Human Glycated Albumin Affects Glucose Metabolism in L6 Skeletal Muscle Cells by Impairing Insulin-induced Insulin Receptor Substrate (IRS) Signaling through a Protein Kinase Cα-mediated Mechanism
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m301088200
ISSN1083-351X
AutoresClaudia Miele, Audrey Riboulet, Maria Alessandra Maitan, Francesco Oriente, Chiara Romano, Pietro Formisano, Jean Giudicelli, Francesco Béguinot, Emmanuel Van Obberghen,
Tópico(s)Natural Antidiabetic Agents Studies
ResumoNonenzymatic 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 GSK3α. 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 PKCα, 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. 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 GSK3α. 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 PKCα, 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. Insulin plays a major role in regulating metabolic pathways associated with energy storage and utilization and with cellular proliferation. Following insulin binding, insulin receptor tyrosine kinase is activated, leading to the phosphorylation of several intracellular protein substrates, including IRS-1/2/3/4 proteins and Shc 1The abbreviations used are: ShcSrc-Homology-CollagenAGEsadvanced glycation end productsBDMbisindolylmaleimideCM-DCFchloro-methyl-2′7′-dichlorofluorescein diacetateERKextracellular-regulated kinaseGSKglycogen synthase kinaseHGAhuman glycated albuminHAhuman albumin (nonglycated)IRSinsulin receptor substratePDTCpyrrolidonedithiocarbamatePI3Kphosphoinositide 3-kinasePKBprotein kinase BPKCprotein kinase CROSreactive oxygen speciesUDPGuridine 5′-diphosphate-glucosephosphoYphosphotyrosine2-DG2-deoxy-d-glucose.1The abbreviations used are: ShcSrc-Homology-CollagenAGEsadvanced glycation end productsBDMbisindolylmaleimideCM-DCFchloro-methyl-2′7′-dichlorofluorescein diacetateERKextracellular-regulated kinaseGSKglycogen synthase kinaseHGAhuman glycated albuminHAhuman albumin (nonglycated)IRSinsulin receptor substratePDTCpyrrolidonedithiocarbamatePI3Kphosphoinositide 3-kinasePKBprotein kinase BPKCprotein kinase CROSreactive oxygen speciesUDPGuridine 5′-diphosphate-glucosephosphoYphosphotyrosine2-DG2-deoxy-d-glucose. proteins. These initial events generate multiple signaling cascades that mediate the final cellular responses to insulin (1Pessin J.E. Saltiel A.R. J. Clin. Investig. 2000; 106: 165-169Crossref PubMed Scopus (659) Google Scholar, 2Van Obberghen E. Baron V. Delahaye L. Emanuelli B. Filippa N. Giorgetti-Peraldi S. Lebrun P. Mothe-Satney I. Peraldi P. Rocchi S. Sawka-Verhelle D. Tartare-Deckert S. Giudicelli J. Eur J. Clin. Investig. 2001; 31: 966-977Crossref PubMed Scopus (78) Google Scholar). Insulin-activated signaling modules include the Ras/ERK (3White M.F. Mol. Cell Biochem. 1998; 182: 3-11Crossref PubMed Scopus (622) Google Scholar), the PI3K/PKB (4Alessi D.R. Cohen P. Curr. Opin. Genet. Dev. 1998; 8: 55-62Crossref PubMed Scopus (673) Google Scholar), and the PKC pathways (5Standaert M.L. Galloway L. Karnam P. Bandyopadhyay G. Moscat J. Farese R.V. J. Biol. Chem. 1997; 272: 30075-30082Abstract Full Text Full Text PDF PubMed Scopus (408) Google Scholar, 6Farese R.V. Am. J. Physiol. Endocrinol. Metab. 2002; 283: E1-E11Crossref PubMed Scopus (144) Google Scholar). Src-Homology-Collagen advanced glycation end products bisindolylmaleimide chloro-methyl-2′7′-dichlorofluorescein diacetate extracellular-regulated kinase glycogen synthase kinase human glycated albumin human albumin (nonglycated) insulin receptor substrate pyrrolidonedithiocarbamate phosphoinositide 3-kinase protein kinase B protein kinase C reactive oxygen species uridine 5′-diphosphate-glucose phosphotyrosine 2-deoxy-d-glucose. Src-Homology-Collagen advanced glycation end products bisindolylmaleimide chloro-methyl-2′7′-dichlorofluorescein diacetate extracellular-regulated kinase glycogen synthase kinase human glycated albumin human albumin (nonglycated) insulin receptor substrate pyrrolidonedithiocarbamate phosphoinositide 3-kinase protein kinase B protein kinase C reactive oxygen species uridine 5′-diphosphate-glucose phosphotyrosine 2-deoxy-d-glucose. Resistance to insulin action is a common abnormality present in major human diseases such as diabetes mellitus and obesity. Insulin resistance in diabetes is genetically determined, but its incidence is also affected by environmental conditions and by factors secondary to diabetes itself (7Kahn B.B. Flier J.S. J. Clin. Investig. 2000; 106: 473-481Crossref PubMed Scopus (2390) Google Scholar). These acquired and secondary factors further impair insulin action in the diabetic individual. For instance, chronic hyperglycemia per se promotes insulin resistance (8Hager S.R. Jochen A.L. Kalkhoff R.K. Am. J. Physiol. Endocrinol. Metab. 1991; 260: E353-E362Crossref PubMed Google Scholar, 9Davidson M.B. Bouch C. Venkatesan N. Karjala R.G. Am. J. Physiol. Endocrinol. Metab. 1994; 267: E808-E813Crossref PubMed Google Scholar). A number of mechanisms have been proposed to explain hyperglycemia-induced insulin resistance. These include abnormalities in the PKC signaling system (10Idris I. Gray S. Donnelly R. Ann. N. Y. Acad. Sci. 2002; 967: 176-182Crossref PubMed Scopus (15) Google Scholar) and activation of the NF-κB transcription factors by chronically elevated glucose concentrations (11Yerneni K.K. Bai W. Khan B.V. Medford R.M. Natarajan R. Diabetes. 1999; 48: 855-864Crossref PubMed Scopus (257) Google Scholar, 12Nishikawa T. Edelstein D. Du X.L. Yamagishi S. Matsumura T. Kaneda Y. Yorek M.A. Beebe D. Oates P.J. Hammes H.P. Giardino I. Brownlee M. Nature. 2000; 404: 787-790Crossref PubMed Scopus (3583) Google Scholar). However, the molecular mechanism(s) through which hyperglycemia exacerbates insulin resistance in diabetes have only partially been elucidated. An additional deleterious effect of chronic hyperglycemia is the increased production of advanced glycation end products (AGEs). Chronic high intracellular glucose concentrations cause an increase in both intracellular and extracellular AGEs (13Degenhardt T.P. Thorpe S.R. Baynes J.W. Cell Mol. Biol. 1998; 44: 1139-1145PubMed Google Scholar, 14Brownlee M. Metabolism. 2000; 49: 9-13Abstract Full Text PDF PubMed Scopus (268) Google Scholar). AGE precursors are formed by several reactions including intracellular auto-oxidation of glucose to glyoxal (15Wells-Knecht K.J. Zyzak D.V. Litchfield J.E. Thorpe S.R. Baynes J.W. Biochemistry. 1995; 34: 3702-3709Crossref PubMed Scopus (548) Google Scholar) and breakdown of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate to methylglyoxal (16Thornalley P.J. Biochem. J. 1990; 269: 1-11Crossref PubMed Scopus (672) Google Scholar). AGEs are generated through the interaction of these intracellular α-dicarbonyl precursors with the amino groups of both intra- and extracellular proteins (17Brownlee M. Nature. 2001; 414: 813-820Crossref PubMed Scopus (6864) Google Scholar). Chronic hyperglycemia also leads to the production of Amadori products through the nonenzymatic glycation reactions between glucose and reactive amino groups of serum proteins. Depending on the protein turnover rate and glucose concentration, these Amadori products undergo further irreversible reactions to form AGEs. The modifications of proteins that lead to their glycation induce alterations in their biological properties as compared with their non-glycated counterparts. Several studies have shown that elevated concentrations of Amadori products such as glycated albumin are associated with diabetic atherogenesis by activating vascular smooth muscle cells (18Hattori Y. Suzuki M. Hattori S. Kasai K. Hypertension. 2002; 39: 22-28Crossref PubMed Scopus (122) Google Scholar). Glycated albumin has also been implicated in the development of diabetic retinopathy (19Ruggiero-Lopez D. Rellier N. Lecomte M. Lagarde M. Wiernsperger N. Diabetes Res. Clin. Pract. 1997; 34: 135-142Abstract Full Text PDF PubMed Scopus (31) Google Scholar) by induction of vascular endothelial growth factor expression (20Treins C. Giorgetti-Peraldi S. Murdaca J. Van Obberghen E. J. Biol. Chem. 2001; 276: 43836-43841Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar, 21Yamagishi S.-i. Inagaki Y. Okamoto T. Amano S. Koga K. Takeuchi M. Makita Z. J. Biol. Chem. 2002; 277: 20309-20315Abstract Full Text Full Text PDF PubMed Scopus (301) Google Scholar) and the stimulation of choroidal endothelial cell proliferation (22Hoffmann S. Friedrichs U. Eichler W. Rosenthal A. Wiedemann P. Graefes Arch Clin Exp Ophthalmol. 2002; 240: 996-1002Crossref PubMed Scopus (69) Google Scholar). Finally, glycated albumin has been shown to participitate in the development of diabetic nephropathy by the induction of the cellular formation of cytokines and growth factors (23Makita Z. Yanagisawa K. Kuwajima S. Yoshioka N. Atsumi T. Hasunuma Y. Koike T. J. Diabetes Complic. 1995; 9: 265-268Crossref PubMed Scopus (37) Google Scholar), which may themselves contribute to diabetic renal disease (24Flyvbjerg A. Diabetologia. 2000; 43: 1205-1223Crossref PubMed Scopus (195) Google Scholar). Glycated albumin and AGEs exert their effects through specific cellular receptors found in different cell types and through the activation of several signaling pathways (25Schmidt A.M. Yan S.D. Yan S.F. Stern D.M. Biochim. Biophy. Acta, Mol. Cell Res. 2000; 1498: 99-111Crossref PubMed Scopus (595) Google Scholar, 26Wu V.-Y. Shearman C.W. Cohen M.P. Biochem. Biophys. Res. Commun. 2001; 284: 602-606Crossref PubMed Scopus (17) Google Scholar). Thus, glycated albumin-receptor binding elicits a signal transduction pathway leading to the generation of oxygen free radicals. These reactive oxygen species activate the redox-sensitive transcription factor NF-κB (18Hattori Y. Suzuki M. Hattori S. Kasai K. Hypertension. 2002; 39: 22-28Crossref PubMed Scopus (122) Google Scholar), a pleiotropic regulator of many genes. Recently, Naitoh et al. (27Naitoh T. Kitahara M. Tsuruzoe N. Cell. Signal. 2001; 13: 331-334Crossref PubMed Scopus (24) Google Scholar) demonstrated that, in human monocytic cells, glycated albumin induces the release of TNF-α, a factor involved in insulin resistance (28Peraldi P. Spiegelman B. Mol. Cell Biochem. 1998; 182: 169-175Crossref PubMed Scopus (236) Google Scholar). Although glycated albumin has been linked to the vascular complications of diabetes, it is presently unclear whether exposure to glycated albumin induces resistance to insulin and at which step in the insulin-signaling cascade this may occur. We hypothesized that glycated albumin may be involved in the modulation of insulin signaling and hence in the generation of insulin resistance. To investigate this hypothesis, we analyzed the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. We demonstrated that, in these cells, exposure to human glycated albumin selectively inhibits the PI3K/PKB branch of the insulin signaling cascade, while leaving the Ras-ERK pathway and mitogenic action of the hormone unaltered. Mechanistically, HGA-mediated PI3K/PKB inhibition is dependent on a PKCα-mediated serine/threonine phosphorylation of IRS-1/2 proteins that leads to a strong decrease in insulin-regulated metabolic responses, such as glucose uptake and glycogen synthesis. Interestingly, activation of PKCα by chronic HGA treatment appears to be independent of ROS production. Thus, by deregulating intracellular insulin signaling, human glycated albumin exacerbates the insulin-resistant state. General—Media and sera for tissue culture were from Invitrogen. Phospho-PKB, phospho-ERK, IRS-1, and IRS-2 antibodies were purchased from Cell Signaling Technology (Beverly MA). PI3K and phospho-GSK-3 antibodies were from Upstate Biotechnology Inc. (Lake Placid, NY). Antibodies directed against PKC isoforms were used as previously described (29Formisano P. Oriente F. Fiory F. Caruso M. Miele C. Maitan M.A. Andreozzi F. Vigliotta G. Condorelli G. Beguinot F. Mol. Cell Biol. 2000; 20: 6323-6333Crossref PubMed Scopus (67) Google Scholar). All other antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). Electrophoresis and Western blot reagents were from Bio-Rad (Richmond, VA), chloro-methyl-2′7′-dichlorofluorescein diacetate was a gift from C. Maziere (Faculté de Médecine, Amiens, France). [γ-32P]ATP (3,000 Ci/mmol), [14C]UDPG, [3H]thymidine, 2-deoxy-d-[3H]glucose, [32P]orthophosphate, and ECL reagents were from Amersham Biosciences. Other reagents were from Sigma. Characterization of Glycated Human Serum Albumin—Glycated and nonglycated human serum albumin were purchased from Sigma Chemical Co. The glycated serum albumin contained 2.7–3.5 mol of fructosamine per mol of albumin. The human glycated and nonglycated albumin preparations were tested for (i) fluorescent advanced glycation end products concentrations, determined by fluorescence assays (from 360 to 600 nm) upon excitation at 370 nm or 350 nm (30Pongor S. Ulrich P.C. Bencsath F.A. Cerami A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2684-2688Crossref PubMed Scopus (367) Google Scholar), (ii) CML concentrations were determined by using a CML-ELISA kit which uses carboxymethyl caproate as standards as described in (31Morcos M. Sayed A.A.R. Bierhaus A. Yard B. Waldherr R. Merz W. Kloeting I. Schleicher E. Mentz S. Abd el Baki R.F. Tritschler H. Kasper M. Schwenger V. Hamann A. Dugi K.A. Schmidt A.-M. Stern D. Ziegler R. Haering H.U. Andrassy M. van der Woude F. Nawroth P.P. Diabetes. 2002; 51: 3532-3544Crossref PubMed Scopus (145) Google Scholar), and (iii) the extent of lysine and arginine modifications, measured using the 2,4,6-trinitrobenzen-suffonic acid and 9,10-phenanthrenequinone methods, respectively (32Fields R. Biochem. J. 1971; 124: 581-590Crossref PubMed Scopus (428) Google Scholar, 33Smith R. MacQuarrie R. Anal. Biochem. 1978; 90: 246-255Crossref PubMed Scopus (98) Google Scholar). Each batch was tested for possible insulin-like growth factor-I contamination by IGF-I-D-RIA-CT (BioSource Europe, Nivelles, Belgium) and the absence of endotoxin (LPS) by the use of Limulus amebocyte lysates assay (Sigma). Each human glycated albumin batch was reconstituted at 10 mg/ml with sterile PBS, and in order to avoid glycoxydation, immediately thereafter frozen at —30 °C until use (34Smith P.R. Thornalley P.J. Eur. J. Biochem. 1992; 210: 729-739Crossref PubMed Scopus (111) Google Scholar). Cell Culture—The L6 skeletal muscle cells were plated (6 × 103 cells/cm2) and grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 2% (v/v) fetal bovine serum and 2 mm glutamine. Cultures were maintained at 37 °C, in a humidified atmosphere containing 5% (v/v) CO2. Under these culture conditions, L6 myoblasts spontaneously differentiate into myotubes upon confluence. For PKC inhibition studies, cells were pretreated with 100 nm BDM for 30 min followed by combined pretreatment with HGA and BDM. Unless otherwise stated all experiments were performed with L6 cells at the myotube stage of differentiation. Immunoblot Analysis—Cells were solubilized for 20 min at 4 °C with lysis buffer containing 50 mm HEPES, 150 mm NaCl, 10 mm EDTA, 10 mm Na4P2O7, 2 mm sodium orthovanadate, 50 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, pH 7.4, and 1% (v/v) Triton X-100 (TAT buffer). The lysates were clarified by centrifugation at 12,000 × g for 15 min at 4 °C, and aliquots were either directly separated by SDS-PAGE or incubated with the indicated antibodies for 18 h at 4 °C. Immune complexes were then precipitated with protein A beads, and resuspended in Laemmli buffer. Proteins were separated by SDS-PAGE and blotted on Immobilon-P membranes (Millipore Corp., Bedford, MA). Membranes were blocked for 1 h in TBS (10 mm Tris-HCl, pH 7.4, 140 mm NaCl), containing 4% (w/v) bovine serum albumin and then incubated with the indicated antibodies. Detection of blotted proteins was performed by ECL according to the manufacturer's instruction. In Vivo Phosphorylation Experiments—Myotubes pretreated or not with HGA for 24 h were washed twice with phosphate-free DMEM and labeled for 12 h in this medium containing 500 μCi of [32P]orthophosphate (1.7 mCi/ml). At the end of labeling, after three washes with ice-cold PBS, proteins were solubilized for 15 min at 4 °C in TAT buffer. The lysates were clarified by centrifugation at 12,000 × g for 15 min at 4 °C and subjected to immunoprecipitation with anti-IRS-1 or IRS-2 antibodies. Immunocomplexes were washed four times with solubilization buffer, boiled in Laemmli sample buffer, and separated by SDS-PAGE. For KOH treatment, the gels were incubated for 1 h at 55 °C in 1 m KOH as described (35Scimeca J.C. Ballotti R. Nguyen T.T. Filloux C. Van Obberghen E. Biochemistry. 1991; 30: 9313-9319Crossref PubMed Scopus (15) Google Scholar). [32P]orthophosphate-labeled IRS-1/2 fractions were detected with a PhosphorImager (Molecular Dynamics) and quantified (ImageQuant software). 2-Deoxy-d-glucose Uptake, Glycogen Synthase Activity, and Thymidine Incorporation Assays—These assays were performed in L6 myotubes as described in (36Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (293) Google Scholar). Briefly, cells were incubated in serum-free α-Dulbecco's modified Eagle's medium supplemented with 0.2% (w/v) bovine serum albumin for 24 h in the presence or absence of 0.1 mg/ml HGA. Cells were incubated in glucose-free 20 mm HEPES, pH 7.4, 140 mm NaCl, 2.5 mm MgSO4, 5 mm KCl, 1 mm CaCl2 (HEPES buffer) and then exposed or not to 100 nm insulin for 10 min. Glucose uptake was measured by incubating cells with 20 μm 2-deoxy-d-[3H]glucose (1 μCi/assay) for 10 min in HEPES buffer. The reaction was terminated by the addition of 10 μm cytochalasin B, and the cells were washed three times with ice-cold isotonic saline solution prior to lysis in 0.1 m NaOH. Incorporated radioactivity was measured in a liquid scintillation counter. Glycogen synthase activity was assayed by a modification of the described method (36Hajduch E. Alessi D.R. Hemmings B.A. Hundal H.S. Diabetes. 1998; 47: 1006-1013Crossref PubMed Scopus (293) Google Scholar). Briefly, L6 myotubes were incubated in HEPES buffer for 3 h before the assay, the cells were then stimulated with 100 nm insulin, resuspended in 10 mm EDTA and sonicated for 10 s at 300 watts. The cell suspension was centrifuged for 10 min at 2,000 × g, and 20-μl aliquots of the supernatants (20 μg of cell protein) were added to 60 μl of a reaction mixture containing 40 mm Tris-HCl, pH 7.8, 25 mm NaF, 20 mm EDTA, 10 mg/ml glycogen, and 7.2 mm uridine 5′-diphosphate-glucose (UDPG) and 0.05 mCi [14C]UDPG in the absence or the presence of 6.7 mm glucose 6-phosphate. The supernatant reaction mixture was incubated for 20 min at 30 °C and terminated by spotting on p81 phosphocellulose filter followed by precipitation with ice-cold ethanol, and the radioactivity was counted by liquid scintillation. Enzyme activity was expressed as percent of the glucose-6-phosphate independent form (% I form). Thymidine incorporation assays were performed as previously described (37Formisano P. Sohn K. Miele C. Di Finizio B. Petruzziello A. Riccardi G. Beguinot L. Beguinot F. J. Biol. Chem. 1993; 268: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Briefly, cells were seeded in 6-well plates at a plating density of 105 cells/well. After 24 h, the culture medium was replaced with serum-free Dulbecco's modified Eagle's medium containing 0.2% (w/v) bovine serum albumin in the presence or absence of HGA, as specified for an additional 24 h. 100 nm insulin and [3H]thymidine were added, and the cells were incubated for an additional 16 h. Kinase Assays—L6 myotubes were deprived of serum for 24 h in the presence or absence of HGA and then exposed to 100 nm insulin for 10 min. For PI3K assays, after insulin stimulation, the cells were solubilized for 30 min at 4 °C in TAT buffer. After lysate clarification by centrifugation at 12,000 × g for 15 min at 4 °C, aliquots of the lysates were subjected to immunoprecipitation with anti-IRS-1 or anti-IRS-2 antibodies coupled to protein A-Sepharose for 2 h at 4 °C. PI3K activity was determined in IRS-1/2 immunoprecipitates as described in Filippa et al. (38Filippa N. Sable C.L. Filloux C. Hemmings B. Van Obberghen E. Mol. Cell Biol. 1999; 19: 4989-5000Crossref PubMed Scopus (230) Google Scholar). For PKB assays, after insulin stimulation, the cells were solubilized for 30 min at 4 °C in TAT buffer. The lysates were clarified by centrifugation at 12,000 × g for 15 min at 4 °C, and PKB was immunoprecipitated using antibodies to PKB. The immune complexes were washed and assayed for PKB activity using Crosstide substrate as previously described (38Filippa N. Sable C.L. Filloux C. Hemmings B. Van Obberghen E. Mol. Cell Biol. 1999; 19: 4989-5000Crossref PubMed Scopus (230) Google Scholar). PKC activity was assayed as previously described (29Formisano P. Oriente F. Fiory F. Caruso M. Miele C. Maitan M.A. Andreozzi F. Vigliotta G. Condorelli G. Beguinot F. Mol. Cell Biol. 2000; 20: 6323-6333Crossref PubMed Scopus (67) Google Scholar). Briefly, cells were solubilized in 20 mm Tris-HCl, pH 7.5, 0.5 mm EDTA, 0.5 mm EGTA, 0.5% (v/v) Triton X-100, 25 μg/ml aprotinin, 25 μg/ml leupeptin. Cell lysates were clarified by centrifugation at 12,000 × g for 20 min and then supplemented with lipid activators (10 μm phorbol 12-myristate 13-acetate, 0.28 mg/ml phosphatidylserine and 4 mg/ml dioleine, final concentrations). Phosphorylation reactions were initiated by addition of 50 μm acetylated myelin basic protein (residues 4–14) substrate in 4 mm Tris, pH 7.5, 1 mm CaCl2, 20 mm MgCl2, 20 μm ATP, and 10 μCi of (3,000 Ci/mmol) [γ-32P]ATP per ml (final concentrations). The reaction mixtures were further incubated for 10 min at room temperature, and then rapidly cooled on ice and spotted onto phosphocellulose discs. Disc-bound radioactivity was quantitated by liquid scintillation counting. PKC activity was calculated by subtracting the nonspecific kinase activity obtained in the absence of lipid activators. Activity of the specific PKC isoforms was assayed as described above, after immunoprecipitation using specific PKC isoform antibodies. Assay for Reactive Oxygen Species Production—Intracellular production of ROS was measured by CM-DCF fluorescence as described (39Maziere C. Floret S. Santus R. Morliere P. Marcheux V. Maziere J.-C. Free Radic. Biol. Med. 2003; 34: 629-636Crossref PubMed Scopus (39) Google Scholar). The cells were treated with H2O2 (500 μm) for 15 min in the absence or presence of the antioxidant PDTC (200 μm), and then exposed to 10—5m CM-DCF in phosphate buffered saline for 45 min. The cells were washed three times in PBS, solubilized in H20, and sonicated. The fluorescence was determined at 503/529 nm, normalized on a protein basis and expressed as percentages of control. Statistical Analysis—Results are expressed as means ± S.D. Statistical significance was evaluated using the Student's t test for unpaired comparison. A value of p < 0.05 was considered statistically significant. Physicochemical Properties of HGA—Table I summarizes the physicochemical properties of the human glycated albumin used in this report. We find that HGA does not contain measurable amounts of fluorescent advanced glycation end products. The CML concentrations observed in our HGA preparations are lower than the level of CML observed in diabetic plasma (32.6 ± 8.3 μg CML/ml) (40Xu B. Chibber R. Ruggerio D. Kohner E. Ritter J. Ferro A. FASEB J. 2003; 17: 1289-1291Crossref PubMed Scopus (85) Google Scholar). Nagai et al. (41Nagai R. Matsumoto K. Ling X. Suzuki H. Araki T. Horiuchi S. Diabetes. 2000; 49: 1714-1723Crossref PubMed Scopus (150) Google Scholar) have observed that methylglyoxal and glyoxal preferentially modified arginine rather than lysine residues. To detect the presence of modified aldehyde in human albumin, the extent of lysine and arginine modifications in human glycated and nonglycated albumin were determined. The results obtained are summarized in Table I and show the absence of significant modifications in the extent of free lysine and arginine residues between the two preparations. Lastly, the batches were found not to contain IGF-I and to be bacterial endotoxin-free. To summarize, the physicochemical properties determined for our HGA preparation demonstrate that this preparation does not contain significant AGEs products and that therefore the effects observed in L6 myotubes after 24 h of preincubation with 0.1 mg/ml of HGA are the consequence of glycated albumin present essentially as a glycated Amadori product.Table IPhysicochemical properties of glycated human albuminNon-glycated human albuminGlycated human albuminFluorescent AGEsUndetectableUndetectableCML/mg protein60 ng170 ngLys modification (%)10091.9 ± 5.2Arg modification (%)10098.6 ± 0.5IGF-1UndetectableUndetectableLPSUndetectableUndetectable Open table in a new tab Effect of HGA on Insulin Action—Insulin-resistant states are associated with alterations affecting glucose metabolism of muscle cells (42Kurowski T. Lin Y. Luo Z. Tsichlis P. Buse M. Heydrick S. Ruderman N. Diabetes. 1999; 48: 658-663Crossref PubMed Scopus (95) Google Scholar, 43Kim Y. Peroni O. Franke T. Kahn B. Diabetes. 2000; 49: 847-856Crossref PubMed Scopus (86) Google Scholar). To determine whether HGA could induce insulin resistance in L6 myotubes, the effect of preincubation for 24 h with 0–0.2 mg/ml HGA on glucose transport was studied. Control cells were treated with nonglycated albumin. In control myotubes not treated with HGA or treated for 24 h with 0.1 mg/ml HA, insulin induced a 2-fold 2-DG uptake. HGA decreased insulin-stimulated glucose uptake in a dose-dependent manner without affecting basal glucose uptake. Maximal inhibition was achieved starting from 0.1 mg/ml HGA (Fig. 1A). To study the time course for HGA-induced insulin resistance in myotubes, cells were preincubated with 0.1 mg/ml HGA for 0–24 h. Insulin-induced glucose uptake was reduced by 60 and 75% upon HGA incubation for 4 and 8 h, respectively (decreases significant at the p < 0.001 level). A 12-h preincubation with 0.1 mg/ml HGA completely abolished insulin stimulation of 2-DG uptake in L6 myotubes, an inhibition conserved for at least 12 additional hours (Fig. 1B). Glycogen synthase activation by insulin was also inhibited by preincubation with HGA (300% increase in the absence of HGA and only 50% upon 24 h HGA preincubation, as shown in Fig. 1C). Similar to 2-DG uptake, the HGA-induced inhibition of glycogen synthase activation was also time- and dose-dependent, reaching a maximum after 12 h of exposure (data not shown). On the c
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