title: Effect of high glucose on mesangial cell protein kinase C-delta and -epsilon is polyol pathway-dependent.
abstract: In diabetes mellitus, enhanced activity of mesangial cell protein kinase C (PKC) may contribute to nephropathy. The purpose of this study was to determine whether high glucose alters mesangial cell diacylglycerol-sensitive PKC-alpha, -beta2, -delta, and -epsilon content, cellular distribution, and activity through polyol pathway activation. Primary cultured rat mesangial cells (passage 10) were growth-arrested in 0.5% fetal bovine serum and cultured in 5.6 mM glucose (NG) or 30 mM glucose (HG) for 48 h, with or without the aldose reductase inhibitor tolrestat or ARI-509. PKC isoform content in total cell lysates, or cytosol, membrane (Triton X-soluble), and particulate (sodium dodecyl sulfate-soluble) fractions was analyzed by immunoblotting, and band density in HG was expressed as a percentage of corresponding NG values. In HG at 48 h, increased total PKC-alpha (222 +/- 17% of NG, P < 0.001), -beta2 (209 +/- 12%, P < 0.001), and -epsilon (195 +/- 19%, P < 0.001) were observed. L-Glucose had no effect on total PKC isoform content. HG caused increased membrane- and particulate-associated PKC-alpha (257 +/- 87 and 327 +/- 66%, respectively, P < 0.05), membrane-associated PKC-delta (143 +/- 10%, P < 0.05), and membrane-associated PKC-epsilon (186 +/- 11%, P < 0.001), with no change in cytosol contents. The HG effects were not mimicked by L-glucose. In NG or HG, PKC-beta2 was not detected in the cytosol fraction, and membrane and particulate association were unchanged with phorbol ester stimulation. Confocal immunofluorescence imaging revealed that in HG, PKC-alpha, -delta, and -epsilon translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate increased from 18 +/- 1 pmol/min per mg cell protein in NG to 33 +/- 3 pmol/min per mg cell protein in HG (P < 0.002 versus NG). In NG, tolrestat and ARI-509 exposure caused increased PKC activity, enhanced accumulation of total PKC-alpha and -beta2, with no change in total or fractional recovery of PKC-delta or -epsilon. In HG, tolrestat and ARI-509 prevented the increase in total PKC-epsilon and membrane-associated PKC-delta and -epsilon. It is concluded that within 48 h of HG, enhanced mesangial cell PKC activity is associated with accumulation and cellular redistribution of diacylglycerol-sensitive PKC isoforms, and that increased PKC-epsilon content and membrane-associated PKC-delta and -epsilon are dependent on polyol pathway activation.
authors.affiliation: Institute of Medical Science and Department of Medicine, University of Toronto, Ontario, Canada.
authors.forename: J
authors.initials: J
authors.lastname: Kapor-Drezgic
authors.forename: X
authors.initials: X
authors.lastname: Zhou
authors.forename: T
authors.initials: T
authors.lastname: Babazono
authors.forename: J A
authors.initials: JA
authors.lastname: Dlugosz
authors.forename: T
authors.initials: T
authors.lastname: Hohman
authors.forename: C
authors.initials: C
authors.lastname: Whiteside
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005920:Glomerular Mesangium / Q000166:cytology / Q000201:enzymology*; D005947:Glucose / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000378:metabolism*; D008297:Male; D018613:Microscopy, Confocal; D009281:Naphthalenes / Q000494:pharmacology; D011108:Polymers / Q000378:metabolism* / Q000494:pharmacology; D011493:Protein Kinase C / Q000378:metabolism*; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D051381:Rats; D017207:Rats, Sprague-Dawley; D012016:Reference Values; D012680:Sensitivity and Specificity; D013012:Sorbitol / Q000378:metabolism / Q000494:pharmacology
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D009281:Naphthalenes; D011108:Polymers; C024617:polyol; C040550:tolrestat; D013012:Sorbitol; C497260:Prkcd protein, rat; C497257:Prkce protein, rat; D011493:Protein Kinase C; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D005947:Glucose