Upregulation of the AMPK-FOXO1-PDK4 pathway is a primary mechanism of pyruvate dehydrogenase activity reduction in tafazzin-deficient cells.

Barth syndrome (BTHS) is a rare disorder caused by mutations in the TAFAZZIN gene. Previous studies from both patients and model systems have established metabolic dysregulation as a core component of BTHS pathology. In particular, features such as lactic acidosis, pyruvate dehydrogenase (PDH) deficiency, and aberrant fatty acid and glucose oxidation have been identified. However, the lack of a mechanistic understanding of what causes these conditions in the context of BTHS remains a significant knowledge gap, and this has hindered the development of effective therapeutic strategies for treating the associated metabolic problems. In the current study, we utilized tafazzin-knockout C2C12 mouse myoblasts (TAZ-KO) and cardiac and skeletal muscle tissue from tafazzin-knockout mice to identify an upstream mechanism underlying impaired PDH activity in BTHS. This mechanism centers around robust upregulation of pyruvate dehydrogenase kinase 4 (PDK4), resulting from hyperactivation of AMP-activated protein kinase (AMPK) and subsequent transcriptional upregulation by forkhead box protein O1 (FOXO1). Upregulation of PDK4 in tafazzin-deficient cells causes direct phospho-inhibition of PDH activity accompanied by increased glucose uptake and elevated intracellular glucose concentration. Collectively, our findings provide a novel mechanistic framework whereby impaired tafazzin function ultimately results in robust PDK4 upregulation, leading to impaired PDH activity and likely linked to dysregulated metabolic substrate utilization. This mechanism may underlie previously reported findings of BTHS-associated metabolic dysregulation.

The paradoxical role of inositol in cancer: a consequence of the metabolic state of a tumor.

Inositol is an essential nutrient, obtained either by uptake from the environment or by de novo synthesis from glucose. Inositol and its derivatives exhibit tumor-suppressive effects, potentially mediated by inhibition of the ERK-MAPK or PI3K-Akt pathways. Accordingly, many cancers have been documented to silence expression of the ISYNA1 gene, which encodes the rate-limiting enzyme of inositol synthesis. Paradoxically, recent studies have also reported upregulation of ISYNA1 in some cancers. Upregulation may reflect a compensatory response brought about by defective inositol uptake or oncogenic mutations that preclude its tumor-suppressive effects. In these scenarios, de novo synthesis of inositol may be upregulated to promote cell proliferation. The role of inositol in cancer is further complicated by its ability to inhibit the master metabolic regulator AMPK, which upon activation can either decrease cell proliferation and metastasis or promote cell survival. Due to its potential dual role in cancer, inositol homeostasis must be tightly regulated in tumor cells. Thus, whether inositol acts to suppress or promote tumor progression is determined by the metabolic profile and oncogenic background of the cancer.

Valproate activates the Snf1 kinase in Saccharomyces cerevisiae by decreasing the cytosolic pH.

Valproate (VPA) is a widely used mood stabilizer, but its therapeutic mechanism of action is not understood. This knowledge gap hinders the development of more effective drugs with fewer side effects. Using the yeast model to elucidate the effects of VPA on cellular metabolism, we determined that the drug upregulated expression of genes normally repressed during logarithmic growth on glucose medium and increased levels of activated (phosphorylated) Snf1 kinase, the major metabolic regulator of these genes. VPA also decreased the cytosolic pH (pHc) and reduced glycolytic production of 2/3-phosphoglycerate. ATP levels and mitochondrial membrane potential were increased, and glucose-mediated extracellular acidification decreased in the presence of the drug, as indicated by a smaller glucose-induced shift in pH, suggesting that the major P-type proton pump Pma1 was inhibited. Interestingly, decreasing the pHc by omeprazole-mediated inhibition of Pma1 led to Snf1 activation. We propose a model whereby VPA lowers the pHc causing a decrease in glycolytic flux. In response, Pma1 is inhibited and Snf1 is activated, resulting in increased expression of normally repressed metabolic genes. These findings suggest a central role for pHc in regulating the metabolic program of yeast cells.

Cardiolipin-deficient cells have decreased levels of the iron-sulfur biogenesis protein frataxin.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays an important role in mitochondrial bioenergetics. Previous studies in the yeast model have indicated that CL is required for optimal iron homeostasis, which is disrupted by a mechanism not yet determined in the yeast CL mutant, crd1Δ. This finding has implications for the severe genetic disorder, Barth syndrome (BTHS), in which CL metabolism is perturbed because of mutations in the CL-remodeling enzyme, tafazzin. Here, we investigate the effects of tafazzin deficiency on iron homeostasis in the mouse myoblast model of BTHS tafazzin knockout (TAZ-KO) cells. Similarly to CL-deficient yeast cells, TAZ-KO cells exhibited elevated sensitivity to iron, as well as to H2O2, which was alleviated by the iron chelator deferoxamine. TAZ-KO cells exhibited increased expression of the iron exporter ferroportin and decreased expression of the iron importer transferrin receptor, likely reflecting a regulatory response to elevated mitochondrial iron. Reduced activities of mitochondrial iron-sulfur cluster enzymes suggested that the mechanism underlying perturbation of iron homeostasis was defective iron-sulfur biogenesis. We observed decreased levels of Yfh1/frataxin, an essential component of the iron-sulfur biogenesis machinery, in mitochondria from TAZ-KO mouse cells and in CL-deleted yeast crd1Δ cells, indicating that the role of CL in iron-sulfur biogenesis is highly conserved. Yeast crd1Δ cells exhibited decreased processing of the Yfh1 precursor upon import, which likely contributes to the iron homeostasis defects. Implications for understanding the pathogenesis of BTHS are discussed.

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.

Valproate inhibits glucose-stimulated insulin secretion in beta cells.

Valproate (VPA), an FDA approved anti-epileptic drug with a half-life of 12-18 h in humans, has been shown to perturb the vacuolar proton pump (vH+-ATPase) function in yeasts by inhibiting myo-inositol phosphate synthase, the first and rate-limiting enzyme in inositol biosynthesis, thereby resulting in inositol depletion. vH+-ATPase transfers protons (H+) across cell membranes, which help maintain pH gradients within cells necessary for various cellular functions including secretion. This proton pump has a membrane (V0) and a soluble cytosolic (V1) domain, with C-subunit associated with V1. In secretory cells such as neurons and insulin-secreting beta cells, vH+-ATPase acidifies vesicles essential for secretion. In this study, we demonstrate that exposure of insulin-secreting Min6 cells to a clinical dose of VPA results in inositol depletion and loss of co-localization of subunit C of vH+-ATPase with insulin-secreting granules. Consequently, a reduction of glucose-stimulated insulin secretion is observed following VPA exposure. These results merit caution and the reassessment of the clinical use of VPA.

Perturbation of the Vacuolar ATPase: A NOVEL CONSEQUENCE OF INOSITOL DEPLETION.

Depletion of inositol has profound effects on cell function and has been implicated in the therapeutic effects of drugs used to treat epilepsy and bipolar disorder. We have previously shown that the anticonvulsant drug valproate (VPA) depletes inositol by inhibiting myo-inositol-3-phosphate synthase, the enzyme that catalyzes the first and rate-limiting step of inositol biosynthesis. To elucidate the cellular consequences of inositol depletion, we screened the yeast deletion collection for VPA-sensitive mutants and identified mutants in vacuolar sorting and the vacuolar ATPase (V-ATPase). Inositol depletion caused by starvation of ino1Δ cells perturbed the vacuolar structure and decreased V-ATPase activity and proton pumping in isolated vacuolar vesicles. VPA compromised the dynamics of phosphatidylinositol 3,5-bisphosphate (PI3,5P2) and greatly reduced V-ATPase proton transport in inositol-deprived wild-type cells. Osmotic stress, known to increase PI3,5P2 levels, did not restore PI3,5P2 homeostasis nor did it induce vacuolar fragmentation in VPA-treated cells, suggesting that perturbation of the V-ATPase is a consequence of altered PI3,5P2 homeostasis under inositol-limiting conditions. This study is the first to demonstrate that inositol depletion caused by starvation of an inositol synthesis mutant or by the inositol-depleting drug VPA leads to perturbation of the V-ATPase.

Inositol synthesis regulates the activation of GSK-3α in neuronal cells.

The synthesis of inositol provides precursors of inositol lipids and inositol phosphates that are pivotal for cell signaling. Mood stabilizers lithium and valproic acid, used for treating bipolar disorder, cause cellular inositol depletion, which has been proposed as a therapeutic mechanism of action of both drugs. Despite the importance of inositol, the requirement for inositol synthesis in neuronal cells is not well understood. Here, we examined inositol effects on proliferation of SK-N-SH neuroblastoma cells. The essential role of inositol synthesis in proliferation is underscored by the findings that exogenous inositol was dispensable for proliferation, and inhibition of inositol synthesis decreased proliferation. Interestingly, the inhibition of inositol synthesis by knocking down INO1, which encodes inositol-3-phosphate synthase, the rate-limiting enzyme of inositol synthesis, led to the inactivation of GSK-3α by increasing the inhibitory phosphorylation of this kinase. Similarly, the mood stabilizer valproic acid effected transient decreases in intracellular inositol, leading to inactivation of GSK-3α. As GSK-3 inhibition has been proposed as a likely therapeutic mechanism of action, the finding that inhibition of inositol synthesis results in the inactivation of GSK-3α suggests a unifying hypothesis for mechanism of mood-stabilizing drugs. Inositol is an essential metabolite that serves as a precursor for inositol lipids and inositol phosphates. We report that inhibition of the rate-limiting enzyme of inositol synthesis leads to the inactivation of glycogen synthase kinase (GSK) 3α by increasing inhibitory phosphorylation of this kinase. These findings have implications for the therapeutic mechanisms of mood stabilizers and suggest that inositol synthesis and GSK 3α activity are intrinsically related.

Loss of cardiolipin leads to perturbation of mitochondrial and cellular iron homeostasis.

Cardiolipin (CL) is the signature phospholipid of mitochondrial membranes, where it is synthesized locally and plays a critical role in mitochondrial bioenergetic functions. The importance of CL in human health is underscored by the observation that perturbation of CL biosynthesis causes the severe genetic disorder Barth syndrome. To fully understand the cellular response to the loss of CL, we carried out genome-wide expression profiling of the yeast CL mutant crd1Δ. Our results show that the loss of CL in this mutant leads to increased expression of iron uptake genes accompanied by elevated levels of mitochondrial iron and increased sensitivity to iron and hydrogen peroxide. Previous studies have shown that increased mitochondrial iron levels result from perturbations in iron-sulfur (Fe-S) cluster biogenesis. Consistent with an Fe-S defect, deletion of ISU1, one of two ISU genes that encode the mitochondrial Fe-S scaffolding protein essential for the synthesis of Fe-S clusters, led to synthetic growth defects with the crd1Δ mutant. We further show that crd1Δ cells have reduced activities of mitochondrial Fe-S enzymes (aconitase, succinate dehydrogenase, and ubiquinol-cytochrome c oxidoreductase), as well as cytosolic Fe-S enzymes (sulfite reductase and isopropylmalate isomerase). Increased expression of ATM1 or YAP1 did not rescue the Fe-S defects in crd1Δ. These findings show for the first time that CL is required for Fe-S biogenesis to maintain mitochondrial and cellular iron homeostasis.

Inositol phosphates and phosphoinositides in health and disease.

In the past two decades, considerable progress has been made toward understanding inositol phosphates and PI metabolism. However, there is still much to learn. The present challenge is to understand how inositol phosphates and PIs are compartmentalized, identify new targets of inositol phosphates and PIs, and elucidate the mechanisms underlying spatial and temporal regulation of the enzymes that metabolize inositol phosphates and PIs. Answers to these questions will help clarify the mechanisms of the diseases associated with these molecules and identify new possibilities for drug design.

The human TAZ gene complements mitochondrial dysfunction in the yeast taz1Delta mutant. Implications for Barth syndrome.

Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1Delta mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1Delta mutant. Energy coupling in taz1Delta mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1Delta mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1Delta also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1Delta cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.