Increased core body temperature exacerbates defective protein prenylation in mouse models of mevalonate kinase deficiency.

Mevalonate kinase deficiency (MKD) is characterized by recurrent fevers and flares of systemic inflammation, caused by biallelic loss-of-function mutations in MVK. The underlying disease mechanisms and triggers of inflammatory flares are poorly understood because of the lack of in vivo models. We describe genetically modified mice bearing the hypomorphic mutation p.Val377Ile (the commonest variant in patients with MKD) and amorphic, frameshift mutations in Mvk. Compound heterozygous mice recapitulated the characteristic biochemical phenotype of MKD, with increased plasma mevalonic acid and clear buildup of unprenylated GTPases in PBMCs, splenocytes, and bone marrow. The inflammatory response to LPS was enhanced in compound heterozygous mice and treatment with the NLRP3 inflammasome inhibitor MCC950 prevented the elevation of circulating IL-1ß, thus identifying a potential inflammasome target for future therapeutic approaches. Furthermore, lines of mice with a range of deficiencies in mevalonate kinase and abnormal prenylation mirrored the genotype-phenotype relationship in human MKD. Importantly, these mice allowed the determination of a threshold level of residual enzyme activity, below which protein prenylation is impaired. Elevated temperature dramatically but reversibly exacerbated the deficit in the mevalonate pathway and the defective prenylation in vitro and in vivo, highlighting increased body temperature as a likely trigger of inflammatory flares.

Analysing intracellular isoprenoid metabolites in diverse prokaryotic and eukaryotic microbes.

Isoprenoids, also known as terpenes or terpenoids, are a very large and diverse group of natural compounds. These compounds fulfil a myriad of critical roles in biology as well as having a wide range of industrial uses. Isoprenoids are produced via two chemically distinct metabolic pathways, the mevalonate (MVA) pathway and the methylerythritol phosphate (MEP) pathway. Downstream of these two pathways is the shared prenyl phosphate pathway. Because of their importance in both basic physiology and industrial biotechnology, extraction, identification, and quantification of isoprenoid pathway intermediates is an important protocol. Here we describe methods for extraction and analysis of intracellular metabolites from the MVA, MEP, and prenyl phosphate pathways for five key model microbes: the yeast Saccharomyces cerevisiae, the bacterium Escherichia coli, the diatom Phaeodactylum tricornutum, the green algae Chlamydomonas reinhardtii, and the cyanobacterium Synechocystis sp. PCC 6803. These methods also detect several central carbon intermediates. These protocols will likely work effectively, or be readily adaptable, to a variety of related microorganisms and metabolic pathways.

Elevation of fatty acid desaturase 2 in esophageal adenocarcinoma increases polyunsaturated lipids and may exacerbate bile acid-induced DNA damage.

BACKGROUND: The risk of esophageal adenocarcinoma (EAC) is associated with gastro-esophageal reflux disease (GERD) and obesity. Lipid metabolism-targeted therapies decrease the risk of progressing from Barrett's esophagus (BE) to EAC, but the precise lipid metabolic changes and their roles in genotoxicity during EAC development are yet to be established. METHODS: Esophageal biopsies from the normal epithelium (NE), BE, and EAC, were analyzed using concurrent lipidomics and proteomics (n = 30) followed by orthogonal validation on independent samples using RNAseq transcriptomics (n = 22) and immunohistochemistry (IHC, n = 80). The EAC cell line FLO-1 was treated with FADS2 selective inhibitor SC26196, and/or bile acid cocktail, followed by immunofluorescence staining for γH2AX. RESULTS: Metabolism-focused Reactome analysis of the proteomics data revealed enrichment of fatty acid metabolism, ketone body metabolism, and biosynthesis of specialized pro-resolving mediators in EAC pathogenesis. Lipidomics revealed progressive alterations (NE-BE-EAC) in glycerophospholipid synthesis with decreasing triglycerides and increasing phosphatidylcholine and phosphatidylethanolamine, and sphingolipid synthesis with decreasing dihydroceramide and increasing ceramides. Furthermore, a progressive increase in lipids with C20 fatty acids and polyunsaturated lipids with ≥4 double bonds were also observed. Integration with transcriptome data identified candidate enzymes for IHC validation: Δ4-Desaturase, Sphingolipid 1 (DEGS1) which desaturates dihydroceramide to ceramide, and Δ5 and Δ6-Desaturases (fatty acid desaturases, FADS1 and FADS2), responsible for polyunsaturation. All three enzymes showed significant increases from BE through dysplasia to EAC, but transcript levels of DEGS1 were decreased suggesting post-translational regulation. Finally, the FADS2 selective inhibitor SC26196 significantly reduced polyunsaturated lipids with three and four double bonds and reduced bile acid-induced DNA double-strand breaks in FLO-1 cells in vitro. CONCLUSIONS: Integrated multiomics revealed sphingolipid and phospholipid metabolism rewiring during EAC development. FADS2 inhibition and reduction of the high polyunsaturated lipids effectively protected EAC cells from bile acid-induced DNA damage in vitro, potentially through reduced lipid peroxidation.

Bisphosphonate drugs have actions in the lung and inhibit the mevalonate pathway in alveolar macrophages.

Bisphosphonates drugs target the skeleton and are used globally for the treatment of common bone disorders. Nitrogen-containing bisphosphonates act by inhibiting the mevalonate pathway in bone-resorbing osteoclasts but, surprisingly, also appear to reduce the risk of death from pneumonia. We overturn the long-held belief that these drugs act only in the skeleton and show that a fluorescently labelled bisphosphonate is internalised by alveolar macrophages and large peritoneal macrophages in vivo. Furthermore, a single dose of a nitrogen-containing bisphosphonate (zoledronic acid) in mice was sufficient to inhibit the mevalonate pathway in tissue-resident macrophages, causing the build-up of a mevalonate metabolite and preventing protein prenylation. Importantly, one dose of bisphosphonate enhanced the immune response to bacterial endotoxin in the lung and increased the level of cytokines and chemokines in bronchoalveolar fluid. These studies suggest that bisphosphonates, as well as preventing bone loss, may boost immune responses to infection in the lung and provide a mechanistic basis to fully examine the potential of bisphosphonates to help combat respiratory infections that cause pneumonia.

Simultaneous quantification of 26 NAD-related metabolites in plasma, blood, and liver tissue using UHPLC-MS/MS.

Nicotinamide adenine dinucleotide (NAD) is a key metabolic intermediate found in all cells and involved in numerous cellular functions. Perturbances in the NAD metabolome are linked to various diseases such as diabetes and schizophrenia, and to congenital malformations and recurrent miscarriage. Mouse models are central to the investigation of these and other NAD-related conditions because mice can be readily genetically modified and treated with diets with altered concentrations of NAD precursors. Simultaneous quantification of as many metabolites of the NAD metabolome as possible is required to understand which pathways are affected in these disease conditions and what are the functional consequences. Here, we report the development of a fit-for-purpose method to simultaneously quantify 26 NAD-related metabolites and creatinine in mouse plasma, whole blood, and liver tissue using ultra-high performance liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). The included metabolites represent dietary precursors, intermediates, enzymatic cofactors, and excretion products. Sample preparation was optimized for each matrix and included 21 isotope-labeled internal standards. The method reached adequate precision and accuracy for the intended context of use of exploratory pathway-related biomarker discovery in mouse models. The method was tested by determining metabolite concentrations in mice fed a special diet with defined precursor content.

Krüppel-like factor 1 is a core cardiomyogenic trigger in zebrafish.

Cardiac regeneration requires dedifferentiation and proliferation of mature cardiomyocytes, but the mechanisms underlying this plasticity remain unclear. Here, we identify a potent cardiomyogenic role for Krüppel-like factor 1 (Klf1/Eklf), which is induced in adult zebrafish myocardium upon injury. Myocardial inhibition of Klf1 function does not affect heart development, but it severely impairs regeneration. Transient Klf1 activation is sufficient to expand mature myocardium in uninjured hearts. Klf1 directs epigenetic reprogramming of the cardiac transcription factor network, permitting coordinated cardiomyocyte dedifferentiation and proliferation. Myocardial expansion is supported by Klf1-induced rewiring of mitochondrial metabolism from oxidative respiration to anabolic pathways. Our findings establish Klf1 as a core transcriptional regulator of cardiomyocyte renewal in adult zebrafish hearts.

Characterization and validation of a preventative therapy for hypertrophic cardiomyopathy in a murine model of the disease.

Currently there is an unmet need for treatments that can prevent hypertrophic cardiomyopathy (HCM). Using a murine model we previously identified that HCM causing cardiac troponin I mutation Gly203Ser (cTnI-G203S) is associated with increased mitochondrial metabolic activity, consistent with the human condition. These alterations precede development of the cardiomyopathy. Here we examine the efficacy of in vivo treatment of cTnI-G203S mice with a peptide derived against the α-interaction domain of the cardiac L-type calcium channel (AID-TAT) on restoring mitochondrial metabolic activity, and preventing HCM. cTnI-G203S or age-matched wt mice were treated with active or inactive AID-TAT. Following treatment, targeted metabolomics was utilized to evaluate myocardial substrate metabolism. Cardiac myocyte mitochondrial metabolic activity was assessed as alterations in mitochondrial membrane potential and flavoprotein oxidation. Cardiac morphology and function were examined using echocardiography. Cardiac uptake was assessed using an in vivo multispectral imaging system. We identified alterations in six biochemical intermediates in cTnI-G203S hearts consistent with increased anaplerosis. We also reveal that AID-TAT treatment of precardiomyopathic cTnI-G203S mice, but not mice with established cardiomyopathy, restored cardiac myocyte mitochondrial membrane potential and flavoprotein oxidation, and prevented myocardial hypertrophy. Importantly, AID-TAT was rapidly targeted to the heart, and not retained by the liver or kidneys. Overall, we identify biomarkers of HCM resulting from the cTnI mutation Gly203Ser, and present a safe, preventative therapy for associated cardiomyopathy. Utilizing AID-TAT to modulate cardiac metabolic activity may be beneficial in preventing HCM in "at risk" patients with identified Gly203Ser gene mutations.

Chronic High-Fat Diet Induces Early Barrett's Esophagus in Mice through Lipidome Remodeling.

Esophageal adenocarcinoma (EAC) incidence has been rapidly increasing, potentially associated with the prevalence of the risk factors gastroesophageal reflux disease (GERD), obesity, high-fat diet (HFD), and the precursor condition Barrett's esophagus (BE). EAC development occurs over several years, with stepwise changes of the squamous esophageal epithelium, through cardiac metaplasia, to BE, and then EAC. To establish the roles of GERD and HFD in initiating BE, we developed a dietary intervention model in C57/BL6 mice using experimental HFD and GERD (0.2% deoxycholic acid, DCA, in drinking water), and then analyzed the gastroesophageal junction tissue lipidome and microbiome to reveal potential mechanisms. Chronic (9 months) HFD alone induced esophageal inflammation and metaplasia, the first steps in BE/EAC pathogenesis. While 0.2% deoxycholic acid (DCA) alone had no effect on esophageal morphology, it synergized with HFD to increase inflammation severity and metaplasia length, potentially via increased microbiome diversity. Furthermore, we identify a tissue lipid signature for inflammation and metaplasia, which is characterized by elevated very-long-chain ceramides and reduced lysophospholipids. In summary, we report a non-transgenic mouse model, and a tissue lipid signature for early BE. Validation of the lipid signature in human patient cohorts could pave the way for specific dietary strategies to reduce the risk of BE in high-risk individuals.

Multi-omic profiling reveals associations between the gut mucosal microbiome, the metabolome, and host DNA methylation associated gene expression in patients with colorectal cancer.

BACKGROUND: The human gut microbiome plays a critical role in the carcinogenesis of colorectal cancer (CRC). However, a comprehensive analysis of the interaction between the host and microbiome is still lacking. RESULTS: We found correlations between the change in abundance of microbial taxa, butyrate-related colonic metabolites, and methylation-associated host gene expression in colonic tumour mucosa tissues compared with the adjacent normal mucosa tissues. The increase of genus Fusobacterium abundance was correlated with a decrease in the level of 4-hydroxybutyric acid (4-HB) and expression of immune-related peptidase inhibitor 16 (PI16), Fc Receptor Like A (FCRLA) and Lymphocyte Specific Protein 1 (LSP1). The decrease in the abundance of another potentially 4-HB-associated genus, Prevotella 2, was also found to be correlated with the down-regulated expression of metallothionein 1 M (MT1M). Additionally, the increase of glutamic acid-related family Halomonadaceae was correlated with the decreased expression of reelin (RELN). The decreased abundance of genus Paeniclostridium and genus Enterococcus were correlated with increased lactic acid level, and were also linked to the expression change of Phospholipase C Beta 1 (PLCB1) and Immunoglobulin Superfamily Member 9 (IGSF9) respectively. Interestingly, 4-HB, glutamic acid and lactic acid are all butyrate precursors, which may modify gene expression by epigenetic regulation such as DNA methylation. CONCLUSIONS: Our study identified associations between previously reported CRC-related microbial taxa, butyrate-related metabolites and DNA methylation-associated gene expression in tumour and normal colonic mucosa tissues from CRC patients, which uncovered a possible mechanism of the role of microbiome in the carcinogenesis of CRC. In addition, these findings offer insight into potential new biomarkers, therapeutic and/or prevention strategies for CRC.

Adaptation of hydroxymethylbutenyl diphosphate reductase enables volatile isoprenoid production.

Volatile isoprenoids produced by plants are emitted in vast quantities into the atmosphere, with substantial effects on global carbon cycling. Yet, the molecular mechanisms regulating the balance between volatile and non-volatile isoprenoid production remain unknown. Isoprenoids are synthesised via sequential condensation of isopentenyl pyrophosphate (IPP) to dimethylallyl pyrophosphate (DMAPP), with volatile isoprenoids containing fewer isopentenyl subunits. The DMAPP:IPP ratio could affect the balance between volatile and non-volatile isoprenoids, but the plastidic DMAPP:IPP ratio is generally believed to be similar across different species. Here we demonstrate that the ratio of DMAPP:IPP produced by hydroxymethylbutenyl diphosphate reductase (HDR/IspH), the final step of the plastidic isoprenoid production pathway, is not fixed. Instead, this ratio varies greatly across HDRs from phylogenetically distinct plants, correlating with isoprenoid production patterns. Our findings suggest that adaptation of HDR plays a previously unrecognised role in determining in vivo carbon availability for isoprenoid emissions, directly shaping global biosphere-atmosphere interactions.

Class IIa Histone Deacetylases Drive Toll-like Receptor-Inducible Glycolysis and Macrophage Inflammatory Responses via Pyruvate Kinase M2.

Histone deacetylases (HDACs) drive innate immune cell-mediated inflammation. Here we identify class IIa HDACs as key molecular links between Toll-like receptor (TLR)-inducible aerobic glycolysis and macrophage inflammatory responses. A proteomic screen identified the glycolytic enzyme pyruvate kinase M isoform 2 (Pkm2) as a partner of proinflammatory Hdac7 in murine macrophages. Myeloid-specific Hdac7 overexpression in transgenic mice amplifies lipopolysaccharide (LPS)-inducible lactate and promotes a glycolysis-associated inflammatory signature. Conversely, pharmacological or genetic targeting of Hdac7 and other class IIa HDACs attenuates LPS-inducible glycolysis and accompanying inflammatory responses in macrophages. We show that an Hdac7-Pkm2 complex acts as an immunometabolism signaling hub, whereby Pkm2 deacetylation at lysine 433 licenses its proinflammatory functions. Disrupting this complex suppresses inflammatory responses in vitro and in vivo. Class IIa HDACs are thus pivotal intermediates connecting TLR-inducible glycolysis to inflammation via Pkm2.

Triheptanoin alters [U-13C6]-glucose incorporation into glycolytic intermediates and increases TCA cycling by normalizing the activities of pyruvate dehydrogenase and oxoglutarate dehydrogenase in a chronic epilepsy mouse model.

Triheptanoin is anticonvulsant in several seizure models. Here, we investigated changes in glucose metabolism by triheptanoin interictally in the chronic stage of the pilocarpine mouse epilepsy model. After injection of [U-13C6]-glucose (i.p.), enrichments of 13C in intermediates of glycolysis and the tricarboxylic acid (TCA) cycle were quantified in hippocampal extracts and maximal activities of enzymes in each pathway were measured. The enrichment of 13C glucose in plasma was similar across all groups. Despite this, we observed reductions in incorporation of 13C in several glycolytic intermediates compared to control mice suggesting glucose utilization may be impaired and/or glycogenolysis increased in the untreated interictal hippocampus. Triheptanoin prevented the interictal reductions of 13C incorporation in most glycolytic intermediates, suggesting it increased glucose utilization or - as an additional astrocytic fuel - it decreased glycogen breakdown. In the TCA cycle metabolites, the incorporation of 13C was reduced in the interictal state. Triheptanoin restored the correlation between 13C enrichments of pyruvate relative to most of the TCA cycle intermediates in "epileptic" mice. Triheptanoin also prevented the reductions of hippocampal pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities. Decreased glycogen breakdown and increased glucose utilization and metabolism via the TCA cycle in epileptogenic brain areas may contribute to triheptanoin's anticonvulsant effects.

Barocycler-Based Concurrent Multiomics Method To Assess Molecular Changes Associated with Atherosclerosis Using Small Amounts of Arterial Tissue from a Single Mouse.

Atherosclerosis is a complex, multifactorial disease characterized by the buildup of plaque in the arterial wall. Apolipoprotein E gene deficient (Apoe-/-) mice serve as a commonly used tool to elucidate the pathophysiology of atherosclerosis because of their propensity to spontaneously develop arterial lesions. To date, however, an integrated omics assessment of atherosclerotic lesions in individual Apoe-/- mice has been challenging because of the small amount of diseased and nondiseased tissue available. To address this current limitation, we developed a multiomics method (Multi-ABLE) based on the proteomic method called accelerated Barocycler lysis and extraction (ABLE) to assess the depth of information that can be obtained from arterial tissue derived from a single mouse by splitting ABLE to allow for a combined proteomics-metabolomics-lipidomics analysis (Multi-ABLE). The new method includes tissue lysis via pressure cycling technology (PCT) in a Barocycler, followed by proteomic analysis of half the sample by nanoLC-MS and sequential extraction of lipids (organic extract) and metabolites (aqueous extract) combined with HILIC and reversed phase chromatography and time-of-flight mass spectrometry on the other half. Proteomic analysis identified 845 proteins, 93 of which were significantly altered in lesion-containing arteries. Lipidomic and metabolomic analyses detected 851 lipid and 362 metabolite features, which included 215 and 65 identified lipids and metabolites, respectively. The Multi-ABLE method is the first to apply a concurrent multiomics pipeline to cardiovascular disease using small (<5 mg) tissue samples, and it is applicable to other diseases where limited size samples are available at specific points during disease progression.

Impaired Pentose Phosphate Pathway in the Spinal Cord of the hSOD1G93A Mouse Model of Amyotrophic Lateral Sclerosis.

Impairments in energy metabolism in amyotrophic lateral sclerosis (ALS) have long been known. However, the changes in the energy-producing pathways in ALS are not comprehensively understood. To investigate specific alterations in glucose metabolism in glycolytic, pentose phosphate, and TCA cycle pathways, we injected uniformly labeled [U-13C]glucose to wild-type and hSOD1G93A mice at symptom onset (80 days). Using liquid chromatography-tandem mass spectrometry (LC-MS/MS), levels of metabolites were determined in extracts of the cortex and spinal cord. In addition, the activities of several enzymes involved in glucose metabolism were quantified. In the spinal cord, the levels of pentose phosphate pathway (PPP) intermediate ribose 5-phosphate (p = 0.037) were reduced by 37% in hSOD1G93A mice, while the % 13C enrichments in glucose 6-phosphate were increased threefold. The maximal activities of the enzyme glucose 6-phosphate dehydrogenase were decreased by 24% in the spinal cord (p = 0.005), suggesting perturbations in the PPP. The total amount of pyruvate in the cortex (p = 0.039) was reduced by 20% in hSOD1G93A mice. Also, the activities of the glycolytic enzyme pyruvate kinase were reduced in the cortex by 31% (p = 0.002), indicating alterations in glycolysis. No significant differences were seen in the total amounts as well as % 13C enrichments in most TCA cycle intermediates, suggesting largely normal TCA cycle function. On the other hand, oxoglutarate dehydrogenase activity was decreased in the cortex, which may indicate increased oxidative stress. Overall, this study revealed decreased activity of the PPP in the spinal cord and alterations in glycolysis in hSOD1G93A mouse CNS tissues at the early symptomatic stage of disease.

Systems-level engineering and characterisation of Clostridium autoethanogenum through heterologous production of poly-3-hydroxybutyrate (PHB).

Gas fermentation is emerging as an economically attractive option for the sustainable production of fuels and chemicals from gaseous waste feedstocks. Clostridium autoethanogenum can use CO and/or CO2 + H2 as its sole carbon and energy sources. Fermentation of C. autoethanogenum is currently being deployed on a commercial scale for ethanol production. Expanding the product spectrum of acetogens will enhance the economics of gas fermentation. To achieve efficient heterologous product synthesis, limitations in redox and energy metabolism must be overcome. Here, we engineered and characterised at a systems-level, a recombinant poly-3-hydroxybutyrate (PHB)-producing strain of C. autoethanogenum. Cells were grown in CO-limited steady-state chemostats on two gas mixtures, one resembling syngas (20% H2) and the other steel mill off-gas (2% H2). Results were characterised using metabolomics and transcriptomics, and then integrated using a genome-scale metabolic model reconstruction. PHB-producing cells had an increased expression of the Rnf complex, suggesting energy limitations for heterologous production. Subsequent optimisation of the bioprocess led to a 12-fold increase in the cellular PHB content. The data suggest that the cellular redox state, rather than the acetyl-CoA pool, was limiting PHB production. Integration of the data into the genome-scale metabolic model showed that ATP availability limits PHB production. Altogether, the data presented here advances the fundamental understanding of heterologous product synthesis in gas-fermenting acetogens.

Systems-based approaches enable identification of gene targets which improve the flavour profile of low-ethanol wine yeast strains.

Metabolic engineering has been vital to the development of industrial microbes such as the yeast Saccharomyces cerevisiae. However, sequential rounds of modification are often needed to achieve particular industrial design targets. Systems biology approaches can aid in identifying genetic targets for modification through providing an integrated view of cellular physiology. Recently, research into the generation of commercial yeasts that can produce reduced-ethanol wines has resulted in metabolically-engineered strains of S. cerevisiae that are less efficient at producing ethanol from sugar. However, these modifications led to the concomitant production of off-flavour by-products. A combination of transcriptomics, proteomics and metabolomics was therefore used to investigate the physiological changes occurring in an engineered low-ethanol yeast strain during alcoholic fermentation. Integration of 'omics data identified several metabolic reactions, including those related to the pyruvate node and redox homeostasis, as being significantly affected by the low-ethanol engineering methodology, and highlighted acetaldehyde and 2,4,5-trimethyl-1,3-dioxolane as the main off-flavour compounds. Gene remediation strategies were then successfully applied to decrease the formation of these by-products, while maintaining the 'low-alcohol' phenotype. The data generated from this comprehensive systems-based study will inform wine yeast strain development programmes, which, in turn, could potentially play an important role in assisting winemakers in their endeavour to produce low-alcohol wines with desirable flavour profiles.

Metabolites Identified during Varied Doses of Aspergillus Species in Zea mays Grains, and Their Correlation with Aflatoxin Levels.

Aflatoxin contamination is associated with the development of aflatoxigenic fungi such as Aspergillus flavus and A. parasiticus on food grains. This study was aimed at investigating metabolites produced during fungal development on maize and their correlation with aflatoxin levels. Maize cobs were harvested at R3 (milk), R4 (dough), and R5 (dent) stages of maturity. Individual kernels were inoculated in petri dishes with four doses of fungal spores. Fungal colonisation, metabolite profile, and aflatoxin levels were examined. Grain colonisation decreased with kernel maturity: milk-, dough-, and dent-stage kernels by approximately 100%, 60%, and 30% respectively. Aflatoxin levels increased with dose at dough and dent stages. Polar metabolites including alanine, proline, serine, valine, inositol, iso-leucine, sucrose, fructose, trehalose, turanose, mannitol, glycerol, arabitol, inositol, myo-inositol, and some intermediates of the tricarboxylic acid cycle (TCA—also known as citric acid or Krebs cycle) were important for dose classification. Important non-polar metabolites included arachidic, palmitic, stearic, 3,4-xylylic, and margaric acids. Aflatoxin levels correlated with levels of several polar metabolites. The strongest positive and negative correlations were with arabitol (R = 0.48) and turanose and (R = −0.53), respectively. Several metabolites were interconnected with the TCA; interconnections of the metabolites with the TCA cycle varied depending upon the grain maturity.

Improved performance of Pseudomonas putida in a bioelectrochemical system through overexpression of periplasmic glucose dehydrogenase.

It was recently demonstrated that a bioelectrochemical system (BES) with a redox mediator allowed Pseudomonas putida to perform anoxic metabolism, converting sugar to sugar acids with high yield. However, the low productivity currently limits the application of this technology. To improve productivity, the strain was optimized through improved expression of glucose dehydrogenase (GCD) and gluconate dehydrogenase (GAD). In addition, quantitative real-time RT-PCR analysis revealed the intrinsic self-regulation of GCD and GAD. Utilizing this self-regulation system, the single overexpression strain (GCD) gave an outstanding performance in the electron transfer rate and 2-ketogluconic acid (2KGA) productivity. The peak anodic current density, specific glucose uptake rate and 2KGA producing rate were 0.12 mA/cm2 , 0.27 ± 0.02 mmol/gCDW /hr and 0.25 ± 0.02 mmol/gCDW /hr, which were 327%, 477%, and 644% of the values of wild-type P. putida KT2440, respectively. This work demonstrates that expression of periplasmic dehydrogenases involved in electron transfer can significantly improve productivity in the BES.

Quantitative analysis of tetrahydrofolate metabolites from clostridium autoethanogenum.

INTRODUCTION: Quantification of tetrahydrofolates (THFs), important metabolites in the Wood-Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen. OBJECTIVE: To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors. METHODS: Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC-MS/MS was used for quantification. RESULTS: Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP. CONCLUSION: Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

Dynamic Metabolomics Reveals that Insulin Primes the Adipocyte for Glucose Metabolism.

Insulin triggers an extensive signaling cascade to coordinate adipocyte glucose metabolism. It is considered that the major role of insulin is to provide anabolic substrates by activating GLUT4-dependent glucose uptake. However, insulin stimulates phosphorylation of many metabolic proteins. To examine the implications of this on glucose metabolism, we performed dynamic tracer metabolomics in cultured adipocytes treated with insulin. Temporal analysis of metabolite concentrations and tracer labeling revealed rapid and distinct changes in glucose metabolism, favoring specific glycolytic branch points and pyruvate anaplerosis. Integrating dynamic metabolomics and phosphoproteomics data revealed that insulin-dependent phosphorylation of anabolic enzymes occurred prior to substrate accumulation. Indeed, glycogen synthesis was activated independently of glucose supply. We refer to this phenomenon as metabolic priming, whereby insulin signaling creates a demand-driven system to "pull" glucose into specific anabolic pathways. This complements the supply-driven regulation of anabolism by substrate accumulation and highlights an additional role for insulin action in adipocyte glucose metabolism.