scholarly journals The effects of trypsin and phospholipase C on insulin binding and action in the isolated adipocyte

1980 ◽  
Vol 186 (2) ◽  
pp. 535-540 ◽  
Author(s):  
S Clark ◽  
R G Larkins ◽  
M De Luise ◽  
R A Melick
Keyword(s):  
Insulin Receptor ◽  
Phospholipase C ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Biological Action ◽  
Enzyme Treatment ◽  
Oxidation Rates ◽  
Significant Difference ◽  
High Insulin ◽  
Induced Changes

The effect of alterations to the insulin receptor on the insulin sensitivity of isolated adipocytes was studied. Receptor changes were induced by treatment of adipocytes with either phospholipase C or trypsin. After enzyme treatment, binding of insulin to insulin receptors and insulin-mediated glucose metabolism were examined. Exposure of adipocytes to phospholipase C (2 units/ml) significantly increased insulin binding to the cells, but destroyed the ability of the cells to oxidize glucose. After treatment with trypsin (500 micrograms/ml) for 5 min, insulin binding to the adipocytes was significantly increased. This was shown to be due to an increase in insulin-receptor affinity. Metabolic studies showed that trypsin treatment led to an increase in basal glucose transport but markedly decreased the response to insulin at all concentrations tested. Adipocytes treated with trypsin showed no significant difference in basal glucose oxidation rates when compared with controls, but were less sensitive to insulin at low insulin concentrations, and showed a decreased maximum response at high insulin concentrations. In conclusion, these findings indicate a dissociation between induced changes in binding of insulin to insulin receptors and subsequent hormone action. The importance of post-receptor events in the biological action of insulin is highlighted.

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 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.

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 Insulin receptors internalize by a rapid, saturable pathway requiring receptor autophosphorylation and an intact juxtamembrane region.

1991 ◽  
Vol 115 (6) ◽  
pp. 1535-1545 ◽  
Author(s):  
J M Backer ◽  
S E Shoelson ◽  
E Haring ◽  
M F White
Keyword(s):  
Insulin Receptor ◽  
Cho Cells ◽  
Insulin Receptors ◽  
Receptor Occupancy ◽  
Wild Type ◽  
Coated Pits ◽  
Intracellular Potassium ◽  
Juxtamembrane Region ◽  
High Insulin ◽  
Mutant Receptors

The effect of receptor occupancy on insulin receptor endocytosis was examined in CHO cells expressing normal human insulin receptors (CHO/IR), autophosphorylation- and internalization-deficient receptors (CHO/IRA1018), and receptors which undergo autophosphorylation but lack a sequence required for internalization (CHO/IR delta 960). The rate of [125I]insulin internalization in CHO/IR cells at 37 degrees C was rapid at physiological concentrations, but decreased markedly in the presence of increasing unlabeled insulin (ED50 = 1-3 nM insulin, or 75,000 occupied receptors/cell). In contrast, [125I]insulin internalization by CHO/IRA1018 and CHO/IR delta 960 cells was slow and was not inhibited by unlabeled insulin. At saturating insulin concentrations, the rate of internalization by wild-type and mutant receptors was similar. Moreover, depletion of intracellular potassium, which has been shown to disrupt coated pit formation, inhibited the rapid internalization of [125I]insulin at physiological insulin concentrations by CHO/IR cells, but had little or no effect on [125I]insulin uptake by CHO/IR delta 960 and CHO/IRA1018 cells or wild-type cells at high insulin concentrations. These data suggest that the insulin-stimulated entry of the insulin receptor into a rapid, coated pit-mediated internalization pathway is saturable and requires receptor autophosphorylation and an intact juxtamembrane region. Furthermore, CHO cells also contain a constitutive nonsaturable pathway which does not require receptor autophosphorylation or an intact juxtamembrane region; this second pathway is unaffected by depletion of intracellular potassium, and therefore may be independent of coated pits. Our data suggest that the ligand-stimulated internalization of the insulin receptor may require specific saturable interactions between the receptor and components of the endocytic system.

scholarly journals Insulin causes insulin-receptor internalization in human erythrocyte ghosts

1987 ◽  
Vol 241 (1) ◽  
pp. 93-97 ◽  
Author(s):  
R S Kelleher ◽  
E F Murray ◽  
S W Peterson
Keyword(s):  
Insulin Receptor ◽  
Receptor Binding ◽  
Human Erythrocyte ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Erythrocyte Ghost ◽  
Receptor Internalization ◽  
Erythrocyte Ghosts ◽  
And Control ◽  
Vesicular Compartment

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.

In vitro regulation of human monocyte insulin receptors by insulin and 1-methyl-3-isobutylxanthine

1983 ◽  
Vol 245 (5) ◽  
pp. E494-E501
Author(s):  
R. H. Whitson ◽  
S. A. Kaplan
Keyword(s):  
Insulin Receptor ◽  
Cultured Cells ◽  
Phosphodiesterase Inhibitor ◽  
Insulin Receptors ◽  
Human Monocyte ◽  
Insulin Binding ◽  
Receptor Downregulation ◽  
Independent Process ◽  
Dose Dependent

Monocytes separated from human blood by Ficoll-Hypaque and adherence to polystyrene flasks were maintained successfully in culture for 7 days. The cultured cells showed normal morphology and good viability. The insulin binding properties of the cultured monocytes were also identical to those of fresh monocytes. In vitro pretreatment of the monocytes with insulin decreased both the number and affinity of insulin receptors, resulting in a 72% reduction in the binding of tracer quantities of 125I-insulin. Insulin-induced receptor down regulation was dose-dependent and specific to the insulin receptor. Monocytes pretreated with insulin in the presence of the cyclic nucleotide phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) lost significantly fewer insulin receptors than monocytes treated with insulin alone. Tracer binding to these cells was 62% of control levels. MIX had no effect on basal insulin binding. The cAMP analogues N6,O2'-dibutyryl cAMP and 8-bromo-cAMP did not counteract insulin-induced receptor downregulation by themselves and did not significantly enhance the effects of MIX. These results indicate that MIX may counteract insulin receptor downregulation by a cAMP-independent process.

scholarly journals Tyrosine kinase activity of insulin receptors from human placenta. Effects of autophosphorylation and cyclic AMP-dependent protein kinase

1986 ◽  
Vol 233 (3) ◽  
pp. 677-681 ◽  
Author(s):  
H G Joost ◽  
H J Steinfelder ◽  
C Schmitz-Salue
Keyword(s):  
Protein Kinase ◽  
Cyclic Amp ◽  
Insulin Receptor ◽  
Human Placenta ◽  
Kinase Activity ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Dependent Protein Kinase ◽  
Dependent Protein

The kinase activity of partially purified insulin receptor obtained from human placenta was studied. When autophosphorylation of the beta-subunit of the receptor was initiated by ATP prior to the addition of the exogenous substrate, both basal and insulin-stimulated kinase activity was increased. However, half-maximum effective insulin concentrations were unchanged. Insulin receptor autophosphorylation as stimulated by ATP and insulin failed to affect significantly 125I-insulin binding to partially purified insulin receptor from human placenta. It is concluded that autophosphorylation of the insulin receptors regulates its kinase activity but not its affinity for insulin. The catalytic subunit of cyclic AMP-dependent protein kinase failed to phosphorylate either subunit of the insulin receptor, and each kinase failed to affect the affinity of the other one. Thus no functional interaction between cyclic AMP-dependent protein kinase and insulin receptors was observed in the in vitro system.

Nutritional state regulates insulin receptor and IRS-1 phosphorylation and expression in chicken

1998 ◽  
Vol 274 (2) ◽  
pp. E309-E316 ◽  
Author(s):  
Joëlle Dupont ◽  
Michel Derouet ◽  
Jean Simon ◽  
Mohammed Taouis
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Nutritional State ◽  
Insulin Resistant ◽  
Liver Membrane ◽  
Enhanced Expression ◽  
Plasma Insulin Levels ◽  
Polymerase Chain

After insulin binding, insulin receptors (IR) phosphorylate the insulin receptor substrate 1 (IRS-1) on specific motifs and thereby initiate insulin action. The interaction between IR and IRS-1 and their expression were studied in vivo in two target tissues (muscle and liver) in chickens, a species that is insulin resistant. To induce extreme changes in plasma insulin levels, chickens were subjected to three different nutritional states (ad libitum fed, fasted for 48 h, and refed for 30 min after 48-h fast). Liver membrane IR number was significantly increased in fasted compared with fed chickens. This upregulation of IR number was concomitant with the an enhanced expression of IR mRNA as determined by reverse transcription-polymerase chain reaction. In leg muscle, IR mRNA was not altered by the nutritional state. Using specific antibodies directed toward human IR, anti-phosphotyrosines, or mouse IRS-1, we demonstrated that IR and IRS-1 are associated in vivo in liver and muscles. Tyrosine phosphorylation of liver IR and IRS-1 were significantly decreased by prolonged fasting and restored by 30-min refeeding. These alterations were not observed in muscle. Fasting increased IRS-1 mRNA expression in liver but not in muscle. These results are the first evidence showing that chicken liver and muscle express IRS-1. Therefore, the chicken insulin resistance is not accounted for by the lack of IRS-1. The differences observed for the regulation of IR and IRS-1 messengers and phosphorylation between liver and muscle in response to alterations of the nutritional state remain to be explained.

THE INSULIN RECEPTOR IN NORMAL AND OBESE PERSONS

1976 ◽  
Vol 83 (3) ◽  
pp. 565-575 ◽  
Author(s):  
Henning Beck-Nielsen ◽  
Oluf Pedersen ◽  
Jens Peder Bagger ◽  
Niels Schwartz Sørensen
Keyword(s):  
Plasma Insulin ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Fat Cell ◽  
Ideal Weight ◽  
Comparable Group ◽  
Significant Difference ◽  
Before And After ◽  
Obese Subjects ◽  
Mononuclear Leucocytes

ABSTRACT Using [125I]insulin at 172 pmol/l (1 ng/ml) the binding of insulin to mononuclear leucocytes isolated from peripheral blood was studied. Our present study comprised 21 healthy subjects (22–33 years old, 90–110% of ideal weight) and a comparable group of 22 obese subjects (20–37 years old, minimum 150% of ideal weight). A significant difference in insulin binding was found between the two groups, the mean specific insulin binding fraction in normals being 1.92 ± 0.58 (s) × 10−2 and that for the obese 1.19 ± 0.41 (s) × 10−2 (P < 0.01). No correlation was found between body weight and the number of insulin receptors in the obese subjects. However, the number of insulin receptors was negatively correlated to fat cell size (P < 0.05). Insulin receptors in subjects were also negatively correlated to fasting plasma insulin (P < 0.05). Insulin receptors were studied in 11 obese subjects before and after 10 days of fasting. A significant increase in the number of insulin receptors was observed with a simultaneous decrease in plasma insulin to normal values. The results indicate that obesity complicated by hyperinsulinism is associated with a decrease in the number of insulin receptors compared with the normal. This finding may in part explain the decreased insulin sensitivity of the hyperinsulinaemic obese.

scholarly journals Changes in insulin-receptor structure associated with trypsin-induced activation of the receptor tyrosine kinase

1991 ◽  
Vol 276 (1) ◽  
pp. 27-33 ◽  
Author(s):  
S Clark ◽  
G Eckardt ◽  
K Siddle ◽  
L C Harrison
Keyword(s):  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Receptor Tyrosine Kinase ◽  
Insulin Receptors ◽  
Insulin Binding ◽  
Alpha Subunit ◽  
Beta Subunits ◽  
Receptor Kinase ◽  
Trypsin Treatment ◽  
Receptor Structure

The tyrosine kinase of the insulin receptor can be activated by trypsin treatment. The concomitant abolition of insulin binding has been postulated to result from proteolytic destruction of the receptor. A discrepancy between the decrease in insulin binding and receptor immunoreactivity after trypsin treatment led us to investigate more closely the structure of the trypsin-treated receptor. After trypsin treatment of the CHOT cell line, which over-expresses transfected human insulin receptors, insulin binding was significantly decreased, but reactivity with five alpha-subunit monoclonal antibodies was either unaffected or only moderately decreased, indicating that the alpha-subunit was substantially intact. Examination of receptor structure after trypsin treatment, receptor autophosphorylation and gel electrophoresis revealed a single band at 110 kDa in non-reduced gels, comprising a small fragment (21 kDa) of the alpha-subunit linked to the beta-subunit by class II disulphides. When the receptor was radio-labelled with 125I, two additional alpha-subunit bands of 142 kDa and 81 kDa (composed of identical reduced bands) were observed on non-reduced gels, which contained disulphide-linked (class I) fragments. All fragments could be precipitated by antibodies to both alpha- and beta-subunits. However, only antibodies directed towards the N-terminus of the receptor could immunoblot trypsin-treated fragments. Thus activation of the receptor tyrosine kinase by trypsin occurs after cleavage, but not loss of the alpha-subunit. This finding has implications for the mechanism of transmembrane activation of the receptor kinase by insulin.

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