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（主要通过新生葡萄糖合成和糖异生）以及脂肪和肌肉中葡萄糖处理减少。我们最近发现MAP激酶磷酸酶3 （MKP- 3）是拮抗胰岛素对肝脏糖异生抑制作用的首选候选。我们证明MKP-3在肥胖小鼠的肝脏中表达升高，MKP-3的过表达足以促进肝细胞的糖异生，并提高瘦小鼠的空腹血糖水平。通过RNA干扰减少MKP-3的表达可降低肥胖小鼠肝细胞的糖异生，降低空腹血糖水平。虽然MKP-3唯一已知的底物是ERK，并且ERK通路的激活可以部分抑制糖异生基因的表达，但ERK功能的破坏并不能逆转胰岛素对糖异生的抑制作用。这表明MKP-3通过一种新机制减弱胰岛素信号。叉头转录因子fox01可以通过去磷酸化激活，导致核易位启动糖异生程序，是胰岛素抑制葡萄糖产生的关键因素。我们最近的数据表明，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磷酸化并增加其核易位。此外，显性阴性FOXO1过表达可阻断MKP-3刺激的糖异生基因表达，原代肝细胞中葡萄糖和细胞核FOXO1通过肝脏MKP-3敲低而减少。有趣的是，mgp -3的糖异生作用在原代肝细胞中是显著的，而在缺乏糖异生放大器PGC-11的Fao细胞中是轻微的。抑制原代肝细胞中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。我们假设mgp -3通过去磷酸化激活fox01来拮抗胰岛素对糖异生的抑制作用，而PGC-11是一个关键的共激活因子。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表达，评价其对糖异生和血糖控制的影响；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细胞测定和腺病毒介导的功能获得和功能丧失研究来实现。本研究结果将为了解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型糖尿病的新靶点的候选性。公共卫生相关性：肥胖相关的2型糖尿病，以身体对胰岛素的反应降低为特征，可归因于肝脏葡萄糖生成增加和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，拮抗肝脏胰岛素的作用，促进肝脏葡萄糖的产生，并在肥胖的高血糖中起重要作用。本提案中概述的研究结果将为了解该蛋白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.