Illuminating novel roles for the Ldb1 co-regulator in transcriptional regulation of brown adipose function

项目摘要/摘要 棕色脂肪组织(BAT)对产热和糖脂平衡起着关键作用。蝙蝠利用脂肪酸 和葡萄糖通过线粒体解偶联产生热量，因此是有吸引力的治疗靶点 与肥胖作斗争。利用这种组织独特的能量去偶联能力需要更大的 了解BAT潜在的转录机制。我们最近报道了一个转录联合- 调节因子LIM结构域结合蛋白1(LDB1)，它似乎在蝙蝠生物学中具有新的作用。LDB1动作 作为一种二聚化支架，允许转录复合体的组装，对 许多代谢组织的发育和功能，包括脑和胰岛。但是，直接 BAT表达的LDB1的作用尚未阐明。我将测试LDB1直接影响 BAT功能。分离的前脂肪细胞中LDB1缺失导致Ucp1表达降低 成熟的脂肪细胞。此外，我还开发了一种在生热脂肪细胞中缺失LDB1的小鼠模型 使用UCP1驱动的Cre重组酶，称为LDB1ΔBAT。这些基因敲除的小鼠的蝙蝠数量减少了- 选择性的mRNAs包括Ucp1和Elovl3，这一结果类似于在缺乏LDB1的X9米色细胞系中观察到的结果。 在寒冷的挑战中，ldb1Δ蝙蝠小鼠不能保持体温，这表明是产热的。 缺陷。我们还观察了LDB1Δ蝙蝠小鼠通过腹腔葡萄糖攻击造成的葡萄糖耐量。至 剖析LDB1在调节全身生理中的作用，将评估LDB1ΔBAT的能量变化 消耗、呼吸商、生热作用、摄食行为、活动能力和燃料利用率 综合实验室动物监测系统。葡萄糖代谢将通过以下变化进行监测 空腹血糖、使用BAT激动剂治疗的腹膜糖耐量、胰岛素耐量试验和 胰岛素信号。为了确定LDB1的缺失如何影响棕色脂肪细胞的功能，脂质含量将 通过油红O染色和实时定量聚合酶链式反应检测脂肪生成标志物。蝙蝠组织学将 确定细胞大小和脂质含量的变化。细胞呼吸测量将决定 线粒体功能、脂肪酸氧化和糖酵解通量。为了阐明转录因子的全球转录作用 在BAT基因的表达上，LDB1ΔBAT小鼠将与一只NuTRAP报告鼠(简称 LDB1nbat)，以便对脂肪细胞核和Δ进行分选。将检查这些排序的分数是否具有全局 通过RNA-Seq转录变化，并通过ChIP-Seq结合靶标，结果基于以下因素进行优先排序 与产热、糖脂代谢和线粒体功能相关的基因。结果来自 这项建议将为未来评估LDB1在高脂肪饮食等代谢压力下BAT中的作用的研究提供信息。 (HFD)以及阐明新的LDB1相互作用的转录调控因子。这项提案的结果和 未来的HFD研究将加强我们对维持BAT功能的转录机制的理解 在基础和代谢压力条件下，这将有利于未来的肥胖症治疗。

Project Summary/Abstract Brown adipose tissue (BAT) is critical for thermogenesis and glucose/lipid homeostasis. BAT utilizes fatty acids and glucose for heat production via mitochondrial uncoupling and is thus an attractive therapeutic target for combatting obesity. Exploiting the unique energy uncoupling capacity of this tissue requires a greater understanding of underlying BAT transcriptional mechanisms. We recently reported on a transcriptional co- regulator, LIM domain binding protein 1 (Ldb1), which appears to have novel roles in BAT biology. Ldb1 acts as a dimerized scaffold allowing for the assembly of transcriptional complexes and is important for the development and function of many metabolic tissues, including the brain and pancreatic islets. However, direct roles for BAT-expressed Ldb1 have not been elucidated. I will test the hypothesis that Ldb1 directly impacts BAT function. Ldb1 deletion in isolated preadipocytes resulted in reduced Ucp1 expression upon induction to mature adipocytes. Additionally, I developed a mouse model which deleted Ldb1 in thermogenic adipocytes using a Ucp1-driven Cre recombinase, termed Ldb1ΔBAT. These knockout mice have reductions in BAT- selective mRNAs including Ucp1 and Elovl3, a result similarly observed in an X9 beige cell line lacking Ldb1. Ldb1ΔBAT mice were unable to defend body temperature during a cold challenge, suggesting thermogenic defects. We also observed glucose intolerance in Ldb1ΔBAT mice via intraperitoneal glucose challenge. To dissect the role of Ldb1 in regulating whole-body physiology, Ldb1ΔBAT will be assessed for changes in energy expenditure, respiratory quotient, thermogenesis, feeding behavior, locomotor activity, and fuel utilization using the Comprehensive Lab Animal Monitoring System. Glucose metabolism will be monitored through changes in fasting blood glucose, intraperitoneal glucose tolerance with BAT-agonist treatment, insulin tolerance test, and insulin signaling. To determine how the loss of Ldb1 affects brown adipocyte function, lipid content will be assessed via Oil Red O staining and quantitative real-time PCR for lipogenesis markers. BAT histology will determine changes to cell size and lipid content. Cellular respiration measurements will determine changes in mitochondrial function, fatty acid oxidation and glycolytic flux. To elucidate the global transcriptional role of Ldb1 in BAT gene expression, Ldb1ΔBAT mice will be crossed with a NuTRAP reporter mouse (termed Ldb1ΔNuBAT) to allow sorting of adipocyte nuclei and mRNA. These sorted fractions will be examined for global transcriptional changes via RNA-Seq, and bound targets via ChIP-Seq, with the results prioritized based on genes associated with thermogenesis, glucose and lipid metabolism, and mitochondrial function. Results from this proposal will inform future studies assessing Ldb1 roles in BAT under metabolic stresses, like high-fat diet (HFD) as well as elucidating novel Ldb1-interacting transcriptional regulators. Outcomes from this proposal and future HFD studies will enhance our understanding of transcriptional mechanisms maintaining BAT function under basal and metabolic stress conditions, which will benefit future obesity therapies.