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

描述(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。在胰岛素抵抗的糖尿病状态下，血糖水平升高是由于肝脏产生的葡萄糖增加(主要是通过从头合成葡萄糖、糖异生)和减少脂肪和肌肉中葡萄糖的处置。我们最近确定MKP-3(MKP-3)是对抗胰岛素对肝脏糖异生抑制作用的首选候选蛋白。我们证明MKP-3在肥胖小鼠的肝脏中的表达增加，并且MKP-3的过度表达足以促进肝细胞中的糖异生和提高瘦小鼠的空腹血糖水平。通过RNA干扰降低MKP-3的表达减少了肝细胞中的糖异生，并降低了肥胖小鼠的空腹血糖水平。尽管MKP-3的唯一已知底物是ERK，激活ERK通路可以部分抑制糖异生基因的表达，但阻断ERK功能不能逆转胰岛素对糖异生的抑制作用。这表明MKP-3通过一种新的机制减弱胰岛素信号。叉头转录因子FOXO1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而被激活，导致随后的核转位启动糖异生程序。我们最近的研究表明，MKP-3与FOXO1相互作用，与FOXO1协同转录PEPCK和G6Pase基因，减少胰岛素刺激的FOXO1磷酸化，增加其核转位。此外，过表达显性负性FOXO1可以阻断MKP-3刺激的原代肝细胞葡萄糖异生基因的表达，而肝MKP-3基因敲除可降低核FOXO1的表达。有趣的是，MKP-3在原代肝细胞中的糖异生作用是显著的，而在FAO细胞中则是轻微的，这些细胞缺乏糖异生放大器PGC-11。在原代肝细胞中下调PGC-11的表达，基本上消除了MKP-3对糖异生基因表达和葡萄糖输出的影响。我们推测MKP-3通过去磷酸化激活FOXO1来拮抗胰岛素对糖异生的抑制作用，而PGC-11是关键的共激活剂。其具体目的是：1)明确FOXO1在MKP-3介导的糖异生中的作用；2)调节肝脏MKP-3的表达，评价其对糖异生和血糖控制的作用；3)了解MKP-3促进体内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分析和腺病毒介导的功能获得和丧失研究来实现。这项提议产生的结果将为了解MKP-3的作用机制和评估其作为治疗肥胖相关2型糖尿病的新治疗靶点的可能性提供关键信息。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，肌肉和脂肪对葡萄糖的利用减少。最近发现了一种新的因子，可以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，并显著导致肥胖的高血糖。这项提案中概述的研究结果将提供关键信息，以了解这种蛋白质的作用机制，并评估其作为治疗肥胖相关2型糖尿病的新治疗目标的可能性。

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.