Cytosolic Redox Research Articles

Salicylic acid-independent role of NPR1 is required for protection from proteotoxic stress in the plant endoplasmic reticulum.

The unfolded protein response (UPR) is an ancient signaling pathway designed to protect cells from the accumulation of unfolded and misfolded proteins in the endoplasmic reticulum (ER). Because misregulation of the UPR is potentially lethal, a stringent surveillance signaling system must be in place to modulate the UPR. The major signaling arms of the plant UPR have been discovered and rely on the transcriptional activity of the transcription factors bZIP60 and bZIP28 and on the kinase and ribonuclease activity of IRE1, which splices mRNA to activate bZIP60. Both bZIP28 and bZIP60 modulate UPR gene expression to overcome ER stress. In this study, we demonstrate at a genetic level that the transcriptional role of bZIP28 and bZIP60 in ER-stress responses is antagonized by nonexpressor of PR1 genes 1 (NPR1), a critical redox-regulated master regulator of salicylic acid (SA)-dependent responses to pathogens, independently of its role in SA defense. We also establish that the function of NPR1 in the UPR is concomitant with ER stress-induced reduction of the cytosol and translocation of NPR1 to the nucleus where it interacts with bZIP28 and bZIP60. Our results support a cellular role for NPR1 as well as a model for plant UPR regulation whereby SA-independent ER stress-induced redox activation of NPR1 suppresses the transcriptional role of bZIP28 and bZIP60 in the UPR.

Adaptive remodeling of skeletal muscle energy metabolism in high-altitude hypoxia: Lessons from AltitudeOmics

Metabolic responses to hypoxia play important roles in cell survival strategies and disease pathogenesis in humans. However, the homeostatic adjustments that balance changes in energy supply and demand to maintain organismal function under chronic low oxygen conditions remain incompletely understood, making it difficult to distinguish adaptive from maladaptive responses in hypoxia-related pathologies. We integrated metabolomic and proteomic profiling with mitochondrial respirometry and blood gas analyses to comprehensively define the physiological responses of skeletal muscle energy metabolism to 16 days of high-altitude hypoxia (5260 m) in healthy volunteers from the AltitudeOmics project. In contrast to the view that hypoxia down-regulates aerobic metabolism, results show that mitochondria play a central role in muscle hypoxia adaptation by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This directs increases in muscle glucose toward pentose phosphate and one-carbon metabolism pathways that support cytosolic redox balance and help mitigate the effects of increased protein and purine nucleotide catabolism in hypoxia. Muscle accumulation of free amino acids favor these adjustments by coordinating cytosolic and mitochondrial pathways to rid the cell of excess nitrogen, but might ultimately limit muscle oxidative capacity in vivo Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress.

Development of an enzymatic assay to measure lactate in perchloric acid-precipitated cerebrospinal fluid

BackgroundIndividuals with inherited deficiencies of the pyruvate dehydrogenase complex or the respiratory chain complex can have increased concentrations of cerebrospinal fluid (CSF) lactate. Such measurements are clinical useful when measured in conjunction with pyruvate in order to calculate the lactate:pyruvate (L:P) ratio, a useful surrogate of cytosolic redox status. CSF pyruvate is measured in a protein-free supernatant prepared by the addition of CSF to perchloric acid while lactate is measured in untreated CSF. Utilizing the same sample for both lactate and pyruvate measurements is desirable. ObjectiveTo develop a method to measure lactate in perchloric-acid precipitated CSF and validate the L:P ratio as calculated from the analysis of both analytes in the same sample. MethodsSamples were prepared by the addition of 1 mL CSF to 2 mL 8% (w/v) cold perchloric acid, incubated on ice for 10 min, then centrifuged to obtain a protein-free supernatant. Lactate was measured by its oxidation to pyruvate and hydrogen peroxide using lactate oxidase and the absorbance of the resulting chromogen determined at 540 nm on a Roche cobas c501 chemistry analyzer. Method accuracy, linearity, imprecision and sensitivity were determined and a reference interval was verified. ResultsTo assess accuracy, this method was compared to lactate determined in unaltered CSF at another laboratory using 41 specimens with lactate concentrations from 0.6–11.9 mmol/L. Linear regression produced a slope of 1.09 and y-intercept of 0.26 (R2 = 1.00). Recovery was performed by ad-mixes of a high lactate standard and a CSF pool in different ratios to create a set of 19 samples prior to preparing protein-free supernatants. Recovery was 94.6–100% (mean ± SD was 97.4 ± 1.4%) at lactate concentrations of 2.68 to 12.63 mmol/L. Linearity was determined by combining two supernatants with low and high lactate concentrations in different ratios to create a set of six samples (0.15–12.70 mmol/L) that were tested in duplicate. Linear regression generated a slope of 1.01, y-intercept of −0.04 (R2 = 1.00). Precision was verified by analyzing quality control materials (acid-treated lactate standard) in 3 replicates each day for 5 days. Within-laboratory imprecision was 2.3% at 1.5 mmol/L and 1.5% at 10.5 mmol/L. The limit of blank was 0.05 mmol/L as determined by the mean added to three standard deviations determined from 10 replicates of perchloric-acid treated saline pool. The limit of detection was determined to be 0.12 mmol/L calculated from 10 replicates of a patient sample treated with perchloric-acid. The manufacturer's reference interval of 1.1–2.4 mmol/L was verified using 20 residual patient CSF samples. ConclusionsCSF lactate can be measured with accuracy and precision using the same perchloric-acid treated sample that is used for pyruvate.

Redox properties of Cys2His2 and Cys4 zinc fingers determined by electrospray ionization mass spectrometry.

Zinc finger (ZF) protein motifs, stabilized by binding of Zn(II), typically function as interaction modules that bind nucleic acids, proteins and other molecules. The elucidation of the redox states of ZF proteins in cellular conditions, which depend on their midpoint redox potentials, is important for understanding of ZF functioning. In the present study we determined the midpoint redox potentials for representatives of Cys2His2 and Cys4 types of ZF proteins in apo and Zn(II)‐bound forms using electrospray ionization mass spectrometry. The midpoint redox potentials of the apo forms of Cys2His2 and Cys4 ZF proteins were −326 and −365 mV (pH 7.5), respectively. These values are close to the cytosolic redox potential of approx. −350 mV (pH 7.5) and thus we can conclude that the apo form of Cys2His2‐type ZF proteins is predominantly reduced but apo forms of Cys4‐type ZF proteins should be substantially oxidized in the cytoplasm. As expected, Zn(II) binding stabilized the reduced forms of both ZF proteins: the corresponding redox potential values were −284 and −301 mV, respectively. Consequently, binding of Zn(II) ions to ZF motifs can act as a sensitive switch that activates the functioning of the ZF motifs within the cell, and also protects them from oxidation and can function as part of a redox‐sensitive regulation mechanism of cellular functions.

PhoPR Positively Regulates whiB3 Expression in Response to Low pH in Pathogenic Mycobacteria.

During infection, Mycobacterium tuberculosis colonizes macrophages or necrotic granulomas, in which low pH is one of the major challenges. The PhoPR two-component regulatory system and the cytosolic redox sensor WhiB3 both play important roles in the response to low pH by M. tuberculosis However, whether close association exists between PhoPR and WhiB3 remains unclear. In this study, the positive regulation of whiB3 by PhoPR in mycobacteria was characterized. We observed that the expression patterns of the whiB3 gene under acidic conditions are different among mycobacterial species, suggesting that the regulation of whiB3 differs among mycobacteria. A sequence analysis of the whiB3 promoters (whiB3p) from M. tuberculosis and two closely related species, namely, M. marinum and M. smegmatis, showed that the whiB3p regions from M. tuberculosis and M. marinum contain a new type of PhoP box that is absent in the M. smegmatiswhiB3p Direct binding of PhoP to whiB3p from M. tuberculosis and M. marinum but not that from M. smegmatis was validated by in vitro protein-DNA binding assays. The direct activation of whiB3 by PhoPR under acidic conditions was further verified by reverse transcription-quantitative PCR (qRT-PCR) analysis in M. marinum Moreover, mutating the residues important for the phosphorylation pathway of PhoPR in M. marinum abolished the activation of whiB3 expression by PhoPR under acidic conditions, suggesting that low pH triggers the phosphorylation of PhoPR, which in turn activates the transcription of whiB3 Since the PhoP box was only identified in whiB3p of pathogenic mycobacteria, we suggest that the PhoPR-whiB3 regulatory pathway may have evolved to facilitate mycobacterial infection.IMPORTANCE The low pH in macrophages is an important barrier for infection by microbes. The PhoPR two-component regulatory system is required for the response to low pH and plays a role in redox homeostasis in Mycobacterium tuberculosis WhiB3, a cytosolic redox-sensing transcriptional regulator, is also involved in these processes. However, there is no direct evidence to demonstrate the regulation of WhiB3 by PhoPR. In this study, we found that PhoPR directly activates whiB3 expression in response to low pH. An atypical PhoP box in the whiB3 promoters has been identified and is only found in pathogenic mycobacteria, which suggests that the PhoPR-whiB3 regulatory pathway may facilitate mycobacterial infection. This study provides novel information for further characterization of the PhoPR regulon.

Fuels derived from starch digestion have different effects on energy intake and metabolic responses of cows in the postpartum period

Absorbed fuels from the digestion of starch include propionic acid (PA) produced by ruminal fermentation and glucose (GLU) from intestinal digestion, which may be partially metabolized to lactic acid (LA) by intestinal tissues. Our objective was to evaluate the effects of these fuels on dry matter intake (DMI) and feeding behavior of cows in the postpartum period. We hypothesized that these fuels affect feed intake differently and that their effects are related to differences in their hepatic metabolism. Glucose was expected to have little effect on feed intake because little or no GLU is extracted from the blood by the liver. Whereas both LA and PA are anaplerotic and can stimulate oxidation of acetyl CoA in hepatocytes, hepatic extraction of PA is greater than LA, which depends on cytosolic redox state. Continuous isoenergetic infusions (150 kcal of ME/h) of PA, LA, or GLU or no infusion were administered abomasally to 8 ruminally cannulated multiparous Holstein cows (12.4 ± 6.2 d postpartum) in a duplicate 4 × 4 Latin square design experiment, with four 1-d infusion periods, balanced for carry-over effects. Treatment sequences were assigned to cows randomly, and treatments included control (CON, no infusion), PA (0.41 mol/h), LA (0.46 mol/h), and GLU (0.22 mol/h). Solutions containing treatments were infused at 500 mL/h for 22 h/d and provided ∼3.3 Mcal/d. Feeding behavior was recorded by a computerized data acquisition system. Gross energy digestibility of the diet was determined for each cow and used to calculate metabolizable energy intake (MEI) from the diet. Total MEI was calculated as the sum of MEI from the diet plus energy from infusions. Data were analyzed statistically with a mixed model including the fixed effect of treatment and random effects of block and cow within block. Each treatment was compared with CON by contrasts. Compared with CON, PA decreased DMI by 24% (14.3 vs. 18.9 kg/d) and total MEI by 13% (34.8 vs. 40.2 Mcal/d) with a tendency to decrease meal frequency. Lactic acid decreased DMI by 14% (16.3 vs. 18.9 kg/d) compared with CON by decreasing meal size 20% but did not affect MEI. Glucose infusion did not affect DMI or MEI. Treatment effects on DMI and MEI were consistent with their expected effects on hepatic oxidation. Depression of feed intake in diets containing highly fermentable starch is likely because of differences in hepatic metabolism.

Biomarkers of tumour redox status in response to modulations of glutathione and thioredoxin antioxidant pathways

The ability of certain cancer cells to maintain a highly reduced intracellular environment is correlated with aggressiveness and drug resistance. Since the glutathione (GSH) and thioredoxin (TRX) systems cooperate to a tight regulation of ROS in cell physiology, and to a stimulation of tumour initiation and progression, modulation of the GSH and TRX pathways are emerging as new potential targets in cancer. In vivo methods to assess changes in tumour redox status are critically needed to assess the relevance of redox-targeted agents. The current study assesses in vitro and in vivo biomarkers of tumour redox status in response to treatments targeting the GSH and TRX pathways, by comparing cytosolic and mitochondrial redox nitroxide electron paramagnetic resonance (EPR) probes, and cross-validation with redox dynamic fluorescent measurement. For that purpose, the effect of the GSH modulator buthionine sulfoximine (BSO) and of the TRX reductase inhibitor auranofin were measured in vitro using both cytosolic and mitochondrial EPR and roGFP probes in breast and cervical cancer cells. In vivo, mice bearing breast or cervical cancer xenografts were treated with the GSH or TRX modulators and monitored using the mito-TEMPO spin probe. Our data highlight the importance of using mitochondria-targeted spin probes to assess changes in tumour redox status induced by redox modulators. Further in vivo validation of the mito-tempo spin probe with alternative in vivo methods should be considered, yet the spin probe used in vivo in xenografts demonstrated sensitivity to the redox status modulators.

Lack of the NAD+-dependent glycerol 3-phosphate dehydrogenase impairs the function of transcription factors Sip4 and Cat8 required for ethanol utilization in Kluyveromyces lactis

The NAD+-dependent glycerol 3-phosphate dehydrogenase (KlGpd1) is an important enzyme for maintenance of the cytosolic redox balance in the milk yeast Kluyveromyces lactis. The enzyme is localized in peroxisomes and in the cytosol, indicating its requirement for the oxidation of NADH in both compartments. Klgpd1 mutants grow more slowly on glucose than wild-type cells and do not grow on ethanol as a sole carbon source. We studied the molecular basis of the latter phenotype and found that Gpd1 is required for high expression of KlICL1 and KlMLS1 which encode the key enzymes of the glyoxylate pathway isocitrate lyase and malate synthase, respectively. This regulation is mediated by CSRE elements in the promoters of these genes and the Snf1-regulated transcription factors KlCat8 and KlSip4. To study the transactivation function of these factors we developed a modified yeast one-hybrid system for K. lactis, using the endogenous ß-galactosidase gene LAC4 as a reporter in a lac9 deletion background. In combination with ChIP analyses we discovered that Gpd1 controls both the specific binding of Cat8 and Sip4 to the target promoters and the capacity of these factors to activate the reporter gene expression. We propose a model in which KlGpd1 activity is required for maintenance of the redox balance. In its absence, genes which function in generating redox balance instabilities are not expressed. A comparison of mutant phenotypes further indicates, that this system not only operates in K. lactis, but also in Saccharomyces cerevisiae.

Cytosolic Redox Status of Wine Yeast (Saccharomyces Cerevisiae) under Hyperosmotic Stress during Icewine Fermentation

Acetic acid is undesired in Icewine. It is unclear whether its production by fermenting yeast is linked to the nicotinamide adenine dinucleotide (NAD+/NADH) system or the nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) system. To answer this question, the redox status of yeast cytosolic NAD(H) and NADP(H) were analyzed along with yeast metabolites to determine how redox status differs under Icewine versus table wine fermentation. Icewine juice and dilute Icewine juice were inoculated with commercial wine yeast Saccharomyces cerevisiae K1-V1116. Acetic acid was 14.3-fold higher in Icewine fermentation than the dilute juice condition. The ratio of NAD+ to total NAD(H) was 24-fold higher in cells in Icewine fermentation than the ratio from the dilute juice condition. Conversely, the ratio of NADP+ to total NADP(H) from the dilute fermentation was 2.9-fold higher than that in the Icewine condition. These results support the hypothesis that in Icewine, increased NAD+ triggered the catalysis of NAD+-dependent aldehyde dehydrogenase(s) (Aldp(s)), which led to the elevated level of acetic acid in Icewine, whereas, in the dilute condition, NADP+ triggered NADP+-dependent Aldp(s), resulting in a lower level of acetic acid. This work, for the first time, analyzed the yeast cytosolic redox status and its correlation to acetic acid production, providing a more comprehensive understanding of the mechanism of acetic acid production in Icewine.

Severe Redox Stress Correlates with pH Changes in the Cytosol and Mitochondria of S. Cerevisiae

Maintenance of thiol redox balance is essential in both prokaryotes and eukaryotes. Glutathione (GSH) has been shown to be a major cellular redox buffer and is critical for mitochondrial functions such as Fe-S cluster biogenesis, oxidative phosphorylation and mitochondrial protein import. Since thiol-disulfide exchange reactions involve the transfer of protons as well as electrons, their redox potentials vary with pH. These pH differences should be taken into consideration while calculating the cytosol, matrix and IMS redox potentials. Previous studies in our group have suggested that GSH:GSSG redox state in subcellular compartments such as the cytosol, mitochondrial matrix and intermembrane space (IMS) is maintained separately [1], but it is still unclear how perturbations of the GSH:GSSG couple influence the distribution of GSH:GSSG between these compartments. Our goal is to understand the mechanisms for maintaining thiol redox equilibrium in different compartments under severe redox stress and determine how GSH:GSSG is exchanged between the cytosol, mitochondrial matrix and IMS under these conditions. Using a targeted GFP-based pH sensor in yeast strains overexpressing a plasma membrane GSH:GSSG transporter, we examined the changes in pH in the cytosol, mitochondrial matrix and IMS upon addition of GSH or GSSG to the growth medium. GSH and GSSG over accumulation were found to directly impact changes in pH in the subcellular compartments, suggesting concomitant changes in redox potentials as well.

Multifunctional Cytochrome c: Learning New Tricks from an Old Dog.

Cytochrome c (cyt c) is a small soluble heme protein characterized by a relatively flexible structure, particularly in the ferric form, such that it is able to sample a broad conformational space. Depending on the specific conditions, interactions, and cellular localization, different conformations may be stabilized, which differ in structure, redox properties, binding affinities, and enzymatic activity. The primary function is electron shuttling in oxidative phosphorylation, and is exerted by the so-called native cyt c in the intermembrane mitochondrial space of healthy cells. Under pro-apoptotic conditions, however, cyt c gains cardiolipin peroxidase activity, translocates into the cytosol to engage in the intrinsic apoptotic pathway, and enters the nucleus where it impedes nucleosome assembly. Other reported functions include cytosolic redox sensing and involvement in the mitochondrial oxidative folding machinery. Moreover, post-translational modifications such as nitration, phosphorylation, and sulfoxidation of specific amino acids induce alternative conformations with differential properties, at least in vitro. Similar structural and functional alterations are elicited by biologically significant electric fields and by naturally occurring mutations of human cyt c that, along with mutations at the level of the maturation system, are associated with specific diseases. Here, we summarize current knowledge and recent advances in understanding the different structural, dynamic, and thermodynamic factors that regulate the primary electron transfer function, as well as alternative functions and conformations of cyt c. Finally, we present recent technological applications of this moonlighting protein.

Activating de novo mutations in NFE2L2 encoding NRF2 cause a multisystem disorder

Transcription factor NRF2, encoded by NFE2L2, is the master regulator of defense against stress in mammalian cells. Somatic mutations of NFE2L2 leading to NRF2 accumulation promote cell survival and drug resistance in cancer cells. Here we show that the same mutations as inborn de novo mutations cause an early onset multisystem disorder with failure to thrive, immunodeficiency and neurological symptoms. NRF2 accumulation leads to widespread misregulation of gene expression and an imbalance in cytosolic redox balance. The unique combination of white matter lesions, hypohomocysteinaemia and increased G-6-P-dehydrogenase activity will facilitate early diagnosis and therapeutic intervention of this novel disorder.

Acute renal metabolic effect of metformin assessed with hyperpolarised MRI in rats.

Metformin inhibits hepatic mitochondrial glycerol phosphate dehydrogenase, thereby increasing cytosolic lactate and suppressing gluconeogenesis flux in the liver. This inhibition alters cytosolic and mitochondrial reduction-oxidation (redox) potential, which has been reported to protect organ function in several disease states including diabetes. In this study, we investigated the acute metabolic and functional changes induced by metformin in the kidneys of both healthy and insulinopenic Wistar rats used as a model of diabetes. Diabetes was induced by intravenous injection of streptozotocin, and kidney metabolism in healthy and diabetic animals was investigated 4weeks thereafter using hyperpolarised 13C-MRI, Clark-type electrodes and biochemical analysis. Metformin increased renal blood flow, but did not change total kidney oxygen consumption. In healthy rat kidneys, metformin increased [1-13C]lactate production and reduced mitochondrial [1-13C]pyruvate oxidation (decreased the 13C-bicarbonate/[1-13C]pyruvate ratio) within 30min of administration. Corresponding alterations to indices of mitochondrial, cytosolic and whole-cell redox potential were observed. Pyruvate oxidation was maintained in the diabetic rats, suggesting that the diabetic state abrogates metabolic reprogramming caused by metformin. This study demonstrates that metformin-induced acute metabolic alterations in healthy kidneys favoured anaerobic metabolism at the expense of aerobic metabolism. The results suggest that metformin directly alters the renal redox state, with elevated renal cytosolic redox states as well as decreased mitochondrial redox state. These findings suggest redox biology as a novel target to eliminate the renal complications associated with metformin treatment in individuals with impaired renal function.

Cytosolic redox components regulate protein homeostasis via additional localisation in the mitochondrial intermembrane space.

Oxidative protein folding is confined to the bacterial periplasm, endoplasmic reticulum and the mitochondrial intermembrane space. Maintaining a redox balance requires the presence of reductive pathways. The major thiol‐reducing pathways engage the thioredoxin and the glutaredoxin systems which are involved in removal of oxidants, protein proofreading and folding. Alterations in redox balance likely affect the flux of these redox pathways and are related to ageing and diseases such as neurodegenerative disorders and cancer. Here, we first review the well‐studied oxidative and reductive processes in the bacterial periplasm and the endoplasmic reticulum, and then discuss the less understood process in the mitochondrial intermembrane space, highlighting its importance for the proper function of the cell.

Circulating acylcarnitine profile in human heart failure: a surrogate of fatty acid metabolic dysregulation in mitochondria and beyond.

Heart failure (HF) is associated with metabolic perturbations, particularly of fatty acids (FAs), which remain to be better understood in humans. This study aimed at testing the hypothesis that HF patients with reduced ejection fraction display systemic perturbations in levels of energy-related metabolites, especially those reflecting dysregulation of FA metabolism, namely, acylcarnitines (ACs). Circulating metabolites were assessed using mass spectrometry (MS)-based methods in two cohorts. The main cohort consisted of 72 control subjects and 68 HF patients exhibiting depressed left ventricular ejection fraction (25.9 ± 6.9%) and mostly of ischemic etiology with ≥2 comorbidities. HF patients displayed marginal changes in plasma levels of tricarboxylic acid cycle-related metabolites or indexes of mitochondrial or cytosolic redox status. They had, however, 22-79% higher circulating ACs, irrespective of chain length (P < 0.0001, adjusted for sex, age, renal function, and insulin resistance, determined by shotgun MS/MS), which reflects defective mitochondrial β-oxidation, and were significantly associated with levels of NH2-terminal pro-B-type natriuretic peptide levels, a disease severity marker. Subsequent extended liquid chromatography-tandem MS analysis of 53 plasma ACs in a subset group from the primary cohort confirmed and further substantiated with a comprehensive lipidomic analysis in a validation cohort revealed in HF patients a more complex circulating AC profile. The latter included dicarboxylic-ACs and dihydroxy-ACs as well as very long chain (VLC) ACs or sphingolipids with VLCFAs (>20 carbons), which are proxies of dysregulated FA metabolism in peroxisomes. Our study identified alterations in circulating ACs in HF patients that are independent of biological traits and associated with disease severity markers. These alterations reflect dysfunctional FA metabolism in mitochondria but also beyond, namely, in peroxisomes, suggesting a novel mechanism contributing to global lipid perturbations in human HF.NEW & NOTEWORTHY Mass spectrometry-based profiling of circulating energy metabolites, including acylcarnitines, in two cohorts of heart failure versus control subjects revealed multiple alterations in fatty acid metabolism in peroxisomes in addition to mitochondria, thereby highlighting a novel mechanism contributing to global lipid perturbations in heart failure.Listen to this article's corresponding podcast at http://ajpheart.podbean.com/e/acylcarnitines-in-human-heart-failure/.

Metabolic correction by pyruvate halts acquired epilepsy in multiple rodent models

Metabolic intervention strategy of epilepsy treatment has been gaining broader attention due to accumulated evidence that hypometabolism, manifested in humans as reduced brain glucose consumption, is a principal factor in acquired epilepsy. Therefore, targeting deficient energy metabolism may be an effective approach for treating epilepsy. To confront this pathology we utilized pyruvate, which besides being an anaplerotic mitochondrial fuel possesses a unique set of neuroprotective properties as it: (i) is a potent reactive oxygen species scavenger; (ii) abates overactivation of Poly [ADP-ribose] polymerase 1 (PARP-1); (iii) facilitates glutamate efflux from the brain; (iv) augments brain glycogen stores; (v) is anti-inflammatory; (vi) prevents neuronal hyperexcitability; and (vii) normalizes the cytosolic redox state. In vivo, chronic oral pyruvate administration completely abolished established epileptic phenotypes in three accepted and fundamentally different rodent acquired epilepsy models. Our study reports metabolic correction by pyruvate as a potentially highly effective treatment of acquired epilepsies.

G6pd Deficiency Does Not Affect the Cytosolic Glutathione or Thioredoxin Antioxidant Defense in Mouse Cochlea.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd-deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1, but not G6pd, decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in Caenorhabditis elegans.

Mitochondrial dysfunction can increase oxidative stress and extend lifespan in Caenorhabditis elegans. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPRmt) and extends lifespan. Here we report that transaldolase (tald-1) deficiency impairs mitochondrial function in vivo, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H2O2 levels. Lifespan extension from knockdown of tald-1 is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (fmo-2). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.

The desert gerbil Psammomys obesus as a model for metformin-sensitive nutritional type 2 diabetes to protect hepatocellular metabolic damage: Impact of mitochondrial redox state.

IntroductionWhile metformin (MET) is the most widely prescribed antidiabetic drug worldwide, its beneficial effects in Psammomys obesus (P. obesus), a rodent model that mimics most of the metabolic features of human diabetes, have not been explored thoroughly. Here, we sought to investigate whether MET might improve insulin sensitivity, glucose homeostasis, lipid profile as well as cellular redox and energy balance in P. obesus maintained on a high energy diet (HED).Materials and methodsP. obesus gerbils were randomly assigned to receive either a natural diet (ND) consisting of halophytic plants (control group) or a HED (diabetic group) for a period of 24 weeks. MET (50 mg/kg per os) was administered in both animal groups after 12 weeks of feeding, i.e., the time required for the manifestation of insulin resistance in P. obesus fed a HED. Parallel in vitro experiments were conducted on isolated hepatocytes that were shortly incubated (30 min) with MET and energetic substrates (lactate + pyruvate or alanine, in the presence of octanoate).ResultsIn vivo, MET lowered glycemia, glycosylated haemoglobin, circulating insulin and fatty acid levels in diabetic P. obesus. It also largely reversed HED-induced hepatic lipid alterations. In vitro, MET increased glycolysis but decreased both gluconeogenesis and ketogenesis in the presence of glucogenic precursors and medium-chain fatty acid. Importantly, these changes were associated with an increase in cytosolic and mitochondrial redox states along with a decline in respiration capacity.ConclusionsMET prevents the progression of insulin resistance in diabetes-prone P. obesus, possibly through a tight control of gluconeogenesis and fatty acid β-oxidation depending upon mitochondrial function. While the latter is increasingly becoming a therapeutic issue in diabetes, the gut microbiota is another promising target that would need to be considered as well.

Lipid production in Yarrowia lipolytica is maximized by engineering cytosolic redox metabolism.

Microbial factories have been engineered to produce lipids from carbohydrate feedstocks for production of biofuels and oleochemicals. However, even the best yields obtained to date are insufficient for commercial lipid production. To maximize the capture of electrons generated from substrate catabolism and thus increase substrate-to-product yields, we engineered 13 strains of Yarrowia lipolytica with synthetic pathways converting glycolytic NADH into the lipid biosynthetic precursors NADPH or acetyl-CoA. A quantitative model was established and identified the yield of the lipid pathway as a crucial determinant of overall process yield. The best engineered strain achieved a productivity of 1.2 g/L/h and a process yield of 0.27 g-fatty acid methyl esters/g-glucose, which constitutes a 25% improvement over previously engineered yeast strains. Oxygen requirements of our highest producer were reduced owing to decreased NADH oxidization by aerobic respiration. We show that redox engineering could enable commercialization of microbial carbohydrate-based lipid production.