Protein targeting to glycogen overexpression results in the specific enhancement of glycogen storage in 3T3-L1 adipocytes

J Biol Chem. 2003 Aug 15;278(33):30835-42. doi: 10.1074/jbc.M303846200. Epub 2003 Jun 12.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 3T3 Cells
  • Adenoviridae / genetics
  • Adipocytes / cytology
  • Adipocytes / drug effects
  • Adipocytes / metabolism*
  • Animals
  • Antibodies
  • Carrier Proteins / genetics*
  • Carrier Proteins / metabolism*
  • Gene Expression
  • Glycogen / metabolism*
  • Glycogen Synthase / immunology
  • Glycogen Synthase / metabolism
  • Hypoglycemic Agents / pharmacology
  • Insulin / pharmacology
  • Intracellular Signaling Peptides and Proteins*
  • Kidney / cytology
  • Mice
  • Phosphoprotein Phosphatases / metabolism
  • Phosphorylation
  • Protein Phosphatase 1
  • Rabbits
  • Substrate Specificity

Substances

  • Antibodies
  • Carrier Proteins
  • Hypoglycemic Agents
  • Insulin
  • Intracellular Signaling Peptides and Proteins
  • Ppp1r3c protein, mouse
  • Glycogen
  • Glycogen Synthase
  • Phosphoprotein Phosphatases
  • Protein Phosphatase 1