Itoh Yumi, Sanosaka Masato, Fuchino Hiroyuki, Yahara Yasuhito, Kumagai Ayako, Takemoto Daisaku, Kagawa Mai, Doi Junko, Ohta Miho, Tsumaki Noriyuki, Kawahara Nobuo, Takemori Hiroshi
Laboratory of Cell Signaling and Metabolic Disease, National Institute of Biomedical Innovation, Osaka, 567-0085, Japan.
Research Center for Medicinal Plant Resources, Tsukuba Division, Ibaraki, 305-0843, Japan.
J Biol Chem. 2015 Jul 17;290(29):17879-17893. doi: 10.1074/jbc.M115.640821. Epub 2015 Jun 5.
Salt-inducible kinases (SIKs), members of the 5'-AMP-activated protein kinase (AMPK) family, are proposed to be important suppressors of gluconeogenic programs in the liver via the phosphorylation-dependent inactivation of the CREB-specific coactivator CRTC2. Although a dramatic phenotype for glucose metabolism has been found in SIK3-KO mice, additional complex phenotypes, dysregulation of bile acids, cholesterol, and fat homeostasis can render it difficult to discuss the hepatic functions of SIK3. The aim of this study was to examine the cell autonomous actions of SIK3 in hepatocytes. To eliminate systemic effects, we prepared primary hepatocytes and screened the small compounds suppressing SIK3 signaling cascades. SIK3-KO primary hepatocytes produced glucose more quickly after treatment with the cAMP agonist forskolin than the WT hepatocytes, which was accompanied by enhanced gluconeogenic gene expression and CRTC2 dephosphorylation. Reporter-based screening identified pterosin B as a SIK3 signaling-specific inhibitor. Pterosin B suppressed SIK3 downstream cascades by up-regulating the phosphorylation levels in the SIK3 C-terminal regulatory domain. When pterosin B promoted glucose production by up-regulating gluconeogenic gene expression in mouse hepatoma AML-12 cells, it decreased the glycogen content and stimulated an association between the glycogen phosphorylase kinase gamma subunit (PHKG2) and SIK3. PHKG2 phosphorylated the peptides with sequences of the C-terminal domain of SIK3. Here we found that the levels of active AMPK were higher both in the SIK3-KO hepatocytes and in pterosin B-treated AML-12 cells than in their controls. These results suggest that SIK3, rather than SIK1, SIK2, or AMPKs, acts as the predominant suppressor in gluconeogenic gene expression in the hepatocytes.
盐诱导激酶（SIKs）是5'-AMP激活蛋白激酶（AMPK）家族的成员，被认为是肝脏中糖异生程序的重要抑制因子，通过依赖磷酸化使CREB特异性共激活因子CRTC2失活。尽管在SIK3基因敲除（KO）小鼠中发现了显著的葡萄糖代谢表型，但其他复杂表型，如胆汁酸、胆固醇和脂肪稳态的失调，使得讨论SIK3的肝脏功能变得困难。本研究的目的是检测SIK3在肝细胞中的细胞自主作用。为了消除全身影响，我们制备了原代肝细胞，并筛选了抑制SIK3信号级联反应的小分子化合物。与野生型（WT）肝细胞相比，用环磷酸腺苷（cAMP）激动剂福斯高林处理后，SIK3-KO原代肝细胞更快地产生葡萄糖，同时糖异生基因表达增强，CRTC2去磷酸化。基于报告基因的筛选确定蝶酸B为SIK3信号特异性抑制剂。蝶酸B通过上调SIK3 C末端调节域的磷酸化水平来抑制SIK3下游级联反应。当蝶酸B通过上调小鼠肝癌AML-12细胞中的糖异生基因表达来促进葡萄糖生成时，它降低了糖原含量，并刺激糖原磷酸化酶激酶γ亚基（PHKG2）与SIK3之间的结合。PHKG2使具有SIK3 C末端结构域序列的肽磷酸化。我们发现，SIK3-KO肝细胞和经蝶酸B处理的AML-12细胞中活性AMPK的水平均高于各自的对照。这些结果表明，在肝细胞糖异生基因表达中，起主要抑制作用的是SIK3，而不是SIK1、SIK2或AMPKs。
