Dynamic regulation of hepatic SIK1 during fasting and feeding

描述（由申请人提供）：空腹期间肝葡萄糖输出量升高是2型糖尿病的主要原因，影响10-25%的美国人。cAMP反应元件结合蛋白CREB及其共激活因子CRTC 2通过在禁食期间转录诱导限速性促血糖生成酶来调节肝葡萄糖输出。鉴于CREB活性在糖尿病啮齿动物中异常激活，我们的目标是确定抑制该途径的分子机制。最近在小鼠模型和培养肝细胞中的研究表明，AMPK相关激酶家族（称为盐诱导激酶（SIK 1 -3））在禁食和再喂养期间控制CRTC共激活因子功能。其中，SIK 1是独特的，因为禁食刺激诱导肝脏SIK 1 mRNA转录。然后，SIK 1蛋白反馈抑制CREB/CRTC 2和餐后葡萄糖输出。这个循环在餐后期间重置，在此期间，SIK 1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SIK 1表达的时间性质及其对肝脏葡萄糖输出的有效抑制作用，我们假设SIK 1活性的时间受到禁食至进食过渡期间调节降解的限制。我们建议使用各种分子和遗传技术来测试这一假设。我们将研究肝细胞和肝组织中SIK 1活性的动态，并探索SIK 1调节其降解的结构决定因素。我们还将测试候选E3泛素连接酶在餐后期间限制SIK 1活性的作用。最后，我们将表征小鼠肝脏特异性Sik 1基因缺失导致的代谢表型。我们已经为这些研究产生了几种转基因动物品系，包括转基因荧光素酶报告小鼠，以在体内观察CREB依赖性转录。这项工作的目标是确定如何动态调节SIK 1有助于维持葡萄糖稳态，并确定新的分子机制，其中SIK 1活性的调节。对这一通路的分子深入了解将揭示抑制糖尿病患者肝葡萄糖输出的新治疗策略。 公共卫生相关性：血糖调节不当是糖尿病的标志，糖尿病是美国日益严重的公共卫生问题。肝脏是发生葡萄糖调节的主要器官。该项目涉及一种酶的分子调节，该酶通常在肝脏中起作用以关闭葡萄糖的产生。本研究旨在确定治疗糖尿病的新治疗靶点。

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.