Overexpression of catalytic subunit p110alpha of phosphatidylinositol 3-kinase increases glucose transport activity with translocation of glucose transporters in 3T3-L1 adipocytes.
To elucidate the mechanisms of phosphatidylinositol (PI) 3-kinase involvement in insulin-stimulated glucose transport activity, the epitope-tagged p110alpha subunit of PI 3-kinase was overexpressed in 3T3-L1 adipocytes using an adenovirus-mediated gene transduction system. Overexpression of p110alpha was confirmed by immunoblot using anti-tagged epitope antibody. p110alpha overexpression induced a 2.5-fold increase in PI 3-kinase activity associated with its regulatory subunits in the basal state, an increase exceeding that of the maximally insulin-stimulated control cells, while PI 3-kinase activity associated with phosphotyrosyl protein was only modestly elevated. Overexpression of p110alpha induced an approximately 14-fold increase in the basal glucose transport rate, which was also greater than that observed in the stimulated control. No apparent difference was observed in the cellular expression level of either GLUT1 or GLUT4 proteins between control and p110alpha-overexpressing 3T3-L1 adipocytes. Subcellular fractionation revealed translocation of glucose transporters from intracellular to plasma membranes in basal p110alpha-overexpressing cells. The translocation of GLUT4 protein to the plasma membrane was further confirmed using a membrane sheet assay. These findings indicate that an increment in PI 3-kinase activity induced by overexpression of p110alpha of PI 3-kinase stimulates glucose transport activity with translocation of glucose transporters, i.e., mimics the effect of insulin.
Katagiri
H
H
Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan.
Asano
T
T
Ishihara
H
H
Inukai
K
K
Shibasaki
Y
Y
Kikuchi
M
M
Yazaki
Y
Y
Oka
Y
Y
D016475:3T3 Cells; D017667:Adipocytes / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D006801:Humans; D007328:Insulin / Q000494:pharmacology; D046911:Macromolecular Substances; D051379:Mice; D009004:Monosaccharide Transport Proteins / Q000378:metabolism*; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D046911:Macromolecular Substances; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011994:Recombinant Proteins; C495677:SLC2A1 protein, human; C495692:SLC2A4 protein, human; C495678:Slc2a1 protein, mouse; C495679:Slc2a1 protein, rat; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat