Gabbay R A, Sutherland C, Gnudi L, Kahn B B, O'Brien R M, Granner D K, Flier J S
Charles A. Dana Laboratories, Harvard-Thorndike Department of Medicine, Beth Israel Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1996 Jan 26;271(4):1890-7. doi: 10.1074/jbc.271.4.1890.
Expression of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting step in hepatic gluconeogenesis, is primarily regulated at the level of gene transcription. Insulin and phorbol esters inhibit basal PEPCK transcription and antagonize the induction of PEPCK gene expression by glucocorticoids and glucagon (or its second messenger cAMP). Insulin activates a signaling cascade involving Ras --> Raf --> p42/p44 mitogen-activated protein (MAP) kinase kinase (MEK) --> p42/p44 MAP kinase (ERK 1 and 2). Recent reports suggest that activation of this Ras/MAP kinase pathway is critical for the effects of insulin on mitogenesis and c-fos transcription but is not required for insulin action on metabolic processes such as glycogen synthesis, lipogenesis, and Glut-4-mediated glucose transport. We have used three distinct approaches to examine the role of the Ras/MAP kinase pathway in the regulation of PEPCK transcription by insulin in H4IIE-derived liver cells: (i) chemical inhibition of Ras farnesylation, (ii) infection of cells with an adenovirus vector encoding a dominant-negative mutant of Ras, and (iii) use of a chemical inhibitor of MEK. Although each of these methods blocks insulin activation of MAP kinase, none alters insulin antagonism of cAMP- and glucocorticoid-stimulated PEPCK transcription. Although phorbol esters activate MAP kinase and mimic the effects of insulin on PEPCK gene transcription, inhibition of MEK has no effect on phorbol ester inhibition of PEPCK gene transcription. Using the structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin and LY 294002, we provide further evidence supporting a role for PI 3-kinase activation in the regulation of PEPCK gene transcription by insulin. We conclude that neither insulin nor phorbol ester regulation of PEPCK gene transcription requires activation of the Ras/MAP kinase pathway and that insulin signaling to the PEPCK promoter is dependent on PI 3-kinase activation.
磷酸烯醇式丙酮酸羧激酶（PEPCK）是肝脏糖异生作用中的限速步骤，其表达主要在基因转录水平受到调控。胰岛素和佛波酯可抑制基础PEPCK转录，并拮抗糖皮质激素和胰高血糖素（或其第二信使cAMP）对PEPCK基因表达的诱导作用。胰岛素激活了一条信号级联反应，涉及Ras→Raf→p42/p44丝裂原活化蛋白（MAP）激酶激酶（MEK）→p42/p44 MAP激酶（ERK 1和2）。最近的报道表明，这条Ras/MAP激酶途径的激活对于胰岛素在有丝分裂和c-fos转录方面的作用至关重要，但对于胰岛素在诸如糖原合成、脂肪生成和Glut-4介导的葡萄糖转运等代谢过程中的作用并非必需。我们采用了三种不同的方法来研究Ras/MAP激酶途径在H4IIE衍生的肝细胞中胰岛素对PEPCK转录调控中的作用：（i）化学抑制Ras法尼基化；（ii）用编码Ras显性负性突变体的腺病毒载体感染细胞；（iii）使用MEK的化学抑制剂。尽管这些方法中的每一种都能阻断胰岛素对MAP激酶的激活，但没有一种能改变胰岛素对cAMP和糖皮质激素刺激的PEPCK转录的拮抗作用。尽管佛波酯激活MAP激酶并模拟胰岛素对PEPCK基因转录的影响，但抑制MEK对佛波酯抑制PEPCK基因转录没有影响。使用结构和作用机制不同的磷脂酰肌醇3激酶（PI 3激酶）抑制剂渥曼青霉素和LY 294002，我们提供了进一步的证据，支持PI 3激酶激活在胰岛素对PEPCK基因转录调控中的作用。我们得出结论，胰岛素和佛波酯对PEPCK基因转录的调控均不需要激活Ras/MAP激酶途径，并且胰岛素向PEPCK启动子的信号传导依赖于PI 3激酶的激活。
