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 st imulates a membranous protein phosphatase-l (PP-I) in cultured rat ske letal 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-1(G)). 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-1(G) in the sense and antisen se orientations. Transfection with the vector containing the PP-1(G) g ene in the sense orientation yielded three stable clones with a 4- to 6-fold increase in PP-1(G) protein expression compared to those of wil d-type L6 cells and neo control cells harboring an empty expression ve ctor. Compared to the neo central, overexpression of PP-1(G) resulted in a 3-fold increase in insulin-stimulated PP-I catalytic activity bou nd to PP-1(G) immunoprecipitates. These cell lines were examined for i nsulin's effect on glucose uptake, glycogen synthase activity, and gly cogen synthesis, insulin treatment resulted in an approximately 2-fold increase in 2-deoxyglucose uptake in recombinant cells compared to co ntrol cells (P < 0.05). This increase in P-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 inc rease in insulin-stimulated glycogen synthesis compared to control cel ls. In conjunction with these observations, we found that an 85% deple tion of endogenous PP-1(G), using antisense constructs, resulted in a complete lack of PP-1 activation and an inhibition of basal and insuli n-stimulated glucose transport. We conclude that the PP-1G holoenzyme is the major phosphatase regulated by insulin in vivo and plays an imp ortant role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.