Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. myo-Inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. scyllo-Inositol is the second most abundant inositol isomer in the human brain, and aberrant concentrations are associated with various diseases; however, its biological functions remain unclear. Here, the development and application of [13C6]scyllo-inositol as an isotopic tracer to study its metabolism are reported. A concise and robust synthetic route was established to obtain [13C6]scyllo-inositol from [13C6]myo-inositol in good yield. The uptake of [13C6]scyllo-inositol at concentrations used in clinical trials and the responses of endogenous inositol isomers were measured in multiple immortalized mammalian cell lines by hydrophilic liquid interaction (HILIC)-MS/MS. [13C6]scyllo-Inositol proved to be a versatile isotopic tracer when coupled with mass spectrometry (MS)-based lipidomics and 2D nuclear magnetic resonance (NMR) experiments. These experiments provide evidence that scyllo-inositol is incorporated into phosphatidylinositols in different immortalized mammalian cell lines and suggest a previously underappreciated role of scyllo-inositol. Future research utilizing [13C6]scyllo-inositol can elucidate scyllo-inositol metabolism and its role in physiological and diseased states.

ABSTRACT Inositol phosphates (InsPs) and inositol pyrophosphates (PP‐InsPs) constitute a remarkably diverse yet evolutionarily conserved signaling network generated through combinatorial phosphorylation of myo‐inositol. Despite their widespread presence across eukaryotes, progress in understanding their biology was long constrained by major analytical challenges arising from their extreme charge density, structural similarity, and isomer diversity. The advent of new analytical approaches is now transforming the field. High‐resolution analytical platforms, including capillary‐electrophoresis mass spectrometry, liquid‐chromatography mass spectrometry, and inductively coupled plasma‐based detection, enable sensitive quantification and isomer discrimination. Parallel developments in chemical synthesis, stable‐isotope labeling, and NMR spectroscopy provide essential reference standards and permit investigation of their metabolic turnover in living systems. These developments have uncovered unrecognized InsP and PP‐InsP isomers, revealed substantial variation in metabolic networks across organisms and cell types, and exposed unexpectedly dynamic turnover. Emerging biological insights highlight InsPs and PP‐InsPs as versatile cellular regulators that link metabolism to cellular signaling, influencing cellular energetics and phosphate homeostasis. These messengers are involved in diverse processes both in the cytoplasm and in the nucleus, controlling protein complex assembly and protein pyrophosphorylation. Together, these advances shed light on InsPs and PP‐InsPs as adaptable signaling molecules and open new avenues for deciphering their physiological roles in health and disease.

Inositol is an essential metabolite required for membrane biogenesis, cell signaling and trafficking, and gene expression. Its importance is underscored by the fact that inositol depletion leads to death in eukaryotic cells ranging from yeast to human. Perturbation of inositol homeostasis is implicated in numerous human disorders. In yeast, inositol is synthesized de novo from glucose-6-phosphate (G-6-P) through a two-step pathway controlled by the rate-limiting enzyme myo-inositol phosphate synthase (MIPS), encoded by INO1. INO1 expression is tightly controlled by the Henry regulatory circuit in response to inositol and by the Reg1-Snf1 pathway in response to glucose. As both inositol synthesis and glycolysis utilize the same precursor, G-6-P, the current study tested the hypothesis that inositol synthesis is regulated by glycolytic activity. Genetic and pharmacological approaches were used to alter glycolysis. Inositol synthesis was determined by liquid chromatography mass spectrometry analysis of [U-13C]-glucose incorporation into inositol and by assaying INO1 mRNA and MIPS protein expression. In rho0 cells and wild type cells treated with the electron transport inhibitor potassium cyanide, both of which exhibit increased glycolytic activity, inositol levels were reduced by 47.5% and 57.9%, respectively, compared to controls. INO1 mRNA and MIPS protein levels were also decreased in these cells. The effect of downregulation of glycolysis was determined using the humanized yeast strain HHK2, which expresses a decreased rate of glycolysis due to the less active human hexokinase. Consistent with our hypothesis, HHK2 cells exhibited a 41.5% increase in inositol synthesis. Dysregulation of inositol synthesis did not affect glycolysis. These results identify altered glycolytic activity as a mechanism of inositol regulation and demonstrate the role of metabolic crosstalk in controlling these biochemical pathways.

Adenosine-to-inosine (A-to-I) RNA editing by ADAR1 is a key post-transcriptional modification, and mutations in ADAR1 lead to Aicardi-Goutières syndrome (AGS), an autoimmune disorder. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Using a combination of biochemical approaches, inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, molecular dynamics simulations, and a cell-permeable inositol hexakisphosphate (IP6) prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. Furthermore, we identify the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly coordinates a Zn²⁺-ion. Finally, we show that the AGS-associated ADAR1 mutation N907S impairs RNA editing activity, most likely by altering the hydrogen-bond interaction network linking IP6 to the ADAR1 catalytic center. Together, these findings identify IP6 as an essential cofactor and regulator of ADAR1 activity and highlight cofactor availability and interaction networks as strategies for therapeutically modulating RNA editing.

Inositol pyrophosphates are conserved signaling molecules synthesized by the bifunctional PPIP5K enzymes, but how their cellular functions diversify across species remains poorly understood. Here, we compared the PPIP5K enzyme Asp1 in the fission yeasts Schizosaccharomyces pombe and Schizosaccharomyces japonicus and in the distantly related fungus Ustilago maydis. All three homologs retained a conserved kinase-phosphatase architecture and catalytic activity. However, whereas Asp1 produced broadly similar effects on actin organization and morphogenesis in S. pombe and U. maydis, its regulatory output was reversed in S. japonicus. In S. pombe and U. maydis, Asp1 positively supported Arp2/3-dependent actin functions, as loss of Asp1 increased sensitivity to the Arp2/3 inhibitor CK666. In contrast, deletion of asp1 in S. japonicus conferred strong CK666 resistance and caused excessive, spatially deregulated actin-patch organization. This opposing cytoskeletal phenotype was mirrored at the level of morphogenesis: Asp1 restricted the yeast-to-hypha transition in S. japonicus, whereas Asp1 was required for pseudohyphal growth in S. pombe and for filamentous development in U. maydis. However, the negative regulatory activity observed in S. japonicus was not an intrinsic property of the SjAsp1 protein. When expressed in S. pombe, SjAsp1 promoted invasive pseudohyphal growth, reproducing the regulatory output of the S. pombe Asp1 morphogenesis pathway rather than that of its native species. Similarly, SjAsp1 supported Arp2/3 functions when expressed in S. pombe. Thus, SjAsp1 adopted the functional behavior imposed by the host cellular environment. S. pombe Asp1 was originally identified as a suppressor of Arp2/3-complex mutant phenotypes, establishing a genetic connection between Asp1 and the actin nucleator. Extending this link, affinity enrichment with inositol pyrophosphates reagents recovered all seven subunits of the S. pombe Arp2/3 complex, providing biochemical support for a potential association between inositol pyrophosphate and Arp2/3. Together, these findings identify S. japonicus as a functional outlier in which a conserved PPIP5K pathway produces an opposing biological output. They further demonstrate that this divergence is determined primarily by species-specific cellular networks rather than by intrinsic differences in the Asp1 protein.

Adenosine-to-inosine (A-to-I) RNA editing, catalyzed by adenosine deaminases acting on RNA (ADARs), is a key post-transcriptional modification that regulates RNA splicing, stability, and translation. Dysregulation of ADAR activity caused by mutations in ADAR1 leads to Aicardi-Goutieres syndrome (AGS), an autoimmune disorder characterized by aberrant activation of Melanoma differentiation-associated protein 5 (MDA5) by self RNA and excessive type I interferon production. Despite its biological and clinical relevance, the regulation of ADAR1 activity remains incompletely understood. Here, we show that ADAR1 protein levels and RNA editing activity in mammalian cells critically depend on the cofactor inositol hexakisphosphate (IP6). Using Inositol-pentakisphosphate 2-kinase (IPPK)-knockout cells, next-generation sequencing (NGS), and a cell-permeable IP6 prodrug (Pro-IP6), we demonstrate that IP6 depletion drastically reduces global RNA editing, while supplementation with Pro-IP6 restores and even enhances editing levels. In vitro ADAR1 translation and RNA editing assays revealed that IP6 contributes to the folding and full catalytic activity of ADAR1, and that inositol pentakisphosphate (1,3,4,5,6-IP5) can partially substitute IP6 as a cofactor. Molecular dynamics simulations and biochemical analyses identified the C6-phosphate of IP6 as a critical determinant of ADAR1 catalytic efficiency, functioning within a hydrogen-bonding network that indirectly governs Zn2+-ion positioning through interactions with key residues, including K1039 and N907. Notably, the AGS-associated N907S mutation impairs RNA editing, by altering IP6 coordination and introducing a more dynamic situation in the hydrogen-bonding network that linked IP6 and Zn2+-ion. Together, these findings identifies IP6 as an essential cofactor and regulator of ADAR1 activity and highlights cofactor availability and interaction networks as potential strategies for therapeutically modulating RNA editing in disease. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, Project-ID 369799452— TRR237 - A02, Project-ID 548714673, CIBSS – EXC-2189 – Project ID 390939984 Volkswagen Foundation, VW Momentum Grant 98604 Israel Science Foundation, 2637/23 Medical Research Council, MR/T028904/1

Summary Inositols are a family of cyclic sugar alcohols comprising nine stereoisomers. Myo -inositol is the most abundant isomer found in humans and has been studied most extensively. It plays an important role in osmoregulation and is incorporated into membrane-anchored phosphatidylinositols. Scyllo -inositol is the second most abundant inositol isomer in the human brain and aberrant concentrations are associated with various diseases; however, its biological functions remain poorly understood. Here, the development and application of [ 13 C 6 ] scyllo -inositol as an isotopic tracer to study its metabolism is reported. A concise and robust synthetic route was established to obtain [ 13 C 6 ] scyllo -inositol from [ 13 C 6 ] myo -inositol in good yield. The uptake of [ 13 C 6 ] scyllo -inositol and responses of endogenous inositol isomers were measured in multiple cell lines by HILIC-MS/MS, showcasing the advantages of isotopic tracing. [ 13 C 6 ] scyllo -inositol proved to be a versatile isotopic tracer, when coupled with MS-based lipidomics and 2D NMR experiments. These experiments provide evidence that scyllo -inositol is incorporated into phosphatidylinositols in different cell lines. The results suggest a previously underappreciated role of scyllo -inositol in mammalian cells. The utilization of [ 13 C 6 ] scyllo -inositol will help to elucidate the role of scyllo -inositol metabolism in healthy and diseased states. Significance Scyllo -inositol is a cyclic sugar alcohol found predominantly in the human brain. Changes in its concentration are associated with different diseases, and scyllo -inositol has been investigated as a potential drug against Alzheimer’s disease in clinical trials. However, its metabolic fate in mammalian cells is not well understood. We report here a synthetic strategy to obtain [ 13 C 6 ] scyllo -inositol and demonstrate, through isotopic tracing, its incorporation into phosphatidylinositols in different human-derived cell lines. This new stable isotopic tracer enables the investigation of the biological role of scyllo -inositol in mammals and beyond. Highlights Concise synthesis of [ 13 C 6 ] scyllo -inositol [ 13 C 6 ] scyllo -inositol uptake and response of endogenous inositol isomers studied in multiple cell lines Use of [ 13 C 6 ] scyllo -inositol as an isotopic tracer in metabolomics and lipidomics experiments Evidence for scyllo -inositol incorporation into phosphatidylinositol in mammalian cells

Nuclear speckles (NS) are membraneless nuclear organelles that act as critical hubs for pre-messenger RNA splicing. Defects in splicing are linked to several human diseases, including cancer, Alzheimer's disease, and dystrophies. While CLK kinases regulate the mobilization of splicing factors from NS, the molecular mechanisms underlying NS assembly and dissolution remain unclear. Using an adaptation of the Biotinylation by Antibody Recognition technique, we identified polyphosphate (polyP) as a novel and essential regulator of NS dynamics. Polyphosphate, a highly conserved polyanion composed of a chain of phosphate molecules, is involved in several functions in mammalian cells. Here, we show that polyP interacts with the NS core component SRRM2, and its depletion disrupts NS organization releasing splicing factors into the nucleoplasm. RNA-seq analysis reveals that polyP depletion increases exon exclusion, particularly in transcripts with multiple isoforms, highlighting its role in splicing regulation. Mechanistically, we demonstrate that polyP acts as a physiological inhibitor of CLK3 kinase, preventing the phosphorylation of SR proteins and thereby maintaining NS stability. Our findings not only expand our understanding of NS biology but also provide new insights into the polyP involvement in splicing-related diseases.

HDAC inhibition shows promise in cancer treatment but pan-HDAC inhibitors cause gastrointestinal issues in 48% of patients. Understanding HDAC activation mechanisms is crucial to treating diverse diseases beyond cancer. Our study reveals that inositol polyphosphate multikinase (IPMK) and inositol hexakisphosphate (InsP6 or phytic acid), enriched in vegan diets, play essential roles in activating the HDAC3 epigenetic axis and maintaining intestinal barrier integrity. IPMK binds to HDAC3 and drives InsP6 synthesis, which selectively activates HDAC3 at a 10 nM concentration by recruiting the DAD domain of its corepressor protein. IPMK deletion diminishes HDAC3 activation, leading to histone hyperacetylation and MMP gene transcription that compromise intestinal barrier integrity. InsP6 treatment is sufficient to rescue these effects. In inflammatory bowel disease, diminished IPMK levels exacerbate intestinal permeability, while oral InsP6 treatment mitigates leaky gut effects by restoring the HDAC3 epigenetic axis, highlighting the clinical significance of the IPMK-HDAC3 pathway and the therapeutic potential of phytic acid.

Inositol is an essential nutrient for most living organisms, as combinatorial phosphorylation on this cyclic sugar generates key cellular messengers, such as lipid-bound phosphoinositides (PtdInsPs), water-soluble inositol phosphates (InsPs), and high-energy inositol pyrophosphates (PP-InsPs). Although the kinases and phosphatases modifying inositol-derived molecules are well-characterised, the molecular pathways controlling the cellular homeostasis of the inositol backbone and transport carriers remain unclear. Using a combination of LC-MS analysis and a screen based on the inositol-exporting opi1Δ mutant yeast, we here discovered that inositol export is tightly regulated by PP-InsPs and that the high-affinity phosphate transporter Pho84 also acts as an inositol exporter. We further expanded these observations to the mammalian system and revealed that inositol export in human cells is similarly controlled by PP-InsPs, and that the human homolog of the yeast Pho84, GLUT2, contributes to inositol export. In summary, we discovered an evolutionarily conserved crosstalk pathway linking PP-InsPs to both phosphate and inositol homeostasis. Inositol provides the backbone for phosphoinositide and inositol phosphate signalling molecules, yet how cells regulate their pool of free inositol remain ill-defined. This study identifies yeast Pho84 and its human homologue GLUT2 as inositol exporters and uncovers inositol pyrophosphates as novel regulators of inositol homeostasis. An evolutionarily conserved transport circuit links inositol pyrophosphate signalling to cellular inositol and phosphate homeostasis.

Inositol polyphosphates are ubiquitously present in eukaryotic cells and play important roles in diverse cellular processes such as protein folding, signal transduction, and phosphate homeostasis. Although these negatively charged small metabolites are easily stripped from metals/proteins and extracted under strong acidic conditions, their low intracellular concentrations in some biological settings command a further enrichment step to facilitate downstream analyses. Here, we describe the use of titanium dioxide beads to enrich for inositol polyphosphate extracted from cultured mammalian cells. This protocol not only concentrates the diluted inositol phosphates but also removes salts and proteins that would otherwise interfere with postpurification analyses.

The diverse chemical structures of inositol pyrophosphates attract growing interest toward this class of small molecule messengers. However, their highly charged nature, the lack of a chromophore or a fluorophore, their close structural relatedness, and the complexity of their metabolism pose serious challenges to inositol pyrophosphates studies. Here, we summarize how researchers have begun to overcome these challenges and how recent experimental advances are propelling inositol pyrophosphate research into the future.

Inositol phosphate (InsP) and diphosphoinositol phosphate (PP-InsP) analysis in tissues is plagued by multiple difficulties of sensitivity, regioisomer resolution and the need for radiolabelling with metabolic precursors. We describe a liquid chromatography (LC) inductively coupled plasma (ICP) mass spectrometry (MS) method (LC-ICP-MS) that addresses all such issues and use LC-ICP-MS to analyse InsPs in avian tissues. The highly sensitive technique tolerates complex matrices and, by powerful chromatography, resolves in a single run multiple non-enantiomeric myo-inositol tetrakisphosphates, myo-inositol pentakisphosphates and all inositol hexakisphosphates, including myo-inositol 1,2,3,4,5,6-hexakisphosphate (phytate), known in nature. It also separates and quantifies diphospho myo-inositol pentakisphosphate (PP-InsP5) isomers from their biological precursors and from 1,5-bis-diphospho myo-inositol 2,3,4,6 tetrakisphosphate (1,5-[PP]2- InsP4). Gut tissue InsPs, belonging to a non-canonical, lipid-independent pathway, are shown to differ from phytate digestion products and to be responsive to diet.

Chromosome transmission fidelity is vital for organism fitness. Yet, extrinsic and intrinsic changes can affect this process, leading to aneuploidy, the loss/gain of chromosomes, which is a hallmark of cancer. Here, using a haploid fission yeast Schizosaccharomyces pombe strain with a segmental aneuploidy, we assayed genome stability under different temperatures and altered gene dosage. We find that S. pombe genome stability is temperature-dependent and is unexpectedly modulated by intracellular levels of inorganic polyphosphate polymers (polyP). The vtc4+ gene, encoding a subunit of the polyP-generating VTC complex, is present twice due to the segmental aneuploidy resulting in a gene-dosage-coupled increase in polyP. Using strains with different amounts of polyP, we find a direct negative correlation between polyP and chromosome segregation fidelity. PolyP modulates the function of the conserved CCAN kinetochore subcomplex, as the abnormal growth phenotype caused by the mutant CCAN protein Fta2-291 was rescued in the absence of polyP, while extra polyP had the opposite effect. Importantly, this appears to occur in part by modulation of the nucleolin Gar2. Gar2 is the functional homolog of the Saccharomyces cerevisiae Nsr1 protein, whose function is modulated by posttranslational polyP-mediated polyphosphorylation. Thus, polyP modulates genome stability, linking cellular metabolism to chromosome transmission fidelity.

Inositol phosphates are involved in a myriad of biological roles and activities such as Ca2+ signaling, phosphate homeostasis, energy metabolism, and disease pathogenicity. In Saccharomyces cerevisiae, synthesis of inositol phosphates occurs through the phosphoinositide phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol and further IP3 phosphorylation by additional kinases that leads to the formation of highly phosphorylated inositol derivatives, known as inositol pyrophosphates. Inositol-tetrakisphosphate 1-kinase (ITPK1) is an enzyme that mediates a PLC-independent inositol polyphosphate synthesis through phosphorylation of inositol monophosphates and other intermediates in the cytosol. In this work, we identified and characterized a Trypanosoma cruzi ITPK1 (TcITPK1) homolog. The ability of TcITPK1 to act as the mediator for this alternative pathway was established through plc1Δ and plc1Δ isc1Δ yeast complementation assays and SAX-HPLC analyses of radioactively labeled inositol. TcITPK1 localizes to the cytosol, and knockout attempts of TcITPK1 revealed that only one allele was replaced by the DNA donor cassette at the specific locus, suggesting that null alleles may have lethal effects in epimastigotes. Ablation of T. cruzi phosphoinositide phospholipase C 1 (TcPI-PLC1) affected the synthesis of IP3 from glucose 6-phosphate but did not affect the synthesis of inositol polyphosphates, while ablation of inositol phosphosphingolipid phospholipase (TcISC1) affected the synthesis of inositol polyphosphates, thus revealing that the PLC-independent pathway using either glucose 6-phosphate or inositol phosphoceramide is involved in the synthesis of inositol polyphosphates, while the PLC-dependent pathway is involved in IP3 formation needed for Ca2+ signaling. Millions of people are infected with Trypanosoma cruzi, and the current treatment is not satisfactory. Inositol pyrophosphates have been established as important signaling molecules. Our work demonstrates the presence of a phospholipase C-independent pathway for the synthesis of inositol pyrophosphates in T. cruzi. Furthermore, we demonstrate that this pathway starts with the synthesis of inositol monophosphates from glucose 6-phosphate or from inositol phosphoceramide, linking it to carbohydrate and sphingolipid metabolism. The essentiality of the pathway for the survival of T. cruzi infective stages makes it an ideal drug target for treating American trypanosomiasis.

Polyacrylamide gel electrophoresis (PAGE) is a versatile technique widely used in molecular biology for the separation of biomolecules based on size and charge. While it is traditionally applied to proteins and nucleic acids, recently, PAGE has been adapted for the analysis of inositol polyphosphates and their pyrophosphate derivatives, which play multiple roles in cellular signaling. Studying these molecules presents analytical challenges due to their small size, high charge density, and low concentrations in biological samples. In this work, we describe an optimized PAGE protocol for the separation and detection of inositol polyphosphates and inositol pyrophosphate. The method uses high-concentration polyacrylamide gels and toluidine blue staining to achieve sensitive detection at nanomolar levels. The simplicity and cost-efficiency of the approach make it accessible to most laboratories. This method provides a reliable tool for investigating inositol-phosphate-based signaling pathways and their involvement in cellular processes.

Inorganic polyphosphate (polyP) is a ubiquitous biopolymer composed of multiple orthophosphates connected by energy-rich phosphoanhydride bonds. In organisms, polyP is digested by two types of enzymes: exopolyphosphatases, which shorten the chain from the ends by cleaving off monophosphate units, and endopolyphosphatases, which cut the chain internally. While several continuous methods are available to monitor exopolyphosphatase activity, endopolyphosphatase activity assays are less common and typically involve multiple tedious steps. Here, we introduce FRET-polyP8, a novel probe for real-time detection of endopolyphosphatase activity. The FRET assay enabled rapid, highly sensitive, single-step detection of specific endopolyphosphatase activity both from isolated proteins and cell extracts. The simple read-out additionally enabled enzyme inhibitor screening. Furthermore, a novel Mn2+-dependent endopolyphosphatase activity in baker's yeast was detected in a quadruple mutant, highlighting the ability to screen for metal-dependence of new endopolyphosphatase activity. This approach thus represents a significant addition to existing methodologies, facilitating the discovery and classification of new endopolyphosphatases and their inhibitors to advance our understanding of polyP metabolism and regulation.

Inositol phosphates control many central processes in eukaryotic cells including nutrient availability, growth, and motility. Kinetic resolution of a key modulator of their signaling functions, the turnover of the phosphate groups on the inositol ring, has been hampered by slow uptake, high dilution, and constraining growth conditions in radioactive pulse-labeling approaches. Here, we demonstrate a rapid (seconds to minutes) and nonradioactive labeling strategy of inositol polyphosphates through 18O-water in yeast, human cells, and amoeba, which can be applied in any media. In combination with capillary electrophoresis and mass spectrometry, 18O-water labeling simultaneously dissects the in vivo phosphate group dynamics of a broad spectrum of even rare inositol phosphates. The good temporal resolution allowed us to discover vigorous phosphate group exchanges in some inositol polyphosphates and pyrophosphates, whereas others remain remarkably inert. We propose a model in which the biosynthetic pathway of inositol polyphosphates and pyrophosphates is organized in distinct, kinetically separated pools. While transfer of compounds between those pools is slow, each pool undergoes rapid internal phosphate cycling. This might enable the pools to perform distinct signaling functions while being metabolically connected.

Inositol plays key roles in many cellular processes. Several studies focussed on the quantitative analysis of phosphorylated forms of inositol, enabled by analytical tools developed to detect these highly charged molecules. Direct measurement of free inositol however has been challenging, because the molecule is uncharged and polar. As a result, the mechanisms maintaining the homeostasis of the inositol remains poorly understood. In this study, we overcome these challenges by developing a quantitative liquid chromatography - mass spectrometry (LC-MS) protocol that can resolve and quantify the three main sugar molecules present inside cells: glucose, fructose, and inositol, as well as distinguish the clinically relevant isomers of inositol: myo-, scyllo-, and chiro-inositol. The quantitative power of the new method was validated by accurately monitoring the changes of inositol levels under well-established conditions in Saccharomyces cerevisiae, where the endogenous synthesis of inositol is increased in the transcription repressor OPI1 knockout opi1D and decreased when wild type yeast is fed with exogenous inositol. The method also revealed a new layer of regulation that takes place when exogenous inositol is added to further boost endogenous inositol synthesis in opi1D in a positive feedback loop. Analyses of mammalian cell lines provided many new insights into inositol metabolism. First, different cell lines displayed distinct sugar profiles and inositol concentrations and responded differently to inositol starvation. Second, mammalian cells can synthesize and import scyllo- but not chiro-inositol. Importantly, our method lent direct evidence to the previous hypothesis that lithium treatment could significantly reduce inositol levels in primary cortical neurons, thus diminishing the pool of free inositol available to the phosphoinositide cycle.