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18 pages, 5769 KiB

Open AccessArticle

A Single LC-MS/MS Analysis to Quantify CoA Biosynthetic Intermediates and Short-Chain Acyl CoAs

by Anthony E. Jones, Nataly J. Arias, Aracely Acevedo, Srinivasa T. Reddy, Ajit S. Divakaruni and David Meriwether

Metabolites 2021, 11(8), 468; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11080468 - 21 Jul 2021

Cited by 9 | Viewed by 4135

Abstract

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify [...] Read more.

Coenzyme A (CoA) is an essential cofactor for dozens of reactions in intermediary metabolism. Dysregulation of CoA synthesis or acyl CoA metabolism can result in metabolic or neurodegenerative disease. Although several methods use liquid chromatography coupled with mass spectrometry/mass spectrometry (LC-MS/MS) to quantify acyl CoA levels in biological samples, few allow for simultaneous measurement of intermediates in the CoA biosynthetic pathway. Here we describe a simple sample preparation and LC-MS/MS method that can measure both short-chain acyl CoAs and biosynthetic precursors of CoA. The method does not require use of a solid phase extraction column during sample preparation and exhibits high sensitivity, precision, and accuracy. It reproduces expected changes from known effectors of cellular CoA homeostasis and helps clarify the mechanism by which excess concentrations of etomoxir reduce intracellular CoA levels. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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15 pages, 3514 KiB

Open AccessArticle

Mitochondrial Fission Governed by Drp1 Regulates Exogenous Fatty Acid Usage and Storage in Hela Cells

by Jae-Eun Song, Tiago C. Alves, Bernardo Stutz, Matija Šestan-Peša, Nicole Kilian, Sungho Jin, Sabrina Diano, Richard G. Kibbey and Tamas L. Horvath

Metabolites 2021, 11(5), 322; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11050322 - 18 May 2021

Cited by 16 | Viewed by 3603

Abstract

In the presence of high abundance of exogenous fatty acids, cells either store fatty acids in lipid droplets or oxidize them in mitochondria. In this study, we aimed to explore a novel and direct role of mitochondrial fission in lipid homeostasis in HeLa [...] Read more.

In the presence of high abundance of exogenous fatty acids, cells either store fatty acids in lipid droplets or oxidize them in mitochondria. In this study, we aimed to explore a novel and direct role of mitochondrial fission in lipid homeostasis in HeLa cells. We observed the association between mitochondrial morphology and lipid droplet accumulation in response to high exogenous fatty acids. We inhibited mitochondrial fission by silencing dynamin-related protein 1(DRP1) and observed the shift in fatty acid storage-usage balance. Inhibition of mitochondrial fission resulted in an increase in fatty acid content of lipid droplets and a decrease in mitochondrial fatty acid oxidation. Next, we overexpressed carnitine palmitoyltransferase-1 (CPT1), a key mitochondrial protein in fatty acid oxidation, to further examine the relationship between mitochondrial fatty acid usage and mitochondrial morphology. Mitochondrial fission plays a role in distributing exogenous fatty acids. CPT1A controlled the respiratory rate of mitochondrial fatty acid oxidation but did not cause a shift in the distribution of fatty acids between mitochondria and lipid droplets. Our data reveals a novel function for mitochondrial fission in balancing exogenous fatty acids between usage and storage, assigning a role for mitochondrial dynamics in control of intracellular fuel utilization and partitioning. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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10 pages, 1304 KiB

Open AccessFeature PaperEditor’s ChoiceArticle

Correcting for Naturally Occurring Mass Isotopologue Abundances in Stable-Isotope Tracing Experiments with PolyMID

by Heesoo Jeong, Yan Yu, Henrik J. Johansson, Frank C. Schroeder, Janne Lehtiö and Nathaniel M. Vacanti

Metabolites 2021, 11(5), 310; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11050310 - 12 May 2021

Cited by 4 | Viewed by 3930

Abstract

Stable-isotope tracing is a method to measure intracellular metabolic pathway utilization by feeding a cellular system a stable-isotope-labeled tracer nutrient. The power of the method to resolve differential pathway utilization is derived from the enrichment of metabolites in heavy isotopes that are synthesized [...] Read more.

Stable-isotope tracing is a method to measure intracellular metabolic pathway utilization by feeding a cellular system a stable-isotope-labeled tracer nutrient. The power of the method to resolve differential pathway utilization is derived from the enrichment of metabolites in heavy isotopes that are synthesized from the tracer nutrient. However, the readout is complicated by the presence of naturally occurring heavy isotopes that are not derived from the tracer nutrient. Herein we present an algorithm, and a tool that applies it (PolyMID-Correct, part of the PolyMID software package), to computationally remove the influence of naturally occurring heavy isotopes. The algorithm is applicable to stable-isotope tracing data collected on low- and high- mass resolution mass spectrometers. PolyMID-Correct is open source and available under an MIT license. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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19 pages, 1113 KiB

Open AccessArticle

Establishment, Validation, and Initial Application of a Sensitive LC-MS/MS Assay for Quantification of the Naturally Occurring Isomers Itaconate, Mesaconate, and Citraconate

by Moritz Winterhoff, Fangfang Chen, Nishika Sahini, Thomas Ebensen, Maike Kuhn, Volkhard Kaever, Heike Bähre and Frank Pessler

Metabolites 2021, 11(5), 270; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11050270 - 26 Apr 2021

Cited by 12 | Viewed by 4041

Abstract

Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that [...] Read more.

Itaconate is derived from the tricarboxylic acid (TCA) cycle intermediate cis-aconitate and links innate immunity and metabolism. Its synthesis is altered in inflammation-related disorders and it therefore has potential as clinical biomarker. Mesaconate and citraconate are naturally occurring isomers of itaconate that have been linked to metabolic disorders, but their functional relationships with itaconate are unknown. We aimed to establish a sensitive high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay for the quantification of itaconate, mesaconate, citraconate, the pro-drug 4-octyl-itaconate, and selected TCA intermediates. The assay was validated for itaconate, mesaconate, and citraconate for intra- and interday precision and accuracy, extended stability, recovery, freeze/thaw cycles, and carry-over. The lower limit of quantification was 0.098 µM for itaconate and mesaconate and 0.049 µM for citraconate in 50 µL samples. In spike-in experiments, itaconate remained stable in human plasma and whole blood for 24 and 8 h, respectively, whereas spiked-in citraconate and mesaconate concentrations changed during incubation. The type of anticoagulant in blood collection tubes affected measured levels of selected TCA intermediates. Human plasma may contain citraconate (0.4–0.6 µM, depending on the donor), but not itaconate or mesaconate, and lipopolysaccharide stimulation of whole blood induced only itaconate. Concentrations of the three isomers differed greatly among mouse organs: Itaconate and citraconate were most abundant in lymph nodes, mesaconate in kidneys, and only citraconate occurred in brain. This assay should prove useful to quantify itaconate isomers in biomarker and pharmacokinetic studies, while providing internal controls for their effects on metabolism by allowing quantification of TCA intermediates. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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15 pages, 4395 KiB

Open AccessArticle

Itaconate Alters Succinate and Coenzyme A Metabolism via Inhibition of Mitochondrial Complex II and Methylmalonyl-CoA Mutase

by Thekla Cordes and Christian M. Metallo

Metabolites 2021, 11(2), 117; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11020117 - 18 Feb 2021

Cited by 30 | Viewed by 5399

Abstract

Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing [...] Read more.

Itaconate is a small molecule metabolite that is endogenously produced by cis-aconitate decarboxylase-1 (ACOD1) in mammalian cells and influences numerous cellular processes. The metabolic consequences of itaconate in cells are diverse and contribute to its regulatory function. Here, we have applied isotope tracing and mass spectrometry approaches to explore how itaconate impacts various metabolic pathways in cultured cells. Itaconate is a competitive and reversible inhibitor of Complex II/succinate dehydrogenase (SDH) that alters tricarboxylic acid (TCA) cycle metabolism leading to succinate accumulation. Upon activation with coenzyme A (CoA), itaconyl-CoA inhibits adenosylcobalamin-mediated methylmalonyl-CoA (MUT) activity and, thus, indirectly impacts branched-chain amino acid (BCAA) metabolism and fatty acid diversity. Itaconate, therefore, alters the balance of CoA species in mitochondria through its impacts on TCA, amino acid, vitamin B₁₂, and CoA metabolism. Our results highlight the diverse metabolic pathways regulated by itaconate and provide a roadmap to link these metabolites to potential downstream biological functions. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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Review

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17 pages, 3223 KiB

Open AccessReview

Coenzyme Q Biosynthesis: An Update on the Origins of the Benzenoid Ring and Discovery of New Ring Precursors

by Lucía Fernández-del-Río and Catherine F. Clarke

Metabolites 2021, 11(6), 385; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11060385 - 14 Jun 2021

Cited by 23 | Viewed by 3316

Abstract

Coenzyme Q (ubiquinone or CoQ) is a conserved polyprenylated lipid essential for mitochondrial respiration. CoQ is composed of a redox-active benzoquinone ring and a long polyisoprenyl tail that serves as a membrane anchor. A classic pathway leading to CoQ biosynthesis employs 4-hydroxybenzoic acid [...] Read more.

Coenzyme Q (ubiquinone or CoQ) is a conserved polyprenylated lipid essential for mitochondrial respiration. CoQ is composed of a redox-active benzoquinone ring and a long polyisoprenyl tail that serves as a membrane anchor. A classic pathway leading to CoQ biosynthesis employs 4-hydroxybenzoic acid (4HB). Recent studies with stable isotopes in E. coli, yeast, and plant and animal cells have identified CoQ intermediates and new metabolic pathways that produce 4HB. Stable isotope labeling has identified para-aminobenzoic acid as an alternate ring precursor of yeast CoQ biosynthesis, as well as other natural products, such as kaempferol, that provide ring precursors for CoQ biosynthesis in plants and mammals. In this review, we highlight how stable isotopes can be used to delineate the biosynthetic pathways leading to CoQ. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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14 pages, 782 KiB

Open AccessEditor’s ChoiceReview

Mitochondrial Lipid Signaling and Adaptive Thermogenesis

by Helaina Von Bank, Mae Hurtado-Thiele, Nanami Oshimura and Judith Simcox

Metabolites 2021, 11(2), 124; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11020124 - 22 Feb 2021

Cited by 20 | Viewed by 3827

Abstract

Thermogenesis is an energy demanding process by which endotherms produce heat to maintain their body temperature in response to cold exposure. Mitochondria in the brown and beige adipocytes play a key role in thermogenesis, as the site for uncoupling protein 1 (UCP1), which [...] Read more.

Thermogenesis is an energy demanding process by which endotherms produce heat to maintain their body temperature in response to cold exposure. Mitochondria in the brown and beige adipocytes play a key role in thermogenesis, as the site for uncoupling protein 1 (UCP1), which allows for the diffusion of protons through the mitochondrial inner membrane to produce heat. To support this energy demanding process, the mitochondria in brown and beige adipocytes increase oxidation of glucose, amino acids, and lipids. This review article explores the various mitochondria-produced and processed lipids that regulate thermogenesis including cardiolipins, free fatty acids, and acylcarnitines. These lipids play a number of roles in thermogenic adipose tissue including structural support of UCP1, transcriptional regulation, fuel source, and activation of cell signaling cascades. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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27 pages, 2779 KiB

Open AccessEditor’s ChoiceReview

Transporters at the Interface between Cytosolic and Mitochondrial Amino Acid Metabolism

by Keeley G. Hewton, Amritpal S. Johal and Seth J. Parker

Metabolites 2021, 11(2), 112; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11020112 - 16 Feb 2021

Cited by 17 | Viewed by 8186

Abstract

Mitochondria are central organelles that coordinate a vast array of metabolic and biologic functions important for cellular health. Amino acids are intricately linked to the bioenergetic, biosynthetic, and homeostatic function of the mitochondrion and require specific transporters to facilitate their import, export, and [...] Read more.

Mitochondria are central organelles that coordinate a vast array of metabolic and biologic functions important for cellular health. Amino acids are intricately linked to the bioenergetic, biosynthetic, and homeostatic function of the mitochondrion and require specific transporters to facilitate their import, export, and exchange across the inner mitochondrial membrane. Here we review key cellular metabolic outputs of eukaryotic mitochondrial amino acid metabolism and discuss both known and unknown transporters involved. Furthermore, we discuss how utilization of compartmentalized amino acid metabolism functions in disease and physiological contexts. We examine how improved methods to study mitochondrial metabolism, define organelle metabolite composition, and visualize cellular gradients allow for a more comprehensive understanding of how transporters facilitate compartmentalized metabolism. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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21 pages, 1982 KiB

Open AccessEditor’s ChoiceReview

L-Carnitine and Acylcarnitines: Mitochondrial Biomarkers for Precision Medicine

by Marc R. McCann, Mery Vet George De la Rosa, Gus R. Rosania and Kathleen A. Stringer

Metabolites 2021, 11(1), 51; https://0-doi-org.brum.beds.ac.uk/10.3390/metabo11010051 - 14 Jan 2021

Cited by 134 | Viewed by 9170

Abstract

Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or [...] Read more.

Biomarker discovery and implementation are at the forefront of the precision medicine movement. Modern advances in the field of metabolomics afford the opportunity to readily identify new metabolite biomarkers across a wide array of disciplines. Many of the metabolites are derived from or directly reflective of mitochondrial metabolism. L-carnitine and acylcarnitines are established mitochondrial biomarkers used to screen neonates for a series of genetic disorders affecting fatty acid oxidation, known as the inborn errors of metabolism. However, L-carnitine and acylcarnitines are not routinely measured beyond this screening, despite the growing evidence that shows their clinical utility outside of these disorders. Measurements of the carnitine pool have been used to identify the disease and prognosticate mortality among disorders such as diabetes, sepsis, cancer, and heart failure, as well as identify subjects experiencing adverse drug reactions from various medications like valproic acid, clofazimine, zidovudine, cisplatin, propofol, and cyclosporine. The aim of this review is to collect and interpret the literature evidence supporting the clinical biomarker application of L-carnitine and acylcarnitines. Further study of these metabolites could ultimately provide mechanistic insights that guide therapeutic decisions and elucidate new pharmacologic targets. Full article

(This article belongs to the Special Issue Mitochondrial Metabolism and Bioenergetics)

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