中國橄欖萃取物對脂質與醣類代謝相關機制的探討

Abstract

流行病學和許多研究發現長期攝取富含多酚的食物可以預防代謝異常，而中國橄欖含有大量的多酚類物質，並擁有多種生理保健功能，包含保肝、抗發炎和抗腫瘤增生等功效。本研究的第一部分，我們旨在探究中國橄欖果實甲醇萃取物-乙酸乙酯區分層(CO-EtOAc)對小鼠肝臟細胞FL83B脂質堆積的影響。結果發現CO-EtOAc可減緩油酸誘導的脂質堆積、ROS含量和MDA含量，亦由實驗結果顯示了CO-EtOAc對FL83B脂質代謝的調控機制，CO-EtOAc抑制脂肪酸轉運蛋白基因(CD36和FABP)和脂肪生成基因(SREBP-1c，FAS和ACC1)，但增加了脂肪分解(HSL)和脂質氧化基因(PPARα，CPT-1和ACOX)。此外，CO-EtOAc也會增加磷酸化AMPK及ACC1，CPT-1和PPAR的蛋白質表現，但減少SREBP-1c和FAS的表達。由添加AMPK抑制劑證實其在CO-EtOAc抑制脂質堆積中扮演重要角色。因此，我們認為中國橄欖果實可以改善與脂質堆積相關的代謝異常。第二部分，我們評估CO-EtOAc對HFD和STZ誘導的糖尿病大鼠之生理調控。管餵CO-EtOAc可顯著降低體重和附睾脂肪量。此外， CO-EtOAc會增加血清HDL-C濃度、肝臟中GSH含量和抗氧化酶活性(SOD，CAT和GPx)，CO-EtOAc也會改善高血糖和減少血清中TC、TG、膽酸和TNFα濃度，並減少肝臟中TC和TG的含量。我們進一步證明CO-EtOAc會抑制肝臟IRS-1磷酸化，TNF-α和IL-6表現量，但增加Akt之磷酸化。我們評估CO-EtOAc可透過調控膽固醇轉運、生物合成和降解的基因表現進而影響膽固醇代謝機制。CO-EtOAc不僅抑制SREBP-2，HMG-CoAR，SR-B1和CYP7A1的基因表現，而且還增加了膽固醇釋出轉運蛋白ABCA1和膽固醇接受器LDLR的基因表現。這些研究結果顯示，中國橄欖果實成份可以改善高脂飲食糖尿病大鼠的代謝功能失衡。第三部分，是透過 L6肌管細胞探討CO-EtOAc改善葡萄糖恆定的可能機制。CO-EtOAc會刺激葡萄糖轉運蛋白轉位至細胞膜促進葡萄糖攝取，同時增加p-GSK3α/β的蛋白質表現量進而增加肝醣儲存含量。此外，我們也發現CO-EtOAc使線粒體膜去極化、降低粒線體氧消耗並抑制粒線體NADH脫氫酶酵素活性，導致ADP / ATP比率增加，然後促使AMPK活化。最後，CO-EtOAc能夠透過活化AMPK刺激葡萄糖攝取和促進線粒體分裂，這些顯示CO-EtOAc在治療或預防HFD誘導的高血糖症中具有潛在的保健功能。總體而言，實驗結果充分顯示中國橄欖果實可做為抗肥胖和相關疾病的健康食品。

Epidemiological numerous studies show that polyphenols contribute various beneficial effects on hepatic protection, anti-inflammation, and anti-cancer. In the first part of the study, we aimed to investigate the role of the ethyl acetate fraction of Chinese olive fruit extract (CO-EtOAc) in suppression of lipid accumulation, ROS and MDA levels in FL83B mouse hepatocytes. CO-EtOAc inhibited the mRNA levels of fatty acid transporter genes (CD36 and FABP) and lipogenesis genes (SREBP-1c, FAS, and ACC1), but upregulated genes that govern lipolysis (HSL) and lipid oxidation (PPARα, CPT-1, and ACOX). Moreover, CO-EtOAc increased the protein expression of phosphorylated AMPK, ACC1, CPT-1, and PPAR, but downregulated the expression of SREBP-1c and FAS. Adding AMPK inhibitor, attenuated the role of in CO-EtOAc-mediated amelioration of lipid accumulation. Therefore, Chinese olive fruits may have the potential to improve the metabolic abnormalities associated with hepatic lipid accumulation. The second part of the study, we evaluated the effect of CO-EtOAc on HFD and STZ-induced diabetes. CO-EtOAc could re-markedly decreased the body weight and epididymal adipose mass. In addition, CO-EtOAc increased serum HDL-C levels, hepatic GSH levels, and antioxidant enzyme activities (GSH, CAT, and GPx). Furthermore, CO-EtOAc lowered blood glucose, serum levels of TC, TG, bile acid, and TNFα; and reduced TC and TG in liver. We further demonstrated that CO-EtOAc also mildly suppressed hepatic levels of phosphorylated IRS-1, TNF-α, and IL-6, but enhanced Akt phosphorylation. The possible mechanisms of CO-EtOAc on cholesterol metabolism were assessed by determining the expression of genes involved in cholesterol transportation, biosynthesis, and degradation. It was found that CO-EtOAc not only inhibited mRNA levels of SREBP-2, HMG-CoAR, SR-B1, and CYP7A1 but also increased the expression of genes, such as ABCA1 and LDLR that governed cholesterol efflux and cholesterol uptake, respectively. Moreover, the protein expressions of ABCA1 and LDLR were also significantly increased in the liver of rats supplemented with CO-EtOAc. These findings suggest that Chinese olive fruit may improve the metabolic dysfunction in diabetic rats under HFD challenge. The third part of the study, we aimed to investigate the potential mechanisms in regulation of glucose homeostasis in L6 myotubes. CO-EtOAc effectively increased glycogen content via increased p-GSK3α/β and enhanced glucose uptake by stimulating glucose transporter 4 membraned translocation. In addition, we found that CO-EtOAc depolarized the mitochondrial membrane, decreased mitochondrial oxygen consumption and inhibited mitochondrial NADH dehydrogenase enzyme activities leading to increases in the ADP/ATP ratio, which then triggered AMPK activation. We also found CO-EtOAc is capable of stimulating glucose uptake and promoting mitochondrial fission through activation of AMPK. We thus suggest that CO-EtOAc has the therapeutic potential in the prevention of HFD-induced hyperglycemia. Overall, the experimental evidence highlight the Chinese olive fruit as a new health food in the prevention of obesity and its related metabolic disorders.