MAP Kinase Phosphatase 3 (MKP-3) and Obesity-related Gluconeogenesis

描述（由申请人提供）：肥胖相关的2型糖尿病，以胰岛素抵抗为特征，目前影响全球1.5亿人。在胰岛素抵抗性糖尿病状态中，血糖水平升高是由于肝脏产生的葡萄糖增加（主要通过从头葡萄糖合成，葡萄糖异生）和脂肪和肌肉中葡萄糖处置减少。我们最近发现MAP激酶磷酸酶3（MKP- 3）是拮抗胰岛素对肝脏新生抑制作用的最佳候选者。我们证明了MKP-3在肥胖小鼠肝脏中的表达升高，并且MKP-3过表达足以促进肝细胞中的肝细胞生成并升高瘦小鼠的空腹血糖水平。通过RNA干扰减少MKP-3表达可降低肥胖小鼠肝细胞中的新生血管生成并降低空腹血糖水平。虽然MKP-3的唯一已知底物是ERK，并且ERK通路的激活可以部分抑制促凋亡基因的表达，但是ERK功能的破坏不能逆转胰岛素对促凋亡基因表达的抑制作用。这表明MKP-3通过一种新的机制减弱胰岛素信号传导。叉头转录因子FOXO 1是胰岛素抑制葡萄糖生成的关键因子，它可以通过去磷酸化激活，导致随后的核转位，从而启动促胰岛素生成程序。我们最近的数据表明，MKP-3与FOXO 1相互作用，与FOXO 1协同转录PEPCK和G6 β基因，减少胰岛素刺激的FOXO 1磷酸化，并增加其核转位。此外，显性失活FOXO 1的过表达可以阻断MKP-3刺激的原代肝细胞中的致凋亡基因表达和葡萄糖，并且通过肝MKP-3敲低降低核FOXO 1。有趣的是，MKP-3的促凋亡作用在原代肝细胞中是显著的，而在Fao细胞中是轻度的，Fao细胞缺乏促凋亡放大器PGC-11。在原代肝细胞中敲低PGC-11表达基本上消除了MKP-3对致炎基因表达和葡萄糖输出的影响。我们假设MKP-3通过去磷酸化激活FOXO 1拮抗胰岛素对胚胎发生的抑制作用，PGC-11是关键的共激活剂。具体目标是：1）确定FOXO 1在MKP-3介导的肝脏新生中的作用; 2）调节肝脏MKP-3表达并评估其对新生和血糖控制的影响; 3）了解MKP-3在体内促进空腹高血糖的下游靶点。这些目标将通过在培养的肝细胞、原代肝细胞以及正常和肥胖动物的肝脏中进行报告基因测定和腺病毒介导的功能获得和丧失研究来实现。该提案产生的结果将为理解MKP-3的作用机制提供关键信息，并评估其作为治疗肥胖相关2型糖尿病的新治疗靶点的候选资格。 公共卫生相关性：肥胖相关的2型糖尿病，其特征是身体对胰岛素的反应降低，可归因于肝脏葡萄糖产生增加以及肌肉和脂肪对葡萄糖的利用减少。最近发现一种新的因子在肥胖啮齿动物的肝脏中升高，拮抗胰岛素在肝脏中的作用，促进肝脏葡萄糖的产生，并显著促进肥胖症的高血糖。本提案中概述的研究结果将为了解这种蛋白质的作用机制提供关键信息，并评估其作为治疗肥胖相关2型糖尿病的新治疗靶点的候选资格。

DESCRIPTION (provided by applicant): Obesity-related type 2 diabetes mellitus, characterized by insulin resistance, currently affects 150 million people worldwide. In the insulin resistant diabetic state, blood glucose levels are elevated due to increased glucose production from the liver (mainly through de novo glucose synthesis, gluconeogenesis) and decreased glucose disposal in fat and muscle. We recently identified MAP kinase phosphatase 3 (MKP- 3) as the top candidate for antagonizing the repressive effect of insulin on liver gluconeogenesis. We demonstrated that expression of MKP-3 is elevated in the liver of obese mice and MKP-3 overexpression is sufficent to promote gluconeogenesis in hepatocytes and elevate fasting blood glucose levels in lean mice. Reduction of MKP-3 expression by RNA interference decreases gluconeogenesis in hepatocytes and lowers fasting blood glucose levels in obese mice. Although the only known substrate of MKP-3 is ERK and activation of the ERK pathway can partially repress gluconeogenic gene expression, disruption of ERK function can not reverse the repressive effect of insulin on gluconeogenesis. This indicates that MKP-3 attenuates insulin signaling through a novel mechanism. Forkhead transcription factor FOXO1, which can be activated through dephosphorylation that leads to consequent nuclear translocatin to initiate the gluconeogenic program, is a critical factor for insulin to repress glucose production. Our recent data indicate that MKP-3 interacts with FOXO1, synergizes with FOXO1 to transcribe PEPCK and G6Pase genes, decreases insulin stimulated FOXO1 phosphorylation and increases its nuclear translocation. Furthermore, overexpression of the dominant negative FOXO1 can block MKP-3 stimulated gluconeogenic gene expression and glucose in primary hepatocytes and nuclear FOXO1 is decreased by hepatic MKP-3 knockdown. Interestingly, the gluconeogenic effect of MKP-3 is dramatic in primary hepatocyes and mild in Fao cells, which are devoid of gluconeogenic amplifier PGC-11. Knocking down PGC-11 expression in primary hepatocytes essentially abolished the effect of MKP-3 on gluconeogenic gene expression and glucose output. We hypothesize that MKP-3 antagonizes the inhibitory effect of insulin on gluconeogenesis through activation of FOXO1 by dephosphorylation and PGC-11 is a critical co- activator. The specific aims are: 1) Define the role of FOXO1 in MKP-3-mediated gluconeogenesis; 2) Modulate hepatic MKP-3 expression and evaluate the effect on gluconeogenesis and glycemic control; 3) Understand the downstream target of MKP-3 promoted fasting hyperglycemia in vivo. The goals will be achieved through reporter assays and adenovirus-mediated gain and loss-of-function studies in cultured liver cells, primary liver cells and in the liver of normal as well as obese animals. Results generated from this proposal will provide key information to understand the mechanism of action of MKP-3 and evaluate its candidacy as a new therapeutic target for treating obesity-related type 2 diabetes. PUBLIC HEALTH RELEVANCE: Obesity-related type 2 diabetes, featured with decreased body response to insulin, is attributable to increased liver glucose production and decreased glucose utilization by muscle and fat. A novel factor was recently found to elevate in the liver of obese rodents, antagonize the action of insulin in the liver, promote liver glucose production and contribute significantly to hyperglycemia in obesity. Results generated from research outlined in this proposal will provide key information to understand the mechanism of action of this protein and evaluate its candidacy as a new therapeutic target for treating obesity-related type 2 diabetes.