Taipei Medical University College of Medicine International Ph. Program in Medicine Ph. Dissertation Effects of SW20.1 on suppressing Adipogenesis in 3T3- L1 Adipocytes via the ATF3-Resistin Pathway and attenuating obesity-induced metabolic dysfunction in High Fat diet-induced Obese Mice Advisor: Hsi-Hsien Chen, Ph. Co-Advisor: Heng Lin, Ph.
Student: Tran Tuan Tu Student ID: D142109017 June, 2023 Declaration I hereby declare that the research titled "Effects of SW20.1 on Suppressing Adipogenesis in 3T3-L1 Adipocytes via the ATF3-Resistin Pathway and Attenuating Obesity-induced Metabolic Dysfunction in High Fat Diet- induced Obese Mice" is my original work conducted under the supervision of Professor Hsi-Hsien Chen and Professor Heng Lin. I affirm that this research project was conducted with integrity, adhering to the highest ethical standards and in compliance with the guidelines and regulations set forth by the relevant authorities. The data presented in this study is accurate and has been obtained through rigorous experimentation and analysis. I declare that any external sources or references used in this research have been appropriately cited and acknowledged.
All intellectual property rights of third-party materials have been respected and upheld. I take full responsibility for the content and findings presented in this research. The conclusions drawn and the interpretations made are based on the data collected and analyzed during the course of this study. Any limitations or uncertainties in the research methodology and results have been duly acknowledged and discussed.
I am committed to sharing the findings of this research with the scientific community and ensuring the transparency and reproducibility of the study. I am open to further discussions, collaborations, and peer review to enhance the validity and impact of this research. Sincerely, Tran Tuan Tu, Taipei, 2023 II Acknowledgment Firstly, I would like to express my heartfelt gratitude to my first supervisor, Prof. Hsi-Hsien Chen, and my co-supervisor, Prof.
Heng Lin, for their invaluable guidance and support throughout my project. Their expertise and mentorship have been instrumental in shaping the success of my research. I am also deeply grateful to all the members of my lab who have generously shared their knowledge and expertise with me. Their teachings in techniques and data analysis have greatly contributed to the quality of my projects.
I recognize that the support and encouragement from my family and friends have played a crucial role in my achievements. I extend my special thanks to my wife, whose unwavering presence and constant motivation have been a driving force behind my accomplishments. Lastly, I express my gratitude to the esteemed members of the committee, namely Prof Dean-Mo Liu, Prof Wen-Shan Li, and Prof Hsin-Yi Hung, for their valuable comments, insightful questions, and helpful suggestions during my thesis defense. III Abbreviations ATF3 Activating transcription factor 3 HFD High fat diet WT Wild type WAT White adipose tissue BAT Brown adipose tissue DMEM Dulbecco modified Eagle medium FBS Fetal bovine serum IBMX Isobutyl methylxanthine MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5- Diphenyltetrazolium Bromide ChIP Chromatin Immunoprecipitation DMSO Dimethyl sulfoxide iWAT Inguinal white adipose tissue C/EBPD CCAAT/enhancer-binding protein D PPARJ2 Peroxisome proliferator-activated receptor J2 ChREBP Carbohydrate-responsive element-binding protein SCD1 Stearoyl-CoA desaturase-1 FABP4 Fatty acid binding protein 4 HSP70 Heat shock proteins 70 PGC1D Peroxisome proliferator-activated receptor- gamma coactivator -1alpha GAPDH Glyceraldehyde 3-phosphate dehydrogenase IV Table of Contents Declaration.
IV List of Figures. VIII List of Tables. 5 Chapter 2: Literature Review. The functions of ATF3.
The effectiveness of ATF3- inducer drugs. The functions of adipokines. 15 Chapter 3: Study Design and Methods. Material and Methods.
The Chemical Structure of SW20.1 and other Derivates. Cytotoxicity of SW20.1 in differentiated 3T3-L1 cells. The effects of SW20.1 on intracellular Lipid accumulation during differentiation of 3T3-L1 preadipocytes. The ATF3 expression was induced by SW20.1 and other derivatives.1 suppress 3T3-L1 pre-adipocyte differentiation .1 inhibits adipogenesis directly through the ATF3-Resistin pathway.1 Treatment ameliorates obesity and obesity-related metabolic syndrome obesity induced by HFD in mice .1 treatment inhibits Adipogenesis in HFD- fed Mice.
81 VI Figure S1: The chemical structure of ATF3- inducer compound (cont). 81 Figure S2: The cardiovascular index. 82 Figure S3: NMR results of SW20. 83 VII List of Figures Figure 1: Chemical structures formula of major ATF3 synthetic compounds.
27 Figure 2: The cytotoxicity of SW20.1 in differentiated 3T3-L1 adipocyte. 31 Figure 3: The effect of SW20.1 on lipid accumulation during differentiation of 3T3-L1 preadipocytes. 34 Figure 4: Identify the function of SW20.1 that can induce ATF3 on preadipocytes (3T3-L1 Cell).1 suppresses lipid accumulation and inhibits adipogenesis/lipogenesis through the ATF3- Resistin pathway. 40 Figure 6: Molecular mechanism of SW20.
43 Figure 7: Results of treatment with or without SW20.1 or ST32db in wild- type mice with high-fat diet (HFD)-induced obesity and metabolic syndrome. 48 Figure 8: Biochemical parameters of mice treated to the DMSO, ST32db or SW20.1 treatment suppresses adipogenesis in HFD-fed mice. 53 VIII List of Tables Table 1: Primer sequence in the study. 25 IX Abstract Obesity has become an emerging problem globally because of its increasing prevalence.
Obesity is associated with many diseases, such as cardiovascular disease, dyslipidemia, and cancer. Thus, effective new anti- obesity drugs should be urgently developed. We synthesized SW20.1, a single compound that can induce activating transcription factor 3(ATF3) expression. The results of Oil-red O staining and quantitative real-time polymerase chain reaction revealed that SW20.1 was more effective in reducing lipid accumulation in 3T3-L1 preadipocytes than the previously synthesized ST32db, and that it inhibited the expression of genes involved in adipogenesis and lipogenesis.
The chromatin immunoprecipitation assay indicated that SW20.1 inhibited adipogenesis/lipogenesis by binding to the upstream promoter region of resistin at two sites (−2861/−2854 and −241/−234). In mice, the intraperitoneal administration of SW20.1 reduced body weight, white adipocyte weight in different regions, serum cholesterol levels, adipogenesis-related gene expression, hepatic-steatosis, and serum resistin levels.1 has an anti-obesity effect by inhibiting resistin via the ATF3 pathway. Our study results indicated that SW20.1 is a promising therapeutic drug for diet-induced obesity. Keywords: activating transcription factor 3; obesity; resistin, SW20.
Background Obesity is one of the most prevalent metabolic disorders worldwide, affecting 61 percent of adults, and is associated with various medical illnesses, such as dyslipidemia, diabetes mellitus, hypertension, malignancies, and osteoarthritis [1, 2]. These associations are contingent upon the excessive differentiation and proliferation of adipocytes. Adipogenesis is characterized by structural alterations, lipid accumulation, lipogenic enzyme production, and the storage of excess energy as triglyceride (TG) in adipocytes [3-5]. Inhibition of adipogenesis and TG deposition in adipocytes may therefore be of critical therapeutic value in drugs designed to treat or prevent Obesity.
The primary treatment for Obesity is a change in lifestyle [6]. Nevertheless, pharmacotherapy is recommended when these methods are ineffective or when Obesity is severe. Although certain anti-obesity medications have been approved and marketed, however, some of them have been discontinued due to severe adverse effects, such as: Amphetamines, rimonabant, and sibutramine [7, 8]. Therefore, there is an urgent need to produce effective and safe anti-obesity medications.
Adipose tissue consists of white adipose tissue (WAT), brown adipose tissue (BAT), and brite adipose tissue and is involved in both energy storage and thermogenesis regulation. Adipose tissue also secretes numerous hormones, cytokines, and metabolites, which are known as the adipokines [9]. Resistin is an adipose tissue-derived signaling cysteine-rich protein. An elevated plasma resistin level is associated with various dysfunctions, 2 including altered lipid and carbohydrate metabolism, inflammation, and insulin resistance [10].
Furthermore, resistin is related to knee osteoarthritis, ovarian cancer, and acute pancreatitis [11-13]. A human study revealed the association of resistin with obesity and impaired insulin sensitivity [14]. Salvia miltiorrhiza (or S. miltiorrhiza radix) is a member of the Labiatae family.
Because of its beneficial ability to enhance cardiovascular health and reduce platelet aggregation, S. miltiorrhiza is a well-known herb used to treat numerous ailments in traditional Chinese medicine [15]. miltiorrhiza contains multiple chemical substances. More than 50 water-soluble components and 30 liposoluble components have been identified in S.
Most water-soluble components of S. miltiorrhiza exhibit antioxidative, anticoagulant, and myocardial protective effects, which are mainly attributed to blood activation and blood clot elimination [15, 17]. Primary tanshinones, which are liposoluble components of S. miltiorrhiza, exhibit antibacterial, anti-inflammatory, and myocardial protective effects and protective effects for endothelial cells [18].
In addition to tanshinone I and tanshinone IIA, several specific liposoluble components of S. miltiorrhiza have been determined to exert antiobesity effects [19, 20]. Activating transcription factor 3 (ATF3), a member of the ATF/cAMP response element-binding (CREB) family, binds to the cyclic AMP response element in promoters containing the consensus sequence TGACGTCA [21, 22]. ATF3 is a homodimer-forming transcriptional repressor.
It forms a heterodimer with the members of the ATF/CERB or CCAAT/enhancer- binding protein (C/EBP) family to exert suppressive or stimulatory effects [23, 24]. Ultraviolet radiation, cAMP, calcium influx, and cytokines are stress signals that can increase the generation of ATF3, which is normally 3 produced at low levels in normal or quiescent cells [25, 26]. ATF3 can inhibit the mRNA expression of peroxisome proliferator-activated receptor J (PPARJ) and C/EBPα, thus suppressing adipocyte development [27]. High- fat diet (HFD)-fed ATF3−/− mice gained more weight than did their littermate wild-type control mice and developed impaired glucose metabolism and hyperlipidemia [28, 29].
We examined ATF3 inducers from a modified Chinese herb single- compound library utilizing ATF3-specific promoter-screening techniques.1, a synthetic molecule similar to neo-tanshinlactone, was derived from Salvia miltiorrhiza. ST32da and ST32db, which are derivatives of SW20.1, have been demonstrated to have anti-obesity effects in mice that were fed high-fat diets, known for inducing obesity [28, 31] [32]. Therefore, we hypothesized that SW20.1 might have an excellent antiobesity and anti- metabolic syndrome via inducing ATF3 expression. Objectives In our study, we aimed to investigate the anti-obesity and anti- metabolic syndrome effects of SW20.1 and ST32db in mice that were induced to become obese through a high-fat diet (HFD).
Additionally, we sought to explore whether the molecular mechanism underlying the effects of SW20.1 involved the ATF3-mediated signaling pathway. 5 Chapter 2: Literature Review 2. The functions of ATF3 ATF3 is a member of the ATF/cAMP response element-binding (CREB) family and binds to the cyclin AMP response element (CRE) in promoters containing the consensus sequence TGACGTCA [33, 34]. This factor is a homodimer-forming transcriptional repressor.
It forms a heterodimer with members of the ATF/CERB or CCAAT/enhancer binding protein (C/EBP) families to exert suppressive or stimulatory actions [23, 24]. UV radiation, cAMP, calcium influx, and cytokines are among the stress signals that can increase ATF3, which is produced at low levels in normal and quiescent cells [25, 26]. ATF3 and ATF3-regulated signals were related to metabolic, immunity, and oncogenesis [35]. In addition, ATF3 has been shown to play crucial functions throughout postnatal cortical growth and damage recovery.
In early developing cortical neuronal cultures, the inhibition of ATF3 specifically inhibited neurite outgrowth and differentiation and caused increased cell death [36]. Regarding cancer, ATF3 is considered a regulator for prostate, breast, colon, lung, and liver cancers [35]. In prostate cancer, ATF3 is a tumor suppression linked to ATF3 and AKT signaling through the loss of phosphatase and tension homolog protein ( Pten) [37]. ATF3 binds to the androgen receptor (AR) and inhibits the androgen signaling that is essential for the proliferation and survival of prostatic epithelial cells [38].
Besides, ATF3 can also suppress the p53 mutation, which serves for chemoresistance, migration, and invasion [39, 40]. The upregulation of ATF3 expression is observed in metastatic breast cancer. However, in contrast to its transient 6 expression in normal mammary epithelial cells, TGFβ plays a critical role in sustaining and prolonging ATF3 expression [41-44]. ATF3 serves a dual function by promoting apoptosis in normal mammary epithelial cells and stimulating proliferation in breast cancer cells, thereby contributing to metastasis.
This dual role is reminiscent of the dichotomy exhibited by TGFβ [45, 46], which induces epithelial to mesenchymal transition through ATF3. The involvement of the Wnt and p53 signaling pathways have been identified in the regulation of the ATF3 expression [43, 47].