School of Health Sciences, Purdue University, West Lafayette, IN 47907, USA.
Exponent Inc., Alexandria, VA 22314, USA.
Biomolecules. 2023 Aug 8;13(8):1229. doi: 10.3390/biom13081229.
The insulin-like growth factor (IGF)/insulin signaling (IIS) pathway is involved in cellular responses against intracellular divalent manganese ion (Mn) accumulation. As a pathway where multiple nodes utilize Mn as a metallic co-factor, how the IIS signaling patterns are affected by Mn overload is unresolved. In our prior studies, acute Mn exposure potentiated IIS kinase activity upon physiological-level stimulation, indicated by elevated phosphorylation of protein kinase B (PKB, also known as AKT). AKT phosphorylation is associated with IIS activity; and provides direct signaling transduction input for the mammalian target of rapamycin complex 1 (mTORC1) and its downstream target ribosomal protein S6 (S6). Here, to better define the impact of Mn exposure on IIS function, Mn-induced IIS activation was evaluated with serial concentrations and temporal endpoints. In the wild-type murine striatal neuronal line ST, the acute treatment of Mn with IGF induced a Mn concentration-sensitive phosphorylation of S6 at Ser235/236 to as low as 5 μM extracellular Mn. This effect required both the essential amino acids and insulin receptor (IR)/IGF receptor (IGFR) signaling input. Similar to simultaneous stimulation of Mn and IGF, when a steady-state elevation of Mn was established via a 24-h pre-exposure, phosphorylation of S6 also displayed higher sensitivity to sub-cytotoxic Mn when compared to AKT phosphorylation at Ser473. This indicates a synergistic effect of sub-cytotoxic Mn on IIS and mTORC1 signaling. Furthermore, elevated intracellular Mn, with both durations, led to a prolonged activation in AKT and S6 upon stimulation. Our data demonstrate that the downstream regulator S6 is a highly sensitive target of elevated Mn and is well below the established acute cytotoxicity thresholds (<50 μM). These findings indicate that the IIS/mTORC1 pathways, in which Mn normally serves as an essential co-factor, are dually responsible for the cellular changes in exposures to real-world Mn concentrations.
胰岛素样生长因子（IGF）/胰岛素信号（IIS）通路参与细胞对细胞内二价锰离子（Mn）积累的反应。作为一个利用 Mn 作为金属辅因子的多个节点的通路，IIS 信号模式如何受到 Mn 过载的影响尚未解决。在我们之前的研究中，急性 Mn 暴露在生理水平的刺激下增强了 IIS 激酶活性，表现为蛋白激酶 B（PKB，也称为 AKT）的磷酸化水平升高。AKT 磷酸化与 IIS 活性相关；并为雷帕霉素复合物 1（mTORC1）及其下游靶标核糖体蛋白 S6（S6）提供直接的信号转导输入。在这里，为了更好地定义 Mn 暴露对 IIS 功能的影响，我们评估了 Mn 诱导的 IIS 激活与连续浓度和时间终点的关系。在野生型小鼠纹状体神经元系 ST 中，IGF 急性处理 Mn 会导致 S6 在 Ser235/236 上发生 Mn 浓度敏感的磷酸化，其最低 Mn 浓度低至 5 μM 细胞外 Mn。这种效应需要必需氨基酸和胰岛素受体（IR）/胰岛素样生长因子受体（IGFR）信号输入。与同时刺激 Mn 和 IGF 类似，当通过 24 小时预暴露建立稳定的 Mn 升高时，与 Ser473 处的 AKT 磷酸化相比，S6 的磷酸化对亚细胞毒性 Mn 也显示出更高的敏感性。这表明亚细胞毒性 Mn 对 IIS 和 mTORC1 信号具有协同作用。此外，无论持续时间长短，升高的细胞内 Mn 都会导致刺激后 AKT 和 S6 的持续激活。我们的数据表明，下游调节剂 S6 是升高的 Mn 的高度敏感靶标，远低于既定的急性细胞毒性阈值（<50 μM）。这些发现表明，IIS/mTORC1 通路通常将 Mn 作为必需的辅因子，在暴露于实际 Mn 浓度时，对细胞变化具有双重责任。
