Major Metabolic Pathways Research Articles

Up-regulated succinylation modifications induce a senescence phenotype in microglia by altering mitochondrial energy metabolism.

The aging of the central nervous system(CNS) is a primary contributor to neurodegenerative diseases in older individuals and significantly impacts their quality of life. Neuroinflammation, characterized by activation of microglia(MG) and release of cytokines, is closely associated with the onset of these neurodegenerative diseases. The activated status of MG is modulated by specifically programmed metabolic changes under various conditions. Succinylation, a novel post-translational modification(PTM) mainly involved in regulating mitochondrial energy metabolism pathways, remains unknown in its role in MG activation and aging. In the present study, we found that succinylation levels were significantly increased both during aging and upon lipopolysaccharide-induced(LPS-induced) MG activation undergoing metabolic reprogramming. Up-regulated succinylation induced by sirtuin 5 knockdown(Sirt5 KD) in microglial cell line BV2 resulted in significant up-regulation of aging-related genes, accompanied by impaired mitochondrial adaptability and a shift towards glycolysis as a major metabolic pathway. Furthermore, after LPS treatment, Sirt5 KD BV2 cells exhibited increased generation of reactive oxygen species(ROS), accumulation of lipid droplets, and elevated levels of lipid peroxidation. By employing immunoprecipitation, introducing point mutation to critical succinylation sites, and conducting enzyme activity assays for succinate dehydrogenase(SDH) and trifunctional enzyme subunit alpha(ECHA), we demonstrated that succinylation plays a regulatory role in modulating the activities of these mitochondrial enzymes. Finally, down-regulation the succinylation levels achieved through administration of succinyl phosphonate(SP) led to amelioration of MG senescence in vitro and neuroinflammation in vivo. To our knowledge, our data provide preliminary evidence indicating that up-regulated succinylation modifications elicit a senescence phenotype in MG through alterations in energy metabolism. Moreover, these findings suggest that manipulation of succinylation levels may offer valuable insights into the treatment of aging-related neuroinflammation.

Unveiling the Antibacterial Mechanism of Gold Nanoparticles by Analyzing Bacterial Metabolism at the Molecular Level.

The threat of drug-resistant bacteria is challenging, and it is urgent to explore new antibiotics. Gold nanoparticles (AuNPs) are known to be a group of promising antibacterial agents for replacing conventional antibiotics. Nevertheless, their antibacterial mechanism remains to be elucidated. Herein, we directly observed the interaction between antibacterial AuNPs and bacteria at the molecular level using neutral desorption extractive electrospray ionization mass spectrometry (ND-EESI-MS). We monitored and analyzed the dynamic changes of bacterial metabolites in real time after AuNP treatment. Ten substances representing 3 major metabolic pathways, including protein and nucleic acid synthesis, energy metabolism, and quorum sensing, were identified, indicating that AuNPs may exert antibacterial effects through multiple mechanisms influencing bacterial metabolism and communication. This study deepens the understanding of the antibacterial mechanism of AuNPs and is insightful for designing and screening new antibacterial agents.

Molecular insights into acetyl triethyl citrate (ATEC) induced toxic effect in HepG2 cells based on multi‐omics integrative analysis

AbstractCitrate esters have become the main alternatives to traditional plasticizers in food packaging materials. However, there is a lack of understanding of their toxic effects, particularly the combined effects and inner mechanisms has not been well studied. Our group pioneered the study on combined toxicity of tributyl citrate (TBC) and acetyl triethyl citrate (ATEC), two commonly co‐used citrate esters in food packaging materials. The results showed that exposure to TBC and ATEC can decrease the viability of HepG2 cells in a dose dependent manner. When the mixtures of ATEC and TBC exposed to HepG2 cells, they exhibited antagonism effect. Therefore, ATEC was selected to investigate the molecular mechanisms with multi‐omics techniques at its 1/4 EC50 concentration. A total of 31 metabolites with significant changes were found as potential biomarkers. The LIPG (Lipase G, endothelial type) and GCLM (glutamate‐cysteine ligase modifier subunit) were identified as differentially expressed genes based on transcriptomic analysis. Moreover, downregulated l‐glutamate and l‐glutamine which participate in TCA cycle, resulting in the collapse of energy production and cytotoxicity. These findings on major metabolic pathways will provide insight into the mechanism of cytotoxicity of HepG2 cells after ATEC exposure.

Metabolic regulation of 5-oxoproline for enhanced heat tolerance in perennial ryegrass

Pyroglutamic acid [(5-oxoproline (5-oxp)], a non-protein amino acid, can be converted to glutamate to regulate amino acid metabolism in plants. Its roles in plant adaptation to abiotic stresses, including heat stress, are not well understood. The objectives of this study were to determine whether exogenous application of 5-oxp could promote heat tolerance in cool-season perennial grass species and identify the major metabolic pathways that could be activated or responsive to 5-oxp for enhancing heat tolerance. Perennial ryegrass (Lolium perenne L.) plants were foliar-sprayed with 5-oxp or water (untreated control) prior to and during the exposure to heat stress (35/33 ℃, day/night temperature) or ambient temperature (25/22 ℃, day/night temperature, non-stress control) in controlled-environment growth chambers. Application of 5-oxp improved the heat tolerance of perennial ryegrass, as manifested by the chlorophyll content, photochemical efficiency, cell membrane stability, and antioxidant enzyme activities increasing by 31.2%, 25.7%, 37.2%, and 57.1-258.3%, as well as the reduction in hydrogen peroxide production by 36.8%. Metabolic profiling identified metabolites up-regulated by 5-oxp that are involved in the metabolic pathways of carbon assimilation in photosynthesis, glycolysis and the tricarboxylic acid cycle of respiration, proteinogenic amino acid metabolism, glutathione metabolism, and nucleotide metabolism for DNA or RNA synthesis and ATP generation. The up-regulation or activation of those metabolic processes could contribute to 5-oxp-mediated enhancement in the heat tolerance of perennial ryegrass.

The ‘photosynthetic C1 pathway’ links carbon assimilation and growth in California poplar

Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO2 fertilization responses by linking CO2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a ‘photosynthetic C1 pathway’ and demonstrate its high activity in foliage of a C3 tree where it rapidly integrates photosynthesis and C1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13C-depleted signature. Using 13CO2-labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO2 fertilization under a changing climate.

Biological Properties of S. warneri KYS-164 Isolated from Kefir Grains.

Staphylococcus worderi KYS-164, isolated from homemade Tibetan kefir grains, produces bacteriocin-like inhibitory substances (BLIS), which are peptides with antimicrobial properties, but have not been fully characterized. The research on BLIS will lay the foundation for mining new bacteriocins. In this study, the optimal culture conditions for the production of highly active BLIS were found to be incubation at 30 °C and 120 rpm, and the most effective extraction method was ammonium sulfate precipitation (ASP) using ammonium sulfate at 80% saturation. The postantibiotic effect (PAE) of BLIS on Staphylococcus aureus CICC 10384 is significant, with a 4 × MIC BLIS concentration able to prolong the PAE to 2.39 h. BLIS has excellent biosafety, with no deleterious effects observed at 8 × MIC concentration. Gas chromatography-ion mobility spectrometry (GC-IMS) was used to analyze the volatile compounds synthesized by Staphylococcus warneri KYS-164 during its growth. Hydroxycitronellal, ethyl pyruvate, and α-pinene were found to be unique substances produced by this strain, which can provide fresh, refreshing floral and fruity aromas as well as strong pine and resinous aromas in the process of kefir grain fermentation of milk. Analysis of the S. warneri KYS-164 genome provided insights into the major metabolic pathways in which genes expressed in this strain are involved. This study represents the first isolation of S. warneri KYS-164 from kefir grains prepared by Tibetan families, and provides a comprehensive analysis of its physicochemical properties. This research provides a solid foundation for better understanding and utilization of S. warneri KYS-164.

Re-aerobic treatment and dissolved oxygen regulation in full-scale aerobic-hydrolysis and denitrification-aerobic process for achieving simultaneous detoxification and nitrification of coking wastewater

The biological treatment of coking wastewater is a challenge. The application of prepositioned aerobic process has rarely been systematically reported, among which the detoxification and nitrification performance of the prepositioned aerobic unit (O1) is worthy of investigation. Results indicate that O1 achieves stable simultaneous detoxification and nitrification by regulating the dissolved oxygen, effectively maintaining ammonification, nitrosation, and complete nitrification phases. Microbial community structure, metabolic pathways and functional genes showed different preferences at different phases. High dissolved oxygen concentrations (2.20–3.00 mg/L) benefited the enrichment of carbon and nitrogen related major metabolic pathways and functional genes. BOD5/CODCr ratio, dissolved oxygen and toxic pollutants together shaped microbial community structure and nitrogen transformation processes. Based on the principle of DO regulation, it could assemble a biotransformation compartment for nitrogen removal from complex wastewaters through a pollutant detoxification mechanism of rapid microbial proliferation，and provides a promising approach for toxic industrial wastewater.

Metabolomics analysis of germinated sesame (Sesamum indicum L.) seeds: Possibility of incorporating short-term germination treatments into the sesame production process

Germination is an effective means to improve the nutritional value of sesame seeds. However, it is not clear whether it would be feasible to improve the nutritional value of sesame seeds by short-term germination treatments without causing substantial loss of oil. In this study, changes in the metabolite profiles of sesame seeds during short-term germination were analyzed by UPLC-MS/MS-based metabolomics. After 20 h of germination, the total phenolic and total flavonoid contents of sesame seeds were elevated by 2.16 and 1.53 folds, respectively, and the antioxidant capacity of their extracts was also significantly increased. At the same time, the oil content was not reduced. In metabolomics analysis, 48 bioactive compounds (19 terpenoids, 10 amino acids and derivatives, 7 flavonoids, 8 cinnamic acid derivatives, and 4 organic acids) were identified as key metabolites associated with elevated antioxidant capacity. Amino acid metabolism and the tricarboxylic acid cycle were the major metabolic pathways during sesame seed germination. Overall, it was possible to use short-term germination treatments as a means of pretreatment during the processing of sesame seeds products.

Yeast Solutions and Hyperpolarization Enable Real-Time Observation of Metabolized Substrates Even at Natural Abundance.

Metabolic changes in an organism often occur much earlier than macroscopic manifestations of disease, such as invasive tumors. Therefore, noninvasive tools to monitor metabolism are fundamental as they provide insights into in vivo biochemistry. NMR represents one of the gold standards for such insights by observing metabolites. Using nuclear spin hyperpolarization greatly increases the NMR sensitivity, enabling μmol/L sensitivity with a time resolution of about one second. However, a metabolic phantom with reproducible, rapid, and human-like metabolism is needed to progress research in this area. Using baker's yeast as a convenient metabolic factory, we demonstrated in a single study that yeast cells provide a robust and rapidly metabolizing phantom for pyruvate and fumarate, including substrates with a natural abundance of 13C: we observed the production of ethanol, carbon dioxide, bicarbonate, lactate, alanine from pyruvate, malate, and oxaloacetate from fumarate. For observation, we hyperpolarized pyruvate and fumarate via the dissolution dynamic nuclear polarization (dDNP) technique to about 30% 13C polarization that is equivalent to 360,000 signal enhancement at 1 T and 310 K. Major metabolic pathways were observed using tracers at a natural abundance of 13C, demonstrating that isotope labeling is not always essential in vitro. Enriched [1-13C]pyruvate revealed minor lactate production, presumably via the D-lactate dehydrogenase (DLD) enzyme pathway, demonstrating the sensitivity gain using a dense yeast solution. We foresee that yeast as a metabolic factory can find application as an abundant MRI phantom standard to calibrate and optimize molecular MRI protocols. Our study highlights the potential of using hyperpolarization to probe the metabolism of yeast and other microorganisms even with naturally abundant substrates, offering valuable insights into their response to various stimuli such as drugs, treatment, nourishment, and genetic modification, thereby advancing drug development and our understanding of biochemical processes.

Comparative profiling of the absorbed compounds and metabolites, and pharmacokinetic studies of Danshen-Chuanxiong herb pair in rat plasma and brain using liquid chromatography-tandem mass spectrometry

Danshen-Chuanxiong (DS-CX) was a classic herb pair commonly used to treat ischemic stroke. Nevertheless, the metabolic conversion and pharmacokinetic behavior of DS-CX in vivo remains unclear. This work aimed to reveal the in vivo metabolic behavior of DS-CX through establishing metabolic profiles and performing multicomponent pharmacokinetics analysis. The mass defect filtering (MDF) strategy integrated with UHPLC-QTOF-MS was firstly developed to characterize the metabolites of DS-CX in rats’ plasma and brain. Moreover, a sensitive UHPLC-QQQ-MS method was utilized to perform the comparative pharmacokinetic studies of major active ingredients of DS-CX in rats’ plasma. A total of 111 exogenous compounds (29 prototype compounds and 82 metabolites) were identified in rat biological samples. The major metabolic pathways were hydroxylation, methylation, deoxidation, dehydration, hydrogenation, demethylation, hydrolysis, decarboxylation and glucuronidation binding reactions. According to the results of metabolites profiling, sixteen active compounds (8 phenolic acids, 5 phthalides and 3 tanshinones) were selected as markers for further comparative pharmacokinetics study. Compared with the oral administration of DS or CX alone, the higher Cmax of salvianolic acid B, crytotanshinone and tanshinone IIA; the shorter Tmax of lithospermic acid, rosmarinic acid and tanshinone IIA; as well as the higher AUC0−∞ of ferulic acid, rosmarinic acid, salvianolic acid B, senkyunolide I and crytotanshinone, could be found after co-administration of DS-CX (P < 0.05). This study provided the overall knowledge of metabolites profiling of DS-CX in vivo, which would help to understand the effective material basis and promote the clinical application of DC-CX herb pair.

ZnR/GPR39 regulates hepatic insulin signaling, tunes liver bioenergetics and ROS production, and mitigates liver fibrosis and injury

Adequate supply of zinc is essential for hepatic function and its deficiency is associated with acute liver injury (ALI) and chronic nonalcoholic fatty liver disease (NAFLD). However, how zinc controls hepatic function is unknown. We found that the zinc sensitive ZnR/GPR39, a mediator of zinc signaling, enhances hepatic phosphorylation of ERK1/2, which is reduced in ZnR/GPR39 deficient livers. Surprisingly, livers from ZnR/GPR39 knockout (KO) mice exhibited elevated insulin receptor expression and downstream AKT activation. Moreover, ZnR/GPR39 KO mice had higher blood fasting glucose level, pronounced hepatic lipid accumulation, increased hepatocyte oxygen consumption rate (OCR) and reactive oxygen species (ROS) levels. These data suggest that ZnR/GPR39 modulates insulin receptor signaling, a major pathway in hepatic metabolism. Associated with the impaired signaling, ZnR/GPR39 KO livers exhibited increased tissue fibrosis, manifested by marked elevation of collagen expression, compared to wildtype (WT). Additionally, we found alteration of hepatocyte junctional proteins that was accompanied by increased macrophage infiltration and higher liver inflammation in ZnR/GPR39 KO mice. To determine the role of ZnR/GPR39 in ALI, we applied a mild LPS challenge that induced profound decrease in hepatic OCR, also leading to higher ROS generation in ZnR/GPR39 KO hepatocytes, but not in WT. We further found increased serum IL-2 and AST/ALT ratio only in ZnR/GPR39 KO mice. Our findings reveal a role of ZnR/GPR39 in controlling hepatic insulin receptor signaling and mitigating liver fibrosis and inflammation, thus underscoring the important role of ZnR/GPR39 in liver signaling and function.

Effect of the quorum sensing signal molecule auto-inducer-2 on fermentation performance of Lactobacillus delbrueckii subsp. Bulgaricus DPUL-F36

Fermented milk is a typically product of co-fermentation by a variety of lactic acid bacteria (LAB). Quorum sensing (QS), as an intercellular communication mechanism of bacteria, may serve as a key factor influencing the quality of fermented milk. In this study, the fermentation performance of Lactobacillus delbrueckii subsp. bulgaricus DPUL-F36 were evaluated after supplementation with auto-inducer-2 (AI-2). The protein-protein interaction network (PPIN) was used to retrieve the interactions between QS genes and four major metabolic pathway genes. The expression of luxS gene in DPUL-F36 may affect amino acid metabolism, carbohydrate metabolism and cell wall formation. The key differential metabolites were identified by metabolomics analysis. A variety of amino acids including L-Glutamic, L-Tryptophan, L-Threonine, L-Histidine and L-Glutamine were found to be up-regulated in response to the addition of AI-2. Malic acid and succinate gradually accumulate in the tricarboxylic acid cycle. The consumption of citric acid increased with the fermentation process. In terms of rheological and texture characteristics of fermented milk, addition of AI-2 could effectively improve its shear stress resistance, firmness and index of viscosity. Our research provided a theoretical basis for improving the fermentation performance of DPUL-F36 strains, and support for the screening of excellent composite starter culture.

Abstract C091: Uncovering genetic regulators of metabolic plasticity in renal cell carcinoma

Abstract We aim to offer new insights on the regulation of metabolic plasticity in renal cell carcinoma (RCC) and identify novel metabolic regulatory targets that can be used for potential treatment therapies of the disease. RCC affects an estimated 400,000 people worldwide each year with over 100,00 deaths annually that disproportionately affects racial/ethnic minorities in the United States. RCC is a term that covers an array of kidney cancer subtypes differing in incidence, histopathology, genetic and molecular alterations, as well as in clinical outcomes and prognoses. However, a common characteristic of many RCCs is that they are driven by metabolic rewiring, due to a high frequency of mutations in genes that regulate major metabolic processes in the cell. It was recently discovered that patient overall survival and prognosis has been linked to the expression of certain metabolic signatures in RCC tumor cells. Metabolic plasticity allows cancer cells to survive and adapt to different environments and metabolic stress conditions, such as nutrient deprivation. Therefore, exploring and exploiting metabolic rewiring in these cells may reveal genetic vulnerabilities and dependencies. This could lead to discovery of novel biomarkers and molecular targets that can be implicated as future therapeutic interventions for this disease. To this end, we applied functional genomic screening to expose genetic dependencies and vulnerabilities under different physiologic and metabolic conditions in patient derived medullary (PDM) RCC cells. The medullary RCC is a rare and aggressive RCC subtype that disproportionately affects the African American/Black population. We applied a CRISPR- based combinatorial screening platform that is based on co-expression of Cas9 and Cas12a nucleases and libraries of hybrid guide RNAs (hgRNA) to achieve ultra-efficient gene knockout (Aregger et al., 2021; Gonatopoulos-Pournatzis et al., 2020) in PDM RCC cells. Additionally, cells were screened under different media conditions that mimic human physiological nutrient conditions and a media deprived of lipids, one of the major metabolic substrates of the cell. By utilizing a genome-wide pooled CRISPR gene knockout screen we were able to systematically uncover genes required for cell survival and proliferation in a panel of patient-derived RCCs. We have begun to uncover genetic dependencies and major metabolic pathways that can be elucidated and exploited for future studies, such as those involving ferroptosis which is a targetable pathway for the development of cancer treatment therapies. Our findings demonstrate some of the key genetic drivers underlying cellular adaptations to different physiological environments and nutrient deprivation. In future studies we will further explore the relationship between different key metabolic regulatory genes in RCC and map for genetic interactions in major metabolic pathways. Citation Format: Chelsee Holloway, Josef Horak, Youngkyu Jeon, Michael Aregger. Uncovering genetic regulators of metabolic plasticity in renal cell carcinoma [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C091.

Abstract C016: Optical Techniques to Democratize Intravital Quantification of Metabolic Heterogeneity

Abstract Since metabolic and vascular adaptations are essential for tumor cell survival, a technique capable of tracking metabolic heterogeneity at both the bulk tumor and cellular scales over extended timelines is necessary. To address this technological need, we have developed a multi-scale optical microscope relying on exogenous fluorescent reporters for dynamic spatiotemporal imaging of major metabolic pathways in vivo. This microscope utilizes a low-cost CMOS detector and LEDs for excitation to enable fluorescence microscopy in resource-limited settings. Here, we implement our system to assess changes in bulk tumor and cellular metabolic heterogeneity as vasculature is perturbed. 4T1 breast tumor-bearing mice were treated with combretastatin A-1 (a vascular disruptor), implanted with window chambers, and temporally imaged (n=5/group). Fluorescence imaging and Gabor filter/Djikstra segmentation approaches enabled metabolic and vascular analyses, respectively. We demonstrated the system’s ability to acquire co-registered images of vasculature, morphology, and distinct metabolic endpoints reporting on glucose uptake, fatty acid uptake, and mitochondrial metabolism at both wide-field and high resolutions. At 12 hours post-treatment, the tumors’ metabolism shifted from mitochondrial respiration to glycolysis in avascular regions. Further, we report a higher average vascular density in untreated versus treated mice at 108 hours post-treatment (p&amp;lt;0.05, Kolmogorov-Smirnov test). On exploring the relationship between metabolic activity and vasculature, regions with high local tortuosity in the untreated group exhibited a higher reliance on fatty acid uptake than high tortuosity regions in the treatment group, indicating these cells’ reliance on fatty acids within a complex vascular network. Our multiplexed imaging strategy enables the quantification of tumor metabolism and vasculature over multiple length scales, which could be leveraged to study adaptations following clinical therapies. Citation Format: Enakshi Sunassee. Optical Techniques to Democratize Intravital Quantification of Metabolic Heterogeneity [abstract]. In: Proceedings of the 17th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2024 Sep 21-24; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2024;33(9 Suppl):Abstract nr C016.

An in vitro evaluation on metabolism of mitragynine to 9-O-demethylmitragynine

Kratom (Mitragyna Speciosa Korth.) is an indigenous tree native to Southeast Asia whose leaves have been traditionally ingested as a tea and has seen its popularity increase in the United States. Although kratom and its constituents presently have no approved uses by the Food and Drug Administration, its major alkaloids (e.g., mitragynine) have psychoactive properties that may hold promise for the treatment of opioid cessation, pain management, and other indications. 9-O-demethylmitragynine is a major metabolite formed from mitragynine metabolism (36 % total metabolism) and displays similar pharmacologic activity. Cytochrome P450 (CYP) 3A4 has been identified as a major enzyme involved in mitragynine metabolism; however, the in vitro metabolism parameters of 9-O-demethylmitragynine formation are not well defined and a risk of potential drug interactions exists. Using human liver S9 fractions, 9-O-demethylmitragynine formation was generally linear for enzyme concentrations of 0–0.25 mg/mL and incubation times of 5–20 min. 9-O-demethylmitragynine displayed a Km 1.37 μM and Vmax of 0.0931 nmol/min/mg protein. Known CYP inhibitors and compounds that might be concomitantly used with kratom were assessed for inhibition of 9-O-demethylmitragynine formation. Ketoconazole, a CYP3A index inhibitor, demonstrated a significant effect on 9-O-demethylmitragynine formation, further implicating CYP3A4 as a major metabolic pathway. Major cannabinoids (10 μg/mL) displayed minor inhibition of 9-O-demethylmitragynine formation, while all other compounds had minimal effects. Mixtures of physiological achievable cannabinoid concentrations also displayed minor effects on 9-O-demethylmitragynine formation, making a metabolic drug interaction unlikely; however, further in vitro, in vivo, and clinical studies are necessary to fully exclude any risk.

Metabolic Stability and Metabolite Identification of CYP450 Probe Substrates in Ferret Hepatocytes.

Ferrets exhibit similar lung physiology to humans and display similar clinical signs following influenza infection, making them a valuable model for studying high susceptibility and infection patterns. However, the metabolic fate of several common human CYP450 probe substrates in ferrets is still unknown and has not been studied. The purpose of this study was to investigate the metabolism of nine human CYP450 probe substrates in ferret hepatocytes and explore their metabolic rate differences between ferrets and other species. Nine substrates were individually incubated in ferret hepatocytes for up to 120 min. At each time point, 30 μL mixtures were extracted for stability analysis using LC-MS/MS methods. After a 120-minute incubation period, 400 μL of the mixtures were extracted for metabolite identification using UHPLC-QExactive Plus. The metabolic clearance was determined as follows: diclofenac > taxol > chlorzoxazone > dextromethorphan > midazolam > omeprazole > bupropion > phenacetin > testosterone. Seven metabolites were identified from phenacetin. Deethylation was found to be the major pathway, and the major metabolite was matched with acetaminophen as probed with the CYP1A2 enzyme. Six metabolites were identified from diclofenac. Glucuronidation was the primary pathway, and a metabolite was found to match 4-OH-diclofenac as probed with the CYP2C9 enzyme. Twenty-two metabolites were identified from omeprazole. The major metabolic pathways included mono-oxygenation and sulfoxide to thioether conversion. No metabolite was found to match with the 5-OH-omeprazole as probed with the CYP2C19 enzyme. Twenty-two metabolites were identified from dextromethorphan. Demethylation was found to be the major metabolic pathway, and one demethylation metabolite was matched with dextrorphan as probed with CYP2D6. Fourteen metabolites were identified from midazolam. Mono-oxygenation was found to be the primary metabolic pathway, and one of the mono-oxygenation metabolites was matched with 1-OH-midazolam as probed with the CYP3A4 enzyme. Eight metabolites were identified from testosterone. Mono-oxygenation and glucuronidation were identified as the major metabolic pathways. One mono-oxygenation was matched with 6-β-testosterone as probed with CYP3A4 enzyme. Six metabolites were identified from taxol. Hydrolysis and mono-oxygenation were the top two metabolic pathways. No metabolite was matched with 6-α-OH-taxol as probed with the CYP2C8 enzyme. Ten metabolites were identified from bupropion. Mono-oxygenation and hydrogenation were identified as the top two metabolic pathways. No mono-oxygenation metabolite was matched with hydroxy-bupropion as probed with the CYP2B6 enzyme. Nine metabolites were identified from chlorzoxazone. Monooxygenation and sulfation were the top two metabolic pathways. One mono-oxygenation metabolite was matched with 6-OH-chlorzoxazone as probed with the CYP2E1 enzyme. Nine human CYP probe substrates were clearly metabolized in ferret hepatocytes, demonstrating substrate-dependent metabolic rates in ferret hepatocytes and species-dependent metabolic rates in mouse, rat, dog, monkey, and human hepatocytes. Except for 6-a-5-OH-omeprazole, 6-α-OH-taxol, and hydroxy-bupropion, specific metabolites of other six probe substrates in ferret hepatocytes were detected and identified as probed with six human CYP enzymes, respectively.

Study on metabolic variation reveals metabolites important for flavor development and antioxidant property of Hainan Dayezhong black tea

To illustrate the development of chemical properties and characteristic flavor of Hainan Dayezhong black tea, the tea shoots under various manufacturing process were sampled and applied to targeted/widely-targeted metabolomic, transcriptomic, chemometric, and electronic sensory determinations. Totally, 2419 metabolites were identified in this study, of which 20 metabolites were selected as the biomarkers, mainly including amino acids, lipids, and pyrimidine derivatives. The metabolomic-transcriptomic integrated analysis indicated carbon fixation, flavonoid biosynthesis and amino acid metabolism were the major metabolic pathways over manufacturing process of Hainan Dayezhong black tea. The targeted metabolomic detection indicated the accumulations of free amino acids and reduction of total catechins, flavonol glycosides collectively contributed to the development of black tea taste; additionally, the antioxidative properties were decreasing along the production process. These results suggest that the tradeoff between bioactivity components and antioxidative capacity contribute to the characteristic flavor of Hainan Dayezhong black tea.

Metabolites profiling, in-vitro and molecular docking studies of five legume seeds for Alzheimer’s disease

Even though legumes are valuable medicinal plants with edible seeds that are extensively consumed worldwide, there is little information available on the metabolic variations between different dietary beans and their influence as potential anti-cholinesterase agents. High-resolution liquid chromatography coupled with mass spectrometry in positive and negative ionization modes combined with multivariate analysis were used to explore differences in the metabolic profiles of five commonly edible seeds, fava bean, black-eyed pea, kidney bean, red lentil, and chickpea. A total of 139 metabolites from various classes were identified including saponins, alkaloids, phenolic acids, iridoids, and terpenes. Chickpea showed the highest antioxidant and anti-cholinesterase effects, followed by kidney beans. Supervised and unsupervised chemometric analysis determined that species could be distinguished by their different discriminatory metabolites. The major metabolic pathways in legumes were also studied. Glycerophospholipid metabolism was the most significantly enriched KEGG pathway. Pearson’s correlation analysis pinpointed 18 metabolites that were positively correlated with the anti-cholinesterase activity. Molecular docking of the biomarkers to the active sites of acetyl- and butyryl-cholinesterase enzymes revealed promising binding scores, validating the correlation results. The present study will add to the metabolomic analysis of legumes and their nutritional value and advocate their inclusion in anti-Alzheimer’s formulations.

Impact of different processing methods of Ligustrum lucidum Ait. on kidney-yin deficiency: a study based on pharmacodynamics and metabolomics research.

This study aimed to explore the pharmacodynamics and mechanisms of different processing methods of Ligustrum lucidum Ait. (LLA) in addressing kidney-yin deficiency (KYD). Forty-eight Sprague-Dawley rats were divided into eight groups based on their weight. The KYD model was established by intragastric administration of levothyroxine sodium. Each group was administered the corresponding treatment for 15 consecutive days. The general condition of the rats during the treatment period was observed. In addition, the levels of cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), and the ratio of cAMP to cGMP in the serum of rats from different groups were measured. Serum samples were analyzed using the ultra-performance liquid chromatography (UPLC)-Orbitrap Fusion MS technique for metabolomics analysis. Compared with the model group, the general condition of the rats in the wine-steamed L. lucidum group (WL) and salt-steamed L. lucidum group (SSL) groups showed significant improvement. The serum levels of cAMP, cGMP, and the cAMP-to-cGMP ratio tended to return to normal. Metabolic analysis identified 38 relevant biomarkers and revealed 3 major metabolic pathways: phenylalanine, tyrosine, and tryptophan biosynthesis; phenylalanine metabolism; and sphingolipid metabolism. The different processing methods of LLA demonstrated therapeutic effects on KYD in rats, likely related to the restoration of disturbed metabolism by adjusting the levels of endogenous metabolites in the kidney. The SSL demonstrated significantly superior effects compared with the other four types of LLA processed products.

Modulation of brain energy metabolism in hepatic encephalopathy: impact of glucose metabolic dysfunction.

Cerebral function is linked to a high level of metabolic activity and relies on glucose as its primary energy source. Glucose aids in the maintenance of physiological brain activities; as a result, a disruption in metabolism has a significant impact on brain function, launching a chain of events that leads to neuronal death. This metabolic insufficiency has been observed in a variety of brain diseases and neuroexcitotoxicity disorders, including hepatic encephalopathy. It is a significant neurological complication that develops in people with liver disease, ranging from asymptomatic abnormalities to coma. Hyperammonemia is the main neurotoxic villain in the development of hepatic encephalopathy and induces a wide range of complications in the brain. The neurotoxic effects of ammonia on brain function are thought to be mediated by impaired glucose metabolism. Accordingly, in this review, we provide an understanding of deranged brain energy metabolism, emphasizing the role of glucose metabolic dysfunction in the pathogenesis of hepatic encephalopathy. We also highlighted the differential metabolic profiles of brain cells and the status of metabolic cooperation between them. The major metabolic pathways that have been explored are glycolysis, glycogen metabolism, lactate metabolism, the pentose phosphate pathway, and the Krebs cycle. Furthermore, the lack of efficacy in current hepatic encephalopathy treatment methods highlights the need to investigate potential therapeutic targets for hepatic encephalopathy, with regulating deficient bioenergetics being a viable alternative in this case. This review also demonstrates the importance of the development of glucose metabolism-focused disease diagnostics and treatments, which are now being pursued for many ailments.