Fish Macrophages Show Distinct Metabolic Signatures Upon Polarization
Wentzel, Annelieke S. ; Janssen, Joëlle J.E. ; Boer, Vincent C.J. de; Veen, Wouter G. van; Forlenza, Maria ; Wiegertjes, Geert F. - \ 2020
Frontiers in Immunology 11 (2020). - ISSN 1664-3224 - 1 p.
extracellular flux analysis - glycolysis - M1 M2 macrophage polarization - metabolic reprogramming - oxidative metabolism - oxidative phosphorylation (OXPHOS) - Seahorse - teleost
Macrophages play important roles in conditions ranging from host immune defense to tissue regeneration and polarize their functional phenotype accordingly. Next to differences in the use of L-arginine and the production of different cytokines, inflammatory M1 macrophages and anti-inflammatory M2 macrophages are also metabolically distinct. In mammals, M1 macrophages show metabolic reprogramming toward glycolysis, while M2 macrophages rely on oxidative phosphorylation to generate energy. The presence of polarized functional immune phenotypes conserved from mammals to fish led us to hypothesize that a similar metabolic reprogramming in polarized macrophages exists in carp. We studied mitochondrial function of M1 and M2 carp macrophages under basal and stressed conditions to determine oxidative capacity by real-time measurements of oxygen consumption and glycolytic capacity by measuring lactate-based acidification. In M1 macrophages, we found increased nitric oxide production and irg1 expression in addition to altered oxidative phosphorylation and glycolysis. In M2 macrophages, we found increased arginase activity, and both oxidative phosphorylation and glycolysis were similar to control macrophages. These results indicate that M1 and M2 carp macrophages show distinct metabolic signatures and indicate that metabolic reprogramming may occur in carp M1 macrophages. This immunometabolic reprogramming likely supports the inflammatory phenotype of polarized macrophages in teleost fish such as carp, similar to what has been shown in mammals.
In vitro and in vivo Effects of Lactate on Metabolism and Cytokine Production of Human Primary PBMCs and Monocytes
Ratter, Jacqueline M. ; Rooijackers, Hanne M.M. ; Hooiveld, Guido J. ; Hijmans, Anneke G.M. ; Galan, Bastiaan E. de; Tack, Cees J. ; Stienstra, Rinke - \ 2018
Frontiers in Immunology 9 (2018). - ISSN 1664-3224
cytokines - glycolysis - immunometabolism - innate immune cells - lactate - monocytes
Lactate, the end product of anaerobic glycolysis, is produced in high amounts by innate immune cells during inflammatory activation. Although immunomodulating effects of lactate have been reported, evidence from human studies is scarce. Here we show that expression of genes involved in lactate metabolism and transport is modulated in human immune cells during infection and upon inflammatory activation with TLR ligands in vitro, indicating an important role for lactate metabolism in inflammation. Extracellular lactate induces metabolic reprogramming in innate immune cells, as evidenced by reduced glycolytic and increased oxidative rates of monocytes immediately after exposure to lactate. A short-term infusion of lactate in humans in vivo increased ex vivo glucose consumption of PBMCs, but effects on metabolic rates and cytokine production were limited. Interestingly, long-term treatment with lactate ex vivo, reflecting pathophysiological conditions in local microenvironments such as tumor or adipose tissue, significantly modulated cytokine production with predominantly anti-inflammatory effects. We found time- and stimuli-dependent effects of extracellular lactate on cytokine production, further emphasizing the complex interplay between metabolism and immune cell function. Together, our findings reveal lactate as a modulator of immune cell metabolism which translates to reduced inflammation and may ultimately function as a negative feedback signal to prevent excessive inflammatory responses.
Glutaminolysis and Fumarate Accumulation Integrate Immunometabolic and Epigenetic Programs in Trained Immunity
Arts, Rob J.W. ; Novakovic, Boris ; Horst, Rob ter; Carvalho, Agostinho ; Bekkering, Siroon ; Lachmandas, Ekta ; Rodrigues, Fernando ; Silvestre, Ricardo ; Cheng, Shih Chin ; Wang, Shuang Yin ; Habibi, Ehsan ; Gonçalves, Luís G. ; Mesquita, Inês ; Cunha, Cristina ; Laarhoven, Arjan van; Veerdonk, Frank L. van de; Williams, David L. ; Meer, Jos W.M. van der; Logie, Colin ; O'Neill, Luke A. ; Dinarello, Charles A. ; Riksen, Niels P. ; Crevel, Reinout van; Clish, Clary ; Notebaart, Richard A. ; Joosten, Leo A.B. ; Stunnenberg, Hendrik G. ; Xavier, Ramnik J. ; Netea, Mihai G. - \ 2016
Cell Metabolism 24 (2016)6. - ISSN 1550-4131 - p. 807 - 819.
cholesterol metabolism - epigenetics - glutamine metabolism - glycolysis - trained immunity
Induction of trained immunity (innate immune memory) is mediated by activation of immune and metabolic pathways that result in epigenetic rewiring of cellular functional programs. Through network-level integration of transcriptomics and metabolomics data, we identify glycolysis, glutaminolysis, and the cholesterol synthesis pathway as indispensable for the induction of trained immunity by β-glucan in monocytes. Accumulation of fumarate, due to glutamine replenishment of the TCA cycle, integrates immune and metabolic circuits to induce monocyte epigenetic reprogramming by inhibiting KDM5 histone demethylases. Furthermore, fumarate itself induced an epigenetic program similar to β-glucan-induced trained immunity. In line with this, inhibition of glutaminolysis and cholesterol synthesis in mice reduced the induction of trained immunity by β-glucan. Identification of the metabolic pathways leading to induction of trained immunity contributes to our understanding of innate immune memory and opens new therapeutic avenues.
Dynamic changes in energy metabolism upon embryonic stem cell differentiation support developmental toxicant identification
Dartel, D.A.M. van; Schulpen, S.H. ; Theunissen, P.T. ; Bunschoten, A. ; Piersma, A.H. ; Keijer, J. - \ 2014
Toxicology 324 (2014). - ISSN 0300-483X - p. 76 - 87.
gene-expression - neural differentiation - proliferation - toxicity - cardiomyocytes - normalization - glycolysis - genomics - protocol - biology
Embryonic stem cells (ESC) are widely used to study embryonic development and to identify developmental toxicants. Particularly, the embryonic stem cell test (EST) is well known as in vitro model to identify developmental toxicants. Although it is clear that energy metabolism plays a crucial role in embryonic development, the modulation of energy metabolism in in vitro models, such as the EST, is not yet described. The present study is among the first studies that analyses whole genome expression data to specifically characterize metabolic changes upon ESC early differentiation. Our transcriptomic analyses showed activation of glycolysis, truncated activation of the tricarboxylic acid (TCA) cycle, activation of lipid synthesis, as well as activation of glutaminolysis during the early phase of ESC differentiation. Taken together, this energy metabolism profile points towards energy metabolism reprogramming in the provision of metabolites for biosynthesis of cellular constituents. Next, we defined a gene set that describes this energy metabolism profile. We showed that this gene set could be successfully applied in the EST to identify developmental toxicants known to modulate cellular biosynthesis (5-fluorouracil and methoxyacetic acid), while other developmental toxicants or the negative control did not modulate the expression of this gene set. Our description of dynamic changes in energy metabolism during early ESC differentiation, as well as specific identification of developmental toxicants modulating energy metabolism, is an important step forward in the definition of the applicability domain of the EST.
Moisture absorption early postmortem predicts ultimate drip loss
Kapper, C. ; Walukonis, C.J. ; Scheffler, T.L. ; Scheffler, J.M. ; Don, C. ; Morgan, M.T. ; Forrest, J.C. ; Gerrard, D.E. - \ 2014
Meat Science 96 (2014)2A. - ISSN 0309-1740 - p. 971 - 976.
water-holding capacity - m-longissimus-dorsi - electrical-stimulation - meat quality - porcine muscle - pigs - ph - halothane - temperature - glycolysis
Water-holding capacity is the ability of meat to hold moisture and is subject to postmortem metabolism. The objective of this study was to characterize the loss of moisture from muscle postmortem and investigate whether these losses are useful in predicting the ultimate drip loss of fresh pork. Cotton–rayon absorptive-based devices were inserted in the longissimus dorsi muscles of pork carcasses (n = 51) postmortem and removed at various intervals for 24 h. Greatest moisture absorption was observed at 105 min post exsanguination. Drip loss varied (0.6–15.3%) across carcasses. Individual absorption at 75 min correlated (r = 0.33) with final drip loss. Correlations improved using individual absorption values at 90 min (r = 0.48) and accumulated absorption values at 150 min (r = 0.41). Results show that significant moisture is lost from muscle tissue early postmortem and suggest that capture of this moisture may be useful in predicting final drip loss of fresh meat.
A translational study “case report” on the small molecule “energy blocker” 3-bromopyruvate (3BP) as a potent anticancer agent: from bench side to bedside
Ko, Y.H. ; Verhoeven, H.A. ; Lee, M.J. ; Corbin, D.J. ; Vogl, T.J. ; Pedersen, P.L. - \ 2012
Journal of Bioenergetics and Biomembranes 44 (2012). - ISSN 0145-479X - p. 163 - 170.
cancer-cells - escherichia-coli - isocitrate lyase - mitochondria - warburg - hexokinase - glycolysis - therapy
The small alkylating molecule, 3-bromopyruvate (3BP), is a potent and specific anticancer agent. 3BP is different in its action from most currently available chemo-drugs. Thus, 3BP targets cancer cells’ energy metabolism, both its high glycolysis (“Warburg Effect”) and mitochondrial oxidative phosphorylation. This inhibits/ blocks total energy production leading to a depletion of energy reserves. Moreover, 3BP as an “Energy Blocker”, is very rapid in killing such cells. This is in sharp contrast to most commonly used anticancer agents that usually take longer to show a noticeable effect. In addition, 3BP at its effective concentrations that kill cancer cells has little or no effect on normal cells. Therefore, 3BP can be considered a member, perhaps one of the first, of a new class of anticancer agents. Following 3BP’s discovery as a novel anticancer agent in vitro in the Year 2000 (Published in Ko et al. Can Lett 173:83–91, 2001), and also as a highly effective and rapid anticancer agent in vivo shortly thereafter (Ko et al. Biochem Biophys Res Commun 324:269–275, 2004), its efficacy as a potent anticancer agent in humans was demonstrated. Here, based on translational research, we report results of a case study in a young adult cancer patient with fibrolamellar hepatocellular carcinoma. Thus, a bench side discovery in the Department of Biological Chemistry at Johns Hopkins University, School of Medicine was taken effectively to bedside treatment at Johann Wolfgang Goethe University Frankfurt/Main Hospital, Germany. The results obtained hold promise for 3BP as a future cancer therapeutic without apparent cyto-toxicity when formulated properly.
Functional analysis of thermostable proteins involved in carbohydrate metabolism
Akerboom, A.P. - \ 2007
Wageningen University. Promotor(en): Willem de Vos; John van der Oost. - [S.l.] : S.n. - ISBN 9789085047124 - 174
thermofiele bacteriën - fermentatie - glycolyse - regulatory sequences - thermophilic bacteria - fermentation - glycolysis - regulatory sequences
Thermostable proteins can resist temperature stress whilst keeping their integrity and functionality. In many cases, thermostable proteins originate from hyperthermophilic microorganisms that thrive in extreme environments. These systems are generally located close to geothermal (volcanic) activity, such as in smoking vents on the bottom of the ocean or hot springs at the earth’s surface. Thermostable proteins, especially enzymes, have found extensive use in various biotechnological processes as their stability makes them ideal catalysts at a variety of temperatures and in harsh solvents. Carbohydrates are one of the main sources of energy for many living organisms. To gain insight into the carbohydrate metabolism of hyperthermophilic organisms, as well as the potential applications of thermostable proteins, several proteins involved in different aspects of carbohydrate metabolism were selected for detailed molecular genetic and biochemical analysis, as described below. The glycolytic enzyme phosphoglucose isomerase from the hyperthermophilic archaeon Pyrococcus furiosus was overproduced, purified, crystallized and biochemically characterized. Several structures together with electron paramagnetic resonance data, nuclear magnetic resonance spectroscopy data and enzyme inhibition analysis were used to elucidate the reaction mechanism of this enzyme. Computational analysis of the cupin superfamily, to which this protein belongs, allowed the essential residues for this protein’s integrity to be predicted. Subsequently, these predictions were verified by testing the activity of several generated mutants. To study regulation of carbohydrate metabolism in two related hyperthermophilic organisms; P. furiosus and Thermococcus kodakaraensis, the transcription initiation sites of glycolytic genes in P. furiosus were mapped and analysed. The obtained promoters were compared computationally to upstream regions of non-glycolytic genes. This allowed for the identification of a potential palindromic transcription factor binding site present in many genes involved in glycolysis and starch metabolism (Thermococcales-Glycolytic-Motif, TGM). Subsequently, two putative regulators of this regulon were selected for further analysis. One of them, the sugar fermentation stimulation protein SfsA, appeared to be an endonuclease. SfsA from both the thermophilic archaeon P. furiosus and the mesophilic bacterium E. coli were further characterized. Both SfsAs appeared to bind nucleotides with a distinct specificity with the P. furiosus SfsA appearing to bind RNA while the E. coli SfsA appeared to bind DNA. The P. furiosus SfsA showed hydrolytic activity on dsDNA. Genomic context analysis together with Histidine-tag pull-downs of E. coli SfsA indicated a possible role in RNA/DNA/nucleotide turnover. The other putative regulator of glycolytic genes in P. furiosus and T. kodakaraensis, Tgr, was over-expressed, purified and binding to the Thermococcales glycolytic promoter motif (TGM) by gel retardation assays was established. Furthermore, whole-genome DNA microarray analysis of T. kodakaraensis and an isogenic tgr knockout of this organism revealed that transcriptional regulation of genes containing a TGM in their promoter region in the knockout of tgr was severely affected, indicating that Tgr was regulating these genes in vivo. Co-factor analysis with several maltooligosaccharides proved that maltotriose was the effector of Tgr. To study the applicability of thermostable proteins, a biosensor was constructed based on the maltotriose binding protein from the ABC-transporter of P. furiosus. Several amino acids of this binding protein were mutated to cysteines and tagged with a fluorescent probe. Addition of maltotriose resulted in a change in fluorescence intensity in some of the biosensors. Tests at 20 and 60 degrees Celsius indicated that the affinity of the sensor was dependent on the temperature of the environment, and proved that the protein could be used at higher temperatures. The work described in this thesis is a contribution to the understanding of how thermostable proteins function in vivo, and how these proteins can be employed in a practical setup. The presented studies have revealed molecular details on enzymatic performance, transcription factor dependent regulation of glycolysis and the use of a thermostable protein in a biosensor setup.
Functional ingredient production: application of global metabolic models
Smid, E.J. ; Molenaar, D. ; Hugenholtz, J. ; Vos, W.M. de; Teusink, B. - \ 2005
Current Opinion in Biotechnology 16 (2005)2. - ISSN 0958-1669 - p. 190 - 197.
lactic-acid bacteria - escherichia-coli - corynebacterium-glutamicum - saccharomyces-cerevisiae - lactococcus-lactis - industrial-production - pathway analysis - glycolysis - kinetics - networks
The biotechnology industry continuously explores new ways to improve the performance of microbial strains in fermentation processes. Recent focus has been on new genome-wide modelling approaches in functional genomics, which aim to take full advantage of genome sequence data, transcription profiling, proteomics and metabolite profiling for strain improvement. The integration of global metabolic models with genetic and regulatory models will be essential for the practice of metabolic engineering for strain improvement to move forward, simply because we cannot rely on our intuition to grasp the complexity of the biological systems involved
Preslaughter stress and muscle energy largely determine pork quality at two commercial processing plants
Hambrecht, E. ; Eissen, J.J. ; Nooijen, R.I.J. ; Ducro, B.J. ; Smits, C.H.M. ; Hartog, L.A. den; Verstegen, M.W.A. - \ 2004
Journal of Animal Science 82 (2004). - ISSN 0021-8812 - p. 1401 - 1409.
meat quality - pre-slaughter - longissimus muscle - stunning method - hampshire pigs - plasma - indicators - glycolysis - cortisol - populations
The objective of the present experiment was to study physiological changes elicited in response to stress in the immediate preslaughter period and to link them to pork quality characteristics. Crossbred, halothane-free pigs (n = 192) were processed in eight groups (24 pigs per group) on various days at one of two commercial processing plants operating different stunning systems (electrical and CO2 stunning in Plants A and B, respectively). In each group, half the pigs were exposed to either minimal or high preslaughter stress. Blood samples were taken at exsanguination, and lactate, cortisol, and catecholamines, as well as blood pH and temperature, were assessed and linked to various longissimus muscle quality attributes. Additionally, muscle pH and temperature were measured 30 min postmortem, and muscle glycolytic potential was determined 22 h postmortem. At both processing plants, high preslaughter stress resulted in higher (P <0.05) blood cortisol and lactate; however, the effects of preslaughter stress on catecholamines and blood pH were believed to be biased by the different stunning methods employed at the plants. High preslaughter stress increased (P <0.05) blood temperature at Plant A but not at Plant B. At both plants, high stress increased (P <0.05) 30-min muscle temperature and decreased (P <0.05) 30-min muscle pH. Ultimate pH was increased (P <0.05) and muscle glycolytic potential was decreased (P <0.05) by high preslaughter stress. At both plants, high stress resulted in inferior pork quality attributes (P <0.05), including reflectance, electrical conductivity, filter paper moisture, drip loss, and L* value. The effect of stress was greater on water-holding capacity than on pork color, with drip losses increased by 56%. Of all stress indicators measured at exsanguination, only blood lactate was strongly correlated with pork quality attributes. Regression analyses revealed that blood lactate and glycolytic potential accounted for 52 and 48% of the variation in drip loss and L* value, respectively. In combination with high preslaughter stress, high glycolytic potentials were related to increased drip losses. We conclude that high preslaughter stress leads to impaired pork quality, with high muscle energy levels aggravating the negative effects of preslaughter stress. Monitoring stress level by blood lactate measurement in combination with strategies to control muscle energy present at slaughter may help to improve meat quality.
Increased exopolysaccharide production in Lactococcuc lactis due to increased levels of expression of the NIZO B40 eps gene cluster
Boels, I.C. ; Kranenburg, R. van; Kanning, M.W. ; Chong, B.F. ; Vos, W.M. de; Kleerebezem, M. - \ 2003
Applied and Environmental Microbiology 69 (2003). - ISSN 0099-2240 - p. 5029 - 5031.
rheological properties - functional-analysis - sugar nucleotide - biosynthesis - glycolysis
Exopolysaccharides (EPS) play an important role in the rheology and texture of fermented food products. This is the first report demonstrating that homologous overexpression of a complete eps gene cluster in Lactococcus lactis leads to increased EPS production levels. A ninefold-elevated EPS plasmid copy number led to an almost threefold increase in the eps expression level, resulting in an almost fourfold increase in the NIZO B40 EPS production level. It was previously reported that increased EPS precursor levels did not influence NIZO B40 EPS production levels. However, the present results indicate that the maximal NIZO B40 EPS production level is limited by the activity level of the expression products of the eps gene cluster rather than by the level of EPS precursors.