Exploring the protective mechanisms of insulin receptor substrate-1 under conditions of hypoxic-ischemic injury

Abstract

Stroke is a major cause of death and disability worldwide, with neuronal death as a key factor. IRS-1, a core insulin signaling protein, may support neuronal survival under hypoxic conditions. In this study, primary cortical neurons were subjected to oxygen-glucose deprivation (OGD) followed by 24 hours of reoxygenation to mimic ischemic injury. Cell viability was significantly reduced, with approximately 50% neuronal death observed under these conditions. Interestingly, while IRS-1 mRNA levels remained relatively stable, a marked and time-dependent decrease in IRS-1 protein was detected, suggesting post-transcriptional regulation. Immunofluorescence confirmed the loss of IRS-1 expression in neurons after OGD. To explore the mechanism of IRS-1 degradation, we applied MG132 (a proteasome inhibitor), 3-MA (an autophagy inhibitor), and Rapamycin (an autophagy activator via mTOR inhibition). While MG132 improved cell viability, it did not restore IRS-1 levels, indicating that proteasomal degradation was not the dominant pathway. In contrast, inhibition of autophagy via 3-MA significantly increased IRS-1 expression and decreased LC3-II levels, a marker of autophagic flux. Rapamycin, surprisingly, also partially restored IRS-1 protein levels despite promoting autophagy, suggesting a modulatory effect through the mTOR/S6K1 pathway. Furthermore, Western blot analysis revealed a substantial increase in Ser636/639 phosphorylation of IRS-1 under OGD, a modification known to target IRS-1 for degradation. Both 3-MA and Rapamycin significantly reduced this phosphorylation, while simultaneously enhancing Tyr608 phosphorylation, a site associated with IRS-1 activation. These findings suggest that under ischemic stress, IRS-1 undergoes degradation primarily through autophagy linked with stress-induced phosphorylation at Ser636/639. Modulation of autophagy pathways or phosphorylation status could thus be a promising strategy to preserve IRS-1 function and support neuronal survival. Altogether, this study highlights IRS-1 as a potential neuroprotective factor and therapeutic target for the treatment of ischemic stroke.