Dzyakanchuk Anna A, Balázs Zoltán, Nashev Lyubomir G, Amrein Kurt E, Odermatt Alex
Division of Molecular and Systems Toxicology, Department of Pharmaceutical Sciences, University of Basel, Klingelbergstrasse 50, CH-4056 Basel, Switzerland.
Mol Cell Endocrinol. 2009 Mar 25;301(1-2):137-41. doi: 10.1016/j.mce.2008.08.009. Epub 2008 Aug 20.
To assess the impact of the NADPH/NADP(+) ratio and the influence of extracellular glucose on 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity, we applied microsomal preparations and intact HEK-293 cells expressing 11beta-HSD1 in the presence or absence of hexose-6-phosphate dehydrogenase (H6PDH). A NADPH/NADP(+) ratio of ten or higher was required for efficient microsomal 11beta-HSD1 reductase activity. Measurements in intact cells suggested that the ER-luminal NADPH concentration is highly sensitive to fluctuating extracellular glucose levels. Lowering glucose in the culture medium dose-dependently decreased 11beta-HSD1 reductase activity and diminished the cortisol/cortisone ratio measured after 24h of incubation. Coexpression with H6PDH potentiated 11beta-HSD1 reductase activity at high glucose. This effect was significantly decreased at low glucose, with concomitantly increased 11beta-HSD1 dehydrogenase activity. In contrast, 11beta-HSD1 reductase activity in H4IIE liver cells and in 3T3-L1 adipocytes was less sensitive to changes in the medium. 11beta-HSD1 dehydrogenase activity was observed in H4IIE cells only at subphysiological glucose levels, indicating a highly efficient supply of substrate for H6PDH and NADPH generation in the ER-lumen. Our results suggest significant cell type-specific differences in ER-luminal NADPH generation that might allow a fine-tuned regulation of glucocorticoid action.
为了评估NADPH/NADP(+)比值的影响以及细胞外葡萄糖对11β-羟基类固醇脱氢酶1（11β-HSD1）活性的影响，我们应用微粒体制剂和表达11β-HSD1的完整HEK-293细胞，分别在存在或不存在己糖-6-磷酸脱氢酶（H6PDH）的情况下进行实验。高效的微粒体11β-HSD1还原酶活性需要NADPH/NADP(+)比值达到10或更高。对完整细胞的测量表明，内质网腔的NADPH浓度对细胞外葡萄糖水平的波动高度敏感。降低培养基中的葡萄糖剂量依赖性地降低了11β-HSD1还原酶活性，并降低了孵育24小时后测得的皮质醇/可的松比值。与H6PDH共表达可增强高葡萄糖条件下的11β-HSD1还原酶活性。在低葡萄糖条件下，这种作用显著降低，同时11β-HSD1脱氢酶活性增加。相比之下，HepG2肝细胞和3T3-L1脂肪细胞中的11β-HSD1还原酶活性对培养基变化的敏感性较低。仅在亚生理葡萄糖水平下在HepG2细胞中观察到11β-HSD1脱氢酶活性，这表明内质网腔中为H6PDH和NADPH生成提供了高效的底物供应。我们的结果表明，内质网腔中NADPH生成存在显著的细胞类型特异性差异，这可能允许对糖皮质激素作用进行微调。
