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1.
PeerJ ; 11: e16335, 2023.
Article in English | MEDLINE | ID: mdl-38025761

ABSTRACT

Hepatocellular carcinoma (HCC) remains a global challenge as it is the sixth most common neoplasm worldwide and the third leading cause of cancer-related death. A key feature of HCC is abnormal metabolism, which promotes cancer cell proliferation, survival, invasion, and metastasis. However, the significance of metabolism-related genes (MRGs) in HCC remains to be elucidated. Here, we aim to establish a novel metabolism-related prognostic signature for the prediction of patient outcomes and to investigate the value of MRG expression in the prognostic prediction of HCC. In our research, a Metabolism-Related Risk Score (MRRS) model was constructed using 14 MRGs (DLAT, SEPHS1, ACADS, UCK2, GOT2, ADH4, LDHA, ME1, TXNRD1, B4GALT2, AK2, PTDSS2, CSAD, and AMD1). The Kaplan-Meier curve confirmed that the MRRS has a high accuracy in predicting the prognosis of HCC patients (p < 0.001). According to the MRRS model, the area under the curve (AUC) values for predicting the prognosis of patients with hepatocellular carcinoma at 1, 3, and 5 years reached 0.829, 0.760, and 0.739, respectively. Functional analyses revealed that signaling pathways associated with the cell cycle were largely enriched by differential genes between high and low-risk groups. In addition, dendritic cells (DCs) (p < 0.001), CD4+ T cells (p < 0.01), CD8+ T cells (p < 0.001), B cells (p < 0.001), neutrophils (p < 0.001), macrophages (p < 0.001) had a higher proportion of infiltrates in high-risk populations. Low GOT2 expression is associated with poor prognosis in patients with hepatocellular carcinoma. Knockdown of GOT2 significantly increased the migration capacity of the Huh7 and MHCC97H hepatocellular carcinoma lines. Our research reveals that GOT2 is negatively related to the survival of patients with hepatocellular carcinoma and GOT2 may contribute to tumor progression by inhibiting the ability of tumor cells to migrate.


Subject(s)
Carcinoma, Hepatocellular , Liver Neoplasms , Humans , Carcinoma, Hepatocellular/genetics , Liver Neoplasms/genetics , Area Under Curve , B-Lymphocytes , Butyryl-CoA Dehydrogenase
2.
Metabolism ; 146: 155661, 2023 09.
Article in English | MEDLINE | ID: mdl-37454871

ABSTRACT

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide; however, the underlying mechanisms remain poorly understood. FAM3D is a member of the FAM3 family; however, its role in hepatic glycolipid metabolism remains unknown. Serum FAM3D levels are positively correlated with fasting blood glucose levels in patients with diabetes. Hepatocytes express and secrete FAM3D, and its expression is increased in steatotic human and mouse livers. Hepatic FAM3D overexpression ameliorated hyperglycemia and steatosis in obese mice, whereas FAM3D-deficient mice exhibited exaggerated hyperglycemia and steatosis after high-fat diet (HFD)-feeding. In cultured hepatocytes, FAM3D overexpression or recombinant FAM3D protein (rFAM3D) treatment reduced gluconeogenesis and lipid deposition, which were blocked by anti-FAM3D antibodies or inhibition of its receptor, formyl peptide receptor 1 (FPR1). FPR1 overexpression suppressed gluconeogenesis and reduced lipid deposition in wild hepatocytes but not in FAM3D-deficient hepatocytes. The addition of rFAM3D restored FPR1's inhibitory effects on gluconeogenesis and lipid deposition in FAM3D-deficient hepatocytes. Hepatic FPR1 overexpression ameliorated hyperglycemia and steatosis in obese mice. RNA sequencing and DNA pull-down revealed that the FAM3D-FPR1 axis upregulated the expression of heterogeneous nuclear ribonucleoprotein U (hnRNP U), which recruits the glucocorticoid receptor (GR) to the promoter region of the short-chain acyl-CoA dehydrogenase (SCAD) gene, promoting its transcription to enhance lipid oxidation. Moreover, FAM3D-FPR1 axis also activates calmodulin-Akt pathway to suppress gluconeogenesis in hepatocytes. In conclusion, hepatocyte-secreted FAM3D activated the FPR1-hnRNP U-GR-SCAD pathway to enhance lipid oxidation in hepatocytes. Under obesity conditions, increased hepatic FAM3D expression is a compensatory mechanism against dysregulated glucose and lipid metabolism.


Subject(s)
Hyperglycemia , Non-alcoholic Fatty Liver Disease , Animals , Humans , Mice , Butyryl-CoA Dehydrogenase/metabolism , Diet, High-Fat , Hepatocytes/metabolism , Heterogeneous-Nuclear Ribonucleoprotein U/metabolism , Hyperglycemia/metabolism , Lipid Metabolism , Lipids , Liver/metabolism , Mice, Inbred C57BL , Mice, Obese , Non-alcoholic Fatty Liver Disease/metabolism , Receptors, Formyl Peptide/metabolism , Receptors, Glucocorticoid/metabolism
3.
Br J Pharmacol ; 180(23): 3024-3044, 2023 12.
Article in English | MEDLINE | ID: mdl-37377111

ABSTRACT

BACKGROUND AND PURPOSE: Our recent studies have shown that flavin adenine dinucleotide (FAD) exerts cardiovascular protective effects by supplementing short-chain acyl-CoA dehydrogenase (SCAD). The current study aimed to elucidate whether riboflavin (the precursor of FAD) could improve heart failure via activating SCAD and the DJ-1-Keap1-Nrf2 signalling pathway. EXPERIMENTAL APPROACH: Riboflavin treatment was given to the mouse transverse aortic constriction (TAC)-induced heart failure model. Cardiac structure and function, energy metabolism and apoptosis index were assessed, and relevant signalling proteins were analysed. The mechanisms underlying the cardioprotection by riboflavin were analysed in the cell apoptosis model induced by tert-butyl hydroperoxide (tBHP). KEY RESULTS: In vivo, riboflavin ameliorated myocardial fibrosis and energy metabolism, improved cardiac dysfunction and inhibited oxidative stress and cardiomyocyte apoptosis in TAC-induced heart failure. In vitro, riboflavin ameliorated cell apoptosis in H9C2 cardiomyocytes by decreasing reactive oxygen species (ROS). At the molecular level, riboflavin significantly restored FAD content, SCAD expression and enzymatic activity, activated DJ-1 and inhibited the Keap1-Nrf2/HO1 signalling pathway in vivo and in vitro. SCAD knockdown exaggerated the tBHP-induced DJ-1 decrease and Keap1-Nrf2/HO1 signalling pathway activation in H9C2 cardiomyocytes. The knockdown of SCAD abolished the anti-apoptotic effects of riboflavin on H9C2 cardiomyocytes. DJ-1 knockdown hindered SCAD overexpression anti-apoptotic effects and regulation on Keap1-Nrf2/HO1 signalling pathway in H9C2 cardiomyocytes. CONCLUSIONS AND IMPLICATIONS: Riboflavin exerts cardioprotective effects on heart failure by improving oxidative stress and cardiomyocyte apoptosis via FAD to stimulate SCAD and then activates the DJ-1-Keap1-Nrf2 signalling pathway.


Subject(s)
Butyryl-CoA Dehydrogenase , Heart Failure , Animals , Mice , Butyryl-CoA Dehydrogenase/metabolism , NF-E2-Related Factor 2/metabolism , Flavin-Adenine Dinucleotide/metabolism , Flavin-Adenine Dinucleotide/pharmacology , Kelch-Like ECH-Associated Protein 1/metabolism , Heart Failure/drug therapy , Heart Failure/metabolism , Oxidative Stress , Apoptosis , Myocytes, Cardiac/metabolism
4.
Eur J Pharmacol ; 954: 175849, 2023 Sep 05.
Article in English | MEDLINE | ID: mdl-37331684

ABSTRACT

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. FAD, a coenzyme of SCAD, participates in the electron transfer of SCAD-catalyzed fatty acid ß-oxidation, which plays a crucial role in maintaining the balance of myocardial energy metabolism. Insufficient riboflavin intake can lead to symptoms similar to short-chain acyl-CoA dehydrogenase (SCAD) deficiency or flavin adenine dinucleotide (FAD) gene abnormality, which can be alleviated by riboflavin supplementation. However, whether riboflavin can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of riboflavin on pathological cardiac hypertrophy and fibrosis. In vitro experiments, riboflavin increased SCAD expression and the content of ATP, decreased the free fatty acids content and improved PE-induced cardiomyocytes hypertrophy and AngⅡ-induced cardiac fibroblasts proliferation by increasing the content of FAD, which were attenuated by knocking down the expression of SCAD using small interfering RNA. In vivo experiments, riboflavin significantly increased the expression of SCAD and the energy metabolism of the heart to improve TAC induced pathological myocardial hypertrophy and fibrosis in mice. The results demonstrate that riboflavin improves pathological cardiac hypertrophy and fibrosis by increasing the content of FAD to activate SCAD, which may be a new strategy for treating pathological cardiac hypertrophy and fibrosis.


Subject(s)
Butyryl-CoA Dehydrogenase , Flavin-Adenine Dinucleotide , Animals , Mice , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Flavin-Adenine Dinucleotide/pharmacology , Riboflavin/pharmacology , Cardiomegaly/pathology , Fatty Acids, Nonesterified , Fibrosis
5.
J Biol Chem ; 299(7): 104853, 2023 07.
Article in English | MEDLINE | ID: mdl-37220854

ABSTRACT

We have investigated the equilibrium properties and rapid-reaction kinetics of the isolated butyryl-CoA dehydrogenase (bcd) component of the electron-bifurcating crotonyl-CoA-dependent NADH:ferredoxin oxidoreductase (EtfAB-bcd) from Megasphaera elsdenii. We find that a neutral FADH• semiquinone accumulates transiently during both reduction with sodium dithionite and with NADH in the presence of catalytic concentrations of EtfAB. In both cases full reduction of bcd to the hydroquinone is eventually observed, but the accumulation of FADH• indicates that a substantial portion of reduction occurs in sequential one-electron processes rather than a single two-electron event. In rapid-reaction experiments following the reaction of reduced bcd with crotonyl-CoA and oxidized bcd with butyryl-CoA, long-wavelength-absorbing intermediates are observed that are assigned to bcdred:crotonyl-CoA and bcdox:butyryl-CoA charge-transfer complexes, demonstrating their kinetic competence in the course of the reaction. In the presence of crotonyl-CoA there is an accumulation of semiquinone that is unequivocally the anionic FAD•- rather than the neutral FADH• seen in the absence of substrate, indicating that binding of substrate/product results in ionization of the bcd semiquinone. In addition to fully characterizing the rapid-reaction kinetics of both the oxidative and reductive half-reactions, our results demonstrate that one-electron processes play an important role in the reduction of bcd in EtfAB-bcd.


Subject(s)
Butyryl-CoA Dehydrogenase , Megasphaera elsdenii , Oxidoreductases , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/metabolism , Electrons , Ferredoxins/metabolism , Kinetics , Megasphaera elsdenii/enzymology , NAD/metabolism , Oxidation-Reduction , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Electron Spin Resonance Spectroscopy , Protein Structure, Tertiary , Models, Molecular
6.
J Hypertens ; 41(5): 775-793, 2023 05 01.
Article in English | MEDLINE | ID: mdl-36883465

ABSTRACT

OBJECTIVES: Short-chain acyl-CoA dehydrogenase (SCAD), a key enzyme in the fatty acid oxidation process, is not only involved in ATP synthesis but also regulates the production of mitochondrial reactive oxygen species (ROS) and nitric oxide synthesis. The purpose of this study was to investigate the possible role of SCAD in hypertension-associated vascular remodelling. METHODS: In-vivo experiments were performed on spontaneously hypertensive rats (SHRs, ages of 4 weeks to 20 months) and SCAD knockout mice. The aorta sections of hypertensive patients were used for measurement of SCAD expression. In-vitro experiments with t-butylhydroperoxide (tBHP), SCAD siRNA, adenovirus-SCAD (MOI 90) or shear stress (4, 15 dynes/cm 2 ) were performed using human umbilical vein endothelial cells (HUVECs). RESULTS: Compared with age-matched Wistar rats, aortic SCAD expression decreased gradually in SHRs with age. In addition, aerobic exercise training for 8 weeks could significantly increase SCAD expression and enzyme activity in the aortas of SHRs while decreasing vascular remodelling in SHRs. SCAD knockout mice also exhibited aggravated vascular remodelling and cardiovascular dysfunction. Likewise, SCAD expression was also decreased in tBHP-induced endothelial cell apoptosis models and the aortas of hypertensive patients. SCAD siRNA caused HUVEC apoptosis in vitro , whereas adenovirus-mediated SCAD overexpression (Ad-SCAD) protected against HUVEC apoptosis. Furthermore, SCAD expression was decreased in HUVECs exposed to low shear stress (4 dynes/cm 2 ) and increased in HUVECs exposed to 15 dynes/cm 2 compared with those under static conditions. CONCLUSION: SCAD is a negative regulator of vascular remodelling and may represent a novel therapeutic target for vascular remodelling.


Subject(s)
Butyryl-CoA Dehydrogenase , Hypertension , Rats , Animals , Mice , Humans , Infant, Newborn , Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Vascular Remodeling , Rats, Inbred SHR , Rats, Wistar , Human Umbilical Vein Endothelial Cells/metabolism , RNA, Small Interfering/metabolism , Mice, Knockout
8.
Int J Med Sci ; 18(16): 3631-3643, 2021.
Article in English | MEDLINE | ID: mdl-34790035

ABSTRACT

Background: Acyl-CoA dehydrogenase short-chain (ACADS) is a crucial enzyme in the fatty acid metabolism pathway located in mitochondria. However, the expression level and prognostic value of ACADS in colorectal cancer (CRC) remain unclear. Methods: The mRNA and protein expression data of ACADS was obtained from The Cancer Genome Atlas (TCGA), Clinical Proteomic Tumor Analysis Consortium (CPTAC), and Oncomine. Prognostic values of ACADS were calculated using Kaplan-Meier survival analysis. Correlations between ACADS and immune infiltration were estimated using TIMER, CIBERSORT, EPIC, quanTIseq, and xCell. The UALCAN and MEXPRESS databases were utilized for Methylation analysis. The co-expression analysis based on mRNA expression and interaction network of ACADS were performed via several online tools. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis on ACADS co-expressed genes were performed using the Metascape. Results: The expression analysis demonstrated that ACADS was down-regulated in CRC tissues compared with paired normal tissue. Expression of ACADS was found to be significantly associated with clinical cancer stages and the consensus molecular subgroups (CMS) constituent ratio in CRC patients. Besides, lower ACADS expression was found to predict poor prognosis and be significantly associated with common immune checkpoint genes and MMR genes in CRC. ACADS expression levels were positively related to B cells, CD4+ T cells, CD8+ T cells, M1 macrophages, neutrophils, and Tregs, while negatively correlated with M0 macrophages, M2 macrophages. The methylation level of ACADS in normal tissues was significantly higher than that in tumor tissues, and several methylation sites were identified. The enrichment analysis suggested the co-expressed genes mainly enriched in cell mitochondrial metabolism. Conclusions: The present study provided multilevel evidences for expression of ACADS in CRC and the function of ACADS in prognostic prediction, immune infiltration, and methylation. ACADS might have the potential as the novel biomarker and therapeutic target in CRC patients.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Butyryl-CoA Dehydrogenase/metabolism , Colorectal Neoplasms/diagnosis , Adenocarcinoma/diagnosis , Adenocarcinoma/genetics , Adenocarcinoma/metabolism , Adenocarcinoma/mortality , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Carcinoma/diagnosis , Carcinoma/genetics , Carcinoma/metabolism , Carcinoma/mortality , Cell Line, Tumor , Chemotaxis, Leukocyte/genetics , Chemotaxis, Leukocyte/immunology , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/mortality , Fatty Acids/metabolism , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Lipid Metabolism/genetics , Predictive Value of Tests , Prognosis , Proteomics , Survival Analysis , Tumor Microenvironment/genetics , Tumor Microenvironment/immunology
9.
FEBS J ; 288(3): 1008-1026, 2021 02.
Article in English | MEDLINE | ID: mdl-32329961

ABSTRACT

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, ß-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces ß-FAD to ß-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of ß-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from ß-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.


Subject(s)
Acidaminococcus/metabolism , Bacterial Proteins/metabolism , Benzoquinones/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Electron-Transferring Flavoproteins/metabolism , Flavins/metabolism , Acyl Coenzyme A/chemistry , Acyl Coenzyme A/metabolism , Bacterial Proteins/chemistry , Benzoquinones/chemistry , Butyryl-CoA Dehydrogenase/chemistry , Electron Transport , Electron-Transferring Flavoproteins/chemistry , Electrons , Ferredoxins/chemistry , Ferredoxins/metabolism , Flavin-Adenine Dinucleotide/chemistry , Flavin-Adenine Dinucleotide/metabolism , Models, Biological , Oxidation-Reduction , Protein Binding , Spectrophotometry
10.
Biochem Pharmacol ; 178: 114100, 2020 08.
Article in English | MEDLINE | ID: mdl-32540485

ABSTRACT

Short-chain acyl-CoA dehydrogenase (SCAD), the rate-limiting enzyme for fatty acid ß-oxidation, has a negative regulatory effect on pathological cardiac hypertrophy and fibrosis. Furthermore, flavin adenine dinucleotide (FAD) can enhance the expression and enzyme activity of SCAD. However, whether FAD can inhibit pathological cardiac hypertrophy and fibrosis remains unclear. Therefore, we observed the effect of FAD on pathological cardiac hypertrophy and fibrosis. FAD significantly inhibited PE-induced cardiomyocyte hypertrophy and AngII-induced cardiac fibroblast proliferation. In addition, FAD ameliorated pathological cardiac hypertrophy and fibrosis in SHR. FAD significantly increased the expression and enzyme activity of SCAD. Meanwhile, ATP content was increased, the content of free fatty acids and reactive oxygen species were decreased by FAD in vivo and in vitro. In addition, molecular dynamics simulations were also used to provide insights into the structural stability and dynamic behavior of SCAD. The results demonstrated that FAD may play an important structural role on the SCAD dimer stability and maintenance of substrate catalytic pocket to increase the expression and enzyme activity of SCAD. In conclusion, FAD can inhibit pathological cardiac hypertrophy and fibrosis through activating SCAD, which may be a novel effective treatment for pathological cardiac hypertrophy and fibrosis, thus prevent them from developing into heart failure.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Cardiomegaly/prevention & control , Cardiotonic Agents/pharmacology , Fibroblasts/drug effects , Flavin-Adenine Dinucleotide/pharmacology , Gene Expression Regulation/drug effects , Adenosine Triphosphate/biosynthesis , Animals , Binding Sites , Butyryl-CoA Dehydrogenase/metabolism , Cardiomegaly/enzymology , Cardiomegaly/genetics , Cardiomegaly/pathology , Cell Proliferation/drug effects , Energy Metabolism/drug effects , Energy Metabolism/genetics , Enzyme Stability , Fatty Acids, Nonesterified/antagonists & inhibitors , Fatty Acids, Nonesterified/metabolism , Fibroblasts/metabolism , Fibroblasts/pathology , Fibrosis , Heart Failure/prevention & control , Male , Molecular Dynamics Simulation , Myocardium/enzymology , Myocardium/pathology , Protein Binding , Protein Conformation, alpha-Helical , Protein Conformation, beta-Strand , Protein Interaction Domains and Motifs , Protein Multimerization , Rats , Rats, Inbred SHR , Rats, Wistar , Reactive Oxygen Species/antagonists & inhibitors , Reactive Oxygen Species/metabolism
11.
Br J Nutr ; 122(3): 241-251, 2019 08 14.
Article in English | MEDLINE | ID: mdl-31475655

ABSTRACT

For the same BMI, South Asians have a higher body fat percentage than Caucasians. There might be differences in the fatty acid (FA) handling in adipose tissue when both ethnicities are exposed to high-fat overfeeding. The objective of the present study was to investigate the molecular adaptation in relation to FA metabolism in response to overfeeding with a high-fat diet (OHFD) in South Asian and Caucasian men. Ten South Asian men (BMI 18-29 kg/m2) and ten Caucasian men (BMI 22-33 kg/m2), matched for body fat percentage, aged 20-40 years were included. A weight-maintenance diet (30 % fat, 55 % carbohydrate and 15 % protein) was given for 3 d followed by 3 d of overfeeding (150 % energy requirement) with a high-fat diet (60 % fat, 25 % carbohydrate and 15 % protein) while staying in a respiration chamber. Before and after overfeeding, abdominal subcutaneous fat biopsies were taken. Proteins were isolated, analysed and quantified for short-chain 3-hydroxyacyl-CoA dehydrogenase (HADH), carnitine palmitoyl-transferase 1α (CPT1a), adipose TAG lipase, perilipin A (PLINA), perilipin B, lipoprotein lipase and fatty acid binding protein 4 using Western blotting. OHFD decreased the HADH level (P < 0·05) in Caucasians more than in Asians (P < 0·05), but the baseline and after intervention HADH level was relatively higher in Caucasians. The level of CPT1a decreased in South Asians and increased in Caucasians (P < 0·05). PLINA did not change with diet but the level was higher in South Asians (P < 0·05). The observed differences in HADH and PLINA levels as well as in CPT1a response may be important for differences in the long-term regulation of energy (fat) metabolism in these populations.


Subject(s)
Adipose Tissue/metabolism , Adiposity , Diet, High-Fat , Energy Intake , Adaptation, Physiological , Adult , Asian People , Biopsy , Body Composition , Body Mass Index , Body Weight , Butyryl-CoA Dehydrogenase/metabolism , Carnitine O-Palmitoyltransferase/metabolism , Dietary Carbohydrates , Dietary Fats , Dietary Proteins , Energy Metabolism , Exercise , Fatty Acids/metabolism , Humans , Lipase/metabolism , Male , Mitochondria/metabolism , Nutrients , Perilipin-1/metabolism , White People , Young Adult
12.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 31(6): 756-761, 2019 Jun.
Article in Chinese | MEDLINE | ID: mdl-31315737

ABSTRACT

OBJECTIVE: To observe the changes of short-chain acyl-CoA dehydrogenase (SCAD) expression on human umbilical vein endothelial cell (HUVEC) apoptosis and investigate its relationship with apoptosis. METHODS: The HUVEC was cultured normally for 2-3 days. The apoptotic model of HUVEC was established by tert-butyl hydrogen peroxide (tBHP). The HUVEC was treated by different concentrations of tBHP (0, 10, 20, 30, 40, 50 µmol/L) for 12 hours and different time (0, 3, 6, 9, 12 hours) with 50 µmol/L tBHP to establish the apoptotic model of HUVEC. The cell viability was detected by methyl thiazolyl tetrazolium (MTT), the mRNA expression of SCAD was determined by real-time polymerase chain reaction (PCR), the protein expression of SCAD was achieved by Western Blot. The best concentrate and time were determined to interfere the HUVEC to achieve the apoptotic model of HUVEC. The SCAD gene of HUVEC was knocked down by RNA interference sequence (siRNA274, siRNA414, siRNA679). The mRNA expression of SCAD, the protein expression of SCAD and the activity of SCAD enzyme were detected to achieve the best RNA interference sequence. The HUVEC was intervened by the best RNA interference sequence and tBHP. The cell activity and apoptosis rate, the enzyme activity of SCAD, the mRNA and protein expression of SCAD, the contents of reactive oxygen species (ROS), aderosine triphosphate (ATP) and free fatty acid (FFA) were detected to observe the effect of SCAD on apoptosis of HUVEC. RESULTS: (1) The cell viability, the mRNA expression and the protein expression of SCAD were decreased gradually in a concentration and time dependent manner with the increase of tBHP concentration and the prolongation of intervention time. The decline was most significant in the group of the 50 µmol/L tBHP to interfere HUVEC for 12 hours. (2) The siRNA679 transfection was the most significant in reducing SCAD mRNA and protein expressions among the three interference sequences (siRNA274, siRNA414, siRNA679). (3) Compare with blank control group, the cell viability was significantly decreased in the siRNA679 group (A value: 0.48±0.09 vs. 1.00±0.09, P < 0.01), the apoptotic rate of HUVEC was significantly increased [(29.96±2.09)% vs. (2.90±1.90)%, P < 0.01], the expression of SCAD mRNA and SCAD protein, the activity of SCAD enzyme and the content of ATP were significantly decreased [SCAD mRNA (2-ΔΔCt): 0.50±0.16 vs. 1.34±0.12, SCAD/α-Tubulin: 0.67±0.11 vs. 1.00±0.06, the activity of SCAD enzyme (kU/g): 0.38±0.04 vs. 0.53±0.04, the content of ATP (µmol/g): 0.14±0.02 vs. 0.19±0.01, all P < 0.05], the contents of FFA and ROS were significantly increased [FFA (nmol/g): 0.84±0.07 vs. 0.47±0.04, ROS (average fluorescence intensity): 647.5±23.7 vs. 434.2±46.5, both P < 0.01]. Meanwhile, SCAD siRNA treatment triggered the same apoptosis as HUVEC treated with tBHP. CONCLUSIONS: Down-regulation of SCAD may play an important role in HUVEC apoptosis. Increase in the expression of SCAD may become an important part in intervening HUVEC apoptosis.


Subject(s)
Apoptosis , Butyryl-CoA Dehydrogenase/metabolism , Human Umbilical Vein Endothelial Cells , Cell Survival , Humans , Reactive Oxygen Species
13.
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue ; 31(2): 172-177, 2019 Feb.
Article in Chinese | MEDLINE | ID: mdl-30827304

ABSTRACT

OBJECTIVE: To Study the changes of short-chain acyl-CoA dehydrogenase (SCAD) in heart failure (HF) after myocardial infarction (MI), and the effect of aerobic exercise on SCAD. METHODS: Healthy male Sprague-Dawley (SD) rats were divided into sham operation group (Sham group), sham operation swimming group (Sham+swim group), HF model group (LAD group) and HF swimming group (LAD+swim group) by random number table method, with 9 rats in each group. The left anterior descending branch of coronary artery (LAD) was ligated to establish a rat model of HF after MI. In Sham group, only one loose knot was threaded under the left coronary artery, and the rest operations were the same as those in LAD group. Rats in Sham+swim group and LAD+swim group were given swimming test for 1 week after operation (from 15 minutes on the 1st day to 60 minutes on the 5th day). Then they were given swimming endurance training (from the 2nd week onwards, 60 minutes daily, 6 times weekly, 10 weeks in a row). Tail artery systolic pressure (SBP) was measured before swimming endurance training and every 2 weeks until the end of the 10th week. Ten weeks after swimming training, echocardiography was performed to measure cardiac output (CO), stroke volume (SV), left ventricular ejection fraction (LVEF), shortening fraction (FS), left ventricular end-systolic diameter (LVESD), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV). Morphological changes of heart were observed by Masson staining. Apoptosis of myocardial cells was detected by transferase-mediated deoxyuridine triphosphate-biotin nick end labeling stain (TUNEL) and apoptosis index (AI) was calculated. Reverse transcription-polymerase chain reaction (RT-PCR) and Western Blot were used to detect the mRNA and protein expression of myocardial SCAD respectively. In addition, the enzyme activity of SCAD, the content of adenosine triphosphate (ATP) and free fatty acid (FFA) in serum and myocardium were detected according to the kit instruction steps. RESULTS: Compared with Sham group, Sham+swim group showed SBP did not change significantly, with obvious eccentric hypertrophy and increased myocardial contractility, and LAD group showed persistent hypotension, obvious MI, thinning of left ventricle, and decreased myocardial systolic/diastolic function. Compared with LAD group, SBP, systolic/diastolic function and MI in LAD+swim group were significantly improved [SBP (mmHg, 1 mmHg = 0.133 kPa): 119.5±4.4 vs. 113.2±4.5 at 4 weeks, 120.3±4.0 vs. 106.5±3.7 at 6 weeks, 117.4±1.3 vs. 111.0±2.3 at 8 weeks, 126.1±1.6 vs. 119.4±1.9 at 10 weeks; CO (mL/min): 59.10±6.31 vs. 33.19±4.76, SV (µL): 139.42±17.32 vs. 84.02±14.26, LVEF: 0.523±0.039 vs. 0.309±0.011, FS: (28.17±2.57)% vs. (15.93±3.64)%, LVEDD (mm): 8.80±0.19 vs. 9.35±0.30, LVESD (mm): 5.90±0.77 vs. 7.97±0.60, LVEDV (µL): 426.57±20.84 vs. 476.24±25.18, LVESV (µL): 209.50±25.18 vs. 318.60±16.10; AI: (20.4±1.4)% vs. (31.2±4.6)%; all P < 0.05]. Compared with Sham group, the mRNA and protein expression of myocardium SCAD, the activity of SCAD in Sham+swim group were significantly increased, the content of ATP was slightly increased, the content of serum FFA was significantly decreased, and the content of myocardial FFA was slightly decreased; conversely, the mRNA and protein expression of myocardium SCAD, the activity of SCAD and the content of ATP in LAD group were significantly decreased, the content of serum and myocardial FFA were significantly increased. Compared with LAD group, the mRNA and protein expression of myocardium SCAD, the content of ATP were significantly increased in LAD+swim group [SCAD mRNA (2-ΔΔCt): 0.52±0.16 vs. 0.15±0.01, SCAD/GAPDH (fold increase from Sham group): 0.94±0.08 vs. 0.60±0.11, ATP content (µmol/g): 52.8±10.1 vs. 14.7±6.1, all P < 0.05], the content of serum and myocardial FFA were significantly decreased [serum FFA (nmol/L): 0.11±0.03 vs. 0.29±0.04, myocardial FFA (nmol/g): 32.7±8.2 vs. 59.7±10.7, both P < 0.05], and the activity of SCAD was slightly increased (kU/g: 12.3±4.3 vs. 8.9±5.8, P > 0.05). CONCLUSIONS: The expression of SCAD in HF was significantly down-regulated, and the expression was significantly up-regulated after aerobic exercise intervention, indicating that swimming may improve the severity of HF by up-regulating the expression of SCAD.


Subject(s)
Butyryl-CoA Dehydrogenase/metabolism , Heart Failure/metabolism , Animals , Heart Failure/etiology , Male , Myocardial Infarction/complications , Physical Conditioning, Animal , Random Allocation , Rats , Rats, Sprague-Dawley
14.
Fungal Genet Biol ; 127: 23-34, 2019 06.
Article in English | MEDLINE | ID: mdl-30822500

ABSTRACT

Short-chain acyl-CoA dehydrogenase (Scad) mediated ß-oxidation serves as the fastest route for generating essential energies required to support the survival of organisms under stress or starvation. In this study, we identified three putative SCAD genes in the genome of the globally destructive rice blast pathogen Magnaporthe oryzae, named as MoSCAD1, MoSCAD2, and MoSCAD3. To elucidate their function, we deployed targeted gene deletion strategy to investigate individual and the combined influence of MoSCAD genes on growth, stress tolerance, conidiation and pathogenicity of the rice blast fungus. First, localization and co-localization results obtained from this study showed that MoScad1 localizes to the endoplasmic reticulum (ER), MoScad2 localizes exclusively to the mitochondria while MoScad3 partially localizes to the mitochondria and peroxisome at all developmental stages of M. oryzae. Results obtained from this investigation showed that the deletion of MoSCAD1 and MoSCAD2 caused a minimal but significant reduction in the growth of ΔMoscad1 and ΔMoscad2 strains, while, growth characteristics exhibited by the ΔMoscad3 strain was similar to the wild-type strain. Furthermore, we observed that deletion of MoSCAD2 resulted in drastic reduction in conidiation, delayed conidia germination, triggered the development of abnormal appressorium and suppressed host penetration and colonization efficiencies of the ΔMoscad1 strain. This study provides first material evidence confirming the possible existence of ER ß-oxidation pathway in M. oryzae. We also infer that mitochondria ß-oxidation rather than peroxisomal and ER ß-oxidation play an essential role in the vegetative growth, conidiation, appressorial morphogenesis and progression of pathogenesis in M. oryzae.


Subject(s)
Butyryl-CoA Dehydrogenase/genetics , Fungal Proteins/genetics , Magnaporthe/genetics , Magnaporthe/pathogenicity , Spores, Fungal/growth & development , Endoplasmic Reticulum , Free Radicals/metabolism , Gene Deletion , Gene Expression Regulation, Fungal , Magnaporthe/enzymology , Mitochondria/metabolism , Oryza/microbiology , Oxidation-Reduction , Peroxisomes/metabolism , Plant Diseases/microbiology , Spores, Fungal/genetics
15.
Biol Trace Elem Res ; 190(1): 87-94, 2019 Jul.
Article in English | MEDLINE | ID: mdl-30251228

ABSTRACT

Twenty four rats were divided into four groups (6 rats/group): 1-control group; 2-licorice (Glycyrrhiza glabra) extract: rats administered with an oral dose of licorice (3 mg/ml/kg/day) using stomach tube for 4 weeks; 3-cadmium chloride group: rats administered with an oral dose of CdCl2 (10 mg/kg/day) using stomach tube for 4 weeks; and 4-licorice extract + CdCl2 group: rats administered orally with both licorice (3 mg/ml/kg/day) and CdCl2 (10 mg/kg/day) using stomach tube for 4 weeks. Administration of CdCl2 induced significant increase in thiobarbituric acid reactive substance (TBARS), paraoxonase-1 (proxon-1), caspase-3 (casp-3) activities, and significant decrease in superoxide dismutase (SOD), catalase (CAT) activities, and glutathione (GSH) content in hepatic tissue. Significant increase in TBARS and kidney injury molecule-1 (KIM-1) and significant decrease in SOD, CAT activities, and GSH content in renal tissue were recorded. Significant increase in alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma glutamyl transferase (GGT) activities, urea, creatinine, and significant decrease in butyryl choline esterase (BChE), total triiodothyronine (T3), and total thyroxin (T4) were recorded in serum. Histological investigation of renal cells showed vacuolations of endothelium lining glomerular tuffs and vacuolations of epithelium lining renal tubules. Investigation of ovarian tissue showed dilatation of interstitial blood vessels and congestion of multiple corpus luteum in CdCl2-treated rats. Significant improvements in the biochemical and histological changes were observed in CdCl2 + licorice extract group. It could be concluded that licorice extract alleviates the hazardous effects of cadmium chloride, which may be attributed to its antioxidant properties.


Subject(s)
Cadmium Chloride/pharmacology , Glycyrrhiza/chemistry , Plant Extracts/pharmacology , Water/chemistry , Alanine Transaminase/blood , Alkaline Phosphatase/blood , Animals , Aspartate Aminotransferases/blood , Butyryl-CoA Dehydrogenase/blood , Female , Kidney/drug effects , Kidney/metabolism , Liver/drug effects , Liver/metabolism , Rats , Thyroid Hormones/blood , Thyroxine/blood , Triiodothyronine/blood , gamma-Glutamyltransferase/blood
16.
BMC Med Genet ; 19(1): 64, 2018 04 20.
Article in English | MEDLINE | ID: mdl-29678161

ABSTRACT

BACKGROUND: Short-chain acyl-CoA dehydrogenase deficiency (SCADD) represents a rare autosomal recessive inborn metabolic disorder of mitochondrial ß-oxidation of monocarboxylic acids. Clinical symptoms can vary from a severe life-threatening condition to an asymptomatic state, reported in the majority of cases. Since the expansion of newborn screenings, more than three hundred probands were admitted for molecular-genetic analysis, most selected because of elevated values of C4-acylcarnitine detected in newborn screenings in Slovakia. Searching for the principal genomic changes led us to the selection of sixty-two patients in whom the presence of sequence variants in the ACADS gene was analysed and correlated with the available biochemical and clinical data. METHODS: Biochemical and molecular genetic tests were performed. Acylcarnitine profiles focused on an elevated level of C4-acylcarnitine, which was analysed via tandem mass spectrometry. Urinary organic acids, specifically a quantity of ethylmalonic acid, were determined by gas chromatography/mass spectrometry. The entire coding region of the ACADS gene was sequenced. A low-cost restriction fragment length polymorphism of PCR amplified fragments analysis (PCR-RFLP) of pathogenic variants was introduced and implemented for the molecular-genetic algorithm appropriate for the Slovak population. RESULTS: Our molecular genetic study was performed on sixty-two patients with a pathological biochemical pattern related to short-chain acyl-CoA dehydrogenase deficiency. In this cohort, we discovered a high occurrence of two rare pathogenic variants-the deletion c.310_312delGAG and the substitution c.1138C>T, with allelic frequencies of 64% and 31%, respectively. Up to 86% of investigated individuals belong to the Roma ethnic group. CONCLUSIONS: Analogous to other countries, SCADD is not included in the newborn screening programme. Based on the exceeded levels of the specific biomarker C4-acylcarnitine as well as ethylmalonic acid, we revealed a high prevalence of short-chain acyl-CoA dehydrogenase deficiency cases, confirmed by the findings of two rare pathogenic variants. A deletion c.310_312delGAG and c.1138C > T substitution in the ACADS gene appear with a high frequency in the Roma ethnic group of Slovakia. Due to the uncertainty of the pathogenicity and clinical consequences, it is important to follow up the morbidity and mortality in these patients over time and evaluate SCADD in relation to clinical outcomes and preventive healthcare recommendations.


Subject(s)
Acyl-CoA Dehydrogenase/deficiency , Butyryl-CoA Dehydrogenase/genetics , Carnitine/analogs & derivatives , Ethnicity/genetics , Lipid Metabolism, Inborn Errors/genetics , Mutation , Acyl-CoA Dehydrogenase/genetics , Acyl-CoA Dehydrogenase/metabolism , Carnitine/metabolism , Female , Gene Frequency , Genetic Testing , Humans , Infant, Newborn , Lipid Metabolism, Inborn Errors/ethnology , Lipid Metabolism, Inborn Errors/metabolism , Male , Neonatal Screening/methods , Polymorphism, Restriction Fragment Length , Sequence Analysis, DNA , Slovakia/ethnology
17.
Nat Commun ; 8(1): 1577, 2017 11 17.
Article in English | MEDLINE | ID: mdl-29146947

ABSTRACT

The electron transferring flavoprotein/butyryl-CoA dehydrogenase (EtfAB/Bcd) catalyzes the reduction of one crotonyl-CoA and two ferredoxins by two NADH within a flavin-based electron-bifurcating process. Here we report on the X-ray structure of the Clostridium difficile (EtfAB/Bcd)4 complex in the dehydrogenase-conducting D-state, α-FAD (bound to domain II of EtfA) and δ-FAD (bound to Bcd) being 8 Å apart. Superimposing Acidaminococcus fermentans EtfAB onto C. difficile EtfAB/Bcd reveals a rotation of domain II of nearly 80°. Further rotation by 10° brings EtfAB into the bifurcating B-state, α-FAD and ß-FAD (bound to EtfB) being 14 Å apart. This dual binding mode of domain II, substantiated by mutational studies, resembles findings in non-bifurcating EtfAB/acyl-CoA dehydrogenase complexes. In our proposed mechanism, NADH reduces ß-FAD, which bifurcates. One electron goes to ferredoxin and one to α-FAD, which swings over to reduce δ-FAD to the semiquinone. Repetition affords a second reduced ferredoxin and δ-FADH-, which reduces crotonyl-CoA.


Subject(s)
Acyl Coenzyme A/chemistry , Butyryl-CoA Dehydrogenase/chemistry , Clostridioides difficile/enzymology , Ferredoxins/chemistry , Flavin-Adenine Dinucleotide/chemistry , NAD/chemistry , Acidaminococcus/enzymology , Acyl Coenzyme A/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Clostridioides difficile/metabolism , Crystallography, X-Ray , Electron Transport , Oxidation-Reduction
18.
FEBS Lett ; 591(12): 1785-1797, 2017 06.
Article in English | MEDLINE | ID: mdl-28524228

ABSTRACT

To investigate the function of the pa4079 gene from the opportunistic pathogen Pseudomonas aeruginosa PAO1, we determined its crystal structure and confirmed it to be a NAD(P)-dependent short-chain dehydrogenase/reductase. Structural similarity and activity for a broad range of substrates indicate that PA4079 functions as a carbonyl reductase. Comparison of apo- and holo-PA4079 shows that NADP stabilizes the active site specificity loop, and small molecule binding induces rotation of the Tyr183 side chain by approximately 90° out of the active site. Quantitative real-time PCR results show that pa4079 maintains high expression levels during antibiotic exposure. This work provides a starting point for understanding substrate recognition and selectivity by PA4079, as well as its possible reduction of antimicrobial drugs. DATABASE: Structural data are available in the Protein Data Bank (PDB) under the following accession numbers: apo PA4079 (condition I), 5WQM; apo PA4079 (condition II), 5WQN; PA4079 + NADP (condition I), 5WQO; PA4079 + NADP (condition II), 5WQP.


Subject(s)
Aldehyde Reductase/metabolism , Bacterial Proteins/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Models, Molecular , NADP/metabolism , Pseudomonas aeruginosa/metabolism , Aldehyde Reductase/chemistry , Aldehyde Reductase/genetics , Aldo-Keto Reductases , Amino Acid Sequence , Amino Acid Substitution , Anti-Bacterial Agents/pharmacology , Apoenzymes/chemistry , Apoenzymes/genetics , Apoenzymes/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Binding Sites , Butyryl-CoA Dehydrogenase/chemistry , Butyryl-CoA Dehydrogenase/genetics , Catalytic Domain , Conserved Sequence , Crystallography, X-Ray , Enzyme Stability , Gene Expression Regulation, Bacterial/drug effects , Holoenzymes/chemistry , Holoenzymes/genetics , Holoenzymes/metabolism , Ligands , Mutation , NADP/chemistry , Protein Conformation , Pseudomonas aeruginosa/drug effects , Pseudomonas aeruginosa/growth & development , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sequence Alignment , Structural Homology, Protein , Substrate Specificity
19.
J Biol Chem ; 291(23): 11993-2002, 2016 Jun 03.
Article in English | MEDLINE | ID: mdl-27048649

ABSTRACT

Electron-transferring flavoprotein (Etf) and butyryl-CoA dehydrogenase (Bcd) from Acidaminococcus fermentans catalyze the endergonic reduction of ferredoxin by NADH, which is also driven by the concomitant reduction of crotonyl-CoA by NADH, a process called electron bifurcation. Here we show that recombinant flavodoxin from A. fermentans produced in Escherichia coli can replace ferredoxin with almost equal efficiency. After complete reduction of the yellow quinone to the blue semiquinone, a second 1.4 times faster electron transfer affords the colorless hydroquinone. Mediated by a hydrogenase, protons reoxidize the fully reduced flavodoxin or ferredoxin to the semi-reduced species. In this hydrogen-generating system, both electron carriers act catalytically with apparent Km = 0.26 µm ferredoxin or 0.42 µm flavodoxin. Membrane preparations of A. fermentans contain a highly active ferredoxin/flavodoxin-NAD(+) reductase (Rnf) that catalyzes the irreversible reduction of flavodoxin by NADH to the blue semiquinone. Using flavodoxin hydroquinone or reduced ferredoxin obtained by electron bifurcation, Rnf can be measured in the forward direction, whereby one NADH is recycled, resulting in the simple equation: crotonyl-CoA + NADH + H(+) = butyryl-CoA + NAD(+) with Km = 1.4 µm ferredoxin or 2.0 µm flavodoxin. This reaction requires Na(+) (Km = 0.12 mm) or Li(+) (Km = 0.25 mm) for activity, indicating that Rnf acts as a Na(+) pump. The redox potential of the quinone/semiquinone couple of flavodoxin (Fld) is much higher than that of the semiquinone/hydroquinone couple. With free riboflavin, the opposite is the case. Based on this behavior, we refine our previous mechanism of electron bifurcation.


Subject(s)
Bacterial Proteins/metabolism , Electron-Transferring Flavoproteins/metabolism , NAD/metabolism , Oxidoreductases/metabolism , Sodium/metabolism , Acidaminococcus/enzymology , Acidaminococcus/genetics , Acidaminococcus/metabolism , Acyl Coenzyme A/metabolism , Benzoquinones/metabolism , Butyryl-CoA Dehydrogenase/metabolism , Catalysis , Electron Transport , Electron-Transferring Flavoproteins/genetics , Electrons , Hydrogen/metabolism , Hydroquinones/metabolism , Kinetics , Oxidation-Reduction , Recombinant Proteins/metabolism , Riboflavin/metabolism , Spectrophotometry
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