Alterations In Metabolite Levels Research Articles

Molecular Insights of Drug Resistance in Epilepsy: Multi-omics Unveil.

Epilepsy is a devastating neurological disorder mainly associated with impaired synchronic discharge that leads to sensory, motor, and psychomotor impairments. Till now, about 30 anti-seizure medications (ASMs) have been approved for the management of epilepsy, yet one-third of individuals still have uncontrollable epilepsy and develop resistance. Drug resistance epilepsy (DRE) is defined as the condition where two ASMs fail to control the seizure in epileptic patients. The leading cause of the resistance was the extended use of ASMs. According to various studies, alterations in some genes and their expressions, along with specific metabolic impairments, are suggested to be associated with ASMs resistance and DRE pathophysiology. Several factors aid in the pathophysiology of DRE, such as alterations in protein-encoding genes such as neurotransmitter receptors, drug transporters, ion channels, and drug targets. Furthermore, the altered metabolite levels of metabolites implicated in neurotransmitter signaling, energetic pathways, oxidative stress, and neuroinflammatory signaling differentiate the epileptic patient from the DRE patient. Various DRE biomarkers can be identified using the "integrated omics approach," which includes the study of genomics, transcriptomics, and metabolomics. The current review has been compiled to understand the pathophysiological mechanisms of DRE by focusing on genomics, transcriptomics, and metabolomics. An effort has also been made to identify the therapeutic targets based on identifying significant markers by a multi-omics approach. This has the potential to develop novel therapeutic interventions in the future.

Aberrant auditory metabolite levels and topological properties are associated with cognitive decline in presbycusis patients.

Presbycusis has been reported as related to cognitive decline, but its underlying neurophysiological mechanism is still unclear. This study aimed to investigate the relationship between metabolite levels, cognitive function, and node characteristics in presbycusis based on graph theory methods. Eighty-four elderly individuals with presbycusis and 63 age-matched normal hearing controls underwent magnetic resonance spectroscopy, functional magnetic resonance imaging scans, audiological assessment, and cognitive assessment. Compared with the normal hearing group, presbycusis patients exhibited reduced gamma-aminobutyric acid and glutamate levels in the auditory region, increased nodal characteristics in the temporal lobe and precuneus, as well as decreased nodal characteristics in the superior occipital gyrus and medial orbital. The right gamma-aminobutyric acid levels were negatively correlated with the degree centrality in the right precuneus and the executive function. Degree centrality in the right precuneus exhibited significant correlations with information processing speed and executive function, while degree centrality in the left medial orbital demonstrated a negative association with speech recognition ability. The degree centrality and node efficiency in the superior occipital gyrus exhibited a negative association with hearing loss and speech recognition ability, respectively. These observed changes indicate alterations in metabolite levels and reorganization patterns at the brain network level after auditory deprivation.

NMR-based metabolomic analysis of plasma from elderly patients with CVD before and after using contrast media

Contrast-induced acute kidney injury (CI-AKI) is a growingly common kidney problem caused by medical procedures involving contrast media (CM), especially in older patients with existing health issues. It is crucial to pinpoint potential biomarkers for the early detection of CI-AKI. Previously, we observed that iodixanol affects glucose, choline, and glutathione metabolism in endothelial cells under laboratory conditions. In this study, we used 1H NMR-based metabolomics to examine the metabolic changes in the blood plasma of elderly patients with cardiovascular disease (CVD) before and after receiving iodixanol. We identified altered metabolites in plasma 24 and 48 h after iodixanol injection compared to levels before injection. Notably, metabolites such as glucose, unsaturated fatty acids (UFA), low-density lipoprotein (LDL)/very low-density lipoprotein (VLDL), pyruvate, choline, and glycine showed potential as biomarkers at 24 h post-injection compared to levels before injection. Similarly, glucose, pyruvate, lactate, choline, and glycine in plasma could serve as potential biomarkers at 48 h post-injection. Iodixanol notably affected pathways related to glycolysis, fatty acid breakdown, and amino acid metabolism according to our metabolic pathway analysis. The altered levels of specific metabolites in plasma could be indicative of CM-induced kidney injury. Overall, this research aids in understanding the physiological mechanisms involved and in identifying early biomarkers and prevention strategies for CI-AKI.

Dopamine‑iron homeostasis interaction rescues mitochondrial fitness in Parkinson's disease

Imbalances of iron and dopamine metabolism along with mitochondrial dysfunction have been linked to the pathogenesis of Parkinson's disease (PD). We have previously suggested a direct link between iron homeostasis and dopamine metabolism, as dopamine can increase cellular uptake of iron into macrophages thereby promoting oxidative stress responses. In this study, we investigated the interplay between iron, dopamine, and mitochondrial activity in neuroblastoma SH-SY5Y cells and human induced pluripotent stem cell (hiPSC)-derived dopaminergic neurons differentiated from a healthy control and a PD patient with a mutation in the α-synuclein (SNCA) gene. In SH-SY5Y cells, dopamine treatment resulted in increased expression of the transmembrane iron transporters transferrin receptor 1 (TFR1), ferroportin (FPN), and mitoferrin2 (MFRN2) and intracellular iron accumulation, suggesting that dopamine may promote iron uptake. Furthermore, dopamine supplementation led to reduced mitochondrial fitness including decreased mitochondrial respiration, increased cytochrome c control efficiency, reduced mtDNA copy number and citrate synthase activity, increased oxidative stress and impaired aconitase activity. In dopaminergic neurons derived from a healthy control individual, dopamine showed comparable effects as observed in SH-SY5Y cells. The hiPSC-derived PD neurons harboring an endogenous SNCA mutation demonstrated altered mitochondrial iron homeostasis, reduced mitochondrial capacity along with increased oxidative stress and alterations of tricarboxylic acid cycle linked metabolic pathways compared with control neurons. Importantly, dopamine treatment of PD neurons promoted a rescue effect by increasing mitochondrial respiration, activating antioxidant stress response, and normalizing altered metabolite levels linked to mitochondrial function. These observations provide evidence that dopamine affects iron homeostasis, intracellular stress responses and mitochondrial function in healthy cells, while dopamine supplementation can restore the disturbed regulatory network in PD cells.

Brain metabolism response to intrahospital transfers in neurocritical ill patients and the impact of microdialysis probe location

Intrahospital transfer (IHT), a routine in the management of neurocritical patients requiring imaging or interventions, might affect brain metabolism. Studies about IHT effects using microdialysis (MD) have produced conflicting results. In these studies, only the most damaged hemisphere was monitored, and those may not reflect the impact of IHT on overall brain metabolism, nor do they address differences between the hemispheres. Herein we aimed to quantify the effect of IHT on brain metabolism by monitoring both hemispheres with bilateral MD. In this study, 27 patients with severe brain injury (10 traumatic brain injury and 17 subarachnoid hemorrhage patients) were included, with a total of 67 IHT. Glucose, glycerol, pyruvate and lactate were measured by MD in both hemispheres for 10 h pre- and post-IHT. Alterations in metabolite levels after IHT were observed on both hemispheres; although these changes were more marked in hemisphere A (most damaged) than B (less damaged). Our results suggest that brain metabolism is altered after an IHT of neurocritical ill patients particularly but not limited to the damaged hemisphere. Bilateral monitorization may be more sensitive than unilateral monitorization for detecting metabolic disturbances not directly related to the course of the disease.

Metabolomics analysis reveals novel serum metabolite alterations in cancer cachexia.

Cachexia is a body wasting syndrome that significantly affects well-being and prognosis of cancer patients, without effective treatment. Serum metabolites take part in pathophysiological processes of cancer cachexia, but apart from altered levels of select serum metabolites, little is known on the global changes of the overall serum metabolome, which represents a functional readout of the whole-body metabolic state. Here, we aimed to comprehensively characterize serum metabolite alterations and analyze associated pathways in cachectic cancer patients to gain new insights that could help instruct strategies for novel interventions of greater clinical benefit. Serum was sampled from 120 metastatic cancer patients (stage UICC IV). Patients were grouped as cachectic or non-cachectic according to the criteria for cancer cachexia agreed upon international consensus (main criterium: weight loss adjusted to body mass index). Samples were pooled by cachexia phenotype and assayed using non-targeted gas chromatography-mass spectrometry (GC-MS). Normalized metabolite levels were compared using t-test (p < 0.05, adjusted for false discovery rate) and partial least squares discriminant analysis (PLS-DA). Machine-learning models were applied to identify metabolite signatures for separating cachexia states. Significant metabolites underwent MetaboAnalyst 5.0 pathway analysis. Comparative analyses included 78 cachectic and 42 non-cachectic patients. Cachectic patients exhibited 19 annotable, significantly elevated (including glucose and fructose) or decreased (mostly amino acids) metabolites associating with aminoacyl-tRNA, glutathione and amino acid metabolism pathways. PLS-DA showed distinct clusters (accuracy: 85.6%), and machine-learning models identified metabolic signatures for separating cachectic states (accuracy: 83.2%; area under ROC: 88.0%). We newly identified altered blood levels of erythronic acid and glucuronic acid in human cancer cachexia, potentially linked to pentose-phosphate and detoxification pathways. We found both known and yet unknown serum metabolite and metabolic pathway alterations in cachectic cancer patients that collectively support a whole-body metabolic state with impaired detoxification capability, altered glucose and fructose metabolism, and substrate supply for increased and/or distinct metabolic needs of cachexia-associated tumors. These findings together imply vulnerabilities, dependencies and targets for novel interventions that have potential to make a significant impact on future research in an important field of cancer patient care.

The Deletion of LeuRS Revealed Its Important Roles in Osmotic Stress Tolerance, Amino Acid and Sugar Metabolism, and the Reproduction Process of Aspergillus montevidensis.

Aspergillus montevidensis is an important domesticated fungus that has been applied to produce many traditional fermented foods under high osmotic conditions. However, the detailed mechanisms of tolerance to osmotic stress remain largely unknown. Here, we construct a target-deleted strain (ΔLeuRS) of A. montevidensis and found that the ΔLeuRS mutants grew slowly and suppressed the development of the cleistothecium compared to the wide-type strains (WT) under salt-stressed and non-stressed conditions. Furthermore, differentially expressed genes (p < 0.001) governed by LeuRS were involved in salt tolerance, ABC transporter, amino acid metabolism, sugar metabolism, and the reproduction process. The ΔLeuRS strains compared to WT strains under short- and long-term salinity stress especially altered accumulation levels of metabolites, such as amino acids and derivatives, carbohydrates, organic acids, and fatty acids. This study provides new insights into the underlying mechanisms of salinity tolerance and lays a foundation for flavor improvement of foods fermented with A. montevidensis.

A metabolomics study in aqueous humor discloses altered arginine metabolism in Parkinson’s disease

BackgroundThe lack of accessible and informative biomarkers results in a delayed diagnosis of Parkinson’s disease (PD), whose symptoms appear when a significant number of dopaminergic neurons have already disappeared. The retina, a historically overlooked part of the central nervous system (CNS), has gained recent attention. It has been discovered that the composition of cerebrospinal fluid influences the aqueous humor composition through microfluidic circulation. In addition, alterations found in the brain of patients with PD have a correlate in the retina. This new paradigm highlights the potential of the aqueous humor as a sample for identifying differentially concentrated metabolites that could, eventually, become biomarkers if also found altered in blood or CSF of patients. In this research we aim at analyzing the composition of the aqueous humor from healthy controls and PD patients.MethodsA targeted metabolomics approach with concentration determination by mass spectrometry was used. Statistical methods including principal component analysis and linear discriminants were used to select differentially concentrated metabolites that allow distinguishing patients from controls.ResultsIn this first metabolomics study in the aqueous humor of PD patients, elevated levels of 16 compounds were found; molecules differentially concentrated grouped into biogenic amines, amino acids, and acylcarnitines. A biogenic amine, putrescine, alone could be a metabolite capable of differentiating between PD and control samples. The altered levels of the metabolites were correlated, suggesting that the elevations stem from a common mechanism involving arginine metabolism.ConclusionsA combination of three metabolites, putrescine, tyrosine, and carnitine was able to correctly classify healthy participants from PD patients. Altered metabolite levels suggest altered arginine metabolism. The pattern of metabolomic disturbances was not due to the levodopa-based dopamine replacement medication because one of the patients was not yet taking levodopa but a dopamine receptor agonist.

Abstract A011: NRBF2 induces radioresistance by increasing autophagy mediated metabolite replenishment in glioblastoma

Abstract Glioblastoma (GBM) is the most lethal brain tumor. Despite the standard of care using radiotherapy and temozolomide, the 5-year recurrence rate of GBM after the treatments is nearly 90% and, notably, about 80% of GBM recurrences occur within radiation treatment fields. Cancer cells possess dynamical metabolic reprogramming to evade cancer therapy and promote their survival. For enhancing metabolic reprogramming and metabolic plasticity, cancer cells need diverse metabolic fuel sources. In this study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon ionizing radiation (IR) treatment on GBM cells. Nuclear receptor binding factor 2 (NRBF2), which is a positive regulator of the autophagy initiation step, was found to be upregulated in a GBM cells and GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. To inhibit NRBF2 mediated GBM radioresistance, we found that druggable inhibition with lidoflazine, thereby suppressing IR-induced migration, invasion, proliferation, autophagic flux and improving the survival of orthotopic GBM-bearing mice. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy. [This research was supported by National Research Foundation of Korea (NRF) funded by the Ministry of Science and ICT (2020M2D9A2094156) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (RS-2023-00207904).] Citation Format: Eunguk Shin, Hyunkoo Kang, Haksoo Lee, BuHyun Youn. NRBF2 induces radioresistance by increasing autophagy mediated metabolite replenishment in glioblastoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A011.

Distinct Temporal Dynamic Serum Metabolite Profiles Characterize Treatment Response and Early Toxicity after B-Cell Lymphoma Directed CAR-T-Cell Therapy

Introduction: CD19 chimeric antigen receptor T-cell therapy (CD19.CAR-T) has profoundly improved treatment options for patients with relapsed/refractory B-cell lymphomas (r/r BCL), but is also associated with distinct toxicities. To elucidate host-intrinsic immunometabolic factors contributing to clinical benefit or immunotoxicity, we recently demonstrated that an increase of metabolically high-risk adipose tissue mass is associated with an elevated cytokine release syndrome (CRS) risk but also with improved survival (Haematologica 2022, CIR 2023). To gain further insights into the underlying immunometabolic mechanisms, we analyzed temporal dynamic changes of serum metabolites in r/r BCL patients during the first 14 days of CD19.CAR-T treatment. Methods: In this single-center analysis, we studied patients receiving CD19.CAR-T for r/r large BCL or mantle cell lymphoma (MCL) between 01/2019 and 12/2021 in a real-world setting. All patients were consecutively treated in our department (LMU University Hospital) and serum was prospectively collected in the morning hours from fasted patients beginning at the day of CAR-T infusion (D0), and 3-5 and 14 days later. Using liquid chromatography mass-spectrometry, we quantitatively analyzed a total of 305 water-soluble metabolites and 734 lipids. Metabolomic data was integrated with clinical patient records including CRS, 1-year progression-free survial (PFS) and amount of visceral adipose tissue (VAT). Results: In total, we measured 170 serum samples from 55 r/r LBCL and 4 r/r MCL patients, of which 52 had serial samples at all three time points (D0: 55, D3-5: 59, D14: 56). The following CAR-T products were administered: axi-cel (n = 26), tisa-cel (n = 29) and brexu-cel (n = 4). 25 (42.4%) patients developed a CRS grade ≥ 2, while 34 patients had a CRS grade 0-1. The best 90-day overall response rate was 52.5%, with a complete remission rate of 42.4%. At 1 year, 67.8% of patients progressed. From the 1039 metabolites measured, 525 metabolites passed quality control. Metabolite values were normalized and auto-scaled for further analysis. A partial least square differential analysis based on the collected time points led to a clear separation of samples with uric acid, desoxy-ribose-5-phosphate (dRibose-P), essential amino acids and (phospho)lipids having the strongest impact. Whereas 56 metabolites showed a significant increase from D0 to D14 led by uric and essential amino acids, 34 metabolites decreased over the course of treatment with the strongest reduction in dRibose-P and sphingosine phosphate. Performing a metabolic pathway enrichment analysis based on the most altered metabolite levels, we detected 22 significantly affected metabolic pathways and programs including aerobic glycolysis, urea cycle, amino acid and lipid metabolism (FDR &amp;lt; 0.1, Fig. 1A). To further delineate metabolite changes in regards to CRS, 1-year PFS and amount of VAT, we conducted a multivariate empirical bayes analysis of variance to specifically detect metabolites with differential temporal dynamics and considered all metabolites with an FDR &amp;lt; 0.1. Patients who developed a CRS ≥ 2° showed increased levels of kynurenine, phosphatidylethanolamides and lysophosphatidylserines, and decreased levels of ornithine and phosphatidylinositols. 1-year progressors showed a significant increase of several phosphatidylinositols beginning at D3-5 after CAR-T infusion, and decreased levels of phosphatidylcholines. For VAT, we used a threshold of 144 cm² which was derived from previously conducted computer tomography segmentation analyses. As expected, VAT cohorts mostly differed in fatty acid metabolism-associated metabolites including (acyl)carnitines, cholesterol esters, triglycerides and xanthine. In order to identify metabolites being associated with multiple outcomes, we looked for mutual metabolites among distinct cohort profiles. Here, one example is phosphatidylinositol-(22:5/16:0), which was increased in CRS 0-1° and 1y-progressors ( Fig. 1B). Conclusions: Our study highlights that CD19.CAR-T treatment is accompanied by broad changes in the serum metabolome of r/r BCL patients, which can be used to profile patients being affected by CAR-T-related immunotoxicity and their response to treatment. In ongoing analyses, we evaluate the use of these profiles for predictive models, and assess their impact on CAR-T cell function in-vitro.

UHPLC-MS/MS-based central carbon metabolism unveils the biomarkers related to colon cancer.

Even though colon cancer ranks among the leading causes of cancer mortality, early detection dramatically increases survival rates. Many studies have been conducted to determine whether altered metabolite levels may serve as a potential biomarker of cancer that affects key metabolic pathways. The goal of the study was to detect metabolic biomarkers in patients with colon cancer using liquid chromatography-mass spectrometry (LC-MS). This study consisted of 30 patients with colon cancer. An analysis of the metabolomes of cancer samples and para-carcinoma tissues was conducted. We identified a series of important metabolic changes in colon cancer by analyzing metabolites in cancerous tissues compared to their normal counterparts. They are mainly involved in the pentose phosphate pathway, the TCA cycle, glycolysis, galactose metabolism, and butanoate metabolism. As well, we observed dysregulation of AMP, dTMP, fructose, and D-glucose in colon cancer. Additionally, the AUCs for AMP, dTMP, fructose, and D-glucose were greater than 0.7 for the diagnosis of colon cancer. In conclusion, AMP, dTMP, fructose, and D-glucose showed excellent diagnostic performance and could serve as novel disease biomarkers for colon cancer diagnosis.

Co-exposure to Fe, Zn, and Cu induced neuronal ferroptosis with associated lipid metabolism disorder via the ERK/cPLA2/AA pathway

Excessive amounts of iron (Fe), zinc (Zn), and copper (Cu) can be toxic to neuronal cells, even though these are essential trace elements for animals and humans. However, the precise mechanisms underlying the neurotoxicity of exposure to mixtures of Fe, Zn, and Cu are still mostly unclear. The research aimed to investigate the influence of co-exposure to iron, zinc and copper and the related mechanisms in HT22 murine hippocampal neuronal cells. Intracellular metal content, markers of oxidative damage, and biomarkers of ferroptosis were respectively detected. Afterward, metabolomic analyses were performed to obtain a comprehensive understanding of the metal mixtures on metabolism, and the functions of key enzymes on metabolic pathways were validated. The results showed that metal co-exposure resulted in cellular iron overload and increased lipid peroxidation, accompanied by significant pathological damage and mitochondrial abnormalities in HT22 cells. Meanwhile, it was found that GSH depletion, decreased GPX4, and increased expression of the lipid metabolism gene ACSL4 play important roles in ferroptosis induced by metal mixture. Further, metabolomic analysis revealed metal co-exposure induced significant alterations in metabolite levels, especially in the glycerophospholipid metabolism pathway and the arachidonic acid metabolism pathway. The levels of cPLA2 and its metabolite, arachidonic acid, were significantly increased after metal co-exposure. Then, inhibition of cPLA2 decreased the level of arachidonic acid and attenuated ferroptosis in neuronal cells. Collectively, our findings unveiled ferroptosis induced by metal co-exposure associated with crucial molecular changes in neuronal cells, providing a novel perspective on the comprehensive toxicity risk assessment of metal mixtures.

Effects on Diabetic Mice of Consuming Lipid Extracted from Foxtail Millet (Setaria italica): Gut Microbiota Analysis and Serum Metabolomics.

Millet and its components have received much extensive attention for their health benefits in mitigating metabolic diseases. Foxtail millet is rich in phytochemicals, including oil. However, the hypoglycemic capacity of foxtail millet oil has yet to be fully investigated. The present study explored the effects of consuming this oil as the lipid extract of foxtail millet (LEFM) on intestinal microbiota composition and metabolic function in diabetic mice. After eight weeks of LEFM supplementation, the blood glucose, insulin resistance index, and lipid accumulation of diabetic mice were significantly decreased. In addition, LEFM feeding modulated gut microbiota composition, reduced the abundance of harmful bacteria (Escherichia-Shigella, Peptococcus, and norank_f_Oscillospiraceae), induced a bloom of probiotics, especially short-chain fatty acid (SCFA)-producing bacteria (Adlercreutzia, Faecalibaculum, and Bifidobacterium), and increased SCFAs concentration. LEFM treatment altered serum metabolite levels, for instance, greatly increasing the levels of l-carnitine and l-glutamine and reducing S-acetyldihydrolipoamide-E and sphingosine. Overall, improvements in gut microbiota and metabolic function were associated with the hypoglycemic potential of LEFM.

Connecting Gut Microbial Diversity with Plasma Metabolome and Fecal Bile Acid Changes Induced by the Antibiotics Tobramycin and Colistin Sulfate.

The diversity of microbial species in the gut has a strong influence on health and development of the host. Further, there are indications that the variation in expression of gut bacterial metabolic enzymes is less diverse than the taxonomic profile, underlying the importance of microbiome functionality, particularly from a toxicological perspective. To address these relationships, the gut bacterial composition of Wistar rats was altered by a 28 day oral treatment with the antibiotics tobramycin or colistin sulfate. On the basis of 16S marker gene sequencing data, tobramycin was found to cause a strong reduction in the diversity and relative abundance of the microbiome, whereas colistin sulfate had only a marginal impact. Associated plasma and fecal metabolomes were characterized by targeted mass spectrometry-based profiling. The fecal metabolome of tobramycin-treated animals had a high number of significant alterations in metabolite levels compared to controls, particularly in amino acids, lipids, bile acids (BAs), carbohydrates, and energy metabolites. The accumulation of primary BAs and significant reduction of secondary BAs in the feces indicated that the microbial alterations induced by tobramycin inhibit bacterial deconjugation reactions. The plasma metabolome showed less, but still many alterations in the same metabolite groups, including reductions in indole derivatives and hippuric acid, and furthermore, despite marginal effects of colistin sulfate treatment, there were nonetheless systemic alterations also in BAs. Aside from these treatment-based differences, we also uncovered interindividual differences particularly centering on the loss of Verrucomicrobiaceae in the microbiome, but with no apparent associated metabolite alterations. Finally, by comparing the data set from this study with metabolome alterations in the MetaMapTox database, key metabolite alterations were identified as plasma biomarkers indicative of altered gut microbiomes resulting from a wide activity spectrum of antibiotics.

A systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions.

Metabolic reprogramming has been coined as a hallmark of cancer, accompanied by which the alterations in metabolite levels have profound effects on gene expression, cellular differentiation, and the tumor environment. Yet a systematic evaluation of quenching and extraction procedures for quantitative metabolome profiling of tumor cells is currently lacking. To achieve this, this study is aimed at establishing an unbiased and leakage-free metabolome preparation protocol for HeLa carcinoma cell. We evaluated 12 combinations of quenching and extraction methods from three quenchers (liquid nitrogen, -40°C 50% methanol, 0.5°C normal saline) and four extractants (-80°C 80% methanol, 0.5°C methanol/chloroform/water [1:1:1 v/v/v], 0.5°C 50% acetonitrile, 75°C 70% ethanol) for global metabolite profiling of adherent HeLa carcinoma cells. Based on the isotope dilution mass spectrometry (IDMS) method, gas/liquid chromatography in tandem with mass spectrometry was used to quantitatively determine 43 metabolites including sugar phosphates, organic acids, amino acids (AAs), adenosine nucleotides, and coenzymes involved in central carbon metabolism. The results showed that the total amount of the intracellular metabolites in cell extracts obtained using different sample preparation procedures with the IDMS method ranged from 21.51 to 295.33nmol per million cells. Among 12 combinations, cells that washed twice with phosphate buffered saline (PBS), quenched with liquid nitrogen, and then extracted with 50% acetonitrile were found to be the most optimal method to acquire intracellular metabolites with high efficiency of metabolic arrest and minimal loss during sample preparation. In addition, the same conclusion was drawn as these 12 combinations were applied to obtain quantitative metabolome data from three-dimensional (3D) tumor spheroids. Furthermore, a case study was carried out to evaluate the effect of doxorubicin (DOX) on both adherent cells and 3D tumor spheroids using quantitative metabolite profiling. Pathway enrichment analysis using targeted metabolomics data showed that DOX exposure would significantly affect AA metabolism-related pathways, which might be related to the mitigation of redox stress. Strikingly, our data suggested that compared to two-dimensional (2D) cells the increased intracellular glutamine level in 3D cells benefited replenishing the tricarboxylic acid (TCA) cycle when the glycolysis was limited after dosing with DOX. Taken together, this study provides a well-established quenching and extraction protocol for quantitative metabolome profiling of HeLa carcinoma cell under 2D and 3D cell culture conditions. Based on this, quantitative time-resolved metabolite data can serve to the generation of hypotheses on metabolic reprogramming to reveal its important role in tumor development and treatment.

DOP66 Ileal resections from fibrotic-CD patients present microbiota dysbiosis, altered metabolomic profiles and metabolite-sensing GPCRs expression

Abstract Background Crohn’s Disease (CD) is a subtype of IBD characterized by a chronic transmural inflammation of the gastrointestinal tract associated with several complications being intestinal fibrosis the most frequent. CD patients present microbiota dysbiosis and altered metabolomic profiles. GPCRs constitute a family of receptors which could be involved in inflammatory and fibrotic processes associated to CD. We aim to characterize microbiota composition, tissue metabolomic profile and metabolite-sensing GPCRs expression in ileal resections from fibrotic CD patients. Methods Ileal resections from B2-CD (n=21) and non-IBD (n=13) patients were obtained. Microbiota characterization was performed by 16S rRNA gene Illumina Miseq sequencing. Bioinformatic analysis of sequencing data was performed using constrained correspondence analysis and non-parametric Wilcoxon test to compare species proportions. Bacterial load was estimated by qPCR. Metabolomic analysis was performed by NMR. Results are expressed as μg metabolite/g tissue. Murine intestinal fibrosis was induced in C67BL/6 mice by: a) the heterotopic intestinal transplant model and b) chronic administration of 4 cycles of increasing DSS percentages. Gene expression of GPCRs was analyzed by qPCR. Data were expressed as fold induction vs control (mean±SEM) and compared by a t-test. Correlations were analyzed with the Spearman coefficient. Results First, microbiota analysis revealed a reduction in bacterial diversity and load in fibrotic CD patients. Then, in B2-CD samples we found at genus level Enterococcus genera significantly decreased and at species level Ruminococcus bromii and Faecalibacterium prausnitzii also reduced compared to controls. From the metabolomic analysis, altered levels of metabolites were found in ileal resections from fibrotic CD patients as summarized in Table 1. Next, B2-CD patients exhibited differential expression of metabolite-sensing GPCRs vs non-IBD as shown in Table 2. Moreover, gene expression of fibrotic markers was analyzed in B2-CD patients and significantly increased levels of COL1A1 (13.22±4.38), COL3A1 (1.84±0.52), and COL4A1(7.75±2.19), were found vs controls. Of interest, GPR81, GPR84, GPR4 and GPR68 positively correlated with profibrotic markers, specifically with COL1A1 and COL4A1. Finally, in line with human results, we also analyzed the expression of metabolite-sensing GPCRs in two different murine colitis models and results obtained are represented in Table 3. Conclusion Fibrotic CD patients exhibit microbial dysbiosis, joined with altered levels of metabolites and gene expression of metabolite-sensing GPCRs, which are also affected in murine colitis models. Their correlation with profibrotic markers points them as protagonists of intestinal fibrosis.

L-Theanine attenuates heat stress-induced proteotoxicity and alterations in carbohydrate and lipid metabolism via heat shock factor 1.

Extreme heat caused by global warming accelerated the frequency of heat stress (HS). Proteotoxic stress induced by the aggregation of misfolded proteins and metabolic stress triggered by alterations in the metabolism were observed during HS. The activation of heat shock factor 1 (Hsf1) and its interaction with adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) are critical in addressing proteotoxicity and metabolic stress in heat-stressed organisms. Previous studies have shown that L-theanine (LTA) can regulate nutrient metabolism through the AMPK pathway and can alleviate HS. Therefore, we hypothesize that LTA may help in restoring homeostasis by regulating nutrient metabolism under HS. Here, we investigated the effects of LTA on nutrient metabolism in heat-stressed rats and characterized the underlying mechanisms using RNA sequencing and metabonomics. The results showed that LTA alleviated HS-induced liver damage, promoted body weight gain, decreased serum cortisol and enhanced the total protein content. Besides, it regulated the expression of genes related to carbohydrate, lipid and amino acid metabolism and altered metabolite levels. Moreover, LTA inhibited the expression of Hsf1 and heat shock protein 70 (Hsp70), promoted AMPK phosphorylation and the expression of glucose-6-phosphatase catalytic subunit 1 (G6pc), and inhibited the phosphorylation of acetyl-CoA carboxylase 1 (ACC1) in heat-stressed rats. Mechanistically, LTA alleviated HS-induced proteotoxic stress by acting on Hsf1/Hsp70; simultaneously, it promoted AMPK phosphorylation by suppressing Hsf1 expression, which in turn inhibited fatty acid synthesis and hepatic gluconeogenesis, thus alleviating HS-induced metabolic stress. These results suggest that LTA regulates nutrient metabolism through Hsf1/AMPK and alleviates HS-induced proteotoxicity via Hsf1/Hsp70.

Impact of broad-spectrum antibiotics on the gut–microbiota–spleen–brain axis

The spleen is a key immune-related organ that plays a role in communication between the brain and the immune system through the brain–spleen axis and brain–gut–microbiota axis. However, how the gut microbiota affects spleen and brain function remains unclear. Here, we investigated whether microbiome depletion induced by administration of an antibiotic cocktail (ABX) affects spleen and brain function. Treatment with ABX for 14 days resulted in a significant decrease in spleen weight and significant alterations in splenic functions, including the percentage of neutrophils, NK cells, macrophages, and CD8+ T cells. Furthermore, ABX treatment resulted in the depletion of a large portion of the gut microbiota. Untargeted metabolomics analysis showed that ABX treatment caused alterations in the levels of certain compounds in the plasma, spleen, and brain. Moreover, ABX treatment decreased the expression of microglia marker Iba1 in the cerebral cortex. Interestingly, correlations were found between the abundance of different microbiome components and metabolites in various tissues, as well as splenic cell populations and spleen weight. These findings suggest that ABX-induced microbiome depletion and altered metabolite levels may affect spleen and brain function through the gut–microbiota–spleen–brain axis.

Severe COVID-19 in pregnancy has a distinct serum profile, including greater complement activation and dysregulation of serum lipids.

Pregnancies complicated by Coronavirus Disease 2019 (COVID-19) are at an increased risk of severe morbidity due to physiologic changes in immunologic, cardiovascular, and respiratory function. There is little is known about how severity of COVID-19 changes protein and metabolite expression in pregnancy. This study aims to investigate the pathophysiology behind various clinical trajectories in pregnant patients diagnosed with COVID-19 using multi-omics profiling. This is a prospective cohort study of 30 pregnant patients at a single tertiary care center. Participants were categorized by severity of COVID-19 disease (control, asymptomatic, mild/moderate, or severe). Maternal serum samples underwent LC-MS-based multiomics analysis for profiling of proteins, lipids, electrolytes, and metabolites. Linear regression models were used to assess how disease severity related to analyte levels. Reactome pathway enrichment analysis was conducted on differential analytes. Of 30 participants, 25 had confirmed diagnosis of COVID-19 (6 asymptomatic (one post-infection), 13 mild/moderate (all post-infection), 6 severe), and 5 participants were controls. Severe COVID-19 was associated with distinct profiles demonstrating significant proteomic and lipidomic signatures which were enriched for annotations related to complement and antibody activity. (FDR < 0.05). Downregulated analytes were not significantly enriched but consisted of annotation terms related to lipoprotein activity (FDR > 0.2). Post-infection mild/moderate COVID-19 did not have significantly altered serum protein, metabolite, or lipid metabolite levels compared to controls. Pregnancies with severe COVID-19 demonstrate greater inflammation and complement activation and dysregulation of serum lipids. This altered multiomic expression provides insight into the pathophysiology of severe COVID-19 in pregnancy and may serve as potential indicators for adverse pregnancy outcomes.

NRBF2-mediated autophagy contributes to metabolite replenishment and radioresistance in glioblastoma

Overcoming therapeutic resistance in glioblastoma (GBM) is an essential strategy for improving cancer therapy. However, cancer cells possess various evasion mechanisms, such as metabolic reprogramming, which promote cell survival and limit therapy. The diverse metabolic fuel sources that are produced by autophagy provide tumors with metabolic plasticity and are known to induce drug or radioresistance in GBM. This study determined that autophagy, a common representative cell homeostasis mechanism, was upregulated upon treatment of GBM cells with ionizing radiation (IR). Nuclear receptor binding factor 2 (NRBF2)—a positive regulator of the autophagy initiation step—was found to be upregulated in a GBM orthotopic xenograft mouse model. Furthermore, ATP production and the oxygen consumption rate (OCR) increased upon activation of NRBF2-mediated autophagy. It was also discovered that changes in metabolic state were induced by alterations in metabolite levels caused by autophagy, thereby causing radioresistance. In addition, we found that lidoflazine—a vasodilator agent discovered through drug repositioning—significantly suppressed IR-induced migration, invasion, and proliferation by inhibiting NRBF2, resulting in a reduction in autophagic flux in both in vitro models and in vivo orthotopic xenograft mouse models. In summary, we propose that the upregulation of NRBF2 levels reprograms the metabolic state of GBM cells by activating autophagy, thus establishing NRBF2 as a potential therapeutic target for regulating radioresistance of GBM during radiotherapy.