Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Discovery of natural alkaloid bouchardatine as a novel inhibitor of adipogenesis/lipogenesis in 3T3-L1 adipocytes Yong Rao a, Hong Liu a, Lin Gao a, Hong Yu a, Jia-Heng Tan a, Tian-Miao Ou a, Shi-Liang Huang a, Lian-Quan Gu a, Ji-Ming Ye b, Zhi-Shu Huang a,⇑ a b
School of Pharmaceutical Sciences, Yat-sen Sun University, Guangzhou 510006, PR China Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of Health Sciences, RMIT University, Melbourne, VIC, Australia
a r t i c l e
i n f o
Article history: Received 24 March 2015 Revised 27 May 2015 Accepted 28 May 2015 Available online xxxx Keywords: Bouchardatine 3T3-L1 adipocytes Lipid lowering Adipogenesis/lipogenesis Anti-obesity
a b s t r a c t Bouchardatine (1), a naturally occurring b-indoloquinazoline alkaloid, was synthesized. For the first time, the lipid-lowering effect and mechanism of 1 was investigated in 3T3-L1 adipocytes. Our study showed that 1 could significantly reduce lipid accumulation without cytotoxicity and mainly inhibited early differentiation of adipocyte through proliferation inhibition and cell cycle arrested in dose-dependent manner. Furthermore, the inhibition of early differentiation was reflected by down-regulation of key regulators of adipogenesis/lipogenesis, including CCAAT enhancer binding proteins (C/EBPb, C/EBPd, C/EBPa), peroxisome proliferator-activated receptors c (PPARc) and sterol-regulatory element binding protein-1c (SREBP-1c), in both of mRNA and protein levels. Subsequently decreasing the protein levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and stearyl coenzyme A desaturated enzyme 1 (SCD-1), the rate-limited metabolic enzymes of fatty acid synthesis, were also observed. Further studies revealed that 1 persistently activated adenosine 50 -monophosphate (AMP)-activated protein kinase (AMPK) during differentiation, suggesting that the AMPK may be an upstream mechanism for the effect of 1 on adipogenesis and lipogenesis. Our data suggest that 1 can be a candidate for the development of new therapeutic drugs against obesity and related metabolic disorders. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Obesity has become one of the most common metabolic syndromes, that pose a considerable threat to human health. Obesity is a serious chronic metabolic health problem that is closely related to type 2 diabetes, hypertension, cardiovascular diseases, and cancer.1–3 In vivo studies have demonstrated that the development of obesity is characterized by the increase in number and size of mature adipocytes, which originated from pre-adipocytes and fibroblasts.4,5 The 3T3-L1 cell line is a well-established model for in vitro studies on the metabolism of fatty acids and obesity.6 3T3-L1 cell-based screening for beneficial compounds with lipid-lowering effect has been showed to be an efficacious tool for identification of anti-obesity compounds.7 Meanwhile, differentiation of pre-adipocytes into mature adipocytes is accompanied by sequential expression and activation of transcription factors governing the expression of adipocyte-specific markers, such as C/EBPa, PPAR and SREBP-1. These factors have important function in regulation ⇑ Corresponding author. Tel./fax: +86 20 39943056. E-mail address: [email protected] (Z.-S. Huang).
of adipogenesis by modulating the expression of their target genes in a coordinated manner.8–12 Moreover, AMPK is one of the most well-characterized and important targets for the prevention and treatment of obesity.13–16 The AMPK complex is a heterotrimer composed of catalytic a subunit and regulatory b, c subunits. Each of these three subunits plays a difference important role in both the stability and activity AMPK.17 It is in the catalytic domain of a subunit where AMPK becomes activated when phosphorylation takes place at threonine-172 by an upstream AMPK kinase (AMPKK). And AMPK b plays a pivotal role in phosphorylated AMPK at threonine-172 and a phosphorylation at Ser108 of the b subunit seems to be required for the activation of AMPK enzyme.18 Once AMPK activated, its functions as a cellular energy sensor and has been shown to be positively correlated with glucose and lipid homeostasis in adipocytes.19 Bouchardatia neurococca is known to provide bioactive compounds, including alkaloids, b-indoloquinazoline, furoquinoline, terpenoids, and sesquiterpene, with valuable biomedical and pharmaceutical potential.20 Bouchardatine (1, Fig. 1), a b-indoloquinazoline alkaloid isolated from B. neurococca (Rutaecae), exhibits a variety of biological effects, such as anti-cancer, anti-inflammatory, and anti-tuberculosis effects.21,22 In the present study, we evaluated,
http://dx.doi.org/10.1016/j.bmc.2015.05.057 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rao, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.057
2
Y. Rao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. Chemical structure of bouchardatine (1).
for the first time, the inhibitory effects of 1, which was synthesized using a method in the literature (Scheme 1) on adipogenesis and lipogenesis in 3T3-L1 cell. In addition, we investigated the molecular mechanisms of 1 by analyzing the expression of adipogenic factors at mRNA and protein levels.
Red O staining and TG analysis. As shown in Figure 2B, 1 decreased lipid accumulation in 3T3-L1 adipocytes in a dose-dependent manner, as evidenced by the decrease in cell size and the number of lipid droplets in mature adipocytes as depicted in the microscopy image (Fig. 2C). As for cell size, after 3T3-L1 differentiated into mature adipocytes, cells morphology was changed from a fibroblastic shape to a spherical shape caused by TG accumulation, like a ‘finger ring’ in cells. After 1 treatment, we can see the diameter of finger ring was smaller compared with control group, especially at higher concentrations. Most notably, almost 50% lipid decrease was observed at the concentration of 25 lM after 1 treatment. Moreover, we also examined the lipid-lowering effect of 1 in HepG-2 cells. The substance 1 also blocked lipid accumulation in HepG-2 induction by 0.5 mM oleic acid sodium in a dose-dependent manner without cytotoxic effect compared with the control group (Fig. 2D and E).
2. Results and discussion 2.1. Chemistry The synthetic route of 1 was shown in Scheme 1. The key intermediate 2-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-indole-3carbaldehyde (d) was prepared according to the literature procedure.23,24 Anthranilamide was coupled with triethyl orthopropionate to yield compound a at 155 °C, then it was converted into the brominated compound b using bromine in acid condition. The reaction of compound b with 3.5 equiv phenylhydrazine afforded the compound c, followed by the condensation reaction with PPA (polyphosphoric acid) at 150 °C producing the intermediate d by classical Fischer reaction. The intermediate d reacted with ammonium acetate and DMSO-water to give the target compound 1.
2.3. Effect of 1 on lipid accumulation at the early stages of differentiation Several stages are involved in adipogenesis/lipogenesis of preadipocytes differentiation into mature adipocytes. To determine whether 1 reduced lipid accumulation by suppressing adipogenesis and/or lipogenesis, we treated 3T3-L1 cells with 1 (25 lM) for various periods, namely, days 0 to 3, 3 to 6, 6 to 9, 0 to 6, 3 to 6, and 0 to 9, which represent different stages of 3T3-L1 adipocyte differentiation (Fig. 3A). On day 9, the lipid content in cells was determined by a TG assay, and representative microscopy images were captured. Our results showed that 1 treatment during days 0 to 3, 3 to 6, and 6 to 9 resulted in 50%, 30%, and
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc
Discovery of natural alkaloid bouchardatine as a novel inhibitor of adipogenesis/lipogenesis in 3T3-L1 adipocytes Yong Rao a, Hong Liu a, Lin Gao a, Hong Yu a, Jia-Heng Tan a, Tian-Miao Ou a, Shi-Liang Huang a, Lian-Quan Gu a, Ji-Ming Ye b, Zhi-Shu Huang a,⇑ a b
School of Pharmaceutical Sciences, Yat-sen Sun University, Guangzhou 510006, PR China Molecular Pharmacology for Diabetes Group, Health Innovations Research Institute and School of Health Sciences, RMIT University, Melbourne, VIC, Australia
a r t i c l e
i n f o
Article history: Received 24 March 2015 Revised 27 May 2015 Accepted 28 May 2015 Available online xxxx Keywords: Bouchardatine 3T3-L1 adipocytes Lipid lowering Adipogenesis/lipogenesis Anti-obesity
a b s t r a c t Bouchardatine (1), a naturally occurring b-indoloquinazoline alkaloid, was synthesized. For the first time, the lipid-lowering effect and mechanism of 1 was investigated in 3T3-L1 adipocytes. Our study showed that 1 could significantly reduce lipid accumulation without cytotoxicity and mainly inhibited early differentiation of adipocyte through proliferation inhibition and cell cycle arrested in dose-dependent manner. Furthermore, the inhibition of early differentiation was reflected by down-regulation of key regulators of adipogenesis/lipogenesis, including CCAAT enhancer binding proteins (C/EBPb, C/EBPd, C/EBPa), peroxisome proliferator-activated receptors c (PPARc) and sterol-regulatory element binding protein-1c (SREBP-1c), in both of mRNA and protein levels. Subsequently decreasing the protein levels of acetyl CoA carboxylase (ACC), fatty acid synthase (FAS), and stearyl coenzyme A desaturated enzyme 1 (SCD-1), the rate-limited metabolic enzymes of fatty acid synthesis, were also observed. Further studies revealed that 1 persistently activated adenosine 50 -monophosphate (AMP)-activated protein kinase (AMPK) during differentiation, suggesting that the AMPK may be an upstream mechanism for the effect of 1 on adipogenesis and lipogenesis. Our data suggest that 1 can be a candidate for the development of new therapeutic drugs against obesity and related metabolic disorders. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction Obesity has become one of the most common metabolic syndromes, that pose a considerable threat to human health. Obesity is a serious chronic metabolic health problem that is closely related to type 2 diabetes, hypertension, cardiovascular diseases, and cancer.1–3 In vivo studies have demonstrated that the development of obesity is characterized by the increase in number and size of mature adipocytes, which originated from pre-adipocytes and fibroblasts.4,5 The 3T3-L1 cell line is a well-established model for in vitro studies on the metabolism of fatty acids and obesity.6 3T3-L1 cell-based screening for beneficial compounds with lipid-lowering effect has been showed to be an efficacious tool for identification of anti-obesity compounds.7 Meanwhile, differentiation of pre-adipocytes into mature adipocytes is accompanied by sequential expression and activation of transcription factors governing the expression of adipocyte-specific markers, such as C/EBPa, PPAR and SREBP-1. These factors have important function in regulation ⇑ Corresponding author. Tel./fax: +86 20 39943056. E-mail address: [email protected] (Z.-S. Huang).
of adipogenesis by modulating the expression of their target genes in a coordinated manner.8–12 Moreover, AMPK is one of the most well-characterized and important targets for the prevention and treatment of obesity.13–16 The AMPK complex is a heterotrimer composed of catalytic a subunit and regulatory b, c subunits. Each of these three subunits plays a difference important role in both the stability and activity AMPK.17 It is in the catalytic domain of a subunit where AMPK becomes activated when phosphorylation takes place at threonine-172 by an upstream AMPK kinase (AMPKK). And AMPK b plays a pivotal role in phosphorylated AMPK at threonine-172 and a phosphorylation at Ser108 of the b subunit seems to be required for the activation of AMPK enzyme.18 Once AMPK activated, its functions as a cellular energy sensor and has been shown to be positively correlated with glucose and lipid homeostasis in adipocytes.19 Bouchardatia neurococca is known to provide bioactive compounds, including alkaloids, b-indoloquinazoline, furoquinoline, terpenoids, and sesquiterpene, with valuable biomedical and pharmaceutical potential.20 Bouchardatine (1, Fig. 1), a b-indoloquinazoline alkaloid isolated from B. neurococca (Rutaecae), exhibits a variety of biological effects, such as anti-cancer, anti-inflammatory, and anti-tuberculosis effects.21,22 In the present study, we evaluated,
http://dx.doi.org/10.1016/j.bmc.2015.05.057 0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Rao, Y.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2015.05.057
2
Y. Rao et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. Chemical structure of bouchardatine (1).
for the first time, the inhibitory effects of 1, which was synthesized using a method in the literature (Scheme 1) on adipogenesis and lipogenesis in 3T3-L1 cell. In addition, we investigated the molecular mechanisms of 1 by analyzing the expression of adipogenic factors at mRNA and protein levels.
Red O staining and TG analysis. As shown in Figure 2B, 1 decreased lipid accumulation in 3T3-L1 adipocytes in a dose-dependent manner, as evidenced by the decrease in cell size and the number of lipid droplets in mature adipocytes as depicted in the microscopy image (Fig. 2C). As for cell size, after 3T3-L1 differentiated into mature adipocytes, cells morphology was changed from a fibroblastic shape to a spherical shape caused by TG accumulation, like a ‘finger ring’ in cells. After 1 treatment, we can see the diameter of finger ring was smaller compared with control group, especially at higher concentrations. Most notably, almost 50% lipid decrease was observed at the concentration of 25 lM after 1 treatment. Moreover, we also examined the lipid-lowering effect of 1 in HepG-2 cells. The substance 1 also blocked lipid accumulation in HepG-2 induction by 0.5 mM oleic acid sodium in a dose-dependent manner without cytotoxic effect compared with the control group (Fig. 2D and E).
2. Results and discussion 2.1. Chemistry The synthetic route of 1 was shown in Scheme 1. The key intermediate 2-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-indole-3carbaldehyde (d) was prepared according to the literature procedure.23,24 Anthranilamide was coupled with triethyl orthopropionate to yield compound a at 155 °C, then it was converted into the brominated compound b using bromine in acid condition. The reaction of compound b with 3.5 equiv phenylhydrazine afforded the compound c, followed by the condensation reaction with PPA (polyphosphoric acid) at 150 °C producing the intermediate d by classical Fischer reaction. The intermediate d reacted with ammonium acetate and DMSO-water to give the target compound 1.
2.3. Effect of 1 on lipid accumulation at the early stages of differentiation Several stages are involved in adipogenesis/lipogenesis of preadipocytes differentiation into mature adipocytes. To determine whether 1 reduced lipid accumulation by suppressing adipogenesis and/or lipogenesis, we treated 3T3-L1 cells with 1 (25 lM) for various periods, namely, days 0 to 3, 3 to 6, 6 to 9, 0 to 6, 3 to 6, and 0 to 9, which represent different stages of 3T3-L1 adipocyte differentiation (Fig. 3A). On day 9, the lipid content in cells was determined by a TG assay, and representative microscopy images were captured. Our results showed that 1 treatment during days 0 to 3, 3 to 6, and 6 to 9 resulted in 50%, 30%, and
