Effect of High Glucose on Mesangial Cell Protein Kinase C-δ and -𝛆 is Polyol Pathway-Dependent

Abstract

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-α, -β2, -δ, and -𝛆 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-α (222 ± 17% of NG, P < 0.001), -β2 (209 ± 12%, P < 0.001), and -𝛆 (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-α (257 ± 87 and 327 ± 66%, respectively, P < 0.05), membrane-associated PKC-δ (143 ± 10%, P < 0.05), and membrane-associated PKC-𝛆 (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-β2 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-α, -δ, and -𝛆 translocate to the nucleus and plasma membrane. Total PKC activity measured by in situ32P-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-α and -β2, with no change in total or fractional recovery of PKC-δ or -𝛆. In HG, tolrestat and ARI-509 prevented the increase in total PKC-𝛆 and membrane-associated PKC-δ and -𝛆. 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-𝛆 content and membrane-associated PKC-δ and -𝛆 are dependent on polyol pathway activation.