Metabolic Responses to Redox Stress in Vascular Cells.

Significance: Redox stress underlies numerous vascular disease mechanisms. Metabolic adaptability is essential for vascular cells to preserve energy and redox homeostasis. Recent Advances: Single-cell technologies and multiomic studies demonstrate significant metabolic heterogeneity among vascular cells in health and disease. Increasing evidence shows that reductive or oxidative stress can induce metabolic reprogramming of vascular cells. A recent example is intracellular L-2-hydroxyglutarate accumulation in response to hypoxic reductive stress, which attenuates the glucose flux through glycolysis and mitochondrial respiration in pulmonary vascular cells and provides protection against further reductive stress. Critical Issues: Regulation of cellular redox homeostasis is highly compartmentalized and complex. Vascular cells rely on multiple metabolic pathways, but the precise connectivity among these pathways and their regulatory mechanisms is only partially defined. There is also a critical need to understand better the cross-regulatory mechanisms between the redox system and metabolic pathways as perturbations in either systems or their cross talk can be detrimental. Future Directions: Future studies are needed to define further how multiple metabolic pathways are wired in vascular cells individually and as a network of closely intertwined processes given that a perturbation in one metabolic compartment often affects others. There also needs to be a comprehensive understanding of how different types of redox perturbations are sensed by and regulate different cellular metabolic pathways with specific attention to subcellular compartmentalization. Lastly, integration of dynamic changes occurring in multiple metabolic pathways and their cross talk with the redox system is an important goal in this multiomics era.

Exercise mitigates reductive stress-induced cardiac remodeling in mice.

The endoplasmic reticulum (ER) regulates protein folding and maintains proteostasis in cells. We observed that the ER transcriptome is impaired during chronic reductive stress (RS) in cardiomyocytes. Here, we hypothesized that a prolonged moderate treadmill exercise mitigates the RS-induced ER dysfunction and cardiac remodeling in cardiac-specific constitutively active Nrf2 mice (CaNrf2-TG). RNA sequencing showed notable alterations in the ER transcriptome of TG hearts at 4, 12, and 24 weeks (16, 28, and 35 genes, respectively). Notably, the downregulation of ER genes was significant at 12 weeks, and further pronounced at 24 weeks, at which the cardiac pathology is evident. We also observed increased levels of ubiquitinated proteins in CaNrf2-TG hearts across all ages, along with VCP, a marker of ERAD function, at 24 weeks. These findings indicate that constitutive Nrf2 activation and RS impair protein-folding activity and augments ERAD function over time. Exercise intervention for 20 weeks (beginning at 6 weeks of age), reduced cardiomyocyte hypertrophy (from 448 µm2 to 280 µm2) in TG mice, through adaptive remodeling, and preserved the cardiac function. However, while exercise did not influence antioxidants or ER stress protein levels, it significantly improved ERAD function and autophagy flux (LC-I to LC-II) in the TG-EXE hearts. Collectively, our findings underscore the prophylactic potential of exercise in mitigating RS-associated pathology, highlighting its essential role in maintaining cellular proteostasis through ER-independent mechanisms.

Multi-omic Analysis of Human B-cell Activation Reveals a Key Lysosomal BCAT1 Role in mTOR Hyperactivation by B-cell receptor and TLR9.

B-lymphocytes play major adaptive immune roles, producing antibody and driving T-cell responses. However, how immunometabolism networks support B-cell activation and differentiation in response to distinct receptor stimuli remains incompletely understood. To gain insights, we systematically investigated acute primary human B-cell transcriptional, translational and metabolomic responses to B-cell receptor (BCR), Toll-like receptor 9 (TLR9), CD40-ligand (CD40L), interleukin-4 (IL4) or combinations thereof. T-independent BCR/TLR9 co-stimulation, which drives malignant and autoimmune B-cell states, jointly induced PD-L1 plasma membrane expression, supported by NAD metabolism and oxidative phosphorylation. BCR/TLR9 also highly induced the transaminase BCAT1, which localized to lysosomal membranes to support branched chain amino acid synthesis and mTORC1 hyperactivation. BCAT1 inhibition blunted BCR/TLR9, but not CD40L/IL4-triggered B-cell proliferation, IL10 expression and BCR/TLR pathway-driven lymphoma xenograft outgrowth. These results provide a valuable resource, reveal receptor-mediated immunometabolism remodeling to support key B-cell phenotypes including PD-L1 checkpoint signaling, and identify BCAT1 as a novel B-cell therapeutic target.

Transcriptional and biochemical changes in mouse liver following exposure to a metal/drug cocktail. Attenuating effect of a selenium-enriched diet.

Real-life pollution usually involves simultaneous co-exposure to different chemicals. Metals and drugs are frequently and abundantly released into the environment, where they interact and bioaccumulate. Few studies analyze potential interactions between metals and pharmaceuticals in these mixtures, although their joint effects cannot be inferred from their individual properties. We have previously demonstrated that the mixture (PC) of the metals Cd and Hg, the metalloid As and the pharmaceuticals diclofenac (DCF) and flumequine (FLQ) impairs hepatic proteostasis. To gain a deeper vision of how PC affects mouse liver homeostasis, we evaluated here the effects of PC exposure upon some biochemical and morphometric parameters, and on the transcriptional profiles of selected group of genes. We found that exposure to PC caused oxidative damage that exceeded the antioxidant capacity of cells. The excessive oxidative stress response resulted in an overabundance of reducing equivalents, which hindered the metabolism and transport of metabolites, including cholesterol and bile acids, between organs. These processes have been linked to metabolic and inflammatory disorders, cancer, and neurodegenerative diseases. Therefore, our findings suggest that unintended exposure to mixtures of environmental pollutants may underlie the etiology of many human diseases. Fortunately, we also found that a diet enriched with selenium mitigated the harmful effects of this combination of toxicants.

An essential role for EROS in redox-dependent endothelial signal transduction.

The chaperone protein EROS ("Essential for Reactive Oxygen Species") was recently discovered in phagocytes. EROS was shown to regulate the abundance of the ROS-producing enzyme NADPH oxidase isoform 2 (NOX2) and to control ROS-mediated cell killing. Reactive oxygen species are important not only in immune surveillance, but also modulate physiological signaling responses in multiple tissues. The roles of EROS have not been previously explored in the context of oxidant-modulated cell signaling. Here we show that EROS plays a key role in ROS-dependent signal transduction in vascular endothelial cells. We used siRNA-mediated knockdown and developed CRISPR/Cas9 knockout of EROS in human umbilical vein endothelial cells (HUVEC), both of which cause a significant decrease in the abundance of NOX2 protein, associated with a marked decrease in RAC1, a small G protein that activates NOX2. Loss of EROS also attenuates receptor-mediated hydrogen peroxide (H2O2) and Ca2+ signaling, disrupts cytoskeleton organization, decreases cell migration, and promotes cellular senescence. EROS knockdown blocks agonist-modulated eNOS phosphorylation and nitric oxide (NO●) generation. These effects of EROS knockdown are strikingly similar to the alterations in endothelial cell responses that we previously observed following RAC1 knockdown. Proteomic analyses following EROS or RAC1 knockdown in endothelial cells showed that reduced abundance of these two distinct proteins led to largely overlapping effects on endothelial biological processes, including oxidoreductase, protein phosphorylation, and endothelial nitric oxide synthase (eNOS) pathways. These studies demonstrate that EROS plays a central role in oxidant-modulated endothelial cell signaling by modulating NOX2 and RAC1.

NAD(P)H-quinone oxidoreductase 1 induces complicated effects on mitochondrial dysfunction and ferroptosis in an expression level-dependent manner.

NAD(P)H-quinone oxidoreductase 1 (NQO1) is an essential redox enzyme responsible for redox balance and energy metabolism. Despite of its importance, the brain contains high capacity of polyunsaturated fatty acids and maintains low levels of NQO1 expression. In this study, we examined how levels of NQO1 expression affects cell survival in response to toxic insults causing mitochondrial dysfunction and ferroptosis, and whether NQO1 has a potential as a biomarker in different stressed conditions. Following treatment with rotenone, overexpressed NQO1 in SH-SY5Y cells improved cell survival by reducing mitochondrial reductive stress via increased NAD+ supply without mitochondrial biogenesis. However, NQO1 overexpression boosted lipid peroxidation following treatment with RSL3 and erastin. A lipid droplet staining assay showed increased lipid droplets in cells overexpressing NQO1. In contrast, NQO1 knockdown protected cells against ferroptosis by increasing GPX4, xCT, and the GSH/GSSG system. Also, NQO1 knockdown showed lower iron contents and lipid droplets than non-transfectants and cells overexpressing NQO1, even though it could not attenuate cell death when exposed to rotenone. In summary, our study suggests that different NQO1 levels may have advantages and disadvantages depending on the surrounding environments. Thus, regulating NQO1 expression could be a potential supplementary tool when treating neuronal diseases.

Cullin 3 RING E3 ligase inactivation causes NRF2-dependent NADH reductive stress, hepatic lipodystrophy, and systemic insulin resistance.

Cullin RING E3 ligases (CRL) have emerged as key regulators of disease-modifying pathways and therapeutic targets. Cullin3 (Cul3)-containing CRL (CRL3) has been implicated in regulating hepatic insulin and oxidative stress signaling. However, CRL3 function in liver pathophysiology is poorly defined. Here, we report that hepatocyte Cul3 knockout results in rapid resolution of steatosis in obese mice. However, the remarkable resistance of hepatocyte Cul3 knockout mice to developing steatosis does not lead to overall metabolic improvement but causes systemic metabolic disturbances. Liver transcriptomics analysis identifies that CRL3 inactivation causes persistent activation of the nuclear factor erythroid 2-related factor 2 (NRF2) antioxidant defense pathway, which also reprograms the lipid transcriptional network to prevent TG storage. Furthermore, global metabolomics reveals that NRF2 activation induces numerous NAD+-consuming aldehyde dehydrogenases to increase the cellular NADH/NAD+ ratio, a redox imbalance termed NADH reductive stress that inhibits the glycolysis-citrate-lipogenesis axis in Cul3 knockout livers. As a result, this NRF2-induced cellular lipid storage defect promotes hepatic ceramide accumulation, elevates circulating fatty acids, and worsens systemic insulin resistance in a vicious cycle. Hepatic lipid accumulation is restored, and liver injury and hyperglycemia are attenuated when NRF2 activation and NADH reductive stress are abolished in hepatocyte Cul3/Nrf2 double-knockout mice. The resistance to hepatic steatosis, hyperglycemia, and NADH reductive stress are observed in hepatocyte Keap1 knockout mice with NRF2 activation. In summary, our study defines a critical role of CRL3 in hepatic metabolic regulation and demonstrates that the CRL3 downstream NRF2 overactivation causes hepatic metabolic maladaptation to obesity and insulin resistance.

A recurrent missense variant in the E3 ubiquitin ligase substrate recognition subunit FEM1B causes a rare syndromic neurodevelopmental disorder.

PURPOSE: Fem1 homolog B (FEM1B) acts as a substrate recognition subunit for ubiquitin ligase complexes belonging to the CULLIN 2-based E3 family. Several biological functions have been proposed for FEM1B, including a structurally resolved function as a sensor for redox cell status by controlling mitochondrial activity, but its implication in human disease remains elusive. METHODS: To understand the involvement of FEM1B in human disease, we made use of Matchmaker exchange platforms to identify individuals with de novo variants in FEM1B and performed their clinical evaluation. We performed functional validation using primary neuronal cultures and in utero electroporation assays, as well as experiments on patient's cells. RESULTS: Five individuals with a recurrent de novo missense variant in FEM1B were identified: NM_015322.5:c.377G>A NP_056137.1:p.(Arg126Gln) (FEM1BR126Q). Affected individuals shared a severe neurodevelopmental disorder with behavioral phenotypes and a variable set of malformations, including brain anomalies, clubfeet, skeletal abnormalities, and facial dysmorphism. Overexpression of the FEM1BR126Q variant but not FEM1B wild-type protein, during mouse brain development, resulted in delayed neuronal migration of the target cells. In addition, the individuals' cells exhibited signs of oxidative stress and induction of type I interferon signaling. CONCLUSION: Overall, our data indicate that p.(Arg126Gln) induces aberrant FEM1B activation, resulting in a gain-of-function mechanism associated with a severe syndromic developmental disorder in humans.

Hypoxic adaptation of mitochondria and its impact on tumor cell function.

Mitochondria are the major sink for oxygen in the cell, consuming it during ATP production. Therefore, when environmental oxygen levels drop in the tumor, significant adaptation is required. Mitochondrial activity is also a major producer of biosynthetic precursors and a regulator of cellular oxidative and reductive balance. Because of the complex biochemistry, mitochondrial adaptation to hypoxia occurs through multiple mechanisms and has significant impact on other cellular processes such as macromolecule synthesis and gene regulation. In tumor hypoxia, mitochondria shift their location in the cell and accelerate the fission and quality control pathways. Hypoxic mitochondria also undergo significant changes to fundamental metabolic pathways of carbon metabolism and electron transport. These metabolic changes further impact the nuclear epigenome because mitochondrial metabolites are used as enzymatic substrates for modifying chromatin. This coordinated response delivers physiological flexibility and increased tumor cell robustness during the environmental stress of low oxygen.

NADP+/NADPH in Metabolism and its Relation to Cardiovascular Pathologies.

The nicotinamide adenine dinucleotide phosphate (NADP+/NADPH) redox couple serves as a substrate or cofactor for many enzymes to maintain cellular redox homeostasis as well as to regulate biosynthetic metabolism. The deficiency or imbalance of NADP+/NADPH redox couple is strongly associated with cardiovascular-related pathologies. An imbalance in the NADP+/NADPH ratio can lead to either oxidative or reductive stress. Reductive stress complicates the cellular redox environment and provides new insights into the cellular redox state. Newly discovered biosynthetic enzymes and developed genetically encoded biosensors provide technical support for studying how cells maintain a compartmentalized NADP(H) pool. NADP(H) plays an important role in cardiovascular pathologies. However, whether NADP(H) is injurious or protective in these diseases is uncertain, as either deficiency or excess NADP(H) levels can lead to imbalances in cellular redox state and metabolic homeostasis, resulting in energy stress, redox stress, and ultimately disease state. Additional study of the replicative regulatory network of NADP(H) metabolism in different compartments, and the mechanisms by which NADP(H) regulates redox state and metabolism under normal and pathological conditions, will develop the targeted and novel therapies based on NADP(H) metabolism.

Carbon-Coated Iron Oxide Nanoparticles Promote Reductive Stress-Mediated Cytotoxic Autophagy in Drug-Induced Senescent Breast Cancer Cells.

The surface modification of magnetite nanoparticles (Fe3O4 NPs) is a promising approach to obtaining biocompatible and multifunctional nanoplatforms with numerous applications in biomedicine, for example, to fight cancer. However, little is known about the effects of Fe3O4 NP-associated reductive stress against cancer cells, especially against chemotherapy-induced drug-resistant senescent cancer cells. In the present study, Fe3O4 NPs in situ coated by dextran (Fe3O4@Dex) and glucosamine-based amorphous carbon coating (Fe3O4@aC) with potent reductive activity were characterized and tested against drug-induced senescent breast cancer cells (Hs 578T, BT-20, MDA-MB-468, and MDA-MB-175-VII cells). Fe3O4@aC caused a decrease in reactive oxygen species (ROS) production and an increase in the levels of antioxidant proteins FOXO3a, SOD1, and GPX4 that was accompanied by elevated levels of cell cycle inhibitors (p21, p27, and p57), proinflammatory (NFκB, IL-6, and IL-8) and autophagic (BECN1, LC3B) markers, nucleolar stress, and subsequent apoptotic cell death in etoposide-stimulated senescent breast cancer cells. Fe3O4@aC also promoted reductive stress-mediated cytotoxicity in nonsenescent breast cancer cells. We postulate that Fe3O4 NPs, in addition to their well-established hyperthermia and oxidative stress-mediated anticancer effects, can also be considered, if modified using amorphous carbon coating with reductive activity, as stimulators of reductive stress and cytotoxic effects in both senescent and nonsenescent breast cancer cells with different gene mutation statuses.

Cysteine induces mitochondrial reductive stress in glioblastoma through hydrogen peroxide production.

Glucose and amino acid metabolism are critical for glioblastoma (GBM) growth, but little is known about the specific metabolic alterations in GBM that are targetable with FDA-approved compounds. To investigate tumor metabolism signatures unique to GBM, we interrogated The Cancer Genome Atlas for alterations in glucose and amino acid signatures in GBM relative to other human cancers and found that GBM exhibits the highest levels of cysteine and methionine pathway gene expression of 32 human cancers. Treatment of patient-derived GBM cells with the FDA-approved single cysteine compound N-acetylcysteine (NAC) reduced GBM cell growth and mitochondrial oxygen consumption, which was worsened by glucose starvation. Normal brain cells and other cancer cells showed no response to NAC. Mechanistic experiments revealed that cysteine compounds induce rapid mitochondrial H2O2 production and reductive stress in GBM cells, an effect blocked by oxidized glutathione, thioredoxin, and redox enzyme overexpression. From analysis of the clinical proteomic tumor analysis consortium (CPTAC) database, we found that GBM cells exhibit lower expression of mitochondrial redox enzymes than four other cancers whose proteomic data are available in CPTAC. Knockdown of mitochondrial thioredoxin-2 in lung cancer cells induced NAC susceptibility, indicating the importance of mitochondrial redox enzyme expression in mitigating reductive stress. Intraperitoneal treatment of mice bearing orthotopic GBM xenografts with a two-cysteine peptide induced H2O2 in brain tumors in vivo. These findings indicate that GBM is uniquely susceptible to NAC-driven reductive stress and could synergize with glucose-lowering treatments for GBM.

Redox-associated changes in healthy individuals at risk of Alzheimer's disease. A ten-year follow-up study.

Carrying an allele 4 of the apolipoprotein E (ApoE) is the best-established genetic risk factor to develop Alzheimer's disease (AD). Fifty percent of ApoE4/4 individuals develop the disease at 70 years of age. ApoE3/4 carriers have a lower risk of developing the disease, still 50% of them suffer AD at around 80 years. In a previous study we showed that healthy young individuals, who had a parent with AD and were carriers of at least one ApoE4 allele displayed reductive stress. This was evidenced as a decrease in oxidative markers, such as oxidized glutathione, p-p38, and NADP+/NADPH ratio, and an increase of antioxidant enzymes, such as glutathione peroxidase (Gpx1) and both the catalytic and regulatory subunits of glutamyl-cysteinyl (GCLM and GCLC). Moreover, we found an increase in stress-related proteins involved in tau physiopathology. Now, 10 years later, we have conducted a follow-up study measuring the same parameters in the same cohort. Our results show that reductive stress has reversed, as we could now observe an increase in lipid peroxidation and in the oxidation of glutathione along with a decrease in the expression of Gpx1 and SOD1 antioxidant enzymes in ApoE4 carriers. Furthermore, we found an increase in plasma levels of IL1ß levels and in PKR (eukaryotic translation initiation factor 2 alpha kinase 2) gene expression in isolated lymphocytes. Altogether, our results suggest that, in the continuum of Alzheimer's disease, people at risk of developing the disease go through different redox phases, from stablished reductive stress to oxidative stress.

Nanomaterials-Induced Redox Imbalance: Challenged and Opportunities for Nanomaterials in Cancer Therapy.

Cancer cells typically display redox imbalance compared with normal cells due to increased metabolic rate, accumulated mitochondrial dysfunction, elevated cell signaling, and accelerated peroxisomal activities. This redox imbalance may regulate gene expression, alter protein stability, and modulate existing cellular programs, resulting in inefficient treatment modalities. Therapeutic strategies targeting intra- or extracellular redox states of cancer cells at varying state of progression may trigger programmed cell death if exceeded a certain threshold, enabling therapeutic selectivity and overcoming cancer resistance to radiotherapy and chemotherapy. Nanotechnology provides new opportunities for modulating redox state in cancer cells due to their excellent designability and high reactivity. Various nanomaterials are widely researched to enhance highly reactive substances (free radicals) production, disrupt the endogenous antioxidant defense systems, or both. Here, the physiological features of redox imbalance in cancer cells are described and the challenges in modulating redox state in cancer cells are illustrated. Then, nanomaterials that regulate redox imbalance are classified and elaborated upon based on their ability to target redox regulations. Finally, the future perspectives in this field are proposed. It is hoped this review provides guidance for the design of nanomaterials-based approaches involving modulating intra- or extracellular redox states for cancer therapy, especially for cancers resistant to radiotherapy or chemotherapy, etc.

Addition of Vitamin C Mitigates the Loss of Antioxidant Capacity, Vitality and DNA Integrity in Cryopreserved Human Semen Samples.

Cryopreservation of human spermatozoa is a necessity for males suffering from infertility who cannot produce fresh semen for insemination. However, current ART cryopreservation protocols are associated with losses of sperm motility, vitality and DNA integrity, which are thought to be linked to the induction of oxidative damage and the toxic properties of commercial cryoprotectants (CPAs). Preventing or mitigating these losses would be hugely beneficial to sperm survival during ART. Therefore, in this in vitro investigation, lipid peroxidation, production of reactive oxygen species, movement characteristics, antioxidant capacity, vitality, and DNA integrity were examined in semen samples both pre- and post-cryopreservation with CPA supplementation. The findings revealed a 50% reduction in antioxidant capacity with CPA addition, which was accompanied by significant increases in generation of reactive oxygen species and formation of lipid aldehydes. These changes were, in turn, correlated with reductions in sperm viability, motility and DNA integrity. Antioxidant supplementation generated bell-shaped dose-response curves with both resveratrol and vitamin C, emphasising the vulnerability of these cells to both oxidative and reductive stress. At the optimal dose, vitamin C was able to significantly enhance vitality and reduce DNA damage recorded in cryopreserved human spermatozoa. An improvement in sperm motility did not reach statistical significance, possibly because additional pathophysiological mechanisms limit the potential effectiveness of antioxidants in rescuing this aspect of sperm function. The vulnerability of human spermatozoa to reductive stress and the complex nature of sperm cryoinjury will present major challenges in creating the next generation of cryoprotective media.

Reductive stress in cancer: coming out of the shadows.

Redox imbalance is defined by disruption in oxidative and reductive pathways and has a central role in cancer initiation, development, and treatment. Although redox imbalance has traditionally been characterized by high levels of oxidative stress, emerging evidence suggests that an overly reductive environment is just as detrimental to cancer proliferation. Reductive stress is defined by heightened levels of antioxidants, including glutathione and elevated NADH, compared with oxidized NAD, which disrupts central biochemical pathways required for proliferation. With the advent of new technologies that measure and manipulate reductive stress, the sensors and drivers of this overlooked metabolic stress are beginning to be revealed. In certain genetically defined cancers, targeting reductive stress pathways may be an effective strategy. Redox-based pathways are gaining recognition as essential 'regulatory hubs,' and a broader understanding of reductive stress signaling promises not only to reveal new insights into metabolic homeostasis but also potentially to transform therapeutic options in cancer.

Adaptive changes in tumor cells in response to reductive stress.

Reductive stress is characterized by an excess of cellular electron donors and can be linked with various human pathologies including cancer. We developed melanoma cell lines resistant to reductive stress agents: rotenone (ROTR), n-acetyl-L-cysteine, (NACR), or dithiothreitol (DTTR). Resistant cells divided more rapidly and had intracellular homeostatic redox-couple ratios that were shifted towards the reduced state. Resistance caused alterations in general cell morphology, but only ROTR cells had significant changes in mitochondrial morphology with higher numbers that were more isolated, fragmented and swollen, with greater membrane depolarization and decreased numbers of networks. These changes were accompanied by lower basal oxygen consumption and maximal respiration rates. Whole cell flux analyses and mitochondrial function assays showed that NACR and DTTR preferentially utilized tricarboxylic acid (TCA) cycle intermediates, while ROTR used ketone body substrates such as D, L-ß-hydroxybutyric acid. NACR and DTTR cells had constitutively decreased levels of reactive oxygen species (ROS), although this was accompanied by activation of nuclear factor erythroid 2-related factor 2 (Nrf2), with concomitant increased expression of the downstream gene products such as glutathione S-transferase P (GSTP). Further adaptations included enhanced expression of endoplasmic reticulum proteins controlling the unfolded protein response (UPR). Although expression patterns of these UPR proteins were distinct between the resistant cells, a trend implied that resistance to reductive stress is accompanied by a constitutively increased UPR phenotype in each line. Overall, tumor cells, although tolerant of oxidative stress, can adapt their energy and survival mechanisms in lethal reductive stress conditions.

Oxidative versus Reductive Stress in Breast Cancer Development and Cellular Mechanism of Alleviation: A Current Perspective with Anti-breast Cancer Drug Resistance.

Redox homeostasis is essential for keeping our bodies healthy, but it also helps breast cancer cells grow, stay alive, and resist treatment. Changes in the redox balance and problems with redox signaling can make breast cancer cells grow and spread and make them resistant to chemotherapy and radiation therapy. Reactive oxygen species/reactive nitrogen species (ROS/RNS) generation and the oxidant defense system are out of equilibrium, which causes oxidative stress. Many studies have shown that oxidative stress can affect the start and spread of cancer by interfering with redox (reduction-oxidation) signaling and damaging molecules. The oxidation of invariant cysteine residues in FNIP1 is reversed by reductive stress, which is brought on by protracted antioxidant signaling or mitochondrial inactivity. This permits CUL2FEM1B to recognize its intended target. After the proteasome breaks down FNIP1, mitochondrial function is restored to keep redox balance and cell integrity. Reductive stress is caused by unchecked amplification of antioxidant signaling, and changes in metabolic pathways are a big part of breast tumors' growth. Also, redox reactions make pathways like PI3K, PKC, and protein kinases of the MAPK cascade work better. Kinases and phosphatases control the phosphorylation status of transcription factors like APE1/Ref-1, HIF-1, AP-1, Nrf2, NF-B, p53, FOXO, STAT, and - catenin. Also, how well anti-breast cancer drugs, especially those that cause cytotoxicity by making ROS, treat patients depends on how well the elements that support a cell's redox environment work together. Even though chemotherapy aims to kill cancer cells, which it does by making ROS, this can lead to drug resistance in the long run. The development of novel therapeutic approaches for treating breast cancer will be facilitated by a better understanding of the reductive stress and metabolic pathways in tumor microenvironments.

ChREBP is activated by reductive stress and mediates GCKR-associated metabolic traits.

Common genetic variants in glucokinase regulator (GCKR), which encodes GKRP, a regulator of hepatic glucokinase (GCK), influence multiple metabolic traits in genome-wide association studies (GWASs), making GCKR one of the most pleiotropic GWAS loci in the genome. It is unclear why. Prior work has demonstrated that GCKR influences the hepatic cytosolic NADH/NAD+ ratio, also referred to as reductive stress. Here, we demonstrate that reductive stress is sufficient to activate the transcription factor ChREBP and necessary for its activation by the GKRP-GCK interaction, glucose, and ethanol. We show that hepatic reductive stress induces GCKR GWAS traits such as increased hepatic fat, circulating FGF21, and circulating acylglycerol species, which are also influenced by ChREBP. We define the transcriptional signature of hepatic reductive stress and show its upregulation in fatty liver disease and downregulation after bariatric surgery in humans. These findings highlight how a GCKR-reductive stress-ChREBP axis influences multiple human metabolic traits.

The PNPLA3 I148M variant increases ketogenesis and decreases hepatic de novo lipogenesis and mitochondrial function in humans.

The PNPLA3 I148M variant is the major genetic risk factor for all stages of fatty liver disease, but the underlying pathophysiology remains unclear. We studied the effect of this variant on hepatic metabolism in homozygous carriers and non-carriers under multiple physiological conditions with state-of-the-art stable isotope techniques. After an overnight fast, carriers had higher plasma ß-hydroxybutyrate concentrations and lower hepatic de novo lipogenesis (DNL) compared to non-carriers. After a mixed meal, fatty acids were channeled toward ketogenesis in carriers, which was associated with an increase in hepatic mitochondrial redox state. During a ketogenic diet, carriers manifested increased rates of intrahepatic lipolysis, increased plasma ß-hydroxybutyrate concentrations, and decreased rates of hepatic mitochondrial citrate synthase flux. These studies demonstrate that homozygous PNPLA3 I148M carriers have hepatic mitochondrial dysfunction leading to reduced DNL and channeling of carbons to ketogenesis. These findings have implications for understanding why the PNPLA3 variant predisposes to progressive liver disease.