Central role for protein targeting to glycogen in the maintenance of cellular glycogen stores in 3T3-L1 adipocytes.
Overexpression of the protein phosphatase 1 (PP1) subunit protein targeting to glycogen (PTG) markedly enhances cellular glycogen levels. In order to disrupt the endogenous PTG-PP1 complex, small interfering RNA (siRNA) constructs against PTG were identified. Infection of 3T3-L1 adipocytes with PTG siRNA adenovirus decreased PTG mRNA and protein levels by >90%. In parallel, PTG reduction resulted in a >85% decrease in glycogen levels 4 days after infection, supporting a critical role for PTG in glycogen metabolism. Total PP1, glycogen synthase, and GLUT4 levels, as well as insulin-stimulated signaling cascades, were unaffected. However, PTG knockdown reduced glycogen-targeted PP1 protein levels, corresponding to decreased cellular glycogen synthase- and phosphorylase-directed PP1 activity. Interestingly, GLUT1 levels and acute insulin-stimulated glycogen synthesis rates were increased two- to threefold, and glycogen synthase activation in the presence of extracellular glucose was maintained. In contrast, glycogenolysis rates were markedly increased, suggesting that PTG primarily acts to suppress glycogen breakdown. Cumulatively, these data indicate that disruption of PTG expression resulted in the uncoupling of PP1 activity from glycogen metabolizing enzymes, the enhancement of glycogenolysis, and a dramatic decrease in cellular glycogen levels. Further, they suggest that reduction of glycogen stores induced cellular compensation by several mechanisms, but ultimately these changes could not overcome the loss of PTG expression.
Greenberg
Cynthia C
CC
Department of Medicine, University of Chicago, MC1027, 5841 S. Maryland Ave., Chicago, IL 60637-1470, USA.
Danos
Arpad M
AM
Brady
Matthew J
MJ
D041721:3T3-L1 Cells; D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D020868:Gene Silencing; D005947:Glucose / Q000378:metabolism; D051272:Glucose Transporter Type 1 / Q000378:metabolism; D051275:Glucose Transporter Type 4 / Q000378:metabolism; D006003:Glycogen / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000235:genetics / Q000378:metabolism*; D054645:Protein Phosphatase 1; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology
D051272:Glucose Transporter Type 1; D051275:Glucose Transporter Type 4; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C104748:Ppp1r3c protein, mouse; D034741:RNA, Small Interfering; C495678:Slc2a1 protein, mouse; C495693:Slc2a4 protein, mouse; D006003:Glycogen; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1; D005947:Glucose