Fukuda H, Noguchi T, Iritani N
Tezukayama Gakuin University, 4-cho, Harumidai, Sakai, Osaka, Japan.
FEBS Lett. 1999 Dec 31;464(3):113-7. doi: 10.1016/s0014-5793(99)01700-7.
When two copies of the sequences spanning -57 to -35 of the fatty acid synthase (FAS) or -64 to -41 of the ATP citrate-lyase (ACL) gene linked to a reporter gene were transfected into primary cultured hepatocytes, the reporter activities significantly increased in response to insulin/glucose treatment. In cotransfection experiments of the FAS(-57/-35) with the Sp1 or Sp3 expression vector, the reporter activities of transcription were suppressed by Sp1 and stimulated by Sp3. In the cotransfection experiments of ACL(-64/-41), the activities were suppressed by Sp1 but were unchanged by Sp3. A similar effect of Sp1 and Sp3 on transcription was seen in mRNA concentrations and enzyme activities of endogenous FAS and ACL. Moreover, the mRNA concentrations and enzyme activities of endogenous acetyl-CoA carboxylase were suppressed by Sp1 and greatly increased by Sp3. Gel mobility super shift assays using antibodies against Sp1 or Sp3 revealed the binding of the transcription factors Sp1 and Sp3 with the GC rich regions located within FAS(-57/-35) and ACL(-64/-41) genes. The formation of DNA-protein complexes was decreased in rats fed a high-carbohydrate diet in comparison with that in fasted rats, but feeding the corn oil diet inhibited this decrease. In Western immunoblotting assay, however, the amount of Sp1 and Sp3 remained unchanged in the dietary conditions. Therefore, the binding of DNA-protein complexes was not due to changes in the amount of Sp1 and Sp3 but to changes in the binding activity, suggesting that these transcription factors may be an important determinant of lipogenic enzyme expression.
当将与报告基因相连的脂肪酸合酶（FAS）-57至-35或ATP柠檬酸裂解酶（ACL）基因-64至-41的两个序列拷贝转染到原代培养的肝细胞中时，报告基因活性在胰岛素/葡萄糖处理后显著增加。在FAS（-57 / -35）与Sp1或Sp3表达载体的共转染实验中，Sp1抑制转录的报告基因活性，而Sp3则刺激该活性。在ACL（-64 / -41）的共转染实验中，Sp1抑制活性，但Sp3对其无影响。Sp1和Sp3对转录的类似作用在内源性FAS和ACL的mRNA浓度及酶活性中也可见到。此外，Sp1抑制内源性乙酰辅酶A羧化酶的mRNA浓度和酶活性，而Sp3则使其大幅增加。使用抗Sp1或Sp3抗体的凝胶迁移超迁移分析揭示了转录因子Sp1和Sp3与位于FAS（-57 / -35）和ACL（-64 / -41）基因内富含GC区域的结合。与禁食大鼠相比，高碳水化合物饮食喂养的大鼠中DNA-蛋白质复合物的形成减少，但玉米油饮食喂养可抑制这种减少。然而，在蛋白质免疫印迹分析中，Sp1和Sp3的量在不同饮食条件下保持不变。因此，DNA-蛋白质复合物的结合不是由于Sp1和Sp3量的变化，而是由于结合活性的变化，这表明这些转录因子可能是脂肪生成酶表达的重要决定因素。
