GLP-1 작용제(GLP-1RA)로서의 기능성 저분자 물질: 3T3- L1 지방세포에서의 크립토탄시논의 항비만 기전 연구 = Functional small molecules as GLP-1 receptor agonists (GLP-1RAs) : Study on the anti-obesity mechanisms of cryptotanshinone in 3T3-L1 adipocytes

다국어 초록 (Multilingual Abstract)

Functional small molecules as GLP-1 receptor agonists (GLP-1RAs): Study on the anti-obesity mechanisms of cryptotanshinone in 3T3-L1 adipocytes MD. ABDUR RAKIB Department of Microbiology, School of Medicine, Soonchunhyang University, Republic of Korea Supervised by: Professor Yong-Sik Kim Obesity and its related metabolic disorders pose significant global health challenges, necessitating the development of novel therapeutic strategies targeting adipose tissue biology and energy metabolism. Current therapy alternatives are nevertheless limited by undesirable effects, high costs, or poor long-term efficacy, creating a need for new medicines capable of modifying adipose tissue biology. This thesis explores the anti- adipogenic and browning potential of bioactive small molecules in 3T3-L1 adipocytes, emphasizing their capacity to influence glucagon-like peptide-1 receptor (GLP-1R)– mediated signaling. This study elucidated the molecular mechanisms by which CT modulates adipogenesis by focusing on its interaction with the glucagon-like peptide- 1 receptor (GLP-1R). Cryptotanshinone (CT), a bioactive compound derived from Salvia miltiorrhiza, has recently been investigated for its anti-adipogenic and metabolic regulatory effects. The molecular mechanism by which CT inhibits adipogenesis remains unknown and requires further elucidation. Molecular docking and surface plasmon resonance investigations were employed to examine CT’s direct binding affinity to GLP-1R specifically targeting potential allosteric binding sites. Expression analyses were conducted to evaluate changes in GLP-1R and downstream signalling components. Functional assays, including cAMP measurements, Western blotting, and gene expression studies, were performed. Additionally, pharmacological inhibitor and siRNA-mediated knockdown of GLP-1R were performed to assess the dependency of CT’s effects on GLP-1R signaling. Our findings demonstrated that CT treatment upregulates GLP-1R expression early in adipogenesis, preceding the induction of the transcription factor CCAAT/enhancer-binding protein beta (C/EBPβ), and shifts G protein coupling by increasing Gαq and Gαs while reducing Gαi, leading to elevated cAMP levels and activation of the Protein Kinase A (PKA) pathway. This cascade robustly activates p38-mitogen-activated protein kinase and AMP-activated protein kinase phosphorylation (AMPK), resulting in suppressed expression of key adipogenic markers, Peroxisome proliferator-activated receptor gamma (PPARγ), C/EBPβ, Adipocyte protein 2 (aP2). Importantly, the antiadipogenic effects were repealed by GLP-1R inhibition, confirming its GLP-1R dependent mechanism of action. These findings reveal a novel mechanism by which CT regulates adipogenesis via GLP-1R signaling, highlighting its potential as a small-molecule therapeutic agent for obesity. CT suppresses adipogenesis by acting as a small-molecule modulator of GLP-1R, altering G protein coupling by inducing cAMP, PKA, p38-MAPK, and AMPK signaling. These findings establish CT’s GLP-1R–dependent anti-adipogenic mechanism and highlight its potential as a therapeutic candidate for obesity.

목차 (Table of Contents)

• Table of Contents i
• List of tables iii
• List of Figures iii
• ABSTRACT iv
• Abbreviations vii
• Chapter One 1
• 1. General Introduction 2
• 1.1. Obesity and Its Global Overview 2
• 1.2. Adipose Tissue Biology and Energy Metabolism 3
• 1.3. Approaches for Studying Anti-Obesity Mechanisms 6
• 1.4. GLP-1 Receptor Signaling and Metabolic Regulation in obesity 8
• 1.5 Bioactive Small Molecules with Anti-Obesity Effects 12
• 1.6. Small Molecules Targeting GLP-1 Receptor–Mediated Signaling 14
• 1.7. Scope of This Thesis 16
• Chapter Two 17
• 2. Anti-adipogenic effect of cryptotanshinone in 3T3-L1 preadipocytes via binding to glucagon-like peptide 1 receptor and its signaling 17
• 2.1. Introduction 18
• 2.2. Materials and Methods 20
• 2.2.1. Chemicals, Reagents, and Antibodies 20
• 2.2.2. Cell Culture and Differentiation 21
• 2.2.3. Oil Red O (ORO) Staining 21
• 2.2.4. qRT-PCR Analyses 22
• 2.2.5. Western Blot Analysis 22
• 2.2.6. Computational Analysis 24
• 2.2.7. Surface Plasma Resonance (SPR) Analysis 26
• 2.2.8. cAMP Assay 27
• 2.2.9. Pertussis Toxin (PTX) Treatment 27
• 2.2.10. GLP-1R Knockdown 28
• 2.1.11. Densitometry in the ImageJ Software 28
• 2.1.12. Statistical Analysis 29
• 2.3. Results 29
• 2.3.1. CT Modulates GLP-1R Signaling and Early Adipogenic Pathway via β-Arrestins and G Protein Subunits 29
• 2.3.2 CT Suppresses Adipocyte Differentiation via cAMP-Mediated Activation of p38-MAPK and AMPK in 3T3-L1 Preadipocytes 37
• 2.3.3 GLP-1R is Essential for CT-Mediated Inhibition of Adipogenesis 39
• 2.4. Discussion 45
• Chapter Three 53
• Summary 54
• Appendix 55
• References 61
• Research achievements 74
• Publications 74
• Korean Abstract 75
• Acknowledgement 77
• Dedication 78