Rinninger F, Jaeckle S, Pittman R C
University Hospital Hamburg Eppendorf, Department of Medicine, Germany.
Biochim Biophys Acta. 1993 Feb 24;1166(2-3):275-83. doi: 10.1016/0005-2760(93)90108-l.
Selective uptake of high-density lipoprotein (HDL) cholesteryl esters without parallel uptake of HDL apolipoproteins occurs by a non-endocytotic pathway that results in net delivery of cholesteryl esters to cells. With respect to the cellular mechanism of this pathway, previous studies with adrenal cells showed a cholesteryl ester pool that is reversibly associated with cells and which appears to mediate irreversible selective uptake. A cholesteryl ester pool with similar properties was observed in plasma membranes isolated from adrenal cells, suggesting that this is the site of the cellular pool. Human Hep G2 hepatoma cells also selectively take up HDL cholesteryl esters. Therefore we asked if these cells have a reversibly cell-associated cholesteryl ester pool as well that could mediate irreversible selective uptake. To do this, human HDL3 (d = 1.125-1.21 g/ml) was labeled in both its protein and cholesteryl ester moieties. Uptake of HDL3 tracers by Hep G2 cells was then studied. After an uptake incubation in the presence of labeled HDL3, either cellular uptake of tracers was immediately determined or cells were 'chase' incubated in the presence of unlabeled HDL before determination of cellular tracer content. Hep G2 cells selectively took up HDL3 cholesteryl esters under these conditions. However, a fraction of cholesteryl ester tracer selectively taken up was chased from the cells by subsequent incubation in the presence of unlabeled HDL. This reversible pool of cholesteryl ester tracer was distinct from that irreversibly internalized, and in excess of that accounted for by dissociation of labeled HDL3 particles bound to the cell surface. Selective uptake was down-regulated by prior incubation with LDL, and cholesteryl ester tracer in the reversible pool was down-regulated in parallel. Plasma membranes were isolated from Hep G2 cells and incubated with doubly labeled HDL3. HDL3 particles bound to these membranes, as indicated by the apolipoprotein tracer. However, HDL cholesteryl esters associated with plasma membranes in excess on that accounted for by HDL3 particles. This selective association of HDL3 cholesteryl ester tracer with membranes was reversible, and the tracer was chased during incubation in the presence of unlabeled HDL. These results suggest that, as with steroidogenic cells, a reversible pool of cholesteryl esters localized in the plasma membrane is involved in selective uptake of HDL3 cholesteryl esters by hepatic cells at a step prior to irreversible internalization.
高密度脂蛋白（HDL）胆固醇酯的选择性摄取，而没有同时摄取HDL载脂蛋白，是通过一种非内吞途径发生的，该途径导致胆固醇酯向细胞的净递送。关于该途径的细胞机制，先前对肾上腺细胞的研究显示存在一个与细胞可逆结合的胆固醇酯池，它似乎介导了不可逆的选择性摄取。在从肾上腺细胞分离的质膜中观察到了具有相似性质的胆固醇酯池，这表明这就是细胞内池的所在位置。人肝癌Hep G2细胞也选择性摄取HDL胆固醇酯。因此，我们询问这些细胞是否也有一个与细胞可逆结合的胆固醇酯池，它可以介导不可逆的选择性摄取。为此，用人HDL3（d = 1.125 - 1.21 g/ml）对其蛋白质和胆固醇酯部分进行了标记。然后研究了Hep G2细胞对HDL3示踪剂的摄取。在存在标记的HDL3的情况下进行摄取孵育后，立即测定示踪剂的细胞摄取量，或者在测定细胞示踪剂含量之前，在存在未标记的HDL的情况下对细胞进行“追踪”孵育。在这些条件下，Hep G2细胞选择性摄取HDL3胆固醇酯。然而，随后在存在未标记的HDL的情况下孵育，会使一部分选择性摄取的胆固醇酯示踪剂从细胞中被追踪出来。这个可逆的胆固醇酯示踪剂池与不可逆内化的池不同，并且超过了与细胞表面结合的标记HDL3颗粒解离所导致的量。预先用低密度脂蛋白（LDL）孵育会下调选择性摄取，并且可逆池中胆固醇酯示踪剂也会相应下调。从Hep G₂细胞中分离出质膜，并与双重标记的HDL3一起孵育。载脂蛋白示踪剂表明HDL3颗粒与这些质膜结合。然而，与质膜相关的HDL胆固醇酯超过了HDL3颗粒所导致的量。HDL3胆固醇酯示踪剂与质膜的这种选择性结合是可逆的，并且在存在未标记的HDL的孵育过程中示踪剂会被追踪出来。这些结果表明，与类固醇生成细胞一样，位于质膜中的可逆胆固醇酯池在肝细胞不可逆内化之前的步骤中参与了HDL3胆固醇酯的选择性摄取。
