TBK1 Regulates Regeneration of Pancreatic b-cells

b-cell proliferation induction is one of the most tangible therapeutic strategies to restore b-cell mass. However, this approach has proven challenging due to a remarkable resistance of adult human b-cells to proliferation. Here we aim to unravel the role of a non-canonical IkB kinase TBK1 (TANK-binding kinase 1), which is predominantly expressed in b-cells in mammalian islets, in regulating cell cycle progression. Genetic silencing of TBK1 in INS-1 832/13 rat b-cell line promoted proliferation of b-cells. Proteomic and transcriptome analyses further revealed changes of proteins and genes critical for cell growth and proliferation, including upregulation of ribosomal proteins and cell cycle regulators, upon depletion of TBK1. TBK1 overexpression decreased sensitivity of b-cells to the elevation of cAMP levels and reduced proliferation of b-cells in a manner dependent on the activity of phosphodiesterase 3 (PDE3). Importantly, pharmacological inhibition of TBK1 using (E)3-(3-phenylbenzo[c]isoxazol-5-yl) acrylic acid (PIAA) augmented proliferation and function of rat and human embryonic stem cell (hESC)-derived insulin-producing b-cells under basal conditions. Diabetogenic insults further induced TBK1 expression and accordingly, PIAA protected b-cells against cytokine- and streptozotocin-induced diabetogenic challenges and promoted b-cell replication. Furthermore, PIAA increased proliferation of β-cells in normal and type 2 diabetic human islets with elevation in insulin secretion. Altogether, these data unveil novel and essential function of TBK1 as a key cell-autonomous negative regulator of b-cell replication and presenting PIAA as a valid therapeutic strategy for augmenting proliferation and function of b-cells. Transfected TBK1 siGENOME SMARTpool siRNA or negative control non-targeting siRNA into INS-1 832/13 beta-cells for 36 hours.

诱导β细胞增殖是恢复β细胞总量的最切实可行的治疗策略之一。然而，成年人类β细胞对增殖存在显著抵抗特性，使得该方法极具挑战性。本研究旨在阐明非经典IκB激酶TBK1（TANK结合激酶1，TANK-binding kinase 1）在调控细胞周期进程中的功能——该激酶在哺乳动物胰岛的β细胞中呈高表达状态。在INS-1 832/13大鼠β细胞系中对TBK1进行基因沉默，可显著促进β细胞增殖。蛋白质组学与转录组学分析进一步揭示，敲低TBK1后，与细胞生长及增殖密切相关的蛋白质与基因发生表达改变，包括核糖体蛋白与细胞周期调控因子的上调。TBK1过表达会降低β细胞对环磷酸腺苷（cAMP）水平升高的敏感性，并以依赖磷酸二酯酶3（PDE3）活性的方式抑制β细胞增殖。尤为关键的是，使用(E)-3-(3-苯基苯并[c]异恶唑-5-基)丙烯酸（PIAA）对TBK1进行药理学抑制，可在基础培养条件下增强大鼠及人类胚胎干细胞（hESC）诱导的胰岛素分泌β细胞的增殖能力与功能活性。致糖尿病应激刺激可进一步上调TBK1的表达，相应地，PIAA可保护β细胞免受细胞因子及链脲佐菌素诱导的致糖尿病损伤，并促进β细胞复制。此外，在正常及2型糖尿病患者的人类胰岛中，PIAA可提升β细胞的增殖能力，同时增加胰岛素分泌量。综上，本研究揭示了TBK1作为细胞自主性负调控β细胞复制的关键因子的全新且不可或缺的功能，并证实PIAA可作为增强β细胞增殖与功能的有效治疗策略。将TBK1的siGENOME SMARTpool小干扰RNA（siRNA）或阴性对照非靶向siRNA转染至INS-1 832/13 β细胞，培养36小时。