臺灣博碩士論文加值系統

肥胖是胰島素阻抗以及胰島素阻抗併發相關疾病的危險因子。在過去
的研究中指出功能異常的脂肪細胞會進一步導致胰島素阻抗，除此之外其他許多證據也顯示相較於瘦者體內的脂肪細胞，肥胖的個體體內脂肪細胞處於低氧的環境之中。另一方面，過去的研究中也指出運動會重新分配組織當中的血流，使細胞處於低氧的狀態並且增加胰島素敏感性。根據以上的發現，低氧對於胰島素敏感性的影響仍然是具有爭議性。本篇論文最主要的目標為探討低氧對於3T3-L1前脂肪細胞分化以及分化完全脂肪細胞胰島素敏感性的影響及相關機轉。在實驗中先以低氧處理3T3-L1前脂肪細胞6小時再繼續於一般氧濃度下完成分化作用以觀察低氧對於脂肪細胞合成的影響，接著藉由檢測完全分化的脂肪細胞中三酸甘油酯 (Triglyceride; TG)累積以及脂肪細胞脂質結合蛋白 (Adipocyte lipid binding protein; ALBP/aP2) 表現量來檢測脂肪細胞分化的效能。其中利用檢測細胞中的三酸甘油酯含量以及油紅染色法來檢測細胞中脂質的累積，並進一步利用西方墨點法檢測重要的細胞分化轉錄因子前脂肪細胞因子 (Preadipocytefactor; Pref-1)、CCAAT/enhancer binding protein b (C/EBPb)、Peroxisome proliferator-activated receptor-g (PPARg)�n表現情形。另一部分的實驗則是利用分化完全的3T3-L1脂肪細胞經過低氧前處理6小時後再給予胰島素刺激，並檢測胰島素訊息傳遞分子的磷酸化以及胰島素所誘發的葡萄糖攝取。更進一步在實驗中利用低氧誘發因子 (Hypoxia-inducible factor-1a;
HIF-1a)�n抑制劑了解 HIF-1a 在脂肪細胞合成以及胰島素敏感度上參與的角色。實驗結果顯示相較於正常氧濃度的對照組，低氧會降低脂肪細胞分化效能，顯著的降低脂質的累積和脂肪細胞脂質結合蛋白的表現量。低氧所抑制的脂肪細胞生合成則是透過增加 Pref-1 表現量及抑制 PPARg�n的表現量。並且，相較於正常氧濃度環境，低氧會增加 3T3-L1 脂肪細胞胰島素所誘發的胰島素受器自主磷酸化表現及 Akt 的磷酸化和葡萄糖的攝取。而當 HIF-1a�n被其抑制劑　YC-1
所抑制時，低氧所抑制的脂肪細胞生成以及低氧所促進的胰島素受器磷酸化都會恢復，因此本實驗認為，低氧所抑制的3T3-L1脂肪細胞合成以及胰島素敏感度的增加是透過 HIF-1a�n所調控。

Obesity is a risk factor for development of insulin resistance (IR) and IR-associated metabolic disorders. Studies also demonstrated that adipose tissue dysfunction might cause insulin resistance in obesity. Besides, several
lines of evidence suggested that adipose tissues of obese individuals were in a low oxygen status compared with the lean control. On the other hand, studies demonstrated that exercise re-arranged tissue blood flow disposing cells to low oxygen concentration and increased insulin sensitivity. According to above findings, regulation of hypoxia on insulin sensitivity is still controversial. The purpose of present study was to investigate the regulatory mechanism of
hypoxia on adipogenesis and insulin sensitivity in 3T3-L1 adipocytes. To explore the effect of hypoxia on adiponenesis, 3T3-L1 preadipocytes were preincubated in hypoxia status for 6 hours and then induced adipocyte differentiation. Differentiation efficiency was determined by lipid accumulation and expression of adipocyte lipid-binding protein (ALBP/aP2). Lipid accumulation was valuated by measuring intracellular triglyceride content and oil red O staining. Furthermore, expressions of key transcriptional regulators, including C/EBPa, PPARg, and preadipocyte factor-1 (Pref-1), were also measured by immunobloting. To explore the effect of hypoxia on insulin sensitivity, fully differentiated 3T3-L1 adipocytes were preincubated in hypoxia status for 6 hours and then the insulin-stimulated phosphorylation of insulin signaling molecules and glucose uptake were measured. To further elucidate the the underlying mechanism, we clarify the involvement of hypoxia inducible factor-1a (HIF-1a) on hypoxia-regulated adipogenesis and insulin sensitivity by using HIF-1a inhibitor. Our results showed that hypoxia significantly decreased lipid accumulation and aP2 expression compared with normoxia control. Inhibitory effects of hypoxia on adipocyte differentiation were associated with increased Pref-1 expression and decreased PPARg�nexpression. In addition, hypoxia treatment increased insulinstimulated
insulin receptor autophosphorylation and Akt phosphorylation and glucose uptake in 3T3-L1 adipocytes compared with normoxia control. Both hypoxia-inhibited adipogenesis and hypoxia-enhanced insulin sensitivity could
be prevented by pretreatment with HIF-1a inhibitor. In conclusion, hypoxia suppressed adipogenesis and enhanced insulin sensitivity through HIF-1a-dependent pathway in 3T3-L1 adipocytes.

目錄 1
重要中英文名詞與縮寫 3
中文摘要 4
Abstract 6
第一章、前言 8
第二章、文獻回顧 9
1. 低氧簡介 9
a. 何謂低氧 9
b. HIF-1簡介 9
2. 脂肪組織 10
a. 脂肪組織的簡介 10
b. 脂肪組織的合成 11
c. 脂肪組織的內分泌器官功能 12
3. 胰島素 13
a. 胰島素作用 13
b. 胰島素訊息傳遞 14
c. 胰島素阻抗 16
第三章、材料與方法 17
1. 實驗設計 17
2. 藥品及抗體來源 19
3. 3T3-L1 纖維母細胞之培養與分化 20
4. 低氧處理 20
5. MTT 細胞存活率檢測 21
6. 油紅染色法 (Oil red O staining) 21
7. 三酸甘油酯含量測定 22
8. 葡萄糖攝取測定法 22
9. 蛋白質樣本製備 23
10. 西方墨點法 23
11. 核蛋白萃取 24
12. 統計方法 25
第四章、結果 26
1. 低氧環境的基本檢測 26
(1) 實驗中的低氧環境 26
(2) 低氧刺激後的細胞存活率 26
2. 短時間低氧誘發 HIF-1�� 表現量上升 26
3.低氧處理抑制3T3-L1脂肪細胞分化 27
4.低氧刺激對於脂肪細胞分化效能的影響 27
(1)低氧刺激降低脂肪細胞分化效能 27
(2) 三酸甘油酯的累積顯著少於一般分化的細胞 28
(3) 成熟3T3-L1脂肪細胞的ALBP/aP2表現量下降 28
5. HIF-1���n抑制劑 YC-1 預防低氧刺激而下降的分化效能 28
6. 低氧處理增加胰島素刺激的胰島素受器磷酸化 29
7. 低氧處理增加胰島素刺激的葡萄糖攝取 29
8. HIF-1���n抑制劑 YC-1 降低低氧增加的胰島素受器敏感性 29
第五章 討論 31
參考文獻 34
圖與圖誌 38

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