臺灣博碩士論文加值系統

Cisd3（CDGSH iron sulfur domain）是演化上高度保留的基因，其中包含兩個參與粒線體呼吸，氧化壓力和鐵平衡的CDGSH iron sulfur doamins。然而，Cisd3蛋白的功能尚不清楚，因此，我們將探討Cisd3蛋白是否參與在代謝途徑中。我們的結果顯示，中年（16 mo）Cisd3 KO小鼠表現出更高的能量消耗，伴隨著體脂肪減少。有趣的是，脂質滴積累在7個月大的Cisd3 KO小鼠的iBAT中，但在8個月大的小鼠則較少。然而，我們對Cisd3 KO小鼠的iBAT功能的進行寒冷環境的壓力測試，顯示與WT小鼠相比沒有顯著差異。我們還餵食Cisd3 KO小鼠導致肥胖的高脂肪食物，然而，我們在Cisd3 KO和WT小鼠之間分析的所有方面沒有顯著差異。另外，我們檢測了肝臟脂質體內平衡，鐵代謝和肝癌發生相關的線粒體功能是否也受到影響。首先，Cisd3缺陷似乎不會加速或緩解高脂肪食物和果糖所誘導引發的脂肪肝炎。其次，在Cisd3 KO小鼠腹腔注射鐵葡聚醣，發現鐵沉積增加，導致肝損傷標誌物升高。這表明Cisd3蛋白可能有助於鐵代謝。最後，Cisd3 KO中N-亞硝基二乙胺（DEN）誘導的肝細胞癌（HCC）發生率高於WT小鼠。總結上述結果，我們的研究顯示，Cisd3可能有助於維持能量消耗，並參與調控肝臟在鐵代謝上的功能。此外，Cisd3缺乏可能促進由DEN誘導所產生肝細胞癌的發生率。

Cisd3 (CDGSH iron sulfur domain 3), an evolutionarily conserved gene, contains two CDGSH iron sulfur domains which are involved in mitochondrial respiration, oxidative stress and iron homeostasis. However, the functions of Cisd3 protein are unclear. We asked whether Cisd3 protein plays a role in the metabolism. Our result revealed that middle-aged (16-mo) Cisd3 KO mice exhibit higher energy expenditure, accompanying by a decreased body fat. Interestingly, the lipid droplets accumulated in iBAT of Cisd3 KO mice at 7-mo but less at 8-mo. However, our cold stress tests on the iBAT function of the Cisd3 KO mice revealed no significant difference compared to WT mice. We also introduced the dietary-associated obesity (high-fat diet) to the Cisd3 KO mice; however, there are no significant differences in all the aspects we analyzed between the Cisd3 KO and WT mice. In addition, the mitochondrial functions related to homeostasis of lipid metabolism, iron metabolism and carcinogenesis in liver were analyzed. Firstly, the Cisd3 deficiency seems not to exaggerate or alleviate HFD-induced and Fructose-induced hepatosteatosis. Secondly, we intraperitoneally injected iron-dextran and found the increase of iron deposition, leading to the elevation of liver damage markers, in the Cisd3 KO mice. It suggested that the Cisd3 protein may contribute to the iron metabolism. Lastly, the incidence of hepatocellular carcinoma (HCC) induced by the N-Nitrosodiethylamine (DEN) in the Cisd3 KO compared to the WT mice were increased. Taken together, our results suggest that Cisd3 may help maintain energy expenditure, and participate in the hepatic iron metabolism. Furthermore, Cisd3 deficiency may promote DEN-induced HCC in mice.

致謝 i
Abstract ii
摘要 iii
I. Introduction 1
II. Materials and Methods 4
II-1. Animal 4
II-2. Mouse genomic DNA extraction 4
II-3. Genotyping by polymerase chain reaction (PCR) 4
II-4. RNA extraction 4
II-5. Reverse transcription and quantitative real-time PCR 5
II-6. Whole-body energy metabolism and body composition 5
II-7. High-fat diet treatment 5
II-8. Fructose-drinking treatment 6
II-9. Iron-dextran injection treatment 6
II-10. Diethylnitrosamine (DEN) injection treatment 6
II-11. In vivo metabolic studies 6
II-12. Acute cold exposure and Brown adipose tissue surgery 7
II-13. Transmission Electron Microscopy 7
II-14. Tissue processing and paraffin-embedding 7
II-15. Hematoxylin and eosin (H&E) staining 7
II-16. Prussian’s blue staining 8
II-17. Tissue iron measurement 8
II-18. Western blotting 8
II-19. Serum biochemical analysis 9
II-20. Statistical analysis 9
III. Results 10
III-1. Cisd3 deficiency seems to promote longevity 10
III-2. Cisd3 KO mice have high energy expenditure at middle-aged 10
III-3. Cisd3 protein might not participate in thermogenesis 10
III-4. Cisd3 KO mice do not have an amelioration of metabolic syndrome in dietary-induced obesity 12
III-5. Cisd3 KO mice do not have an amelioration of metabolic syndrome in fructose-drinking induced non-alcoholic fatty liver disease (NAFLD) 13
III-6. Cisd3 KO mice might increase iron content and iron deposition-induced liver damage. 14
III-7. Disruption of Cisd3 protein might promote hepatocellular carcinoma (HCC) with diethylnitrosamine (DEN) treatment 15
III-8. The Cisd3 gene expression are upregulated in methionine-choline deficient diet (MCD) and downregulated in 3,5-Diethoxy-carbonyl-1,4-dihydrocollidine diet (DDC) 16
IV. Discussion 17
IV-1. Cisd3 deficiency might trigger the protect effect to maintain the energy expenditure and promote the long life span. 17
IV-2. Cisd3 might not involve in adaptive thermogenesis during acute cold stress challenge. 18
IV-3. Cisd3 might involve in lipid metabolism. 18
IV-4. Cisd3 might involve in iron homeostasis. 19
IV-5. Cisd3 deficiency might increase the hepatocellular carcinoma (HCC) incidence. 19
V. Reference 20
VI. Figures 26
Figure 1 26
Figure 2 27
Figure 3 28
Figure 4 30
Figure 5 31
Figure 5 continued 32
Figure 6 34
Figure 6 continued 35
Figure 7 37
Figure 7 continued 38
Figure 8 40
Figure 9 42
Figure 10 44
Figure 11 46
Figure 11 continued 47
Figure 12 49
Figure 13 51
Figure 14 53
Figure 15 54
Figure 16 56
Figure 17 58
VII. Table 60
Table 1 Primer sequence for genotyping 60
Table 2 Primer sequence for RT-PCR 60
VIII.Appendix 61
Appendix 1 61

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