Insulin receptors prepared with iodoacetamide show enhanced autophosphorylation and receptor kinase activity.

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.

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Insulin receptor kinase is hyperresponsive in adipocytes of young obese Zucker rats

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1987.252.2.e273 ◽

1987 ◽

Vol 252 (2) ◽

pp. E273-E278 ◽

Cited By ~ 1

Author(s):

A. Debant ◽

M. Guerre-Millo ◽

Y. Le Marchand-Brustel ◽

P. Freychet ◽

M. Lavau ◽

...

Keyword(s):

Skeletal Muscle ◽

Insulin Receptor ◽

Insulin Action ◽

Kinase Activity ◽

Insulin Receptors ◽

Zucker Rats ◽

Receptor Kinase ◽

Insulin Receptor Tyrosine Kinase ◽

Obese Zucker Rats ◽

Insulin Receptor Kinase

Thirty-day-old obese Zucker rats have hyperresponsive adipose tissue, whereas their skeletal muscle normally responds to insulin in vitro. To further substantiate the role of insulin receptor tyrosine kinase in insulin action, we have studied the kinase activity of receptors obtained from adipocytes and skeletal muscle of these young obese Zucker rats. Insulin receptors, partially purified by wheat germ agglutinin agarose chromatography from plasma membranes of isolated adipocytes or from skeletal muscles, were studied in a cell-free system for auto-phosphorylation and for their ability to phosphorylate a synthetic glutamate-tyrosine copolymer. For an identical amount of receptors, the insulin stimulatory action on its beta-subunit receptor phosphorylation was markedly augmented in preparations from hyperresponsive adipocytes of obese animals compared with lean rats. Basal phosphorylation of adipocyte insulin receptors was nearly identical in lean and obese animals. Similarly the capacity of adipocyte insulin receptors to catalyze the phosphorylation of the synthetic substrate in response to insulin was increased. By contrast, the kinase activity of insulin receptors prepared from normally insulin-responsive skeletal muscle was similar in preparations of lean and obese rats. These results show that a state of hyperresponsiveness to insulin is correlated with a parallel increase of insulin receptor kinase activity suggesting an important role for this activity in insulin action.

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Hydrogen peroxide stimulates tyrosine phosphorylation of the insulin receptor and its tyrosine kinase activity in intact cells

Biochemical Journal ◽

10.1042/bj2500095 ◽

1988 ◽

Vol 250 (1) ◽

pp. 95-101 ◽

Cited By ~ 75

Author(s):

O Koshio ◽

Y Akanuma ◽

M Kasuga

Keyword(s):

Insulin Receptor ◽

Kinase Activity ◽

Intact Cell ◽

Insulin Receptors ◽

Beta Subunit ◽

Receptor Kinase ◽

Intact Cells ◽

Tyrosine Residues ◽

Insulin Receptor Kinase ◽

Insulin Receptor Kinase Activity

H-35 rat hepatoma cells were labelled with [32P]orthophosphate and their insulin receptors isolated on wheat germ agglutinin (WGA)-agarose and anti-(insulin receptor) serum. The incubation of these cells with 10 mM-H2O2 for 10 min increased the phosphorylation of both the serine and tyrosine residues of the beta subunit of the insulin receptor. Next, insulin receptors were purified on WGA-agarose from control and H2O2-treated H-35 cells and the purified fractions incubated with [gamma-32P]ATP and Mn2+. Phosphorylation of the beta subunit of insulin receptors obtained from H2O2-treated cells was 150% of that of control cells. The kinase activity of the WGA-purified receptor preparation obtained from H2O2-treated cells, as measured by phosphorylation of src-related synthetic peptide, was increased about 4-fold over control cells. These data suggest that in intact cell systems, H2O2 may increase the insulin receptor kinase activity by inducing phosphorylation of the beta subunit of insulin receptor.

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Increased in-vivo insulin sensitivity but normal liver insulin receptor kinase activity in dwarf chickens

Journal of Endocrinology ◽

10.1677/joe.0.1260067 ◽

1990 ◽

Vol 126 (1) ◽

pp. 67-74 ◽

Cited By ~ 3

Author(s):

I. Guéritault ◽

J. Simon ◽

B. Chevalier ◽

M. Derouet ◽

M. Tixier-Boichard ◽

...

Keyword(s):

Insulin Sensitivity ◽

Tyrosine Kinase ◽

Insulin Receptor ◽

Kinase Activity ◽

Insulin Receptors ◽

Oral Glucose ◽

Receptor Kinase ◽

Tyrosine Kinase Activity ◽

Insulin Receptor Kinase

ABSTRACT The effects of the recessive and sex-linked dw gene on insulin sensitivity and liver insulin receptors were compared in normal (Dw-dw) and dwarf (dw-dw) brother or half-brother chickens. At 3·5 weeks of age, following an overnight fast, exogenous insulin (0–6·9 nmol/kg body weight) was slightly but significantly more hypoglycaemic in dwarf chickens. At 4 weeks of age, following an oral glucose load (2 g/kg), glucose tolerance was the same in both genotypes, whereas plasma insulin levels were greatly decreased in dwarf chickens. At 5 weeks of age, plasma concentrations of glucose and insulin were the same in both genotypes in the fasting state and decreased in the fed state in dwarf chickens. In liver membranes prepared from fasted chickens, insulin binding was increased in dwarf chickens, while the affinity of insulin receptors and the insulin-degrading activity of the membranes were the same in both genotypes. Following solubilization with Triton X-100, liver receptors were successively purified on lentil then wheat germ lectins. Autophosphorylation of the β-subunit did not differ between either the genotype or the nutritional (fed or fasted) state. In the basal state (in the absence of insulin) the tyrosine kinase activity of the receptor towards artificial substrate poly(Glu,Tyr)4:1 was significantly decreased in dwarf chickens by fasting. However, the change in tyrosine kinase activity of the receptor in response to insulin was similar, irrespective of the genotype and the nutritional state. Therefore, the slight increase in insulin sensitivity observed in vivo in dwarf chickens is accounted for, at least partly, by a slight increase in liver insulin receptor number, but not by a change in the kinase activity of liver insulin receptors. In addition, post-insulin receptor kinase events and/or GH-dependent counter-regulatory mechanisms may superimpose and increase the insulin sensitivity of dwarf chickens. Journal of Endocrinology (1990) 126, 67–74

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Effect of a thermogenic agent, BRL 26830A, on insulin receptors in obese mice

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1988.255.2.e101 ◽

1988 ◽

Vol 255 (2) ◽

pp. E101-E109 ◽

Cited By ~ 1

Author(s):

N. Rochet ◽

J. F. Tanti ◽

T. Gremeaux ◽

E. Van Obberghen ◽

Y. Le Marchand-Brustel

Keyword(s):

Skeletal Muscle ◽

Adipose Tissue ◽

Brown Adipose Tissue ◽

Insulin Receptor ◽

Kinase Activity ◽

Insulin Receptors ◽

Receptor Kinase ◽

Obese Mice ◽

Brown Adipose ◽

Receptor Number

The effect of a new type of antidiabetic agent, BRL 26830A, has been tested in obese mice. Since this drug increases thermogenesis, insulin receptor binding and kinase activity were studied in brown adipose tissue and skeletal muscle of mice made obese by gold thioglucose. At 1 mg.kg-1.day-1, a 3-wk treatment normalized the glycemia and increased the uncoupling protein content of brown adipose tissue. The insulin receptor number and its associated kinase activity increased only in brown adipose tissue. At 2 mg.kg-1.day-1, additional effects, i.e., a 20% reduction in body weight and a normalization of insulin receptor number both in brown adipose tissue and in skeletal muscle, were observed. All those results were obtained even though hyperinsulinemia was not corrected. At the higher drug dosage, insulin receptor kinase activity evolved in direct proportion to the receptor number in brown adipose tissue. By contrast, in skeletal muscle, the receptor kinase activity toward exogenous substrates increased more than the receptor number, suggesting that the alteration of insulin receptor kinase activity previously reported in skeletal muscle of obese mice was partly reversed by BRL 26830A. None of these parameters was modified by the drug in lean mice. These results show that, even without affecting obesity, BRL 26830A improves insulin resistance in obese mice, probably through its effect on insulin receptors. This action prevails in brown adipose tissue, supporting the idea that this tissue plays an important role in glucose homeostasis. Thermogenic drugs could thus be powerful agents for the treatment of noninsulin-dependent diabetics.

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Insulin receptor kinase activity in muscles and white adipose tissue during course of VMH obesity

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1992.262.2.e161 ◽

1992 ◽

Vol 262 (2) ◽

pp. E161-E166 ◽

Cited By ~ 1

Author(s):

A. Pujol ◽

B. Cousin ◽

A. F. Burnol ◽

M. Loizeau ◽

L. Picon ◽

...

Keyword(s):

Adipose Tissue ◽

Insulin Receptor ◽

White Adipose Tissue ◽

Kinase Activity ◽

Insulin Receptors ◽

Ventromedial Hypothalamus ◽

Receptor Kinase ◽

Insulin Receptor Tyrosine Kinase ◽

Insulin Receptor Kinase ◽

Insulin Receptor Kinase Activity

Early after lesion of the ventromedial hypothalamus nuclei (VMH), insulin-induced glucose utilization is increased in white adipose tissue (WAT), whereas oxidative and glycolytic muscles are, respectively, normoresponsive or resistant to insulin. Five weeks later, all of the muscles are resistant, whereas WAT returns to normal responsiveness. The aim of this study was to characterize the insulin receptor kinase activity in WAT and muscles 1 and 6 wk after lesion. The number and affinity of insulin receptors were not modified in any of the tissues studied. Autophosphorylation and phosphorylation of an exogenous substrate were similar in oxidative and glycolytic muscles of VMH and control rats both 1 and 6 wk after the lesion. Insulin receptors from WAT of 1-wk VMH rats exhibited a 2.5-fold increase in insulin-stimulated autophosphorylation and phosphorylation. Six weeks after the lesion, both autophosphorylation and phosphorylation returned to normal values. This suggests that insulin receptor tyrosine kinase activity does not play a significant role in the insulin resistance of skeletal muscles but has a crucial role in mediating the variations of insulin action on WAT observed during the development of VMH obesity.

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Phospholipid environment alters hormone-sensitivity of the purified insulin receptor kinase

Biochemical Journal ◽

10.1042/bj2480829 ◽

1987 ◽

Vol 248 (3) ◽

pp. 829-836 ◽

Cited By ~ 21

Author(s):

R E Lewis ◽

M P Czech

Keyword(s):

Insulin Sensitivity ◽

Insulin Receptor ◽

Kinase Activity ◽

Insulin Receptors ◽

Complete Loss ◽

Detergent Solution ◽

Receptor Kinase ◽

Apparent Affinity ◽

Reconstituted System ◽

Insulin Receptor Kinase

Insulin receptor kinase, affinity-purified by adsorption and elution from immobilized insulin, is stimulated 2-3-fold by insulin in detergent solution. Reconstitution of the receptor kinase into leaky vesicles containing phosphatidylcholine and phosphatidylethanolamine (1:1, w/w) by detergent removal on Sephadex G-50 results in the complete loss of receptor kinase sensitivity to activation by insulin. Insulin receptors in these vesicles also exhibit an increase in their apparent affinity for 125I-insulin (Kd = 0.12 nM versus 0.76 nM). Inclusion of 8.3-16.7% phosphatidylserine into the reconstituted vesicles restores 40-50% of the insulin-sensitivity to the receptor kinase. An elevated apparent affinity for 125I-insulin of insulin receptors in vesicles containing phosphatidylcholine and phosphatidylethanolamine is also restored to the value observed in detergent solution by the inclusion of phosphatidylserine in the reconstituted system. The effect of phosphatidylserine on insulin receptor kinase appears specific, because cholesterol, phosphatidylinositol and phosphatidic acid are all unable to restore insulin-sensitivity to the receptor kinase. Autophosphorylation sites on the insulin receptor as analysed by h.p.l.c. of tryptic 32P-labelled receptor phosphopeptides are not different for insulin receptors autophosphorylated in detergent solution or for the reconstituted vesicles in the presence or absence of phosphatidylserine. These data indicate that the phospholipid environment of insulin receptors can modulate its binding and kinase activity, and phosphatidylserine acts to restore insulin-sensitivity to the receptor kinase incorporated into phosphatidylcholine/phosphatidylethanolamine vesicles.

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Functional characteristics of insulin receptors with a Thr-->Ser1200 mutation overexpressed in Chinese hamster ovary cells

Acta Endocrinologica ◽

10.1530/eje.0.1430125 ◽

2000 ◽

pp. 125-131 ◽

Cited By ~ 1

Author(s):

M Schutt ◽

H Benecke ◽

M Drenckhan ◽

HH Klein

Keyword(s):

Insulin Receptor ◽

Kinase Activity ◽

Chinese Hamster Ovary Cells ◽

Chinese Hamster ◽

Insulin Receptors ◽

Receptor Kinase ◽

Ovary Cells ◽

Insulin Receptor Kinase

OBJECTIVE: To investigate the functional properties of insulin receptors with a Thr-->Ser(1200)-mutation that is associated with severe insulin resistance in humans. DESIGN AND METHODS: The effect of in situ insulin-stimulation on insulin receptor kinase activity was studied in Chinese hamster ovary cells with overexpressed human Ser(1200)-mutated, non-mutated, and ATP-binding site-mutated (Lys-->Arg(1030)) receptors using a microwell-based assay that only detects human (and not hamster) insulin receptors. Moreover, the fraction of anti-phosphotyrosine antibody-binding receptors following in situ stimulation was separated, and autophosphorylation and kinase activity resulting from in situ and/or in vitro activation evaluated in this fraction. RESULTS: Although insulin-stimulated kinase activity of human-specific anti-insulin receptor antibody-binding receptors in cells with Ser(1200)-mutated insulin receptors represented only 3.3% of that reached in cells with non-mutated receptors, a clear insulin-induced increase in kinase activity was observed (3.4-fold; P<0.05). This increase was associated with a 2.3+/-0.6% (P<0.05) increase in anti-phosphotyrosine-binding receptors with a kinase activity representing 43+/-8% of that found in activated non-mutated receptors. In vitro autophosphorylation and kinase activation proceeded much more slowly in Ser(1200)-mutated receptors (t(1/2)): 100 min) compared with non-mutated receptors (t(1/2)): 1 min) and were inhibitable by lower alkaline phosphatase concentrations (EC(50): 3 U/ml and 70 U/ml respectively). No activation of insulin receptor kinase was observed with Arg(1030)-mutated receptors. CONCLUSIONS: Overexpressed Ser(1200)-mutated human insulin receptors possess insulin-stimulated kinase activity and can be activated in situ and in vitro. They are characterized by a markedly slower autophosphorylation reaction, which, in a phosphatase-containing environment, results in a small fraction of phosphorylated and activated receptors.

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