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Glucose-6-phosphate dehydrogenase (G6PD) deficiency affects 500 million people globally, impacting red blood cell (RBC) antioxidant pathways and increasing susceptibility to hemolysis under oxidative stress. Despite the systemic generation of reactive oxygen species during exercise, the effects of exercise on individuals with G6PD deficiency remain poorly understood This study utilized humanized mouse models expressing the G6PD Mediterranean variant (S188F, with 10% enzymatic activity) to investigate exercise performance and molecular outcomes. Surprisingly, despite decreased enzyme activity, G6PD-deficient mice have faster critical speed (CS) compared to mice expressing human canonical G6PD. Post-exercise, deficient mice did not exhibit differences in RBC morphology or hemolysis, but had improved cardiac function, including cardiac output, stroke volume, sarcomere length and mitochondrial content. Proteomics analyses of cardiac and skeletal muscles (gastrocnemius, soleus) from G6PD deficient compared to sufficient mice revealed improvements in mitochondrial function and increased protein turnover via ubiquitination, especially for mitochondrial and structural myofibrillar proteins. Mass spectrometry-based metabolomics revealed alterations in energy metabolism and fatty acid oxidation. These findings challenge the traditional assumptions regarding hemolytic risk during exercise in G6PD deficiency, suggesting a potential metabolic advantage in exercise performance for individuals carrying non-canonical G6PD variants., (Copyright © 2024 American Society of Hematology.)

Preclinical rodent models are essential research tools for improving understanding of physiological aging processes in humans. However, the translatability of findings obtained leveraging rodent models to humans is limited, likely due in part to differences in macronutrient composition of the diets. Here, we investigated the impact of a 3-month diet intervention in old male C57BL/6JN mice in which the macronutrient composition was aligned with that of a midlife/older adult in the United States, compared to a traditional rodent diet, and assessed various phenotypes that are typically altered with aging. Following the diet period, mice fed the human macronutrient-matched diet had greater quadricep and subcutaneous adipose and visceral adipose tissue masses compared to animals fed a traditional mouse diet. Frailty, assessed using a clinical frailty index, was lower, while grip strength was higher in mice fed the human-matched diet. Circulating metabolite and inflammatory cytokine profiles were altered in mice fed the human-matched diet. Notably, mortality rate (assessed in animals who died or were euthanized per veterinary recommendation before the pre-determined end of study euthanasia), tended to be lower in mice fed the human-matched diet. The present study underscores the importance of diet in rodent studies of aging, as differences in macronutrient composition can affect various physiological processes in old mice that are relevant to aging research., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Senescent neural progenitor cells have been identified in brain lesions of people with progressive multiple sclerosis (PMS). However, their role in disease pathobiology and contribution to the lesion environment remains unclear. By establishing directly induced neural stem/progenitor cell (iNSC) lines from PMS patient fibroblasts, we studied their senescent phenotype in vitro. Senescence was strongly associated with inflammatory signaling, hypermetabolism, and the senescence-associated secretory phenotype (SASP). PMS-derived iNSCs displayed increased glucose-dependent fatty acid and cholesterol synthesis, which resulted in the accumulation of lipid droplets. A 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A (CoA) reductase (HMGCR)-mediated lipogenic state was found to induce a SASP in PMS iNSCs via cholesterol-dependent transcription factors. SASP from PMS iNSC lines induced neurotoxicity in mature neurons, and treatment with the HMGCR inhibitor simvastatin altered the PMS iNSC SASP, promoting cytoprotective qualities and reducing neurotoxicity. Our findings suggest a disease-associated, cholesterol-related, hypermetabolic phenotype of PMS iNSCs that leads to neurotoxic signaling and is rescuable pharmacologically., Competing Interests: Declaration of interests S.P. is founder, CSO, and shareholder (>5%) of CITC Ltd. A.D. is a founder of Omix Technologies Inc, Altis Biosciences LLC, and an advisory board member for Hemanext Inc and Macopharma Inc., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Copper (Cu) is a cofactor of cytochrome c oxidase (CuCOX), indispensable for aerobic mitochondrial respiration. This study reveals that advanced clear cell renal cell carcinomas (ccRCCs) accumulate Cu, allocating it to CuCOX. Using a range of orthogonal approaches, including metabolomics, lipidomics, isotope-labeled glucose and glutamine flux analysis, and transcriptomics across tumor samples, cell lines, xenografts, and PDX models, combined with genetic and pharmacological interventions, we explored Cu's role in ccRCC. Elevated Cu levels stimulate CuCOX biogenesis, providing bioenergetic and biosynthetic benefits that promote tumor growth. This effect is complemented by glucose-dependent glutathione production, which facilitates detoxification and mitigates Cu-H2O2 toxicity. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics reveal increased oxidative metabolism, altered glutathione and Cu metabolism, and diminished HIF activity during ccRCC progression. Thus, Cu drives an integrated oncogenic remodeling of bioenergetics, biosynthesis, and redox homeostasis, fueling ccRCC growth, which can be targeted for new therapeutic approaches.

Increasing global life expectancy motivates investigations of molecular mechanisms of aging and age-related diseases. This study examines age-associated changes in red blood cells (RBCs), the most numerous host cell in humans. Four cohorts, including healthy individuals and patients with sickle cell disease, were analyzed to define age-dependent changes in RBC metabolism. Over 15,700 specimens from 13,757 humans were examined, a major expansion over previous studies of RBCs in aging. Multi-omics approaches identified chronological age-related alterations in the arginine pathway with increased arginine utilization in RBCs from older individuals. These changes were consistent across healthy and sickle cell disease cohorts and were influenced by genetic variation, sex, and body mass index. Integrating multi-omics data and metabolite quantitative trait loci (mQTL) in humans and 525 diversity outbred mice functionally linked metabolism of arginine during RBC storage to increased vesiculation-a hallmark of RBC aging-and lower post-transfusion hemoglobin increments. Thus, arginine metabolism is a biomarker of RBC and organismal aging, suggesting potential new targets for addressing sequelae of aging., (© 2024 The Author(s). Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

2-Aminoethanethiol dioxygenase (ADO) is a thiol dioxygenase that sulfinylates cysteamine and amino-terminal cysteines in polypeptides. The pathophysiological roles of ADO remain largely unknown. Here, we demonstrate that ADO expression represents a vulnerability in cancer cells, as ADO depletion led to loss of proliferative capacity and survival in cancer cells and reduced xenograft growth. In contrast, generation of the ADO knockout mouse revealed high tolerance for ADO depletion in adult tissues. To understand the mechanism underlying ADO's essentiality in cancer cells, we characterized the cell proteome and metabolome following depletion of ADO. This revealed that ADO depletion leads to toxic levels of polyamines which can be driven by ADO's substrate cysteamine. Polyamine accumulation in turn stimulated expression of proline dehydrogenase (PRODH) which resulted in mitochondrial hyperactivity and ROS production, culminating in cell toxicity. This work identifies ADO as a unique vulnerability in cancer cells, due to its essential role in maintenance of redox homeostasis through restraining polyamine levels and proline catabolism.

Significance: TDO2 is more highly expressed than the nonhomologous TRP-catabolizing enzyme IDO1 in TNBC. We find that TDO2 knockdown can lead to a compensatory increase in IDO1. Therefore, we tested a newly developed TDO2/IDO1 dual inhibitor and found that it decreases TRP catabolism, anchorage-independent survival, and invasive capacity., (©2024 The Authors; Published by the American Association for Cancer Research.)

High-altitude (HA) hypoxia lowers uterine artery (UtA) blood flow during pregnancy and birth weight. Adenosine monophosphate kinase (AMPK) activation has selective, uteroplacental vasodilator effects that lessen hypoxia-associated birth weight reductions. In this study, we determined the relationship between AMPK-pathway gene expression and metabolites in the maternal circulation during HA pregnancy as well as with the maintenance of UtA blood flow and birth weight at HA. Residents at HA (2,793 m) versus low altitude (LA; 1,640 m) had smaller UtA diameters at weeks 20 and 34 , lower UtA blood flow at week 20 , and lower birth weight babies. At week 34 , women residing at HA versus women residing at LA had decreased expression of upstream and downstream AMPK-pathway genes. Expression of the α 1 -AMPK catalytic subunit, PRKAA1 , correlated positively with UtA diameter and blood flow at weeks 20 (HA) and 34 (LA). Downstream AMPK-pathway gene expression positively correlated with week 20 fetal biometry at both altitudes and with UtA diameter and birth weight at LA. Reduced gene expression of AMPK activators and downstream targets in women residing at HA versus women residing at LA, together with positive correlations between PRKAA1 gene expression, UtA diameter, and blood flow suggest that greater sensitivity to AMPK activation at midgestation at HA may help offset later depressant effects of hypoxia on fetal growth. NEW & NOTEWORTHY Fetal growth restriction (FGR) is increased and uterine artery (UtA) blood flow is lower at high altitudes (HA) but not all HA pregnancies have FGR. Here we show that greater UtA diameter and blood flow at week 20 are positively correlated with higher expression of the gene encoding the α 1 -catalytic subunit of AMP protein kinase, PRKAA1 , suggesting that increased AMPK activation may help to prevent the detrimental effects of chronic hypoxia on fetal growth.

Introduction: Females suffer greater lifetime risk of stroke and greater morbidity and mortality from stroke compared with males. This study's objective was to identify differences in metabolomic profiling of females and males with stroke and which differences were associated with neurological outcome. Methods: Females and males with acute ischemic stroke enrolled in the Emergency Medicine Specimen Bank at a comprehensive stroke center provided whole blood samples upon arrival for mass spectrometry-based metabolomics. We used descriptive statistics to characterize the cohort. A linear regression model was fit for individual metabolites to determine differences in relative abundance between males and females while controlling for covariates (age, race/ethnicity, postmenopausal status, cardiovascular risk factors, depression, time between sample collection and last known well, and initial National Institutes of Health Stroke Scale [NIHSS] score). For each differentially expressed metabolite, a linear regression model was fit to determine the association between the metabolite and NIHSS at 24 hours after admission while controlling for the covariates and acute treatments. Results: After adjusting for covariates, eight metabolites differed in females and males with a stroke. These included amino acids or their metabolites (proline and tryptophan), nucleotides (guanosine diphosphate [GDP], and inosine-3',5'-cyclic monophosphate), citrate, dehydroascorbate, choline, and acylcarnitine-(5-OH). GDP and dehydroascorbate were significantly associated with 24-hour NIHSS ( p = 0.0991). Conclusions: Few metabolites were differentially abundant in blood after a stroke when comparing females with males and controlling for confounders, but the interactions between biological sex and GDP, as well as biological sex and dehydroascorbate, were associated with 24-hour neurological function. This has important implications for future studies that evaluate the therapeutic potential of these metabolites in ischemic stroke.

Acute myeloid leukemia (AML) is a devastating disease initiated and maintained by a rare subset of cells called leukemia stem cells (LSCs). LSCs are responsible for driving disease relapse, making the development of new therapeutic strategies to target LSCs urgently needed. The use of mass spectrometry-based metabolomics profiling has enabled the discovery of unique and targetable metabolic properties in LSCs. However, we do not have a comprehensive understanding of metabolite differences between LSCs and their normal counterparts, hematopoietic stem and progenitor cells (HSPCs). In this study, we used an unbiased mass spectrometry-based metabolomics analysis to define differences in metabolites between primary human LSCs and HSPCs, which revealed that LSCs have a distinct metabolome. Spermidine was the most enriched metabolite in LSCs compared with HSPCs. Pharmacological reduction of spermidine concentrations decreased LSC function but spared normal HSPCs. Polyamine depletion also decreased leukemic burden in patient-derived xenografts. Mechanistically, spermidine depletion induced LSC myeloid differentiation by decreasing eIF5A-dependent protein synthesis, resulting in reduced expression of a select subset of proteins. KAT7, a histone acetyltransferase, was one of the top candidates identified to be down-regulated by spermidine depletion. Overexpression of KAT7 partially rescued polyamine depletion-induced decreased colony-forming ability, demonstrating that loss of KAT7 is an essential part of the mechanism by which spermidine depletion targets AML clonogenic potential. Together, we identified and mechanistically dissected a metabolic vulnerability of LSCs that has the potential to be rapidly translated into clinical trials to improve outcomes for patients with AML.

Primaquine and Tafenoquine are the only approved drugs that can achieve a radical cure for Plasmodium vivax malaria but are contraindicated in patients who are deficient in glucose 6-phosphate dehydrogenase (G6PDd) due to risk of severe hemolysis from reactive oxygen species generated by redox cycling of drug metabolites. 5-hydroxyprimaquine and its quinoneimine cause robust redox cycling in red blood cells (RBCs) but are so labile as to not be detected in blood or urine. Rather, the quinoneimine is rapidly converted into primaquine-5,6-orthoquinone (5,6-POQ) that is then excreted in the urine. The extent to which 5,6-POQ contributes to hemolysis remains unclear, although some have suggested that it is a minor toxin that should be used predominantly as a surrogate to infer levels of 5-hydroxyprimaquine. In this report, we describe a novel humanized mouse model of the G6PD Mediterranean variant (hG6PD Med- ) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PD ND In vitro challenge of RBCs with 5,6-POQ causes increased generation of superoxide and methemoglobin. Infusion of treated RBCs shows that 5,6-POQ selectively causes in vivo clearance of older hG6PD Med- RBCs. These findings support the hypothesis that 5,6-POQ directly induces hemolysis and challenges the notion that 5,6-POQ is an inactive metabolic waste product. Indeed, given the extreme lability of 5-hydroxyprimaquine and the relative stability of 5,6-POQ, these data raise the possibility that 5,6-POQ is a major hemolytic primaquine metabolite in vivo. SIGNIFICANCE STATEMENT: These findings demonstrate that 5,6-POQ, which has been considered an inert waste product of primaquine metabolism, directly induces ROS that cause clearance of older G6PDd RBCs. As 5,6-POQ is relatively stable compared with other active primaquine metabolites, these data support the hypothesis that 5,6-POQ is a major toxin in primaquine induced hemolysis. The findings herein also establish a new model of G6PDd and provide the first direct evidence, to our knowledge, that young G6PDd RBCs are resistant to primaquine-induced hemolysis., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Mature red blood cells (RBCs) lack mitochondria and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo or storage in blood banks. Here, we leveraged 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study to identify associations between end-of-storage levels of glycolytic metabolites and donor age, sex, and ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (detected in mature RBCs); hexokinase 1 (HK1); and ADP-ribosyl cyclase 1 and 2 (CD38/BST1). Gene-metabolite associations were validated in fresh and stored RBCs from 525 Diversity Outbred mice and via multi-omics characterization of 1,929 samples from 643 human RBC units during storage. ATP and hypoxanthine (HYPX) levels-and the genetic traits linked to them-were associated with hemolysis in vitro and in vivo, both in healthy autologous transfusion recipients and in 5,816 critically ill patients receiving heterologous transfusions, suggesting their potential as markers to improve transfusion outcomes., Competing Interests: Declaration of interests A.D’A., K.C.H., and T.N. are founders of Omix Technologies Inc. and Altis Biosciences LLC. A.D’A., S.L.S., and T.N. are Scientific Advisory Board (SAB) members for Hemanext Inc. A.D’A. is an SAB member for Macopharma Inc. S.L.S. is an SAB member for Alcor, Inc.; a consultant for Team Conveyer Intellectual Properties; executive director for Worldwide Initiative for Rh Disease Eradication (WIRhE); and CEO for Ferrous Wheel Consultants, LLC. J.C.Z. is a founder of Svalinn Therapeutics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The genetic landscape of cancer cells can lead to specific metabolic dependencies for tumor growth. Dietary interventions represent an attractive strategy to restrict the availability of key nutrients to tumors. In this study, we identified that growth of a subset of melanoma was severely restricted by a rationally designed combination therapy of a stearoyl-CoA desaturase (SCD) inhibitor with an isocaloric low-oleic acid diet. Despite its importance in oncogenesis, SCD underwent monoallelic codeletion along with PTEN on chromosome 10q in approximately 47.5% of melanoma, and the other SCD allele was methylated, resulting in very low-SCD expression. Although this SCD-deficient subset was refractory to SCD inhibitors, the subset of PTEN wild-type melanoma that retained SCD was sensitive. As dietary oleic acid could potentially blunt the effect of SCD inhibitors, a low oleic acid custom diet was combined with an SCD inhibitor. The combination reduced monounsaturated fatty acids and increased saturated fatty acids, inducing robust apoptosis and growth suppression and inhibiting lung metastasis with minimal toxicity in preclinical mouse models of PTEN wild-type melanoma. When combined with anti-PD1 immunotherapy, the SCD inhibitor improved T-cell functionality and further constrained melanoma growth in mice. Collectively, these results suggest that optimizing SCD inhibitors with diets low in oleic acid may offer a viable and efficacious therapeutic approach for improving melanoma treatment. Significance: Blockade of endogenous production of fatty acids essential for melanoma combined with restriction of dietary intake blocks tumor growth and enhances response to immunotherapy, providing a rational drug-diet treatment regimen for melanoma., (©2024 American Association for Cancer Research.)

Acute kidney injury (AKI) is a systemic disease that affects energy metabolism in various remote organs in murine models of ischemic AKI. However, AKI-mediated effects in the liver have not been comprehensively assessed. After inducing ischemic AKI in 8-10-week-old, male C57BL/6 mice, mass spectrometry metabolomics revealed that the liver had the most distinct phenotype 24 h after AKI versus 4 h and 7 days. Follow up studies with in vivo [ 13 C 6 ]-glucose tracing on liver and kidney 24 h after AKI revealed 4 major findings: (1) increased flux through glycolysis and the tricarboxylic (TCA) cycle in both kidney and liver; (2) depleted hepatic glutathione levels and its intermediates despite unchanged level of reactive oxygen species, suggesting glutathione consumption exceeds production due to systemic oxidative stress after AKI; (3) hepatic ATP depletion despite unchanged rate of mitochondrial respiration, suggesting increased ATP consumption relative to production; (4) increased hepatic and renal urea cycle intermediates suggesting hypercatabolism and upregulation of the urea cycle independent of impaired renal clearance of nitrogenous waste. Taken together, this is the first study to describe the hepatic metabolome after ischemic AKI in a murine model and demonstrates that there is significant liver-kidney crosstalk after AKI., (© 2024. The Author(s).)

Abstract: Recent large-scale multiomics studies suggest that genetic factors influence the chemical individuality of donated blood. To examine this concept, we performed metabolomics analyses of 643 blood units from volunteers who donated units of packed red blood cells (RBCs) on 2 separate occasions. These analyses identified carnitine metabolism as the most reproducible pathway across multiple donations from the same donor. We also measured l-carnitine and acyl-carnitines in 13 091 packed RBC units from donors in the Recipient Epidemiology and Donor Evaluation study. Genome-wide association studies against 879 000 polymorphisms identified critical genetic factors contributing to interdonor heterogeneity in end-of-storage carnitine levels, including common nonsynonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, and SLC16A9); carnitine synthesis (FLVCR1 and MTDH) and metabolism (CPT1A, CPT2, CRAT, and ACSS2), and carnitine-dependent repair of lipids oxidized by ALOX5. Significant associations between genetic polymorphisms on SLC22 transporters and carnitine pools in stored RBCs were validated in 525 Diversity Outbred mice. Donors carrying 2 alleles of the rs12210538 SLC22A16 single-nucleotide polymorphism exhibited the lowest l-carnitine levels, significant elevations of in vitro hemolysis, and the highest degree of vesiculation, accompanied by increases in lipid peroxidation markers. Separation of RBCs by age, via in vivo biotinylation in mice, and Percoll density gradients of human RBCs, showed age-dependent depletions of l-carnitine and acyl-carnitine pools, accompanied by progressive failure of the reacylation process after chemically induced membrane lipid damage. Supplementation of stored murine RBCs with l-carnitine boosted posttransfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality., (© 2024 American Society of Hematology. Published by Elsevier Inc. All rights are reserved, including those for text and data mining, AI training, and similar technologies.)

Declines in physiological function with aging are strongly linked to age-related diseases. Lifelong voluntary aerobic exercise (LVAE) preserves physiological function with aging, possibly by increasing cellular quality control processes, but the circulating molecular transducers mediating these processes are incompletely understood. The plasma metabolome may predict biological aging and is impacted by a single bout of aerobic exercise. Here, we conducted an ancillary analysis using plasma samples, and physiological function data, from previously reported studies of LVAE in male C57BL/6N mice randomized to LVAE (wheel running) or sedentary (SED) (n = 8-9/group) to determine if LVAE alters the plasma metabolome and whether these changes correlated with preservation of physiological function with LVAE. Physical function (grip strength, coordination, and endurance) was assessed at 3 and 18 months of age; vascular endothelial function and the plasma metabolome were assessed at 19 months. Physical function was preserved (%decline; mean ± SEM) with LVAE vs SED (all p < 0.05)-grip strength, 0.4 ± 1.7% vs 12 ± 4.0%; coordination, 10 ± 4% vs 73 ± 10%; endurance, 1 ± 15% vs 61 ± 5%. Vascular endothelial function with LVAE (88.2 ± 2.0%) was higher than SED (79.1 ± 2.5%; p = 0.03) and similar to the young controls (91.4 ± 2.9%). Fifteen metabolites were different with LVAE compared to SED (FDR < 0.05) and correlated with the preservation of physiological function. Plasma spermidine, a polyamine that increases cellular quality control (e.g., autophagy), correlated with all assessed physiological indices. Autophagy (LC3A/B abundance) was higher in LVAE skeletal muscle compared to SED (p < 0.01) and inversely correlated with plasma spermidine (r =  - 0.5297; p = 0.054). These findings provide novel insight into the circulating molecular transducers by which LVAE may preserve physiological function with aging., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Background: Red blood cell (RBC) storage promotes biochemical and morphological alterations, collectively referred to as storage lesions (SLs). Studies in humans have identified leukoreduction (LR) as a critical processing step that mitigates SLs. To date no study has evaluated the impact of LR on metabolic SLs in canine blood units using omics technologies., Objective: Compare the lipid and metabolic profiles of canine packed RBC (pRBC) units as a function of LR in fresh and stored refrigerated (up to 42 days) units., Animals: Packed RBC units were obtained from 8 donor dogs enrolled at 2 different Italian veterinary blood banks., Study Design and Methods: Observational study. A volume of 450 mL of whole blood was collected using Citrate-Phosphate-Dextrose-Saline-Adenine-Glucose-Mannitol (CPD-SAGM) transfusion bags with a LR filter to produce 2 pRBC units for each donor, without (nLR-pRBC) and with (LR-pRBC) LR. Units were stored in the blood bank at 4 ± 2°C. Sterile weekly samples were obtained from each unit for omics analyses., Results: A significant effect of LR on fresh and stored RBC metabolic phenotypes was observed. The nLR-pRBC were characterized by higher concentrations of free short and medium-chain fatty acids, carboxylic acids (pyruvate, lactate), and amino acids (arginine, cystine). The LR-pRBC had higher concentrations of glycolytic metabolites, high energy phosphate compounds (adenosine triphosphate [ATP]), and antioxidant metabolites (pentose phosphate, total glutathione)., Conclusion and Clinical Importance: Leukoreduction decreases the metabolic SLs of canine pRBC by preserving energy metabolism and preventing oxidative lesions., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Background: Platelet (PLT) product transfusion is a life-saving therapy for actively bleeding patients. There is an urgent need to maintain PLT function and extend shelf life to improve outcomes in these patients. Cold-stored PLT (CS-PLT) maintain hemostatic potential better than room temperature-stored PLT (RT-PLT). However, whether function in long-term CS-PLT is maintained under physiological flow regimes and/or determined by cold-induced metabolic changes is unknown., Objectives: This study aimed to (i) compare the function of RT-PLT and CS-PLT under physiological flow conditions, (ii) determine whether CS-PLT maintain function after 3 weeks of storage, and (iii) identify metabolic pathways associated with the CS-PLT lesion., Methods: We performed phenotypic and functional assessments of RT- and CS-PLT (22 °C and 4 °C storage, respectively; N = 10 unique donors) at storage days 0, 5, and/or 21 via metabolomics, flow cytometry, aggregation, thrombin generation, viscoelastic testing, and a microfluidic assay to measure primary hemostatic function., Results: Day 21 4 °C PLT formed an occlusive thrombus under arterial shear at a similar rate to day 5 22 °C PLT. Day 21 4 °C PLTs had enhanced thrombin generation capacity compared with day 0 PLT and maintained functionality comparable to day RT-PLT across all assays performed. Key metrics from microfluidic assessment, flow cytometry, thrombin generation, and aggregation were associated with 4 °C storage, and metabolites involved in taurine and purine metabolism significantly correlated with these metrics. Taurine supplementation of PLT during storage improved hemostatic function under flow., Conclusion: CS-PLT stored for 3 weeks maintain hemostatic activity, and storage-induced phenotype and function are associated with taurine and purine metabolism., Competing Interests: Declaration of competing interests P.C.S. is a consultant for Cerus Corporation and Haima Therapeutics. All other authors have no competing interests to disclose., (Copyright © 2023 International Society on Thrombosis and Haemostasis. Published by Elsevier Inc. All rights reserved.)

Sustained smouldering, or low-grade activation, of myeloid cells is a common hallmark of several chronic neurological diseases, including multiple sclerosis 1 . Distinct metabolic and mitochondrial features guide the activation and the diverse functional states of myeloid cells 2 . However, how these metabolic features act to perpetuate inflammation of the central nervous system is unclear. Here, using a multiomics approach, we identify a molecular signature that sustains the activation of microglia through mitochondrial complex I activity driving reverse electron transport and the production of reactive oxygen species. Mechanistically, blocking complex I in pro-inflammatory microglia protects the central nervous system against neurotoxic damage and improves functional outcomes in an animal disease model in vivo. Complex I activity in microglia is a potential therapeutic target to foster neuroprotection in chronic inflammatory disorders of the central nervous system 3 ., (© 2024. The Author(s).)

Background: Donor genetic variation is associated with red blood cell (RBC) storage integrity and post-transfusion recovery. Our previous large-scale genome-wide association study demonstrated that the African G6PD deficient A- variant (rs1050828, Val68Met) is associated with higher oxidative hemolysis after cold storage. Despite a high prevalence of X-linked G6PD mutation in African American population (>10%), blood donors are not routinely screened for G6PD status and its importance in transfusion medicine is relatively understudied., Study Design and Methods: To further evaluate the functional effects of the G6PD A- mutation, we created a novel mouse model carrying this genetic variant using CRISPR-Cas9. We hypothesize that this humanized G6PD A- variant is associated with reduced G6PD activity with a consequent effect on RBC hemolytic propensity and post-transfusion recovery., Results: G6PD A- RBCs had reduced G6PD protein with ~5% residual enzymatic activity. Significantly increased in vitro hemolysis induced by oxidative stressors was observed in fresh and stored G6PD A- RBCs, along with a lower GSH:GSSG ratio. However, no differences were observed in storage hemolysis, osmotic fragility, mechanical fragility, reticulocytes, and post-transfusion recovery. Interestingly, a 14% reduction of 24-h survival following irradiation was observed in G6PD A- RBCs compared to WT RBCs. Metabolomic assessment of stored G6PD A- RBCs revealed an impaired pentose phosphate pathway (PPP) with increased glycolytic flux, decreasing cellular antioxidant capacity., Discussion: This novel mouse model of the common G6PD A- variant has impaired antioxidant capacity like humans and low G6PD activity may reduce survival of transfused RBCs when irradiation is performed., (© 2024 The Authors. Transfusion published by Wiley Periodicals LLC on behalf of AABB.)

Mechanisms governing the maintenance of blood-producing hematopoietic stem and multipotent progenitor cells (HSPCs) are incompletely understood, particularly those regulating fate, ensuring long-term maintenance, and preventing aging-associated stem cell dysfunction. We uncovered a role for transitory free cytoplasmic iron as a rheostat for adult stem cell fate control. We found that HSPCs harbor comparatively small amounts of free iron and show the activation of a conserved molecular response to limited iron-particularly during mitosis. To study the functional and molecular consequences of iron restriction, we developed models allowing for transient iron bioavailability limitation and combined single-molecule RNA quantification, metabolomics, and single-cell transcriptomic analyses with functional studies. Our data reveal that the activation of the limited iron response triggers coordinated metabolic and epigenetic events, establishing stemness-conferring gene regulation. Notably, we find that aging-associated cytoplasmic iron loading reversibly attenuates iron-dependent cell fate control, explicating intervention strategies for dysfunctional aged stem cells., Competing Interests: Declaration of interests B.W. and U.S. have received funds for research projects and for serving on the advisory board of Novartis Pharmaceuticals., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Understanding of the biochemical and morphological lesions associated with storage of equine blood is limited., Objective: To demonstrate the temporal sequences of lipid and metabolic profiles of equine fresh and stored (up to 42 days) and leukoreduced packed red blood cells (LR-pRBC) and non-leukoreduced packed RBC (nLR-pRBC)., Animals: Packed RBC units were obtained from 6 healthy blood donor horses enrolled in 2 blood banks., Methods: Observational study. Whole blood was collected from each donor using transfusion bags with a LR filter. Leukoreduction pRBC and nLR-pRBC units were obtained and stored at 4°C for up 42 days. Sterile weekly sampling was performed from each unit for analyses., Results: Red blood cells and supernatants progressively accumulated lactate products while high-energy phosphate compounds (adenosine triphosphate and 2,3-Diphosphoglycerate) declined. Hypoxanthine, xanthine, and free fatty acids accumulated in stored RBC and supernatants. These lesions were exacerbated in non-LR-pRBC., Conclusion and Clinical Importance: Leukoreduction has a beneficial effect on RBC energy and redox metabolism of equine pRBC and the onset and severity of the metabolic storage lesions RBC., (© 2024 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals LLC on behalf of American College of Veterinary Internal Medicine.)

Chronic, systemic inflammation is a pathophysiological manifestation of metabolic disorders. Inflammatory signaling leads to elevated glycolytic flux and a metabolic shift towards aerobic glycolysis and lactate generation. This rise in lactate corresponds with increased generation of lactoylLys modifications on histones, mediating transcriptional responses to inflammatory stimuli. Lactoylation is also generated through a non-enzymatic S-to-N acyltransfer from the glyoxalase cycle intermediate, lactoylglutathione (LGSH). Here, we report a regulatory role for LGSH in mediating histone lactoylation and inflammatory signaling. In the absence of the primary LGSH hydrolase, glyoxalase 2 (GLO2), RAW264.7 macrophages display significant elevations in LGSH and histone lactoylation with a corresponding potentiation of the inflammatory response when exposed to lipopolysaccharides. An analysis of chromatin accessibility shows that lactoylation is associated with more compacted chromatin than acetylation in an unstimulated state; upon stimulation, however, regions of the genome associated with lactoylation become markedly more accessible. Lastly, we demonstrate a spontaneous S-to-S acyltransfer of lactate from LGSH to CoA, yielding lactoyl-CoA. This represents the first known mechanism for the generation of this metabolite. Collectively, these data suggest that LGSH, and not intracellular lactate, is the primary driving factor facilitating histone lactoylation and a major contributor to inflammatory signaling., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Purpose: This study aimed to determine if time-efficient, high-resistance inspiratory muscle strength training (IMST), comprising 30 inhalation-resisted breaths per day, improves cardiorespiratory fitness, exercise tolerance, physical function, and/or regional body composition in healthy midlife and older adults., Methods: We performed a double-blind, randomized, sham-controlled clinical trial (NCT03266510) testing 6 wk of IMST (30 breaths per day, 6 d·wk -1 , 55%-75% maximal inspiratory pressure) versus low-resistance sham training (15% maximal inspiratory pressure) in healthy men and women 50-79 yr old. Subjects performed a graded treadmill exercise test to exhaustion, physical performance battery (e.g., handgrip strength, leg press), and body composition testing (dual x-ray absorptiometry) at baseline and after 6 wk of training., Results: Thirty-five participants (17 women, 18 men) completed high-resistance IMST ( n = 17) or sham training ( n = 18). Cardiorespiratory fitness (V̇O 2peak ) was unchanged, but exercise tolerance, measured as treadmill exercise time during a graded exercise treadmill test, increased with IMST (baseline, 539 ± 42 s; end intervention, 606 ± 42 s; P = 0.01) but not sham training (baseline, 562 ± 39 s; end intervention, 553 ± 38 s; P = 0.69). IMST increased peak RER (baseline, 1.09 ± 0.02; end intervention, 1.13 ± 0.02; P = 0.012), peak ventilatory efficiency (baseline, 25.2 ± 0.8; end intervention, 24.6 ± 0.8; P = 0.036), and improved submaximal exercise economy (baseline, 23.5 ± 1.1 mL·kg -1 ⋅min -1 ; end intervention, 22.1 ± 1.1 mL·kg -1 ⋅min -1 ; P < 0.001); none of these factors were altered by sham training (all P > 0.05). Changes in plasma acylcarnitines (targeted metabolomics analysis) were consistently positively correlated with changes in exercise tolerance after IMST but not sham training. IMST was associated with regional increases in thorax lean mass (+4.4%, P = 0.06) and reductions in trunk fat mass (-4.8%, P = 0.04); however, peripheral muscle strength, muscle power, dexterity, and mobility were unchanged., Conclusions: These data suggest that high-resistance IMST is an effective, time-efficient lifestyle intervention for improving exercise tolerance in healthy midlife and older adults., (Copyright © 2023 by the American College of Sports Medicine.)

Biomanufacturing via microorganisms relies on carbon substrates for molecular feedstocks and a source of energy to carry out enzymatic reactions. This creates metabolic bottlenecks and lowers the efficiency for substrate conversion. Nanoparticle biohybridization with proteins and whole cell surfaces can bypass the need for redox cofactor regeneration for improved secondary metabolite production in a non-specific manner. Here we propose using nanobiohybrid organisms (Nanorgs), intracellular protein-nanoparticle hybrids formed through the spontaneous coupling of core-shell quantum dots (QDs) with histidine-tagged enzymes in non-photosynthetic bacteria, for light-mediated control of bacterial metabolism. This proved to eliminate metabolic constrictions and replace glucose with light as the source of energy in Escherichia coli, with an increase in growth by 1.7-fold in 75 % reduced nutrient media. Metabolomic tracking through carbon isotope labeling confirmed flux shunting through targeted pathways, with accumulation of metabolites downstream of respective targets. Finally, application of Nanorgs with the Ehrlich pathway improved isobutanol titers/yield by 3.9-fold in 75 % less sugar from E. coli strains with no genetic alterations. These results demonstrate the promise of Nanorgs for metabolic engineering and low-cost biomanufacturing., (© 2023 Wiley-VCH GmbH.)

Pyruvate kinase (Pyk) is a rate-limiting enzyme that catalyzes the final metabolic reaction in glycolysis. The importance of this enzyme, however, extends far beyond ATP production, as Pyk is also known to regulate tissue growth, cell proliferation, and development. Studies of this enzyme in Drosophila melanogaster are complicated by the fact that the fly genome encodes 6 Pyk paralogs whose functions remain poorly defined. To address this issue, we used sequence distance and phylogenetic approaches to demonstrate that the gene Pyk encodes the enzyme most similar to the mammalian Pyk orthologs, while the other 5 Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of 2 different Pyk mutant strains revealed that larvae lacking Pyk exhibit a severe block in glycolysis, with a buildup of glycolytic intermediates upstream of pyruvate. However, our analysis also unexpectedly reveals that pyruvate levels are unchanged in Pyk mutants, indicating that larval metabolism maintains pyruvate pool size despite severe metabolic limitations. Consistent with our metabolomic findings, a complementary RNA-seq analysis revealed that genes involved in lipid metabolism and protease activity are elevated in Pyk mutants, again indicating that loss of this glycolytic enzyme induces compensatory changes in other aspects of metabolism. Overall, our study provides both insight into how Drosophila larval metabolism adapts to disruption of glycolytic metabolism as well as immediate clinical relevance, considering that Pyk deficiency is the most common congenital enzymatic defect in humans., Competing Interests: Conflicts of interest The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Di-2-ethylhexyl phthalate (DEHP) is commonly used in the manufacturing of plastic materials, including intravenous bags, blood storage bags, and medical-grade tubing. DEHP can leach from plastic medical products, which can result in inadvertent patient exposure. DEHP concentrations were measured in red blood cell units stored between 7 and 42 days (17-119 μg/ml). Using these concentrations as a guide, Langendorff-perfused rat heart preparations were acutely exposed to DEHP. Sinus activity remained stable with lower doses of DEHP (25-50 μg/ml), but sinus rate declined by 43% and sinus node recovery time (SNRT) prolonged by 56.5% following 30-min exposure to 100 μg/ml DEHP. DEHP exposure also exerted a negative dromotropic response, as indicated by a 69.4% longer PR interval, 108.5% longer Wenckebach cycle length (WBCL), and increased incidence of atrioventricular (AV) uncoupling (60-min exposure). Pretreatment with doxycycline partially rescued the effects of DEHP on sinus activity, but did not ameliorate the effects on AV conduction. DEHP exposure also prolonged the ventricular action potential and effective refractory period, but had no measurable effect on intracellular calcium transient duration. Follow-up studies using human-induced pluripotent stem cell-derived cardiomyocytes confirmed that DEHP slows electrical conduction in a time (15 min-3 h) and dose-dependent manner (10-100 μg/ml). Previous studies have suggested that phthalate toxicity is specifically attributed to metabolites of DEHP, including mono-2-ethylhexylphthalate. This study demonstrates that DEHP exposure also contributes to cardiac dysfunction in a dose- and time-dependent manner. Future work is warranted to investigate the impact of DEHP (and its metabolites) on human health, with special consideration for clinical procedures that employ plastic materials., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Metabolomics studies in sickle cell disease (SCD) have been so far limited to tens of samples, owing to technical and experimental limitations. To overcome these limitations, we performed plasma metabolomics analyses on 596 samples from patients with SCD enrolled in the WALK-PHaSST study (clinicaltrials gov. Identifier: NCT00492531). Clinical covariates informed the biological interpretation of metabolomics data, including genotypes (hemoglobin [Hb] SS, hemoglobin SC), history of recent transfusion (HbA%), response to hydroxyurea treatment (fetal Hb%). We investigated metabolic correlates to the degree of intravascular hemolysis, cardiorenal function, as determined by tricuspid regurgitation velocity (TRV), estimated glomerular filtration rate (eGFR), and overall hazard ratio (unadjusted or adjusted by age). Recent transfusion events or hydroxyurea treatment were associated with elevation in plasma-free fatty acids and decreases in acyl-carnitines, urate, kynurenine, indoles, carboxylic acids, and glycine- or taurine-conjugated bile acids. High levels of these metabolites, along with low levels of plasma S1P and L-arginine were identified as top markers of hemolysis, cardiorenal function (TRV, eGFR), and overall hazard ratio. We thus uploaded all omics and clinical data on a novel online portal that we used to identify a potential mechanism of dysregulated red cell S1P synthesis and export as a contributor to the more severe clinical manifestations in patients with the SS genotype compared to SC. In conclusion, plasma metabolic signatures - including low S1P, arginine and elevated kynurenine, acyl-carnitines and bile acids - are associated with clinical manifestation and therapeutic efficacy in SCD patients, suggesting new avenues for metabolic interventions in this patient population.

Cancer stem cells (CSCs) drive tumor growth, metastasis, and chemoresistance. While emerging evidence suggests that CSCs have a unique dependency on lipid metabolism, the functions and regulation of distinct lipid species in CSCs remain poorly understood. Here, we developed a stem cell factor SOX9-based reporter for isolating CSCs in primary tumors and metastases of spontaneous mammary tumor models. Transcriptomic analyses uncover that SOX9 high CSCs up-regulate the ABCA12 lipid transporter. ABCA12 down-regulation impairs cancer stemness and chemoresistance. Lipidomic analyses reveal that ABCA12 maintains cancer stemness and chemoresistance by reducing intracellular ceramide abundance, identifying a CSC-associated function of ABCA subfamily transporter. Ceramide suppresses cancer stemness by inhibiting the YAP-SOX9 signaling pathway in CSCs. Increasing ceramide levels in tumors enhances their sensitivity to chemotherapy and prevents the enrichment of SOX9 high CSCs. In addition, SOX9 high and ABCA12 high cancer cells contribute to chemoresistance in human patient-derived xenografts. These findings identify a CSC-suppressing lipid metabolism pathway that can be exploited to inhibit CSCs and overcome chemoresistance.

Loss of protein kinase Cδ (PKCδ) activity renders cells resistant to DNA damaging agents, including irradiation; however, the mechanism(s) underlying resistance is poorly understood. Here, we have asked if metabolic reprogramming by PKCδ contributes to radioprotection. Analysis of global metabolomics showed that depletion of PKCδ affects metabolic pathways that control energy production and antioxidant, nucleotide, and amino acid biosynthesis. Increased NADPH and nucleotide production in PKCδ-depleted cells is associated with upregulation of the pentose phosphate pathway (PPP) as evidenced by increased activation of G6PD and an increase in the nucleotide precursor, 5-phosphoribosyl-1-pyrophosphate. Stable isotope tracing with U-[ 13 C 6 ] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[ 13 C 6 ] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [ 13 C 5 , 15 N 2 ] glutamine showed increased utilization of glutamine for synthesis of nucleotides, glutathione, and tricarboxylic acid intermediates and increased incorporation of labeled glutamine into pyruvate and lactate. Using a glycolytic rate assay, we confirmed that anaerobic glycolysis is increased in PKCδ-depleted cells; this was accompanied by a reduction in oxidative phosphorylation, as assayed using a mitochondrial stress assay. Importantly, pretreatment of cells with specific inhibitors of the PPP or glutaminase prior to irradiation reversed radioprotection in PKCδ-depleted cells, indicating that these cells have acquired codependency on the PPP and glutamine for survival. Our studies demonstrate that metabolic reprogramming to increase utilization of glutamine and nucleotide synthesis contributes to radioprotection in the context of PKCδ inhibition., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

RNA-binding proteins (RBPs) are key regulators of gene expression. Small molecules targeting these RBP-RNA interactions are a rapidly emerging class of therapeutics for treating a variety of diseases. Ro-08-2750 (Ro) is a small molecule identified as a competitive inhibitor of Musashi (MSI)-RNA interactions. Here, we show that multiple Ro-dependent cellular phenotypes, specifically adrenocortical steroid production and cell viability, are Musashi-2 (MSI2)-independent. Using an unbiased proteome-wide approach, we discovered Ro broadly interacts with RBPs, many containing RRM domains. To confirm this finding, we leveraged the large-scale ENCODE data to identify a subset of RBPs whose depletion phenocopies Ro inhibition, indicating Ro is a promiscuous inhibitor of multiple RBPs. Consistent with broad disruption of ribonucleoprotein complexes, Ro treatment leads to stress granule formation. This strategy represents a generalizable framework for validating the specificity and identifying targets of RBP inhibitors in a cellular context., (© 2023 Walters et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.)

Recent studies illustrate the importance of regulation of cellular metabolism, especially glycolysis and pathways branching from glycolysis, during vertebrate embryo development. For example, glycolysis generates cellular energy ATP. Glucose carbons are also directed to the pentose phosphate pathway, which is needed to sustain anabolic processes in the rapidly growing embryos. However, our understanding of the exact status of glycolytic metabolism as well as genes that regulate glycolytic metabolism are still incomplete. Sall4 is a zinc finger transcription factor that is highly expressed in undifferentiated cells in developing mouse embryos, such as blastocysts and the post-implantation epiblast. TCre; Sall4 conditional knockout mouse embryos exhibit various defects in the posterior part of the body, including hindlimbs. Using transcriptomics approaches, we found that many genes encoding glycolytic enzymes are upregulated in the posterior trunk, including the hindlimb-forming region, of Sall4 conditional knockout mouse embryos. In situ hybridization and qRT-PCR also confirmed upregulation of expression of several glycolytic genes in hindlimb buds. A fraction of those genes are bound by SALL4 at the promoters, gene bodies or distantly-located regions, suggesting that Sall4 directly regulates expression of several glycolytic enzyme genes in hindlimb buds. To further gain insight into the metabolic status associated with the observed changes at the transcriptional level, we performed a comprehensive analysis of metabolite levels in limb buds in wild type and Sall4 conditional knockout embryos by high-resolution mass spectrometry. We found that the levels of metabolic intermediates of glycolysis are lower, but glycolytic end-products pyruvate and lactate did not exhibit differences in Sall4 conditional knockout hindlimb buds. The increased expression of glycolytic genes would have caused accelerated glycolytic flow, resulting in low levels of intermediates. This condition may have prevented intermediates from being re-directed to other pathways, such as the pentose phosphate pathway. Indeed, the change in glycolytic metabolite levels is associated with reduced levels of ATP and metabolites of the pentose phosphate pathway. To further test whether glycolysis regulates limb patterning downstream of Sall4, we conditionally inactivated Hk2, which encodes a rate-limiting enzyme gene in glycolysis and is regulated by Sall4. The TCre; Hk2 conditional knockout hindlimb exhibited a short femur, and a lack of tibia and anterior digits in hindlimbs, which are defects similarly found in the TCre; Sall4 conditional knockout. The similarity of skeletal defects in Sall4 mutants and Hk2 mutants suggests that regulation of glycolysis plays a role in hindlimb patterning. These data suggest that Sall4 restricts glycolysis in limb buds and contributes to patterning and regulation of glucose carbon flow during development of limb buds., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans (∼5% of all individuals). G6PD deficiency (G6PDd) is caused by an unstable enzyme and manifests most strongly in red blood cells (RBCs) that cannot synthesize new protein. G6PDd RBCs have decreased ability to mitigate oxidative stress due to lower levels of NADPH, as a result of a defective pentose phosphate pathway. Accordingly, oxidative drugs can result in hemolysis and potentially life-threatening anemia in G6PDd patients. Dapsone is a highly useful drug for treating a variety of pathologies but oral dapsone is contraindicated in patients with G6PDd due to oxidative stress-induced anemia. Dapsone must be metabolized to become hemolytic. Dapsone hydroxylamine (DDS-NOH) has been implicated as the major hemolytic dapsone metabolite, but this has never been tested on G6PDd RBCs with in vivo circulation as a metric. Moreover, the metabolic lesion caused by DDS-NOH is unknown. We report that RBCs from a novel humanized mouse expressing the human Mediterranean G6PD-deficient variant have increased sensitivity to DDS-NOH. In addition, we show that DDS-NOH damaged RBCs can either undergo sequestration (with subsequent return to circulation) or permanent removal in a dose-dependent manner, with G6PD-sufficient RBCs mostly being sequestered, and G6PDd RBCs mostly being permanently removed. Finally, we characterize the metabolic lesion caused by DDS-NOH in G6PDd RBCs and report a blockage in terminal glycolysis resulting in a cellular accumulation of pyruvate. These findings confirm DDS-NOH as a hemolytic metabolite and elucidate metabolic effects of DDS-NOH on G6PDd RBCs. SIGNIFICANCE STATEMENT: These findings confirm that dapsone hydroxylamine, an active metabolite of dapsone, causes in vivo clearance of murine red blood cells expressing a human variant of deficient glucose 6-phosphate dehydrogenase (G6PD), an enzymopathy that affects half a billion individuals (G6PD deficiency). Both cellular mechanisms of clearance (sequestration versus destruction) and specific metabolic disturbances caused by dapsone hydroxylamine are elucidated, providing novel mechanistic understanding., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Outcomes for patients with acute myeloid leukemia (AML) remain poor due to the inability of current therapeutic regimens to fully eradicate disease-initiating leukemia stem cells (LSC). Previous studies have demonstrated that oxidative phosphorylation (OXPHOS) is an essential process that is targetable in LSC. Sirtuin 3 (SIRT3), a mitochondrial deacetylase with a multi-faceted role in metabolic regulation, has been shown to regulate OXPHOS in cancer models; however, it has not yet been studied in the context of LSC. Thus, we sought to identify if SIRT3 is important for LSC function. Using RNAi and a SIRT3 inhibitor (YC8-02), we demonstrate that SIRT3 is a critical target for the survival of primary human LSC but is not essential for normal human hematopoietic stem and progenitor cell function. In order to elucidate the molecular mechanisms by which SIRT3 is essential in LSC we combined transcriptomic, proteomic, and lipidomic approaches, showing that SIRT3 is important for LSC function through the regulation of fatty acid oxidation (FAO) which is required to support OXPHOS and ATP production in human LSC. Further, we discovered two approaches to further sensitize LSC to SIRT3 inhibition. First, we found that LSC tolerate the toxic effects of fatty acid accumulation induced by SIRT3 inhibition by upregulating cholesterol esterification. Disruption of cholesterol homeostasis sensitizes LSC to YC8-02 and potentiates LSC death. Second, SIRT3 inhibition sensitizes LSC to the BCL-2 inhibitor venetoclax. Together, these findings establish SIRT3 as a regulator of lipid metabolism and potential therapeutic target in primitive AML cells.

Pulmonary microvascular endothelial cells contribute to the integrity of the lung gas exchange interface, and they are highly glycolytic. Although glucose and fructose represent discrete substrates available for glycolysis, pulmonary microvascular endothelial cells prefer glucose over fructose, and the mechanisms involved in this selection are unknown. 6-Phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) is an important glycolytic enzyme that drives glycolytic flux against negative feedback and links glycolytic and fructolytic pathways. We hypothesized that PFKFB3 inhibits fructose metabolism in pulmonary microvascular endothelial cells. We found that PFKFB3 knockout cells survive better than wild-type cells in fructose-rich medium under hypoxia. Seahorse assays, lactate and glucose measurements, and stable isotope tracing showed that PFKFB3 inhibits fructose-hexokinase-mediated glycolysis and oxidative phosphorylation. Microarray analysis revealed that fructose upregulates PFKFB3, and PFKFB3 knockout cells increase fructose-specific GLUT5 (glucose transporter 5) expression. Using conditional endothelial-specific PFKFB3 knockout mice, we demonstrated that endothelial PFKFB3 knockout increases lung tissue lactate production after fructose gavage. Last, we showed that pneumonia increases fructose in BAL fluid in mechanically ventilated ICU patients. Thus, PFKFB3 knockout increases GLUT5 expression and the hexokinase-mediated fructose use in pulmonary microvascular endothelial cells that promotes their survival. Our findings indicate that PFKFB3 is a molecular switch that controls glucose versus fructose use in glycolysis and help better understand lung endothelial cell metabolism during respiratory failure.

Lesch-Nyhan syndrome (LN) is an is an X-linked recessive inborn error of metabolism that arises from a deficiency of purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). The disease manifests severely, causing intellectual deficits and other neural abnormalities, hypercoagulability, uncontrolled self-injury, and gout. While allopurinol is used to alleviate gout, other symptoms are less understood, impeding treatment. Herein, we present a high-throughput multi-omics analysis of red blood cells (RBCs) from three pediatric siblings carrying a novel S162N HPRT1 mutation. RBCs from both parents-the mother, a heterozygous carrier, and the father, a clinically healthy control-were also analyzed. Global metabolite analysis of LN RBCs shows accumulation of glycolytic intermediates upstream of pyruvate kinase, unsaturated fatty acids, and long chain acylcarnitines. Similarly, highly unsaturated phosphatidylcholines are also elevated in LN RBCs, while free choline is decreased. Intracellular iron, zinc, selenium, and potassium are also decreased in LN RBCs. Global proteomics documented changes in RBC membrane proteins, hemoglobin, redox homeostasis proteins, and the enrichment of coagulation proteins. These changes were accompanied by elevation in protein glutamine deamidation and methylation in the LN children and carrier mother. Treatment with allopurinol incompletely reversed the observed phenotypes in the two older siblings currently on this treatment. This unique data set provides novel opportunities for investigations aimed at potential therapies for LN-associated sequelae.

Background: Acetaminophen (APAP) is the most common cause liver injury following alcohol in US patients. Predicting liver injury and subsequent hepatic regeneration in patients taking therapeutic doses of APAP may be possible using new 'omic methods such as metabolomics and genomics. Multi'omic techniques increase our ability to find new mechanisms of injury and regeneration., Methods: We used metabolomic and genomic data from a randomized controlled trial of patients administered 4 g of APAP per day for 14 days or longer with blood samples obtained at 0 (baseline), 4, 7, 10, 13 and 16 days. We used the highest ALT as the clinical outcome to be predicted in our integrated analysis. We used penalized regression to model the relationship between genetic variants and day 0 metabolite level, and then performed a metabolite-wide colocalization scan to associate the genetically regulated component of metabolite expression with ALT elevation. Genome-wide association study (GWAS) analyses were conducted for ALT elevation and metabolite level using linear regression, with age, sex, and the first five principal components included as covariates. Colocalization was tested via a weighted sum test., Results: Out of the 164 metabolites modeled, 120 met the criteria for predictive accuracy and were retained for genetic analyses. After genomic examination, eight metabolites were found to be under genetic control and predictive of ALT elevation due to therapeutic acetaminophen. The metabolites were: 3-oxalomalate, allantoate, diphosphate, L-carnitine, L-proline, maltose, and ornithine. These genes are important in the tricarboxylic acid cycle (TCA), urea breakdown pathway, glutathione production, mitochondrial energy production, and maltose metabolism., Conclusions: This multi'omic approach can be used to integrate metabolomic and genomic data allowing identification of genes that control downstream metabolites. These findings confirm prior work that have identified mitochondrial energy production as critical to APAP induced liver injury and have confirmed our prior work that demonstrate the importance of the urea cycle in therapeutic APAP liver injury., (© 2023. American College of Medical Toxicology.)

Despite a wealth of exploratory plasma metabolomics studies in sickle cell disease (SCD), no study to date has evaluate a large and well phenotyped cohort to compare the primary erythrocyte metabolome of hemoglobin SS, SC and transfused AA red blood cells (RBCs) in vivo. The current study evaluates the RBC metabolome of 587 subjects with sickle cell sickle cell disease (SCD) from the WALK-PHaSST clinical cohort. The set includes hemoglobin SS, hemoglobin SC SCD patients, with variable levels of HbA related to RBC transfusion events. Here we explore the modulating effects of genotype, age, sex, severity of hemolysis, and transfusion therapy on sickle RBC metabolism. Results show that RBCs from patients with Hb SS genotypes-compared to AA RBCs from recent transfusion events or SC RBCs-are characterized by significant alterations of RBC acylcarnitines, pyruvate, sphingosine 1-phosphate, creatinine, kynurenine and urate metabolism. Surprisingly, the RBC metabolism of SC RBCs is dramatically different from SS, with all glycolytic intermediates significantly elevated in SS RBCs, with the exception of pyruvate. This result suggests a metabolic blockade at the ATP-generating phosphoenolpyruvate to pyruvate step of glycolysis, which is catalyzed by redox-sensitive pyruvate kinase. Metabolomics, clinical and hematological data were collated in a novel online portal. In conclusion, we identified metabolic signatures of HbS RBCs that correlate with the degree of steady state hemolytic anemia, cardiovascular and renal dysfunction and mortality., (© 2023 Wiley Periodicals LLC.)

Red blood cells (RBC) are the most abundant cell in the human body, with a central role in oxygen transport and its delivery to tissues. However, omics technologies recently revealed the unanticipated complexity of the RBC proteome and metabolome, paving the way for a reinterpretation of the mechanisms by which RBC metabolism regulates systems biology beyond oxygen transport. The new data and analytical tools also informed the dissection of the changes that RBCs undergo during refrigerated storage under blood bank conditions, a logistic necessity that makes >100 million units available for life-saving transfusions every year worldwide. In this narrative review, we summarize the last decade of advances in the field of RBC metabolism in vivo and in the blood bank in vitro, a narrative largely influenced by the authors' own journeys in this field. We hope that this review will stimulate further research in this interesting and medically important area or, at least, serve as a testament to our fascination with this simple, yet complex, cell.

The interferon-induced transmembrane proteins (IFITM) are implicated in several biological processes, including antiviral defense, but their modes of action remain debated. Here, taking advantage of pseudotyped viral entry assays and replicating viruses, we uncover the requirement of host co-factors for endosomal antiviral inhibition through high-throughput proteomics and lipidomics in cellular models of IFITM restriction. Unlike plasma membrane (PM)-localized IFITM restriction that targets infectious SARS-CoV2 and other PM-fusing viral envelopes, inhibition of endosomal viral entry depends on lysines within the conserved IFITM intracellular loop. These residues recruit Phosphatidylinositol 3,4,5-trisphosphate (PIP3) that we show here to be required for endosomal IFITM activity. We identify PIP3 as an interferon-inducible phospholipid that acts as a rheostat for endosomal antiviral immunity. PIP3 levels correlated with the potency of endosomal IFITM restriction and exogenous PIP3 enhanced inhibition of endocytic viruses, including the recent SARS-CoV2 Omicron variant. Together, our results identify PIP3 as a critical regulator of endosomal IFITM restriction linking it to the Pi3K/Akt/mTORC pathway and elucidate cell-compartment-specific antiviral mechanisms with potential relevance for the development of broadly acting antiviral strategies., (© 2023 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Conditions related to cardiometabolic disease, including metabolic syndrome and type 2 diabetes, are common among men with Klinefelter syndrome (KS). The molecular mechanisms underlying this aberrant metabolism in KS are largely unknown, although there is an assumption that chronic testosterone deficiency plays a role. This cross-sectional study compared plasma metabolites in 31 pubertal adolescent males with KS to 32 controls of similar age (14 ± 2 years), pubertal stage, and body mass index z-score of 0.1 ± 1.2 and then between testosterone-treated (n = 16) and untreated males with KS. The plasma metabolome in males with KS was distinctly different from that in controls, with 22% of measured metabolites having a differential abundance and seven metabolites nearly completely separating KS from controls (area under the curve > 0.9, P < 0.0001). Multiple saturated free fatty acids were higher in KS, while mono- and polyunsaturated fatty acids were lower, and the top significantly enriched pathway was mitochondrial β-oxidation of long-chain saturated fatty acids (enrichment ratio 16, P < 0.0001). In contrast, there were no observed differences in metabolite concentrations between testosterone-treated and untreated individuals with KS. In conclusion, the plasma metabolome profile in adolescent males with KS is distinctly different from that in males without KS independent of age, obesity, pubertal development, or testosterone treatment status and is suggestive of differences in mitochondrial β-oxidation.

Background: We compared plasma metabolites of amino acid oxidation and the tricarboxylic acid (TCA) cycle in youth with and without type 1 diabetes mellitus (T1DM) and related the metabolites to glomerular filtration rate (GFR), renal plasma flow (RPF), and albuminuria. Metabolites associated with impaired kidney function may warrant future study as potential biomarkers or even future interventions to improve kidney bioenergetics., Methods: Metabolomic profiling of fasting plasma samples using a targeted panel of 644 metabolites and an untargeted panel of 19,777 metabolites was performed in 50 youth with T1DM ≤ 10 years and 20 controls. GFR and RPF were ascertained by iohexol and p-aminohippurate clearance, and albuminuria calculated as urine albumin to creatinine ratio. Sparse partial least squares discriminant analysis and moderated t tests were used to identify metabolites associated with GFR and RPF., Results: Adolescents with and without T1DM were similar in age (16.1 ± 3.0 vs. 16.1 ± 2.9 years) and BMI (23.4 ± 5.1 vs. 22.7 ± 3.7 kg/m 2 ), but those with T1DM had higher GFR (189 ± 40 vs. 136 ± 22 ml/min) and RPF (820 ± 125 vs. 615 ± 65 ml/min). Metabolites of amino acid oxidation and the TCA cycle were significantly lower in adolescents with T1DM vs. controls, and the measured metabolites were able to discriminate diabetes status with an AUC of 0.82 (95% CI: 0.71, 0.93) and error rate of 0.21. Lower glycine (r:-0.33, q = 0.01), histidine (r:-0.45, q < 0.001), methionine (r: -0.29, q = 0.02), phenylalanine (r: -0.29, q = 0.01), serine (r: -0.42, q < 0.001), threonine (r: -0.28, q = 0.02), citrate (r: -0.35, q = 0.003), fumarate (r: -0.24, q = 0.04), and malate (r: -0.29, q = 0.02) correlated with higher GFR. Lower glycine (r: -0.28, q = 0.04), phenylalanine (r:-0.3, q = 0.03), fumarate (r: -0.29, q = 0.04), and malate (r: -0.5, q < 0.001) correlated with higher RPF. Lower histidine (r: -0.28, q = 0.02) was correlated with higher mean ACR., Conclusions: In conclusion, adolescents with relatively short T1DM duration exhibited lower plasma levels of carboxylic acids that associated with hyperfiltration and hyperperfusion., Trial Registration: ClinicalTrials.gov NCT03618420 and NCT03584217 A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s), under exclusive licence to International Pediatric Nephrology Association.)

Objectives: Identify the metabolites that are increased in the plasma of severely injured patients that developed ARDS versus severely injured patients that did not, and assay if these increased metabolites prime pulmonary sequestration of neutrophils (PMNs) and induce pulmonary sequestration in an animal model of ARDS. We hypothesize that metabolic derangement due to advanced shock in critically injured patients leads to the PMNs, which serves as the first event in the ARDS. Summary of Background Data: Intracellular metabolites accumulate in the plasma of severely injured patients., Methods: Untargeted metabolomics profiling of 67 critically injured patients was completed to establish a metabolic signature associated with ARDS development. Metabolites that significantly increased were assayed for PMN priming activity in vitro. The metabolites that primed PMNs were tested in a 2-event animal model of ARDS to identify a molecular link between circulating metabolites and clinical risk for ARDS., Results: After controlling for confounders, 4 metabolites significantly increased: creatine, dehydroascorbate, fumarate, and succinate in trauma patients who developed ARDS ( P < 0.05). Succinate alone primed the PMN oxidase in vitro at physiologically relevant levels. Intravenous succinate-induced PMN sequestration in the lung, a first event, and followed by intravenous lipopolysaccharide, a second event, resulted in ARDS in vivo requiring PMNs. SUCNR1 inhibition abrogated PMN priming, PMN sequestration, and ARDS. Conclusion: Significant increases in plasma succinate post-injury may serve as the first event in ARDS. Targeted inhibition of the SUCNR1 may decrease ARDS development from other disease states to prevent ARDS globally., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)