| 1. |
- Cataldo, Luis Rodrigo, et al.
(author)
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The human batokine EPDR1 regulates β-cell metabolism and function
- 2022
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In: Molecular Metabolism. - : Elsevier BV. - 2212-8778. ; 66
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Journal article (peer-reviewed)abstract
- Objective: Ependymin-Related Protein 1 (EPDR1) was recently identified as a secreted human batokine regulating mitochondrial respiration linked to thermogenesis in brown fat. Despite that EPDR1 is expressed in human pancreatic β-cells and that glucose-stimulated mitochondrial metabolism is critical for stimulus-secretion coupling in β-cells, the role of EPDR1 in β-cell metabolism and function has not been investigated. Methods: EPDR1 mRNA levels in human pancreatic islets from non-diabetic (ND) and type 2 diabetes (T2D) subjects were assessed. Human islets, EndoC-βH1 and INS1 832/13 cells were transfected with scramble (control) and EPDR1 siRNAs (EPDR1-KD) or treated with human EPDR1 protein, and glucose-stimulated insulin secretion (GSIS) assessed by ELISA. Mitochondrial metabolism was investigated by extracellular flux analyzer, confocal microscopy and mass spectrometry-based metabolomics analysis. Results: EPDR1 mRNA expression was upregulated in human islets from T2D and obese donors and positively correlated to BMI of donors. In T2D donors, EPDR1 mRNA levels negatively correlated with HbA1c and positively correlated with GSIS. EPDR1 silencing in human islets and β-cell lines reduced GSIS whereas treatment with human EPDR1 protein increased GSIS. Epdr1 silencing in INS1 832/13 cells reduced glucose- and pyruvate- but not K+-stimulated insulin secretion. Metabolomics analysis in Epdr1-KD INS1 832/13 cells suggests diversion of glucose-derived pyruvate to lactate production and decreased malate-aspartate shuttle and the tricarboxylic acid (TCA) cycle activity. The glucose-stimulated rise in mitochondrial respiration and ATP/ADP-ratio was impaired in Epdr1-deficient cells. Conclusion: These results suggests that to maintain glucose homeostasis in obese people, upregulation of EPDR1 may improve β-cell function via channelling glycolysis-derived pyruvate to the mitochondrial TCA cycle.
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| 2. |
- Green, Alanna C., et al.
(author)
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Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells
- 2023
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In: Nature Metabolism. - : Springer Nature. - 2522-5812. ; 5:4, s. 642-659
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Journal article (peer-reviewed)abstract
- Cancer cells fuel their increased need for nucleotide supply by upregulating one-carbon (1C) metabolism, including the enzymes methylenetetrahydrofolate dehydrogenase–cyclohydrolase 1 and 2 (MTHFD1 and MTHFD2). TH9619 is a potent inhibitor of dehydrogenase and cyclohydrolase activities in both MTHFD1 and MTHFD2, and selectively kills cancer cells. Here, we reveal that, in cells, TH9619 targets nuclear MTHFD2 but does not inhibit mitochondrial MTHFD2. Hence, overflow of formate from mitochondria continues in the presence of TH9619. TH9619 inhibits the activity of MTHFD1 occurring downstream of mitochondrial formate release, leading to the accumulation of 10-formyl-tetrahydrofolate, which we term a ‘folate trap’. This results in thymidylate depletion and death of MTHFD2-expressing cancer cells. This previously uncharacterized folate trapping mechanism is exacerbated by physiological hypoxanthine levels that block the de novo purine synthesis pathway, and additionally prevent 10-formyl-tetrahydrofolate consumption for purine synthesis. The folate trapping mechanism described here for TH9619 differs from other MTHFD1/2 inhibitors and antifolates. Thus, our findings uncover an approach to attack cancer and reveal a regulatory mechanism in 1C metabolism.
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| 3. |
- Hodek, Ondrej, et al.
(author)
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Mixed-mode chromatography-mass spectrometry enables targeted and untargeted screening of carboxylic acids in biological samples
- 2022
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In: Analytical Methods. - : Royal Society of Chemistry (RSC). - 1759-9660 .- 1759-9679. ; 14, s. 1015-1022
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Journal article (peer-reviewed)abstract
- Carboxylic acids are crucial metabolites in the tricarboxylic acid (TCA) cycle and thus participate in central carbon metabolism (CCM). Research dependent on the analysis of metabolites involved in central carbon metabolism requires fast separation and sensitive detection of carboxylic acids using liquid chromatography-mass spectrometry (LC-MS). However, successful separation of all carboxylic acids from the TCA cycle by liquid chromatography remains a challenging task because of their high polarity and thus low retention on the conventional reversed-phase columns. In this study, we tested a reversed-phase/anion exchange mixed-mode stationary phase (Waters BEH C-18 AX) using liquid chromatography-tandem mass spectrometry (LC-MS/MS). We developed and optimized a method that enables a 10 minute separation of all carboxylic acids from the TCA cycle and lactic acid without prior derivatization or addition of ion-pair reagents in the mobile phase. The developed method was validated for quantification of 8 acids in murine brown preadipocytes, 5 acids in human plasma and 6 acids in Arabidopsis thaliana leaves with limits of quantification ranging from 0.1 mu M for malic acid to 10 mu M for isocitric acid. Moreover, the mixed-mode chromatography enabled untargeted screening of medium- to long-chain fatty acids in murine brown preadipocytes, Arabidopsis thaliana, and human plasma, where 23 fatty acids were identified by using liquid chromatography with high-resolution mass spectrometry (HRMS).
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| 4. |
- Hodek, Ondrej, et al.
(author)
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Structural elucidation of 3-nitrophenylhydrazine derivatives of tricarboxylic acid cycle acids and optimization of their fragmentation to boost sensitivity in liquid chromatography-mass spectrometry
- 2023
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In: Journal of Chromatography B. - 1570-0232 .- 1873-376X. ; 1222
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Journal article (peer-reviewed)abstract
- Carboxylic acids participate in many metabolic pathways including tricarboxylic acid (TCA) cycle. Therefore, there have been ongoing attempts to develop sensitive liquid chromatography-mass spectrometry methods over the last decades. Derivatization of the carboxylic acids with 3-nitrophenylhydrazine presents a well-established methodology, and yet the derivatized species of polycarboxylic acids and their fragmentation in collision-induced dissociation have not been fully studied before. In our study, we elucidated how annotation of most abundant 3-nitrophenylhydrazine derivatives and optimization of their fragmentation in multiple reaction monitoring can boost the sensitivity, especially for polycarboxylic acids. Finally, the optimized liquid chromatography-tandem mass spectrometry method allowed for low detection limits ranging from 10 pM for 2-oxoglutaric acid to 800 pM for pyruvic acid. All TCA carboxylates were quantified in 20 mu L of human plasma and the targeted method was validated in the same matrix. The same methodology with a modified gradient elution was also applied to untargeted screening of fatty acids by using high-resolution mass spectrometry enabling identification of 29 medium-to long-chain fatty acids in human plasma. The TCA carboxylates were also quantified in 105 of C2C12 mouse myuotube cells grown under different treatments to proof applicability of the methodology to biological studies in a wider sense. However, unfortunately all the TCA carboxylates were also found in the derivatized blanks in substantial amounts, which prevents from using the methodology for quan-tification of the carboxylates in less than 105 cells.
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| 5. |
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| 6. |
- Wang, Wei, et al.
(author)
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Sucrose synthase activity is not required for cellulose biosynthesis in Arabidopsis
- 2022
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In: Plant Journal. - : Wiley. - 0960-7412 .- 1365-313X. ; 110, s. 1493-1497
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Journal article (peer-reviewed)abstract
- Biosynthesis of plant cell walls requires UDP-glucose as the substrate for cellulose biosynthesis, and as an intermediate for the synthesis of other matrix polysaccharides. The sucrose cleaving enzyme sucrose synthase (SUS) is thought to have a central role in UDP-glucose biosynthesis, and a long-held and much debated hypothesis postulates that SUS is required to supply UDP-glucose to cellulose biosynthesis. To investigate the role of SUS in cellulose biosynthesis of Arabidopsis thaliana we characterized mutants in which four or all six Arabidopsis SUS genes were disrupted. These sus mutants showed no growth phenotypes, vascular tissue cell wall defects, or changes in cellulose content. Moreover, the UDP-glucose content of rosette leaves of the sextuple sus mutants was increased by approximately 20% compared with wild type. It can thus be concluded that cellulose biosynthesis is able to employ alternative UDP-glucose biosynthesis pathway(s), and thereby the model of SUS requirements for cellulose biosynthesis in Arabidopsis can be refuted.
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