Cytosolic Redox Research Articles

REDD1 Is a Promising Therapeutic Target to Combat the Development of Diabetes Complications: A Report on Research Supported by Pathway to Stop Diabetes.

The stress response protein regulated in development and DNA damage response 1 (REDD1) has emerged as a key player in the pathogenesis of diabetes. Diabetes upregulates REDD1 in a variety of insulin-sensitive tissues, where the protein acts to inhibit signal transduction downstream of the insulin receptor. REDD1 functions as a cytosolic redox sensor that suppresses Akt/mTORC1 signaling to reduce energy expenditure in response to cellular stress. Whereas a transient increase in REDD1 contributes to an adaptive cellular response, chronically elevated REDD1 levels are implicated in disease progression. Recent studies highlight the remarkable benefits of both whole-body and tissue-specific REDD1 deletion in preclinical models of type 1 and type 2 diabetes. In particular, REDD1 is necessary for the development of glucose intolerance and the consequent rise in oxidative stress and inflammation. Herein, we review studies that support a role for chronically elevated REDD1 levels in the development of diabetic complications, reflect on limitations of prior therapeutic approaches targeting REDD1 in patients, and discuss potential opportunities for future interventions to improve the lives of people living with diabetes. This article is part of a series of Perspectives that report on research funded by the American Diabetes Association Pathway to Stop Diabetes program.

Importance of Michaelis Constants for Cancer Cell Redox Balance and Lactate Secretion-Revisiting the Warburg Effect.

Cancer cells metabolize a large fraction of glucose to lactate, even under a sufficient oxygen supply. This phenomenon-the "Warburg Effect"-is often regarded as not yet understood. Cancer cells change gene expression to increase the uptake and utilization of glucose for biosynthesis pathways and glycolysis, but they do not adequately up-regulate the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). Thereby, an increased glycolytic flux causes an increased production of cytosolic NADH. However, since the corresponding gene expression changes are not neatly fine-tuned in the cancer cells, cytosolic NAD+ must often be regenerated by loading excess electrons onto pyruvate and secreting the resulting lactate, even under sufficient oxygen supply. Interestingly, the Michaelis constants (KM values) of the enzymes at the pyruvate junction are sufficient to explain the priorities for pyruvate utilization in cancer cells: 1. mitochondrial OXPHOS for efficient ATP production, 2. electrons that exceed OXPHOS capacity need to be disposed of and secreted as lactate, and 3. biosynthesis reactions for cancer cell growth. In other words, a number of cytosolic electrons need to take the "emergency exit" from the cell by lactate secretion to maintain the cytosolic redox balance.

Uncoupling of nutrient metabolism and cellular redox by cytosolic routing of the mitochondrial G-3-P dehydrogenase Gpd2 causes loss of conidiation and pathogenicity in Pyricularia oryzae

Oxidation of self-stored carbohydrates and lipids provides the energy for the rapid morphogenetic transformation during asexual and infection-related development in Pyricularia oryzae, which results in intracellular accumulation of reducing equivalents NADH and FADH2, requiring a cytosolic shuttling machinery towards mitochondria. Our previous studies identified the mitochondrial D-lactate dehydrogenase MoDld1 as a regulator to channel the metabolite flow in conjunction with redox homeostasis. However, the regulator(s) facilitating the cytosolic redox balance and the importance in propelling nutrient metabolite flow remain unknown. The G-3-P shuttle is a conserved machinery transporting the cytosolic reducing power to mitochondria. In P. oryzae, the mitochondrial G-3-P dehydrogenase Gpd2 was required for cellular NAD+/NADH balance and fungal virulence. In this study, we re-locate the mitochondrial G-3-P dehydrogenase Gpd2 to the cytosol for disturbing cytosolic redox status. Our results showed overexpression of cytosolic gpd2Δmts without the mitochondrial targeted signal (MTS) driven by Ribosomal protein 27 promoter (PR27) exerted conflicting regulation of cellular oxidoreductase activities compared to the ΔModld1 deletion mutant by RNA-seq and prevented the conidiation and pathogenicity of P. oryzae. Moreover, overexpression of gpd2Δmts caused defects in glycogen and lipid mobilization underlying asexual and infectious structural development associated with decreased cellular NADH production and weakened anti-oxidation activities. RNA-seq and non-targeted metabolic profiling revealed down-regulated transcriptional activities of carbohydrate metabolism and lower abundance of fatty acids and secondary metabolites in RP27:gpd2Δmts. Thus, our studies indicate the essential role of cytosolic redox control in nutrient metabolism fueling the asexual and infection-related development in P. oryzae.

Analysis of thioredoxin 1 levels in a mouse model of unilateral renal ischemia-reperfusion injury

Kidney disease, acute and chronic, is associated with high morbidity and mortality. Currently, a major obstacle in the field of nephrology is the lack of novel and more sensitive biomarkers that would allow an early diagnose and a proper monitoring of disease progression. Oxidative stress and the increased production of reactive oxygen species (ROS) have been implicated in the pathophysiology of a variety of renal diseases. Proteins of the thioredoxin (Trx) family, among others thioredoxins (Trxs) and glutaredoxins (Grxs), represent one of the most important protective systems in the defense against ROS as well as in the repair of their intracellular effects. Thioredoxin 1 (Trx1) is a cytosolic redox protein that catalyzes the reduction of protein disulfides using the thiol groups of two cysteine residues on its CGPC active site. Trx1 is abundantly expressed in different segments of the nephron and has been shown to be released from cells under conditions of oxidative stress in vitro and in vivo. We hypothesized that renal IRI induces the release of Trx1 from cells into extracellular fluids such as serum and tubular fluid/urine and that Trx1 increase may correlate with the degree of damage in the kidney. We established an effective mouse model of unilateral renal ischemia-reperfusion injury (IRI) and studied Trx1 levels in the serum, urine, as well as in various nephron segments of mice following IRI or sham surgery. We compared different reperfusion times (4h, 8h, 16h, 24h, 48h, 96h, and 168h) to track Trx1 levels fluctuation over time. Serum levels of Trx1 increased 4h after renal IRI, while other markers such as blood urea nitrogen and creatinine remained low. Serum Trx1 showed a positive correlation with tubular injury score helping predict intrarenal damage over time. Additionally, immunohistochemistry studies revealed an increase in Trx1 signals in the proximal tubules and collecting ducts as well as a strong nuclear localization in the same nephron segments that became more intense by 96h, a time point characterized by an intense mitotic activity in the tubules. Between 16 and 24h, Trx1 appeared in the lumen of proximal tubules, however urinary levels of Trx1 did not significantly increase. Based on these findings we conclude that serum Trx1 may represent a novel biomarker for assessing renal damage. In addition to this, we hypothesize that intrarenal changes in Trx1 expression and localization might be related to the activation of regeneration mechanisms in the renal tubular epithelium. Further studies are needed to understand the role of Trx1 in the regeneration process after renal IRI. This work was funded by the Center of Integrative Mammalian Research of Ross University School of Veterinary Medicine and by a Startup funding from Long Island University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Systems Metabolic Engineering for Efficient Violaxanthin Production in Yeast.

Violaxanthin is a plant-derived orange xanthophyll with remarkable antioxidant activity that has wide applications in various industries, such as food, agriculture, and cosmetics. In addition, it is the key precursor of important substances such as abscisic acid and fucoxanthin. Saccharomyces cerevisiae, as a GRAS (generally regarded as safe) chassis, provides a good platform for producing violaxanthin production with a yield of 7.3 mg/g DCW, which is far away from commercialization. Herein, an integrated strategy involving zeaxanthin epoxidase (ZEP) source screening, cytosol redox state engineering, and nicotinamide adenine dinucleotide phosphate (NADPH) regeneration was implemented to enhance violaxanthin production in S. cerevisiae. 58aa-truncated ZEP from Vitis vinifera exhibited optimal efficiency in an efficient zeaxanthin-producing strain. The titer of violaxanthin gradually increased by 17.9-fold (up to 119.2 mg/L, 15.19 mg/g DCW) via cytosol redox state engineering and NADPH supplementation. Furthermore, balancing redox homeostasis considerably improved the zeaxanthin concentration by 139.3% (up to 143.9 mg/L, 22.06 mg/g DCW). Thus, the highest reported titers of violaxanthin and zeaxanthin in S. cerevisiae were eventually achieved. This study not only builds an efficient platform for violaxanthin biosynthesis but also serves as a useful reference for the microbial production of xanthophylls.

Specificity and dynamics of H2O2 detoxification by the cytosolic redox regulatory network as revealed by in vitro reconstitution

The thiol redox state is a decisive functional characteristic of proteins in cell biology. Plasmatic cell compartments maintain a thiol-based redox regulatory network linked to the glutathione/glutathione disulfide couple (GSH/GSSG) and the NAD(P)H system. The basic network constituents are known and in vivo cell imaging with gene-encoded probes have revealed insight into the dynamics of the [GSH]2/[GSSG] redox potential, cellular H2O2 and NAD(P)H+H+ amounts in dependence on metabolic and environmental cues. Less understood is the contribution and interaction of the network components, also because of compensatory reactions in genetic approaches. Reconstituting the cytosolic network of Arabidopsis thaliana in vitro from fifteen recombinant proteins at in vivo concentrations, namely glutathione peroxidase-like (GPXL), peroxiredoxins (PRX), glutaredoxins (GRX), thioredoxins, NADPH-dependent thioredoxin reductase A and glutathione reductase and applying Grx1-roGFP2 or roGFP2-Orp1 as dynamic sensors, allowed for monitoring the response to a single H2O2 pulse. The major change in thiol oxidation as quantified by mass spectrometry-based proteomics occurred in relevant peptides of GPXL, and to a lesser extent of PRX, while other Cys-containing peptides only showed small changes in their redox state and protection. Titration of ascorbate peroxidase (APX) into the system together with dehydroascorbate reductase lowered the oxidation of the fluorescent sensors in the network but was unable to suppress it. The results demonstrate the power of the network to detoxify H2O2, the partially independent branches of electron flow with significance for specific cell signaling and the importance of APX to modulate the signaling without suppressing it and shifting the burden to glutathione oxidation.

Protective Effect of Knee Postoperative Fluid on Oxidative-Induced Damage in Human Knee Articular Chondrocytes.

The oxidative-stress-elicited deterioration of chondrocyte function is the initial stage of changes leading to the disruption of cartilage homeostasis. These changes entail a series of catabolic damages mediated by proinflammatory cytokines, MMPs, and aggrecanases, which increase ROS generation. Such uncontrolled ROS production, inadequately balanced by the cellular antioxidant capacity, eventually contributes to the development and progression of chondropathies. Several pieces of evidence show that different growth factors, single or combined, as well as anti-inflammatory cytokines and chemokines, can stimulate chondrogenesis and improve cartilage repair and regeneration. In this view, hypothesizing a potential growth-factor-associated action, we investigate the possible protective effect of post-operation knee fluid from patients undergoing prosthesis replacement surgery against ROS-induced damage on normal human knee articular chondrocytes (HKACs). To this end, HKACs were pre-treated with post-operation knee fluid and then exposed to H2O2 to mimic oxidative stress. Intracellular ROS levels were measured by using the molecular probe H2DCFDA; cytosolic and mitochondrial oxidative status were assessed by using HKACs infected with lentiviral particles harboring the redox-sensing green fluorescent protein (roGFP); and cell proliferation was determined by measuring the rate of DNA synthesis with BrdU incorporation. Moreover, superoxide dismutase (SOD), catalase, and glutathione levels from the cell lysates of treated cells were also measured. Postoperative peripheral blood sera from the same patients were used as controls. Our study shows that post-operation knee fluid can counteract H2O2-elicited oxidative stress by decreasing the intracellular ROS levels, preserving the cytosolic and mitochondrial redox status, maintaining the proliferation of oxidatively stressed HKACs, and upregulating chondrocyte antioxidant defense. Overall, our results support and propose an important effect of post-operation knee fluid substances in maintaining HKAC function by mediating cell antioxidative system upregulation and protecting cells from oxidative stress.

Contrasting cytosolic glutathione redox dynamics under abiotic and biotic stress in barley as revealed by the biosensor Grx1-roGFP2.

Barley is a staple crop of major global importance and relatively resilient to a wide range of stress factors in the field. Transgenic reporter lines to investigate physiological parameters during stress treatments remain scarce. We generated and characterized transgenic homozygous barley lines (cv. Golden Promise Fast) expressing the genetically encoded biosensor Grx1-roGFP2, which indicates the redox potential of the major antioxidant glutathione in the cytosol. Our results demonstrated functionality of the sensor in living barley plants. We determined the glutathione redox potential (EGSH) of the cytosol to be in the range of -308 mV to -320 mV. EGSH was robust against a combined NaCl (150 mM) and water deficit treatment (-0.8 MPa) but responded with oxidation to infiltration with the phytotoxic secretome of the necrotrophic fungus Botrytis cinerea. The generated reporter lines are a novel resource to study biotic and abiotic stress resilience in barley, pinpointing that even severe abiotic stress leading to a growth delay does not automatically induce cytosolic EGSH oxidation, while necrotrophic pathogens can undermine this robustness.

HFR1 antagonizes ABI4 to coordinate cytosolic redox status for seed germination under high-temperature stress.

Seed germination and dormancy represent critical phases in the life cycle of plants, tightly regulated by endogenous phytochrome levels and environment signals. High temperatures (HT) induce the overaccumulation of reactive oxygen species (ROS) and increase abscisic acid (ABA), thereby inhibiting seed germination. Our previous findings showed that HT induced the burst of reactive nitrogen species (RNS), increasing the S-nitrosylation modification of HFR1, which effectively blocks seed germination. Importantly, stabilizing HFR1 has been shown to significantly mitigate the inhibitory effect of HT on seed germination. In this study, we reported that HT increased the protein abundance of ABI4, a crucial component in ABA signaling, which in turn activates the expression of RbohD while suppressing the expression of VTC2. This leads to the rapid generation of ROS, thereby inhibiting seed germination. Consistently, the seed germination of abi4 mutant showed insensitivity to HT with lower ROS level during seed germination, whereas transgenic lines overexpressing ABI4 showed reduced germination rates accompanied by elevated ROS levels. Furthermore, we noted that HFR1 interacts with ABI4 to sequester its activity under normal conditions. However, HT-induced ROS triggered the degradation of HFR1, consequently releasing ABI4 activity. This activation of ABI4 promotes RbohD expression, culminating in a ROS burst that suppresses seed germination. Thus, our study unveils a novel function for ABI4 in regulating ROS level and seed germination under HT stress. Throughout this process, HFR1 plays a critical role in restraining ABI4 activity to maintain an optimal endogenous ROS level, thereby ensuring seed germination under favorable environmental conditions.

A role and mechanism for redox sensing by SENP1 in β-cell responses to high fat feeding

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in β-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted β-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased β-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or β-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained β-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

Pitfalls of Mitochondrial Redox Signaling Research.

Redox signaling from mitochondria (mt) to the cytosol and plasma membrane (PM) has been scarcely reported, such as in the case of hypoxic cell adaptation or (2-oxo-) 2-keto-isocaproate (KIC) β-like-oxidation stimulating insulin secretion in pancreatic β-cells. Mutual redox state influence between mitochondrial major compartments, the matrix and the intracristal space, and the cytosol is therefore derived theoretically in this article to predict possible conditions, when mt-to-cytosol and mt-to-PM signals may occur, as well as conditions in which the cytosolic redox signaling is not overwhelmed by the mitochondrial antioxidant capacity. Possible peroxiredoxin 3 participation in mt-to-cytosol redox signaling is discussed, as well as another specific case, whereby mitochondrial superoxide release is diminished, whereas the matrix MnSOD is activated. As a result, the enhanced conversion to H2O2 allows H2O2 diffusion into the cytosol, where it could be a predominant component of the H2O2 release. In both of these ways, mt-to-cytosol and mt-to-PM signals may be realized. Finally, the use of redox-sensitive probes is discussed, which disturb redox equilibria, and hence add a surplus redox-buffering to the compartment, where they are localized. Specifically, when attempts to quantify net H2O2 fluxes are to be made, this should be taken into account.

1558-P: The Mitochondrial Calcium Uniporter Regulates Hepatic Mitochondrial Oxidation and Intracellular Redox In Vivo

The mitochondrial calcium uniporter (MCU) facilitates mitochondrial calcium influx, and several enzymes that catalyze key steps of the TCA cycle require calcium for their activation. As such, it is generally accepted that rates of mitochondrial oxidation correlate with mitochondrial calcium activity. Decreased rates of hepatic mitochondrial oxidation are associated with metabolic dysfunction and ectopic lipid accumulation as seen with type 2 diabetes and nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis. Despite this, a role for MCU and mitochondrial calcium in regulating hepatic mitochondrial oxidation is still unclear. To address this question, we generated a liver-specific MCU knockout (MCU KO) mouse model which displayed reduced mitochondrial calcium ([Ca2+]mt; p<0.05) and increased cytosolic calcium ([Ca2+]cyt; p<0.01). Despite decreased [Ca2+]mt, we found that deletion of hepatic MCU increased rates of isocitrate dehydrogenase flux (VIDH; p<0.01), α-ketoglutarate dehydrogenase flux (VOGDH; p<0.01), and succinate dehydrogenase flux (VSDH; p<0.05) in vivo, using a novel [13C5]glutamine tracer infusion technique (Q-Flux). Further, we show that rates of [14C16]palmitate oxidation (p<0.01) and intrahepatic lipolysis (p<0.05) were significantly increased in liver slices collected from MCU KO mice compared to WT mice, and were associated with reductions in hepatic triacylglycerol content in vivo (p<0.01). Hepatocellular redox state was also altered with MCU KO, as indicated by a decreased cytosolic redox ratio ([lactate]:[pyruvate]; p<0.05) and increased mitochondrial redox ratio ([β-hydroxybutyrate]:[acetoacetate]; p<0.01). Conclusion: Here we show that increases in [Ca2+]cyt potently increase hepatic mitochondrial fat oxidation and alters hepatic redox balance to reduce hepatic lipid content independently of [Ca2+]mt in vivo and targeting [Ca2+]cyt may represent a novel therapeutic target to treat NAFLD. Disclosure T.E.Lamoia: None. B.T.Hubbard: None. M.Guerra: None. R.Goodman: None. M.H.Nathanson: None. G.I.Shulman: Consultant; Novo Nordisk, DiCerna Pharmaceuticals, Inc., Bayer Consumer Care AG, Kriya Therapeutics, Arrowhead Pharmaceuticals, Inc., ESPERION Therapeutics, Inc., Other Relationship; AstraZeneca, Merck & Co., Inc., Janssen Research & Development, LLC, iMetabolic Biopharma Corporation, Maze Therapeutics, 89bio, Inc., Equator Therapeutics, Inc., Generian Pharmaceuticals, Fortress Biotech, Inc., OrsoBio, Aro Biotherapeutics Company, Stock/Shareholder; Levels Health, Inc. Funding National Institutes of Health (F31DK126362, T32GM007324)

The mitochondrial calcium uniporter (MCU) activates mitochondrial respiration and enhances mobility by regulating mitochondrial redox state

Regulation of mitochondrial redox balance is emerging as a key event for cell signaling in both physiological and pathological conditions. However, the link between the mitochondrial redox state and the modulation of these conditions remains poorly defined. Here, we discovered that activation of the evolutionary conserved mitochondrial calcium uniporter (MCU) modulates mitochondrial redox state. By using mitochondria-targeted redox and calcium sensors and genetic MCU-ablated models, we provide evidence of the causality between MCU activation and net reduction of mitochondrial (but not cytosolic) redox state. Redox modulation of redox-sensitive groups via MCU stimulation is required for maintaining respiratory capacity in primary human myotubes and C. elegans, and boosts mobility in worms. The same benefits are obtained bypassing MCU via direct pharmacological reduction of mitochondrial proteins. Collectively, our results demonstrate that MCU regulates mitochondria redox balance and that this process is required to promote the MCU-dependent effects on mitochondrial respiration and mobility.

β-lapachone-mediated WST1 Reduction as Indicator for the Cytosolic Redox Metabolism of Cultured Primary Astrocytes

Electron cycler-mediated extracellular reduction of the water-soluble tetrazolium salt 1 (WST1) is frequently used as tool for the determination of cell viability. We have adapted this method to monitor by determining the extracellular WST1 formazan accumulation the cellular redox metabolism of cultured primary astrocytes via the NAD(P)H-dependent reduction of the electron cycler β-lapachone by cytosolic NAD(P)H:quinone oxidoreductase 1 (NQO1). Cultured astrocytes that had been exposed to β-lapachone in concentrations of up to 3 µM remained viable and showed an almost linear extracellular accumulation of WST1 formazan for the first 60 min, while higher concentrations of β-lapachone caused oxidative stress and impaired cell metabolism. β-lapachone-mediated WST1 reduction was inhibited by the NQO1 inhibitors ES936 and dicoumarol in a concentration-dependent manner, with half-maximal inhibition observed at inhibitor concentrations of about 0.3 µM. β-lapachone-mediated WST1 reduction depended strongly on glucose availability, while mitochondrial substrates such as lactate, pyruvate or ketone bodies allowed only residual β-lapachone-mediated WST1 reduction. Accordingly, the mitochondrial respiratory chain inhibitors antimycin A and rotenone hardly affected astrocytic WST1 reduction. Both NADH and NADPH are known to supply electrons for reactions catalysed by cytosolic NQO1. Around 60% of the glucose-dependent β-lapachone-mediated WST1 reduction was prevented by the presence of the glucose-6-phosphate dehydrogenase inhibitor G6PDi-1, while the glyceraldehyde-3-phosphate dehydrogenase inhibitor iodoacetate had only little inhibitory potential. These data suggest that pentose phosphate pathway-generated NADPH, and not glycolysis-derived NADH, is the preferred electron source for cytosolic NQO1-catalysed reductions in cultured astrocytes.

Real-time simultaneous imaging of temporal alterations in cytoplasmic and mitochondrial redox in single cells during cell division and cell death.

Cytosolic and organelle redox are highly interrelated, and their alterations play critical roles in both physiological and pathological cell states. This highly regulated process is crucial in life-death decisions of cells. Among organelles, the mitochondrion is the major source of intracellular-ROS and contributes to oxidation damage-induced cell death. Increase in cytosolic-redox and mitochondrial-redox is evident in cells undergoing diverse forms of cell death, such as apoptosis, necrosis, and necroptosis. The hierarchical profiling of redox signaling at the cytosol and mitochondria in a single cell is important to understand the relative contribution of each species in the initiation and shaping of cell death. Here, we demonstrate the potential application of ratiometric redox GFP (roGFP) and intensity-based redox-sensitive RFP (rxRFP) targeted to mitochondria in revealing both rapid and slow progressing changes in redox during cell division and in cells undergoing multiple modes of cell death. To generate imaging quality signal, single-cell clones stably expressing both roGFP at the cytosol and rxRFP probes targeted to mitochondria were generated. The cells provided sufficient temporal resolution with imaging-ready signal for the real-time visualization of rapidly progressing redox alterations at the cytosol and mitochondria. The long-time imaging of the cells revealed that a moderate increase in cytosolic ROS marks the division stage. Similarly, distinct forms of cell death trigger a unique and temporally regulated redox change at the cytosol and mitochondria, suggesting the potential utility of the sensor cells to dissect the nature of cell death pathways induced by specific forms of stress.

Reversal of mitochondrial malate dehydrogenase 2 enables anaplerosis via redox rescue in respiration-deficient cells

Inhibition of the electron transport chain (ETC) prevents the regeneration of mitochondrial NAD+, resulting in cessation of the oxidative tricarboxylic acid (TCA) cycle and a consequent dependence upon reductive carboxylation for aspartate synthesis. NAD+ regeneration alone in the cytosol can rescue the viability of ETC-deficient cells. Yet, how this occurs and whether transfer of oxidative equivalents to the mitochondrion is required remain unknown. Here, we show that inhibition of the ETC drives reversal of the mitochondrial aspartate transaminase (GOT2) as well as malate and succinate dehydrogenases (MDH2 and SDH) to transfer oxidative NAD+ equivalents into the mitochondrion. This supports the NAD+-dependent activity of the mitochondrial glutamate dehydrogenase (GDH) and thereby enables anaplerosis-the entry of glutamine-derived carbon into the TCA cycle and connected biosynthetic pathways. Thus, under impaired ETC function, the cytosolic redox state is communicated into the mitochondrion and acts as a rheostat to support GDH activity and cell viability.

Dissecting the dynamics of major cytosolic redox systems under low oxygen stress

Dissecting the dynamics of major cytosolic redox systems under low oxygen stress

Mitochondrial GCN5L1 regulates cytosolic redox state and hepatic gluconeogenesis via glycerol phosphate shuttle GPD2

Hepatic gluconeogenesis is crucial for maintaining blood glucose during starvation, and a major contributor for hyperglycemia. Cellular redox state is related to mitochondrial biology and regulates conversion of specific metabolites to glucose. General control of amino acid synthesis 5 (GCN5) like-1 (GCN5L1) is a mitochondria-enriched protein which modulates glucose and amino acid metabolism. Here we show a new regulatory mode of GCN5L1 on gluconeogenesis using lactate and glycerol. We observed GCN5L1 deletion dramatically inhibited glucose production derived from glycerol and lactate, due to increased cytosolic redox state. The underlying mechanism is that GCN5L1 directly binds to the key component of mitochondrial shuttle glycerol phosphate dehydrogenase 2 (GPD2) and modulates its activity. These results have significant implications for understanding the physiological role and regulatory mechanism of mitochondrial shuttle in diabetes development and provide a novel therapeutic potential for diabetes.

Pyridine nucleotide redox potential in coronary smooth muscle couples myocardial blood flow to cardiac metabolism

Adequate oxygen delivery to the heart during stress is essential for sustaining cardiac function. Acute increases in myocardial oxygen demand evoke coronary vasodilation and enhance perfusion via functional upregulation of smooth muscle voltage-gated K+ (Kv) channels. Because this response is controlled by Kv1 accessory subunits (i.e., Kvβ), which are NAD(P)(H)-dependent aldo-keto reductases, we tested the hypothesis that oxygen demand modifies arterial [NAD(H)]i, and that resultant cytosolic pyridine nucleotide redox state influences Kv1 activity. High-resolution imaging mass spectrometry and live-cell imaging reveal cardiac workload-dependent increases in NADH:NAD+ in intramyocardial arterial myocytes. Intracellular NAD(P)(H) redox ratios reflecting elevated oxygen demand potentiate native coronary Kv1 activity in a Kvβ2-dependent manner. Ablation of Kvβ2 catalysis suppresses redox-dependent increases in Kv1 activity, vasodilation, and the relationship between cardiac workload and myocardial blood flow. Collectively, this work suggests that the pyridine nucleotide sensitivity and enzymatic activity of Kvβ2 controls coronary vasoreactivity and myocardial blood flow during metabolic stress.

Metformin, phenformin, and galegine inhibit complex IV activity and reduce glycerol-derived gluconeogenesis.

SignificanceMetformin is the most commonly prescribed drug for the treatment of type 2 diabetes mellitus, yet the mechanism by which it lowers plasma glucose concentrations has remained elusive. Most studies to date have attributed metformin's glucose-lowering effects to inhibition of complex I activity. Contrary to this hypothesis, we show that inhibition of complex I activity invitro and invivo does not reduce plasma glucose concentrations or inhibit hepatic gluconeogenesis. We go on to show that metformin, and the related guanides/biguanides, phenformin and galegine, inhibit complex IV activity at clinically relevant concentrations, which, in turn, results in inhibition of glycerol-3-phosphate dehydrogenase activity, increased cytosolic redox, and selective inhibition of glycerol-derived hepatic gluconeogenesis both invitro and invivo.