Publications by authors named "James A West"

32 Publications

Early detection of doxorubicin-induced cardiotoxicity in rats by its cardiac metabolic signature assessed with hyperpolarized MRI.

Commun Biol 2020 Nov 19;3(1):692. Epub 2020 Nov 19.

Oxford Centre for Clinical Magnetic Resonance Research, John Radcliffe Hospital, Headington, Oxford, OX3 9DU, UK.

Doxorubicin (DOX) is a widely used chemotherapeutic agent that can cause serious cardiotoxic side effects culminating in congestive heart failure (HF). There are currently no clinical imaging techniques or biomarkers available to detect DOX-cardiotoxicity before functional decline. Mitochondrial dysfunction is thought to be a key factor driving functional decline, though real-time metabolic fluxes have never been assessed in DOX-cardiotoxicity. Hyperpolarized magnetic resonance imaging (MRI) can assess real-time metabolic fluxes in vivo. Here we show that cardiac functional decline in a clinically relevant rat-model of DOX-HF is preceded by a change in oxidative mitochondrial carbohydrate metabolism, measured by hyperpolarized MRI. The decreased metabolic fluxes were predominantly due to mitochondrial loss and additional mitochondrial dysfunction, and not, as widely assumed hitherto, to oxidative stress. Since hyperpolarized MRI has been successfully translated into clinical trials this opens up the potential to test cancer patients receiving DOX for early signs of cardiotoxicity.
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http://dx.doi.org/10.1038/s42003-020-01440-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7678845PMC
November 2020

Consequences of Lipid Remodeling of Adipocyte Membranes Being Functionally Distinct from Lipid Storage in Obesity.

J Proteome Res 2020 10 31;19(10):3919-3935. Epub 2020 Aug 31.

Department of Biochemistry & Cambridge Systems Biology Centre, University of Cambridge, Tennis Court Road, Cambridge CB2 1GA, U.K.

Obesity is a complex disorder where the genome interacts with diet and environmental factors to ultimately influence body mass, composition, and shape. Numerous studies have investigated how bulk lipid metabolism of adipose tissue changes with obesity and, in particular, how the composition of triglycerides (TGs) changes with increased adipocyte expansion. However, reflecting the analytical challenge posed by examining non-TG lipids in extracts dominated by TGs, the glycerophospholipid composition of cell membranes has been seldom investigated. Phospholipids (PLs) contribute to a variety of cellular processes including maintaining organelle functionality, providing an optimized environment for membrane-associated proteins, and acting as pools for metabolites (e.g. choline for one-carbon metabolism and for methylation of DNA). We have conducted a comprehensive lipidomic study of white adipose tissue in mice which become obese either through genetic modification (/), diet (high fat diet), or a combination of the two, using both solid phase extraction and ion mobility to increase coverage of the lipidome. Composition changes in seven classes of lipids (free fatty acids, diglycerides, TGs, phosphatidylcholines, lyso-phosphatidylcholines, phosphatidylethanolamines, and phosphatidylserines) correlated with perturbations in one-carbon metabolism and transcriptional changes in adipose tissue. We demonstrate that changes in TGs that dominate the overall lipid composition of white adipose tissue are distinct from diet-induced alterations of PLs, the predominant components of the cell membranes. PLs correlate better with transcriptional and one-carbon metabolism changes within the cell, suggesting that the compositional changes that occur in cell membranes during adipocyte expansion have far-reaching functional consequences. Data are available at MetaboLights under the submission number: MTBLS1775.
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http://dx.doi.org/10.1021/acs.jproteome.9b00894DOI Listing
October 2020

Effects of Germline VHL Deficiency on Growth, Metabolism, and Mitochondria.

N Engl J Med 2020 02;382(9):835-844

From the Departments of Woman, Child, and General and Specialized Surgery (S.P., D.R., M. Caiazza, S.S., I.T., M. Casale), Precision Medicine (D.B., E.S., A.B., F.D.R.), and Advanced Medical and Surgical Sciences (A.A.S.), University of Campania Luigi Vanvitelli, Naples, and the Departments of Pharmacology and Biomolecular Science, University of Milan, Milan (P.C., A.M.R.) - both in Italy; the Departments of Physiology, Development, and Neuroscience (K.A.O., A.J.M.) and Biochemistry (J.A.W., J.L.G.), University of Cambridge, Cambridge, the Centre for Ultrastructural Imaging (L.A., R.A.F.) and the Centre for Human and Applied Physiological Sciences, Faculty of Life Sciences and Medicine (F.F.), King's College London, London, and the Department of Physiology, Anatomy, and Genetics (P.A.R., F.F.) and Nuffield Division of Anaesthetics (F.F.), University of Oxford, Oxford - all in the United Kingdom; and the Department of Cell and Developmental Biology, University of Pennsylvania, Philadelphia (C.F.-A.).

Mutations in , which encodes von Hippel-Lindau tumor suppressor (VHL), are associated with divergent diseases. We describe a patient with marked erythrocytosis and prominent mitochondrial alterations associated with a severe germline VHL deficiency due to homozygosity for a novel synonymous mutation (c.222C→A, p.V74V). The condition is characterized by early systemic onset and differs from Chuvash polycythemia (c.598C→T) in that it is associated with a strongly reduced growth rate, persistent hypoglycemia, and limited exercise capacity. We report changes in gene expression that reprogram carbohydrate and lipid metabolism, impair muscle mitochondrial respiratory function, and uncouple oxygen consumption from ATP production. Moreover, we identified unusual intermitochondrial connecting ducts. Our findings add unexpected information on the importance of the VHL-hypoxia-inducible factor (HIF) axis to human phenotypes. (Funded by Associazione Italiana Ricerca sul Cancro and others.).
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http://dx.doi.org/10.1056/NEJMoa1907362DOI Listing
February 2020

FAMIN Is a Multifunctional Purine Enzyme Enabling the Purine Nucleotide Cycle.

Cell 2020 01;180(2):278-295.e23

Cambridge Institute of Therapeutic Immunology and Infectious Disease, Jeffrey Cheah Biomedical Centre, University of Cambridge, Cambridge CB2 0AW, UK; Division of Gastroenterology and Hepatology, Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0QQ, UK. Electronic address:

Mutations in FAMIN cause arthritis and inflammatory bowel disease in early childhood, and a common genetic variant increases the risk for Crohn's disease and leprosy. We developed an unbiased liquid chromatography-mass spectrometry screen for enzymatic activity of this orphan protein. We report that FAMIN phosphorolytically cleaves adenosine into adenine and ribose-1-phosphate. Such activity was considered absent from eukaryotic metabolism. FAMIN and its prokaryotic orthologs additionally have adenosine deaminase, purine nucleoside phosphorylase, and S-methyl-5'-thioadenosine phosphorylase activity, hence, combine activities of the namesake enzymes of central purine metabolism. FAMIN enables in macrophages a purine nucleotide cycle (PNC) between adenosine and inosine monophosphate and adenylosuccinate, which consumes aspartate and releases fumarate in a manner involving fatty acid oxidation and ATP-citrate lyase activity. This macrophage PNC synchronizes mitochondrial activity with glycolysis by balancing electron transfer to mitochondria, thereby supporting glycolytic activity and promoting oxidative phosphorylation and mitochondrial H and phosphate recycling.
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http://dx.doi.org/10.1016/j.cell.2019.12.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6978800PMC
January 2020

A model for determining cardiac mitochondrial substrate utilisation using stable C-labelled metabolites.

Metabolomics 2019 11 26;15(12):154. Epub 2019 Nov 26.

Department of Biochemistry and the Cambridge Systems Biology Centre, University of Cambridge, Cambridge, UK.

Introduction: Relative oxidation of different metabolic substrates in the heart varies both physiologically and pathologically, in order to meet metabolic demands under different circumstances. C labelled substrates have become a key tool for studying substrate use-yet an accurate model is required to analyse the complex data produced as these substrates become incorporated into the Krebs cycle.

Objectives: We aimed to generate a network model for the quantitative analysis of Krebs cycle intermediate isotopologue distributions measured by mass spectrometry, to determine the C labelled proportion of acetyl-CoA entering the Krebs cycle.

Methods: A model was generated, and validated ex vivo using isotopic distributions measured from isolated hearts perfused with buffer containing 11 mM glucose in total, with varying fractions of universally labelled with C. The model was then employed to determine the relative oxidation of glucose and triacylglycerol by hearts perfused with 11 mM glucose and 0.4 mM equivalent Intralipid (a triacylglycerol mixture).

Results: The contribution of glucose to Krebs cycle oxidation was measured to be 79.1 ± 0.9%, independent of the fraction of buffer glucose which was U-C labelled, or of which Krebs cycle intermediate was assessed. In the presence of Intralipid, glucose and triglyceride were determined to contribute 58 ± 3.6% and 35.6 ± 0.8% of acetyl-CoA entering the Krebs cycle, respectively.

Conclusion: These results demonstrate the accuracy of a functional model of Krebs cycle metabolism, which can allow quantitative determination of the effects of therapeutics and pathology on cardiac substrate metabolism.
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http://dx.doi.org/10.1007/s11306-019-1618-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6892366PMC
November 2019

Cytosine-5 RNA methylation links protein synthesis to cell metabolism.

PLoS Biol 2019 06 14;17(6):e3000297. Epub 2019 Jun 14.

Department of Genetics, University of Cambridge, Cambridge, United Kingdom.

Posttranscriptional modifications in transfer RNA (tRNA) are often critical for normal development because they adapt protein synthesis rates to a dynamically changing microenvironment. However, the precise cellular mechanisms linking the extrinsic stimulus to the intrinsic RNA modification pathways remain largely unclear. Here, we identified the cytosine-5 RNA methyltransferase NSUN2 as a sensor for external stress stimuli. Exposure to oxidative stress efficiently repressed NSUN2, causing a reduction of methylation at specific tRNA sites. Using metabolic profiling, we showed that loss of tRNA methylation captured cells in a distinct catabolic state. Mechanistically, loss of NSUN2 altered the biogenesis of tRNA-derived noncoding fragments (tRFs) in response to stress, leading to impaired regulation of protein synthesis. The intracellular accumulation of a specific subset of tRFs correlated with the dynamic repression of global protein synthesis. Finally, NSUN2-driven RNA methylation was functionally required to adapt cell cycle progression to the early stress response. In summary, we revealed that changes in tRNA methylation profiles were sufficient to specify cellular metabolic states and efficiently adapt protein synthesis rates to cell stress.
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http://dx.doi.org/10.1371/journal.pbio.3000297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594628PMC
June 2019

A randomized 3-way crossover study indicates that high-protein feeding induces de novo lipogenesis in healthy humans.

JCI Insight 2019 06 30;4(12). Epub 2019 May 30.

Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom.

BACKGROUNDDietary changes have led to the growing prevalence of type 2 diabetes and nonalcoholic fatty liver disease. A hallmark of both disorders is hepatic lipid accumulation, derived in part from increased de novo lipogenesis. Despite the popularity of high-protein diets for weight loss, the effect of dietary protein on de novo lipogenesis is poorly studied. We aimed to characterize the effect of dietary protein on de novo lipid synthesis.METHODSWe use a 3-way crossover interventional study in healthy males to determine the effect of high-protein feeding on de novo lipogenesis, combined with in vitro models to determine the lipogenic effects of specific amino acids. The primary outcome was a change in de novo lipogenesis-associated triglycerides in response to protein feeding.RESULTSWe demonstrate that high-protein feeding, rich in glutamate, increases de novo lipogenesis-associated triglycerides in plasma (1.5-fold compared with control; P < 0.0001) and liver-derived very low-density lipoprotein particles (1.8-fold; P < 0.0001) in samples from human subjects (n = 9 per group). In hepatocytes, we show that glutamate-derived carbon is incorporated into triglycerides via palmitate. In addition, supplementation with glutamate, glutamine, and leucine, but not lysine, increased triglyceride synthesis and decreased glucose uptake. Glutamate, glutamine, and leucine increased activation of protein kinase B, suggesting that induction of de novo lipogenesis occurs via the insulin signaling cascade.CONCLUSIONThese findings provide mechanistic insight into how select amino acids induce de novo lipogenesis and insulin resistance, suggesting that high-protein feeding to tackle diabetes and obesity requires greater consideration.FUNDINGThe research was supported by UK Medical Research Council grants MR/P011705/1, MC_UP_A090_1006 and MR/P01836X/1. JLG is supported by the Imperial Biomedical Research Centre, National Institute for Health Research (NIHR).
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http://dx.doi.org/10.1172/jci.insight.124819DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629161PMC
June 2019

A dietary pattern derived using B-vitamins and its relationship with vascular markers over the life course.

Clin Nutr 2019 06 28;38(3):1464-1473. Epub 2018 Jun 28.

MRC Elsie Widdowson Laboratory, Cambridge CB1 9NL, United Kingdom; NNEdPro Global Centre for Nutrition and Health (Affiliated with: Cambridge University Health Partners, Wolfson College Cambridge and the British Dietetic Association), St John's Innovation Centre, Cowley Road, Cambridge CB4 0WS, United Kingdom. Electronic address:

Background: Diet may influence vascular function through elevated homocysteine (Hcy) concentrations. However the relationship between dietary patterns (DP), characterised by Hcy and its associated nutrients is unknown.

Objective: To identify a DP characterised by plasma Hcy, dietary folate and dietary vitamin B12, and examine its associations with two markers of vascular function: carotid intima-media thickness (cIMT) and pulse wave velocity (PWV).

Methods: 1562 participants of the MRC National Survey of Health and Development (NSHD), a British birth cohort, with dietary data measured at least once between 36 and 60-64 years, and cIMT or PWV measured at 60-64 years were included. DPs were derived using reduced rank regression with three intermediate variables: 1) plasma Hcy (μmol/L) 2) folate intake (μg/1000 kcal) 3) vitamin B12 intake (μg/1000 kcal). Multiple regression models assessed associations between the derived DP z-scores and vascular function adjusting for dietary misreporting, socioeconomic position, BMI, smoking, physical activity and diabetes.

Results: A DP explaining the highest amount of shared variation (4.5%) in plasma Hcy, dietary folate and dietary vitamin B12 highly correlated with folate (r = 0.96), moderately correlated with vitamin B12 (r = 0.27), and weakly correlated with Hcy (r = 0.10). This "high B-vitamin" DP (including folate) was characterised by high intakes of vegetables, fruit and low fibre breakfast cereal, and low intakes of processed meat, white bread, sugar and preserves. No associations were observed between DP z-scores and vascular function at any time point following adjustment for covariates.

Conclusion: This study explored a specific hypothesised pathway linking diet to vascular function. Although we found no consistent evidence for an association between a high B-vitamin DP and vascular function, we did observe an association with CRP and triglycerides in secondary analyses. Further analyses using strongly correlated and biologically relevant intermediate variables are required to refine investigations into diet and CVD in longitudinal cohort data.
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http://dx.doi.org/10.1016/j.clnu.2018.06.969DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546956PMC
June 2019

Cyclooxygenase-2, Asymmetric Dimethylarginine, and the Cardiovascular Hazard From Nonsteroidal Anti-Inflammatory Drugs.

Circulation 2018 11;138(21):2367-2378

Department of Systems Pharmacology and Translational Therapeutics and the Institute for Translational Medicine and Therapeutics, Perelman School of Medicine, Philadelphia, PA (E.R., S.Y.T., D.M., S.D.R., H.M., X.L., N.F.L., A.M.W., G.A.F.).

Background: Large-scale, placebo-controlled trials established that nonsteroidal anti-inflammatory drugs confer a cardiovascular hazard: this has been attributed to depression of cardioprotective products of cyclooxygenase (COX)-2, especially prostacyclin. An alternative mechanism by which nonsteroidal anti-inflammatory drugs might constrain cardioprotection is by enhancing the formation of methylarginines in the kidney that would limit the action of nitric oxide throughout the vasculature.

Methods: Targeted and untargeted metabolomics were used to investigate the effect of COX-2 deletion or inhibition in mice and in osteoarthritis patients exposed to nonsteroidal anti-inflammatory drugs on the l-arginine/nitric oxide pathway.

Results: Analysis of the plasma and renal metabolome was performed in postnatal tamoxifen-inducible Cox-2 knockout mice, which exhibit normal renal function and blood pressure. This revealed no changes in arginine and methylarginines compared with their wild-type controls. Moreover, the expression of genes in the l-arginine/nitric oxide pathway was not altered in the renal medulla or cortex of tamoxifen inducible Cox-2 knockout mice. Therapeutic concentrations of the selective COX-2 inhibitors, rofecoxib, celecoxib, and parecoxib, none of which altered basal blood pressure or renal function as reflected by plasma creatinine, failed to elevate plasma arginine and methylarginines in mice. Finally, plasma arginine or methylarginines were not altered in osteoarthritis patients with confirmed exposure to nonsteroidal anti-inflammatory drugs that inhibit COX-1 and COX-2. By contrast, plasma asymmetrical dimethylarginine was increased in mice infused with angiotensin II sufficient to elevate blood pressure and impair renal function. Four weeks later, blood pressure, plasma creatinine, and asymmetrical dimethylarginine were restored to normal levels. The increase in asymmetrical dimethylarginine in response to infusion with angiotensin II in celecoxib-treated mice was also related to transient impairment of renal function.

Conclusions: Plasma methylarginines are not altered by COX-2 deletion or inhibition but rather are elevated coincident with renal compromise.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.118.033540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6291350PMC
November 2018

Nox4 reprograms cardiac substrate metabolism via protein O-GlcNAcylation to enhance stress adaptation.

JCI Insight 2017 12 21;2(24). Epub 2017 Dec 21.

Cardiovascular Division, King's College London British Heart Foundation Centre of Excellence, London, United Kingdom.

Cardiac hypertrophic remodeling during chronic hemodynamic stress is associated with a switch in preferred energy substrate from fatty acids to glucose, usually considered to be energetically favorable. The mechanistic interrelationship between altered energy metabolism, remodeling, and function remains unclear. The ROS-generating NADPH oxidase-4 (Nox4) is upregulated in the overloaded heart, where it ameliorates adverse remodeling. Here, we show that Nox4 redirects glucose metabolism away from oxidation but increases fatty acid oxidation, thereby maintaining cardiac energetics during acute or chronic stresses. The changes in glucose and fatty acid metabolism are interlinked via a Nox4-ATF4-dependent increase in the hexosamine biosynthetic pathway, which mediates the attachment of O-linked N-acetylglucosamine (O-GlcNAcylation) to the fatty acid transporter CD36 and enhances fatty acid utilization. These data uncover a potentially novel redox pathway that regulates protein O-GlcNAcylation and reprograms cardiac substrate metabolism to favorably modify adaptation to chronic stress. Our results also suggest that increased fatty acid oxidation in the chronically stressed heart may be beneficial.
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http://dx.doi.org/10.1172/jci.insight.96184DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5752273PMC
December 2017

Metabolic basis to Sherpa altitude adaptation.

Proc Natl Acad Sci U S A 2017 06 22;114(24):6382-6387. Epub 2017 May 22.

Department of Physiology, Development & Neuroscience, University of Cambridge, Cambridge CB2 3EG, United Kingdom;

The Himalayan Sherpas, a human population of Tibetan descent, are highly adapted to life in the hypobaric hypoxia of high altitude. Mechanisms involving enhanced tissue oxygen delivery in comparison to Lowlander populations have been postulated to play a role in such adaptation. Whether differences in tissue oxygen utilization (i.e., metabolic adaptation) underpin this adaptation is not known, however. We sought to address this issue, applying parallel molecular, biochemical, physiological, and genetic approaches to the study of Sherpas and native Lowlanders, studied before and during exposure to hypobaric hypoxia on a gradual ascent to Mount Everest Base Camp (5,300 m). Compared with Lowlanders, Sherpas demonstrated a lower capacity for fatty acid oxidation in skeletal muscle biopsies, along with enhanced efficiency of oxygen utilization, improved muscle energetics, and protection against oxidative stress. This adaptation appeared to be related, in part, to a putatively advantageous allele for the peroxisome proliferator-activated receptor A () gene, which was enriched in the Sherpas compared with the Lowlanders. Our findings suggest that metabolic adaptations underpin human evolution to life at high altitude, and could have an impact upon our understanding of human diseases in which hypoxia is a feature.
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http://dx.doi.org/10.1073/pnas.1700527114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5474778PMC
June 2017

Inhibition of sarcolemmal FAT/CD36 by sulfo-N-succinimidyl oleate rapidly corrects metabolism and restores function in the diabetic heart following hypoxia/reoxygenation.

Cardiovasc Res 2017 Jun;113(7):737-748

Department of Physiology, Anatomy and Genetics, University of Oxford, Parks Road, Oxford, OX1 3PT, UK.

Aims: The type 2 diabetic heart oxidizes more fat and less glucose, which can impair metabolic flexibility and function. Increased sarcolemmal fatty acid translocase (FAT/CD36) imports more fatty acid into the diabetic myocardium, feeding increased fatty acid oxidation and elevated lipid deposition. Unlike other metabolic modulators that target mitochondrial fatty acid oxidation, we proposed that pharmacologically inhibiting fatty acid uptake, as the primary step in the pathway, would provide an alternative mechanism to rebalance metabolism and prevent lipid accumulation following hypoxic stress.

Methods And Results: Hearts from type 2 diabetic and control male Wistar rats were perfused in normoxia, hypoxia and reoxygenation, with the FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) infused 4 min before hypoxia. SSO infusion into diabetic hearts decreased the fatty acid oxidation rate by 29% and myocardial triglyceride concentration by 48% compared with untreated diabetic hearts, restoring fatty acid metabolism to control levels following hypoxia-reoxygenation. SSO infusion increased the glycolytic rate by 46% in diabetic hearts during hypoxia, increased pyruvate dehydrogenase activity by 53% and decreased lactate efflux rate by 56% compared with untreated diabetic hearts during reoxygenation. In addition, SSO treatment of diabetic hearts increased intermediates within the second span of the Krebs cycle, namely fumarate, oxaloacetate, and the FAD total pool. The cardiac dysfunction in diabetic hearts following decreased oxygen availability was prevented by SSO-infusion prior to the hypoxic stress. Infusing SSO into diabetic hearts increased rate pressure product by 60% during hypoxia and by 32% following reoxygenation, restoring function to control levels.

Conclusions: Diabetic hearts have limited metabolic flexibility and cardiac dysfunction when stressed, which can be rapidly rectified by reducing fatty acid uptake with the FAT/CD36 inhibitor, SSO. This novel therapeutic approach not only reduces fat oxidation but also lipotoxicity, by targeting the primary step in the fatty acid metabolism pathway.
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http://dx.doi.org/10.1093/cvr/cvx045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5437367PMC
June 2017

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance.

Sci Rep 2017 03 23;7:44845. Epub 2017 Mar 23.

Medical Research Council Human Nutrition Research, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge, CB1 9NL. Affiliated with the University of Cambridge, United Kingdom.

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1 mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1 only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.
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http://dx.doi.org/10.1038/srep44845DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5362956PMC
March 2017

Integration of metabolomics, lipidomics and clinical data using a machine learning method.

BMC Bioinformatics 2016 Nov 22;17(Suppl 15):440. Epub 2016 Nov 22.

Medical Research Council, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge, CB1 9NL, UK.

Background: The recent pandemic of obesity and the metabolic syndrome (MetS) has led to the realisation that new drug targets are needed to either reduce obesity or the subsequent pathophysiological consequences associated with excess weight gain. Certain nuclear hormone receptors (NRs) play a pivotal role in lipid and carbohydrate metabolism and have been highlighted as potential treatments for obesity. This realisation started a search for NR agonists in order to understand and successfully treat MetS and associated conditions such as insulin resistance, dyslipidaemia, hypertension, hypertriglyceridemia, obesity and cardiovascular disease. The most studied NRs for treating metabolic diseases are the peroxisome proliferator-activated receptors (PPARs), PPAR-α, PPAR-γ, and PPAR-δ. However, prolonged PPAR treatment in animal models has led to adverse side effects including increased risk of a number of cancers, but how these receptors change metabolism long term in terms of pathology, despite many beneficial effects shorter term, is not fully understood. In the current study, changes in male Sprague Dawley rat liver caused by dietary treatment with a PPAR-pan (PPAR-α, -γ, and -δ) agonist were profiled by classical toxicology (clinical chemistry) and high throughput metabolomics and lipidomics approaches using mass spectrometry.

Results: In order to integrate an extensive set of nine different multivariate metabolic and lipidomics datasets with classical toxicological parameters we developed a hypotheses free, data driven machine learning approach. From the data analysis, we examined how the nine datasets were able to model dose and clinical chemistry results, with the different datasets having very different information content.

Conclusions: We found lipidomics (Direct Infusion-Mass Spectrometry) data the most predictive for different dose responses. In addition, associations with the metabolic and lipidomic data with aspartate amino transaminase (AST), a hepatic leakage enzyme to assess organ damage, and albumin, indicative of altered liver synthetic function, were established. Furthermore, by establishing correlations and network connections between eicosanoids, phospholipids and triacylglycerols, we provide evidence that these lipids function as a key link between inflammatory processes and intermediary metabolism.
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http://dx.doi.org/10.1186/s12859-016-1292-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133491PMC
November 2016

Metabolomics dataset of PPAR-pan treated rat liver.

Data Brief 2016 Sep 10;8:196-202. Epub 2016 May 10.

Medical Research Council, Human Nutrition Research, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge CB1 9NL, UK; The Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK; The Cambridge Systems Biology Centre (CSBC), University of Cambridge, Cambridge CB2 1QR, UK.

This article contains mass spectrometry (MS) data investigating small molecule changes as an effect of a triple peroxisome proliferator-activated receptor (PPAR-pan) agonist GW625019 in the liver as described in the manuscript (Ament et al., 2016) [1]. Samples were measured using gas chromatography-mass spectrometry (GC-MS) for total fatty acid content, and liquid chromatography-mass spectrometry (LC-MS) to measure intact lipids, carnitines and selected aqueous metabolites and eicosanoids. Data files comprise of Excel (Microsoft, WA, USA) spreadsheets of identified metabolites and their area ratio values for total fatty acids, carnitines, aqueous metabolites, and eicosanoids where the intensity of the analytes were normalised to the intensity of the internal standard. In the case of open profiling intact lipid data, the Excel file contains area ratio values of retention time and mass to charge ratio pairs; again, the area ratio values were calculated by normalising to the intensity of the internal standard. It should be noted that several metabolic changes are potentially indirect (secondary, tertiary and ensuing changes).
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http://dx.doi.org/10.1016/j.dib.2016.05.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898904PMC
September 2016

A targeted metabolomics assay for cardiac metabolism and demonstration using a mouse model of dilated cardiomyopathy.

Metabolomics 2016;12:59. Epub 2016 Mar 7.

The Department of Biochemistry & The Cambridge Systems Biology Centre, University of Cambridge, Tennis Court Road, Cambridge, CB2 1GA UK ; The Elsie Widdowson Laboratory, Medical Research Council Human Nutrition Research, 120 Fulbourn Road, Cambridge, CB1 9NL UK.

Metabolomics can be performed either as an 'open profiling' tool where the aim is to measure, usually in a semi-quantitative manner, as many metabolites as possible or perform 'closed' or 'targeted' analyses where instead a pre-defined set of metabolites are measured. Targeted methods can be designed to be more sensitive and quantitative and so are particularly appropriate to systems biology for quantitative models of systems or when metabolomics is performed in a hypothesis driven manner to test whether a particular pathway is perturbed. We describe a targeted metabolomics assay that quantifies a broad range of over 130 metabolites relevant to cardiac metabolism including the pathways of the citric acid cycle, fatty acid oxidation, glycolysis, the pentose phosphate pathway, amino acid metabolism, the urea cycle, nucleotides and reactive oxygen species using tandem mass spectrometry to produce quantitative, sensitive and robust data. This assay is illustrated by profiling cardiac metabolism in a lamin A/C (Lmna) mouse model of dilated cardiomyopathy (DCM). The model of DCM was characterised by increases in concentrations of proline and methyl-histidine suggestive of increased myofibrillar and collagen degradation, as well as decreases in a number of citric acid cycle intermediates and carnitine derivatives indicating reduced energy metabolism in the dilated heart. These assays could be used for any other cardiac or cardiovascular disease in that they cover central core metabolism and key pathways involved in cardiac metabolism, and may provide a general start for many mammalian systems.
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http://dx.doi.org/10.1007/s11306-016-0956-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4781888PMC
March 2016

Nitrate enhances skeletal muscle fatty acid oxidation via a nitric oxide-cGMP-PPAR-mediated mechanism.

BMC Biol 2015 Dec 22;13:110. Epub 2015 Dec 22.

Department of Physiology, Development & Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, UK.

Background: Insulin sensitivity in skeletal muscle is associated with metabolic flexibility, including a high capacity to increase fatty acid (FA) oxidation in response to increased lipid supply. Lipid overload, however, can result in incomplete FA oxidation and accumulation of potentially harmful intermediates where mitochondrial tricarboxylic acid cycle capacity cannot keep pace with rates of β-oxidation. Enhancement of muscle FA oxidation in combination with mitochondrial biogenesis is therefore emerging as a strategy to treat metabolic disease. Dietary inorganic nitrate was recently shown to reverse aspects of the metabolic syndrome in rodents by as yet incompletely defined mechanisms.

Results: Herein, we report that nitrate enhances skeletal muscle FA oxidation in rodents in a dose-dependent manner. We show that nitrate induces FA oxidation through a soluble guanylate cyclase (sGC)/cGMP-mediated PPARβ/δ- and PPARα-dependent mechanism. Enhanced PPARβ/δ and PPARα expression and DNA binding induces expression of FA oxidation enzymes, increasing muscle carnitine and lowering tissue malonyl-CoA concentrations, thereby supporting intra-mitochondrial pathways of FA oxidation and enhancing mitochondrial respiration. At higher doses, nitrate induces mitochondrial biogenesis, further increasing FA oxidation and lowering long-chain FA concentrations. Meanwhile, nitrate did not affect mitochondrial FA oxidation in PPARα(-/-) mice. In C2C12 myotubes, nitrate increased expression of the PPARα targets Cpt1b, Acadl, Hadh and Ucp3, and enhanced oxidative phosphorylation rates with palmitoyl-carnitine; however, these changes in gene expression and respiration were prevented by inhibition of either sGC or protein kinase G. Elevation of cGMP, via the inhibition of phosphodiesterase 5 by sildenafil, also increased expression of Cpt1b, Acadl and Ucp3, as well as CPT1B protein levels, and further enhanced the effect of nitrate supplementation.

Conclusions: Nitrate may therefore be effective in the treatment of metabolic disease by inducing FA oxidation in muscle.
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http://dx.doi.org/10.1186/s12915-015-0221-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4688964PMC
December 2015

PPAR-pan activation induces hepatic oxidative stress and lipidomic remodelling.

Free Radic Biol Med 2016 06 30;95:357-68. Epub 2015 Nov 30.

Medical Research Council, Human Nutrition Research, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge CB1 9NL, UK; The Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK; The Cambridge Systems Biology Centre (CSBC), University of Cambridge, Cambridge CB2 1QR, UK. Electronic address:

The peroxisome proliferator-activated receptors (PPARs) are ligand activated nuclear receptors that regulate cellular homoeostasis and metabolism. PPARs control the expression of genes involved in fatty-acid and lipid metabolism. Despite evidence showing beneficial effects of their activation in the treatment of metabolic diseases, particularly dyslipidaemias and type 2 diabetes, PPAR agonists have also been associated with a variety of side effects and adverse pathological changes. Agonists have been developed that simultaneously activate the three PPAR receptors (PPARα, γ and δ) in the hope that the beneficial effects can be harnessed while avoiding some of the negative side effects. In this study, the hepatic effects of a discontinued PPAR-pan agonist (a triple agonist of PPAR-α, -γ, and -δ), was investigated after dietary treatment of male Sprague-Dawley (SD) rats. The agonist induced liver enlargement in conjunction with metabolomic and lipidomic remodelling. Increased concentrations of several metabolites related to processes of oxidation, such as oxo-methionine, methyl-cytosine and adenosyl-methionine indicated increased stress and immune status. These changes are reflected in lipidomic changes, and increased energy demands as determined by free fatty acid (decreased 18:3 n-3, 20:5 n-3 and increased ratios of n-6/n-3 fatty acids) triacylglycerol, phospholipid (decreased and increased bulk changes respectively) and eicosanoid content (increases in PGB2 and 15-deoxy PGJ2). We conclude that the investigated PPAR agonist, GW625019, induces liver enlargement, accompanied by lipidomic remodelling, oxidative stress and increases in several pro-inflammatory eicosanoids. This suggests that such pathways should be monitored in the drug development process and also outline how PPAR agonists induce liver proliferation.
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http://dx.doi.org/10.1016/j.freeradbiomed.2015.11.033DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4891066PMC
June 2016

Mechanistic insights revealed by lipid profiling in monogenic insulin resistance syndromes.

Genome Med 2015 28;7:63. Epub 2015 Jun 28.

MRC Human Nutrition Research, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge, CB1 9NL UK ; Department of Biochemistry and the Cambridge Systems Biology Centre, University of Cambridge, Tennis Court Road, Cambridge, UK.

Background: Evidence from several recent metabolomic studies suggests that increased concentrations of triacylglycerols with shorter (14-16 carbon atoms), saturated fatty acids are associated with insulin resistance and the risk of type 2 diabetes. Although causality cannot be inferred from association studies, patients in whom the primary cause of insulin resistance can be genetically defined offer unique opportunities to address this challenge.

Methods: We compared metabolite profiles in patients with congenital lipodystrophy or loss-of-function insulin resistance (INSR gene) mutations with healthy controls.

Results: The absence of significant differences in triacylglycerol species in the INSR group suggest that changes previously observed in epidemiological studies are not purely a consequence of insulin resistance. The presence of triacylglycerols with lower carbon numbers and high saturation in patients with lipodystrophy suggests that these metabolite changes may be associated with primary adipose tissue dysfunction. The observed pattern of triacylglycerol species is indicative of increased de novo lipogenesis in the liver. To test this we investigated the distribution of these triacylglycerols in lipoprotein fractions using size exclusion chromatography prior to mass spectrometry. This associated these triacylglycerols with very low-density lipoprotein particles, and hence release of triacylglycerols into the blood from the liver. To test further the hepatic origin of these triacylglycerols we induced de novo lipogenesis in the mouse, comparing ob/ob and wild-type mice on a chow or high fat diet, confirming that de novo lipogenesis induced an increase in relatively shorter, more saturated fatty acids.

Conclusions: Overall, these studies highlight hepatic de novo lipogenesis in the pathogenesis of metabolic dyslipidaemia in states where energy intake exceeds the capacity of adipose tissue.
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http://dx.doi.org/10.1186/s13073-015-0179-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4535665PMC
May 2016

Solitary supratentorial Listeria monocytogenes brain abscess in an immunocompromised patient.

Proc (Bayl Univ Med Cent) 2015 Jul;28(3):337-9

Department of Diagnostic Radiology (West, Onofrio, Opatowsky, Layton), the Department of Internal Medicine (Martinez), and the Division of Infectious Diseases (Spak), Baylor University Medical Center at Dallas.

We describe an 81-year-old man receiving azacitidine monotherapy for myelodysplastic syndrome who was improving from Listeria monocytogenes bacteremia after receiving antibiotic therapy during an earlier hospital admission. Shortly after discharge he developed new-onset seizure activity, with brain imaging on subsequent admissions demonstrating a posterior right frontal lobe mass. Specimen cultures after resection of the mass revealed this to be a cerebral abscess related to L. monocytogenes. Brain abscesses related to this organism are rare.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462214PMC
http://dx.doi.org/10.1080/08998280.2015.11929266DOI Listing
July 2015

Radiographic findings in the nail-patella syndrome.

Proc (Bayl Univ Med Cent) 2015 Jul;28(3):334-6

Department of Diagnostic Radiology, Baylor University Medical Center at Dallas, Dallas, Texas.

Nail-patella syndrome is a rare disorder characterized classically by the tetrad of nail hypoplasia or aplasia, aplastic or hypoplastic patellae, elbow dysplasia, and the presence of iliac horns. Iliac horns are considered pathognomonic, and the presence of hypoplastic or aplastic patellae in conjunction with nail abnormalities is a cardinal feature of diagnosis. Elbow dysplasia is present in most cases and can exhibit features typical of the syndrome. Herein we present the radiographic findings of the elbows, knees, and pelvis of a woman with nail-patella syndrome.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4462213PMC
http://dx.doi.org/10.1080/08998280.2015.11929265DOI Listing
July 2015

Comprehensive Metabolic Profiling of Age-Related Mitochondrial Dysfunction in the High-Fat-Fed ob/ob Mouse Heart.

J Proteome Res 2015 Jul 22;14(7):2849-62. Epub 2015 Jun 22.

‡MRC, Human Nutrition Research, Elsie Widdowson Laboratory, 120 Fulbourn Road, Cambridge, CB1 9NL, U.K.

The ectopic deposition of fat is thought to lead to lipotoxicity and has been associated with mitochondrial dysfunction and diabetic cardiomyopathy. We have measured mitochondrial respiratory capacities in the hearts of ob/ob and wild-type mice on either a regular chow (RCD) or high-fat (HFD) diet across four age groups to investigate the impact of diet and age on mitochondrial function alongside a comprehensive strategy for metabolic profiling of the tissue. Myocardial mitochondrial dysfunction was only evident in ob/ob mice on RCD at 14 months, but it was detectable at 3 months on the HFD. Liquid chromatography-mass spectrometry (LC-MS) was used to study the profiles of acylcarnitines and the accumulation of triglycerides, but neither class of lipid was associated with mitochondrial dysfunction. However, a targeted LC-MS/MS analysis of markers of oxidative stress demonstrated increases in GSSG/GSH and 8-oxoguanine, in addition to the accumulation of diacylglycerols, which are lipid species linked to lipotoxicity. Our results demonstrate that myocardial mitochondria in ob/ob mice on RCD maintained a similar respiratory capacity to that of wild type until a late stage in aging. However, on a HFD, unlike wild-type mice, ob/ob mice failed to increase mitochondrial respiration, which may be associated with a complex I defect following increased oxidative damage.
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http://dx.doi.org/10.1021/acs.jproteome.5b00128DOI Listing
July 2015

A review of odd-chain fatty acid metabolism and the role of pentadecanoic Acid (c15:0) and heptadecanoic Acid (c17:0) in health and disease.

Molecules 2015 Jan 30;20(2):2425-44. Epub 2015 Jan 30.

MRC HNR, Elsie Widdowson Laboratory, Fulbourn Road, Cambridge CB1 9NL, UK.

The role of C17:0 and C15:0 in human health has recently been reinforced following a number of important biological and nutritional observations. Historically, odd chain saturated fatty acids (OCS-FAs) were used as internal standards in GC-MS methods of total fatty acids and LC-MS methods of intact lipids, as it was thought their concentrations were insignificant in humans. However, it has been thought that increased consumption of dairy products has an association with an increase in blood plasma OCS-FAs. However, there is currently no direct evidence but rather a casual association through epidemiology studies. Furthermore, a number of studies on cardiometabolic diseases have shown that plasma concentrations of OCS-FAs are associated with lower disease risk, although the mechanism responsible for this is debated. One possible mechanism for the endogenous production of OCS-FAs is α-oxidation, involving the activation, then hydroxylation of the α-carbon, followed by the removal of the terminal carboxyl group. Differentiation human adipocytes showed a distinct increase in the concentration of OCS-FAs, which was possibly caused through α-oxidation. Further evidence for an endogenous pathway, is in human plasma, where the ratio of C15:0 to C17:0 is approximately 1:2 which is contradictory to the expected levels of C15:0 to C17:0 roughly 2:1 as detected in dairy fat. We review the literature on the dietary consumption of OCS-FAs and their potential endogenous metabolism.
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http://dx.doi.org/10.3390/molecules20022425DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6272531PMC
January 2015

Impaired in vivo mitochondrial Krebs cycle activity after myocardial infarction assessed using hyperpolarized magnetic resonance spectroscopy.

Circ Cardiovasc Imaging 2014 Nov 8;7(6):895-904. Epub 2014 Sep 8.

Department of Physiology, Anatomy, and Genetics, University of Oxford, Oxford, United Kingdom (M.S.D., H.J.A., C.A.C., G.K.R., K.C., L.C.H., D.J.T.); Centre for Advanced Biomedical Imaging, University College London, London, United Kingdom (D.J.S.); and Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom (J.A.W., J.L.G.).

Background: Myocardial infarction (MI) is one of the leading causes of heart failure. An increasing body of evidence links alterations in cardiac metabolism and mitochondrial function with the progression of heart disease. The aim of this work was to, therefore, follow the in vivo mitochondrial metabolic alterations caused by MI, thereby allowing a greater understanding of the interplay between metabolic and functional abnormalities.

Methods And Results: Using hyperpolarized carbon-13 ((13)C)-magnetic resonance spectroscopy, in vivo alterations in mitochondrial metabolism were assessed for 22 weeks after surgically induced MI with reperfusion in female Wister rats. One week after MI, there were no detectable alterations in in vivo cardiac mitochondrial metabolism over the range of ejection fractions observed (from 28% to 84%). At 6 weeks after MI, in vivo mitochondrial Krebs cycle activity was impaired, with decreased (13)C-label flux into citrate, glutamate, and acetylcarnitine, which correlated with the degree of cardiac dysfunction. These changes were independent of alterations in pyruvate dehydrogenase flux. By 22 weeks, alterations were also seen in pyruvate dehydrogenase flux, which decreased at lower ejection fractions. These results were confirmed using in vitro analysis of enzyme activities and metabolomic profiles of key intermediates.

Conclusions: The in vivo decrease in Krebs cycle activity in the 6-week post-MI heart may represent an early maladaptive phase in the metabolic alterations after MI in which reductions in Krebs cycle activity precede a reduction in pyruvate dehydrogenase flux. Changes in mitochondrial metabolism in heart disease are progressive and proportional to the degree of cardiac impairment.
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http://dx.doi.org/10.1161/CIRCIMAGING.114.001857DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4450075PMC
November 2014

Dietary nitrate increases arginine availability and protects mitochondrial complex I and energetics in the hypoxic rat heart.

J Physiol 2014 Nov 28;592(21):4715-31. Epub 2014 Aug 28.

Department of Physiology, Development & Neuroscience, University of Cambridge, UK

Hypoxic exposure is associated with impaired cardiac energetics in humans and altered mitochondrial function, with suppressed complex I-supported respiration, in rat heart. This response might limit reactive oxygen species generation, but at the cost of impaired electron transport chain (ETC) activity. Dietary nitrate supplementation improves mitochondrial efficiency and can promote tissue oxygenation by enhancing blood flow. We therefore hypothesised that ETC dysfunction, impaired energetics and oxidative damage in the hearts of rats exposed to chronic hypoxia could be alleviated by sustained administration of a moderate dose of dietary nitrate. Male Wistar rats (n = 40) were given water supplemented with 0.7 mmol l(-1) NaCl (as control) or 0.7 mmol l(-1) NaNO3, elevating plasma nitrate levels by 80%, and were exposed to 13% O2 (hypoxia) or normoxia (n = 10 per group) for 14 days. Respiration rates, ETC protein levels, mitochondrial density, ATP content and protein carbonylation were measured in cardiac muscle. Complex I respiration rates and protein levels were 33% lower in hypoxic/NaCl rats compared with normoxic/NaCl controls. Protein carbonylation was 65% higher in hearts of hypoxic rats compared with controls, indicating increased oxidative stress, whilst ATP levels were 62% lower. Respiration rates, complex I protein and activity, protein carbonylation and ATP levels were all fully protected in the hearts of nitrate-supplemented hypoxic rats. Both in normoxia and hypoxia, dietary nitrate suppressed cardiac arginase expression and activity and markedly elevated cardiac l-arginine concentrations, unmasking a novel mechanism of action by which nitrate enhances tissue NO bioavailability. Dietary nitrate therefore alleviates metabolic abnormalities in the hypoxic heart, improving myocardial energetics.
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http://dx.doi.org/10.1113/jphysiol.2014.275263DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4253472PMC
November 2014

Clinical and morphologic findings in disseminated Scedosporium apiospermum infections in immunocompromised patients.

Proc (Bayl Univ Med Cent) 2014 Jul;27(3):253-6

Departments of Pathology (Campa-Thompson, Guileyardo, Beal), Radiology (West), and Internal Medicine, Division of Infectious Diseases (Spak, Sloan), Baylor University Medical Center at Dallas; and med fusion Laboratory (Campa-Thompson, Beal), Lewisville, Texas.

Scedosporium apiospermum is a ubiquitous, saprophytic, filamentous mold that may cause localized, subcutaneous infections in immunocompetent hosts, but disseminated infection in severely immunocompromised patients. This mold is often highly resistant to multiple commonly used antifungal drugs. Even with treatment, there is a high mortality rate. We present two patients with fatal disseminated S. apiospermum infections after bone marrow and lung transplantation. This infection can be rapidly fatal, and survival may be improved by early recognition.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4059584PMC
http://dx.doi.org/10.1080/08998280.2014.11929129DOI Listing
July 2014

Methods for performing lipidomics in white adipose tissue.

Methods Enzymol 2014 ;538:211-31

MRC Human Nutrition Research, The Elsie Widdowson Laboratory, Cambridge, United Kingdom; Department of Biochemistry and Cambridge Systems Biology Centre, University of Cambridge, Cambridge, United Kingdom. Electronic address:

Lipid metabolism is central to the function of white adipose tissue, with the tissue having a central role in storing triacylglycerides following feeding and releasing free fatty acids and monoacylglycerides during periods of fasting. In addition, lipid species have been suggested to play a role in lipotoxicity and as signaling molecules during adipose tissue inflammation. This chapter details how mass spectrometry (MS) can be used to profile a range of lipid species found in adipose tissue. The initial step required in any MS-based approach is to extract the lipid fraction from the tissue. We detail one commonly used method based on the Folch extraction procedure. The total fatty acid composition of the lipid fraction can readily be defined using gas chromatography-MS, and we provide a method routinely used for rodent and human adipose tissue samples. However, such approaches do not provide insight into what lipid classes the various fatty acids are associated with. To better understand the global lipid profile of the tissue, we provide a general-purpose liquid chromatography-MS-based approach useful for processing phospholipids, free fatty acids, and triacylglycerides. In addition, we provide a method for profiling eicosanoids, a class of important lipid-signaling molecules, which have been implicated in white adipose tissue inflammation in rodent models of obesity, insulin resistance, and type 2 diabetes.
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http://dx.doi.org/10.1016/B978-0-12-800280-3.00012-8DOI Listing
October 2014

A metabolomics investigation of non-genotoxic carcinogenicity in the rat.

J Proteome Res 2013 Dec 7;12(12):5775-90. Epub 2013 Nov 7.

Medical Research Council Human Nutrition Research (MRC HNR), Elsie Widdowson Laboratory , 120 Fulbourn Road, Cambridge CB1 9NL, U.K. , The Department of Biochemistry, University of Cambridge , 80 Tennis Court Road, Cambridge CB2 1GA, U.K. , and Cambridge Systems Biology Centre (CSBC), University of Cambridge , Cambridge CB2 1QR, U.K.

Non-genotoxic carcinogens (NGCs) promote tumor growth by altering gene expression, which ultimately leads to cancer without directly causing a change in DNA sequence. As a result NGCs are not detected in mutagenesis assays. While there are proposed biomarkers of carcinogenic potential, the definitive identification of non-genotoxic carcinogens still rests with the rat and mouse long-term bioassay. Such assays are expensive and time-consuming and require a large number of animals, and their relevance to human health risk assessments is debatable. Metabolomics and lipidomics in combination with pathology and clinical chemistry were used to profile perturbations produced by 10 compounds that represented a range of rat non-genotoxic hepatocarcinogens (NGC), non-genotoxic non-hepatocarcinogens (non-NGC), and a genotoxic hepatocarcinogen. Each compound was administered at its maximum tolerated dose level for 7, 28, and 91 days to male Fisher 344 rats. Changes in liver metabolite concentration differentiated the treated groups across different time points. The most significant differences were driven by pharmacological mode of action, specifically by the peroxisome proliferator activated receptor alpha (PPAR-α) agonists. Despite these dominant effects, good predictions could be made when differentiating NGCs from non-NGCs. Predictive ability measured by leave one out cross validation was 87% and 77% after 28 days of dosing for NGCs and non-NGCs, respectively. Among the discriminatory metabolites we identified free fatty acids, phospholipids, and triacylglycerols, as well as precursors of eicosanoid and the products of reactive oxygen species linked to processes of inflammation, proliferation, and oxidative stress. Thus, metabolic profiling is able to identify changes due to the pharmacological mode of action of xenobiotics and contribute to early screening for non-genotoxic potential.
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http://dx.doi.org/10.1021/pr4007766DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3980845PMC
December 2013

Hypoxic regulation of hand1 controls the fetal-neonatal switch in cardiac metabolism.

PLoS Biol 2013 Sep 24;11(9):e1001666. Epub 2013 Sep 24.

Developmental Biology, MRC-National Institute for Medical Research, London, United Kingdom ; Division of Medicine, University College London, London, United Kingdom.

Cardiomyocytes are vulnerable to hypoxia in the adult, but adapted to hypoxia in utero. Current understanding of endogenous cardiac oxygen sensing pathways is limited. Myocardial oxygen consumption is determined by regulation of energy metabolism, which shifts from glycolysis to lipid oxidation soon after birth, and is reversed in failing adult hearts, accompanying re-expression of several "fetal" genes whose role in disease phenotypes remains unknown. Here we show that hypoxia-controlled expression of the transcription factor Hand1 determines oxygen consumption by inhibition of lipid metabolism in the fetal and adult cardiomyocyte, leading to downregulation of mitochondrial energy generation. Hand1 is under direct transcriptional control by HIF1α. Transgenic mice prolonging cardiac Hand1 expression die immediately following birth, failing to activate the neonatal lipid metabolising gene expression programme. Deletion of Hand1 in embryonic cardiomyocytes results in premature expression of these genes. Using metabolic flux analysis, we show that Hand1 expression controls cardiomyocyte oxygen consumption by direct transcriptional repression of lipid metabolising genes. This leads, in turn, to increased production of lactate from glucose, decreased lipid oxidation, reduced inner mitochondrial membrane potential, and mitochondrial ATP generation. We found that this pathway is active in adult cardiomyocytes. Up-regulation of Hand1 is protective in a mouse model of myocardial ischaemia. We propose that Hand1 is part of a novel regulatory pathway linking cardiac oxygen levels with oxygen consumption. Understanding hypoxia adaptation in the fetal heart may allow development of strategies to protect cardiomyocytes vulnerable to ischaemia, for example during cardiac ischaemia or surgery.
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http://dx.doi.org/10.1371/journal.pbio.1001666DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3782421PMC
September 2013