Peng Qianyi, Jia Song Hui, Parodo Jean, Ai Yuhang, Marshall John C
Keenan Research Centre for Biomedical Science, St. Michael's Hospital, and the Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Ontario, Canada; and Department of Critical Care Medicine, Xiangya Hospital, Central South University, Changsha, China.
Department of Surgery, Keenan Research Centre for Biomedical Science, St. Michael's Hospital, and the Interdepartmental Division of Critical Care Medicine, University of Toronto, Toronto, Ontario, Canada; and.
Am J Physiol Endocrinol Metab. 2015 Feb 15;308(4):E324-33. doi: 10.1152/ajpendo.00006.2014. Epub 2014 Dec 16.
Pre-B cell colony-enhancing factor (PBEF) is a highly conserved pleiotropic protein reported to be an alternate ligand for the insulin receptor (IR). We sought to clarify the relationship between PBEF and insulin signaling by evaluating the effects of PBEF on the localization of the IRβ chain to lipid rafts in A549 epithelial cells. We isolated lipid rafts from A549 cells and detected the IR by immunoprecipitation from raft fractions or whole cell lysates. Cells were treated with rPBEF, its enzymatic product nicotinamide adenine dinucleotide (NAD), or the Nampt inhibitor daporinad to study the effect of PBEF on IRβ movement. We used coimmunoprecipitation studies in cells transfected with PBEF and IRβ constructs to detect interactions between PBEF, the IRβ, and caveolin-1 (Cav-1). PBEF was present in both lipid raft and nonraft fractions, whereas the IR was found only in lipid raft fractions of resting A549 cells. The IR-, PBEF-, and Cav-1-coimmunoprecipitated rPBEF treatment resulted in the movement of IRβ- and tyrosine-phosphorylated Cav-1 from lipid rafts to nonrafts, an effect that could be blocked by daporinad, suggesting that this effect was facilitated by the Nampt activity of PBEF. The addition of PBEF to insulin-treated cells resulted in reduced Akt phosphorylation of both Ser⁴⁷³ and Thr³⁰⁸. We conclude that PBEF can inhibit insulin signaling through the IR by Nampt-dependent promotion of IR translocation into the nonraft domains of A549 epithelial cells. PBEF-induced alterations in the spatial geometry of the IR provide a mechanistic explanation for insulin resistance in inflammatory states associated with upregulation of PBEF.
前B细胞集落增强因子（PBEF）是一种高度保守的多效性蛋白，据报道它是胰岛素受体（IR）的一种替代配体。我们试图通过评估PBEF对A549上皮细胞中IRβ链定位于脂筏的影响，来阐明PBEF与胰岛素信号传导之间的关系。我们从A549细胞中分离出脂筏，并通过对脂筏组分或全细胞裂解物进行免疫沉淀来检测IR。用重组PBEF、其酶促产物烟酰胺腺嘌呤二核苷酸（NAD）或Nampt抑制剂达泊西汀处理细胞，以研究PBEF对IRβ移动的影响。我们在转染了PBEF和IRβ构建体的细胞中进行共免疫沉淀研究，以检测PBEF、IRβ和小窝蛋白-1（Cav-1）之间的相互作用。PBEF存在于脂筏和非脂筏组分中，而IR仅存在于静息A549细胞的脂筏组分中。IR、PBEF和Cav-1共免疫沉淀的重组PBEF处理导致IRβ和酪氨酸磷酸化的Cav-1从脂筏移动到非脂筏，达泊西汀可以阻断这种效应，这表明这种效应是由PBEF的Nampt活性促进的。向胰岛素处理的细胞中添加PBEF导致Ser⁴⁷³和Thr³⁰⁸的Akt磷酸化降低。我们得出结论，PBEF可以通过Nampt依赖性促进IR易位到A549上皮细胞的非脂筏结构域来抑制通过IR的胰岛素信号传导。PBEF诱导的IR空间几何结构改变为与PBEF上调相关的炎症状态下的胰岛素抵抗提供了一种机制解释。
