ABSTRACT

Macroautophagy/autophagy is critical for the regulation of pancreatic β-cell mass and its deregulation has been implicated in the pathogenesis of type 2 diabetes (T2D). We have previously shown that treatment of pancreatic β-cells with the GLP1R (glucagon like peptide 1 receptor) agonist exendin-4 stimulates autophagic flux in a setting of chronic nutrient excess. The aim of this study was to identify the underlying pathways contributing to enhanced autophagic flux.

Pancreatic β-cells (INS-1E),mouse and human islets were treated with glucolipotoxic stress (0.5 mM palmitate and 25 mM glucose) in the presence of exendin-4. Consistent with our previous work, exendin-4 stimulated autophagic flux. Using chemical inhibitors and siRNA knockdown, we identified RAPGEF4/EPAC2 (Rap guanine nucleotide exchange factor 4) and downstream calcium signaling to be essential for regulation of autophagic flux by exendin-4. This pathway was independent of AMPK and MTOR signaling. Further analysis identified PPP3/calcineurin and its downstream regulator TFEB (transcription factor EB) as key proteins mediating exendin-4 induced autophagy. Importantly, inhibition of this pathway prevented exendin-4-mediated cell survival and overexpression of TFEB mimicked the cell protective effects of exendin-4 in INS-1E and human islets. Moreover, treatment of db/db mice with exendin-4 for 21 days increased the expression of lysosomal markers within the pancreatic islets. Collectively our data identify the RAPGEF4/EPAC2-calcium-PPP3/calcineurin-TFEB axis as a key mediator of autophagic flux, lysosomal function and cell survival in pancreatic β-cells. Pharmacological modulation of this axis may offer a novel therapeutic target for the treatment of T2D.

Abbreviations: AKT1/protein kinase B: AKT serine/threonine kinase 1; AMPK: 5’ AMP-activated protein kinase; CAMKK: calcium/calmodulin-dependent protein kinase kinase; cAMP: cyclic adenosine monophosphate; CASP3: caspase 3; CREB: cAMP response element-binding protein; CTSD: cathepsin D; Ex4: exendin-4(1-39); GLP-1: glucagon like peptide 1; GLP1R: glucagon like peptide 1 receptor; GLT: glucolipotoxicity; INS: insulin; MTOR: mechanistic target of rapamycin kinase; NFAT: nuclear factor of activated T-cells; PPP3/calcineurin: protein phosphatase 3; PRKA/PKA: protein kinase cAMP activated; RAPGEF3/EPAC1: Rap guanine nucleotide exchange factor 3; RAPGEF4/EPAC2: Rap guanine nucleotide exchange factor 4; SQSTM1/p62: sequestosome 1; T2D: type 2 diabetes; TFEB: transcription factor EB

摘要

巨自噬/自噬对于胰腺 β 细胞团的调节至关重要，其失调与 2 型糖尿病 (T2D) 的发病机制有关。我们之前已经表明，在慢性营养过剩的情况下，用 GLP1R（胰高血糖素样肽 1 受体）激动剂 exendin-4 治疗胰腺 β 细胞会刺激自噬通量。本研究的目的是确定有助于增强自噬通量的潜在途径。

在 exendin-4 存在下，用糖脂毒性应激（0.5 mM 棕榈酸酯和 25 mM 葡萄糖）处理胰腺 β 细胞 (INS-1E)、小鼠和人类胰岛。与我们之前的工作一致，exendin-4 刺激了自噬通量。使用化学抑制剂和 siRNA 敲低，我们确定了 RAPGEF4/EPAC2（Rap 鸟嘌呤核苷酸交换因子 4）和下游钙信号传导对于 exendin-4 调节自噬通量至关重要。该途径独立于 AMPK 和 MTOR 信号传导。进一步分析确定 PPP3/钙调神经磷酸酶及其下游调节因子 TFEB（转录因子 EB）是介导 exendin-4 诱导的自噬的关键蛋白。重要的是，抑制该途径阻止了 exendin-4 介导的细胞存活，并且 TFEB 的过表达模拟了 exendin-4 在 INS-1E 和人类胰岛中的细胞保护作用。而且，使用 exendin-4 的db/db小鼠 21 天增加了胰岛内溶酶体标志物的表达。总的来说，我们的数据确定 RAPGEF4/EPAC2-钙-PPP3/钙调神经磷酸酶-TFEB 轴是胰腺 β 细胞中自噬通量、溶酶体功能和细胞存活的关键介质。该轴的药理学调节可能为治疗 T2D 提供新的治疗靶点。

缩写：AKT1/蛋白激酶 B：AKT 丝氨酸/苏氨酸激酶 1；AMPK：5' AMP 活化蛋白激酶；CAMKK：钙/钙调蛋白依赖性蛋白激酶激酶；cAMP：环磷酸腺苷；CASP3：半胱天冬酶 3；CREB：cAMP反应元件结合蛋白；CTSD：组织蛋白酶 D；Ex4：exendin-4(1-39)；GLP-1：胰高血糖素样肽 1；GLP1R：胰高血糖素样肽 1 受体；GLT：糖脂毒性；INS：胰岛素；MTOR：雷帕霉素激酶的机制靶点；NFAT：活化T细胞的核因子；PPP3/钙调神经磷酸酶：蛋白磷酸酶 3；PRKA/PKA：蛋白激酶 cAMP 激活；RAPGEF3/EPAC1：说唱鸟嘌呤核苷酸交换因子 3；RAPGEF4/EPAC2：说唱鸟嘌呤核苷酸交换因子 4；SQSTM1/p62：隔离体 1；T2D：2型糖尿病；TFEB：转录因子EB