基于JNK-IRS1信号通路研究长期服用奥氮平诱导胰岛素抵抗的机制

Numerous studies show that chronic olanzapine treatment induced insulin resistance. JNK is closely related to insulin resistance. Inflammatory cytokines could activate JNK. Our previous study found that patients who developed insulin resistance after long-term olanzapine treatment showed significantly elevated inflammatory cytokines in plasma. Animal experiments showed that mice developed insulin resistance after long-term administration of olanzapine. And inflammatory cytokines were significantly increased in plasma and adipose tissue in mice. Further study found that phosphorylated JNK and IRS1 were significantly increased in adipose tissue in mice. We put forward the hypothesis that long-term treatment of olanzapine may inhibit insulin signaling and induce insulin resistance via JNK activation and IRS1 phosphorylation. First we intend to establish a cellular model to clarify the effects of olanzapine on inflammatory cytokines, JNK and IRS1, and then investigate the effect of olanzapine on the insulin-stimulated glucose uptake after JNK inhibition. Finally, we intend to establish a mouse model to investigate the effect of JNK inhibitor on long-term olanzapine-induced insulin resistance in order to reveal the role of JNK-IRS1 signaling pathway in long-term olanzapine-induced insulin resistance. It may provide a scientific basis for new strategy to alleviate olanzapine-induced insulin resistance with important clinical significance.

大量研究表明长期服用奥氮平显著诱导胰岛素抵抗的发生。JNK与胰岛素抵抗密切相关。炎症因子能够激活JNK。我们研究发现长期服用奥氮平发生胰岛素抵抗的患者血浆中炎症因子显著升高。动物实验结果显示长期给药奥氮平后小鼠出现胰岛素抵抗，血浆和脂肪组织中炎症因子显著升高。进一步研究发现小鼠脂肪组织中磷酸化JNK和IRS1表达显著上调。我们据此假设长期服用奥氮平可能激活JNK磷酸化IRS1，抑制胰岛素信号传导，诱导胰岛素抵抗。我们拟首先建立细胞模型，明确细胞水平上奥氮平对炎症因子、JNK和IRS1的影响，然后研究用抑制剂抑制JNK后奥氮平对细胞胰岛素刺激的葡萄糖吸收的影响，最后建立小鼠模型研究JNK抑制剂对长期给药奥氮平诱导胰岛素抵抗的影响，以期揭示JNK-IRS1信号通路在长期服用奥氮平诱导胰岛素抵抗中的作用和分子机制，为寻找缓解奥氮平诱导的胰岛素抵抗的新策略提供科学依据，具有重要临床意义。

本研究揭示了JNK在奥氮平诱导胰岛素抵抗中的作用，初步阐明了JNK1与IRS1相互作用诱导IRS1Ser307磷酸化介导长期给药奥氮平诱导胰岛素抵抗的分子机制。小鼠长期给药奥氮平后，空腹血糖和胰岛素水平显著升高，产生胰岛素抵抗状态。同时，血浆和eWAT中炎症因子（TNF-α, IL-6和IL-1β）水平显著升高，巨噬细胞浸润增加，M1巨噬细胞极化增加。Wb结果显示长期给药奥氮平激活eWAT中JNK1，p-JNK1表达显著上调。eWAT中JNK1敲除小鼠模型结果显示JNK1敲除小鼠长期给药奥氮平后，和对照组小鼠比较胰岛素抵抗显著改善，p-AKT和p-GSK3β显著上调，胰岛素敏感性增强。此外，JNK1敲除小鼠血浆和eWAT中炎症因子显著降低。流式细胞实验表明JNK敲除小鼠eWAT中巨噬细胞浸润减少，CD68和F4/80表达水平显著降低，SVF中M1巨噬细胞比例降低而M2巨噬细胞比例升高。基于3T3-L1脂肪细胞模型开展JNK1和IRS1过表达和敲减实验，结果显示JNK1过表达加剧奥氮平对胰岛素信号通路的抑制作用和炎症反应，而JNK1敲除后奥氮平对胰岛素信号通路的抑制作用减弱，炎症反应降低。JNK1过表达显著上调p-IRS1的表达而JNK1敲除则显著抑制p-IRS1的表达。Co-IP实验显示在奥氮平诱导胰岛素抵抗中，JNK1与IRS1存在密切相互作用。突变细胞实验显示奥氮平干预IRS1S307A转染细胞后，和对照组比较p-IRS1没有显著变化，且p-AKT和p-GSK3β也没有显著变化，表明IRS1Ser307在JNK1介导奥氮平诱导胰岛素抵抗中具有关键作用。此外，动物模型结果显示二甲双胍可以显著缓解奥氮平诱导的胰岛素抵抗，可能与其缓解奥氮平引起的eWAT炎症反应有关。我们的实验结果表明JNK和JNK-IRS相互作用可能会成为干预奥氮平等抗精神分裂药物诱导胰岛素抵抗的新靶点。