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

点击翻译按钮获取中文摘要

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.