Fukuda H, Iritani N
Faculty of Human and Cultural Studies, Tezukayama Gakuin University, Sakai, Osaka, Japan.
FEBS Lett. 1999 Jul 16;455(1-2):165-9. doi: 10.1016/s0014-5793(99)00877-7.
To investigate the DNA regulatory sequences required for stimulation and suppression of leptin gene expression, primary cultured hepatocytes and adipocytes of rats were transfected with plasmids containing the 5'-flanking sequences of the rat leptin gene fused to the luciferase gene. When two copies of the sequences spanning nucleotides -101 to -83 of the leptin promoter were used for transfection, the reporter activity significantly increased in the presence of glucose/insulin in comparison with glucose alone. The glucose/insulin stimulation of the transcription was inhibited by addition of polyunsaturated fatty acids. These results were similar to those found earlier for the transcription of the fatty acid synthase, FAS(-57/-35) and ATP citrate-lyase, ACL(-64/-41) genes. Cotransfection studies in the cells with a Sp1 expression vector and leptin (-101/-83) constructs showed the inactivation of the leptin promoter by Sp1. Gel mobility shift assays using an end-labeled leptin (-101/-83) construct as a probe revealed that nuclear factor(s) from rat liver or adipose tissue specifically formed complexes with the sequence. The DNA-protein complexes were common to the glucose/insulin-responsive regions of the leptin, ACL and FAS genes, suggesting that these genes are coordinately regulated. In addition, by antibody supershift assays, the transcription factor Sp1 was found to bind the GC-rich region located between nucleotides -101 and -83 of the leptin gene. Mutational analysis of this region showed that the sequence of the region was critical for glucose/insulin stimulation of transcription. Thus, we postulated that the region from -101 to -83 of the leptin gene is responsible for glucose/insulin stimulation of transcription, and that Sp1 is somehow involved in this regulation.
为了研究刺激和抑制瘦素基因表达所需的DNA调控序列，将含有与荧光素酶基因融合的大鼠瘦素基因5'侧翼序列的质粒转染到原代培养的大鼠肝细胞和脂肪细胞中。当使用跨越瘦素启动子核苷酸-101至-83的两个拷贝的序列进行转染时，与单独的葡萄糖相比，在葡萄糖/胰岛素存在下报告基因活性显著增加。添加多不饱和脂肪酸可抑制转录的葡萄糖/胰岛素刺激。这些结果与早期脂肪酸合酶FAS（-57 / -35）和ATP柠檬酸裂解酶ACL（-64 / -41）基因转录的结果相似。用Sp1表达载体和瘦素（-101 / -83）构建体在细胞中进行的共转染研究表明Sp1使瘦素启动子失活。使用末端标记的瘦素（-101 / -83）构建体作为探针的凝胶迁移率变动分析表明，来自大鼠肝脏或脂肪组织的核因子与该序列特异性形成复合物。DNA-蛋白质复合物在瘦素、ACL和FAS基因的葡萄糖/胰岛素反应区域中是常见的，表明这些基因是协同调节的。此外，通过抗体超迁移分析，发现转录因子Sp1与位于瘦素基因核苷酸-101和-83之间的富含GC的区域结合。该区域的突变分析表明该区域的序列对于转录的葡萄糖/胰岛素刺激至关重要。因此，我们推测瘦素基因的-101至-83区域负责转录的葡萄糖/胰岛素刺激，并且Sp1以某种方式参与这种调节。
