11b-HSD1 is essential for human adipogenesis . I J BUJALSKA and others 299
oncoprotein (HPV-E7) genes. The cell line has an unlimited life
span and the capacity to accumulate lipid without chromosomal
alteration. Confluent Chub-S7 cells were cultured in 75 cm2
TC flasks in DMEM/Ham’s F-12 medium supplemented
with 10% FBS. For experiments, cells were seeded in 24-well
plates at density 105 cells/well. Chub-S7 were differentiated (up
to 21 days) according to Hauner et al. (1987) with 166 nM
human insulin (I-9278 Sigma), 1 mM PPARg agonist
(GW1929, Camlab, Cambridge, UK) and, where specified,
with the addition of 1 mM cortisol (F). For inhibition studies,
Chub-S7 cells were differentiated with 0.5 mM cortisone (E)
and an optimised concentration of the selective 11b-HSD1
inhibitor, PF-877423, at a final concentration of 100 nM.
Human subcutaneous stromal-vascular cell differentiation
Confluent human subcutaneous stromal-vascular (s—v) cells
(96-well plates) were obtained from Zen-Bio Inc. (Research
Triangle Park, NC, USA) and allowed to recover overnight at
37 8C 5% CO2. The following day, day 0, differentiation was
initiated by incubating cells in differentiating media, DM2(E)
(Zen-Bio Inc.), which consists of DMEM/Ham’s F-12
medium containing FBS, l-glutamine, penicillin/streptomy-
cin, insulin, indomethacin, 3-isobutyl-1-methylxanthine
with 1 mM E and vehicle (DMSO not exceeding 0.1%). To
define the effect of the PF-877423 inhibitor upon the
differentiation of primary preadipocytes, cultures were
supplemented with 300 nM PF-877423 (in DMSO). Control
cells cultured without E. Media were replenished and
PF-877423 or vehicle treatment repeated every 2—3 days,
except when 11b-HSD1 activity was assessed.
Measuring lipid content in human subcutaneous s—v cells
Lipogenesis in human subcutaneous s—v cells was measured as
triglyceride accumulation. Cells were carefully washed with
PBS and lysed in situ by adding 50 ml/well of Hecameg (10%
solution in water — Calbiochem, Nottingham, UK). After gentle
shaking at room temperature for 10 min, 200 ml triglyceride
(Infinity) reagent (Thermo DMA, Louisville, CO, USA) was
added to each well. Plates were read after 10—20 min at
500 nm with correction at 660 nm (Spectra MAX PLUS —
Molecular Devices Corporation, Sunnyvale, CA, USA).
Results were expressed as optical density (OD) values.
HEK293 and Chubb-S7 11b-HSD assay
Cells were washed and incubated with 100 nM F (for
dehydrogenase activity) or E (for oxo-reductase activity) with
appropriate tritiated tracer — 3H F (Du Pont, Stevenage, UK) or
3H E (0.02 mCi/reaction; Bujalska et al. 1999). PF-877423
specificity upon 11b-HSD1 was carried out with the addition of
100 nM inhibitor to HEK293T1 and HEK293T2 cells 24h
before and during the enzyme assay. After 3 h incubation with
substrate, media was removed and steroids extracted with 4 ml
dichloromethane, evaporated under the air and reconstituted
with 70 ml dichloromethane, then spotted on silica plates
(Sigma-Aldrich). Steroidswere separated by thin-layer chroma-
tography in chloroform and ethanol (92:8) and steroid
conversion was quantified using a LabLogic AR-200 scanner
(LabLogic, Sheffield, UK). Cells were washed and protein
concentration was measured using colorimetric 96-well plate
assay (Bio-Rad) and total RNA was extracted.
Human subcutaneous s—v cells 11b-HSD1 assay
During 11b-HSD1 activity assessment in human subcutaneous
s-v cells, the medium was changed to basal medium (BM-1,
Zen-Bio Inc.) containing only antibiotics, and cells were cultured
for 24 h in the presence of PF-877423 with or without 0.5 mM
E. At the end of the incubation, the cortisol signal was
quantitatively determined in the 100 ml cell supernatant with
the Correlate-Enzyme Immunoassay Cortisol kit (Assay Designs
Inc. Ann Arbor, MI, USA) following the manufacturers’
instructions. Plates were read on a plate reader (Spectra MAX
PLUS - Molecular Devices Corporation) at 405 nm, with
correction at 580 nm. Activity has been expressed as cortisol
production in pg/ml per 24 h.
Total RNA extraction and RT reaction
Total RNA was extracted from cells using TriReagent
(Sigma) according to the manufacturer’s protocol. Integrity
and concentration of RNA were assessed by electrophoresis
and spectrophotometry respectively. Reverse transcription
was carried out using AMV and random primers at 37 8C for
1 h. All RT reagents were purchased from Promega.
PCR
Expression studies were carried out using gene specific primers
for human 11 b-HSD1, hexose 6-phosphate dehydrogenase
(H6PDH), glucocorticoid receptor a (GRa, FABP4), G3PD,
glucose transporter 4 (GLUT-4) and PPARg. All primers were
designed using primer 3 software (http://frodo.wi.mit.edu/cgi-
bin/primer3/primer3.cgi) and their sequences from 50 to 30 are
shown in Table 1. Primers for 18S as internal control were
purchased from Ambion (Quantum RNATM Classic 18S
Internal Standard #1716; Ambion). PCRs were carried out at
95 8C for 30 s, 60 8C for 30 s and 72 8C for 30 s in 20 ml final
volume for 30 cycles.
Real-time PCR
Quantitative mRNA expression levels of 11b-HSD1, H6PDH,
GRa,FABP4,G3PD, GLUT-4 andPPARg2 were measured by
real-time PCR using an ABI 7500 system (Perkin-Elmer,
Biosystems, Warrington, UK). PCR was performed in 25 ml
reactions on 96-well plates. Reactions contained TaqMan
universal PCR master mix (Applied Biosystems, Foster City,
CA, USA), 900 nmol primers, 100-200 nmol TaqMan probe
and 25-50 ng cDNA. All reactions were correlated to expression
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Journal of Endocrinology (2008) 197, 297-307