title: Impaired phosphorylation and insulin-stimulated translocation to the plasma membrane of protein kinase B/Akt in adipocytes from Type II diabetic subjects.
abstract: OBJECTIVE
  To examine protein kinase B/Akt distribution and phosphorylation in response to insulin in different subcellular fractions of human fat cells from healthy subjects and subjects with Type II (non-insulin-dependent) diabetes mellitus.
  
  
  METHODS
  We prepared subcellular fractions of plasma membranes (PM), low density microsomes and cytosol and examined gene and protein expression as well as serine and threonine phosphorylation in response to insulin.
  
  
  RESULTS
  Protein kinase B/Akt mRNA as well as total protein kinase B/Akt protein in whole-cell lysate and cytosol were similar in both groups. Insulin increased protein kinase B/Akt translocation to the the plasma membrane about twofold [(p < 0.03) in non-diabetic cells but this effect was impaired in diabetic cells (approximately 30%; p > 0.1)]. In both groups, protein kinase B/Akt threonine phosphorylation considerably increased in low density microsomes and cytosol whereas serine phosphorylation was predominant in the plasma membrane. Phosphatidylinositol-dependent kinase 1, which partially activates and phosphorylates protein kinase B/Akt on the specific threonine site, was predominant in cytosol but it was also recovered in low density microsomes. Serine phosphorylation in response to insulin was considerably reduced (50-70 %; p < 0.05) in diabetic cells but threonine phosphorylation was less reduced (approximately 20%). Wortmannin inhibited these effects of insulin supporting a role for PI3-kinase activation.
  
  
  CONCLUSIONS
  Insulin stimulates a differential subcellular pattern of phosphorylation of protein kinase B/Akt. Furthermore, insulin-stimulated translocation of protein kinase B/Akt to the plasma membrane, where serine phosphorylation and full activation occurs, is impaired in Type II diabetes. Threonine phosphorylation was much less reduced. This discrepancy may be related to differential activation of phosphatidylinositol 3-kinase in the different subcellular compartments and phosphatidylinositol-dependent kinase 1 having high affinity for phosphatidylinositol phosphate 3.
authors.affiliation: Department of Internal Medicine, Gothenburg University, Sahlgrenska University Hospital, Sweden.
authors.forename: E
authors.initials: E
authors.lastname: Carvalho
authors.forename: B
authors.initials: B
authors.lastname: Eliasson
authors.forename: C
authors.initials: C
authors.lastname: Wesslau
authors.forename: U
authors.initials: U
authors.lastname: Smith
mesh_terms: D017667:Adipocytes / Q000187:drug effects / Q000201:enzymology*; D000273:Adipose Tissue / Q000201:enzymology; D002462:Cell Membrane / Q000201:enzymology*; D003924:Diabetes Mellitus, Type 2 / Q000201:enzymology*; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000494:pharmacology*; D008861:Microsomes / Q000201:enzymology; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt; D012333:RNA, Messenger / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D007328:Insulin; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt