A novel selective 11b-hydroxysteroid dehydrogenase type 1 inhibitor prevents human adipogenesis

11b-HSD1 is essential for human adipogenesis . I J BUJALSKA and others 301

Cortisone (nM)

Figure 1 Effect of cortisone concentration upon the apparent
inhibition constant Ki_app of the inhibitor PF-877423: a value for
the true inhibition constant Ki (0∙2 G0^.04 nM) and the Michaelis-
Menten constant Km (333.4G109.2 nM) is calculated by fitting the
experimental data using equation (2).

Characterisation of chub-S7 cells

At confluence (day 0), Chub-S7 cells did not accumulate lipid
droplets (Fig. 3A); however, they readily underwent adipogen-
esis (shown as oil red O staining) when cultured for 21 days in
chemically-defined, serum-free media (166 nM insulin, 1 mM
PPARg agonist and 1 mM F; Fig. 3B). As demonstrated
by conventional PCR, confluent undifferentiated Chub-S7
cells expressed GRa H6PDH and PPARg1 mRNA but not

Figure 2 (A) PF-877423 inhibits 11b-HSD1 enzyme activity
(dehydrogenase: 12.4G1.0 vs 0.2G0.01, % cortisol to cortisone
conversion, and oxo-reductase: 34.7G0.6 vs 0.4G0.1, % cortisone
to cortisol conversion, meanGS.D.) as measured in HEK293T1
(HEK293 cells stably transfected with human 11b-HSD type 1
cDNA), nZ3 but not (B) 11b-HSD2 enzyme activity (63.6G4.0 vs
62.2G4.4, % cortisol to cortisone conversion, meanGS.D., control
versus PF-9Z877423 respectively) as measured in HEK293T2 (cells
stably transfected with human 11b-HSD type 2 cDNA), nZ3.

P values: **P!0.01, ***P!0.001.

11b-HSD1, PPARg2, GLUT-4, G3PD or FABP4 mRNA
(Fig. 3C). In the differentiated Chub-S7 cells, increased
expression of adipogenic markers including G3PD and
FABP4 was observed. This process resulted in an increase in
11b-HSD1, GLUT-4 and PPARg2 mRNA levels (Fig. 3D).

Across differentiation, 11b-HSD1 oxo-reductase activity
increased significantly; from 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., nZ7, all
P!0.01 versus previous time point; Fig. 4A). Conventional
PCR findings were endorsed and quantified by real-time PCR.
Expression of 11b-HSD1 mRNA increased 2.9-fold on day 5,
3.6-fold on day 7, 3.4-fold on day 9 (P!0.01) and 38.1-fold on
day 16 (P!0.001) when compared with day 3, nZ4 (Fig. 4B).

We observed a transient increase in H6PDH mRNA levels
(11b-HSD1 co-factor provider) — 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, P!0^.01 (Fig. 4C) — but there was no
significant change in GRa mRNA during Chub-S7
differentiation (Fig. 4D).

Significant increases in differentiation markers FABP4 (2-fold
on day 5 (P!0.01), 38-fold on day 7, 142-fold on day 9 and
870-fold on day 16 versus day 3, P!0.001) and G3PD (4.5-fold
on day 7 (P!0.01), 22-fold on day 9 and 380-fold on day 16
versus day 5, P!0.001) were also observed (Fig. 5A and B
respectively). When compared with day 7, the expression of
adipocyte-specific genes including GLUT-4 and PPARg2 also
increased — 24-fold on day 9 and 9^.8-fold on day 16, P!0^.01
(GLUT-4) and 1.3-fold on day 7 and 2.2-fold on day 16,
P!0.01 (PPARg2; Fig. 5C andD respectively).

Glucocorticoid metabolism and adipogenesis in Chub-S7 cells
incubated with PF-877423

Chub-S7 cells differentiated for 10 days with 500 nM
cortisone showed increased 11b-HSD1 oxo-reductase
activity: 14.6G2.3 (E) versus 3.4G1.3 (control), pmol/mg
per h meanGS.E.M., P!0.001 (Fig. 6A), and mRNA
expression (14.1-fold versus control; Fig. 6B). Co-incubation
with 100 nM PF-877423 abolished this effect: 14.6G2.3 (E)
versus 1.3G1.1 (ECPF-877423) vs 0.6G0.5 (PF-877423)
pmol/mg per h, meanGS.E.M. (Fig. 6A) and 14.1-fold (E)
versus 1.2-fold (ECPF-877423), 11b-HSD1 activity and
mRNA respectively (Fig. 6B). Differentiated Chub-S7 cells
with E showed increased expression of the adipogenic
markers FABP4 (312-fold versus control, P!0.001) and
G3PD (47-fold versus control, P!0^.001) — an effect that was
completely abolished by co-incubation with PF-877423 (1.3-
and 0.7-fold, FABP4 and G3PD respectively; Fig. 6C and D).
The change in adipogenesis following incubation with the
11b-HSD1-specific inhibitor was confirmed visually through
staining the cells with oil red O after 21 days of differentiation.
A marked increase in the number of red-stained cells was
observed in cells differentiated with E or F but not in the
presence of PF-877423 (Fig. 6E).

Journal of Endocrinology (2008) 197, 297-307

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