Title Page
국문요약
Contents
1. Introduction 12
1.1. Insulin signaling pathway and insulin resistance in obesity. 12
1.2. Dysregulation of Calcium homeostasis in metabolic diseases 14
1.3. NEDD4-2 E3 ligase and ubiquitin-proteasome degradation system 17
2. Materials and Methods 19
2.1. Materials 19
2.2. Animals 20
2.3. Cell Culture and Transfection 20
2.4. Western blotting 21
2.5. RNA isolation 23
2.6. Synthesis of cDNA 24
2.7. Quantitative real-time PCR 24
2.8. Cloning of the PMCA2, NEDD4-2 and AKT. 26
2.9. Enzyme and Reagents 26
2.10. Immunoprecipitation 28
2.11. Fluorescence microscopy of intracellular calcium flux and translocation of NEDD4-2 and NEDD4-2 C2 domain or AKT PH domain in CHO/IR/IRS1 and HepG2 cells 29
2.12. Statistical analyses 29
3. Results 30
3.1. Obesity and hyperlipidemia elevated lipid accumulation and intracellular Ca2+ levels, which impair insulin signaling.[이미지참조] 30
3.2. Dysregulation of intracellular Ca2+ homeostasis in obesity and hyperlipidemia.[이미지참조] 38
3.3. Obesity and hyperlipidemia impaired insulin signaling and decreased the phosphorylation of NEDD4-2 at Ser342 and Ser448 45
3.4. PMCA2 is a direct target of NEDD4-2 for proteasome-dependent degradation 55
3.5. Higher intracellular Ca2+ levels stimulate membrane localization of the C2 domain of NEDD4-2 and cause ubiquitin-dependent proteasomal degradation of PMCA2 in obesity or hyperlipidemia.[이미지참조] 63
4. Conclusion 71
5. Refereces 77
Abstract 83
Table 1. List of primers used for qRT-PCR. 25
Table 2. List of primers used for cloning. 27
Figure 1. Insulin signaling pathway in the liver. 13
Figure 2. Regulation of intracellular Ca2+ homeostasis by calcium channels, transporters, and pumps.[이미지참조] 16
Figure 3. Immunoblot analyses of the phosphrylation states of AKT, FOXO3A, and AS160 in the liver lysates from mice fed a normal chow diet (NCD), high-fat dietary (HFD) and genetic mouse models of obesity... 33
Figure 4. Immunoblot analyses of the phosphorylation states of AKT, FOXO3A, and AS160 in PA-treated HepG2 cells. 35
Figure 5. The effects of PA treatment on intracellular Ca2+ and lipid accumulation in HepG2 cells.[이미지참조] 37
Figure 6. Immunoblot analyses of various calcium channels in liver lysates of obese mice models. 41
Figure 7. mRNA expression level of various calcium in PA-treated HepG2 cells. 42
Figure 8. Immunoblot analyses of various calcium channels in PA-treated HepG2 cells. 44
Figure 9. Protein degradation by the Ubiquitin-dependent proteasome system. 48
Figure 10. RNA-sequencing analysis of NEDD4 family in obese mouse models. 49
Figure 11. Immunoblot analyses of various E3 ligases in liver lysates of obese mice models. 51
Figure 12. Immunoblot analyses of various E3 ligases in PA-treated HepG2 cells. 53
Figure 13. Immunoprecipitation analyses 54
Figure 14. The effects of NEDD4-2 overexpression on various calcium channels and pumps in PA-treated HepG2 cells. 58
Figure 15. The effects of MG132 on various calcium channels and pumps in NEDD4-2 overexpression in PA-treated HepG2 cells. 60
Figure 16. Physical interaction between NEDD4-2 and PMCA2. 62
Figure 17. Higher intracellular Ca2+ levels stimulate membrane localization of NEDD4-2.[이미지참조] 66
Figure 18. Higher intracellular Ca2+ levels stimulate membrane localization of the mCherry C2 domain NEDD4-2.[이미지참조] 68
Figure 19. Proposed model of obesity-associated NEDD4-2 activation for aggravating intracellular Ca2+ homeostasis by proteasomal degradation of plasma membrane Ca2+ ATPase.[이미지참조] 70