pig insulin, and the association rate constant is about one eighth. A longer time is, therefore, required for hagfish insulin to reach a steady state of binding. Failure to reach steady state is the probable reason why some previous results suggested a relative binding affinity of hagfish insulin of the same magnitude as the relative biologic potency.

Adipose Tissue↗
PubMed1979

Receptor binding studies on A1-(2-nitro-4-trimethylammoniophenyl)insulin.

The results are presented from receptor binding studies and isolated fat cell assays on A1-(2-nitro-4-trimethylammoniophenyl)insulin. In the former tests, the derivative is shown to have properties similar to native insulin and the fat cell assays afford a value of 76 +/- 4%. The importance of a charged, hydrophilic moiety attached at A1-glycine is discussed in the light of the above findings.

Adipose Tissue↗
PubMed1979

Isolation of a crystalline A14-(N-methylpyridinium) derivative of bovine insulin.

Reaction of insulin with 2-chloro- or 2-iodo-N-methylpyridinium iodide affords, amongst others, an insulin analogue modified at tha A14-tyrosine residue. Degradative procedures which pinpoint the exact position of modification are described. Highest yields of the derivative are obtained using the 2-iodo-N-methylpyridinium iodide as reagent. In the mouse convulsion assay this derivative retains 72% of its activity.

Amino Acids↗
PubMed1979

Receptor-bound insulin as a substrate for insulin degradation in adipocytes.

Isolated rat adipocytes were incubated with 125I-labeled insulin (about 100 pmoles/liter) at 37 degrees C, pH 7.4, and the label associated with the cells was measured by a modified oil-flotation method. The binding of tracer alone was at least 10 times higher than the binding in the presence of insulin (1 mumole/liter.) Under conditions with low rates of insulin degradation (4% per hour or less) the amount of label associated with the cells was constant from 40--180 min and about 80% of the label was extracted (acetic acid 3 moles/liter, urea 6 moles/liter) as iodoinsulin as judged by gel filtration followed by titration with antiinsulin antibody. The remaining 20% coeluted with iodotyrosine. At steady state about half of the label dissociated from the cells as iodoinsulin and about half as iodotyrosine, as judged by gel filtration and paper chromatography in two solvent systems. The degradation of receptor-bound insulin was independent of the receptor occupancy. "Nonspecifically" bound labeled insulin (ie, in the presence of unlabeled insulin 1 mumole/liter) was not degraded. It is concluded that receptor-bound insulin is a substrate for degradation in adipocytes and about half of the molecules are degraded at 37 degrees, pH 7.4. The only identified labeled degradation product is iodotyrosine and the pool size within the cell seems to be about 20%.

Adipose Tissue↗
PubMed1977

[Insulin receptors (author's transl)].

The saturable insulin binding is linked to insulin degradation in two important target cells: The hepatocyte and the adipocyte. One of the consequences is that the changes in binding which are observed under various conditions cannot a priori be regarded as caused by either increased number of receptors or increased affinity of the binding site. This observation raises new questions. For instance, could the effect of insulin be mediated by a fragment of the molecule? No evidence which is available for the moment seems to rule out this hypothesis. The findings with insulin analogues, the kinetics of insulin binding and activation and the effect of mild trypsin treatment, would equally well support the hypothesis that the binding itself causes activation of hexose transport and that degradation secondary to binding mediates the activation.

Adipose Tissue↗