Field F J, Albright E, Mathur S N
Department of Internal Medicine, University of Iowa, University of Iowa Hospitals and Clinics, Iowa City 52242.
J Lipid Res. 1987 Sep;28(9):1057-66.
The regulation of acylcoenzyme A:cholesterol acyltransferase (ACAT) activity by cholesterol was studied in an established enterocyte cell line. CaCo-2 cells were grown in culture to confluency and dome formation. They were characterized morphologically by light and transmission electron microscopy. During the culture period, ACAT activity remained stable while the activities of the brush border enzymes sucrase and alkaline phosphatase progressively increased with time and plateaued 12 days after plating. As determined by the rate of incorporation of oleic acid into the individual lipid classes, the rate of triglyceride synthesis was twice that of phospholipid and 15 times that of cholesteryl ester synthesis in these cells. Incubating CaCo-2 cells with cholesterol solubilized in taurocholate micelles resulted in a significant increase in ACAT activity (149 +/- 5 pmol/dish per 2 hr vs. 366 +/- 5, (P less than 0.001) without changing the rates of triglyceride or phospholipid synthesis. The stimulation of ACAT activity by micellar cholesterol was rapid, occurring within 5 min and reaching a maximal effect by 2 hr. The regulation of ACAT activity by cholesterol was directly dependent upon the concentration of cholesterol solubilized in the micelle and was independent of protein synthesis. Incubating CaCo-2 cells with micellar cholesterol did not increase the esterification of, nor did the cholesterol enter the pool of, newly synthesized or performed cholesterol within 2 hr. The micellar cholesterol that was taken up by the cells was esterified within 5 min after starting the incubation. Progesterone, a known ACAT inhibitor, significantly decreased the rate of esterification of intracellular micellar cholesterol proving that the cholesterol taken up by CaCo-2 cells was indeed entering the ACAT pool. Despite increasing amounts of unesterified cholesterol entering the cells via micelles, the percent of cholesterol that was esterified at any one time remained constant at 1%. The results suggest that ACAT activity in CaCo-2 cells is stimulated by cholesterol delivered to the cells by way of taurocholate micelles. The rapid entry of this sterol into the ACAT substrate pool suggests that ACAT activity in CaCo-2 cells is regulated by the expansion of the cholesterol substrate pool that is being utilized by an unsaturated ACAT enzyme.
在一个已建立的肠上皮细胞系中研究了胆固醇对酰基辅酶A：胆固醇酰基转移酶（ACAT）活性的调节作用。将CaCo-2细胞培养至汇合并形成穹窿。通过光学显微镜和透射电子显微镜对其进行形态学表征。在培养期间，ACAT活性保持稳定，而刷状缘酶蔗糖酶和碱性磷酸酶的活性随时间逐渐增加，并在接种后12天达到平台期。通过油酸掺入各个脂质类别的速率测定，在这些细胞中甘油三酯合成速率是磷脂合成速率的两倍，是胆固醇酯合成速率的15倍。用牛磺胆酸盐微胶粒中溶解的胆固醇孵育CaCo-2细胞导致ACAT活性显著增加（每2小时每培养皿149±5 pmol对366±5，（P＜0.001）），而甘油三酯或磷脂合成速率未改变。微胶粒胆固醇对ACAT活性的刺激迅速，在5分钟内发生，并在2小时达到最大效应。胆固醇对ACAT活性的调节直接取决于微胶粒中溶解的胆固醇浓度，且与蛋白质合成无关。用微胶粒胆固醇孵育CaCo-2细胞在2小时内既未增加新合成或已存在胆固醇的酯化，胆固醇也未进入该池中。细胞摄取的微胶粒胆固醇在开始孵育后5分钟内被酯化。孕酮，一种已知的ACAT抑制剂，显著降低细胞内微胶粒胆固醇的酯化速率，证明CaCo-2细胞摄取的胆固醇确实进入了ACAT池。尽管通过微胶粒进入细胞的未酯化胆固醇量增加，但在任何一个时间点被酯化的胆固醇百分比保持恒定在1%。结果表明，CaCo-2细胞中的ACAT活性受到通过牛磺胆酸盐微胶粒递送至细胞的胆固醇的刺激。这种固醇迅速进入ACAT底物池表明，CaCo-2细胞中的ACAT活性受不饱和ACAT酶所利用的胆固醇底物池扩张的调节。
