Hilaire N, Salvayre R, Thiers J C, Bonnafé M J, Nègre-Salvayre A
Department of Biochemistry, Faculty of Medicine in Rangueil, University Paul Sabatier, Toulouse, France.
J Biol Chem. 1995 Nov 10;270(45):27027-34. doi: 10.1074/jbc.270.45.27027.
Cultured fibroblasts from patients affected with the genetic metabolic disorder named neutral lipid storage disease (NLSD) exhibit a dramatic accumulation of cytoplasmic triacylglycerols (Radom, J., Salvayre, R., Nègre, A., Maret, A., and Douste-Blazy, L. (1987) Eur. J. Biochem. 164, 703-708). We compared here the metabolism of radiolabeled short-, medium- and long-chain fatty acids in these cells. Short/medium-chain fatty acids (C4-C10) were incorporated into polar lipids (60-80%) and triacylglycerols (20-40%) at a lower rate (5-10 times lower) than long-chain fatty acids. Pulse-chase experiments allowed to evaluate the degradation rate of cytoplasmic triacylglycerols in normal and NLSD fibroblasts and to discriminate between two catabolic pathways of cytoplasmic triacylglycerols. Short/medium-chain (C4-C10) triacylglycerols were degraded at a normal rate in NLSD fibroblasts, whereas long-chain (C12 and longer) triacylglycerols remained undegraded. These data are confirmed by mass analysis. The use of diethylparanitrophenyl phosphate (E600) and parachloromercuribenzoate (PCMB) inhibitors allows to discriminate between the two triacylglycerol degradation pathways. E600 inhibited selectively the in situ degradation of short/medium-chain triacylglycerols without inhibition of the degradation of long-chain triacylglycerols, whereas PCMB inhibited selectively the in situ hydrolysis of long-chain triacylglycerols without affecting the degradation of long-chain triacylglycerols. This was correlated with the in vitro properties of cellular triacylglycerol-hydrolyzing enzymes characterized by their substrate specificity and their susceptibility to inhibitors; the neutral lipase specific to long-chain triacylglycerols is inhibited by PCMB, but not by E600, in contrast to short/medium-chain lipase, which is inhibited by E600 but not by PCMB. The data of in vitro and in situ experiments suggest the existence in fibroblasts of two separate acyl chain length-dependent pathways involved in the degradation of cytoplasmic triacylglycerols, one mediated by a neutral long-chain lipase and another one mediated by a short/medium-chain lipase.
来自患有名为中性脂质贮积病（NLSD）的遗传性代谢紊乱患者的培养成纤维细胞表现出细胞质三酰甘油的显著积累（拉多姆，J.，萨尔韦尔，R.，内格雷，A.，马雷，A.，和杜斯特 - 布拉齐，L.（1987年）《欧洲生物化学杂志》164卷，703 - 708页）。我们在此比较了这些细胞中放射性标记的短链、中链和长链脂肪酸的代谢情况。短/中链脂肪酸（C4 - C10）掺入极性脂质（60 - 80%）和三酰甘油（20 - 40%）的速率比长链脂肪酸低（低5 - 10倍）。脉冲追踪实验能够评估正常和成纤维细胞中细胞质三酰甘油的降解速率，并区分细胞质三酰甘油的两种分解代谢途径。短/中链（C4 - C10）三酰甘油在NLSD成纤维细胞中以正常速率降解，而长链（C12及更长）三酰甘油仍未降解。这些数据通过质谱分析得到证实。使用对氧磷（E600）和对氯汞苯甲酸（PCMB）抑制剂能够区分两种三酰甘油降解途径。E600选择性抑制短/中链三酰甘油的原位降解，而不抑制长链三酰甘油的降解，而PCMB选择性抑制长链三酰甘油的原位水解，同时不影响短/中链三酰甘油的降解。这与细胞三酰甘油水解酶的体外特性相关，这些酶的特征在于它们的底物特异性及其对抑制剂的敏感性；与短/中链脂肪酶相反，对长链三酰甘油特异的中性脂肪酶被PCMB抑制，但不被E600抑制，短/中链脂肪酶被E600抑制，但不被PCMB抑制。体外和原位实验的数据表明，成纤维细胞中存在两种独立的、依赖酰基链长度的途径参与细胞质三酰甘油的降解，一种由中性长链脂肪酶介导，另一种由短/中链脂肪酶介导。
