Insulin Signaling and Metabolic Effects through CLK2 Kinase

描述（由申请人提供）：通过控制代谢基因的信号传导和转录组分形成的复杂调控网络维持营养稳态。肝脏是机体营养平衡的关键组织之一，其功能取决于生理/病理条件。胰岛素是影响营养调节的最强有力的激素之一，临床上，胰岛素抵抗是代谢综合征包括2型糖尿病的标志。作为胰岛素抵抗的结果，有助于维持糖尿病状态的代谢过程之一是在转录水平上至少部分地控制的肝葡萄糖产生。PI 3 K/Akt通路是胰岛素代谢作用的主要效应子之一。Akt通过直接磷酸化和叉头转录因子FoxO 1和辅激活因子如PGC-11和CRCT 2（转录致突变程序的关键组分）的负调节来控制代谢基因的表达。虽然Akt可以直接介导这种作用，但在某些情况下，如晚期再喂养或糖尿病状态，活性Akt与抑制肝葡萄糖产生并不完全相关。这表明，额外的关键调控成分，可能是激酶，可以介导这种抑制。沿着这些路线，我们最近确定Cdc 2样激酶2（Clk 2）作为胰岛素/Akt下游的一种新的组分，并作为肝脏摄食反应的一部分发挥作用。值得注意的是，Clk 2控制致炎基因的表达、肝葡萄糖输出和血糖水平。此外，肥胖/糖尿病db/db小鼠具有较低量的Clk 2蛋白，并且水平的恢复校正高血糖症。这一结果表明，Clk 2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Clk 2激酶活性的分子机制，并在体外和体内小鼠模型中测试Clk 2代谢功能。我们有三个具体目标：目的1是进行胰岛素如何控制Clk 2激酶活性的分子和功能分析，重点是通过泛素连接酶调节Clk 2蛋白降解。目的二是对Clk 2介导的肝细胞凋亡抑制作用进行分子和功能分析。我们专注于Clk 2诱导的PGC-11的磷酸化和抑制。目的3将通过在禁食/喂养周期中肝脏Clk 2的功能丧失以及遗传和饮食诱导的肥胖来确定Clk 2对小鼠肝脏葡萄糖和脂质代谢的影响。这些研究的结果将提供胰岛素通过Clk 2激酶控制葡萄糖和脂质代谢作用的分子机制的鉴定。由于胰岛素抵抗以及因此增加和不受控制的肝脏葡萄糖输出是2型糖尿病中发生的主要缺陷，因此我们对Clk 2和PGC-11调节的研究可能转化为治疗这种疾病的潜在疗法。 公共卫生相关性：胰岛素抵抗和肝葡萄糖产生增加是肥胖和2型糖尿病等代谢疾病的标志，因此这项资助提案中的研究重点是这些过程的关键调节因子，包括新型激酶Clk 2和PGC-11，可能会转化为潜在的治疗方法。

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. PUBLIC HEALTH RELEVANCE: Insulin resistance and increased hepatic glucose production are hallmarks of metabolic diseases such as obesity and type 2 diabetes, thus studies in this grant proposal focusing on key regulators of these processes including a novel kinase Clk2 and PGC-11 might translate into potential therapies.