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The Effect of Modulating the Glycogen-Associated Regulatory Subunit of Protein Phosphatase-1 on Insulin Action in Rat Skeletal Muscle Cells¹

Diabetes Research Laboratory, Winthrop University Hospital, Mineola, New York 11501; and the School of Medicine, State University of New York, Stony Brook, New York 11794

Address all correspondence and requests for reprints to: Dr. Najma Begum, Diabetes Research Laboratory, Winthrop University Hospital, 259 First Street, Mineola, New York 11501. E-mail: diabetes96{at}aol

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 vivo and plays an important role in insulin-stimulated glycogen synthesis by regulating the catalytic activity of bound PP-1.

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