The effect of modulating the glycogen-associated regulatory subunit of protein phosphatase-1 on insulin action in rat skeletal muscle cells.
Recent studies from this laboratory have shown that insulin rapidly stimulates a membranous protein phosphatase-1 (PP-1) in cultured rat skeletal muscle cells and isolated rat adipocytes. Stimulation of PP-1 is accompanied by the phosphorylation of a 160-kDa regulatory subunit of PP-1 (PP-1G). To further evaluate the exact role of this subunit in insulin action, L6 rat skeletal muscle cells were stably transfected with a vector containing the gene for PP-1G in the sense and antisense orientations. Transfection with the vector containing the PP-1G gene in the sense orientation yielded three stable clones with a 4- to 6-fold increase in PP-1G protein expression compared to those of wild-type L6 cells and neo control cells harboring an empty expression vector. Compared to the neo control, overexpression of PP-1G resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity bound to PP-1G immunoprecipitates. These cell lines were examined for insulin's effect on glucose uptake, glycogen synthase activity, and glycogen synthesis. Insulin treatment resulted in an approximately 2-fold increase in 2-deoxyglucose uptake in recombinant cells compared to control cells (P < 0.05). This increase in 2-deoxyglucose transport was accompanied by an approximately 2-fold increase in insulin-stimulated glycogen synthase fractional activity (P < 0.05) and a 2- to 4-fold increase in insulin-stimulated glycogen synthesis compared to control cells. In conjunction with these observations, we found that an 85% depletion of endogenous PP-1G, using antisense constructs, resulted in a complete lack of PP-1 activation and an inhibition of basal and insulin-stimulated glucose transport. We conclude that the PP-1G holoenzyme is the major phosphatase regulated by insulin in vivo and plays an important role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.
Ragolia
L
L
Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501, USA.
Begum
N
N
D017667:Adipocytes / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D001692:Biological Transport; D002459:Cell Fusion; D002460:Cell Line; D002478:Cells, Cultured; D003847:Deoxyglucose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D006006:Glycogen Synthase / Q000378:metabolism; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D046911:Macromolecular Substances; D018482:Muscle, Skeletal / Q000187:drug effects / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D014162:Transfection
D007328:Insulin; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D006003:Glycogen; D003847:Deoxyglucose; D006006:Glycogen Synthase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D005947:Glucose