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  • 學位論文

脂締素受體一藉由PI3K/AKT路徑促進肝細胞脂肪酸代謝及細胞存活率

Adiponectin receptor 1 enhances fatty acid metabolism and cell viability in HepG2 cells through PI3K/AKT pathway

指導教授 : 丁詩同
共同指導教授 : 陳靜宜

摘要


肝臟中油脂累積過量會引起發炎反應、細胞傷害、肝纖維化甚至肝硬化,亦即非酒精性脂肪肝之症狀。棕櫚酸為血清中含量最多之飽和脂肪酸,且廣泛存在於動植物及及人類日常飲食中,然而其已被證實在多種細胞中會引起細胞毒性,肝細胞亦在其中。 以棕櫚酸(palmitate)處理人類肝臟細胞HepG2,發現細胞內油滴累積上升,ATP產生量下降以及細胞存活率下降,亦即棕櫚酸會造成細胞功能缺損(cell dysfunction),更進一步使細胞死亡。觀察細胞之基因表現,促進脂肪酸進行氧化之基因,包含carnitine palmitoyltransferase I (CPT1),acyl-coA oxidase (ACO),cytochrome c oxidase,peroxisome-proliferator-activated receptor-co-activator 1-alpha(PGC1-alpha),peroxisome-proliferator- activated receptor-alpha(PPAR-alpha),以及促進脂肪酸合成之基因,包含:acetyl-CoA carboxylase (ACC)和fatty acid synthase (FAS)表現均顯著提高,而促進棕櫚酸合成ceramide之基因serine palmitoyltransferase (SPTLC)表現沒有顯著增加,此顯示ceramide可能並非棕櫚酸造成HepG2細胞凋亡之主要原因,另外引起細胞凋亡之caspase 3之基因表現則在棕櫚酸處理後顯著增加。 脂締素受體一(adiponectin receptor 1)為脂締素(adiponectin)之受體,此廣泛地在身體各組織被表現,並且被認為和脂肪酸與糖類代謝有密切之關係;然而其作用機制尚未完全明瞭。此外,目前並沒有此受體和細胞凋亡(apoptosis)之關係之研究。故在本研究中,我們將細胞中脂締素受體一過度表現,以研究脂締素受體一對脂肪代謝及對於細胞凋亡之作用機轉。 在HepG2中過度表現脂締素受體一,再以棕櫚酸處理後,細胞內油滴累積下降,ATP產生量上升,且細胞存活率上升。觀察其基因表現,CPT1,ACO,cytochrome c oxidase,PGC1-alpha和PPAR-alpha表現量均顯著上升,推測可能導致脂肪酸氧化增加,進而使能量產生增加。而ACC和FAS基因表現則顯著下降,和細胞油滴減少之結果一致。此外,caspase 3表現下降,均可能是脂締素受體一保護細胞免於死亡之機轉。 同時,我們觀察到在過度表現脂締素受體一的細胞中,磷酸化之AKT含量增加,且抑制其磷酸化後,脂締素受體一的保護作用消失,也就是ATP產生量再度下降,細胞存活率再度降低。綜合以上所述,脂締素受體一對於細胞在棕櫚酸處理之下具有保護效果,其效果部分來自於促進磷酸化AKT增加,除此之外,於基因之調控也為其作用之一。此研究成果對於肝臟之細胞功能缺失及死亡有進一步的了解,亦即在非酒精性脂肪肝中,脂締素受體一為一有潛力之治療標的。

並列摘要


Hepatic lipid overloading mainly in the form of triglyceride accumulation, which can result in the inflammatory response, cause hepatocyte damage, fibrosis, and eventually progress to cirrhosis, is associated with nonalcoholic fatty liver disease. The most abundant saturated fatty acid in plasma is palmitate, which is also common in a wide range of plant and animal sources. However, it is found lipotoxic in a variety of cell types, including hepatocytes. Exposure of human hepatocytes HepG2 to 0.2 mM or 0.4 mM palmitate for 24 hours resulted in raising lipid accumulation and decreasing ATP production and cell viability, suggesting the lipotoxicity of palmitate. Expression of genes related to fatty acid metabolism and cell apoptosis was affected by palmitate treatment. Expression of rate-limiting regulator in fatty acid oxidation carnitine palmitoyltransferase I (CPT1) was elevated in transcription level, and other genes, such as acyl-coA oxidase (ACO), cytochrome c oxidase, peroxisome-proliferator-activated receptor-co-activator 1-alpha(PGC1-alpha), peroxisome-proliferator- activated receptor-alpha(PPAR-alpha) and acetyl-CoA carboxylase (ACC) were increased slightly, while fatty acid synthase (FAS) was increased significantly. On the other hand, the mRNA expression of caspase 3 was increased significantly. In brief, palmitate contributed to cell dysfunction, and led to cell death ultimately. Adiponectin and its receptors play important roles in regulating lipid metabolism. In the second experiment, we over-expressed adiponectin receptor 1 in HepG2 cells by lentivirus system to define its role in the development of fatty liver syndrome. First of all, mRNA level of adiponectin receptor 1 in adiponectin receptor 1 transfected-HepG2 (AdipoR1) cells was 1.5-fold higher than the mock vector transfected-HepG2 cells (mock). Meanwhile, the protein expression of FLAG, which was constructed at the adiponectin receptor 1 vector, was 5-fold higher than the mock. When AdipoR1 cells were cultured with palmitate, fatty acid transport protein 2 (FATP2), ACO, CPT1, cytochrome c oxidase, and PPAR-alpha were significantly increased, but FAS, ACC, and PGC1-alpha were significantly decreased compared to mock group. In addition, ATP production was increased whereas the lipid accumulation was reduced in AdipoR1 transfected-HepG2 cells. These results suggest that adiponectin receptor 1 improves fatty acid metabolism, by increasing fatty acid up take and oxidation. We then investigated the cell death of palmitate-treated AdipoR1 cells. At 20 hours, cell viability of AdipoR1 cells was significantly higher than that of mock group, furthermore, annexin V positive cells of which was much lower. Additionally, the gene expression of caspase 3 was decreased significantly in AdipoR1 cells. Namely, adiponectin receptor 1 ameliorates palmitate-induced apoptosis in HepG2 cells. Meanwhile the phosphorylation of AKT was increased in AdipoR1 cells, and when PI3 kinase inhibitor was applied, the protective effect of adiponectin receptor 1 was absent, with which ATP production was decreased again and so did the cell viability by palmitate. Taken together, adiponectin receptor 1 enhances fatty acid metabolism and cell viability when treated with palmitate partially by activating AKT signaling. Therefore adiponectin receptor 1 serves a potential therapeutic target in nonalcoholic fatty liver disease.

並列關鍵字

adiponectin receptor HepG2 apoptosis cytotoxicity

參考文獻


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