Anti-Obesity Mechanisms of Ethanolic Extract and Two Meroterpenoids from Sargassum serratifolium Using In Vitro and In Vivo Model
- Abstract
- Sargassum serratifolium (C. Agardh) C. Agardh, a brown alga, has diverse health-promoting effects, including anti-inflammatory, anti-oxidant, and anti-diabetic activities with high levels of meroterpenoids. Thus, meroterpinoid-rich fraction of the ethanolic extract from Sargassum serratifolium (MES) was prepared with improved preparation methods and active components, sargaquinoic acid (SQA) and sargahydroquinoic acid (SHQA) were isolated from MES. This study aims to investigate the inhibitory effects of MES on adipogenesis and the anti-obesity mechanisms in HFD-induced obese mice. Additionally, SQA and SHQA from S. serratifolium were investigated to understand molecular mechanism of browning using differentiated 3T3-L1 cells.
3T3-L1 cells were treated with 3-isobutyl-1-methyxanthine, dexametha-sone, insulin (MDI) and MES (5 and 10 μg/mL) to investigate the inhibition of adipogenesis and lipid accumulation in 3T3-L1 preadipocytes. MES suppressed the accumulation of lipid droplets and triacylglycerol (TG), while enhancing the lipolysis of intracellular TG. Results showed that MES induced cell cycle arrest at the G1/S phase via suppressing proteins expression of cyclin-dependent kinases (CDK) 2, CDK4, CDK6, and cyclin E. Moreover, MES reduced the expression of CCAAT/enhancer-binding protein (C/EBP) β during the early stage of adipogenesis, led to suppressed expression of both mRNA and protein of C/EBPα and peroxisome proliferator–activated receptor γ (PPARγ). As a result, MES reduced mRNA and protein level of lipogenic transcription factors such as retinoid X receptor α (RXRα), and sterol regulatory element–binding protein 1c (SREBP1c) and their target genes, such as fatty acid synthase (FAS) and stearoyl-CoA desaturase 1 (SCD-1). Overall, MES inhibited adipocyte differentiation in early stage and lipid accumulation during adipogenesis through down-regulation of adipogenic and lipogenic transcription factors, providing the anti-obesity mechanism of MES.
The effects of MES on obesity and obesity-related hepatic steatosis were investigated using HFD-fed C57BL/6J mice. MES attenuated body weight gain, visceral adiposity, lipid accumulation in liver, serum TG, and inflammation in C57BL/6J mice fed a HFD (60% kcal from fat) for 8 weeks, whereas it increased plasma adiponectin level and HDL-cholesterol without changes in food intake. The consumption of MES suppressed nonalcoholic fatty liver disease (NAFLD) through phosphorylation of AMP-activated protein kinase (AMPK), which elevated expression of hepatic fatty acid oxidation factors [perosixome proliferator-activated receptor (PPARα) acyl-CoA synthetase long-chain family member 1 (ASCL1), and carnitine palmitoyltrasferase 1 (CPT1)]. Additionally, MES attenuated the expression of SREBP2, HMG-CoA reductase (HMGCR), SREBP1c, FAS, and SCD-1 in liver tissue of HFD-induced mice. Notably, when compared to HF-fed mice, the MES alleviated HFD-induced inflammation and suppressed lipogenesis in white adipocyte through suppressing fatty acid synthesis and increasing lipolysis. Furthermore, MES notably increased uncoupling protein 1 (UCP-1)-positive cells and decreased lipid droplet in subcutaneous tissues. This result indicates that MES exerted the anti-obesity and lipid-lowering effects by activating AMPK-related fatty acid oxidation signaling and inhibiting SREBP1c-related lipogenesis signaling in liver and adipose tissues.
Brown and beige/brite adipocytes were known as discouraging obesity and hyperlipidemia by escalating energy expenditure through thermogenesis and fatty acid oxidation. To confirm anti-obesity effects of MES, SQA, and SHQA, major components of MES were investigated for the differentiation of preadipocytes to beige/brite adipocyte phenotype. SQA and SHQA induced the differentiation of white adipotye to beige/brite adipocytes through increasing multilocular lipid droplets and mitochondrial dense, brown adipocyte markers: PR domain-containing 16 (PRDM16), peroxisome proliferator-activated receptor gamma coactivator 1α (PGC1α), and UCP-1. Moreover, SQA and SHQA increased expression levels of hormone-sensitive lipase (HSL) and perilipin (PLIN) which play key roles in lipolysis. Notably, increased fatty acid oxidation by SQA and SHQA was determined by high expression levels of CPT1 and ASCL1. SQA and SHQA directly activated AMPK and led to induce expression of brown adipocyte markers including PGC1α, PRDM16, and UCP-1. Additionally, SQA and SHQA treatment alleviated the inhibition of AMPK phosphorylation and the expression of PGC1α, PRDM16, and UCP-1 by AMPK inhibitor (dorsomorphin), suggesting both components mediated the conversion of WAT to beige adipocytes by AMPK pathway. These findings indicate that SQA and SHQA play a role in the induction of preadipocytes into beige/brite adipocyte phenotype, suggesting a potential therapeutic agent to treat or prevent obesity. Taken together, these findings indicate that MES, SQA, and SHQA may be used as a dietary supplement to treat obesity and related metabolic syndrome.
- Issued Date
- 2018
- Awarded Date
- 2018. 8
- Type
- Dissertation
- Keyword
- Sargassum serratifolium Adipogenesis Lipogenesis Cell cycle Meroterpinoids MES high-fat diet-induced obese mice AMPKα nonalcoholic fatty liver disease browning
- Publisher
- 부경대학교
- Affiliation
- 부경대학교 대학원
- Department
- 대학원 식품생명과학과
- Advisor
- 김형락
- Table Of Contents
- Contents i
List of Tables v
List of Figures vi
Abbreviations viii
Abstract x
Chapter I. Introduction 1
1. Bioactive compounds from Sargassum 2
2. Obesity 4
3. Non-alcoholic fatty liver disease (NAFLD) 6
4. Browning 8
4.1. Adipocytes 8
4.2. Mitochondrial uncoupling protein-1 (UCP-1) 12
Chapter II. Meroterpenoid-rich fraction of an ethanolic extract from Sargassum serratifolium inhibits adipogenesis in 3T3-L1 preadipocytes by cell cycle arrest 13
1. Introduction 14
2. Materials and Methods 16
2.1. Materials 16
2.2. Preparation of ethanolic extract and isolation of chemical components 16
2.3. Cell culture and treatment 17
2.4. Oil red O staining 17
2.5. Measurement of intracellular triacylglycerol (TG) 18
2.6. Measurement of free glycerol in culture media 18
2.7. Analysis of cell cycle progression 18
2.8. Western blot analysis 19
2.9. RNA isolation and real-time PCR 19
2.10. Statistical analysis 20
3. Results 23
3.1. MES suppresses TG accumulation during adipogenesis 23
3.2. MES blocks cell cycle progression during adipocyte differentiation 23
3.3. MES inhibits the production of adipogenic transcription factors 27
3.4. MES inhibits the production of lipogenic transcription factors 31
3.5. MES suppresses enzyme related to lipid accumulation 31
3.6. Identification of anti-adipogenic compounds in MES 35
4. Discussion 40
Chapter III. Meroterpenoid-rich fraction of an ethanolic extract from Sargassum serratifolium alleviates obesity and non-alcoholic fatty liver disease in high fat-fed C57BL/6J mice 44
1. Introduction 45
2. Materials and Methods 47
2.1. Materials and Reagents 47
2.2. Preparation of MES and analysis of chemical components 47
2.3. Animals 48
2.4. Measurement of plasma parameters 49
2.5. Western blot analysis 49
2.6. Hematoxylin and eosin (H&E) and Oil red O staining 51
2.7. TG assay of liver lipid 51
2.8. Cholesterol assay of liver lipid 51
2.9. Statistical analysis 52
3. Results 54
3.1. MES suppressed body, liver, and epididymal tissue weight in HFD-fed mice 54
3.2. MES ameliorated glucose, TG, free fatty acid, and cholesterol levels 58
3.3. MES reduced spleen, liver tissue weight, AST, and AST levels in HFD-fed mice 58
3.4. MES inhibited lipid accumulation in the liver via inhibition of lipogenesis and activation of lipolysis 64
3.5. MES ameliorated hepatic steatosis partially by activating AMPK and deactivating SREBP1c signaling 64
3.6. MES prevented lipid accumulation and inflammation in epididymal tissue 73
3.7. MES suppressed lipid accumulation in epididymal tissue via inhibition of lipogenesis and activation of lipolysis 77
3.8. MES reduced lipid accumulation in brown adipose tissues and regulated subcutaneous tissues 80
4. Discussion 86
Chapter IV. Effects of sargaquinoic acid and sargahydroquinoic acid on inducing brown fat-like phenotype in 3T3-L1 adipocytes 90
1. Introduction 91
2. Materials and Methods 94
2.1. Reagents 94
2.2. Isolation of SQA and SHQA 95
2.3. Cell culture and treatment 96
2.4. Immunofluorescence analysis 96
2.5. Oil red O staining 97
2.6. Determination of TG concentration 97
2.7. Determination of free glycerol concentration 97
2.8. Intracellular reactive oxygen species (ROS) measurement 97
2.9. Determination of ATP concentration 97
2.10. Western blot analysis 98
2.11. Mitochondrial DNA (MtDNA) isolation and real-time PCR 98
2.12. RNA isolation and real-time PCR 99
2.13. Molecular docking 99
2.14. Statistical analysis 100
3. Results 102
3.1. SQA and SHQA inhibit TG and lipid accumulation during browning 102
3.2. SQA and SHQA induce the expression of brown-like phenotype factors 107
3.3. SQA and SHQA regulated lipid metabolism 116
3.4. SQA and SHQA regulate browning through p-AMPK pathway 122
3.5. SQA and SHQA suppress the accumulation of TG and lipid 126
3.6. SQA and SHQA induced beige adipocyte markers through controlling the preadipocytes differentiation 129
3.7. Analysis of binding mode of activators for PPARγ, PPARα, PPARγ/α, and AMPK 135
4. Discussion 150
V. References 155
- Degree
- Doctor
- Files in This Item:
-
-
Download
Anti-Obesity Mechanisms of Ethanolic Extract and Two Meroterpenoids from Sargassum serratifolium Usi.pdf
기타 데이터 / 7.01 MB / Adobe PDF
-
Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.