脂肪组织中p53与NF-κB失衡在高脂诱导胰岛素抵抗过程中的作用

Chronic metabolic and cardiovascular diseases, described as the epidemics of the 21st century, are connected to the resistance of peripheral tissues, such as liver, muscle and fat, to insulin. Insulin resistance can precede the development of type 2 diabetes and cardiovascular diseases by several years. Currently, an attractive hypothesis describing the mechanism of insulin resistance may be the increase of adipose tissue inflammation in obese subjects. .The transcription factors Nuclear factor-κB (NF-κB) and p53 are separately linked to chronic inflammation and insulin resistance. Both factors can cross-inhibit each other's actions and maintain a balance of their activity in normal state. We have previously found that impaired p53 and NF-κB balance existed in the adipose tissue of insulin resistant mice induced with high-fat diet, which is associated with the dysregulation of inflammatory cytokine. The observation suggest a possible role for an imbalance between NF-κB and p53 in the development and progression of insulin resistance. It remains to be demonstrated whether imbalance between p53 and NF-κB contribute to high-fat diet-induced insulin resistance via inflammation. In addition, the interplay between p53 and NF-κB family members and the molecular mechanism of their interactions are still to be elucidated. Hence, our study was designed to l) determine the correlation between p53/NF-κB imbalance and high-fat diet-induced insulin resistance by examining the expression and activity of p53 and NF-κB signaling in vitro and in an animal model, 2) elucidate the interplay between p53 and NF-κB family members and the molecular mechanism of their interactions by introduction of p53, NF-κB recombinant plasmid or small interfering RNA or their specific antagonist and agonist, 3)examine the effect of p53 over-expression or RNA silencing of NF-κB on insulin sensitivity. Elucidating these questions not only will uncover a novel molecular targets for pharmacological intervention against insulin resistance but also may provide important fundamental knowledge of the relation between cancer and diabetes.

脂肪炎性反应异常是导致胰岛素抵抗发生的重要因素。p53、NF-κB分别与炎性反应及胰岛素抵抗相关，两者间存在负向调控关系，在正常生理状态下处于一种平衡关系。我们的前期研究结果表明了高脂诱导胰岛素抵抗过程中p53和NF-κB间的失衡，并伴有炎症反应失调，提示p53和NF-κB间的失衡很可能在高脂诱导炎性反应及胰岛素抵抗过程中起重要作用，但这种失衡与胰岛素抵抗的关系以及p53和NF-κB之间的调控作用及机制尚不清楚。本课题拟采用高脂诱导胰岛素抵抗3T3-L1细胞模型和小鼠模型相结合的方法，探讨p53、NF-κB通路在胰岛素抵抗过程中的失衡现象，利用特异性抑制剂、激活剂及基因转染技术研究p53、NF-κB间的调控作用及机制，观察上调p53或下调NF-κB活性后对胰岛素敏感性的影响。旨在探索高脂诱导胰岛素抵抗的新的分子机制并为寻找新的药理靶点提供理论依据。

目的：探讨p53和NF-κB及MAPK通路在胰岛素抵抗过程中的变化，并通过上调或下调p53表达，研究其对胰岛素敏感性的影响以及对NF-κB和MAPK通路的调控作用。方法：利用Western blot方法检测体内、体外胰岛素抵抗模型中p53和NF-κB、MAPK信号通路中相关蛋白的表达水平，确定其负相关性。（2）用p53激动剂Nutlin-3和p53抑制剂pifithrin-α(PFT)分别处理细胞，观察其对葡萄糖摄取量的影响及NF-κB p65，p-p38和p-ERK1/2 MAPK的表达情况。（3）用p53激动剂Nutlin-3与棕榈酸（PA）联合处理HepG2细胞，观察对葡萄糖摄取量的影响及NF-κBp65，p-p38和p-ERK1/2 MAPK的表达情况。（4）用p53重组腺病毒处理ob/ob小鼠，观察对胰岛素敏感性的影响，检测肝脏p53的表达水平及NF-κBp65，p-p38和p-ERK1/2 MAPK的表达情况。结果：（1）在胰岛素抵抗细胞模型及动物模型中，p53蛋白表达下降，p-p65表达升高，同时p-p38和p-ERK1/2表达升高。（2）用Nutlin-3 处理细胞后，p53表达水平上调，激活胰岛素信号通路，p65和p38、ERK1/2磷酸化水平下降。用PFT处理HepG2细胞后，结果相反。（3）用Nutlin-3和PA联合处理HepG2细胞后，Nutlin-3可以上调胰岛素抵抗HepG2细胞中p53的表达，改善胰岛素抵抗，同时下调p-p65和p-p38及p-ERK1/2的表达。（4）用p53重组腺病毒注射ob/ob小鼠后，血糖、血胰岛素水平升高，p53表达水平上升，胰岛素信号通路关键分子IR、AKT磷酸化水平下降，p70s6k磷酸化水平上升，p65和p38、ERK1/2的磷酸化水平升高。结论：（1）在胰岛素抵抗的细胞模型和动物模型中，p53与NF-κB呈负相关关系。（2）上调p53表达能抑制NF-κB和MAPK通路，激活胰岛素信号通路；而下调p53表达则激活NF-κB和MAPK通路，抑制胰岛素通路，引起胰岛素抵抗。上调p53表达后能改善胰岛素信号通路，逆转由棕榈酸诱导的胰岛素抵抗。总之，p53可通过调节NF-κB及p38/ERK1/2 MAPK信号通路相关信号分子，改善胰岛素抵抗。