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 Insulin activates GTP binding to a 40 kDa protein in fat cells

1991 ◽  
Vol 276 (1) ◽  
pp. 103-108 ◽  
Author(s):  
M Kellerer ◽  
B Obermaier-Kusser ◽  
A Pröfrock ◽  
E Schleicher ◽  
E Seffer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Binding Site ◽  
G Protein ◽  
Plasma Membranes ◽  
Fat Cells ◽  
Dependent Manner ◽  
Receptor Kinase ◽  
Gtp Binding ◽  
Insulin Receptor Kinase

The first steps in insulin action are binding of insulin to its receptor and activation of the insulin receptor kinase. As there is indirect evidence that further signal transduction might involve a guanine-nucleotide-binding protein (G-protein), we studied whether insulin modulates GTP binding to plasma membrane proteins of fat cells and skeletal muscle. We found that insulin rapidly increased (30 s) binding of guanosine 5′-[gamma-thio]triphosphate (GTP[S]) in a dose dependent manner (0.03-2.0 nM). This effect was not altered by pertussis toxin, but it was abolished by cholera toxin treatment of fat cells. Scatchard analysis of the binding data showed that the increased GTP[S] binding is due to a decrease in the Kd for GTP from 100 nM to 50 nM. Furthermore, binding of GTP to these plasma membranes inhibited both the binding of 125I-insulin to the insulin receptor and the stimulation of the insulin receptor kinase, suggesting a feedback interaction between the insulin-stimulated GTP-binding site and the insulin receptor. In order to identify this insulin-stimulated GTP-binding site, plasma membranes were labelled with the photoreactive GTP analogue [alpha-32P]GTP gamma-azidoanilide. We found that insulin selectively stimulated GTP binding to a 40 kDa protein. In conclusion, in plasma membranes of fat cells and skeletal muscle, the insulin receptor interacts with a 40 kDa GTP-binding site. We speculate that this 40 kDa GTP-binding site might be a G-protein which is involved in insulin signal transmission.

scholarly journals Insulin activation of insulin receptor kinase in erythrocytes is not altered in non-insulin-dependent diabetes and not influenced by hyperglycemia

2000 ◽  
Vol 166 (2) ◽  
pp. 275-281 ◽  
Author(s):  
HH Klein ◽  
R Muller ◽  
M Drenckhan ◽  
M Schutt ◽  
B Batge ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Diabetic Control ◽  
Insulin Dependent Diabetes ◽  
Receptor Kinase ◽  
Kinase Activation ◽  
Insulin Dependent ◽  
Insulin Receptor Kinase

Recent studies suggest that high glucose concentrations impair insulin receptor phosphorylation and kinase activation in certain cell models. To examine whether such an effect of glucose can also be demonstrated in vivo, insulin receptor kinase activation was studied in erythrocytes from 11 patients with non-insulin-dependent diabetes (NIDDM), before and after reduction of hyperglycemia (from 14.6+/-1.6 to 6.6+/-0.5 mmol/l fasting plasma glucose within 8.6+/-0.6 days). For the measurement of receptor kinase activation, cells were incubated with insulin (0-400 nmol/l), solubilized and insulin receptors immobilized to microwells coated with anti-insulin receptor antibody. Kinase activity towards insulin receptor substrate-1 and insulin binding were then measured in these wells. Kinase activities (expressed as amol phosphate transferred per min and per fmol insulin binding activity) were similar before (2.4+/-0.4 and 32.2+/-2.0 amol/min per fmol with 0 and 400 nmol/l insulin, respectively) and after improvement of metabolic control (2.4+/-0.5 and 32.0+/-2.3 amol/min per fmol with 0 and 400 nmol/l insulin, respectively). Moreover, activities were also similar in 22 hyperglycemic patients with NIDDM (2.1+/-0.3 and 35.1+/-1.4 amol/min per fmol with 0 and 400 nmol/l insulin, respectively) compared with those in 21 non-diabetic control individuals (2.1+/-0.3 and 34.2+/-1.2 amol/min per fmol with 0 and 400 nmol/l insulin, respectively). We conclude that insulin activation of erythrocyte insulin receptor kinase is not impaired in NIDDM and is not influenced by hyperglycemia.

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.

Mechanisms by Which Insulin and Muscle Contraction Stimulate Glucose Transport

1997 ◽  
Vol 22 (6) ◽  
pp. 519-530 ◽  
Author(s):  
Ronald N. Cortright ◽  
G. Lynis Dohm
Keyword(s):  
Insulin Receptor ◽  
Glucose Uptake ◽  
Glucose Transport ◽  
Initial Step ◽  
Insulin Binding ◽  
Channel Blockers ◽  
Receptor Kinase ◽  
Increase Insulin Sensitivity ◽  
Key Words Insulin ◽  
Insulin Receptor Kinase

Insulin binding to its receptor activates a tyrosine kinase that initiates a cascade of signaling events, the initial step being the tyrosine phosphorylation of insulin receptor substrate 1 (IRS-1). Subsequent IRS-1 association and activation of phosphatidylinositiol 3-kinase (PI 3-kinase) is believed to be involved in the events leading to the translocation of glucose transporters (GLUT4) to the plasma membrane resulting in uptake of glucose into the cell. Muscle contractions increase insulin sensitivity, but also stimulate muscle glucose uptake independent of insulin. The contraction signaling pathway is distinct from the insulin pathway because the effect of insulin and contractions on glucose uptake are additive, and contractions do not increase insulin receptor kinase or PI 3-kinase activity. In contrast, studies indicating that contractions cause the translocation of GLUT4 and that both contractions and insulin-stimulated glucose transport can be blocked by calcium channel blockers suggest that the two pathways may converge. However, the possibility that two distinct GLUT4 pools may be targeted, one by insulin the other by contractions, indicates that additional research is needed to better define the mechanisms by which glucose transport is stimulated in muscle. Key words: insulin signaling, exercise

scholarly journals ATP-dependent Desensitization of Insulin Binding and Tyrosine Kinase Activity of the Insulin Receptor Kinase

1998 ◽  
Vol 273 (34) ◽  
pp. 22007-22013 ◽  
Author(s):  
Jean-Olivier Contreres ◽  
Robert Faure ◽  
Gerardo Baquiran ◽  
John J. Bergeron ◽  
Barry I. Posner
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Binding ◽  
Receptor Kinase ◽  
Tyrosine Kinase Activity ◽  
Insulin Receptor Kinase

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

1987 ◽  
Vol 252 (2) ◽  
pp. E273-E278 ◽  
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.

scholarly journals Lipid-induced insulin resistance in cultured hepatoma cells is associated with a decreased insulin receptor tyrosine kinase activity.

1991 ◽  
Vol 2 (1) ◽  
pp. 65-72 ◽  
Author(s):  
P Hubert ◽  
C Bruneau-Wack ◽  
G Cremel ◽  
Y Le Marchand-Brustel ◽  
C Staedel
Keyword(s):  
Insulin Resistance ◽  
Insulin Receptor ◽  
Kinase Activity ◽  
Insulin Binding ◽  
Hepatoma Cells ◽  
Receptor Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Normal Medium ◽  
Insulin Receptor Kinase ◽  
Insulin Receptor Kinase Activity

We have shown previously that experimental modifications of the cellular lipid composition of an insulin-sensitive rat hepatoma cell line (Zajdela Hepatoma Culture, ZHC) affect both binding and biological actions of insulin. Discrepancies between insulin binding and actions implied a postbinding defect, responsible for the observed insulin resistance in lipid-treated cells. To elucidate the mechanism for this defect, we have studied insulin binding and insulin receptor kinase activity in partially purified receptor preparations from ZHC cells grown either in normal medium or in medium supplemented with linoleic acid or 25-hydroxycholesterol. Insulin binding to the lectin-purified insulin receptor showed only a small alteration in receptor affinity for the preparations from lipid-treated cells. Insulin-stimulated autophosphorylation of the beta-subunit of the insulin receptor, as well as insulin-induced phosphorylation of the artificial substrate poly(Glu,Tyr)4:1, was significantly decreased in the preparations from lipid-modified cells. Although differences in basal levels were observed, the magnitude of the insulin-stimulated kinase activity was significantly decreased in receptor preparations from lipid-treated cells. These findings indicate that experimental modification of the lipids of cultured hepatoma cells can produce in insulin receptor kinase activity changes that are proportional to the reduced insulin action observed in these cells.

In vivo insulin mimetic effects of pV compounds: role for tissue targeting in determining potency

1995 ◽  
Vol 268 (1) ◽  
pp. E60-E66 ◽  
Author(s):  
A. P. Bevan ◽  
J. W. Burgess ◽  
J. F. Yale ◽  
P. G. Drake ◽  
D. Lachance ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Insulin Receptor ◽  
Glycogen Synthesis ◽  
Insulin Receptors ◽  
Receptor Kinase ◽  
Rat Diaphragm ◽  
Insulin Mimetic ◽  
Integrated Response ◽  
Insulin Receptor Kinase

Peroxovanadium (pV) compounds activate the insulin receptor kinase in hepatocytes and inhibit the dephosphorylation of insulin receptors in hepatic endosomes with highly correlated potencies (Posner, B. I., R. Faure, J. W. Burgess, A. P. Bevan, D. Lachance, G. Zhang-Sun, J. B. Ng, D. A. Hall, B. S. Lum, and A. Shaver J. Biol. Chem. 269: 4596–4604, 1994). After intravenous administration, K2[VO(O2)2(picolinato)].2H2O [bpV(pic)], VO(O2) (picolinato) (H2O)2 [mpV(pic)], K[VO(O2)2(picolinato)].3H2O [bpV(phen)], and K[VO(O2)2(4,7-dimethyl-1,10-phenanthroline)].1/2H2O [bpV(Me2phen)] produced 50% of their maximal hypoglycemic effect at doses of 0.04, 0.04, 0.32, and 0.65 mumol/100 g body wt, respectively. In contrast, their potencies as inhibitors of dephosphorylation were bpV(pic) = bpV(phen) > mpV(pic) = bpV(Me2phen). bpV(pic) stimulated [14C]glucose incorporation into rat diaphragm glycogen in vivo, and its effect was dose dependent, synergistic with insulin, and evident in other skeletal muscles. In contrast, bpV(phen) displayed no effect on glycogen synthesis in skeletal muscle. mpV(pic) stimulated and bpV(Me2phen) had no effect on glycogen synthesis in the diaphragm. bpV(pic) augmented rat diaphragm insulin receptor kinase 2.2-fold with a time-integrated response 70% that of insulin. In contrast, the effect of bpV(phen) was delayed and much reduced. Thus, the in vivo potencies of pV compounds reflect differing capacities to act on skeletal muscle. The ancillary ligand within the pV complex may target one tissue in preference to another.

scholarly journals Tyrosine phosphorylation of two cytosolic proteins of 50 kDa and 35 kDa in rat liver by insulin-receptor kinase in vitro

1987 ◽  
Vol 248 (1) ◽  
pp. 27-33 ◽  
Author(s):  
Y C Kwok ◽  
C C Yip
Keyword(s):  
Insulin Receptor ◽  
Rat Liver ◽  
Growth Factor Receptor ◽  
Insulin Receptors ◽  
Artificial Substrates ◽  
Receptor Kinase ◽  
Insulin Receptor Tyrosine Kinase ◽  
Cytosolic Proteins ◽  
Insulin Receptor Kinase

Insulin-receptor tyrosine kinase can phosphorylate a variety of artificial substrates in vitro. Its physiological substrate(s), however, remains unknown. In the present study, we show that immobilized insulin receptors phosphorylate tyrosine residues of two cytosolic proteins of 50 kDa and 35 kDa in rat liver. Phosphorylation of these two proteins required Mn2+- or Mg2+-ATP as the phosphate donor. Phosphorylation was time- and temperature-dependent. Furthermore, the rate of phosphorylation of the two proteins was related to the autophosphorylated state of the insulin receptor. The pI of the phosphorylated 50 kDa and 35 kDa proteins was 5.4 and 5.6 respectively. These proteins were present in low abundance. They were not related to each other, nor to the insulin receptor, as demonstrated by in-gel proteolytic digestion and by immunoprecipitation using antibodies produced against them. They were specific substrates for the insulin receptor kinase, since they were not phosphorylated by epidermal-growth-factor-receptor kinase. These observations suggest that the 50 kDa and 35 kDa cytosolic proteins may be endogenous substrates for the insulin-receptor kinase.

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