O'Brien R M, Noisin E L, Granner D K
Department of Molecular Physiology and Biophysics, Vanderbilt University Medical School, Nashville 37232-0615.
Biochem J. 1994 Nov 1;303 ( Pt 3)(Pt 3):737-42. doi: 10.1042/bj3030737.
Many hormones regulate the rate of synthesis of phosphoenolpyruvate carboxykinase (PEPCK), the enzyme that governs the rate-limiting step in gluconeogenesis. In H4IIE rat hepatoma cells, glucocorticoids, retinoic acid and cyclic AMP (cAMP) increase PEPCK gene transcription whereas insulin and phorbol esters have the opposite effect. Insulin and phorbol esters are dominant as they prevent cAMP- and glucocorticoid-stimulated PEPCK gene transcription. In contrast, insulin and phorbol esters both stimulate transcription of gene 33 in the same H4IIE cells, with the same time course as seen for their inhibitory effect on PEPCK gene transcription. We now report that the protein phosphatase inhibitor, okadaic acid, mimics the action of insulin and phorbol esters on expression of both gene 33 and PEPCK gene in H4IIE cells. Okadaic acid stimulates gene 33 mRNA accumulation whereas it inhibits cAMP- and glucocorticoid-stimulated PEPCK mRNA accumulation. The effect of okadaic acid on the PEPCK gene is mediated through the PEPCK promoter as, in a cell line, HL1C, stably transfected with a PEPCK-chloramphenicol acetyltransferase (CAT) fusion gene, okadaic acid inhibits cAMP- and glucocorticoid-stimulated CAT expression. Desensitization of the protein kinase C pathway by exposure to phorbol 12-myristate 13-acetate for 16 h abolishes the subsequent action of the phorbol ester but does not markedly affect the inhibition of cAMP- and glucocorticoid-stimulated CAT expression by insulin or okadaic acid. Even though insulin and okadaic acid appear to repress PEPCK gene expression through a pathway initially distinct from that used by phorbol esters, transient-transfection studies show that the final target of the action of okadaic acid, insulin and phorbol ester is the same DNA element.
许多激素调节磷酸烯醇丙酮酸羧激酶（PEPCK）的合成速率，该酶控制糖异生过程中的限速步骤。在H4IIE大鼠肝癌细胞中，糖皮质激素、视黄酸和环磷酸腺苷（cAMP）可增加PEPCK基因转录，而胰岛素和佛波酯则具有相反作用。胰岛素和佛波酯起主导作用，因为它们可阻止cAMP和糖皮质激素刺激的PEPCK基因转录。相比之下，胰岛素和佛波酯在相同的H4IIE细胞中均刺激33号基因的转录，其时间进程与它们对PEPCK基因转录的抑制作用相同。我们现在报告，蛋白磷酸酶抑制剂冈田酸可模拟胰岛素和佛波酯对H4IIE细胞中33号基因和PEPCK基因表达的作用。冈田酸刺激33号基因mRNA积累，而抑制cAMP和糖皮质激素刺激的PEPCK mRNA积累。冈田酸对PEPCK基因的作用是通过PEPCK启动子介导的，因为在稳定转染了PEPCK-氯霉素乙酰转移酶（CAT）融合基因的HL1C细胞系中，冈田酸抑制cAMP和糖皮质激素刺激的CAT表达。通过暴露于佛波醇12-肉豆蔻酸酯13-乙酸酯16小时使蛋白激酶C途径脱敏，可消除随后佛波酯的作用，但对胰岛素或冈田酸抑制cAMP和糖皮质激素刺激的CAT表达没有明显影响。尽管胰岛素和冈田酸似乎通过一条最初不同于佛波酯所使用的途径来抑制PEPCK基因表达，但瞬时转染研究表明，冈田酸、胰岛素和佛波酯作用的最终靶点是相同的DNA元件。
