Dehydroepiandrosterone Down-Regulates the Expression of Peroxisome Proliferator-Activated Receptor in Adipocytes
Abstract6uj, http://www.100md.com
Dehydroepiandrosterone (DHEA) is expected to have a weight-reducing effect. In this study, we evaluated the effect of DHEA on genetically obese Otsuka Long Evans Fatty rats (OLETF) compared with Long-Evans Tokushima rats (LETO) as control. Feeding with 0.4% DHEA-containing food for 2 wk reduced the weight of sc, epididymal, and perirenal adipose tissue in association with decreased plasma leptin levels in OLETF. Adipose tissue from OLETF showed increased expression of peroxisome proliferator-activated receptor (PPAR) protein, which was prevented by DHEA treatment. Further, we examined the effect of DHEA on PPAR in primary cultured adipocytes and monolayer adipocytes differentiated from rat preadipocytes. PPAR protein level was decreased in a time- and concentration-dependent manner, and DHEA significantly reduced mRNA levels of PPAR{gamma} , adipocyte lipid-binding protein, and sterol regulatory element-binding protein, but not CCAAT/enhancer binding protein {alpha} . DHEA-sulfate also reduced the PPAR{gamma} protein, but dexamethasone, testosterone, or androstenedione did not alter its expression. In addition, treatment with DHEA for 5 d reduced the triglyceride content in monolayer adipocytes. These results suggest that DHEA down-regulates adiposity through the reduction of PPAR{gamma} in adipocytes.
Introductionhg1)h, 百拇医药
MUCH EVIDENCE SUGGESTS that adrenal androgen, dehydroepiandrosterone (DHEA), or its sulfate metabolite DHEA sulfate (DHEA-S), ameliorate glucose metabolism in diabetic animals, but the underlying mechanisms are still controversial. Coleman et al. (1) reported that DHEA administration to db/db mice prevented the development of diabetes mellitus. Kimura et al. (2) reported that DHEA decreases serum TNF{alpha} in Zucker fatty rats , whereas Aoki et al. (3) indicated that DHEA suppresses the elevated glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice. These authors suggested that administration of DHEA prevented insulin resistance. We reported previously that oral administration of DHEA for 2 wk to Otsuka Long Evans Fatty (OLETF) rats, an animal model of obese type 2 diabetes, but not Goto-Kakizaki rats, an animal model of lean type 2 diabetes, decreased the elevated plasma glucose (4). We remarked that DHEA improved plasma glucose only in the obese animal models but did not alter their body weight within 2 wk administration. Animal studies indicate that DHEA has antiobesity effect (5, 6, 7, 8, 9, 10, 11). Moreover, DHEA prevents proliferation and differentiation of 3T3-L1 preadipocytes (12). Accordingly, we assumed that DHEA might have some effect on adipose tissue, which results in the prevention of insulin resistance.
Recent studies have indicated that transcription factor, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma} ), and CCAAT/enhancer binding protein {alpha} (C/EBP{alpha} ) play a critical role in promoting adipocyte differentiation. PPAR{gamma} is expressed in the early phase of adipocyte differentiation (13), whereas C/EBP{alpha} appears in a relatively late phase (14). The fact that cotransfection of PPAR{gamma} and C/EBP{alpha} was sufficient to induce adipogenesis in the absence of exogenous ligands (15) suggests that they act synergistically in the differentiation process. On the other hand, PPAR{gamma} expression is necessary for the maintenance of the adipocyte phenotype (16), although the regulation mechanisms of these transcription factors in mature adipocytes are uncertain. In this study, we investigated whether DHEA reduces the adiposity in OLETF rats, and whether DHEA modulates the expression of PPAR{gamma} and C/EBP{alpha} in adipocytes.;d, 百拇医药
Materials and Methods
Materialsif7s, 百拇医药
DHEA, DHEA-S, dexamethasone, androstenedione, and testosterone were purchased from Sigma (St. Louis, MO). Pork insulin was obtained from Novo Nordisk (Copenhagen, Denmark). Anti-PPAR{gamma} antibody, anti-CEBP{alpha} and ß antibody, and antiactin antibody were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Rat epididymal fat derived preadipocytes were purchased from Toyobo (Osaka, Japan). All other chemicals were of reagent grade or better.if7s, 百拇医药
Methodsif7s, 百拇医药
Animal treatment.if7s, 百拇医药
Long-Evans Tokushima (LETO rat, control) rats and OLETF rats (18 wk of age) were fed CE2 (Japan Clea, Tokyo, Japan) ad libitum, treated with CE2 powder containing 0.4% DHEA for 2 wk, and then killed by decapitation. Epididymal, sc, periintestinal, perirenal, and brown adipose tissues were collected. After wet weight was measured, they were homogenized in buffer 1 containing 20 mM Tris/HCl (pH 7.5), 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 0.2 mM Na2VO4, 0.1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin.
Primary culture.):, 百拇医药
To obtain the primary culture of rat adipocyte, Male Wistar rats weighing 200 g were killed by decapitation. Isolated adipocytes were obtained by collagenase digestion of epididymal fat pads in DMEM containing 5% calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Adipocytes were washed three times and incubated with various concentrations of DHEA, 1 µM DHEA-S, 1 µM androstendione, 1 µM testosterone, and 100 nM insulin at 37 C in 5% CO2+ 95% O2. After 24 or 48 h incubation, adipocytes were collected and homogenized in buffer 1.):, 百拇医药
Monolayer culture.):, 百拇医药
Rat epididymal fat derived preadipocytes were maintained in DMEM containing 5% calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 7 d (growth medium). After they reached confluency, the medium was replaced with differentiation medium containing 5 µg/ml insulin, 100 nM dexamethasone, 0.2 nM T3, and 10 µg/ml transferrin in growth medium. The differentiation medium was changed every 2 d until the cells were differentiated. After 10–14 d, differentiation was morphologically ascertained, and then the differentiation medium was replaced with growth medium containing 100 mM DHEA or DHEAS. The medium was changed every 2 d, and cells were collected after 3 and 5 d.
PPAR{gamma} and CEBP protein assay.m, 百拇医药
Equal amounts of cell lysate (50 µg protein) were subjected to SDS-PAGE, and transferred onto nitrocellulose paper. The paper was blocked with 5% skim milk TBS, and incubated with polyclonal anti-PPAR{gamma} antibody and anti-Glut4 antibody. Protein bands were visualized with an electrochemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan), and quantified by laser densitometry. The specific band was ascertained with negative control such as cell lysate of preadipocytes. The triglyceride content was measured with a triglyceride assay kit (Lipldos Lipid, Toyobo, Japan). Plasma leptin concentration was measured with a Rat Leptin RIA Kit (Linco Research, Inc., Tokyo, Japan).m, 百拇医药
Quantitative real-time-PCR for PPAR, C/EBP, adipocyte lipid-binding protein (aP2), PPAR, and sterol regulatory element binding protein (SREBP).m, 百拇医药
The mRNA levels of PPAR, C/EBP, aP2, PPAR, SREBP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by a real-time PCR following reverse transcription. Total RNA was prepared from the primary culture of rat adipocyte using Isogen (Nippon Gene, Toyama, Japan), and 2.2 µg of total RNA was treated with deoxyribonuclease I (Life Technologies, Inc., Gaithersburg, MD) and reverse transcribed with 160 U Superscript II reverse transcriptase (Life Technologies, Inc.) in a 20-µl reaction volume containing 2.5 mM random 9-oligomers, 1 mM each deoxy-NTP, 8 U placental ribonuclease inhibitor, and the manufacturer’s buffer. Each reaction was allowed to proceed at room temperature for 15 min followed by incubation at 42 C for 1.5 h. Aliquots (2 µl) of the 10 times-diluted reverse-transcribed samples were used for quantitative real-time PCRs in the LightCycler System using the FastStart DNA Master SYBR Green I kit (both from Roche, Arlington Heights, IL). The PCR products were detected via intercalation of the fluorescent dye SYBR Green. The sense and antisense primers used (GenBank accession numbers are in parentheses) with the concentrations of Mg2+/dimethylsulfoxide (DMSO) in buffer and the annealing temperature were as follows: GAPDH (), nucleotides (nt) 370–389 and 724–743 with 4 mM Mg2+/5% DMSO at 56 C; PPAR{gamma} (NM_013124), nt 944–963 and 1233–1253 with 3 mM Mg2+/4% DMSO at 58 C; C/EBP{alpha} (X12752), nt 854–872 and 1050–1070 with 4 mM Mg2+ at 56 C; aP2 (AF144756), nt 231–250 and 383–401 with 4 mM Mg2+/5% DMSO at 59 C; PPAR{alpha} (NM_013196), nt 1001–1020 and 1349–1369 with 3 mM Mg2+/2% DMSO at 59 C; SREBP (AF286470), nt 2550–2567 and 2820–2839 with 4 mM Mg2+/5% DMSO at 66 C. Corresponding fragments amplified in the 2 mM Mg2+ PCR buffer with Ex Taq DNA polymerase (TaKaRa Co., Osaka, Japan) were gel purified and quantitated using a DC120 digital camera and Kodak Digital Science 1D Image Analysis Software (Eastman Kodak Co., Rochester, NY), yielding each standard to calculate the exact copy number of each mRNA in the samples. Standards were prepared by 10-fold serial dilutions and used over the range of 10–100,000 copies/µl. The protocol included a 5-min denaturation at 95 C, followed by 45 cycles consisting of denaturation at 95 C for 10 sec, annealing at above temperature for 10 sec, and an extension phase at 72 C for 15 sec. Fluorescence was measured at the end of the 72 C extension phase. The quality of the RT-PCR products was controlled by melting point curve analysis (see representatives shown in Fig. 5). The mRNA values calculated as copy numbers in each samples were normalized for GAPDH, a housekeeping gene, to minimize variations in deoxyribonuclease digestion and RT between samples.
fig.ommitteed94#{i^3, 百拇医药
Figure 5. Effect of DHEA on PPAR, PPAR, and C/EBP mRNA expression in adipocyte primary culture. Primary cultured adipocytes obtained from Wistar rat epididymal fat pads were incubated with or without (control) 1 µM DHEA for 24 and 48 h. Real-time RT-PCR was performed to quantitate PPAR, PPAR, and C/EBP mRNA levels (see Materials and Methods). Each value was normalized against the level of GAPDH mRNA, and expression levels of mRNA are represented as percent of value found in control (100%) of each time. Melting point analysis demonstrated single peaks and specific products (upper). DHEA treatment for 24 h reduced PPAR mRNA to 20.0% (lower). Results shown are means ± SE of eight determinations. White bar, Control; black bar, DHEA. *, P < 0.05, vs. control by ANOVA.94#{i^3, 百拇医药
Statistical comparisons were performed using ANOVA followed up by Fisher’s protected least significant difference test. Unless otherwise stated, all data are expressed as the mean ± SE.94#{i^3, 百拇医药
Results94#{i^3, 百拇医药
Effect of DHEA treatment on body weight, adipose tissue weight, and plasma leptin concentration
Although DHEA treatment did not alter the body weight of OLETF, it reduced the weight of sc, epididymal, and perirenal adipose tissue significantly (Fig. 1). It also repressed the weight of epididymal fat in LETO rats. This effect was not observed in brown adipose tissue of both strains. These results indicate that DHEA treatment results in reduction of white adipose tissue weight. Plasma leptin concentration decreased after 2 wk treatment with DHEA in OLETF (Fig. 2). It was possible that DHEA might modulate transcriptional factors in adipocytes, so that we measured the expression of PPAR{gamma} protein in adipose tissue. As shown in Fig. 3, the expression of PPAR{gamma} protein in adipose tissue was increased in OLETF compared with LETO. DHEA treatment prevented the increase of PPAR{gamma} in OLETF.vwu, 百拇医药
fig.ommitteedvwu, 百拇医药
Figure 1. Effect of DHEA treatment on the weight of adipose tissue. LETO rats and OLETF rats (18 wk of age) were fed with or without (control) CE2 powder containing 0.4% DHEA for 2 wk. Epididymal, sc (anterior abdominal area) periintestinal, perirenal, and brown adipose tissues (BAT) were collected, and their wet weight was measured. White bar represents control rats and black bar DHEA-treated ones. n = 10; *, P < 0.05, by ANOVA.
fig.ommitteedj?, http://www.100md.com
Figure 2. Effect of DHEA treatment on plasma leptin level. LETO rats and OLETF rats were fed with or without 0.4% DHEA-containing feed. After 2 wk treatment, animals were killed and plasma was collected. Leptin concentration was measured with a rat leptin RIA kit. Results shown are means ± SE of six determinations. *, P < 0.05, by ANOVA.j?, http://www.100md.com
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Figure 3. PPAR protein expression in adipose tissue from LETO and OLETF rats. After the adipose tissues were collected, they were homogenized in buffer 1 (see Materials and Methods). Cell lysate of preadipocyte was applied as a negative control. Actin protein levels were simultaneously measured as control. Typical result is shown in A, and quantification of PPAR is shown in B. Data are represented LETO as 100%. Results shown are means ± SE of five determinations. A, White bar, LETO without DHEA treatment. B, Black bar, OLETF without DHEA treatment. C, Gray bar, OLETF with DHEA treatment. *, P < 0.05; **, P < 0.01 by ANOVA.j?, http://www.100md.com
Effect of DHEA on the expression of PPAR in cultured adipocytes
To examine the possibility that DHEA might directly act on adipose tissue, we assessed its effects on cultured adipocytes. DHEA induced a decrease in PPAR protein level in a time- and concentration-dependent manner (Fig. 4). Treatment with 1 µM DHEA in primary adipocyte culture for 48 h, and in monolayer adipocyte culture for 5 d reduced PPAR to 61.4% and 62.9% of the control, respectively. Moreover, exposure to 1 µM DHEA for 24 h markedly reduced mRNA expression of PPAR in primary adipocyte culture, although it did not affect slightly expressed C/EBP and PPAR mRNA levels (Fig. 5). Treatment with 1 µM dexamethasone, 1 µM testosterone, 1 µM androstenedione, and 100 nM insulin for 48 h did not change the PPAR and C/EBP{alpha} protein level in primary adipocyte culture, whereas 1 µM DHEA-S as well as DHEA significantly reduced the expression of PPAR protein levels (Fig. 6). To ascertain the reduced activity of PPAR{gamma} , we measured mRNA level of aP2 and SREBP, of which expression are regulated by PPAR (15), in primary adipocyte culture. Both adipocyte specific mRNAs, aP2 and lipogenetic enzyme, SREBP, were repressed to 28.6% and 33.1%, respectively, with DHEA treatment for 48 h (Fig. 7). We also found that protein level of Glut 4, but not Glut 1, was reduced with DHEA (data not shown). Furthermore, treatment with DHEA or DHEA-S for 5 d suppressed triglyceride content to 80.5% and 89.5% in monolayer adipocyte culture, respectively (Fig. 8).
fig.ommitteednq/ms*, 百拇医药
Figure 4. Effect of DHEA on PPAR protein level in adipocyte primary culture, and monolayer adipocyte differentiated from rat preadipocyte. Isolated adipocytes obtained by from epididymal fat pads of Wistar rat by collagenase digestion were cultured in DMEM with or without (control) DHEA for 24 and 48 h. Treatment with 1 µM DHEA for 24 h and 48 h reduced PPAR protein level to 73.5% and 61.4%, respectively, in primary culture cells (n = 7) (A). Rat epididymal fat-derived preadipocytes, fully differentiated to adipocytes, were incubated with or without (control) DHEA or DHEAS for 3 d and 5 d. PPAR protein level decreased to 62.9% and 71.0%, respectively, with 1 µM DHEA or DHEA-S treatment for 5 d (n = 6) (B). Data are represented control value as 100%. White bar, Control; black bar, DHEA; gray bar, DHEAS. *, P < 0.05, **, P < 0.01 vs. control by ANOVA.nq/ms*, 百拇医药
fig.ommitteednq/ms*, 百拇医药
Figure 6. Effect of various hormones on of PPAR protein expression. Isolated adipocytes obtained by collagenase digestion of epididymal fat pads from Wistar rat was cultured in DMEM with or without (control) 1 µM DHEA, 1 µM DHEA-S, 1 µM dexamethasone (Dexa), 1 µM testosterone (Testo), 1 µM androstenedione (Andro), and 100 nM insulin (Ins) for 48 h. The expression of PPAR protein was measured with immunoblotting. Only DHEA and DHEA-S suppressed PPAR protein expression to 74% and 86%, respectively. Results shown are means ± SE of five determinations. **, P < 0.01 vs. control by ANOVA.
fig.ommitteedl4!10, 百拇医药
Figure 7. Effect of DHEA on aP2 and SREBP expression. Primary adipocyte culture was treated with or without (control) 1 µM DHEA for 24 h and 48 h. Real-time PCR was used to measure aP2 and SREBP mRNA levels. Each value was normalized against the level of GAPDH mRNA. Expression levels of mRNA are represented as percent of the values found in control (100%) at each time. Results shown are means ± SE of four determinations. White bar, Control; black bar, DHEA. *, P < 0.05, vs. control by ANOVA.l4!10, 百拇医药
fig.ommitteedl4!10, 百拇医药
Figure 8. Effect of DHEA and DHEAS on triglyceride content in monolayer adipocytes. Adipocytes were differentiated from preadipocytes by incubation with differentiation medium containing 510 nM insulin, 100 nM dexamethasone, 0.2 nM triiodothyronine, and 10 µg/ml transferrin for 10–14 d. After the differentiation was ascertained, the medium was exchanged to DMEM with or without 1 µM DHEA or DHEAS. These treatments did not induce any significant morphological changes (left); however, DHEA and DHEA-S prevented triglyceride accumulation (right). Triglyceride content was measured with a triglyceride assay kit (Lipldos Lipid), and adjusted for the protein concentration of each cell lysate. Results shown are means ± SE of five determinations. **, P < 0.01 vs. control by ANOVA.
Discussiongk\l, 百拇医药
Although DHEA and DHEA-S are relatively abundant adrenal steroid hormones, their physiologic roles are unclear. Pharmacological effects of DHEA such as preventing diabetes, obesity, cancer, or modulation of the immune system have been postulated (17). Especially, many authors (6, 7, 8, 9, 10, 11) have described the antiobesity effect of DHEA. Some of them attributed this effect to diminished appetite (6, 7, 8), whereas others claimed that DHEA suppresses weight gain without changing food intake (5, 9, 10, 11). In this study, DHEA treatment for 2 wk in genetically obese OLETF rats reduced the weight of sc and visceral adipose tissues, but not total body weight. Although a little effect was observed also in control rats, DHEA decreased adipose tissues of broad range in fatty ones. We also revealed that DHEA treatment significantly reduced plasma leptin level. Leptin is thought to correlate with the amount of body fat stores. Therefore, DHEA-induced reduction in adipose tissue mass might contribute to the decrease in plasma leptin concentration. Moreover, we found that DHEA treatment prevented the elevation of PPAR{gamma} expression in adipose tissue from OLETF rats. The present study could not ascertain whether the expression of PPAR{gamma} in adipose tissue is genetically determined or not. However, considering the fact that an increased amount of PPAR{gamma} expression in obese human adipose tissue, which is proportional to body mass index, can be reduced by a low calorie diet (18), our result seems to suggest an acquired origin.
We questioned whether these effects of DHEA on adipose tissue are direct or not. Interestingly, DHEA-S cannot stimulate peroxisomal gene induction in the liver of PPAR{alpha} knockout mice, suggesting that some of the DHEA or DHEA-S actions are mediated via the PPAR family (19). We examined the effect of DHEA on the expression of PPAR{gamma} and C/EBP{alpha} using primary culture from rat adipose tissue, and monolayer adipocytes differentiated from rat preadipocytes. Primary cultured cells exhibited a native response to hormones, but it was difficult to obtain stable viability of the cells. Therefore, we used cultured monolayer adipocytes to examine the effect of DHEA treatment for more than 3 d. DHEA down-regulated the expression of PPAR protein in both cell culture, and there was an effect on mRNA expression. Although the concentrations of DHEA and DHEA-S we examined were pharmacological doses, significant effects were observed within 2 d in primary cultured cells. Our in vivo and in vitro study induced 30–40% and 25–40% decrease in PPAR protein level, respectively. These results are supposed to be biologically significant, because Pro 12 Ala substitution in human PPAR gene, which shows 30–45% less transactivation capacity, leads to prevention of obesity and insulin resistance (20). Our recent preliminary study revealed that the effect of DHEA on PPAR was enhanced in adipocytes with overexpression of protein kinase C (PKC) (data not shown). We previously reported that DHEA activates phosphatidylinositol 3-kinase and atypical PKC (21). In addition, TNF-, another PPAR-reducing agonist, also activated atypical PKC (22). Taken together, we hypothesized that PPAR-reducing effect of DHEA might depend on the expression level of atypical PKC. These effects depend on specific DHEA action, as shown in Fig. 6. DHEA treatment decreased other adipocyte specific phenotypes including triglyceride content and expression of aP2 and SREBP. The reduction of mRNA level of PPAR during DHEA treatment was significant at 24 h, whereas those of aP2 and SREBP were at 48 h. Because the promoter of aP2 contains peroxisome proliferator response element (15), decreased PPAR activity might lead to reduction in aP2 expression. On the other hand, expression of SREBP, a transcriptional factor associated with lipid metabolism, is known to be regulated by several factors, such as insulin and cholesterol. It also correlates with PPAR{gamma} activity (23), which explains our result.
DHEA treatment suppressed plasma leptin concentration in OLETF rats. In our preliminary experiment (data not shown), DHEA decreased leptin secretion in primary adipocyte culture, consistent with our present animal study and those of other authors (24, 25). C/EBP{alpha} is implicated as a transactivator of the leptin promoter through a consensus binding site at nt -55 to -47 in the proximal promoter region (26). PPAR{gamma} mediates down-regulation of the leptin promoter by inhibiting C/EBP{alpha} -mediated transactivation (27). If plasma leptin concentration was regulated solely by PPAR activity, DHEA administration would lead to the up-regulation of leptin expression. Accordingly, we speculated that the reduced C/EBP expression might lead to suppression of leptin secretion. However, DHEA did not change the expression of C/EBP mRNA in our experiment. Possible explanations are that DHEA might increase degradation of C/EBP, or that DHEA-induced phosphatidylinositol 3- kinase reduced C/EBP activity like as insulin (28). In fact, leptin expression does not always relate to C/EBP expression level (29). Further study will be necessary to understand the effect of DHEA on leptin secretion.
In conclusion, DHEA down-regulates adiposity through the reduction of PPAR expression, which will contribute to the amelioration of insulin sensitivity.]6g3%, 百拇医药
Received January 14, 2002.]6g3%, 百拇医药
Accepted for publication September 6, 2002.]6g3%, 百拇医药
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Sloop KW, Surface PL, Heiman ML, Slieker LJ 1998 Changes in leptin expression are not associated with corresponding changes in CCAAT/Enhancer binding protein-. Biochem Biophys Res Commun 251:142–147(Kazuo Kajita Tatsuo Ishizuka Tomoatsu Mune Atsushi Miura Masayoshi Ishizawa Yoshinori Kanoh Yasunori)
Dehydroepiandrosterone (DHEA) is expected to have a weight-reducing effect. In this study, we evaluated the effect of DHEA on genetically obese Otsuka Long Evans Fatty rats (OLETF) compared with Long-Evans Tokushima rats (LETO) as control. Feeding with 0.4% DHEA-containing food for 2 wk reduced the weight of sc, epididymal, and perirenal adipose tissue in association with decreased plasma leptin levels in OLETF. Adipose tissue from OLETF showed increased expression of peroxisome proliferator-activated receptor (PPAR) protein, which was prevented by DHEA treatment. Further, we examined the effect of DHEA on PPAR in primary cultured adipocytes and monolayer adipocytes differentiated from rat preadipocytes. PPAR protein level was decreased in a time- and concentration-dependent manner, and DHEA significantly reduced mRNA levels of PPAR{gamma} , adipocyte lipid-binding protein, and sterol regulatory element-binding protein, but not CCAAT/enhancer binding protein {alpha} . DHEA-sulfate also reduced the PPAR{gamma} protein, but dexamethasone, testosterone, or androstenedione did not alter its expression. In addition, treatment with DHEA for 5 d reduced the triglyceride content in monolayer adipocytes. These results suggest that DHEA down-regulates adiposity through the reduction of PPAR{gamma} in adipocytes.
Introductionhg1)h, 百拇医药
MUCH EVIDENCE SUGGESTS that adrenal androgen, dehydroepiandrosterone (DHEA), or its sulfate metabolite DHEA sulfate (DHEA-S), ameliorate glucose metabolism in diabetic animals, but the underlying mechanisms are still controversial. Coleman et al. (1) reported that DHEA administration to db/db mice prevented the development of diabetes mellitus. Kimura et al. (2) reported that DHEA decreases serum TNF{alpha} in Zucker fatty rats , whereas Aoki et al. (3) indicated that DHEA suppresses the elevated glucose-6-phosphatase and fructose-1,6-bisphosphatase activities in C57BL/Ksj-db/db mice. These authors suggested that administration of DHEA prevented insulin resistance. We reported previously that oral administration of DHEA for 2 wk to Otsuka Long Evans Fatty (OLETF) rats, an animal model of obese type 2 diabetes, but not Goto-Kakizaki rats, an animal model of lean type 2 diabetes, decreased the elevated plasma glucose (4). We remarked that DHEA improved plasma glucose only in the obese animal models but did not alter their body weight within 2 wk administration. Animal studies indicate that DHEA has antiobesity effect (5, 6, 7, 8, 9, 10, 11). Moreover, DHEA prevents proliferation and differentiation of 3T3-L1 preadipocytes (12). Accordingly, we assumed that DHEA might have some effect on adipose tissue, which results in the prevention of insulin resistance.
Recent studies have indicated that transcription factor, peroxisome proliferator-activated receptor {gamma} (PPAR{gamma} ), and CCAAT/enhancer binding protein {alpha} (C/EBP{alpha} ) play a critical role in promoting adipocyte differentiation. PPAR{gamma} is expressed in the early phase of adipocyte differentiation (13), whereas C/EBP{alpha} appears in a relatively late phase (14). The fact that cotransfection of PPAR{gamma} and C/EBP{alpha} was sufficient to induce adipogenesis in the absence of exogenous ligands (15) suggests that they act synergistically in the differentiation process. On the other hand, PPAR{gamma} expression is necessary for the maintenance of the adipocyte phenotype (16), although the regulation mechanisms of these transcription factors in mature adipocytes are uncertain. In this study, we investigated whether DHEA reduces the adiposity in OLETF rats, and whether DHEA modulates the expression of PPAR{gamma} and C/EBP{alpha} in adipocytes.;d, 百拇医药
Materials and Methods
Materialsif7s, 百拇医药
DHEA, DHEA-S, dexamethasone, androstenedione, and testosterone were purchased from Sigma (St. Louis, MO). Pork insulin was obtained from Novo Nordisk (Copenhagen, Denmark). Anti-PPAR{gamma} antibody, anti-CEBP{alpha} and ß antibody, and antiactin antibody were purchased from Santa Cruz Biotechnologies, Inc. (Santa Cruz, CA). Rat epididymal fat derived preadipocytes were purchased from Toyobo (Osaka, Japan). All other chemicals were of reagent grade or better.if7s, 百拇医药
Methodsif7s, 百拇医药
Animal treatment.if7s, 百拇医药
Long-Evans Tokushima (LETO rat, control) rats and OLETF rats (18 wk of age) were fed CE2 (Japan Clea, Tokyo, Japan) ad libitum, treated with CE2 powder containing 0.4% DHEA for 2 wk, and then killed by decapitation. Epididymal, sc, periintestinal, perirenal, and brown adipose tissues were collected. After wet weight was measured, they were homogenized in buffer 1 containing 20 mM Tris/HCl (pH 7.5), 145 mM NaCl, 10% glycerol, 5 mM EDTA, 1% Triton X-100, 0.5% Nonidet P-40, 0.2 mM Na2VO4, 0.1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, and 10 µg/ml aprotinin.
Primary culture.):, 百拇医药
To obtain the primary culture of rat adipocyte, Male Wistar rats weighing 200 g were killed by decapitation. Isolated adipocytes were obtained by collagenase digestion of epididymal fat pads in DMEM containing 5% calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin. Adipocytes were washed three times and incubated with various concentrations of DHEA, 1 µM DHEA-S, 1 µM androstendione, 1 µM testosterone, and 100 nM insulin at 37 C in 5% CO2+ 95% O2. After 24 or 48 h incubation, adipocytes were collected and homogenized in buffer 1.):, 百拇医药
Monolayer culture.):, 百拇医药
Rat epididymal fat derived preadipocytes were maintained in DMEM containing 5% calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin for 7 d (growth medium). After they reached confluency, the medium was replaced with differentiation medium containing 5 µg/ml insulin, 100 nM dexamethasone, 0.2 nM T3, and 10 µg/ml transferrin in growth medium. The differentiation medium was changed every 2 d until the cells were differentiated. After 10–14 d, differentiation was morphologically ascertained, and then the differentiation medium was replaced with growth medium containing 100 mM DHEA or DHEAS. The medium was changed every 2 d, and cells were collected after 3 and 5 d.
PPAR{gamma} and CEBP protein assay.m, 百拇医药
Equal amounts of cell lysate (50 µg protein) were subjected to SDS-PAGE, and transferred onto nitrocellulose paper. The paper was blocked with 5% skim milk TBS, and incubated with polyclonal anti-PPAR{gamma} antibody and anti-Glut4 antibody. Protein bands were visualized with an electrochemiluminescence system (Amersham Pharmacia Biotech, Tokyo, Japan), and quantified by laser densitometry. The specific band was ascertained with negative control such as cell lysate of preadipocytes. The triglyceride content was measured with a triglyceride assay kit (Lipldos Lipid, Toyobo, Japan). Plasma leptin concentration was measured with a Rat Leptin RIA Kit (Linco Research, Inc., Tokyo, Japan).m, 百拇医药
Quantitative real-time-PCR for PPAR, C/EBP, adipocyte lipid-binding protein (aP2), PPAR, and sterol regulatory element binding protein (SREBP).m, 百拇医药
The mRNA levels of PPAR, C/EBP, aP2, PPAR, SREBP and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were measured by a real-time PCR following reverse transcription. Total RNA was prepared from the primary culture of rat adipocyte using Isogen (Nippon Gene, Toyama, Japan), and 2.2 µg of total RNA was treated with deoxyribonuclease I (Life Technologies, Inc., Gaithersburg, MD) and reverse transcribed with 160 U Superscript II reverse transcriptase (Life Technologies, Inc.) in a 20-µl reaction volume containing 2.5 mM random 9-oligomers, 1 mM each deoxy-NTP, 8 U placental ribonuclease inhibitor, and the manufacturer’s buffer. Each reaction was allowed to proceed at room temperature for 15 min followed by incubation at 42 C for 1.5 h. Aliquots (2 µl) of the 10 times-diluted reverse-transcribed samples were used for quantitative real-time PCRs in the LightCycler System using the FastStart DNA Master SYBR Green I kit (both from Roche, Arlington Heights, IL). The PCR products were detected via intercalation of the fluorescent dye SYBR Green. The sense and antisense primers used (GenBank accession numbers are in parentheses) with the concentrations of Mg2+/dimethylsulfoxide (DMSO) in buffer and the annealing temperature were as follows: GAPDH (), nucleotides (nt) 370–389 and 724–743 with 4 mM Mg2+/5% DMSO at 56 C; PPAR{gamma} (NM_013124), nt 944–963 and 1233–1253 with 3 mM Mg2+/4% DMSO at 58 C; C/EBP{alpha} (X12752), nt 854–872 and 1050–1070 with 4 mM Mg2+ at 56 C; aP2 (AF144756), nt 231–250 and 383–401 with 4 mM Mg2+/5% DMSO at 59 C; PPAR{alpha} (NM_013196), nt 1001–1020 and 1349–1369 with 3 mM Mg2+/2% DMSO at 59 C; SREBP (AF286470), nt 2550–2567 and 2820–2839 with 4 mM Mg2+/5% DMSO at 66 C. Corresponding fragments amplified in the 2 mM Mg2+ PCR buffer with Ex Taq DNA polymerase (TaKaRa Co., Osaka, Japan) were gel purified and quantitated using a DC120 digital camera and Kodak Digital Science 1D Image Analysis Software (Eastman Kodak Co., Rochester, NY), yielding each standard to calculate the exact copy number of each mRNA in the samples. Standards were prepared by 10-fold serial dilutions and used over the range of 10–100,000 copies/µl. The protocol included a 5-min denaturation at 95 C, followed by 45 cycles consisting of denaturation at 95 C for 10 sec, annealing at above temperature for 10 sec, and an extension phase at 72 C for 15 sec. Fluorescence was measured at the end of the 72 C extension phase. The quality of the RT-PCR products was controlled by melting point curve analysis (see representatives shown in Fig. 5). The mRNA values calculated as copy numbers in each samples were normalized for GAPDH, a housekeeping gene, to minimize variations in deoxyribonuclease digestion and RT between samples.
fig.ommitteed94#{i^3, 百拇医药
Figure 5. Effect of DHEA on PPAR, PPAR, and C/EBP mRNA expression in adipocyte primary culture. Primary cultured adipocytes obtained from Wistar rat epididymal fat pads were incubated with or without (control) 1 µM DHEA for 24 and 48 h. Real-time RT-PCR was performed to quantitate PPAR, PPAR, and C/EBP mRNA levels (see Materials and Methods). Each value was normalized against the level of GAPDH mRNA, and expression levels of mRNA are represented as percent of value found in control (100%) of each time. Melting point analysis demonstrated single peaks and specific products (upper). DHEA treatment for 24 h reduced PPAR mRNA to 20.0% (lower). Results shown are means ± SE of eight determinations. White bar, Control; black bar, DHEA. *, P < 0.05, vs. control by ANOVA.94#{i^3, 百拇医药
Statistical comparisons were performed using ANOVA followed up by Fisher’s protected least significant difference test. Unless otherwise stated, all data are expressed as the mean ± SE.94#{i^3, 百拇医药
Results94#{i^3, 百拇医药
Effect of DHEA treatment on body weight, adipose tissue weight, and plasma leptin concentration
Although DHEA treatment did not alter the body weight of OLETF, it reduced the weight of sc, epididymal, and perirenal adipose tissue significantly (Fig. 1). It also repressed the weight of epididymal fat in LETO rats. This effect was not observed in brown adipose tissue of both strains. These results indicate that DHEA treatment results in reduction of white adipose tissue weight. Plasma leptin concentration decreased after 2 wk treatment with DHEA in OLETF (Fig. 2). It was possible that DHEA might modulate transcriptional factors in adipocytes, so that we measured the expression of PPAR{gamma} protein in adipose tissue. As shown in Fig. 3, the expression of PPAR{gamma} protein in adipose tissue was increased in OLETF compared with LETO. DHEA treatment prevented the increase of PPAR{gamma} in OLETF.vwu, 百拇医药
fig.ommitteedvwu, 百拇医药
Figure 1. Effect of DHEA treatment on the weight of adipose tissue. LETO rats and OLETF rats (18 wk of age) were fed with or without (control) CE2 powder containing 0.4% DHEA for 2 wk. Epididymal, sc (anterior abdominal area) periintestinal, perirenal, and brown adipose tissues (BAT) were collected, and their wet weight was measured. White bar represents control rats and black bar DHEA-treated ones. n = 10; *, P < 0.05, by ANOVA.
fig.ommitteedj?, http://www.100md.com
Figure 2. Effect of DHEA treatment on plasma leptin level. LETO rats and OLETF rats were fed with or without 0.4% DHEA-containing feed. After 2 wk treatment, animals were killed and plasma was collected. Leptin concentration was measured with a rat leptin RIA kit. Results shown are means ± SE of six determinations. *, P < 0.05, by ANOVA.j?, http://www.100md.com
fig.ommitteedj?, http://www.100md.com
Figure 3. PPAR protein expression in adipose tissue from LETO and OLETF rats. After the adipose tissues were collected, they were homogenized in buffer 1 (see Materials and Methods). Cell lysate of preadipocyte was applied as a negative control. Actin protein levels were simultaneously measured as control. Typical result is shown in A, and quantification of PPAR is shown in B. Data are represented LETO as 100%. Results shown are means ± SE of five determinations. A, White bar, LETO without DHEA treatment. B, Black bar, OLETF without DHEA treatment. C, Gray bar, OLETF with DHEA treatment. *, P < 0.05; **, P < 0.01 by ANOVA.j?, http://www.100md.com
Effect of DHEA on the expression of PPAR in cultured adipocytes
To examine the possibility that DHEA might directly act on adipose tissue, we assessed its effects on cultured adipocytes. DHEA induced a decrease in PPAR protein level in a time- and concentration-dependent manner (Fig. 4). Treatment with 1 µM DHEA in primary adipocyte culture for 48 h, and in monolayer adipocyte culture for 5 d reduced PPAR to 61.4% and 62.9% of the control, respectively. Moreover, exposure to 1 µM DHEA for 24 h markedly reduced mRNA expression of PPAR in primary adipocyte culture, although it did not affect slightly expressed C/EBP and PPAR mRNA levels (Fig. 5). Treatment with 1 µM dexamethasone, 1 µM testosterone, 1 µM androstenedione, and 100 nM insulin for 48 h did not change the PPAR and C/EBP{alpha} protein level in primary adipocyte culture, whereas 1 µM DHEA-S as well as DHEA significantly reduced the expression of PPAR protein levels (Fig. 6). To ascertain the reduced activity of PPAR{gamma} , we measured mRNA level of aP2 and SREBP, of which expression are regulated by PPAR (15), in primary adipocyte culture. Both adipocyte specific mRNAs, aP2 and lipogenetic enzyme, SREBP, were repressed to 28.6% and 33.1%, respectively, with DHEA treatment for 48 h (Fig. 7). We also found that protein level of Glut 4, but not Glut 1, was reduced with DHEA (data not shown). Furthermore, treatment with DHEA or DHEA-S for 5 d suppressed triglyceride content to 80.5% and 89.5% in monolayer adipocyte culture, respectively (Fig. 8).
fig.ommitteednq/ms*, 百拇医药
Figure 4. Effect of DHEA on PPAR protein level in adipocyte primary culture, and monolayer adipocyte differentiated from rat preadipocyte. Isolated adipocytes obtained by from epididymal fat pads of Wistar rat by collagenase digestion were cultured in DMEM with or without (control) DHEA for 24 and 48 h. Treatment with 1 µM DHEA for 24 h and 48 h reduced PPAR protein level to 73.5% and 61.4%, respectively, in primary culture cells (n = 7) (A). Rat epididymal fat-derived preadipocytes, fully differentiated to adipocytes, were incubated with or without (control) DHEA or DHEAS for 3 d and 5 d. PPAR protein level decreased to 62.9% and 71.0%, respectively, with 1 µM DHEA or DHEA-S treatment for 5 d (n = 6) (B). Data are represented control value as 100%. White bar, Control; black bar, DHEA; gray bar, DHEAS. *, P < 0.05, **, P < 0.01 vs. control by ANOVA.nq/ms*, 百拇医药
fig.ommitteednq/ms*, 百拇医药
Figure 6. Effect of various hormones on of PPAR protein expression. Isolated adipocytes obtained by collagenase digestion of epididymal fat pads from Wistar rat was cultured in DMEM with or without (control) 1 µM DHEA, 1 µM DHEA-S, 1 µM dexamethasone (Dexa), 1 µM testosterone (Testo), 1 µM androstenedione (Andro), and 100 nM insulin (Ins) for 48 h. The expression of PPAR protein was measured with immunoblotting. Only DHEA and DHEA-S suppressed PPAR protein expression to 74% and 86%, respectively. Results shown are means ± SE of five determinations. **, P < 0.01 vs. control by ANOVA.
fig.ommitteedl4!10, 百拇医药
Figure 7. Effect of DHEA on aP2 and SREBP expression. Primary adipocyte culture was treated with or without (control) 1 µM DHEA for 24 h and 48 h. Real-time PCR was used to measure aP2 and SREBP mRNA levels. Each value was normalized against the level of GAPDH mRNA. Expression levels of mRNA are represented as percent of the values found in control (100%) at each time. Results shown are means ± SE of four determinations. White bar, Control; black bar, DHEA. *, P < 0.05, vs. control by ANOVA.l4!10, 百拇医药
fig.ommitteedl4!10, 百拇医药
Figure 8. Effect of DHEA and DHEAS on triglyceride content in monolayer adipocytes. Adipocytes were differentiated from preadipocytes by incubation with differentiation medium containing 510 nM insulin, 100 nM dexamethasone, 0.2 nM triiodothyronine, and 10 µg/ml transferrin for 10–14 d. After the differentiation was ascertained, the medium was exchanged to DMEM with or without 1 µM DHEA or DHEAS. These treatments did not induce any significant morphological changes (left); however, DHEA and DHEA-S prevented triglyceride accumulation (right). Triglyceride content was measured with a triglyceride assay kit (Lipldos Lipid), and adjusted for the protein concentration of each cell lysate. Results shown are means ± SE of five determinations. **, P < 0.01 vs. control by ANOVA.
Discussiongk\l, 百拇医药
Although DHEA and DHEA-S are relatively abundant adrenal steroid hormones, their physiologic roles are unclear. Pharmacological effects of DHEA such as preventing diabetes, obesity, cancer, or modulation of the immune system have been postulated (17). Especially, many authors (6, 7, 8, 9, 10, 11) have described the antiobesity effect of DHEA. Some of them attributed this effect to diminished appetite (6, 7, 8), whereas others claimed that DHEA suppresses weight gain without changing food intake (5, 9, 10, 11). In this study, DHEA treatment for 2 wk in genetically obese OLETF rats reduced the weight of sc and visceral adipose tissues, but not total body weight. Although a little effect was observed also in control rats, DHEA decreased adipose tissues of broad range in fatty ones. We also revealed that DHEA treatment significantly reduced plasma leptin level. Leptin is thought to correlate with the amount of body fat stores. Therefore, DHEA-induced reduction in adipose tissue mass might contribute to the decrease in plasma leptin concentration. Moreover, we found that DHEA treatment prevented the elevation of PPAR{gamma} expression in adipose tissue from OLETF rats. The present study could not ascertain whether the expression of PPAR{gamma} in adipose tissue is genetically determined or not. However, considering the fact that an increased amount of PPAR{gamma} expression in obese human adipose tissue, which is proportional to body mass index, can be reduced by a low calorie diet (18), our result seems to suggest an acquired origin.
We questioned whether these effects of DHEA on adipose tissue are direct or not. Interestingly, DHEA-S cannot stimulate peroxisomal gene induction in the liver of PPAR{alpha} knockout mice, suggesting that some of the DHEA or DHEA-S actions are mediated via the PPAR family (19). We examined the effect of DHEA on the expression of PPAR{gamma} and C/EBP{alpha} using primary culture from rat adipose tissue, and monolayer adipocytes differentiated from rat preadipocytes. Primary cultured cells exhibited a native response to hormones, but it was difficult to obtain stable viability of the cells. Therefore, we used cultured monolayer adipocytes to examine the effect of DHEA treatment for more than 3 d. DHEA down-regulated the expression of PPAR protein in both cell culture, and there was an effect on mRNA expression. Although the concentrations of DHEA and DHEA-S we examined were pharmacological doses, significant effects were observed within 2 d in primary cultured cells. Our in vivo and in vitro study induced 30–40% and 25–40% decrease in PPAR protein level, respectively. These results are supposed to be biologically significant, because Pro 12 Ala substitution in human PPAR gene, which shows 30–45% less transactivation capacity, leads to prevention of obesity and insulin resistance (20). Our recent preliminary study revealed that the effect of DHEA on PPAR was enhanced in adipocytes with overexpression of protein kinase C (PKC) (data not shown). We previously reported that DHEA activates phosphatidylinositol 3-kinase and atypical PKC (21). In addition, TNF-, another PPAR-reducing agonist, also activated atypical PKC (22). Taken together, we hypothesized that PPAR-reducing effect of DHEA might depend on the expression level of atypical PKC. These effects depend on specific DHEA action, as shown in Fig. 6. DHEA treatment decreased other adipocyte specific phenotypes including triglyceride content and expression of aP2 and SREBP. The reduction of mRNA level of PPAR during DHEA treatment was significant at 24 h, whereas those of aP2 and SREBP were at 48 h. Because the promoter of aP2 contains peroxisome proliferator response element (15), decreased PPAR activity might lead to reduction in aP2 expression. On the other hand, expression of SREBP, a transcriptional factor associated with lipid metabolism, is known to be regulated by several factors, such as insulin and cholesterol. It also correlates with PPAR{gamma} activity (23), which explains our result.
DHEA treatment suppressed plasma leptin concentration in OLETF rats. In our preliminary experiment (data not shown), DHEA decreased leptin secretion in primary adipocyte culture, consistent with our present animal study and those of other authors (24, 25). C/EBP{alpha} is implicated as a transactivator of the leptin promoter through a consensus binding site at nt -55 to -47 in the proximal promoter region (26). PPAR{gamma} mediates down-regulation of the leptin promoter by inhibiting C/EBP{alpha} -mediated transactivation (27). If plasma leptin concentration was regulated solely by PPAR activity, DHEA administration would lead to the up-regulation of leptin expression. Accordingly, we speculated that the reduced C/EBP expression might lead to suppression of leptin secretion. However, DHEA did not change the expression of C/EBP mRNA in our experiment. Possible explanations are that DHEA might increase degradation of C/EBP, or that DHEA-induced phosphatidylinositol 3- kinase reduced C/EBP activity like as insulin (28). In fact, leptin expression does not always relate to C/EBP expression level (29). Further study will be necessary to understand the effect of DHEA on leptin secretion.
In conclusion, DHEA down-regulates adiposity through the reduction of PPAR expression, which will contribute to the amelioration of insulin sensitivity.]6g3%, 百拇医药
Received January 14, 2002.]6g3%, 百拇医药
Accepted for publication September 6, 2002.]6g3%, 百拇医药
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