Fatty acid transport and metabolism in HepG2 cells
Fatty acid transport and metabolism in HepG2 cells
复制标题
DOI：
10.1152/ajpgi.00386.2005
复制
发表时间：
2006-03-01
影响因子：
4.5
通讯作者：
Hamilton, JA
中科院分区：
文献类型：
--
作者：
Guo, W;Huang, N;Hamilton, JA
肝细胞摄取脂肪酸的机制尚未完全了解。我们采用新的方法来解决长期存在的争议脂肪酸摄取和区分扩散和蛋白质为基础的机制。使用含有包埋的pH敏感荧光染料的HepG 2细胞，我们表明，向外部缓冲液中加入油酸酯（未结合或结合环糊精）会导致细胞内pH值（pH（in））快速（秒）和剂量依赖性降低，表明脂肪酸扩散穿过质膜。pH值（in）恢复至其初始值，时间过程（min）可抑制放射性标记油酸盐的代谢。用抑制剂根皮素或三胞苷C预孵育细胞对加入油酸后的快速pH（in）下降没有影响，但大大抑制了pH（in）恢复。使用放射性标记的油酸酯，我们发现，它的酯化几乎完全抑制根皮素或三胞苷C，支持pH（在）恢复和代谢之间的相关性。然后，我们使用双荧光测定来研究HepG 2细胞与顺式-parinaric酸（PA）（一种天然荧光但代谢缓慢的脂肪酸）之间的相互作用。PA的荧光迅速增加后，其添加到细胞，表明快速结合到质膜; pH值（在）迅速下降，同时，但没有恢复5分钟内。根皮素没有影响PA介导的pH值（在）下降或其缓慢恢复，但降低了膜结合PA的绝对荧光。我们的研究结果表明，天然脂肪酸迅速结合，并通过质膜扩散，而不受代谢抑制剂或抑制剂的假定膜结合脂肪酸转运。
The mechanism(s) of fatty acid uptake by liver cells is not fully understood. We applied new approaches to address long-standing controversies of fatty acid uptake and to distinguish diffusion and protein-based mechanisms. Using HepG2 cells containing an entrapped pH-sensing fluorescence dye, we showed that the addition of oleate (unbound or bound to cyclodextrin) to the external buffer caused a rapid (seconds) and dose-dependent decrease in intracellular pH (pH(in)), indicating diffusion of fatty acids across the plasma membrane. pH(in) returned to its initial value with a time course (in min) that paralleled the metabolism of radiolabeled oleate. Preincubation of cells with the inhibitors phloretin or triacsin C had no effect on the rapid pH(in) drop after the addition of oleate but greatly suppressed pH(in) recovery. Using radiolabeled oleate, we showed that its esterification was almost completely inhibited by phloretin or triacsin C, supporting the correlation between pH(in) recovery and metabolism. We then used a dual-fluorescence assay to study the interaction between HepG2 cells and cis-parinaric acid (PA), a naturally fluorescent but slowly metabolized fatty acid. The fluorescence of PA increased rapidly upon its addition to cells, indicating rapid binding to the plasma membrane; pH(in) decreased rapidly and simultaneously but did not recover within 5 min. Phloretin had no effect on the PA-mediated pH(in) drop or its slow recovery but decreased the absolute fluorescence of membrane-bound PA. Our results show that natural fatty acids rapidly bind to, and diffuse through, the plasma membrane without hindrance by metabolic inhibitors or by an inhibitor of putative membrane-bound fatty acid transporters.