Serum metabolomic analysis of feline mammary carcinomas based on LC-MS and MRM techniques

AbstractIntroductionTo date, there have been no panoramic studies of the serum metabolome in feline mammary carcinoma. As the first such study, metabolomics techniques were used to analyse the serum of cats with these tumours. Three important metabolic pathways of screened differential metabolites closely related to feline mammary carcinomas were analysed to lay a theoretical basis for further study of the pathogenesis of these carcinomas.Material and MethodsBlood in a 5–8 mL volume was sampled from twelve cats of the same breed and similar age (close to nine years on average). Six were feline mammary carcinoma patients and six were healthy. L glutamate, L alanine, succinate, adenine, hypoxanthine, and inosine were screened as were alanine, aspartate, and glutamate metabolism, the tricarboxylid acid (TCA) cycle, and purine metabolism. Data were acquired with LC-MS non-target metabolomics, multiple reaction monitoring target metabolomics, and multivariate statistical and bioinformatic analysis.ResultsExpression of five of the metabolites was upregulated and only inosine expression was downregulated. Up- and downregulation of metabolites related to glycometabolism, potentiation of the TCA cycle, greater content of lipid mobilisation metabolites, and abnormality of amino acid metabolism were closely related to the occurrence of the carcinomas.ConclusionThese findings provide a new direction for further study of the mechanisms associated with cat mammary neoplasms.

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The Metabolic Fates of Pyruvate in Normal and Neoplastic Cells

Cells ◽

10.3390/cells10040762 ◽

2021 ◽

Vol 10 (4) ◽

pp. 762

Author(s):

Edward V. Prochownik ◽

Huabo Wang

Keyword(s):

Metabolic Pathways ◽

Redox State ◽

Pyruvate Dehydrogenase Complex ◽

Tca Cycle ◽

Vascular Supply ◽

Extrinsic Factors ◽

The Tca Cycle ◽

Initial Substrate ◽

Numerous Cell ◽

Bio Synthesis

Pyruvate occupies a central metabolic node by virtue of its position at the crossroads of glycolysis and the tricarboxylic acid (TCA) cycle and its production and fate being governed by numerous cell-intrinsic and extrinsic factors. The former includes the cell’s type, redox state, ATP content, metabolic requirements and the activities of other metabolic pathways. The latter include the extracellular oxygen concentration, pH and nutrient levels, which are in turn governed by the vascular supply. Within this context, we discuss the six pathways that influence pyruvate content and utilization: 1. The lactate dehydrogenase pathway that either converts excess pyruvate to lactate or that regenerates pyruvate from lactate for use as a fuel or biosynthetic substrate; 2. The alanine pathway that generates alanine and other amino acids; 3. The pyruvate dehydrogenase complex pathway that provides acetyl-CoA, the TCA cycle’s initial substrate; 4. The pyruvate carboxylase reaction that anaplerotically supplies oxaloacetate; 5. The malic enzyme pathway that also links glycolysis and the TCA cycle and generates NADPH to support lipid bio-synthesis; and 6. The acetate bio-synthetic pathway that converts pyruvate directly to acetate. The review discusses the mechanisms controlling these pathways, how they cross-talk and how they cooperate and are regulated to maximize growth and achieve metabolic and energetic harmony.

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Comprehensive Plasma Metabolomic Profile of Patients with Advanced Neuroendocrine Tumors (NETs). Diagnostic and Biological Relevance

Cancers ◽

10.3390/cancers13112634 ◽

2021 ◽

Vol 13 (11) ◽

pp. 2634

Author(s):

Beatriz Soldevilla ◽

Angeles López-López ◽

Alberto Lens-Pardo ◽

Carlos Carretero-Puche ◽

Angeles Lopez-Gonzalvez ◽

...

Keyword(s):

Metabolic Pathways ◽

Metabolic Networks ◽

Tca Cycle ◽

Metabolomic Profile ◽

Diagnostic Potential ◽

The Tca Cycle ◽

Novel Biomarkers ◽

Potential Methods ◽

Clinical Potential ◽

First Time

Purpose: High-throughput “-omic” technologies have enabled the detailed analysis of metabolic networks in several cancers, but NETs have not been explored to date. We aim to assess the metabolomic profile of NET patients to understand metabolic deregulation in these tumors and identify novel biomarkers with clinical potential. Methods: Plasma samples from 77 NETs and 68 controls were profiled by GC−MS, CE−MS and LC−MS untargeted metabolomics. OPLS-DA was performed to evaluate metabolomic differences. Related pathways were explored using Metaboanalyst 4.0. Finally, ROC and OPLS-DA analyses were performed to select metabolites with biomarker potential. Results: We identified 155 differential compounds between NETs and controls. We have detected an increase of bile acids, sugars, oxidized lipids and oxidized products from arachidonic acid and a decrease of carnitine levels in NETs. MPA/MSEA identified 32 enriched metabolic pathways in NETs related with the TCA cycle and amino acid metabolism. Finally, OPLS-DA and ROC analysis revealed 48 metabolites with diagnostic potential. Conclusions: This study provides, for the first time, a comprehensive metabolic profile of NET patients and identifies a distinctive metabolic signature in plasma of potential clinical use. A reduced set of metabolites of high diagnostic accuracy has been identified. Additionally, new enriched metabolic pathways annotated may open innovative avenues of clinical research.

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Intracellular Fate of Universally Labelled 13C Isotopic Tracers of Glucose and Xylose in Central Metabolic Pathways of Xanthomonas oryzae

Metabolites ◽

10.3390/metabo8040066 ◽

2018 ◽

Vol 8 (4) ◽

pp. 66 ◽

Cited By ~ 4

Author(s):

Manu Shree ◽

Shyam K. Masakapalli

Keyword(s):

Amino Acids ◽

Metabolic Pathways ◽

Metabolic Networks ◽

Tca Cycle ◽

Xanthomonas Oryzae ◽

Flux Analysis ◽

Isotopic Tracers ◽

Feeding Experiments ◽

The Tca Cycle ◽

Metabolic Route

The goal of this study is to map the metabolic pathways of poorly understood bacterial phytopathogen, Xanthomonas oryzae (Xoo) BXO43 fed with plant mimicking media XOM2 containing glutamate, methionine and either 40% [13C5] xylose or 40% [13C6] glucose. The metabolic networks mapped using the KEGG mapper and the mass isotopomer fragments of proteinogenic amino acids derived from GC-MS provided insights into the activities of Xoo central metabolic pathways. The average 13C in histidine, aspartate and other amino acids confirmed the activities of PPP, the TCA cycle and amino acid biosynthetic routes, respectively. The similar labelling patterns of amino acids (His, Ala, Ser, Val and Gly) from glucose and xylose feeding experiments suggests that PPP would be the main metabolic route in Xoo. Owing to the lack of annotated gene phosphoglucoisomerase in BXO43, the 13C incorporation in alanine could not be attributed to the competing pathways and hence warrants additional positional labelling experiments. The negligible presence of 13C incorporation in methionine brings into question its potential role in metabolism and pathogenicity. The extent of the average 13C labelling in several amino acids highlighted the contribution of pre-existing pools that need to be accounted for in 13C-flux analysis studies. This study provided the first qualitative insights into central carbon metabolic pathway activities in Xoo.

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Disruption of the TCA cycle reveals an ATF4-dependent integration of redox and amino acid metabolism

eLife ◽

10.7554/elife.72593 ◽

2021 ◽

Vol 10 ◽

Author(s):

Dylan Gerard Ryan ◽

Ming Yang ◽

Hiran A Prag ◽

Giovanny Rodriguez Blanco ◽

Efterpi Nikitopoulou ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Metabolic Response ◽

Tricarboxylic Acid Cycle ◽

Fumarate Hydratase ◽

Tca Cycle ◽

Comparative Approach ◽

Redox Biology ◽

The Tca Cycle

The Tricarboxylic Acid Cycle (TCA) cycle is arguably the most critical metabolic cycle in physiology and exists as an essential interface coordinating cellular metabolism, bioenergetics, and redox homeostasis. Despite decades of research, a comprehensive investigation into the consequences of TCA cycle dysfunction remains elusive. Here, we targeted two TCA cycle enzymes, fumarate hydratase (FH) and succinate dehydrogenase (SDH), and combined metabolomics, transcriptomics, and proteomics analyses to fully appraise the consequences of TCA cycle inhibition (TCAi) in murine kidney epithelial cells. Our comparative approach shows that TCAi elicits a convergent rewiring of redox and amino acid metabolism dependent on the activation of ATF4 and the integrated stress response (ISR). Furthermore, we also uncover a divergent metabolic response, whereby acute FHi, but not SDHi, can maintain asparagine levels via reductive carboxylation and maintenance of cytosolic aspartate synthesis. Our work highlights an important interplay between the TCA cycle, redox biology and amino acid homeostasis.

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Glutamate availability is important in intramuscular amino acid metabolism and TCA cycle intermediates but does not affect peak oxidative metabolism

Journal of Applied Physiology ◽

10.1152/japplphysiol.90394.2008 ◽

2008 ◽

Vol 105 (2) ◽

pp. 547-554 ◽

Cited By ~ 8

Author(s):

M. Mourtzakis ◽

T. E. Graham ◽

J. González-Alonso ◽

B. Saltin

Keyword(s):

Oxidative Metabolism ◽

Acid Metabolism ◽

Glutamate Uptake ◽

Knee Extensor ◽

Tca Cycle ◽

Peak Oxygen Consumption ◽

Peak Exercise ◽

Early Exercise ◽

The Tca Cycle ◽

Tca Cycle Intermediates

Muscle glutamate is central to reactions producing 2-oxoglutarate, a tricarboxylic acid (TCA) cycle intermediate that essentially expands the TCA cycle intermediate pool during exercise. Paradoxically, muscle glutamate drops ∼40–80% with the onset of exercise and 2-oxoglutarate declines in early exercise. To investigate the physiological relationship between glutamate, oxidative metabolism, and TCA cycle intermediates (i.e., fumarate, malate, 2-oxoglutarate), healthy subjects trained (T) the quadriceps of one thigh on the single-legged knee extensor ergometer (1 h/day at 70% maximum workload for 5 days/wk), while their contralateral quadriceps remained untrained (UT). After 5 wk of training, peak oxygen consumption (V̇o2peak) in the T thigh was greater than that in the UT thigh ( P < 0.05); V̇o2peak was not different between the T and UT thighs with glutamate infusion. Peak exercise under control conditions revealed a greater glutamate uptake in the T thigh compared with rest (7.3 ± 3.7 vs. 1.0 ± 0.1 μmol·min−1·kg wet wt−1, P < 0.05) without increase in TCA cycle intermediates. In the UT thigh, peak exercise (vs. rest) induced an increase in fumarate (0.33 ± 0.07 vs. 0.02 ± 0.01 mmol/kg dry wt (dw), P < 0.05) and malate (2.2 ± 0.4 vs. 0.5 ± 0.03 mmol/kg dw, P < 0.05) and a decrease in 2-oxoglutarate (12.2 ± 1.6 vs. 32.4 ± 6.8 μmol/kg dw, P < 0.05). Overall, glutamate infusion increased arterial glutamate ( P < 0.05) and maintained this increase. Glutamate infusion coincided with elevated fumarate and malate ( P < 0.05) and decreased 2-oxoglutarate ( P < 0.05) at peak exercise relative to rest in the T thigh; there were no further changes in the UT thigh. Although glutamate may have a role in the expansion of the TCA cycle, glutamate and TCA cycle intermediates do not directly affect V̇o2peak in either trained or untrained muscle.

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Xuebijing Injection Affects the Dynamic Change of Metabolism in CLP-Induced Septic Rats

10.21203/rs.3.rs-526582/v1 ◽

2021 ◽

Author(s):

Yanjuan Liu ◽

Qi Zeng ◽

Wen Xiao ◽

Fang Chen ◽

Lianhong Zou ◽

...

Keyword(s):

Amino Acid ◽

Metabolic Pathways ◽

Amino Acid Metabolism ◽

Acid Metabolism ◽

Dynamic Change ◽

Sepsis Group ◽

Control Group ◽

Multivariate Statistical ◽

Xuebijing Injection ◽

Kegg Pathway Analysis

Abstract Xuebijing injection has been widely applied to treat sepsis. However, its roles in the dynamic change of metabolism in sepsis are still unknown. In our study, Gas chromatography-mass spectrometer (GC-MS) combined with multivariate statistical techniques was used to detect the metabolic change in septic rats with or without XBJ injection treatment. The KEGG pathway analysis was used to further analyze the related metabolic pathways in which the identified metabolites were involved. Based on the fold change, variable important in projection, and P value, we found 11, 33 and 26 differential metabolites in the sepsis group at 2, 6 and 12 hours post CLP, compared with the control group. Besides, we also found 32, 23 and 28 differential metabolites in the XBJ group at 2, 6 and 12 hours post CLP. The related pathways of differential metabolites were glycometabolism at 2h, glycometabolism and amino acid metabolism at 6h and amino acid metabolism at 12h post CLP in the sepsis group compared with the control group. Besides, glycometabolism, amino acid metabolism and lipid metabolism changed markedly after XBJ injection for 2 hours; while only amino acid metabolism changed significantly with the treatment of XBJ injection for 6 and 12 hours, compared with the sepsis group. Further analysis showed 3, 6 and 6 differential metabolites were overlapped in the sepsis group and XBJ group at 2, 6 and 12 hours post CLP. These identified differential metabolites were majorly involved in arginine and proline metabolism, suggesting that XBJ injection is capable of improving metabolic disorders in CLP-induced septic rat to a certain extent.

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Metabolic Constants and Plasticity of Cancer Cells in a Limiting Glucose and Glutamine Microenvironment—A Pyruvate Perspective

Frontiers in Oncology ◽

10.3389/fonc.2020.596197 ◽

2020 ◽

Vol 10 ◽

Author(s):

Angela M. Otto

Keyword(s):

Cancer Cells ◽

Metabolic Network ◽

Metabolic Pathways ◽

Cancer Cell Line ◽

Tca Cycle ◽

Reaction Rates ◽

Diagnostic Methods ◽

Metabolic Reprogramming ◽

Amino Acid Synthesis ◽

The Tca Cycle

The metabolism of cancer cells is an issue of dealing with fluctuating and limiting levels of nutrients in a precarious microenvironment to ensure their vitality and propagation. Glucose and glutamine are central metabolites for catabolic and anabolic metabolism, which is in the limelight of numerous diagnostic methods and therapeutic targeting. Understanding tumor metabolism in conditions of nutrient depletion is important for such applications and for interpreting the readouts. To exemplify the metabolic network of tumor cells in a model system, the fate 13C6-glucose was tracked in a breast cancer cell line growing in variable low glucose/low glutamine conditions. 13C-glucose-derived metabolites allowed to deduce the engagement of metabolic pathways, namely glycolysis, the TCA-cycle including glutamine and pyruvate anaplerosis, amino acid synthesis (serine, glycine, aspartate, glutamate), gluconeogenesis, and pyruvate replenishment. While the metabolic program did not change, limiting glucose and glutamine supply reduced cellular metabolite levels and enhanced pyruvate recycling as well as pyruvate carboxylation for entry into the TCA-cycle. Otherwise, the same metabolic pathways, including gluconeogenesis, were similarly engaged with physiologically saturating as with limiting glucose and glutamine. Therefore, the metabolic plasticity in precarious nutritional microenvironment does not require metabolic reprogramming, but is based on dynamic changes in metabolite quantity, reaction rates, and directions of the existing metabolic network.

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Host nutrient milieu drives an essential role for aspartate biosynthesis during invasiveStaphylococcus aureusinfection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1922211117 ◽

2020 ◽

Vol 117 (22) ◽

pp. 12394-12401 ◽

Cited By ~ 2

Author(s):

Aimee D. Potter ◽

Casey E. Butrico ◽

Caleb A. Ford ◽

Jacob M. Curry ◽

Irina A. Trenary ◽

...

Keyword(s):

Metabolic Pathways ◽

Aerobic Glycolysis ◽

Bacterial Pathogen ◽

Tca Cycle ◽

Acid Synthesis ◽

Tricarboxylic Acid ◽

Staphylococcal Infection ◽

Amino Acid Synthesis ◽

The Tca Cycle ◽

Host Tissues

The bacterial pathogenStaphylococcus aureusis capable of infecting a broad spectrum of host tissues, in part due to flexibility of metabolic programs.S. aureus, like all organisms, requires essential biosynthetic intermediates to synthesize macromolecules. We therefore sought to determine the metabolic pathways contributing to synthesis of essential precursors during invasiveS. aureusinfection. We focused specifically on staphylococcal infection of bone, one of the most common sites of invasiveS. aureusinfection and a unique environment characterized by dynamic substrate accessibility, infection-induced hypoxia, and a metabolic profile skewed toward aerobic glycolysis. Using a murine model of osteomyelitis, we examined survival ofS. aureusmutants deficient in central metabolic pathways, including glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, and amino acid synthesis/catabolism. Despite the high glycolytic demand of skeletal cells, we discovered thatS. aureusrequires glycolysis for survival in bone. Furthermore, the TCA cycle is dispensable for survival during osteomyelitis, andS. aureusinstead has a critical need for anaplerosis. Bacterial synthesis of aspartate in particular is absolutely essential for staphylococcal survival in bone, despite the presence of an aspartate transporter, which we identified as GltT and confirmed biochemically. This dependence on endogenous aspartate synthesis derives from the presence of excess glutamate in infected tissue, which inhibits aspartate acquisition byS. aureus. Together, these data elucidate the metabolic pathways required for staphylococcal infection within bone and demonstrate that the host nutrient milieu can determine essentiality of bacterial nutrient biosynthesis pathways despite the presence of dedicated transporters.

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Reconstruction of Sugar Metabolic Pathways of Giardia lamblia

International Journal of Proteomics ◽

10.1155/2012/980829 ◽

2012 ◽

Vol 2012 ◽

pp. 1-9 ◽

Cited By ~ 8

Author(s):

Jian Han ◽

Lesley J. Collins

Keyword(s):

Giardia Lamblia ◽

Metabolic Pathways ◽

Drug Targets ◽

Sugar Metabolism ◽

Tca Cycle ◽

The Tca Cycle ◽

Glycolysis Pathway ◽

Bioinformatic Approaches ◽

Novel Drug ◽

Novel Drug Targets

Giardia lamblia is an “important” pathogen of humans, but as a diplomonad excavate it is evolutionarily distant from other eukaryotes and relatively little is known about its core metabolic pathways. KEGG, the widely referenced site for providing information of metabolism, does not yet include many enzymes from Giardia species. Here we identify Giardia’s core sugar metabolism using standard bioinformatic approaches. By comparing Giardia proteomes with known enzymes from other species, we have identified enzymes in the glycolysis pathway, as well as some enzymes involved in the TCA cycle and oxidative phosphorylation. However, the majority of enzymes from the latter two pathways were not identifiable, indicating the likely absence of these functionalities. We have also found enzymes from the Giardia glycolysis pathway that appear more similar to those from bacteria. Because these enzymes are different from those found in mammals, the host organisms for Giardia, we raise the possibility that these bacteria-like enzymes could be novel drug targets for treating Giardia infections.

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PD-1 and PD-L1 are diagnostic biomarkers and PD-L1 a promising therapeutic target in HER2-positive and triple negative normal-like feline mammary carcinoma subtypes

10.1101/2020.04.25.061655 ◽

2020 ◽

Author(s):

Catarina Nascimento ◽

Ana Catarina Urbano ◽

Andreia Gameiro ◽

João Ferreira ◽

Jorge Correia ◽

...

Keyword(s):

Cancer Cells ◽

Mammary Carcinoma ◽

Triple Negative ◽

Strong Positive Correlation ◽

Her2 Positive ◽

Diagnostic Biomarkers ◽

Mammary Carcinomas ◽

Tumor Subtypes ◽

Promising Therapeutic Target ◽

Feline Mammary Carcinoma

AbstractTumor microenvironment has gained great relevance due to its ability to regulate distinct checkpoints mediators, orchestrating tumor progression. In order to understand the role of the PD- 1/PD-L1 axis in cats with mammary carcinoma, serum PD-1 and PD-L1 levels were compared with healthy controls and with serum CTLA-4 and TNF-α levels. PD-1 and PD-L1 expression was evaluated in TILs and cancer cells, as the presence of somatic mutations. Results showed that serum PD-1 and PD-L1 levels were significantly higher in cats with HER2-positive (p=0.017; p=0.032) and triple negative (TN) normal-like mammary carcinomas (p=0.004; p=0.015). Besides, cats presenting these tumor subtypes showed a strong positive correlation between serum PD-1, PD-L1, CTLA-4 and TNF-α levels. In tumors, PD-L1 expression in cancer cells was significantly higher in HER2-positive samples than in TN normal-like tumors (p=0.010), as the percentage of PD-L1-positive TILs (p=0.038). Results from the PD-L1 gene sequencing identified two heterozygous mutations in exon 4 (A245T, 3.8%; V252M, 42.3%) and one in exon 5 (T267S, 3.8%). In summary, results support that serum PD-1 and PD-L1 levels can be used as diagnostic biomarkers of HER2-positive and TN normal-like feline mammary carcinomas and suggest that the development of monoclonal antibodies may be a good therapeutic strategy.

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