title: Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes.
abstract: Protein phosphatase-1 (PP1) plays an important role in the regulation of glycogen synthesis by insulin. Protein targeting to glycogen (PTG) enhances glycogen accumulation by increasing PP1 activity against glycogen-metabolizing enzymes. However, the specificity of PTG's effects on cellular dephosphorylation and glucose metabolism is unclear. Overexpression of PTG in 3T3-L1 adipocytes using a doxycycline-controllable adenoviral construct resulted in a 10-20-fold increase in PTG levels and an 8-fold increase in glycogen levels. Inclusion of 1 microg/ml doxycycline in the media suppressed PTG expression, and fully reversed all PTG-dependent effects. Infection of 3T3-L1 adipocytes with the PTG adenovirus caused a marked dephosphorylation and activation of glycogen synthase. The effects of PTG seemed specific, because basal and insulin-stimulated phosphorylation of a variety of signaling proteins was unaffected. Indeed, glycogen synthase was the predominant protein whose phosphorylation state was decreased in 32P-labeled cells. PTG overexpression did not alter PP1 protein levels but increased PP1 activity 6-fold against phosphorylase in vitro. In contrast, there was no change in PP1 activity measured using myelin basic protein, suggesting that PTG overexpression specifically directed PP1 activity against glycogen-metabolizing enzymes. To investigate the metabolic consequences of altering PTG levels, glucose uptake and storage in 3T3-L1 adipocytes was measured. PTG overexpression did not affect 2-deoxy-glucose transport rates in basal and insulin-stimulated cells but dramatically enhanced glycogen synthesis rates under both conditions. Despite the large increases in cellular glucose flux upon PTG overexpression, basal and insulin-stimulated glucose incorporation into lipid were unchanged. Cumulatively, these data indicate that PTG overexpression in 3T3-L1 adipocytes discretely stimulates PP1 activity against glycogen synthase and phosphorylase, resulting in a marked and specific increase in glucose uptake and storage as glycogen.
authors.affiliation: Department of Medicine, The University of Chicago, Chicago, Illinois 60637, USA.
authors.forename: Cynthia C
authors.initials: CC
authors.lastname: Greenberg
authors.forename: Kimberly N
authors.initials: KN
authors.lastname: Meredith
authors.forename: Limei
authors.initials: L
authors.lastname: Yan
authors.forename: Matthew J
authors.initials: MJ
authors.lastname: Brady
mesh_terms: D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D000906:Antibodies; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D006003:Glycogen / Q000378:metabolism*; D006006:Glycogen Synthase / Q000276:immunology / Q000378:metabolism; D007004:Hypoglycemic Agents / Q000494:pharmacology; D007328:Insulin / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D007668:Kidney / Q000166:cytology; D051379:Mice; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010766:Phosphorylation; D054645:Protein Phosphatase 1; D011817:Rabbits; D013379:Substrate Specificity
chemical_list: D000906:Antibodies; D002352:Carrier Proteins; D007004:Hypoglycemic Agents; D007328:Insulin; D047908:Intracellular Signaling Peptides and Proteins; C104748:Ppp1r3c protein, mouse; D006003:Glycogen; D006006:Glycogen Synthase; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1