Inositol Depletion Regulates Phospholipid Metabolism And Activates Stress Signaling In Hek293t Cells

Inositol is a cyclical six-carbon sugar that plays an essential role in human health and disease. Inositol metabolism has been associated with several human disorders such as bipolar disorder (BD), Alzheimer\u27s disease, Lowe syndrome, polycystic ovary syndrome, cancer, and Charcot-Marie-Tooth. Furthermore, inositol-less death studies in small eukaryotes suggest that inositol is a critically important metabolite. In yeast, it has been shown to be a major metabolic regulator, its synthesis is highly regulated, and it affects the expression of hundreds of genes. Despite its importance, almost nothing is known about the regulation of inositol synthesis and the effects of inositol depletion in human cells. Therefore, the current thesis aimed to address this knowledge gap. I developed a simple and reliable method for assaying inositol levels, which utilizes an inositol auxotrophic strain of Saccharomyces cerevisiae. This assay will contribute to the research into inositol metabolism and function. I generated a HEK293T ISYNA1-KO human cell line that is deficient in the first step of inositol synthesis. Lipidomic analysis identified inositol depletion as a major regulator of lipid metabolism, including a decrease in phosphatidylinositol (PI) levels, upregulation of ceramides, and perturbation of synthesis of phosphatidylglycerol (PG)/cardiolipin (CL)-derived lipids. Additionally, a transcriptomic analysis demonstrated that inositol depletion induces profound changes in gene transcription and cell signaling, including the activation of the integrated stress response, upregulation of amino acid metabolism, and autophagy. Interestingly, the mood stabilizing drug valproate (VPA) was also found to regulate lipid metabolism, increase ceramide levels and activate endoplasmic reticulum stress elements (ERSE). These findings will provide a foundation to understand disease processes in human disorders that stem from perturbation of inositol homeostasis, including BD and cancer. Additionally, it will improve our understanding of the therapeutic mechanism of action of inositol-depleting drugs