The effect of modulating the glycogen-associated regulatory subunit of protein phosphatase-1 on insulin action in rat skeletal muscle cells

Abstract

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-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 antisense orientations. Transfection with the vector containing the PP-1(G) gene 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 wild-type L6 cells and neo control cells harboring an empty expression vector. Compared to the neo control, overexpression of PP- 1(G) resulted in a 3-fold increase in insulin-stimulated PP-1 catalytic activity bound to PP-1(G) 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-1(G), 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 vive and plays an important role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.