Received for publication, April 27, 2000, and in revised form, December 1, 2000
Peroxisome proliferator-activated
receptor-
(PPAR
) has been shown to play an important role in the
regulation of expression of a subclass of adipocyte genes and to serve
as the molecular target of the thiazolidinedione (TZD) and certain
non-TZD antidiabetic agents. Hypercorticosteroidism leads to insulin
resistance, a variety of metabolic dysfunctions typically seen in
diabetes, and hypertrophy of visceral adipose tissue. In adipocytes,
the enzyme 11
-hydroxysteroid dehydrogenase type 1 (11
-HSD-1)
converts inactive cortisone into the active glucocorticoid cortisol and thereby plays an important role in regulating the actions of
corticosteroids in adipose tissue. Here, we show that both TZD and
non-TZD PPAR
agonists markedly reduced 11
-HSD-1 gene expression
in 3T3-L1 adipocytes. This diminution correlated with a significant
decrease in the ability of the adipocytes to convert cortisone to
cortisol. The half-maximal inhibition of 11
-HSD-1 mRNA
expression by the TZD, rosiglitazone, occurred at a concentration that
was similar to its Kd for binding PPAR
and
EC50 for inducing adipocyte differentiation thereby
indicating that this action was PPAR
-dependent. The time
required for the inhibitory action of the TZD was markedly greater for
11
-HSD-1 gene expression than for leptin, suggesting that these
genes may be down-regulated by different molecular mechanisms.
Furthermore, whereas regulation of PPAR
-inducible genes such as
phosphoenolpyruvate carboxykinase was maintained when cellular protein
synthesis was abrogated, PPAR
agonist inhibition of 11
-HSD-1 and
leptin gene expression was ablated, thereby supporting the conclusion
that PPAR
affects the down-regulation of 11
-HSD-1 indirectly.
Finally, treatment of diabetic db/db mice with rosiglitazone inhibited
expression of 11
-HSD-1 in adipose tissue. This decrease in enzyme
expression correlated with a significant decline in plasma
corticosterone levels. In sum, these data indicate that some of the
beneficial effects of PPAR
antidiabetic agents may result, at least
in part, from the down-regulation of 11
-HSD-1 expression in adipose tissue.
 |
INTRODUCTION |
Obesity has been shown to be a major risk factor in the
development of a group of maladies, including insulin resistance, noninsulin-dependent diabetes mellitus
(NIDDM),1 hyperlipidemia, and
hypertension, that results in premature mortality. In particular, a
number of epidemiological studies have suggested that for a given body
mass index, central obesity (visceral or omental), as opposed to
general obesity, is highly associated with these disorders that are
collectively known as "Syndrome X" or the "Metabolic Syndrome"
(1-3). Adipose tissue can increase in mass by the hypertrophy of
existing adipocytes and/or by the adipogenic differentiation of
preadipocytes. Two nuclear receptors, the glucocorticoid receptor and
PPAR
, and their ligands play important roles in adipocyte
differentiation and metabolism (4, 5).
Glucocorticoids have been shown to potentiate the adipogenic process
and anabolic lipid metabolism in adipocytes (6, 7). Patients suffering
from hypercorticosteroidism due to Cushing's syndrome or those
undergoing corticosteroid therapy demonstrate specific increases in
visceral adiposity (8, 9). In addition, they suffer metabolic and
signal transduction disturbances mirroring those observed in NIDDM,
including insulin resistance, hyperglycemia, and hyperlipidemia (10).
Corticosteroid activity is regulated, in part, by the enzyme
11
-hydroxysteroid dehydrogenase (11
-HSD) that exists as two
isozymes (as reviewed in Ref. 11). 11
-HSD-1 is predominantly
expressed in adipose, liver, gonadal, and central nervous system tissue
where it mainly serves as a reductase that converts inactive cortisone
to the active glucocorticoid receptor agonist cortisol. 11
-HSD-2 is
expressed primarily in aldosterone target cells such as kidney and
colon where it inactivates cortisol via its dehydrogenase activity,
thereby preventing excessive activation of the mineralocorticoid
receptor and sequelae that include hypertension. It has recently been
shown that omental adipose tissue contains significantly more
11
-HSD-1 activity than subcutaneous adipose tissue and that
preadipocytes from the former tissue can be more readily differentiated
into adipocytes by cortisone than those from the latter tissue (12).
Previous studies suggested that the effect of glucocorticoids on body
fat distribution may be regulated locally through the metabolism of
glucocorticoids within adipose tissue itself (12). Furthermore,
11
-HSD-1 knockout mice have been shown to resist diet-induced
insulin resistance and hyperglycemia (13). Such observations have led
to the suggestion that 11
-HSD-1 may play a key role in the
dysregulation of metabolism observed in NIDDM and other metabolic
disorders linked to central obesity (14).
The peroxisome proliferator-activated receptors (PPARs) are a subclass
of the nuclear receptor gene family that serves as ligand-regulated
transcription factors. Three PPAR isoforms have been identified and
shown to be encoded by separate genes (15, 16). PPAR
is expressed
predominantly in liver, kidney, and monocytes (17, 18). It has been
shown to play an important regulatory role in the catabolic metabolism
of lipids and, perhaps, inflammatory responses (19-21). PPAR
is
ubiquitously expressed (22, 23); its major physiological action has yet
to be determined. PPAR
exists as two isoforms, PPAR
1 and
PPAR
2, due to alternative promoter usage and differential RNA
splicing (24-26). PPAR
1 is expressed in a number of tissues
including adipose, liver, skeletal and cardiac muscle, and macrophages.
PPAR
2 is highly and specifically expressed in adipocytes. Ectopic
expression of PPAR
has been shown to promote adipogenesis (27, 28).
PPAR
knockout mice display decreased adiposity and greater insulin
sensitivity relative to their wild-type littermates (29-32).
Thiazolidinediones and a subclass of nonthiazolidinedione
insulin-sensitizing agents have been shown to be potent PPAR
ligands
(33-35). The in vivo antidiabetic activities of these
pharmacological agents correlate well with their PPAR
agonist
activity in vitro. The mechanism(s) by which these
beneficial actions are attained is not fully understood at present.
Activation of PPAR
potentiates adipocyte differentiation and
anabolic lipid metabolism. In addition, PPAR
has been shown to
transactivate directly a number of genes in adipocytes that contain
peroxisome proliferator response elements (PPREs) in their promoters,
including adipocyte fatty acid-binding protein (aP2) (24) and
phosphoenolpyruvate carboxykinase (PEPCK) (36). PPAR
agonists have
also been show to inhibit expression of the leptin (ob) gene via a
receptor-mediated mechanism that may also involve the adipogenic
transcription factor CCAAT/enhancer-binding protein
(37, 38).
Several recent observations have suggested a role for PPAR
in
controlling the expression of 11
-HSD-1 in adipocytes. Expression of
11
-HSD-1 increases in 3T3-L1 cells as they proceed through the
differentiation process in a manner similar to other late PPAR
-sensitive adipocyte differentiation markers (39). Moreover, we
have recently observed in DNA chip studies that hepatic expression of
11
-HSD-1 may be regulated by PPAR
agonists (47). Here, we
demonstrate that both thiazolidinedione and nonthiazolidinedione PPAR
agonists markedly inhibited 11
-HSD-1 gene expression in 3T3-L1 adipocytes. A decrease in conversion of radiolabeled cortisone to cortisol by the cells was also observed after PPAR
agonist treatment. The time course of the diminution in 11
-HSD-1 expression was significantly greater than that required to reduce expression of
the leptin gene. Half-maximal inhibition of 11
-HSD-1 expression by
the thiazolidinedione rosiglitazone occurred at a concentration that
was similar to ED50 values the compound previously
displayed for potentiating adipocyte differentiation and decreasing
leptin expression as well as the Kd value reported
for its binding to PPAR
(33, 37). Thus, it appears that ligand
activation of PPAR
down-regulates expression of 11
-HSD-1 in
3T3-L1 adipocytes. A similar diminution in adipose tissue 11
-HSD-1
expression was also observed in diabetic mice treated with
rosiglitazone. This decline may mediate some of the antidiabetic
actions of PPAR
agonists in vivo.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Cell culture reagents were obtained from
Life Technologies, Inc. All other reagent grade chemicals were from
Sigma. The thiazolidinediones, rosiglitazone
(((±)-5-(4-(2-(methyl-2-pyridinylamino)ethoxy)phenyl)methyl)-2,4-thiazolidinedione) and TZD2
(5-[4-[2-(5-methyl-2-phenyl-4-oxazoly)-2-hydroxyethoxy]benzyl]2,4-thiazolidinedione) were chosen for use in these studies. In addition, a novel
indole-acetic acid PPAR
agonist, L-805645
(2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid), was
kindly provided by Drs. Derek Von Langen and Michael Kress of Merck.
Cell Culture and Treatment--
3T3-L1 cells (ATCC, Manassas,
VA; passages 3-9) were grown to confluence in medium A (Dulbecco's
modified Eagle's medium with 10% fetal calf serum, 100 units/ml
penicillin, and 100 µg/ml streptomycin) at 37 °C in 5%
CO2 and induced to differentiate as described previously (40). Briefly, differentiation was induced by incubating the cells with
medium A supplemented with methylisobutylxanthine, dexamethasone, and
insulin for 2 days, followed by another 2-day incubation with medium A
supplemented with insulin. The cells were further incubated in medium A
for an additional 4 days to complete the adipocyte conversion. At day 8 following the initiation of differentiation, cells were incubated in
medium A +/
compounds for the times and at the concentrations
indicated in the figure legends.
RNA Isolation and Analysis--
Total RNA was prepared from
cells and tissue using the Ultraspec RNA isolation kit (Biotecx,
Houston, TX), and RNA concentration was estimated from absorbance at
260 nm. Expression levels of specific mRNAs were quantitated using
quantitative fluorescent real time polymerase chain reaction (PCR). RNA
was first reverse-transcribed using random hexamers in a protocol
provided by the manufacturer (PE Applied Biosystems, Foster City, CA).
Amplification of each target cDNA was then performed with TaqMan®
PCR Reagent Kits in the ABI Prism 7700 Sequence Detection System
according to the protocols provided by the manufacturer (PE Applied
Biosystems, Foster City, CA). The primer/probe sets used for the
amplification step are shown in Table
I.
The levels of mRNA were normalized to the amount of 18 S ribosomal
RNA (primers and probes commercially available from PE Biosystems)
detected in each sample. The chance of amplifying contaminating genomic
DNA in our RNA samples was minimized in two ways as follows: first, by
using primer/probe sets that span intron/exon junctions where possible
(not shown), and second, by demonstrating that no significant signal
was obtained in control PCRs performed with samples obtained from
reverse transcription reactions carried out in the absence of reverse
transcriptase and the primer/probe sets presented above (not shown).
Assay of 11
-HSD-1 Activity--
11
-HSD activity was
measured in intact cells cultured and treated in 6-well tissue culture
dishes as described above by measuring the rate of conversion of
[3H]cortisone to [3H]cortisol. Briefly,
after treatment of the cells, medium was removed and replaced with 1 ml
of medium A containing 15 nM [3H]cortisone
(specific activity 50 Ci/mmol; American Radiolabeled Chemicals, St.
Louis, MO). Medium was removed in intervals between 30 min and 24 h after the addition of steroids. Steroids were extracted with 3 ml of
ethyl acetate. The organic phase was collected, evaporated to dryness,
and reconstituted in dimethyl sulfoxide containing 16 µg/ml each of
unlabeled cortisone and cortisol. The samples were injected into a
Waters HPLC system using an Inertsil 5-µm ODS2 column
(Metachem Technologies, Torrence, CA) and eluted using a gradient of
70% solvent A (water/methanol/trifluoroacetic acid, 90:10:0.05,
v/v/v), 30% solvent B (water/methanol/trifluoroacetic acid,
10:90:0.05, v/v/v) to 40% solvent A, 60% solvent B. Eluted tritiated
steroids were detected using a
-RAM scintillation counter. The
conversion of [3H]cortisone to [3H]cortisol
was calculated as an index of activity.
In Vivo Studies--
Specific pathogen-free, 10-11-week-old
male db/db (C57BL6/J +/+Leprdb) or lean control
heterozygous mice (Jackson Laboratories) were housed in static
microisolators and allowed ad libitum access to pelleted
chow (Purina 5001, Richmond, IN) and water. The animal room was
maintained on a 12:12 h light/dark cycle. The Institutional Animal Care
and Use Committee of Merck reviewed and approved all animal use, and
all animals were cared for in accordance with the Guide for the Care
and Use of Laboratory Animals.
The animals were dosed daily for 11 days by oral gavage with vehicle
(0.5% carboxymethyl cellulose) with or without rosiglitazone (10 mg/kg; n = 7). On day 11 of treatment, blood samples
were collected into lithium heparin microtainer tubes, and epididymal white adipose tissue was removed and snap-frozen in liquid nitrogen. Tissue samples were further processed for RNA isolation as described above. Glucose and triglyceride levels were determined by hexokinase and glycerophosphate oxidase methods, respectively (Hitachi 911, Roche
Molecular Biochemicals). Plasma corticosterone levels were quantitated
by radioimmunoassay (Linco Research, St. Charles, MO).
Statistical Analysis--
Data are expressed as the mean ± S.E. of several determinations. Statistical significance was determined
using Student's t test.
 |
RESULTS |
It has previously been shown by qualitative Northern blot analysis
that 11
-HSD-1 mRNA expression increases dramatically when 3T3-L1
preadipocytes are differentiated into terminally differentiated adipocytes (39). To quantify the relative increase in 11
-HSD-1 gene
expression resulting from the adipogenesis process, confluent 3T3-L1
preadipocytes were allowed to differentiate by a standard protocol
described under "Experimental Procedures." Total RNA was then
isolated from preadipocytes and adipocytes, and the level of
11
-HSD-1 mRNA was determined using quantitative real time PCR.
As shown in Fig. 1A, the level
of expression of 11
-HSD-1 increased almost 500-fold upon
differentiation of the cells. By using the same RNA samples and
techniques, the relative level of the adipocyte fatty acid binding
protein, aP2, was found to be ~30-fold greater in 3T3-L1 adipocytes
than preadipocytes (Fig. 1A). We have previously observed
similar increases in aP2 mRNA levels using quantitative slot-blot
analysis (41).

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|
Fig. 1.
Expression of
11 -HSD-1 and aP2 mRNA and activity of
11 -HSD-1 in 3T3-L1 pre-adipocytes and
adipocytes. Cells were grown to confluence as pre-adipocytes or
were differentiated into adipocytes with methylisobutylxanthine,
dexamethasone, and insulin as described under "Experimental
Procedures." A, the levels of 11 -HSD-1 and aP2 mRNA
were determined by quantitative real time PCR and are expressed
relative to the amounts of mRNA found in preadipocytes.
B, 11 -HSD-1 activity was determined as the percent
conversion of cortisone into cortisol in medium overlying intact cells
at 5 h after addition of [3H]cortisone. The data are
shown as the means + S.E. (A) or S.D. (B) from
triplicate samples of a representative experiment.
|
|
In further experiments, we demonstrated that protein synthesis was
required for PPAR
-dependent inhibition of 11
-HSD-1
gene expression. When 3T3-L1 adipocytes were treated with the protein synthesis inhibitor cycloheximide, we observed that the basal level of
11
-HSD-1 mRNA decreased. This nonspecific,
cycloheximide-produced diminution in basal mRNA expression observed
here is likely to be caused, at least in part, by an overall decrease
in the level of proteins that compose the general transcriptional
machinery of the cell. More importantly, however, inhibition of protein synthesis ablated the ability of the PPAR
agonist rosiglitazone to
inhibit expression of the 11
-HSD-1 mRNA. Similar results were obtained with leptin. In contrast, when genes that are induced by
PPAR
agonists were examined, the results differed, in part, from
those obtained above for genes whose expression was blocked by such
compounds. Although basal PEPCK and aP2 mRNA levels declined with
cycloheximide treatment, as was the case with 11
-HSD-1 and leptin,
the ability of rosiglitazone to induce PEPCK and aP2 gene expression
was maintained when protein synthesis was abrogated.
It has been shown previously that elevated levels of glucocorticoids
result in marked increases in visceral adipose tissue (8, 9). In
addition, patients with hypercorticosteroidism demonstrate marked
elevations in blood insulin, glucose, and lipids levels similar to
those suffering from insulin resistance and NIDDM (10). In contrast to
the effects of glucocorticoids, it has been shown that antidiabetic
PPAR
agonists sensitize peripheral tissue to the actions of insulin
resulting in decreases in the hyperglycemia, hyperlipidemia, and
hyperinsulinemia seen in type 2 diabetes. Also, it has recently been
observed that activation of PPAR
results in a decrease in
intra-abdominal and an increase in subcutaneous adiposity in diabetic
animals and NIDDM subjects (42, 43). This redistribution of adipose
tissue may be at least partially responsible for the
insulin-sensitizing effects of PPAR
agonists since visceral fat is
closely associated with the development of insulin resistance and other
metabolic disturbances associated with NIDDM (44). Here we demonstrated
that treatment of db/db mice with rosiglitazone resulted in a
diminution of 11
-HSD-1 expression in adipose tissue. We suggest that
the ability of PPAR
agonists to reduce visceral adipose tissue
hypertrophy and insulin resistance may result, at least in part, from
their ability to inhibit expression of 11
-HSD-1 and, as a result,
decrease the level of active glucocorticoid within adipocytes. In fact,
chronic treatment of db/db mice with rosiglitazone resulted in a
significant decrease of plasma corticosterone levels. A decrease in
adipocyte corticosterone levels, we speculate, may diminish
glucocorticoid induction of adipogenesis, as well as its potentiating
effects on anabolic lipid metabolism in mature visceral adipocytes. In this regard it is worth noting that administration of carbenoxolone, a
well described inhibitor of 11
-HSD-1, was shown to increase insulin
sensitivity in human subjects (45). Wang et al. (46) have
shown that chronic treatment with rosiglitazone (at 1 mg/kg/day) caused an increase in corticosterone levels in fatty Zucker rats. This
apparent contradiction with the data presented here may be due to
differences in species or drug dosage.
In summary, we have demonstrated that thiazolidinedione and
nonthiazolidinedione agonists of PPAR
markedly inhibit expression of
the 11
-HSD-1 gene in 3T3-L1 adipocytes. This decrease in expression led to a significant diminution in the cellular enzyme activity that
converts cortisone to active cortisol. The half-maximal inhibitory effect of the thiazolidinedione rosiglitazone occurred at a
concentration that supported a PPAR
-mediated mechanism of action. As
was reported previously for leptin, the inhibitory action of PPAR
agonists on 11
-HSD-1 mRNA expression appears to take place at
the level of transcription. In addition, it was found that while
activation of expression of PPRE-containing genes by PPAR
can occur
when protein synthesis is blocked, the inhibitory action of the
receptor on 11
-HSD-1 and leptin gene expression was ablated, thereby
leading to the hypothesis that such inhibitory actions may be mediated by PPAR
-regulated trans-acting factors. Finally, we showed that treatment of diabetic db/db mice with the PPAR
agonist rosiglitazone diminished expression of 11
-HSD-1 mRNA in adipose tissue and reduced plasma corticosterone levels. Since elevated corticosteroid levels have been shown to induce visceral adiposity and insulin resistance, our results support the conclusion that the antidiabetic actions of PPAR
agonists may be mediated, in part, by their ability to inhibit adipocyte expression of 11
-HSD-1. To our knowledge, regulation of 11
-HSD-1 represents the first case of a bona
fide transcriptional consequence of PPAR
activation that can
also be strongly implicated (via prior validation of the role of
11
-HSD-1 in knockout mice) as a contributor to the
insulin-sensitizing effects of compounds acting through this mechanism.
We are indebted to Derek Von Langen and
Michael Kress for providing the novel indole-acetic acid
PPAR
agonist, L-805645
(2-(2-(4-phenoxy-2-propylphenoxy)ethyl)indole-5-acetic acid). We thank
Thomas W. Doebber, Margaret Wu, Alison Strack, Ramon Camacho, Roger
Meurer, and Xiaoying Shi for their critical assistance in the
performance of the db/db mouse experiments.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M003592200
The abbreviations used are:
NIDDM, noninsulin-dependent diabetes mellitus;
PPAR
, peroxisome
proliferator-activated receptor-
;
TZD, thiazolidinedione;
11
-HSD-1, 11
-hydroxysteroid dehydrogenase type 1;
PPREs, peroxisome proliferator response elements;
PEPCK, phosphoenolpyruvate
carboxykinase;
PCR, polymerase chain reaction.
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