scholarly journals Identification and characterization of insulin receptors on foetal-mouse brain-cortical cells

1984 ◽  
Vol 220 (1) ◽  
pp. 165-172 ◽  
Author(s):  
C F H Van Schravendijk ◽  
E L Hooghe-Peters ◽  
P De Meyts ◽  
D G Pipeleers
Keyword(s):  
Mouse Brain ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Types ◽  
Receptor Interaction ◽  
Target Cells ◽  
Scatchard Analysis ◽  
Cortical Cells ◽  
Foetal Brain ◽  
Binding Cell

The occurrence of insulin receptors was investigated in freshly dissociated brain-cortical cells from mouse embryos. By analogy with classical insulin-binding cell types, binding of 125I-insulin to foetal brain-cortical cells was time- and pH-dependent, only partially reversible, and competed for by unlabelled insulin and closely related peptides. Desalanine-desasparagine-insulin, pig proinsulin, hagfish insulin and turkey insulin were respectively 2%, 4%, 2% and 200% as potent as bovine insulin in inhibiting 125I-insulin binding to brain-cortical cells, which corresponds to their relative biological potencies in classical insulin-target cells; no competition was observed with glucagon and nerve growth factor, even at high concentrations. Scatchard analysis of competitive-binding data resulted in curvilinear plots with a high-affinity binding of Ka = 3.6 X 10(8) M-1. Insulin binding to foetal brain-cortical cells differed, however, in two distinct aspects from that to classical insulin-binding cell types. Firstly, dilution of 125I-insulin-bound cells in the presence of unlabelled insulin did not accelerate dissociation of the labelled hormone. Secondly, exposure of brain-cortical cells to insulin before the binding assay enhanced insulin binding, suggesting up-regulation of insulin receptors in response to insulin. In conclusion, foetal-mouse brain-cortical cells bear specific binding sites for insulin. Their insulin receptor shows a marked specificity and affinity for insulin, but differs in at least two properties from most classical insulin receptors. These differences in hormone-receptor interaction could reflect structural differences between insulin receptors on embryonic and differentiated cells.

Insulin receptors on Xenopus laevis oocytes: effects of injection of ob/ob mouse liver mRNA

1991 ◽  
Vol 100 (1) ◽  
pp. 167-171
Author(s):  
D.A. Diss ◽  
B.D. Greenstein
Keyword(s):  
Xenopus Laevis ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Types ◽  
Follicle Cells ◽  
Vitelline Membrane ◽  
Xenopus Laevis Oocytes ◽  
Endogenous Insulin ◽  
Order Of Magnitude

We describe here conditions for the detection of insulin binding sites on Xenopus laevis oocytes. The binding of 125I-labelled insulin displayed sigmoidal behaviour, which is characteristic of the binding relationship between insulin and its receptor. Resolution of the resulting curvilinear Scatchard plot into two components revealed KD values of 8.86 × 10(−10) +/− 1.9 × 10(−10) and 5.32 × 10(−9) +/− 2.4 × 10(−9) M and n values of 9.7 × 10(7) +/− 0.4 × 10(7) and 3.3 × 10(8) +/− 0.5 × 10(8) binding sites per oocyte, respectively. The possibility cannot be excluded, however, that receptors for IGF-1 were also being detected. Also described are conditions for the rapid and efficient removal of all tissues surrounding the oocyte, including the vitelline membrane. We could not detect any specific 125I-labelled insulin binding to oocytes that had their follicle cells or vitelline membrane removed and this was not due to the enzymic treatment used in the process. Microinjection of oocytes without follicular layers did not result in the appearance of any detectable insulin binding sites, which were, however, observed if oocytes were first stripped of the vitelline membrane. We suggest that oocytes may possess endogenous insulin receptors on their surface in numbers of the same order of magnitude as those present on somatic cells. The removal of tissues surrounding the oocyte should facilitate studies aimed at determining functional interactions of the various cell types during oocyte development and for studying insulin receptors on the oocyte-follicular cell complex.

Adult cardiac myocytes in primary culture: cell characteristics and insulin-receptor interaction

1985 ◽  
Vol 249 (2) ◽  
pp. H212-H221 ◽  
Author(s):  
J. Eckel ◽  
G. van Echten ◽  
H. Reinauer
Keyword(s):  
Cardiac Myocytes ◽  
Structural Integrity ◽  
Cultured Cells ◽  
Insulin Binding ◽  
Receptor Interaction ◽  
Scatchard Analysis ◽  
Apparent Dissociation Constant ◽  
Adult Cardiac Myocytes ◽  
Receptor Number

Calcium-tolerant adult cardiac myocytes were kept in culture under serum-free conditions in the presence of physiological concentrations of insulin. Up to 4 days, 70% of cells retained their in vivo rodshaped morphology without gross structural alterations. During that period a constant ATP-to-ADP ratio was observed with a mean value of 10.6 +/- 0.5 (n = 4). The rate of [14C]phenylalanine incorporation remained unaltered up to 63 h in culture. Insulin binding to cultured cells was found to be time-and temperature-dependent, reversible, and highly specific. Scatchard analysis of equilibrium binding data showed a curvilinear plot with a high-affinity segment yielding an apparent dissociation constant of 4.5 X 10(-10) mol/l and a receptor number of 125,000 sites/cell. Both affinity and receptor number remained unaltered between 18 and 66 h in culture. [14C]phenylalanine incorporation was stimulated by 108% in cardiocytes cultured in the presence of high concentrations of insulin (1.7 X 10(-7) mol/l) for 63 h, when compared with control cells cultured in the absence of insulin. These data demonstrate the retention of structural integrity, insulin receptors, and insulin responsiveness in primary cultured adult cardiac myocytes and provide a useful model for long-term studies on the regulation of insulin action on the heart.

scholarly journals Insertion of isolated insulin receptors into placental membrane vesicles

1992 ◽  
Vol 281 (2) ◽  
pp. 425-430 ◽  
Author(s):  
K Christiansen ◽  
J Carlsen
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Insulin Receptors ◽  
Membrane Vesicles ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Plasma Membrane Vesicles ◽  
Intact Cells ◽  
Receptor Number ◽  
Triton X

Purified human insulin receptors were inserted into placental plasma-membrane vesicles by fusion of membranes with receptor-lysophosphatidylcholine micelles. Scatchard analysis of insulin binding showed that about 10-15% of the added receptors became inserted into the membrane. The receptor number could be increased about 3-fold, corresponding to approx. 5 pmol of receptor/mg of membrane protein. The receptors became firmly bound to the membrane, as they could not be removed by extensive wash. The insertion of exogenous receptors could be demonstrated by immunoblotting. The inserted insulin receptor had the same insulin-binding affinity as the isolated receptor and the endogenous receptor of the membrane. Insulin binding in the presence or absence of Triton X-100 revealed that more than 80% of the exogenous receptors had a right-side-out orientation. Function of the inserted receptors, as observed by insulin-stimulated autophosphorylation, could be demonstrated. About 80% of the added lysophospholipid, corresponding to approx. 160 nmol of lysophospholipid/mg of membrane protein, became integrated into the membrane and was partly metabolized to phospholipid and to non-esterified fatty acid. The method of insertion of isolated insulin receptors using the natural detergent, lysophospholipid, may be a method for insertion of receptors into intact cells, where the lysophospholipid, as in the plasma-membrane vesicles, will be acylated to phospholipid.

INSULIN RECEPTORS ARE NOT COUPLED TO THE PHOSPHOINOSITIDE OR ADENYLCYCLASE MESSENGER SYSTEMS IN HUMAN PLATELETS

1987 ◽  
Author(s):  
G Pfliegler ◽  
J Arnout ◽  
J Kienast ◽  
K Wittevrongel ◽  
J Vermylen
Keyword(s):  
Cyclic Amp ◽  
Inositol Phosphate ◽  
Binding Capacity ◽  
Physiological Role ◽  
Insulin Receptors ◽  
Cell Types ◽  
Human Platelets ◽  
Target Cells ◽  
Diabetic Patients ◽  
Thrombotic Complications

Insulin receptors have been found not only on its “target Cells” but also on several other cell-types, including human platelets. From studies on adipocytes and liver cells it seems that they are coupled both to the adenylate cyclase-cyclic AMP and the polyphosphoinositide messenger systems. Circulating blood cells might faithfully reflect the insulin receptor state of target organ tissues. Impaired platelet function has an important role in the pathogenesis of vascular and thrombotic complications in diabetes mellitus, and insulin seems to act directly on platelets. A reduction in the number and binding capacity of platelet insulin receptors in diabetic patients (Udvardy et al. 1986) suggested a (patho)physiological role for these receptors. In our studies, insulin (1 × 10-9 - 1 × 10-6 M) did not affect basal platelet cyclic AMP levels, as measured following incorporation of [3H] adenine. Insulin did not prevent PGI2 (25-75 nmol/L) induced cyclic AMP formation in platelets. Insulin did not modify the basal levels of inositol phosphate (IP), IP2 or IP3 in platelets, as measured following incorporation of [3H] inositol. Insulin did not affect formation of IP, IP2 or IP3 by thrombin. No changes in cytosolic free Ca2+ (Quin 2 method) were detected in the presence of insulin. Sodium nitroprusside on the other hand, which is known to mimic several effects of insulin on adipocytes, inhibited IP formation induced by threshold concentrations of thrombin.On the basis of our results the insulin receptors in human platelets seem to be “non-functional” insofar as their occupancy is not accompanied by the stimulation or inhibition of phospho-inositide breakdown or cyclic AMP formation. Similarly, “silent” muscarinic-cholinergic receptors have recently been reported in human erythrocytes (Sehar et al. 1986).

Insulin binding, internalization, and receptor regulation in cultured human fibroblasts

1981 ◽  
Vol 241 (3) ◽  
pp. E251-E260
Author(s):  
D. Baldwin ◽  
M. Prince ◽  
P. Tsai ◽  
C. Johnson ◽  
R. Lotan ◽  
...  
Keyword(s):  
Incubation Temperature ◽  
Degradation Products ◽  
Human Fibroblasts ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Insulin Concentration ◽  
Target Cells ◽  
Kinetic Properties ◽  
Insulin Degradation ◽  
Normal Human

Insulin binding to receptors was studied using monolayers of cultured normal human fibroblasts. Binding was rapid and inversely related to the incubation temperature; prolonged periods of steady-state binding were achieved at all temperatures studied and the amount of degradation of extracellular insulin was minimal. Competition curves demonstrated half-maximal inhibition of 125I-insulin binding at an unlabeled insulin concentration of 125I-insulin binding at an unlabeled insulin concentration of 7 ng/ml. Scatchard plots of the binding data were curvilinear and revealed that fibroblasts contained about 7,000 receptor sites per cell. Bound 125I-insulin dissociated from fibroblasts with a t 1/2 of 10 min at 30 degrees C and 35 min at 16 degrees C. After 60 min dissociation at 30 degrees C, 45% of the dissociated radioactivity consisted of 125I-insulin degradation products, whereas only 8% of the dissociated material was in the form of degraded products after 60 min of dissociation at 16 degrees C. This indicates that fibroblasts possess a temperature-sensitive receptor-mediated process for insulin degradation. Preincubation of the monolayers with insulin led to a hormone-induced loss of insulin receptors. Thus, incubating cells with 25 ng/ml insulin for 6 h at 37 degrees C caused a 50% reduction in subsequently measured 125I-insulin binding. This hormone-induced receptor loss was sensitive to physiologic insulin levels, with approximately 5 ng/ml causing a half-maximal receptor loss. When monolayers were treated with the lysosomotropic agent chloroquine and subsequently incubated with 5 X 10(-11) M 125I-insulin, a 130% increase in cell-associated radioactivity was observed after 120 min at 30 degrees C. In summary, 1) cultured normal human fibroblasts possess insulin receptors that exhibit kinetic properties and specificity identical to that of other insulin target cells; 2) incubation of fibroblasts with physiologic concentrations of insulin causes a marked loss of cell-surface insulin receptors; and 3) receptor-bound 125I-insulin is internalized through an energy-dependent endocytotic pathway and subsequently degraded by a chloroquine-sensitive reaction.

A STUDY OF INSULIN RECEPTORS IN HUMAN MONONUCLEAR LEUCOCYTES

1976 ◽  
Vol 83 (3) ◽  
pp. 556-564 ◽  
Author(s):  
Oluf Pedersen ◽  
Henning Beck-Nielsen
Keyword(s):  
Silicone Oil ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Cell Number ◽  
Receptor Interaction ◽  
Specific Cell ◽  
Cell Binding ◽  
Temperature Dependent ◽  
Normal Body Weight ◽  
Mononuclear Leucocytes

ABSTRACT Insulin binding sites were demonstrated in human mononuclear leucocytes by use of a technique which includes isolation of mononuclear leucocytes from defibrinated blood and separation of cell bound and free [125I] insulin with silicone oil. The binding was time and temperature dependent. At 15°C equilibrium was reached after 90 min and a plateau maintained for at least 50 min. Incubations were carried out at 4°C, 15°C and 37°C. Maximal binding was obtained at 15°C. The optimum pH for insulin receptor interaction occurred at about 8. [125I] insulin binding to mononuclear leucocytes was demonstrated to be a linear function of cell number concentration over a range of 17–70× 106×ml−1. The binding was a displaceable function of native insulin concentration. In a group of 21 young healthy persons with normal body weight we found a mean specific cell binding fraction of 1.92 ± 0.58 (s) × 10−2. Analysis of the equilibrium between insulin and its receptor revealed an apparent heterogeneity of insulin receptors.

scholarly journals Guanosine nucleotides regulate hormone binding of insulin receptors

1992 ◽  
Vol 281 (3) ◽  
pp. 735-743 ◽  
Author(s):  
E R Mortensen ◽  
J Drachman ◽  
G Guidotti
Keyword(s):  
Binding Sites ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Hormone Binding ◽  
Temperature Dependent ◽  
High Affinity ◽  
Mild Reduction ◽  
Adipocyte Plasma Membranes

Insulin receptors in turkey erythrocyte and rat adipocyte plasma membranes display non-linear hormone binding by Scatchard analysis. This result is consistent with evidence that the insulin-binding sites are heterogeneous and have at least two affinities for the hormone. Mild reduction of plasma membranes with dithiothreitol, before insulin binding, increased the fraction of hormone binding with high affinity without significantly changing the total number of receptor-binding sites. In the presence of guanosine 5′-[gamma-thio]triphosphate, the amount of receptor with high affinity for insulin in the reduced membranes decreased to that present in the absence of reduction; the effect of the nucleotide was concentration- and temperature-dependent. This decrease in insulin binding was specific for guanine nucleotides.

Comparative studies of insulin binding to receptor from adipocytes, hepatocytes, monocytes and erythrocytes from the pig

1988 ◽  
Vol 118 (1) ◽  
pp. 59-67 ◽  
Author(s):  
Elisabeth Hjøllund ◽  
Bjørn Richelsen ◽  
Oluf Pedersen
Keyword(s):  
Steady State ◽  
Receptor Binding ◽  
Specific Binding ◽  
Insulin Binding ◽  
Cell Types ◽  
Target Cells ◽  
Suitable Model ◽  
The Individual ◽  
Different Cell Types ◽  
Specific Insulin

Abstract. We have described the receptor binding of A 14-labelled [125I]insulin to viable adipocytes, hepatocytes, monocytes and erythrocytes from the pig. For all cell types the binding was of high affinity, specific for insulin, the non-specific binding low and degradation of insulin in the medium was minimal. At 24°C, steady state insulin binding was achieved in all four cell types. At 37°C, steady state insulin binding could be measured to adipocytes and hepatocytes. Specific insulin binding levels and receptor affinity for blood and fat cells from the pig are comparable to that in human cells, whereas differences, especially according to affinity, exist between pig and rat cell insulin receptor binding. It is therefore concluded that the pig is a more suitable model for studies of insulin binding in man than rodents. Finally, no correlations between the individual binding levels to the different cell types were observed. Hence, measurement of insulin binding to the easier available blood cells cannot replace studies of insulin binding to target cells of insulin.

scholarly journals Presence of insulin receptors in cultured glial C6 cells. Regulation by butyrate

1989 ◽  
Vol 258 (1) ◽  
pp. 147-155 ◽  
Author(s):  
F Montiel ◽  
J Ortiz-Caro ◽  
A Villa ◽  
A Pascual ◽  
A Aranda
Keyword(s):  
Fatty Acids ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Short Chain Fatty Acids ◽  
Short Chain ◽  
Scatchard Analysis ◽  
Maximal Effect ◽  
C6 Cells ◽  
Receptor Affinity ◽  
Chain Fatty Acids

The presence of insulin receptor and its regulation by butyrate and other short-chain fatty acids was studied in C6 cells, a rat glioma cell line. Intact C6 cells bind 125I-insulin in a rapid, reversible and specific manner. Scatchard analysis of the binding data gives typical curvilinear plots with apparent affinities of approx. 6 nM and 70 nM for the low-affinity (approx. 90% of total) and high-affinity (approx. 10% of total) sites respectively. Incubation with butyrate results in a time- and dose-dependent decrease of insulin binding to C6 cells. A maximal effect was found with 2 mM-butyrate that decreased the receptor by 40-70% after 48 h. Butyrate decreased numbers of receptors of both classes, but did not significantly alter receptor affinity. Other short-chain fatty acids, as well as keto acids, had a similar effect, but with a lower potency. Cycloheximide caused an accumulation of insulin receptors at the cell surface, since insulin binding increased and receptor affinity did not change after incubation with the inhibitor. Simultaneous addition of butyrate and cycloheximide abolished the loss of receptors produced by the fatty acid. In cells preincubated with butyrate, cycloheximide also produced a large increase in receptor numbers, showing that in the absence of new receptor synthesis a large pool of receptors re-appears at the surface of butyrate-treated cells.

Chronic and endogenous regulation of insulin receptors by catecholamines in adipocytes from patients with a phaeochromocytoma

1990 ◽  
Vol 10 (2) ◽  
pp. 201-207 ◽  
Author(s):  
M. C. Carranza ◽  
M. A. Simón ◽  
A. Torres ◽  
C. Calle
Keyword(s):  
Adipose Tissue ◽  
Binding Sites ◽  
Insulin Receptors ◽  
Human Adipose Tissue ◽  
Insulin Binding ◽  
Scatchard Analysis ◽  
Cooperative Model ◽  
Receptor Affinity ◽  
Site Model ◽  
Binding Data

Insulin binding in adipocytes from patients with a phaeochromocytoma (PH) approached that of the controls (C) at low and higher concentrations of unlabeled insulin. The apparent receptor affinity was unchanged (ED50: PH 0.50×10−9M and C0.60×10−9M). Scatchard analysis of the binding data using the negative cooperative model revealed a 46% decrease in the total number of receptors together with no changes in both K−e (PH 0.55×109M−1 and C 0.36×109M−1) and K−f (PH 0.13×109 M−1 and C 0.07×109 M−1). According to the two site model, an altered proportion in the two classes of insulin binding sites was detected. This was accompanied by a catecholamine-desensitization of the adipocytes to the antilipolytic action of insulin. These events could represent a final situation of a chronic and endogeneous regulation by high levels of catecholamines of insulin receptors in human adipose tissue.

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