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Agrimol B suppresses adipogenesis through modulation of SIRT1-PPAR gamma signal pathway
Biochemical and Biophysical Research Communications 477 (2016) 454e460
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Agrimol B suppresses adipogenesis through modulation of SIRT1-PPAR gamma signal pathway Shifeng Wang a, Qiao Zhang a, Yuxin Zhang a, Cheng Shen a, Zhen Wang b, Qinghua Wu c, Yanling Zhang a, Shiyou Li b, *, Yanjiang Qiao a, ** a b c
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China HD Biosciences Co., Ltd, Shanghai 201201, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2016 Accepted 15 June 2016 Available online 16 June 2016
Studies of human genetics have implicated the role of SIRT1 in regulating obesity, insulin resistance, and longevity. These researches motivated the identification of novel SIRT1 activators. The current study aimed to investigate the potential efficacy of agrimol B, a polyphenol derived from Agrimonia pilosa Ledeb., on mediating SIRT1 activity and fat metabolism. Results showed that agrimol B significantly induced cytoplasm-to-nucleus shuttle of SIRT1. Furthermore, we confirmed that agrimol B dramatically inhibited 3T3-L1 adipocyte differentiation by reducing PPARg, C/EBPa, FAS, UCP-1, and apoE expression. Consequently, adipogenesis was blocked by treatment of agrimol B at the early stage of differentiation in a dose-dependent manner, the IC50 value was determined as 3.35 ± 0.32 mM. Taken together, our data suggest a therapeutic potential of agrimol B on alleviating obesity, through modulation of SIRT1-PPARg signal pathway. © 2016 Published by Elsevier Inc.
Keywords: Agrimol B Agrimonia pilosa Ledeb. Lipid droplet Obesity PPARg SIRT1
1. Introduction Adipocyte plays a key role in regulating energy homeostasis. Deficiency of adipose tissue may cause severe hyperlipidemia as well as atherosclerosis since it contains the largest pool of free cholesterol [1,2]. Adipose tissue also serves as an endocrine organ that controls inflammatory responses through secreting hormones, such as adiponectin, leptin, tumor necrosis factor a (TNFa) [3]. Latent inflammation in adipose tissue is a major negative impact of metabolic syndrome that promotes atherosclerosis, myocarditis [4],
Abbreviations: ApoE, apolipoprotein E; BSA, bovine serum albumin; C/EBPb, CCAAT/enhancer-binding protein b; FAS, fatty acid synthesis; IBMX, 3-isobutyl-1methylxanthine; MCE, mitotic clonal expansion; PBS, phosphate buffer saline solution; SIRT1, silent information regulator 2 homolog 1; TNFa, tumor necrosis factor a; UCP-1, uncoupling protein-1. * Corresponding author. Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. ** Corresponding author. Key Laboratory of TCM-Information Engineer of State Administration of TCM, School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 6 Wangjing Zhong Huan South Road, Chaoyang District, Beijing 100102, China. E-mail addresses: [email protected] (S. Li), [email protected] (Y. Qiao). http://dx.doi.org/10.1016/j.bbrc.2016.06.078 0006-291X/© 2016 Published by Elsevier Inc.
and non-alcoholic fatty liver disease [5]. Meanwhile, dyslipidemia may contribute to early neurological deterioration after ischemic stroke [6]. Importantly, metabolic disturbance of lipid and cholesterol were associated with higher incidence of cancer [7,8]. Thus, maintaining fat homeostasis may provide an alternative avenue for therapies of metabolic syndromes, cardiovascular diseases, and tumors. Caloric restriction has been intimately related to obesity and longevity [9,10]. Silent information regulator 2 homolog 1 (SIRT1) is a critical factor that influences obesity and life span. It controls adipocyte fat storage [11] and glucose homeostasis through modulation of PGC-1a activity [12]. White adipocyte is remodeled by SIRT1 through interaction with other epigenetic regulators, such as PPARY [13,14]. The first SIRT1 activator isolated from the peer of grapes, resveratrol, has been proved to be efficient in alleviating insulin resistance and prolonging life span [15]. However, the liability of resveratrol as a SIRT1 activator lies in its lack of specificity, known as “off target effects” [16]. With the prevalence of obesity and ageing, there is an increasing demand for novel SIRT1 activators. Agrimonia pilosa Ledeb. (Xianhecao in Chinese) is an herbal medicine listed in Pharmacopoeia of China [17]. It possesses broad range of pharmaceutical activities, such anti-oxidant [18] and anti-
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inflammation [19]. Agrimol B is considered as an appropriate indicator for the quality control of Agrimonia pilosa Ledeb. [20]. Many molecular chaperones have been observed anti-obesity activities [21]. Nonetheless, the function of agrimol B on fat metabolism and the underlying molecular mechanism remained poorly understood. Therefore, to systematically assess the potency of agrimol B on lipid metabolism, we developed an in situ fluorescence imaging assay, investigated the role of agrimol B on SIRT1 activation and lipid droplet accumulation. 2. Materials and methods 2.1. Materials
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were rinsed with PBS for three times. Thereafter, 10 mg/mL Hoechst 33342 was added to cells for nuclei staining as described above. For co-staining of SIRT1 and lipid droplet, 1 mg/mL Bodipy 493/503 (Sigma) was performed. 2.5. Imaging and analysis The fluorescent images were yielded on ImageXpress Micro XLS System (MD, CA, USA). Signals of the biomarkers, such as PPARY, C/ EBPa, FAS, UCP-1, and apoE were yielded using a 20 objective. Lipid droplet fluorescent signal was acquired by 10 objective. The images were analyzed using a custom module of cell scoring protocol.
Agrimol B was provided by Dalian Institute of Chemical Physics (Dalian, China). Ingredients for adipocyte differentiation were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies were acquired as follows: rabbit anti-SIRT1 (sc-15404) and mouse anti-PPARY (E-8, sc-7273) were purchased from Santa Cruz Biotechnology Inc. (CA, USA); rabbit anti-C/EBPa (1704-1) and antiFAS (3655-1) were acquired from Epitomics Inc. (CA, USA); Rabbit polyclonal to apolipoprotein E (ab20874); anti-UCP-1 (U6382), anti-rabbit TRITC (T6778), and anti-rabbit FITC IgG (F7512) were obtained from Sigma-Aldrich. Goat anti-mouse IgG (H þ L) secondary antibody (35502) was purchased from Thermo Fisher Scientific (Waltham, USA).
2.6. Statistical analysis
2.2. Adipocyte differentiation and compound treatment
To determine if agrimol B could promote SIRT1 activity, we measured the ability of agrimol B on inducing SIRT1 translocation and expression. Results showed that SIRT1 was absent in the nuclei of mature adipocytes contained visible lipid droplets, but localized in the cytoplasmic area by the form of small spots (Fig. 1A). In contrast, with administration of agrimol B, more nuclear SIRT1 were observed. The phenotype of agrimol B treated cells was in accordance with that of resveratrol, a well-known SIRT1 activator [24]. Normalized responses showed that agrimol B significantly increased nuclear positive rate of SIRT1 compared with vehicle control (P < 0.0001). Moreover, the effect of agrimol B was stronger than that of resveratrol (Fig. 1B), showing significant differences between the responses of resveratrol and agrimol B (P < 0.005 for 10 mM, P < 0.01 for 3 mM). Analysis of integrated intensity (Fig. 1C) showed that Agrimol B markedly increased SIRT1 expression at 10 mM (P < 0.01), the effect vanished at 3 mM (P > 0.05). These observations suggest that agrimol B activates SIRT1 signal by stimulating it importation and expression.
3T3-L1 preadipocyte was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were incubated in DMEM with 10% FBS at 37 C with 5% CO2. The adipocyte differentiation was conducted according to the procedures reported previously [22]. Briefly, 3T3-L1 preadipocytes were seeded into 96-well microplate (Frickenhausen, Germany) to allow cell confluence. On day 4, hormone cocktail was performed, which contained 0.5 mM IBMX, 1 mg/mL insulin, 0.25 mM dexamethasone, and 2 mM rosiglitazone. On day 6, cell culture medium was replaced with 1 mg/mL insulin for promotion. On day 8, refreshed the medium with complete DMEM and cultured for another two days. For detection of SIRT1, PPARg, C/EBPa, FAS, UCP-1, and apoE expression, adipocytes were continuously incubated with agrimol B for 6 days during differentiation. Resveratrol (50 mM) and berberine (10 mM) were performed as positive controls. In determination of the stages of adipocyte differentiation most sensitive to agrimol B, it was diluted to various doses and added to the cells at different time points.
The data indicated mean ± SEM from at least three independent experiments. The figures were drawn with Graphpad Prism 6.0 software (La Jolla, CA, USA). Comparison of fold changes of SIRT1 was performed by One-way ANOVA; Statistic analysis of PPARg, C/ EBPa, FAS, UCP-1, and apoE were conducted with Two-way ANOVA. *P < 0. 05 was defined as statistically significant. 3. Results 3.1. Agrimol B induces SIRT1 translocation and expression
3.2. Agrimol B blocks PPARg and C/EBPa expression 2.3. Immunofluorescent staining assay Adipocytes were fixed in 4% (wt/vol) paraformaldehyde for 20 min, then immunofluorescent staining was performed according to the procedures described previously [23]. Briefly, permeabilized the cells with 0.1% (vol/vol) Triton X-100 at room temperature for 30 min. Then, blocked the cells with 5% (wt/vol) bovine serum albumin (BSA) for 1 h. The primary antibodies were used at recommended concentrations, incubated at 4 C overnight. On the following day, the cells were incubated with second antibodies (TRITC or FITC) for 1 h at room temperature. Finally, 10 mg/ mL Hoechst 33342 was added to cells for DNA staining. 2.4. Lipid droplet staining The adipocytes were fixed and replenished with 5 mg/mL freshly prepared Nile red solution for lipid droplet labeling. After incubated 10 min at RT, the staining solution was discarded and the plates
To assess the effect of agrimol B on adipocyte differentiation, we next titrated the expression of PPARg and C/EBPa. Heterogeneous phenotypes were observed in differentiated adipocytes (Fig. 2A). The results suggested that agrimol B significantly decreased the positive staining of PPARg and C/EBPa (Fig. 2B). The differences of percentages were measurable between vehicle control and agrimol B treatment (P < 0.0001 at 10 mM, P < 0.01 mM at 3 mM). Additionally, statistical analysis of integrated intensity per cell confirmed that agrimol B down-regulated PPARg and C/EBPa expression (Fig. 2C). Efficacy of agrimol B was as strong as berberine at 10 mM (positive control). These results indicate that agrimol B reverses 3T3-L1 adipocyte differentiation at the transcriptional level. 3.3. Regulation of FAS, UCP-1, and apoE expression by agrimol B Subsequently,
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S. Wang et al. / Biochemical and Biophysical Research Communications 477 (2016) 454e460
Fig. 1. Agrimol B induced SIRT1 translocation and expression in 3T3-L1 adipocytes. (A) Representative images of SIRT1, lipid droplet, and nuclei staining. The images were acquired using a 20 objective on ImageXpress Micro XLS System. Scale bar: 100 mm. (B) Analysis of positive rate of nuclear SIRT1. (C) Integrated intensity per cell of SIRT1 expression. 3T3-L1 adipocytes were differentiated in the presence or absence of agrimol B for 6 days. The date represented mean ± SEM (n ¼ 6). ****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05, ns, no significant differences, one-way ANOVA with Turkey’s multiple comparisons test.
triglyceride formation and lipid droplet metabolism. PPARY targeted lipogenic genes, including fatty acid synthesis (FAS), uncoupling protein-1 (UCP1), and apolipoprotein (apo) E were assessed. Fluorescent staining of these three markers were significantly elevated after differentiation in mature adipocytes (Fig. 3A). Meanwhile, treatment with 10 mM agrimol B significantly decreased FAS, UCP-1, and apoE (P < 0.0001) expression (Fig. 3B). As
FAS controls free fatty acid production, these results confirm the inhibitory effect of agrimol B on adipogenesis. In view of the facts that UCP-1 and apoE represent the level of fat metabolism and in situ fat genesis [25,26], decreased UCP-1 and apoE expression indicate that agrimol B declines energy consumption in 3T3-L1 adipocytes.
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Fig. 2. Agrimol B blocked PPARY and C/EBPa expression. (A) Representative images of PPARY and C/EBPa immunofluorescent labeling. The images were acquired using a 20 objective on ImageXpress Micro XLS System. Scale bar: 100 mm. (B) Quantitative analysis of positive PPARg and C/EBPa staining. (C) Fold changes of integrated intensity per cell for PPARY and C/EBPa expression, the responses were normalized to DM vehicle control. Data represented mean ± SEM (n ¼ 6) of at least three independent experiments. ****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05, ns, no significant differences, two-way ANOVA with Turkey’s multiple comparisons test.
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Fig. 3. Effect of agrimol B on FAS, UCP-1, and apoE expression. (A) Fluorescence staining of FAS, UCP-1, and apoE in 3T3-L1 adipocytes, undifferentiated preadipocytes (UDM, panel 1) and mature adipocytes treated with vehicle (DM, panel 2), agrimol B (panel 3), and berberine (panel 4), respectively. The fluorescent signal was acquired using TRITC channel, to distinguish with each other, the images of FAS, UCP-1, and apoE were masked with green, red, and yellow. (B) Fold changes of FAS, UCP-1, and apoE expression in the presence of agrimol B. Data represented mean ± SEM (n ¼ 6). ****P < 0.0001, **P < 0.01, *P < 0.05, ns, no significant differences, two-way ANOVA with Turkey’s multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Agrimol B suppresses early stage of 3T3-L1 adipocyte differentiation Lipid droplet inhibition may be caused at the initial stage of cell differentiation, at the middle phase promotion, or at the terminal adipogenesis. To differentiate these three events, 3T3-L1 adipocytes were administrated with agrimol B at different time points following induction of differentiation. The indicated time points were shown in Fig. 4A, S1, S2, and S3 represented early, intermediate, and terminal phases, respectively. Whereas adipocytes treated with agrimol B during the early stage exhibited similar inhibition to the efficacy as cells treated for the early-intermediate
and complete periods (Fig. 4B). To assess these visual phenotypic modifications, lipid droplet content was measured by integrated intensity of lipid droplet per cell (Fig. 4C). Consistently, dose response curves of the three effective treatments were quite adjacent. The IC50 value was determined as 3.35 ± 0.32 mM. Meanwhile, agrimol B induced mild adipocyte cell loss by early phase treatment. In parallel, the cell number was not reduced by treatment merely at S2 or S3 phase (Fig. 4D). A possible explanation for this was that agrimol B blocked mitotic clonal expansion (MCE) of preadipocytes. These findings strongly indicate that the inhibitory efficacy of agrimol B on adipogenesis is mainly attributed to its impact at the early stage.
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Fig. 4. Agrimol B dose-dependently reduced lipid droplet accumulation by treatment at early phase differentiation. (A) Schematic time points of agrimol B administration, S1, S2, and S3 indicated that agrimol B was performed at early, intermediate, and terminal phases, respectively. (B) Nile red staining of 3T3-L1 adipocytes, scale bar: 100 mm. (C) Lipid droplet analysis by quantification of integrated intensity per cell. (D) Cell count of adipocytes treated with or without agrimol B. Data indicated mean ± SEM (n ¼ 3) from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion and conclusion The obesity epidemic has caused considerable expenditures and burden to the whole society [27], since it has been increasingly associated with some symptoms, such as metabolic syndromes, cardiovascular diseases, and even cognitive impairments [28]. Our current study provides a novel anti-adipogenic candidate for alleviating obesity from the herbal medicine Agrimonia pilosa Ledeb. The beauty of agrimol B maybe lie in its high efficacy compared with other active ingredients reported previously, such as resveratrol, berberine, and lovastatin. Recent studies demonstrated that nuclear but not cytoplasmic SIRT1 possessed deacetylation efficacy [29]. Nucleocytoplasmic shuttling of SIRT1 would impair the deacetylation activity and permit the initiation of differentiation [30]. In agreement with our study, we observed that agrimol B increased SIRT1 expression at high concentration (10 mM), while it imported cytoplasmic SIRT1 into the nuclei and blocked adipocyte differentiation by 3 mM. This indicated that nuclear SIRT1 silenced the PPARg signal by deacetylation and inhibition of its expression. Additionally, it has been reported by Li, et al. [31] that gain of function of SIRT1 would stimulate “browning” of WAT by deacetylation of PPARg on Lys268 and Lys293. However, the brown adipocyte biomarker UCP-1 was decreased by agrimol B in our experiment. A possible explanation for this was that agrimol B prevented 3T3-L1 preadipocyte being differentiated into white adipocyte, thus brown adipocyte was not induced. Agrimol B administration at early stage of adipocyte differentiation inhibited cell growth and the molecular mechanism remained unclear. However, a recent work by Abdesselem et al. [32] may provide insight for explanation of how agrimol B regulates adipocyte differentiation. It was observed that CCAAT/enhancerbinding protein b (C/EBPb) and c-Myc were elevated in SIRT1 knockout preadipocytes, suggesting that SIRT1 controls adipocyte proliferation. Therefore, agrimol B may attenuate adipocyte
hyperplasia through increasing SIRT1 expression. Besides therapeutic potential in metabolic dysfunctions, SIRT1 activators have also been identified effective for many other pathologies [33]. Examples included as a regulator of cardiomyopathy through attenuating cardiac fibrosis [34] and for attenuating vascular diseases through alleviating vascular ageing [35]. Recently, the importance of SIRT1 in neurodegenerative diseases of ageing were recognized, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis [36]. Thus it demands further investigations for identifying the activity of agrimol B on cardiovascular and neurodegenerative disorders by activating SIRT1. Collectively, using in situ fluorescence imaging assay, we demonstrate novel effect of agrimol B on SIRT1 activation in the context of transcriptional regulation of adipogenesis. These results suggest a therapeutic potential of agrimol B on alleviating obesity and related dysfunctions. Acknowledgement The authors would like to thank Haili Guo (Molecular Devices) for technical assistance with HCS imaging and analysis. This work was supported by a grant from the National Natural Science Foundation of China (No. 81430094). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.078. References [1] M. Wang, M. Gao, J. Liao, Y. Qi, X. Du, Y. Wang, et al., Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice, Bba-Mol. Cell Biol. L 1861 (2016) 410e418. [2] B.L. Yu, S.P. Zhao, J.R. Hu, Cholesterol imbalance in adipocytes: a possible mechanism of adipocytes dysfunction in obesity, Obes. Rev. 11 (2010)
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Agrimol B suppresses adipogenesis through modulation of SIRT1-PPAR gamma signal pathway Shifeng Wang a, Qiao Zhang a, Yuxin Zhang a, Cheng Shen a, Zhen Wang b, Qinghua Wu c, Yanling Zhang a, Shiyou Li b, *, Yanjiang Qiao a, ** a b c
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, China Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing 100101, China HD Biosciences Co., Ltd, Shanghai 201201, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 June 2016 Accepted 15 June 2016 Available online 16 June 2016
Studies of human genetics have implicated the role of SIRT1 in regulating obesity, insulin resistance, and longevity. These researches motivated the identification of novel SIRT1 activators. The current study aimed to investigate the potential efficacy of agrimol B, a polyphenol derived from Agrimonia pilosa Ledeb., on mediating SIRT1 activity and fat metabolism. Results showed that agrimol B significantly induced cytoplasm-to-nucleus shuttle of SIRT1. Furthermore, we confirmed that agrimol B dramatically inhibited 3T3-L1 adipocyte differentiation by reducing PPARg, C/EBPa, FAS, UCP-1, and apoE expression. Consequently, adipogenesis was blocked by treatment of agrimol B at the early stage of differentiation in a dose-dependent manner, the IC50 value was determined as 3.35 ± 0.32 mM. Taken together, our data suggest a therapeutic potential of agrimol B on alleviating obesity, through modulation of SIRT1-PPARg signal pathway. © 2016 Published by Elsevier Inc.
Keywords: Agrimol B Agrimonia pilosa Ledeb. Lipid droplet Obesity PPARg SIRT1
1. Introduction Adipocyte plays a key role in regulating energy homeostasis. Deficiency of adipose tissue may cause severe hyperlipidemia as well as atherosclerosis since it contains the largest pool of free cholesterol [1,2]. Adipose tissue also serves as an endocrine organ that controls inflammatory responses through secreting hormones, such as adiponectin, leptin, tumor necrosis factor a (TNFa) [3]. Latent inflammation in adipose tissue is a major negative impact of metabolic syndrome that promotes atherosclerosis, myocarditis [4],
Abbreviations: ApoE, apolipoprotein E; BSA, bovine serum albumin; C/EBPb, CCAAT/enhancer-binding protein b; FAS, fatty acid synthesis; IBMX, 3-isobutyl-1methylxanthine; MCE, mitotic clonal expansion; PBS, phosphate buffer saline solution; SIRT1, silent information regulator 2 homolog 1; TNFa, tumor necrosis factor a; UCP-1, uncoupling protein-1. * Corresponding author. Key Laboratory of Genomic and Precision Medicine, Beijing Institute of Genomics, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing 100101, China. ** Corresponding author. Key Laboratory of TCM-Information Engineer of State Administration of TCM, School of Chinese Materia Medica, Beijing University of Chinese Medicine, No. 6 Wangjing Zhong Huan South Road, Chaoyang District, Beijing 100102, China. E-mail addresses: [email protected] (S. Li), [email protected] (Y. Qiao). http://dx.doi.org/10.1016/j.bbrc.2016.06.078 0006-291X/© 2016 Published by Elsevier Inc.
and non-alcoholic fatty liver disease [5]. Meanwhile, dyslipidemia may contribute to early neurological deterioration after ischemic stroke [6]. Importantly, metabolic disturbance of lipid and cholesterol were associated with higher incidence of cancer [7,8]. Thus, maintaining fat homeostasis may provide an alternative avenue for therapies of metabolic syndromes, cardiovascular diseases, and tumors. Caloric restriction has been intimately related to obesity and longevity [9,10]. Silent information regulator 2 homolog 1 (SIRT1) is a critical factor that influences obesity and life span. It controls adipocyte fat storage [11] and glucose homeostasis through modulation of PGC-1a activity [12]. White adipocyte is remodeled by SIRT1 through interaction with other epigenetic regulators, such as PPARY [13,14]. The first SIRT1 activator isolated from the peer of grapes, resveratrol, has been proved to be efficient in alleviating insulin resistance and prolonging life span [15]. However, the liability of resveratrol as a SIRT1 activator lies in its lack of specificity, known as “off target effects” [16]. With the prevalence of obesity and ageing, there is an increasing demand for novel SIRT1 activators. Agrimonia pilosa Ledeb. (Xianhecao in Chinese) is an herbal medicine listed in Pharmacopoeia of China [17]. It possesses broad range of pharmaceutical activities, such anti-oxidant [18] and anti-
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inflammation [19]. Agrimol B is considered as an appropriate indicator for the quality control of Agrimonia pilosa Ledeb. [20]. Many molecular chaperones have been observed anti-obesity activities [21]. Nonetheless, the function of agrimol B on fat metabolism and the underlying molecular mechanism remained poorly understood. Therefore, to systematically assess the potency of agrimol B on lipid metabolism, we developed an in situ fluorescence imaging assay, investigated the role of agrimol B on SIRT1 activation and lipid droplet accumulation. 2. Materials and methods 2.1. Materials
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were rinsed with PBS for three times. Thereafter, 10 mg/mL Hoechst 33342 was added to cells for nuclei staining as described above. For co-staining of SIRT1 and lipid droplet, 1 mg/mL Bodipy 493/503 (Sigma) was performed. 2.5. Imaging and analysis The fluorescent images were yielded on ImageXpress Micro XLS System (MD, CA, USA). Signals of the biomarkers, such as PPARY, C/ EBPa, FAS, UCP-1, and apoE were yielded using a 20 objective. Lipid droplet fluorescent signal was acquired by 10 objective. The images were analyzed using a custom module of cell scoring protocol.
Agrimol B was provided by Dalian Institute of Chemical Physics (Dalian, China). Ingredients for adipocyte differentiation were purchased from Sigma-Aldrich (St. Louis, MO, USA). The antibodies were acquired as follows: rabbit anti-SIRT1 (sc-15404) and mouse anti-PPARY (E-8, sc-7273) were purchased from Santa Cruz Biotechnology Inc. (CA, USA); rabbit anti-C/EBPa (1704-1) and antiFAS (3655-1) were acquired from Epitomics Inc. (CA, USA); Rabbit polyclonal to apolipoprotein E (ab20874); anti-UCP-1 (U6382), anti-rabbit TRITC (T6778), and anti-rabbit FITC IgG (F7512) were obtained from Sigma-Aldrich. Goat anti-mouse IgG (H þ L) secondary antibody (35502) was purchased from Thermo Fisher Scientific (Waltham, USA).
2.6. Statistical analysis
2.2. Adipocyte differentiation and compound treatment
To determine if agrimol B could promote SIRT1 activity, we measured the ability of agrimol B on inducing SIRT1 translocation and expression. Results showed that SIRT1 was absent in the nuclei of mature adipocytes contained visible lipid droplets, but localized in the cytoplasmic area by the form of small spots (Fig. 1A). In contrast, with administration of agrimol B, more nuclear SIRT1 were observed. The phenotype of agrimol B treated cells was in accordance with that of resveratrol, a well-known SIRT1 activator [24]. Normalized responses showed that agrimol B significantly increased nuclear positive rate of SIRT1 compared with vehicle control (P < 0.0001). Moreover, the effect of agrimol B was stronger than that of resveratrol (Fig. 1B), showing significant differences between the responses of resveratrol and agrimol B (P < 0.005 for 10 mM, P < 0.01 for 3 mM). Analysis of integrated intensity (Fig. 1C) showed that Agrimol B markedly increased SIRT1 expression at 10 mM (P < 0.01), the effect vanished at 3 mM (P > 0.05). These observations suggest that agrimol B activates SIRT1 signal by stimulating it importation and expression.
3T3-L1 preadipocyte was obtained from American Type Culture Collection (Manassas, VA, USA). The cells were incubated in DMEM with 10% FBS at 37 C with 5% CO2. The adipocyte differentiation was conducted according to the procedures reported previously [22]. Briefly, 3T3-L1 preadipocytes were seeded into 96-well microplate (Frickenhausen, Germany) to allow cell confluence. On day 4, hormone cocktail was performed, which contained 0.5 mM IBMX, 1 mg/mL insulin, 0.25 mM dexamethasone, and 2 mM rosiglitazone. On day 6, cell culture medium was replaced with 1 mg/mL insulin for promotion. On day 8, refreshed the medium with complete DMEM and cultured for another two days. For detection of SIRT1, PPARg, C/EBPa, FAS, UCP-1, and apoE expression, adipocytes were continuously incubated with agrimol B for 6 days during differentiation. Resveratrol (50 mM) and berberine (10 mM) were performed as positive controls. In determination of the stages of adipocyte differentiation most sensitive to agrimol B, it was diluted to various doses and added to the cells at different time points.
The data indicated mean ± SEM from at least three independent experiments. The figures were drawn with Graphpad Prism 6.0 software (La Jolla, CA, USA). Comparison of fold changes of SIRT1 was performed by One-way ANOVA; Statistic analysis of PPARg, C/ EBPa, FAS, UCP-1, and apoE were conducted with Two-way ANOVA. *P < 0. 05 was defined as statistically significant. 3. Results 3.1. Agrimol B induces SIRT1 translocation and expression
3.2. Agrimol B blocks PPARg and C/EBPa expression 2.3. Immunofluorescent staining assay Adipocytes were fixed in 4% (wt/vol) paraformaldehyde for 20 min, then immunofluorescent staining was performed according to the procedures described previously [23]. Briefly, permeabilized the cells with 0.1% (vol/vol) Triton X-100 at room temperature for 30 min. Then, blocked the cells with 5% (wt/vol) bovine serum albumin (BSA) for 1 h. The primary antibodies were used at recommended concentrations, incubated at 4 C overnight. On the following day, the cells were incubated with second antibodies (TRITC or FITC) for 1 h at room temperature. Finally, 10 mg/ mL Hoechst 33342 was added to cells for DNA staining. 2.4. Lipid droplet staining The adipocytes were fixed and replenished with 5 mg/mL freshly prepared Nile red solution for lipid droplet labeling. After incubated 10 min at RT, the staining solution was discarded and the plates
To assess the effect of agrimol B on adipocyte differentiation, we next titrated the expression of PPARg and C/EBPa. Heterogeneous phenotypes were observed in differentiated adipocytes (Fig. 2A). The results suggested that agrimol B significantly decreased the positive staining of PPARg and C/EBPa (Fig. 2B). The differences of percentages were measurable between vehicle control and agrimol B treatment (P < 0.0001 at 10 mM, P < 0.01 mM at 3 mM). Additionally, statistical analysis of integrated intensity per cell confirmed that agrimol B down-regulated PPARg and C/EBPa expression (Fig. 2C). Efficacy of agrimol B was as strong as berberine at 10 mM (positive control). These results indicate that agrimol B reverses 3T3-L1 adipocyte differentiation at the transcriptional level. 3.3. Regulation of FAS, UCP-1, and apoE expression by agrimol B Subsequently,
we
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Fig. 1. Agrimol B induced SIRT1 translocation and expression in 3T3-L1 adipocytes. (A) Representative images of SIRT1, lipid droplet, and nuclei staining. The images were acquired using a 20 objective on ImageXpress Micro XLS System. Scale bar: 100 mm. (B) Analysis of positive rate of nuclear SIRT1. (C) Integrated intensity per cell of SIRT1 expression. 3T3-L1 adipocytes were differentiated in the presence or absence of agrimol B for 6 days. The date represented mean ± SEM (n ¼ 6). ****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05, ns, no significant differences, one-way ANOVA with Turkey’s multiple comparisons test.
triglyceride formation and lipid droplet metabolism. PPARY targeted lipogenic genes, including fatty acid synthesis (FAS), uncoupling protein-1 (UCP1), and apolipoprotein (apo) E were assessed. Fluorescent staining of these three markers were significantly elevated after differentiation in mature adipocytes (Fig. 3A). Meanwhile, treatment with 10 mM agrimol B significantly decreased FAS, UCP-1, and apoE (P < 0.0001) expression (Fig. 3B). As
FAS controls free fatty acid production, these results confirm the inhibitory effect of agrimol B on adipogenesis. In view of the facts that UCP-1 and apoE represent the level of fat metabolism and in situ fat genesis [25,26], decreased UCP-1 and apoE expression indicate that agrimol B declines energy consumption in 3T3-L1 adipocytes.
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Fig. 2. Agrimol B blocked PPARY and C/EBPa expression. (A) Representative images of PPARY and C/EBPa immunofluorescent labeling. The images were acquired using a 20 objective on ImageXpress Micro XLS System. Scale bar: 100 mm. (B) Quantitative analysis of positive PPARg and C/EBPa staining. (C) Fold changes of integrated intensity per cell for PPARY and C/EBPa expression, the responses were normalized to DM vehicle control. Data represented mean ± SEM (n ¼ 6) of at least three independent experiments. ****P < 0.0001, ***P < 0.005, **P < 0.01, *P < 0.05, ns, no significant differences, two-way ANOVA with Turkey’s multiple comparisons test.
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Fig. 3. Effect of agrimol B on FAS, UCP-1, and apoE expression. (A) Fluorescence staining of FAS, UCP-1, and apoE in 3T3-L1 adipocytes, undifferentiated preadipocytes (UDM, panel 1) and mature adipocytes treated with vehicle (DM, panel 2), agrimol B (panel 3), and berberine (panel 4), respectively. The fluorescent signal was acquired using TRITC channel, to distinguish with each other, the images of FAS, UCP-1, and apoE were masked with green, red, and yellow. (B) Fold changes of FAS, UCP-1, and apoE expression in the presence of agrimol B. Data represented mean ± SEM (n ¼ 6). ****P < 0.0001, **P < 0.01, *P < 0.05, ns, no significant differences, two-way ANOVA with Turkey’s multiple comparisons test. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3.4. Agrimol B suppresses early stage of 3T3-L1 adipocyte differentiation Lipid droplet inhibition may be caused at the initial stage of cell differentiation, at the middle phase promotion, or at the terminal adipogenesis. To differentiate these three events, 3T3-L1 adipocytes were administrated with agrimol B at different time points following induction of differentiation. The indicated time points were shown in Fig. 4A, S1, S2, and S3 represented early, intermediate, and terminal phases, respectively. Whereas adipocytes treated with agrimol B during the early stage exhibited similar inhibition to the efficacy as cells treated for the early-intermediate
and complete periods (Fig. 4B). To assess these visual phenotypic modifications, lipid droplet content was measured by integrated intensity of lipid droplet per cell (Fig. 4C). Consistently, dose response curves of the three effective treatments were quite adjacent. The IC50 value was determined as 3.35 ± 0.32 mM. Meanwhile, agrimol B induced mild adipocyte cell loss by early phase treatment. In parallel, the cell number was not reduced by treatment merely at S2 or S3 phase (Fig. 4D). A possible explanation for this was that agrimol B blocked mitotic clonal expansion (MCE) of preadipocytes. These findings strongly indicate that the inhibitory efficacy of agrimol B on adipogenesis is mainly attributed to its impact at the early stage.
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Fig. 4. Agrimol B dose-dependently reduced lipid droplet accumulation by treatment at early phase differentiation. (A) Schematic time points of agrimol B administration, S1, S2, and S3 indicated that agrimol B was performed at early, intermediate, and terminal phases, respectively. (B) Nile red staining of 3T3-L1 adipocytes, scale bar: 100 mm. (C) Lipid droplet analysis by quantification of integrated intensity per cell. (D) Cell count of adipocytes treated with or without agrimol B. Data indicated mean ± SEM (n ¼ 3) from three independent experiments. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
4. Discussion and conclusion The obesity epidemic has caused considerable expenditures and burden to the whole society [27], since it has been increasingly associated with some symptoms, such as metabolic syndromes, cardiovascular diseases, and even cognitive impairments [28]. Our current study provides a novel anti-adipogenic candidate for alleviating obesity from the herbal medicine Agrimonia pilosa Ledeb. The beauty of agrimol B maybe lie in its high efficacy compared with other active ingredients reported previously, such as resveratrol, berberine, and lovastatin. Recent studies demonstrated that nuclear but not cytoplasmic SIRT1 possessed deacetylation efficacy [29]. Nucleocytoplasmic shuttling of SIRT1 would impair the deacetylation activity and permit the initiation of differentiation [30]. In agreement with our study, we observed that agrimol B increased SIRT1 expression at high concentration (10 mM), while it imported cytoplasmic SIRT1 into the nuclei and blocked adipocyte differentiation by 3 mM. This indicated that nuclear SIRT1 silenced the PPARg signal by deacetylation and inhibition of its expression. Additionally, it has been reported by Li, et al. [31] that gain of function of SIRT1 would stimulate “browning” of WAT by deacetylation of PPARg on Lys268 and Lys293. However, the brown adipocyte biomarker UCP-1 was decreased by agrimol B in our experiment. A possible explanation for this was that agrimol B prevented 3T3-L1 preadipocyte being differentiated into white adipocyte, thus brown adipocyte was not induced. Agrimol B administration at early stage of adipocyte differentiation inhibited cell growth and the molecular mechanism remained unclear. However, a recent work by Abdesselem et al. [32] may provide insight for explanation of how agrimol B regulates adipocyte differentiation. It was observed that CCAAT/enhancerbinding protein b (C/EBPb) and c-Myc were elevated in SIRT1 knockout preadipocytes, suggesting that SIRT1 controls adipocyte proliferation. Therefore, agrimol B may attenuate adipocyte
hyperplasia through increasing SIRT1 expression. Besides therapeutic potential in metabolic dysfunctions, SIRT1 activators have also been identified effective for many other pathologies [33]. Examples included as a regulator of cardiomyopathy through attenuating cardiac fibrosis [34] and for attenuating vascular diseases through alleviating vascular ageing [35]. Recently, the importance of SIRT1 in neurodegenerative diseases of ageing were recognized, such as Alzheimer’s disease, Parkinson’s disease, and Amyotrophic Lateral Sclerosis [36]. Thus it demands further investigations for identifying the activity of agrimol B on cardiovascular and neurodegenerative disorders by activating SIRT1. Collectively, using in situ fluorescence imaging assay, we demonstrate novel effect of agrimol B on SIRT1 activation in the context of transcriptional regulation of adipogenesis. These results suggest a therapeutic potential of agrimol B on alleviating obesity and related dysfunctions. Acknowledgement The authors would like to thank Haili Guo (Molecular Devices) for technical assistance with HCS imaging and analysis. This work was supported by a grant from the National Natural Science Foundation of China (No. 81430094). Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2016.06.078. References [1] M. Wang, M. Gao, J. Liao, Y. Qi, X. Du, Y. Wang, et al., Adipose tissue deficiency results in severe hyperlipidemia and atherosclerosis in the low-density lipoprotein receptor knockout mice, Bba-Mol. Cell Biol. L 1861 (2016) 410e418. [2] B.L. Yu, S.P. Zhao, J.R. Hu, Cholesterol imbalance in adipocytes: a possible mechanism of adipocytes dysfunction in obesity, Obes. Rev. 11 (2010)
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