The regulation of glycogen synthase by protein phosphatase 1 in 3T3-L1 adipocytes. Evidence for a potential role for DARPP-32 in insulin action.
The stimulation of glycogen-targeted protein phosphatase 1 (PP1), glycogen synthase, and glycogen synthesis by insulin was examined during the differentiation of 3T3-L1 fibroblasts into adipocytes. Insulin treatment barely changed the low levels of glycogen synthesis measured in fibroblasts. Following differentiation into adipocytes, insulin increased glycogen synthesis up to 40-fold. After further culturing of the adipocytes for a week, insulin stimulated glycogen accumulation 700-fold. Differentiation of 3T3-L1 cells also resulted in the increased expression of glycogen synthase and in increases in both total glycogen synthase activity and -fold stimulation by insulin. While the levels of PP1 protein were unchanged by differentiation, PP1 specific activity decreased over 60%, although sensitivity to insulin treatment was augmented. Concurrently, levels of the PP1 inhibitor protein DARPP-32 were dramatically induced upon 3T3-L1 adipogenesis. DARPP-32 in both 3T3-L1 and primary rat adipocytes was exclusively localized to the particulate fractions, including the glycogen-enriched pellet. PP1 activity from 3T3-L1 adipocytes exhibited a kinetic lag in vitro, which was not present in fibroblast extracts. Insulin pretreatment of the adipocyte cells overcame the in vitro lag in PP1 activity, resulting in up to 5-fold stimulation of PP1 activity being measured at early assay time points. These results suggest that in 3T3-L1 adipocytes, DARPP-32 may maintain glycogen-targeted PP1 activity in a low basal state, priming the phosphatase for stimulation by insulin.
Brady
M J
MJ
Department of Cell Biology, Parke-Davis Pharmaceutical Research Division, Ann Arbor, Michigan 48105, USA.
Nairn
A C
AC
Saltiel
A R
AR
D016475:3T3 Cells; D017667:Adipocytes / Q000166:cytology / Q000201:enzymology*; D000818:Animals; D002454:Cell Differentiation; D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004791:Enzyme Inhibitors / Q000378:metabolism*; D006003:Glycogen / Q000096:biosynthesis; D006006:Glycogen Synthase / Q000378:metabolism*; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D054645:Protein Phosphatase 1; D051381:Rats; D015398:Signal Transduction
D051937:Dopamine and cAMP-Regulated Phosphoprotein 32; D004791:Enzyme Inhibitors; D007328:Insulin; D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D006003:Glycogen; D006006:Glycogen Synthase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1