Title Page
ABSTRACT
Contents
INTRODUCTION 12
MATERIALS AND METHODS 24
1. Constructs 24
2. Animals 24
3. Cell culture studies 25
4. Generation of CRISPR/Cas9-mediated knockout cells 26
5. Tet-on-inducible stable cell construct 26
6. Transfection and conditioned media 26
7. Cell Isolation and glucose production 27
8. RNA interference 27
9. RNA extraction and quantitative PCR 28
10. Ubiquitination assays 28
11. Western blots and immunoprecipitation 29
12. Dual luciferase reporter assay 30
13. Metabolic analyses 30
14. Hematoxylin-eosin staining (H&E) and immunohistochemistry (IHC) 30
15. Alanine aminotransferase assay (ALT) 31
16. Enzyme-linked immunosorbent assay (ELISA) 31
17. Immunofluorescence (IF) 32
18. Sirius red staining 32
19. Human transcriptomic analysis 33
20. Antisense oligonucleotide (ASO) 34
21. Statistical analysis 34
LIST OF ABBREVIATION 35
CHAPTER 1. Hepatocyte Kctd17 inhibition ameliorates glucose intolerance and hepatic steatosis caused by obesity-induced Chrebp stabilization 37
RESULTS 38
1.1. Hepatocyte-specific knockout of Kctd17 ameliorates HFD-induced liver steatosis 38
1.2. Hepatocyte-specific knockout of Kctd17 ameliorates HFD-induced glucose homeostasis 42
1.3. Hepatocyte-specific knockdown of Kctd17 ameliorates HFD-induced liver steatosis and glucose homeostasis 45
1.4. Forced hepatocyte Kctd17 expression provokes liver steatosis 48
1.5. Forced hepatocyte Kctd17 expression provokes glucose homeostasis 51
1.6. Kctd17 provokes Chrebp expression 54
1.7. Oga-Chrebp pathway mediates the lipogenic action of Kctd17 57
1.8. Oga deletion negates Kctd17 effects on glucose and lipid metabolism 60
1.9. Kctd17 expression is dependent on hepatocyte Srebp1c activity 63
1.10. Liver KCTD17 expression tracks with ChREBP activity in patients with NAFLD 67
1.11. Kctd17 ASO protects from diet-induced glucose intolerance 70
1.12. Kctd17 ASO protects from diet-induced liver steatosis 72
CHAPTER 2. Hepatocyte Kctd17 promotes liver fibrosis by inhibiting the secretion of hepatocyte Serpina3k in nonalcoholic steatohepatitis (NASH) 75
RESULTS 76
2.1. KCTD17 is increased in the liver of NASH patients 76
2.2. Hepatocyte-specific Kctd17 depletion alleviates FPC diet-induced liver fibrosis 78
2.3. Hepatocyte-specific Kctd17 depletion alleviates CDAHFD-induced liver fibrosis 82
2.4. Serpina3 isoform expression and identification by RNA-Seq analysis in NASH-induced mice 85
2.5. Serpina3k is decreased in the liver from NASH-provoked mice 87
2.6. Kctd17 mediates Sepina3k expression and secretion in NASH-induced liver 90
2.7. Serpina3k expression in CDAHFD-fed mice for 4 weeks 92
2.8. Hepatocyte-specific Serpina3k overexpression restores CDAHFD-induced liver fibrosis 94
2.9. Kctd17 prevents Zbtb7b-mediated Serpina3k expression 97
2.10. Serpina3k/SERPINA3 attenuates hepatic stellate cell activity by reducing TGFβ1 and Par2 signaling pathways 101
2.11. Liver-selective Kctd17 inhibition protects from FPC-induced liver fibrosis 105
2.12. Liver-selective Kctd17 inhibition protects from CDAHFD-induced liver fibrosis 108
2.13. Schematic diagram of the role of Kctd17 in regulation of liver fibrosis through inhibition of hepatic secreted Serpina3k in NASH 111
DISCUSSION 113
CHAPTER 1 113
CHAPTER 2 116
REFERENCE 121
Table 1. Information of primer for qPCR 20
Table 2. Information of antibody for Western blots, immunoprecipitation and immunohistochemistry 21
Table 3. Information of sequences for chromatin immunoprecipitation 22
Table 4. Information of single guide RNA sequence for Kctd17 and Oga knockout in vivo 23
Table 5. Information of ASO sequence for Kctd17 knockdown in vivo 23
CHAPTER 1 17
Figure 1. Hepatocyte-specific KO of Kctd17 ameliorates HFD-induced liver steatosis 41
Figure 2. Hepatocyte-specific KO of Kctd17 ameliorates HFD-induced glucose homeostasis 44
Figure 3. Hepatocyte-specific knockdown of Kctd17 ameliorates HFD-induced liver steatosis and glucose homeostasis 47
Figure 4. Forced hepatocyte Kctd17 expression provokes liver steatosis 50
Figure 5. Forced hepatocyte Kctd17 expression provokes glucose homeostasis 53
Figure 6. Kctd17 regulates Chrebp protein stability 56
Figure 7. Kctd17 prevents Oga-mediated Chrebp degradation 59
Figure 8. Oga deletion negates Kctd17 effects on glucose and lipid metabolism 62
Figure 9. Kctd17 is activated by hepatocyte Srebp1c 66
Figure 10. KCTD17 expression tracks with hepatic ChREBP activity and NAFLD severity in patients 69
Figure 11. Kctd17 ASO protects from diet-induced glucose intolerance 71
Figure 12. Kctd17 ASO protects from diet-induced liver steatosis 74
CHAPTER 2 18
Figure 13. Kctd17 is increased in the liver of NASH patients 77
Figure 14. Hepatocyte-specific Kctd17 depletion alleviates FPC diet-induced NASH liver fibrosis 81
Figure 15. Hepatocyte-specific Kctd17 depletion alleviates CDAHFD-induced NASH liver fibrosis 84
Figure 16. Sepina3 isoform expression and identification by RNA-Seq analysis in NASH-induced mice 86
Figure 17. Sepina3k is decreased in the liver from NASH-provoked mice 89
Figure 18. Kctd17 mediates Sepina3k expression and secretion in vitro 91
Figure 19. Serpina3k expression in CDAHFD-fed mice for 4 weeks 93
Figure 20. Hepatocyte-specific serpina3k overexpression restores CDAHFD-induced NASH liver fibrosis 96
Figure 21. Kctd17 prevents Zbtb7b mediated Serpina3k expression 100
Figure 22. Serpina3k/SERPINA3 attenuates hepatic stellate cell activity by reducing Par2-mediated TGFβ1 signaling pathways 104
Figure 23. Liver-selective Kctd17 inhibition protects from FPC-induced liver fibrosis 107
Figure 24. Liver-selective Kctd17 inhibition protects from CDAHFD-induced liver fibrosis 110
Figure 25. Schematic diagram of the role of Kctd17 in regulation of liver fibrosis through inhibition of hepatic secreted Serpina3k in NASH 112