کد مقاله کد نشریه سال انتشار مقاله انگلیسی نسخه تمام متن
1174019 961722 2009 7 صفحه PDF دانلود رایگان
عنوان انگلیسی مقاله ISI
Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors
موضوعات مرتبط
مهندسی و علوم پایه شیمی شیمی آنالیزی یا شیمی تجزیه
پیش نمایش صفحه اول مقاله
Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors
چکیده انگلیسی

11β-Hydroxysteroid dehydrogenase 1 (11β-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11β-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11β-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11β-HSD1 inhibitor, exhibited an IC50 value similar to that in in vitro microsomal assay (IC50 = 0.3 μM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11β-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.

ناشر
Database: Elsevier - ScienceDirect (ساینس دایرکت)
Journal: Analytical Biochemistry - Volume 392, Issue 2, 15 September 2009, Pages 110–116
نویسندگان
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