Role of the nuclear estrogen-related receptor alpha in mitochondrial function

在小鼠中的敲除研究表明，CB 1 R的缺失防止了饮食诱导的肥胖，从而使这种GPCR连接的受体成为新治疗方法的有吸引力的靶点。CB 1 R反向激动剂，如伊比那班，AM 251和临床类似物利莫那班，在动物中产生有效的厌食作用。然而，这些化合物也通过未知的机制发挥不依赖于CB 1 R的脱靶效应。在这里，我们证明了所有测试的CB 1 R反向激动剂（例如，AM 251、利莫那班和伊比那班）在三种不同的CB 1 R阴性培养细胞系中诱导ERR降解。先前的研究表明，合成的非甾体雌激素，己烯雌酚，通过其与ERR的配体结合结构域的相互作用拮抗ERR信号转导。因此，我们假设己烯雌酚和经典的ERR反向激动剂XCT 790可能具有AM 251模拟作用，而选择性ERR激动剂可能阻断AM 251信号传导。发现所有这些预测都是正确的，因此证实了ERR不稳定在AM 251的细胞作用中的重要性。另外的实验表明，AM 251促进ERR蛋白的蛋白水解降解，半衰期为7小时，而不损失相应的mRNA。此外，用蛋白酶体抑制剂MG 132预处理细胞，阻断了AM 251诱导的ERR降解，并导致孤儿核受体的核蓄积，从而证实了AM 251诱导ERR蛋白降解。 为了进一步阐明这种降解的机制，目前正在开展工作，以确定负责ERR去稳定化的假定翻译后修饰以及随后靶基因表达响应于AM 251和相关化合物的改变。先前的研究已经证明ERR蛋白是翻译后修饰的靶点，如磷酸化、乙酰化和类小泛素化，这些修饰改变了随后的转录活性。 这些翻译后修饰也可能改变核共调节分子的募集（例如，PGC-1和RIP 140）和其它DNA结合蛋白在靶基因启动子区与ERR结合。 因此，正在进行DNA下拉试验，以测量在不存在和存在AM 251的情况下溶液中的ERR-DNA结合，并在一组用CB 1 R反向激动剂处理的细胞培养模型中通过亲和纯化和质谱法绘制相互作用的多蛋白复合物。类似的方法将用于组织（例如，肝脏、骨骼肌、脂肪细胞）。 该项目的第二个方面集中在ERR/PGC-1复合物的作用是调节能量代谢和线粒体功能。我们假设AM 251和相关的CB 1 R反向激动剂使这一关键复合物不稳定，从而可能改变线粒体生物合成和呼吸。我们的初步体外实验表明，线粒体活性响应于AM 251而降低。使用共聚焦显微镜，AM 251处理减少ERR染色，MitoTracker Red CMXRos染色相应减少，表明线粒体膜电位损失。这些发现得到了初步流式细胞术结果的支持，表明在AM 251处理的细胞中，通过TMRM染色线粒体膜去极化增加。此外，所有测试的CB 1 R反向激动剂均降低壬基吖啶橙子染色，表明这些化合物改变了线粒体总质量。目前正在进行使用DNA下拉测定的研究，以确定这种线粒体改变是否确实依赖于ERR/PGC-1复合物的不稳定。 线粒体生物发生和膜电位的这些变化特别令人感兴趣，因为ERR缺陷的小鼠表现出脂肪酸代谢缺陷，并且对饮食诱导的肥胖具有抗性。与Rafael deCabo和Julie Mattison合作，我们对中年雄性恒河猴进行了全球基因表达谱分析，这些猴子在不存在或存在白藜芦醇的情况下喂食高脂肪高糖（HFS）饮食2年，白藜芦醇是一种已知促进长寿和改善线粒体功能的植物分子。结果显示，与对照组相比，HFS喂养动物骨骼肌中ERR基因表达显著增加，但肝脏或脂肪组织中没有。此外，白藜芦醇阻断了HFS诱导的ERR表达增加，同时抵消了HFS对PGC-1 mRNA水平的消耗。随后的蛋白质印迹分析证实了这些组织特异性变化，由于HSF饮食，骨骼肌中的ERR蛋白表达增加，但肝脏中的ERR蛋白表达减少。有趣的是，白藜芦醇补充剂也具有组织特异性作用，使值恢复到对照动物的值。在大鼠中的其他研究进一步证明了饮食和年龄两者影响骨骼肌中ERR和PGC-1表达的能力。具体而言，虽然肌肉PGC-1表达随年龄而降低，但由于热量限制，ERR和PGC-1均增加。因此，可以合理地得出结论，旨在靶向ERR稳定性和/或功能的药理学干预可能有助于对抗代谢疾病和改善氧化还原状态，最终不仅延长寿命，而且延长健康寿命。

Knockout studies in mice demonstrated that deletion of the CB1R prevented diet-induced obesity, thereby making this GPCR-linked receptor an attractive target for novel therapeutic approaches. CB1R inverse agonists, such as ibipinabant, AM251 and the clinical analog rimonabant, produce potent anorectic effects in animals. However, these compounds also exert off-target effects independent of the CB1R through unknown mechanisms. Here, we demonstrate that all CB1R inverse agonists tested (e.g., AM251, rimonabant and ibipinabant) induced ERRα degradation in three different CB1R-negative cultured cell lines. Previous studies have shown that the synthetic nonsteroidal estrogen, diethylstilbestrol, antagonizes ERRα signaling through its interaction with the ligand-binding domain of ERRα. We therefore hypothesized that diethylstilbestrol and the classical ERRα inverse agonist, XCT790, might have AM251-mimicking actions, whereas selective ERRα agonists might block AM251 signaling. All of these predictions were found to be correct, hence confirming the importance of ERRα destabilization in the cellular actions of AM251. Additional experiments demonstrated that AM251 promoted the proteolytic degradation of ERRα protein, with a half-life of 7 hours, without loss of the corresponding mRNA. Moreover, cell pretreatment with the proteasome inhibitor, MG132, blocked AM251-induced ERRα degradation and resulted in nuclear accumulation of the orphan nuclear receptor, thereby confirming that AM251 induces ERRα protein degradation. In order to further elucidate the mechanism for such degradation, work is currently underway to identify the putative posttranslational modification(s) responsible for the destabilization of ERRα and subsequent alterations in target gene expression in response to AM251 and related compounds. Previous studies have demonstrated that ERRα protein is a target for posttranslational modifications such as phosphorylation, acetylation and sumoylation, with such modifications altering subsequent transcriptional activity. These posttranslational modifications are also likely to alter the recruitement of nuclear co-regulatory molecules (e.g., PGC-1α and RIP140) and other DNA-binding proteins to ERRα at the target gene promoter region. Therefore, DNA pull-down assays are being carried out to measure ERRα-DNA binding in solution in the absence and presence of AM251, and mapping the interacting multiprotein complexes by affinity purification and mass spectrometry in a panel of cell culture models treated with CB1R inverse agonists. A similar approach will be used in tissues (e.g., liver, skeletal muscle, adipocytes) of animal models that received peripherally active CB1R inverse agonists. A second aspect of the project focuses on the role of the ERRα/PGC-1α complex is regulating energy metabolism and mitochondrial function. We hypothesize that AM251 and related CB1R inverse agonists destabilize this critical complex and thereby may alter mitochondrial biogenesis and respiration. Our preliminary in vitro experiments indicate a decrease in mitochondrial activity in response to AM251. Using confocal microscopy, AM251 treatment decreased ERRα staining with a corresponding reduction in MitoTracker Red CMXRos staining, suggesting a loss of mitochondrial membrane potential. These findings are supported by initial flow cytometry results demonstrating an increase in mitochondrial membrane depolarization via TMRM staining in AM251-treated cells. Additionally, all CB1R inverse agonists tested decrease nonyl acridine orange staining, suggesting alterations in total mitochondrial mass by these compounds. Studies are currently underway using DNA pull-down assays to determine if such mitochondrial alterations are indeed dependent on the destabilization of the ERRα/PGC-1α complex. These changes in mitochondrial biogenesis and membrane potential are particularly interesting since mice deficient in ERRα exhibit defects in fatty acid metabolism and are resistant to diet-induced obesity. In collaboration with Rafael deCabo and Julie Mattison, we performed a global gene expression profiling of middle-aged male Rhesus monkeys fed a high fat-high sugar (HFS) diet for 2 years in the absence or presence of resveratrol, a plant molecule known to promote longevity and improves mitochondrial function. The results showed significant increase in ERRα gene expression in skeletal muscle of HFS-fed animals as compared to controls, but not in liver or adipose tissue. Moreover, resveratrol blocked the HFS-inducible increase in ERRα expression while counteracting the depletion in PGC-1α mRNA levels by HFS. Subsequent Western blot analyses confirmed these tissue-specific changes, with ERRα protein expression increasing in skeletal muscle and, yet, decreasing in the liver as a result of the HSF diet. Interestingly, resveratrol supplementation also had tissue-specific effects that restored values to that of control animals. Additional studies in rats have further demonstrated the ability of both diet and age to influence ERRα and PGC-1α expression in skeletal muscle. Specifically, while muscle PGC-1α expression decreases with age, both ERRα and PGC-1α are increased as a result of caloric restriction. It is therefore reasonable to conclude that pharmacological interventions aimed at targeting ERRα stability and/or function may prove useful in fighting metabolic diseases and improving redox status, ultimately extending not only lifespan, but healthspan as well.