Kempen H J, Imbach A P, Giller T, Neumann W J, Hennes U, Nakada N
F. Hoffmann-La Roche Ltd., Pharma Division Preclinical Research, Basel, Switzerland.
J Lipid Res. 1995 Aug;36(8):1796-806.
It was the aim of this study to i) compare the effects of glucose and other hexoses with that of oleate on secretion of apolipoproteins (apos) A-I and B by HepG2 cells, and ii) document the effect of various metabolic inhibitors on the secretion of these apos in the absence or presence of extra glucose/oleate. i) The addition of 10 mM glucose increased secretion of apoA-I and apoB, as measured by enzyme immunoassay, by about 60% when cells were incubated for 48 h in DMEM + 10% fetal calf serum. The addition of extra glucose also increased the mRNA levels for these apos. Increased radioactivity was also found in these apolipoproteins by immunoprecipitation after metabolic labeling with [35S]methionine for 48 h. However, in a pulse-chase experiment (15 min labeling, 2 h chase), glucose was found to increase apoA-I synthesis but not apoB synthesis. More labeled apoB appeared in the medium during the chase because glucose inhibited its intracellular degradation. The effect of glucose on secretion of these apos could be mimicked by fructose and mannose but not by 6-deoxyglucose, showing that the hexoses must enter the cells and be phosphorylated. In contrast, the addition of 0.5 mM oleate had a weak inhibitory effect on secretion of apoA-I whereas it increased the secretion of apoB by more than twofold. The combination of 10 mM glucose and 0.5 mM oleate had no greater effect than glucose alone on apoA-I secretion but increased apoB secretion by fourfold. ii) Inhibiting glycolysis (by glucosamine) lowered secretion of both apoA-I and apoB, while inhibiting lipogenesis (using 8-Br-cyclic AMP or 5-(tetradecyloxy)-2-furancarboxylic acid (TOFA)) did not affect apoA-I secretion but clearly decreased that of apoB. However, the inhibitory effect of TOFA on apoB secretion was much smaller in the presence of 0.5 mM oleate instead of extra glucose. Actinomycin-D and cycloheximide strongly suppressed the stimulatory effect of glucose on secretion of both apolipoproteins. Actinomycin-D also suppressed basal secretion of apoA-I but surprisingly stimulated that of apoB. These observations indicate that in HepG2 cells secretion of apoA-I is strongly dependent on ongoing protein synthesis and can be boosted by glucose, whereas that of apoB is primarily driven by internal (via lipogenesis from glucose) or external supply of fatty acyl-residues.
i）比较葡萄糖和其他己糖与油酸对HepG2细胞载脂蛋白（apo）A-I和B分泌的影响；ii）记录各种代谢抑制剂在有无额外葡萄糖/油酸存在时对这些载脂蛋白分泌的影响。i）通过酶免疫测定法测得，当细胞在含10%胎牛血清的DMEM中孵育48小时时，添加10 mM葡萄糖可使apoA-I和apoB的分泌增加约60%。额外添加葡萄糖也会增加这些载脂蛋白的mRNA水平。在用[35S]甲硫氨酸代谢标记48小时后，通过免疫沉淀法在这些载脂蛋白中也发现放射性增加。然而，在脉冲追踪实验（15分钟标记，2小时追踪）中，发现葡萄糖可增加apoA-I的合成，但不增加apoB的合成。在追踪过程中，更多标记的apoB出现在培养基中，因为葡萄糖抑制了其细胞内降解。果糖和甘露糖可模拟葡萄糖对这些载脂蛋白分泌的影响，但6-脱氧葡萄糖则不能，这表明己糖必须进入细胞并被磷酸化。相比之下，添加0.5 mM油酸对apoA-I的分泌有微弱的抑制作用，而对apoB的分泌则增加了两倍多。10 mM葡萄糖和0.5 mM油酸的组合对apoA-I分泌的影响并不比单独使用葡萄糖时更大，但使apoB的分泌增加了四倍。ii）抑制糖酵解（通过氨基葡萄糖）会降低apoA-I和apoB的分泌，而抑制脂肪生成（使用8-溴环磷酸腺苷或5-（十四烷氧基）-2-呋喃羧酸（TOFA））并不影响apoA-I的分泌，但明显降低了apoB的分泌。然而，在存在0.5 mM油酸而非额外葡萄糖的情况下，TOFA对apoB分泌的抑制作用要小得多。放线菌素-D和环己酰亚胺强烈抑制葡萄糖对两种载脂蛋白分泌的刺激作用。放线菌素-D也抑制apoA-I的基础分泌，但令人惊讶的是，它刺激了apoB的基础分泌。这些观察结果表明，在HepG2细胞中，apoA-I的分泌强烈依赖于正在进行的蛋白质合成，并且可被葡萄糖促进，而apoB的分泌主要由内部（通过葡萄糖的脂肪生成）或外部脂肪酸残基供应驱动。
