Reconstitution of Insulin Signaling Pathways in Rat 3Y1 Cells Lacking Insulin Receptor and Insulin Receptor Substrate-1: EVIDENCE THAT ACTIVATION OF Akt IS INSUFFICIENT FOR INSULIN-STIMULATED GLYCOGEN SYNTHESIS OR GLUCOSE UPTAKE IN RAT 3Y1 CELLS

Takanobu Imanaka,Hideki Hayashi,Kazuhiro Kishi,Lihong Wang,Kazuo Ishii,Osamu Hazeki,Toshiaki Katada,Yousuke Ebina

doi:10.1074/jbc.273.39.25347

Takanobu Imanaka, Hideki Hayashi + Show 6 more

Open Access

https://doi.org/10.1074/jbc.273.39.25347

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Abstract

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1. To investigate the role of IR and IRS-1 in effects of insulin, we transfected IR and IRS-1 expression plasmids into cells and reconstituted the insulin signaling pathways. 3Y1 cells stably expressing the c-myc epitope-tagged glucose transporter type 4 (3Y1-GLUT4myc) exhibit no effects of insulin, at physiological concentrations. The 3Y1-GLUT4myc-IR cells expressing GLUT4myc and IR responded to phosphatidylinositol 3,4, 5-trisphosphate (PI-3,4,5-P3) accumulation, Akt activation, the stimulation of DNA synthesis, and membrane ruffling but not to glycogen synthesis, glucose uptake, or GLUT4myc translocation. The further expression of IRS-1 in 3Y1-GLUT4myc-IR cells led to stimulation of glycogen synthesis but not to glucose uptake or GLUT4myc translocation in response to insulin, although NaF or phorbol 12-myristate 13-acetate did trigger GLUT4myc translocation in the cells. These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for GLUT4 translocation.

Highlights

Rat 3Y1 cells have endogenous insulin-like growth factor-1 receptors and insulin receptor substrate (IRS)-2, but lack both insulin receptor (IR) and IRS-1
These results suggest that, in rat 3Y1 cells, (i) IRS-1 is essential for insulin-stimulated glycogen synthesis but not for DNA synthesis, PI-3,4,5-P3 accumulation, Akt phosphorylation, or membrane ruffling, and (ii) the accumulation of PI-3,4,5-P3 and activation of Akt are insufficient for glycogen synthesis, glucose uptake or for glucose transporter type 4 (GLUT4) translocation
Proteins were visualized using ECL (Amersham Pharmacia Biotech). Lack of both IR and IRS-1 in 3Y1 Cells—We found that rat 3Y1 cells have no detectable amounts of IR and IRS-1

Summary

EXPERIMENTAL PROCEDURES

Immunoblotting—Cell lysates or the immunoprecipitated proteins described above were boiled for 3 min in Laemmli sample buffer and subjected to SDS-polyacrylamide gel electrophoresis; the separated proteins were transferred to nitrocellulose filter and were probed with the indicated antibodies and detected using horseradish peroxidaseconjugated anti-mouse or anti-rabbit antibodies and ECL systems (Amersham Pharmacia Biotech). The cells were further incubated with 0.5 ␮Ci of [methyl-3H]thymidine (Amersham Pharmacia Biotech)/well for 1 h, washed twice with ice-cold phosphate-buffered saline (PBS, pH 7.4), and precipitated with cold 10% trichloroacetic acid. Cell Surface Anti-c-myc Antibody Binding Assay (GLUT4 Translocation)—Cells in 24-well plates were incubated in 500 ␮l of Krebs-RingerHepes buffer for 30 min at 37 °C and stimulated with the indicated ligands for 30 min at 37 °C.

RESULTS

DISCUSSION

Glucose uptake