本文選題：十五烷酸　切入點：3T3-L1成熟脂肪細(xì)胞　出處：《華東師范大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：糖代謝紊亂是引起糖尿病的一個關(guān)鍵的危險因素。飽和長鏈脂肪酸棕櫚酸和硬脂酸對糖代謝產(chǎn)生不利影響,增加2型糖尿病的發(fā)病危險性,但不同的脂肪酸對糖代謝及糖尿病的發(fā)生和發(fā)展產(chǎn)生不同影響,與其鏈長度及飽和度相關(guān)。通過前期對降糖植物中活性天然產(chǎn)物的篩選,我們發(fā)現(xiàn)十五烷酸對成熟脂肪細(xì)胞具有非常顯著的促進(jìn)葡萄糖消耗的作用,提示十五烷酸可能對糖代謝起到一定的調(diào)節(jié)作用。屬于長鏈脂肪酸的十五烷酸對糖代謝的影響尚不清楚,其作用機(jī)制也不明確。為此本研究采用脂肪細(xì)胞、骨骼肌細(xì)胞以及實驗性類似2型糖尿病小鼠,在細(xì)胞和整體水平系統(tǒng)研究了十五烷酸對糖代謝的影響,并深入探究了其作用機(jī)制,旨在明確十五烷酸對糖代謝的調(diào)節(jié)作用及其作用靶點,為全面認(rèn)識飽和長鏈脂肪酸的功能提供實驗依據(jù),并對合理攝入膳食脂肪酸提供了一定的理論依據(jù)。在細(xì)胞模型方面,不同濃度的十五烷酸能夠顯著促進(jìn)基礎(chǔ)狀態(tài)3T3-L1成熟脂肪細(xì)胞和C2C12骨骼肌細(xì)胞對葡萄糖的消耗。在3T3-L1成熟脂肪細(xì)胞或者是C2C12骨骼肌細(xì)胞中十五烷酸與胰島素聯(lián)合給藥,促進(jìn)葡萄糖消耗的作用均顯著強(qiáng)于胰島素單獨作用。進(jìn)一步的機(jī)制探究,發(fā)現(xiàn)在基礎(chǔ)狀態(tài)下,十五烷酸對2種細(xì)胞中CAP、ERK、PPARγ、PPARβ、PPARα的蛋白表達(dá)無明顯的影響。但能明顯降低P-ERK、P-PPARγ(Ser112)的表達(dá),并且在與胰島素的聯(lián)合作用下,十五烷酸同樣可降低骨骼肌細(xì)胞中P-ERK、P-PPARy(Ser112)的表達(dá),說明十五烷酸可以通過降低ERK的磷酸化來提高PPARγ的活性,這可能為促進(jìn)細(xì)胞糖消耗起到了一定的幫助作用。此外,在2種細(xì)胞中十五烷酸都會降低基礎(chǔ)狀態(tài)及胰島素聯(lián)合處理下胰島素信號通路中IRS、PI3K、AKT的磷酸化水平;但同時十五烷酸可增強(qiáng)基礎(chǔ)狀態(tài)及胰島素聯(lián)合處理下AMPK的磷酸化及其下游P38的磷酸化水平,提高AKT下游的AS160的磷酸化和膜蛋白Glut4的表達(dá)。不同的是,在骨骼肌細(xì)胞中十五烷酸能夠明顯提高P-PKC(ζ/λ)的表達(dá),但是在脂肪細(xì)胞中卻沒有發(fā)現(xiàn)這一現(xiàn)象。上述結(jié)果提示,十五烷酸促進(jìn)細(xì)胞糖消耗可能是通過提高AMPK的活性,激活P38的磷酸化進(jìn)而促進(jìn)下游的AS160的磷酸化,最終導(dǎo)致膜蛋白Glut4的轉(zhuǎn)運。采用AMPK的抑制劑Compound C進(jìn)行驗證,發(fā)現(xiàn)Compound C可逆轉(zhuǎn)十五烷酸促進(jìn)的糖消耗,并能逆轉(zhuǎn)十五烷酸促進(jìn)的AMPK及AMPK下游的AS16O、aPKC、P38的磷酸化,證明十五烷酸是通過AMPK信號通路發(fā)揮促進(jìn)糖消耗的作用。進(jìn)一步采用高熱量飲食聯(lián)合STZ誘導(dǎo)的2型糖尿病小鼠模型,觀察十五烷酸對2型糖尿病小鼠糖代謝的影響情況。十五烷酸對2型糖尿病小鼠的飲水、飲食、體重?zé)o明顯影響;對小鼠空腹血糖、糖基化血紅蛋白和胰島素抵抗指數(shù)也無明顯的影響,但十五烷酸能降低2型糖尿病小鼠異常升高的血清胰島素水平,改善高胰島素血癥,降低血清炎癥因子TNF-α平,通過HE染色觀察到,十五烷酸阻止糖尿病對胰島細(xì)胞的破壞作用。對于與糖代謝紊亂密切相關(guān)的脂代謝方面,十五烷酸對模型小鼠血清甘油三酯、高、低密度脂蛋白膽固醇、游離脂肪酸水平均無明顯的影響,但它能顯著性的減少附睪脂肪墊的重量,可降低血清總膽固醇含量。綜上所述,十五烷酸主要通過AMPK途徑促進(jìn)脂肪細(xì)胞和骨骼肌細(xì)胞對葡萄糖的攝取。十五烷酸不影響2型糖尿病小鼠空腹血糖水平,但能改善高胰島素血癥,保護(hù)胰島細(xì)胞,并能降低糖尿病小鼠血清膽固醇,減少脂肪的積累,改善糖尿病小鼠的炎癥狀態(tài)。
[Abstract]:Glucose metabolism is a key risk factor for diabetes. Saturated long-chain fatty acid palmitic acid and stearic acid on glucose metabolism adversely affected, increases the risk of type 2 diabetes, but have different effects of different fatty acids on glucose metabolism and the occurrence and development of diabetes, and the chain length and saturation. Through the screening of hypoglycemic activity. Plant natural products early, we found that fifteen alkyl acid has a very significant role in promoting the consumption of glucose on mature adipocytes, suggesting that fifteen alkyl acid may regulate the glucose metabolism. The effects of fifteen belong to acid and long chain fatty acid on glucose metabolism is unclear, the mechanism is not clear. This study uses fat cells, skeletal muscle cells and similar experimental type 2 diabetic mice, in the cell and the overall level of fifteen alkyl acid The influence on glucose metabolism, and explores its mechanism, to clear the fifteen alkyl acid on glucose metabolism regulation and its targets, and provide the experimental basis for the comprehensive understanding of saturated long-chain fatty acids, and certain theoretical basis for the reasonable intake of dietary fatty acids. In the cell model, fifteen acid of different concentrations to consumption based 3T3-L1 adipocytes and C2C12 skeletal muscle cells to glucose significantly enhanced. In 3T3-L1 adipocytes or C2C12 skeletal muscle cells in fifteen acetic acid combined with insulin administration, to promote the consumption of glucose effect was significantly stronger than insulin alone. Further study the mechanism, found in the baseline state, fifteen alkyl acid of 2 kinds of cells in CAP, ERK, PPAR gamma, PPAR beta, PPAR alpha protein expression had no significant effect. But it can significantly reduce P-ERK, P-PPAR gamma (Ser112). And in the expression, and the joint action of insulin, fifteen acid also reduced P-ERK in skeletal muscle cells, the expression of P-PPARy (Ser112), fifteen acid can improve PPAR gamma activity by inhibiting ERK phosphorylation, which may promote cell sugar consumption played a helpful role in addition. In fifteen, the 2 kinds of cells are under the acid combined treatment of insulin signaling and insulin IRS, reducing PI3K, AKT phosphorylation; but at the same time, fifteen alkyl acid combined treatment of AMPK and its downstream phosphorylation of P38 enhanced the basic condition and the phosphorylation level of insulin, increase the expression of AKT downstream the phosphorylation of AS160 and membrane protein Glut4. The difference is, fifteen alkyl acid can significantly improve P-PKC in skeletal muscle cells (zeta / lambda) expression, but in the fat cells but did not find this phenomenon. These results suggest that fifteen Acid sugar consumption may promote cell by increasing the activity of AMPK, activation of P38 phosphorylation and promote downstream phosphorylation of AS160, resulting in membrane protein translocation of Glut4. Using the AMPK inhibitor Compound C verifies that Compound C can reverse the fifteen alkyl acid promoted sugar consumption, and can reverse the fifteen alkyl acid to promote the downstream AMPK and AMPK AS16O, aPKC, P38 phosphorylation, fifteen alkyl acid is proved to play a role in promoting the consumption of glucose through the AMPK signaling pathway. The high calorie diet in type 2 diabetic mice model induced with STZ, observe fifteen alkyl acid on glucose metabolism in mice with type 2 diabetes diet for fifteen. Acid on mice with type 2 diabetes mellitus, drinking water, no significant effect on body weight in mice; fasting blood glucose, glycosylated hemoglobin and insulin resistance index have no obvious effect, but the fifteen alkyl acid can reduce type 2 diabetes. Serum insulin levels in mice increased abnormally, hyperinsulinemia, decreased serum inflammatory factor TNF- alpha level was observed by HE staining, fifteen damage acid to prevent diabetes on islet cells. The glucose metabolism and lipid metabolism disorders are closely related, fifteen alkyl acid on mice serum triglyceride, high and low the LDL had no significant effect of free fatty acid levels, but it can reduce the weight of epididymal fat pad significantly, can reduce serum total cholesterol content. To sum up, fifteen alkyl acid mainly through the AMPK pathway to promote glucose uptake in adipocytes and skeletal muscle cells. Fifteen acid does not affect the blood glucose level of 2 diabetic mice fasting, but it can improve the hyperinsulinemia, protect the islet cells, and can reduce the serum cholesterol in diabetic mice, reduce fat accumulation, improve diabetic mice A state of inflammation.

【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R587.1

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