目的:肥胖是一个以慢性低度炎症和代谢失调为特征的全球性健康挑战。下丘脑,能量稳态的关键调节器,特别容易受到肥胖的有害影响。这项研究调查了免疫蛋白酶体的作用,一种与炎症和细胞稳态有关的特殊的蛋白酶体复合物,在代谢性疾病期间。
方法:通过免疫染色分析免疫蛋白酶体β5i亚基的水平,西方印迹,在高脂肪饮食(HFD)或常规饮食(CHOW)喂养的小鼠中进行蛋白酶体活性测定。我们还表征了自噬抑制对免疫蛋白酶体β5i亚基水平和AKT途径激活的影响。最后,通过共聚焦显微镜,我们通过流式细胞术和线粒体自噬分析分析了β5i亚基抑制对线粒体功能的贡献。
结果:使用HFD喂养的肥胖小鼠模型,我们发现下丘脑POMC神经元的免疫蛋白酶体水平升高。此外,我们观察到棕榈酸(PA),在HFD中发现的饱和脂肪的主要成分,增加了下丘脑神经元细胞中免疫蛋白酶体β5i亚基的水平。值得注意的是,免疫蛋白酶体表达的增加与自噬的减少有关,维持体内平衡和抑制炎症的关键细胞过程。功能上,PA破坏了胰岛素-葡萄糖轴,由于免疫蛋白酶体的上调,导致响应胰岛素的AKT磷酸化减少和细胞内葡萄糖水平增加。机械上,我们发现PTEN蛋白,胰岛素信号的关键调节器,以免疫蛋白酶体依赖性方式减少。为了进一步研究这些发现的潜在治疗意义,我们使用了ONX-0914,一种特异性的免疫蛋白酶体抑制剂。我们证明该抑制剂可防止PA诱导的胰岛素-葡萄糖轴失衡。鉴于线粒体功能障碍和代谢紊乱之间的相互作用,我们探讨了ONX-0914对线粒体功能的影响。值得注意的是,ONX-0914在PA存在下保留了线粒体膜电位并减弱了线粒体ROS的产生。此外,我们发现ONX-0914在PA存在下减少线粒体自噬。
结论:我们的研究结果强烈支持在HFD诱导的肥胖和代谢紊乱的背景下,下丘脑神经元中免疫蛋白酶体的致病参与。靶向免疫蛋白酶体强调了一种有希望的治疗策略,以减轻肥胖对胰岛素-葡萄糖轴和细胞稳态的有害影响。这项研究为肥胖相关代谢性疾病的驱动机制提供了有价值的见解,并为开发新的治疗干预措施提供了潜在的途径。
OBJECTIVE: Obesity represents a significant global health challenge characterized by chronic low-grade inflammation and metabolic dysregulation. The hypothalamus, a key regulator of energy homeostasis, is particularly susceptible to obesity\'s deleterious effects. This study investigated the role of the
immunoproteasome, a specialized proteasomal complex implicated in inflammation and cellular homeostasis, during metabolic diseases.
METHODS: The levels of the
immunoproteasome β5i subunit were analyzed by immunostaining, western blotting, and proteasome activity assay in mice fed with either a high-fat diet (HFD) or a regular diet (CHOW). We also characterized the impact of autophagy inhibition on the levels of the
immunoproteasome β5i subunit and the activation of the AKT pathway. Finally, through confocal microscopy, we analyzed the contribution of β5i subunit inhibition on mitochondrial function by flow cytometry and mitophagy assay.
RESULTS: Using an HFD-fed obese mouse model, we found increased
immunoproteasome levels in hypothalamic POMC neurons. Furthermore, we observed that palmitic acid (PA), a major component of saturated fats found in HFD, increased the levels of the β5i subunit of the immunoproteasome in hypothalamic neuronal cells. Notably, the increase in
immunoproteasome expression was associated with decreased autophagy, a critical cellular process in maintaining homeostasis and suppressing inflammation. Functionally, PA disrupted the insulin-glucose axis, leading to reduced AKT phosphorylation and increased intracellular glucose levels in response to insulin due to the upregulation of the immunoproteasome. Mechanistically, we identified that the protein PTEN, a key regulator of insulin signaling, was reduced in an immunoproteasome-dependent manner. To further investigate the potential therapeutic implications of these findings, we used ONX-0914, a specific immunoproteasome inhibitor. We demonstrated that this inhibitor prevents PA-induced insulin-glucose axis imbalance. Given the interplay between mitochondrial dysfunction and metabolic disturbances, we explored the impact of ONX-0914 on mitochondrial function. Notably, ONX-0914 preserved mitochondrial membrane potential and attenuated mitochondrial ROS production in the presence of PA. Moreover, we found that ONX-0914 reduced mitophagy in the presence of PA.
CONCLUSIONS: Our findings strongly support the pathogenic involvement of the immunoproteasome in hypothalamic neurons in the context of HFD-induced obesity and metabolic disturbances. Targeting the immunoproteasome highlights a promising therapeutic strategy to mitigate the detrimental effects of obesity on the insulin-glucose axis and cellular homeostasis. This study provides valuable insights into the mechanisms driving obesity-related metabolic diseases and offers potential avenues for developing novel therapeutic interventions.