Bilateral remote ischemic conditioning in children: A two-center, double-blind, randomized controlled trial in young children undergoing cardiac surgery.

Objective: The study objective was to determine whether adequately delivered bilateral remote ischemic preconditioning is cardioprotective in young children undergoing surgery for 2 common congenital heart defects with or without cyanosis. Methods: We performed a prospective, double-blind, randomized controlled trial at 2 centers in the United Kingdom. Children aged 3 to 36 months undergoing tetralogy of Fallot repair or ventricular septal defect closure were randomized 1:1 to receive bilateral preconditioning or sham intervention. Participants were followed up until hospital discharge or 30 days. The primary outcome was area under the curve for high-sensitivity troponin-T in the first 24 hours after surgery, analyzed by intention-to-treat. Right atrial biopsies were obtained in selected participants. Results: Between October 2016 and December 2020, 120 eligible children were randomized to receive bilateral preconditioning (n = 60) or sham intervention (n = 60). The primary outcome, area under the curve for high-sensitivity troponin-T, was higher in the preconditioning group (mean: 70.0 ± 50.9 µg/L/h, n = 56) than in controls (mean: 55.6 ± 30.1 µg/L/h, n = 58) (mean difference, 13.2 µg/L/h; 95% CI, 0.5-25.8; P = .04). Subgroup analyses did not show a differential treatment effect by oxygen saturations (pinteraction = .25), but there was evidence of a differential effect by underlying defect (pinteraction = .04). Secondary outcomes and myocardial metabolism, quantified in atrial biopsies, were not different between randomized groups. Conclusions: Bilateral remote ischemic preconditioning does not attenuate myocardial injury in children undergoing surgical repair for congenital heart defects, and there was evidence of potential harm in unstented tetralogy of Fallot. The routine use of remote ischemic preconditioning cannot be recommended for myocardial protection during pediatric cardiac surgery.

Lipidome characterisation and sex-specific differences in type 1 and type 2 diabetes mellitus.

BACKGROUND: In this study, we evaluated the lipidome alterations caused by type 1 diabetes (T1D) and type 2 diabetes (T2D), by determining lipids significantly associated with diabetes overall and in both sexes, and lipids associated with the glycaemic state. METHODS: An untargeted lipidomic analysis was performed to measure the lipid profiles of 360 subjects (91 T1D, 91 T2D, 74 with prediabetes and 104 controls (CT)) without cardiovascular and/or chronic kidney disease. Ultra-high performance liquid chromatography-electrospray ionization mass spectrometry (UHPLC-ESI-MS) was conducted in two ion modes (positive and negative). We used multiple linear regression models to (1) assess the association between each lipid feature and each condition, (2) determine sex-specific differences related to diabetes, and (3) identify lipids associated with the glycaemic state by considering the prediabetes stage. The models were adjusted by sex, age, hypertension, dyslipidaemia, body mass index, glucose, smoking, systolic blood pressure, triglycerides, HDL cholesterol, LDL cholesterol, alternate Mediterranean diet score (aMED) and estimated glomerular filtration rate (eGFR); diabetes duration and glycated haemoglobin (HbA1c) were also included in the comparison between T1D and T2D. RESULTS: A total of 54 unique lipid subspecies from 15 unique lipid classes were annotated. Lysophosphatidylcholines (LPC) and ceramides (Cer) showed opposite effects in subjects with T1D and subjects with T2D, LPCs being mainly up-regulated in T1D and down-regulated in T2D, and Cer being up-regulated in T2D and down-regulated in T1D. Also, Phosphatidylcholines were clearly down-regulated in subjects with T1D. Regarding sex-specific differences, ceramides and phosphatidylcholines exhibited important diabetes-associated differences due to sex. Concerning the glycaemic state, we found a gradual increase of a panel of 1-deoxyceramides from normoglycemia to prediabetes to T2D. CONCLUSIONS: Our findings revealed an extensive disruption of lipid metabolism in both T1D and T2D. Additionally, we found sex-specific lipidome changes associated with diabetes, and lipids associated with the glycaemic state that can be linked to previously described molecular mechanisms in diabetes.

Determining the role of novel metabolic pathways in driving intracranial pressure reduction after weight loss.

Idiopathic intracranial hypertension, a disease classically occurring in women with obesity, is characterized by raised intracranial pressure. Weight loss leads to the reduction in intracranial pressure. Additionally, pharmacological glucagon-like peptide-1 agonism reduces cerebrospinal fluid secretion and intracranial pressure. The potential mechanisms by which weight loss reduces intracranial pressure are unknown and were the focus of this study. Meal stimulation tests (fasted plasma sample, then samples at 15, 30, 60, 90 and 120 min following a standardized meal) were conducted pre- and post-bariatric surgery [early (2 weeks) and late (12 months)] in patients with active idiopathic intracranial hypertension. Dynamic changes in gut neuropeptides (glucagon-like peptide-1, gastric inhibitory polypeptide and ghrelin) and metabolites (untargeted ultra-high performance liquid chromatography-mass spectrometry) were evaluated. We determined the relationship between gut neuropeptides, metabolites and intracranial pressure. Eighteen idiopathic intracranial hypertension patients were included [Roux-en-Y gastric bypass (RYGB) n = 7, gastric banding n = 6 or sleeve gastrectomy n = 5]. At 2 weeks post-bariatric surgery, despite similar weight loss, RYGB had a 2-fold (50%) greater reduction in intracranial pressure compared to sleeve. Increased meal-stimulated glucagon-like peptide-1 secretion was observed after RYGB (+600%) compared to sleeve (+319%). There was no change in gastric inhibitory polypeptide and ghrelin. Dynamic changes in meal-stimulated metabolites after bariatric surgery consistently identified changes in lipid metabolites, predominantly ceramides, glycerophospholipids and lysoglycerophospholipids, which correlated with intracranial pressure. A greater number of differential lipid metabolites were observed in the RYGB cohort at 2 weeks, and these also correlated with intracranial pressure. In idiopathic intracranial hypertension, we identified novel changes in lipid metabolites and meal-stimulated glucagon-like peptide-1 levels following bariatric surgery which were associated with changes in intracranial pressure. RYGB was most effective at reducing intracranial pressure despite analogous weight loss to gastric sleeve at 2 weeks post-surgery and was associated with more pronounced changes in these metabolite pathways. We suggest that these novel perturbations in lipid metabolism and glucagon-like peptide-1 secretion are mechanistically important in driving a reduction in intracranial pressure following weight loss in patients with idiopathic intracranial hypertension. Therapeutic targeting of these pathways, for example with glucagon-like peptide-1 agonist infusion, could represent a therapeutic strategy.

Oxidative stress from DGAT1 oncoprotein inhibition in melanoma suppresses tumor growth when ROS defenses are also breached.

Dysregulated cellular metabolism is a cancer hallmark for which few druggable oncoprotein targets have been identified. Increased fatty acid (FA) acquisition allows cancer cells to meet their heightened membrane biogenesis, bioenergy, and signaling needs. Excess FAs are toxic to non-transformed cells but surprisingly not to cancer cells. Molecules underlying this cancer adaptation may provide alternative drug targets. Here, we demonstrate that diacylglycerol O-acyltransferase 1 (DGAT1), an enzyme integral to triacylglyceride synthesis and lipid droplet formation, is frequently up-regulated in melanoma, allowing melanoma cells to tolerate excess FA. DGAT1 over-expression alone transforms p53-mutant zebrafish melanocytes and co-operates with oncogenic BRAF or NRAS for more rapid melanoma formation. Antagonism of DGAT1 induces oxidative stress in melanoma cells, which adapt by up-regulating cellular reactive oxygen species defenses. We show that inhibiting both DGAT1 and superoxide dismutase 1 profoundly suppress tumor growth through eliciting intolerable oxidative stress.

Cytoglobin protects cancer cells from apoptosis by regulation of mitochondrial cardiolipin.

Cytoglobin is important in the progression of oral squamous cell carcinoma but the molecular and cellular basis remain to be elucidated. In the current study, we develop a new cell model to study the function of cytoglobin in oral squamous carcinoma and response to cisplatin. Transcriptomic profiling showed cytoglobin mediated changes in expression of genes related to stress response, redox metabolism, mitochondrial function, cell adhesion, and fatty acid metabolism. Cellular and biochemical studies show that cytoglobin expression results in changes to phenotype associated with cancer progression including: increased cellular proliferation, motility and cell cycle progression. Cytoglobin also protects cells from cisplatin-induced apoptosis and oxidative stress with levels of the antioxidant glutathione increased and total and mitochondrial reactive oxygen species levels reduced. The mechanism of cisplatin resistance involved inhibition of caspase 9 activation and cytoglobin protected mitochondria from oxidative stress-induced fission. To understand the mechanism behind these phenotypic changes we employed lipidomic analysis and demonstrate that levels of the redox sensitive and apoptosis regulating cardiolipin are significantly up-regulated in cells expressing cytoglobin. In conclusion, our data shows that cytoglobin expression results in important phenotypic changes that could be exploited by cancer cells in vivo to facilitate disease progression.

Bilateral Remote Ischaemic Conditioning in Children (BRICC) trial: protocol for a two-centre, double-blind, randomised controlled trial in young children undergoing cardiac surgery.

INTRODUCTION: Myocardial protection against ischaemic-reperfusion injury is a key determinant of heart function and outcome following cardiac surgery in children. However, with current strategies, myocardial injury occurs routinely following aortic cross-clamping, as demonstrated by the ubiquitous rise in circulating troponin. Remote ischaemic preconditioning, the application of brief, non-lethal cycles of ischaemia and reperfusion to a distant organ or tissue, is a simple, low-risk and readily available technique which may improve myocardial protection. The Bilateral Remote Ischaemic Conditioning in Children (BRICC) trial will assess whether remote ischaemic preconditioning, applied to both lower limbs immediately prior to surgery, reduces myocardial injury in cyanotic and acyanotic young children. METHODS AND ANALYSIS: The BRICC trial is a two-centre, double-blind, randomised controlled trial recruiting up to 120 young children (age 3 months to 3 years) undergoing primary repair of tetralogy of Fallot or surgical closure of an isolated ventricular septal defect. Participants will be randomised in a 1:1 ratio to either bilateral remote ischaemic preconditioning (3×5 min cycles) or sham immediately prior to surgery, with follow-up until discharge from hospital or 30 days, whichever is sooner. The primary outcome is reduction in area under the time-concentration curve for high-sensitivity (hs) troponin-T release in the first 24 hours after aortic cross-clamp release. Secondary outcome measures include peak hs-troponin-T, vasoactive inotrope score, arterial lactate and central venous oxygen saturations in the first 12 hours, and lengths of stay in the paediatric intensive care unit and the hospital. ETHICS AND DISSEMINATION: The trial was approved by the West Midlands-Solihull National Health Service Research Ethics Committee (16/WM/0309) on 5 August 2016. Findings will be disseminated to the academic community through peer-reviewed publications and presentation at national and international meetings. Parents will be informed of the results through a newsletter in conjunction with a local charity. TRIAL REGISTRATION NUMBER: ISRCTN12923441.

Metabolic engineering against the arginine microenvironment enhances CAR-T cell proliferation and therapeutic activity.

Hematological and solid cancers catabolize the semiessential amino acid arginine to drive cell proliferation. However, the resulting low arginine microenvironment also impairs chimeric antigen receptor T cells (CAR-T) cell proliferation, limiting their efficacy in clinical trials against hematological and solid malignancies. T cells are susceptible to the low arginine microenvironment because of the low expression of the arginine resynthesis enzymes argininosuccinate synthase (ASS) and ornithine transcarbamylase (OTC). We demonstrate that T cells can be reengineered to express functional ASS or OTC enzymes, in concert with different chimeric antigen receptors. Enzyme modifications increase CAR-T cell proliferation, with no loss of CAR cytotoxicity or increased exhaustion. In vivo, enzyme-modified CAR-T cells lead to enhanced clearance of leukemia or solid tumor burden, providing the first metabolic modification to enhance CAR-T cell therapies.

Urinary biomonitoring of subjects with different smoking habits. Part II: an untargeted metabolomic approach and the comparison with the targeted measurement of mercapturic acids.

BACKGROUND: Although thousands of different chemicals have been identified in cigarette smoke, the characterization of their urinary metabolites still requires significant research. The aim of this work was to perform an untargeted metabolomic approach to a pilot cross-sectional study conducted on subjects with different smoking habits and to compare the results with those of the targeted measurement of mercapturic acids. METHODS: Urine samples from 67 adults, including 38 non-smokers, 7 electronic cigarette users, and 22 traditional tobacco smokers were collected. Samples were analysed by liquid chromatography/time-of flight mass spectrometry. Data were processed using the R-packages IPO and XCMS to perform feature detection, retention time correction and alignment. One-way ANOVA test was used to identify different features among groups. Quantitative determination of 17 mercapturic acids was available from a previous study. RESULTS: One hundred and seventeen features, out of 3613, were different among groups. They corresponded to 91 potential metabolites, 5 of which were identified vs authentic standards, 43 were putatively annotated and 13 were attributed to chemical classes. Among identified compounds there were the mercapturic acids of acrolein, 1,3-butadiene, and crotonaldehyde; among putatively annotated compounds there were the glucuronide conjugated of 3-hydroxycotinine and the sulfate conjugate of methoxyphenol; with the lowest degree of confidence several sulfate conjugates of small molecules were annotated. Considering mercapturic acids, the coherence between the targeted and untargeted approach was found for a limited number of chemicals, typically the most abundant. CONCLUSIONS: Differences in the urinary levels of several compounds were associated to the different smoking habits, suggesting that the proposed approach is useful for the investigation of the metabolite patterns related to the exposure to toxicants. However, limitations were highlighted, in particular regarding the identification of low concentration compounds.

Systematic Review: Clinical Metabolomics to Forecast Outcomes in Liver Transplantation Surgery.

Liver transplantation is an effective intervention for end-stage liver disease, fulminant hepatic failure, and early hepatocellular carcinoma. Yet, there is marked patient-to-patient variation in liver transplantation outcomes. This calls for novel diagnostics to enable rational deployment of donor livers. Metabolomics is a postgenomic high-throughput systems biology approach to diagnostic innovation in clinical medicine. We report here an original systematic review of the metabolomic studies that have identified putative biomarkers in the context of liver transplantation. Eighteen studies met the inclusion criteria that involved sampling of blood (n = 4), dialysate fluid (n = 4), bile (n = 5), and liver tissue (n = 5). Metabolites of amino acid and nitrogen metabolism, anaerobic glycolysis, lipid breakdown products, and bile acid metabolism were significantly different in transplanted livers with and without graft dysfunction. However, criteria for defining the graft dysfunction varied across studies. This systematic review demonstrates that metabolomics can be deployed in identification of metabolic indicators of graft dysfunction with a view to implicated molecular mechanisms. We conclude the article with a horizon scanning of metabolomics technology in liver transplantation and its future prospects and challenges in research and clinical practice.

Bidirectional Cross-Talk between Biliary Epithelium and Th17 Cells Promotes Local Th17 Expansion and Bile Duct Proliferation in Biliary Liver Diseases.

There is no effective treatment for autoimmune biliary diseases. Therefore, understanding their immunopathology is crucial. The biliary epithelial cells (BEC), expressing TLR-4, are constantly exposed to gut microbes and bacterial wall LPS, and in settings of inflammation, the immune infiltrate is dense within the peribiliary region of human liver. By dual immunohistochemistry, we affirm human intrahepatic T cell infiltrate includes CCR6+CD4+ and AhR+CD4+ T cells with potential for plasticity to Th17 phenotype. Mechanistically, we demonstrate that Th1 and Th17 inflammatory cytokines and LPS enhance human primary BEC release of the CCR6 ligand CCL20 and BEC secretion of Th17-polarizing cytokines IL-6 and IL-1ß. Cell culture assays with human BEC secretome showed that secretome polarizes CD4 T cells toward a Th17 phenotype and supports the survival of Th17 cells. BEC secretome did not promote Th1 cell generation. Additionally, we give evidence for a mutually beneficial feedback of the type 17 cell infiltrate on BEC, showing that treatment with type 17 cytokines increases BEC proliferation, as monitored by Ki67 and activation of JAK2-STAT3 signaling. This study identifies human BEC as active players in determining the nature of the intrahepatic immune microenvironment. In settings of inflammation and/or infection, biliary epithelium establishes a prominent peribiliary type 17 infiltrate via recruitment and retention and enhances polarization of intrahepatic CD4 cells toward Th17 cells via type 17 cytokines, and, reciprocally, Th17 cells promote BEC proliferation for biliary regeneration. Altogether, we provide new insight into cross-talk between Th17 lymphocytes and human primary biliary epithelium in biliary regenerative pathologies.

Towards quality assurance and quality control in untargeted metabolomics studies.

We describe here the agreed upon first development steps and priority objectives of a community engagement effort to address current challenges in quality assurance (QA) and quality control (QC) in untargeted metabolomic studies. This has included (1) a QA and QC questionnaire responded to by the metabolomics community in 2015 which recommended education of the metabolomics community, development of appropriate standard reference materials and providing incentives for laboratories to apply QA and QC; (2) a 2-day 'Think Tank on Quality Assurance and Quality Control for Untargeted Metabolomic Studies' held at the National Cancer Institute's Shady Grove Campus and (3) establishment of the Metabolomics Quality Assurance and Quality Control Consortium (mQACC) to drive forward developments in a coordinated manner.

Enhanced Fatty Acid Scavenging and Glycerophospholipid Metabolism Accompany Melanocyte Neoplasia Progression in Zebrafish.

Alterations in lipid metabolism in cancer cells impact cell structure, signaling, and energy metabolism, making lipid metabolism a potential diagnostic marker and therapeutic target. In this study, we combined PET, desorption electrospray ionization-mass spectrometry (DESI-MS), nonimaging MS, and transcriptomic analyses to interrogate changes in lipid metabolism in a transgenic zebrafish model of oncogenic RAS-driven melanocyte neoplasia progression. Exogenous fatty acid uptake was detected in melanoma tumor nodules by PET using the palmitic acid surrogate tracer 14(R,S)-18F-fluoro-6-thia-heptadecanoic acid ([18F]-FTHA), consistent with upregulation of genes associated with fatty acid uptake found through microarray analysis. DESI-MS imaging revealed that FTHA uptake in tumors was heterogeneous. Transcriptome and lipidome analyses further highlighted dysregulation of glycerophospholipid pathways in melanoma tumor nodules, including increased abundance of phosphatidyl ethanolamine and phosphatidyl choline species, corroborated by DESI-MS, which again revealed heterogeneous phospholipid composition in tumors. Overexpression of the gene encoding lipoprotein lipase (LPL), which was upregulated in zebrafish melanocyte tumor nodules and expressed in the majority of human melanomas, accelerated progression of oncogenic RAS-driven melanocyte neoplasia in zebrafish. Depletion or antagonism of LPL suppressed human melanoma cell growth; this required simultaneous fatty acid synthase (FASN) inhibition when FASN expression was also elevated. Collectively, our findings implicate fatty acid acquisition as a possible therapeutic target in melanoma, and the methods we developed for monitoring fatty acid uptake have potential for diagnosis, patient stratification, and monitoring pharmacologic response. SIGNIFICANCE: These findings demonstrate the translational potential of monitoring fatty acid uptake and identify lipoprotein lipase as a potential therapeutic target in melanoma.

Nicotinamide Nucleotide Transhydrogenase as a Novel Treatment Target in Adrenocortical Carcinoma.

Adrenocortical carcinoma (ACC) is an aggressive malignancy with poor response to chemotherapy. In this study, we evaluated a potential new treatment target for ACC, focusing on the mitochondrial reduced form of NAD phosphate (NADPH) generator nicotinamide nucleotide transhydrogenase (NNT). NNT has a central role within mitochondrial antioxidant pathways, protecting cells from oxidative stress. Inactivating human NNT mutations result in congenital adrenal insufficiency. We hypothesized that NNT silencing in ACC cells will induce toxic levels of oxidative stress. To explore this, we transiently knocked down NNT in NCI-H295R ACC cells. As predicted, this manipulation increased intracellular levels of oxidative stress; this resulted in a pronounced suppression of cell proliferation and higher apoptotic rates, as well as sensitization of cells to chemically induced oxidative stress. Steroidogenesis was paradoxically stimulated by NNT loss, as demonstrated by mass spectrometry-based steroid profiling. Next, we generated a stable NNT knockdown model in the same cell line to investigate the longer lasting effects of NNT silencing. After long-term culture, cells adapted metabolically to chronic NNT knockdown, restoring their redox balance and resilience to oxidative stress, although their proliferation remained suppressed. This was associated with higher rates of oxygen consumption. The molecular pathways underpinning these responses were explored in detail by RNA sequencing and nontargeted metabolome analysis, revealing major alterations in nucleotide synthesis, protein folding, and polyamine metabolism. This study provides preclinical evidence of the therapeutic merit of antioxidant targeting in ACC as well as illuminating the long-term adaptive response of cells to oxidative stress.

Modelling non-alcoholic fatty liver disease in human hepatocyte-like cells.

Non-alcoholic fatty liver disease (NAFLD) is the most common cause of liver disease in developed countries. An in vitro NAFLD model would permit mechanistic studies and enable high-throughput therapeutic screening. While hepatic cancer-derived cell lines are a convenient, renewable resource, their genomic, epigenomic and functional alterations mean their utility in NAFLD modelling is unclear. Additionally, the epigenetic mark 5-hydroxymethylcytosine (5hmC), a cell lineage identifier, is rapidly lost during cell culture, alongside expression of the Ten-eleven-translocation (TET) methylcytosine dioxygenase enzymes, restricting meaningful epigenetic analysis. Hepatocyte-like cells (HLCs) derived from human embryonic stem cells can provide a non-neoplastic, renewable model for liver research. Here, we have developed a model of NAFLD using HLCs exposed to lactate, pyruvate and octanoic acid (LPO) that bear all the hallmarks, including 5hmC profiles, of liver functionality. We exposed HLCs to LPO for 48 h to induce lipid accumulation. We characterized the transcriptome using RNA-seq, the metabolome using ultra-performance liquid chromatography-mass spectrometry and the epigenome using 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) sequencing. LPO exposure induced an NAFLD phenotype in HLCs with transcriptional and metabolomic dysregulation consistent with those present in human NAFLD. HLCs maintain expression of the TET enzymes and have a liver-like epigenome. LPO exposure-induced 5hmC enrichment at lipid synthesis and transport genes. HLCs treated with LPO recapitulate the transcriptional and metabolic dysregulation seen in NAFLD and additionally retain TET expression and 5hmC. This in vitro model of NAFLD will be useful for future mechanistic and therapeutic studies.This article is part of the theme issue 'Designer human tissue: coming to a lab near you'.

Exercise and high-fat feeding remodel transcript-metabolite interactive networks in mouse skeletal muscle.

Enhanced coverage and sensitivity of next-generation 'omic' platforms has allowed the characterization of gene, metabolite and protein responses in highly metabolic tissues, such as, skeletal muscle. A limitation, however, is the capability to determine interaction between dynamic biological networks. To address this limitation, we applied Weighted Analyte Correlation Network Analysis (WACNA) to RNA-seq and metabolomic datasets to identify correlated subnetworks of transcripts and metabolites in response to a high-fat diet (HFD)-induced obesity and/or exercise. HFD altered skeletal muscle lipid profiles and up-regulated genes involved in lipid catabolism, while decreasing 241 exercise-responsive genes related to skeletal muscle plasticity. WACNA identified the interplay between transcript and metabolite subnetworks linked to lipid metabolism, inflammation and glycerophospholipid metabolism that were associated with IL6, AMPK and PPAR signal pathways. Collectively, this novel experimental approach provides an integrative resource to study transcriptional and metabolic networks in skeletal muscle in the context of health and disease.

AKR1C3-Mediated Adipose Androgen Generation Drives Lipotoxicity in Women With Polycystic Ovary Syndrome.

Context: Polycystic ovary syndrome (PCOS) is a prevalent metabolic disorder occurring in up to 10% of women of reproductive age. PCOS is associated with insulin resistance and cardiovascular risk. Androgen excess is a defining feature of PCOS and has been suggested as causally associated with insulin resistance; however, mechanistic evidence linking both is lacking. We hypothesized that adipose tissue is an important site linking androgen activation and metabolic dysfunction in PCOS. Methods: We performed a human deep metabolic in vivo phenotyping study examining the systemic and intra-adipose effects of acute and chronic androgen exposure in 10 PCOS women, in comparison with 10 body mass index-matched healthy controls, complemented by in vitro experiments. Results: PCOS women had increased intra-adipose concentrations of testosterone (P = 0.0006) and dihydrotestosterone (P = 0.01), with increased expression of the androgen-activating enzyme aldo-ketoreductase type 1 C3 (AKR1C3) (P = 0.04) in subcutaneous adipose tissue. Adipose glycerol levels in subcutaneous adipose tissue microdialysate supported in vivo suppression of lipolysis after acute androgen exposure in PCOS (P = 0.04). Mirroring this, nontargeted serum metabolomics revealed prolipogenic effects of androgens in PCOS women only. In vitro studies showed that insulin increased adipose AKR1C3 expression and activity, whereas androgen exposure increased adipocyte de novo lipid synthesis. Pharmacologic AKR1C3 inhibition in vitro decreased de novo lipogenesis. Conclusions: These findings define an intra-adipose mechanism of androgen activation that contributes to adipose remodeling and a systemic lipotoxic metabolome, with intra-adipose androgens driving lipid accumulation and insulin resistance in PCOS. AKR1C3 represents a promising therapeutic target in PCOS.

Carbohydrate and fatty acid perturbations in the amniotic fluid of the recipient twin of pregnancies complicated by twin-twin transfusion syndrome in relation to treatment and fetal cardiovascular risk.

INTRODUCTION: Twin-twin transfusion syndrome (TTTS) complicates 15% of monochorionic twin pregnancies, often being associated with recipient cardiac dysfunction. Untreated, it has a fetal mortality rate of at least 90%; although treatment by fetoscopic laser coagulation significantly improves prognosis. Measurement of recipient amniotic fluid metabolites, such as cardiac Troponin T and atrial natriuretic polypeptide, correlate with cardiac function in this fetus. The aim of this study is to describe the amniotic fluid metabolomic profile in TTTS, relate this to fetal recipient cardiac function and assess the metabolomic changes induced by fetoscopic laser coagulation. METHODS: Prospective single centre cohort study. The metabolomics profile of the amniotic fluid from the recipient sac of TTTS pregnancies was assessed using ultra high performance liquid chromatography-mass spectrometry. Profiles were compared pre- and post-laser coagulation and related to fetal recipient cardiac function, as assessed using Doppler ultrasound within 4 h of treatment. RESULTS: Eleven metabolites had significant associations with recipient fetal right and left ventricular myocardial performance index pre-laser. 200 metabolites in recipient amniotic fluid demonstrated a change in relative concentrations when comparing pre- and post-laser coagulation (p < 0.005). The most prominent change is in the balance of carbohydrate and fatty acid metabolic profile contributing to fetal or placental energy metabolism. These changes were also associated with the echocardiographic measures of recipient cardiac function. DISCUSSION: Changes in carbohydrate and fatty acid metabolic profiles are noted in recipients with cardiac dysfunction, and further changes are noted after treatment. Validation and investigation may identify targets for potential pharmacological treatment.

Metabolites involved in glycolysis and amino acid metabolism are altered in short children born small for gestational age.

BACKGROUND: Later life metabolic dysfunction is a well-recognized consequence of being born small for gestational age (SGA). This study has applied metabolomics to identify whether there are changes in these pathways in prepubertal short SGA children and aimed to compare the intracellular and extracellular metabolome in fibroblasts derived from healthy children and SGA children with postnatal growth impairment. METHODS: Skin fibroblast cell lines were established from eight SGA children (age 1.8-10.3 y) with failure of catch-up growth and from three healthy control children. Confluent cells were incubated in serum-free media and the spent growth medium (metabolic footprint), and intracellular metabolome (metabolic fingerprint) were analyzed by gas-chromatography mass spectrometry. RESULTS: Nineteen metabolites were significantly altered between SGA and control cell lines. The greatest fold difference (FD) was seen for alanine (fingerprint FD, SGA: control 0.3, P = 0.01 and footprint FD = 0.19, P = 0.01), aspartic acid (fingerprint FD = 5.21, P = 0.01), and cystine (footprint FD = 1.66, P = 0.02). Network analysis of the differentially expressed metabolites predicted inhibition of insulin as well as growth (ERK) signaling in SGA cells. CONCLUSION: This study indicates that changes in cellular metabolism associated with both growth failure and insulin insensitivity are present in prepubertal short children born SGA.

Metabolomics reveals the physiological response of Pseudomonas putida KT2440 (UWC1) after pharmaceutical exposure.

Human pharmaceuticals have been detected in wastewater treatment plants, rivers, and estuaries throughout Europe and the United States. It is widely acknowledged that there is insufficient information available to determine whether prolonged exposure to low levels of these substances is having an impact on the microbial ecology in such environments. In this study we attempt to measure the effects of exposing cultures of Pseudomonas putida KT2440 (UWC1) to six pharmaceuticals by looking at differences in metabolite levels. Initially, we used Fourier transform infrared (FT-IR) spectroscopy coupled with multivariate analysis to discriminate between cell cultures exposed to different pharmaceuticals. This suggested that on exposure to propranolol there were significant changes in the lipid complement of P. putida. Metabolic profiling with gas chromatography-mass spectrometry (GC-MS), coupled with univariate statistical analyses, was used to identify endogenous metabolites contributing to discrimination between cells exposed to the six drugs. This approach suggested that the energy reserves of exposed cells were being expended and was particularly evident on exposure to propranolol. Adenosine triphosphate (ATP) concentrations were raised in P. putida exposed to propranolol. Increased energy requirements may be due to energy dependent efflux pumps being used to remove propranolol from the cell.

Metabolic profiling reveals potential metabolic markers associated with Hypoxia Inducible Factor-mediated signalling in hypoxic cancer cells.

Hypoxia inducible factors (HIFs) plays an important role in oxygen compromised environments and therefore in tumour survival. In this research, metabolomics has been applied to study HIFs metabolic function in two cell models: mouse hepatocellular carcinoma and human colon carcinoma, whereby the metabolism has been profiled for a range of oxygen potentials. Wild type cells have been compared to cells deficient in HIF signalling to reveal its effect on cellular metabolism under normal oxygen conditions as well as low oxygen, hypoxic and anoxic environments. Characteristic responses to hypoxia that were conserved across both cell models involved the anti-correlation between 2-hydroxyglutarate, 2-oxoglutarate, fructose, hexadecanoic acid, hypotaurine, pyruvate and octadecenoic acid with 4-hydroxyproline, aspartate, cysteine, glutamine, lysine, malate and pyroglutamate. Further to this, network-based correlation analysis revealed HIF specific pathway responses to each oxygen condition that were also conserved between cell models. From this, 4-hydroxyproline was revealed as a regulating hub in low oxygen survival of WT cells while fructose appeared to be in HIF deficient cells. Pathways surrounding these hubs were built from the direct connections of correlated metabolites that look beyond traditional pathways in order to understand the mechanism of HIF response to low oxygen environments.