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.
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.
Imanaka
T
T
Division of Molecular Genetics, Institute for Enzyme Research, University of Tokushima, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan.
Hayashi
H
H
Kishi
K
K
Wang
L
L
Ishii
K
K
Hazeki
O
O
Katada
T
T
Ebina
Y
Y
D000595:Amino Acid Sequence; D000818:Animals; D029681:Arabidopsis Proteins; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D004261:DNA Replication; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism*; D051275:Glucose Transporter Type 4; D006003:Glycogen / Q000096:biosynthesis*; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D015221:Potassium Channels / Q000378:metabolism; D051381:Rats; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction
D029681:Arabidopsis Proteins; D051275:Glucose Transporter Type 4; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D018129:Phosphatidylinositol Phosphates; D010750:Phosphoproteins; D010940:Plant Proteins; D015221:Potassium Channels; C495694:Slc2a4 protein, rat; C074500:AKT1 protein, Arabidopsis; D006003:Glycogen; D011972:Receptor, Insulin; D005947:Glucose