Insulin Signaling and Metabolic Effects through CLK2 Kinase

描述（由申请人提供）：营养稳态是通过控制代谢基因的信号和转录成分形成的复杂调控网络来维持的。肝脏是根据生理/病理条件缓冲全身营养平衡的关键组织之一。胰岛素是影响营养调节的最强大的激素之一，在临床上，胰岛素抵抗是包括2型糖尿病在内的代谢综合征的标志。作为胰岛素抵抗的后果之一，维持糖尿病状态的代谢过程之一是肝脏葡萄糖的产生，至少部分是在转录水平上控制的。PI3K/Akt通路是胰岛素代谢作用的主要效应器之一。Akt通过直接磷酸化和负调控叉头转录因子FoxO1和共激活因子PGC-11和CRCT2（转录糖异生程序的关键组分）来控制代谢基因的表达。虽然Akt可以直接介导这一作用，但在晚期再进食或糖尿病状态等情况下，活性Akt并不完全与抑制肝糖生成相关。这表明额外的关键调控成分，如激酶，可能介导这种抑制。沿着这些思路，我们最近发现了cdc2样激酶2 （Clk2）作为胰岛素/Akt下游的一个新成分，并作为肝脏摄食反应的一部分发挥作用。值得注意的是，Clk2控制糖异生基因的表达、肝糖输出和血糖水平。此外，肥胖/糖尿病db/db小鼠的Clk2蛋白含量较低，恢复水平可纠正高血糖。这一结果表明，Clk2可能在肥胖/糖尿病的条件下失调，并有助于临床表现。基于这些发现，本提案的主要目标是确定胰岛素控制Clk2激酶活性的分子机制，并在体外和体内小鼠模型中测试Clk2代谢功能。我们有三个具体目标：目标1是进行胰岛素如何控制Clk2激酶活性的分子和功能分析，重点是通过泛素连接酶调节Clk2蛋白降解。目的2致力于Clk2介导的肝脏糖异生抑制的分子和功能分析。我们关注的是clk2诱导的PGC-11的磷酸化和抑制。Aim 3将通过禁食/喂养周期中肝脏Clk2功能丧失以及遗传和饮食诱导的肥胖来确定Clk2对小鼠肝脏糖脂代谢的影响。这些研究的结果将提供胰岛素通过Clk2激酶控制糖脂代谢作用的分子机制的鉴定。由于胰岛素抵抗以及由此导致的肝糖输出增加和不受控制是2型糖尿病的主要缺陷，我们对Clk2和PGC-11调节的研究可能转化为治疗这种疾病的潜在疗法。

DESCRIPTION (provided by applicant): Nutrient homeostasis is maintained through a complex regulatory network formed by signaling and transcriptional components that control metabolic genes. The liver is one of the key tissues that depending of the physio/pathological conditions buffers whole body nutrient homeostasis. Insulin is one of the most powerful hormones that affect nutrient regulation and clinically, insulin resistance is a hallmark of the metabolic syndrome including type 2 diabetes. As a consequence of insulin resistance one of the metabolic processes that contribute to maintain the diabetic state is hepatic glucose production that is controlled, at least in part, at the transcriptional level. The PI3K/Akt pathway is one of the main effectors of insulin metabolic action. Akt controls expression of metabolic genes through direct phosphorylation and negative regulation of the forkhead transcription factor FoxO1 and coactivators such as PGC-11 and CRCT2, key components of the transcriptional gluconeogenic program. Although Akt can directly mediate this action, there are conditions such as late refeeding or diabetic states where active Akt does not entirely correlate with suppression hepatic glucose production. This indicates that additional key regulatory components, likely kinases, could mediate this repression. Along these lines, we have recently identified Cdc2-like kinase 2 (Clk2) as a novel component downstream of insulin/Akt and functions as part of the hepatic feeding response. Notably, Clk2 controls expression of gluconeogenic genes, hepatic glucose output and blood glucose levels. Moreover, obese/diabetic db/db mice have lower amounts of Clk2 protein and restoration of the levels corrects hyperglycemia. This result suggests that Clk2 might be dysregulated in conditions of obesity/diabetes and contributes to the clinical manifestations. Based on these findings, the major goal of this proposal is to identify the molecular mechanisms by which insulin controls Clk2 kinase activity and to test Clk2 metabolic functionality in-vitro and in in-vivo mouse models. We have three Specific Aims: Aim 1 is to perform molecular and functional analysis of how insulin controls Clk2 kinase activity focusing on regulation of Clk2 protein degradation through ubiquitin ligases. Aim 2 is devoted to carry out molecular and functional analysis of Clk2- mediated suppression of hepatic gluconeogenesis. We focus on the Clk2-induced phosphorylation and repression of PGC-11. Aim 3 will determine the effects of Clk2 on hepatic glucose and lipid metabolism in mice through loss-of-function of hepatic Clk2 in fasting/feeding cycles and genetic and diet-induced obesity. The outcomes of these studies will provide the identification of the molecular mechanisms by which insulin controls glucose and lipid metabolic effects through Clk2 kinase. Since insulin resistance and as a consequence increased and uncontrolled hepatic glucose output is a major defect that occurs in type 2 diabetes, our investigation on the regulation of Clk2 and PGC-11 might translate into potential therapies to treat this disease.