臺灣博碩士論文加值系統

Naa10p (N-α-acetyltransferase 10 protein) 作為主要的乙醯基轉移酶，可於超過50%的哺乳類蛋白質加上乙醯基。此外，Naa10p也是個具有DNA結合能力的蛋白質，能夠促進甲基轉移酶DNA methyltransferase 1 (DNMT1)與DNA結合，調控基因甲基化。已知Naa10p在癌症形成扮演重要角色，Naa10p突變則造成嚴重發育遲緩及與X染色體關聯人類疾病。我們實驗室研究更指出，Naa10p剔除小鼠較正常小鼠瘦小，平均體重減少百分之二十。本篇論文，主要研究Naa10p在脂肪生成與代謝上所扮演的角色。本篇研究發現，長期餵食高脂飼料的Naa10基因剔除鼠比正常小鼠能抵抗飲食引起的肥胖與胰島素抗性。長期餵食高脂飼料的Naa10基因剔除鼠的皮下脂肪較少，並伴隨著較強的生理代謝率以及脂肪棕化現象。此外，缺少Naa10p的白脂肪前驅細胞3T3-L1在脂肪生成的能力上有所缺損。這個現象可以透過重新表現正常Naa10p或是給與過氧化體增生活化接受器(Pparγ)之促進劑troglitazone而恢復。透過表現不同Naa10p突變蛋白，我們也證實此現象主要是受到Naa10p乙醯基轉移酶活性的調控。除此之外，我們也提出Naa10p能夠直接與Pparγ交互作用的證據。Pparγ在脂肪生成中扮演著核心的調控角色，同時也在白色脂肪組織中生成似棕色脂肪細胞的過程中扮演決定性的角色，且此現象受到Pparγ上離胺酸乙醯化的調控。因此，Naa10p或許是透過改變Pparγ的乙醯化來抑制脂肪細胞的棕化現象並促進前驅細胞分化為白色脂肪的品系。根據我們的研究，我們提出Naa10p在脂肪生成與代謝上所扮演的新角色，並提出一個具有潛力針對飲食引起的肥胖的治療策略。

Naa10p (N-α-acetyltransferase 10 protein) is the major N-α-acetyltransferase that acetylates more than 50% of proteins in mammalian cells. Naa10p is also a DNA binding protein which mediates gene-specific DNA methylation by recruiting DNMT1 (DNA methyltransferase 1). Studies have indicated that Naa10p plays an important role in cancer formation and Naa10p mutation causes severe developmental delay in human. Our laboratory also found that Naa10p knockout mice showed 20% reduction in body weight and size. This thesis aims to understand the role of Naa10p in adipogenesis and metabolism. We found that Naa10 knockout (Naa10-KO) mice were protected from high fat diet (HFD)-induced obesity and insulin resistance. HFD-fed Naa10-KO mice had less subcutaneous fat which is also accompanied with increased energy expenditure and browning of preadipocyte. In addition, white preadipocyte 3T3-L1 with Naa10p depletion led to impaired adipogenesis which could be rescued by adding back WT Naa10p or treating with Pparγ agonist troglitazone. We also demonstrated that this phenotype is acetyltransferase activity-dependent as adding back enzyme-dead Naa10 mutant protein could not rescue the phenotype. Furthermore, we provided evidence that Naa10p directly interacts with Pparγ. Pparγ plays a central role in adipogenesis and determination of white-to-brown plasticity in white adipose tissue which is regulated by lysine acetylation. Thus, Naa10p might inhibit browning and promote white lineage differentiation through mediating acetylation of Pparγ. These results reveal a novel role of Naa10p in adipogenesis and metabolism which provide a potential new therapeutic strategy against diet-induce obesity.

Abstract………………………………………………………………………………..1
中文摘要……………………………………………………………………………...2
Introduction……………………………………………………………………………3
Obesity is one of the prevalent diseases in the world.…………………………….3
Adipogenesis is separated into two phases and regulated by a transcriptional
cascade…………………………………………………………………………….3
Brown adipocyte can reduce obesity through the dissipation of energy as heat....…5
PPARγ and PGC1α determine adipocyte fate to white or brown by acetylation…...6
PPARγ agonist TZDs promote browning by increasing the stability of PRDM16
and enhancing the interaction of Prdm16 with SIRT1-dependent deacetylation of
PPARγ……………………………………………………………………………..7
N-α-acetyltransferase 10 protein acetylates more than 80% of proteins in human…8
Naa10p has DNA-binding ability………………………………………...…………9
Human Naa10p has an important role in cancer formation…………………...10
Human Naa10p deficiency results in developmental delays and abnormally low
subcutaneous fat………………………………………………………………….10
Naa10-KO mice have lower body weight and resist to diet-induced obesity…..…11
Study Purposes…………………………………………………...…………………..13
Materials and Methods……………………………………………...………………..14
Animals………………………………………………………...………………….14
Cell culture and differentiation………………………..…………………………...14
Plasmids and antibodies…………………………………………………………16
Lentiviral transductions………………………………………………………….17
RNA purification, reverse transcription, and quantitative PCR……….…………..17
Histology and blood biochemistry analysis……………………………………..18
Cell proliferation rate……………………………………………………………18
Immunofluorescence………………………………………………………………19
Glucose and insulin tolerance test…………………………………………………19
Body composition and indirect calorimetric analysis……………………………19
Cold exposure and body temperature monitoring………………………………....20
Affymetrix microarray hybridization and data analysis……………………..20
Primers…………………………………………………………………………….21
Statistical analysis……………………………………………………………….21
Result…………………………………………………………………………………22
Naa10p knockout protects mice from high-fat diet induced obesity due to
subcutaneous adipocyte hypotrophy…………………………………………….22
Naa10p knockout increases energy expenditure…………………………………24
Naa10p knockout alters glucose homeostasis and protects mice from insulin
resistance…………………………………………………………………………24
Naa10p knockout promotes browning in subcutaneous fat………………………..25
Naa10p knockout promotes skeletal muscle-like signature in inguinal white
adipose tissue…………………………………………………………………..…26
Adipogenesis is impaired in Naa10-KO MEFs and 3T3-L1………………………27
Impaired adipogenesis in Naa10-KO 3T3-L1 could be fully rescued by Pparγ
agonist……………………………………………………………………………28
Adipogenesis is Naa10p acetyltransferase activity-dependent…………………....29
Discussion………………………………………………………………………….31
Naa10p knockout protects mice from diet-induced obesity and insulin resistance.31
Naa10p regulates adipogenesis by acetylation-dependent manner………………..32
Naa10p depletion promotes browning in subcutaneous fat………………………..33
Pparγ is a potential acetylation target of Naa10p………………………………….34
References………………………………………………………………………….35
Figure 1. Naa10p knockout protects mice from high fat diet-induced obesity………48
Figure 2. Naa10p knockout protects mice from high-fat diet-induced liver steatosis.50
Figure 3. HFD fed Naa10-KO mice show less lipid accumulation in skeletal muscle51
Figure 4. HFD fed Naa10-KO mice show less lipid accumulation in brown adipose tissue………………………………………………………………………………..52
Figure 5. Naa10-KO mice with HFD-treatment show no significant difference of eWAT…………………………………………………………………….…………53
Figure 6. Naa10-KO mice with HFD-treatment have smaller inguinal white
adipocytes…………………………………………………………………………….54
Figure 7. Subcutaneous fat on Naa10-KO mice show adipocyte hypotrophy……….55
Figure 8. High fat diet fed Naa10-KO mice show increased energy expenditure…56
Figure 9. High fat diet fed Naa10-KO mice maintained higher core body temperature after cold exposure………………………………………………………………….57
Figure 10. Naa10p knockout alters glucose homeostasis and ameliorated insulin resistance on HFD treatment……………………………………………………….58
Figure 11. Naa10p knockout alters plasma lipid profile on HFD treatment……….59
Figure 12. Naa10p knockout promotes white adipocyte browning in subcutaneous fat……………………………………………………………………………………..60
Figure 13. Naa10p knockout does not change Pgc1α protein level in skeletal muscle………………………………………………………………………………...61
Figure 14. Naa10p knockout promotes skeletal muscle-like signature in inguinal white adipose tissue……………………………………………………………………...….62
Figure 15. Naa10-KO 3T3-L1 have various proliferation rate…………………….64
Figure 16. Naa10-KO/KD 3T3-L1 show impaired terminal differentiation……….65
Figure 17. Impaired terminal differentiation in Naa10-KO/KD 3T3-L1 can be rescued by troglitazone treatment……………………………………………………….…….66
Figure 18. Impaired terminal differentiation in Naa10-KO/KD 3T3-L1 can be rescued by troglitazone treatment…………………………………………………………..…67
Figure 19. WT and Naa10-KO 3T3-L1 show similar Pparγ and Cebpα gene expression pattern…………………………………………………………………..68
Figure 20. Naa10p knockout lead to higher Pparγ expression in differentiated cells, but decreased number of Pparγ-positive cells………………………………………..69
Figure 21. Naa10-KO 3T3-L1 show down-regulation in white genes and up-regulation in part of brown genes……………………………………………….70
Figure 22. Impaired adipogenesis can be rescued by overexpressing both Naa10p variant 1 and 2………………………………………………………………………..71
Figure 23. Adipogenesis is Naa10p acetyltransferase activity-dependent…………72
Figure 24. Naa10p can directly interact with and Pparγ isoform 2…………………..73
Table 1. Summary of CRISPR Naa10-KO 3T3-L1……………………….……….74
Table 2. Primer sequence…………………………………………………….……….75
Primer for genotyping……………………………………………………………75
Primers for quantitative PCR……………………………………………………..75
Appendix 1. Naa10p is highly conserved in eukaryotes from yeast to human…….78
Appendix 2. hNaa10 S37P mutation cause developmental delay and abnormally low subcutaneous fat in human………………………………………………………….79
Appendix 3. Acetyltransferase domain locates on N-terminal of Naa10p, where DNA-binding domain on C-terminal………………………………………………80
Appendix 4. Construction of Naa10-KO mice……………………………………….81
Appendix 5. Naa10 knockout in oocyte causes embryonic lethality……………….82
Appendix 6. Naa10p knockout protects mice from high-fat diet induced obesity…83
Appendix 7. The number of adipocytes in Naa10-KO MEFs was fewer than
Naa10 X/Y after adipogenic induction……………………………………………..…84
Appendix 8. Mouse fat can classify into brown fat, subcutaneous white fat and visceral white fat……………………………………………………………………85
Appendix 9. The schematic illustration of CRISPR Naa10-KO 3T3-L1 generation...86
Appendix 10. Naa10p K165A mutation shows similar acetyltransferase activity with WT………………………………………………………………………………….87

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