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.
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.
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.
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.
•EPDR1 expression is upregulated in pancreatic islets from obese and T2D donors.•EPDR1 positively regulates insulin secretion in human pancreatic islets and β-cell lines.•EPDR1 promotes the coupling of glycolisis to mitochondrial TCA cycle.•EPDR1 is required for the glucose-stimulated increases of cytosolic ATP/ADP-ratio.
•3-NPH derivatization of tricarboxylates forms mostly doubly derivatized species.•Isomers of 3-NPH derivatives efficiently separated on HSS-T3 column.•Quantification of TCA carboxylates in human ...plasma and C2C12 myotubes.•All 3-NPH derivatives detected in derivatized blanks.
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 µL 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 quantification of the carboxylates in less than 105 cells.
This work focuses on the preparation and application of supramolecular structures based on mono-cinnamyl-α-cyclodextrins (Cin-α-CD). Pure regioisomers of Cin-α-CD having the cinnamyl moiety at the ...2-O- or at the 3-O-position, respectively, were prepared, characterized and applied in capillary electrophoresis as additives to the background electrolyte. These new monomer units with a potential to self-organize into supramolecular structures were synthesized via a straightforward one-step synthetic procedure and purified using preparative reversed-phase chromatography allowing a large scale separation of the regioisomers. The ability of the monomers to self-assemble was proved by various methods including NMR spectroscopy and dynamic light scattering (DLS). The light scattering experiments showed that the monomer units have distinguishable ability to form supramolecular structures in different solvents and the size distribution of the aggregates in water can be easily modulated using different external stimuli, such as temperature or competitive guest molecules. The obtained results indicated that the two regioisomers of Cin-α-CD formed different supramolecular assemblies highlighting the fact that the position of the cinnamyl group plays an important role in the intermolecular complex formation.
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
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
leaves with limits of quantification ranging from 0.1 μM for malic acid to 10 μM for isocitric acid. Moreover, the mixed-mode chromatography enabled untargeted screening of medium- to long-chain fatty acids in murine brown preadipocytes,
, and human plasma, where 23 fatty acids were identified by using liquid chromatography with high-resolution mass spectrometry (HRMS).
Pancreatic α-amylase plays an important role in dietary starch hydrolysis in the small intestine and participates in enhanced glucose concentration after meals. It seems to be a problem for diabetic ...patients, who suffer from longer postprandial hyperglycemia after meal consumption than healthy people. There are commercially available drugs that inhibit α-amylase and thus reduce the postprandial hyperglycemia effect. However, these drugs may cause severe side effects. Conversely, some naturally occurring flavonoids were suggested to have an α-amylase-inhibiting effect without any side effects. There had been no rapid, undemanding method in terms of sample and reagent preparation that would enable screening of many potential inhibitors. Therefore, we developed an online capillary electrophoresis method to monitor α-amylase activity in the presence of an inhibitor. Each reaction constituent was introduced separately, directly into a capillary where the reagents were mixed by diffusion, which resulted in a 5-min analysis including conditioning of the capillary. We applied the method to test the inhibitory effect of flavonoid standards and their mixture and we investigated the inhibitory effect of ethanolic extract from
Betula pendula
bark. The developed method presents a faster and less expensive alternative to previously described offline methods.
Graphical abstract
Online CE screening of α-amylase inhibitors
In contrast to inorganic nitrogen (N) assimilation, the role of organic N forms, such as proteins and peptides, as sources of N and their impact on plant metabolism remains unclear. Simultaneously, ...organic biostimulants are used as priming agents to improve plant defense response. Here, we analysed the metabolic response of tobacco plants grown in vitro with casein hydrolysate or protein. As the sole source of N, casein hydrolysate enabled tobacco growth, while protein casein was used only to a limited extent. Free amino acids were detected in the roots of tobacco plants grown with protein casein but not in the plants grown with no source of N. Combining hydrolysate with inorganic N had beneficial effects on growth, root N uptake and protein content. The metabolism of casein‐supplemented plants shifted to aromatic (Trp), branched‐chain (Ile, Leu, Val) and basic (Arg, His, Lys) amino acids, suggesting their preferential uptake and/or alterations in their metabolic pathways. Complementarily, proteomic analysis of tobacco roots identified peptidase C1A and peptidase S10 families as potential key players in casein degradation and response to N starvation. Moreover, amidases were significantly upregulated, most likely for their role in ammonia release and impact on auxin synthesis. In phytohormonal analysis, both forms of casein influenced phenylacetic acid and cytokinin contents, suggesting a root system response to scarce N availability. In turn, metabolomics highlighted the stimulation of some plant defense mechanisms under such growth conditions, that is, the high concentrations of secondary metabolites (e.g., ferulic acid) and heat shock proteins.
Fermentation of undigested carbohydrates and amino acids in the large intestine leads to the production of short-chain fatty acids (SCFAs). Increasing attention has been recently paid to ...investigating the effect of the presence of SCFAs in the mammalian large intestine on the colon health. Thus, clinical practice requires analytical methods capable of fast, reliable and inexpensive quantification of SCFAs. Capillary electrophoresis meets such requirements, and hence a capillary electrophoresis method was developed to quantify acetate, propionate, butyrate and isobutyrate in human and canine feces. Benzoic acid in the background electrolyte was selected as a suitable indirect probe for indirect UV-VIS detection. A considerable advantage of the proposed method is the determination of butyric acid as well as isobutyric acid in one analytical run. The separation of these isomers was achieved through addition of α-cyclodextrin to the background electrolyte. The optimized method offers quantification of acetate at concentrations above 10 μM, and propionate, butyrate and isobutyrate at concentrations above 8 μM with acceptable precision, accuracy and with no significant matrix effects.
A capillary electrophoresis method enables quantification of acetate, propionate, isobutyrate and butyrate in human and canine feces.
In this study, we optimized a method for the determination of free amino acids in
Nicotiana tabacum
leaves. Capillary electrophoresis with contactless conductivity detector was used for the ...separation of 20 proteinogenic amino acids in acidic background electrolyte. Subsequently, the conditions of extraction with HCl were optimized for the highest extraction yield of the amino acids because sample treatment of plant materials brings some specific challenges. Central composite face-centered design with fractional factorial design was used in order to evaluate the significance of selected factors (HCl volume, HCl concentration, sonication, shaking) on the extraction process. In addition, the composite design helped us to find the optimal values for each factor using the response surface method. The limits of detection and limits of quantification for the 20 proteinogenic amino acids were found to be in the order of 10
−5
and 10
−4
mol l
−1
, respectively. Addition of acetonitrile to the sample was tested as a method commonly used to decrease limits of detection. Ambiguous results of this experiment pointed out some features of plant extract samples, which often required specific approaches. Suitability of the method for metabolomic studies was tested by analysis of a real sample, in which all amino acids, except for
L
-methionine and
L
-cysteine, were successfully detected. The optimized extraction process together with the capillary electrophoresis method can be used for the determination of proteinogenic amino acids in plant materials. The resulting inexpensive, simple, and robust method is well suited for various metabolomic studies in plants. As such, the method represents a valuable tool for research and practical application in the fields of biology, biochemistry, and agriculture.
SUMMARY
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.
Significance Statement
Many models of cellulose biosynthesis in plants imply that sucrose synthase channels the substrate UDP‐glucose to the plasma membrane localized cellulose synthase complex. Here we used sextuple Arabidopsis mutants devoid of sucrose synthase activity to test this hypothesis directly. The results show that sucrose synthase is not required for cellulose biosynthesis in Arabidopsis.
All four isomeric series of novel 4‐substituted pyrido‐fused 7‐deazapurine ribonucleosides possessing the pyridine nitrogen atom at different positions were designed and synthesized. The total ...synthesis of each isomeric fused heterocycle through multistep heterocyclization was followed by glycosylation and derivatization at position 4 by cross‐coupling reactions or nucleophilic substitutions. All compounds were tested for cytostatic and antiviral activity. The most active were pyrido4′,3′:4,5pyrimidine nucleosides bearing MeO, NH2, MeS, or CH3 groups at position 4, which showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism involved activation by phosphorylation and incorporation to DNA where the presence of the modified ribonucleosides causes double‐strand breaks and apoptosis.
Isomers matter: All four isomeric series of novel 4‐substituted pyrido‐fused 7‐deazapurine ribonucleosides possessing pyridine nitrogen at different positions were designed, synthesized, and tested for cytostatic and antiviral activity. The most active showed submicromolar cytotoxic effects and good selectivity for cancer cells. The mechanism of action was also investigated.
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