臺灣博碩士論文加值系統

油炸食物因具有特殊之色、香、味，而廣受人們喜愛，但攝食炸油的安全性也備受關注。從我們過去的研究發現，攝食炸油會影響體內脂質與葡萄糖代謝，除了活化 peroxisome proliferator-activated receptor α ，促進脂肪酸氧化，也觀察到炸油飲食導致大小鼠葡萄糖不耐（glucose intolerance），在口服葡萄糖耐受性試驗時可見血清胰島素與 C-peptide 濃度降低。猜測炸油導致葡萄糖不耐之原因可能為胰臟小島受到氧化壓力或發炎傷害，導致胰島素分泌不足。本研究目的在探討炸油降低胰島素分泌原因及機制，為瞭解炸油效應是否與促進胰臟小島過氧化壓力有關，實驗一將 C57BL/6J mice 分為三組，分別餵以正常含量新鮮油(4 %;LF)、20 %新鮮油(HF)及20 %炸油(HO)飼料，待2個月炸油組出現 glucose intolerance 及 hypoinsulinemia 現象後，分離胰臟小島進行體外糖水培養實驗觀察胰島素分泌，證實 HO 組不論第一期還是第二期的葡萄糖刺激胰島素分泌(GSIS)皆降低(P<0.05 及 P<0.01)，而 HO 組胰臟小島的 total insulin content 雖和 HF 組相當，但與 LF 組相比仍有顯著較低(P<0.01)。在氧化壓力方面， HO 組的肝臟相較它組有顯著較低的維生素 E (P<0.001)及顯著較高的 TBARS (P<0.0001)，但 glutathione (GSH)與 LF 組相比則有顯著較高(P=0.01)。HO組不只肝臟，在胰臟小島亦可觀察到高氧化壓力：HO 組的胰臟小島相較它組有較高的 lipid hydroperoxide 及顯著較低的 glutathione peroxidase-1(P<0.05)與 total antioxidant capacity (HO vs. LF, P<0.05)。另外，HO 組的胰臟維生素 E 含量也顯著的低於 HF 組(P<0.0001)。在發炎因子方面， HO 和 HF 組胰臟小島的 TNF-α 含量並無顯著差異。利用 Western blot 證實，HO 組其磷酸化的 c-Jun NH2-terminal kinase (JNK)有大於 LF 與 HF 組傾向 (p=0.06)，而 Pancreatic duodenal homeobox-1 (PDX-1) 的表現量則為 HO 組低於 LF 及 HF 組(p=0.055)。在 mRNA 方面， HO 組除了 UCP2 的表現量與 HF 組一樣皆顯著高於 LF 組外，其他 PDX-1 的下游基因 mRNA 表現量(preproinsulin-1 及 2、glucokinase 及 GLUT2)則皆無差異。在實驗二則進一步觀察，炸油所導致的胰島素分泌減少是否可藉由體外或體內補充維生素 E 來克服。在體外補充的實驗，將 LF、HF 和 HO 三組胰臟小島分離後與 100 μM 劑量維生素 E 培養，發現除 LF 外，體外補充維生素 E 並不能改善其受損的 GSIS 。在體內補充的實驗，將 C57BL/6J mice 分為四組，HO、HF、HO+E 及 HF+E ，後兩組飼料為添加十倍 AIN-76 建議量之維生素 E ，發現在給予飲食補充維生素 E 後， HO+E 組其肝臟及胰臟內維生素 E 含量有顯著增加(P<0.0001)，而炸油所致的 hypoinsulinemia 也被克服(P<0.005) ，並有改善 GSIS 的趨勢。總和以上所知，炸油飲食導致的葡萄糖不耐及低胰島素血症與胰臟小島過氧化傷害有關，使得 GSIS 受損，而口服補充維生素E可部分恢復 GSIS 。

Being crisp and aromatic, fried foods are popular with consumers world-wide while the safety concerns about the ingestion of oxidized frying oil (OFO) are still raised. We had previously shown that OFO ingestion can influence lipid and glucose metabolism. Dietary OFO increases fatty acids oxidation in liver by activating peroxisome proliferator-activated receptor α. However, accompanied with TG reductions, glucose intolerance is observed in OFO-fed rodents. Since the serum levels of insulin and C-peptide in oral glucose tolerance test were both decreased in OFO-fed mice, we speculated the reduced ability to secrete insulin caused by dietary OFO is associated with oxidative damages and/or inflammatory responses of the pancreas. Accordingly, this study was aimed at investigating the underlying mechanisms of insulin secretion impairment associated with OFO consumption. To test the involvement of oxidative stress in pancreatic islets, Experiment I was conducted by dividing C57BL/6J mice into LF, HF and HO groups which received a basal diet containing 5 g/100 g fresh soybean oil or high fat diets containing 20 g/100 g of fresh soybean oil or OFO respectively. When glucose intolerance and hypoinsulinemia were observed in HO group (2 months of feeding), all mice were killed and their pancreatic islets were isolated. Results showed glucose stimulated insulin secretion (GSIS) of islets, both in the first phase and second phase, was significantly reduced in HO group compared with the other two groups (P<0.05),. The total insulin content in islets of HO group was not different from that of HF group, but was significantly lower than that of LF group (P<0.01). For oxidative indexes, livers vitamin E and TBARS in HO group were significantly lower (P<0.001) and higher (P<0.0001), respectively, than those of other groups. A higher oxidative stress was observed not only in livers, but also in pancreatic islets of HO group. Compared to HF or LF groups, the lipid hydroperoxide was increased, and the glutathione peroxidase-1 activity and total antioxidant capacity (HO vs. LF, P<0.05) was decreased in islets of HO groups. Moreover, the vitamin E levels in the pancreas of HO group were significantly lower than that of HF group (P<0.0001). For inflammatory indexes, there was no significant difference between HO and HF groups in islets of TNF-α levels. The immunoblotting showed, the protein levels of phosphorylated c-Jun NH2-terminal kinase (JNK) tended to be higher (p=0.06), and the protein levels of pancreatic duodenal homeobox-1 (PDX-1) significantly lowered in HO group compared with the other two groups (p=0.055). Using qRT-PCR, the mRNA levels of UCP2 in islets of HO and HF groups were significantly higher than those of LF group, while no difference in mRNA levels of PDX-1 downstream genes (preproinsulin-1 and 2、glucokinase and GLUT2) was observed between groups. In Experiment II, we tried to investigate whether the impairment of insulin secretion by pancreatic islets associated with dietary OFO consumption could be reversed by supplementation of vitamin E in vitro or in vivo. For the in vitro supplementation study, islets separated from LF, HF and HO groups were incubated with or without the addition of vitamin E (100 μM). The improvement of GSIS due to vitamin E supplementation was only observed in LF group, but not in HF and HO group. In in vivo supplementation study, mice were separated into HO, HF, HO+E and HF+E groups, with a dietary supplementation of 10-fold higher AIN-76 recommended vitamin E for the latter two groups. Results showed that the vitamin E supplementation not only corrected the vitamin E status in livers and pancreas of OFO-treated mice (P<0.0001), but also alleviated the reduction of serum levels of insulin, and GSIS caused by dietary OFO. In conclusion, this study provided evidences that dietary OFO-induced glucose intolerance and hypoinsulinemia was associated with oxidative damages of pancreatic islets, thus leading to GSIS impairment. Oral supplementation of vitamin E can partly overcome the OFO mediated GSIS impairment.

縮寫對照表 I
中文摘要 II
英文摘要 IV

第一章 前言 1
第二章 文獻探討 3
一、氧化炸油 3
二、胰臟胰島素分泌 8
第三章 材料與方法 20
第一節 實驗設計與假說 20
第二節 實驗一 材料方法 23
一、氧化炸油的製備 23
二、油脂品質分析 24
三、試驗飼料配製 26
四、動物飼養 28
五、口服葡萄糖耐受試驗 29
六、動物犧牲與樣品收集 31
七、血糖分析 31
八、禁食血清胰島素含量測定 32
九、分離胰臟小島 34
十、葡萄糖刺激胰島素分泌試驗 (GSIS) 35
十一、Islet 總胰島素含量 36
十二、抗氧化與過氧化物質測定 36
十三、蛋白質定量 46
十四、西方墨點法 (Western blot) 46
十五、統計分析 51
第三節 實驗二 材料方法 52
一、前言 52
二、試驗飼料配製 52
三、動物飼養 54
四、肝臟脂質萃取 55
五、統計分析 56
第四章 結果 57
第五章 討論 87
第六章 結論 96
第七章 參考文獻 98

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