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Abstract After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.

Research Infrastructures (RI) and Core Facilities (CF) are strong drivers for generating research results and, thus, knowledge at European Universities. In this paper, we provide insight into different features of RI and CF, their organisational structure and governance, funding mechanisms, critical factors for success, and challenges and opportunities associated with implementing and operating these research support structures. Our results are based on a comparative analysis across six European universities from the 4EU + University Alliance. Due to the lack of a clear definition of RI and CF, we provide a variety of indicators and criteria attributed to such facilities and highlight differences between CF and RI in terms of goals and objectives. If establishing Core Facilities is seen as a response to the evolving needs and challenges of researchers, a centralised management and collaborative governance model can provide a practical solution. We explore the legal framework, organisation, access, users and charge rate models at Core Facilities. We also identify several challenges in setting up and maintaining Core Facilities. Special attention is directed towards staff challenges, including introducing the staff category of ‘scientific technical experts’. Finally, we present a Core Facility Manifesto and share our conclusions.

Abstract Microglia, brain-resident macrophages, can acquire distinct functional phenotypes, which are supported by differential reprogramming of cell metabolism. These adaptations include remodeling in glycolytic and mitochondrial metabolic fluxes, potentially altering energy substrate availability at the tissue level. This phenomenon may be highly relevant in the brain, where metabolism must be precisely regulated to maintain appropriate neuronal excitability and synaptic transmission. Direct evidence that microglia can impact on neuronal energy metabolism has been widely lacking, however. Combining molecular profiling, electrophysiology, oxygen microsensor recordings and mathematical modeling, we investigated microglia-mediated disturbances in brain energetics during neuroinflammation. Our results suggest that proinflammatory microglia showing enhanced nitric oxide release and decreased CX3CR1 expression transiently increase the tissue lactate/glucose ratio that depends on transcriptional reprogramming in microglia, not in neurons. In this condition, neuronal network activity such as gamma oscillations (30–70 Hz) can be fueled by increased ATP production in mitochondria, which is reflected by elevated oxygen consumption. During dysregulated inflammation, high energy demand and low glucose availability can be boundary conditions for neuronal metabolic fitness as revealed by kinetic modeling of single neuron energetics. Collectively, these findings indicate that metabolic flexibility protects neuronal network function against alterations in local substrate availability during moderate neuroinflammation.

Abstract Omega‐6 fatty acids are the primary polyunsaturated fatty acids in most Western diets, while their role in diabetes remains controversial. Exposure of omega‐6 fatty acids to an oxidative environment results in the generation of a highly reactive carbonyl species known as trans, trans‐2,4‐decadienal (tt‐DDE). The timely and efficient detoxification of this metabolite, which has actions comparable to other reactive carbonyl species, such as 4‐hydroxynonenal, acrolein, acetaldehyde, and methylglyoxal, is essential for disease prevention. However, the detoxification mechanism for tt‐DDE remains elusive. In this study, the enzyme Aldh9a1b is identified as having a key role in the detoxification of tt‐DDE. Loss of Aldh9a1b increased tt‐DDE levels and resulted in an abnormal retinal vasculature and glucose intolerance in aldh9a1b−/− zebrafish. Transcriptomic and metabolomic analyses revealed that tt‐DDE and aldh9a1b deficiency in larval and adult zebrafish induced insulin resistance and impaired glucose homeostasis. Moreover, alterations in hyaloid vasculature is induced by aldh9a1b knockout or by tt‐DDE treatment can be rescued by the insulin receptor sensitizers metformin and rosiglitazone. Collectively, these results demonstrated that tt‐DDE is the substrate of Aldh9a1b which causes microvascular damage and impaired glucose metabolism through insulin resistance.

Abstract Background Increased circulating uric acid (UA) concentration may disrupt cardiac function in heart failure patients, but the specific mechanism remains unclear. Here, we postulate that hyperuremia induces sterol regulatory element binding protein 1 (SREBP1), which in turn activate hepatic fatty acid biosynthesis response, leading to cardiac dysfunction. Methods and results Increased circulating uric acid was observed in heart failure patients and inversely correlated to cardiac function. Besides, uric acid correlated to circulating lipids profile based on metabolomics in heart failure patients. Using cultured human hepatoellular carcinomas (HepG2) and Tg(myl7:egfp) zebrafish, we demonstrated that UA regulated fatty acid synthase (FASN) via SREBP1 signaling pathway, leading to FFA accumulation and impaired energy metabolism, which could be rescued via SREBP1 knockdown. In ISO treated zebrafish, UA aggravated heart failure via increased cardiovascular cavity size, decreased heart beats, pericardial edema and long-stretched heart deformation. Conclusions Our findings suggest that UA-SREBP1-FASN signaling exacerbates cardiac dysfunction during FFA accumulation. Identification of this mechanism may help in treatment and prevention of heart failure.

Exercise and increased physical activity are vital components of the standard treatment guidelines for many chronic diseases such as diabetes, obesity and cardiovascular disease. Although strenuous exercise cannot be recommended to people with numerous chronic conditions, walking is something most people can perform. In comparison to high-intensity training, the metabolic consequences of low-intensity walking have been less well studied. We present here a feasibility study of a subject who performed an exercise intervention of low-intensity, non-fatiguing walking on a deskmill/treadmill for 200 min daily, approximately the average time a German spends watching television per day. This low-impact physical activity has the advantages that it can be done while performing other tasks such as reading or watching TV, and it can be recommended to obese patients or patients with heart disease. We find that this intervention led to substantial weight loss, comparable to that of bariatric surgery. To study the metabolic changes caused by this intervention, we performed an in-depth metabolomic profiling of the blood both directly after walking to assess the acute changes, as well as 1.5 days after physical activity to identify the long-term effects that persist. We find changes in acylcarnitine levels suggesting that walking activates fatty acid beta oxidation, and that this mitochondrial reprogramming is still visible 1.5 days post-walking. We also find that walking mildly increases gut permeability, leading to increased exposure of the blood to metabolites from the gut microbiome. Overall, these data provide a starting point for designing future intervention studies with larger cohorts.

Diabetic peripheral neuropathy (DPN) is the prevalent type of peripheral neuropathy; it primarily impacts extremity nerves. Its multifaceted nature makes the molecular mechanisms of diabetic neuropathy intricate and incompletely elucidated. Several types of post-translational modifications (PTMs) have been implicated in the development and progression of DPN, including phosphorylation, glycation, acetylation and SUMOylation. SUMOylation involves the covalent attachment of small ubiquitin-like modifier (SUMO) proteins to target proteins, and it plays a role in various cellular processes, including protein localization, stability, and function. While the specific relationship between high blood glucose and SUMOylation is not extensively studied, recent evidence implies its involvement in the development of DPN in type 1 diabetes. In this study, we investigated the impact of SUMOylation on the onset and progression of DPN in a type 2 diabetes model using genetically modified mutant mice lacking SUMOylation, specifically in peripheral sensory neurons (SNS-Ubc9−/−). Behavioural measurement for evoked pain, morphological analyses of nerve fibre loss in the epidermis, measurement of reactive oxygen species (ROS) levels, and antioxidant molecules were analysed over several months in SUMOylation-deficient and control mice. Our longitudinal analysis at 30 weeks post-high-fat diet revealed that SNS-Ubc9−/− mice exhibited earlier and more pronounced thermal and mechanical sensation loss and accelerated intraepidermal nerve fibre loss compared to control mice. Mechanistically, these changes are associated with increased levels of ROS both in sensory neuronal soma and in peripheral axonal nerve endings in SNS-Ubc9−/− mice. In addition, we observed compromised detoxifying potential, impaired respiratory chain complexes, and reduced levels of protective lipids in sensory neurons upon deletion of SUMOylation in diabetic mice. Importantly, we also identified mitochondrial malate dehydrogenase (MDH2) as a SUMOylation target, the activity of which is negatively regulated by SUMOylation. Our results indicate that SUMOylation is an essential neuroprotective mechanism in sensory neurons in type 2 diabetes, the deletion of which causes oxidative stress and an impaired respiratory chain, resulting in energy depletion and subsequent damage to sensory neurons.

Abstract Blood vessel formation is dependent on metabolic adaption in endothelial cells. Glucose and fatty acids are essential substrates for ATP and biomass production; however, the metabolism of other substrates remains poorly understood. Ketone bodies are important nutrients for cardiomyocytes during starvation or consumption of carbohydrate‐restrictive diets. This raises the question whether cardiac endothelial cells would not only transport ketone bodies but also consume some of these to achieve their metabolic needs. Here, we report that cardiac endothelial cells are able to oxidize ketone bodies and that this enhances cell proliferation, migration, and vessel sprouting. Mechanistically, this requires succinyl‐CoA:3‐oxoacid‐CoA transferase, a key enzyme of ketone body oxidation. Targeted metabolite profiling revealed that carbon from ketone bodies got incorporated into tricarboxylic acid cycle intermediates as well as other metabolites fueling biomass production. Elevation of ketone body levels by a high‐fat, low‐carbohydrate ketogenic diet transiently increased endothelial cell proliferation in mouse hearts. Notably, in a mouse model of heart hypertrophy, ketogenic diet prevented blood vessel rarefication. This suggests a potential beneficial role of dietary intervention in heart diseases.

Glyoxalase 2 is the second enzyme of the glyoxalase system, catalyzing the detoxification of methylglyoxal to d-lactate via SD-Lactoylglutathione. Recent in vitro studies have suggested Glo2 as a regulator of glycolysis, but if Glo2 regulates glucose homeostasis and related organ specific functions in vivo has not yet been evaluated. Therefore, a CRISPR-Cas9 knockout of glo2 in zebrafish was created and analyzed. Consistent with its function in methylglyoxal detoxification, SD-Lactoylglutathione, but not methylglyoxal accumulated in glo2−/− larvae, without altering the glutathione metabolism or affecting longevity. Adult glo2−/− livers displayed a reduced hexose concentration and a reduced postprandial P70–S6 kinase activation, but upstream postprandial AKT phosphorylation remained unchanged. In contrast, glo2−/− skeletal muscle remained metabolically intact, possibly compensating for the dysfunctional liver through increased glucose uptake and glycolytic activity. glo2−/− zebrafish maintained euglycemia and showed no damage of the retinal vasculature, kidney, liver and skeletal muscle. In conclusion, the data identified Glo2 as a regulator of cellular energy metabolism in liver and skeletal muscle, but the redox state and reactive metabolite accumulation were not affected by the loss of Glo2.

IntroductionDiabetes mellitus (DM) is associated with physiological disorders such as delayed wound healing, diabetic retinopathy, diabetic nephropathy, and diabetic peripheral neuropathy (DPN). Over 50% of diabetic patients will develop DPN, characterized by motor dysfunction and impaired sensory nerve function. In a previous study, we have uncovered acrolein (ACR) as an upstream initiator which induced impaired glucose homeostasis and microvascular alterations in zebrafish. Whether ACR has specific effects on peripheral neurogenesis and mediates DPN, is still waiting for clarification.MethodsTo evaluate the function of ACR in peripheral nerve development, in vivo experiments were performed in Tg(hb9:GFP) zebrafish. In addition, a series of rescue experiments, metabolomics assessment, and bioinformatics analysis was performed aimed at identifying the molecular mechanisms behind ACR’s function and impaired neurogenesis.ResultsImpaired motor neuron development was confirmed in wild-type embryos treated with external ACR. ACR treated embryos displayed ferroptosis and reduction of several amino acids and increased glutathione (GSH). Furthermore, ferroptosis inducer caused similarly suppressed neurogenesis in zebrafish embryos, while anti-ACR treatment or ferroptosis inhibitor could successfully reverse the detrimental phenotypes of ACR on neurogenesis in zebrafish.DiscussionOur data indicate that ACR could directly activate ferroptosis and impairs peripheral neurogenesis. The data strongly suggest ACR and activated ferroptosis as inducers and promising therapeutic targets for future DPN studies.

Metabolomic and proteomic analyses of human plasma and serum samples harbor the power to advance our understanding of disease biology. Pre-analytical factors may contribute to variability and bias in the detection of analytes, especially when multiple labs are involved, caused by sample handling, processing time, and differing operating procedures. To better understand the impact of pre-analytical factors that are relevant to implementing a unified proteomic and metabolomic approach in a clinical setting, we assessed the influence of temperature, sitting times, and centrifugation speed on the plasma and serum metabolomes and proteomes from six healthy volunteers. We used targeted metabolic profiling (497 metabolites) and data-independent acquisition (DIA) proteomics (572 proteins) on the same samples generated with well-defined pre-analytical conditions to evaluate criteria for pre-analytical SOPs for plasma and serum samples. Time and temperature showed the strongest influence on the integrity of plasma and serum proteome and metabolome. While rapid handling and low temperatures (4°C) are imperative for metabolic profiling, the analyzed proteomics data set showed variability when exposed to temperatures of 4°C for more than 2 h, highlighting the need for compromises in a combined analysis. We formalized a quality control scoring system to objectively rate sample stability and tested this score using external data sets from other pre-analytical studies. Stringent and harmonized standard operating procedures (SOPs) are required for pre-analytical sample handling when combining proteomics and metabolomics of clinical samples to yield robust and interpretable data on a longitudinal scale and across different clinics. To ensure an adequate level of practicability in a clinical routine for metabolomics and proteomics studies, we suggest keeping blood samples up to 2 h on ice (4°C) prior to snap-freezing as a compromise between stability and operability. Finally, we provide the methodology as an open-source R package allowing the systematic scoring of proteomics and metabolomics data sets to assess the stability of plasma and serum samples.

Little is known about the metabolic differences between endurance and strength athletes in comparison with sedentary subjects under controlled conditions and about variation of the metabolome throughout one year. We hypothesized that (1) the resting metabolic profile differs between sedentary subjects and athletes and between perennially endurance- and strength-trained athletes and (2) varies throughout one year of training. We performed quantitative, targeted metabolomics (Biocrates MxP® Quant 500, Biocrates Life Sciences AG, Innsbruck, Austria) in plasma samples at rest in three groups of male adults, 12 strength-trained (weightlifters, 20 ± 3 years), 10 endurance-trained athletes (runners, 24 ± 3 years), and 12 sedentary subjects (25 ± 4 years) at the end of three training phases (regeneration, preparation, and competition) within one training year. Performance and anthropometric data showed significant (p < 0.05) differences between the groups. Metabolomic analysis revealed different resting metabolic profiles between the groups with acetylcarnitines, di- and triacylglycerols, and glycerophospho- and sphingolipids, as well as several amino acids as the most robust metabolites. Furthermore, we observed changes in free carnitine and 3-methylhistidine in strength-trained athletes throughout the training year. Regular endurance or strength training induces changes in the concentration of several metabolites associated with adaptations of the mitochondrial energy and glycolytic metabolism with concomitant changes in amino acid metabolism and cell signaling.

Analyses of metabolic compounds inside cells or tissues provide high information content since they represent the endpoint of biological information flow and are a snapshot of the integration of many regulatory processes. However, quantification of the abundance of metabolites requires their careful extraction. We present a comprehensive study comparing ten extraction protocols in four human sample types (liver tissue, bone marrow, HL60, and HEK cells) aiming to detect and quantify up to 630 metabolites of different chemical classes. We show that the extraction efficiency and repeatability are highly variable across protocols, tissues, and chemical classes of metabolites. We used different quality metrics including the limit of detection and variability between replicates as well as the sum of concentrations as a global estimate of analytical repeatability of the extraction. The coverage of extracted metabolites depends on the used solvents, which has implications for the design of measurements of different sample types and metabolic compounds of interest. The benchmark dataset can be explored in an easy-to-use, interactive, and flexible online resource (R/shiny app MetaboExtract: http://www.metaboextract.shiny.dkfz.de) for context-specific selection of the optimal extraction method. Furthermore, data processing and conversion functionality underlying the shiny app are accessible as an R package: https://cran.r-project.org/package=MetAlyzer.

The ELMO protein family consists of the homologues ELMO1, ELMO2 and ELMO3. Several studies have shown that the individual ELMO proteins are involved in a variety of cellular and developmental processes. However, it has poorly been understood whether the Elmo proteins show similar functions and act redundantly. To address this question, elmo1−/−, elmo2−/− and elmo3−/− zebrafish were generated and a comprehensive comparison of the phenotypic changes in organ morphology, transcriptome and metabolome was performed in these mutants. The results showed decreased fasting and increased postprandial blood glucose levels in adult elmo1−/−, as well as a decreased vascular formation in the adult retina in elmo1−/−, but an increased vascular formation in the adult elmo3−/− retina. The phenotypical comparison provided few similarities, as increased Bowman space areas in adult elmo1−/− and elmo2−/− kidneys, an increased hyaloid vessel diameter in elmo1−/− and elmo3−/− and a transcriptional downregulation of the vascular development in elmo1−/−, elmo2−/−, and elmo3−/− zebrafish larvae. Besides this, elmo1−/−, elmo2−/−, and elmo3−/− zebrafish exhibited several distinct changes in the vascular and glomerular structure and in the metabolome and the transcriptome. Especially, elmo3−/− zebrafish showed extensive differences in the larval transcriptome and an impaired survivability. Together, the data demonstrated that the three zebrafish Elmo proteins regulate not only similar but also divergent biological processes and mechanisms and show a low functional redundancy.

Carnosine and anserine supplementation markedLy reduce diabetic nephropathy in rodents. The mode of nephroprotective action of both dipeptides in diabetes, via local protection or improved systemic glucose homeostasis, is uncertain. Global carnosinase-1 knockout mice (Cndp1-KO) and wild-type littermates (WT) on a normal diet (ND) and high fat diet (HFD) (n = 10/group), with streptozocin (STZ)-induced type-1 diabetes (n = 21–23/group), were studied for 32 weeks. Independent of diet, Cndp1-KO mice had 2- to 10-fold higher kidney anserine and carnosine concentrations than WT mice, but otherwise a similar kidney metabolome; heart, liver, muscle and serum anserine and carnosine concentrations were not different. Diabetic Cndp1-KO mice did not differ from diabetic WT mice in energy intake, body weight gain, blood glucose, HbA1c, insulin and glucose tolerance with both diets, whereas the diabetes-related increase in kidney advanced glycation end-product and 4-hydroxynonenal concentrations was prevented in the KO mice. Tubular protein accumulation was lower in diabetic ND and HFD Cndp1-KO mice, interstitial inflammation and fibrosis were lower in diabetic HFD Cndp1-KO mice compared to diabetic WT mice. Fatalities occurred later in diabetic ND Cndp1-KO mice versus WT littermates. Independent of systemic glucose homeostasis, increased kidney anserine and carnosine concentrations reduce local glycation and oxidative stress in type-1 diabetic mice, and mitigate interstitial nephropathy in type-1 diabetic mice on HFD.

Metabolic profiling harbors the potential to better understand various disease entities such as cancer, diabetes, Alzheimer’s, Parkinson’s disease or COVID-19. To better understand such diseases and their intricate metabolic pathways in human studies, model animals are regularly used. There, standardized rearing conditions and uniform sampling strategies are prerequisites towards a successful metabolomic study that can be achieved through model organisms. Although metabolomic approaches have been employed on model organisms before, no systematic assessment of different conditions to optimize metabolite extraction across several organisms and sample types has been conducted. We address this issue using a highly standardized metabolic profiling assay analyzing 630 metabolites across three commonly used model organisms (Drosophila, mouse, and zebrafish) to find an optimal extraction protocol for various matrices. Focusing on parameters such as metabolite coverage, concentration and variance between replicates we compared seven extraction protocols. We found that the application of a combination of 75% ethanol and methyl tertiary-butyl ether (MTBE), while not producing the broadest coverage and highest concentrations, was the most reproducible extraction protocol. We were able to determine up to 530 metabolites in mouse kidney samples, 509 in mouse liver, 422 in zebrafish and 388 in Drosophila and discovered a core overlap of 261 metabolites in these four matrices. To enable other scientists to search for the most suitable extraction protocol in their experimental context and interact with this comprehensive data, we have integrated our data set in the open-source shiny app “MetaboExtract”. Hereby, scientists can search for metabolites or compound classes of interest, compare them across the different tested extraction protocols and sample types as well as find reference concentration values.

Objective: Glucosamine, an intermetabolite of the hexosamine biosynthesis pathway (HBP), is a widely used nutritional supplement in osteoarthritis patients, a subset of whom also suffer from diabetes. HBP is activated in diabetic retinopathy (DR). The aim of this study is to investigate the yet unclear effects of glucosamine on DR. Methods: In this study, we tested the effect of glucosamine on vascular and neuronal pathology in a mouse model of streptozotocin-induced DR in vivo and on cultured endothelial and Müller cells to elucidate the underlying mechanisms of action in vitro. Results: Glucosamine did not alter the blood glucose or HbA1c levels in the animals, but induced body weight gain in the non-diabetic animals. Interestingly, the impaired neuronal function in diabetic animals could be prevented by glucosamine treatment. Correspondingly, the activation of Müller cells was prevented in the retina as well as in cell culture. Conversely, glucosamine administration in the normal retina damaged the retinal vasculature by increasing pericyte loss and acellular capillary formation, likely by interfering with endothelial survival signals as seen in vitro in cultured endothelial cells. Nevertheless, under diabetic conditions, no further increase in the detrimental effects were observed. Conclusions: In conclusion, the effects of glucosamine supplementation in the retina appear to be a double-edged sword: neuronal protection in the diabetic retina and vascular damage in the normal retina. Thus, glucosamine supplementation in osteoarthritis patients with or without diabetes should be taken with care.

Targeting cancer metabolism has become one of the strategies for a rational anti-tumor therapy. However, cellular plasticity, driven by a major regulator of cellular growth and metabolism, mTORC1, often leads toward treatment resistance. Sestrin2, a stress-inducible protein, has been described as an mTORC1 inhibitor upon various types of stress signals. Immune assays and online measurements of cellular bioenergetics were employed to investigate the nature of Sestrin2 regulation, and finally, by silencing the SESN2 gene, to identify the role of induced Sestrin2 upon a single amino acid deprivation in cancer cells of various origins. Our data suggest that a complex interplay of either oxidative, energetic, nutritional stress, or in combination, play a role in Sestrin2 regulation upon single amino acid deprivation. Therefore, cellular metabolic background and sequential metabolic response dictate Sestrin2 expression in the absence of an amino acid. While deprivations of essential amino acids uniformly induce Sestrin2 levels, non-essential amino acids regulate Sestrin2 differently, drawing a characteristic Sestrin2 expression fingerprint, which could serve as a first indication of the underlying cellular vulnerability. Finally, we show that canonical GCN2-ATF4-mediated Sestrin2 induction leads to mTORC1 inhibition only in amino acid auxotroph cells, where the amino acid cannot be replenished by metabolic reprogramming.

The continuous global increase in population and consumption of resources due to human activities has had a significant impact on the environment. Therefore, assessment of environmental exposure to toxic chemicals as well as their impact on biological systems is of significant importance. Freshwater systems are currently under threat and monitored; however, current methods for pollution assessment can neither provide mechanistic insight nor predict adverse effects from complex pollution. Using daphnids as a bioindicator, we assessed the impact in acute exposures of eight individual chemicals and specifically two metals, four pharmaceuticals, a pesticide and a stimulant, and their composite mixture combining phenotypic, biochemical and metabolic markers of physiology. Toxicity levels were in the same order of magnitude and significantly enhanced in the composite mixture. Results from individual chemicals showed distinct biochemical responses for key enzyme activities such as phosphatases, lipase, peptidase, β-galactosidase and glutathione-S-transferase. Following this, a more realistic mixture scenario was assessed with the aforementioned enzyme markers and a metabolomic approach. A clear dose-dependent effect for the composite mixture was validated with enzyme markers of physiology, and the metabolomic analysis verified the effects observed, thus providing a sensitive metrics in metabolite perturbations. Our study highlights that sensitive enzyme markers can be used in advance on the design of metabolic and holistic assays to guide the selection of chemicals and the trajectory of the study, while providing mechanistic insight. In the future this could prove to become a useful tool for understanding and predicting freshwater pollution.

The liver is the central hub for processing and maintaining homeostatic levels of dietary nutrients especially essential amino acids such as tryptophan (Trp). Trp is required not only to sustain protein synthesis but also as a precursor for the production of NAD, neurotransmitters and immunosuppressive metabolites. In light of these roles of Trp and its metabolic products, maintaining homeostatic levels of Trp is essential for health and well-being. The liver regulates global Trp supply by the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2), which degrades Trp down the kynurenine pathway (KP). In the current study, we show that isolated primary hepatocytes when exposed to hypoxic environments, extensively rewire their Trp metabolism by reducing constitutive Tdo2 expression and differentially regulating other Trp pathway enzymes and transporters. Mathematical modelling of Trp metabolism in liver cells under hypoxia predicted decreased flux through the KP while metabolic flux through the tryptamine branch significantly increased. In line, the model also revealed an increased accumulation of tryptamines under hypoxia, at the expense of kynurenines. Metabolic measurements in hypoxic hepatocytes confirmed the predicted reduction in KP metabolites as well as accumulation of tryptamine. Tdo2 expression in cultured primary hepatocytes was reduced upon hypoxia inducible factor (HIF) stabilisation by dimethyloxalylglycine (DMOG), demonstrating that HIFs are involved in the hypoxic downregulation of hepatic Tdo2. DMOG abrogated hepatic luciferase signals in Tdo2 reporter mice, indicating that HIF stability also recapitulates hypoxic rewiring of Trp metabolism in vivo. Also in WT mice HIF stabilization drove homeostatic Trp metabolism away from the KP towards enhanced tryptamine production, leading to enhanced levels of tryptamine in liver, serum and brain. As tryptamines are the most potent hallucinogens known, the observed upregulation of tryptamine in response to hypoxic exposure of hepatocytes may be involved in the generation of hallucinations occurring at high altitude. KP metabolites are known to activate the aryl hydrocarbon receptor (AHR). The AHR-activating properties of tryptamines may explain why immunosuppressive AHR activity is maintained under hypoxia despite downregulation of the KP. In summary our results identify hypoxia as an important factor controlling Trp metabolism in the liver with possible implications for immunosuppressive AHR activation and mental disturbances.

Summary: Regulation of glucose homeostasis is a fundamental process to maintain blood glucose at a physiological level, and its dysregulation is associated with the development of several metabolic diseases. Here, we report on a zebrafish mutant for Aldo-keto-reductase 1a1b (akr1a1b) as a regulator of gluconeogenesis. Adult akr1a1b−/− mutant zebrafish developed fasting hypoglycemia, which was caused by inhibiting phosphoenolpyruvate carboxykinase (PEPCK) expression as rate-limiting enzyme of gluconeogenesis. Subsequently, glucogenic amino acid glutamate as substrate for gluconeogenesis accumulated in the kidneys, but not in livers, and induced structural and functional pronephros alterations in 48-hpf akr1a1b−/− embryos. Akr1a1b−/− mutants displayed increased nitrosative stress as indicated by increased nitrotyrosine, and increased protein-S-nitrosylation. Inhibition of nitrosative stress using the NO synthase inhibitor L-NAME prevented kidney damage and normalized PEPCK expression in akr1a1b−/− mutants. Thus, the data have identified Akr1a1b as a regulator of gluconeogenesis in zebrafish and thereby controlling glucose homeostasis.

The plant kingdom contains an enormous diversity of bioactive compounds which regulate plant growth and defends against biotic and abiotic stress. Some of these compounds, like flavonoids, have properties which are health supporting and relevant for industrial use. Many of these valuable compounds are synthesized in various pepper (Capsicum sp.) tissues. Further, a huge amount of biomass residual remains from pepper production after harvest, which provides an important opportunity to extract these metabolites and optimize the utilization of crops. Moreover, abiotic stresses induce the synthesis of such metabolites as a defense mechanism. Two different Capsicum species were therefore exposed to chilling temperature (24/18 ℃ vs. 18/12 ℃), to salinity (200 mM NaCl), or a combination thereof for 1, 7 and 14 days to investigate the effect of these stresses on the metabolome and transcriptome profiles of their leaves. Both profiles in both species responded to all stresses with an increase over time. All stresses resulted in repression of photosynthesis genes. Stress involving chilling temperature induced secondary metabolism whereas stresses involving salt repressed cell wall modification and solute transport. The metabolome analysis annotated putatively many health stimulating flavonoids (apigetrin, rutin, kaempferol, luteolin and quercetin) in the Capsicum biomass residuals, which were induced in response to salinity, chilling temperature or a combination thereof, and supported by related structural genes of the secondary metabolism in the network analysis.

Background: Deficient glucocorticoid biosynthesis leading to adrenal insufficiency is life-threatening and is associated with significant co-morbidities. The affected pathways underlying the pathophysiology of co-morbidities due to glucocorticoid deficiency remain poorly understood and require further investigation. Methods: To explore the pathophysiological processes related to glucocorticoid deficiency, we have performed global transcriptional, post-transcriptional and metabolic profiling of a cortisol-deficient zebrafish mutant with a disrupted ferredoxin (fdx1b) system. Findings: fdx1b−/− mutants show pervasive reprogramming of metabolism, in particular of glutamine-dependent pathways such as glutathione metabolism, and exhibit changes of oxidative stress markers. The glucocorticoid-dependent post-transcriptional regulation of key enzymes involved in de novo purine synthesis was also affected in this mutant. Moreover, fdx1b−/− mutants exhibit crucial features of primary adrenal insufficiency, and mirror metabolic changes detected in primary adrenal insufficiency patients. Interpretation: Our study provides a detailed map of metabolic changes induced by glucocorticoid deficiency as a consequence of a disrupted ferredoxin system in an animal model of adrenal insufficiency. This improved pathophysiological understanding of global glucocorticoid deficiency informs on more targeted translational studies in humans suffering from conditions associated with glucocorticoid deficiency. Fund: Marie Curie Intra-European Fellowships for Career Development, HGF-programme BIFTM, Deutsche Forschungsgemeinschaft, BBSRC. Keywords: Zebrafish, Ferredoxin, Adrenal insufficiency, Oxidative stress, Purine metabolism

Obesity is a worldwide public health problem with increasing prevalence and affects 80% of diabetes mellitus type 2 cases. Zebrafish (Danio rerio) is an established model organism for studying obesity and diabetes including diabetic microvascular complications. We aimed to determine whether physical activity is an appropriate tool to examine training effects in zebrafish and to analyse metabolic and transcriptional processes in trained zebrafish. A 2- and 8-week experimental training phase protocol with adult zebrafish in a swim tunnel system was established. We examined zebrafish basic characteristics before and after training such as body weight, body length and maximum speed and considered overfeeding as an additional parameter in the 8-weeks training protocol. Ultimately, the effects of training and overfeeding on blood glucose, muscle core metabolism and liver gene expression using RNA-Seq were investigated. Zebrafish maximum speed was correlated with body length and was significantly increased after 2 weeks of training. Maximum swim speed further increased after 8 weeks of training in both the normal-fed and the overfed groups, but training was found not to be sufficient in preventing weight gain in overfed fish. Metabolome and transcriptome profiling in trained fish exhibited increased blood glucose levels in the short-term and upregulated energy supply pathways as well as response to oxidative stress in the long-term. In conclusion, swim training is a valuable tool to study the effects of physical activity in zebrafish, which is accompanied by metabolic and transcriptional adaptations.

Abstract Growth of eukaryotic cells is regulated by the target of rapamycin (TOR). The strongest activator of TOR in metazoa is amino acid availability. The established transducers of amino acid sensing to TOR in metazoa are absent in plants. Hence, a fundamental question is how amino acid sensing is achieved in photo-autotrophic organisms. Here we demonstrate that the plant Arabidopsis does not sense the sulfur-containing amino acid cysteine itself, but its biosynthetic precursors. We identify the kinase GCN2 as a sensor of the carbon/nitrogen precursor availability, whereas limitation of the sulfur precursor is transduced to TOR by downregulation of glucose metabolism. The downregulated TOR activity caused decreased translation, lowered meristematic activity, and elevated autophagy. Our results uncover a plant-specific adaptation of TOR function. In concert with GCN2, TOR allows photo-autotrophic eukaryotes to coordinate the fluxes of carbon, nitrogen, and sulfur for efficient cysteine biosynthesis under varying external nutrient supply.

Abstract The citric acid cycle intermediate 2-oxoglutarate (2-OG, a.k.a. alpha-ketoglutarate) links the carbon and nitrogen metabolic pathways and can provide information on the metabolic status of cells. In recent years, it has become exceedingly clear that 2-OG also acts as a master regulator of diverse biologic processes in all domains of life. Consequently, there is a great demand for time-resolved data on 2-OG fluctuations that can’t be adequately addressed using established methods like mass spectrometry-based metabolomics analysis. Therefore, we set out to develop a novel intramolecular 2-OG FRET sensor based on the signal transduction protein PII from Synechococcus elongatus PCC 7942. We created two variants of the sensor, with a dynamic range for 2-OG from 0.1 µM to 0.1 mM or from 10 µM to 10 mM. As proof of concept, we applied the sensors to determine in situ glutamine:2-oxoglutarate aminotransferase (GOGAT) activity in Synechococcus elongatus PCC 7942 cells and measured 2-OG concentrations in cell extracts from Escherichia coli in vitro. Finally, we could show the sensors’ functionality in living human cell lines, demonstrating their potential in the context of mechanistic studies and drug screening.

Abnormal circulation in solid tumors results in hypoxia, which modulates both tumor intrinsic malignant properties as well as anti-tumor immune responses. Given the importance of hypoxia in glioblastoma (GBM) biology and particularly in shaping anti-tumor immunity, we analyzed which immunomodulatory genes are differentially regulated in response to hypoxia in GBM cells. Gene expression analyses identified the immunosuppressive enzyme tryptophan-2,3-dioxygenase (TDO2) as the second most downregulated gene in GBM cells cultured under hypoxic conditions. TDO2 catalyses the oxidation of tryptophan to N-formyl kynurenine, which is the first and rate-limiting step of Trp degradation along the kynurenine pathway (KP). In multiple GBM cell lines hypoxia reduced TDO2 expression both at mRNA and protein levels. The downregulation of TDO2 through hypoxia was reversible as re-oxygenation rescued TDO2 expression. Computational modeling of tryptophan metabolism predicted reduced flux through the KP and lower intracellular concentrations of kynurenine and its downstream metabolite 3-hydroxyanthranilic acid under hypoxia. Metabolic measurements confirmed the predicted changes, thus demonstrating the ability of the mathematical model to infer intracellular tryptophan metabolite concentrations. Moreover, we identified hypoxia inducible factor 1α (HIF1α) to regulate TDO2 expression under hypoxic conditions, as the HIF1α-stabilizing agents dimethyloxalylglycine (DMOG) and cobalt chloride reduced TDO2 expression. Knockdown of HIF1α restored the expression of TDO2 upon cobalt chloride treatment, confirming that HIF1α controls TDO2 expression. To investigate the immunoregulatory effects of this novel mechanism of TDO2 regulation, we co-cultured isolated T cells with TDO2-expressing GBM cells under normoxic and hypoxic conditions. Under normoxia TDO2-expressing GBM cells suppressed T cell proliferation, while hypoxia restored the proliferation of the T cells, likely due to the reduction in kynurenine levels produced by the GBM cells. Taken together, our data suggest that the regulation of TDO2 expression by HIF1α may be involved in modulating anti-tumor immunity in GBM.

Analysis of metabolomics has been suggested as a promising approach for early detection of colorectal cancer and advanced adenomas. We investigated and compared the metabolomics profile in blood, stool, and urine samples of screening colonoscopy participants and aimed to evaluate differences in metabolite concentrations between people with advanced colorectal neoplasms and those without neoplasms. Various types of bio-samples (plasma, feces, and urine) from 400 participants of screening colonoscopy were investigated using the MxP® Quant 500 kit (Biocrates, Innsbruck, Austria). We detected a broad range of metabolites in blood, stool, and urine samples (504, 331, and 131, respectively). Significant correlations were found between concentrations in blood and stool, blood and urine, and stool and urine for 93, 154, and 102 metabolites, of which 68 (73%), 126 (82%), and 39 (38%) were positive correlations. We found significant differences between participants with and without advanced colorectal neoplasms for concentrations of 123, 49, and 28 metabolites in blood, stool and urine samples, respectively. We detected mostly positive correlations between metabolite concentrations in blood samples and urine or stool samples, and mostly negative correlations between urine and stool samples. Differences between subjects with and without advanced colorectal neoplasms were found for metabolite concentrations in each of the three bio-fluids.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Altered daily patterns of hormone action are suspected to contribute to metabolic disease. It is poorly understood how the adrenal glucocorticoid hormones contribute to the coordination of daily global patterns of transcription and metabolism. Here, we examined diurnal metabolite and transcriptome patterns in a zebrafish glucocorticoid deficiency model by RNA-Seq, NMR spectroscopy and liquid chromatography-based methods. We observed dysregulation of metabolic pathways including glutaminolysis, the citrate and urea cycles and glyoxylate detoxification. Constant, non-rhythmic glucocorticoid treatment rescued many of these changes, with some notable exceptions among the amino acid related pathways. Surprisingly, the non-rhythmic glucocorticoid treatment rescued almost half of the entire dysregulated diurnal transcriptome patterns. A combination of E-box and glucocorticoid response elements is enriched in the rescued genes. This simple enhancer element combination is sufficient to drive rhythmic circadian reporter gene expression under non-rhythmic glucocorticoid exposure, revealing a permissive function for the hormones in glucocorticoid-dependent circadian transcription. Our work highlights metabolic pathways potentially contributing to morbidity in patients with glucocorticoid deficiency, even under glucocorticoid replacement therapy. Moreover, we provide mechanistic insight into the interaction between the circadian clock and glucocorticoids in the transcriptional regulation of metabolism.

Background Metabolomics analysis of human stool samples is of great interest for a broad range of applications in biomedical research including early detection of colorectal neoplasms. However, due to the complexity of metabolites there is no consensus on how to process samples for stool metabolomics measurements to obtain a broad coverage of hydrophilic and hydrophobic substances.Methods We used frozen stool samples (50 mg) from healthy study participants. Stool samples were processed after thawing using eight different processing protocols and different solvents (solvents such as phosphate-buffered saline, isopropanol, methanol, ethanol, acetonitrile and solvent mixtures with or without following evaporation and concentration steps). Metabolites were measured afterwards using the MxP Quant 500 kit (Biocrates). The best performing protocol was subsequently applied to compare stool samples of participants with different dietary habits.Results In this study, we were able to determine up to 340 metabolites of various chemical classes extracted from stool samples of healthy study participants with eight different protocols. Polar metabolites such as amino acids could be measured with each method while other metabolite classes, particular lipid species (better with isopropanol and ethanol or methanol following a drying step), are more dependent on the solvent or combination of solvents used. Only a small number of triglycerides or acylcarnitines were detected in human faeces. Extraction efficiency was higher for protocols using isopropanol (131 metabolites>limit of detection (LOD)) or those using ethanol or methanol and methyl tert-butyl ether (MTBE) including an evaporation and concentration step (303 and 342 metabolites>LOD, respectively) than for other protocols. We detected significant faecal metabolite differences between vegetarians, semivegetarians and non-vegetarians.Conclusion For the evaluation of metabolites in faecal samples, we found protocols using solvents like isopropanol and those using ethanol or methanol, and MTBE including an evaporation and concentration step to be superior regarding the number of detected metabolites of different chemical classes over others tested in this study.