Inflammation promotes aging-associated oncogenesis in the lung.

Background: Lung cancer is the leading cause of cancer death in the world. While cigarette smoking is the major preventable factor for cancers in general and lung cancer in particular, old age is also a major risk factor. Aging-related chronic, low-level inflammation, termed inflammaging, has been widely documented; however, it remains unclear how inflammaging contributes to increased lung cancer incidence. Aim: To establish connections between aging-associated changes in the lungs and cancer risk. Methods: We analyzed public databases of gene expression for normal and cancerous human lungs and used mouse models to understand which changes were dependent on inflammation, as well as to assess the impact on oncogenesis. Results: Analyses of GTEx and TCGA databases comparing gene expression profiles from normal lungs, lung adenocarcinoma, lung squamous cell carcinoma of subjects across age groups revealed upregulated pathways such as inflammatory response, TNFA signaling via NFκB, and interferon-gamma response. Similar pathways were identified comparing the gene expression profiles of young and old mouse lungs. Transgenic expression of alpha 1 antitrypsin (AAT) partially reverses increases in markers of aging-associated inflammation and immune deregulation. Using an orthotopic model of lung cancer using cells derived from EML4-ALK fusion-induced adenomas, we demonstrated an increased tumor outgrowth in lungs of old mice while NLRP3 knockout in old mice decreased tumor volumes, suggesting that inflammation contributes to increased lung cancer development in aging organisms. Conclusions: These studies reveal how expression of an anti-inflammatory mediator (AAT) can reduce some but not all aging-associated changes in mRNA and protein expression in the lungs. We further show that aging is associated with increased tumor outgrowth in the lungs, which may relate to an increased inflammatory microenvironment.

Genetic regulation of carnitine metabolism controls lipid damage repair and aging RBC hemolysis in vivo and in vitro.

Recent large-scale multi-omics 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 two 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 (REDS) study. Genome wide association studies against 879,000 polymorphisms identified critical genetic factors contributing to inter-donor heterogeneity in end-of-storage carnitine levels, including common non-synonymous polymorphisms in genes encoding carnitine transporters (SLC22A16, SLC22A5, SLC16A9); carnitine synthesis (FLVCR1, MTDH) and metabolism (CPT1A, CPT2, CRAT, 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 two 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 following chemically induced membrane lipid damage. Supplementation of stored murine RBCs with L-carnitine boosted post-transfusion recovery, suggesting this could represent a viable strategy to improve RBC storage quality.

A Multiomics Assessment of Preoperative Exercise in Pancreatic Cancer Survivors Receiving Neoadjuvant Therapy: A Case Series.

To molecularly characterize the impact of exercise on mitigating neoadjuvant treatment (NAT)-induced physical decline in pancreatic ductal adenocarcinoma (PDAC) patients, a multi-omics approach was employed for the analysis of plasma samples before and after a personalized exercise intervention. Consisting of personalized aerobic and resistance exercises, this intervention was associated with significant molecular changes that correlated with improvements in lean mass, appendicular skeletal muscle index (ASMI), and performance in the 400-m walk test (MWT) and sit-to-stand test. These alterations indicated exercise-induced modulation of inflammation and mitochondrial function markers. This case study provides proof-of-principal application for multiomics-based assessments of supervised exercise, thereby supporting this intervention as a feasible and beneficial intervention for PDAC patients to potentially enhance treatment response and patient quality of life. The molecular changes observed here underscore the importance of physical activity in cancer treatment protocols, advocating for the development of accessible multiomics-guided exercise programs for cancer patients.

Biological and Genetic Determinants of Glycolysis: Phosphofructokinase Isoforms Boost Energy Status of Stored Red Blood Cells and Transfusion Outcomes.

Mature red blood cells (RBCs) lack mitochondria, and thus exclusively rely on glycolysis to generate adenosine triphosphate (ATP) during aging in vivo and during storage in vitro in the blood bank. Here we identify an association between blood donor age, sex, ethnicity and end-of-storage levels of glycolytic metabolites in 13,029 volunteers from the Recipient Epidemiology and Donor Evaluation Study. Associations were also observed to ancestry-specific genetic polymorphisms in regions encoding phosphofructokinase 1, platelet (which we detected in mature RBCs), hexokinase 1, 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 levels, breakdown, and deamination into hypoxanthine 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. Highlights: Blood donor age and sex affect glycolysis in stored RBCs from 13,029 volunteers;Ancestry, genetic polymorphisms in PFKP, HK1, CD38/BST1 influence RBC glycolysis;RBC PFKP boosts glycolytic fluxes when ATP is low, such as in stored RBCs;ATP and hypoxanthine are biomarkers of hemolysis in vitro and in vivo.

Auxin exposure disrupts feeding behavior and fatty acid metabolism in adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here, we show that auxin delays larval development in another widely used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin-fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Time-Efficient, High-Resistance Inspiratory Muscle Strength Training Increases Exercise Tolerance in Midlife and Older Adults.

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.

Regulation of kynurenine metabolism by blood donor genetics and biology impacts red cell hemolysis in vitro and in vivo.

ABSTRACT: In the field of transfusion medicine, the clinical relevance of the metabolic markers of the red blood cell (RBC) storage lesion is incompletely understood. Here, we performed metabolomics of RBC units from 643 donors enrolled in the Recipient Epidemiology and Donor Evaluation Study, REDS RBC Omics. These units were tested on storage days 10, 23, and 42 for a total of 1929 samples and also characterized for end-of-storage hemolytic propensity after oxidative and osmotic insults. Our results indicate that the metabolic markers of the storage lesion poorly correlated with hemolytic propensity. In contrast, kynurenine was not affected by storage duration and was identified as the top predictor of osmotic fragility. RBC kynurenine levels were affected by donor age and body mass index and were reproducible within the same donor across multiple donations from 2 to 12 months apart. To delve into the genetic underpinnings of kynurenine levels in stored RBCs, we thus tested kynurenine levels in stored RBCs on day 42 from 13 091 donors from the REDS RBC Omics study, a population that was also genotyped for 879 000 single nucleotide polymorphisms. Through a metabolite quantitative trait loci analysis, we identified polymorphisms in SLC7A5, ATXN2, and a series of rate-limiting enzymes (eg, kynurenine monooxygenase, indoleamine 2,3-dioxygenase, and tryptophan dioxygenase) in the kynurenine pathway as critical factors affecting RBC kynurenine levels. By interrogating a donor-recipient linkage vein-to-vein database, we then report that SLC7A5 polymorphisms are also associated with changes in hemoglobin and bilirubin levels, suggestive of in vivo hemolysis in 4470 individuals who were critically ill and receiving single-unit transfusions.

Genetic polymorphisms and expression of Rhesus blood group RHCE are associated with 2,3-bisphosphoglycerate in humans at high altitude.

Red blood cell (RBC) metabolic reprogramming upon exposure to high altitude contributes to physiological human adaptations to hypoxia, a multifaceted process critical to health and disease. To delve into the molecular underpinnings of this phenomenon, first, we performed a multi-omics analysis of RBCs from six lowlanders after exposure to high-altitude hypoxia, with longitudinal sampling at baseline, upon ascent to 5,100 m and descent to sea level. Results highlighted an association between erythrocyte levels of 2,3-bisphosphoglycerate (BPG), an allosteric regulator of hemoglobin that favors oxygen off-loading in the face of hypoxia, and expression levels of the Rhesus blood group RHCE protein. We then expanded on these findings by measuring BPG in RBCs from 13,091 blood donors from the Recipient Epidemiology and Donor Evaluation Study. These data informed a genome-wide association study using BPG levels as a quantitative trait, which identified genetic polymorphisms in the region coding for the Rhesus blood group RHCE as critical determinants of BPG levels in erythrocytes from healthy human volunteers. Mechanistically, we suggest that the Rh group complex, which participates in the exchange of ammonium with the extracellular compartment, may contribute to intracellular alkalinization, thus favoring BPG mutase activity.

Metabolomic analysis of Drosophila melanogaster larvae lacking pyruvate kinase.

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.

Spenito-dependent metabolic sexual dimorphism intrinsic to fat storage cells.

Metabolism in males and females is distinct. Differences are usually linked to sexual reproduction, with circulating signals (e.g. hormones) playing major roles. In contrast, sex differences prior to sexual maturity and intrinsic to individual metabolic tissues are less understood. We analyzed Drosophila melanogaster larvae and find that males store more fat than females, the opposite of the sexual dimorphism in adults. We show that metabolic differences are intrinsic to the major fat storage tissue, including many differences in the expression of metabolic genes. Our previous work identified fat storage roles for Spenito (Nito), a conserved RNA-binding protein and regulator of sex determination. Nito knockdown specifically in the fat storage tissue abolished fat differences between males and females. We further show that Nito is required for sex-specific expression of the master regulator of sex determination, Sex-lethal (Sxl). "Feminization" of fat storage cells via tissue-specific overexpression of a Sxl target gene made larvae lean, reduced the fat differences between males and females, and induced female-like metabolic gene expression. Altogether, this study supports a model in which Nito autonomously controls sexual dimorphisms and differential expression of metabolic genes in fat cells in part through its regulation of the sex determination pathway.

Auxin Exposure Disrupts Feeding Behavior and Fatty Acid Metabolism in Adult Drosophila.

The ease of genetic manipulation in Drosophila melanogaster using the Gal4/UAS system has been beneficial in addressing key biological questions. Current modifications of this methodology to temporally induce transgene expression require temperature changes or exposure to exogenous compounds, both of which have been shown to have detrimental effects on physiological processes. The recently described auxin-inducible gene expression system (AGES) utilizes the plant hormone auxin to induce transgene expression and is proposed to be the least toxic compound for genetic manipulation, with no obvious effects on Drosophila development and survival in one wild-type strain. Here we show that auxin delays larval development in another widely-used fly strain, and that short- and long-term auxin exposure in adult Drosophila induces observable changes in physiology and feeding behavior. We further reveal a dosage response to adult survival upon auxin exposure, and that the recommended auxin concentration for AGES alters feeding activity. Furthermore, auxin fed male and female flies exhibit a significant decrease in triglyceride levels and display altered transcription of fatty acid metabolism genes. Although fatty acid metabolism is disrupted, auxin does not significantly impact adult female fecundity or progeny survival, suggesting AGES may be an ideal methodology for studying limited biological processes. These results emphasize that experiments using temporal binary systems must be carefully designed and controlled to avoid confounding effects and misinterpretation of results.

Red Blood Cell Metabolism In Vivo and In Vitro.

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.

Dasatinib and Trametinib Promote Anti-Tumor Metabolic Activity.

Thyroid cancer is the most common endocrine neoplasm, and despite its overall high survival rate, patients with metastatic disease or tumors that resist radioactive iodine experience a significantly worse prognosis. Helping these patients requires a better understanding of how therapeutics alter cellular function. Here, we describe the change in metabolite profiles after treating thyroid cancer cells with the kinase inhibitors dasatinib and trametinib. We reveal alterations to glycolysis, the TCA cycle, and amino acid levels. We also highlight how these drugs promote short-term accumulation of the tumor-suppressive metabolite 2-oxoglutarate, and demonstrate that it reduces the viability of thyroid cancer cells in vitro. These results show that kinase inhibition profoundly alters the metabolome of cancer cells and highlight the need to better understand how therapeutics reprogram metabolic processes, and ultimately, cancer cell behavior.

Metabolomic analysis of Drosophila melanogaster larvae lacking Pyruvate kinase.

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 , however, are complicated by the fact that the fly genome encodes six 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 five Drosophila Pyk paralogs have significantly diverged from the canonical enzyme. Consistent with this observation, metabolomic studies of two different Pyk mutant backgrounds 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 steady state 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 peptidase 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.

Metabolic Profile of Individuals with and without Type 2 Diabetes from Sub-Saharan Africa.

Epidemiological data predicts that sub-Saharan Africa will have the largest increase in type 2 diabetes (T2D) prevalence over the next two decades. Metabolomics studies have identified biomarkers that could improve T2D diagnosis and follow-up. However, no studies have characterized the metabolome of people from sub-Saharan Africa. Plasma samples from Senegalese individuals with T2D (n = 31) or without T2D (n = 34) were compared using measures of oxidative stress damage and plasma antioxidant enzyme activity and mass-spectrometry-based metabolomics analyses. Results showed that glucose, lactate, and tricarboxylic acid metabolites (fumarate, malate, and succinate) were increased in the T2D group, suggesting alterations in glycolysis and mitochondrial dysfunction. Several amino acids (leucine, isoleucine, valine, and tryptophan) and long-to-very-long-chain fatty acids were higher in the T2D group. Finally, elevated levels of ketone bodies and acylcarnitines were observed along with increased levels of oxidative stress damage and antioxidant activity. In conclusion, the T2D group exhibited modifications in metabolites previously shown to be associated with T2D risk in populations from other areas of the world. Future studies should seek to test whether these metabolites could be used as predictors for T2D-related complications in people from sub-Saharan Africa.

Metabolic Signatures of Performance in Elite World Tour Professional Male Cyclists.

BACKGROUND AND OBJECTIVE: Metabolomics studies of recreational and elite athletes have been so far limited to venipuncture-dependent blood sample collection in the setting of controlled training and medical facilities. However, limited to no information is currently available to determine if findings in laboratory settings are translatable to a real-world scenario in elite competitions. The goal of this study was to define molecular signatures of exertion under controlled exercise conditions and use these signatures as a framework for assessing cycling performance in a World Tour competition. METHODS: To characterize molecular profiles of exertion in elite athletes during cycling, we performed metabolomics analyses on blood isolated from 28 international-level, elite, World Tour professional male athletes from a Union Cycliste Internationale World Team taken before and after a graded exercise test to volitional exhaustion and before and after a long aerobic training session. Moreover, established signatures were then used to characterize the metabolic physiology of five of these cyclists who were selected to represent the same Union Cycliste Internationale World Team during a seven-stage elite World Tour race. RESULTS: Using dried blood spot collection to circumvent logistical hurdles associated with field sampling, these studies defined metabolite signatures and fold change ranges of anaerobic or aerobic exertion in elite cyclists, respectively. Blood profiles of lactate, carboxylic acids, fatty acids, and acylcarnitines differed between exercise modes. The graded exercise test elicited significant two- to three-fold accumulations in lactate and succinate, in addition to significant elevations in free fatty acids and acylcarnitines. Conversely, the long aerobic training session elicited a larger magnitude of increase in fatty acids and acylcarnitines without appreciable increases in lactate or succinate. Comparable signatures were revealed after sprinting and climbing stages, respectively, in a World Tour race. In addition, signatures of elevated fatty acid oxidation capacity correlated with competitive performance. CONCLUSIONS: Collectively, these studies provide a unique view of alterations in the blood metabolome of elite athletes during competition and at the peak of their performance capabilities. Furthermore, they demonstrate the utility of dried blood sampling for omics analysis, thereby enabling molecular monitoring of athletic performance in the field during training and competition.

Platelets from blood diversion pouches (DPs) are a suitable alternative for functional, bioenergetic, and metabolomic analyses.

BACKGROUND: The collection of the first blood flow into a diversion pouch (DP) has become widely adopted in blood donation systems to reduce whole-blood unit contamination from skin bacteria. The strict control of pre-analytical variables, such as blood collection and proper anticoagulant selection, is critical to diminish experimental variability when studying different aspects of platelet biology. We hypothesize that the functional, mitochondrial, and metabolomic profiles of platelets isolated from the DP are not different from the ones isolated from standard venipuncture (VP), thus representing a suitable collection method of platelets for experimental purposes. MATERIALS AND METHODS: Whole blood from the blood DP or VP was collected. Platelets were subsequently isolated and washed following standard protocols. Platelet function was assessed by flow cytometry, light transmission aggregometry, clot retraction, and under flow conditions using the total thrombus formation analyzer (T-TAS). Mitochondrial function and the platelet metabolome profiles were determined by the Seahorse extracellular flux analyzer (Agilent, Santa Clara, CA, USA) and ultra-high-pressure liquid chromatography-mass spectrometry metabolomics, respectively. RESULTS: Platelets isolated from VP and the DP have similar functional, mitochondrial, and metabolic profiles with no significant differences between both groups at baseline and upon activation by any of the assays mentioned above. DISCUSSION: The findings of our study support the use of platelets from the DP for performing functional and metabolic studies on platelets from a wide range of blood donors. The DP may serve as an alternative blood collection method to standard VP, allowing the study of diverse aspects of platelet biology, such as age, sex, race, and ethnicity, in many eligible individuals for blood donation.

Metabolic correlates to critical speed in murine models of sickle cell disease.

Introduction: Exercise intolerance is a common clinical manifestation in patients with sickle cell disease (SCD), though the mechanisms are incompletely understood. Methods: Here we leverage a murine mouse model of sickle cell disease, the Berkeley mouse, to characterize response to exercise via determination of critical speed (CS), a functional measurement of mouse running speed upon exerting to exhaustion. Results: Upon observing a wide distribution in critical speed phenotypes, we systematically determined metabolic aberrations in plasma and organs-including heart, kidney, liver, lung, and spleen-from mice ranked based on critical speed performances (top vs. bottom 25%). Results indicated clear signatures of systemic and organ-specific alterations in carboxylic acids, sphingosine 1-phosphate and acylcarnitine metabolism. Metabolites in these pathways showed significant correlations with critical speed across all matrices. Findings from murine models were thus further validated in 433 sickle cell disease patients (SS genotype). Metabolomics analyses of plasma from 281 subjects in this cohort (with HbA < 10% to decrease confounding effects of recent transfusion events) were used to identify metabolic correlates to sub-maximal exercise test performances, as measure by 6 min walking test in this clinical cohort. Results confirmed strong correlation between test performances and dysregulated levels of circulating carboxylic acids (especially succinate) and sphingosine 1-phosphate. Discussion: We identified novel circulating metabolic markers of exercise intolerance in mouse models of sickle cell disease and sickle cell patients.

pH regulates hematopoietic stem cell potential via polyamines.

Upon ex vivo culture, hematopoietic stem cells (HSCs) quickly lose potential and differentiate into progenitors. The identification of culture conditions that maintain the potential of HSCs ex vivo is therefore of high clinical interest. Here, we demonstrate that the potential of murine and human HSCs is maintained when cultivated for 2 days ex vivo at a pH of 6.9, in contrast to cultivation at the commonly used pH of 7.4. When cultivated at a pH of 6.9, HSCs remain smaller, less metabolically active, less proliferative and show enhanced reconstitution ability upon transplantation compared to HSC cultivated at pH 7.4. HSCs kept at pH 6.9 show an attenuated polyamine pathway. Pharmacological inhibition of the polyamine pathway in HSCs cultivated at pH 7.4 with DFMO mimics phenotypes and potential of HSCs cultivated at pH 6.9. Ex vivo exposure to a pH of 6.9 is therefore a positive regulator of HSC function by reducing polyamines. These findings might improve HSC short-term cultivation protocols for transplantation and gene therapy interventions.

Vitamin B5 and succinyl-CoA improve ineffective erythropoiesis in SF3B1-mutated myelodysplasia.

Patients with myelodysplastic syndrome and ring sideroblasts (MDS-RS) present with symptomatic anemia due to ineffective erythropoiesis that impedes their quality of life and increases morbidity. More than 80% of patients with MDS-RS harbor splicing factor 3B subunit 1 (SF3B1) mutations, the founder aberration driving MDS-RS disease. Here, we report how mis-splicing of coenzyme A synthase (COASY), induced by mutations in SF3B1, affects heme biosynthesis and erythropoiesis. Our data revealed that COASY was up-regulated during normal erythroid differentiation, and its silencing prevented the formation of erythroid colonies, impeded erythroid differentiation, and precluded heme accumulation. In patients with MDS-RS, loss of protein due to COASY mis-splicing led to depletion of both CoA and succinyl-CoA. Supplementation with COASY substrate (vitamin B5) rescued CoA and succinyl-CoA concentrations in SF3B1mut cells and mended erythropoiesis differentiation defects in MDS-RS primary patient cells. Our findings reveal a key role of the COASY pathway in erythroid maturation and identify upstream and downstream metabolites of COASY as a potential treatment for anemia in patients with MDS-RS.