title: Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism.
abstract: Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.
authors.affiliation: Dipartimento di Biologia e Patologia Cellulare e Molecolare & Centro di Endocrinologia ed Oncologia Sperimentale del Consiglio Nazionale delle Ricerche, Università di Napoli Federico II, Via S. Pansini, 5, 80131 Naples, Italy.
authors.forename: Claudia
authors.initials: C
authors.lastname: Miele
authors.forename: Audrey
authors.initials: A
authors.lastname: Riboulet
authors.forename: Maria Alessandra
authors.initials: MA
authors.lastname: Maitan
authors.forename: Francesco
authors.initials: F
authors.lastname: Oriente
authors.forename: Chiara
authors.initials: C
authors.lastname: Romano
authors.forename: Pietro
authors.initials: P
authors.lastname: Formisano
authors.forename: Jean
authors.initials: J
authors.lastname: Giudicelli
authors.forename: Francesco
authors.initials: F
authors.lastname: Beguinot
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Van Obberghen
mesh_terms: D000818:Animals; D000963:Antimetabolites / Q000494:pharmacology; D003847:Deoxyglucose / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005947:Glucose / Q000378:metabolism*; D017127:Glycation End Products, Advanced; D006006:Glycogen Synthase / Q000378:metabolism; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism; D000071679:Glycogen Synthase Kinase 3 beta; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D008239:Lysine / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018482:Muscle, Skeletal / Q000166:cytology*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D051381:Rats; D017382:Reactive Oxygen Species; D012709:Serum Albumin / Q000378:metabolism*; D015398:Signal Transduction; D013936:Thymidine / Q000378:metabolism; D013997:Time Factors; D000093423:Glycated Serum Albumin
chemical_list: D000963:Antimetabolites; D017127:Glycation End Products, Advanced; C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011518:Proto-Oncogene Proteins; D017382:Reactive Oxygen Species; D012709:Serum Albumin; D003847:Deoxyglucose; D006006:Glycogen Synthase; D000071679:Glycogen Synthase Kinase 3 beta; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D038362:Glycogen Synthase Kinase 3; C448021:glycogen synthase kinase 3 alpha; D005947:Glucose; D008239:Lysine; D013936:Thymidine; D000093423:Glycated Serum Albumin