scholarly journals Insulin stimulates tyrosine phosphorylation of its receptor β-subunit in intact rat hepatocytes

1987 ◽  
Vol 241 (1) ◽  
pp. 99-104 ◽  
Author(s):  
R Ballotti ◽  
A Kowalski ◽  
M F White ◽  
Y Le Marchand-Brustel ◽  
E Van Obberghen
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Biological Effects ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Enzymic Activity ◽  
Beta Subunit ◽  
Isolated Rat Hepatocytes ◽  
Receptor Phosphorylation ◽  
Insulin Receptor Kinase

We studied the phosphorylation of the beta subunit of the insulin receptor in intact freshly isolated rat hepatocytes, labelled with [32P]Pi. Insulin receptors partially purified by wheat-germ agglutinin chromatography were immunoprecipitated with either antibodies to insulin receptor or antibodies to phosphotyrosine. Receptors derived from cells incubated in the absence of insulin contained only phosphoserine. Addition of insulin to hepatocytes led to a dose-dependent increase in receptor beta-subunit phosphorylation, with half-maximal stimulation being observed at 2 nM-insulin. Incubation of cells with 100 nM-insulin showed that, within 1 min of exposure to the hormone, maximal receptor phosphorylation occurred, which was followed by a slight decrease and then a plateau. This insulin-induced stimulation of its receptor phosphorylation was largely accounted for by phosphorylation on tyrosine residues. Sequential immunoprecipitation of receptor with anti-phosphotyrosine antibodies and with anti-receptor antibodies, and phosphoamino acid analysis of the immunoprecipitated receptors, revealed that receptors that failed to undergo tyrosine phosphorylation were phosphorylated on serine residues. The demonstration of a functional hormone-sensitive insulin-receptor kinase in normal cells strongly supports a role for this receptor enzymic activity in mediating biological effects of insulin.

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

1988 ◽  
Vol 250 (1) ◽  
pp. 95-101 ◽  
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.

scholarly journals Interaction between cAMP-dependent and insulin-dependent signal pathways in tyrosine phosphorylation in primary cultures of rat hepatocytes

1997 ◽  
Vol 324 (2) ◽  
pp. 379-388 ◽  
Author(s):  
Yoshiaki ITO ◽  
Yasunobu UCHIJIMA ◽  
Miyako ARIGA ◽  
Taiichiro SEKI ◽  
Asako TAKENAKA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Β Subunit ◽  
Receptor Kinase ◽  
Dibutyryl Camp ◽  
Insulin Dependent ◽  
Insulin Receptor Kinase

The present studies were undertaken to determine whether the interaction between cAMP-dependent and insulin-dependent pathways in primary cultures of rat hepatocytes affects biological functions and tyrosine phosphorylation. Quiescent hepatocytes were pretreated with dibutyryl cAMP or cAMP-generating agents such as glucagon, and then treated or not with insulin. Preincubation for 6 h with dibutyryl cAMP or glucagon enhanced the effect of insulin on DNA synthesis, but not the effect of insulin on amino acid transport or glycogen and protein synthesis. Tyrosine phosphorylation of intracellular proteins was determined by immunoblot analysis using an anti-phosphotyrosine antibody. Maximum tyrosine phosphorylation of a 195 kDa protein, which may be a substrate of insulin receptor kinase, of 175–180 kDa proteins, including insulin receptor substrate (IRS)-1, and of 90–95 kDa proteins, including the insulin receptor β-subunit, was reached within 30 s of incubation with insulin. Pretreatment for about 3 h with dibutyryl cAMP or cAMP-generating agents clearly increased insulin-dependent tyrosine phosphorylation of the 195 kDa protein, but not IRS-1, IRS-2 or the insulin receptor β-subunit. Because dibutyryl cAMP and cAMP-generating agents did not increase insulin receptor number or its kinase activity, the effect of cAMP on this potentiation of tyrosine phosphorylation is assumed to be exerted at a step distal to insulin receptor kinase activation. The potentiation by cAMP pretreatment of insulin-stimulated tyrosine phosphorylation may in part be secondary to inhibition of phosphotyrosine phosphatase activity, because cAMP pretreatment blunted the effect of Na3VO4 on the net tyrosine phosphorylation of the 195 kDa protein as compared with cells pretreated with no additive. In summary, the interactions between cAMP-dependent and insulin-dependent pathways that lead to augmentation of DNA synthesis appear to parallel the changes in tyrosine phosphorylation. Further studies will be required to determine whether there is a causal relationship between these phenomena.

scholarly journals Two different protein kinase activities are associated with the insulin receptor

1983 ◽  
Vol 216 (3) ◽  
pp. 575-582 ◽  
Author(s):  
H Gazzano ◽  
A Kowalski ◽  
M Fehlmann ◽  
E Van Obberghen
Keyword(s):  
Insulin Receptor ◽  
Insulin Receptors ◽  
Rat Hepatocytes ◽  
Beta Subunit ◽  
Stimulatory Action ◽  
Tyrosine Residues ◽  
Binding Event ◽  
Tyrosine Specific Kinase ◽  
Receptor Antibodies ◽  
Receptor Beta

In intact rat hepatocytes insulin stimulates the phosphorylation of the beta-subunit of its receptor exclusively on serine residues, which are also phosphorylated in the absence of insulin. In contrast, in partially purified insulin receptors derived from these same cells and in highly purified insulin receptors obtained by immunoprecipitation with anti-receptor antibodies, the receptor beta-subunit is phosphorylated solely on tyrosine residues. For both cell-free systems, insulin's stimulatory action on receptor phosphorylation leads to an increase in phosphotyrosine. When partially purified receptors were used to phosphorylate two exogenous substrates, casein and histone, insulin was found to stimulate the phosphorylation of both tyrosine and serine. However, the basal and insulin-stimulated kinase activity of immunoprecipitated receptors was only tyrosine-specific. From these observations we propose that the insulin-receptor complex consists of two different insulin-stimulatable kinase activities: (1) a tyrosine-specific kinase, which is a constituent of the insulin-receptor structure and whose activation is likely to be the first post-binding event in insulin action; and (2) a serine-specific kinase, which is closely associated with the receptor in the cell membrane.

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 Insulin receptor substrate 1 rescues insulin action in CHO cells expressing mutant insulin receptors that lack a juxtamembrane NPXY motif.

1995 ◽  
Vol 15 (9) ◽  
pp. 4711-4717 ◽  
Author(s):  
D Chen ◽  
D J Van Horn ◽  
M F White ◽  
J M Backer
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Glycogen Synthesis ◽  
Insulin Receptors ◽  
Insulin Receptor Substrate ◽  
Insulin Stimulation ◽  
Insulin Receptor Substrate 1 ◽  
Npxy Motif ◽  
Stimulation Of

Insulin signals are mediated through tyrosine phosphorylation of specific proteins such as insulin receptor substrate 1 (IRS-1) and Shc by the activated insulin receptor (IR). Phosphorylation of both proteins is nearly abolished by an alanine substitution at Tyr-960 (A960) in the beta-subunit of the receptor. However, overexpression of IRS-1 in CHO cells expressing the mutant receptor (A960 cells) restored sufficient tyrosine phosphorylation of IRS-1 to rescue IRS-1/Grb-2 binding and phosphatidylinositol 3' kinase activation during insulin stimulation. Shc tyrosine phosphorylation and its binding to Grb-2 were impaired in the A960 cells and were unaffected by overexpression of IRS-1. Although overexpression of IRS-1 increased IRS-1 binding to Grb-2, ERK-1/ERK-2 activation was not rescued. These data suggest that signaling molecules other than IRS-1, perhaps including Shc, are critical for insulin stimulation of p21ras. Interestingly, overexpression of IRS-1 in the A960 cells restored insulin-stimulated mitogenesis and partially restored insulin stimulation of glycogen synthesis. Thus, IRS-1 tyrosine phosphorylation is sufficient to increase the mitogenic response to insulin, whereas insulin stimulation of glycogen synthesis appears to involve other factors. Moreover, IRS-1 phosphorylation is either not sufficient or not involved in insulin stimulation of ERK.

Tissue specific differences in insulin receptor characteristics in the mature bovine animal

1997 ◽  
Vol 1997 ◽  
pp. 51-51
Author(s):  
P.D. McGrattan ◽  
A.R.G. Wylie ◽  
R.W.J. Steen ◽  
J. Nelson
Keyword(s):  
Insulin Receptor ◽  
Biological Effects ◽  
Insulin Receptors ◽  
Plasma Insulin Concentration ◽  
Protein Deposition ◽  
Wheatgerm Agglutinin ◽  
Receptor Number ◽  
Tracer Amount ◽  
Triton X ◽  
Bovine Animal

Insulin is believed to play a key role in the partitioning of nutrients towards fat and protein deposition. The biological effects of insulin are a function of plasma insulin concentration, insulin-receptor concentration and the affinity of the receptor. The number and affinity of insulin receptors on adipocytes differs between genetically lean and obese pigs (Camara and Mourot, 1996) while receptor number, but not affinity, was shown to differ in a variety of bovine muscles (Boge et al., 1995). The objective of the current work was to determine if insulin receptor affinity and number vary between the principal target tissues (i.e. liver, muscle and adipose tissue) in the bovine animal.Five Charolais-cross steers were offered grass silage ad libitum from 18 months of age until slaughter, at 701± 22.9 kg, at a commercial abattoir. Samples of liver [L], of two skeletal muscles [Ml, M3] and of subcutaneous [S], omental [0]and renal [R] fats were taken as soon as possible after slaughter (typically <30min for L, M3, S and O and <45min for Mland R), wrapped and snap frozen in liquid nitrogen prior to storage at -70 °. Insulin receptors were partially purified by solubilising tissues in 1% Triton X-100 for 24 hours at 4 °C followed by centrifugation (100000 x g, lh, 4°) and affinity chromatography of the supernatants on 0.5 ml wheatgerm agglutinin Sepharose 6MB columns. Bound receptors were eluted from the column with 0.3M N-acetyl-D-glucosamine. Receptor binding was assessed using a tracer amount of A-14 125I-insulin (∼35 pM) and increasing concentrations of unlabelled insulin (0-10 μM) in a total volume of 150 μl of pH 7.4 buffer as described by Magri et al (1990).

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