Peroxisome Proliferator-activated Receptor-gamma Ligands Inhibit Adipocyte 11beta -Hydroxysteroid Dehydrogenase Type 1 Expression and Activity*

Joel BergerDagger, Michael Tanen, Alex Elbrecht, Anne Hermanowski-Vosatka§, David E. Moller, Samuel D. Wright§, and Rolf Thieringer§

From the Departments of Molecular Endocrinology and § Atherosclerosis and Endocrinology, Merck Research Laboratories, Rahway, New Jersey 07065

Received for publication, April 27, 2000, and in revised form, December 1, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Peroxisome proliferator-activated receptor-gamma (PPARgamma ) 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 11beta -hydroxysteroid dehydrogenase type 1 (11beta -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 PPARgamma agonists markedly reduced 11beta -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 11beta -HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its Kd for binding PPARgamma and EC50 for inducing adipocyte differentiation thereby indicating that this action was PPARgamma -dependent. The time required for the inhibitory action of the TZD was markedly greater for 11beta -HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARgamma -inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARgamma agonist inhibition of 11beta -HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARgamma affects the down-regulation of 11beta -HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11beta -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 PPARgamma antidiabetic agents may result, at least in part, from the down-regulation of 11beta -HSD-1 expression in adipose tissue.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PPARgamma , 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 11beta -hydroxysteroid dehydrogenase (11beta -HSD) that exists as two isozymes (as reviewed in Ref. 11). 11beta -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. 11beta -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 11beta -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, 11beta -HSD-1 knockout mice have been shown to resist diet-induced insulin resistance and hyperglycemia (13). Such observations have led to the suggestion that 11beta -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). PPARalpha 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). PPARdelta is ubiquitously expressed (22, 23); its major physiological action has yet to be determined. PPARgamma exists as two isoforms, PPARgamma 1 and PPARgamma 2, due to alternative promoter usage and differential RNA splicing (24-26). PPARgamma 1 is expressed in a number of tissues including adipose, liver, skeletal and cardiac muscle, and macrophages. PPARgamma 2 is highly and specifically expressed in adipocytes. Ectopic expression of PPARgamma has been shown to promote adipogenesis (27, 28). PPARgamma 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 PPARgamma ligands (33-35). The in vivo antidiabetic activities of these pharmacological agents correlate well with their PPARgamma agonist activity in vitro. The mechanism(s) by which these beneficial actions are attained is not fully understood at present. Activation of PPARgamma potentiates adipocyte differentiation and anabolic lipid metabolism. In addition, PPARgamma 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). PPARgamma 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 alpha  (37, 38).

Several recent observations have suggested a role for PPARgamma in controlling the expression of 11beta -HSD-1 in adipocytes. Expression of 11beta -HSD-1 increases in 3T3-L1 cells as they proceed through the differentiation process in a manner similar to other late PPARgamma -sensitive adipocyte differentiation markers (39). Moreover, we have recently observed in DNA chip studies that hepatic expression of 11beta -HSD-1 may be regulated by PPARalpha agonists (47). Here, we demonstrate that both thiazolidinedione and nonthiazolidinedione PPARgamma agonists markedly inhibited 11beta -HSD-1 gene expression in 3T3-L1 adipocytes. A decrease in conversion of radiolabeled cortisone to cortisol by the cells was also observed after PPARgamma agonist treatment. The time course of the diminution in 11beta -HSD-1 expression was significantly greater than that required to reduce expression of the leptin gene. Half-maximal inhibition of 11beta -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 PPARgamma (33, 37). Thus, it appears that ligand activation of PPARgamma down-regulates expression of 11beta -HSD-1 in 3T3-L1 adipocytes. A similar diminution in adipose tissue 11beta -HSD-1 expression was also observed in diabetic mice treated with rosiglitazone. This decline may mediate some of the antidiabetic actions of PPARgamma agonists in vivo.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 PPARgamma 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.

                              
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Table I
Primer/probe sets

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 11beta -HSD-1 Activity-- 11beta -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 beta -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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

It has previously been shown by qualitative Northern blot analysis that 11beta -HSD-1 mRNA expression increases dramatically when 3T3-L1 preadipocytes are differentiated into terminally differentiated adipocytes (39). To quantify the relative increase in 11beta -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 11beta -HSD-1 mRNA was determined using quantitative real time PCR. As shown in Fig. 1A, the level of expression of 11beta -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 11beta -HSD-1 and aP2 mRNA and activity of 11beta -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 11beta -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, 11beta -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.

To examine the effect of 3T3-L1 cell differentiation on 11beta -HSD-1 activity, the ability of cells to convert radiolabeled cortisone to cortisol was determined before and after differentiation as described under "Experimental Procedures." As the data presented in Fig. 1B demonstrate, adipocytes possess dramatically more 11beta -HSD-1 activity than undifferentiated preadipocytes. These results correlated well with the increased levels of enzyme mRNA described in Fig. 1A. We were not able to measure any conversion of tritiated cortisol to cortisone in intact preadipocyte even after extended (24 h) incubations with the substrate, indicating that 11beta -HSD-1 functions almost exclusively as a reductase in 3T3-L1 adipocytes and that 11beta -HSD-2 activity is absent from these cells. We were able to demonstrate very small amounts of 11beta -HSD-2 mRNA in the preadipocytes by quantitative real time PCR which, upon differentiation to adipocytes, decreased to undetectable levels (data not shown).

To determine the effect of PPARgamma activation on 11beta -HSD-1 gene expression, 3T3-L1 adipocytes were incubated for 48 h alone or in the presence of the PPARgamma agonists rosiglitazone, TZD2, or L-805645. The first two compounds are thiazolidinediones and the third is a nonthiazolidinedione carboxylic acid-containing compound. All three compounds caused marked reductions in expression of 11beta -HSD-1 mRNA (Fig. 2A) and leptin mRNA (Fig. 3B and data not shown). In contrast, all of the agonists increased expression of the aP2 gene 2-3-fold (Fig. 2B), thereby demonstrating that the observed inhibition in 11beta -HSD-1 mRNA expression did not result from general cytotoxic effects of the compounds.


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Fig. 2.   Inhibition of 11beta -HSD-1 and activation of aP2 mRNA expression by PPARgamma agonists in 3T3-L1 adipocytes. 3T3-L1 cells were grown to confluence and then differentiated into adipocytes. At 8 days post-confluence, the cells were incubated for a further 48 h in media alone or containing 10 µM rosiglitazone (rosi), TZD2, or L-805645. The levels of 11beta -HSD-1 mRNA expression (A) or aP2 mRNA expression (B) were quantified by quantitative real time PCR and are expressed relative to the amount of mRNA found in the untreated adipocytes. The data are shown as the means ± S.E. from triplicate samples of a representative experiment.


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Fig. 3.   Time courses of inhibition of 11beta -HSD-1 and leptin mRNA expression in 3T3-L1 adipocytes by rosiglitazone. 3T3-L1 cells were grown to confluence and subsequently differentiated into adipocytes. At 8 days post-confluence, the cells were incubated for varying lengths of time with 10 µM rosiglitazone. The levels of 11beta -HSD-1 mRNA expression (A) and leptin mRNA expression (B) at each time point were quantified by quantitative real time PCR and are expressed relative to the amount of mRNA found in untreated adipocytes. The data are shown as the means ± S.E. from triplicate samples of a representative experiment.

Next, we examined the time course of inhibition of 11beta -HSD-1 mRNA expression by rosiglitazone. As depicted in Fig. 3A, the mRNA level of the enzyme decreased over an extended period with half-maximal inhibition occurring after more than 10 h and maximal diminution after 24 h. This result contrasted with that observed for leptin mRNA levels which, in accordance with previously published results (37), dropped precipitously with half-maximal inhibition requiring less than 2 h and the maximal decrease ~4 h (Fig. 3B). It is also worth noting that leptin mRNA levels consistently declined to a greater extent than 11beta -HSD-1 mRNA levels. The above temporal differences may be explained, at least in part, by a disparity in mRNA stability since leptin mRNA demonstrated a shorter half-life than 11beta -HSD-1 mRNA in rosiglitazone-treated adipocytes when actinomycin was used to inhibit RNA transcription (data not shown).

Rosiglitazone has been shown to bind PPARgamma with a Kd ~40 nM (33). When 3T3-L1 adipocytes were incubated with various doses of this compound, expression of 11beta -HSD-1 mRNA was inhibited in a concentration-dependent manner (Fig. 4). Notably, the half-maximal decrease in expression of the enzyme occurred at a concentration ~40 nM. The PPARgamma agonist has displayed similar potency in decreasing leptin expression, increasing aP2 expression in adipocytes, and potentiating adipocyte differentiation (37) (data not shown).


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Fig. 4.   Dose response of inhibition of 11beta -HSD-1 mRNA expression in 3T3-L1 adipocytes by rosiglitazone. 3T3-L1 cells were grown to confluence and differentiated into adipocytes. At 8 days post-confluence, the cells were incubated for a further 48 h in media alone or containing varying concentrations of rosiglitazone. The levels of 11beta -HSD-1 mRNA expression at each time point were quantified by quantitative real time PCR and are expressed relative to the amount of mRNA found in untreated adipocytes. The data are shown as the means ± S.E. from triplicate samples of a representative experiment.

It has been demonstrated previously that activation of PPARgamma inhibits the steady-state level of leptin mRNA not by altering its stability but by decreasing its transcription (37, 38). Similarly, we found that the half-life of 11beta -HSD-1 mRNA was not significantly decreased by rosiglitazone after RNA transcription was inhibited by treatment of adipocytes with actinomycin D (not shown). These results suggest that activation of PPARgamma inhibited transcription rather than increasing degradation of the mRNA of the enzyme. To determine whether the synthesis of new protein was required for inhibition of 11beta -HSD-1 expression by PPARgamma activation, 3T3-L1 adipocytes were incubated in the absence or presence of the protein synthesis inhibitor cycloheximide with or without rosiglitazone cotreatment. As demonstrated in Fig. 5A, inhibition of protein synthesis by cycloheximide diminished the basal level of 11beta -HSD-1 mRNA and ablated the ability of the PPARgamma agonist to inhibit expression of the 11beta -HSD-1 gene. Similar results were obtained with leptin (not shown). In contrast, whereas the protein synthesis inhibitor still decreased the basal level of PEPCK gene expression, it did not abrogate the ability of rosiglitazone to induce PEPCK expression (Fig. 5B). Likewise, cycloheximide did not prevent the activation of aP2 expression (not shown).


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Fig. 5.   Inhibition of 11beta -HSD-1 and activation of PEPCK mRNA expression in 3T3-L1 adipocytes by rosiglitazone in the absence or presence of cycloheximide. Confluent 3T3-L1 cells were differentiated into adipocytes. At 8 days post-confluence, the cells were incubated for a further 48 h in media with or without 5 µg/ml cycloheximide alone or containing 10 µM rosiglitazone. The levels of 11beta -HSD-1 mRNA expression (A) or PEPCK mRNA expression (B) were quantified by quantitative real time PCR and are expressed in arbitrary units. The data are shown as the means ± S.E. from triplicate samples of a representative experiment. *, p < 0.005; **, p < 0.001; ***, p < 0.01 by Student's t test.

By having demonstrated that PPARgamma agonists reduce expression of the 11beta -HSD-1 gene in adipocytes, we examined the effect of PPARgamma activation on enzyme activity in the cells. Conversion of radiolabeled cortisone to cortisol was assayed in 3T3-L1 adipocytes incubated with or without rosiglitazone. As shown in Fig. 6, 11beta -HSD-1 activity was significantly decreased (by 29.2%) in cells treated with the PPARgamma agonist for 48 h relative to control cells. A greater fractional decline (41.4%) in cellular reductase activity was seen after 96 h of treatment with rosiglitazone.


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Fig. 6.   Effect of rosiglitazone on 11beta -HSD-1 activity in 3T3-L1 adipocytes. 3T3-L1 cells were grown to confluence and differentiated into adipocytes. At 8 days post-confluence, the adipocytes were treated for an additional 48 or 96 h with media alone or containing 10 µM rosiglitazone. 11beta -HSD-1 activity was determined by adding medium containing [3H]cortisone and determining the levels of radiolabeled steroids in medium harvested 30 min after the addition of the substrate. The data are shown as the means ± S.D. from triplicate samples of a representative experiment. *, p = 0.03; **, p < 0.001 by Student's t test.

To examine the regulation of 11beta -HSD-1 expression in adipose tissue in vivo, we treated obese diabetic db/db mice with rosiglitazone (10 mg/kg for 11 days). This regimen resulted in a 56 and a 61% correction in glucose and triglyceride levels, respectively. Such treatment also caused a 52% decline in 11beta -HSD-1 mRNA levels in epididymal white adipose tissue (Fig. 7). Furthermore, serum corticosterone levels were significantly decreased in treated animals compared with untreated controls (262 ± 26 versus 376 ± 38 ng/ml; p = 0.03).


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Fig. 7.   Inhibition of 11beta -HSD-1 mRNA expression by rosiglitazone in db/db mice. Rosiglitazone was administered to db/db mice for 11 days. Total RNA was isolated from epididymal white adipose tissue of the lean controls, db/db controls, and db/db treated with rosiglitazone (rosi). The levels of 11beta -HSD-1 mRNA expression were quantified by quantitative real time PCR and are expressed relative to the amount of mRNA found in the lean controls. The data are shown as the means ± S.E. of seven individual samples from each treatment group. *, p < 0.01 comparing db/db + rosiglitazone to db/db controls by Student's t test.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we demonstrate that PPARgamma agonists inhibit expression of 11beta -HSD-1 in 3T3-L1 adipocytes. This observation provides a possible novel mechanism that explains the actions of such compounds on adipose tissue redistribution and insulin sensitization in vivo. Our data support the conclusion that the ligands manifest their effect via a PPARgamma -dependent mechanism. First, structurally distinct PPARgamma effectors (TZD and non-TZD compounds) were able to inhibit 11beta -HSD-1 expression to a similar extent. Second, the half-maximal inhibition of 11beta -HSD-1 expression occurred at a ligand concentration that was very similar to the Kd for PPARgamma binding, the EC50 of PPARgamma -induced adipogenesis, and the IC50 for the inhibition of leptin expression (33, 37).

The PPARgamma agonist rosiglitazone did not induce degradation of 11beta -HSD-1 mRNA in adipocytes treated with actinomycin D, thereby suggesting that the inhibitory effects of PPARgamma activation were occurring at the level of 11beta -HSD-1 gene transcription. Similar results have previously been reported for leptin (37). It has previously been shown that activation of PPARgamma rapidly inhibits expression of the leptin gene (37). Our data, demonstrating that leptin mRNA expression declines over a period of t1/2 ~4 h, confirmed that previous observation. In contrast, we found that half-maximal inhibition of 11beta -HSD-1 by the potent PPARgamma agonist rosiglitazone occurred after more than 10 h of treatment and, in general, was not maximized until >= 24 h. Such differences can be explained, at least partially, by our observation that leptin mRNA has a shorter half-life than 11beta -HSD-1 mRNA in actinomycin D-treated adipocytes.

In further experiments, we demonstrated that protein synthesis was required for PPARgamma -dependent inhibition of 11beta -HSD-1 gene expression. When 3T3-L1 adipocytes were treated with the protein synthesis inhibitor cycloheximide, we observed that the basal level of 11beta -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 PPARgamma agonist rosiglitazone to inhibit expression of the 11beta -HSD-1 mRNA. Similar results were obtained with leptin. In contrast, when genes that are induced by PPARgamma 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 11beta -HSD-1 and leptin, the ability of rosiglitazone to induce PEPCK and aP2 gene expression was maintained when protein synthesis was abrogated.

The fact that inhibition of protein synthesis abolishes the ability of PPARgamma activation to inhibit expression of certain genes while not affecting the ability of the receptor to induce expression of others suggests these two phenomena occur via different molecular mechanisms. The induction of aP2 and PEPCK gene transcription is mediated, as demonstrated previously (24, 36), through a direct interaction of the ligand-bound PPARgamma /retinoid X receptor heterodimer with the PPREs located in the promoters of the genes. Protein synthesis is, apparently, not required for such an interaction. In contrast, since PPARgamma inhibition of 11beta -HSD-1 and leptin mRNA expression appears to require active protein synthesis, the action of the receptor on the promoters of these genes may not be direct but mediated by transacting factors whose expression is up-regulated by activation of PPARgamma . It is worth noting here that Hollenberg et al. (38) could demonstrate the PPARgamma /retinoid X receptor-mediated inhibition of leptin gene transcription in adipocytes was not mediated by a direct interaction with a PPRE found in the promoter of the gene.

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 PPARgamma 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 PPARgamma 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 PPARgamma 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 11beta -HSD-1 expression in adipose tissue. We suggest that the ability of PPARgamma agonists to reduce visceral adipose tissue hypertrophy and insulin resistance may result, at least in part, from their ability to inhibit expression of 11beta -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 11beta -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 PPARgamma markedly inhibit expression of the 11beta -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 PPARgamma -mediated mechanism of action. As was reported previously for leptin, the inhibitory action of PPARgamma agonists on 11beta -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 PPARgamma can occur when protein synthesis is blocked, the inhibitory action of the receptor on 11beta -HSD-1 and leptin gene expression was ablated, thereby leading to the hypothesis that such inhibitory actions may be mediated by PPARgamma -regulated trans-acting factors. Finally, we showed that treatment of diabetic db/db mice with the PPARgamma agonist rosiglitazone diminished expression of 11beta -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 PPARgamma agonists may be mediated, in part, by their ability to inhibit adipocyte expression of 11beta -HSD-1. To our knowledge, regulation of 11beta -HSD-1 represents the first case of a bona fide transcriptional consequence of PPARgamma activation that can also be strongly implicated (via prior validation of the role of 11beta -HSD-1 in knockout mice) as a contributor to the insulin-sensitizing effects of compounds acting through this mechanism.

    ACKNOWLEDGEMENTS

We are indebted to Derek Von Langen and Michael Kress for providing the novel indole-acetic acid PPARgamma 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.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: RY80N-C31, Merck Research Laboratories, 126 E. Lincoln Ave., Rahway, NJ 07065. Tel.: 732-594-4738; Fax: 732-594-3925; E-mail: joel_berger@merck.com.

Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M003592200

    ABBREVIATIONS

The abbreviations used are: NIDDM, noninsulin-dependent diabetes mellitus; PPARgamma , peroxisome proliferator-activated receptor-gamma ; TZD, thiazolidinedione; 11beta -HSD-1, 11beta -hydroxysteroid dehydrogenase type 1; PPREs, peroxisome proliferator response elements; PEPCK, phosphoenolpyruvate carboxykinase; PCR, polymerase chain reaction.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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