Suppression of ITPK1 and IPMK activities impairs mTORC1 signaling due to accelerated signal termination and prevents pancreatic ß-cell metabolic adaptation to excess glucose

Abstract mTORC1 integrates growth factor and nutrient signals to regulate cellular metabolism, yet there are no metabolites known to directly regulate mTORC1 activity in cells. Cryo-EM studies revealed that inositol hexakisphosphate (IP 6 ) associates with the FAT domain of mTOR, suggesting that inositol phosphates may directly modulate mTOR activity. We previously showed that higher-order inositol phosphates enhance mTORC1 kinase activity and stability in vitro . Here, we investigated whether inositol phosphate metabolism regulates mTORC1 signaling in HEK293 and pancreatic β-cells. Suppression or acute inhibition of inositol phosphate multikinase (IPMK) selectively reduced cellular IP 5 levels without altering IP 6 and resulted in reduced basal and insulin-stimulated mTORC1 signaling, without decreasing mTORC1 complex stability or lysosomal localization. ITPK1 knockdown phenocopied IPMK inhibition by reducing IP 5 and basal mTORC1. Combined inhibition of IPMK and ITPK1 nearly abolished IP 5 and reduced IP 6 , demonstrating that these enzymes compensate to supply IP 5 for IP 6 synthesis. Importantly, depletion of IP 5 did not impair PI3K/Akt activation but accelerated termination of the mTORC1 signal, indicating a role for IP 5 in stabilizing the active mTORC1 complex. Reduction of IP 5 levels did not prevent insulin- or glucose-induced mTORC1 activation, revealing that inositol phosphates primarily regulate signal persistence rather than initiation. Lastly, we show that suppression of IPMK or ITPK1 in ß-cells delayed the mTORC1-dependent metabolic acceleration in response to excess glucose. Together, these findings identify higher order inositol phosphates as metabolic regulators that prolong mTORC1 activity, providing a potential strategy for modulating mTORC1 signaling in cells. Significance Statement mTORC1 is a central metabolic regulator whose chronic activation contributes to metabolic disease, yet mechanisms that sustain mTORC1 activity after its activation are poorly understood. We show that enzymes controlling inositol phosphate metabolism regulate the persistence of mTORC1 signaling in pancreatic β-cells by maintaining cellular levels of inositol pentakisphosphate (IP 5 ). Reducing IP 5 impairs basal and sustained mTORC1 signaling without affecting upstream growth factor or energy-sensing pathways, revealing a mechanism that controls signal duration rather than activation. Reduction in IP 5 levels also delayed the mTORC1-dependent ß-cell metabolic acceleration in response to elevated glucose. These findings identify IP 5 as a metabolic regulator of mTORC1 and suggest that targeting inositol phosphate metabolism may provide a strategy to modulate mTORC1 activity in metabolic disease.