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Intracellular Na elevation in the heart is a hallmark of pathologies where both acute and chronic metabolic remodelling occurs. Here, we assess whether acute (75 μM ouabain 100 nM blebbistatin) or chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and whether the failing heart shares a common Na-mediated metabolic ‘fingerprint’. Control (PLMWT), transgenic (PLM3SA), ouabain-treated and hypertrophied Langendorff-perfused mouse hearts are studied by 23Na, 31P, 13C NMR followed by 1H-NMR metabolomic profiling. Elevated Nai leads to common adaptive metabolic alterations preceding energetic impairment: a switch from fatty acid to carbohydrate metabolism and changes in steady-state metabolite concentrations (glycolytic, anaplerotic, Krebs cycle intermediates). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 ameliorates the metabolic changes. In silico modelling indicates altered metabolic fluxes (Krebs cycle, fatty acid, carbohydrate, amino acid metabolism). Prevention of Nai overload or inhibition of Na/Camito may be a new approach to ameliorate metabolic dysregulation in heart failure., The failing heart is characterised by both alterations in mitochondrial metabolism and an elevation of cytosolic sodium. Here, the authors use 23Na NMR and metabolic profiling to show these are related, and that elevation in intracellular Na reprograms cardiac substrate utilisation via effects on mitochondrial Na/Ca exchange.

Background The molecular profiling of glioblastoma (GBM) based on transcriptomic analysis could provide precise treatment and prognosis. However, current subtyping (classic, mesenchymal, neural, proneural) is time-consuming and cost-intensive hindering its clinical application. A simple and efficient method for classification was imperative. Methods In this study, to simplify GBM subtyping more efficiently, we applied a random forest algorithm to conduct 26 genes as a cluster featured with hub genes, OLIG2 and CD276. Functional enrichment analysis and Protein–protein interaction were performed using the genes in this gene cluster. The classification efficiency of the gene cluster was validated by WGCNA and LASSO algorithms, and tested in GSE84010 and Gravandeel’s GBM datasets. Results The gene cluster (n = 26) could distinguish mesenchymal and proneural excellently (AUC = 0.92), which could be validated by multiple algorithms (WGCNA, LASSO) and datasets (GSE84010 and Gravandeel’s GBM dataset). The gene cluster could be functionally enriched in DNA elements and T cell associated pathways. Additionally, five genes in the signature could predict the prognosis well (p = 0.0051 for training cohort, p = 0.065 for test cohort). Conclusions Our study proved the accuracy and efficiency of random forest classifier for GBM subtyping, which could provide a convenient and efficient method for subtyping Proneural and Mesenchymal GBM.

Mutations in succinate dehydrogenase (SDH) lead to the development of tumors in a restricted subset of cell types, including chromaffin cells and paraganglia. The molecular basis for this specificity is currently unknown. We show that loss of SDH activity in a chromaffin cell model does not perturb complex I function, retaining the ability to oxidize NADH within the electron transport chain. This activity supports continued oxidation of substrates within the tricarboxylic acid (TCA) cycle. However, due to the block in the TCA cycle at SDH, the high glutamine oxidation activity is only maintained through an efflux of succinate. We also show that although the mitochondria of SDH-deficient cells are less active per se, their higher mass per cell results in an overall respiratory rate that is comparable with wild-type cells. Finally, we observed that when their mitochondria are uncoupled, SDH-deficient cells are unable to preserve their viability, suggesting that the mitochondrial metabolic network is unable to compensate when exposed to additional stress. We therefore show that in contrast to models of SDH deficiency based on epithelial cells, a chromaffin cell model retains aspects of metabolic "health," which could form the basis of cell specificity of this rare tumor type.

Purpose: Intratumoral hypoxia and immunity have been correlated with patient outcome in various tumor settings. However, these factors are not currently considered for treatment selection in head and neck cancer (HNC) due to lack of validated biomarkers. Here we sought to develop a hypoxia-immune classifier with potential application in patient prognostication and prediction of response to targeted therapy. Experimental Design: A 54-gene hypoxia-immune signature was constructed on the basis of literature review. Gene expression was analyzed in silico using the The Cancer Genome Atlas (TCGA) HNC dataset (n = 275) and validated using two independent cohorts (n = 130 and 123). IHC was used to investigate the utility of a simplified protein signature. The spatial distribution of hypoxia and immune markers was examined using multiplex immunofluorescence staining. Results: Unsupervised hierarchical clustering of TCGA dataset (development cohort) identified three patient subgroups with distinct hypoxia-immune phenotypes and survival profiles: hypoxialow/immunehigh, hypoxiahigh/immunelow, and mixed, with 5-year overall survival (OS) rates of 71%, 51%, and 49%, respectively (P = 0.0015). The prognostic relevance of the hypoxia-immune gene signature was replicated in two independent validation cohorts. Only PD-L1 and intratumoral CD3 protein expression were associated with improved OS on multivariate analysis. Hypoxialow/immunehigh and hypoxiahigh/immunelow tumors were overrepresented in “inflamed” and “immune-desert” microenvironmental profiles, respectively. Multiplex staining demonstrated an inverse correlation between CA-IX expression and prevalence of intratumoral CD3+ T cells (r = −0.5464; P = 0.0377), further corroborating the transcription-based classification. Conclusions: We developed and validated a hypoxia-immune prognostic transcriptional classifier, which may have clinical application to guide the use of hypoxia modification and targeted immunotherapies for the treatment of HNC.

Brain tumours are the most common cause of cancer death in children. Molecular studies have greatly improved our understanding of these tumours but tumour metabolism is underexplored. Metabolites measured in vivo have been reported as prognostic biomarkers of these tumours but analysis of surgically resected tumour tissue allows a more extensive set of metabolites to be measured aiding biomarker discovery and providing validation of in vivo findings. In this study, metabolites were quantified across a range of paediatric brain tumours using 1H-High-Resolution Magic Angle Spinning nuclear magnetic resonance spectroscopy (HR-MAS) and their prognostic potential investigated. HR-MAS was performed on pre-treatment frozen tumour tissue from a single centre. Univariate and multivariate Cox regression was used to examine the ability of metabolites to predict survival. The models were cross validated using C-indices and further validated by splitting the cohort into two. Higher concentrations of glutamine were predictive of a longer overall survival, whilst higher concentrations of lipids were predictive of a shorter overall survival. These metabolites were predictive independent of diagnosis, as demonstrated in multivariate Cox regression models. Whilst accurate quantification of metabolites such as glutamine in vivo is challenging, metabolites show promise as prognostic markers due to development of optimised detection methods and increasing use of 3 T clinical scanners.

Succinate dehydrogenase is a key enzyme in the tricarboxylic acid cycle and the electron transport chain. All four subunits of succinate dehydrogenase are tumor suppressor genes predisposing to paraganglioma, but only mutations in the SDHB subunit are associated with increased risk of metastasis. Here we generated an Sdhd knockout chromaffin cell line and compared it with Sdhb-deficient cells. Both cell types exhibited similar SDH loss of function, metabolic adaptation, and succinate accumulation. In contrast, Sdhb−/− cells showed hallmarks of mesenchymal transition associated with increased DNA hypermethylation and a stronger pseudo-hypoxic phenotype compared with Sdhd−/− cells. Loss of SDHB specifically led to increased oxidative stress associated with dysregulated iron and copper homeostasis in the absence of NRF2 activation. High-dose ascorbate exacerbated the increase in mitochondrial reactive oxygen species, leading to cell death in Sdhb−/− cells. These data establish a mechanism linking oxidative stress to iron homeostasis that specifically occurs in Sdhb-deficient cells and may promote metastasis. They also highlight high-dose ascorbate as a promising therapeutic strategy for SDHB-related cancers. Significance: Loss of different succinate dehydrogenase subunits can lead to different cell and tumor phenotypes, linking stronger 2-OG–dependent dioxygenases inhibition, iron overload, and ROS accumulation following SDHB mutation.

BACKGROUND Idiopathic intracranial hypertension (IIH) is a condition predominantly affecting obese women of reproductive age. Recent evidence suggests that IIH is a disease of metabolic dysregulation, androgen excess, and an increased risk of cardiovascular morbidity. Here we evaluate systemic and adipose specific metabolic determinants of the IIH phenotype. METHODS In fasted, matched IIH (n = 97) and control (n = 43) patients, we assessed glucose and insulin homeostasis and leptin levels. Body composition was assessed along with an interrogation of adipose tissue function via nuclear magnetic resonance metabolomics and RNA sequencing in paired omental and subcutaneous biopsies in a case-control study. RESULTS We demonstrate an insulin- and leptin-resistant phenotype in IIH in excess of that driven by obesity. Adiposity in IIH is preferentially centripetal and is associated with increased disease activity and insulin resistance. IIH adipocytes appear transcriptionally and metabolically primed toward depot-specific lipogenesis. CONCLUSION These data show that IIH is a metabolic disorder in which adipose tissue dysfunction is a feature of the disease. Managing IIH as a metabolic disease could reduce disease morbidity and improve cardiovascular outcomes. FUNDING This study was supported by the UK NIHR (NIHR-CS-011-028), the UK Medical Research Council (MR/K015184/1), Diabetes UK, Wellcome Trust (104612/Z/14/Z), the Sir Jules Thorn Award, and the Midlands Neuroscience Teaching and Research Fund.

Summary Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent form of liver disease worldwide. This term encompasses a spectrum of pathologies, from benign hepatic steatosis to non-alcoholic steatohepatitis, which have, to date, been challenging to model in the laboratory setting. Here, we present a human pluripotent stem cell (hPSC)-derived model of hepatic steatosis, which overcomes inherent challenges of current models and provides insights into the metabolic rewiring associated with steatosis. Following induction of macrovesicular steatosis in hepatocyte-like cells using lactate, pyruvate, and octanoate (LPO), respirometry and transcriptomic analyses revealed compromised electron transport chain activity. 13C isotopic tracing studies revealed enhanced TCA cycle anaplerosis, with concomitant development of a compensatory purine nucleotide cycle shunt leading to excess generation of fumarate. This model of hepatic steatosis is reproducible, scalable, and overcomes the challenges of studying mitochondrial metabolism in currently available models., Graphical Abstract, Highlights • LPO-exposed hPSC-derived hepatocyte-like cells resemble human hepatic steatosis • Steatosis is associated with decreased mitochondrial maximal respiration • Purine nucleotide cycle activity increases in steatotic hepatocytes • Excess fumarate production is associated with induction of macrovesicular steatosis, Endocrine System Physiology; Stem Cells Research; Transcriptomics

SummaryNonalcoholic fatty liver disease (NAFLD) affects ~88% of obese individuals and is characterised by hepatic lipid accumulation. Mitochondrial metabolic dysfunction is a feature of NAFLD. We used a human pluripotent stem cell-based system to determine how mitochondrial dysfunction is linked to hepatic lipid accumulation. We induced lipid accumulation in hepatocyte-like cells (HLCs) using lactate, pyruvate and octanoate (LPO). Transcriptomic analysis revealed perturbation of mitochondrial respiratory pathways in LPO exposed cells. Using13C isotopic tracing, we identified truncation of the TCA cycle in steatotic HLCs. We show that increased purine nucleotide cycle (PNC) activity fuels fumarate accumulation and drives lipid accumulation in steatotic cells. These findings provide new insights into the pathogenesis of hepatic steatosis and may lead to an improved understanding of the metabolic and transcriptional rewiring associated with NAFLD.

The alpha ketoglutarate-dependent dioxygenase, prolyl-4-hydroxylase 3 (PHD3), is a Hypoxia-Inducible Factor (HIF) target that uses molecular oxygen to hydroxylate peptidyl prolyl residues. While PHD3 has been reported to influence cancer cell metabolism and liver insulin sensitivity, relatively little is known about effects of this highly conserved enzyme in insulin-secreting β-cells in vivo. Here, we show that deletion of PHD3 specifically in β-cells (βPHD3KO) is associated with impaired glucose homeostasis in mice fed high fat diet. In the early stages of dietary fat excess, βPHD3KO islets energetically rewire, leading to defects in the management of pyruvate fate and a shift from glycolysis to increased fatty acid oxidation (FAO). However, under more prolonged metabolic stress, this switch to preferential FAO in βPHD3KO islets is associated with impaired glucose-stimulated ATP/ADP rises, Ca2+ fluxes and insulin secretion. Thus, PHD3 might be a pivotal component of the β-cell glucose metabolism machinery in mice by suppressing the use of fatty acids as a primary fuel source during the early phases of metabolic stress.

Advancements in prevention, detection and treatment over the last 40 years have significantly transformed cancer healthcare however there are a few cancers, such as brain tumours, which are consistently lagging behind. The most common adult brain tumour is glioma; a highly aggressive cancer that invades deep into the surrounding brain consequently making treatment challenging. The severe hypoxic nature of glioma adds further complications to therapeutic efficacy as hypoxia limits efficient drug delivery as well as increasing treatment resistance. Therapies that therefore target both the hypoxic tumour microenvironment and metabolic pathways that sustain growth have significant potential to improve patient prognosis. It is well known that cancer cells demonstrate an abnormal metabolism, resulting in an altered requirement for amino acids to aid uncontrolled proliferation. Furthermore, tumour metabolism can also be influenced by this hostile hypoxic microenvironment, leading to a more malignant phenotype. We are therefore interested in a family of mitochondrial transporters, SLC25A, which translocate numerous solutes across the mitochondrial membrane and are crucial for many metabolic reactions. TCGA analysis has shown that many of these amino acid carriers are upregulated in glioma. Remarkably however, around 23 of the 53 mammalian SLC25A members lack defined substrate selectivity and so we are interested in identifying which transporters are particularly important in the metabolic adaptation to hypoxia. Using CRISPR and siRNA technologies we have identified transporters that are functionally required to maintain cell proliferation of glioma cell lines and patient tumour cells. Furthermore, using stable isotope-enriched nutrients, we have identified novel means by which glioma cell metabolism can be perturbed by inhibition of these transporters. Characterising which SLC25A transporters are important for hypoxic tumour metabolism could therefore expose a way to exploit these hypoxic areas subsequently making them more vulnerable to treatment and thus impacting patient survival.

Glioblastoma is the most common primary brain tumour and has a poor prognosis. The median survival is less than two years despite clinical intervention that usually involves the resection of the tumour volume, chemotherapy and radiotherapy. Achieving gross-total resection is challenging due to poorly defined boundaries as a result of tumour infiltration. Fluorescence-guided surgery (FGS) utilises an apparently selective accumulation of protoporphyrin IX (PPIX) that occurs in areas of glioblastoma after administration of the metabolite, 5-aminolevulinic acid (5-ALA). 5-ALA and the fluorescent metabolite, PPIX, sit within the endogenous heme biosynthetic pathway, which suggests that FGS is not only an important clinical tool, but also highlights differing metabolic phenotypes naturally present throughout the tumour. Genetic and mechanistic studies into this phenomenon have shown that differential expression of metabolite transporters, altered activity of the heme pathway enzymes and variable nutrient availability are all factors in the accumulation of PPIX. However, little is known about the cellular driving force for the uptake of 5-ALA and subsequent conversion into PPIX. Our data suggest that different microenvironments within the tumour alter the activity of the heme biosynthetic pathway, resulting in differential fluorescence in glioblastoma. It paves the way for work to alter the glioblastoma microenvironment in order to further improve the use of FGS in guiding surgery across these devastating tumours.

Aims Glioblastoma is the most common primary brain tumour and has a poor prognosis. Standard clinical intervention involves the resection of the tumour volume, chemotherapy and radiotherapy. However, achieving gross-total resection is challenging due to poorly defined boundaries as a result of tumour infiltration. Fluorescence-guided surgery (FGS) utilises an apparently selective accumulation of protoporphyrin IX (PPIX) that occurs in areas of glioblastoma after systemic administration of the metabolite 5-aminolevulinic acid (5-ALA). We have investigated the metabolic basis for the heterogeneity of the PPIX fluorescent signal, and its implications for glioma biology. Method Using glioblastoma cell lines and patient-derived primary cells, we have monitored the uptake of 5-ALA and conversion to the fluorescent molecule PPIX. Stable isotope tracing coupled with GCMS and LCMS was used to analyse intra- and extracellular metabolite levels arising from exogenous 5-ALA administration under both normoxic (21% O2) and hypoxic (1% O2) conditions. Results Uptake of exogenous 5-ALA from culture media and conversion to PPIX is observed in a time and dose-dependent manner in both normoxia and hypoxia. High levels of PPIX accumulation are associated with reduced cell proliferation despite the majority of the PPIX synthesised not being retained within the tumour cell, but exported into the medium. Under hypoxic conditions, reduced fluorescence is observed as a result of the decrease in oxygen availability likely affecting the oxygen-dependent enzymes. Stable isotope tracing experiments indicate an increase in the glutamine-derived succinate pool in response to exogenous 5-ALA, which is dependent on flux through the heme pathway. Conclusion Our data suggest that different microenvironments within the tumour alter the activity of the heme biosynthetic pathway, resulting in differential fluorescence in glioblastoma. It paves the way by which we could work to alter the glioblastoma microenvironment in order to further improve the use of FGS in guiding surgery across these devastating tumours.

Background Hexose-6-Phosphate Dehydrogenase (H6PD) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PD with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PD in ER/SR NAD(P)(H) homeostasis is incomplete. Lack of H6PD results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PD deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation. Methods We analysed skeletal muscle from H6PD knockout (H6PDKO), H6PD and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subjected to biochemical analysis including NAD(H) measurement, LC-MS based metabolomics, Western blotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO. Results H6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis, H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl-CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside elevated NAD+/NADH but had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability. Conclusions These findings suggest a complex metabolic response to changes in muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PD. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.

SUMMARYNAD+ is modulated by conditions of metabolic stress and has been reported to decline with aging, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome, and questioned if tissue NAD+ levels are depressed with aging. We supplemented 12 aged men with NR 1g per day for 21-days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways. NR also depressed levels of circulating inflammatory cytokines. In an additional study, 31P magnetic resonance spectroscopy-based NAD+ measurement in muscle and brain showed no difference between young and aged individuals. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR, while suggesting that NAD+ decline is not associated with chronological aging per se in human muscle or brain.

An abundant supply of amino acids is important for cancers to sustain their proliferative drive. Alongside their direct role as substrates for protein synthesis, they can have roles in energy generation, driving the synthesis of nucleosides and maintenance of cellular redox homoeostasis. As cancer cells exist within a complex and often nutrient-poor microenvironment, they sometimes exist as part of a metabolic community, forming relationships that can be both symbiotic and parasitic. Indeed, this is particularly evident in cancers that are auxotrophic for particular amino acids. This review discusses the stromal/cancer cell relationship, by using examples to illustrate a number of different ways in which cancer cells can rely on and contribute to their microenvironment – both as a stable network and in response to therapy. In addition, it examines situations when amino acid synthesis is driven through metabolic coupling to other reactions, and synthesis is in excess of the cancer cell’s proliferative demand. Finally, it highlights the understudied area of non-proteinogenic amino acids in cancer metabolism and their potential role.

BackgroundHexose-6-Phosphate Dehydrogenase (H6PDH) is a generator of NADPH in the Endoplasmic/Sarcoplasmic Reticulum (ER/SR). Interaction of H6PDH with 11β-hydroxysteroid dehydrogenase type 1 provides NADPH to support oxo-reduction of inactive to active glucocorticoids, but the wider understanding of H6PDH in ER/SR NAD(P)(H) homeostasis is incomplete. Muscle specific lack of H6PDH results in a deteriorating skeletal myopathy, altered glucose homeostasis, ER stress and activation of the unfolded protein response. Here we further assess muscle responses to H6PDH deficiency to delineate pathways that may underpin myopathy and link SR redox status to muscle wide metabolic adaptation.MethodsWe analysed skeletal muscle from H6PDH knockout (H6PDKO), H6PDH and NRK2 double knockout (DKO) and wild-type (WT) mice. H6PDKO mice were supplemented with the NAD+ precursor nicotinamide riboside. Skeletal muscle samples were subject to biochemical analysis including NAD(H) measurement, LC/MS based metabolomics, Western immunoblotting, and high resolution mitochondrial respirometry. Genetic and supplement models were assessed for degree of myopathy compared to H6PDKO.ResultsH6PDKO skeletal muscle showed adaptations in the routes regulating nicotinamide and NAD+ biosynthesis, with significant activation of the Nicotinamide Riboside Kinase 2 (NRK2) pathway. Associated with changes in NAD+ biosynthesis H6PDKO muscle had impaired mitochondrial respiratory capacity with altered mitochondrial acylcarnitine and acetyl CoA metabolism. Boosting NAD+ levels through the NRK2 pathway using the precursor nicotinamide riboside had no effect to mitigate ER stress and dysfunctional mitochondrial respiratory capacity or Acetyl-CoA metabolism. Similarly, H6PDKO/NRK2 double KO mice did not display an exaggerated timing or severity of myopathy or overt change in mitochondrial metabolism despite depression of NAD+ availability.ConclusionsThese findings suggest a complex metabolic response to changes to muscle SR NADP(H) redox status that result in impaired mitochondrial energy metabolism and activation of cellular NAD+ salvage pathways. It is possible that SR can sense and signal perturbation in NAD(P)(H) that cannot be rectified in the absence of H6PDH. Whether NRK2 pathway activation is a direct response to changes in SR NAD(P)(H) availability or adaptation to deficits in metabolic energy availability remains to be resolved.

Graphical abstract, Highlights • Dendritic cells (DC) treated with 1,25-dihydroxyvitamin D (1,25D) alone show a tolerogenic DC phenotype. • 1,25D-treated DC show altered oxidative phosphorylation and electron transport. • 1,25D-treated DC show altered glycolysis and TCA cycle metabolism. • TCA effects of 1,25D enhance fatty acid synthesis by DC. • Fatty acid synthesis appears to be essential for DC tolerogenic effects of 1,25D., The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D) is a potent regulator of immune function, promoting anti-inflammatory, tolerogenic T cell responses by modulating antigen presentation by dendritic cells (DC). Transcriptomic analyses indicate that DC responses to 1,25D involve changes in glycolysis, oxidative phosphorylation, electron transport and the TCA cycle. To determine the functional impact of 1,25D-mediated metabolic remodelling, human monocyte-derived DC were differentiated to immature (+vehicle, iDC), mature (+LPS, mDC), and immature tolerogenic DC (+1,25D, itolDC) and characterised for metabolic function. In contrast to mDC which showed no change in respiration, itolDC showed increased basal and ATP-linked respiration relative to iDC. Tracer metabolite analyses using 13C -labeled glucose showed increased lactate and TCA cycle metabolites. Analysis of lipophilic metabolites of 13C-glucose revealed significant incorporation of label in palmitate and palmitoleate, indicating that 1,25D promotes metabolic fatty acid synthesis in itolDC. Inhibition of fatty acid synthesis in itolDC altered itolDC morphology and suppressed expression of CD14 and IL-10 by these cells. These data indicate that the ability of 1,25D to induce tolerogenic DC involves metabolic remodelling leading to synthesis of fatty acids.

Hypoxia is a common feature of glioblastoma, and a known driver of therapy resistance in brain tumours. Understanding the metabolic adaptations to hypoxia is key to develop new effective treatments for patients. A recent screening study highlighted Pyrroline-5-carboxylate reductase-like (PYCRL) as one of the top three genes that allowed tumour survival in hypoxia. PYCRL is one of the three enzymes involved in proline biosynthesis along with the mitochondrial pyrroline-5-carboxylate reductase 1 and 2 (PYCR1/2). The latter use glutamine as the carbon source to fuel the pyrroline-5-carboxylate (P5C)-to-proline reaction, whereas the cytosolic PYCRL is known to use ornithine to produce proline. Our investigations have shown that PYCRL differs from PYCR1 and 2 in the impact on cellular redox, which is a critical factor in hypoxic survival. Our data suggest that PYCRL activity is required for normal regulation of glioblastoma cell growth and the ability to deal with cellular stress, and that this enzyme may therefore represent a novel target in the treatment of these devastating tumours. Importantly, our study also begins to provide much-needed clarity over the network surrounding proline metabolism and redox maintenance.

Hypothermic machine perfusion (HMP) and static cold storage (SCS) are the two methods used to preserve deceased donor kidneys prior to transplant. This study seeks to characterise the metabolic profile of HMP and SCS porcine kidneys in a cardiac death donor model. Twenty kidneys were cold flushed and stored for two hours following retrieval. Paired kidneys then underwent 24 h of HMP or SCS or served as time zero controls. Metabolite quantification in both storage fluid and kidney tissue was performed using one dimensional 1H NMR spectroscopy. For each metabolite, the net gain for each storage modality was determined by comparing the total amount in each closed system (i.e. total amount in storage fluid and kidney combined) compared with controls. 26 metabolites were included for analysis. Total system metabolite quantities following HMP or SCS were greater for 14 compared with controls (all p The metabolic environments of both perfusion fluid and the kidney tissue are strikingly different between SCS and HMP systems in this animal model. The total amount of central metabolites such as lactate and glutamate observed in the HMP kidney system suggests a greater degree of de novo metabolic activity than in the SCS system. Maintenance of central metabolic pathways may contribute to the clinical benefits of HMP.

In the early 1920s Otto Warburg observed that cancer cells have altered metabolism and from this, posited that mitochondrial dysfunction underpinned the aetiology of cancers. The more recent identification of mutations of mitochondrial metabolic enzymes in a wide range of human cancers has now provided a direct link between metabolic alterations and cancer. In this review we discuss the consequences of dysfunction of three metabolic enzymes involved in or associated with the tricarboxylic acid (TCA) cycle: succinate dehydrogenase (SDH), fumarate hydratase (FH) and isocitrate dehydrogenase (IDH) focusing on the similarity between the phenotypes of cancers harbouring these mutations.

Medulloblastoma (MB) is the most common malignant brain tumour arising in paediatric patients. These young patients are typically subjected to harsh regimens of treatments such as chemotherapy and radiotherapy, with varying degrees of success. The stratification of patients has been steadily improving, where patient tumours can be divided into four main groups (WNT, SHH, group 3 and group 4), each having differing prognostic implications, but does not accurately predict response to treatment. Previous work on this cancer, through the use of magnetic resonance spectroscopy (MRS), has uncovered a link between high glutamate concentrations and poor patient survival. Cellular metabolism is now accepted as a hallmark of cancer biology, where tumours are able to reprogram aspects of their metabolism to aid the biosynthetic, bioenergetic and redox burdens associated with increased growth and treatment resistance. We have shown that the medulloblastoma cell lines (ONS-76, DAOY and UW228.3) are addicted to glutamine, which is a precursor metabolite to glutamate. Furthermore, we have elucidated the importance of glutamate in treatment resistance and show the significance of glutathione (GSH) synthesis in this disease model. GSH is a tripeptide consisting of glutamate, glycine and cysteine. Therefore, glutamate has a potent role in the production of GSH, where it can be directly and indirectly utilised. Our research, through RNA inhibition of glutaminase 1 and 2, and the chemical inhibition of the xCT transporter, has pinpointed this metabolism as a potential therapeutic target and a means of sensitising MB cells to chemotherapy and irradiation.

Metabolic profiling by Magnetic Resonance Spectroscopy has shown the three most common paediatric cerebellar tumours are metabolically distinct; however, these techniques are limited by their sensitivity. Mass spectrometry is more sensitive and able to detect many more metabolites. In this study, metabolic phenotyping was performed to investigate the water-soluble metabolome and the lipidome of the three most common paediatric cerebellar tumours with the aim of informing a detailed metabolic pathway analysis. A discovery based experiment was performed on 60 surgically resected snap frozen pilocytic astrocytomas, ependymomas and medulloblastomas using UPLC-MS assays to detect water soluble and lipid metabolites. Principal component analysis demonstrates separation of the tumours based on the relative concentration of thousands of detected metabolites. The relative concentrations of glucose and glycolytic intermediates varied between the tumour types, as did metabolites involved in purine and pyrimidine metabolism. Key metabolites within the taurine metabolism pathway were found to be higher in medulloblastoma. Alterations were observed in lipid metabolism, with a wide range of relative concentration differences in di- and triacylglycerides, sphingolipids, ceramides, cardiolipins and glycerophospholipids. This study has shown that these tumours are metabolically distinct and offer the opportunities for stratified diagnosis and treatment. Some of the observed changes in relative concentration between the tumours reflect findings from spectroscopic studies, particularly with regards to taurine which is known to be higher in medulloblastoma. The ability of mass spectrometry to identify and measure lipid species and low concentration metabolites from tissue could allow targetable metabolic differences to be characterised and exploited.

Rationale Intracellular Na elevation is a hallmark of the ischaemic and failing heart – pathologies in which both acute and chronic metabolic remodelling occur. Objective To assess whether acute (75 µM ouabain 100 nM blebbistatin) and chronic myocardial Nai load (PLM3SA mouse) are causally linked to metabolic remodelling and if the failing heart shares a common Na-mediated metabolic ‘fingerprint’. Methods 23Na, 31P and 13C NMRS were performed in normal and hypertrophied (pressure overload) Langendorff perfused mouse hearts followed by 1 hour NMRS metabolomic profiling, mass spec and in silico modelling. Results Na overload (acute, chronic (PLM3SA), and hypertrophy 2, 1.3 and 1.4-fold respectively) resulted in common metabolic perturbations: substrate switch (palmitate 35% reduction, glucose 58% increase), flux (TCA cycle, OXPHOS, glycolysis) and metabolomic profile (TCA cycle, glycolysis, anaplerosis) without energetic impairment (PCr/ATP 1.5±0.1 vs control 1.3±0.1). Inhibition of mitochondrial Na/Ca exchanger by CGP37157 during both acute and chronic Na load ameliorated the metabolic changes. Conclusion Elevated Na leads to complex metabolic alterations preceding any energetic and functional impairment. Early prevention of Na overload and inhibition of Na/Camito could ameliorate metabolic dysregulation in hypertrophy and failure.

Hypoxia is a critical driver of cancer pathogenesis, directly inducing malignant phenotypes such as epithelial-mesenchymal transition, stem cell-like characteristics and metabolic transformation. However, hypoxia-associated phenotypes are often observed in cancer in the absence of hypoxia, a phenotype known as pseudohypoxia, which is very well documented in specific tumour types, including in paraganglioma/pheochromocytoma (PPGL). Approximately 40% of the PPGL tumours carry a germ line mutation in one of a number of susceptibility genes of which those that are found in succinate dehydrogenase (SDH) or in von Hippel-Lindau (VHL) genes manifest a strong pseudohypoxic phenotype. Mutations in SDH are oncogenic, forming tumours in a select subset of tissues, but the cause for this remains elusive. Although elevated succinate levels lead to increase in hypoxia-like signalling, there are other phenotypes that are being increasingly recognised in SDH-mutated PPGL, such as DNA hypermethylation. Further, recently unveiled changes in metabolic re-wiring of SDH-deficient cells might help to decipher cancer related roles of SDH in the future. In this review, we will discuss the various implications that the malfunctioning SDH can have and its impact on cancer development.

Summary: Nicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR. : Elhassan et al. show that oral nicotinamide riboside increases the NAD+ metabolome in aged human skeletal muscle, without apparently altering mitochondrial bioenergetics. Measures of muscle and whole-body metabolism are also unchanged. Nicotinamide riboside reduces the levels of circulating inflammatory cytokines. Studies in relevant human disease models are warranted. Keywords: nicotinamide adenine dinucleotide, metabolism, aging, inflammation, cell adhesion

Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD+, which was rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.

Pilocytic astrocytomas are the most common brain tumours arising in children, accounting for ~18% of primary brain tumours. 42% are cerebellar in origin and, whilst they have an excellent 10-year overall survival rate of >90%, progression-free survival for these tumours is variable. Identifying tumours more likely to progress will help determine those requiring more aggressive treatment. Metabolite profiles were obtained from 23 cerebellar pilocytic astrocytomas using High Resolution Magic Angle Spinning NMR (HR-MAS). Cox regression tested the ability of individual metabolites to predict progression-free survival. Progression was defined as the time point at which the local multi-disciplinary team decided to further treat the tumour. Low normalised tissue glutamine concentration was found to be associated with a worse progression-free survival (HR=4.58; P

6049Background: Genomic characterisation of head and neck cancer (HNC) has identified 3-5 subgroups with distinct biological properties, including metabolic profile and immune status. Both facets c...

Many solid tumors consist of large regions of poorly perfused cells, resulting in areas of low oxygen (hypoxia) throughout the cell mass. Cells subjected to hypoxia turn on a complex set of responses that alter their metabolism, rebalance their survival mechanisms, increase their invasive capacity, and stimulate angiogenesis. This allows them to at least temporarily escape the nutrient starvation and cell death resulting from this hostile environment. Accordingly, the hypoxic regions of tumors are often sources of the most aggressive and therapy-resistant cells, and therefore those cells that drive tumorigenesis. The hypoxia inducible factor (HIF) prolyl hydroxylases (PHDs) are enzymes that are functionally inactivated in hypoxia, as they use both oxygen and alpha-ketoglutarate as substrates to hydroxylate target prolyl residues. Although HIF1alpha, the most highly characterized PHD target, orchestrates many of the cellular responses to hypoxia observed in tumors, PHDs themselves have previously been shown to regulate some hypoxia responses, including apoptosis, in a HIF-independent mechanism. We have previously shown that PHDs can be reactivated under hypoxia and that this results in a metabolic defect, both in vitro and in vivo. This led us to investigate whether chronic reactivation of these enzymes may inhibit tumor progression. We show here that esterified alpha-ketoglutarate given daily will induce apoptosis and inhibit tumor growth, in vivo. The effects are independent of HIF1alpha but dependent on the presence of PHD3. These data suggest that PHD3 may be a valid target in vivo for anti-tumor therapy.

Cells exposed to low-oxygen conditions (hypoxia) alter their metabolism to survive. This response, although vital during development and high-altitude survival, is now known to be a major factor in the selection of cells with a transformed metabolic phenotype during tumorigenesis. It is thought that hypoxia-selected cells have increased invasive capacity and resistance to both chemo- and radiotherapies, and therefore represent an attractive target for antitumor therapy. Hypoxia inducible factors (HIFs) are responsible for the majority of gene expression changes under hypoxia, and are themselves controlled by the oxygen-sensing HIF prolyl hydroxylases (PHDs). It was previously shown that mutations in succinate dehydrogenase lead to the inactivation PHDs under normoxic conditions, which can be overcome by treatment with alpha-ketoglutarate derivatives. Given that solid tumors contain large regions of hypoxia, the reactivation of PHDs in these conditions could induce metabolic catastrophe and therefore prove an effective antitumor therapy. In this report we demonstrate that derivatized alpha-ketoglutarate can be used as a strategy for maintaining PHD activity under hypoxia. By increasing intracellular alpha-ketoglutarate and activating PHDs we trigger PHD-dependent reversal of HIF1 activation, and PHD-dependent hypoxic cell death. We also show that derivatized alpha-ketoglutarate can permeate multiple layers of cells, reducing HIF1alpha levels and its target genes in vivo.

In 2000, Douglas Hanahan and Robert Weinberg published a review detailing the six hallmarks of cancer. These are six phenotypes that a tumour requires in order to become a fully fledged malignancy: persistent growth signals, evasion of apoptosis, insensitivity to anti-growth signals, unlimited replicative potential, angiogenesis and invasion and metastasis. However, it is becoming increasingly clear that these phenotypes do not portray the whole story and that other hallmarks are necessary: one of which is a shift in cellular metabolism. The tumour environment creates a unique collection of stresses to which cells must adapt in order to survive. This environment is formed by the uncontrolled proliferation of cells, which ignore the cues that would create normal tissue architecture. As a result, the cells forming the tumour are exposed to low oxygen and nutrient levels, as well as high levels of toxic cellular waste products, which is thought to propel cells towards a more transformed phenotype, resistant to cell death and pro-metastatic.

Renal transplantation is the gold standard treatment for end stage renal disease; however, there are currently not enough suitable organs to meet a growing demand. The Hypoxia Inducible Factor (HIF) pathway comprises a number of oxygen sensitive transcription factors which activate cellular protective functions. Allografts from cadaveric organs make up a large proportion of the organ pool. These organs are subjected to hypoxic and ischemic conditions during the harvesting and transplant process. Manipulation of the HIF pathway may reduce organ damage and improve outcomes. This review evaluates the current evidence for the use of the HIF pathway in renal transplantation and also adds new research conducted into the effect on organ metabolism.

INTRODUCTION: The recent identification of recurrent heterozygous somatic mutations in the isocitrate dehydrogenase (IDH1) gene in low-grade gliomas and secondary glioblastoma, provides a much-needed advance in our understanding of the drivers of these tumours. Patients with this mutation are generally younger and have improved prognosis. Mutations in IDH1 occur at an arginine residue at codon 132 (R132) and result in production of the putative oncometabolite - (R)-2-hydroxyglutarate (2HG). (R)-2HG production has subsequently been shown to induce changes in DNA and histone methylation. However, there are conflicting reports regarding the metabolic role of IDH1 mutations and subsequent production of (R)-2HG in hypoxia, which are yet to be reconciled. METHOD: To define the hypoxic metabolic phenotype of IDH1 mutations, we applied complementary techniques - gas chromatography mass spectrometry (GC-MS), and nuclear magnetic resonance (NMR) spectroscopy to probe differences in the metabolic adaptation to hypoxia using [13C6]-glucose and [13C5]-glutamine tracers. RESULTS: Employing a human oligodendroglioma (HOG) cell line model, infected with mutant IDH1 by lentivirus, we show that IDH1 mutations metabolically compromise the adaptation to hypoxia, despite observable HIF1alpha stabilisation. CONCLUSION: We hypothesise that the adaptive transcriptional responses of hypoxia is blunted in IDH1 mutants resulting in the inability ofHIF1alpha to fully regulate downstream targets and ultimately cell metabolism. These results may account for the improved prognosis associated with this subset of glial tumours. Increasing our understanding of metabolic redundancies in tumour cells will aid the development of more effective treatments.

Gliomas are the most common type of brain tumour, yet their biology is still poorly understood. Despite recent advances in our understanding of low grade gliomas through the discovery of mutations in isocitrate dehydrogenase (IDH) enzymes 1&2 in the majority of patients, this has only provided us with a single line of sight to new therapies to treat these devastating tumours. To further unpick the biology of grade 2 gliomas, we used an unbiased consensus clustering approach to identify biologically similar groups of tumours based on their gene expression profiles. Through this, we identified three clusters of tumours with significantly differing survival rates, which did not always correlate with IDH status. Importantly, this method of tumour stratification identified a population of patients with an overall survival of 6 years. We therefore report three novel biological groups of grade 2 tumours, and in doing so identified a high risk subgroup of patients who would likely benefit from aggressive early intervention.

Since the description of mutations in isocitrate dehydrogenase 1 (IDH1) in adult glial tumours in 2008, many studies have focused on the role of the oncometabolite 2-hydroxyglutarate in determining the pheontype of affected gliomas. However, another aspect of IDH1 activity - its redox-dependency - may also represent an important determinant of IDH1-mutant glioma phenotype. To examine the effect of IDH1 mutations on redox homeostasis, we traced their use of redox-active metabolic pathways using nutrients enriched in the stable isotope, 13C. We found that cells expressing the IDH1 R132H mutant demonstrated enhanced proline synthesis, but that unexpectedly the proline produced was excreted rather than used for biosynthetic processes. As proline synthesis is a highly redox-dependent pathway, we hypothesised that this represented a metabolic response to altered isocitrate metabolism by the IDH1 mutant enzyme. We furthermore showed that metabolic response was dependent on pyrroline 5-carboxylate reductase 1 (PYCR1), and was important for the oxidation of mitochondrial NADH. Consistent with this, knockdown of PYCR1 resulted in a compensatory increase in cell respiration. We therefore show that cells with IDH1 mutations require proline synthesis in order to retain normal TCA cycle activity while using less oxygen. This may mean that IDH1 mutant tumours are in general less hypoxic, and therefore more responsive to treatment.

INTRODUCTION: Low socioeconomic status is associated with increased frequency of chronic conditions, a higher frequency of mental health illness, greater use of primary care and hospital services and a lower relative cancer survival for some of the commonest cancers. The impact of social deprivation on the survival of patents with glial tumours however is poorly understood. We examine the role of social deprivation on the survival of patients with glial tumours METHOD: We performed a retrospective analysis of clinical records of patients with histologically confirmed glial tumours treated at our institution between 2009 and February 2013. The overall survival (from time of tissue diagnosis) and index of multiple deprivation (IMD - 2010) were obtained. Potential confounding factors incorporated into a multivariable Cox regression model included age, gender, ethnicity, tumour location, neurosurgical procedure, antiepileptic drug taken and past medical history. RESULTS: 485 patients with WHO Grade II, III and IV glioma were identified with all five quintiles of deprivation classes represented. 96 patients fell into the most deprived class (1), while 49 patients were found to be within the least deprived class (5). Patients in the lowest socioeconomic class had a significantly worse survival compared with those in the least deprived class. (p = 0.03) The significance of this finding persisted even after incorporation into a cox multivariable regression model to take account for other factors likely to affect survival. CONCLUSION:This study highlights the importance of addressing social deprivation and its effects in the effort to improve patient outcomes for patients with high grade glioma.

The metabolic processes occurring within the preserved kidney during hypothermic machine perfusion (HMP) are not well characterized. The aim of this study was to use nuclear magnetic resonance (NMR) spectroscopy to examine the metabolomic profile of HMP perfusate from human cadaveric kidneys awaiting transplantation and to identify possible discriminators between the profiles of kidneys with delayed graft function (DGF) and immediate graft function (IGF).Perfusates from HMP kidneys were sampled at 45 min and 4 hr of preservation with the LifePort Kidney Transporter 1.0 (Organ Recovery Systems, Chicago, IL) using KPS-1. Prepared samples underwent 1-D Proton-NMR spectroscopy, and resultant spectra were analyzed. Clinical parameters were collected prospectively.Perfusate of 26 transplanted cadaveric kidneys was analyzed; 19(73%) with IGF and 7(27%) with DGF. Glucose concentrations were significantly lower in DGF kidneys compared to those with IGF at both 45 min (7.772 vs. 9.459 mM, P = 0.006) and 4 hr (8.202 vs. 10.235 mM, P = 0.003). Concentrations of inosine and leucine were significantly different between DGF and IGF kidneys at 45 min (0.002 vs. 0.013 mM, P = 0.009 and 0.011 vs. 0.006 mM, P = 0.036), and gluconate levels were also significantly different between DGF and IGF kidneys at 4 hr (49.099 vs. 59.513 mM, P = 0.009).Significant metabolic activity may be occurring in kidneys during HMP. The NMR spectroscopy of the perfusate can identify differences in the metabolomic profiles of DGF and IGF kidneys that might have a predictive role in viability assessment. Modification of harmful metabolic processes may improve outcomes for HMP kidneys.

The reduction or cessation of the blood supply to an organ results in tissue ischemia. Ischemia can cause significant tissue damage, and is observed as a result of a thrombosis, as part of a disease process, and during surgery. However, the restoration of the blood supply often causes more damage to the tissue than the ischemic episode itself. Research is therefore focused on identifying the cellular pathways involved in the protection of organs from the damage incurred by this process of ischemia reperfusion (I/R). The hypoxia-inducible factors (HIFs) are a family of heterodimeric transcription factors that are stabilized during ischemia. The genes that are expressed downstream of HIF activity enhance oxygen-independent ATP generation, cell survival, and angiogenesis, amongst other phenotypes. They are, therefore, important factors in the protection of tissues from I/R injury. Interestingly, a number of the mechanisms already known to induce organ protection against I/R injury, including preconditioning, postconditioning, and activation of signaling pathways such as adenosine receptor signaling, converge on the HIF system. This review describes the evidence for HIFs playing a role in I/R protection mediated by these factors, highlights areas that require further study, and discuss whether HIFs themselves are good therapeutic targets for protecting tissues from I/R injury.