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Sildenafil promotes adipogenesis through a PKG pathway
Biochemical and Biophysical Research Communications 396 (2010) 1054–1059
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Sildenafil promotes adipogenesis through a PKG pathway Xiaodong Zhang a, Jun Ji a,b, Guirui Yan a, Jingwei Wu a, Xiaoyun Sun a, Jingshan Shen a, Hualiang Jiang a, Heyao Wang a,* a b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang Jiang Hi-Tech Park, Pudong, Shanghai 201203, China School of Pharmacy, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 7 May 2010 Available online 21 May 2010 Keywords: Adipogenesis PDE5 inhibitor PKG Adipocytes
a b s t r a c t Sildenafil is the first oral PDE5 inhibitor for the treatment of erectile dysfunction and pulmonary arterial hypertension. In the present study, we investigated the effect of sildenafil on adipogenesis in 3T3L1 preadipocytes. Treatment with sildenafil for 8 days significantly promoted adipogenesis characterized by increased lipid droplet and triglyceride content in 3T3L1 cells. Meanwhile, sildenafil induced a pronounced up-regulation of the expression of adipocyte-specific genes, such as aP2 and GLUT4. The results by RT-PCR and Western blotting further showed that sildenafil increased the sequential expression of C/ EBPb, PPARc and C/EBPa. Additionally, we found that the other two PDE5 inhibitors (vardenafil and tadalafil) and the cGMP analog 8-pCPT-cGMP also increased adipogenesis. Likewise, 8-pCPT-cGMP could upregulate the expression of adipogenic and adipocyte-specific genes. Importantly, the PKG inhibitor Rp-8pCPT-cGMP was able to inhibit both sildenafil and 8-pCPT-cGMP-induced adipogenesis. Furthermore, sildenafil promoted basal and insulin-mediated glucose uptake in 3T3L1 cells, which was counteracted by Rp-8-pCPT-cGMP. These results indicate that sildenafil could promote adipogenesis accompanied by increased glucose uptake through a PKG pathway at least partly. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Adipose tissue, as a metabolic and endocrine organ, plays critical roles in the regulation of energy balance, lipid metabolism and insulin action [1]. Obesity is a major public health problem with increased risks of diabetes and cardiovascular disease in many countries [2,3], however, lipoatrophy, the lack of adipose tissue, is also associated with diabetes and other metabolic abnormalities [4]. Adipocytes have been emerging as a potential pharmacological target for obesity, diabetes and cardiovascular diseases [5]. Adipogenesis is a process from fibroblast-like preadipocytes to mature adipocytes regulated by two key families of adipogenic transcription factors, CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor c (PPARc). In response to hormonal stimuli of adipogenesis, C/EBPb is rapidly and transiently induced, followed by the expression of PPARc and C/EBPa [6]. Then PPARc and C/EBPa act synergistically to induce the expression of genes that are necessary for the generation and maintenance of the adipogenic phenotype such as lipid accumulation and insulin sensitivity [7]. Nitric oxide (NO), a bioactive free radical messenger molecule synthesized by endothelial, neuronal or inducible forms of NO synthases (NOSs), is involved in various physiological and pathological processes in organ systems [8,9]. The second messenger cyclic guanosine 30 ,50 -monophosphate (cGMP) is a pivotal intracellular medi* Corresponding author. Fax: +86 21 50807088. E-mail address: [email protected] (H. Wang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.064
ator of NO signaling [10,11]. cGMP is produced by NO-sensitive, soluble or particulate guanylyl cyclase (sGC) [12,13]. In addition to cGMP formation, degradation of cGMP by cyclic nucleotide phosphodiesterases (PDEs) determines intracellular cGMP levels [14]. PDE5 is the predominant enzyme responsible for cGMP hydrolysis in various types of tissues or cells [15]. Sildenafil is a potent specific inhibitor of PDE5, which ultimately increases intracellular cGMP concentration and activates cGMP-dependent kinase (PKG). Sildenafil has been approved for the treatment of erectile dysfunction (ED) and is also effective in the treatment for pulmonary arterial hypertension (PAH) [16,17]. It is reported that NO can promote adipogenesis in cultured preadipocytes derived from rat white adipose tissue [18], but the effect of sildenafil and cGMP on adipogenesis has not been examined. The 3T3L1 cell line is commonly used as an adipocyte differentiation model to investigate the regulation of adipogenesis. The aim of the present study is to determine whether sildenafil could promote adipogenesis and elucidate the molecular mechanism for that in 3T3L1 cells. 2. Materials and methods 2.1. Reagents Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and 8pCPT-cGMP were purchased from Sigma. Dulbecco’s modified
X. Zhang et al. / Biochemical and Biophysical Research Communications 396 (2010) 1054–1059
Eagle’s medium (DMEM) and newborn calf serum (NCS) were purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Hyclone. Rp-8-pCPT-cGMP was purchased from Biolog. 2deoxy-D-[2-3H] glucose was purchased from Perkin–Elmer. 2.2. Cell culture and differentiation 3T3L1 mouse fibroblasts were maintained in DMEM with 10% NCS in a humidified atmosphere containing 5% CO2 in air at 37 °C. Two days after confluence, adipogenesis was induced by a differentiation mixture containing 2 lg/ml insulin, 1 lM dexamethasone and 0.1 mM 3-isobutyl-1-methylxanthine in DMEM with 10% FBS. Thereafter, the medium was changed with 10% FBS/DMEM containing 2 lg/ml insulin every two days. The test compounds were administered at the initiation of differentiation and with every medium change for 8 days. 2.3. Triglyceride measurement For measurement of triglyceride content, cells were lysed by freezing/thawing three times, and then triglyceride content was determined using a commercial enzyme assay kit (Rongsheng, Shanghai, China). The concentration of cellular protein was determined using the BCA protein assay kit (Pierce) after solubilizing with phosphate-buffered saline (PBS) containing 1% Triton X-100. Results are expressed as milligrams of triglyceride per milligram of cellular protein. 2.4. Oil Red O staining Eight days after the induction of differentiation, cells were washed twice with PBS and fixed with 10% formalin for 1 h. After washed twice with PBS, cells were stained with freshly diluted Oil Red O (six parts of 0.6% Oil Red O dye in isopropanol and four parts of water; Sakura Finetek USA Inc.) for 0.5 h. Then cells were washed three times with water to remove unbound dye and photographed. 2.5. Gene expression analysis Total RNA from 3T3L1 cells was extracted using TRIzol reagent (Invitrogen). For gene expression analysis, the cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Invitrogen). Semi-quantitative real-time PCR (RT-PCR) was performed using SYBRÒ Premix Ex Taq™ II (Takara) according to the manufacturer’s instruction on the 7500 Fast Real-Time PCR System (Applied Biosystems). The primer sequences were as follows: aP2 (forward, 50 - GCGTAAATGGGGATTTGGTC-30 , and reverse 50 -CTCCTGTCGTCTG CGGTGATT-30 ); adiponectin (forward, 50 -CGCACTGGCAAGTTCTAC TGCAA-30 , and reverse 50 -CGTAGGTGAAGAGAACGGCCTTGT-30 ); C/EBPa (forward, 50 -CACTCGCTCCTTTTCCTACCG-30 , and reverse 50 CCCCCAACACCTAAGTCCCTC-30 ); C/EBPb (forward, 50 -CGGGGTTG TTGATGTTTTTGG-30 , and reverse 50 -CCGAAACGGAAAAGGTTCTCA30 ); FAS (fatty acid synthase) (forward, 50 -CGCTGGCATTCGTGATGGAGT-30 , and reverse 50 -CTGGGCAGAAGGTCTTGGAGA-30 ); GLUT4 (forward, 50 -CTCCTTCTATTTGCCGTCCTC-30 , and reverse 50 -CTGT TTTGCCCCTCAGTCATT-30 ); PPARc (forward, 50 -CTTTTCAAGGGTGCCAGTTTC-30 , and reverse 50 -CAATCCTTGGCCCTCTGAGAT-30 ); b-actin (forward, 50 -CCACGATGGAGGGGCCGGACTCATC-30 , and reverse 50 -CTAAAGACCTCTATGCCAACACAGT-30 ). The mRNA levels were normalized relative to the amount of b-actin mRNA and presented as arbitrary units.
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1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) with protease inhibitor cocktail (Sigma). Proteins were separated by SDS– PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Then membranes were incubated with primary antibodies for aP2 (Santa Cruz), CEBPa (CST), CEBPb (Santa Cruz), Glut4 (CST), PPARc (CST) and b-actin (Sigma), followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The resulting immunoblots were visualized using ECL substrate (Pierce). 2.7. Glucose uptake assay After treatment, adipocytes were serum starved for 4 h in DMEM containing 0.2% bovine serum albumin (BSA). Then cells were washed twice with Krebs-Ringer Hepes buffer (KRH, 136 mM NaCl, 4.7 mM KCl2, 1.25 mM CaCl2, 1.25 mM MgSO4, 20 mM Hepes, pH 7.4), and incubated in KRH buffer with or without 10 ng/ml insulin for 15 min. Uptake was initiated by addition of 0.5 lCi/ml 2-deoxy3 D-[2- H] glucose (final concentration) in KRH buffer. After 10 min, the reaction was terminated by quickly washed with ice-cold KRH buffer. The cells were lysed with 0.1 N NaOH and the radioactivity was measured in a scintillation counter (Beckman Instruments). 2.8. Statistical analysis All data are expressed as means ± SD. The comparison of different groups was assessed by two-tailed unpaired Student’s t test. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. Sildenafil promoted adipogenesis in 3T3L1 cells To explore the potential effect of sildenafil on adipogenesis, 3T3L1 cells were treated with sildenafil for 8 days. Adipogenesis was assessed by triglyceride content measurement and Oil Red O staining. Compared with the untreated control, sildenafil apparently enhanced the accumulation of intracellular lipid droplets in a dose-dependent manner (Fig. 1A). Fig. 1B showed that triglyceride content in 3T3L1 cells was significantly increased by sildenafil at the concentrations of 10–40 lM. Adipogenesis is accompanied by increased expression of various transcription factors and adipocyte-specific genes [6,7]. Treatment with 10 lM sildenafil induced a significant up-regulation of the expression of genes including PPARc, FAS, adiponectin, CEBPa, aP2 and GLUT4 by RT-PCR analysis (Fig. 1C). The results by Western blotting further showed that sildenafil dose-dependently increased the protein levels of GLUT4, aP2, PPARc and CEBPa. These results indicate that sildenafil could promote adipogenesis in 3T3L1 cells. 3.2. Sildenafil-induced adipogenesis may be associated with the PKG pathway Next we examined whether PKG is involved in sildenafil-induced adipogenesis. As shown in Fig. 2A, the other two PDE5 inhibitors tadalafil and vardenafil also significantly increased triglyceride content in 3T3L1 cells. In addition, the cGMP analog 8-pCPT-cGMP was able to increase triglyceride content as PDE5 inhibitors did. Furthermore, the special PKG inhibitor Rp-8-pCPT-cGMP reduced both sildenafil and 8-pCPT-cGMP-induced increase in triglyceride content in 3T3L1 cells (Fig. 2B). Thus cGMP and PDE5 inhibitors promoted adipocyte differentiation in a PKG-dependent manner.
2.6. Western blotting
3.3. Sildenafil increased the expression of adipogenic genes at early stage
Cells were washed twice with PBS and harvested in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS,
As described in Fig. 1C and D, PPARc and CEBPa expressions were up-regulated by sildenafil. The temporal expression patters
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Fig. 1. The effect of sildenafil on adipogenesis in 3T3L1 cells. Confluent cells were treated with various concentrations of sildenafil for 8 days. (A) Oil Red O staining. (B) Triglyceride content measurement in 3T3L1 cells. (C) Gene expression analysis by RT-PCR. (D) Gene expression analysis by Western blotting. Differences statistically significant from the untreated control: *p < 0.05. Si, sildenafil; Adip, adiponectin.
of PPARc and CEBPa in sildenafil-treated cells were further examined. As shown in Fig. 3A and B, a significant increase of the mRNA levels of PPARc and CEBPa in sildenafil-treated cells was observed after 2 days of treatment. What is more, sildenafil significantly increased the expression of CEBPb after 24 h of treatment (Fig. 3C), which is the upstream regulator of PPARc and CEBPa [19]. Next we performed immunoblot analysis to confirm the protein levels of CEBPb and PPARc. Fig. 3D showed that treatment with sildenafil dramatically increased the protein levels of CEBPb after 2 days and PPARc after 3 days, respectively. Therefore, sildenafil could increase the sequential expression of CEBPb, PPARc and CEBPa in 3T3L1 cells.
out sildenafil for 8 days. Sildenafil induced a significant increase of basal glucose uptake at the concentration of 20 or 40 lM (Fig. 2C). Insulin-mediated glucose uptake was about 3-fold of basal glucose uptake in differentiated adipocytes without sildenafil treatment (Fig. 2C). Sildenafil further enhanced insulin-mediated glucose uptake at the concentrations of 10–40 lM. Additionally, Fig. 2D showed that the PKG inhibitor Rp-8-pCPT-cGMP significantly decreased sildenafil-induced enhancement of glucose uptake at both basal and insulin-mediated conditions. These results indicate that sildenafil could promote glucose uptake in adipocytes. 4. Discussion
3.4. PKG may be involved in sildenafil-induced expression of adipogenic genes In order to determine whether sildenafil-induced expression of adipogenic genes is mediated through the PKG pathway, 3T3L1 cells were treated with sildenafil or 8-pCPT-cGMP in the presence or absence of Rp-8-pCPT-cGMP. Fig. 4A showed that Rp-8-pCPTcGMP decreased sildenafil-induced up-regulation of CEBPb and PPARc expression (3 days) and aP2 expression (8 days), respectively. Likewise, the cGMP analog 8-pCPT-cGMP increased the expression of CEBPb and PPARc expression (3 days) and aP2 expression (8 days), which were decreased by Rp-8-pCPT-cGMP (Fig. 4B). Thereby sildenafil increased the expression of adipogenic genes through a PKG way. 3.5. Sildenafil promoted basal and insulin-mediated glucose uptake Given that the degree of adipogenesis can affect glucose transport in adipocytes, we also examined basal and insulin-mediated glucose uptake in differentiated 3T3L1 cells treated with or with-
In the present study, we investigated the effect of the PDE5 inhibitor sildenafil on adipogenesis in 3T3L1 preadipocytes. Sildenafil significantly promoted adipogenesis characterized by increased lipid droplet and triglyceride content in 3T3L1 cells. Gene expression profiles in sildenafil-treated adipocytes further demonstrated that sildenafil induced a pronounced up-regulation of adipogenic or adipocyte-specific gene expression, for example aP2 and GLUT4, both of which are important markers of adipogenesis. As the first oral PDE5 inhibitor for the treatment of ED and PAH, sildenafil acts mainly via PKG activation by inhibition of cGMP degradation [16,20]. In order to determine whether sildenafil-induced adipogenesis is related to the PKG pathway, the other two clinically used PDE5 inhibitors (vardenafil and tardalafil) and the cGMP analog 8-pCPT-cGMP were employed. Of note, they all dramatically increased triglyceride content in 3T3L1 cells, implying that they promoted adipogenesis as sildenafil did. To further confirm the involvement of PKG in sildenafil-induced adipogenesis, preadipocytes were stimulated in the presence of Rp-8-pCPTcGMP, which is commonly used as a selective PKG inhibitor
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Fig. 2. The effect of the PKG inhibitor Rp-8-pCPT-cGMP on sildenafil-induced triglyceride accumulation and glucose uptake in 3T3L1 cells. (A) Cells were treated with 8-pCPTcGMP, vardenafil or tadalafil for 8 days, then triglyceride content was detected. (B) Cells were treated with 8-pCPT-cGMP or sildenafil in the presence or absence of Rp-8pCPT-cGMP for 8 days, then triglyceride content was detected. (C, D) Cells were treated with sildenafil in the absence (C) or presence (D) of Rp-8-pCPT-cGMP, then glucose uptake was determined. *p < 0.05, compared to the untreated control; #p < 0.05, compared to sildenafil only. Si, sildenafil; Var, vardenafil; Tad, tadalafil; cGMP, 8-pCPT-cGMP; Rp-cGMP, Rp-8-pCPT-cGMP.
[21,22]. Rp-8-pCPT-cGMP inhibited both sildenafil and 8-pCPTcGMP-induced adipogenesis. These results indicate that sildenafil could promote adipogenesis through a PKG pathway at least partly. Differentiation of preadipocytes into adipocytes is regulated by the coordinated expression of various transcription factors. Among these factors, the most important are the two principal adipogenic factors, PPARc and C/EBPa [7]. Recognition that C/EBPa functions as a principal player in adipogenesis resulted from gain-of-function studies in vitro and ablation of C/EBPa in vivo [6]. Ectopic expression of C/EBPa in fibroblastic cells induces adipogenesis [23]; the phenotype from C/EBPa/ mice shows that C/EBPa is required for formation of white adipose tissue [24]. PPARc in particular is considered the master regulator of adipogenesis, because precursor cells without expression of PPARc would not be differentiated into mature adipocytes [25,26]. In addition, forced expression of PPARc is able to induce adipogenesis even in C/EBPa-deficient cells [27], however, these C/EBPa-deficient cells are insulin resistant. In fact, it is now well accepted that PPARc and C/EBPa function as synergistic regulators of the expression of genes responsible for lipid accumulation and insulin sensitivity in adipocytes [28]. To understand the mechanism by which sildenafil regulates adipogenesis, we analyzed the time-course expression of PPARc and C/EBPa. Our results showed that sildenafil induced a significant up-regulation of the mRNA levels of PPARc and C/EBPa in a dose-dependent manner after 2 days of treatment. Therefore, sildenafil could induce the expression of PPARc and C/EBPa, leading to the differentiation of 3T3L1 cells.
Previous studies provide evidence that the expression of PPARc and C/EBPa is predominantly regulated by C/EBPb, one of the C/EBP family members [19,29]. Compared with the expression of PPARc and C/EBPa, sildenafil increased the expression of C/EBPb at an earlier time (after 1 day of treatment). However, the up-regulation of C/EBPb expression was attenuated after 2 days of treatment, which was consistent with previous reports that the inducement of C/EBPb expression is rapidly and transiently [30]. In order to determine the potential role of PKG in sildenafil-induced up-regulation of the expression of PPARc and C/EBPa in 3T3L1 cells, the cGMP analog 8-pCPT-cGMP and the PKG inhibitor Rp-8-pCPT-cGMP were used. Similar to that of sildenafil, 8-pCPT-cGMP significantly increased the expression of PPARc and C/EBPa, and their upstream regulator, C/EBPb. Furthermore, the up-regulation of the expression of these adipogenic genes was counteracted by the PKG inhibitor Rp-8-pCPT-cGMP. Thus PKG may be involved in the regulative effect of sildenafil on the expression of C/EBPb, PPARc and C/EBPa. GLUT4 expression is an important marker of mature adipocytes, which is regulated by PPARc and C/EBPa [28,31]. It is well known that glucose uptake is regulated by the expression of GLUT4 and translocation of GLUT4 to the plasma membrane [32,33]. In our study, treatment with sildenafil for 8 days significantly increased the expression of GLUT4. As we expected, sildenafil also promoted both basal and insulin-mediated glucose uptake after 8 days of treatment. In the present study, the increase of glucose uptake in sildenafil-treated adipocytes seems to be the consequence of increased expression of GLUT4, because in differentiated adipocytes,
X. Zhang et al. / Biochemical and Biophysical Research Communications 396 (2010) 1054–1059
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Time (days) Fig. 3. The effect of sildenafil on the expression of C/EBPb, PPARc and C/EBPa in 3T3L1 cells. Cells were treated with sildenafil for different time, then the mRNA levels of C/ EBPa (A), PPARc (B) and C/EBPb (C) were detected by RT-PCR. (D) Cells were treated with sildenafil for 1, 2 or 3 days, then the protein levels of PPARc and C/EBPb were detected by Western blotting. Differences statistically significant from the untreated control: *p < 0.05. Si, sildenafil.
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Fig. 4. The effect of Rp-8-pCPT-cGMP on sildenafil and 8-pCPT-cGMP-induced upregulation of adipogenic genes in 3T3L1 cells. Cells were treated with sildenafil or 8-pCPT-cGMP in the presence or absence of Rp-8-pCPT-cGMP for 3 days, and then the protein levels of PPARc and C/EBPb were detected by Western blotting. For analysis of the protein levels of aP2 by Western blotting, cell were treated for 8 days. Si, sildenafil; cGMP, 8-pCPT-cGMP; Rp-cGMP, Rp-8-pCPT-cGMP.
treatment with sildenafil in a short time from 0 to 8 h had no effect on the expression of GLUT4 and did not acutely increase glucose uptake in basal or insulin-mediated conditions (data not shown), excluding the possibility of GLUT4 translocation. Meanwhile, the PKG inhibitor Rp-8-pCPT-cGMP reduced glucose uptake induced by sildenafil, which was in agreement with the inhibitive effect of Rp-8-pCPT-cGMP on sildenafil-induced adipogenesis. In conclusion, we first reported that sildenafil, a potent PDE5 inhibitor, promotes adipogenesis in 3T3L1 cells by up-regulating the expression of C/EBPa and PPARc through a PKG pathway. Silde-
nafil augments basal and insulin-mediated glucose uptake in adipocytes along with increased expression of GLUT4. Therefore, PDE5 inhibitors might be effective for the treatment of type 2 diabetes. Acknowledgments This work was supported by Grant 2007AA02Z301 of the National High Technology Research and Development Program of China (863 Program), by Grant 2009ZX09301-001 of National Science and Technology Major Project ‘‘Key New Drug Creation and Manufacturing Program”, by Grant 08431900800 of Shanghai Science and Technology Innovation Program by Grant 2009CB918502 of the State Key Program of Basic Research of China grant. References [1] W. Kiess, S. Petzold, M. Topfer, A. Garten, S. Bluher, T. Kapellen, A. Korner, J. Kratzsch, Adipocytes and adipose tissue, Best Pract. Res. Clin. Endocrinol. Metab. 22 (2008) 135–153. [2] X. Pi-Sunyer, The medical risks of obesity, Postgrad. Med. 121 (2009) 21–33. [3] R.M. Cooper-DeHoff, S. Wen, A.L. Beitelshees, I. Zineh, J.G. Gums, S.T. Turner, Y. Gong, K. Hall, V. Parekh, A.B. Chapman, E. Boerwinkle, J.A. Johnson, Impact of abdominal obesity on incidence of adverse metabolic effects associated with antihypertensive medications, Hypertension 55 (2010) 61–68. [4] M.L. Reitman, E. Arioglu, O. Gavrilova, S.I. Taylor, Lipoatrophy revisited, Trends Endocrinol. Metab. 11 (2000) 410–416. [5] A.R. Nawrocki, P.E. Scherer, Keynote review: the adipocyte as a drug discovery target, Drug Discov. Today 10 (2005) 1219–1230. [6] S.R. Farmer, Transcriptional control of adipocyte formation, Cell Metab. 4 (2006) 263–273. [7] E.D. Rosen, O.A. MacDougald, Adipocyte differentiation from the inside out, Nat. Rev. Mol. Cell Biol. 7 (2006) 885–896. [8] U. Raff, C. Ott, S. John, B.M. Schmidt, E.H. Fleischmann, R.E. Schmieder, Nitric oxide and reactive hyperemia: role of location and duration of ischemia, Am. J. Hypertens. (2010).
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Sildenafil promotes adipogenesis through a PKG pathway Xiaodong Zhang a, Jun Ji a,b, Guirui Yan a, Jingwei Wu a, Xiaoyun Sun a, Jingshan Shen a, Hualiang Jiang a, Heyao Wang a,* a b
Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 555 Zu Chong Zhi Road, Zhang Jiang Hi-Tech Park, Pudong, Shanghai 201203, China School of Pharmacy, Fudan University, 138 Yi Xue Yuan Road, Shanghai 200032, China
a r t i c l e
i n f o
Article history: Received 7 May 2010 Available online 21 May 2010 Keywords: Adipogenesis PDE5 inhibitor PKG Adipocytes
a b s t r a c t Sildenafil is the first oral PDE5 inhibitor for the treatment of erectile dysfunction and pulmonary arterial hypertension. In the present study, we investigated the effect of sildenafil on adipogenesis in 3T3L1 preadipocytes. Treatment with sildenafil for 8 days significantly promoted adipogenesis characterized by increased lipid droplet and triglyceride content in 3T3L1 cells. Meanwhile, sildenafil induced a pronounced up-regulation of the expression of adipocyte-specific genes, such as aP2 and GLUT4. The results by RT-PCR and Western blotting further showed that sildenafil increased the sequential expression of C/ EBPb, PPARc and C/EBPa. Additionally, we found that the other two PDE5 inhibitors (vardenafil and tadalafil) and the cGMP analog 8-pCPT-cGMP also increased adipogenesis. Likewise, 8-pCPT-cGMP could upregulate the expression of adipogenic and adipocyte-specific genes. Importantly, the PKG inhibitor Rp-8pCPT-cGMP was able to inhibit both sildenafil and 8-pCPT-cGMP-induced adipogenesis. Furthermore, sildenafil promoted basal and insulin-mediated glucose uptake in 3T3L1 cells, which was counteracted by Rp-8-pCPT-cGMP. These results indicate that sildenafil could promote adipogenesis accompanied by increased glucose uptake through a PKG pathway at least partly. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Adipose tissue, as a metabolic and endocrine organ, plays critical roles in the regulation of energy balance, lipid metabolism and insulin action [1]. Obesity is a major public health problem with increased risks of diabetes and cardiovascular disease in many countries [2,3], however, lipoatrophy, the lack of adipose tissue, is also associated with diabetes and other metabolic abnormalities [4]. Adipocytes have been emerging as a potential pharmacological target for obesity, diabetes and cardiovascular diseases [5]. Adipogenesis is a process from fibroblast-like preadipocytes to mature adipocytes regulated by two key families of adipogenic transcription factors, CCAAT/enhancer-binding proteins (C/EBPs) and peroxisome proliferator-activated receptor c (PPARc). In response to hormonal stimuli of adipogenesis, C/EBPb is rapidly and transiently induced, followed by the expression of PPARc and C/EBPa [6]. Then PPARc and C/EBPa act synergistically to induce the expression of genes that are necessary for the generation and maintenance of the adipogenic phenotype such as lipid accumulation and insulin sensitivity [7]. Nitric oxide (NO), a bioactive free radical messenger molecule synthesized by endothelial, neuronal or inducible forms of NO synthases (NOSs), is involved in various physiological and pathological processes in organ systems [8,9]. The second messenger cyclic guanosine 30 ,50 -monophosphate (cGMP) is a pivotal intracellular medi* Corresponding author. Fax: +86 21 50807088. E-mail address: [email protected] (H. Wang). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.064
ator of NO signaling [10,11]. cGMP is produced by NO-sensitive, soluble or particulate guanylyl cyclase (sGC) [12,13]. In addition to cGMP formation, degradation of cGMP by cyclic nucleotide phosphodiesterases (PDEs) determines intracellular cGMP levels [14]. PDE5 is the predominant enzyme responsible for cGMP hydrolysis in various types of tissues or cells [15]. Sildenafil is a potent specific inhibitor of PDE5, which ultimately increases intracellular cGMP concentration and activates cGMP-dependent kinase (PKG). Sildenafil has been approved for the treatment of erectile dysfunction (ED) and is also effective in the treatment for pulmonary arterial hypertension (PAH) [16,17]. It is reported that NO can promote adipogenesis in cultured preadipocytes derived from rat white adipose tissue [18], but the effect of sildenafil and cGMP on adipogenesis has not been examined. The 3T3L1 cell line is commonly used as an adipocyte differentiation model to investigate the regulation of adipogenesis. The aim of the present study is to determine whether sildenafil could promote adipogenesis and elucidate the molecular mechanism for that in 3T3L1 cells. 2. Materials and methods 2.1. Reagents Insulin, dexamethasone, 3-isobutyl-1-methylxanthine, and 8pCPT-cGMP were purchased from Sigma. Dulbecco’s modified
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Eagle’s medium (DMEM) and newborn calf serum (NCS) were purchased from Invitrogen. Fetal bovine serum (FBS) was purchased from Hyclone. Rp-8-pCPT-cGMP was purchased from Biolog. 2deoxy-D-[2-3H] glucose was purchased from Perkin–Elmer. 2.2. Cell culture and differentiation 3T3L1 mouse fibroblasts were maintained in DMEM with 10% NCS in a humidified atmosphere containing 5% CO2 in air at 37 °C. Two days after confluence, adipogenesis was induced by a differentiation mixture containing 2 lg/ml insulin, 1 lM dexamethasone and 0.1 mM 3-isobutyl-1-methylxanthine in DMEM with 10% FBS. Thereafter, the medium was changed with 10% FBS/DMEM containing 2 lg/ml insulin every two days. The test compounds were administered at the initiation of differentiation and with every medium change for 8 days. 2.3. Triglyceride measurement For measurement of triglyceride content, cells were lysed by freezing/thawing three times, and then triglyceride content was determined using a commercial enzyme assay kit (Rongsheng, Shanghai, China). The concentration of cellular protein was determined using the BCA protein assay kit (Pierce) after solubilizing with phosphate-buffered saline (PBS) containing 1% Triton X-100. Results are expressed as milligrams of triglyceride per milligram of cellular protein. 2.4. Oil Red O staining Eight days after the induction of differentiation, cells were washed twice with PBS and fixed with 10% formalin for 1 h. After washed twice with PBS, cells were stained with freshly diluted Oil Red O (six parts of 0.6% Oil Red O dye in isopropanol and four parts of water; Sakura Finetek USA Inc.) for 0.5 h. Then cells were washed three times with water to remove unbound dye and photographed. 2.5. Gene expression analysis Total RNA from 3T3L1 cells was extracted using TRIzol reagent (Invitrogen). For gene expression analysis, the cDNA was synthesized from total RNA using M-MLV reverse transcriptase (Invitrogen). Semi-quantitative real-time PCR (RT-PCR) was performed using SYBRÒ Premix Ex Taq™ II (Takara) according to the manufacturer’s instruction on the 7500 Fast Real-Time PCR System (Applied Biosystems). The primer sequences were as follows: aP2 (forward, 50 - GCGTAAATGGGGATTTGGTC-30 , and reverse 50 -CTCCTGTCGTCTG CGGTGATT-30 ); adiponectin (forward, 50 -CGCACTGGCAAGTTCTAC TGCAA-30 , and reverse 50 -CGTAGGTGAAGAGAACGGCCTTGT-30 ); C/EBPa (forward, 50 -CACTCGCTCCTTTTCCTACCG-30 , and reverse 50 CCCCCAACACCTAAGTCCCTC-30 ); C/EBPb (forward, 50 -CGGGGTTG TTGATGTTTTTGG-30 , and reverse 50 -CCGAAACGGAAAAGGTTCTCA30 ); FAS (fatty acid synthase) (forward, 50 -CGCTGGCATTCGTGATGGAGT-30 , and reverse 50 -CTGGGCAGAAGGTCTTGGAGA-30 ); GLUT4 (forward, 50 -CTCCTTCTATTTGCCGTCCTC-30 , and reverse 50 -CTGT TTTGCCCCTCAGTCATT-30 ); PPARc (forward, 50 -CTTTTCAAGGGTGCCAGTTTC-30 , and reverse 50 -CAATCCTTGGCCCTCTGAGAT-30 ); b-actin (forward, 50 -CCACGATGGAGGGGCCGGACTCATC-30 , and reverse 50 -CTAAAGACCTCTATGCCAACACAGT-30 ). The mRNA levels were normalized relative to the amount of b-actin mRNA and presented as arbitrary units.
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1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) with protease inhibitor cocktail (Sigma). Proteins were separated by SDS– PAGE and transferred to PVDF membranes (Bio-Rad Laboratories). Then membranes were incubated with primary antibodies for aP2 (Santa Cruz), CEBPa (CST), CEBPb (Santa Cruz), Glut4 (CST), PPARc (CST) and b-actin (Sigma), followed by HRP-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). The resulting immunoblots were visualized using ECL substrate (Pierce). 2.7. Glucose uptake assay After treatment, adipocytes were serum starved for 4 h in DMEM containing 0.2% bovine serum albumin (BSA). Then cells were washed twice with Krebs-Ringer Hepes buffer (KRH, 136 mM NaCl, 4.7 mM KCl2, 1.25 mM CaCl2, 1.25 mM MgSO4, 20 mM Hepes, pH 7.4), and incubated in KRH buffer with or without 10 ng/ml insulin for 15 min. Uptake was initiated by addition of 0.5 lCi/ml 2-deoxy3 D-[2- H] glucose (final concentration) in KRH buffer. After 10 min, the reaction was terminated by quickly washed with ice-cold KRH buffer. The cells were lysed with 0.1 N NaOH and the radioactivity was measured in a scintillation counter (Beckman Instruments). 2.8. Statistical analysis All data are expressed as means ± SD. The comparison of different groups was assessed by two-tailed unpaired Student’s t test. Differences were considered statistically significant at p < 0.05. 3. Results 3.1. Sildenafil promoted adipogenesis in 3T3L1 cells To explore the potential effect of sildenafil on adipogenesis, 3T3L1 cells were treated with sildenafil for 8 days. Adipogenesis was assessed by triglyceride content measurement and Oil Red O staining. Compared with the untreated control, sildenafil apparently enhanced the accumulation of intracellular lipid droplets in a dose-dependent manner (Fig. 1A). Fig. 1B showed that triglyceride content in 3T3L1 cells was significantly increased by sildenafil at the concentrations of 10–40 lM. Adipogenesis is accompanied by increased expression of various transcription factors and adipocyte-specific genes [6,7]. Treatment with 10 lM sildenafil induced a significant up-regulation of the expression of genes including PPARc, FAS, adiponectin, CEBPa, aP2 and GLUT4 by RT-PCR analysis (Fig. 1C). The results by Western blotting further showed that sildenafil dose-dependently increased the protein levels of GLUT4, aP2, PPARc and CEBPa. These results indicate that sildenafil could promote adipogenesis in 3T3L1 cells. 3.2. Sildenafil-induced adipogenesis may be associated with the PKG pathway Next we examined whether PKG is involved in sildenafil-induced adipogenesis. As shown in Fig. 2A, the other two PDE5 inhibitors tadalafil and vardenafil also significantly increased triglyceride content in 3T3L1 cells. In addition, the cGMP analog 8-pCPT-cGMP was able to increase triglyceride content as PDE5 inhibitors did. Furthermore, the special PKG inhibitor Rp-8-pCPT-cGMP reduced both sildenafil and 8-pCPT-cGMP-induced increase in triglyceride content in 3T3L1 cells (Fig. 2B). Thus cGMP and PDE5 inhibitors promoted adipocyte differentiation in a PKG-dependent manner.
2.6. Western blotting
3.3. Sildenafil increased the expression of adipogenic genes at early stage
Cells were washed twice with PBS and harvested in RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% SDS,
As described in Fig. 1C and D, PPARc and CEBPa expressions were up-regulated by sildenafil. The temporal expression patters
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Fig. 1. The effect of sildenafil on adipogenesis in 3T3L1 cells. Confluent cells were treated with various concentrations of sildenafil for 8 days. (A) Oil Red O staining. (B) Triglyceride content measurement in 3T3L1 cells. (C) Gene expression analysis by RT-PCR. (D) Gene expression analysis by Western blotting. Differences statistically significant from the untreated control: *p < 0.05. Si, sildenafil; Adip, adiponectin.
of PPARc and CEBPa in sildenafil-treated cells were further examined. As shown in Fig. 3A and B, a significant increase of the mRNA levels of PPARc and CEBPa in sildenafil-treated cells was observed after 2 days of treatment. What is more, sildenafil significantly increased the expression of CEBPb after 24 h of treatment (Fig. 3C), which is the upstream regulator of PPARc and CEBPa [19]. Next we performed immunoblot analysis to confirm the protein levels of CEBPb and PPARc. Fig. 3D showed that treatment with sildenafil dramatically increased the protein levels of CEBPb after 2 days and PPARc after 3 days, respectively. Therefore, sildenafil could increase the sequential expression of CEBPb, PPARc and CEBPa in 3T3L1 cells.
out sildenafil for 8 days. Sildenafil induced a significant increase of basal glucose uptake at the concentration of 20 or 40 lM (Fig. 2C). Insulin-mediated glucose uptake was about 3-fold of basal glucose uptake in differentiated adipocytes without sildenafil treatment (Fig. 2C). Sildenafil further enhanced insulin-mediated glucose uptake at the concentrations of 10–40 lM. Additionally, Fig. 2D showed that the PKG inhibitor Rp-8-pCPT-cGMP significantly decreased sildenafil-induced enhancement of glucose uptake at both basal and insulin-mediated conditions. These results indicate that sildenafil could promote glucose uptake in adipocytes. 4. Discussion
3.4. PKG may be involved in sildenafil-induced expression of adipogenic genes In order to determine whether sildenafil-induced expression of adipogenic genes is mediated through the PKG pathway, 3T3L1 cells were treated with sildenafil or 8-pCPT-cGMP in the presence or absence of Rp-8-pCPT-cGMP. Fig. 4A showed that Rp-8-pCPTcGMP decreased sildenafil-induced up-regulation of CEBPb and PPARc expression (3 days) and aP2 expression (8 days), respectively. Likewise, the cGMP analog 8-pCPT-cGMP increased the expression of CEBPb and PPARc expression (3 days) and aP2 expression (8 days), which were decreased by Rp-8-pCPT-cGMP (Fig. 4B). Thereby sildenafil increased the expression of adipogenic genes through a PKG way. 3.5. Sildenafil promoted basal and insulin-mediated glucose uptake Given that the degree of adipogenesis can affect glucose transport in adipocytes, we also examined basal and insulin-mediated glucose uptake in differentiated 3T3L1 cells treated with or with-
In the present study, we investigated the effect of the PDE5 inhibitor sildenafil on adipogenesis in 3T3L1 preadipocytes. Sildenafil significantly promoted adipogenesis characterized by increased lipid droplet and triglyceride content in 3T3L1 cells. Gene expression profiles in sildenafil-treated adipocytes further demonstrated that sildenafil induced a pronounced up-regulation of adipogenic or adipocyte-specific gene expression, for example aP2 and GLUT4, both of which are important markers of adipogenesis. As the first oral PDE5 inhibitor for the treatment of ED and PAH, sildenafil acts mainly via PKG activation by inhibition of cGMP degradation [16,20]. In order to determine whether sildenafil-induced adipogenesis is related to the PKG pathway, the other two clinically used PDE5 inhibitors (vardenafil and tardalafil) and the cGMP analog 8-pCPT-cGMP were employed. Of note, they all dramatically increased triglyceride content in 3T3L1 cells, implying that they promoted adipogenesis as sildenafil did. To further confirm the involvement of PKG in sildenafil-induced adipogenesis, preadipocytes were stimulated in the presence of Rp-8-pCPTcGMP, which is commonly used as a selective PKG inhibitor
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Fig. 2. The effect of the PKG inhibitor Rp-8-pCPT-cGMP on sildenafil-induced triglyceride accumulation and glucose uptake in 3T3L1 cells. (A) Cells were treated with 8-pCPTcGMP, vardenafil or tadalafil for 8 days, then triglyceride content was detected. (B) Cells were treated with 8-pCPT-cGMP or sildenafil in the presence or absence of Rp-8pCPT-cGMP for 8 days, then triglyceride content was detected. (C, D) Cells were treated with sildenafil in the absence (C) or presence (D) of Rp-8-pCPT-cGMP, then glucose uptake was determined. *p < 0.05, compared to the untreated control; #p < 0.05, compared to sildenafil only. Si, sildenafil; Var, vardenafil; Tad, tadalafil; cGMP, 8-pCPT-cGMP; Rp-cGMP, Rp-8-pCPT-cGMP.
[21,22]. Rp-8-pCPT-cGMP inhibited both sildenafil and 8-pCPTcGMP-induced adipogenesis. These results indicate that sildenafil could promote adipogenesis through a PKG pathway at least partly. Differentiation of preadipocytes into adipocytes is regulated by the coordinated expression of various transcription factors. Among these factors, the most important are the two principal adipogenic factors, PPARc and C/EBPa [7]. Recognition that C/EBPa functions as a principal player in adipogenesis resulted from gain-of-function studies in vitro and ablation of C/EBPa in vivo [6]. Ectopic expression of C/EBPa in fibroblastic cells induces adipogenesis [23]; the phenotype from C/EBPa/ mice shows that C/EBPa is required for formation of white adipose tissue [24]. PPARc in particular is considered the master regulator of adipogenesis, because precursor cells without expression of PPARc would not be differentiated into mature adipocytes [25,26]. In addition, forced expression of PPARc is able to induce adipogenesis even in C/EBPa-deficient cells [27], however, these C/EBPa-deficient cells are insulin resistant. In fact, it is now well accepted that PPARc and C/EBPa function as synergistic regulators of the expression of genes responsible for lipid accumulation and insulin sensitivity in adipocytes [28]. To understand the mechanism by which sildenafil regulates adipogenesis, we analyzed the time-course expression of PPARc and C/EBPa. Our results showed that sildenafil induced a significant up-regulation of the mRNA levels of PPARc and C/EBPa in a dose-dependent manner after 2 days of treatment. Therefore, sildenafil could induce the expression of PPARc and C/EBPa, leading to the differentiation of 3T3L1 cells.
Previous studies provide evidence that the expression of PPARc and C/EBPa is predominantly regulated by C/EBPb, one of the C/EBP family members [19,29]. Compared with the expression of PPARc and C/EBPa, sildenafil increased the expression of C/EBPb at an earlier time (after 1 day of treatment). However, the up-regulation of C/EBPb expression was attenuated after 2 days of treatment, which was consistent with previous reports that the inducement of C/EBPb expression is rapidly and transiently [30]. In order to determine the potential role of PKG in sildenafil-induced up-regulation of the expression of PPARc and C/EBPa in 3T3L1 cells, the cGMP analog 8-pCPT-cGMP and the PKG inhibitor Rp-8-pCPT-cGMP were used. Similar to that of sildenafil, 8-pCPT-cGMP significantly increased the expression of PPARc and C/EBPa, and their upstream regulator, C/EBPb. Furthermore, the up-regulation of the expression of these adipogenic genes was counteracted by the PKG inhibitor Rp-8-pCPT-cGMP. Thus PKG may be involved in the regulative effect of sildenafil on the expression of C/EBPb, PPARc and C/EBPa. GLUT4 expression is an important marker of mature adipocytes, which is regulated by PPARc and C/EBPa [28,31]. It is well known that glucose uptake is regulated by the expression of GLUT4 and translocation of GLUT4 to the plasma membrane [32,33]. In our study, treatment with sildenafil for 8 days significantly increased the expression of GLUT4. As we expected, sildenafil also promoted both basal and insulin-mediated glucose uptake after 8 days of treatment. In the present study, the increase of glucose uptake in sildenafil-treated adipocytes seems to be the consequence of increased expression of GLUT4, because in differentiated adipocytes,
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Time (days) Fig. 3. The effect of sildenafil on the expression of C/EBPb, PPARc and C/EBPa in 3T3L1 cells. Cells were treated with sildenafil for different time, then the mRNA levels of C/ EBPa (A), PPARc (B) and C/EBPb (C) were detected by RT-PCR. (D) Cells were treated with sildenafil for 1, 2 or 3 days, then the protein levels of PPARc and C/EBPb were detected by Western blotting. Differences statistically significant from the untreated control: *p < 0.05. Si, sildenafil.
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Fig. 4. The effect of Rp-8-pCPT-cGMP on sildenafil and 8-pCPT-cGMP-induced upregulation of adipogenic genes in 3T3L1 cells. Cells were treated with sildenafil or 8-pCPT-cGMP in the presence or absence of Rp-8-pCPT-cGMP for 3 days, and then the protein levels of PPARc and C/EBPb were detected by Western blotting. For analysis of the protein levels of aP2 by Western blotting, cell were treated for 8 days. Si, sildenafil; cGMP, 8-pCPT-cGMP; Rp-cGMP, Rp-8-pCPT-cGMP.
treatment with sildenafil in a short time from 0 to 8 h had no effect on the expression of GLUT4 and did not acutely increase glucose uptake in basal or insulin-mediated conditions (data not shown), excluding the possibility of GLUT4 translocation. Meanwhile, the PKG inhibitor Rp-8-pCPT-cGMP reduced glucose uptake induced by sildenafil, which was in agreement with the inhibitive effect of Rp-8-pCPT-cGMP on sildenafil-induced adipogenesis. In conclusion, we first reported that sildenafil, a potent PDE5 inhibitor, promotes adipogenesis in 3T3L1 cells by up-regulating the expression of C/EBPa and PPARc through a PKG pathway. Silde-
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