探討lunasin對3T3-L1脂肪細胞與C2C12肌小管細胞在肥胖相關發炎微環境下對葡萄糖利用之影響

Abstract

肥胖為增加胰島素阻抗、第二型糖尿病，以及心血管疾病罹患機率的主要危險因子之一，肥胖對人體所造成的負面影響，使其成為不可忽略的健康議題。在肥胖狀態下，過多的脂肪堆積所分泌的脂肪激素及游離脂肪酸，造成免疫細胞的浸潤，造成部份組織或全身處於慢性低度發炎的狀態，導致相關代謝功能障礙。種子胜肽 lunasin目前已被證實具有多種生理功能如抗癌、抗發炎、抗氧化、調節免疫功能以及調解血脂質等。本研究設計為將 3T3-L1 脂肪細胞以腫瘤壞死因子-α (tumor necrosis factor-α, TNF-α)；C2C12 肌小管細胞以棕櫚酸 (palmitic acid, PA) 誘導，來模擬肥胖下的發炎微環境，並探討lunasin對於脂肪細胞與肌小管細胞葡萄糖利用的影響。使用 2-NBDG 進行葡萄糖攝取實驗，自然狀態下有胰島素組別中，經處理 10 μM lunasin顯著增加脂肪細胞葡萄糖攝取，處理 25 μM lunasin 顯著增加肌小管細胞葡萄糖攝取。利用免疫螢光染色分析脂肪細胞與肌小管細胞葡萄糖轉運蛋白-4 (glucose transporter type 4, GLUT-4) 與胰島素受體 (insulin receptor) 表現，自然狀態下有胰島素組別中，經處理 50 μM lunasin 可顯著增加脂肪細胞 GLUT-4 表現；經處理 10 與 25 μM lunasin則有增加 GLUT-4表現趨勢。最後利用西方墨點法探討肌小管細胞胰島素受體底物-1 (insulin receptor substrate-1, IRS-1)、蛋白激酶B (protein kinase B, AKT) 與單磷酸腺苷活化蛋白質激酶 (AMP-activated protein kinase, AMPK) 磷酸化表現，自然狀態下有胰島素組別中，經處理 10 μM lunasin 可顯著增加 IRS-1 及 AKT 蛋白磷酸化表現。綜合上述，自然狀態下，lunasin 可能具有調節脂肪細胞與肌小管細胞葡萄糖利用的功效，特別是在無胰島素下更為顯著，顯示 lunasin 可能具有類似胰島素，且可能機制則透過增加胰島素受體路徑訊號來調節肌小管細胞葡萄糖攝入。然而對於 lunasin 在肥胖相關發炎反應下，並未明確顯示可調控葡萄糖利用，針對相關訊息路徑值得更進一步探討。Obesity is one of the major risk factors for diseases including insulin resistance, type 2 diabetes, and cardiovascular disease. Global obesity rates have increased recently, and the negative impact that obesity causes to humans makes it a serious health crisis. In the case of obesity, excessive fat accumulation with adipokines and free fatty acids release, which trigger immune cells recruitment. Obesity is also considered in a chronic low-grade inflammatory state, and the metabolic dysfunction is caused by the persistent low-grade inflammation. It has been established that the seed peptide lunasin has a variety of physiological properties, including anti-cancer, anti-inflammatory, anti-oxidant, and immune regulation. The aim of this study, we investigate the effects of lunasin on glucose utilization in 3T3-L1 adipocytes and C2C12 myotubes. 3T3-L1 adipocytes were treated with tumor necrosis factor-α (TNF-α) and C2C12 myotubes were treated with palmitic acid (PA) to simulate the inflammatory microenvironment associated with obesity. In spontaneous condition, treated with 10 and 25 μM lunasin increase 2-NBDG uptake in 3T3-L1 adipocytes and C2C12 myotubes in the presence of insulin, and the glucose transporter type 4 (GLUT-4) and insulin receptor expressions in 3T3-L1 adipocytes and C2C12 myotubes were used immunofluorescent staining, and the GLUT-4 expression also increased after lunasin treatment. Western bloting results demonstrated that treated with 10 μM lunasin also increase the level of insulin receptor substrate-1 (IRS-1) and AKT protein phosphorylation in C2C12 myotubes, shows that lunasin may improve glucose utilization via regulating insulin receptor signaling. Nevertheless, the signaling pathway of lunasin regulating glucose utilization in obesity-related inflammation is still unclear, and need further study.