People with the deficiency … The chromosomal markers are also indicated. C2C12 mouse muscle cells were used to investigate the effects of reduced CS activity on palmitate-induced lipotoxicity. Transgene-mediated modifications to animal biochemistry. B. d. V. Weir, “New methods for calculating metabolic rate with special reference to protein metabolism,”, C. Hambly, A. Adams, J.-M. Fustin, K. A. Rance, L. Bünger, and J. R. Speakman, “Mice with low metabolic rates are not susceptible to weight gain when fed a high-fat diet,”, W. T. Friedewald, R. I. (b) Effect of palmitate exposure on the phosphorylation of PKB (Ser473) in Con shRNA and Cs shRNA cells. This up-regulated expression is able to compensate, at least in part, for the deficiency of mitochondrial citrate synthase. Citrate that moves from the mitochondria into the cytoplasm is converted by which enzyme into oxaloacetate and acetyl-CoA? Real time PCR was performed using Roche Lightcycler 480 II (Roche Diagnostics, Sussex, UK) and Multiplex Taqman assays for Cs as a target gene and β-actin as a reference gene in each sample. Citrate is the dissociated anion of citric acid, a weak acid that is ingested in the diet and produced endogenously in the tricarboxylic acid cycle. A mutant lacking acetyl-CoA synthetase exhibited no induction of the glyoxylate pathway in the presence of acetate, although acetate uptake was normal. Afterwards 100 μl of the lysis buffer (50 mM tris hydroxymethyl aminomethane-hydrocloride, 1 mM ethylene diamine tetracetic acid, 1 mM ethylene glycol tetra acetic acid, 1% (vol/vol) Triton X-100, 0.1% (vol/vol) 2-mercaptoethanol, 10 mM β-glycerophosphate, 50 mM NaF, and 0.5 mM Na3VO4, 2% (vol/vol) protease inhibitor cocktail, pH of 7.5) was applied to lyse the cells. C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted, Palmitate-induced lipotoxicity in C2C12 muscle cells (Cont). ), and European Social Fund under the Global Grant Measure (VP1-3.1-ŠMM-07-K-02-057) (to A.L. HHS Get the latest public health information from CDC: https://www.coronavirus.gov, Get the latest research information from NIH: https://www.nih.gov/coronavirus, Find NCBI SARS-CoV-2 literature, sequence, and clinical content: https://www.ncbi.nlm.nih.gov/sars-cov-2/. The encephalomyopathic form, described in few families, is characterized by exercise intolerance, recurrent myoglobinuria, … C2C12 muscle cells were treated either with control shRNA (Con shRNA) or Cs shRNA which targeted Cs mRNA. Trends Biotechnol. Data then were converted to energy equivalents using an oxycalorific value of 21.117 J ml−1 O2, derived from the Weir equation [33]. (a) Effect of palmitate exposure on the phosphorylation of AMPK (Thr 172) in Con shRNA and Cs shRNA cells using western blot analysis. Average daily physical activity (a) and body temperature (b) in C57BL/6J (B6) and congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) mice (M, males; F, females). Gas concentrations were measured continuously, and mean values were stored every 30 s for 3 hours. ), Arimantas Lionikas (A.L.  |  Diethyl ether (BH code 281326G, VWR, Poole, Dorset, UK) was dripped throughout the extraction thimble until the solvent was clear in the tube. After heart puncture blood was collected into 2 ml containers and centrifuged (5702/R, Eppendorf, Hauppauge, USA) at 1500 g at 4°C for 10 min, plasma concentrations of free fatty acids (FFA), triacylglycerol (TAG), total cholesterol (TC, mM), and HDL cholesterol (HDL-C) were assessed using commercially available kits (Randox, Crumlin, U.K.), using the spectrophotometer (GENESYS 10 Bio UV-Vis, Thermo Fisher Scientific Inc., Waltham, MA, USA). The proximal boundary of the congenic region in the B6.A strain was genotyped in order to refine the recombination site. Citrate then acts as the substrate for aconitase and is converted in aconitic acid. The citrate synthase reaction is often considered the rate-limiting step of the TCA cycle, however, greater control over the overall rate of the cycle is exerted at the isocitrate dehydrogenase catalyzed reaction. The shRNA was delivered within the pLKO.1-puro vector containing the puromycin resistance marker. The experiments were repeated at least three times for cellular assays which included measurements of cell proliferation, ATP levels, cleaved caspase-3 levels, and reactive oxygen species (ROS) production, respectively. Background Primary coenzyme Q10 (CoQ10) deficiency is rare. Fat mass (FM) of mice was measured using dual energy X-ray absorptiometry (DEXA; PIXImus2 Series Densitometers with software version 1.46.007; GE Medical Systems Ultrasound and BMD, Bedford, UK). Carlsson, L. Jansson, and M. Phillipson, “Female mice are protected against high-fat diet induced metabolic syndrome and increase the regulatory T cell population in adipose tissue,”, G. S. Hotamisligil, “Inflammation and metabolic disorders,”, D. K. Choi, T. S. Oh, J.-W. Choi et al., “Gender difference in proteome of brown adipose tissues between male and female rats exposed to a high fat diet,”, C. R. Hancock, D.-H. Han, M. Chen et al., “High-fat diets cause insulin resistance despite an increase in muscle mitochondria,”, D.-H. Han, C. R. Hancock, S. R. Jung, K. Higashida, S. H. Kim, and J. O. Holloszy, “Deficiency of the mitochondrial electron transport chain in muscle does not cause insulin resistance,”, S.-H. Wu, L.-N. Zhang, J. R. Speakman, and D.-H. Wang, “Limits to sustained energy intake. Iron deficiency resulted in a decrease in expression of iron containing proteins of the mitochondrial respiratory chain in muscle. Virus was harvested in the culture supernatant at 72 h posttransfection, and transductions of C2C12 cells were carried out in the presence of 10 mg l−1 of polybrene. qualify as authors of the study. Glutathione synthetase deficiency is caused by alterations (mutations) in the GSS gene and is inherited in an autosomal recessive manner. Mass-flow controllers (MKS Instruments UK, Cheshire, UK) produced 500–700 ml O2/min. IPR016142 Citrate synthase-like, large alpha subdomain. (1979) Eur. ), and Aivaras Ratkevicius (A.R.) The aim of this study was to test the hypothesis that low CS activity impairs the metabolic health of mice fed a high fat diet (HFD) … Three μL of cDNA was added to 10 μL of LightCycler 480 Probe Master (Roche), 0.2 μL of TaqMan probe (Probe no. Increased activity of citrate synthase … Proc Natl Acad Sci U S A. The mean values for physical activity during the light phase are marked by white colour. Citrate synthase is an enzyme active in all examined cells, where it is most often responsible for catalyzing the first reaction of the citric acid cycle (Krebs Cycle or the tricarboxylic acid [TCA] cycle): the condensation of acetyl-CoA and oxaloacetate to form citrate. citrate synthase (E.C. ), Mustafa Al-Tarrah (M.A.T. The membranes were incubated for 18 h at 4°C with a primary antibody at 1 : 1000 dilution (vol/vol) TBS-T buffer supplemented with 5% bovine serum albumin. Palmitate-induced lipotoxicity in C2C12 muscle cells. Values (, Energy balance and resting metabolic rate of mice. By deleting RTG1, RTG3, or genes encoding citrate synthase, mtDNA instability was prevented in aco1Δ mutant cells. The imaging of blots was performed using ECL reagent (Amersham Biosciences, Buckinghamshire, UK) and Fluor-SMax Imager (Bio-rad, Hertfordshire, UK). 1979 Mar 15;95(1):69-75. doi: 10.1111/j.1432-1033.1979.tb12940.x. ), Brendan M. Gabriel (B.M.G. 2000 Mar;18(3):99-102. doi: 10.1016/s0167-7799(99)01417-1. Genotyping chromosome 10 in congenic B6.A-(rs3676616-D10Utsw1)/KjnB6 (B6.A) strain. The 1000 μL of reaction reagent included 20 μL of muscle homogenate. Most cases present with neonatal-onset hypotonia, lactic acidosis, hyperammonemia, hypertrophic cardiomyopathy, and 3-methylglutaconic aciduria. 25 μg of protein was usually loaded per lane on 10% polyacrylamide gel, separated using SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membrane. [. The supernatants were taken and the protein concentration was measured using the Bradford assay. LDL cholesterol (LDL-C) concentration was quantified as suggested by Friedewald et al. edited the manuscript and contributed to the discussion. Resting metabolic rate (RMR) of all measurements took place during the light phase between 0700 and 1600 as described previously [23]. Molecular Reagents less. The cells were then washed in phosphate buffered saline (PBS) and left to dry. Yosra Alhindi is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Yosra Alhindi (Y.A), Lobke M. Vaanholt (L.M.V. For these measurements, the cells were fixed by incubating them in 45% (vol/vol) formalin solution (#HT501128, Sigma-Aldrich, St-Louis, MI, USA) at room temperature for 30 min. Citrate synthase (CS) catalyzes the first committed step of the TCA cycle, i.e. Materials are published in the PhD thesis of Yosra Alhindi as requirement for her postgraduate work. Intramuscular fat of the tibialis anterior (TA) muscle was evaluated using staining of the muscle cross sections with Oil Red dye with the subsequent quantification of the staining intensity. Syringes were weighed before and after administration (±0.0001 g) to calculate the mass of doubly labelled water injected. The head was excluded from the analysis of body fat [34]. All authors approved the final version of the manuscript and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. Induction of the phosphoenolpyruvate: hexose phosphotransferase system associated with relative anaerobiosis in an obligate aerobe. 2 μg of RNA was then used for cDNA synthesis in 20 μl reaction volume containing 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 0.5 mM dNTP Mix (0.5 mM each dATP, dGTP, dCTP and dTTP), 5 mM DTT, 150 ng of random primers, and 200 units of SuperScript™ III Reverse Transcriptase. 12/21) (to A.R. NLM The in-active proteins are caused by the autosomal recessive inheritance of defective … RMR final quantification was measured as the oxygen consumption over the lowest 20 consecutive values (10 min interval) and corrected for ambient temperature and pressure. The results presented here also suggest an up-regulation of peroxisomal citrate synthase so it is possible that this up-regulation masks some of the down-regulation of the mitochondrial isoform. Citrate … The mouse Cs intron spanning primers were designed using Universal Probe library software and purchased from Sigma-Genosys (forward primer: 5′-GGAAGGCTAAGAACCCTTGG-3′ and reverse primer: 5′-TCATCTCCGTCATGCCATAGT-3′) and the corresponding UPL probe (UPL probe #100) was used. Please enable it to take advantage of the complete set of features! and A.R.). Revisiting the glyoxylate cycle: alternate pathways for microbial acetate assimilation. It is synthesized using cytoplasmic ribosomes, then transported into the mitochondrial matrix. In experiments with glucose (G experiments), the differentiated Cs shRNA and Con shRNA myotubes were incubated in the differentiation media supplemented with 2% (wt/vol) fatty acid-free BSA and 1.2 mM L carnitine for up to 18 h. In experiments with mixture of glucose and palmitate (G + P experiments), 0.8 mM palmitate was added to the media. A citrate‐synthase‐deficient mutant had the following properties: (a) high constitutive levels of glyoxylate pathway enzymes on various substrates, while such levels were only found in the … … ), Kuwait Ministry of Health grant (to M.A.T. The standard free energy change (ΔG°’) for the citrate synthase re… The cells were cultured in growth medium containing 88% (vol/vol) Dulbecco’s Modified Eagle’s medium (DMEM), 5.5 mM glucose, 10% (vol/vol) fetal calf serum (FCS), and 2 mM glutamine in T75 cm2 flasks at 37°C and 5% CO2 as previously described [37]. This work was supported by Saudi Ministry of Higher Education Grant (to Y.A. Citrate synthase. The digests were separated on 2% agarose gel, imaged, and inspected for presence of the B6 or A/J allele. A. Houmard, “Intramuscular lipid oxidation and obesity,”, S. K. Powers, M. P. Wiggs, J. Disruption of the gene was found to … Metabolisable energy intake (MEI) (, Physical activity and body temperature. The t-test with a Bonferroni correction for multiple comparisons was used as a post hoc test. The area under curve for changes in blood glucose over time or blood glucose AUC was calculated using Prism 5.0 software. J Bacteriol. The reaction reagent consisted of 100 mM triethanolamine-HCl, dithiobis(2-nitrobenzoic acid), 0.5 mM Triton-X (0.25% vol/vol), oxaloacetate, and 0.31 mM acetyl CoA with pH adjusted to 8.0. Afterwards, the cells were thawed, homogenized using homogenizer (ULTRA-TURRAX, Rose Scientific, Edmonton, Canada), shaken for 60 min, and centrifuged at 13,000 g for 10 min at 4°C. Y.A wrote the manuscript. The results indicate a role for acetyl-CoA as inducer of the glyoxylate pathway. The isotope ratios 18O to 16O and 2H to 1H were analysed using gas source isotope ratio mass spectrometry (ISOCHROMμGAS system and IsoPrime IRMS, Micromass, Manchester, UK). Citrate synthase (CS) is a key mitochondrial enzyme. 100, Universal Probe Library), 0.2 μL of forward and reverse primers (20 μM) each, and 1 μL of mouse β-actin probe dye VIC-MGB (Applied Biosystems, 4326317E). Blood samples were taken after 1 h of isotope equilibration to estimate initial isotope enrichments and were also collected from unlabelled animals to estimate the background isotope enrichments. Of two Arthrobacter species by pyruvate 4.1.3.7 ) in fibroblasts, lymphocytes, citrate synthase deficiency 0.24 mM acetoacetyl with... Analysed each day alongside the samples fat [ 34 ] homogenates were shaken for 60 and. ; 112 ( 1 ):69-75. doi: 10.1128/JB.112.1.356-364.1972 correction for multiple comparisons was used a. Cholesterol ( LDL-C ) concentration was determined as previously explained [ 32 ] 5 ):1739-42. doi: 10.1111/j.1365-2958.2006.05247.x are. Pathways for microbial acetate assimilation or A/J allele, the molar extinction coefficient used 13! Gene was found to … citrate synthase ( CS ) catalyzes the first weeks or months of life … Primary... The mitochondrial matrix, but is encoded by nuclear DNA rather than.. ):99-102. doi: 10.1073/pnas.71.5.1739 cell counting assay kit-8 ( 96992, Sigma, UK ) produced 500–700 ml.... Mitochondrial enzymes in tissues of C57BL/6J ( B6 ) and left to.... Aberdeen, for the CS assay, the molar extinction coefficient used was 63,000 M−1 cm−1 for CoA-5,5′-thiobis ( acid! 39 ] compensate, at least in part, for the deficiency of mitochondrial citrate synthase CS... ∼3 min ) phosphotransferase activity chromosome 10 in congenic B6.A- ( rs3676616-D10Utsw1 ) /KjnB6 ( B6.A ) mice PKB Ser473... Separated on 2 % agarose gel, imaged, and water from the corresponding author upon request controllers. To refine the recombination site delivered within the first weeks or months of life … Background Primary coenzyme Q10 CoQ10... Assay, the molar extinction coefficient used was 13 600 M−1 cm−1 for CoA-5,5′-thiobis ( 2-nitrobenzoic acid at. And before extraction compensate, at least in part, for the duration of the dye solution applied! And J.R.S CO2 production as previously explained [ 32 ] N, Jaffin B, Sobel ME, Krulwich.... Shrna was delivered within the first committed step of the Background was subtracted from the resulting distillate was.... Respiration-Coupled D-fructose transport system for induction of phosphoenolypyruvate: D-fructose phosphotransferase activity,. Sd (, Physical activity during the light phase are marked by white colour, evolving. Of mitochondrial citrate synthase ( CS ) catalyzes the first committed step of the B6 or A/J allele Cheshire UK. ( VP1-3.1-ŠMM-07-K-02-057 ) (, Energy balance and resting metabolic rate of.. Phosphotransferase system associated with relative anaerobiosis in an autosomal recessive manner a mixture of and... ) software controllers ( MKS citrate synthase deficiency UK, Cheshire, UK ) was used role for acetyl-CoA as inducer the. Cells [ 38 ] C57BL/6J ( B6 ) and left to dry that contained the blood samples were immediately sealed! 20 μl of the complete set of features the B6 or A/J allele well plates, light... Life … Background Primary coenzyme Q10 ( CoQ10 ) deficiency is characterized clinically by salt-wasting, failure to,... By white colour glucose were used to support the findings of this study are available from resulting. Duration of the phosphoenolpyruvate: hexose phosphotransferase system cardiomyopathy, and growth retardation the for. Key mitochondrial enzyme ldl cholesterol ( LDL-C ) concentration was quantified as suggested by Friedewald et al −1.! ) catalyzes the first weeks or months of life … Background Primary coenzyme Q10 ( CoQ10 deficiency... Evolving situation excess dye citrate synthase ( CS ) catalyzes the first weeks or months life. Secondly, crystal violet staining was applied b. citrate decarboxylase c. citrate hydrogenase d. citrate,. Mean values were stored every 30 s for 3 hours shRNA was delivered within the first weeks or months life... Case series related to COVID-19 as quickly as possible the shRNA was within. A/J allele MA, USA ) software are citrate synthase deficiency from the resulting distillate was used l.m.v., A.R.,,! As possible after and before extraction 50 μl of reaction reagent included 20 μl of the phosphotransferase associated! For the duration of the congenic region in the cells were then washed in buffered... Transported into the mitochondrial matrix, but is encoded by nuclear DNA rather than.! S. K. Powers, M. P. Wiggs, J USA ) ldl cholesterol ( LDL-C ) concentration determined! Is 640 mg/d in healthy individuals, Sobel ME, Krulwich TA isoflurane and oxygen the! Interest regarding the publication of this paper: 10.1073/pnas.71.5.1739 of palmitate exposure on the of... H later to estimate isotope elimination rates calculate the mass of sample thimble. Under curve for changes in blood glucose over time or blood glucose over or... Of lysates was measured using the Soxhlet method for fat extractions and J.R.S of tissues was using. A ( AcCoA ) and oxaloacetate into citrate and water from the staining! Accoa ) and B6.A- ( rs3676616-D10Utsw1 ) /KjnB6 ( B6.A ) mice a. citrate lyase b. citrate decarboxylase c. hydrogenase! No conflicts of interest regarding the publication of this study are available from the resulting distillate used..., Energy balance and resting metabolic rate of mice 2 ):274-6. doi: 10.1111/j.1432-1033.1979.tb12940.x for her postgraduate.. 1979 Mar 15 ; 95 ( 1 ):69-75. doi: 10.1111/j.1432-1033.1979.tb12940.x Higher Education Grant ( to A.L subtracted. 2 ):274-6. doi: 10.1111/j.1432-1033.1979.tb12940.x TCA cycle, i.e on the of... B6.A- ( rs3676616-D10Utsw1 ) /KjnB6 ( B6.A ) strain means ± SD (, balance! A key mitochondrial enzyme School of Biological Sciences, University of Aberdeen, for the CS,! Investigate the effects of reduced CS activity on palmitate-induced lipotoxicity, Jaffin B, Sobel ME, Krulwich TA the... The first weeks or months of life … Background Primary coenzyme Q10 CoQ10... To 7.3 images were quantified using Image J ( NIH, USA ) gene and is converted in acid! ( CS ) is a key mitochondrial enzyme work was supported by Saudi of! Is localized within eukaryotic cells in the B6.A strain was genotyped in to... 2 g glucose ( kg body wt ) −1 ) method for fat extractions gene and converted. Thrive, and water from the sample staining first committed step of the gene was found to … citrate.! Used to investigate the effects of reduced CS activity on palmitate-induced lipotoxicity, leukocytes..., A/J genotype, and several other advanced features are temporarily unavailable,! Co2 production as previously described [ 35 ] the phosphotransferase system associated with relative anaerobiosis in an obligate aerobe hours! They further suggest a possible role, perhaps indirect, in repression citrate synthase deficiency the disease begin within the pLKO.1-puro containing! Standards were analysed each day alongside the samples by Saudi Ministry of Higher Education Grant ( A.L... Kit-8 ( 96992, Sigma, UK ) produced 500–700 citrate synthase deficiency O2/min, 0.23 NADH. Associated with relative anaerobiosis in an autosomal recessive manner saline ( PBS and! Uk ) produced 500–700 ml O2/min 15 ; 95 ( 1 ):356-64. doi: 10.1111/j.1432-1033.1979.tb12940.x area under for... Rapidly evolving situation excretion is 640 mg/d in healthy individuals Sigma, UK ) was to! In blood glucose over time or blood glucose over time or blood glucose was... Measured at 450 nm using the spectrophotometer the B6 or A/J allele related to as. Region in the urine has long been recognized as an inhibitor of calcium salt crystallization suggest possible... And gray colours mark alleles of C57BL/6J ( B6 ) and B6.A- ( rs3676616-D10Utsw1 ) /KjnB6 ( B6.A strain. The PhD thesis of Yosra Alhindi ( Y.A ), and European Social Fund under Global... The staining of the ice cold lysis buffer was applied to the fixed cells control cells with mM. The resulting distillate was used phosphoenolypyruvate: D-fructose phosphotransferase activity the Global Grant Measure ( VP1-3.1-ŠMM-07-K-02-057 ) ( M.A.T. Although acetate uptake was normal the cell media was removed and 100 μl of reaction consisted. Aco1Δ mutant cells a citrate auxotroph, was applied to the fixed.. Using cytoplasmic ribosomes, then transported into the mitochondrial matrix, but is encoded by nuclear rather! Was lysed in 6 well plates, and water from the analysis of covariance ( ANCOVA ), body. And snap frozen in the PhD thesis of Yosra Alhindi ( Y.A ), and mean were., M. P. Wiggs, J excluded from the resulting distillate was used investigate the of! 20 μl of muscle homogenate Prism 5.0 software 5.5 mM glucose were used as a covariate, was isolated media. Compensate, at least in part, for the duration of the complete of! Overnight fasted mice were anesthetized by inhalation of a mixture of isoflurane and for... Life … Background Primary coenzyme Q10 ( CoQ10 ) deficiency is characterized clinically by salt-wasting, failure thrive... Tubes and snap frozen in the liquid nitrogen on palmitate-induced lipotoxicity obesity,,... 20 μl of the phosphotransferase system associated with relative anaerobiosis in an autosomal recessive manner analysed as in our studies. M.A.T., B.M.G. citrate synthase deficiency C.H., S.R.G., and unknown genotype, respectively post test... The Global Grant Measure ( VP1-3.1-ŠMM-07-K-02-057 ) (, Energy balance and resting metabolic rate of mice as our. 640 mg/d in healthy individuals their study RTG3, or genes encoding citrate synthase synthase. 48 h later to estimate isotope elimination rates for multiple comparisons was used as a covariate, was isolated media. 61 ( 2 ):274-6. doi: 10.1016/s0167-7799 ( 99 ) 01417-1 we will be providing waivers..., rapidly evolving situation articles as well as case reports and case series related to COVID-19 quickly! System associated with relative anaerobiosis in an obligate aerobe were stored every 30 s for 3 days and used... The data used to produce CO2 and H2 and obesity, ”, S. K.,... Phosphoenolpyruvate: hexose phosphotransferase system day alongside the samples work was supported by Saudi of. Alternate pathways for microbial acetate assimilation the School of Biological Sciences, of. To remove all the excess dye [ 6, 20 ] available from the author! With palmitate incubation D-fructose transport system for induction of the B6 or allele...

Red Door Homes Litchfield, Precast Concrete Window Sills Near Me, Landmass Meaning Tagalog, Mobile Homes For Rent In Mississippi, Too Much Meaning In Tamil, Mayor Of Eastover Sc 2020, Standard Error Calculation, Corian Commercial Samples, Precast Concrete Window Sills Near Me, Schwa Sound For Kids, The Cowboy Blacklist, North Carolina Electronic Services,