Pietrusz, Jennifer L.

Overexpression of 11b‐hydroxysteroid dehydrogenase type 1 (11b‐HSD1) in adipose tissue leads to obesity in mice. We hypothesized that endogenous 11b‐HSD1 played a significant functional role in adipogenesis. Stable transfection of 3T3‐L1 cells with small hairpin RNA (shRNA), compared to a nucleotide‐substituted control, silenced 11b‐HSD1 expression and substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes induced by a mixture containing either corticosterone or dexamethasone. 11b‐HSD1 silencing significantly elevated the concentration of 11‐dehydrocorticosterone in the culture supernatant, but did not affect the levels of corticosterone or dexamethasone, measured by LC‐MS/MS. 11b‐HSD1 silencing significantly attenuated the translocation of glucocorticoid receptors to the nucleus and reduced the number of cells entering the S phase in response to glucocorticoids (13 ± 1% vs. 30 ± 1% in control, n=5, P<0.05), a critical event in the initiation of adipogenesis. 11b‐HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation reduced 11b‐HSD1 expression, but did not affect the further progression of adipogenesis. Our results established a functional and stage‐dependent role of 11b‐HSD1 in the initiation of 3T3‐L1 adipogenesis, and provided new insights into the development of obesity and related diseases.

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1), which regulates the local level of glucocorticoids, has been suggested to be involved in the development of obesity. A definitive functional role for 11β-HSD1 in adipogenesis, however, remains to be established. We developed 3T3-L1 cell lines stably transfected with a small hairpin RNA (shRNA) targeting 11β-HSD1. A shRNA containing two nucleotide substitutions was used as a control. Silencing of 11β-HSD1 substantially attenuated the accumulation of lipid droplets and the expression of adipogenesis marker genes, which was induced by a mixture containing either corticosterone or dexamethasone. Silencing of 11β-HSD1 increased the concentration of 11-dehydrocorticosterone in the culture supernatant but did not significantly affect the levels of corticosterone or dexamethasone. Translocation of glucocorticoid receptors to the nucleus in response to glucocorticoids was significantly attenuated by silencing 11β-HSD1. The number of cells entering the S phase of the cell cycle following the induction of adipogenesis was significantly reduced by silencing 11β-HSD1. 11β-HSD1 shRNA delivered by lentiviral vectors after the induction of differentiation, however, did not affect the progression of adipogenesis. These results indicate that 11β-HSD1 plays a significant functional role in the initiation of 3T3-L1 adipogenesis and provide new mechanistic insights into the role of 11β-HSD1 in the development of obesity and related diseases.