Huff M W, Miller D B, Wolfe B M, Connelly P W, Sawyez C G
Department of Medicine and the Robarts Research Institute, The University of Western Ontario, London, Canada.
J Lipid Res. 1997 Jul;38(7):1318-33.
Hypertriglyceridemic very low density lipoproteins (HTG-VLDL, S(f) 60-400) are not taken up by HepG2 cells. However, addition of bovine milk lipoprotein lipase (LPL) at physiological concentrations markedly stimulates uptake. In the present study, we determined whether: a) LPL catalytic activity is required for uptake, b) LPL functions as a ligand, and c) cell surface hepatic triglyceride lipase (HL) and/or proteoglycans are involved. Incubation of HepG2 cells with HTG-VLDL plus LPL (8 ng/ml) increased cellular cholesteryl ester (CE) 3.5-fold and triglyceride (TG) 6-fold. Heat-inactivation of LPL abolished the effect. Addition of tetrahydrolipstatin (THL, an LPL active-site inhibitor) to HTG-VLDL + LPL, inhibited the cellular increase in both CE and TG by greater than 90%. Co-incubation of HTG-VLDL + LPL with heparin, heparinase, or heparitinase, blocked CE accumulation by 70%, 48%, and 95%, respectively, but had no effect on the increase in cellular TG. Pre-treatment of cells with 1 mM 4-methylumbelliferyl-beta-D-xyloside, (beta-xyloside) to reduce cell surface proteoglycans inhibited the increase in CE induced by HTG-VLDL + LPL by 78%. HTG-VLDL remnants, prepared in vitro and isolated free of LPL activity, stimulated HepG2 cell CE 2.8-fold in the absence of added LPL, a process inhibited with THL by 66%. Addition of LPL (8 ng/ml) to remnants did not further enhance CE accumulation. HepG2 cell HL activity, released by heparin, was inhibited 95% by THL. The amount of HL activity and immunoreactive mass, released by heparin, was reduced 50-60% in beta-xyloside-treated cells. These results indicate that physiological concentrations of LPL promote HepG2 cell uptake of HTG-VLDL primarily due to remnant formation and that LPL does not play a major role as a ligand. HL activity and cell surface proteoglycans significantly enhance the subsequent uptake of VLDL remnants.
高甘油三酯血症性极低密度脂蛋白（HTG-VLDL，S(f) 60 - 400）不会被HepG2细胞摄取。然而，添加生理浓度的牛乳脂蛋白脂肪酶（LPL）能显著刺激其摄取。在本研究中，我们确定了：a）摄取是否需要LPL催化活性，b）LPL是否作为配体发挥作用，以及c）细胞表面肝甘油三酯脂肪酶（HL）和/或蛋白聚糖是否参与其中。用HTG-VLDL加LPL（8 ng/ml）孵育HepG2细胞，使细胞胆固醇酯（CE）增加3.5倍，甘油三酯（TG）增加6倍。LPL热失活消除了该效应。向HTG-VLDL + LPL中添加四氢脂抑素（THL，一种LPL活性位点抑制剂），抑制CE和TG的细胞内增加超过90%。HTG-VLDL + LPL与肝素、肝素酶或类肝素酶共同孵育，分别使CE积累减少70%、48%和95%，但对细胞内TG的增加无影响。用1 mM 4-甲基伞形酮基-β-D-木糖苷（β-木糖苷）预处理细胞以减少细胞表面蛋白聚糖，抑制了HTG-VLDL + LPL诱导的CE增加78%。体外制备且不含LPL活性的HTG-VLDL残余物，在未添加LPL的情况下刺激HepG2细胞CE增加2.8倍，该过程被THL抑制66%。向残余物中添加LPL（8 ng/ml）并未进一步增强CE积累。肝素释放的HepG2细胞HL活性被THL抑制95%。在β-木糖苷处理的细胞中，肝素释放的HL活性量和免疫反应性物质减少了50 - 60%。这些结果表明，生理浓度的LPL促进HepG2细胞摄取HTG-VLDL主要是由于残余物形成，并且LPL作为配体不起主要作用。HL活性和细胞表面蛋白聚糖显著增强了随后VLDL残余物的摄取。
