IGF/mTORC1/S6 Signaling Is Potentiated and Prolonged by Acute Loading of Subtoxicological Manganese Ion

The insulin-like growth factor (IGF)/insulin signaling (IIS) pathway is involved in cellular responses against intracellular divalent manganese ion (Mn²⁺) accumulation. As a pathway where multiple nodes utilize Mn²⁺ as a metallic co-factor, how the IIS signaling patterns are affected by Mn²⁺ overload is unresolved. In our prior studies, acute Mn²⁺ 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 Mn²⁺ exposure on IIS function, Mn²⁺-induced IIS activation was evaluated with serial concentrations and temporal endpoints. In the wild-type murine striatal neuronal line STHdh, the acute treatment of Mn²⁺ with IGF induced a Mn²⁺ concentration-sensitive phosphorylation of S6 at Ser235/236 to as low as 5 μM extracellular Mn²⁺. This effect required both the essential amino acids and insulin receptor (IR)/IGF receptor (IGFR) signaling input. Similar to simultaneous stimulation of Mn²⁺ and IGF, when a steady-state elevation of Mn²⁺ was established via a 24-h pre-exposure, phosphorylation of S6 also displayed higher sensitivity to sub-cytotoxic Mn²⁺ when compared to AKT phosphorylation at Ser473. This indicates a synergistic effect of sub-cytotoxic Mn²⁺ on IIS and mTORC1 signaling. Furthermore, elevated intracellular Mn²⁺, 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 Mn²⁺ and is well below the established acute cytotoxicity thresholds (<50 μM). These findings indicate that the IIS/mTORC1 pathways, in which Mn²⁺ normally serves as an essential co-factor, are dually responsible for the cellular changes in exposures to real-world Mn²⁺ concentrations.