302 I J BUJALSKA and others . 11b-HSD1 is essential for human adipogenesis
Day О — oil red O staining
Day 21 — oil red O staining
DNA NegCtr 18S 11β-HSDI GRa H6PDH PPARgI PPARg2 GLUT4 G3PD FABP4
ladder
Figure 3 Oil red O staining in (A) confluent Chub-S7 cells (day 0) and (B) differentiated Chub-S7 (day 21). RT-PCR analysis (30 cycles) of
nine gene expressions in (C) confluent Chub-S7 cells and (D) in differentiated Chub-S7 cells.
DNA NegCtr 18S 11β-HSDI GRa H6PDH PPARgI PPARg2 GLUT4 G3PD FABP4
ladder
Human subcutaneous s—v cells differentiation
Human subcutaneous s—v cells differentiated with ECPF-
877423 had significantly lower 11b-HSD1 oxo-reductase
activity compared with cells differentiated with E at any time
point studied (day 6: 154G8 vs 5387G182; day 9: 128G1 vs
5489G230; day 14: 174G18 vs 4041G106; day 20: 409G27
vs 10443G78; day 22: 330G7 vs 11218G193 pg/ml per 24 h,
meanGS.D., P!0.001, nZ3, EK or ECPF-877423 treated
respectively (Fig. 7A)). Lipid content in cells differentiated
with E and PF-877423 was significantly lower than in cells
differentiated with E only and similar to undifferentiated cells
(day 16, 0.25G0.03 vs 0.20G0.01; day 20, 0.3G0.02 vs
0.20G0.01; day 22, 0.27G0.01 vs 0.19G0.01; OD (500/
660 nm); meanGS.D., P!0.01, nZ3, EK or ECPF-877423
treated respectively; Fig. 7B).
Discussion
Numerous studies (Hauner et al. 1989, Gregoire et al. 1991, Wolf
1999), including our own (Bujalska et al. 1999, 2002b), have
Figure 4 (A) 11b-HSD1 oxo-reductase activity (cortisone to cortisol conversion); nil on day 0 to 0.4G0.2 on day 3,
5.3G0.7 on day 5, 8.4G0.14 on day 7, 10.5G1.9 on day 9 and 5.9G1.9 on day 16, pmol/mg per h, meanGS.D. versus
previous time point, nZ7 and mRNA levels measured by real-time PCR of (B) 11b-HSD1 (2.9-fold increase on day 5,
3.6-fold on day 7, 3.4-fold on day 9 and 38.1-fold on day 16 when compared with day 3). (C) H6PDH increased 2.9-
fold on day 3, 3.5-fold on day 5, 3.7-fold on day 7, 3.4-fold on day 9 and 0.6-fold on day 16 versus day 0 and (D) GRa
(no significant change) across Chub-S7 differentiation, nZ4. P values: **P!0.01, ***P!0.001.
Journal of Endocrinology (2008) 197, 297-307
www.endocrinology-journals.org
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