제주대학교 Repository

Anti-diabetic and An ti-obesity effects of Sulforaphane in Broccoli leaf extract

Metadata Downloads
Abstract
The present study evaluated the anti-diabetic activity of SFN enriched broccoli leafextract (BLE) on HepG2 cells and ob/ob mice. The treatment of SFN and BLEsignificantly improved glucose uptake in high glucose treated HepG2 cells. Westernblot analysis revealed that SFN and BLE increased phosphorylation levels of bothAkt and GSK3β in high glucose treated HepG2 cells and ob/ob mice liver. SFN andBLE supplemented ob/ob mice showed significantly reduced (P < 0.05) seruminsulin, blood glucose, and HOMA-IR values. Both insulin tolerance and glucosetolerance were significantly improved (P < 0.05) by SFN and BLE in ob/ob mice.Based on differential gene expression analysis, SFN and BLE normalized upregulated11 genes and down-regulated 11 genes related to insulin signaling andglucose metabolism in ob/ob mice liver. The protein-protein interaction network wasevaluated through STRING analysis and revealed functional interaction between thenormalized genes including Atf4, Atf3, Myc, PGC-1α, Phkg1, Phka1, Pygm, andGys1 by SFN and BLE. Our findings suggest that SFN in BLE exerts a potent antidiabeticeffect by normalizing the expression of genes related to insulin signaling andglucose metabolism which are up- or down-regulated in ob/ob mice.
The present study evaluated the anti-obesity effect of sulforaphane (SFN) andglucoraphanin (GRN)-enriched broccoli leaf extract (BLE) on 3T3-L1 adipocyte andob/ob mice. Based on Oil Red O staining and triglyceride (TG) assay, SFN and BLEsignificantly reduced (P < 0.05) both lipid accumulation and TG content in thedifferentiated 3T3-L1 adipocytes. SFN and BLE increased 2-NBDG uptake by 3T3-L1 adipocytes in a dose-dependent manner. Western blot analysis confirmed thatSFN and BLE increased the phosphorylation levels of both AMPK (Thr172) andACC (Ser79) in vitro and in vivo. Both SFN and BLE significantly reduced theexpression of HMGCR in liver and white adipose tissues of ob/ob mice. Histologicalanalysis revealed that SFN and BLE ameliorated hepatic steatosis in ob/ob mice.Treatment with SFN and BLE significantly reduced (P < 0.05) the serum levels ofTG, low-density lipoprotein cholesterol (LDL), total cholesterol (TC), and glucose inob/ob mice. RNA sequencing analysis showed that up- or down-regulation of 32genes related to lipid metabolism was restored to control level in both SFN-treatedob/ob mice group and BLE-treated group. A protein-protein interaction (PPI)network was constructed via STRING analysis, and Srebf2, Pla2g2c, Elovl5, Elovl7,Plb1, Ctp1a, Lipin, Fgfr1, Plcg1, and Plcb4 were located in the functional hubs ofthe PPI network of lipid metabolism. Overall results suggest that the SFN content inBLE exerts a potential anti-obesity effect by normalizing the expression of genesrelated to lipid metabolism, which are up- or down-regulated in ob/ob mice.
Author(s)
Sachithra S. Ranaweera
Issued Date
2021
Awarded Date
2021. 2
Type
Dissertation
URI
https://oak.jejunu.ac.kr/handle/2020.oak/23473
Alternative Author(s)
라나위라 아라크질라그 사치트라 세완디
Affiliation
제주대학교 대학원
Department
대학원 수의학과
Advisor
한창훈
Table Of Contents
List of Tables . vii
List of Figures . viii
List of Abbreviations . x
General Introduction 12
PART-I. 20
Anti-diabetic effect of Sulforaphane in Broccoli leaf extract on HepG2 cells and ob/ob mice 20
1.1. Abstract . 21
1.2. Introduction . 22
1.3. Materials and Method . 24
1.3.1. Materials and chemicals. 24
1.3.2. Preparation of BLE 24
1.3.3. Broccoli extract UPLC standardization . 25
1.3.4. HepG2 cell culture and treatment 25
1.3.5. Cell viability assay . 26
1.3.6. 2-NBDG glucose uptake assay in insulin resistance HepG2 cells. 26
1.3.7. Animals 27
1.3.8. Sample treatment . 28
1.3.9. Insulin tolerance test (ITT) 29
1.3.10. Glucose tolerance test (GTT) . 29
1.3.11. Blood glucose, serum insulin, and HOMA-IR 29
1.3.12. Western blot . 30
1.3.13. RNA isolation, library preparation, sequencing, and data analysis . 30
1.3.14. Statistical analysis 32
1.4. Results . 33
1.4.1. UPLC analysis of BLE 33
1.4.2. Cytotoxicity of BLE on HepG2 cells . 34
1.4.3. Glucose uptake in high glucose treated HepG2 cells 34
1.4.4. The phosphorylation of Akt and GSK3β in high glucose treated HepG 2cells . 36
1.4.5. Insulin sensitivity in ob/ob mice 38
1.4.6. Glucose tolerance in ob/ob mice 40
1.4.7. Serum parameters in ob/ob mice . 42
1.4.8. The phosphorylation levels of Akt and GSK3β in ob/ob mice liver . 44
1.4.9. Differential gene expression in ob/ob mice liver . 46
1.5. Discussion . 50
PART-II . 56
Anti-obesity effect of Sulforaphane in Broccoli leaf extract on 3T3-L1 adipocyte and
ob/ob mice 56
2.1. Abstract . 57
2.2. Introduction . 58
2.3. Materials and methods 61
2.3.1. Material and Chemicals . 61
2.3.2. Preparation of broccoli leaf extract 61
2.3.3. Broccoli extract UPLC standardization. 62
2.3.4. Cell culture and differentiation 62
2.3.5. Cytotoxicity assay 63
2.3.6. Oil Red O staining . 64
2.3.7. Measurement of triglyceride (TG) content in 3T3-L1 adipocytes . 64
2.3.8. 2-NBDG uptake assay . 65
2.3.9. Animals 65
2.3.10. Sample treatment . 66
2.3.11. Blood glucose 66
2.3.12. Serum parameters in ob/ob mice . 67
2.3.13. Tissue processing and hematoxylin & eosin (H&E) staining 67
2.3.14. Western blot analysis . 68
2.3.15. RNA sequencing analysis 68
2.3.16. Statistical analysis 69
2.4. Results . 70
2.4.1. UPLC analysis of BLE 70
2.4.2. Cytotoxicity of BLE in 3T3-L1 cells . 70
2.4.3. Lipid accumulation and TG content in 3T3-L1 adipocytes . 71
2.4.4. Glucose uptake in 3T3-L1 adipocytes . 73
2.4.5. Phosphorylation of AMPK and ACC in 3T3-L1 adipocytes . 75
2.4.6. Hepatic lipid accumulation in ob/ob mouse liver 77
2.4.7. AMPK signaling pathway in white adipose tissue of ob/ob mice . 80
2.4.8. Serum parameters of ob/ob mice . 82
2.4.9. Blood glucose concentrations of ob/ob mice . 84
2.4.10. Hepatic gene expression analysis 85
2.5. Discussion . 89
PART-III 95
Anti-inflammatory effects of Sulforaphane in Broccoli leaf extract on LPSstimulated RAW 264.7 cells and ob/ob mice 95
3.1. Abstract . 96
3.2. Introduction . 97
3.3. Materials and Methods 100
3.3.1. Materials 100
3.3.2. Preparation of broccoli leaf extract 100
3.3.3. DPPH radical scavenging activity . 101
3.3.4. RAW 264.7 cell culture and treatment 101
3.3.5. Cell viability assay . 102
3.3.6. Measurement of NO production 102
3.3.7. Enzyme-linked immunosorbent assay (ELISA) 103
3.3.8. Measurement of COX-2 and iNOS protein expression . 103
3.3.9. Animals experiments . 104
3.3.10. Sample treatment . 104
3.3.11. Total RNA isolation, library preparation, sequencing, and data analysis . 105
3.3.12. Statistical analysis 106
3.4. Results . 107
3.4.1. DPPH radical scavenging activity . 107
3.4.2. Effects of BLE on cell viability in RAW 264.7 cells 108
3.4.3. Inhibition of LPS-stimulated NO production in RAW 264.7 cells 109
3.4.4. Inhibition of LPS-stimulated COX-2 and iNOS production in RAW 264.7 cells . 111
3.4.5. Inhibitory effect of SFN and BLE on TNF-α, IL-6, and IL-1β
production . 112
3.4.6. Differential gene expression analysis of ob/ob mice liver . 115
3.5. Discussion . 120
General Conclusion 125
References 128
Acknowledgement . 149
Degree
Doctor
Publisher
제주대학교 대학원
Citation
Sachithra S. Ranaweera. (2021). Anti-diabetic and An ti-obesity effects of Sulforaphane in Broccoli leaf extract
Appears in Collections:
General Graduate School > Veterinary Medicine
공개 및 라이선스
  • 공개 구분공개
파일 목록

Items in Repository are protected by copyright, with all rights reserved, unless otherwise indicated.