Mechanisms and significance of tissue-specific MICU regulation of the mitochondrial calcium uniporter complex.

Mitochondrial Ca2+ uptake, mediated by the mitochondrial Ca2+ uniporter, regulates oxidative phosphorylation, apoptosis, and intracellular Ca2+ signaling. Previous studies suggest that non-neuronal uniporters are exclusively regulated by a MICU1-MICU2 heterodimer. Here, we show that skeletal-muscle and kidney uniporters also complex with a MICU1-MICU1 homodimer and that human/mouse cardiac uniporters are largely devoid of MICUs. Cells employ protein-importation machineries to fine-tune the relative abundance of MICU1 homo- and heterodimers and utilize a conserved MICU intersubunit disulfide to protect properly assembled dimers from proteolysis by YME1L1. Using the MICU1 homodimer or removing MICU1 allows mitochondria to more readily take up Ca2+ so that cells can produce more ATP in response to intracellular Ca2+ transients. However, the trade-off is elevated ROS, impaired basal metabolism, and higher susceptibility to death. These results provide mechanistic insights into how tissues can manipulate mitochondrial Ca2+ uptake properties to support their unique physiological functions.

Interferon-inducible phospholipids govern IFITM3-dependent endosomal antiviral immunity.

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.

Chaperone-mediated autophagy sustains haematopoietic stem-cell function.

The activation of mostly quiescent haematopoietic stem cells (HSCs) is a prerequisite for life-long production of blood cells1. This process requires major molecular adaptations to allow HSCs to meet the regulatory and metabolic requirements for cell division2-4. The mechanisms that govern cellular reprograming upon stem-cell activation, and the subsequent return of stem cells to quiescence, have not been fully characterized. Here we show that chaperone-mediated autophagy (CMA)5, a selective form of lysosomal protein degradation, is involved in sustaining HSC function in adult mice. CMA is required for protein quality control in stem cells and for the upregulation of fatty acid metabolism upon HSC activation. We find that CMA activity in HSCs decreases with age and show that genetic or pharmacological activation of CMA can restore the functionality of old mouse and human HSCs. Together, our findings provide mechanistic insights into a role for CMA in sustaining quality control, appropriate energetics and overall long-term HSC function. Our work suggests that CMA may be a promising therapeutic target for enhancing HSC function in conditions such as ageing or stem-cell transplantation.

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

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.

Small-molecule Ro-08-2750 interacts with many RNA-binding proteins and elicits MUSASHI2-independent phenotypes.

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.

Specialized interferon action in COVID-19.

The impacts of interferon (IFN) signaling on COVID-19 pathology are multiple, with both protective and harmful effects being documented. We report here a multiomics investigation of systemic IFN signaling in hospitalized COVID-19 patients, defining the multiomics biosignatures associated with varying levels of 12 different type I, II, and III IFNs. The antiviral transcriptional response in circulating immune cells is strongly associated with a specific subset of IFNs, most prominently IFNA2 and IFNG. In contrast, proteomics signatures indicative of endothelial damage and platelet activation associate with high levels of IFNB1 and IFNA6. Seroconversion and time since hospitalization associate with a significant decrease in a specific subset of IFNs. Additionally, differential IFN subtype production is linked to distinct constellations of circulating myeloid and lymphoid immune cell types. Each IFN has a unique metabolic signature, with IFNG being the most associated with activation of the kynurenine pathway. IFNs also show differential relationships with clinical markers of poor prognosis and disease severity. For example, whereas IFNG has the strongest association with C-reactive protein and other immune markers of poor prognosis, IFNB1 associates with increased neutrophil to lymphocyte ratio, a marker of late severe disease. Altogether, these results reveal specialized IFN action in COVID-19, with potential diagnostic and therapeutic implications.

Sall4 restricts glycolytic metabolism in limb buds through transcriptional regulation of glycolytic enzyme genes.

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.

Metabolic reprogramming contributes to radioprotection by protein kinase Cδ.

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-[13C6] glucose showed reduced utilization of glucose for glycolysis in PKCδ-depleted cells and no increase in U-[13C6] glucose incorporation into purines or pyrimidines. In contrast, isotope tracing with [13C5, 15N2] 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.

Maternal AMPK pathway activation with uterine artery blood flow and fetal growth maintenance during hypoxia.

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 α1-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 α1-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.

Primaquine-5,6-Orthoquinone Is Directly Hemolytic to Older G6PD Deficient RBCs in a Humanized Mouse Model.

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 (hG6PDMed-) that recapitulates the human biology of RBC age-dependent enzyme decay, as well as an isogenic matched control mouse with human nondeficient G6PD hG6PDND 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 hG6PDMed- 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.

Light-Driven Metabolic Pathways in Non-Photosynthetic Biohybrid Bacteria.

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.

Functional effects of an African glucose-6-phosphate dehydrogenase (G6PD) polymorphism (Val68Met) on red blood cell hemolytic propensity and post-transfusion recovery.

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.

Erythrocyte adenosine A2B receptor prevents cognitive and auditory dysfunction by promoting hypoxic and metabolic reprogramming.

Hypoxia drives aging and promotes age-related cognition and hearing functional decline. Despite the role of erythrocytes in oxygen (O2) transport, their role in the onset of aging and age-related cognitive decline and hearing loss (HL) remains undetermined. Recent studies revealed that signaling through the erythrocyte adenosine A2B receptor (ADORA2B) promotes O2 release to counteract hypoxia at high altitude. However, nothing is known about a role for erythrocyte ADORA2B in age-related functional decline. Here, we report that loss of murine erythrocyte-specific ADORA2B (eAdora2b-/-) accelerates early onset of age-related impairments in spatial learning, memory, and hearing ability. eAdora2b-/- mice display the early aging-like cellular and molecular features including the proliferation and activation of microglia and macrophages, elevation of pro-inflammatory cytokines, and attenuation of hypoxia-induced glycolytic gene expression to counteract hypoxia in the hippocampus (HIP), cortex, or cochlea. Hypoxia sufficiently accelerates early onset of cognitive and cochlear functional decline and inflammatory response in eAdora2b-/- mice. Mechanistically, erythrocyte ADORA2B-mediated activation of AMP-activated protein kinase (AMPK) and bisphosphoglycerate mutase (BPGM) promotes hypoxic and metabolic reprogramming to enhance production of 2,3-bisphosphoglycerate (2,3-BPG), an erythrocyte-specific metabolite triggering O2 delivery. Significantly, this finding led us to further discover that murine erythroblast ADORA2B and BPGM mRNA levels and erythrocyte BPGM activity are reduced during normal aging. Overall, we determined that erythrocyte ADORA2B-BPGM axis is a key component for anti-aging and anti-age-related functional decline.

Divergent Genetic Regulation of Nitric Oxide Production between C57BL/6J and Wild-Derived PWD/PhJ Mice Controls Postactivation Mitochondrial Metabolism, Cell Survival, and Bacterial Resistance in Dendritic Cells.

Dendritic cell (DC) activation is characterized by sustained commitment to glycolysis that is a requirement for survival in DC subsets that express inducible NO synthase (Nos2) due to NO-mediated inhibition of mitochondrial respiration. This phenomenon primarily has been studied in DCs from the classic laboratory inbred mouse strain C57BL/6J (B6) mice, where DCs experience a loss of mitochondrial function due to NO accumulation. To assess the conservation of NO-driven metabolic regulation in DCs, we compared B6 mice to the wild-derived genetically divergent PWD/PhJ (PWD) strain. We show preserved mitochondrial respiration and enhanced postactivation survival due to attenuated NO production in LPS-stimulated PWD DCs phenocopying human monocyte-derived DCs. To genetically map this phenotype, we used a congenic mouse strain (B6.PWD-Chr11.2) that carries a PWD-derived portion of chromosome 11, including Nos2, on a B6 background. B6.PWD-Chr11.2 DCs show preserved mitochondrial function and produce lower NO levels than B6 DCs. We demonstrate that activated B6.PWD-Chr11.2 DCs maintain mitochondrial respiration and TCA cycle carbon flux, compared with B6 DCs. However, reduced NO production by the PWD Nos2 allele results in impaired cellular control of Listeria monocytogenes replication. These studies establish a natural genetic model for restrained endogenous NO production to investigate the contribution of NO in regulating the interplay between DC metabolism and immune function. These findings suggest that reported differences between human and murine DCs may be an artifact of the limited genetic diversity of the mouse models used, underscoring the need for mouse genetic diversity in immunology research.

Multiomic Analysis Reveals Disruption of Cholesterol Homeostasis by Cannabidiol in Human Cell Lines.

The nonpsychoactive cannabinoid, cannabidiol (CBD), is Food and Dug Administration approved for treatment of two drug-resistant epileptic disorders and is seeing increased use among the general public, yet the mechanisms that underlie its therapeutic effects and side-effect profiles remain unclear. Here, we report a systems-level analysis of CBD action in human cell lines using temporal multiomic profiling. FRET-based biosensor screening revealed that CBD elicits a sharp rise in cytosolic calcium, and activation of AMP-activated protein kinase in human keratinocyte and neuroblastoma cell lines. CBD treatment leads to alterations in the abundance of metabolites, mRNA transcripts, and proteins associated with activation of cholesterol biosynthesis, transport, and storage. We found that CBD rapidly incorporates into cellular membranes, alters cholesterol accessibility, and disrupts cholesterol-dependent membrane properties. Sustained treatment with high concentrations of CBD induces apoptosis in a dose-dependent manner. CBD-induced apoptosis is rescued by inhibition of cholesterol synthesis and potentiated by compounds that disrupt cholesterol trafficking and storage. Our data point to a pharmacological interaction of CBD with cholesterol homeostasis pathways, with potential implications in its therapeutic use.

PFKFB3 Inhibits Fructose Metabolism in Pulmonary Microvascular Endothelial 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.

Hemolysis and Metabolic Lesion of G6PD Deficient RBCs in Response to Dapsone Hydroxylamine in a Humanized Mouse Model.

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.

Metabolic signatures of cardiorenal dysfunction in plasma from sickle cell patients as a function of therapeutic transfusion and hydroxyurea treatment.

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.

Simultaneous inhibition of Sirtuin 3 and cholesterol homeostasis targets acute myeloid leukemia stem cells by perturbing fatty acid ß-oxidation and inducing lipotoxicity.

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.

In vivo evaluation of the effect of sickle cell hemoglobin S, C and therapeutic transfusion on erythrocyte metabolism and cardiorenal dysfunction.

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.