Pentose Phosphate Pathway Intermediates Research Articles

Insulin and glycolysis dependency of cardioprotection by nicotinamide riboside

Decreased nicotinamide adenine dinucleotide (NAD+) levels contribute to various pathologies such as ageing, diabetes, heart failure and ischemia–reperfusion injury (IRI). Nicotinamide riboside (NR) has emerged as a promising therapeutic NAD+ precursor due to efficient NAD+ elevation and was recently shown to be the only agent able to reduce cardiac IRI in models employing clinically relevant anesthesia. However, through which metabolic pathway(s) NR mediates IRI protection remains unknown. Furthermore, the influence of insulin, a known modulator of cardioprotective efficacy, on the protective effects of NR has not been investigated. Here, we used the isolated mouse heart allowing cardiac metabolic control to investigate: (1) whether NR can protect the isolated heart against IRI, (2) the metabolic pathways underlying NR-mediated protection, and (3) whether insulin abrogates NR protection. NR protection against cardiac IRI and effects on metabolic pathways employing metabolomics for determination of changes in metabolic intermediates, and 13C-glucose fluxomics for determination of metabolic pathway activities (glycolysis, pentose phosphate pathway (PPP) and mitochondrial/tricarboxylic acid cycle (TCA cycle) activities), were examined in isolated C57BL/6N mouse hearts perfused with either (a) glucose + fatty acids (FA) (“mild glycolysis group”), (b) lactate + pyruvate + FA (“no glycolysis group”), or (c) glucose + FA + insulin (“high glycolysis group”). NR increased cardiac NAD+ in all three metabolic groups. In glucose + FA perfused hearts, NR reduced IR injury, increased glycolytic intermediate phosphoenolpyruvate (PEP), TCA intermediate succinate and PPP intermediates ribose-5P (R5P) / sedoheptulose-7P (S7P), and was associated with activated glycolysis, without changes in TCA cycle or PPP activities. In the “no glycolysis” hearts, NR protection was lost, whereas NR still increased S7P. In the insulin hearts, glycolysis was largely accelerated, and NR protection abrogated. NR still increased PPP intermediates, with now high 13C-labeling of S7P, but NR was unable to increase metabolic pathway activities, including glycolysis. Protection by NR against IRI is only present in hearts with low glycolysis, and is associated with activation of glycolysis. When activation of glycolysis was prevented, through either examining “no glycolysis” hearts or “high glycolysis” hearts, NR protection was abolished. The data suggest that NR’s acute cardioprotective effects are mediated through glycolysis activation and are lost in the presence of insulin because of already elevated glycolysis.

Deciphering Metabolomic Signatures Associated with Sickle Cell Disease Using Mouse Models

Introduction: Sickle cell disease (SCD) is debilitating and affects millions of people worldwide. Although SCD is caused by a single-point mutation of the β-globin chain of hemoglobin, the clinical manifestations are heterogeneous, and there is a lack of well-characterized predictive biomarkers of disease progression. Metabolomics is a promising tool to interrogate disease pathophysiology and underlying molecular mechanisms. Although prior metabolomic studies have been performed on human and murine red blood cells (RBCs) in the context of SCD, no systematic investigation across commonly used mouse models of SCD has been published. In the present study, we examined the blood cell metabolome of 2 widely used SCD mouse models to study and compare the metabolomic changes in disease models. Methods: Blood samples were collected from 5 groups of mice (10-14 weeks old, n=6-7 per group): wild-type (WT) C57BL/6J mice; Townes AA, AS, and SS mice; and Berkeley SCD mice. Direct injection high-resolution mass spectrometry was used to generate untargeted metabolomics data. Three matrices (RBC, plasma, and whole blood) were isolated from each mouse group. The RBC fraction, containing peripheral blood mononuclear cells, was separated from plasma via centrifugation of whole blood. The current study focuses on the RBC dataset.Raw profile data were centroided, merged, and recalibrated using methods previously described (Fuhrer et al. Anal Chem 2011). Putative annotations were generated based on compounds contained in the Human Metabolome Database, KEGG, and ChEBI databases using accurate mass per charge (tolerance 0.001 m/z) and isotopic correlation patterns. Z-score normalization was applied across all samples for unsupervised clustering using principal component analysis (PCA) and to identify metabolites with significant changes between groups. Metabolites were mapped to central RBC metabolic pathways using MetaboAnalyst (Xia et al. Nat Protoc 2011) and manual curation. Results: Unsupervised PCA that used all detectable RBC metabolites resulted in clear separation of mouse groups by disease status (Figure 1), suggesting robust metabolomic changes in mouse models of SCD. Focusing on the top divergent metabolites across the mouse groups, several enriched pathways were identified, such as amino acid metabolism, pyrimidine and purine metabolism, and aminoacyl-tRNA biosynthesis, all potentially attributable to the buildup of RBC precursors. Metabolites characteristic of immature RBCs were further examined, with elevated hexokinase activity (hexose:hexose-6-phosphate ratio), ATP levels, and mitochondrial metabolites found in RBCs from SCD, in line with the significantly elevated reticulocyte content in SCD mice. Metabolites were curated from additional relevant RBC metabolic pathways; some showed significant differences among mouse groups, and others were significantly elevated in sickle mice (Townes SS and Berkeley SCD) compared with non-SCD controls (WT and Townes AA), including some glycolytic and pentose phosphate pathway (PPP) intermediates (Figure 2). The product-to-substrate ratios of metabolite levels from glycolytic and oxidative PPP (oxPPP) steps suggested glycolysis was favored and the later stage of the oxPPP was reduced in mouse models of SCD. There was also a higher metabolic turnover of glutathione, which is characteristic of SCD in humans, but we observed divergent metabolomic signatures between the disease models in our study (Figure 2). Conclusions: To our knowledge, this is the most comprehensive metabolomics study in mouse models of SCD. It provides a foundation to assess disease biology and response to treatment in future studies. Untargeted RBC metabolomics revealed metabolomic signatures that are consistent with human disease pathology in both murine SCD models, including reticulocytosis, hemolysis, and mitochondria retention in RBCs, and signatures that are unique to the murine models. Of the 2 main RBC metabolic pathways, glycolysis was favored over the oxPPP in SCD mice. This is the first step towards understanding the impact of SCD on RBC carbohydrate metabolism.

Nicotinamide riboside cardioprotection is mediated through glycolysis activation

Abstract Purpose Low NAD+ levels are observed in pathologies such as ageing, diabetes, heart failure and ischemia-reperfusion (IR) injury. Nicotinamide riboside (NR) has emerged as promising therapeutic NAD+ precursor [1,2]. NR was recently reported to protect against in vivo cardiac IR injury [3], but underlying mechanisms of protection were unexplored. Here we used the isolated mouse heart allowing metabolic control to examine NR’s metabolic mechanisms of cardiac protection. Methods Isolated C57BL/6N hearts were Langendorff-perfused with 5.5 mM glucose and 0.2 mM fatty acids (FA), and subjected to 35 min global ischemia and 90 min reperfusion. NR (50 mg/L) or vehicle were administered for 25 min before ischemia, and during the first 25 min of reperfusion. Cardiac functional recovery and cell death parameters (lactate dehydrogenase (LDH) release, % infarct size) were determined. To examine NR’s effects on cardiac metabolism, hearts were perfused with 5.5 mM 13C-glucose and 0.2 mM FA and analyzed by metabolomics and fluxomics using LC-MS techniques before ischemia. The dependency of NR’s protection on glycolysis was tested in hearts perfused with glucose replaced by pyruvate and lactate ("no glycolysis" hearts). Finally, NR’s protection and effects on cardiac metabolism were also examined in "high glycolysis" hearts, by adding insulin to the perfusate. Results NR reduced infarct size, LDH release and improved cardiac functional recovery for glucose/FA perfused hearts, demonstrating NR’s cardioprotection. In these glucose/FA hearts, metabolomics analysis showed NR increased NAD+, glycolytic intermediate phosphoenolpyruvate (PEP), Krebs cycle intermediate succinate and pentose phosphate pathway (PPP) intermediates ribose-5P (R5P) and sedoheptulose-7P (S7P) (Fig. 1). Fluxomics analysis demonstrated that NR’s protection was associated with activated glycolysis, without changes in tricarboxylic acid (TCA) cycle or PPP activities. No 13C-labeling of S7P was observed (Fig. 1). NR protection was lost in the "no glycolysis" hearts. In the "high glycolysis" hearts, NR was unable to protect the heart and increase glycolysis and reduced anaerobic glycolysis and ATP with increases in AMP, indicative of NR-induced energetic stress (Fig. 2). Remarkably, in these insulin perfused hearts, NR still increased PPP intermediates, with high 13C-labeling of S7P (Fig. 2). Conclusions NR’s protection against IR injury is only present in hearts with low glycolysis, associated with activation of pre-ischemic glycolysis. When activation of glycolysis was prevented, through either examining "no glycolysis" hearts or "high glycolysis" hearts, NR protection was abolished. NR treatment increased pre-ischemic PPP intermediates in either low or high glycolysis hearts, whereby insulin was necessary to incorporate 13C-glucose into S7P. The data suggest that NR’s acute cardioprotective effects are mediated through the activation of glycolysis.NR effects in low-glycolysis heartsNR effects in high-glycolysis hearts

Metabolic Hijacking of Hexose Metabolism to Ascorbate Synthesis Is the Unifying Biochemical Basis of Murine Liver Fibrosis.

We wished to understand the metabolic reprogramming underlying liver fibrosis progression in mice. Administration to male C57BL/6J mice of the hepatotoxins carbon tetrachloride (CCl4), thioacetamide (TAA), or a 60% high-fat diet, choline-deficient, amino-acid-defined diet (HF-CDAA) was conducted using standard protocols. Livers collected at different times were analyzed by gas chromatography-mass spectrometry-based metabolomics. RNA was extracted from liver and assayed by qRT-PCR for mRNA expression of 11 genes potentially involved in the synthesis of ascorbic acid from hexoses, Gck, Adpgk, Hk1, Hk2, Ugp2, Ugdh, Ugt1a1, Akr1a4, Akr1b3, Rgn and Gulo. All hepatotoxins resulted in similar metabolic changes during active fibrogenesis, despite different etiology and resultant scarring pattern. Diminished hepatic glucose, galactose, fructose, pentose phosphate pathway intermediates, glucuronic acid and long-chain fatty acids were compensated by elevated ascorbate and the product of collagen prolyl 4-hydroxylase, succinate and its downstream metabolites fumarate and malate. Recovery from the HF-CDAA diet challenge (F2 stage fibrosis) after switching to normal chow was accompanied by increased glucose, galactose, fructose, ribulose 5-phosphate, glucuronic acid, the ascorbate metabolite threonate and diminished ascorbate. During the administration of CCl4, TAA and HF-CDAA, aldose reductase Akr1b3 transcription was induced six- to eightfold, indicating increased conversion of glucuronic acid to gulonic acid, a precursor of ascorbate synthesis. Triggering hepatic fibrosis by three independent mechanisms led to the hijacking of glucose and galactose metabolism towards ascorbate synthesis, to satisfy the increased demand for ascorbate as a cofactor for prolyl 4-hydroxylase for mature collagen production. This metabolic reprogramming and causal gene expression changes were reversible. The increased flux in this pathway was mediated predominantly by increased transcription of aldose reductase Akr1b3.

Up-regulation of thioesterase superfamily member 2 in skeletal muscle promotes hepatic steatosis and insulin resistance in mice.

Thioesterase superfamily member 2 (Them2) is highly expressed in liver and oxidative tissues, where it hydrolyzes long-chain fatty acyl-CoA esters to free fatty acids and CoA. Although mice globally lacking Them2 (Them2-/- ) are protected against diet-induced obesity, hepatic steatosis (HS), and insulin resistance (IR), liver-specific Them2-/- mice remain susceptible. The aim of this study was to test whether Them2 activity in extrahepatic oxidative tissues is a primary determinant of HS and IR. Upon observing IR and up-regulation of Them2 in skeletal, but not cardiac, muscle of high-fat-diet (HFD)-fed wild-type compared to Them2-/- mice, we created mice with Them2 specifically deleted in skeletal (S-Them2-/- ) and cardiac muscle (C-Them2-/- ), as well as in adipose tissue (A-Them2-/- ). When fed an HFD, S-Them2-/- , but not C-Them2-/- or A-Them2-/- , mice exhibited reduced weight gain and improved glucose homeostasis and insulin sensitivity. Reconstitution of Them2 expression in skeletal muscle of global Them2-/- mice, using adeno-associated virus, was sufficient to restore excess weight gain. Increased rates of fatty acid oxidation in skeletal muscle of S-Them2-/- mice contributed to protection from HFD-induced HS by increasing VLDL triglyceride secretion rates in response to greater demand. Increases in insulin sensitivity were further attributable to alterations in production of skeletal muscle metabolites, including short-chain fatty acids, branched-chain amino acids, and pentose phosphate pathway intermediates, as well as in expression of myokines that modulate insulin responsiveness. These results reveal a key role for skeletal muscle Them2 in the pathogenesis of HS and IR and implicate it as a target in the management of NAFLD.

1249. Metabolomic Profile of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus (hVISA) in Latin-American MRSA Isolates

BackgroundVancomycin (VAN) is a first-line therapeutic option in severe infections caused by MRSA in Latin-America (LA). Development of reduced susceptibility to VAN has been associated with multiple changes in genes encoding pathways for cell wall metabolism and envelope stress responses. Nevertheless, a detailed and coherent mechanistic model to explain the phenotype remains elusive. To gain further insights into the hVISA phenotype, we sought to explore the metabolomic profile of hVISA isolates from LA. MethodsThe undirected profile of intracellular S. aureus metabolites was analysed in four clinical isolates (two hVISA and two VSSA [Vancomycin susceptible S. aureus]) belonging to the Chilean/Cordobes clone-ST5 (the predominant hVISA lineage in LA), and two reference strains Mu3 and N315. The metabolites were obtained in mid-exponential growth phase in trypticase soy broth in five independent replicates. The metabolites were determined by reverse phase liquid chromatography and hydrophilic interaction. The metabolic profile was determined by variable importance in the projection score (VIP > 1). The differences between hVISA and VSSA were maximized by orthogonal partial least squares discriminant analysis (OPLS-DA) and the affected metabolic pathways were identified with MetaboAnalyst.ResultsAmong the differences identified in the metabolic profiles of hVISA respect to VSSA, 69 metabolites were relevant. Of these, 47 were fatty acids (including glycerol), 7 amino acids and 6 nucleosides (Table 1). These changes mainly impact the biosynthesis of amino acids derived from pyruvate since tyrosine, valine and leucine, had a reduction of 34%, 57% and 41% in hVISA compared to VSSA, respectively, which suggests alterations of the acid cycle tricarboxylic (TCA). Additionally, a reduction in purine and pyrimidine metabolism in hVISA was identified with reduction of nucleosides and dinucleotides derived from the pentose phosphate pathway.Table 1. Metabolites with higher VIP scores in the comparison of the hVISA and VSSA profile ConclusionWe were able to observe metabolic alterations in TCA, pentose phosphate pathway and purine intermediates in hVISA-ST5 isolates. Our results support that gluconeogenesis and biosynthesis of carbohydrates and nucleic acids are the main pathways involved in the reduced susceptibility to VAN as reported in VISA isolates.Disclosures Cesar A. Arias, M.D., MSc, Ph.D., FIDSA, Entasis Therapeutics (Grant/Research Support)MeMed Diagnostics (Grant/Research Support)Merk (Grant/Research Support) Lorena Diaz, PhD , Nothing to disclose

Metabolomics Analysis Discovers Estrogen Altering Cell Proliferation via the Pentose Phosphate Pathway in Infertility Patient Endometria.

Estrogen therapy is widely used as a supplementary treatment after hysteroscopy for female infertility patients owing to its protective function that improves endometrial regeneration and menstruation, inhibits recurrent adhesions, and improves subsequent conception rate. The endometrial protective function of such estrogen administration pre-surgery is still controversial. In the current study, 12 infertility patients were enrolled, who were treated with estrogen before hysteroscopy surgery. Using cutting-edge metabolomic analysis, we observed alterations in the pentose phosphate pathway (PPP) intermediates of the patient’s endometrial tissues. Furthermore, using Ishikawa endometrial cells, we validated our clinical discovery and identified estrogen–ESR–G6PD–PPP axial function, which promotes estrogen-induced cell proliferation.

Enhanced glycolysis and GSK3 inactivation promote brain metabolic adaptations following neuronal mitochondrial stress

We analyzed early brain metabolic adaptations in response to mitochondrial dysfunction in a mouse model of mitochondrial encephalopathy with complex IV deficiency [neuron-specific COX10 knockout (KO)]. In this mouse model, the onset of the mitochondrial defect did not coincide with immediate cell death, suggesting early adaptive metabolic responses to compensate for the energetic deficit. Metabolomic analysis in the KO mice revealed increased levels of glycolytic and pentose phosphate pathway intermediates, amino acids and lysolipids. Glycolysis was modulated by enhanced activity of glycolytic enzymes, and not by their overexpression, suggesting the importance of post-translational modifications in the adaptive response. Glycogen synthase kinase 3 inactivation was the most upstream regulation identified, implying that it is a key event in this adaptive mechanism. Because neurons are thought not to rely on glycolysis for adenosine triphosphate production in normal conditions, our results indicate that neurons still maintain their ability to upregulate this pathway when under mitochondrial respiration stress.

The impact of donor sex and age on stored platelet metabolism and post-transfusion recovery.

The impact of donor biology on blood component storability is increasingly appreciated as a determinant of the storage lesion and post-transfusion performances. Platelet metabolism is affected by age and it is critical to platelet responses to activating stimuli in an age-dependent manner. Sex has been previously highlighted as a contributing factor to the platelet proteomics lesion. However, little is known about the impact of donor sex and age on stored platelet metabolism and post-transfusion capacity to circulate. Apheresis platelets were donated via apheresis by 21 healthy volunteers (12 males and 9 females; ages 20 to 59). Metabolomics analyses were performed at day 0 and after 5 days of storage at 22+2 °C, along with autologous post-transfusion recovery and survival studies with 51Cr and 111In. Sex and age significantly impacted platelet metabolism at baseline and upon storage. Platelets from older, male donors were characterised by higher levels of Krebs cycle metabolites, pentose phosphate pathway intermediates and byproducts, deaminated purines and long chain fatty acids. These metabolites ranked amongst the top significant correlates to post-transfusion recoveries. Glutathione homeostasis and sphingosine 1-phosphate were the top positive correlates to long term survival, which was lower in platelets from older, male donors - without reaching statistical significance. In this study we report that donor sex and age have a significant impact on platelet metabolism. Novel metabolic correlates to platelet post-transfusion performances (24 h recovery and long-term survival) were identified through high-resolution, stable isotope-labeled internal standard-assisted metabolomics approach.

Guanine-Based Purines as an Innovative Target to Treat Major Depressive Disorder.

Guanine-Based Purines as an Innovative Target to Treat Major Depressive Disorder.

Non-canonical metabolic pathways in the malaria parasite detected by isotope-tracing metabolomics.

The malaria parasite, Plasmodium falciparum, proliferates rapidly in human erythrocytes by actively scavenging multiple carbon sources and essential nutrients from its host cell. However, a global overview of the metabolic capacity of intraerythrocytic stages is missing. Using multiplex 13C‐labelling coupled with untargeted mass spectrometry and unsupervised isotopologue grouping, we have generated a draft metabolome of P. falciparum and its host erythrocyte consisting of 911 and 577 metabolites, respectively, corresponding to 41% of metabolites and over 70% of the metabolic reaction predicted from the parasite genome. An additional 89 metabolites and 92 reactions were identified that were not predicted from genomic reconstructions, with the largest group being associated with metabolite damage‐repair systems. Validation of the draft metabolome revealed four previously uncharacterised enzymes which impact isoprenoid biosynthesis, lipid homeostasis and mitochondrial metabolism and are necessary for parasite development and proliferation. This study defines the metabolic fate of multiple carbon sources in P. falciparum, and highlights the activity of metabolite repair pathways in these rapidly growing parasite stages, opening new avenues for drug discovery.

Abstract PO-039: Combined in vivo 13C-metabolomics and proteomics approach to optimise immunotherapy response in malignant melanoma

Abstract Metabolic reprogramming is a common feature during tumourigenesis that allows tumours to adapt to nutrient-poor microenvironments, thereby maintaining cell viability and produce biomass for cell proliferation. Increased aerobic lactate fermentation, known as the "Warburg effect" is a well-studied metabolic alteration in melanoma, as well as other cancer types, that renders the tumour microenvironment hypoglycaemic and acidic. Preclinical in vitro and in vivo data show that this phenomenon has immunosuppressive effects and may as well attenuate patient response to immunotherapy. Interestingly, this metabolic alteration distinguishes a tumour and its corresponding microenvironment from healthy tissue and make their metabolic processes susceptible to drug targeting. We hypothesise that drugs normalising tumour metabolism may revert metabolic-induced immunosuppression and increase patient response to immunotherapy. In this study, we use a combined proteomics and 13C-metabolomics approach to investigate the effect of dichloroacetate (DCA) on normalizing tumour metabolism in vivo. DCA reroutes the pyruvate produced in glycolysis to be oxidized in the mitochondria, thereby reducing the flux to lactic acid and neutralising the tumour microenvironment. A seven-day, phase 2 clinical trial of DCA has been planned in 36 patients with malignant melanoma prior to immunotherapy. Pre- and post-DCA treatment biopsies will be taken after intravenous [U-13C]glucose infusion in isotopic steady-state. We present the development of GC-EI-MS(MS) methods for quantitative analysis of -13C-label incorporation in glycolytic, TCA cycle and pentose phosphate pathway intermediates. In addition we have developed targeted proteomics for the absolute quantification of glycolytic and mitochondrial metabolic enzymes using in-house designed -13C-labelled peptide standards based on QConCat technology. These methods will be applied to the respective paired biopsies. Combing the patient-specific response to immunotherapy and DCA with subsequent analysis and computational modelling will enable detailed characterisation of metabolic activity and give insight into metabolic regulation upon treatment in vivo in melanoma patients. Thus, we expect to provide an unprecedented insight into melanoma tumour metabolism and proof-of-concept that targeting metabolism improves immunotherapy response in malignant melanoma patients. Citation Format: Bernardus Evers, Albert Gerding, Jiske Tiersma, Karen van Eunen, Justina C. Wolters, Dirk-Jan Reijngoud, Mathilde Jalving, Barbara M. Bakker. Combined in vivo 13C-metabolomics and proteomics approach to optimise immunotherapy response in malignant melanoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-039.

Two high-rate pentose-phosphate pathways in cancer cells

The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and d-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.

An Integrative Unbiased Global Analysis of the Metabolic Adaptive Response to Tryptophan Deprivation in Classical Hodgkin Lymphoma

Background Tryptophan (trp) is an essential aminoacid, required for de novo NAD+ synthesis. Our previous work showed that the microenvironment in classical Hodgkin Lymphoma is characterized by dysfunctional neutrophils and myeloid derived suppressor cells that produce a trp-degrading enzyme indoelamine deoxygenase (IDO-1), lowering the amount of trp. Aims To detect metabolomics changes in human cHL cell lines exposed in vitro to tryptophan deprivation, an amino acid involved in immune dysregulation and generation of anergic and tolerogenic T- cells. Methods In order to better understand the impact of extra-cellular IDO1 increase on the metabolome of human cHL cells, three human cHL cell lines (L428, L540 and KM-H2) were individually cultured with customized complete media or media lacking tryptophan (W0), +10% dyalised fetal bovum serum, in six independent experiments. After 48 hours of culture the cells were collected for global metabolomic analysis, by gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) platforms by Metabolon Inc. Following normalization to DNA concentration, log transformation and imputation of missing values, if any, with the minimum observed value for each compound, Welch's two-sample t-test was used to identify biochemicals that differed significantly between experimental groups (Table 1). Results The lack of tryptophan in media had a profound effect on the cell metabolome in 2 cell lines, KMH2 and L428 cells, while L540 cell line was pretty resilient (Table 1). In all cell lines, the removal of tryptophan from the media resulted in significantly lower levels of tryptophan. Kynurenine, the metabolic product of IDO-1 action on tryptophan, was lower in all cells, but did not reach significance in the L540 cells, whereas it trended lower in L428 and was significantly lower in the KMH2 compared to controls. Indolelactate, another major tryptophan metabolite was also significantly lower in the L428 and KMH2 cells lines compared to controls. Glucose uptake and aerobic glycolysis are frequently upregulated in tumor cells to support energy needs and provide biosynthetic precursors (e.g. pentose phosphate pathway intermediates for nucleotide synthesis). Known as the Warburg effect, this process of reliance on glucose for energy results in high levels of lactate production. We found that trp deficiency lead to lower levels of the hexose diphosphates (fructose 1,6-diphosphate/glucose 1,6-diphosphate/myo-inositol diphosphates isobar) and dihydroxyacetone phosphate, suggesting the revert of Warburg effect due to reduced bio-energetic requirements for proliferation. In line with this observation, culture in trp deficient media resulted in increased levels of long chain saturated fatty acids and long chain polyunsaturated fatty acids (PUFAs), suggesting that specific-amino acid deficiency leads to an increase in uptake of free fatty acids from the media, to preserve membrane dynamics. Since prolonged trp deprivation (up to 10 days) delayed cell cycle length without affecting proliferation or changes in intracellular amount of NAD+, we investigated changes in mitochondrial membranes network to explain these findings. Trp deprivation induced the rearrangement of the mitochondrial network at 48 hours, with more fission than fusion, as suggested by increased expression of Fis1 and Drp1 and reduced expression of Tfam and Opa1, without affecting significantly mitochondrial mass and depolarization. This adaptive response was associated to increased oxidative stress, as suggested by of reduced glutathione (GSH) and oxidized glutathione (GSSG) in the L540 and KMH2 cells, depletion of gamma-glutamylcysteine, increased cystine, the oxidative product of cysteine, and methionine sulfoxide (an oxidation product of methionine). Conclusions The removal of trp from L428 and KMH2 resulted in changes in the specific-amino acid related metabolites. The adaptive response to trp-depleted microenvironment can revert the Warburg effect, promoting a shift in the glucose use in the futile attempt to preserve mitochondrial function, and increase oxidative stress. Quantity and function of mitochondria network can play a major role in selecting the fittest clones, a metabolic pathway that should be explored as novel non -synthetic lethal targets. Disclosures Puglisi: Amgen:Honoraria.Di Raimondo:Takeda:Consultancy, Honoraria;GILEAD, Incyte:Research Funding;Amgen, Takeda, Novartis:Honoraria;Celgene:Consultancy, Honoraria;Janssen:Consultancy, Honoraria;GSK:Consultancy, Honoraria;Amgen:Consultancy, Honoraria.Romano:Takeda:Honoraria;Novartis:Honoraria.

Mitochondrial pyruvate carrier abundance mediates pathological cardiac hypertrophy.

Cardiomyocytes rely on metabolic substrates, not only to fuel cardiac output, but also for growth and remodeling during stress. Here we show that Mitochondrial Pyruvate Carrier (MPC) abundance mediates pathological cardiac hypertrophy. MPC abundance was reduced in failing hypertrophic human hearts, as well as the myocardium of mice induced to fail by angiotensin II or transverse-aortic constriction-induced. Constitutive knockout of cardiomyocyte MPC1/2 in mice resulted in cardiac hypertrophy and reduced survival, while tamoxifen-induced cardiomyocyte-specific reduction of MPC1/2 to the attenuated levels observed during pressure-overload was sufficient to induce hypertrophy with impaired cardiac function. Failing hearts from cardiomyocyte-restricted knockout mice displayed increased abundance of anabolic metabolites, including amino acids and pentose phosphate pathway intermediates and reducing cofactors. These hearts showed a concomitant decrease in carbon flux into mitochondrial tricarboxylic acid cycle intermediates, as corroborated by complementary 1,2-13C2-glucose tracer studies. In contrast, inducible cardiomyocyte overexpression of MPC1/2 resulted in increased tricarboxylic acid cycle intermediates, and sustained carrier expression during transverse-aortic constriction protected against cardiac hypertrophy and failure. Collectively, we demonstrate that loss of the MPC1/2 causally mediates adverse cardiac remodelling.

Differential Metabolomics and Lipidomics of Hypoxic/Hypocapnic Long-Term Stored Red Cells Associate with Improved 24-Hour Recovery after Transfusion

Storage lesion accumulates during refrigerated storage of RBC, and development of novel methods and/or additive solutions are needed for more efficacious RBC with reduced burden of harmful byproducts. Autologous 24-hr post-transfusion recovery (PTR24) at the end of storage measured in healthy volunteers is the only in vivo practical metrics to evaluate consequences of RBC storage. PTR24 studies are logistically and economically demanding and as such, the identification of easier biomarkers of stored blood quality can expedite the development and testing of novel storage strategies. We performed a prospective, randomized, dual‐arm, crossover, two‐center radio labeled 24-hr PTR24 study was conducted as a part of the pivotal study to evaluate Hemanext® hypoxic/hypocapnic processing system for meeting standard FDA acceptability criteria for RBC storage. Subjects donated CP2D whole blood (500 ± 50 mL). The Test arm consisted of whole blood‐derived RBC, leukoreduced, and processed to achieve hypoxic/hypocapnic state in AS-3 additive within 12 hours of phlebotomy then stored for 42 days at 1‐6°C under hypoxic conditions (Test). Unprocessed normal atmosphere stored units (Control) were stored within 8 hours under conventional storage condition. PTR24 data was obtained from 19 test subjects and 21 control subjects (14 paired) by infusion of 51‐Cr/99‐Tc(m) radiolabeled RBCs*. At day 0, 21 and 42, in vitro metrics, including conventional quality parameters (%hemolysis, ATP, 2,3-DPG, morphology, microparticles and deformability), as well as a large panel of metabolites and lipids were analyzed for correlations with PTR24. Bioactive lipids were quantified by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring (LC-MS/MS-MRM). Pearson's correlation coefficients for PTR24 and analytes were examined; correlation pattern differed significantly between normal air Control and hypoxic Test RBCs and thus analyzed separately. Among the conventional RBC quality metrics, ATP as well as ability to rejuvenate ATP and 2,3-DPG at end of storage correlated with PTR24. RBC deformability correlated only with Control, while reduced oxidative damage in Test removed its dependency. Test showed stronger correlations with ATP and other metabolic parameters. 302 metabolites and lipids were analyzed by qualitative metabolomics workflow, and significant correlations were observed with |R| ≥ 0.5 for 37 compounds in normal air stored RBCs (15 with p ≤ 0.05), and 41 compounds in hypoxic RBCs (19 with p ≤ 0.05). The top 5 correlating and inversely correlating compounds for each condition are reported along with p-values in Table 1. For RBC stored in normal air, strong correlations were primarily seen with membrane lipids and associated fatty acids, amino acids, and the Pentose Phosphate Pathway intermediates. Hypoxic storage PTR24 values primarily correlated with alternative membrane components, amino acids, and Pentose Phosphate Pathway intermediates (Table 2). Among 67 bio-reactive lipids analyzed quantitatively, negative correlations of oxylipins (9- or 13-HODEs, 5-, 8-, 11-, 12-HETEs) to PTR24 were observed under normal conditions, but not under hypoxic condition (Table 3). In summary, RBC deformability correlated only with PTR24 of normal air stored RBC, while reduced oxidative damage in hypoxic/hypocapnic conditions removed this dependency. Although normal air and hypoxic stored RBCs show differing metabolite correlates with PTR24, significant associations tend to occur within similar overall pathways. Comparing the PTR24-metabolite correlations between normal air and hypoxic/hypocapnic RBC storage reveals that intermediates of oxidative stress management and membrane homeostasis are potential predictors of post-transfusion recovery. Detailed analysis of lipids supports that hypoxic/hypocapnic storage may reduce membrane lipid degradation and generates the hypothesis that the metabolism of bioactive lipids under hypoxia/hypocapnia may be different from the one found in RBC stored under normal air storage conditions. * Radio-labeling for 14 subjects occurred without prior RDRC approval; one site did not meet GMP standards. Disclosures Yoshida: New Health Sciences Inc.: Employment, Equity Ownership. D'Alessandro:Hemanext, Inc.: Membership on an entity's Board of Directors or advisory committees; Omix Technologies, Inc.: Other: Founder. Nemkov:Omix Technologies, Inc.: Equity Ownership, Other: Founder. Dioguardi:Hemanext: Employment. Iselin:Hemanext: Employment. Dunham:Hemanext: Employment. Cancelas:Hemanext: Consultancy, Research Funding; Fresenius-Kabi: Research Funding; Cerus Co.: Research Funding; TerumoBCT: Consultancy, Research Funding; Macopharma Inc: Research Funding; Cytosorbents: Research Funding; Cellphire: Research Funding; Velico: Consultancy, Research Funding.

Hemoglobin S Polymerization Can be Inhibited By Alterations in Erythrocyte Glycolysis

Sickle cell disease (SCD) is caused by the polymerization of deoxy Hemoglobin S (HbS) resulting in lifelong complications including anemia, infections, stroke, tissue damage, organ failure, intense painful episodes, and premature death. Each year more than 300,000 children are born with SCD, and greater than 95% of these children live in resource-poor countries. Although bone marrow transplantation and/or gene therapy are curative or potentially curative treatments for SCD, these options are not available for the vast majority of people with SCD. The most effective palliative treatment for SCD is hydroxyurea, a drug that was approved by the FDA in 1998. Clearly there is a global need for low cost, portable treatments for SCD. We have previously shown that erythrocytes sense oxygen through reversible binding of deoxy Hb to the cytoplasmic domain of Band 3 using 3 lines of transgenic mice: 1) a mouse in which the mouse Hb/glycolytic enzyme binding region of Band 3 is replaced with the equivalent human sequences (humanized), 2) a humanized mouse lacking the Band 3 Hb binding site (-Hb) and 3) a humanized mouse with a deletion of glycolytic enzyme binding sites, resulting in increased affinity for deoxy Hb (HI affinity). Complete blood counts of these animals demonstrated only a mild increase in the MCV for the -Hb mice (p<0.03) compared to the humanized control. In the humanized control mouse, under oxygenated conditions glycolytic enzymes associate with Band 3, inhibiting glycolysis. In deoxy conditions, deoxy Hb displaces the glycolytic enzymes, activating glycolysis. In the -Hb mouse, the glycolytic enzymes were permanently bound to Band 3 (inactive glycolysis), while in the HI affinity mouse, the glycolytic enzymes were constitutively in the cytoplasm. (Chu et al. Blood 128:2708-16, 2016, Zhang et al. JBC 284:2519-25, 2019, Zhou et al. Sci Adv. 5:4466, 2019). To determine whether the alteration in glycolysis had effects on the polymerization of HbS, each of the mutant mouse strains were crossed to the Townes SCD mouse model. The end result was 3 different strains of animals that were 1) homozygous for one of the Band 3 mutations, 2) homozygous for the human alpha globin genes and 3) either HbA/HbA, HbA/HbS or HbS/HbS. Changes in CBC were related to the beta globin genotype. An elevated MCV was observed in the -Hb animals of all 3 genotypes (p<0.02). We compared the rate of sickling of S/S cells (Li et al. PNAS 114 (5) E689-E696, 2017) and showed that the -Hb S/S mice had a 16% increase in both the absolute number of sickled cells and the rate of appearance of the sickled cells relative to the humanized control (p<0.02). In contrast, sickling of HI affinity S/S cells was inhibited. We hypothesized that alterations in glycolysis in the -Hb and HI affinity erythrocytes were responsible for the changes in HbS polymerization. To test this hypothesis, we analyzed the metabolites in erythrocytes from our mutant mice, including those with HbA/A, HbA/S and HbS/S. We found that the ADP concentration was 2-fold higher in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). The relative levels of ATP were 1.5 fold lower in -Hb S/S cells compared to HI affinity S/S cells (p <0.05). Pentose phosphate pathway (PPP) intermediates were elevated in HI Affinity S/S cells compared to -Hb S/S cells, indicating that the Hi Affinity S/S mice have recovered PPP activity. To test whether altering glycolysis chemically could alter sickling, we treated humanized S/S cells with phosphoenolpyruvate (PEP), a membrane-permeable, rate-limiting glycolytic substrate that is rapidly metabolized to lactate and/or processed up the glycolysis pathway to generate 2,3-bisphosphoglycerate, which can initiate the PPP. We found that PEP treatment led to a dose dependent suppression of sickling compared to untreated cells (p<10-16). At a PEP concentration of 10mM, sickling was reduced by 15% compared to untreated cells with no effects on either membrane stability or MCV (p<10-15). In summary, we have shown that alterations in glycolysis can enhance or suppresses the sickling of red blood cells in a mouse model. Our results offer the prospect of a non-invasive, dietary therapy to potentially inhibit sickling and improve the outcome for SCD patients in resource poor areas of the world. Disclosures Low: Umoja BioPharma Inc: Other: Stockholder, Founder; Novosteo Inc: Other: Stockholder, Founder; Erythrocure Inc.: Other: Stockholder, Founder; On Target Laboratories Inc: Other: Stockholder, Founder; HuLow LLC: Other: Stockholder, Founder.

Entry of glucose- and glutamine-derived carbons into the citric acid cycle supports early steps of HIV-1 infection in CD4 T cells

The susceptibility of CD4 T cells to human immunodeficiency virus 1 (HIV-1) infection is regulated by glucose and glutamine metabolism, but the relative contributions of these nutrients to infection are not known. Here we show that glutaminolysis is the major pathway fuelling the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS) in T-cell receptor-stimulated naïve, as well as memory CD4, subsets and is required for optimal HIV-1 infection. Under conditions of attenuated glutaminolysis, the α-ketoglutarate (α-KG) TCA rescues early steps in infection; exogenous α-KG promotes HIV-1 reverse transcription, rendering both naïve and memory cells more sensitive to infection. Blocking the glycolytic flux of pyruvate to lactate results in altered glucose carbon allocation to TCA and pentose phosphate pathway intermediates, an increase in OXPHOS and augmented HIV-1 reverse transcription. Moreover, HIV-1 infection is significantly higher in CD4 T cells selected on the basis of high mitochondrial biomass and OXPHOS activity. Therefore, the OXPHOS/aerobic glycolysis balance is a major regulator of HIV-1 infection in CD4 T lymphocytes.

Promotion of Calcium/Calmodulin-Dependent Protein Kinase 4 by GLUT1-Dependent Glycolysis in Systemic Lupus Erythematosus.

To clarify the significance of immunometabolism in systemic lupus erythematosus (SLE), and to determine the effect of calcium/calmodulin-dependent protein kinase 4 (CaMK4) on T cell metabolism. Metabolomic profiling was performed using capillary electrophoresis mass spectrometry in naive T cells from MRL/lpr mice treated with anti-CD3/CD28 antibodies in the absence or presence of a CaMK4 inhibitor (KN-93). The expression of GLUT1 and CaMK4 in CD4+ T cells from healthy controls (n = 16), patients with inactive SLE (n = 13), and patients with active SLE (n = 14) was examined by flow cytometry and quantitative polymerase chain reaction. In vitro experiments were performed to determine the effect of KN-93 on the expression of GLUT1 during Th17 cell differentiation in T cells from patients with SLE. CaMK4 inhibition significantly decreased the levels of glycolytic intermediates such as glucose-6-phosphate, fructose-6-phosphate, fructose-1,6-diphosphate, pyruvate, and lactate (P<0.05), whereas it did not affect the levels of the pentose phosphate pathway intermediates such as 6-phospho-d-gluconate, ribulose-5-phosphate, ribose-5-phosphate, and phosphoribosyl pyrophosphate. The expression levels of GLUT1 and CaMK4 in effector memory CD4+ T cells were significantly higher in patients with active SLE compared to healthy controls (P<0.01 and P<0.05, respectively) and patients with inactive SLE (P<0.05 and P<0.01, respectively). A functional analysis revealed that CaMK4 inhibition decreased the expression of GLUT1 during Th17 cell differentiation (P<0.01), followed by a reduction of interleukin-17 (IL-17) production (P<0.05). The results of the study indicate that the activity of CaMK4 could be responsible for glycolysis, which contributes to the production of IL-17, and CaMK4 may contribute to aberrant expression of GLUT1 in T cells from patients with active SLE.

Glycolysis is important for optimal asexual growth and formation of mature tissue cysts by Toxoplasma gondii

Toxoplasma gondii can grow and replicate using either glucose or glutamine as the major carbon source. Here, we have studied the essentiality of glycolysis in the tachyzoite and bradyzoite stages of T. gondii, using transgenic parasites that lack a functional hexokinase gene (Δhk) in RH (Type-1) and Prugniaud (Type-II) strain parasites. Tachyzoite stage Δhk parasites exhibit a fitness defect similar to that reported previously for the major glucose transporter mutant, and remain virulent in mice. However, although Prugniaud strain Δhk tachyzoites were capable of transforming into bradyzoites in vitro, they were severely compromised in their ability to make mature bradyzoite cysts in the brain tissue of mice. Isotopic labelling studies reveal that glucose-deprived tacyzoites utilise glutamine to replenish glycolytic and pentose phosphate pathway intermediates via gluconeogenesis. Interestingly, while glutamine-deprived intracellular Δhk tachyzoites continued to replicate, extracellular parasites were unable to efficiently invade host cells. Further, studies on mutant tachyzoites lacking a functional phosphoenolpyruvate carboxykinase (Δpepck1) revealed that glutaminolysis is the sole source of gluconeogenic flux in glucose-deprived parasites. In addition, glutaminolysis is essential for sustaining oxidative phosphorylation in Δhk parasites, while wild type (wt) and Δpepck1 parasites can obtain ATP from either glycolysis or oxidative phosphorylation. This study provides insights into the role of nutrient metabolism during asexual propagation and development of T. gondii, and validates the versatile nature of central carbon and energy metabolism in this parasite.