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Biomedical subjects

J Gliemann

Publications and source records attributed to J Gliemann.

At least 145 records · Page 8Linked to original sources
PubMed1977

Degradation, receptor binding affinity, and potency of insulin from the Atlantic hagfish (Myxine glutinosa) determined in isolated rat fat cells.

Insulin from the Atlantic hagfish, Myxine glutinosa, a primitive vertebrate, was studied with respect to degradation, receptor binding, and stimulation of glucose transport and metabolism in isolated rat adipocytes. The degradation was studied in a concentrated suspension with about 100mul of cells/ml of suspension. 125I-labeled hagfish insulin and 125I-labeled pig insulin were degraded at the same rate when present in concentrations of 0.3nM. Native hagfish insulin inhibited the rate of degradation of 125I-labeled pig insulin half-maximally at a concentration of 12+/-2 nM (S.D., n=6) as compared to 130+/-32 nM (S.D.,n=6) for pig insulin. Native hagfish insulin in a concentration of 130 nM was biologically inactivated at a rate several times slower than pig insulin in the same concentration. The results indicate that the maximal velocity (Vmax) of degradation of hagfish insulin as well as the concentration causing half-maximal velocity (Km) are about 10 times lower for hagfish insulin than for pig insulin. The receptor binding and the biological effects of hagfish insulin were studied in dilute cell suspensions where the degradation of hormone in the medium was negligible. The receptor binding affinity of hagfish insulin was 23+/-7 per cent (S.D., n=10) of that of pig insulin. Hagfish insulin was able to elicit the same maximal stimulation of both 3-o-methylglucose exchange and lipogenesis from glucose as pig insulin. However, the potency of hagfish insulin with respect to activation of lipogenesis was only 4.6+/-0.6 per cent (S.D., n=15) of that of pig insulin. Hagfish insulin thus constitutes the first described insulin which exhibits a discrepancy between relative binding affinity and relative potency. This discrepancy was not due to the methionine residue (B31) at the COOH-terminal end of the B chain of hagfish insulin, since removal of this residue caused no marked change in the binding affinity or the potency. The results indicate that the receptor occupancy must be 5 times higher with hagfish insulin than with pig insulin to cause a particular degree of activation of lipogenesis. Hagfish insulin might therefore be characterized as a "partial antagonist" on the receptors. However, it was not possible to demonstrate antagonistic properties of hagfish insulin on the cells. The effect of hagfish insulin plus pig insulin in submaximally stimulating concentrations was additive. Furthermore, the decay of activation of adipocytes after incubation with hagfish insulin followed the same time course as the decay of activation after incubation with pig insulin in a concentration of equal potency. These phenomena are in agreement with the concept that adipocytes possess a large excess of receptors which can mediate the effect of insulin on lipogenesis from glucose.

Adipose Tissue↗
PubMed1977

Receptor binding and biological effect of insulin in human adipocytes.

The binding of 125I-labelled insulin to human adipocytes was studied at 37 degrees C. The precipitability of the 125I-labelled insulin preparation (0.03 nmol/l) in trichloroacetic acid and the concentration of biologically active insulin (7.5 nmol/l) remained constant in buffer incubated with human adipocytes (100 microliter cells/ml suspension) for 30--60 minutes at 37 degrees C, whereas more than half of the insulin was inactivated by rat fat cells under the same conditions. A constant level of binding of 125I-labelled insulin (0.03 nmol/l) to human adipocytes was obtained after 45 minutes. The apparent dissociation constant of receptor binding was about 0.2 nmol/l as compared to about 2 nmol/l for rat adipocytes. Conversion of [UP14C]glucose to lipids was stimulated half-maximally by about 0.05 nmol/l of insulin (similar to rat adipocytes). Thus, half-maximal stimulation of human adipocytes was obtained with a recptor occupancy of about 20--30 per cent.

Adipose Tissue↗
PubMed1977

Degradation, receptor binding affinity and biological potency of monoiodoinsulin in isolated rat fat cells.

The degradation, binding affinity and biological potency of monoiodoinsulin was studied in isolated rat fat cells. The rate of inactivation by a concentrated cell suspension was indistinguishable from that of native insulin, whereas the relative biological potency (increase in lipid synthesis from glucose) and binding affinity (inhibition of receptor binding of 125 I-labelled insulin) was 60-80%. Assuming that the insulin receptor binding is a simple, reversible, bimolecular reaction, the following are the consequences for the interpretation of experiments at equilibrium in which an unlabelled species of insulin competes with (125I) monoiodoinsulin present in a concentration much below the dissociation constant for the insulin: 1. The dissociation constant is estimated without bias. 2. The total number of receptors is slightly underestimated.

Adipose Tissue↗
PubMed1976

The content of water and potassium in fat cells.

The distribution spaces at equilibrium for 3H2O, [14C]urea and s-O-[14C]-methylglucose were measured in white fat cells using centrifugation through silicone oil at 2500 X g; no significant differences were observed. L-[14C]Glucose added immediately before the centrifugation was used as a marker for the extracellular water space. The calculated intracellular water content of the cells after the centrifugation through oil (e.g. 3H2O space minus L-[14C]glucose space) is an unbiased measure of the water content of the fat cells in suspension as judged by the following criteria: (1) The intracellular distribution space for 3-O-[14C]'methylglucose at equilibrium (methylglucose space minus L-glucose space) was not different from that calculated from a methylglucose wash-out curve. (2) The intracellular content of L-[14C]glucose (half time of efflux about 60 min) in cells preloaded during incubation of the tissue with collagenase was not different in cells recovered by (a) centrifugation through oil at 2500 X g, (b) centrifugation through oil at 600 X g, (c) centrifugation at 600 X g in the absence of oil and (d) filtration on Millipore filters. The intracellular content of water determined on cells from single rats weighing 120-150 g was 2.75 +/- 0.55 mul/100 mul fat cells (+/- S.D., n = 30). The intracellular content of potassium, determined on cells from the same rats, was 252 +/- 62 nmols/100 mul fat cells (+/- S.D., n = 30). The concentration of potassium in the intracellular water was calculated as 104 +/- 15 mM (+/- S.D., n = 30).

Animals↗
PubMed1976

Exchange of 3-O-methylglucose in isolated fat cells. Concentration dependence and effect of insulin.

3-O-[14C]Methylglucose was used to study the insulin action on the sugar transport in white fat cells. The experiments comprised determinations of the 3-O-methylglucose space at stationary distribution, of the rate constants for 3-O-methylglucose equilibrium exchange under various conditions, and of the 3-O-methylglucose inhibition of the lipogenesis from glucose. The following was found. The intracellular distribution space for 3-O-methylglucose at equilibrium was unaffected by insulin and was identical with the intracellular 3H2O space. The half-time for the equilibrium exchange of 3-O-methylglucose at a concentration of 25 mM was about 240 s in the absence of insulin and about 15 s with insulin (0.7 muM) present. Addition of phloridzin (5 mM) decreased the rate of the exchange process about 25-fold in both cases. The self-exchange of 3-O-methylglucose (1 mM) was at least 50 times faster than the self-exchange of L-glucose (1 mM). The concentration dependence of the 3-O-methylglucose exchange rate was approximately hyperbolic both in the absence and the presence of insulin, although the saturation of the transport mechanism at high concentrations of sugar was not as complete as predicted. In the absence of insulin the estimate of the half-saturation constant (Kt) was about 5 mM; that of the maximal exchange rate (Vmax) varied from 0.07 mmol s-1/liter of intracellular water to 0.2 mmol s-1 liter-1. In the presence of insulin Kt remained about 5 mM, whereas Vmax was increased to about 1.7 mmol s-1 liter-1. The latter estimate was reproducible within about 20%. The incorporation of trace amounts of [U-14C]glucose into intracellular lipids was inhibited by unlabeled 3-O-methylglucose pre-equilibrated over the membrane. The inhibition constant estimated from such experiments was about 5 mM both in the absence and the presence of insulin, and the insulin-induced increase in the rate of glucose incorporation was similar to the increase in the rate of the 3-O-methylglucose exchange process. It is concluded that exchange of 3-O-methylglucose proceeds via a mechanism which shows stereospecificity and saturability and that insulin acts by increasing the maximal transport capacity without changing the half-saturation constant.

Adipose Tissue↗
PubMed1976

Receptor-binding region of insulin.

X-ray analysis, circular dichroism, receptor binding and biological potencies of chemically modified insulins suggest that the conformation of the insulin molecule is critical to the formation of both the zinc insulin hexamer and the insulin-receptor complex. Results are consistent with an insulin receptor-binding region including many of the hydrophobic residues important to dimerisation in addition to more polar surface residues. There is a further possibility of formation of an antiparallel sheet structure between the insulin and receptor molecules in the complex similar to that between monomers in the insulin dimer.

Adipose Tissue↗
PubMed1976

The excretion of insulin in urine.

The concentration of biologically active insulin was measured by the isolated fat cell method in serum and urine from healthy subjects and compared with the concentration of immunoreactive insulin. In both urine and serum the values obtained by the two methods correlated closely; In addition, there was a close correlation between the concentration of biologically active and immunoreactive insulin in urine from maturity onset diabetics. Therefore, conclusions on the excretion in the urine and the urinary clearance of insulin, which are based on measurements of immunoreactive insulin, are also valid for biologically active insulin.

Adipose Tissue↗
PubMed1975

Time course of insulin-receptor binding and insulin-induced lipogenesis in isolated rat fat cells.

1. Isolated rat fat cells were incubated at 37 degrees with [U-14C]-glucose 0.55 mM and 125I-labeled insulin. The amount of receptor-bound 125I-labeled insulin and the rate of insulin-induced 14C-lipid synthesis were assessed during association and dissociation of 125I-labeled insulin. 2. The rate of 14C-lipid synthesis was constant from zero time in the absence of insulin and in the presence of insulin in a high concentration (0.7 muM). With insulin in a low concentration (56 pM) the insulin-induced rate of 14C-lipid synthesis was proportional to the receptor occupancy; the receptor binding reached equilibrium and the rate of 14C-lipid synthesis reached a constant value after 30 to 45 min. With insulin in a concentration of 0.7 nM the rate of 14C-lipid synthesis reached a steady state before equilibrium of the receptor binding was obtained. 3. Ater preincubation with 56 pM 125I-labeled insulin followed by removal of extracellular insulin the decrease in the rate of insulin induced 14C-lipid synthesis followed the decrease in receptor occupancy with a half-time of about 10 min. After preincubation with insulin in concentrations of 0.28, 0.56, and 1.4 nM a maximum rate of 14C-lipid synthesis was maintained for about 8, 15, and 30 min, respectively. 4. The following model is suggested. Binding of insulin to the previously described receptors with a dissociation constant of about 3 nM (Gammeltoft, S., and Gliemann, J. (1973) Biochim. Biophys Acta 320, 16-32) represents the first step in the action of insulin on lipid synthesis from glucose. The receptor occupancy is rate-determining at low concentrations of insulin, i.e. when the occupancy is small (about 2 percent or less). At higher insulin concentrations some other step becomes rate-determining and the higher occupancy at equilibrium therefore causes no further increase in the steady state lipogenesis. However, a high receptor occupancy causes a prolonged maintenance of a maximal (or near-maximal) effect after removal of insulin from the medium.

Adipose Tissue↗
PubMed1975

Relationships between the exchange of calcium and phosphate in isolated fat-cells.

The uptake and the washout of 45Ca2+ and 32Pi is described in free fat-cells and whole epididymal fat-pads from fed rats. 2. In isolated fat-cells, the uptake of 45Ca2+ proceeds with an initial rapid phase of about 1 min duration, followed by a slower subsequent accumulation. In contrast with the rapid phase, the slow phase is inhibited by 2,4-dinitrophenol, warfarin, oligomycin and verapamil, shows saturation, and presumably represents transport across the plasma membrane. 3. The washout of 45Ca2+ from preloaded cells consists of a rapid (1 min) initial phase and a slow phase which is non-monoexponential, suggesting that the radioactive isotope is released from several cellular pools. 4. When Pi is omitted from the incubation medium, the slow phase of 45Ca uptake is almost abolished, and the washout of 45Ca from preloaded fat-cells is markedly accelerated. At elevated extracellular concentrations of Pi (2,4-6.2mM), the uptake of 45Ca is stimulated by 2-10-fold, and the release of the radioactive isotope from preloaded cells is inhibited. In whole epididymal fat-pads, variations in the extracellular concentration of Pi have no detectable effect on the uptake or the washout of 45Ca. 5. In isolated fat-cells, the accumulation of 32Pi is inhibited by 2,4-dinitrophenol or the omission of glucose from the incubation medium. In a Ca2+-depleted buffer, the uptake of 32Pi is diminished, and hyperosmolarity, which stimulates 45Ca uptake, also accelerates the accumulation of 32Pi. 6. It is concluded that in free fat-cells, the uptake and release of Ca2+ and Pi take place by closely interrelated processes, which are dependent on mitochondrial energy production.

Adipose Tissue↗
PubMed1974

Lipogenesis and insulin sensitivity of single fat cells.

1. A method for measuring the lipogenesis from [(14)C]glucose by single fat cells is described: (i) after incubation with ;carrier-free' [U-(14)C]glucose (0.55 mu-mole/ml.), collagenase-isolated fat cells were fixed with osmium tetroxide; (ii) similarly incubated pieces of epididymal fat pads were treated with osmium tetroxide for 90 sec, whereby only the superficial cells are fixed, and the tissue was then disintegrated by shaking with collagenase. The osmium-fixed free cells were washed, sucked into a micropipette, measured under a microscope and assayed individually for (14)C-activity.2. There was a quantitative recovery of (14)C-lipid activity from osmium-fixed single cells.3. Both collagenase-isolated cells and in situ fixed surface cells were normally distributed with respect to diameters (for both cell groups from ad lib. fed rats of ca. 110 g; mean diameter, about 55 mum; S.D. about 7 mum).4. Frequency distribution curves (number of fat cells versus (14)C-lipogenesis per cell) were asymmetric and very broad (coefficient of variation about 50%) for collagenase-isolated cells incubated with insulin (10(3) mu-u./ml.). Frequency distribution curves for surface cells obtained from similarly incubated pieces of epididymal fat pads showed a coefficient of variation of the same magnitude, whereas the mean lipogenesis of these cells was only about one third of that of the isolated cells.5. Collagenase-isolated cells incubated in the presence of insulin (10(3) mu-u./ml.) showed a weak but highly significant positive correlation between fat cell diameter and (14)C-lipogenesis (eight rats, r about 0.5 and P < 0.001 for each rat). Analysis of the relationship: lipogenesis = k x diameter to the exponent of beta showed that the estimates of beta varied significantly from rat to rat (range: 1.3-2.9). Similar correlations between cell size and lipogenesis were found both for cells incubated with insulin in various submaximal concentrations and for cells incubated without insulin.6. Small and large cells from the same rat were equally sensitive to insulin.7. Statistical analysis of frequency distribution curves (number of cells versus (14)C-lipogenesis per unit surface area) representing cells from the same rat incubated with insulin 0, 2.5, 5, 10, and 10(3) mu-u./ml., respectively, suggests that insulin exerts a graded influence on the lipogenesis of each fat cell.

Adipose Tissue↗