Kinlaw W B, Church J L, Harmon J, Mariash C N
Department of Medicine, Dartmouth Medical School, Lebanon, New Hampshire 03750, USA.
J Biol Chem. 1995 Jul 14;270(28):16615-8. doi: 10.1074/jbc.270.28.16615.
"Spot 14" is a nuclear protein that is rapidly induced by thyroid hormone (T3) and dietary carbohydrate in liver. We used an antisense oligonucleotide to inhibit induction of spot 14 protein by T3 and glucose in primary cultures of rat hepatocytes to test the hypothesis that the protein could function in the regulation of lipid synthesis. Spot 14 protein was undetectable in hepatocytes maintained in 5.5 mM glucose without T3, and was induced within 4 h after addition of 27.5 mM glucose and 50 nM T3 to the culture medium, reaching a maximal level within 24 h. Accumulation of spot 14 protein was markedly inhibited in hepatocytes transfected with a spot 14 antisense oligonucleotide, but not in those treated with a control oligonucleotide. Transfection of the antisense, but not control, oligonucleotide also abrogated the increase in lipogenesis induced by T3 and glucose. Reduced triglyceride formation accounted for the diminished net lipid synthesis. In contrast to lipogenesis, glucose uptake was not significantly affected by the transfections. Antisense transfection inhibited the induction of both ATP-citrate lyase and fatty acid synthase immunoreactivities, as well as malic enzyme activity, indicating that the observed reduction in lipogenesis could be explained by diminished cellular content of lipogenic enzymes. Reduced malic enzyme activity in antisense-transfected hepatocytes was accompanied by lowered relative abundance of malic enzyme mRNA, suggesting that the antisense effects on lipogenic enzymes were mediated at the pretranslational level. The oligonucleotides did not significantly affect lipogenesis in a rat hepatoma cell line that does not express detectable spot 14 mRNA or protein. These data directly implicate the spot 14 protein in the transduction of hormonal and dietary signals for increased lipid metabolism in hepatocytes.
“斑点14”是一种核蛋白，在肝脏中可被甲状腺激素（T3）和膳食碳水化合物快速诱导产生。我们使用反义寡核苷酸抑制原代培养的大鼠肝细胞中T3和葡萄糖对斑点14蛋白的诱导作用，以检验该蛋白可能在脂质合成调节中发挥作用的假说。在不含T3且葡萄糖浓度为5.5 mM的培养基中培养的肝细胞中检测不到斑点14蛋白，而向培养基中添加27.5 mM葡萄糖和50 nM T3后，该蛋白在4小时内被诱导产生，并在24小时内达到最高水平。用斑点14反义寡核苷酸转染的肝细胞中，斑点14蛋白的积累受到显著抑制，但用对照寡核苷酸处理的肝细胞则没有这种现象。反义寡核苷酸（而非对照寡核苷酸）的转染也消除了T3和葡萄糖诱导的脂肪生成增加。甘油三酯形成减少导致净脂质合成减少。与脂肪生成不同，转染对葡萄糖摄取没有显著影响。反义转染抑制了ATP-柠檬酸裂解酶和脂肪酸合酶免疫反应性的诱导以及苹果酸酶活性，这表明观察到的脂肪生成减少可能是由于生脂酶的细胞含量减少所致。反义转染的肝细胞中苹果酸酶活性降低，同时苹果酸酶mRNA的相对丰度也降低，这表明反义对生脂酶的作用是在翻译前水平介导的。这些寡核苷酸对不表达可检测到的斑点14 mRNA或蛋白的大鼠肝癌细胞系中的脂肪生成没有显著影响。这些数据直接表明，斑点14蛋白参与了肝细胞中激素和膳食信号转导以增加脂质代谢的过程。
