scholarly journals Cell-surface insulin receptor cycling and its implication in the glycogenic response in cultured foetal hepatocytes

1986 ◽  
Vol 239 (3) ◽  
pp. 609-615 ◽  
Author(s):  
P Soubigou ◽  
E Pringault ◽  
C Plas
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Polyacrylamide Gel Electrophoresis ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Insulin Binding ◽  
Surface Proteins ◽  
Short Exposure ◽  
Intermittent Exposure ◽  
Initial Binding

The insulin-receptor cycle was investigated in cultured foetal rat hepatocytes by determining the variations in insulin-binding sites at the cell surface after short exposure to the hormone. Binding of 125I-insulin was measured at 4 degrees C after dissociation of prebound native insulin. Two protocols were used: exchange binding assay and binding after acid treatment; both gave the same results. Cell-surface 125I-insulin-receptor binding decreased sharply (by 40%) during the first 5 min of 10 nM-insulin exposure (t1/2 = 2 min) and remained practically constant thereafter; subsequent removal of the hormone restored the initial binding within 10 min. This fall-rise sequence corresponded to variations in the number of insulin receptors at the cell surface, with no detectable change in receptor affinity. The reversible translocation of insulin receptors from the cell surface to a compartment not accessible to insulin at 4 degrees C was hormone-concentration- and temperature-dependent. SDS/polyacrylamide-gel electrophoresis after cross-linking of bound 125I-insulin to cell-surface proteins with disuccinimidyl suberate showed that these variations were not associated with changes in Mr of binding components, in particular for the major labelled band of Mr 130,000. The insulin-receptor cycle could be repeated after intermittent exposure to insulin. Continuous or intermittent exposure to the hormone gave a similar glycogenic response, contrary to the partial effect of a unique short (5-20 min) exposure. A relationship could be established between the repetitive character of the rapid insulin-receptor cycle and the maximal expression of the biological effect in cultured foetal hepatocytes.

Effect of exercise on insulin receptor binding and kinase activity in skeletal muscle

1989 ◽  
Vol 256 (1) ◽  
pp. E138-E144 ◽  
Author(s):  
J. L. Treadway ◽  
D. E. James ◽  
E. Burcel ◽  
N. B. Ruderman
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Insulin Action ◽  
White Muscle ◽  
Kinase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Similar Data

Insulin action in skeletal muscle is markedly enhanced for several hours after an acute bout of exercise. The purpose of this study was to examine the possible involvement of the intrinsic tyrosine kinase activity of the insulin receptor in mediating these effects. Red and white muscles were removed from rats either at rest or following a treadmill run (45 min at 18 m/min), and insulin receptors were isolated in partially purified form. Basal and insulin-stimulated receptor kinase activity was higher in red than in white muscle, in agreement with previous studies (J. Biol. Chem. 261: 14939-14944, 1986). There was no effect of exercise on insulin binding, basal and insulin-stimulated receptor autophosphorylation, or basal and insulin-stimulated exogenous kinase activity, in either red or white muscle. Similar data were obtained when phosphatase inhibitors were used during receptor isolation. The structure of insulin receptors isolated from the muscle of exercised and control rats was similar as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity cross-linked insulin receptors. We conclude that enhanced insulin action in muscle during the postexercise state is not related to increased kinase activity of the insulin receptor.

scholarly journals Quantitative studies of the rate of insulin internalization in isolated rat hepatocytes

1984 ◽  
Vol 218 (2) ◽  
pp. 307-312 ◽  
Author(s):  
B Draznin ◽  
M Trowbridge ◽  
L Ferguson
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Isolated Hepatocytes ◽  
Receptor Internalization ◽  
Insulin Concentration ◽  
Receptor Complex ◽  
Isolated Rat Hepatocytes ◽  
Isolated Rat

We studied internalization of 125I-labelled insulin in isolated rat hepatocytes. Using the acidification technique, we were able to dissociate the ligand from its cell-surface receptors, and thus to separate internalized from surface-bound insulin. Because during the first 5 min of incubation of 125I-labelled insulin with freshly isolated hepatocytes there is no loss of internalized label, the ratio of the amount of internalized ligand to the amount of cell-surface-bound ligand may serve as an index of insulin internalization. Within the first 10 min of insulin's interaction with hepatocytes, the plot of the above ratio as a function of time yields a straight line. The slope of this line is referred to as the endocytic rate constant (Ke) for insulin and denotes the probability with which the insulin-receptor complex is internalized in 1 min. At the insulin concentration of 0.295 ng/ml, the Ke is 0.049 min-1. It is independent of insulin concentration until the latter exceeds 1 ng/ml. At the insulin concentration of 3.2 ng/ml, the Ke accelerates to 0.131 min-1. With the Ke being the probability of insulin-receptor-complex internalization, 4.9% of occupied insulin receptors will be internalized in 1 min at an insulin concentration of 0.295 ng/ml, and 13.1% of occupied insulin receptors will be internalized in 1 min at 3.2 ng/ml. When the insulin concentration decreases from 3.2 to 0.3 ng/ml, the Ke decreases accordingly. The half-time of occupied receptor internalization is 15.4 min at the lower insulin concentration and 5.3 min at the higher insulin concentration.

scholarly journals Glucocorticoids increase insulin binding and the amount of insulin-receptor mRNA in human cultured lymphocytes

1988 ◽  
Vol 249 (3) ◽  
pp. 715-719 ◽  
Author(s):  
Y Shibasaki ◽  
H Sakura ◽  
M Odawara ◽  
M Shibuya ◽  
Y Kanazawa ◽  
...  
Keyword(s):  
Cell Surface ◽  
Insulin Receptor ◽  
De Novo ◽  
Insulin Receptors ◽  
Fold Increase ◽  
Insulin Binding ◽  
Dependent Manner ◽  
Receptor Mrna ◽  
Dose Dependent ◽  
Receptor Mrnas

The effect of steroid hormones on insulin binding and the amount of insulin-receptor mRNA was examined in IM-9 lymphocytes. Cortisol and cortexolone, but not oestrogen, increased both the binding of insulin and the amount of insulin-receptor mRNA in a time- and dose-dependent manner. Cortisol was most potent, and induced a 2-fold increase in insulin binding and a 4-fold increase in mRNA. The elevation in binding was due to an increased number of insulin receptors at the cell surface. The increase in mRNA involved all four of the insulin-receptor mRNAs and could not be inhibited by cycloheximide. The cortisol-induced increase in mRNA was associated with a 3-4-fold increase in the synthesis of pro-receptor. The relative potency of the three steroids indicated that these effects were mediated by an interaction with the glucocorticoid receptor. The results of this study suggest that cortisol can increase the number of insulin receptors at the cell surface by increasing the amounts of insulin-receptor mRNA and the synthesis de novo of insulin receptors.

scholarly journals Tyrosine kinase activity of liver insulin receptor is inhibited in rats at term gestation

1989 ◽  
Vol 263 (1) ◽  
pp. 267-272 ◽  
Author(s):  
C Martínez ◽  
P Ruiz ◽  
A Andrés ◽  
J Satrústegui ◽  
J M Carrascosa
Keyword(s):  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Signalling ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Late Gestation ◽  
Receptor Kinase ◽  
Pregnant Rats ◽  
Insulin Resistant ◽  
32P Incorporation

Late gestation is associated with insulin resistance in rats and humans. It has been reported that rats at term gestation show active hepatic gluconeogenesis and glycogenolysis, and diminished lipogenesis, despite normal or mildly elevated plasma insulin concentrations, indicating a state of resistance to the hormone action. Since autophosphorylation of the insulin receptor has been reported to play a key role in the hormone signal transduction, we have partially purified plasma-membrane liver insulin receptors from virgin and 22-day-pregnant rats and studied their binding and kinase activities. (1) Insulin binding to partially purified receptors does not appear to be influenced by gestation, as indicated by the observed KD and Bmax. values. (2) The rate of autophosphorylation and the maximal 32P incorporation into the receptor beta-subunit from pregnant rats at saturating concentrations of insulin are markedly decreased with respect to the corresponding values for virgin rats. (3) The diminished autophosphorylation rate was due to a decreased responsiveness of the kinase activity to the action of insulin. (4) Phosphorylation of the exogenous substrates casein and poly(Glu80Tyr20) by insulin-receptor kinase was also less when receptors from pregnant rats were used. These results show the existence of an impairment at the receptor kinase level of the insulin signalling mechanism that might be related to the insulin-resistant state characteristic of term gestation in rats.

scholarly journals Cell-surface radioiodination with the sparingly soluble catalyst Iodogen. Difference between the dividing and non-dividing populations of rodent thymocytes

1981 ◽  
Vol 194 (1) ◽  
pp. 351-355 ◽  
Author(s):  
J G Salisbury ◽  
J M Graham
Keyword(s):  
Cell Surface ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Surface Proteins ◽  
New Method

The surface proteins of dividing and non-dividing subpopulations of rat and mouse thymocytes have been labelled by using a new method of radioiodination. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and autoradiography of the labelled proteins shows distinct differences in labelling between the mouse and rat cells and also, in the case of the rat, between the dividing and non-dividing populations.

scholarly journals Insulin receptor function is inhibited by guanosine 5′-[γ-thio]triphosphate (GTP[S])

1990 ◽  
Vol 270 (2) ◽  
pp. 401-407 ◽  
Author(s):  
H W Davis ◽  
J M McDonald
Keyword(s):  
Insulin Receptor ◽  
G Proteins ◽  
Plasma Membranes ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Peptide Sequence ◽  
Receptor Kinase ◽  
Receptor Function ◽  
Gtp Binding ◽  
Insulin Receptor Kinase

The regulatory role of GTP-binding proteins (G-proteins) in insulin receptor function was investigated using isolated insulin receptors and plasma membranes from rat adipocytes. Treatment of isolated insulin receptors with 1 mM-guanosine 5′-[gamma-thio]triphosphate (GTP[S]) inhibited insulin-stimulated phosphorylation of the beta-subunit, histone Hf2b and poly(GluNa4,Tyr1) by 22%, 65% and 65% respectively. Phosphorylation of calmodulin by the insulin receptor kinase was also inhibited by 1 mM-GTP[S] both in the absence (by 88%) and in the presence (by 81%) of insulin. In the absence of insulin, 1 mM-GTP had the same effect on calmodulin phosphorylation as 1 mM-GTP[S]. However, when insulin was present, GTP was less effective than GTP[S] (41% versus 81% inhibition). Concentrations of GTP[S] greater than 250 microM are necessary to inhibit phosphorylation. Although these concentrations are relatively high, the effect of GTP[S] is not due to competition with [32P]ATP for the insulin receptor kinase since (1) other nucleotide triphosphates did not inhibit phosphorylation as much as did GTP[S] (or GTP) and (2) the Vmax of the ATP-dependent kinase reaction was decreased in the presence of GTP[S]. GTP[S] (1 mM) also inhibited insulin binding to isolated receptors and plasma membranes, by 80% and 50% respectively. Finally, an antibody raised to a peptide sequence common to the alpha-subunits of G-proteins Gs, Gi, Go and transducin detected G-proteins in plasma membranes but failed to detect them in the insulin receptor preparation. These results indicate that GTP inhibits insulin receptor function, but does so through a mechanism that does not require a conventional GTP-binding protein.

scholarly journals Identification of the major lectin-binding surface proteins of human neutrophils and alveolar macrophages

Blood ◽  
1988 ◽  
Vol 71 (6) ◽  
pp. 1624-1632
Author(s):  
NP Christiansen ◽  
KM Skubitz
Keyword(s):  
Cell Surface ◽  
Alveolar Macrophages ◽  
Cell Function ◽  
Lectin Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Surface Proteins ◽  
Human Neutrophils ◽  
Lectin Affinity Chromatography ◽  
Con A ◽  
Major Surface

Concanavalin A (Con A) and wheat germ agglutinin (WGA) are frequently used as stimuli of neutrophils and macrophages. While the effects of these lectins on cell function are presumably mediated by interaction with cell-surface molecules, the target structures on the cell surface involved are not well defined. We have used the techniques of lactoperoxidase catalyzed cell-surface iodination, lectin affinity chromatography, monoclonal antibody immunoprecipitation, and NaDodSO4- polyacrylamide gel electrophoresis to study the surface proteins of human neutrophils and alveolar macrophages that react with six lectins including Con A and WGA. We found that several major surface-labeled proteins of neutrophils bound Con A. Four of these proteins were identified by immunoprecipitation as members of the LFA-1/HMac- 1/gp150,95 adhesion glycoprotein family. Con A also bound CR1 and a 135- kd surface-labeled protein recognized by CD15 monoclonal antibodies. WGA also bound many of these proteins, but had a much lower avidity for CR1. All three of the major surface-labeled proteins of human alveolar macrophages bound to Con A, including the 183-kd mannose receptor and the 30-kd smoking-associated protein. WGA also bound the 183-kd macrophage protein, but not the 30-kd protein. These results should aid the understanding of studies using these lectins as stimuli.

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