BET bromodomain proteins Brd2, Brd3 and Brd4 selectively regulate metabolic pathways in the pancreatic β-cell

Displacement of Bromodomain and Extra-Terminal (BET) proteins from chromatin has promise for cancer and inflammatory disease treatments, but roles of BET proteins in metabolic disease remain unexplored. Small molecule BET inhibitors, such as JQ1, block BET protein binding to acetylated lysines, but lack selectivity within the BET family (Brd2, Brd3, Brd4, Brdt), making it difficult to disentangle contributions of each family member to transcriptional and cellular outcomes. Here, we demonstrate multiple improvements in pancreatic β-cells upon BET inhibition with JQ1 or BET-specific siRNAs. JQ1 (50-400 nM) increases insulin secretion from INS-1 cells in a concentration dependent manner. JQ1 increases insulin content in INS-1 cells, accounting for increased secretion, in both rat and human islets. Higher concentrations of JQ1 decrease intracellular triglyceride stores in INS-1 cells, a result of increased fatty acid oxidation. Specific inhibition of both Brd2 and Brd4 enhances insulin transcription, leading to increased insulin content. Inhibition of Brd2 alone increases fatty acid oxidation. Overlapping yet discrete roles for individual BET proteins in metabolic regulation suggest new isoform-selective BET inhibitors may be useful to treat insulin resistant/diabetic patients. Results imply that cancer and diseases of chronic inflammation or disordered metabolism are related through shared chromatin regulatory mechanisms. BET proteins are not redundant in their functions, thus pan-BET inhibitors like JQ1 cannot properly be interpreted without specific RNA knockdown or small molecules of proven selectivity. Here, we expose INS-1 cells, a rat insulinoma model for the pancreatic beta cell, to selective siRNAs to ablate Brd2, Brd3 or Brd4 mRNAs and compared genome-wide changes in transcription to non-targeted siRNA control. As expected, three resolvably different patterns of gene expression were identified, establishing that each BET protein controls its own set of target genes.

溴结构域与额外末端结构域（Bromodomain and Extra-Terminal, BET）蛋白从染色质上的解离，在癌症及炎症性疾病治疗中展现出应用潜力，但BET蛋白在代谢疾病中的作用仍未得到充分探索。小分子BET抑制剂（如JQ1）可阻断BET蛋白与乙酰化赖氨酸的结合，但该类抑制剂在BET家族（Brd2、Brd3、Brd4、Brdt）内缺乏选择性，因此难以厘清各个家族成员对转录及细胞结局的具体贡献。本研究中，我们证实了使用JQ1或BET特异性小干扰RNA（small interfering RNA, siRNA）抑制BET蛋白后，胰腺β细胞可产生多项有益改变。JQ1（50~400 nM）可呈浓度依赖性提升INS-1细胞的胰岛素分泌水平。在大鼠胰岛与人胰岛中，JQ1均可增加INS-1细胞的胰岛素含量，该变化是其胰岛素分泌水平提升的原因。较高浓度的JQ1可降低INS-1细胞内的甘油三酯储存量，这一现象源于脂肪酸氧化水平的提升。同时特异性抑制Brd2与Brd4可增强胰岛素转录，进而提升胰岛素含量。仅抑制Brd2即可提升脂肪酸氧化水平。单个BET蛋白在代谢调控中兼具重叠与独特的功能，这提示新型亚型选择性BET抑制剂或可用于治疗胰岛素抵抗/糖尿病患者。研究结果表明，癌症、慢性炎症性疾病及代谢紊乱性疾病可通过共享的染色质调控机制实现关联。BET蛋白的功能并不冗余，因此若缺乏特异性RNA敲低或经验证具有选择性的小分子工具，无法对JQ1这类泛BET抑制剂的作用进行准确解读。本研究中，我们将胰腺β细胞的大鼠胰岛素瘤模型INS-1细胞暴露于靶向Brd2、Brd3或Brd4 mRNA的特异性siRNA中，以敲低对应基因的表达，并将全基因组转录组的变化与非靶向siRNA对照组进行比较。正如预期，研究共鉴定出三种可明确区分的基因表达模式，证实每个BET蛋白均调控着各自独特的靶基因集。