298 I J BUJALSKA and others . 11b-HSD1 is essential for human adipogenesis
glycerol-3-phosphate dehydrogenase (G3PD) and fatty acid-
binding protein 4 (FABP4; Hotamisligil et al. 1996); many of
these genes are regulated by glucocorticoids (Wu et al. 1996,
Rosen & MacDougald 2006). Previously, we have shown that
non-selective inhibition of 11b-HSD1 can prevent human
adipocyte differentiation in vitro (Bujalska et al. 1999). The
potential for therapeutic intervention has been tested in rodent
models where selective 11b-HSD1 inhibitors lower plasma
glucose, improve insulin sensitivity and in some studies reduce
body weight in mice (Alberts et al. 2002, Kershaw et al. 2005).
However, the potency of these inhibitors has been variable and
there are no data on effcacy in human tissue. We report the
development of a selective inhibitor against human 11b-HSD1,
PF-877423 (Pfzer Global R&D, La Jolla, CA, USA) and the
effect of this compound upon adipogenesis in a well-
characterised differentiating human subcutaneous preadipocyte
cell line (Darimont et al. 2003, Qiao et al. 2005) and in primary
cultures of subcutaneous human preadipocytes.
Research design and methods
Recombinant protein assay
Wild-type recombinant human 11b-HSD1 protein (24—292)
was used for studying the inhibitor kinetics. Radio-labelled
[1,2-3H]-cortisone was purchased from American Radio-
labeled Chemicals Inc (St Louis, MO, USA). NAD (reduced
form; NADPH), glucose-6-phosphate (G6P) and G6P
dehydrogenase (G6PD) were purchased from Sigma—
Aldrich. All the concentrations reported in the following
section are final in the assay buffer. In addition, the enzyme
concentrations represent the active concentrations that were
determined by active-site titration using a tight-binding
inhibitor. The experimental data were fitted by using the
non-linear regression analysis software, Grafit (Leather-
barrow (2001) GraFit Version 5, Erithacus Software Ltd,
Horley, UK).
The measurement of the in vitro 11b-HSD1 activity was
performed in a 100 mM triethanolamine buffer (pH 8.0),
containing 200 mM NaCl, 0.02% n-dodecyl b-D-maltoside,
5% glycerol and 5 mM b-mercaptoethanol. A typical
reaction for the determination of enzyme activity comprised
the following: 5 nM enzyme pre-incubated for at least
30 minutes in the assay buffer in the presence of 500 mM
NADPH in round-bottom 96-well plates (Costar cat #
3365). Next, the reaction was initiated by adding a
regenerating system (consisting of 2 mM G6P, 1 U/ml
G6PD and 6 mM MgCl2) and labelled 3H-cortisone as
substrate. After an incubation period (30—40 min), 100 ml
of the assay mixture were transferred to a second empty
round-bottom 96-well plate and mixed with an equal
volume of dimethylsulphoxide ( DMSO) to quench the
reaction. Then, a 15 ml aliquot of the assay solution was
loaded into a C-18 column (Polaris C18-A, 50!4. 6 mm,
5 u, 180 A, Varian; Polaris, Palo Alto, CA, USA connected
to an automated High-Throughput Liquid Chromatography
instrument (Cohesive Technologies, HTLC, Franklin, MA,
USA). The radioactive material from the column was
detected with a b-RAM model 3 Radio-HPLC detector
(IN/US, Tampa, FL, USA). Substrate and product peaks
were separated by using an isocratic mixture of 38:62
methanol to water (v/v) at a flow rate of 1. 0 ml/min. Under
these experimental conditions, the retention time for
cortisone and cortisol were 4.5 and 5.5 min respectively.
The initial reaction velocities recorded were in the linear
range and were determined by measuring the peak area for
cortisol formation with time.
Recombinant protein kinetic analysis
The inhibition of 11b-HSD1 by PF-877423 was analysed
by fitting to the equation described below (Equation
(1); Morrison 1969) and provided an accurate measure-
ment for the value of Kiapp at a fixed concentration of
cortisone
Vi Z Vo 1 K
[E]o C ½I]o C Kiapp
K q([Eo] C ½I]o C Kiapp)2 K 4½E]o½I]o
2[e]o
(1)
where [E]o and [I]o are the active enzyme and inhibitor
concentration respectively; Vi and Vo are the rates of
cortisone reduction in the presence or in the absence of
inhibitor respectively. Four Kiapp values were determined
by varying the cortisone concentration while keeping
the concentration of NADPH constant at 500 mMin
the assay buffer. The true inhibition constant, Ki, for
PF-877423 was then obtained by plotting the Kiapp
values versus the cortisone concentration, [C]o, and
fitting the data using Equation (2) for a competitive
inhibitor
Kian0 z Ki ∣ 1 C ½Co
app i K
m
(2)
where Km was the Michaelis-Menten constant for
cortisone.
HEK293 and Chubb-S7 cell culture
HEK293 cells stably transfected with human 11b-HSD1
(HEK293T1) or 11b-HSD2 (HEK293T2) cDNA as
described previously (Bujalska et al. 1997a) were used to
study the specificity of inhibitor PF-877423 upon 11b-HSDs.
Cells were cultured in minimum essential medium (MEM)
media supplemented with 10% fetal bovine serum (FBS) and
1% non-essential fatty acids; for experiments, cells were
seeded into 24-well tissue culture dishes and maintained in
MEM with 10% FBS until confluence.
The Chub-S7 cell line was derived from human subcu-
taneous adipose tissue (Darimont et al. 2003) by co-expression of
human telomerase reverse transcriptase and papillomavirus E7
Journal of Endocrinology (2008) 197, 297-307
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