A ferroptosis defense mechanism mediated by glycerol-3-phosphate dehydrogenase 2 in mitochondria.

Mechanisms of defense against ferroptosis (an iron-dependent form of cell death induced by lipid peroxidation) in cellular organelles remain poorly understood, hindering our ability to target ferroptosis in disease treatment. In this study, metabolomic analyses revealed that treatment of cancer cells with glutathione peroxidase 4 (GPX4) inhibitors results in intracellular glycerol-3-phosphate (G3P) depletion. We further showed that supplementation of cancer cells with G3P attenuates ferroptosis induced by GPX4 inhibitors in a G3P dehydrogenase 2 (GPD2)-dependent manner; GPD2 deletion sensitizes cancer cells to GPX4 inhibition-induced mitochondrial lipid peroxidation and ferroptosis, and combined deletion of GPX4 and GPD2 synergistically suppresses tumor growth by inducing ferroptosis in vivo. Mechanistically, inner mitochondrial membrane-localized GPD2 couples G3P oxidation with ubiquinone reduction to ubiquinol, which acts as a radical-trapping antioxidant to suppress ferroptosis in mitochondria. Taken together, these results reveal that GPD2 participates in ferroptosis defense in mitochondria by generating ubiquinol.

Mitochondrial Glycerol-3-Phosphate Dehydrogenase Restricts HBV Replication via the TRIM28-Mediated Degradation of HBx.

Hepatitis B virus (HBV) infection affects hepatic metabolism. Serum metabolomics studies have suggested that HBV possibly hijacks the glycerol-3-phosphate (G3P) shuttle. In this study, the two glycerol-3-phosphate dehydrogenases (GPD1 and GPD2) in the G3P shuttle were analyzed for determining their role in HBV replication and the findings revealed that GPD2 and not GPD1 inhibited HBV replication. The knockdown of GPD2 expression upregulated HBV replication, while GPD2 overexpression reduced HBV replication. Moreover, the overexpression of GPD2 significantly reduced HBV replication in hydrodynamic injection-based mouse models. Mechanistically, this inhibitory effect is related to the GPD2-mediated degradation of HBx protein by recruiting the E3 ubiquitin ligase TRIM28 and not to the alterations in G3P metabolism. In conclusion, this study revealed GPD2, a key enzyme in the G3P shuttle, as a host restriction factor in HBV replication. IMPORTANCE The glycerol-3-phosphate (G3P) shuttle is important for the delivery of cytosolic reducing equivalents into mitochondria for oxidative phosphorylation. The study analyzed two key components of the G3P shuttle and identified GPD2 as a restriction factor in HBV replication. The findings revealed a novel mechanism of GPD2-mediated inhibition of HBV replication via the recruitment of TRIM28 for degrading HBx, and the HBx-GPD2 interaction could be another potential therapeutic target for anti-HBV drug development.

Contribution of GPD2/mGPDH to an alternative respiratory chain of the mitochondrial energy metabolism and the stemness in CD133-positive HuH-7 cells.

HuH-7 cells, derived from human hepatocarcinoma, are known to contain the CD133-positive cancer stem cell populations. HuH-7 cells showed higher ATP synthesis activity through the respiratory chain compared to another human hepatocarcinoma cell line HepG2 and showed an especially higher glycerol-3-phosphate (G3P)-driven ATP synthesis (G3P-ATPase) activity. We found that the CD133-positive HuH-7 cells expressed high levels of GPD2 (glycerol-3-phosphate dehydrogenase or mGPDH) and showed high G3P-ATPase activity. Next, to elucidate the relationship between CD133 and GPD2, we inhibited downstream factors of CD133 and found that a p38 inhibitor decreased the expression of GPD2 and decreased the G3P-ATPase activity. Furthermore, GPD2-knockdown (GPD2-KD) cells exhibited strong reduction of the G3P-ATPase activity and reduction of lactic acid secretion. Finally, we validated the effect of GPD2-KD on tumorigenicity. GPD2-KD cells were found to show decreased anchorage-independent cell proliferation, suggesting the linkage of G3P-ATPase activity to the tumorigenicity of the CD133-positive HuH-7 cells. Inhibition of G3P-ATPase disrupts the homeostasis of energy metabolism and blocks cancer development and progression. Our results suggest inhibitors, targeting GPD2 may be potential new anticancer agents.

Beneficial Effects of Metformin on the Central Nervous System, with a Focus on Epilepsy and Lafora Disease.

Metformin is a drug in the family of biguanide compounds that is widely used in the treatment of type 2 diabetes (T2D). Interestingly, the therapeutic potential of metformin expands its prescribed use as an anti-diabetic drug. In this sense, it has been described that metformin administration has beneficial effects on different neurological conditions. In this work, we review the beneficial effects of this drug as a neuroprotective agent in different neurological diseases, with a special focus on epileptic disorders and Lafora disease, a particular type of progressive myoclonus epilepsy. In addition, we review the different proposed mechanisms of action of metformin to understand its function at the neurological level.

Tumor-associated macrophage interleukin-ß promotes glycerol-3-phosphate dehydrogenase activation, glycolysis and tumorigenesis in glioma cells.

Tumor-immune crosstalk within the tumor microenvironment (TME) occurs at all stages of tumorigenesis. Tumor-associated M2 macrophages play a central role in tumor development, but the molecular underpinnings have not been fully elucidated. We demonstrated that M2 macrophages produce interleukin 1ß (IL-1ß), which activates phosphorylation of the glycolytic enzyme glycerol-3-phosphate dehydrogenase (GPD2) at threonine 10 (GPD2 pT10) through phosphatidylinositol-3-kinase-mediated activation of protein kinase-delta (PKCδ) in glioma cells. GPD2 pT10 enhanced its substrate affinity and increased the catalytic rate of glycolysis in glioma cells. Inhibiting PKCδ or GPD2 pT10 in glioma cells or blocking IL-1ß generated by macrophages attenuated the glycolytic rate and proliferation of glioma cells. Furthermore, human glioblastoma tumor GPD2 pT10 levels were positively correlated with tumor p-PKCδ and IL-1ß levels as well as intratumoral macrophage recruitment, tumor grade and human glioblastoma patient survival. These results reveal a novel tumorigenic role for M2 macrophages in the TME. In addition, these findings suggest possible treatment strategies for glioma patients through blockade of cytokine crosstalk between M2 macrophages and glioma cells.

NR4A2 haploinsufficiency is associated with intellectual disability and autism spectrum disorder.

NR4A2, a member of the nuclear receptor superfamily, is involved in modulation of target gene transcription, regulating several developmental processes such as regulation of cellular homeostasis, neuronal development, inflammation and carcinogenesis. 2q24.1 deletions are extremely rare, and only 1 patient with a de novo deletion encompassing only NR4A2 gene was reported so far. We report 3 additional patients with a de novo deletion encompassing NR4A2: 2 patients have deletions encompassing only NR4A2 gene and 1 patient has a deletion including NR4A2 and the first exon of GPD2. Our patients presented a neurodevelopmental disorder including language impairment, developmental delay, intellectual disability and/or autism spectrum disorder. We suggest that NR4A2 haploinsufficiency is implicated in neurodevelopmental disorder with high penetrance.

Novel mechanism of positive versus negative regulation by thyroid hormone receptor ß1 (TRß1) identified by genome-wide profiling of binding sites in mouse liver.

Triiodothyronine (T3) regulates key metabolic processes in the liver through the thyroid hormone receptor, TRß1. However, the number of known target genes directly regulated by TRß1 is limited, and the mechanisms by which positive and especially negative transcriptional regulation occur are not well understood. To characterize the TRß1 cistrome in vivo, we expressed a biotinylated TRß1 in hypo- and hyperthyroid mouse livers, used ChIP-seq to identify genomic TRß1 targets, and correlated these data with gene expression changes. As with other nuclear receptors, the majority of TRß1 binding sites were not in proximal promoters but in the gene body of known genes. Remarkably, T3 can dictate changes in TRß1 binding, with strong correlation to T3-induced gene expression changes, suggesting that differential TRß1 binding regulates transcriptional outcome. Additionally, DR-4 and DR-0 motifs were significantly enriched at binding sites where T3 induced an increase or decrease in TRß1 binding, respectively, leading to either positive or negative regulation by T3. Taken together, the results of this study provide new insights into the mechanisms of transcriptional regulation by TRß1 in vivo.

GPD2: The relationship with cancer and neural stemness.

We previously reported that knocking down GPD2 (glycerol-3-phosphate dehydrogenase 2), responsible for the glycerol-phosphate shuttle, causes human hepatocarcinoma-derived HuH-7 cells, lowering the cancer stemness. After examining whether GPD2 expression in the other cell lines could affect their cancer stemness, this study showed that human neuroblastoma-derived SH-SY5Y cells also lower the ability of sphere formation by knocking down GPD2. This suggests that GPD2 relates to the common mechanism for maintaining cancer stem cells, as in the cases like SH-SY5Y and HuH-7 cells. In addition, knocking down GPD2 in SH-SY5Y cells showed a morphological change and increasing tendency of neuronal marker genes, including GAP43, NeuN, and TUBB3, indicating that GPD2 may contribute to not only cancer but also neural stem cell maintenance. After all, GPD2 may play a role in maintaining cancer and neural stemness, although further rigorous studies are essential to conclude this. It is expected that GPD2 will be a novel target gene for cancer therapy, stem cell research, and development.

Non-bioenergetic roles of mitochondrial GPD2 promote tumor progression.

Rationale: Despite growing evidence for mitochondria's involvement in cancer, the roles of specific metabolic components outside the respiratory complex have been little explored. We conducted metabolomic studies on mitochondrial DNA (mtDNA)-deficient (ρ0) cancer cells with lower proliferation rates to clarify the undefined roles of mitochondria in cancer growth. Methods and results: Despite extensive metabolic downregulation, ρ0 cells exhibited high glycerol-3-phosphate (G3P) level, due to low activity of mitochondrial glycerol-3-phosphate dehydrogenase (GPD2). Knockout (KO) of GPD2 resulted in cell growth suppression as well as inhibition of tumor progression in vivo. Surprisingly, this was unrelated to the conventional bioenergetic function of GPD2. Instead, multi-omics results suggested major changes in ether lipid metabolism, for which GPD2 provides dihydroxyacetone phosphate (DHAP) in ether lipid biosynthesis. GPD2 KO cells exhibited significantly lower ether lipid level, and their slower growth was rescued by supplementation of a DHAP precursor or ether lipids. Mechanistically, ether lipid metabolism was associated with Akt pathway, and the downregulation of Akt/mTORC1 pathway due to GPD2 KO was rescued by DHAP supplementation. Conclusion: Overall, the GPD2-ether lipid-Akt axis is newly described for the control of cancer growth. DHAP supply, a non-bioenergetic process, may constitute an important role of mitochondria in cancer.

Antioxidant Behavioural Phenotype in the Immp2l Gene Knock-Out Mouse.

Mitochondrial dysfunction is strongly associated with autism spectrum disorder (ASD) and the Inner mitochondrial membrane protein 2-like (IMMP2L) gene is linked to autism inheritance. However, the biological basis of this linkage is unknown notwithstanding independent reports of oxidative stress in association with both IMMP2L and ASD. To better understand IMMP2L's association with behaviour, we developed the Immp2lKD knockout (KO) mouse model which is devoid of Immp2l peptidase activity. Immp2lKD -/- KO mice do not display any of the core behavioural symptoms of ASD, albeit homozygous Immp2lKD -/- KO mice do display increased auditory stimulus-driven instrumental behaviour and increased amphetamine-induced locomotion. Due to reports of increased ROS and oxidative stress phenotypes in an earlier truncated Immp2l mouse model resulting from an intragenic deletion within Immp2l, we tested whether high doses of the synthetic mitochondrial targeted antioxidant (MitoQ) could reverse or moderate the behavioural changes in Immp2lKD -/- KO mice. To our surprise, we observed that ROS levels were not increased but significantly lowered in our new Immp2lKD -/- KO mice and that these mice had no oxidative stress-associated phenotypes and were fully fertile with no age-related ataxia or neurodegeneration as ascertained using electron microscopy. Furthermore, the antioxidant MitoQ had no effect on the increased amphetamine-induced locomotion of these mice. Together, these findings indicate that the behavioural changes in Immp2lKD -/- KO mice are associated with an antioxidant-like phenotype with lowered and not increased levels of ROS and no oxidative stress-related phenotypes. This suggested that treatments with antioxidants are unlikely to be effective in treating behaviours directly resulting from the loss of Immp2l/IMMP2L activity, while any behavioural deficits that maybe associated with IMMP2L intragenic deletion-associated truncations have yet to be determined.

Exogenous aralar/slc25a12 can replace citrin/slc25a13 as malate aspartate shuttle component in liver.

The deficiency of CITRIN, the liver mitochondrial aspartate-glutamate carrier (AGC), is the cause of four human clinical phenotypes, neonatal intrahepatic cholestasis caused by CITRIN deficiency (NICCD), silent period, failure to thrive and dyslipidemia caused by CITRIN deficiency (FTTDCD), and citrullinemia type II (CTLN2). Clinical symptoms can be traced back to disruption of the malate-aspartate shuttle due to the lack of citrin. A potential therapy for this condition is the expression of aralar, the AGC present in brain, to replace citrin. To explore this possibility we have first verified that the NADH/NAD+ ratio increases in hepatocytes from citrin(-/-) mice, and then found that exogenous aralar expression reversed the increase in NADH/NAD+ observed in these cells. Liver mitochondria from citrin (-/-) mice expressing liver specific transgenic aralar had a small (~ 4-6 nmoles x mg prot-1 x min-1) but consistent increase in malate aspartate shuttle (MAS) activity over that of citrin(-/-) mice. These results support the functional replacement between AGCs in the liver. To explore the significance of AGC replacement in human therapy we studied the relative levels of citrin and aralar in mouse and human liver through absolute quantification proteomics. We report that mouse liver has relatively high aralar levels (citrin/aralar molar ratio of 7.8), whereas human liver is virtually devoid of aralar (CITRIN/ARALAR ratio of 397). This large difference in endogenous aralar levels partly explains the high residual MAS activity in liver of citrin(-/-) mice and why they fail to recapitulate the human disease, but supports the benefit of increasing aralar expression to improve the redox balance capacity of human liver, as an effective therapy for CITRIN deficiency.

Estrogen promotes innate immune evasion of Candida albicans through inactivation of the alternative complement system.

Candida albicans is a commensal of the urogenital tract and the predominant cause of vulvovaginal candidiasis (VVC). Factors that increase circulatory estrogen levels such as pregnancy, the use of oral contraceptives, and hormone replacement therapy predispose women to VVC, but the reasons for this are largely unknown. Here, we investigate how adaptation of C. albicans to estrogen impacts the fungal host-pathogen interaction. Estrogen promotes fungal virulence by enabling C. albicans to avoid the actions of the innate immune system. Estrogen-induced innate immune evasion is mediated via inhibition of opsonophagocytosis through enhanced acquisition of the human complement regulatory protein, Factor H, on the fungal cell surface. Estrogen-induced accumulation of Factor H is dependent on the fungal cell surface protein Gpd2. The discovery of this hormone-sensing pathway might pave the way in explaining gender biases associated with fungal infections and may provide an alternative approach to improving women's health.

Taraxasterol suppresses cell proliferation and boosts cell apoptosis via inhibiting GPD2-mediated glycolysis in gastric cancer.

Gastric cancer (GC) is the most common malignant tumor of digestive tract. Taraxasterol (TAX), a kind of phytosterol, has been proved to exert anti-tumor functions in GC. Herein, the current work was carried out to identify the biological role of TAX and molecular mechanisms underlying TAX in the progression of GC. In the present study, CCK-8 assay, Colony formation assay, EDU staining and TUNEL staining were performed to evaluate the malignant behaviors of GC cells. Levels of proliferation and apoptosis-associated proteins were assessed using western blotting analysis. Besides, GPD2 expression in GC cells was presented on CCLE database and the interaction between TAX and GPD2 was obtained from STRING database. The glucose uptake, lactate production, LDH activity, ATP and expressions of glycolysis-associated enzymes were measured to evaluate glycolysis level. Results of the present research revealed that TAX suppressed the proliferative and clone-forming abilities of GC cells and boosted the apoptosis of GC cells. TAX reduced GPD2 expression in GC cells. Furthermore, overexpression of GPD2 reversed the inhibitory effects of TAX on the proliferative and clone-forming abilities of GC cells as well as abolished the promoting effects of TAX on the apoptosis of GC cells. Besides, upregulation of GPD2 abrogated the inhibition of TAX on glycolysis. To conclude, TAX could suppress GC progression via inhibiting GPD2-mediated glycolysis, which helps to develop a promising molecular target for GC therapies.

Role of Mitochondrial Glycerol-3-Phosphate Dehydrogenase in Metabolic Adaptations of Prostate Cancer.

Prostate cancer is one of the most prominent cancers diagnosed in males. Contrasting with other cancer types, glucose utilization is not increased in prostate carcinoma cells as they employ different metabolic adaptations involving mitochondria as a source of energy and intermediates required for rapid cell growth. In this regard, prostate cancer cells were associated with higher activity of mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH), the key rate limiting component of the glycerophosphate shuttle, which connects mitochondrial and cytosolic processes and plays significant role in cellular bioenergetics. Our research focused on the role of mGPDH biogenesis and regulation in prostate cancer compared to healthy cells. We show that the 42 amino acid presequence is cleaved from N-terminus during mGPDH biogenesis. Only the processed form is part of the mGPDH dimer that is the prominent functional enzyme entity. We demonstrate that mGPDH overexpression enhances the wound healing ability in prostate cancer cells. As mGPDH is at the crossroad of glycolysis, lipogenesis and oxidative metabolism, regulation of its activity by intramitochondrial processing might represent rapid means of cellular metabolic adaptations.

Esculetin Inhibits Cancer Cell Glycolysis by Binding Tumor PGK2, GPD2, and GPI.

Glycolysis can improve the tolerance of tissue cells to hypoxia, and its intermediates provide raw materials for the synthesis and metabolism of the tumor cells. If it can inhibit the activity of glycolysis-related enzymes and control the energy metabolism of tumor, it can be targeted for the treatment of malignant tumor. The target proteins phosphoglycerate kinase 2 (PGK2), glycerol-3-phosphate dehydrogenase (GPD2), and glucose-6-phosphate isomerase (GPI) were screened by combining transcriptome, proteomics, and reverse docking. We detected the binding constant of the active compound using microscale thermophoresis (MST). It was found that esculetin bound well with three potential target proteins. Esculetin significantly inhibited the rate of glycolysis, manifested by differences of cellular lactate production and glucose consumption in HepG2 cells with or without esculetin. It was found that GPD2 bound strongly to GPI, revealing the direct interaction between the two glycolysis-related proteins. Animal tests have further demonstrated that esculetin may have anticancer effects by affecting the activity of PGK2, GPD2, and GPI. The results of this study demonstrated that esculetin can affect the glucose metabolism by binding to glycolytic proteins, thus playing an anti-tumor role, and these proteins which have direct interactions are potential novel targets for tumor treatment by esculetin.

Exploring the potential of the glycerol-3-phosphate dehydrogenase 2 (GPD2) promoter for recombinant gene expression in Saccharomyces cerevisiae.

A control point for keeping redox homeostasis in Saccharomyces cerevisiae during fermentative growth is the dynamic regulation of transcription for the glycerol-3-phosphate dehydrogenase 2 (GPD2) gene. In this study, the possibility to steer the activity of the GPD2 promoter was investigated by placing it in strains with different ability to reoxidise NADH, and applying different environmental conditions. Flow cytometric analysis of reporter strains expressing green fluorescent protein (GFP) under the control of the GPD2 promoter was used to determine the promoter activity at the single-cell level. When placed in a gpd1Δgpd2Δ strain background, the GPD2 promoter displayed a 2-fold higher activity as compared to the strong constitutive glyceraldehyde-3-phosphate dehydrogenase (TDH3). In contrast, the GPD2 promoter was found to be inactive when cells were cultivated in continuous mode at a growth rate of 0.3 h-1 and in conditions with excess oxygen (i.e. with an aeration of 2.5 vvm, and a stirring of 800 rpm). In addition, a clear window of operation where the gpd1Δgpd2Δ strain can be grown with the same efficiency as wild type yeast was identified. In conclusion, the flow cytometry mapping revealed conditions where the GPD2 promoter was either completely inactive or hyperactive, which has implications for its implementation in future biotechnological applications such as for process control of heterologous gene expression.

Examination of bioenergetic function in the inner mitochondrial membrane peptidase 2-like (Immp2l) mutant mice.

Inner mitochondrial membrane peptidase 2-like (IMMP2L) protein is a mitochondrial inner membrane peptidase that cleaves the signal peptide sequences of cytochrome c1 (CYC1) and mitochondrial glycerol phosphate dehydrogenase (GPD2). Immp2l mutant mice show infertility and early signs of aging. It is unclear whether mitochondrial respiratory deficiency underlies this phenotype. Here we show that the intermediate forms of GPD2 and CYC1 have normal expression levels and enzymatic function in Immp2l mutants. Mitochondrial respiration is not diminished in isolated mitochondria and cells from mutant mice. Our data suggest that respiratory deficiency is not the cause of the observed Immp2l mutant phenotypes.

The role of nitric oxide signaling in food intake; insights from the inner mitochondrial membrane peptidase 2 mutant mice.

Reactive oxygen species have been implicated in feeding control through involvement in brain lipid sensing, and regulating NPY/AgRP and pro-opiomelanocortin (POMC) neurons, although the underlying mechanisms are unclear. Nitric oxide is a signaling molecule in neurons and it stimulates feeding in many species. Whether reactive oxygen species affect feeding through interaction with nitric oxide is unclear. We previously reported that Immp2l mutation in mice causes excessive mitochondrial superoxide generation, which causes infertility and early signs of aging. In our present study, reduced food intake in mutant mice resulted in significantly reduced body weight and fat composition while energy expenditure remained unchanged. Lysate from mutant brain showed a significant decrease in cGMP levels, suggesting insufficient nitric oxide signaling. Thus, our data suggests that reactive oxygen species may regulate food intake through modulating the bioavailability of nitric oxide.