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.

Impaired insulin action but normal insulin receptor activity in diabetic rat liver: effect of vanadate

1990 ◽  
Vol 258 (3) ◽  
pp. E459-E467 ◽  
Author(s):  
O. Blondel ◽  
J. Simon ◽  
B. Chevalier ◽  
B. Portha
Keyword(s):  
Insulin Receptor ◽  
Insulin Action ◽  
Diabetic Rats ◽  
Receptor Kinase ◽  
Peripheral Tissues ◽  
Impaired Insulin ◽  
Insulin Dependent ◽  
Insulin Receptor Kinase ◽  
Impaired Insulin Action

In vivo insulin resistance is a characteristic of the liver and peripheral tissues in 10-wk-old female rats with non-insulin-dependent diabetes induced by streptozotocin given on day 5 after birth. Oral administration of vanadate (0.2 mg/ml) for 20 days in the diabetic rats lowered their plasma glucose levels to normal values without affecting their basal plasma insulin levels. In the basal state as well as after submaximal or maximal hyperinsulinemia (euglycemic clamp studies), peripheral glucose utilization and hepatic glucose production in vivo were normalized in the diabetic rats after the vanadate treatment. In wheat germ agglutinin purified receptors, 125I-labeled porcine insulin binding, basal and insulin-stimulated insulin receptor kinase activities for both the autophosphorylation of the beta-subunit and the phosphorylation of the artificial substrate poly (Glu-Tyr) 4:1, were found identical in diabetic and control rats, treated or not with vanadate. Liver phosphoenolpyruvate carboxykinase activity was significantly enhanced in untreated diabetic rats (P less than 0.01) as compared with control rats and returned to normal values after the 20-day vanadate treatment. Thus, in that model of non-insulin-dependent diabetes, 1) oral vanadate exerts a corrective insulin-like effect on impaired insulin action both at the level of liver and peripheral tissues, 2) impaired insulin action with no alteration of the insulin receptor tyrosine kinase is observed in the liver of untreated rats, and 3) corrective effect of vanadate on liver glucose metabolism is probably distal to the insulin receptor kinase activity.

scholarly journals The insulinomimetic agents H2O2 and vanadate stimulate tyrosine phosphorylation of potential target proteins for the insulin receptor kinase in intact cells

1992 ◽  
Vol 288 (2) ◽  
pp. 631-635 ◽  
Author(s):  
D Heffetz ◽  
W J Rutter ◽  
Y Zick
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Cho Cells ◽  
Receptor Gene ◽  
Chinese Hamster ◽  
Receptor Kinase ◽  
Intact Cells ◽  
Insulin Receptor Gene ◽  
Protein Tyrosine ◽  
Insulin Receptor Kinase

H2O2 and vanadate are known insulinomimetic agents. Together they induce insulin's bioeffects with a potency which exceeds that seen with insulin, vanadate or H2O2 alone. We have previously shown that a combination of H2O2 and vanadate, when added to intact cells, rapidly stimulates protein tyrosine phosphorylation, owing to the inhibitory effects of these agents on intracellular protein tyrosine phosphatases (PTPases). Employing Western blotting with anti-phosphotyrosine antibodies, we have now identified in Chinese-hamster ovary (CHO) cells transfected with a wild-type insulin-receptor gene (CHO.T cells) several proteins (e.g. pp180, 125, 100, 60 and 52) whose phosphotyrosine content is rapidly increased upon treatment of the cells with a combination of insulin and 3 mM-H2O2. Tyrosine phosphorylation of these and additional proteins was further potentiated when 100 microM-sodium orthovanadate was added together with H2O2. The effects of insulin, insulin/H2O2, and H2O2/vanadate on tyrosine phosphorylation were markedly decreased in CHO cells transfected with an insulin-receptor gene where the twin tyrosines 1162 and 1163 were replaced with phenylalanine (CHO.YF-3 cells). Similarly, most of these proteins failed to undergo enhanced tyrosine phosphorylation in parental CHO cells incubated in the presence of insulin or the insulinomimetic agents. Our findings suggest that inhibition of PTPase activity by H2O2/vanadate augments the autophosphorylation of tyrosines 1162 and 1163 of the insulin receptor kinase, leading to its activation in an insulin-independent manner. As a result, tyrosine phosphorylation of potential targets for this enzyme takes place. Failure of H2O2/vanadate to induce phosphorylation of these proteins in receptor mutants lacking these twin tyrosine residues supports this hypothesis.

scholarly journals Tyrosine Phosphorylation of pp185 by Insulin Receptor Kinase in a Cell-free System

1989 ◽  
Vol 264 (12) ◽  
pp. 6879-6885 ◽  
Author(s):  
Y Tashiro-Hashimoto ◽  
K Tobe ◽  
O Koshio ◽  
T Izumi ◽  
F Takaku ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Tyrosine Phosphorylation ◽  
Receptor Kinase ◽  
Free System ◽  
Cell Free System ◽  
A Cell ◽  
Insulin Receptor Kinase

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.

Rutin potentiates insulin receptor kinase to enhance insulin-dependent glucose transporter 4 translocation

2014 ◽  
Vol 58 (6) ◽  
pp. 1168-1176 ◽  
Author(s):  
Chia-Yu Hsu ◽  
Hung-Yuan Shih ◽  
Yi-Chen Chia ◽  
Chia-Hung Lee ◽  
Hitoshi Ashida ◽  
...  
Keyword(s):  
Insulin Receptor ◽  
Glucose Transporter ◽  
Receptor Kinase ◽  
Glucose Transporter 4 ◽  
Insulin Dependent ◽  
Insulin Receptor Kinase

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

2006 ◽  
Vol 291 (1) ◽  
pp. G16-G25 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Linda Zhang ◽  
Mary C. Stevenson ◽  
Eun Soo Kwak ◽  
William E. Russell
Keyword(s):  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Immunological Methods ◽  
Erbb Receptors ◽  
Erbb2 Expression ◽  
Egfr Expression

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (>40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin β1 (HRG-β1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.

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