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Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, School of Medicine, Tsinghua University, Beijing, P.R. China
Testosterone inhibits the activity of peroxisome proliferator-activated receptor g in a transcriptional transaction assay J. Du, L. Zhang, Z. Wang
Received April 13, 2009, accepted May 5, 2009 Prof. Dr. Zhao Wang, Protein Science Key Laboratory of the Ministry of Education, Department of Biological Sciences and Biotechnology, Medical School, Tsinghua University, Beijing 100084, the People’s Republic of China
[email protected] Pharmazie 64: 692–693 (2009) doi: 10.1691/ph.2009.9606
Emerging evidences suggest that testosterone is an important regulator of body fat mass in men. However, the mechanism underlying the regulation of body composition by testosterone is not well understood. The paper demonstrates testosterone inhibited the activity of peroxisome proliferator-activated receptor g (PPARg), which controls the adipogenic differentiation and lipid metabolism. Pre-inhibition of androgen receptor did not influence the inhibition of PPARg activity by testosterone. Moreover, pre-treatment with testosterone attenuated the insulin-induced up-regulation of PPARg activity. These results provide a novel mechanism for the effect of testosterone on fat mass control.
Emerging evidences suggest that testosterone is an important regulator of body fat mass in men (Wilson 1988; Bhasin et al. 2001a). Androgen deficiency is associated with increased fat mass (Katznelson et al. 1998), while testosterone supplementation decreases fat mass (Snyder et al. 2000; Wang et al. 2000). Although testosterone effects on whole-body and regional fat mass are inversely correlated with testosterone dose and concentrations (Bhasin et al. 2001b), however, the mechanism underlying the regulation
of body composition by testosterone is not well understood. A recent report shows that testosterone down-regulates the expression of peroxisome proliferator-activated receptor g (PPARg) (Singh et al. 2003), a key transcription factor which controls the adipogenic differentiation and lipid metabolism (Gregoire et al. 1998; Rosen and Spiegelman 2000; MacDougald and Mandrup 2002). In the present study, we used an established cell-based transactivation assay to evaluate the regulation of the activity of PPARg by testosterone. An agonist (rosiglitazone) and an antagonist (GW9662) of PPARg were used as the positive and negative reference standards respectively for PPARg in our studies. The results show that treatment with testosterone (10 nM) significantly inhibited the activity of PPARg (0.43-fold, p < 0.001). The inhibitory effect of testosterone on PPARg activity was comparable to the effect of GW9662 (10 mM, 0.48-fold, p < 0.001). Conversely, treatment with rosiglitazone (1 mM) markedly induced PPARg activity (5.3-fold, p < 0.01, Fig. 1A). Next, we investigated whether the effect of testosterone on the activity of PPARg occurs through an androgen receptor (AR)-mediated pathway. An androgen receptor antagonist, flutamide, was used to pre-inhibit AR activity. As shown in Fig. 1B, pre-inhibition of androgen receptor by flutamide (10 mM) did not influence the inhibition of PPARg activity by testosterone. These results indicate that testosterone inhibits the activity of PPARg probably independent of AR activation. According to the previous studies, in addition to regulation of gene transcription via activating AR, testosterone also acts through non-genetic pathways, producing rapid and transcription-independent effects, including activation of second messenger signaling mechanism (Heinlein and Chang 2002). Evidences have shown that insulin signal pathway induces PPARg activity (Al-Rasheed et al. 2004). Thus, we examined the effect of testosterone on the insulin-induced PPARg activation. As shown in Fig. 2, treatment with insulin (20 nM) significantly induced the activity of PPARg (1.7fold, p < 0.01), while pre-treatment with testosterone (10 nM) almost blocked the up-regulation of PPARg activity by insulin. These results indicate that the inhibition of PPARg activity by testosterone probably involves interaction between testosterone and insulin signal pathway. The present study reveals, for the first time, the inhibitory effect of testosterone on PPARg activity, which is probably independent of AR activation. Testosterone is reported to inhibit differentiation of adipocyte precursor cells and suppress lipid uptake and lipoprotein lipase activity in adipocytes (De Pergola 2000). Thus, the inhibi-
Fig. 1: Testosterone inhibited the activity of PPARg. (A) The effects of testosterone (Tes, 10 nM), rosiglitazone (RGZ, 1 mM) and GW9662 (GW, 10 mM) on the activity of PPARg were evaluated by PPARg transaction assay. Data are presented as the mean S.E. Asterisks indicates statistical difference from vehicle (VEH). **p < 0.001, t test, n ¼ 4. (B) The effects of testosterone (Tes, 10 nM), with pre-inhibition of AR by flutamide (10 mM) on the activity of PPARg were evaluated by PPARg transaction assay. Data are presented as the mean S.E. Asterisks indicates statistical difference from vehicle (VEH). **p < 0.001, t-test, n ¼ 4
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4. Statistical analyses Data were presented as group means S.E. Differences between means were determined using the Student’s t-test for paired or unpaired observations. Differences were considered statistically significant when p value < 0.05. Acknowledgements: The wild-type PPARg expression construct and PPRE-TK reporter gene were kindly provided by Professor R. Evans (Salk Institute, La Jolla, CA, USA). This work was financially supported by the National Basic Research Program (973 Project) of China (No. 2007CB507406), the National Natural Science Foundation of China (No. 30572341) and the Tsinghua-Yue-Yuen Medical Sciences Fund (THYY20070008). References
Fig. 2: Testosterone attenuated the up-regulation of PPARg activity induced by insulin. The effect of insulin (20 nM), with or without pre-incubation with testosterone (Tes, 10 nM), on the activity of PPARg were evaluated by PPARg transaction assay. Data are presented as the mean S.E. Asterisks indicates statistical difference from vehicle (VEH). *p < 0.05, t-test, n ¼ 4
tory effect of testosterone on PPARg activity is consistent with previous studies and provides a novel explanation for the effect of testosterone on fat mass control. Experimental 1. Cell culture 293T cells (ATCC, USA) were maintained in Dulbecco’s modified Eagle medium (Hyclone, USA), supplemented with 10% fetal bovine serum (GIBCO, USA), 100 mg/ml streptomycin and 100 units/ml penicillin. 2. In vitro PPARg transaction assay 293T cells were seeded in 24 well plates at a density of 1106 cells/well in 1 ml of medium per well. Cells were transfected using the lipofectamine2000 as recommended by the manufacturer (Invitrogen, USA) with a mixture containing 1 mg of PPRE3-TK-luc reporter plasmid, 5 ng of pRLCMV, and 0.5 mg of pCMV expression vector containing the cDNA of PPARg. After 6 h of transfection, the medium was changed and the cells were treated in serum-free medium in the presence of 10 nM testosterone or other pharmacological agents for 18 h. Firefly and Renilla luciferase activities were measured using the Dual Luciferase Kit (Promega, USA). The firefly luciferase values of each sample were normalized by Renilla luciferase activity and data were reported as relative light units. 3. Chemicals Testosterone, flutamide and insulin were purchased from Sigma-Aldrich (USA). Rosiglitazone and GW9662 were obtained from Cayman Chemical (USA).
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