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



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

Reverse


Table 1 Primer sequences for various human genes for the PCR

Forward

Gene

HSD11B1

ACCAGAGATGCTCCAAGGAA

H6PDH

AGAAGCGAGACAGCTTCCAC

GRa

TCGACCAGTGTTCCAGAGAAC

GLUT-4

GCCATTGTTATCGGCATTCT

PPARg1

TCTCTCCGTAATGGAAGACC

PPARg2

GCGATTCCTTCACTGATAC

G3PD

GGAAGACATTGGAGGCAAAA

FABP4

CATCAGTGTGAATGGGGATG

Primers were designed using Primer3 software (http://frodo.wi.mit.edu/cgi-bin/primer3).


ATGCTTCCATTGCTCTGCTT
GCTGCTGGGAAAAGAACAAC
TTTCGGAACCAACGGGAATTG
CTACCCCTGCTGTCTCGAAG
GCATTATGAGACATCCCCAC
GCATTATGAGACATCCCCAC
CCACGGCCACTACATTCTTT
ATGCGAACTTCAGTCCAGGT


of ribosomal 18S rRNA (provided as a pre-optimized mix;
Perkin-Elmer) as an internal reference. All target gene probes
were labelled with the fluorescent label FAM and the 18S probe
with the fluorescent label VIC. Reactions were as follows: 50
8C
for2 min, 95
8C for 10 min, and then 40 cycles of95 8Cfor15s
and 60
8C for 1 min. Data were analysed according to the
manufacturer’s guidelines and were obtained as
Ct values (the
cycle number at which logarithmic PCR plots cross a calculated
threshold line) and used to determine d
Ct values (dCtZCt of the
target gene minus
Ct of the internal reference, 18S). Primers and
probes for 11
b-HSD1, H6PDH and G3PD were designed using
PrimerExpress 1
.0 software (Applied Biosystems). Sequences
from 5
0 to 30 are shown in Table 2. Expression assay kits were
purchased from Applied Biosystems to measure the gene
expression of GR
a, GLUT-4, PPARg2 and FABP4.

Statistical analysis

Where data were normally distributed, unpaired Student’s t-test
was used to compare single treatments with control. If normality
tests failed, then non-parametric tests were used. One-way
ANOVA on ranks was used to compare multiple treatments
(SigmaStat 3
.1, Systat Software Inc., Point Richmond, CA,
USA). Results were expressed as mean values
GS.D. or s.e.m.
values and a P value of !0.05 was accepted as statistically
significant. Statistical analysis on real-time PCR data was
performed on mean
DCt values and not on fold changes.

Results

Kinetics of PF-877423 upon recombinant 11b-HSD1 protein

The potency for PF-877423 was strongly affected by the
presence of the substrate in the assay buffer (
Fig. 1): Kiapp
values increased at high cortisone concentration, suggesting
that the inhibitor behaved as a reversible and competitive
inhibitor against cortisone. Fitting the experimental data
using equation (2) provided a value of 0
.2G0.04 and 333.4G
109.2 nM for the inhibition constant, Ki, and the apparent
Michaelis-Menten constant,
Km respectively.

Specificity of PF-877423

11b-HSD enzyme assays on HEK293T1 and HEK293T2 cells
showed total abolition of dehydrogenase (12
.4G1.0 vs 0.2G
0.01, % cortisol to cortisone conversion, meanGS.D.) and oxo-
reductase (34
.7G0.6 vs 0.4G0.1, % cortisone to cortisol
conversion, mean
GS.D.) activities of 11b-HSD1 following
incubation with 100 nM PF-877423 for 24 h (
Fig. 2A), but
PF-877423 had no effect on 11
b-HSD2 activity (63.6G4.0 vs
62
.2G4.4, % cortisol to cortisone conversion, meanGS.D.,
control versus PF-877423 respectively;
Fig. 2B). No toxic
effects of PF-877423 were observed up to 10
mM concentrations
using a commercially available assay kit (CellTiter 96 Aqueous,
Promega; data not shown).

Table 2 Primer and probe sequences for various human genes for the real-time PCR

Forward


Reverse


Probe


Gene

HSD11B1

H6PDH

PPARg2

G3PD


AGGAAAGCTCATGGGAGGACTAG

GGGCCTATGGAACCTCCAA

AGAAGAAGGCGGCGTTGTC

CCATCAGTTCATCGGCAAGAT


ATGGTGAATATCATCATGAA AAAGATTC

GACCCACGTTTCTCACTGAC TCT

TCAGGTTGAGGCCACCATC

TCGTCTACCCCCTTAATAAG AGATATG

CATGCTCATTCTCAACCACATCA CCAACA

CCGTGGCGCTACTCATGGACAC A

AGGGGCCACCACAGACTTGCAC AT

AGGGCCATCTGAAGGCAAACGC C

Primers and probes were designed using PrimerExpress software (Applied Biosystems, UK).

Journal of Endocrinology (2008) 197, 297-307

www.endocrinology-journals.org




More intriguing information

1. The name is absent
2. DISCUSSION: ASSESSING STRUCTURAL CHANGE IN THE DEMAND FOR FOOD COMMODITIES
3. Sector Switching: An Unexplored Dimension of Firm Dynamics in Developing Countries
4. Dendritic Inhibition Enhances Neural Coding Properties
5. The name is absent
6. Non Linear Contracting and Endogenous Buyer Power between Manufacturers and Retailers: Empirical Evidence on Food Retailing in France
7. AGRICULTURAL TRADE LIBERALIZATION UNDER NAFTA: REPORTING ON THE REPORT CARD
8. The Structure Performance Hypothesis and The Efficient Structure Performance Hypothesis-Revisited: The Case of Agribusiness Commodity and Food Products Truck Carriers in the South
9. Apprenticeships in the UK: from the industrial-relation via market-led and social inclusion models
10. Education Responses to Climate Change and Quality: Two Parts of the Same Agenda?