Dynamic regulation of hepatic SIK1 during fasting and feeding

描述(由申请人提供)：禁食期间肝脏葡萄糖排出量升高是2型糖尿病的主要诱因，影响10%-25%的美国人。CAMP反应元件结合蛋白CREB及其共激活子CRTC2在禁食期间通过转录诱导限速糖异生酶来调节肝脏的葡萄糖输出。鉴于CREB活性在糖尿病啮齿动物中异常激活，我们的目标是确定这一途径被抑制的分子机制。最近在小鼠模型和培养的肝细胞中的研究表明，AMPK相关的一族称为盐诱导蛋白激酶(SIK1-3)，在禁食和再喂养过程中控制CRTC共激活功能。其中，SIK1的独特之处在于禁食刺激诱导肝脏SIK1基因转录。SIK1蛋白随后反馈抑制CREB/CRTC2和餐后葡萄糖输出。这个循环在餐后期间被重置，在此期间SIK1蛋白水平下降，从而允许CREB在下一次禁食开始时重新激活。基于SIK1表达的时间性及其对肝脏葡萄糖输出的有效抑制作用，我们假设SIK1活性的时间受到禁食到喂养转变过程中调节降解的限制。我们建议使用各种分子和基因技术来检验这一假说。我们将研究肝细胞和肝组织中SIK1活性的动态变化，并探索调节其降解的SIK1结构决定因素。我们还将测试候选E3泛素连接酶在餐后限制SIK1活性方面的作用。最后，我们将描述小鼠肝脏特异性SIK1基因缺失所导致的代谢表型。我们已经为这些研究创造了几个转基因动物品系，包括转基因荧光素酶报告小鼠，以在体内可视化CREB依赖的转录。这项工作的目的是确定SIK1的动态调节如何有助于维持血糖稳态，并确定SIK1活性调节的新的分子机制。对这一途径的分子洞察将揭示抑制糖尿病患者肝脏葡萄糖输出的新治疗策略。 与公共健康相关：血糖调节不当是糖尿病的标志，在美国，糖尿病是一个日益严重的公共健康问题。肝脏是葡萄糖调节的主要器官。这个项目解决了一种酶的分子调控问题，这种酶通常在肝脏中发挥作用，以关闭葡萄糖的产生。本研究旨在寻找治疗糖尿病的新靶点。

DESCRIPTION (provided by applicant): Elevated hepatic glucose output during fasting is a major contributor to type 2 diabetes, which affects 10-25% of Americans. The cAMP response element binding protein CREB and its co-activator CRTC2 regulate hepatic glucose output by transcriptional induction of rate-limiting gluconeogenic enzymes during fasting. Given that CREB activity becomes aberrantly activated in diabetic rodents, we aim to identify molecular mechanisms by which this pathway can be inhibited. Recent studies in mouse models and cultured hepatocytes implicated a family of AMPK-related kinases called Salt-Inducible Kinases (SIK1-3), in the control of CRTC co-activator function during fasting and re-feeding. Among these, SIK1 is unique in that fasting stimuli induce hepatic SIK1 mRNA transcription. SIK1 protein then feeds back to inhibit CREB/CRTC2 and glucose output after a meal. This cycle is reset during the postprandial period, during which SIK1 protein levels decline, thus allowing re- activation of CREB at the onset of the next fast. Based on the temporal nature of SIK1 expression and its potent inhibitory effects on hepatic glucose output, we hypothesize that the timing of SIK1 activity is limited by regulated degradation during the fasting to feeding transition. We propose to test this hypothesis using a variety of molecular and genetic techniques. We will investigate the dynamics of SIK1 activity in hepatocytes and liver tissue and explore the structural determinants of SIK1 that regulate its degradation. We will also test the role of a candidate E3 ubiquitin ligase in limiting SIK1 activity during the postprandial period. Finally, we will characterize metabolic phenotypes resulting from liver-specific deletion of the Sik1 gene in mice. We have generated several transgenic animal strains for these studies, including transgenic luciferase reporter mice to visualize CREB-dependent transcription in vivo. The goal of this work is to determine how dynamic regulation of SIK1 contributes to maintenance of glucose homeostasis and identify new molecular mechanisms by which SIK1 activity is regulated. Molecular insight into this pathway will reveal new therapeutic strategies for inhibition of hepatic glucose output in diabetic patients. PUBLIC HEALTH RELEVANCE: Inappropriate regulation of blood glucose is the hallmark of diabetes mellitus, which is a growing public health problem in the United States. The liver is a major organ in which glucose regulation occurs. This project addresses the molecular regulation of an enzyme that normally functions in the liver to turn off glucose production. This study aims to identify novel therapeutic targets for treatment of diabetes.