title: Adenovirus-mediated expression of a naturally occurring Asp905Tyr variant of the glycogen-associated regulatory subunit of protein phosphatase-1 in L6 myotubes.
abstract: OBJECTIVE
  The glycogen-associated protein phosphatase-1 (PP1G) is thought to play an important part in the regulation of skeletal muscle glycogen content. We have previously identified an Asp905Tyr polymorphism of the glycogen-associated regulatory subunit of the protein phosphatase 1 (PPP1R3) gene which among healthy subjects was associated with decreased insulin stimulated non-oxidative glucose metabolism, i.e. primary glycogen synthesis. In this study, the functional effect of the polymorphism was examined in vitro.
  
  
  METHODS
  Wild type (PPP1R3-Asp905) and mutant (PPP1R3-Tyr905) PPP1R3 were expressed in L6 myotubes using adenovirus-mediated gene transfer. Basal and insulin-stimulated glucose uptake and glycogen synthesis were measured. Furthermore, the sensitivity of glycogen synthesis to a cyclic AMP agonist was measured.
  
  
  RESULTS
  Compared with green fluorescent protein-transduced myotubes and non-transduced myotubes, overexpression of PPP1R3-Asp905 and PPP1R3-Tyr905 increased both basal and insulin-stimulated glycogen synthesis approximately twofold. Treatment of both non-transduced and PPP1R3-transduced L6 myotubes with a cAMP agonist decreased both basal and insulin-stimulated glycogen synthesis by about 40%. Overexpression of PPP1R3 did not affect either basal or insulin-stimulated 2-deoxy-D-glucose uptake compared with green fluorescent protein-transduced cells.
  
  
  CONCLUSIONS
  Results obtained from L6 myotubes transduced with PPP1R3-Asp905 or PPP1R3-Tyr905 showed no statistically significant difference. Therefore, the Asp905Tyr variant alone is unlikely to account for the decreased insulin stimulated non-oxidative glucose metabolism observed in the human study reported previously.
authors.affiliation: Steno Diabetes Center, Gentofte, Denmark.
authors.forename: S K
authors.initials: SK
authors.lastname: Rasmussen
authors.forename: L
authors.initials: L
authors.lastname: Hansen
authors.forename: E U
authors.initials: EU
authors.lastname: Frevert
authors.forename: P T
authors.initials: PT
authors.lastname: Cohen
authors.forename: B B
authors.initials: BB
authors.lastname: Kahn
authors.forename: O
authors.initials: O
authors.lastname: Pedersen
mesh_terms: D000256:Adenoviridae; D000818:Animals; D001224:Aspartic Acid; D001692:Biological Transport; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D003847:Deoxyglucose / Q000378:metabolism; D014644:Genetic Variation; D005822:Genetic Vectors; D006003:Glycogen / Q000096:biosynthesis; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000032:analysis; D018482:Muscle, Skeletal / Q000166:cytology / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000096:biosynthesis / Q000235:genetics*; D011110:Polymorphism, Genetic; D054645:Protein Phosphatase 1; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D014162:Transfection; D014443:Tyrosine
chemical_list: D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D001224:Aspartic Acid; D014443:Tyrosine; D006003:Glycogen; D003847:Deoxyglucose; C114205:PPP1R3A protein, human; D010749:Phosphoprotein Phosphatases; D054645:Protein Phosphatase 1