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Introduction
Obesity is reaching epidemic proportions and has significant health implications. Although many studies have focused
on the role of 11 beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) in the expansion of adipose tissue, the
modulation of 11beta-HSD1 activity affects multiple target tissues, and may promote insulin resistance independent of
obesity[1]. Examining the potential role of 11beta-HSD1 as a therapeutic target is an attractive research area for several reasons.
11beta-HSD1 knockout mice have low intracellular glucocorticoid levels, and are protected from obesity, diabetes, and
dyslipidemia[2,3]. Conversely, the overexpression of 11beta-HSD1 in white adipose tissue results in elevated intracellular glucocorticoid
levels, central obesity, insulin resistance, hypertension, hyperglycemia, and
dyslipidemia in mice[4_6]. In
vivo, 11beta-HSD1 catalyzes the conversion of glucocorticoids from the
inactive to the active form. Glucocorticoids play an important
role in normal physiology by modulating metabolic and
immune responses[7]. Although it is now clear that
glucocorticoids regulate the transcription of a diverse array of target
genes, the precise mechanisms by which glucocorticoid
receptor (GR)-mediated changes in cell proliferation and
differentiation occur are still far from
clear[8].
In this study, we explored the relationship between
11beta-HSD1 and preadipocyte differentiation in an
in vitro 3T3-L1 preadipocyte
model[9]. We constructed an 11beta-HSD1 siRNA vector and stably transfected 3T3-L1 cells to
examine the effect of 11beta-HSD1 on adipocyte
differentia-tion.
Materials and methods
3T3-L1 cell culture and induction of differentiation
3T3-L1 preadipocytes were cultured and induced to differentiate
as previously described[10]. In short, the cells were cultured
in Dulbecco's modified Eagle's medium (DMEM) (Gibco,
Invitrogen Corporation, Carlsbad, CA, USA) containing 10%
fetal bovine serum (FBS) in 5% CO2 at 37 °C for 2 d for
confluence (d 0). Differentiation was induced by changing
the medium to DMEM containing 10% FBS, 0.5 mmol/L
3-isobutyl-1-methyxanthine (Sigma, St Louis, MO, USA), 1
μg/mL insulin (Sigma, St Louis, MO, USA), and 1 mmol/L
dexamethasone (Sigma, St Louis, MO, USA). After 48 h
incubation (d 2), the medium was replaced with DMEM containing
10% FBS and 1 μg/mL insulin. On d 4, the medium was
replaced with DMEM containing only 10% FBS, and then
changed back to the same medium every 2 d for 4 d. The
3T3-L1 cells transfected with 11beta-HSD1 siRNA were
cultured as above, with 200 μg/mL G418 (geneticin) (Sigma, St
Louis, MO, USA).
Oil Red O staining and quantitative determination of
triglyceride accumulation in cells The cells were stained
with Oil Red O. Briefly, the cells were fixed with 10% formalin
(pH 7.4), then stained with Oil Red O (stock solution: 3
mg/mL in isopropanol; working solution: 60% Oil Red O stock
solution and 40% distilled water) for 5 min. The absorbance of
the cell monolayers was spectrophotometrically determined
at 510 nm after Oil Red O staining.
RNA preparation and real-time PCR Total RNA was
isolated from cells using TRIzol (Invitrogen Corporation,
Carlsbad, CA, USA). 1 mg total RNA was reverse
transcribed using 200 U Moloney murine leukemia virus reverse
transcriptase (Promega, Madison, WI, USA) in the presence of
0.5 mmol/L dNTP, 25 U RNase inhibitor, and 0.5 μg N6
random primers in a volume of 25 µL reaction buffer (Promega,
Shanghai, China). Samples without reverse transcriptase
treatment were also examined as controls for genomic DNA
contamination. PCR primers (Table 1) were designed by Primer
Design software Premier 5.0 (PREMIER Biosoft International,
Palo Alto, CA,USA) using GenBank sequences and were
synthesized by Invitrogen (Shanghai, China). Each real-time
PCR reaction was carried out in triplicate in a total volume of
20 μL with Quanti Tect SYBR Green PCR Master Mix (MJ
Research, Waltham, MA, USA) according to the following
conditions: 5 min at 94 ºC, 44 cycles at 94 ºC for 30 s, 58 ºC for
30 s, and 72 ºC for 40 s using the ABI Prism 7700 sequence
detection system (ABI, Oyster Bay, NY, USA). The Ct (cycle
threshold) values (2Ct) for each gene were normalized to the
expression levels of S18 (Ribosomal protein)(Table 1).
Construction of 11beta-HSD1 short interference siRNA
expression plasmids BLAST 2 (Basic Local Alignment Search
Tool) homology searching was used to identify 2 conserved
cDNA fragments for RNA interference (RNAi) within the
coding region of the mouse 11beta-HSD1 gene. The
sequences were as follows:
sense: 5'-GAAGAGTCATGGAGGTCAA-3' and
antisense:5'-AAAGCGAGGTGTACTATGA-3'.
From these sequences, we designed and synthesized 2
pairs of reverse complementary oligonucleotides, each
containing a loop sequence (5'-TTCAAGAGA-3'), the RNA
Pol III terminator (5'-TTTTTT-3') and 5' single-stranded
overhangs for ligation into HindIII and
BamHI-digested pGCsilencer H1/TetO1 RNAi vector (GeneChem, Shanghai,
China). Oligonucleotides were annealed and inserted into
the siRNA expression vectors to form
pGCsilencer H1/TetO1_11beta-HSD1_1 and pGCsilencer
H1/TetO1_11beta-HSD1_2, respectively. A control with limited homology to
the human, mouse, and rat genome sequences was also
created (GeneChem, Shanghai, China).
RNAi For stable expression, 2 mg each of the
lipopolysaccharide-free vectors were transfected into 3T3-L1 cells(50%
confluence) with Fugene 6 (Roche, Shanghai, China). The
3T3-L1 cells were seeded into 6-well culture plates with
complete culture medium. The ratio of Fugene 6 to the vectors
for transfection was 6:1. Two days after transfection, GFP
(Green fluorescent protein) expression was observed. G418
was used for selection by adjusting the concentration of
G418 from 400 to 800 µg/mL every 2 d, maintaining 800 µg/mL
for 1 week. Non-transfected 3T3-L1 cells were used as a
control for G418 sensitivity. G418-resistant cells were
maintained in culture medium supplemented with 200
μg/mL G418 for further analysis.
Western blot analysis Non-transfected and transfected
3T3-L1 cells were collected, and the total proteins were
isolated using the TRIzol method. The protein concentration
was determined by BCA assay (Pierce, Rockford, IL, USA).
The total proteins (10 μg per lane) were subjected to 12%
SDS-PAGE and transferred onto PROTRAN nitrocellulose
membranes (Schleicher and Schuell, Dassel, Germany), then
immunoprobed with rabbit anti-human primary antibody
against 11beta-HSD1 (1:200; Santa Cruz Biotechnology, Santa
Cruz, CA, USA) and horseradish peroxidase-conjugated goat
anti-rabbit IgG as the secondary antibody (1:800; Santa Cruz
Biotechnology, Santa Cruz, USA). Immune bands were
visualized by applying an ECL Western blot analysis system
according to the manufac-turer's instructions (Amersham,
Amersham Pharmacia, Piscataway, NJ, USA). The antibody
against beta-actin, which detects the expression of beta-actin,
was used as an internal control (Santa Cruz, USA). Exposed
film was then analyzed using a densitometer (Furi, Shanghai,
China) to determine the optical densities of each band, and
the density ratio of 11beta-HSD1 to beta-actin bands was
calculated.
Statistical analysis All results are expressed as mean±SD.
Data were analyzed using one-way ANOVA with a
correction for multiple comparisons as appropriate.
Results
Characterization of 11beta-HSD1 stably transfected
cells To further investigate the role of 11beta-HSD1 on
adipocyte differentiation, transfection of the pGCsilencer
H1/TetO1_11beta-HSD1_2 vector remarkably downregulated the
transcription and expression level of mouse 11beta-HSD1,
while no significant differences were detected in the cells
transfected with pGCsilencer H1/TetO1_11beta-HSD1_1
(Figure 1).
3T3-L1 preadipocyte as an in vitro differentiated model
3T3-L1 preadipocyte cells can be stimulated to differentiate
into adipocytes. Differentiation was induced to the normal
and transfected 3T3-L1 cells to examine the role of
11beta-HSD1 in adipocyte differentiation. On d 0, 2, 4, 6, and 8 of
3T3-L1 cell differentiation, Oil Red O staining was used to
detect lipid droplets, which are indicative of adipocyte
differentiation. Lipid droplets accumulated after stimulation
(d 0) and the degree of lipid droplet accumulation increased
over time. However, lipid accumulation was significantly
inhibited in cells transfected with mouse 11beta-HSD1 siRNA
compared with non-transfected 3T3-L1 cells and cells
transfected with pGCsilencer H1/TetO1 (Figure 2A). The
triglyceride content in the cells was measured by the absorbance
of the cell monolayers, which was spectrophotometrically
determined at 510 nm after Oil Red O staining. The
triglyceride decreased in the cells transfected with 11beta-HSD1
siRNA (Figure 2B). These results suggest that 11beta-HSD1
is normally required for 3T3-L1 preadipocyte differentiation.
Expression of 11beta-HSD1 protein During adipocyte
differentiation (d 0, 2, 4, 6, and 8), the expression of the
11beta-HSD1 protein was assessed by Western blot analysis. A
major band migrating at 34 kDa, corresponding to the
11beta-HSD1 protein, was confirmed to be present in the 3T3-L1
cells. 11beta-HSD1 protein expression was upregulated
during the course of adipocyte differentiation (d 8
vs d 0, P<0.01; Figure 3).
mRNA expression of GR GR mRNA was detected by
real-time PCR from both non-transfected 3T3-L1 cells and
11beta-HSD1 siRNA-transfected 3T3-L1 cells during the
course of adipocyte differentiation (d 0, 2, 4, 6, and 8). GR
expression was upregulated on d 4 and 6, but was rapidly
downregulated on d 8 in the control and siRNA-transfected
3T3-L1 cells (Figure 4).
Expression of markers of 3T3-L1
differentiation The expression of several adipocyte differentiation markers, such
as fatty acid synthetase and lipoprotein lipase, was also
upregulated upon stimulation of differentiation in the
non-transfected 3T3-L1 cells and the cells transfected with
pGCsilencer H1/TetO1, but were downregulated in the cells
transfected with 11beta-HSD1-siRNA (Figure 5). Similarly,
the expression of preadipocyte factor-1, an inhibitor of
adipocyte differentiation, was downregulated upon
stimulation of differentiation and had no changes in the cells
transfected with 11beta-HSD1 siRNA (Figure 5).
Discussion
Just as transgenic and knockout models allow
researchers to study gene function in vivo, overexpression and
knocking down with RNA interference are powerful technologies
for exploring gene function in mammalian cultured cells.
11beta-HSD1 is a microsome enzyme located in various
tissues. It interconverts inactive cortisone and active
cortisol, regulating the level of cortisol. Glucocorticoid as a
prereceptor regulator promotes adipocyte differentiation.
Recent studies in mouse models have suggested that
11beta-HSD1 may represent the long-sought link between
obesity and insulin resistance[1,4,11]. 11beta-HSD1 catalyzes
the conversion of glucocorticoid molecules from an inactive
to an active form. There is accumulating evidence
suggesting that glucocorticoids may play a role in adipocyte
maturation, at least in part, via the inhibition of cell
proliferation and the induction of
differentiation[8]. To verify this hypothesis, we
used in vitro RNAi experiments to explore the effect of
11beta-HSD1 on preadipocyte differentiation. We synthesized 2 pairs of
mouse 11beta-HSD1 mRNA-specific interfere plasmids:
pGCsilencer H1/TetO1_11beta-HSD1_1 and pGCsilencer H1/TetO1_11beta-HSD1_2. The
pGCsilencer H1/TetO1_11beta-HSD1_2 showed significant
silencing effects on the expression of 11beta-HSD1 at both
RNA and protein levels (Figure 1). It is not clear
why pGCsilencer H1/TetO1_11beta-HSD1_1 did not show
significant silencing effects.
Compared with non-transfected 3T3-L1 cells, we found
that pGCsilencer H1/TetO1_11beta-HSD1_2-transfected cells
exhibited much less adipoconversion and triglyceride (Figure
2A,B). Small lipid droplets could be observed in transfected
cells as late as d 8. This notable decrease in adipoconversion
was consistent throughout the whole process of
differentia-tion.
The mechanisms by which 11beta-HSD1 regulates adipocyte differentiation are still unclear.
Glucocorticoids inhibit cellular proliferation by inducing cell cycle arrest at
the G1 phase; the prereceptor modulation of cortisol
metabolism has a dramatic effect on the cell proliferation
rate[7,8]. Numerous studies have focused on examining the effects of
various steroid moieties on 11beta-HSD1 activity since
factors that inhibit metabolism of the 11beta-hydroxyl group
increase glucocorticoid potency. Glucocorticoids, the
C/EBP-α (CAATT enhancer binding proteins alpha),
PPAR-gamma (peroxisome proliferator activiated receptor) agonists,
and some pro-inflammatory cytokines (TNF-alpha, interleukin
1-beta) can increase 11beta-HSD1 expression, while GH
(growth hormone), acting via IGF-1 (insulin-like growth
factor-1) and LXR (liver X receptor) agonists can inhibit its
expression[12_14]. A key question remains concerning the
extent to which the upregulation of adipose 11beta-HSD1 in
obesity influences intra-adipose cortisol levels and the
metabolic consequences of obesity.
In the present study, the level of 11beta-HSD1 increased
(Figure 3) in the differentiation of 3T3-L1, but GR was
increased shortly after induced differentiation in the 3T3-L1
cells, even following 11beta-HSD1 knock-down (Figure 4).
In light of the known effect of glucocorticoids on adipose
tissue function and distribution, it has been postulated that
the enhanced conversion of E to F by 11beta-HSD1 within
omental adipose tissue may play an important role in the
pathogenesis of central obesity. Cortisol is essential for
adipocyte differentiation[15], while 11beta-HSD1 promotes
differentiation and may be related to the development of
obesity and insulin-resistance.
Recently, it has been shown that the inhibition of GR
using a 10-fold molar excess of the GR-antagonist
RU38486 (the name of the drug), could block the cortisol-mediated
upregulation of 11beta-HSD1 expression and
11-oxoreductase activity. These results suggest that this effect is mediated
exclusively by GR. However, 11beta-HSD1 is thought to
modulate glucocorticoid hormone action by regulating ligand
supply to GR[16], and our results suggest that it may be an
autoprotected mechanism for the effect of glucocorticoids.
This study provides a useful new model for determining
the relative impact of the divergent GR-mediated pathways
on cell growth. The data presented here emphasize the
pivotal role of 11beta-HSD1 as a prereceptor determinant of
GR-mediated signal transduction. It has recently been shown
that 11beta-HSD1 activity regulates adipocyte
differentiation in an autocrine
manner[17] by supplying adequate amounts of cortisol to support cell differentiation and
preventing excessive and detrimental amounts of cortisol in
mature cells. This study demonstrates the importance of
this autocrine 11beta-HSD1 action for proper cell
differentiation and function. This may explain why the timing of
11beta-HSD1 expression is correlated with that of GR when
stimulated by cortisol[18]. Cortisol induces adipogenesis and
11beta-HSD1 activity in preadipocytes. When adipocyte
differentiation is initiated, 11beta-HSD1 dehydrogenase
activity is switched to reductase activity which generates
cortisol and thereby promotes adipocyte
differentiation[7,19,20].
In summary, our data support the hypothesis that
11beta-HSD1 acts as a prereceptor regulator to stimulate
preadi-pocyte differentiation in an autocrine manner. The level of
triglyceride decreased and the transcription of some marker
genes were downregulated in 11beta-HSD1 siRNA cells
(Figures 2B, 5). Thus, this enzyme may serve as a critical link
between obesity and the development of insulin resistance.
Further studies will be needed to explore the regulation and
function of 11beta-HSD1 at the transcription and expression
levels. A better understanding of the biological pathways
mediated by 11beta-HSD1 may lead to new therapies for
obesity and diabetes mellitus.
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