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Cell-based assay for screening 11beta-hydroxysteroid dehydrogenase 1 inhibitors
Young Sik Cho1, Chi Hyun Kim, Hyae Gyeong Cheon
1Center for Metabolic Syndrome Therapeutics, Korea Research Institute of Chemical Technology, Yuseong-gu, Daejeon 305-600, Republic of Korea.
Analytical Biochemistry
|June 6, 2009
Summary
We developed a cell-based assay to measure 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) activity in skeletal muscle cells. This assay is useful for screening 11beta-HSD1 inhibitors and studying glucocorticoid action.
Area of Science:
- Biochemistry
- Molecular Biology
- Endocrinology
Background:
- 11beta-hydroxysteroid dehydrogenase 1 (11beta-HSD1) is crucial for active glucocorticoid synthesis.
- Tissue-specific dysregulation of 11beta-HSD1 is linked to metabolic syndromes.
- Understanding 11beta-HSD1 activity in relevant cell types is important.
Purpose of the Study:
- To develop and validate a cell-based assay for measuring 11beta-HSD1 activity.
- To investigate 11beta-HSD1 expression and activity during C2C12 myoblast differentiation.
- To assess the utility of the assay for inhibitor screening and mechanistic studies.
Main Methods:
- Utilized murine skeletal muscle cell line C2C12.
- Employed homogeneous time-resolved fluorescence (HTRF) assay for enzyme activity measurement.
- Investigated gene expression, enzyme kinetics, and downstream signaling pathways.
Main Results:
- 11beta-HSD1 mRNA expression and activity increased upon C2C12 cell differentiation.
- Carbenoxolone demonstrated an IC(50) of 0.3 microM, comparable to in vitro assays.
- The cell-based assay does not require exogenous NADPH, suggesting endogenous supply.
- Cortisone treatment modulated glutamine synthase and IL-6 expression, inhibited by carbenoxolone and RU-486.
Conclusions:
- A novel cell-based assay using differentiated skeletal muscle cells effectively measures 11beta-HSD1 activity.
- The assay is suitable for high-throughput screening of 11beta-HSD1 inhibitors.
- This method aids in elucidating the molecular mechanisms of glucocorticoid action in muscle cells.
