Derylo B, Babazono T, Glogowski E, Kapor-Drezgic J, Hohman T, Whiteside C
Department of Medicine and Institute of Medical Science, University of Toronto, Canada.
Diabetologia. 1998 May;41(5):507-15. doi: 10.1007/s001250050939.
Glomerular mesangial cells cultured in high glucose conditions display impaired contractile responsiveness. It was postulated that glucose metabolism through the polyol pathway leads to altered mesangial cell contractility involving protein kinase C. Rat mesangial cells were growth-arrested for 24 h with 0.5% fetal bovine serum in either normal (5.6 mmol/l) or high (30 mmol/l) glucose concentrations or high glucose plus the aldose reductase inhibitor, ARI-509 (100 micromol/l). The reduction of cell planar surface area (contraction) in response to endothelin-1 (0.1 micromol/l), or to phorbol 12-myristate 13-acetate (50 pmol/l), was studied by videomicroscopy. In response to endothelin-1, mesangial cells in normal glucose contracted to 52+/-3% of initial planar area. In high glucose, the significantly (p < 0.05) smaller cell size and no contractile responsiveness to endothelin-1 were normalized with ARI-509. Membrane-associated diacylglycerol, measured by a kinase specific 32P-phosphorylation assay, in high glucose was unchanged after 3 h, but significantly increased (p < 0.05) after 24 h which was normalized with ARI-509. Protein kinase C activity, measured by in situ 32P-phosphorylation of the epidermal growth factor receptor substrate was: increased by 32% at 3 h of high glucose, unchanged by ARI-509; and decreased significantly (p < 0.05) at 24 h compared to cells in normal glucose, normalized by ARI-509. Total cellular protein kinase C-alpha, -delta and -epsilon, analysed by immunoblotting, were unchanged in high glucose at 24 h. Only protein kinase C-epsilon content was reduced by ARI-509 in both normal and high glucose. Therefore, high glucose-induced loss of mesangial cell contractility, diacylglycerol accumulation and altered protein kinase C activity are mediated through activation of the polyol-pathway, although no specific relationship between elevated diacylglycerol and protein kinase C activity was observed. In high glucose, altered protein kinase C function, or another mechanism related to the polyol pathway, contribute to loss of mesangial cell contractile responsiveness.
在高糖条件下培养的肾小球系膜细胞表现出收缩反应性受损。据推测，通过多元醇途径的葡萄糖代谢导致涉及蛋白激酶C的系膜细胞收缩性改变。将大鼠系膜细胞在含有0.5%胎牛血清的条件下生长停滞24小时，分别置于正常（5.6 mmol/L）或高糖（30 mmol/L）浓度环境中，或高糖环境加醛糖还原酶抑制剂ARI-509（100 μmol/L）。通过视频显微镜研究内皮素-1（0.1 μmol/L）或佛波酯12-肉豆蔻酸酯13-乙酸酯（50 pmol/L）刺激后细胞平面表面积的减少（收缩）情况。对内皮素-1的反应中，正常葡萄糖环境中的系膜细胞收缩至初始平面面积的52±3%。在高糖环境中，细胞大小显著减小（p<0.05）且对内皮素-1无收缩反应性，而ARI-509可使其恢复正常。通过激酶特异性32P磷酸化测定法测量的膜相关二酰甘油，在高糖环境中3小时后无变化，但24小时后显著增加（p<0.05），而ARI-509可使其恢复正常。通过表皮生长因子受体底物的原位32P磷酸化测量的蛋白激酶C活性：在高糖3小时时增加32%，ARI-509对此无影响；与正常葡萄糖环境中的细胞相比，24小时时显著降低（p<0.05），ARI-509可使其恢复正常。通过免疫印迹分析的总细胞蛋白激酶C-α、-δ和-ε，在高糖24小时时无变化。仅蛋白激酶C-ε的含量在正常和高糖环境中均被ARI-509降低。因此，高糖诱导的系膜细胞收缩性丧失、二酰甘油积累和蛋白激酶C活性改变是通过多元醇途径的激活介导的，尽管未观察到二酰甘油升高与蛋白激酶C活性之间的特定关系。在高糖环境中，蛋白激酶C功能改变或与多元醇途径相关的另一种机制导致系膜细胞收缩反应性丧失。
