Citric Acid Cycle Intermediates Research Articles

A Lacticaseibacillus rhamnosus secretome induces immunoregulatory transcriptional, functional and immunometabolic signatures in human THP-1 monocytes

Macrophage responses to activation are fluid and dynamic in their ability to respond appropriately to challenges, a role integral to host defence. While bacteria can influence macrophage differentiation and polarization into pro-inflammatory and alternatively activated phenotypes through direct interactions, many questions surround indirect communication mechanisms mediated through secretomes derived from gut bacteria, such as lactobacilli. We examined effects of secretome-mediated conditioning on THP-1 human monocytes, focusing on the ability of the Lacticaseibacillus rhamnosus R0011 secretome (LrS) to drive macrophage differentiation and polarization and prime immune responses to subsequent challenge with lipopolysaccharide (LPS). Genome-wide transcriptional profiling revealed increased M2-associated gene transcription in response to LrS conditioning in THP-1 cells. Cytokine and chemokine profiling confirmed these results, indicating increased M2-associated chemokine and cytokine production (IL-1Ra, IL-10). These cells had increased cell-surface marker expression of CD11b, CD86, and CX3CR1, coupled with reduced expression of the M1 macrophage-associated marker CD64. Mitochondrial substrate utilization assays indicated diminished reliance on glycolytic substrates, coupled with increased utilization of citric acid cycle intermediates, characteristics of functional M2 activity. LPS challenge of LrS-conditioned THP-1s revealed heightened responsiveness, indicative of innate immune priming. Resting stage THP-1 macrophages co-conditioned with LrS and retinoic acid also displayed an immunoregulatory phenotype with expression of CD83, CD11c and CD103 and production of regulatory cytokines. Secretome-mediated conditioning of macrophages into an immunoregulatory phenotype is an uncharacterized and potentially important route through which lactic acid bacteria and the gut microbiota may train and shape innate immunity at the gut-mucosal interface.

Permafrost microbial communities and functional genes are structured by latitudinal and soil geochemical gradients

Permafrost underlies approximately one quarter of Northern Hemisphere terrestrial surfaces and contains 25–50% of the global soil carbon (C) pool. Permafrost soils and the C stocks within are vulnerable to ongoing and future projected climate warming. The biogeography of microbial communities inhabiting permafrost has not been examined beyond a small number of sites focused on local-scale variation. Permafrost is different from other soils. Perennially frozen conditions in permafrost dictate that microbial communities do not turn over quickly, thus possibly providing strong linkages to past environments. Thus, the factors structuring the composition and function of microbial communities may differ from patterns observed in other terrestrial environments. Here, we analyzed 133 permafrost metagenomes from North America, Europe, and Asia. Permafrost biodiversity and taxonomic distribution varied in relation to pH, latitude and soil depth. The distribution of genes differed by latitude, soil depth, age, and pH. Genes that were the most highly variable across all sites were associated with energy metabolism and C-assimilation. Specifically, methanogenesis, fermentation, nitrate reduction, and replenishment of citric acid cycle intermediates. This suggests that adaptations to energy acquisition and substrate availability are among some of the strongest selective pressures shaping permafrost microbial communities. The spatial variation in metabolic potential has primed communities for specific biogeochemical processes as soils thaw due to climate change, which could cause regional- to global- scale variation in C and nitrogen processing and greenhouse gas emissions.

Mycobacterium tuberculosis protein MoxR1 enhances virulence by inhibiting host cell death pathways and disrupting cellular bioenergetics

ABSTRACT Mycobacterium tuberculosis (M. tb) utilizes the multifunctionality of its protein factors to deceive the host. The unabated global incidence and prevalence of tuberculosis (TB) and the emergence of multidrug-resistant strains warrant the discovery of novel drug targets that can be exploited to manage TB. This study reports the role of M. tb AAA+ family protein MoxR1 in regulating host-pathogen interaction and immune system functions. We report that MoxR1 binds to TLR4 in macrophage cells and further reveal how this signal the release of proinflammatory cytokines. We show that MoxR1 activates the PI3K-AKT-MTOR signalling cascade by inhibiting the autophagy-regulating kinase ULK1 by potentiating its phosphorylation at serine 757, leading to its suppression. Using autophagy-activating and repressing agents such as rapamycin and bafilomycin A1 suggested that MoxR1 inhibits autophagy flux by inhibiting autophagy initiation. MoxR1 also inhibits apoptosis by suppressing the expression of MAPK JNK1/2 and cFOS, which play critical roles in apoptosis induction. Intriguingly, MoxR1 also induced robust disruption of cellular bioenergetics by metabolic reprogramming to rewire the citric acid cycle intermediates, as evidenced by the lower levels of citric acid and electron transport chain enzymes (ETC) to dampen host defence. These results point to a multifunctional role of M. tb MoxR1 in dampening host defences by inhibiting autophagy, apoptosis, and inducing metabolic reprogramming. These mechanistic insights can be utilized to devise strategies to combat TB and better understand survival tactics by intracellular pathogens.

Metabolic Characterization and Glyceraldehyde-3-Phosphate Dehydrogenase-Dependent Regulation of Epithelial Sodium Channels in hPheo1 Wild-type and SDHB Knockdown Cells.

Pheochromocytomas (PCC) and paragangliomas (PGL) are rare neuroendocrine tumors with limited curative treatment options outside of surgical resection. Patients with mutations in succinate dehydrogenase subunit B (SDHB) are at an increased risk of malignant and aggressive disease. As cation channels are associated with tumorigenesis, we studied the expression and activity of cation channels from the Degenerin superfamily in a progenitor cell line derived from a human PCC. hPheo1 wild-type (WT) and SDHB knockdown (KD) cells were studied to investigate whether epithelial sodium channels (ENaC) and acid-sensing ion channels (ASIC) are regulated by the activity of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). First, we performed targeted metabolomic studies and quantified changes in glycolysis pathway intermediates and citric acid cycle intermediates using hPheo1 WT cells and SDHB KD cells. Next, we performed protein biochemistry and electrophysiology studies to characterize the protein expression and activity, respectively, of these ion channels. Our western blot experiments show both ENaC alpha and ASIC1/2 are expressed in both hPheo1 WT and SDHB KD cells, with lower levels of a cleaved 60 kDa form of ENaC in SDHB KD cells. Single-channel patch clamp studies corroborate these results and further indicate channel activity is decreased in SDHB KD cells. Additional experiments showed a more significant decreased membrane potential in SDHB KD cells, which were sensitive to amiloride compared to WT cells. We provide evidence for the differential expression and activity of ENaC and ASIC hybrid channels in hPheo1 WT and SDHB KD cells, providing an important area of investigation in understanding SDHB-related disease.

Dynamic Metabolic and Transcriptional Responses of Proteasome-Inhibited Neurons.

Proteasome inhibition is associated with parkinsonian pathology in vivo and degeneration of dopaminergic neurons in vitro. We explored here the metabolome (386 metabolites) and transcriptome (3257 transcripts) regulations of human LUHMES neurons, following exposure to MG-132 [100 nM]. This proteasome inhibitor killed cells within 24 h but did not reduce viability for 12 h. Overall, 206 metabolites were changed in live neurons. The early (3 h) metabolome changes suggested a compromised energy metabolism. For instance, AMP, NADH and lactate were up-regulated, while glycolytic and citric acid cycle intermediates were down-regulated. At later time points, glutathione-related metabolites were up-regulated, most likely by an early oxidative stress response and activation of NRF2/ATF4 target genes. The transcriptome pattern confirmed proteostatic stress (fast up-regulation of proteasome subunits) and also suggested the progressive activation of additional stress response pathways. The early ones (e.g., HIF-1, NF-kB, HSF-1) can be considered a cytoprotective cellular counter-regulation, which maintained cell viability. For instance, a very strong up-regulation of AIFM2 (=FSP1) may have prevented fast ferroptotic death. For most of the initial period, a definite life-death decision was not taken, as neurons could be rescued for at least 10 h after the start of proteasome inhibition. Late responses involved p53 activation and catabolic processes such as a loss of pyrimidine synthesis intermediates. We interpret this as a phase of co-occurrence of protective and maladaptive cellular changes. Altogether, this combined metabolomics-transcriptomics analysis informs on responses triggered in neurons by proteasome dysfunction that may be targeted by novel therapeutic intervention in Parkinson's disease.

AMPK is required for recovery from metabolic stress induced by ultrasound microbubble treatment

Ultrasound-stimulated microbubble (USMB) treatment is a promising strategy for cancer therapy. USMB promotes drug delivery by sonoporation and enhanced endocytosis, and also impairs cell viability. However, USMB elicits heterogeneous effects on cell viability, with apparently minimal effects on a subset of cells. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. Herein, we used several triple negative breast cancer cells to identify the molecular mechanisms of adaptation to USMB-induced stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates, suggesting that USMB imposes metabolic stress on cells. USMB treatment acutely reduces ATP levels and stimulates the phosphorylation and activation of AMP-activated protein kinase (AMPK). AMPK is required to restore ATP levels and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the antineoplastic efficacy of USMB treatment.

TMET-38. LOSS OF FUNCTION OF CDK7 IS SYNTHETICALLY LETHAL WITH FATTY ACID OXIDATION INHIBITION IN GLIOBLASTOMA

Abstract CDK7 has been identified as a potential drug target for glioblastoma (GBM), a highly lethal primary brain tumor. However, resistance to therapy develops quickly, which may be facilitated by drug-induced reprogramming of metabolism. By combination of a transcriptome and metabolite screening analyses followed by carbon tracing (U-13C-Glucose, U-13C-Glutamine and U-13C-Palmitic acid) and extracellular flux analysis we demonstrated that both genetic and pharmacological (YKL-5-124 and THZ1) CDK7 inhibition elicited substantial metabolic reprogramming. Specifically, CDK7i elicited an increase of oxygen consumption rate fueled by enhanced fatty acid oxidation (FAO) manifested by enhanced labeling of citric acid cycle intermediates from palmitic acid. Consistently, the combination treatment of CDK7i inhibitors with blockers of FAO (etomoxir) or cellular respiration (gamitrinib) exerted substantial synergistic growth inhibition in patient derived xenograft as well as neurosphere GBM cultures, which was mainly driven by a collapse of oxidative energy metabolism. In turn, exogenous administration of adenosine triphosphate partially rescued from the cell death induced by the combination treatment. Moreover, the combination treatment activated intrinsic apoptosis through a reduction of both Mcl-1 and Bcl-xL as demonstrated by rescue experiments. Finally, the combined administration of YKL-5-124 and etomoxir extended overall in an orthotopic patient-derived xenograft model of GBM. In summary, these data support that simultaneous targeting of CDK7 and FAO might be a potential novel therapy against GBM.

Redundancy in Citrate and cis-Aconitate Transport in Pseudomonas aeruginosa.

Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the tricarboxylic acid cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Here, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate importer; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to an inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. IMPORTANCE Pseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. Here, we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.

Celecoxib impairs primary human myoblast proliferation and differentiation independent of cyclooxygenase 2 inhibition

The use of non-steroidal anti-inflammatory drugs (NSAIDs) for treatment of musculoskeletal injuries is commonplace in the general, athletic, and military populations. While NSAIDs have been studied in a variety of tissues, the effects of NSAIDs on skeletal muscle have not been fully defined. To address this, we investigated the degree to which the cyclooxygenase (COX)-2-selective NSAID celecoxib affects muscle cell proliferation, differentiation, anabolic signaling, and mitochondrial function in primary human skeletal myoblasts and myotubes. Primary muscle cells were treated with celecoxib or NS-398 (a pharmacological inhibitor of COX-2) as a control. Celecoxib administration significantly reduced myoblast proliferation, viability, fusion, and myotube area in a dose-dependent manner, whereas NS-398 had no effect on any of these outcomes. Celecoxib treatment was also associated with reduced phosphorylation of ribosomal protein S6 in myoblasts, and reduced phosphorylation of AKT, p70S6K, S6, and ERK in myotubes. In contrast, NS-398 did not alter phosphorylation of these molecules in myoblasts or myotubes. In myoblasts, celecoxib significantly reduced mitochondrial membrane potential and respiration, as evidenced by the decreased citric acid cycle (CAC) intermediates cis-aconitic acid, alpha-keto-glutarate acid, succinate acid, and malic acid. Similar results were observed in myotubes, although celecoxib also reduced pyruvic acid, citric acid, and fumaric acid. NS-398 did not affect CAC intermediates in myoblasts or myotubes. Together, these data reveal that celecoxib inhibits proliferation, differentiation, intracellular signaling, and mitochondrial function in primary human myoblasts and myotubes independent of its function as a COX-2 inhibitor.

Changes in metabolic profiling of whiteleg shrimp (Penaeus vannamei) under hypoxic stress

Hypoxia is a common concern in shrimp aquaculture, affecting growth and survival. Although recent studies have revealed important insights into hypoxia in shrimp and crustaceans, knowledge gaps remain regarding this stressor at the molecular level. In the present study, a gas chromatography–mass spectrometry (GC–MS)-based metabolomics approach was employed to characterize the metabolic signatures and pathways underlying responses of Pacific white shrimp (Penaeus vannamei) to hypoxia and to identify associated candidate biomarkers. We compared metabolite profiles of shrimp haemolymph before (0 h) and after exposure to hypoxia (1 & 2 h). Dissolved oxygen levels were maintained above 85 % saturation in the control and before hypoxia, and 15 % saturation in the hypoxic stress treatment. Results showed 44 metabolites in shrimp haemolymph that were significantly different between before and after hypoxia exposure. These metabolites were energy-related metabolites (e.g., intermediates of citric acid cycle, lactic acid, alanine), fatty acids and amino acids. Pathway analysis revealed 17 pathways that were significantly affected by hypoxia. The changes in metabolites and pathways indicate a shift from aerobic to anaerobic metabolism, disturbance in amino acid metabolism, osmoregulation, oxidative damage and Warburg effect-like response caused by hypoxic stress. Among the altered metabolites, lactic acid was most different between before and after hypoxia exposure and had the highest accurate value for biomarker identification. Future investigations may validate this molecule as a stress biomarker in aquaculture. This study contributes to a better understanding of hypoxia in shrimp and crustaceans at the metabolic level and provides a base for future metabolomics investigations on hypoxia.

Metabolic rescue ameliorates mitochondrial encephalo-cardiomyopathy in murine and human iPSC models of Leigh syndrome.

BackgroundMice with deletion of complex I subunit Ndufs4 develop mitochondrial encephalomyopathy resembling Leigh syndrome (LS). The metabolic derangement and underlying mechanisms of cardio‐encephalomyopathy in LS remains incompletely understood.MethodsWe performed echocardiography, electrophysiology, confocal microscopy, metabolic and molecular/morphometric analysis of the mice lacking Ndufs4. HEK293 cells, human iPS cells‐derived cardiomyocytes and neurons were used to determine the mechanistic role of mitochondrial complex I deficiency.ResultsLS mice develop severe cardiac bradyarrhythmia and diastolic dysfunction. Human‐induced pluripotent stem cell‐derived cardiomyocytes (iPS‐CMs) with Ndufs4 deletion recapitulate LS cardiomyopathy. Mechanistically, we demonstrate a direct link between complex I deficiency, decreased intracellular (nicotinamide adenine dinucleotide) NAD+/NADH and bradyarrhythmia, mediated by hyperacetylation of the cardiac sodium channel NaV1.5, particularly at K1479 site. Neuronal apoptosis in the cerebellar and midbrain regions in LS mice was associated with hyperacetylation of p53 and activation of microglia. Targeted metabolomics revealed increases in several amino acids and citric acid cycle intermediates, likely due to impairment of NAD+‐dependent dehydrogenases, and a substantial decrease in reduced Glutathione (GSH). Metabolic rescue by nicotinamide riboside (NR) supplementation increased intracellular NAD+/ NADH, restored metabolic derangement, reversed protein hyperacetylation through NAD+‐dependent Sirtuin deacetylase, and ameliorated cardiomyopathic phenotypes, concomitant with improvement of NaV1.5 current and SERCA2a function measured by Ca2+‐transients. NR also attenuated neuronal apoptosis and microglial activation in the LS brain and human iPS‐derived neurons with Ndufs4 deletion.ConclusionsOur study reveals direct mechanistic explanations of the observed cardiac bradyarrhythmia, diastolic dysfunction and neuronal apoptosis in mouse and human induced pluripotent stem cells (iPSC) models of LS.

Sustained Control of Pyruvate Carboxylase by the Essential Second Messenger Cyclic di-AMP in Bacillus subtilis.

ABSTRACTIn Bacillus subtilis and other Gram-positive bacteria, cyclic di-AMP is an essential second messenger that signals potassium availability by binding to a variety of proteins. In some bacteria, c-di-AMP also binds to the pyruvate carboxylase to inhibit its activity. We have discovered that in B. subtilis the c-di-AMP target protein DarB, rather than c-di-AMP itself, specifically binds to pyruvate carboxylase both in vivo and in vitro. This interaction stimulates the activity of the enzyme, as demonstrated by in vitro enzyme assays and in vivo metabolite determinations. Both the interaction and the activation of enzyme activity require apo-DarB and are inhibited by c-di-AMP. Under conditions of potassium starvation and corresponding low c-di-AMP levels, the demand for citric acid cycle intermediates is increased. Apo-DarB helps to replenish the cycle by activating both pyruvate carboxylase gene expression and enzymatic activity via triggering the stringent response as a result of its interaction with the (p)ppGpp synthetase Rel and by direct interaction with the enzyme, respectively.

Анаплеротичні принципи нутритивної терапії в клініці критичних станів

У статті надані сучасні анаплеротичні концепції нутритивної терапії для модуляції метаболічної відповіді в клініці критичних станів. Метаболічний стрес — універсальна гіперметаболічна гіперкатаболічна відповідь на захворювання, травму з активацією гіпоталамо-гіпофізарно-адреналової системи; виділенням гормонів стресу; катехоламінів; нейропептиду S; фактора, індукованого гіпоксією HIF-1α; експресією генів, що контролюють механізми адаптації до гіпоксії, у тому числі гліколіз і цикл трикарбонових кислот. У статті наведена розроблена нами модель визначення метаболічного стресу і мітохондріальної дисфункції. Анаплеротична терапія базується на концепції використання альтернативних субстратів як для циклу трикарбонових кислот, так і для транспорту електронів у дихальному ланцюзі з метою посилення продукції аденозинтрифосфату. Багато патологічних станів вимагають проведення анаплеротичної терапії або шляхом поповнення пулу анаплеротичних субстратів (піруват, аспартат, аспарагінат та інші джерела, поповнюючи оксалоацетат, глутамін та інші кислоти), або видаленням з тканин продуктів катаплеротичних реакцій — інтермедіатів циклу трикарбонових кислот. Катаплероз врівноважує анаплероз шляхом переміщення надлишку інтермедіаторів циклу трикарбонових кислот з мітохондріального матриксу в цитоплазму, в екстрамітохондріальний простір. Сучасні анаплеротичні концепції для модуляції метаболічної відповіді при критичних станах включають в себе такі позиції: I. Раннє використання розчинів глюкози та інтенсивна інсулінотерапія: мінімальна кількість вуглеводів близько 2 г/кг глюкози/добу; введення інсуліну, якщо два послідовних аналізи показали рівень глюкози > 10 ммоль/л; уникати обов’язкового повноцінного калорійного харчування в перший тиждень, починати з 500 ккал/добу. II. Попередження гіперглікемічного метаболічного стресу — метаболічне прекондиціювання: вживання прозорих рідин припиняється за 2 години до індукції в анестезію, споживання щільної їжі — за 6 годин. Передопераційне призначення вуглеводів застосовується у всіх пацієнтів без цукрового діабету за 2 години до індукції; при наявності протипоказань — в/в інфузія глюкози за 2 години до операції. III. Використання енергосубстратів, що не потребують інсулінової стимуляції для їх входження до клітини: виявлено, що особи, які одержували < 60 г фруктози на добу, мали кращі показники здоров’я, ніж ті, які споживали > 100 г/добу. IV. Амінокислотні суміші: якщо парентеральне харчування показано, доцільно призначати збалансовані амінокислотні суміші, з розрахунку 1,3–1,5 г/кг/добу білка; 0,2–0,4 г/кг/добу L-глутаміну. При тяжких опіках режим введення білка в амінокислотних сумішах зростає до 1,5–2 г/кг/добу. V. Поповнення пулу анаплеротичних жирних кислот: жирові емульсії (LCT, MCT) повинні призначатися в кількості 0,7–1,5 г/кг/добу; ентерально призначається суміш соєвої і оливкової олій, риб’ячий жир, що містять омега-3, -6, -9 жирні кислоти; дієтична анаплеротична терапія з тригептаноїном підвищує виживаність пацієнтів у критичних станах. VI. Використання інтермедіатів циклу Кребса: превентивне або терапевтичне використання сукцинатвмісних препаратів може бути ефективним для активації ургентних адаптивних механізмів. Тіамін є найважливішим вітаміном для підтримки аеробного метаболізму й активності ключових ферментів циклу Кребса, а також чолночного механізму пентозофосфатного циклу.

The Effects of Live Transport on Metabolism and Stress Responses of Abalone (Haliotis iris).

The New Zealand abalone industry relies mostly on the export of processed products to distant Asian markets, notably China. Over the past five years, live export of high quality abalone from New Zealand has proven successful. However, transport of live animals is associated with multiple stressors that affect survival and meat quality at the end of the transport phase. Better understanding of transport-derived stress is needed to improve transport conditions and recovery at destination to ensure high product quality and safety throughout the supply chain. To this end, we applied an untargeted GC–MS-based metabolomics approach to examine the changes in metabolite profiles of abalone after a 2-day transport event and subsequent water re-immersion for 2 days. The results revealed alterations of many metabolites in the haemolymph and muscle of post-transported abalone. Decreased concentrations of many amino acids suggest high energy demands for metabolism and stress responses of transported abalone, while increases of other amino acids may indicate active osmoregulation and/or protein degradation due to oxidative stress and apoptosis. The accumulation of citric acid cycle intermediates and anaerobic end-products are suggestive of hypoxia stress and a shift from aerobic to anaerobic metabolism (resulting from aerial exposure). Interestingly, some features in the metabolite profile of reimmersed abalone resembled those of pre-transported individuals, suggesting progressive recovery after reimmersion in water. Evidence of recovery was observed in the reduction of some stress biomarkers (e.g., lactic acid, succinic acid) following reimmersion. This study revealed insights into the metabolic responses to transport stress in abalone and highlights the importance of reimmersion practices in the supply chain of live animal exports.

Metabolite profiling of the post-ovulatory oocytes of the common carp, Cyprinus carpio: A 1H NMR-based metabolomics approach

A metabolomics study was conducted to investigate the molecular bases of oocyte over-ripening in common carp, Cyprinus carpio from a metabolic point of view. The ovulation was induced in fish brooders by intramuscular injection of pituitary extract and oocytes were collected four times post-ovulation with 30 min intervals. A set of 32 metabolites were identified on the NMR spectra of the oocytes, which mainly included energy-linked metabolites, amino acids, methylated metabolites and citric acid cycle (TCA) intermediates. PCA and PLS-DA models clearly separated the post ovulations times, indicating the effects of post-ovulation time on oocyte metabolome content. Based on the loading plot outputs, 15 metabolites including tryptophan, cysteine, AMP, tyrosine, valine, creatine phosphate (PCr), ATP, leucine, inosine, malate, acetate, TMAO, glucose, fumarate and lysine had more effects on the separation of post ovulation times. According to the results of metabolite profiling, the concentrations of glutamine, alanine, tryptophan, lysine and cysteine mostly significantly (P < 0.01) increased at 90 and 120 min post-ovulation. The concentrations of PCr, ATP, inosine and guanosine were relatively stable until 60 min post-ovulation, while significantly (P < 0.01) decreased at 90 and 120 min post ovulation. The TCA metabolites succinate, malate and fumarate significantly (P < 0.01) elevated at 90 and 120 min post-ovulation. AMP concentrations remained relatively unchanged until 30 min and then progressively decreased with time post ovulation (P < 0.01). The concentrations of lactate showed significant elevations at 90 and 120 min post ovulation (P < 0.01). In conclusion, the energetic potentials of the oocytes reduced with time post ovulation. There were apparent elevations in the concentrations of free amino acids, which may be associated with the onset of proteolytic activities in the post ovulatory oocytes. In addition, we found some changes in the apoptotic-related metabolites, which may support the results of previous studies regarding the oxidative stress and following apoptosis in post ovulatory oocytes of fish.

Serum and urinary metabolites discriminate disease activity in ANCA associated glomerulonephritis in a pilot study.

Renal biopsy is currently the gold standard for diagnosing active renal vasculitis. In this pilot study, metabolomics analysis was used to investigate the differences in metabolic profiles between paired patients' serum and urine samples collected during both the active and the remission phase of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitides (AAV). Ten patients with AAV renal disease were included. Mean age was 61years, with 6 patients each being male and Caucasian. Mean Birmingham Vasculitis Activity Score (BVAS) and mean glomerular filtration rate (GFR) were 17 and 28, respectively. We found that while the citric acid cycle intermediates citrate, iso-citrate and oxaloacetate had lower intensities in the active phase samples as compared to the remission phase samples. The intensities of other metabolites of carbohydrate metabolism, amino acid metabolism, and nucleotide synthesis were significantly higher in the active phase samples, indicating the upregulation of these pathways for the production of energy and other biomolecules such as proteins and nucleic acids during the active phase of AAV. This pilot studysuggests that serum and urinary metabolomic profiling may be usefulto monitor disease activity in renal AAV.

Simultaneous Quantification of the Concentration and Carbon Isotopologue Distribution of Polar Metabolites in a Single Analysis by Gas Chromatography and Mass Spectrometry.

13C-isotope tracing is a frequently employed approach to study metabolic pathway activity. When combined with the subsequent quantification of absolute metabolite concentrations, this enables detailed characterization of the metabolome in biological specimens and facilitates computational time-resolved flux quantification. Classically, a 13C-isotopically labeled sample is required to quantify 13C-isotope enrichments and a second unlabeled sample for the quantification of metabolite concentrations. The rationale for a second unlabeled sample is that the current methods for metabolite quantification rely mostly on isotope dilution mass spectrometry (IDMS) and thus isotopically labeled internal standards are added to the unlabeled sample. This excludes the absolute quantification of metabolite concentrations in 13C-isotopically labeled samples. To address this issue, we have developed and validated a new strategy using an unlabeled internal standard to simultaneously quantify metabolite concentrations and 13C-isotope enrichments in a single 13C-labeled sample based on gas chromatography–mass spectrometry (GC/MS). The method was optimized for amino acids and citric acid cycle intermediates and was shown to have high analytical precision and accuracy. Metabolite concentrations could be quantified in small tissue samples (≥20 mg). Also, we applied the method on 13C-isotopically labeled mammalian cells treated with and without a metabolic inhibitor. We proved that we can quantify absolute metabolite concentrations and 13C-isotope enrichments in a single 13C-isotopically labeled sample.

Impact of Lactation Stage on the Metabolite Composition of Milk

Abstract Objectives Milk and dairy products are a major component of dietary intake, with milk consumption estimated at 6 billion people globally. Adults are reported to consume two or more portions of dairy daily. Cow's milk is composed of a variety of micro and macro nutrients, which is impacted by a range of factors, including lactation stage, and feeding system (indoor vs. pasture). Application of Metabolomics provides in depth analysis of the metabolite composition of milk. The objective was to examine the impact of lactation on bovine milk metabolite levels. Methods Metabolomic analysis of bovine milk powder samples across lactation (N = 18), was performed using 1H-NMR. Metabolites were quantified using Chenomx software (7.7). One-way repeated measures of analysis variance (ANOVA) with Tukeys post hoc test, was preformed on NMR data using SPSS (version 24.0), to assess the impact of lactation stages. P-values were corrected for multiple comparisons using Benjamini and Hochberg false discovery rate (FDR) procedure. Multivariate statistical analysis of the 1H-NMR data was carried out using SIMCA 13 (SIMCA Version 13.0.3.0 Umetrics, AB). Results 42 metabolites were identified and quantified in the 1H-NMR spectra. Statistical analysis revealed that 17 metabolites were significantly different across lactation stages (FDR &amp;lt; 0.05). Of these 17 metabolites, the majority had higher concentrations in early lactation. Examples of metabolites that changed with lactation stage include the tricarboxylic acid cycle intermediates citrate and 2-oxoglutarate, amino acids, choline and phosphocholine. Interestingly choline and phosphocholine levels changed in opposite directions. Conclusions Our results demonstrate significant differences in metabolites across the stages of lactation, with early lactation having a distinct metabolic profile. Future work will explore these compositional differences further, encompassing animal feeding practice. Funding Sources Food for Health Ireland and Enterprise Ireland.

Rhizosphere symbionts improve water stress tolerance in Moldavian balm through modulation of osmolytes

The metabolomics analysis was used in this study to examine the changes in metabolite profile of Moldavian balm under water-deficit stress, symbiosis with arbuscular mycorrhizal fungus, plant growth-promoting rhizobacteria, and their interactions. This project was realized by applying a completely randomized model considering three factors, the arbuscular mycorrhizal fungus Claroideoglomus etunicatum, the rhizobacteria Micrococcus yunnanensis, and irrigation regimes (100, 70, and 40% field capacity). The results showed that the sugar metabolisms are the most affected metabolic pathways under drought stress. Moreover, the increase of some amino acids, citric acid cycle intermediates, and phenolic acid metabolites could contribute to the tolerance mechanisms of non-inoculated plants against water-deficit stress. These results suggested that soil microorganisms could be used to mitigate the negative impacts of drought stress by inducing the accumulation of some of the intermediary metabolites that act as osmolytes or their precursors. Overall, these results indicated that symbiotic association with soil microorganisms leads to metabolic and physiological adaptation which can increase the performance of this important medicinal plant under drought conditions.

A metabolomics approach to investigate the proceedings of mitochondrial dysfunction in rats from prediabetes to diabetes.

BackgroundDiabetes mellitus (DM) is a leading cause of preventable cardiovascular disease, but the metabolic changes from prediabetes to diabetes have not been fully clarified. This study implemented a metabolomics profiling platform to investigate the variations of metabolites and to elucidate their global profiling from metabolic syndrome to DM. Methods: Male Sprague-Dawley rats (n = 44) were divided into four groups. Three groups were separately fed with a normal diet, a high-fructose diet (HF), or a high-fat (HL) diet while one group was treated with streptozotocin. The HF and HL diet were meant to induce insulin resistance, obesity, and dyslipidemia, which known to induce DM. Results: The most significant metabolic variations in the DM group’s urine samples were the reduced release of citric acid cycle intermediates, the increase in acylcarnitines, and the decrease in urea excretion, all of which indicated energy metabolism abnormalities and mitochondrial dysfunction. Overall, the metabolic analysis revealed tryptophan metabolic pathway variations in the prediabetic phase, even though the mitochondrial function remains unaffected. Conclusion: This study show that widespread methylations and impaired tryptophan metabolism occur in metabolic syndrome and are then followed by a decline in citric acid cycle intermediates, indicating mitochondrial dysfunction in diabetes.