Gene expression profiles of SIRT6-deficient pancreatic beta cells from Ngn3-Cre Sirt6 f/f and MIP1-CreERT Sirt6 f/f mice

Purpose: The complete understanding of how genetic and epigenetic components control beta cell differentiation and function is key to the discovery of novel therapeutic approaches to prevent beta cell dysfunction and failure in the progression of type 2 diabetes. Our goal was to elucidate the role of histone deacetylase SIRT6 in beta-cell development and homeostasis. Methods: The Sirt6 endocrine progenitor cell conditional knockout (EKO) and beta-cell-specific knockout (BKO) mice were generated using the Cre-loxP system. Mice were assayed for islet morphology, glucose tolerance, glucose-stimulated insulin secretion, and susceptibility to streptozotocin. Transcriptional regulatory functions of SIRT6 in primary islets were evaluated by RNA-seq analysis. RT-qPCR and immunoblot were used to verify and investigate the gene expression changes. Chromatin occupancies of SIRT6, H3K9Ac, H3K56Ac, and active RNA Polymerase II were evaluated by chromatin immunoprecipitation. Results: Deletion of Sirt6 in pancreatic endocrine progenitor cells did not affect endocrine morphology, beta cell mass, or insulin production, but did result in glucose intolerance and defective glucose-stimulated insulin secretion in mice. Conditional deletion of Sirt6 in adult beta cells reproduced the insulin secretion defect. Loss of Sirt6 resulted in aberrant upregulation of TXNIP. SIRT6 deficiency led to increased accumulations of H3K9Ac, H3K56Ac, and active RNA polymerase II at the promoter region of Txnip. SIRT6-deficient beta cells exhibited a time-dependent increase of H3K9Ac, H3K56Ac, and TXNIP levels. Furthermore, beta-cell-specific SIRT6 deficient mice showed increased sensitivity to streptozotocin. Conclusions: Our results reveal that SIRT6 suppresses Txnip expression in beta-cells via deacetylation of histone H3 and plays a critical role in maintaining beta-cell function and viability. Agents that preserve SIRT6 activity may be beneficial for preventing the progression of type 2 diabetes. Profiles of pancreatic beta cells gene expression from 2-week post tamoxifen MIP1-CreERT Sirt6 f/f and 2-month-old Ngn3-Cre Sirt6 f/f male mice were generated by RNA-sequencing with duplicate using Illumina HiSeq 2000 and 3000. Samples from MIP-CreERT Sirt6 f/+, MIP-CreERT Sirt6 +/+, and Ngn3-Cre Sirt6 +/+, which did not show phenotype, were used as controls for BKO and EKO models, respectively, in the differential gene expression analysis.

目的：完整阐明遗传与表观遗传组分如何调控β细胞分化与功能，是发现新型治疗手段以预防2型糖尿病进程中β细胞功能障碍与衰竭的关键。本研究旨在阐明组蛋白去乙酰化酶SIRT6（histone deacetylase SIRT6）在β细胞发育及稳态维持中的作用。
方法：本研究利用Cre-loxP系统（Cre-loxP system）构建了内分泌前体细胞条件性敲除（endocrine progenitor cell conditional knockout, EKO）及β细胞特异性敲除（beta-cell-specific knockout, BKO）小鼠模型。通过胰岛形态学观察、葡萄糖耐受实验、葡萄糖刺激胰岛素分泌实验及链脲佐菌素（streptozotocin）敏感性实验对小鼠进行表型分析。采用RNA测序（RNA-seq）分析原代胰岛中SIRT6的转录调控功能；通过实时定量聚合酶链反应（RT-qPCR）与免疫印迹实验验证并探究基因表达变化；采用染色质免疫沉淀（chromatin immunoprecipitation）检测SIRT6、H3K9乙酰化修饰（H3K9Ac）、H3K56乙酰化修饰（H3K56Ac）及活性RNA聚合酶II的染色质结合情况。
结果：胰腺内分泌前体细胞中Sirt6的缺失并不会影响内分泌细胞形态、β细胞数量或胰岛素产生，但会导致小鼠出现葡萄糖耐受不良及葡萄糖刺激胰岛素分泌功能缺陷。在成年β细胞中条件性敲除Sirt6可重现上述胰岛素分泌缺陷表型。Sirt6缺失会引发TXNIP基因的异常上调。SIRT6缺陷会使Txnip启动子区域的H3K9Ac、H3K56Ac及活性RNA聚合酶II的富集水平升高。SIRT6缺陷的β细胞中，H3K9Ac、H3K56Ac及TXNIP的表达水平随时间推移逐渐升高。此外，β细胞特异性SIRT6缺陷小鼠对链脲佐菌素的敏感性显著升高。
结论：本研究结果表明，SIRT6可通过组蛋白H3的去乙酰化作用抑制β细胞中Txnip的表达，在维持β细胞功能与存活方面发挥关键作用。维持SIRT6活性的药物或可用于预防2型糖尿病的疾病进展。
本研究通过Illumina HiSeq 2000与3000平台进行双重复RNA测序，获取了他莫昔芬处理2周后的MIP1-CreERT Sirt6 f/f小鼠及2月龄Ngn3-Cre Sirt6 f/f雄性小鼠的胰腺β细胞基因表达谱。以无表型的MIP-CreERT Sirt6 f/+、MIP-CreERT Sirt6 +/+及Ngn3-Cre Sirt6 +/+小鼠样本分别作为BKO与EKO模型的对照，用于差异基因表达分析。