Docosahexaenoic Acid Modulates Myo-Inositol and Myo-Inositol Biosynthetic Genes Expression: Implications for Bipolar Disorder

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Although pharmacological mood stabilizers remain a cornerstone of treatment, their variable efficacy and adverse side effect profiles limit long-term use in many patients. As such, dietary interventions, particularly omega-3 polyunsaturated fatty acids have garnered interest as adjunctive or alternative treatments. Among these, docosahexaenoic acid (DHA) is one of the most extensively studied. While several clinical trials suggest omega-3 fatty acids may alleviate certain symptoms of bipolar disorder, results have been inconsistent, and the mechanisms of action remain unclear. Given that intracellular myo -inositol depletion has been proposed as a common mechanism of action for conventional mood stabilizers we hypothesized that DHA, which modulates components of the phosphatidylinositol pathway, will also deplete intracellular myo -inositol and thus modulate myo -inositol homeostasis. Methodology: Using an enzymatic assay intracellular myo-inositol levels were measured in the extracts of cells grown in the presence of DHA. The effect of DHA on expression of the myo-inositol biosynthetic genes INO1 and INM1 was assessed by RT-qPCR. Results: The results showed DHA alters intracellular myo -inositol in a concentration-dependent manner which paralleled its effect on INO1 and INM1 expression. Conclusion: These findings suggest that the therapeutic potential of DHA in bipolar disorder may be mediated, at least in part, by its regulation of intracellular myo -inositol and its biosynthetic gene network. omega-3 fatty acids docosahexaenoic acid myo-inositol INM1 INO1 bipolar disorder Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Bipolar disorder (BD) is one of the most debilitating and severe mood disorders. It is the 6th leading cause of disability among non-communicable diseases, with an estimated prevalence between 1 and 2% (Merinkgas et al., 2007; Kessler er al., 2005; Bauer and Pfennig, 2005 ). Referred to as manic-depressive illness, it is characterized by recurring episodes of mania and depression, with the possibility of each episode lasting up to several months. While the cause of bipolar disorder remains unknown, dysregulation of the phosphatidylinositol signaling pathway has been implicated. For example, Jope et al. ( 1996 ) observed reduced phosphatidylinositol hydrolysis in the occipital cortex of postmortem bipolar patients, while Brown et al. ( 1993 ) found increased levels of phosphatidylinositol-4,5-bisphosphate in the platelet membranes of unmedicated individuals during the manic phase. Treatment options for bipolar disorder include mood stabilizers, antipsychotics, and antidepressants. Among these, lithium and valproate are the most prescribed mood stabilizers; however, their use is limited by significant side effects and narrow therapeutic windows (Crudup, 2011; Gitlin, 2016 ; Carli et al., 2022 ). Although the precise therapeutic mechanisms of lithium and valproate are not fully understood, one proposed mode of action is the depletion of myo -inositol levels which may help normalize disrupted neuronal circuitry associated with the disorder (Moore et al., 1999 ; O' Donnell et al., 2000; Shaltiel et al., 2004 ; Silverstone et al., 2005 ; Machado-Vieira et al., 2009 ; Bergamelli et al., 2021 ). Preliminary clinical evidence suggests that omega-3 fatty acids, whether administered as monotherapy or as an adjunct to antidepressant treatment, are associated with improvements in depressive symptoms among individuals with bipolar disorder. These effects are demonstrated by reductions in Hamilton Depression Rating Scale scores and enhanced overall clinical evaluations, as measured by the Clinical Global Impression and Global Assessment Scales (Krawczyk & Rybakowski, 2012 ; Frangou et al., 2006 ; Osher, 2005). However, their efficacy in addressing manic symptoms appears limited. For example, Fristad et al. (2015) reported only modest effects on manic symptomatology, as assessed by the Young Mania Rating Scale. Furthermore, a broader review of clinical trials has yielded inconsistent findings regarding the overall effectiveness of omega-3 fatty acids in the treatment of bipolar disorder (Psara et al., 2025 ). While the molecular mechanisms underlying the therapeutic benefits of omega-3-fatty acids remain unclear, there is evidence that like lithium and valproate, omega-3 fatty acids affect the phosphatidylinositol signaling pathway. Specifically, they have been shown to decrease protein kinase C and phospholipase C activity, as well as the production of inositol triphosphate (Mirnikjoo et al., 2001 ; Sperling et al., 1993 ). To date however, there is no evidence of a direct effect of omega-3-fatty acids on myo -inositol levels. Therefore, we hypothesized that the omega-3-fatty acid docosahexaenoic acid (DHA) will deplete intracellular myo -inositol levels, the mechanism by which lithium, valproate and other mood stabilizers attenuate phosphatidylinositol signaling pathway and the proposed mechanism underlying their therapeutic benefit. Myo -inositol depletion has been previously employed as a screen for potential treatments for bipolar disorder using the yeast Saccharomyces cerevisiae as a model system (Ding et al., 2009 ). Thus, in the current study we used S . cerevisiae to examine the effects of DHA on intracellular myo -inositol levels and INO1( inositol-1-phosphate synthase) and INM1 (inositol monophosphatase) — two key genes in the de novo synthesis pathway of myo -inositol. Materials and Methods Strain : The Saccharomyces cerevisiae strain used in this study: SMY7 (derivative of D273-10B/A1, ade5, ura3, MAT a ) was obtained from the laboratory of Miriam Greenberg; Wayne State University. It was maintained on solid YPD agar plates (1% yeast extract; 2% Bacto peptone; 2% glucose; 2% agar) at 30°C. Media and Growth Conditions : Cells were grown in myo -inositol free complete medium [0.069% vitamin-free yeast base, 2% glucose, 0.201% ammonium sulfate, 20mg/l adenine, 20mg/l arginine, 10mg/l histidine, 60mg/l leucine, 20mg/l lysine 20mg/l methionine, 300mg/l threonine, 20mg/l tryptophan, 40mg/l uracil, and vitamins (described in Culbertson and Henry, 1975 )]. Cultures were inoculated to an A 550 of 0.1 and grown for 24 hours at 30 o C in a shaking water bath at 150 rpm. To determine the effect of DHA on intracellular myo -inositol levels, cultures were treated with 0.2mM, 0.4mM and 0.6mM DHA. VPA-treated cultures were used as positive control. Cell extracts : After 24 hours of growth, cultures were centrifuged, and the cell pellets washed twice then resuspended in deionized water to a 1mg/ml concentration. Glass beads were added to ~ 50% of volume, and the mixture vortexed for 10 minutes at 2-minute intervals, alternating with 2-minute incubations on ice. Extracts were clarified by centrifugation for 5 minutes at 2,000 rpm then stored at -80 o C. Protein concentration was determined via the Bradford method. Myo-inositol Assay : Myo -inositol levels in cell extracts were determined using the enzyme-based spectrophotometric assay described by Ashizawa et al. ( 2000 ). Cell extracts were deproteinized with 16% (w/v) perchloric acid and centrifuged at 5,000 rpm for 10 minutes. The resultant supernatant was neutralized with 2M K 2 CO 3 before being phosphorylated with hexokinase reagent (200 mM Tris-HCl buffer, 400 mM adenosine triphosphate disodium (pH adjusted to 8.6 with 10M NaOH) and 115U/ml hexokinase) for 90 minutes at 37 o C. Hexokinase phosphorylates the glucose in the extract, preparing it for the subsequent steps of myo -inositol synthesis. The reaction was stopped by heating for 3 minutes in a boiling water bath then 4.5 M HCl was added to remove endogenous NADH and NADPH. After 10 minutes at 25 o C, 3M K 2 CO 3 was added for neutralization. The neutralized extract was then mixed with myo -inositol reagent (210 mM triethanolamine hydrochloride-32mM K 2 HPO 4 -KOH buffer (pH 8.6), 1.2% (v/v) Triton X-100, 10mM β-NAD, 1.0 U/ml diaphorase, 0.1% (w/v) bovine serum albumin, 60µg/ml iodonitrotetrazolium chloride). The absorbance of the solution was measured at 492 nm with a microplate reader and the reaction initiated by addition of 2.1U/ml myo -inositol dehydrogenase dissolved in 20 mM potassium phosphate buffer (pH 7.0) to each well. After 30 minutes at room temperature, absorbance at 492 nm was again measured. Myo -inositol content was calculated from ΔA as an increase in absorbance during the reaction. A myo -inositol standard curve was used to determine intracellular concentrations. It was obtained by adding myo -inositol of known concentrations to the neutralized perchloric acid solution followed by hexokinase treatment. RNA extraction and cDNA synthesis : Total RNA was extracted from cells using the Aurum ™ Total RNA Mini Kit (Bio-Rad). The spin protocol was employed according to the manufacturer’s instructions. cDNA synthesis was performed using iScript Reverse Transcription Supermix for RTqPCR (Bio-Rad) according to the manufacturer’s instructions. RT-qPCR : The Sso Advanced™ Universal SYBR ® Green Master-Mix and primers (Bio-Rad) were used to measure expression INO1 and INM1 . The transcription factor class C ( TFC1 ) was used as the reference gene. Reactions were performed on the Bio-Rad ® CFX96™ system under the following conditions: 30 seconds at 95 o C for polymerase activation and 40 cycles of 5 seconds at 95 o C and 30 seconds at 62 o C. Melt curves were obtained between 65 o C to 95 o C with a ramp rate of 0.5 o C/second. Relative gene expression was determined by the Livak ( 2 −ΔΔCT ) method (Livak and Schmittgen, 2001 ). Statistical analysis: The significance of the differences between the means of intracellular myo -inositol concentration and relative mRNA expression was determined by One-way ANOVA and Tukey's HSD for post hoc comparison. A p-value < 0.05 was considered significant. Results The myo -inositol standard curve shown in Fig. 1 was obtained by addition of known concentrations of myo -inositol to the neutralized perchloric acid solution followed by hexokinase treatment. Myo -inositol was linear in amounts ranging from 0.1 to 1mM. Effect of DHA on intracellular myo-inositol : As shown in Fig. 2 , treatment with 0.2 mM DHA led to a 3.1-fold decrease in intracellular myo -inositol levels compared to the untreated control. In contrast, exposure to higher DHA concentrations (0.4 mM and 0.6 mM) resulted in approximately 2.3-fold increases in intracellular myo -inositol relative to the control. A one-way ANOVA revealed a statistically significant effect of treatment across all groups ( F ₄,₂₃ = 4.33, p < 0.05), with a partial eta-squared (η²) effect size of 0.43, indicating a large effect. Post hoc comparisons using Tukey’s HSD indicated that while none of the DHA-treated groups differed significantly from each other or the untreated control, the differences between the untreated and VPA-treated controls and the VPA-treated control and the treatment with 0.4 mM DHA were significant ( p < 0.05). Effect of DHA on INO1 and INM1expression : All DHA-treated samples showed increased INO1 expression compared to the untreated control (Fig. 3 ). The fold increases were 1.4, 1.2 and 0.8 in the 0.2mM, 0.4mM and 0.6mM DHA respectively, while the VPA-treated control exhibited the highest increase at 1.8-fold. However, the observed differences in INO1 expression were not significant ( F 3,21 =1.23, ns). Similarly, INM1 expression was higher in all treated samples compared to the untreated control (Fig. 4 ). The greatest fold increases were observed in both the VPA-treated control and the 0.6 mM DHA-treated cultures, each showing approximately a 1.2-fold increase, while the 0.2 mM and 0.4 mM DHA treatments produced smaller increases of about 0.8-fold. Although one-way ANOVA revealed a marginally significant overall effect of treatment on INM1 expression ( F 3,16 =4.29, p < 0.05) with a partial eta-squared (η²) effect size of 0.45 indicating a large effect, post hoc comparisons using the Tukey HSD test indicated that none of the pairwise differences reached statistical significance. The comparison between VPA and 0.2 mM DHA approached significance ( p = 0.068) as did comparisons between VPA and 0.4 mM DHA ( p = 0.096), and 0.6 mM DHA vs. 0.2 mM DHA ( p = 0.089), suggesting a possible trend toward higher INM1 expression at higher DHA concentrations. However, all confidence intervals included zero, indicating no statistically significant differences among treatment groups at the 95% confidence level. Discussion Omega-3 fatty acids are among the most extensively investigated natural compounds for their potential role in the management of bipolar disorder, a chronic and recurrent mood disorder characterized by alternating episodes of mania and depression. Despite decades of clinical research, meta-analyses continue to report mixed findings on their efficacy in the treatment of bipolar disorder. A better understanding of their mechanism of action may help explain these inconsistencies. One longstanding hypothesis proposes that mood stabilizers exert their effects by depleting myo-inositol. This study is the first to directly examine the impact of docosahexaenoic acid (DHA), an omega-3 fatty acid, on intracellular myo -inositol levels and the expression of genes involved in its biosynthesis. Our results demonstrate a biphasic effect of DHA on intracellular myo -inositol levels: treatment with 0.2 mM DHA led to a significant decrease, whereas higher concentrations (0.4 mM and 0.6 mM) resulted in increased myo -inositol levels compared to the untreated control. The observed reduction at low DHA concentrations parallels the effect of VPA and may reflect inhibition of inositol biosynthesis through decreased activity or expression of upstream enzymes such as inositol-1-phosphate synthase. In contrast, the elevation in myo -inositol at higher DHA concentrations suggests the activation of compensatory biosynthetic or transport mechanisms. Notably, the 0.4 mM DHA group showed a significant increase in myo -inositol compared to the VPA-treated control, indicating that DHA may counteract the inositol-depleting effect of VPA at sufficient concentrations. These findings align with the proposed neuroprotective role of DHA in modulating membrane phospholipid composition, cell signaling, and metabolic homeostasis (Lavental et al., 2020; Cheon et al., 2012 ; Lukiw et al., 2005 ). In addition to modulating intracellular myo -inositol, DHA altered the expression of two key biosynthetic genes: INO1 and INM1 . INO1 encodes inositol-1-phosphate synthase, the rate-limiting enzyme in de novo myo-inositol synthesis, while INM1 encodes inositol monophosphatase, which catalyzes the final step in converting myo -inositol-1-phosphate to free myo -inositol. Both genes are regulated by the myo -inositol-sensitive upstream activating sequence (UAS ino ) and have been shown to respond differentially to intracellular myo -inositol concentrations. Specifically, INO1 is typically downregulated in the presence of high myo -inositol, whereas INM1 is upregulated (Vaden et al., 2001 ; Murray & Greenberg, 2000 ). While our findings show general upregulation of both genes relative to the untreated control, a concentration-dependent divergence emerged: INO1 expression decreased at higher DHA concentrations, while INM1 expression increased—mirroring their canonical regulatory patterns and consistent with the observed biphasic trend in intracellular myo -inositol levels. These molecular effects may provide insight into the differential mood-modulating properties of DHA. Elevated myo -inositol has been detected in the anterior cingulate cortex of individuals with bipolar disorder during manic episodes (Davanzo et al., 2001 ), whereas reduced myo -inositol has been observed in the frontal cortex during depressive episodes (Frey et al., 1998 ). The ability of DHA to either reduce or elevate myo -inositol depending on concentration may underlie its reported antidepressant efficacy and limited antimanic effects. In this context, DHA may act more effectively as a treatment for depressive symptoms in bipolar disorder by restoring or enhancing intracellular myo -inositol during the depressive phase. This study provides novel evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) modulates intracellular myo -inositol levels and regulates the expression of key biosynthetic genes, INO1 and INM1 , in a concentration-dependent manner. The observed biphasic response characterized by reduced myo -inositol at low DHA concentrations and elevated levels at higher concentrations, suggests a dose-sensitive mechanism that may underlie DHA’s variable impact on mood regulation. Importantly, treatment with 0.4 mM DHA significantly increased intracellular myo -inositol compared to the VPA-treated control, indicating DHA’s potential to counteract the inositol-depleting effect of VPA. This raises the possibility that DHA may mitigate VPA’s reported depressogenic effects in some patients by restoring or elevating myo -inositol levels. While these findings do not support the hypothesis that DHA acts through myo -inositol depletion, they suggest that DHA may exert its therapeutic, particularly antidepressant effects, by modulating myo -inositol homeostasis. The results also highlight the value of targeting myo -inositol regulation as a mechanistic screen for identifying dietary supplements with potential efficacy in mood disorder management. Further in vivo and clinical investigations are needed to evaluate DHA’s role as a dietary adjunct in bipolar disorder, especially in combination with standard pharmacological treatments. Declarations Conflict of Interest Statement: The authors have no relevant financial or non-financial interests to disclose. The authors have no conflicts of interest to declare that are relevant to the content of this article. All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript. The authors have no financial or proprietary interests in any material discussed in this article. Funding Declaration: This research was supported by a faculty research grant awarded by Andrews University No animals or humans including cells or tissues were used in this research. Ethics and Consent to Participate Declaration : Not applicable Clinical trial number: Not applicable References Merikangas K, Pato M. Recent Developments in the Epidemiology of Bipolar Disorder in Adults and Children: Magnitude, Correlates, and Future Directions. Clin Psychology: Sci Pract. 2009;16(2):121–33. Kessler RC, Chiu WT, Demler O, Merikangas KR, Walters EE. Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry. 2005;62(6):617–27. https://doi.org/10.1001/archpsyc.62.6.617 . Bauer M, Pfennig A. Epidemiology of bipolar disorders. Epilepsia. 2005;46(Suppl 4):8–13. Jope RS, Song L, Li PP, Young LT, Kish SJ, Pacheco MA, et al. The phosphoinositide signal transduction system is impaired in bipolar affective disorder brain. J Neurochem. 1996;66:2402–9. Brown AS, Mallinger AG, Renbaum LC. Elevated platelet membrane phosphatidylinositol-4,5-bisphosphate in bipolar mania. Am J Psychiatry. 1993;150(8):1252–4. Crudup III, Hartley J, Keel N, Panda B, M. Recognizing and Treating Valproic Acid Toxicity: A Case Report. J Med Cases. 2001;2(5):185–7. Gitlin M. Lithium side effects and toxicity: prevalence and management strategies. Int J Bipolar Disord. 2016;4:27. https://doi.org/10.1186/s40345-016-0068-y . Carli M, Risaliti E, Francomano M, Kolachalam S, Longoni B, Bocci G, Maggio R, Scarselli M. A 5-Year Study of Lithium and Valproic Acid Drug Monitoring in Patients with Bipolar Disorders in an Italian Clinical Center. Pharmaceuticals (Basel). 2022;15(1):105. https://doi.org/10.3390/ph15010105 . Moore GJ, Bebchuk JM, Parrish JK, Faulk MW, Arfken CL, Strahl-Bevacqua J, Manji HK. Temporal dissociation between lithium-induced changes in frontal lobe myo-inositol and clinical response in manic-depressive illness. Am J Psychiatry. 1999;156(12):1902–8. https://doi.org/10.1176/ajp.156.12.1902 . O'Donnell T, Rotzinger S, Nakashima TT, Hanstock CC, Ulrich M, Silverstone PH. Chronic lithium and sodium valproate both decrease the concentration of myo-inositol and increase the concentration of inositol monophosphates in rat brain. Brain Res. 2000;880(1–2):84–91. https://doi.org/10.1016/s0006-8993(00)02797-9 . Shaltiel G, Shamir A, Shapiro J, Ding D, Dalton E, Bialer M, et al. Valproate decreases inositol biosynthesis. Biol Psychiatry. 2004;56(11):868–74. Silverstone PH, McGrath BM, Kim H. Bipolar disorder and myo-inositol: a review of the magnetic resonance spectroscopy findings. Bipolar Disord. 2005;7(1):1–10. https://doi.org/10.1111/j.1399-5618.2004.00174.x . Machado-Vieira R, Manji HK, Zarate CA Jr. (2009). The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. Bipolar disorders , 11 Suppl 2 (Suppl 2), 92–109. https://doi.org/10.1111/j.1399-5618.2009.00714.x Bergamelli E, Del Fabro L, Delvecchio G, D'Agostino A, Brambilla P. The Impact of Lithium on Brain Function in Bipolar Disorder: An Updated Review of Functional Magnetic Resonance Imaging Studies. CNS Drugs. 2021;35(12):1275–87. https://doi.org/10.1007/s40263-021-00869-y . Krawczyk K, Rybakowski J. Augmentation of antidepressants with unsaturated fatty acids omega-3 in drug-resistant depression. Psychiatr Pol. 2012;46(4):585–98. Frangou S, Lewis M, McCrone P. Efficacy of ethyl-eicosapentaenoic acid in bipolar depression: randomized double-blind placebo-controlled study. Br J Psychiatry. 2006;188:46–50. Osher Y, Bersudsky Y, Belmaker RH. Omega-3 eicospentaenoic acid in bipolar depression: report of a small open-label study. J Clin Psychiatry. 2005;66(6):726–9. Fristad MA, Young AS, Vesco AT, Nader ES, Healy KZ, Gardner W, Wolfson HL, Arnold LE. A Randomized Controlled Trial of Individual Family Psychoeducational Psychotherapy and Omega-3 Fatty Acids in Youth with Subsyndromal Bipolar Disorder. J Child Adolesc Psychopharmacol. 2015. Dec;25(10):764–74. PMID: 26682997; PMCID: PMC4691654. Psara E, Papadopoulou SK, Mentzelou M, Voulgaridou G, Vorvolakos T, Apostolou T, Giaginis C. Omega-3 Fatty Acids for the Treatment of Bipolar Disorder Symptoms: A Narrative Review of the Current Clinical Evidence. Mar Drugs. 2025;23(2):84. https://doi.org/10.3390/md23020084 . Mirnikjoo B, Brown SE, Kim HF, Marangell LB, Sweatt JD, Weeber EJ. Protein kinase inhibition by omega-3 fatty acids. J Biol Chem. 2001;276(14):10888–96. Sperling RI, Benincaso AI, Knoell CT, Larkin JK, Austen KF, Robinson DR. Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils. J Clin Invest. 1993;91(2):651–60. Ding D, Shi Y, Shaltiel G, Azab AN, Pullumbi E, Campbell A, et al. Yeast bioassay for identification of inositol depleting compounds. World J Biol Psychiatry. 2009;10(4:3):893–9. Culbertson MR, Henry SA. Inositol requiring mutants of Saccharomyces cerevisiae . Genetics. 1975;80(1):23–40. Ashizawa N, Yoshida M, Aotsuka T. An enzymatic assay for myo-inositol in tissue samples. J Biochem Biophys Methods. 2000;44(1–2):89–94. Livak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 –∆∆ C T Method. Methods. 2001;25(4):402–8. Levental KR, Malmberg E, Symons JL, et al. Lipidomic and biophysical homeostasis of mammalian membranes counteracts dietary lipid perturbations to maintain cellular fitness. Nat Commun. 2020;11:1339. https://doi.org/10.1038/s41467-020-15203-1 . Cheon Y, Kim HW, Igarashi M, Modi HR, Chang L, Ma K, Greenstein D, Wohltmann M, Turk J, Rapoport SI, Taha AY. Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)β)-knockout mice. Biochim Biophys Acta. 2012;1821(9):1278–86. https://doi.org/10.1016/j.bbalip.2012.02.003 . Lukiw WJ, Cui JG, Marcheselli VL, Bodker M, Botkjaer A, Gotlinger K, Serhan CN, Bazan NG. A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease. J Clin Investig. 2005;115(10):2774–83. https://doi.org/10.1172/JCI25420 . Vaden DL, Ding D, Peterson B, Greenberg ML. Lithium and valproate decrease inositol mass and increase expression of the yeast INO1 and INO2 genes for inositol biosynthesis. J Biol Chem. 2001;276(19):15466–71. Murray M, Greenberg ML. Expression of yeast INM1 encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate. Mol Micro. 2000;36(3):651–61. Davanzo P, Thomas MA, Yue K, Oshiro T, Belin T, Strober M, McCracken J. Decreased anterior cingulate myo-inositol/creatine spectroscopy resonance with lithium treatment in children with bipolar disorder. Neuropsychopharmacology: official publication Am Coll Neuropsychopharmacol. 2001;24(4):359–69. https://doi.org/10.1016/S0893-133X(00)00207-4 . Frey R, Metzler D, Fischer P, Heiden A, Scharfetter J, Moser E, Kasper S. Myo-inositol in depressive and healthy subjects determined by frontal 1H-magnetic resonance spectroscopy at 1.5 tesla. J Psychiatr Res. 1998;32(6):411–20. https://doi.org/10.1016/s0022-3956(98)00033-8 . Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 05 Jan, 2026 Read the published version in Pharmacological Reports → Version 1 posted Editorial decision: Revision requested 03 Sep, 2025 Reviews received at journal 25 Aug, 2025 Reviews received at journal 21 Aug, 2025 Reviewers agreed at journal 20 Aug, 2025 Reviewers agreed at journal 19 Aug, 2025 Reviewers agreed at journal 18 Aug, 2025 Reviewers invited by journal 13 Aug, 2025 Editor assigned by journal 30 Jul, 2025 Submission checks completed at journal 30 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Murray","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAxklEQVRIiWNgGAWjYBADOSA2gLITiNNiTLqWxAaitehOO2P2uaLmcHr/7OZtHz621THws+cY4NVidjvHeOaZY4dzZ9w5VjxzZtthBsmeN4S1MDawHc5tuJFjzMy77QCDwQ0ibGFs+Hc4XR6k5e+2OgZ7orQ0th1OMABpYdzGzGAgQVBLWjFjY1+64cYbQEbvv8M8EmeeFRDQkryZseGbtbzcjeTNDD/O1MnxtydvwKsFCprhLB5ilINAHbEKR8EoGAWjYCQCAEgESMs5ORKgAAAAAElFTkSuQmCC","orcid":"","institution":"Andrews University","correspondingAuthor":true,"prefix":"","firstName":"Marlene","middleName":"","lastName":"Murray","suffix":""},{"id":502425278,"identity":"233083ef-3ec9-4334-87a4-07cae2cff847","order_by":1,"name":"Haley Kang","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Haley","middleName":"","lastName":"Kang","suffix":""},{"id":502425279,"identity":"b0f69f4e-e3ac-46ed-8d78-7f284cb7b0cf","order_by":2,"name":"Taejun Ok","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Taejun","middleName":"","lastName":"Ok","suffix":""},{"id":502425285,"identity":"dbd32b0e-dc77-4f2b-ae22-1fd0ee7109e5","order_by":3,"name":"Kimberly Park","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Kimberly","middleName":"","lastName":"Park","suffix":""},{"id":502425286,"identity":"094272e0-aea3-4a43-abac-a12821a939c6","order_by":4,"name":"Jee Yeon Lee","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Jee","middleName":"Yeon","lastName":"Lee","suffix":""},{"id":502425287,"identity":"1f2fa594-e42f-46da-9172-c914b160e8e8","order_by":5,"name":"Bomi Kim","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Bomi","middleName":"","lastName":"Kim","suffix":""},{"id":502425288,"identity":"b7865ec9-f4be-419c-b068-9e499647d3c3","order_by":6,"name":"Hyukje Sung","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Hyukje","middleName":"","lastName":"Sung","suffix":""},{"id":502425289,"identity":"ba5acd1f-0992-4235-8066-a73b9f4c8e01","order_by":7,"name":"Talisa Tait","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Talisa","middleName":"","lastName":"Tait","suffix":""},{"id":502425290,"identity":"088119e6-e909-46e4-afeb-239390a79b4a","order_by":8,"name":"Daniel Colon Hidalgo","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Daniel","middleName":"Colon","lastName":"Hidalgo","suffix":""},{"id":502425291,"identity":"d836eaad-5403-41db-8529-984d3ce637f5","order_by":9,"name":"Yudy Guzman","email":"","orcid":"","institution":"Andrews University","correspondingAuthor":false,"prefix":"","firstName":"Yudy","middleName":"","lastName":"Guzman","suffix":""}],"badges":[],"createdAt":"2025-07-24 17:53:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7207940/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7207940/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s43440-025-00815-5","type":"published","date":"2026-01-05T15:58:33+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":89561800,"identity":"16726cf1-b5b7-405b-8476-61d6ef35960d","added_by":"auto","created_at":"2025-08-21 10:23:47","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":25594,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative standard curve used to determine \u003cem\u003emyo\u003c/em\u003e-inositol concentration in cell extracts.\u003c/p\u003e\n\u003cp\u003eData represents the average of five standard curves.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-7207940/v1/e3bf6934a6a328e67a78e05b.png"},{"id":89561857,"identity":"d70b1f12-62a2-459f-98ed-cd5afb17fd94","added_by":"auto","created_at":"2025-08-21 10:23:50","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":115193,"visible":true,"origin":"","legend":"\u003cp\u003eMean ± SE of intracellular \u003cem\u003emyo\u003c/em\u003e-inositol concentration in extracts of cells grown in the absence (untreated control, n= 8) or presence of the indicated concentrations of docosahexaenoic acid (DHA), n= 2-7, and valproate (VPA)-treated control, n= 8. * \u003cem\u003eP\u003c/em\u003e ≤ 0.05 compared with VPA-treated control, ** \u003cem\u003eP\u003c/em\u003e ≤ 0.05 compared with untreated control.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207940/v1/e2c9992cefb6705b750ef8e2.jpeg"},{"id":89561866,"identity":"b404c217-e53d-4dbe-83b5-0606b84c8596","added_by":"auto","created_at":"2025-08-21 10:23:51","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123976,"visible":true,"origin":"","legend":"\u003cp\u003eRelative \u003cem\u003eINO1\u003c/em\u003e gene expression of cells grown for 24 hours in the indicated concentrations of DHA (n =6) and positive control (VPA, n =7). The delta-delta CT method was used to determine fold change. \u003cem\u003ep \u003c/em\u003e\u0026gt; 0.05 by ANOVA.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207940/v1/c8721727d27646fc9ec1217c.jpeg"},{"id":89561924,"identity":"1c478a9b-83f7-4434-ac7d-998bb5bea79a","added_by":"auto","created_at":"2025-08-21 10:23:52","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":109429,"visible":true,"origin":"","legend":"\u003cp\u003eRelative \u003cem\u003eINM1\u003c/em\u003e gene expression of cells grown for 24 hours in the indicated concentrations of DHA (n =5) and positive control (VPA, n =5). The delta-delta CT method was used to determine fold change. \u003cem\u003ep\u0026lt;\u003c/em\u003e 0.05 by ANOVA.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7207940/v1/d9f6b64ae52d226261e4f51b.jpeg"},{"id":100069341,"identity":"f490ff97-f9d9-4425-9bc2-07917d1d94dd","added_by":"auto","created_at":"2026-01-12 16:13:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":882983,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7207940/v1/8505307e-1ba2-4b97-978c-075dadc1f6f4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eDocosahexaenoic Acid Modulates Myo-Inositol and Myo-Inositol Biosynthetic Genes Expression: Implications for Bipolar Disorder\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eBipolar disorder (BD) is one of the most debilitating and severe mood disorders. It is the 6th leading cause of disability among non-communicable diseases, with an estimated prevalence between 1 and 2% (Merinkgas et al., 2007; Kessler er al., 2005; Bauer and Pfennig, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Referred to as manic-depressive illness, it is characterized by recurring episodes of mania and depression, with the possibility of each episode lasting up to several months. While the cause of bipolar disorder remains unknown, dysregulation of the phosphatidylinositol signaling pathway has been implicated. For example, Jope et al. (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e1996\u003c/span\u003e) observed reduced phosphatidylinositol hydrolysis in the occipital cortex of postmortem bipolar patients, while Brown et al. (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e) found increased levels of phosphatidylinositol-4,5-bisphosphate in the platelet membranes of unmedicated individuals during the manic phase.\u003c/p\u003e\u003cp\u003eTreatment options for bipolar disorder include mood stabilizers, antipsychotics, and antidepressants. Among these, lithium and valproate are the most prescribed mood stabilizers; however, their use is limited by significant side effects and narrow therapeutic windows (Crudup, 2011; Gitlin, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Carli et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Although the precise therapeutic mechanisms of lithium and valproate are not fully understood, one proposed mode of action is the depletion of \u003cem\u003emyo\u003c/em\u003e-inositol levels which may help normalize disrupted neuronal circuitry associated with the disorder (Moore et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e1999\u003c/span\u003e; O' Donnell et al., 2000; Shaltiel et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2004\u003c/span\u003e; Silverstone et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Machado-Vieira et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Bergamelli et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e\u003cp\u003ePreliminary clinical evidence suggests that omega-3 fatty acids, whether administered as monotherapy or as an adjunct to antidepressant treatment, are associated with improvements in depressive symptoms among individuals with bipolar disorder. These effects are demonstrated by reductions in Hamilton Depression Rating Scale scores and enhanced overall clinical evaluations, as measured by the Clinical Global Impression and Global Assessment Scales (Krawczyk \u0026amp; Rybakowski, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Frangou et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2006\u003c/span\u003e; Osher, 2005). However, their efficacy in addressing manic symptoms appears limited. For example, Fristad et al. (2015) reported only modest effects on manic symptomatology, as assessed by the Young Mania Rating Scale. Furthermore, a broader review of clinical trials has yielded inconsistent findings regarding the overall effectiveness of omega-3 fatty acids in the treatment of bipolar disorder (Psara et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eWhile the molecular mechanisms underlying the therapeutic benefits of omega-3-fatty acids remain unclear, there is evidence that like lithium and valproate, omega-3 fatty acids affect the phosphatidylinositol signaling pathway. Specifically, they have been shown to decrease protein kinase C and phospholipase C activity, as well as the production of inositol triphosphate (Mirnikjoo et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Sperling et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1993\u003c/span\u003e). To date however, there is no evidence of a direct effect of omega-3-fatty acids on \u003cem\u003emyo\u003c/em\u003e-inositol levels. Therefore, we hypothesized that the omega-3-fatty acid docosahexaenoic acid (DHA) will deplete intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels, the mechanism by which lithium, valproate and other mood stabilizers attenuate phosphatidylinositol signaling pathway and the proposed mechanism underlying their therapeutic benefit.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMyo\u003c/em\u003e-inositol depletion has been previously employed as a screen for potential treatments for bipolar disorder using the yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e as a model system (Ding et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Thus, in the current study we used \u003cem\u003eS\u003c/em\u003e. \u003cem\u003ecerevisiae\u003c/em\u003e to examine the effects of DHA on intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels and \u003cem\u003eINO1(\u003c/em\u003einositol-1-phosphate synthase) and \u003cem\u003eINM1\u003c/em\u003e(inositol monophosphatase) \u0026mdash; two key genes in the de novo synthesis pathway of \u003cem\u003emyo\u003c/em\u003e-inositol.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003e\u003cem\u003eStrain\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eThe \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e strain used in this study: SMY7 (derivative of D273-10B/A1, \u003cem\u003eade5, ura3, MAT a\u003c/em\u003e) was obtained from the laboratory of Miriam Greenberg; Wayne State University. It was maintained on solid YPD agar plates (1% yeast extract; 2% Bacto peptone; 2% glucose; 2% agar) at 30\u0026deg;C.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMedia and Growth Conditions\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eCells were grown in \u003cem\u003emyo\u003c/em\u003e-inositol free complete medium [0.069% vitamin-free yeast base, 2% glucose, 0.201% ammonium sulfate, 20mg/l adenine, 20mg/l arginine, 10mg/l histidine, 60mg/l leucine, 20mg/l lysine 20mg/l methionine, 300mg/l threonine, 20mg/l tryptophan, 40mg/l uracil, and vitamins (described in Culbertson and Henry, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1975\u003c/span\u003e)]. Cultures were inoculated to an A\u003csub\u003e550\u003c/sub\u003e of 0.1 and grown for 24 hours at 30\u003csup\u003eo\u003c/sup\u003eC in a shaking water bath at 150 rpm. To determine the effect of DHA on intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels, cultures were treated with 0.2mM, 0.4mM and 0.6mM DHA. VPA-treated cultures were used as positive control.\u003c/p\u003e\u003cp\u003e\u003cem\u003eCell extracts\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eAfter 24 hours of growth, cultures were centrifuged, and the cell pellets washed twice then resuspended in deionized water to a 1mg/ml concentration. Glass beads were added to ~\u0026thinsp;50% of volume, and the mixture vortexed for 10 minutes at 2-minute intervals, alternating with 2-minute incubations on ice. Extracts were clarified by centrifugation for 5 minutes at 2,000 rpm then stored at -80\u003csup\u003eo\u003c/sup\u003eC. Protein concentration was determined via the Bradford method.\u003c/p\u003e\u003cp\u003e\u003cem\u003eMyo-inositol Assay\u003c/em\u003e:\u003c/p\u003e\u003cp\u003e\u003cem\u003eMyo\u003c/em\u003e-inositol levels in cell extracts were determined using the enzyme-based spectrophotometric assay described by Ashizawa et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Cell extracts were deproteinized with 16% (w/v) perchloric acid and centrifuged at 5,000 rpm for 10 minutes. The resultant supernatant was neutralized with 2M K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e before being phosphorylated with hexokinase reagent (200 mM Tris-HCl buffer, 400 mM adenosine triphosphate disodium (pH adjusted to 8.6 with 10M NaOH) and 115U/ml hexokinase) for 90 minutes at 37\u003csup\u003eo\u003c/sup\u003eC. Hexokinase phosphorylates the glucose in the extract, preparing it for the subsequent steps of \u003cem\u003emyo\u003c/em\u003e-inositol synthesis. The reaction was stopped by heating for 3 minutes in a boiling water bath then 4.5 M HCl was added to remove endogenous NADH and NADPH. After 10 minutes at 25\u003csup\u003eo\u003c/sup\u003eC, 3M K\u003csub\u003e2\u003c/sub\u003eCO\u003csub\u003e3\u003c/sub\u003e was added for neutralization. The neutralized extract was then mixed with \u003cem\u003emyo\u003c/em\u003e-inositol reagent (210 mM triethanolamine hydrochloride-32mM K\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e-KOH buffer (pH 8.6), 1.2% (v/v) Triton X-100, 10mM β-NAD, 1.0 U/ml diaphorase, 0.1% (w/v) bovine serum albumin, 60\u0026micro;g/ml iodonitrotetrazolium chloride). The absorbance of the solution was measured at 492 nm with a microplate reader and the reaction initiated by addition of 2.1U/ml \u003cem\u003emyo\u003c/em\u003e-inositol dehydrogenase dissolved in 20 mM potassium phosphate buffer (pH 7.0) to each well. After 30 minutes at room temperature, absorbance at 492 nm was again measured. \u003cem\u003eMyo\u003c/em\u003e-inositol content was calculated from ΔA as an increase in absorbance during the reaction.\u003c/p\u003e\u003cp\u003eA \u003cem\u003emyo\u003c/em\u003e-inositol standard curve was used to determine intracellular concentrations. It was obtained by adding \u003cem\u003emyo\u003c/em\u003e-inositol of known concentrations to the neutralized perchloric acid solution followed by hexokinase treatment.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRNA extraction and cDNA synthesis\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eTotal RNA was extracted from cells using the Aurum\u003csup\u003e\u0026trade;\u003c/sup\u003e Total RNA Mini Kit (Bio-Rad). The spin protocol was employed according to the manufacturer\u0026rsquo;s instructions. cDNA synthesis was performed using iScript Reverse Transcription Supermix for RTqPCR (Bio-Rad) according to the manufacturer\u0026rsquo;s instructions.\u003c/p\u003e\u003cp\u003e\u003cem\u003eRT-qPCR\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eThe Sso Advanced\u0026trade; Universal SYBR\u003csup\u003e\u0026reg;\u003c/sup\u003e Green Master-Mix and primers (Bio-Rad) were used to measure expression \u003cem\u003eINO1\u003c/em\u003e and \u003cem\u003eINM1\u003c/em\u003e. The transcription factor class C (\u003cem\u003eTFC1\u003c/em\u003e) was used as the reference gene. Reactions were performed on the Bio-Rad\u003csup\u003e\u0026reg;\u003c/sup\u003e CFX96\u0026trade; system under the following conditions: 30 seconds at 95\u003csup\u003eo\u003c/sup\u003eC for polymerase activation and 40 cycles of 5 seconds at 95\u003csup\u003eo\u003c/sup\u003eC and 30 seconds at 62\u003csup\u003eo\u003c/sup\u003eC. Melt curves were obtained between 65 \u003csup\u003eo\u003c/sup\u003eC to 95\u003csup\u003eo\u003c/sup\u003eC with a ramp rate of 0.5\u003csup\u003eo\u003c/sup\u003eC/second. Relative gene expression was determined by the Livak ( 2\u003csup\u003e\u0026minus;ΔΔCT\u003c/sup\u003e) method (Livak and Schmittgen, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2001\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStatistical analysis:\u003c/h2\u003e\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe significance of the differences between the means of intracellular \u003cem\u003emyo\u003c/em\u003e-inositol concentration and relative mRNA expression was determined by One-way ANOVA and Tukey's HSD for post hoc comparison. A p-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered significant.\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eThe \u003cem\u003emyo\u003c/em\u003e-inositol standard curve shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e was obtained by addition of known concentrations of \u003cem\u003emyo\u003c/em\u003e-inositol to the neutralized perchloric acid solution followed by hexokinase treatment. \u003cem\u003eMyo\u003c/em\u003e-inositol was linear in amounts ranging from 0.1 to 1mM.\u003c/p\u003e\u003c/div\u003e\u003cp\u003e\u003cem\u003eEffect of DHA on intracellular myo-inositol\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eAs shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, treatment with 0.2 mM DHA led to a 3.1-fold decrease in intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels compared to the untreated control. In contrast, exposure to higher DHA concentrations (0.4 mM and 0.6 mM) resulted in approximately 2.3-fold increases in intracellular \u003cem\u003emyo\u003c/em\u003e-inositol relative to the control. A one-way ANOVA revealed a statistically significant effect of treatment across all groups (\u003cem\u003eF\u003c/em\u003e₄,₂₃ = 4.33, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with a partial eta-squared (η\u0026sup2;) effect size of 0.43, indicating a large effect. Post hoc comparisons using Tukey\u0026rsquo;s HSD indicated that while none of the DHA-treated groups differed significantly from each other or the untreated control, the differences between the untreated and VPA-treated controls and the VPA-treated control and the treatment with 0.4 mM DHA were significant (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cem\u003eEffect of DHA on INO1\u003c/em\u003e and \u003cem\u003eINM1expression\u003c/em\u003e:\u003c/p\u003e\u003cp\u003eAll DHA-treated samples showed increased \u003cem\u003eINO1\u003c/em\u003e expression compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). The fold increases were 1.4, 1.2 and 0.8 in the 0.2mM, 0.4mM and 0.6mM DHA respectively, while the VPA-treated control exhibited the highest increase at 1.8-fold. However, the observed differences in \u003cem\u003eINO1\u003c/em\u003e expression were not significant (\u003cem\u003eF\u003c/em\u003e \u003csub\u003e3,21\u003c/sub\u003e=1.23, ns).\u003c/p\u003e\u003cp\u003eSimilarly, \u003cem\u003eINM1\u003c/em\u003e expression was higher in all treated samples compared to the untreated control (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). The greatest fold increases were observed in both the VPA-treated control and the 0.6 mM DHA-treated cultures, each showing approximately a 1.2-fold increase, while the 0.2 mM and 0.4 mM DHA treatments produced smaller increases of about 0.8-fold. Although one-way ANOVA revealed a marginally significant overall effect of treatment on \u003cem\u003eINM1\u003c/em\u003e expression (\u003cem\u003eF\u003c/em\u003e\u003csub\u003e3,16\u003c/sub\u003e =4.29, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) with a partial eta-squared (η\u0026sup2;) effect size of 0.45 indicating a large effect, post hoc comparisons using the Tukey HSD test indicated that none of the pairwise differences reached statistical significance. The comparison between VPA and 0.2 mM DHA approached significance (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.068) as did comparisons between VPA and 0.4 mM DHA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.096), and 0.6 mM DHA vs. 0.2 mM DHA (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.089), suggesting a possible trend toward higher \u003cem\u003eINM1\u003c/em\u003e expression at higher DHA concentrations. However, all confidence intervals included zero, indicating no statistically significant differences among treatment groups at the 95% confidence level.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eOmega-3 fatty acids are among the most extensively investigated natural compounds for their potential role in the management of bipolar disorder, a chronic and recurrent mood disorder characterized by alternating episodes of mania and depression. Despite decades of clinical research, meta-analyses continue to report mixed findings on their efficacy in the treatment of bipolar disorder. A better understanding of their mechanism of action may help explain these inconsistencies. One longstanding hypothesis proposes that mood stabilizers exert their effects by depleting myo-inositol. This study is the first to directly examine the impact of docosahexaenoic acid (DHA), an omega-3 fatty acid, on intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels and the expression of genes involved in its biosynthesis.\u003c/p\u003e\u003cp\u003eOur results demonstrate a biphasic effect of DHA on intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels: treatment with 0.2 mM DHA led to a significant decrease, whereas higher concentrations (0.4 mM and 0.6 mM) resulted in increased \u003cem\u003emyo\u003c/em\u003e-inositol levels compared to the untreated control. The observed reduction at low DHA concentrations parallels the effect of VPA and may reflect inhibition of inositol biosynthesis through decreased activity or expression of upstream enzymes such as inositol-1-phosphate synthase. In contrast, the elevation in \u003cem\u003emyo\u003c/em\u003e-inositol at higher DHA concentrations suggests the activation of compensatory biosynthetic or transport mechanisms. Notably, the 0.4 mM DHA group showed a significant increase in \u003cem\u003emyo\u003c/em\u003e-inositol compared to the VPA-treated control, indicating that DHA may counteract the inositol-depleting effect of VPA at sufficient concentrations. These findings align with the proposed neuroprotective role of DHA in modulating membrane phospholipid composition, cell signaling, and metabolic homeostasis (Lavental et al., 2020; Cheon et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Lukiw et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2005\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eIn addition to modulating intracellular \u003cem\u003emyo\u003c/em\u003e-inositol, DHA altered the expression of two key biosynthetic genes: \u003cem\u003eINO1\u003c/em\u003e and \u003cem\u003eINM1\u003c/em\u003e. \u003cem\u003eINO1\u003c/em\u003e encodes inositol-1-phosphate synthase, the rate-limiting enzyme in de novo myo-inositol synthesis, while \u003cem\u003eINM1\u003c/em\u003e encodes inositol monophosphatase, which catalyzes the final step in converting \u003cem\u003emyo\u003c/em\u003e-inositol-1-phosphate to free \u003cem\u003emyo\u003c/em\u003e-inositol. Both genes are regulated by the \u003cem\u003emyo\u003c/em\u003e-inositol-sensitive upstream activating sequence (UAS\u003csub\u003eino\u003c/sub\u003e) and have been shown to respond differentially to intracellular \u003cem\u003emyo\u003c/em\u003e-inositol concentrations. Specifically, \u003cem\u003eINO1\u003c/em\u003e is typically downregulated in the presence of high \u003cem\u003emyo\u003c/em\u003e-inositol, whereas \u003cem\u003eINM1\u003c/em\u003e is upregulated (Vaden et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Murray \u0026amp; Greenberg, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). While our findings show general upregulation of both genes relative to the untreated control, a concentration-dependent divergence emerged: \u003cem\u003eINO1\u003c/em\u003e expression decreased at higher DHA concentrations, while \u003cem\u003eINM1\u003c/em\u003e expression increased\u0026mdash;mirroring their canonical regulatory patterns and consistent with the observed biphasic trend in intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels.\u003c/p\u003e\u003cp\u003eThese molecular effects may provide insight into the differential mood-modulating properties of DHA. Elevated \u003cem\u003emyo\u003c/em\u003e-inositol has been detected in the anterior cingulate cortex of individuals with bipolar disorder during manic episodes (Davanzo et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2001\u003c/span\u003e), whereas reduced \u003cem\u003emyo\u003c/em\u003e-inositol has been observed in the frontal cortex during depressive episodes (Frey et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). The ability of DHA to either reduce or elevate \u003cem\u003emyo\u003c/em\u003e-inositol depending on concentration may underlie its reported antidepressant efficacy and limited antimanic effects. In this context, DHA may act more effectively as a treatment for depressive symptoms in bipolar disorder by restoring or enhancing intracellular \u003cem\u003emyo\u003c/em\u003e-inositol during the depressive phase.\u003c/p\u003e\u003cp\u003eThis study provides novel evidence that the omega-3 fatty acid docosahexaenoic acid (DHA) modulates intracellular \u003cem\u003emyo\u003c/em\u003e-inositol levels and regulates the expression of key biosynthetic genes, \u003cem\u003eINO1\u003c/em\u003e and \u003cem\u003eINM1\u003c/em\u003e, in a concentration-dependent manner. The observed biphasic response characterized by reduced \u003cem\u003emyo\u003c/em\u003e-inositol at low DHA concentrations and elevated levels at higher concentrations, suggests a dose-sensitive mechanism that may underlie DHA\u0026rsquo;s variable impact on mood regulation. Importantly, treatment with 0.4 mM DHA significantly increased intracellular \u003cem\u003emyo\u003c/em\u003e-inositol compared to the VPA-treated control, indicating DHA\u0026rsquo;s potential to counteract the inositol-depleting effect of VPA. This raises the possibility that DHA may mitigate VPA\u0026rsquo;s reported depressogenic effects in some patients by restoring or elevating \u003cem\u003emyo\u003c/em\u003e-inositol levels. While these findings do not support the hypothesis that DHA acts through \u003cem\u003emyo\u003c/em\u003e-inositol depletion, they suggest that DHA may exert its therapeutic, particularly antidepressant effects, by modulating \u003cem\u003emyo\u003c/em\u003e-inositol homeostasis. The results also highlight the value of targeting \u003cem\u003emyo\u003c/em\u003e-inositol regulation as a mechanistic screen for identifying dietary supplements with potential efficacy in mood disorder management. Further in vivo and clinical investigations are needed to evaluate DHA\u0026rsquo;s role as a dietary adjunct in bipolar disorder, especially in combination with standard pharmacological treatments.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of Interest Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003eThe authors have no conflicts of interest to declare that are relevant to the content of this article.\u003c/p\u003e\n\u003cp\u003eAll authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest or non-financial interest in the subject matter or materials discussed in this manuscript.\u003c/p\u003e\n\u003cp\u003eThe authors have no financial or proprietary interests in any material discussed in this article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by a faculty research grant awarded by Andrews University\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNo animals or humans including cells or tissues were used in this research.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics and Consent to Participate Declaration\u003c/strong\u003e:\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eClinical trial number:\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMerikangas K, Pato M. Recent Developments in the Epidemiology of Bipolar Disorder in Adults and Children: Magnitude, Correlates, and Future Directions. Clin Psychology: Sci Pract. 2009;16(2):121\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKessler RC, Chiu WT, Demler O, Merikangas KR, Walters EE. Prevalence, severity, and comorbidity of 12-month DSM-IV disorders in the National Comorbidity Survey Replication. Arch Gen Psychiatry. 2005;62(6):617\u0026ndash;27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1001/archpsyc.62.6.617\u003c/span\u003e\u003cspan address=\"10.1001/archpsyc.62.6.617\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBauer M, Pfennig A. Epidemiology of bipolar disorders. Epilepsia. 2005;46(Suppl 4):8\u0026ndash;13.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eJope RS, Song L, Li PP, Young LT, Kish SJ, Pacheco MA, et al. The phosphoinositide signal transduction system is impaired in bipolar affective disorder brain. J Neurochem. 1996;66:2402\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBrown AS, Mallinger AG, Renbaum LC. Elevated platelet membrane phosphatidylinositol-4,5-bisphosphate in bipolar mania. Am J Psychiatry. 1993;150(8):1252\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCrudup III, Hartley J, Keel N, Panda B, M. Recognizing and Treating Valproic Acid Toxicity: A Case Report. J Med Cases. 2001;2(5):185\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGitlin M. Lithium side effects and toxicity: prevalence and management strategies. Int J Bipolar Disord. 2016;4:27. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s40345-016-0068-y\u003c/span\u003e\u003cspan address=\"10.1186/s40345-016-0068-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCarli M, Risaliti E, Francomano M, Kolachalam S, Longoni B, Bocci G, Maggio R, Scarselli M. A 5-Year Study of Lithium and Valproic Acid Drug Monitoring in Patients with Bipolar Disorders in an Italian Clinical Center. Pharmaceuticals (Basel). 2022;15(1):105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ph15010105\u003c/span\u003e\u003cspan address=\"10.3390/ph15010105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMoore GJ, Bebchuk JM, Parrish JK, Faulk MW, Arfken CL, Strahl-Bevacqua J, Manji HK. Temporal dissociation between lithium-induced changes in frontal lobe myo-inositol and clinical response in manic-depressive illness. Am J Psychiatry. 1999;156(12):1902\u0026ndash;8. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1176/ajp.156.12.1902\u003c/span\u003e\u003cspan address=\"10.1176/ajp.156.12.1902\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eO'Donnell T, Rotzinger S, Nakashima TT, Hanstock CC, Ulrich M, Silverstone PH. Chronic lithium and sodium valproate both decrease the concentration of myo-inositol and increase the concentration of inositol monophosphates in rat brain. Brain Res. 2000;880(1\u0026ndash;2):84\u0026ndash;91. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0006-8993(00)02797-9\u003c/span\u003e\u003cspan address=\"10.1016/s0006-8993(00)02797-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShaltiel G, Shamir A, Shapiro J, Ding D, Dalton E, Bialer M, et al. Valproate decreases inositol biosynthesis. Biol Psychiatry. 2004;56(11):868\u0026ndash;74.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSilverstone PH, McGrath BM, Kim H. Bipolar disorder and myo-inositol: a review of the magnetic resonance spectroscopy findings. Bipolar Disord. 2005;7(1):1\u0026ndash;10. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-5618.2004.00174.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1399-5618.2004.00174.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMachado-Vieira R, Manji HK, Zarate CA Jr. (2009). The role of lithium in the treatment of bipolar disorder: convergent evidence for neurotrophic effects as a unifying hypothesis. \u003cem\u003eBipolar disorders\u003c/em\u003e, \u003cem\u003e11 Suppl 2\u003c/em\u003e(Suppl 2), 92\u0026ndash;109. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1399-5618.2009.00714.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1399-5618.2009.00714.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eBergamelli E, Del Fabro L, Delvecchio G, D'Agostino A, Brambilla P. The Impact of Lithium on Brain Function in Bipolar Disorder: An Updated Review of Functional Magnetic Resonance Imaging Studies. CNS Drugs. 2021;35(12):1275\u0026ndash;87. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s40263-021-00869-y\u003c/span\u003e\u003cspan address=\"10.1007/s40263-021-00869-y\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKrawczyk K, Rybakowski J. Augmentation of antidepressants with unsaturated fatty acids omega-3 in drug-resistant depression. Psychiatr Pol. 2012;46(4):585\u0026ndash;98.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrangou S, Lewis M, McCrone P. Efficacy of ethyl-eicosapentaenoic acid in bipolar depression: randomized double-blind placebo-controlled study. Br J Psychiatry. 2006;188:46\u0026ndash;50.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eOsher Y, Bersudsky Y, Belmaker RH. Omega-3 eicospentaenoic acid in bipolar depression: report of a small open-label study. J Clin Psychiatry. 2005;66(6):726\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFristad MA, Young AS, Vesco AT, Nader ES, Healy KZ, Gardner W, Wolfson HL, Arnold LE. A Randomized Controlled Trial of Individual Family Psychoeducational Psychotherapy and Omega-3 Fatty Acids in Youth with Subsyndromal Bipolar Disorder. J Child Adolesc Psychopharmacol. 2015. Dec;25(10):764\u0026ndash;74. PMID: 26682997; PMCID: PMC4691654.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePsara E, Papadopoulou SK, Mentzelou M, Voulgaridou G, Vorvolakos T, Apostolou T, Giaginis C. Omega-3 Fatty Acids for the Treatment of Bipolar Disorder Symptoms: A Narrative Review of the Current Clinical Evidence. Mar Drugs. 2025;23(2):84. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/md23020084\u003c/span\u003e\u003cspan address=\"10.3390/md23020084\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMirnikjoo B, Brown SE, Kim HF, Marangell LB, Sweatt JD, Weeber EJ. Protein kinase inhibition by omega-3 fatty acids. J Biol Chem. 2001;276(14):10888\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSperling RI, Benincaso AI, Knoell CT, Larkin JK, Austen KF, Robinson DR. Dietary omega-3 polyunsaturated fatty acids inhibit phosphoinositide formation and chemotaxis in neutrophils. J Clin Invest. 1993;91(2):651\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDing D, Shi Y, Shaltiel G, Azab AN, Pullumbi E, Campbell A, et al. Yeast bioassay for identification of inositol depleting compounds. World J Biol Psychiatry. 2009;10(4:3):893\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCulbertson MR, Henry SA. Inositol requiring mutants of \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e. Genetics. 1975;80(1):23\u0026ndash;40.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAshizawa N, Yoshida M, Aotsuka T. An enzymatic assay for myo-inositol in tissue samples. J Biochem Biophys Methods. 2000;44(1\u0026ndash;2):89\u0026ndash;94.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2\u003csup\u003e\u0026ndash;∆∆\u003cem\u003eC\u003c/em\u003e\u003c/sup\u003e\u003csub\u003eT\u003c/sub\u003e Method. Methods. 2001;25(4):402\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLevental KR, Malmberg E, Symons JL, et al. Lipidomic and biophysical homeostasis of mammalian membranes counteracts dietary lipid perturbations to maintain cellular fitness. Nat Commun. 2020;11:1339. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41467-020-15203-1\u003c/span\u003e\u003cspan address=\"10.1038/s41467-020-15203-1\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCheon Y, Kim HW, Igarashi M, Modi HR, Chang L, Ma K, Greenstein D, Wohltmann M, Turk J, Rapoport SI, Taha AY. Disturbed brain phospholipid and docosahexaenoic acid metabolism in calcium-independent phospholipase A(2)-VIA (iPLA(2)β)-knockout mice. Biochim Biophys Acta. 2012;1821(9):1278\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbalip.2012.02.003\u003c/span\u003e\u003cspan address=\"10.1016/j.bbalip.2012.02.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eLukiw WJ, Cui JG, Marcheselli VL, Bodker M, Botkjaer A, Gotlinger K, Serhan CN, Bazan NG. A role for docosahexaenoic acid-derived neuroprotectin D1 in neural cell survival and Alzheimer disease. J Clin Investig. 2005;115(10):2774\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1172/JCI25420\u003c/span\u003e\u003cspan address=\"10.1172/JCI25420\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVaden DL, Ding D, Peterson B, Greenberg ML. Lithium and valproate decrease inositol mass and increase expression of the yeast \u003cem\u003eINO1\u003c/em\u003e and \u003cem\u003eINO2\u003c/em\u003e genes for inositol biosynthesis. J Biol Chem. 2001;276(19):15466\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eMurray M, Greenberg ML. Expression of yeast \u003cem\u003eINM1\u003c/em\u003e encoding inositol monophosphatase is regulated by inositol, carbon source and growth stage and is decreased by lithium and valproate. Mol Micro. 2000;36(3):651\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDavanzo P, Thomas MA, Yue K, Oshiro T, Belin T, Strober M, McCracken J. Decreased anterior cingulate myo-inositol/creatine spectroscopy resonance with lithium treatment in children with bipolar disorder. Neuropsychopharmacology: official publication Am Coll Neuropsychopharmacol. 2001;24(4):359\u0026ndash;69. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S0893-133X(00)00207-4\u003c/span\u003e\u003cspan address=\"10.1016/S0893-133X(00)00207-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFrey R, Metzler D, Fischer P, Heiden A, Scharfetter J, Moser E, Kasper S. Myo-inositol in depressive and healthy subjects determined by frontal 1H-magnetic resonance spectroscopy at 1.5 tesla. J Psychiatr Res. 1998;32(6):411\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0022-3956(98)00033-8\u003c/span\u003e\u003cspan address=\"10.1016/s0022-3956(98)00033-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"omega-3 fatty acids, docosahexaenoic acid, myo-inositol, INM1, INO1, bipolar disorder","lastPublishedDoi":"10.21203/rs.3.rs-7207940/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7207940/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground:\u003c/p\u003e\n\u003cp\u003eBipolar disorder is a serious and recurrent mood disorder marked by alternating episodes of mania and depression, affecting an estimated 2.6% of the adult population and 3.9% across the lifetime. Although pharmacological mood stabilizers remain a cornerstone of treatment, their variable efficacy and adverse side effect profiles limit long-term use in many patients. As such, dietary interventions, particularly omega-3 polyunsaturated fatty acids have garnered interest as adjunctive or alternative treatments. Among these, docosahexaenoic acid (DHA) is one of the most extensively studied. While several clinical trials suggest omega-3 fatty acids may alleviate certain symptoms of bipolar disorder, results have been inconsistent, and the mechanisms of action remain unclear. Given that intracellular \u003cem\u003emyo\u003c/em\u003e-inositol depletion has been proposed as a common mechanism of action for conventional mood stabilizers we hypothesized that DHA, which modulates components of the phosphatidylinositol pathway, will also deplete intracellular \u003cem\u003emyo\u003c/em\u003e-inositol and thus modulate \u003cem\u003emyo\u003c/em\u003e-inositol homeostasis.\u003c/p\u003e\n\u003cp\u003eMethodology:\u003c/p\u003e\n\u003cp\u003eUsing an enzymatic assay intracellular myo-inositol levels were measured in the extracts of cells grown in the presence of DHA. The effect of DHA on expression of the myo-inositol biosynthetic genes INO1 and INM1 was assessed by RT-qPCR.\u003c/p\u003e\n\u003cp\u003eResults:\u003c/p\u003e\n\u003cp\u003eThe results showed DHA alters intracellular \u003cem\u003emyo\u003c/em\u003e-inositol in a concentration-dependent manner which paralleled its effect on \u003cem\u003eINO1\u003c/em\u003e and \u003cem\u003eINM1\u003c/em\u003e expression.\u003c/p\u003e\n\u003cp\u003eConclusion:\u003c/p\u003e\n\u003cp\u003eThese findings suggest that the therapeutic potential of DHA in bipolar disorder may be mediated, at least in part, by its regulation of intracellular \u003cem\u003emyo\u003c/em\u003e-inositol and its biosynthetic gene network.\u003c/p\u003e","manuscriptTitle":"Docosahexaenoic Acid Modulates Myo-Inositol and Myo-Inositol Biosynthetic Genes Expression: Implications for Bipolar Disorder","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-21 10:23:15","doi":"10.21203/rs.3.rs-7207940/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-03T17:28:20+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-25T12:43:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-21T18:29:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"48054285320750911053562020597930729205","date":"2025-08-20T17:23:34+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"129816929898385065734135460214235456446","date":"2025-08-19T23:53:53+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"145732746033813616111198924656456619996","date":"2025-08-18T12:04:47+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-13T06:18:41+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-30T06:27:23+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-30T06:22:20+00:00","index":"","fulltext":""},{"type":"submitted","content":"Pharmacological Reports","date":"2025-07-24T17:40:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"pharmacological-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"prep","sideBox":"Learn more about [Pharmacological Reports](https://link.springer.com/journal/43440)","snPcode":"43440","submissionUrl":"https://submission.springernature.com/new-submission/43440/3","title":"Pharmacological Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"f3f8e61e-b4b9-4d59-a47c-a5b84f289271","owner":[],"postedDate":"August 21st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-12T16:04:18+00:00","versionOfRecord":{"articleIdentity":"rs-7207940","link":"https://doi.org/10.1007/s43440-025-00815-5","journal":{"identity":"pharmacological-reports","isVorOnly":false,"title":"Pharmacological Reports"},"publishedOn":"2026-01-05 15:58:33","publishedOnDateReadable":"January 5th, 2026"},"versionCreatedAt":"2025-08-21 10:23:15","video":"","vorDoi":"10.1007/s43440-025-00815-5","vorDoiUrl":"https://doi.org/10.1007/s43440-025-00815-5","workflowStages":[]},"version":"v1","identity":"rs-7207940","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7207940","identity":"rs-7207940","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}