Changes In Expression Of Enzymes Research Articles

Exploring neuron-specific steroid synthesis and DHEAS therapy in Alzheimer's disease

Alzheimer's Disease (AD) is a neurodegenerative disorder characterized by cognitive decline and memory loss. Recent studies have suggested a potential role for steroid synthesis in AD pathology. This study investigated the co-localization of steroidogenic enzymes in neuronal cells, changes in enzyme expression in an AD mouse model, and steroid expressions in human AD samples. Additionally, we conducted a steroidomic metabolomics analysis and evaluated the effects of dehydroepiandrosterone sulfate (DHEAS) treatment in an AD mouse model. Immunofluorescence analysis revealed significant co-localization of cytochrome P450 family 17 subfamily A member 1 (CYP17A1) and steroidogenic acute regulatory protein (StAR) proteins with α-synuclein in presynaptic neurons, suggesting active steroid synthesis in these cells. Conversely, such co-localization was absent in astrocytes. In the AD mouse model, a marked decrease in the expression of steroidogenic enzymes (Cyp11a1, Cyp17a1, Star) was observed, especially in areas with amyloid beta plaque accumulation. Human AD and MS brain tissues showed similar reductions in StAR and CYP17A1 expressions. Steroidomic analysis indicated a downregulation of key steroids in the serum of AD patients. DHEAS treatment in AD mice resulted in improved cognitive function and reduced Aβ accumulation. Our findings indicate a neuron-specific pathway for steroid synthesis, potentially playing a crucial role in AD pathology. The reduction in steroidogenic enzymes and key steroids in AD models and human samples suggests that impaired steroid synthesis is a feature of neurodegenerative diseases. The therapeutic potential of targeting steroid synthesis pathways, as indicated by the positive effects of DHEAS treatment, warrants further investigation.

Abstract 17042: Altered Myocardial Energy Substrate Utilization in Metabolic Syndrome May Impact Pathways Involved in Contractile Dysfunction in Ischemic Myocardium

Introduction: Metabolic syndrome (MetS) is increasingly prevalent and an independent risk factor for ischemic heart disease (IHD). MetS favors lipids over glucose as the predominant myocardial energy substrate. Similarly, in IHD, energy utilization is dependent on ketone bodies. Evidence is emerging that such changes in cardiac energy metabolism may drive contractile dysfunction, but the underlying processes have not been clarified in the context of coincident MetS and ischemia. Aims: The purpose of this multiomics study was to define the global metabolic state of ischemic myocardium in the setting of MetS, with a focus on energy metabolism and pathways modulating myocardial function and contractility. We hypothesized MetS would further shift substrate utilization away from glycolysis to favor ketone bodies, contributing to pathways of contractile dysfunction in injured myocardium. Methods: We used our well-established porcine model of diet-induced MetS and chronic myocardial ischemia to study effects on left ventricular tissue. Tissue samples from MetS swine and controls were analyzed using targeted LC-MS/MS for polar metabolites, RNA-seq, proteomics, and computational nonnegative matrix factorization. Results: MetS swine had altered bioavailability of 302 polar metabolites together with changes in gene expression of enzymes involved in glycolysis, fatty acid metabolism, ketone body utilization, and abnormal activation of renin-angiotensin-aldosterone system (RAAS). MetS swine also had reduced expression of Apelin protein, a potent stimulator of cardiac contractility (Figure). Conclusions: Our data show MetS enzymatically downregulates glycolysis and upregulates ketone body utilization together with derangement in RAAS in injured myocardium. Decreased Apelin expression may contribute to lipid-induced cardiac dysfunction through maladaptive effects on contractility and remodeling, and thus may serve as a future therapeutic target.

P-299 Influence of one carbon metabolism supplements over developmental dynamics and gene expression of genes related to DNA methylation machinery in mice

Abstract Study question Does betaine and the active metabolite 5-MTHF influence cleavage dynamics and expression of genes related to methylation during preimplantation embryos? Summary answer Supplementation of betaine and 5-MTHF had no influence over morphokinetics of mice preimplantation embryos but induced transcriptional variations over methylation activity related-genes. What is known already After fertilization, the emerging genome is reprogrammed to enable the control of cellular differentiation all over downstream events and lead further developmental progression. One of the most determining processes defining these new genomic states in cell daughter is mediated by methylation. S-adenosylmethionine (SAM) is the main donor of methyl groups to DNA and its availability during highly active conditions can be sensitive to external conditions and dependent on one carbon metabolism (OCM). DNA methyltransferases (DNMT1, DNMT3a and DNTM3b) and methionine adenosyltransferase (MAT2a and MAT2b) are influenced by OCM and key components of DNA methylation machinery. Study design, size, duration This is a prospective experimental study conducted from November 2022 to January 2023. F1 hybrid (B6/CBA) fresh mice embryos were exposed to different culture conditions. A primary analysis consisted of the morphokinetics assessment of embryo development through time-lapse system up to expanded blastocyst (105 hours), including a score assessment based on blastocyst appearance. Subsequently functional analysis based on gene expression of selected genes was carried out over individual embryos. Participants/materials, setting, methods The zygotes were collected from the oviduct, washed and culture in KSOM supplemented with 5-MTHF and betaine at respectively concentrations of 50μM and 50μg/mL (THF/bet(50)), 10μM and 10μg/mL (THF/bet(10)) and no supplements for control. Geri incubator was used for culture at 37 °C, 6%CO2 and 5%O2. In a new assay, 4cells and 8cells embryos exposed to THF/bet(50) and control conditions were individually collected for pre-amplification and subsequent RT-qPCR quantification using GADPH as housekeeping gene. Main results and the role of chance A total of 54 mice embryos were used for the present study. The timings required for embryos to reach different developmental stages (t2, t4, t8 and tB) were manually selected and their comparison did not show any overall differences. A slightly faster trend could be observed for the THF/bet(50) group during the 3 first cleavage events (t2, t4 and t8), this group showed proportionally better morphology of blastocysts. Expression analysis showed no variation for DNMT1; by contrast, greatest changes were observed for DNMT3a and DNMT3b whose relative expressions were downregulated at 4 cells ∼0.5 fold-changes (p < 0.01) and highly increased for more than 2 fold-change in 8 cells in THF/bet(50) group. In addition, early downregulation at 4 cells was observed for either MAT2a and MAT2b with ∼0.5 fold-changes (p < 0.01); however, at 8 cells levels were reestablished in both similar levels than control. One carbon metabolism induced changes in gene expression of enzymes involved in de novo methylation of DNA DNMT3a and DNMT3a. In addition, Methyl adenyl transferases MAT2a and MAT2b were also influenced, particularly MAT2a which has a pivotal role relevant for genome activation in the embryo. Limitations, reasons for caution The translational approach is still limited due to known differences between kinetics of mice and human embryo. Intracellular concentration of folates and betaine in embryos are still unknown and the range of dosage used has been decided accordingly to previous studies showing a response in mice embryos under similar conditions. Wider implications of the findings Folate supplements are widely accepted for pregnant and women attempting to become pregnant, these are also recently incorporated in some IVF mediums. The influence of OCM in surrounding environment during highly active methylation activities of early embryo is still unknown, benefits or risks of these supplies require further assessment. Trial registration number not applicable

Association of histo-blood group antigens and predisposition to gastrointestinal diseases.

Infectious gastroenteritis is a common illness afflicting people worldwide. The two most common etiological agents of viral gastroenteritis, rotavirus and norovirus are known to recognize histo-blood group antigens (HBGAs) as attachment receptors. ABO, Lewis, and secretor HBGAs are distributed abundantly on mucosal epithelia, red blood cell membranes, and also secreted in biological fluids, such as saliva, intestinal content, milk, and blood. HBGAs are fucosylated glycans that have been implicated in the attachment of some enteric pathogens such as bacteria, parasites, and viruses. Single nucleotide polymorphisms in the genes encoding ABO (H), fucosyltransferase gene FUT2 (Secretor/Se), FUT3 (Lewis/Le) have been associated with changes in enzyme expression and HBGAs production. The highly polymorphic HBGAs among different populations and races influence genotype-specific susceptibility or resistance to enteric pathogens and its epidemiology, and vaccination seroconversion. Therefore, there is an urgent need to conduct population-based investigations to understand predisposition to enteric infections and gastrointestinal diseases. This review focuses on the relationship between HBGAs and predisposition to common human gastrointestinal illnesses caused by viral, bacterial, and parasitic agents.

Abstract P1099: Evidence Of Sialylation Pathway Alterations In Peripheral Blood Of Brugada Syndrome Patients

Brugada syndrome (BrS) is a cardiac arrhythmia associated with a higher risk of SCD. BrS is considered a genetic disorder, and the most commonly mutated gene is SCN5A, encoding the alpha subunit of the voltage-gated cardiac sodium channel (NaV1.5). Mutations of SCN5A usually lead to impairment of NaV1.5 functionality, which is considered the main mechanism of the disease. However, SCN5A mutations are responsible for only 30% of BrS cases. Therefore, it is conceivable that other mechanisms such as post-translational modifications (PTMs) affect NaV1.5 activity. Among others, sialylation may alter ion channel activity by carrying a sugar with a negative charge. Alterations in sialylation have been described in several cardiovascular diseases, as myocardial infarction, Chagas disease, and congenital disorders of glycosylation. For these reasons, the aim was to investigate changes in sialylation in BrS patients to obtain new information on the pathogenesis of BrS. Peripheral blood mononuclear cells (PBMCs) were collected from BrS patients and healthy controls. SNA lectin, a sialic acid-binding protein, was used to characterize the protein sialylation status of PBMCs by Western blot and flow cytometry. Gene expression of enzymes involved in the sialic acid pathway was also examined. The results showed a significant decrease in intracellular and extracellular protein sialylation levels in BrS PBMCs compared with controls. In addition, changes in gene expression of enzymes involved in the sialic acid pathway were detected. Moreover, these changes correlated with clinical parameters associated with phenotypic expression of the disease, such as arrhythmogenic BrS substrate area and potential duration. These findings support that BrS should be considered a systemic disease and are consistent with the presence of overlapping non-cardiac pathologies, as epilepsy, cancer, diabetes, skeletal muscle channelopathies, and laminopathies in BrS patients. Moreover, the discovery that molecular alterations can be found in the peripheral blood of BrS patients supports the existence of a biomarker of the disease. It is a challenge to develop an effective diagnostic test to screen broadly for BrS. In this direction, a multiomic study is ongoing in our center.

Differences in Cellular Metabolism and Metabolic Regulation between Non‐diabetic and Diabetic Human Coronary Artery Endothelial Cells

ObjectiveTo determine the differences in cellular metabolism and metabolic regulation in non‐diabetic and diabetic human coronary artery endothelial cells (HCAEC and DM‐HCAEC) to further the understanding of the effects of metabolic syndrome on endothelial dysfunction in the setting of cardiovascular disease.MethodsBoth HCAEC (n = 5) and DM‐HCAEC (n = 5) were cultured to passage 6. The cells were then starved for 18 hours. After treatment, a Reactive Oxygen Species (ROS) Assay was performed, and cell lysates were made for proteomics analysis and western blotting.ResultsThe ROS assay demonstrated that the total ROS was significantly higher in DM‐HCAEC (p<0.0001) than in HCAEC. Proteomics analysis and western blotting showed that DM‐HCAECs had significantly altered cellular transport, RNA processing, protein biogenesis, and antioxidant pathways. Proteomics analysis demonstrated that there were about 1,500 proteins significantly upregulated or downregulated in DM‐HCAEC compared to HCAEC. Analysis using Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed a significant increase in the “vesicle‐mediated transport” pathway and significant decrease in the “mRNA metabolic process” pathway in DM‐HCAEC with false discovery rates (FDR) of 1.40 x 10‐58 and 4.90 x 10‐92, respectively. Western blot analysis showed a significant changes in phosphorylated AMPK/total AMPK, phosphorylated mTOR (serine 2481), total mTOR, and several enzymes responsible for the regulation or elimination of ROS (Figure 1). The increased levels of AMPK activation and dysregulation of mTOR may be due to the excessive ROS in DM‐HCAEC, which are consistent with the ROS assay findings and significant changes in expression of enzymes that control ROS levels. Further research is needed to determine the mechanisms of downstream effects.ConclusionsAMPK and mTOR are key regulators of energy homeostasis, cellular growth and metabolism, and are altered in diabetes in the setting of increased oxidative stress. These findings will guide further experiments to assess the effect of metabolic syndrome and diabetes on therapeutics targeted at improving the outcomes of both acute and chronic myocardial ischemia. This also underlines the need for further development of therapies or the use of current therapies to address the metabolic disturbances found in diabetes to improve clinical outcomes in cardiovascular disease.

Epigenetic Modifier Supplementation Improves Mitochondrial Respiration and Growth Rates and Alters DNA Methylation of Bovine Embryonic Fibroblast Cells Cultured in Divergent Energy Supply.

Epigenetic modifiers (EM; methionine, choline, folate, and vitamin B12) are important for early embryonic development due to their roles as methyl donors or cofactors in methylation reactions. Additionally, they are essential for the synthesis of nucleotides, polyamines, redox equivalents, and energy metabolites. Despite their importance, investigation into the supplementation of EM in ruminants has been limited to one or two epigenetic modifiers. Like all biochemical pathways, one-carbon metabolism needs to be stoichiometrically balanced. Thus, we investigated the effects of supplementing four EM encompassing the methionine–folate cycle on bovine embryonic fibroblast growth, mitochondrial function, and DNA methylation. We hypothesized that EM supplemented to embryonic fibroblasts cultured in divergent glucose media would increase mitochondrial respiration and cell growth rate and alter DNA methylation as reflected by changes in the gene expression of enzymes involved in methylation reactions, thereby improving the growth parameters beyond Control treated cells. Bovine embryonic fibroblast cells were cultured in Eagle’s minimum essential medium with 1 g/L glucose (Low) or 4.5 g/L glucose (High). The control medium contained no additional OCM, whereas the treated media contained supplemented EM at 2.5, 5, and 10 times (×2.5, ×5, and ×10, respectively) the control media, except for methionine (limited to ×2). Therefore, the experimental design was a 2 (levels of glucose) × 4 (levels of EM) factorial arrangement of treatments. Cells were passaged three times in their respective treatment media before analysis for growth rate, cell proliferation, mitochondrial respiration, transcript abundance of methionine–folate cycle enzymes, and DNA methylation by reduced-representation bisulfite sequencing. Total cell growth was greatest in High ×10 and mitochondrial maximal respiration, and reserve capacity was greatest (p < 0.01) for High ×2.5 and ×10 compared with all other treatments. In Low cells, the total growth rate, mitochondrial maximal respiration, and reserve capacity increased quadratically to 2.5 and ×5 and decreased to control levels at ×10. The biological processes identified due to differential methylation included the positive regulation of GTPase activity, molecular function, protein modification processes, phosphorylation, and metabolic processes. These data are interpreted to imply that EM increased the growth rate and mitochondrial function beyond Control treated cells in both Low and High cells, which may be due to changes in the methylation of genes involved with growth and energy metabolism.

Multi-Dimensional Transcriptome Analysis Reveals Modulation of Cholesterol Metabolism as Highly Integrated Response to Brain Injury.

Zebrafish is an attractive model to investigate regeneration of the nervous system. Despite major progress in our understanding of the underlying processes, the transcriptomic changes are largely unknown. We carried out a computational analysis of the transcriptome of the regenerating telencephalon integrating changes in the expression of mRNAs, their splice variants and investigated the putative role of regulatory RNAs in the modulation of these transcriptional changes. Profound changes in the expression of genes and their splice variants engaged in many distinct processes were observed. Differential transcription and splicing are important processes in response to injury of the telencephalon. As exemplified by the coordinated regulation of the cholesterol synthesizing enzymes and transporters, the genome responded to injury of the telencephalon in a multi-tiered manner with distinct and interwoven changes in expression of enzymes, transporters and their regulatory molecules. This coordinated genomic response involved a decrease of the mRNA of the key transcription factor SREBF2, induction of microRNAs (miR-182, miR-155, miR-146, miR-31) targeting cholesterol genes, shifts in abundance of splice variants as well as regulation of long non-coding RNAs. Cholesterol metabolism appears to be switched from synthesis to relocation of cholesterol. Based on our in silico analyses, this switch involves complementary and synergistic inputs by different regulatory principles. Our studies suggest that adaptation of cholesterol metabolism is a key process involved in regeneration of the injured zebrafish brain.

Developmental Programming: Prenatal Bisphenol A Induces Non-Monotonic Changes in Epigenetic Modulators in Metabolic Tissues of Female Sheep

Developmental exposure to endocrine disruptor bisphenol A (BPA) is associated \\with metabolic defects during adulthood in the female sheep. These are characterized by peripheral insulin resistance and increase in negative mediators of insulin sensitivity such as oxidative stress in metabolic tissues, lipotoxicity in liver and muscle and adipocyte hypertrophy in visceral (VAT) and subcutaneous (SAT) adipose tissue. Conceivably, developmental impact of BPA on regulators of insulin sensitivity involves changes in epigenetic machinery and mediated via changes in expression of enzymes that induce covalent modifications of DNA and histone. To determine the impact of prenatal BPA exposure on epigenetic enzymes [DNA methyltransferases (DNMT), histone deacetylases (HDAC), histone acetyl transferase EP300, histone methylases (SUV39H1, SMYD3 and EZH2) and histone demethylase KDM1A], metabolic tissues (liver, muscle, VAT and SAT) were collected from 21-month-old female offspring born to mothers treated with 0, 0.05, 0.5, or 5 mg/kg/day of BPA from days 30-90 of gestation. Data were analyzed by Cohen’s effect size analysis and large magnitude differences (Cohens d>0.8) discussed. In liver, prenatal BPA induced: 1) a decrease in DNMT1 and 3B at all doses and DNMT3A at the highest dose, 2) a decrease in histone deacetylase HDAC3 as opposed to increase in acetylase EP300 at the highest dose, 3) a decrease in SUV39H1 at the two higher doses, and 4) an increase in EZH2 only with 0.5 mg dose. The prenatal BPA-induced changes in muscle include: 1) increases in expression of DNMTs and EP300 at all doses, 2) an increase in SUV39H1 at 0.5 mg dose and EZH2 at 0.05 and 0.5 mg doses, and 3) decreases in SMYD3 at the lowest dose and KDM1A with 0.05 and 5 mg doses. Prenatal BPA treatment also induced depot-specific changes at the adipose tissue level. In the VAT prenatal BPA induced: 1) increases in expression of all DNMTs examined 2) increases in HDAC2 at all doses except HDAC3 only at 0.05 and 0.5mg dose and 3) increases in histone acetylase EP300 at all doses. In SAT BPA induced: 1) decrease in DNMT3A at 0.5mg and increase at 5 mg, 2) decreases in HDAC1 and HDAC2 at the lowest dose, 3) an increase in HDAC3 at the medium dose, and 4) a decrease in EP300 at the lowest dose. Contrasting changes in histone methylation modifying enzymes were also evident between VAT and SAT manifested as increases in SUV39H1 at the two higher doses and SMYD3 at all three doses in the VAT as opposed to decrease in SUV39H1 and SMYD3 at 0.05 and 0.5 mg doses and EZH2 and KDM1A at the lowest dose in the SAT. These findings indicating developmental exposure to BPA induces non-monotonic dose responses in epigenetic modifying enzymes are consistent with the premise that changes in epigenetic machinery underlie the metabolic disruptions induced by prenatal BPA treatment likely accounting for the tissue specific changes in insulin sensitivity. (support by R01-ES-016541)

Short-Chain Fatty Acids Outpace Ketone Oxidation in the Failing Heart

The failing heart is energy starved with impaired oxidation of long-chain fatty acids (LCFAs) at the level of reduced CPT1 (carnitine palmitoyltransferase 1) activity at the outer mitochondrial membrane. Recent work shows elevated ketone oxidation in failing hearts as an alternate carbon source for oxidative ATP generation. We hypothesized that another short-chain carbon source, short-chain fatty acids (SCFAs) that bypass carnitine palmitoyltransferase 1, could similarly support energy production in failing hearts. Cardiac hypertrophy and dysfunction were induced in rats by transverse-aortic constriction (TAC). Fourteen weeks after TAC or sham operation, isolated hearts were perfused with either the 4 carbon, 13C-labeled ketone (D3-hydroxybutyrate) or the 4 carbon, 13C-labeled SCFA butyrate in the presence of glucose and the LCFA palmitate. Oxidation of ketone and SCFA was compared by in vitro 13C nuclear magnetic resonance spectroscopy, as was the capacity for short-chain carbon sources to compensate for impaired LCFA oxidation in the hypertrophic heart. Adaptive changes in enzyme expression and content for the distinct pathways of ketone and SCFA oxidation were examined in both failing rat and human hearts. TAC produced pathological hypertrophy and increased the fractional contributions of ketone to acetyl coenzyme-A production in the tricarboxylic acid cycle (0.60±0.02 sham ketone versus 0.70±0.02 TAC ketone; P<0.05). However, butyrate oxidation in failing hearts was 15% greater (0.803±0.020 TAC SCFA) than ketone oxidation. SCFA was also more readily oxidized than ketone in sham hearts by 15% (0.693±0.020 sham SCFA). Despite greater SFCA oxidation, TAC did not change short-chain acyl coenzyme-A dehydrogenase content. However, failing hearts of humans and the rat model both contain significant increases in acyl coenzyme-A synthetase medium-chain 3 enzyme gene expression and protein content. The increased oxidation of SCFA and ketones occurred at the expense of LCFA oxidation, with LCFA contributing less to acetyl coenzyme-A production in failing hearts perfused with SCFA (0.190±0.012 TAC SCFA versus 0.3163±0.0360 TAC ketone). SCFAs are more readily oxidized than ketones in failing hearts, despite both bypassing reduced CPT1 activity and represent an unexplored carbon source for energy production in failing hearts.

Model Parameterization with Quantitative Proteomics: Case Study with Trehalose Metabolism in Saccharomyces cerevisiae

When Saccharomyces cerevisiae undergoes heat stress it stimulates several changes that are necessary for its survival, notably in carbon metabolism. Notable changes include increase in trehalose production and glycolytic flux. The increase in glycolytic flux has been postulated to be due to the regulatory effects in upper glycolysis, but this has not been confirmed. Additionally, trehalose is a useful industrial compound for its protective properties. A model of trehalose metabolism in S. cerevisiae was constructed using Convenient Modeller, a software that uses a combination of convenience kinetics and a genetic algorithm. The model was parameterized with quantitative omics under standard conditions and validated using data collected under heat stress conditions. The completed model was used to show that feedforward activation of pyruvate kinase by fructose 1,6-bisphosphate during heat stress contributes to the increase in metabolic flux. We were also able to demonstrate in silico that overexpression of enzymes involved in production and degradation of trehalose can lead to higher trehalose yield in the cell. By integrating quantitative proteomics with metabolic modelling, we were able to confirm that the flux increase in trehalose metabolic pathways during heat stress is due to regulatory effects and not purely changes in enzyme expression. The overexpression of enzymes involved in trehalose metabolism is a potential approach to be exploited for trehalose production without need for increasing temperature.

Derangement of hepatic polyamine, folate, and methionine cycle metabolism in cystathionine beta-synthase-deficient homocystinuria in the presence and absence of treatment: Possible implications for pathogenesis

Cystathionine beta-synthase deficient homocystinuria (HCU) is a life-threatening disorder of sulfur metabolism. Our knowledge of the metabolic changes induced in HCU are based almost exclusively on data derived from plasma. In the present study, we present a comprehensive analysis on the effects of HCU upon the hepatic metabolites and enzyme expression levels of the methionine-folate cycles in a mouse model of HCU.HCU induced a 10-fold increase in hepatic total homocysteine and in contrast to plasma, this metabolite was only lowered by approximately 20% by betaine treatment indicating that this toxic metabolite remains unacceptably elevated. Hepatic methionine, S-adenosylmethionine, S-adenosylhomocysteine, N-acetlymethionine, N-formylmethionine, methionine sulfoxide, S-methylcysteine, serine, N-acetylserine, taurocyamine and N-acetyltaurine levels were also significantly increased by HCU while cysteine, N-acetylcysteine and hypotaurine were all significantly decreased. In terms of polyamine metabolism, HCU significantly decreased spermine and spermidine levels while increasing 5′-methylthioadenosine. Betaine treatment restored normal spermine and spermidine levels but further increased 5′-methylthioadenosine.HCU induced a 2-fold induction in expression of both S-adenosylhomocysteine hydrolase and methylenetetrahydrofolate reductase. Induction of this latter enzyme was accompanied by a 10-fold accumulation of its product, 5-methyl-tetrahydrofolate, with the potential to significantly perturb one‑carbon metabolism. Expression of the cytoplasmic isoform of serine hydroxymethyltransferase was unaffected by HCU but the mitochondrial isoform was repressed indicating differential regulation of one‑carbon metabolism in different sub-cellular compartments. All HCU-induced changes in enzyme expression were completely reversed by either betaine or taurine treatment. Collectively, our data show significant alterations of polyamine, folate and methionine cycle metabolism in HCU hepatic tissues that in some cases, differ significantly from those observed in plasma, and have the potential to contribute to multiple aspects of pathogenesis.

Modeling age-dependent developmental changes in the expression of genes involved in citrulline synthesis using pig enteroids.

BackgroundAge‐dependent changes in the intestinal gene expression of enzymes involved in the metabolism of citrulline and arginine are well characterized. Enteroids, a novel ex‐vivo model that recreates the three‐dimensional structure of the intestinal crypt‐villus unit, have shown to replicate molecular and physiological profiles of the intestinal segment from where they originated (“location memory”).ObjectiveThe present study tested the hypothesis that enteroids recapitulate the developmental changes observed in vivo regarding citrulline production in pigs (“developmental memory”).MethodsPreterm (10‐ and 5‐d preterm) and term pigs at birth, together with 7‐ and 35‐d‐old pigs were studied. Gene expression was measured in jejunal samples and in enteroids derived from this segment. Whole body citrulline production was measured by isotope dilution and enteroid citrulline production by accumulation in the media.ResultsWith the exception of arginase I and inducible nitric oxide synthase, all the genes investigated expressed in jejunum were expressed by enteroids. In the jejunum, established markers of development (lactase and sucrase‐isomaltase), as well as genes that code for enzymes involved in the production and utilization of citrulline and arginine, underwent the ontogenic changes described in the literature. However, enteroid expression of these genes, as well as citrulline production, failed to recapitulate the changes observed in vivo.ConclusionsUnder culture conditions used in our study, enteroids derived from jejunal crypts of pigs at different ages failed to replicate the gene expression observed in whole tissue and whole body citrulline production. Additional extracellular cues may be needed to reproduce the age‐dependent phenotype.

Protein O-GlcNAcylation levels are regulated independently of dietary intake in a tissue and time-specific manner during rat postnatal development.

AimMetabolic sources switch from carbohydrates in utero, to fatty acids after birth and then a mix once adults. O‐GlcNAcylation (O‐GlcNAc) is a post‐translational modification considered as a nutrient sensor. The purpose of this work was to assess changes in protein O‐GlcNAc levels, regulatory enzymes and metabolites during the first periods of life and decipher the impact of O‐GlcNAcylation on cardiac proteins.MethodsHeart, brain and liver were harvested from rats before and after birth (D‐1 and D0), in suckling animals (D12), after weaning with a standard (D28) or a low‐carbohydrate diet (D28F), and adults (D84). O‐GlcNAc levels and regulatory enzymes were evaluated by western blots. Mass spectrometry (MS) approaches were performed to quantify levels of metabolites regulating O‐GlcNAc and identify putative cardiac O‐GlcNAcylated proteins.ResultsProtein O‐GlcNAc levels decrease drastically and progressively from D‐1 to D84 (13‐fold, P < .05) in the heart, whereas the changes were opposite in liver and brain. O‐GlcNAc levels were unaffected by weaning diet in any tissues. Changes in expression of enzymes and levels of metabolites regulating O‐GlcNAc were tissue‐dependent. MS analyses identified changes in putative cardiac O‐GlcNAcylated proteins, namely those involved in the stress response and energy metabolism, such as ACAT1, which is only O‐GlcNAcylated at D0.ConclusionOur results demonstrate that protein O‐GlcNAc levels are not linked to dietary intake and regulated in a time and tissue‐specific manner during postnatal development. We have identified by untargeted MS putative proteins with a particular O‐GlcNAc signature across the development process suggesting specific role of these proteins.

Esophageal Squamous Cell Carcinoma Is Accompanied by Local and Systemic Changes in L-arginine/NO Pathway.

The L-arginine/NO pathway holds promise as a source of potential therapy target and biomarker; yet, its status and utility in esophageal squamous cell carcinoma (ESCC) is unclear. We aimed at quantifying pathway metabolites in sera from patients with ESCC (n = 61) and benign conditions (n = 62) using LC-QTOF-MS and enzyme expression in esophageal tumors and matched noncancerous samples (n = 40) using real-time PCR with reference to ESCC pathology and circulating immune/inflammatory mediators, quantified using Luminex xMAP technology. ESCC was associated with elevated systemic arginine and asymmetric dimethylarginine. Citrulline decreased and arginine bioavailability increased along with increasing ESCC advancement. Compared to adjacent tissue, tumors overexpressed ODC1, NOS2, PRMT1, and PRMT5 but had downregulated ARG1, ARG2, and DDAH1. Except for markedly higher NOS2 and lower ODC1 in tumors from M1 patients, the pathology-associated changes in enzyme expression were subtle and present also in noncancerous tissue. Both the local enzyme expression level and systemic metabolite concentration were related to circulating inflammatory and immune mediators, particularly those associated with eosinophils and those promoting viability and self-renewal of cancer stem cells. Metabolic reprogramming in ESCC manifests itself by the altered L-arginine/NO pathway. Upregulation of PRMTs in addition to NOS2 and ODC1 and the pathway link with stemness-promoting cytokines warrants further investigation.

Effect of S-Allyl -L-Cysteine on MCF-7 Cell Line 3-Mercaptopyruvate Sulfurtransferase/Sulfane Sulfur System, Viability and Apoptosis.

The S-Allyl-L-cysteine (SAC) component of aged garlic extract (AGE) is proven to have anticancer, antihepatotoxic, neuroprotective and neurotrophic properties. γ-Cystathionase (CTH), cystathionine β-synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) are involved in H2S/sulfane sulfur endogenous formation from L-cysteine. The aim of the study was to determine the effect of SAC on MCF-7 cells survival and apoptosis, which is a widely known approach to reduce the number of cancer cells. An additional goal of this paper was to investigate the effect of SAC on the activity and expression of enzymes involved in H2S production. The experiments were carried out in the human breast adenocarcinoma cell line MCF-7. Changes in the cell viability were determined by MTT assay. Cell survival was determined by flow cytometry (FC). Changes in enzymes expression were analyzed using Western blot. After 24 h and 48 h incubation with 2245 µM SAC, induction of late apoptosis was observed. A decrease in cell viability was observed with increasing SAC concentration and incubation time. SAC had no significant cytotoxic effect on the MCF-7 cells upon all analyzed concentrations. CTH, MPST and CBS expression were confirmed in non-treated MCF-7 cells. Significant decrease in MPST activity at 2245 µM SAC after 24 h and 48 h incubation vs. 1000 µM SAC was associated with decrease in sulfane sulfur levels. The presented results show promising SAC effects regarding the deterioration of the MCF-7 cells’ condition in reducing their viability through the downregulation of MPST expression and sulfate sulfur level reduction.

Pre-Exposure to Nicotine with Nocturnal Abstinence Induces Epigenetic Changes that Potentiate Nicotine Preference.

Prior exposure to drugs of abuse may facilitate addiction. It has been described that pre-exposure to nicotine can increase or, contrarily, prevent conditioned place preference (CPP). Here, we evaluated the effect of nicotine pre-exposure on CPP performance using an original protocol mimicking smokers' behaviour in zebrafish. We simulated nicotine withdrawal at sleep time by exposing zebrafish to nicotine during daylight but not at night (D/N) for 14days and then performed nicotine-CPP in zebrafish. D/N-nicotine-treated zebrafish obtained the highest CPP score, whereas zebrafish pre-exposed continuously to nicotine did not show nicotine-CPP. Evaluation of locomotor activity, seeking and anxiety-like behaviours supported the CPP findings. Nicotinic receptor subunit gene expression showed significant increases in the brain of zebrafish exposed to nicotine. Zebrafish exposed to D/N-nicotine showed further increases of α6- and α7-subunit expression after CPP establishment. Inhibition of histone acetylation by phenylbutyrate prevented nicotine-CPP. Transcriptional expression of epigenetic enzymes controlling histone acetylation/deacetylation and DNA methylation/demethylation was widely modified in brain portions containing reward areas of zebrafish exposed to D/N-nicotine after CPP. Zebrafish exposed to D/N-nicotine showed high levels of acetylated histone 3 and pCREB immunoreactivity differentially found in nuclei of the dopaminergic reward circuit in zebrafish homologous to the ventral tegmental area, nucleus accumbens and dorsal habenula. Our findings demonstrated that repetitive abstinent periods are risky factors for drug abuse that potentiate nicotine-environment associations and seeking. Brain modifications can persist long after nicotine use and are likely due to changes in the transcriptional expression of enzymes regulating drug reward-related gene expression via epigenetic modifications.

GENETIC AND BIOCHEMICAL ASPECTS OF THE ASSOCIATION OF THE PPD-D1 LOCUS WITH THE RESISTANCE OF WINTER SOFT WHEAT TO LOW TEMPERATURE

The aim of this work was to study the expression of some enzymes under the influence of unfavorable temperature conditions on plants that differ in allelic composition of Ppd genes. To accomplish this goal, the following tasks were set: 1) to carry out qualitative and quantitative analysis of electrophoregrams of multiple forms of antioxidant enzymes (catalase, peroxidase, superoxide dismutase); 2) to establish whether there is a relationship between the spectra of the enzyme under study and the tolerance to low temperatures and allelic composition of the Ppd-D1 gene.Material. Winter wheat varieties: Mironovskaya 808 (recessive alleles of the Ppd-D1 gene) and Tira (the dominant allele of the Ppd-D1 gene). Methods. Electrophoresis. Complex comparative analysis. Methods of descriptive statistics. Computer data processing.Results. It was shown that in varieties that differ in the composition of Ppd-D1 alleles and resistance to hypothermia gene-enzyme systems function in a different, often opposite way. Moreover, the difference was observed both in control conditions and under stress. This indicates that for the formation of adaptation, not only the peculiarities of the reaction of the metabolic reaction to adverse conditions, but also their initial state are important.In Mironovskaya 808 plants, unlike Tire, peroxidase plays a major role in neutralizing peroxide. This is manifested both in higher activity and in features of changes in enzyme expression during hypothermia, as well as in the ratio of peroxidase activity to the activity of other antioxidant enzymes. The leading role of peroxidase, due to its versatility, leads to a finer regulation of cellular metabolism during adaptation. In addition, in Mironovskaya 808, in response to the action of low temperature, the functioning of specific peroxidases, ascorbate peroxidase and ferulic-specific peroxidase, changes in the opposite way, compared to the variety Tira. The expression of such an important antioxidant enzyme as SOD also changes in different ways.Based on the data obtained, it can be assumed that Tira's system of protection against oxidative explosion in stress does not cope with the excess of superoxide radicals formed.

Decreased Expression of CHST-12, CHST-13, and UST in the Proximal Interphalangeal Joint Cartilage of School-Age Children with Kashin-Beck Disease: an Endemic Osteoarthritis in China Caused by Selenium Deficiency.

The objective of this study is to investigate changes in the expression of enzymes involved in chondroitin sulfate (CS) sulfation in distal articular surface of proximal interphalangeal joint isolated from school-age children patients with Kashin-Beck disease (KBD), using normal children as controls. Articular cartilage samples were collected from four normal and four KBD children (7-12years old), and these children were assigned to control and KBD groups. Hematoxylin and eosin (H&E), toluidine blue (TB), and immunohistochemical (IHC) stainings were utilized to evaluate changes in joint pathology and expression of enzymes involved in CS sulfation, including carbohydrate sulfotransferase 12 (CHST-12), carbohydrate sulfotransferase 13 (CHST-13), and uronyl 2-O-sulfotransferase (UST). The correspondence results were examined by semi-quantitative analysis. Compared with the control group, the KBD group showed the following: a significant decrease of total chondrocytes in superficial, middle, and deep layers and deposition of sulfated glycosaminoglycans in extracellular matrix of KBD cartilage were observed; positive staining chondrocytes of CHST-12, CHST-13, and UST were significantly less in superficial zone of KBD cartilage; and CHST-13 positive staining chondrocytes was reduced in deep zone of KBD cartilage. In contrast, the positive staining rates of CHST-12, CHST-13, and UST in KBD were significantly higher than those in the control group. The decreased expression of these enzymes and the physiologic compensatory reaction may be the signs of early-stage KBD. The alterations of CS structure modifying sulfotransferases in finger articular cartilage might play an important role in the onset and pathogenesis of school-age KBD children.

Conjugated estrogens in the endometrium during the estrous cycle in pigs

Estrogen metabolism results in the formation of inactive estrogen sulphates and glucuronides. Despite the lack of receptor binding, circulating conjugated estrogens might serve as a reservoir for the active form through the involvement of specific cleaving enzymes. In order to elucidate the potential role that estrogen conjugates play in the regulation of the estrous cycle, we determined the concentration of progesterone, estrogen and estrogen conjugates in serum and endometrial homogenates of cycling gilts. In addition, we determined the mRNA expression changes of enzymes (UDP glucuronosyltransferase (UGT), β-glucuronidase (GUSB), sulphotransferases (SULT) and steroid sulphatase (STS)) and transporters (multidrug resistance-associated protein (MRP), organic anion-transporting polypeptide (OATPs)) involved in the estrogen metabolism in the endometrium across the estrous cycle. GUSB displayed highest expression at estrous (day 0), decreasing expression during metestrus (day 3 and 6), minimal expression on day 10 and 12, and increasing expression towards proestrus (day 18), suggesting either a stimulation by estrogens or a negative impact of progesterone. The mRNA expression of the influx-transporter OATP1A2 significantly increased from day 0 to 6 and decreased again by day 10, while the efflux-transporters (MRP1, MRP2, and MDR1) displayed minimal expression at day 3 and 6. The mRNA expression of the UDP-glucuronsyltransferases followed a similar pattern, with minimal expression found at day 6. The analyses of the concentration of local and circulating steroid hormones points towards an interaction of the analyzed transporters and enzymes with steroid hormones, thereby possibly regulating the reservoir of active steroids contributing to the endometrial function.