Phosphatidic acid inhibits inositol synthesis by inducing nuclear translocation of kinase IP6K1 and repression of myo-inositol-3-P synthase.

Inositol is an essential metabolite that serves as a precursor for structural and signaling molecules. Although perturbation of inositol homeostasis has been implicated in numerous human disorders, surprisingly little is known about how inositol levels are regulated in mammalian cells. A recent study in mouse embryonic fibroblasts demonstrated that nuclear translocation of inositol hexakisphosphate kinase 1 (IP6K1) mediates repression of myo-inositol-3-P synthase (MIPS), the rate-limiting inositol biosynthetic enzyme. Binding of IP6K1 to phosphatidic acid (PA) is required for this repression. Here, we elucidate the role of PA in IP6K1 repression. Our results indicate that increasing PA levels through pharmacological stimulation of phospholipase D (PLD) or direct supplementation of 18:1 PA induces nuclear translocation of IP6K1 and represses expression of the MIPS protein. We found that this effect was specific to PA synthesized in the plasma membrane, as endoplasmic reticulum-derived PA did not induce IP6K1 translocation. Furthermore, we determined that PLD-mediated PA synthesis can be stimulated by the master metabolic regulator 5' AMP-activated protein kinase (AMPK). We show that activation of AMPK by glucose deprivation or by treatment with the mood-stabilizing drugs valproate or lithium recapitulated IP6K1 nuclear translocation and decreased MIPS expression. This study demonstrates for the first time that modulation of PA levels through the AMPK-PLD pathway regulates IP6K1-mediated repression of MIPS.

NAD supplementation improves mitochondrial performance of cardiolipin mutants.

Cardiolipin (CL) deficiency causes mitochondrial dysfunction and aberrant metabolism that are associated in humans with the severe disease Barth syndrome (BTHS). Several metabolic abnormalities are observed in BTHS patients and model systems, including decreased oxidative phosphorylation, reduced tricarboxylic acid (TCA) cycle flux, and accumulated lactate and D-ß-hydroxybutyrate, which strongly suggests that nicotinamide adenine dinucleotide (NAD) redox metabolism may be altered in CL-deficient cells. In this study, we identified abnormal NAD+ metabolism in multiple BTHS model systems and demonstrate that supplementation of NAD+ precursors such as nicotinamide mononucleotide (NMN) improves mitochondrial function. Improved mitochondrial function in the Drosophila model was associated with restored exercise endurance, which suggests a potential therapeutic benefit of NAD+ precursor supplementation in the management of BTHS patients.

Valproate inhibits mitochondrial bioenergetics and increases glycolysis in Saccharomyces cerevisiae.

The widely used mood stabilizer valproate (VPA) causes perturbation of energy metabolism, which is implicated in both the therapeutic mechanism of action of the drug as well as drug toxicity. To gain insight into these mechanisms, we determined the effects of VPA on energy metabolism in yeast. VPA treatment increased levels of glycolytic intermediates, increased expression of glycolysis genes, and increased ethanol production. Increased glycolysis was likely a response to perturbation of mitochondrial function, as reflected in decreased membrane potential and oxygen consumption. Interestingly, yeast, mouse liver, and isolated bovine cytochrome c oxidase were directly inhibited by the drug, while activities of other oxidative phosphorylation complexes (III and V) were not affected. These findings have implications for mechanisms of therapeutic action and toxicity.

Loss of the mitochondrial lipid cardiolipin leads to decreased glutathione synthesis.

Previous studies demonstrated that loss of CL in the yeast mutant crd1Δ leads to perturbation of mitochondrial iron­sulfur (FeS) cluster biogenesis, resulting in decreased activity of mitochondrial and cytosolic Fe-S-requiring enzymes, including aconitase and sulfite reductase. In the current study, we show that crd1Δ cells exhibit decreased levels of glutamate and cysteine and are deficient in the essential antioxidant, glutathione, a tripeptide of glutamate, cysteine, and glycine. Glutathione is the most abundant non-protein thiol essential for maintaining intracellular redox potential in almost all eukaryotes, including yeast. Consistent with glutathione deficiency, the growth defect of crd1Δ cells at elevated temperature was rescued by supplementation of glutathione or glutamate and cysteine. Sensitivity to the oxidants iron (FeSO4) and hydrogen peroxide (H2O2), was rescued by supplementation of glutathione. The decreased intracellular glutathione concentration in crd1Δ was restored by supplementation of glutamate and cysteine, but not by overexpressing YAP1, an activator of expression of glutathione biosynthetic enzymes. These findings show for the first time that CL plays a critical role in regulating intracellular glutathione metabolism.

MCK1 is a novel regulator of myo-inositol phosphate synthase (MIPS) that is required for inhibition of inositol synthesis by the mood stabilizer valproate.

Myo-inositol, the precursor of all inositol compounds, is essential for the viability of eukaryotes. Identifying the factors that regulate inositol homeostasis is of obvious importance to understanding cell function and the pathologies underlying neurological and metabolic resulting from perturbation of inositol metabolism. The current study identifies Mck1, a GSK3 homolog, as a novel positive regulator of inositol de novo synthesis in yeast. Mck1 was required for normal activity of myo-inositol phosphate synthase (MIPS), which catalyzes the rate-limiting step of inositol synthesis. mck1Δ cells exhibited a 50% decrease in MIPS activity and a decreased rate of incorporation of [13C6]glucose into [13C6]-inositol-3-phosphate and [13C6]-inositol compared to WT cells. mck1Δ cells also exhibited decreased growth in the presence of the inositol depleting drug valproate (VPA), which was rescued by supplementation of inositol. However, in contrast to wild type cells, which exhibited more than a 40% decrease in MIPS activity in the presence of VPA, the drug did not significantly decrease MIPS activity in mck1Δ cells. These findings indicate that VPA-induced MIPS inhibition is Mck1-dependent, and suggest a model that unifies two current hypotheses of the mechanism of action of VPA-inositol depletion and GSK3 inhibition.

Valproate Induces the Unfolded Protein Response by Increasing Ceramide Levels.

Bipolar disorder (BD), which is characterized by depression and mania, affects 1-2% of the world population. Current treatments are effective in only 40-60% of cases and cause severe side effects. Valproate (VPA) is one of the most widely used drugs for the treatment of BD, but the therapeutic mechanism of action of this drug is not understood. This knowledge gap has hampered the development of effective treatments. To identify candidate pathways affected by VPA, we performed a genome-wide expression analysis in yeast cells grown in the presence or absence of the drug. VPA caused up-regulation of FEN1 and SUR4, encoding fatty acid elongases that catalyze the synthesis of very long chain fatty acids (C24 to C26) required for ceramide synthesis. Interestingly, fen1Δ and sur4Δ mutants exhibited VPA sensitivity. In agreement with increased fatty acid elongase gene expression, VPA increased levels of phytoceramide, especially those containing C24-C26 fatty acids. Consistent with an increase in ceramide, VPA decreased the expression of amino acid transporters, increased the expression of ER chaperones, and activated the unfolded protein response element (UPRE), suggesting that VPA induces the UPR pathway. These effects were rescued by supplementation of inositol and similarly observed in inositol-starved ino1Δ cells. Starvation of ino1Δ cells increased expression of FEN1 and SUR4, increased ceramide levels, decreased expression of nutrient transporters, and induced the UPR. These findings suggest that VPA-mediated inositol depletion induces the UPR by increasing the de novo synthesis of ceramide.

Loss of tafazzin in yeast leads to increased oxidative stress during respiratory growth.

The tafazzin (TAZ) gene is highly conserved from yeast to humans, and the yeast taz1 null mutant shows alterations in cardiolipin (CL) metabolism, mitochondrial dysfunction and stabilization of supercomplexes similar to those found in Barth syndrome, a human disorder resulting from loss of tafazzin. We have previously shown that the yeast tafazzin mutant taz1Delta, which cannot remodel CL, is ethanol-sensitive at elevated temperature. In the current report, we show that in response to ethanol, CL mutants taz1Delta as well as crd1Delta, which cannot synthesize CL, exhibited increased protein carbonylation, an indicator of reactive oxygen species (ROS). The increase in ROS is most likely not due to defective oxidant defence systems, as the CL mutants do not display sensitivity to paraquat, menadione or hydrogen peroxide (H2O2). Ethanol sensitivity and increased protein carbonylation in the taz1Delta mutant but not in crd1Delta can be rescued by supplementation with oleic acid, suggesting that oleoyl-CL and/or oleoyl-monolyso-CL enables growth of taz1Delta in ethanol by decreasing oxidative stress. Our findings of increased oxidative stress in the taz1Delta mutant during respiratory growth may have important implications for understanding the pathogenesis of Barth syndrome.

Glycogen synthase kinase-3 is required for optimal de novo synthesis of inositol.

Studies have shown that the inositol biosynthetic pathway and the enzyme glycogen synthase kinase-3 (GSK-3) are targets of the mood-stabilizing drugs lithium and valproate. However, a relationship between these targets has not been previously described. We hypothesized that GSK-3 may play a role in inositol synthesis, and that loss of GSK-3 may lead to inositol depletion, thus providing a mechanistic link between the two drug targets. Utilizing a yeast Saccharomyces cerevisiae gsk-3Delta quadruple-null mutant, in which all four genes encoding homologues of mammalian GSK-3 are disrupted, we tested the hypothesis that GSK-3 is required for de novo inositol biosynthesis. The gsk-3Delta mutant exhibited multiple features of inositol depletion, including defective growth in inositol-lacking medium, decreased intracellular inositol, increased INO1 and ITR1 expression, and decreased levels of phosphatidylinositol. Treatment of wild-type cells with a highly specific GSK-3 inhibitor led to a significant increase in INO1 expression. Supplementation with inositol alleviated the temperature sensitivity of gsk-3Delta. Activity of myo-inositol-3 phosphate synthase, the rate-limiting enzyme in inositol de novo biosynthesis, was decreased in gsk-3Delta. These results demonstrate for the first time that GSK-3 is required for optimal myo-inositol-3 phosphate synthase activity and de novo inositol biosynthesis, and that loss of GSK-3 activity causes inositol depletion.

Post-translational regulation of phosphatidylglycerolphosphate synthase in response to inositol.

Phosphatidylglycerolphosphate synthase (Pgs1p) catalyses the committed step in the synthesis of cardiolipin (CL). This is the only step of CL synthesis that is regulated by inositol. We have shown previously that Pgs1p enzyme activity is decreased within minutes after supplementation with inositol, but PGS1 expression is unaltered. We utilized an epitope-tagged Pgs1p to determine if the rapid decrease in activity following inositol was because of degradation or inactivation of the protein. In this report, we show that, in response to inositol, the decrease in CL content and Pgs1p enzyme activity are associated with increased phosphorylation of Pgs1p, but not with degradation or mislocalization of the protein. This is the first evidence of phosphorylation of a phospholipid biosynthetic enzyme in response to inositol and identifies a new mechanism of inositol-mediated regulation.

Human 1-D-myo-inositol-3-phosphate synthase is functional in yeast.

We have cloned, sequenced, and expressed a human cDNA encoding 1-d-myo-inositol-3-phosphate (MIP) synthase (hINO1). The encoded 62-kDa human enzyme converted d-glucose 6-phosphate to 1-d-myo-inositol 3-phosphate, the rate-limiting step for de novo inositol biosynthesis. Activity of the recombinant human MIP synthase purified from Escherichia coli was optimal at pH 8.0 at 37 degrees C and exhibited K(m) values of 0.57 mm and 8 microm for glucose 6-phosphate and NAD(+), respectively. NH(4)(+) and K(+) were better activators than other cations tested (Na(+), Li(+), Mg(2+), Mn(2+)), and Zn(2+) strongly inhibited activity. Expression of the protein in the yeast ino1Delta mutant lacking MIP synthase (ino1Delta/hINO1) complemented the inositol auxotrophy of the mutant and led to inositol excretion. MIP synthase activity and intracellular inositol were decreased about 35 and 25%, respectively, when ino1Delta/hINO1 was grown in the presence of a therapeutically relevant concentration of the anti-bipolar drug valproate (0.6 mm). However, in vitro activity of purified MIP synthase was not inhibited by valproate at this concentration, suggesting that inhibition by the drug is indirect. Because inositol metabolism may play a key role in the etiology and treatment of bipolar illness, functional conservation of the key enzyme in inositol biosynthesis underscores the power of the yeast model in studies of this disorder.

The effect of lithium on expression of genes for inositol biosynthetic enzymes in mouse hippocampus; a comparison with the yeast model.

In the de novo synthesis of inositol, the conversion of D-glucose-6-phosphate to L-myo-inositol-1-phosphate (MIP) is catalyzed by MIP synthase. Little is known about mammalian MIP synthase and nothing is known about its regulation. The second step in inositol biosynthesis is the conversion of MIP to inositol by inositol-monophosphatase (IMPase), a common step to inositol production via the de novo pathway and its recycling from inositol phosphates. Because lithium inhibits IMPase both in yeast and in mammals, and the drug upregulates yeast MIP synthase (INO1) and downregulates IMPase (INM1), the present study was undertaken to determine whether chronic in vivo therapeutic lithium concentrations affect MIP synthase and IMPase expression in mouse frontal cortex and hippocampus. Mice were treated with food containing LiCl (2.5 g/kg) for 10 days. RNA was purified from the brain areas and mRNA amplified using RT-PCR. Expression of MIP synthase and IMPA1 (one of the genes coding for IMPase) but not IMPA2 was upregulated in mouse hippocampus. None of the genes were affected in the frontal cortex. In yeast, when inositol is limiting, the heterodimeric transcriptional activator Ino2p/Ino4p derepresses expression of INO1 by binding to the upstream activation sequence UAS(INO). Using the TFSEARCH program, we found that the promoter of the virtual human MIP synthase gene contains upstream stimulating factor (USF) elements with a similar core binding sequence. The fact that lithium treatment upregulates both MIP synthase and IMPA1 mRNA levels in mouse hippocampus may reflect a compensatory response of both genes to inositol depletion.