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Chronic stress has been shown to induce hyperglycemia in both peripheral blood and the brain, yet the detailed mechanisms of glucose metabolism under stress remain unclear. Utilizing 13 C 6 -labeled glucose to trace metabolic pathways, our study investigated the impact of stress by chronic social defeat (CSD) on glucose metabolites in the liver and brain one week post-stress. We observed a reduction in 13 C 6 -enrichment of glucose metabolites in the liver, contrasting with unchanged levels in the brain. Notably, hepatic glycogen levels were reduced while lactate concentrations were elevated, suggesting lactate as an alternative energy source during stress. Long-term effects were also examined, revealing normalized blood glucose levels and restored glycogen stores in the liver three weeks post-CSD, despite sustained increases in food intake. This normalization is hypothesized to result from diminished glucagon levels leading to reduced glycogen phosphorylase activity. Our findings highlight a temporal shift in glucose metabolism, with hyperglycemia and glycogen depletion in the liver early after CSD, followed by a later phase of metabolic stabilization. These results underscore the liver's critical role in adapting to CSD and provide insights into the metabolic adjustments that maintain glucose homeostasis under prolonged stress conditions., (© 2024. The Author(s).)

Aims: During embryonic development, arteriovenous (AV) differentiation ensures proper blood vessel formation and maturation. Defects in arterial or venous identity cause inappropriate fusion of vessels, resulting in atypical shunts, so-called arteriovenous malformations (AVM). Currently, the mechanism behind AVM formation remains unclear and treatment options are fairly limited. Mammalian AV differentiation is initiated before the onset of blood flow in the embryo; however, this pre-flow mechanism is poorly understood. Here, we aimed to unravel the role of Smad1/5 signalling in pre-flow arterial identity, and in the process uncovered an unexpected control mechanism of Smad1/5 signalling., Methods and Results: We establish that despite Notch1 being expressed in the pre-flow mouse embryo, it is not activated, nor is it necessary for the expression of the earliest arterial genes in the dorsal aortae (i.e., Hey1 and Gja4). Furthermore, interrupting blood flow by using the Ncx1 KO model completely prevents the activation of Notch1 signalling, suggesting a strong role of shear stress in maintaining arterial identity. We demonstrate that early expression of Hey1 and Gja4 requires SMAD1/5 signalling. Using embryo cultures, we show that Smad1/5 signalling is activated through the Alk1/Alk5/TGFβR2 receptor complex, with TGFβ1 as a necessary ligand. Furthermore, our findings demonstrate that early arterial gene expression requires the acetylation of Smad1/5 proteins, rendering them more sensitive to TGFβ1 stimulation. Blocking acetyl-CoA production prevents pre-flow arterial expression of Hey1 and Gja4, while stabilizing acetylation rescues their expression., Conclusions: Our findings highlight the importance of the acetyl-CoA production in the cell and provide a novel control mechanism of Smad1/5 signalling involving protein acetylation. As disturbed canonical Smad1/5 signalling is involved in several vascular conditions, our results offer new insights in treatment options for circumventing canonical Smad1/5 signalling., (© The Author(s) 2024. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Solid tumors are highly reliant on lipids for energy, growth, and survival. In prostate cancer, the activity of the androgen receptor (AR) is associated with reprogramming of lipid metabolic processes. Here, we identified acyl-CoA synthetase medium chain family members 1 and 3 (ACSM1 and ACSM3) as AR-regulated mediators of prostate cancer metabolism and growth. ACSM1 and ACSM3 were upregulated in prostate tumors compared with nonmalignant tissues and other cancer types. Both enzymes enhanced proliferation and protected prostate cancer cells from death in vitro, whereas silencing ACSM3 led to reduced tumor growth in an orthotopic xenograft model. ACSM1 and ACSM3 were major regulators of the prostate cancer lipidome and enhanced energy production via fatty acid oxidation. Metabolic dysregulation caused by loss of ACSM1/3 led to mitochondrial oxidative stress, lipid peroxidation, and cell death by ferroptosis. Conversely, elevated ACSM1/3 activity enabled prostate cancer cells to survive toxic levels of medium chain fatty acids and promoted resistance to ferroptosis-inducing drugs and AR antagonists. Collectively, this study reveals a tumor-promoting function of medium chain acyl-CoA synthetases and positions ACSM1 and ACSM3 as key players in prostate cancer progression and therapy resistance. Significance: Androgen receptor-induced ACSM1 and ACSM3 mediate a metabolic pathway in prostate cancer that enables the utilization of medium chain fatty acids for energy production, blocks ferroptosis, and drives resistance to clinically approved antiandrogens., (©2024 American Association for Cancer Research.)

Endothelial cells (ECs) are highly glycolytic, but whether they generate glycolytic intermediates via gluconeogenesis (GNG) in glucose-deprived conditions remains unknown. Here, we report that glucose-deprived ECs upregulate the GNG enzyme PCK2 and rely on a PCK2-dependent truncated GNG, whereby lactate and glutamine are used for the synthesis of lower glycolytic intermediates that enter the serine and glycerophospholipid biosynthesis pathways, which can play key roles in redox homeostasis and phospholipid synthesis, respectively. Unexpectedly, however, even in normal glucose conditions, and independent of its enzymatic activity, PCK2 silencing perturbs proteostasis, beyond its traditional GNG role. Indeed, PCK2-silenced ECs have an impaired unfolded protein response, leading to accumulation of misfolded proteins, which due to defective proteasomes and impaired autophagy, results in the accumulation of protein aggregates in lysosomes and EC demise. Ultimately, loss of PCK2 in ECs impaired vessel sprouting. This study identifies a role for PCK2 in proteostasis beyond GNG., (© 2024. The Author(s).)

Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a rare inborn error of metabolism caused by deficiency of the PMM2 enzyme, which leads to impaired protein glycosylation. While the disorder presents with primarily neurological symptoms, there is limited knowledge about the specific brain-related changes caused by PMM2 deficiency. Here, we demonstrate aberrant neural activity in 2D neuronal networks from PMM2-CDG individuals. Utilizing multi-omics datasets from 3D human cortical organoids (hCOs) derived from PMM2-CDG individuals, we identify widespread decreases in protein glycosylation, highlighting impaired glycosylation as a key pathological feature of PMM2-CDG, as well as impaired mitochondrial structure and abnormal glucose metabolism in PMM2-deficient hCOs, indicating disturbances in energy metabolism. Correlation between PMM2 enzymatic activity in hCOs and symptom severity suggests that the level of PMM2 enzyme function directly influences neurological manifestations. These findings enhance our understanding of specific brain-related perturbations associated with PMM2-CDG, offering insights into the underlying mechanisms and potential directions for therapeutic interventions., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

T cell exhaustion is a hallmark of cancer and persistent infections, marked by inhibitory receptor upregulation, diminished cytokine secretion, and impaired cytolytic activity. Terminally exhausted T cells are steadily replenished by a precursor population (Tpex), but the metabolic principles governing Tpex maintenance and the regulatory circuits that control their exhaustion remain incompletely understood. Using a combination of gene-deficient mice, single-cell transcriptomics, and metabolomic analyses, we show that mitochondrial insufficiency is a cell-intrinsic trigger that initiates the functional exhaustion of T cells. At the molecular level, we find that mitochondrial dysfunction causes redox stress, which inhibits the proteasomal degradation of hypoxia-inducible factor 1α (HIF-1α) and promotes the transcriptional and metabolic reprogramming of Tpex cells into terminally exhausted T cells. Our findings also bear clinical significance, as metabolic engineering of chimeric antigen receptor (CAR) T cells is a promising strategy to enhance the stemness and functionality of Tpex cells for cancer immunotherapy., (© 2023. The Author(s).)

Introduction: Naïve T cells remain in an actively maintained state of quiescence until activation by antigenic signals, upon which they start to proliferate and generate effector cells to initiate a functional immune response. Metabolic reprogramming is essential to meet the biosynthetic demands of the differentiation process, and failure to do so can promote the development of hypofunctional exhausted T cells., Methods: Here we used 13C metabolomics and transcriptomics to study the metabolism of CD8+ T cells in their complete course of differentiation from naïve over stem-like memory to effector cells and in exhaustion-inducing conditions., Results: The quiescence of naïve T cells was evident in a profound suppression of glucose oxidation and a decreased expression of ENO1, downstream of which no glycolytic flux was detectable. Moreover, TCA cycle activity was low in naïve T cells and associated with a downregulation of SDH subunits. Upon stimulation and exit from quiescence, the initiation of cell growth and proliferation was accompanied by differential expression of metabolic enzymes and metabolic reprogramming towards aerobic glycolysis with high rates of nutrient uptake, respiration and lactate production. High flux in anabolic pathways imposed a strain on NADH homeostasis, which coincided with engagement of the proline cycle for mitochondrial redox shuttling. With acquisition of effector functions, cells increasingly relied on glycolysis as opposed to oxidative phosphorylation, which was, however, not linked to changes in mitochondrial abundance. In exhaustion, decreased effector function concurred with a reduction in mitochondrial metabolism, glycolysis and amino acid import, and an upregulation of quiescence-associated genes, TXNIP and KLF2, and the T cell suppressive metabolites succinate and itaconate., Discussion: Overall, these results identify multiple metabolic features that regulate quiescence, proliferation and effector function, but also exhaustion of CD8+ T cells during differentiation. Thus, targeting these metabolic checkpoints may be a promising therapeutic strategy for both prevention of exhaustion and promotion of stemness of anti-tumor T cells., Competing Interests: Authors AnS and PH were employed by Tyrol Clinics GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Kirchmair, Nemati, Lamberti, Trefny, Krogsdam, Siller, Hörtnagl, Schumacher, Sopper, Sandbichler, Zippelius, Ghesquière and Trajanoski.)

Intracellular Ca 2+ signals control several physiological and pathophysiological processes. The main tool to chelate intracellular Ca 2+ is intracellular BAPTA (BAPTA i ), usually introduced into cells as a membrane-permeant acetoxymethyl ester (BAPTA-AM). Previously, we demonstrated that BAPTA i enhanced apoptosis induced by venetoclax, a BCL-2 antagonist, in diffuse large B-cell lymphoma (DLBCL). This finding implied a novel interplay between intracellular Ca 2+ signaling and anti-apoptotic BCL-2 function. Hence, we set out to identify the underlying mechanisms by which BAPTA i enhances cell death in B-cell cancers. In this study, we discovered that BAPTA i alone induced apoptosis in hematological cancer cell lines that were highly sensitive to S63845, an MCL-1 antagonist. BAPTA i provoked a rapid decline in MCL-1-protein levels by inhibiting mTORC1-driven Mcl-1 translation. These events were not a consequence of cell death, as BAX/BAK-deficient cancer cells exhibited similar downregulation of mTORC1 activity and MCL-1-protein levels. Next, we investigated how BAPTA i diminished mTORC1 activity and identified its ability to impair glycolysis by directly inhibiting 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase 3 (PFKFB3) activity, a previously unknown effect of BAPTA i . Notably, these effects were also induced by a BAPTA i analog with low affinity for Ca 2+ . Consequently, our findings uncover PFKFB3 inhibition as an Ca 2+ -independent mechanism through which BAPTA i impairs cellular metabolism and ultimately compromises the survival of MCL-1-dependent cancer cells. These findings hold two important implications. Firstly, the direct inhibition of PFKFB3 emerges as a key regulator of mTORC1 activity and a promising target in MCL-1-dependent cancers. Secondly, cellular effects caused by BAPTA i are not necessarily related to Ca 2+ signaling. Our data support the need for a reassessment of the role of Ca 2+ in cellular processes when findings were based on the use of BAPTA i ., (© 2023. The Author(s).)

Protective immunity against pathogens or cancer is mediated by the activation and clonal expansion of antigen-specific naive T cells into effector T cells. To sustain their rapid proliferation and effector functions, naive T cells switch their quiescent metabolism to an anabolic metabolism through increased levels of aerobic glycolysis, but also through mitochondrial metabolism and oxidative phosphorylation, generating energy and signalling molecules 1-3 . However, how that metabolic rewiring drives and defines the differentiation of T cells remains unclear. Here we show that proliferating effector CD8 + T cells reductively carboxylate glutamine through the mitochondrial enzyme isocitrate dehydrogenase 2 (IDH2). Notably, deletion of the gene encoding IDH2 does not impair the proliferation of T cells nor their effector function, but promotes the differentiation of memory CD8 + T cells. Accordingly, inhibiting IDH2 during ex vivo manufacturing of chimeric antigen receptor (CAR) T cells induces features of memory T cells and enhances antitumour activity in melanoma, leukaemia and multiple myeloma. Mechanistically, inhibition of IDH2 activates compensating metabolic pathways that cause a disequilibrium in metabolites regulating histone-modifying enzymes, and this maintains chromatin accessibility at genes that are required for the differentiation of memory T cells. These findings show that reductive carboxylation in CD8 + T cells is dispensable for their effector response and proliferation, but that it mainly produces a pattern of metabolites that epigenetically locks CD8 + T cells into a terminal effector differentiation program. Blocking this metabolic route allows the increased formation of memory T cells, which could be exploited to optimize the therapeutic efficacy of CAR T cells., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Introduction: Malaria remains a widespread health problem with a huge burden. Severe or complicated malaria is highly lethal and encompasses a variety of pathological processes, including immune activation, inflammation, and dysmetabolism. Previously, we showed that adrenal hormones, in particular glucocorticoids (GCs), play critical roles to maintain disease tolerance during Plasmodium infection in mice. Here, GC responses were studied in Cameroon in children with uncomplicated malaria (UM), severe malaria (SM) and asymptomatic controls (AC)., Methods: To determine the sensitivity of leukocytes to GC signaling on a transcriptional level, we measured the ex vivo induction of glucocorticoid induced leucine zipper (GILZ) and FK506-binding protein 5 (FKBP5) by GCs in human and murine leukocytes. Targeted tracer metabolomics on peripheral blood mononuclear cells (PBMCs) was performed to detect metabolic changes induced by GCs., Results: Total cortisol levels increased in patients with clinical malaria compared to AC and were higher in the SM versus UM group, while cortisol binding globulin levels were unchanged and adrenocorticotropic hormone (ACTH) levels were heterogeneous. Induction of both GILZ and FKBP5 by GCs was significantly reduced in patients with clinical malaria compared to AC and in malaria-infected mice compared to uninfected controls. Increased activity in the pentose phosphate pathway was found in the patients, but this was not affected by ex vivo stimulation with physiological levels of hydrocortisone. Interestingly, hydrocortisone induced increased levels of cAMP in AC, but not in clinical malaria patients., Discussion: Altogether, this study shows that patients with SM have increased cortisol levels, but also a decreased sensitivity to GCs, which may clearly contribute to the severity of disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Vandermosten, Prenen, Fogang, Dagneau de Richecour, Knoops, Donkeu, Nguefack, Taguebue, Ndombo, Ghesquière, Ayong and Van den Steen.)

Abnormal polyol metabolism is predominantly associated with diabetes, where excess glucose is converted to sorbitol by aldose reductase (AR). Recently, abnormal polyol metabolism has been implicated in phosphomannomutase 2 congenital disorder of glycosylation (PMM2-CDG) and an AR inhibitor, epalrestat, proposed as a potential therapy. Considering that the PMM2 enzyme is not directly involved in polyol metabolism, the increased polyol production and epalrestat's therapeutic mechanism in PMM2-CDG remained elusive. PMM2-CDG, caused by PMM2 deficiency, presents with depleted GDP-mannose and abnormal glycosylation. Here, we show that, apart from glycosylation abnormalities, PMM2 deficiency affects intracellular glucose flux, resulting in polyol increase. Targeting AR with epalrestat decreases polyols and increases GDP-mannose both in patient-derived fibroblasts and in pmm2 mutant zebrafish. Using tracer studies, we demonstrate that AR inhibition diverts glucose flux away from polyol production toward the synthesis of sugar nucleotides, and ultimately glycosylation. Finally, PMM2-CDG individuals treated with epalrestat show a clinical and biochemical improvement., Competing Interests: Declaration of interests Mayo Clinic and E.M. have a financial interest related to this research. This research has been reviewed by the Mayo Clinic Conflict of Interest Review Board and is being conducted in compliance with Mayo Clinic Conflict of Interest policies. E.M. has the following patents planned, issued, or pending: application title, “Methods and Materials for Treating Glycosylation Disorders”; application # 16/973,210; filing date, 12/08/2020; Mayo Case # 2018-132., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

This scoping review summarizes what is known about kidney metabolism during hypothermic perfusion preservation. Papers studying kidney metabolism during hypothermic (<12 °C) perfusion were identified (PubMed, Embase, Web of Science, Cochrane). Out of 14,335 initially identified records, 52 were included [dog (26/52), rabbit (2/52), pig (20/52), human (7/52)]. These were published between 1970-2023, partially explaining study heterogeneity. There is a considerable risk of bias in the reported studies. Studies used different perfusates, oxygenation levels, kidney injury levels, and devices and reported on perfusate and tissue metabolites. In 11 papers, (non)radioactively labeled metabolites (tracers) were used to study metabolic pathways. Together these studies show that kidneys are metabolically active during hypothermic perfusion, regardless of the perfusion setting. Although tracers give us more insight into active metabolic pathways, kidney metabolism during hypothermic perfusion is incompletely understood. Metabolism is influenced by perfusate composition, oxygenation levels, and likely also by pre-existing ischemic injury. In the modern era, with increasing donations after circulatory death and the emergence of hypothermic oxygenated perfusion, the focus should be on understanding metabolic perturbations caused by pre-existing injury levels and the effect of perfusate oxygen levels. The use of tracers is indispensable to understanding the kidney's metabolism during perfusion, given the complexity of interactions between different metabolites.

Metabolic reprogramming can contribute to colorectal cancer progression and therapy resistance. Identification of key regulators of colorectal cancer metabolism could provide new approaches to improve treatment and reduce recurrence. Here, we demonstrate a critical role for the COP9 signalosome subunit CSN6 in rewiring nucleotide metabolism in colorectal cancer. Transcriptomic analysis of colorectal cancer patient samples revealed a correlation between CSN6 expression and purine and pyrimidine metabolism. A colitis-associated colorectal cancer model established that Csn6 intestinal conditional deletion decreased tumor development and altered nucleotide metabolism. CSN6 knockdown increased the chemosensitivity of colorectal cancer cells in vitro and in vivo, which could be partially reversed with nucleoside supplementation. Isotope metabolite tracing showed that CSN6 loss reduced de novo nucleotide synthesis. Mechanistically, CSN6 upregulated purine and pyrimidine biosynthesis by increasing expression of PHGDH, a key enzyme in the serine synthesis pathway. CSN6 inhibited β-Trcp-mediated DDX5 polyubiquitination and degradation, which in turn promoted DDX5-mediated PHGDH mRNA stabilization, leading to metabolic reprogramming and colorectal cancer progression. Butyrate treatment decreased CSN6 expression and improved chemotherapy efficacy. These findings unravel the oncogenic role of CSN6 in regulating nucleotide metabolism and chemosensitivity in colorectal cancer., Significance: CSN6 deficiency inhibits colorectal cancer development and chemoresistance by downregulating PHGDH to block nucleotide biosynthesis, providing potential therapeutic targets to improve colorectal cancer treatment., (©2022 American Association for Cancer Research.)

Hepatic encephalopathy (HE) is a common complication of chronic liver disease, characterized by an altered mental state and hyperammonemia. Insight into the brain pathophysiology of HE is limited due to a paucity of well-characterized HE models beyond the rat bile duct ligation (BDL) model. Here, we assess the presence of HE characteristics in the mouse BDL model. We show that BDL in C57Bl/6j mice induces motor dysfunction, progressive liver fibrosis, liver function failure and hyperammonemia, all hallmarks of HE. Swiss mice however fail to replicate the same phenotype, underscoring the importance of careful strain selection. Next, in-depth characterisation of metabolic disturbances in the cerebrospinal fluid of BDL mice shows glutamine accumulation and transient decreases in taurine and choline, indicative of brain ammonia overload. Moreover, mouse BDL induces glial cell dysfunction, namely microglial morphological changes with neuroinflammation and astrocyte reactivity with blood-brain barrier (BBB) disruption. Finally, we identify putative novel mechanisms involved in central HE pathophysiology, like bile acid accumulation and tryptophan-kynurenine pathway alterations. Our study provides the first comprehensive evaluation of a mouse model of HE in chronic liver disease. Additionally, this study further underscores the importance of neuroinflammation in the central effects of chronic liver disease., (© 2022. The Author(s).)

Introduction: Primary mitochondrial diseases (PMD) are a large, heterogeneous group of genetic disorders affecting mitochondrial function, mostly by disrupting the oxidative phosphorylation (OXPHOS) system. Understanding the cellular metabolic re-wiring occurring in PMD is crucial for the development of novel diagnostic tools and treatments, as PMD are often complex to diagnose and most of them currently have no effective therapy., Objectives: To characterize the cellular metabolic consequences of OXPHOS dysfunction and based on the metabolic signature, to design new diagnostic and therapeutic strategies., Methods: In vitro assays were performed in skin-derived fibroblasts obtained from patients with diverse PMD and validated in pharmacological models of OXPHOS dysfunction. Proliferation was assessed using the Incucyte technology. Steady-state glucose and glutamine tracing studies were performed with LC-MS quantification of cellular metabolites. The therapeutic potential of nutritional supplements was evaluated by assessing their effect on proliferation and on the metabolomics profile. Successful therapies were then tested in a in vivo lethal rotenone model in zebrafish., Results: OXPHOS dysfunction has a unique metabolic signature linked to an NAD+/NADH imbalance including depletion of TCA intermediates and aspartate, and increased levels of glycerol-3-phosphate. Supplementation with pyruvate and uridine fully rescues this altered metabolic profile and the subsequent proliferation deficit. Additionally, in zebrafish, the same nutritional treatment increases the survival after rotenone exposure., Conclusions: Our findings reinforce the importance of the NAD+/NADH imbalance following OXPHOS dysfunction in PMD and open the door to new diagnostic and therapeutic tools for PMD., (Copyright © 2022 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

A functional electron transport chain (ETC) is crucial for supporting bioenergetics and biosynthesis. Accordingly, ETC inhibition decreases proliferation in cancer cells but does not seem to impair stem cell proliferation. However, it remains unclear how stem cells metabolically adapt. In this study, we show that pharmacological inhibition of complex III of the ETC in skeletal stem and progenitor cells induces glycolysis side pathways and reroutes the tricarboxylic acid (TCA) cycle to regenerate NAD + and preserve cell proliferation. These metabolic changes also culminate in increased succinate and 2-hydroxyglutarate levels that inhibit Ten-eleven translocation (TET) DNA demethylase activity, thereby preserving self-renewal and multilineage potential. Mechanistically, mitochondrial malate dehydrogenase and reverse succinate dehydrogenase activity proved to be essential for the metabolic rewiring in response to ETC inhibition. Together, these data show that the metabolic plasticity of skeletal stem and progenitor cells allows them to bypass ETC blockade and preserve their self-renewal., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Tracer metabolomics is a powerful technology for the biomedical community to study and understand disease-inflicted metabolic mechanisms. However, the interpretation of tracer metabolomics results is highly technical, as the metabolites' abundances, tracer incorporation and positions on the metabolic map all must be jointly interpreted. The field is currently lacking a structured approach to help less experienced researchers start the interpretation of tracer metabolomics datasets. We propose an approach using an intuitive visualization concept aided by a novel open-source tool, and provide guidelines on how researchers can apply the approach and the visualization tool to their own datasets. Using a showcase experiment, we demonstrate that the visualization approach leads to an intuitive interpretation that can ease researchers into understanding their tracer metabolomics data.

Core facilities have a different mission than academic research labs. Accordingly, they require different career paths and structures., (© 2022 The Authors.)

Autophagy has vasculoprotective roles, but whether and how it regulates lymphatic endothelial cells (LEC) homeostasis and lymphangiogenesis is unknown. Here, we show that genetic deficiency of autophagy in LEC impairs responses to VEGF-C and injury-driven corneal lymphangiogenesis. Autophagy loss in LEC compromises the expression of main effectors of LEC identity, like VEGFR3, affects mitochondrial dynamics and causes an accumulation of lipid droplets (LDs) in vitro and in vivo. When lipophagy is impaired, mitochondrial ATP production, fatty acid oxidation, acetyl-CoA/CoA ratio and expression of lymphangiogenic PROX1 target genes are dwindled. Enforcing mitochondria fusion by silencing dynamin-related-protein 1 (DRP1) in autophagy-deficient LEC fails to restore LDs turnover and lymphatic gene expression, whereas supplementing the fatty acid precursor acetate rescues VEGFR3 levels and signaling, and lymphangiogenesis in LEC-Atg5 -/- mice. Our findings reveal that lipophagy in LEC by supporting FAO, preserves a mitochondrial-PROX1 gene expression circuit that safeguards LEC responsiveness to lymphangiogenic mediators and lymphangiogenesis., (© 2022. The Author(s).)

Metabolic reprogramming is a hallmark of activated T cells. The switch from oxidative phosphorylation to aerobic glycolysis provides energy and intermediary metabolites for the biosynthesis of macromolecules to support clonal expansion and effector function. Here, we show that glycolytic reprogramming additionally controls inflammatory gene expression via epigenetic remodeling. We found that the glucose transporter GLUT3 is essential for the effector functions of Th17 cells in models of autoimmune colitis and encephalomyelitis. At the molecular level, we show that GLUT3-dependent glucose uptake controls a metabolic-transcriptional circuit that regulates the pathogenicity of Th17 cells. Metabolomic, epigenetic, and transcriptomic analyses linked GLUT3 to mitochondrial glucose oxidation and ACLY-dependent acetyl-CoA generation as a rate-limiting step in the epigenetic regulation of inflammatory gene expression. Our findings are also important from a translational perspective because inhibiting GLUT3-dependent acetyl-CoA generation is a promising metabolic checkpoint to mitigate Th17-cell-mediated inflammatory diseases., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Molecular drivers of metastasis in patients with high-risk localized prostate cancer are poorly understood. Therefore, we aim to study molecular drivers of metastatic progression in patients with high-risk prostate cancer. A retrospective matched case-control study of two clinico-pathologically identical groups of patients with high-risk prostate cancer was undertaken. One group developed metastatic recurrence (n = 19) while the other did not (n = 25). The primary index tumor was identified by a uro-pathologist, followed by DNA and RNA extraction for somatic copy-number aberration (SCNA) analysis and whole-transcriptome gene expression analysis. In vitro and in vivo studies included cell line manipulation and xenograft models., The integrative CNA and gene expression analyses identified an increase in Antizyme Inhibitor 1 (AZIN1) gene expression within a focal amplification of 8q22.3, which was associated with metastatic recurrence of patients with high-risk prostate cancer in four independent cohorts. The effects of AZIN1 knockdown were evaluated, due to its therapeutic potential. AZIN1 knockdown effected proliferation and metastatic potential of prostate cancer cells and xenograft models. RNA sequencing after AZIN1 knockdown in prostate cancer cells revealed upregulation of genes coding for collagen subunits. The observed effect on cell migration after AZIN1 knockdown was mimicked when exposing prostate cancer cells to bio-active molecules deriving from COL4A1 and COL4A2. Our integrated CNA and gene expression analysis of primary high-risk prostate cancer identified the AZIN1 gene as a novel driver of metastatic progression, by altering collagen subunit expression. Future research should further investigate its therapeutic potential in preventing metastatic recurrence., Implications: AZIN1 was identified as driver of metastatic progression in high-risk prostate cancer through matrikine regulation., (©2022 American Association for Cancer Research.)

Purpose: TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities., Methods: Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics., Results: All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites., Conclusion: We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease., Competing Interests: Conflicts of Interest Authors declare no conflicts of interests., (Copyright © 2021 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Multiple myeloma (MM) remains an incurable cancer despite advances in therapy. Therefore, the search for new targets is still essential to uncover potential treatment strategies. Metabolic changes, induced by the hypoxic bone marrow, contribute to both MM cell survival and drug resistance. Pyrroline-5-carboxylate reductase 1 and 2 (PYCR1 and PYCR2) are two mitochondrial enzymes that facilitate the last step in the glutamine-to-proline conversion. Overexpression of PYCR1 is involved in progression of several cancers, however, its' role in hematological cancers is unknown. In this study, we investigated whether PYCR affects MM viability, proliferation and response to bortezomib., Methods: Correlation of PYCR1/2 with overall survival was investigated in the MMRF CoMMpass trial (653 patients). OPM-2 and RPMI-8226 MM cell lines were used to perform in vitro experiments. RPMI-8226 cells were supplemented with 13 C-glutamine for 48 h in both normoxia and hypoxia (< 1% O 2 , by chamber) to perform a tracer study. PYCR1 was inhibited by siRNA or the small molecule inhibitor pargyline. Apoptosis was measured using Annexin V and 7-AAD staining, viability by CellTiterGlo assay and proliferation by BrdU incorporation. Differential protein expression was evaluated using Western Blot. The SUnSET method was used to measure protein synthesis. All in vitro experiments were performed in hypoxic conditions., Results: We found that PYCR1 and PYCR2 mRNA expression correlated with an inferior overall survival. MM cells from relapsed/refractory patients express significantly higher levels of PYCR1 mRNA. In line with the strong expression of PYCR1, we performed a tracer study in RPMI-8226 cells, which revealed an increased conversion of 13 C-glutamine to proline in hypoxia. PYCR1 inhibition reduced MM viability and proliferation and increased apoptosis. Mechanistically, we found that PYCR1 silencing reduced protein levels of p-PRAS40, p-mTOR, p-p70, p-S6, p-4EBP1 and p-eIF4E levels, suggesting a decrease in protein synthesis, which we also confirmed in vitro. Pargyline and siPYCR1 increased bortezomib-mediated apoptosis. Finally, combination therapy of pargyline with bortezomib reduced viability in CD138 +  MM cells and reduced tumor burden in the murine 5TGM1 model compared to single agents., Conclusions: This study identifies PYCR1 as a novel target in bortezomib-based combination therapies for MM., (© 2022. The Author(s).)

Here, we investigate the impact of hypoxia on the hepatic response of glucocorticoid receptor (GR) to dexamethasone (DEX) in mice via RNA-sequencing. Hypoxia causes three types of reprogramming of GR: (i) much weaker induction of classical GR-responsive genes by DEX in hypoxia, (ii) a number of genes is induced by DEX specifically in hypoxia, and (iii) hypoxia induces a group of genes via activation of the hypothalamic-pituitary-adrenal (HPA) axis. Transcriptional profiles are reflected by changed GR DNA-binding as measured by ChIP sequencing. The HPA axis is induced by hypothalamic HIF1α and HIF2α activation and leads to GR-dependent lipolysis and ketogenesis. Acute inflammation, induced by lipopolysaccharide, is prevented by DEX in normoxia but not during hypoxia, and this is attributed to HPA axis activation by hypoxia. We unfold new physiological pathways that have consequences for patients suffering from GC resistance., (© 2021 The Authors.)

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Exercise is a powerful driver of physiological angiogenesis during adulthood, but the mechanisms of exercise-induced vascular expansion are poorly understood. We explored endothelial heterogeneity in skeletal muscle and identified two capillary muscle endothelial cell (mEC) populations that are characterized by differential expression of ATF3/4. Spatial mapping showed that ATF3/4 + mECs are enriched in red oxidative muscle areas while ATF3/4 low ECs lie adjacent to white glycolytic fibers. In vitro and in vivo experiments revealed that red ATF3/4 + mECs are more angiogenic when compared with white ATF3/4 low mECs. Mechanistically, ATF3/4 in mECs control genes involved in amino acid uptake and metabolism and metabolically prime red (ATF3/4 + ) mECs for angiogenesis. As a consequence, supplementation of non-essential amino acids and overexpression of ATF4 increased proliferation of white mECs. Finally, deleting Atf4 in ECs impaired exercise-induced angiogenesis. Our findings illustrate that spatial metabolic angiodiversity determines the angiogenic potential of muscle ECs., Competing Interests: Declaration of interests These authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Muscle weakness is a complication of critical illness which hampers recovery. In critically ill mice, supplementation with the ketone body 3-hydroxybutyrate has been shown to improve muscle force and to normalize illness-induced hypocholesterolemia. We hypothesized that altered cholesterol homeostasis is involved in development of critical illness-induced muscle weakness and that this pathway can be affected by 3-hydroxybutyrate., Methods: In both human critically ill patients and septic mice, the association between circulating cholesterol concentrations and muscle weakness was assessed. In septic mice, the impact of 3-hydroxybutyrate supplementation on cholesterol homeostasis was evaluated with use of tracer technology and through analysis of markers of cholesterol metabolism and downstream pathways., Results: Serum cholesterol concentrations were lower in weak than in non-weak critically ill patients, and in multivariable analysis adjusting for baseline risk factors, serum cholesterol was inversely correlated with weakness. In septic mice, plasma cholesterol correlated positively with muscle force. In septic mice, exogenous 3-hydroxybutyrate increased plasma cholesterol and altered cholesterol homeostasis, by normalization of plasma mevalonate and elevation of muscular, but not hepatic, expression of cholesterol synthesis genes. In septic mice, tracer technology revealed that 3-hydroxybutyrate was preferentially taken up by muscle and metabolized into cholesterol precursor mevalonate, rather than TCA metabolites. The 3-hydroxybutyrate protection against weakness was not related to ubiquinone or downstream myofiber mitochondrial function, whereas cholesterol content in myofibers was increased., Conclusions: These findings point to a role for low cholesterol in critical illness-induced muscle weakness and to a protective mechanism-of-action for 3-hydroxybutyrate supplementation., (© 2021. The Author(s).)

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Cystic fibrosis (CF) is a life-threatening disorder characterised by decreased pulmonary mucociliary and pathogen clearance, and an exaggerated inflammatory response leading to progressive lung damage. CF is caused by bi-allelic pathogenic variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel. CFTR is expressed in endothelial cells (ECs) and EC dysfunction has been reported in CF patients, but a role for this ion channel in ECs regarding CF disease progression is poorly described.We used an unbiased RNA sequencing approach in complementary models of CFTR silencing and blockade (by the CFTR inhibitor CFTRinh-172) in human ECs to characterise the changes upon CFTR impairment. Key findings were further validated in vitro and in vivo in CFTR-knockout mice and ex vivo in CF patient-derived ECs.Both models of CFTR impairment revealed that EC proliferation, migration and autophagy were downregulated. Remarkably though, defective CFTR function led to EC activation and a persisting pro-inflammatory state of the endothelium with increased leukocyte adhesion. Further validation in CFTR-knockout mice revealed enhanced leukocyte extravasation in lung and liver parenchyma associated with increased levels of EC activation markers. In addition, CF patient-derived ECs displayed increased EC activation markers and leukocyte adhesion, which was partially rescued by the CFTR modulators VX-770 and VX-809.Our integrated analysis thus suggests that ECs are no innocent bystanders in CF pathology, but rather may contribute to the exaggerated inflammatory phenotype, raising the question of whether normalisation of vascular inflammation might be a novel therapeutic strategy to ameliorate the disease severity of CF., Competing Interests: Conflict of interest: M. Declercq has nothing to disclose. Conflict of interest: P. de Zeeuw has nothing to disclose. Conflict of interest: N.V. Conchinha has nothing to disclose. Conflict of interest: V. Geldhof has nothing to disclose. Conflict of interest: A.S. Ramalho has nothing to disclose. Conflict of interest: M. García-Caballero has nothing to disclose. Conflict of interest: K. Brepoels has nothing to disclose. Conflict of interest: M. Ensinck has nothing to disclose. Conflict of interest: M.S. Carlon has nothing to disclose. Conflict of interest: M.J. Bird has nothing to disclose. Conflict of interest: S. Vinckier has nothing to disclose. Conflict of interest: M. Proesmans has nothing to disclose. Conflict of interest: F. Vermeulen has nothing to disclose. Conflict of interest: L. Dupont has nothing to disclose. Conflict of interest: B. Ghesquière has nothing to disclose. Conflict of interest: M. Dewerchin has nothing to disclose. Conflict of interest: P. Carmeliet has nothing to disclose. Conflict of interest: D. Cassiman has nothing to disclose. Conflict of interest: L. Treps has nothing to disclose. Conflict of interest: G. Eelen has nothing to disclose. Conflict of interest: P. Witters has nothing to disclose., (Copyright ©ERS 2021.)

Recessive loss-of-function mutations in ATP13A2 ( PARK9 ) are associated with a spectrum of neurodegenerative disorders, including Parkinson's disease (PD). We recently revealed that the late endo-lysosomal transporter ATP13A2 pumps polyamines like spermine into the cytosol, whereas ATP13A2 dysfunction causes lysosomal polyamine accumulation and rupture. Here, we investigate how ATP13A2 provides protection against mitochondrial toxins such as rotenone, an environmental PD risk factor. Rotenone promoted mitochondrial-generated superoxide (MitoROS), which was exacerbated by ATP13A2 deficiency in SH-SY5Y cells and patient-derived fibroblasts, disturbing mitochondrial functionality and inducing toxicity and cell death. Moreover, ATP13A2 knockdown induced an ATF4-CHOP-dependent stress response following rotenone exposure. MitoROS and ATF4-CHOP were blocked by MitoTEMPO, a mitochondrial antioxidant, suggesting that the impact of ATP13A2 on MitoROS may relate to the antioxidant properties of spermine. Pharmacological inhibition of intracellular polyamine synthesis with α-difluoromethylornithine (DFMO) also increased MitoROS and ATF4 when ATP13A2 was deficient. The polyamine transport activity of ATP13A2 was required for lowering rotenone/DFMO-induced MitoROS, whereas exogenous spermine quenched rotenone-induced MitoROS via ATP13A2. Interestingly, fluorescently labeled spermine uptake in the mitochondria dropped as a consequence of ATP13A2 transport deficiency. Our cellular observations were recapitulated in vivo, in a Caenorhabditis elegans strain deficient in the ATP13A2 ortholog catp-6 These animals exhibited a basal elevated MitoROS level, mitochondrial dysfunction, and enhanced stress response regulated by atfs-1 , the C. elegans ortholog of ATF4, causing hypersensitivity to rotenone, which was reversible with MitoTEMPO. Together, our study reveals a conserved cell protective pathway that counters mitochondrial oxidative stress via ATP13A2-mediated lysosomal spermine export., Competing Interests: Competing interest statement: Patent WO-2018002350-A1 of KU Leuven describes methods for detecting compounds with therapeutic use that target ATP13A2 or related isoforms using biological material and assays described in the current manuscript. A second patent of KU Leuven describing ATP13A2 cell models described in this manuscript has also been filed., (Copyright © 2020 the Author(s). Published by PNAS.)

Cancer cells display DNA hypermethylation at specific CpG islands in comparison to their normal healthy counterparts, but the mechanism that drives this so-called CpG island methylator phenotype (CIMP) remains poorly understood. Here, we show that CpG island methylation in human T-cell acute lymphoblastic leukemia (T-ALL) mainly occurs at promoters of Polycomb Repressor Complex 2 (PRC2) target genes that are not expressed in normal or malignant T-cells and which display a reciprocal association with H3K27me3 binding. In addition, we revealed that this aberrant methylation profile reflects the epigenetic history of T-ALL and is established already in pre-leukemic, self-renewing thymocytes that precede T-ALL development. Finally, we unexpectedly uncover that this age-related CpG island hypermethylation signature in T-ALL is completely resistant to the FDA-approved hypomethylating agent Decitabine. Altogether, we here provide conceptual evidence for the involvement of a pre-leukemic phase characterized by self-renewing thymocytes in the pathogenesis of human T-ALL., Competing Interests: Conflicts of Interest The authors declare no potential conflicts of interest.

Muscle regeneration is sustained by infiltrating macrophages and the consequent activation of satellite cells 1-4 . Macrophages and satellite cells communicate in different ways 1-5 , but their metabolic interplay has not been investigated. Here we show, in a mouse model, that muscle injuries and ageing are characterized by intra-tissue restrictions of glutamine. Low levels of glutamine endow macrophages with the metabolic ability to secrete glutamine via enhanced glutamine synthetase (GS) activity, at the expense of glutamine oxidation mediated by glutamate dehydrogenase 1 (GLUD1). Glud1-knockout macrophages display constitutively high GS activity, which prevents glutamine shortages. The uptake of macrophage-derived glutamine by satellite cells through the glutamine transporter SLC1A5 activates mTOR and promotes the proliferation and differentiation of satellite cells. Consequently, macrophage-specific deletion or pharmacological inhibition of GLUD1 improves muscle regeneration and functional recovery in response to acute injury, ischaemia or ageing. Conversely, SLC1A5 blockade in satellite cells or GS inactivation in macrophages negatively affects satellite cell functions and muscle regeneration. These results highlight the metabolic crosstalk between satellite cells and macrophages, in which macrophage-derived glutamine sustains the functions of satellite cells. Thus, the targeting of GLUD1 may offer therapeutic opportunities for the regeneration of injured or aged muscles.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body 1 , and regulates inflammation, cell proliferation, and tissue regeneration 2-4 . How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.

Profound metabolic changes are characteristic of macrophages during classical activation and have been implicated in this phenotype. Here we demonstrate that nitric oxide (NO) produced by murine macrophages is responsible for TCA cycle alterations and citrate accumulation associated with polarization. 13 C tracing and mitochondrial respiration experiments map NO-mediated suppression of metabolism to mitochondrial aconitase (ACO2). Moreover, we find that inflammatory macrophages reroute pyruvate away from pyruvate dehydrogenase (PDH) in an NO-dependent and hypoxia-inducible factor 1α (Hif1α)-independent manner, thereby promoting glutamine-based anaplerosis. Ultimately, NO accumulation leads to suppression and loss of mitochondrial electron transport chain (ETC) complexes. Our data reveal that macrophages metabolic rewiring, in vitro and in vivo, is dependent on NO targeting specific pathways, resulting in reduced production of inflammatory mediators. Our findings require modification to current models of macrophage biology and demonstrate that reprogramming of metabolism should be considered a result rather than a mediator of inflammatory polarization.

The Serine-Glycine-One-Carbon (SGOC) pathway is pivotal in multiple anabolic processes. Expression levels of SGOC genes are deregulated under tumorigenic conditions, suggesting participation of oncogenes in deregulating the SGOC biosynthetic pathway. However, the underlying mechanism remains elusive. Here, we identified that Interleukin enhancer-binding factor 3 (ILF3) is overexpressed in primary CRC patient specimens and correlates with poor prognosis. ILF3 is critical in regulating the SGOC pathway by directly regulating the mRNA stability of SGOC genes, thereby increasing SGOC genes expression and facilitating tumor growth. Mechanistic studies showed that the EGF-MEK-ERK pathway mediates ILF3 phosphorylation, which hinders E3 ligase speckle-type POZ protein (SPOP)-mediated poly-ubiquitination and degradation of ILF3. Significantly, combination of SGOC inhibitor and the anti-EGFR monoclonal antibody cetuximab can hinder the growth of patient-derived xenografts that sustain high ERK-ILF3 levels. Taken together, deregulation of ILF3 via the EGF-ERK signaling plays an important role in systemic serine metabolic reprogramming and confers a predilection toward CRC development. Our findings indicate that clinical evaluation of SGOC inhibitor is warranted for CRC patients with ILF3 overexpression.

ATP13A2 (PARK9) is a late endolysosomal transporter that is genetically implicated in a spectrum of neurodegenerative disorders, including Kufor-Rakeb syndrome-a parkinsonism with dementia 1 -and early-onset Parkinson's disease 2 . ATP13A2 offers protection against genetic and environmental risk factors of Parkinson's disease, whereas loss of ATP13A2 compromises lysosomes 3 . However, the transport function of ATP13A2 in lysosomes remains unclear. Here we establish ATP13A2 as a lysosomal polyamine exporter that shows the highest affinity for spermine among the polyamines examined. Polyamines stimulate the activity of purified ATP13A2, whereas ATP13A2 mutants that are implicated in disease are functionally impaired to a degree that correlates with the disease phenotype. ATP13A2 promotes the cellular uptake of polyamines by endocytosis and transports them into the cytosol, highlighting a role for endolysosomes in the uptake of polyamines into cells. At high concentrations polyamines induce cell toxicity, which is exacerbated by ATP13A2 loss due to lysosomal dysfunction, lysosomal rupture and cathepsin B activation. This phenotype is recapitulated in neurons and nematodes with impaired expression of ATP13A2 or its orthologues. We present defective lysosomal polyamine export as a mechanism for lysosome-dependent cell death that may be implicated in neurodegeneration, and shed light on the molecular identity of the mammalian polyamine transport system.

Primary mitochondrial disease (PMD) is a large group of genetic disorders directly affecting mitochondrial function. Although next generation sequencing technologies have revolutionized the diagnosis of these disorders, biochemical tests remain essential and functional confirmation of the critical genetic diagnosis. While enzymological testing of the mitochondrial oxidative phosphorylation (OXPHOS) complexes remains the gold standard, oxygraphy could offer several advantages. To this end, we compared the diagnostic performance of both techniques in a cohort of 34 genetically defined PMD patient fibroblast cell lines. We observed that oxygraphy slightly outperformed enzymology for sensitivity (79 ± 17% versus 68 ± 15%, mean and 95% CI), and had a better discriminatory power, identifying 58 ± 17% versus 35 ± 17% as "very likely" for oxygraphy and enzymology, respectively. The techniques did, however, offer synergistic diagnostic prediction, as the sensitivity rose to 88 ± 11% when considered together. Similarly, the techniques offered varying defect specific information, such as the ability of enzymology to identify isolated OXPHOS deficiencies, while oxygraphy pinpointed PDHC mutations and captured POLG mutations that were otherwise missed by enzymology. In summary, oxygraphy provides useful information for the diagnosis of PMD, and should be considered in conjunction with enzymology for the diagnosis of PMD.

Energy metabolism has been repeatedly linked to amyotrophic lateral sclerosis (ALS). Yet, motor neuron (MN) metabolism remains poorly studied and it is unknown if ALS MNs differ metabolically from healthy MNs. To address this question, we first performed a metabolic characterization of induced pluripotent stem cells (iPSCs) versus iPSC-derived MNs and subsequently compared MNs from ALS patients carrying FUS mutations to their CRISPR/Cas9-corrected counterparts. We discovered that human iPSCs undergo a lactate oxidation-fuelled prooxidative metabolic switch when they differentiate into functional MNs. Simultaneously, they rewire metabolic routes to import pyruvate into the TCA cycle in an energy substrate specific way. By comparing patient-derived MNs and their isogenic controls, we show that ALS-causing mutations in FUS did not affect glycolytic or mitochondrial energy metabolism of human MNs in vitro. These data show that metabolic dysfunction is not the underlying cause of the ALS-related phenotypes previously observed in these MNs.

Phosphoglucomutase 1 (PGM1) encodes the metabolic enzyme that interconverts glucose-6-P and glucose-1-P. Mutations in PGM1 cause impairment in glycogen metabolism and glycosylation, the latter manifesting as a congenital disorder of glycosylation (CDG). This unique metabolic defect leads to abnormal N-glycan synthesis in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). On the basis of the decreased galactosylation in glycan chains, galactose was administered to individuals with PGM1-CDG and was shown to markedly reverse most disease-related laboratory abnormalities. The disease and treatment mechanisms, however, have remained largely elusive. Here, we confirm the clinical benefit of galactose supplementation in PGM1-CDG-affected individuals and obtain significant insights into the functional and biochemical regulation of glycosylation. We report here that, by using tracer-based metabolomics, we found that galactose treatment of PGM1-CDG fibroblasts metabolically re-wires their sugar metabolism, and as such replenishes the depleted levels of galactose-1-P, as well as the levels of UDP-glucose and UDP-galactose, the nucleotide sugars that are required for ER- and GA-linked glycosylation, respectively. To this end, we further show that the galactose in UDP-galactose is incorporated into mature, de novo glycans. Our results also allude to the potential of monosaccharide therapy for several other CDG., (Copyright © 2019 American Society of Human Genetics. All rights reserved.)

Endochondral ossification, an important process in vertebrate bone formation, is highly dependent on correct functioning of growth plate chondrocytes 1 . Proliferation of these cells determines longitudinal bone growth and the matrix deposited provides a scaffold for future bone formation. However, these two energy-dependent anabolic processes occur in an avascular environment 1,2 . In addition, the centre of the expanding growth plate becomes hypoxic, and local activation of the hypoxia-inducible transcription factor HIF-1α is necessary for chondrocyte survival by unidentified cell-intrinsic mechanisms 3-6 . It is unknown whether there is a requirement for restriction of HIF-1α signalling in the other regions of the growth plate and whether chondrocyte metabolism controls cell function. Here we show that prolonged HIF-1α signalling in chondrocytes leads to skeletal dysplasia by interfering with cellular bioenergetics and biosynthesis. Decreased glucose oxidation results in an energy deficit, which limits proliferation, activates the unfolded protein response and reduces collagen synthesis. However, enhanced glutamine flux increases α-ketoglutarate levels, which in turn increases proline and lysine hydroxylation on collagen. This metabolically regulated collagen modification renders the cartilaginous matrix more resistant to protease-mediated degradation and thereby increases bone mass. Thus, inappropriate HIF-1α signalling results in skeletal dysplasia caused by collagen overmodification, an effect that may also contribute to other diseases involving the extracellular matrix such as cancer and fibrosis.

The role of fatty acid synthesis in endothelial cells (ECs) remains incompletely characterized. We report that fatty acid synthase knockdown (FASN KD ) in ECs impedes vessel sprouting by reducing proliferation. Endothelial loss of FASN impaired angiogenesis in vivo, while FASN blockade reduced pathological ocular neovascularization, at >10-fold lower doses than used for anti-cancer treatment. Impaired angiogenesis was not due to energy stress, redox imbalance, or palmitate depletion. Rather, FASN KD elevated malonyl-CoA levels, causing malonylation (a post-translational modification) of mTOR at lysine 1218 (K1218). mTOR K-1218 malonylation impaired mTOR complex 1 (mTORC1) kinase activity, thereby reducing phosphorylation of downstream targets (p70S6K/4EBP1). Silencing acetyl-CoA carboxylase 1 (an enzyme producing malonyl-CoA) normalized malonyl-CoA levels and reactivated mTOR in FASN KD ECs. Mutagenesis unveiled the importance of mTOR K1218 malonylation for angiogenesis. This study unveils a novel role of FASN in metabolite signaling that contributes to explaining the anti-angiogenic effect of FASN blockade., (Copyright © 2018 Elsevier Inc. All rights reserved.)