Peroxisome Proliferator-activated Receptor-γ Ligands Inhibit Adipocyte 11β-Hydroxysteroid Dehydrogenase Type 1 Expression and Activity
2001; Elsevier BV; Volume: 276; Issue: 16 Linguagem: Inglês
10.1074/jbc.m003592200
ISSN1083-351X
AutoresJoel P. Berger, Michael Tanen, Alex Elbrecht, Anne Hermanowski‐Vosatka, David E. Moller, Samuel D. Wright, Rolf Thieringer,
Tópico(s)Eicosanoids and Hypertension Pharmacology
ResumoPeroxisome proliferator-activated receptor-γ (PPARγ) 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 11β-hydroxysteroid dehydrogenase type 1 (11β-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 PPARγ agonists markedly reduced 11β-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 11β-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its Kd for binding PPARγ and EC50 for inducing adipocyte differentiation thereby indicating that this action was PPARγ-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11β-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARγ-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARγ agonist inhibition of 11β-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARγ affects the down-regulation of 11β-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11β-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 PPARγ antidiabetic agents may result, at least in part, from the down-regulation of 11β-HSD-1 expression in adipose tissue. Peroxisome proliferator-activated receptor-γ (PPARγ) 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 11β-hydroxysteroid dehydrogenase type 1 (11β-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 PPARγ agonists markedly reduced 11β-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 11β-HSD-1 mRNA expression by the TZD, rosiglitazone, occurred at a concentration that was similar to its Kd for binding PPARγ and EC50 for inducing adipocyte differentiation thereby indicating that this action was PPARγ-dependent. The time required for the inhibitory action of the TZD was markedly greater for 11β-HSD-1 gene expression than for leptin, suggesting that these genes may be down-regulated by different molecular mechanisms. Furthermore, whereas regulation of PPARγ-inducible genes such as phosphoenolpyruvate carboxykinase was maintained when cellular protein synthesis was abrogated, PPARγ agonist inhibition of 11β-HSD-1 and leptin gene expression was ablated, thereby supporting the conclusion that PPARγ affects the down-regulation of 11β-HSD-1 indirectly. Finally, treatment of diabetic db/db mice with rosiglitazone inhibited expression of 11β-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 PPARγ antidiabetic agents may result, at least in part, from the down-regulation of 11β-HSD-1 expression in adipose tissue. 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), 1The abbreviations used are:NIDDMnoninsulin-dependent diabetes mellitusPPARγperoxisome proliferator-activated receptor-γTZDthiazolidinedione11β-HSD-111β-hydroxysteroid dehydrogenase type 1PPREsperoxisome proliferator response elementsPEPCKphosphoenolpyruvate carboxykinasePCRpolymerase chain reaction 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" (1Bjorntorp P. J. Intern. Med. 1991; 230: 195-201Crossref PubMed Scopus (260) Google Scholar, 2Reaven G.M. Hoffman B.B. 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A decrease in conversion of radiolabeled cortisone to cortisol by the cells was also observed after PPARγ agonist treatment. The time course of the diminution in 11β-HSD-1 expression was significantly greater than that required to reduce expression of the leptin gene. Half-maximal inhibition of 11β-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 PPARγ (33Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3460) Google Scholar, 37Kallen C.B. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5793-5796Crossref PubMed Scopus (343) Google Scholar). Thus, it appears that ligand activation of PPARγ down-regulates expression of 11β-HSD-1 in 3T3-L1 adipocytes. A similar diminution in adipose tissue 11β-HSD-1 expression was also observed in diabetic mice treated with rosiglitazone. This decline may mediate some of the antidiabetic actions of PPARγ agonists in vivo. 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 PPARγ 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. 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 (40Zhang B. Berger J. Hu E. Szalkowski D. White-Carrington S. Spiegelman B. Moller D.E. Mol. Endocrinol. 1996; 10: 1457-1466Crossref PubMed Scopus (312) Google Scholar). 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. 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 TableI.Table IPrimer/probe setsGene5′ Primer (5′-3′)3′ Primer (5′-3′)Probe (5′-3′)11β-HSD-1AAGCAGAGCAATGGCAGCATGAGCAATCATAGGCTGGGTCATCGTCATCTCCTCCTTGGCTGGGAAaP2GAATTCGATGAAATCACCGCACTCTTTATTGTGGTCGACTTTCCACGACAGGAAGGTGAAGAAGCATCATAACLeptinCCAAAACCCTCATCAAGACCAAGTCCAAGCCAGTGACCCTCTATTTCACACACGCAGTCGGTATCCGCPEPCKAAATCCGGCAAGGCGCTCTGGTGCCACCTTTCTTCCCAGCGATCTCTGATCCAGACCTTCCAA Open table in a new tab 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). 11β-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 β-RAM scintillation counter. The conversion of [3H]cortisone to [3H]cortisol was calculated as an index of activity. Specific pathogen-free, 10–11-week-old male db/db (C57BL6/J +/+Lepr db) 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). Data are expressed as the mean ± S.E. of several determinations. Statistical significance was determined using Student's t test. It has previously been shown by qualitative Northern blot analysis that 11β-HSD-1 mRNA expression increases dramatically when 3T3-L1 preadipocytes are differentiated into terminally differentiated adipocytes (39Napolitano A. Voice M.W. Edwards C.R. Seckl J.R. Chapman K.E. J. Steroid. Biochem. Mol. Biol. 1998; 64: 251-260Crossref PubMed Scopus (108) Google Scholar). To quantify the relative increase in 11β-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 11β-HSD-1 mRNA was determined using quantitative real time PCR. As shown in Fig. 1 A, the level of expression of 11β-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. 1 A). We have previously observed similar increases in aP2 mRNA levels using quantitative slot-blot analysis (41Zhang B. MacNaul K. Szalkowski D. Li Z. Berger J. Moller D.E. J. Clin. Endocrinol. & Metab. 1999; 84: 4274-4277Crossref PubMed Google Scholar). To examine the effect of 3T3-L1 cell differentiation on 11β-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.1 B demonstrate, adipocytes possess dramatically more 11β-HSD-1 activity than undifferentiated preadipocytes. These results correlated well with the increased levels of enzyme mRNA described in Fig. 1 A. 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 11β-HSD-1 functions almost exclusively as a reductase in 3T3-L1 adipocytes and that 11β-HSD-2 activity is absent from these cells. We were able to demonstrate very small amounts of 11β-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 PPARγ activation on 11β-HSD-1 gene expression, 3T3-L1 adipocytes were incubated for 48 h alone or in the presence of the PPARγ 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 11β-HSD-1 mRNA (Fig. 2 A) and leptin mRNA (Fig. 3 B and data not shown). In contrast, all of the agonists increased expression of the aP2 gene 2–3-fold (Fig. 2 B), thereby demonstrating that the observed inhibition in 11β-HSD-1 mRNA expression did not result from general cytotoxic effects of the compounds.Figure 3Time courses of inhibition of 11β-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 11β-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.View Large Image Figure ViewerDownload (PPT) Next, we examined the time course of inhibition of 11β-HSD-1 mRNA expression by rosiglitazone. As depicted in Fig. 3 A, 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 (37Kallen C.B. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5793-5796Crossref PubMed Scopus (343) Google Scholar), dropped precipitously with half-maximal inhibition requiring less than 2 h and the maximal decrease ∼4 h (Fig. 3 B). It is also worth noting that leptin mRNA levels consistently declined to a greater extent than 11β-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 11β-HSD-1 mRNA in rosiglitazone-treated adipocytes when actinomycin was used to inhibit RNA transcription (data not shown). Rosiglitazone has been shown to bind PPARγ with aKd ∼40 nm (33Lehmann J.M. Moore L.B. Smith-Oliver T.A. Wilkison W.O. Willson T.M. Kliewer S.A. J. Biol. Chem. 1995; 270: 12953-12956Abstract Full Text Full Text PDF PubMed Scopus (3460) Google Scholar). When 3T3-L1 adipocytes were incubated with various doses of this compound, expression of 11β-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 PPARγ agonist has displayed similar potency in decreasing leptin expression, increasing aP2 expression in adipocytes, and potentiating adipocyte differentiation (37Kallen C.B. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5793-5796Crossref PubMed Scopus (343) Google Scholar) (data not shown). It has been demonstrated previously that activation of PPARγ inhibits the steady-state level of leptin mRNA not by altering its stability but by decreasing its transcription (37Kallen C.B. Lazar M.A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5793-5796Crossref PubMed Scopus (343) Google Scholar, 38Hollenbe
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