Metabolism-driven post-translational modifications of H3K9 in early bovine embryos

Reproduction ◽  
2021 ◽  
Vol 162 (3) ◽  
pp. 181-191
Author(s):  
Jessica Ispada ◽  
Aldcejam Martins da Fonseca Junior ◽  
Otávio Luiz Ramos Santos ◽  
Camila Bruna de Lima ◽  
Erika Cristina dos Santos ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Developmental Stages ◽  
De Novo ◽  
Blastocyst Stage ◽  
Developmental Model ◽  
Bovine Embryos ◽  
Acetyl Coa ◽  
Post Translational Modifications ◽  
The Relationship ◽  
Different Levels

Metabolic and molecular profiles were reported as different for bovine embryos with distinct kinetics during the first cleavages. In this study, we used this same developmental model (fast vs slow) to determine if the relationship between metabolism and developmental kinetics affects the levels of acetylation or tri-methylation at histone H3 lysine 9 (H3K9ac and H3K9me3, respectively). Fast and slow developing embryos presented different levels of H3K9ac and H3K9me3 from the earliest stages of development (40 and 96 hpi) and up to the blastocyst stage. For H3K9me3, both groups of embryos presented a wave of demethylation and de novo methylation, although it was more pronounced in fast than slow embryos, resulting in blastocysts with higher levels of this mark. The H3K9ac reprogramming profile was distinct between kinetics groups. While slow embryos presented a wave of deacetylation, followed by an increase in this mark at the blastocyst stage, fast embryos reduced this mark throughout all the developmental stages studied. H3K9me3 differences corresponded to writer and eraser transcript levels, while H3K9ac patterns were explained by metabolism-related gene expression. To verify if metabolic differences could alter levels of H3K9ac, embryos were cultured with sodium-iodoacetate (IA) or dichloroacetate (DCA) to disrupt the glycolytic pathway or increase acetyl-CoA production, respectively. IA reduced H3K9ac while DCA increased H3K9ac in blastocysts. Concluding, H3K9me3 and H3K9ac patterns differ between embryos with different kinetics, the second one explained by metabolic pathways involved in acetyl-CoA production. So far, this is the first study demonstrating a relationship between metabolic differences and histone post-translational modifications in bovine embryos.

138 THE RELATIONSHIP BETWEEN THE NORMALITY OF FIRST CLEAVAGE, THE GENE EXPRESSION IN BLASTOMERES, AND THE ABILITY TO DEVELOP TO THE BLASTOCYST STAGE IN IVF-DERIVED BOVINE 2-CELL STAGE EMBRYOS

2017 ◽  
Vol 29 (1) ◽  
pp. 177
Author(s):  
S. Matoba ◽  
M. Kaneda ◽  
T. Somfai ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Target Genes ◽  
Calf Serum ◽  
Transcript Abundance ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Stage ◽  
Embryonic Cleavage ◽  
The Relationship

Early first and second cleaved embryos after IVF associated with even blastomeres without fragments or protrusions were found to be a potent criterion for the selection of embryos with high developmental competence (Sugimura et al. 2012 PLOS ONE 7, e36627). The aim of this study was to examine the relationship between an early normal first cleavage pattern, the transcript abundance, and their development to the blastocyst stage in each blastomere in 2-cell stage bovine embryos. The IVF-derived bovine embryos were cultured individually in well-of-the-well culture dishes in CR1aa medium supplemented with 5% calf serum and 0.25 mg mL−1 linoleic acid albumin at 38.5°C in 5% CO2, 5% O2, and 90% N2. The first embryonic cleavage was categorized as being either normal (occurring within 28 h after IVF with 2 even blastomeres without fragment or protrusion) or abnormal (2 uneven blastomeres, with/without fragment/protrusion and/or later than 28 h after IVF). Then, cleaved embryos were placed in 0.5% actinase-E in Ca- and Mg-free PBS and blastomeres were separated by pipetting (n = 85; 4 replicates). In each embryo, one blastomere was subjected to quantitative RT-PCR to analyse the expression of developmentally important genes. The remaining blastomere was subsequently cultured in an individually identifiable manner to verify their ability to develop to the blastocyst stage. Primers were designed for 12 target genes related to pluripotency, cell cycle, metabolism, pregnancy reorganization, placentation, and fetal growth (OCT4, ATP1A1, CCNB1, CDH1, COX1, CTNNB1, GLUT8, MNSOD-3, SOX2, DYNLL1, IGFBP3, and PMSB1) and a reference gene (PPIA). Transcript abundance of target genes in individual blastomeres was compared between embryos showing normal and abnormal cleavage. Values were normalized to the average values of the reference genes and all the means were compared by the Student t-test. Blastomeres resulted from normal cleavage developed to the blastocyst stage on Day 7 to 8 (Day 0 = IVF) at significantly higher rates than those resulted from abnormal cleavage (65.7% v. 37.5%, respectively, P < 0.05). Transcript abundance of OCT4 was significantly higher in blastomeres associated with all abnormal cleavage than in those associated with normal cleavage (P < 0.05). The expression of CCNB1, COX1, ATP1A1, GLUT8, and PMSB1 in blastomeres associated with normal cleavage and blastocyst development was higher than that in those of abnormal cleavage (P < 0.05). However, the level of OCT4, CCNB1, COX1, ATP1A1, and PMSB1 was lower in blastomeres associated with normal cleavage but failure of blastocyst development than those in blastomeres showing abnormal cleavage (P < 0.05). Our results reveal that significantly higher expression of CCNB1, COX1, ATP1A1, and PMSB1 in blastomeres at the 2-cell stage in bovine embryos with superior developmental competence compared with those showing abnormal cleavage and low competence. Research was supported by JSPS KAKENHI (26450388).

scholarly journals Anthracyclins increase free PUFAs and etherPEs with PUFAs as potential hallmarks of lipid peroxidation and ferroptosis

2021 ◽  
Author(s):  
David Balgoma ◽  
Fredrik Kullenberg ◽  
Carlemi Calitz ◽  
Maria Kopsida ◽  
Femke Heindryckx ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Cell Death ◽  
Cell Lines ◽  
Metabolic Pathways ◽  
De Novo ◽  
Primary Liver Cancer ◽  
De Novo Lipogenesis ◽  
Liver Cancer Cell ◽  
The Individual ◽  
The Relationship

AbstractMetabolic and personalized interventions in cancer treatment require a better under-standing of the relationship between the induction of cell death and metabolism. Consequently, we treated three primary liver cancer cell lines with two anthracyclins (doxorubicin and idarubin) and studied the changes of the lipidome. We found that both anthracyclins in the three cell lines increased the levels of polyunsaturated fatty acids (PUFAs) and alkylacylglycerophosphoethano-lamines (etherPEs) with PUFAs. As PUFAs and alkylacylglycerophospholipids with PUFAs are fundamental in lipid peroxidation during ferroptotic cell death, our results suggests supplementa-tion with PUFAs and/or etherPEs with PUFAs as a potential general adjuvant of anthracyclins. In contrast, neither the markers of de novo lipogenesis nor cholesterol lipids presented the same trend in all cell lines and treatments. In agreement with previous research, this suggests that modulation of the metabolism of cholesterol could be considered a specific adjuvant of anthracyclins depend-ing on the type of tumor and the individual. Finally, we discuss the changes in the lipidome in re-lation to the endoplasmic reticulum stress and the sensitivity to anthracyclins of the different cells. In conclusion, our results suggest that the modulation of different lipid metabolic pathways may be considered for generalized and personalized metabochemotherapies.

Effect of Two-step Vitrification on Developmental Competence of in vitro and in vivo Produced Bovine Embryos

2010 ◽  
Vol 79 (9) ◽  
pp. S55-S61 ◽  
Author(s):  
Jaroslava Hlavicová ◽  
Miloslava Lopatářová ◽  
Svatopluk Čech
Keyword(s):  
Developmental Stages ◽  
Blastocyst Stage ◽  
Developmental Competence ◽  
Hatching Rate ◽  
Bovine Embryos ◽  
Holstein Friesian ◽  
Quality Grade ◽  
Friesian Cattle

The aim of this study was to establish the effect of two-step vitrification on survival rate of bovine embryos produced in vitro (method A) and in vivo (method B) from Holstein-Friesian cattle. The embryos suitable for vitrification were frozen by a two-step technique, using increasing concentrations of dimethyl sulphoxide (DMSO) and ethylene glycol (EG). After thawing, the quality grade and developmental stage of embryos was assessed. In vitro developmental competence of embryos of different quality grade obtained by method B (n = 82) was significantly higher (p < 0.001) compared to method A (n = 98). The best results were detected when we vitrified the embryos of the grade 1 quality; namely, the hatched blastocyst stage was reached by 6.9% (2/29) of embryos retrieved by method A and by 36.7% (11/30) of embryos retrieved by method B (p < 0.01). In the case of developmental competence of embryos at different developmental stages we reached significantly better results (p < 0.001) when we vitrified the embryos produced by method B (n = 84) in comparison with method A (n = 67). We noted a higher hatching rate at the stage of expanded blastocyst; namely, the hatched blastocyst stage was reached by 7.4% (2/27) of embryos produced by method A and by 30.8% (8/26) of embryos produced by method B (p < 0.05). In general, the hatched blastocyst stage was reached by 15.1% (50/331) of all thawed embryos retrieved by method A and B. In conclusion, when we applied two-step vitrification on the grade 1 quality embryos at the stage of expanded blastocyst produced in vitro or at the stage of morula produced in vivo we achieved the highest hatching rates.

scholarly journals The second lineage differentiation of bovine embryos fails in the absence of OCT4/POU5F1

2021 ◽  
Author(s):  
Kilian Simmet ◽  
Mayuko Kurome ◽  
Valerie Zakhartchenko ◽  
Horst-Dieter Reichenbach ◽  
Claudia Springer ◽  
...  
Keyword(s):  
Developmental Stages ◽  
Inner Cell Mass ◽  
Ex Vivo ◽  
Expression Patterns ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Vitro Fertilization ◽  
Lineage Differentiation

The mammalian blastocyst undergoes two lineage segregations, i.e., formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) the remaining pluripotent lineage. To clarify expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9 and 12 blastocysts completely derived ex vivo by staining for OCT4, NANOG, SOX2 (EPI) and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost of NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show, that OCT4 is required cell-autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

89 Embryonic metabolism orchestrates epigenetic mechanisms: What can we anticipate from the first cleavages?

2020 ◽  
Vol 32 (2) ◽  
pp. 170
Author(s):  
J. Ispada ◽  
A. M. Fonseca Junior ◽  
E. C. dos Santos ◽  
K. Annes ◽  
O. L. R. Santos ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Fluorescence Intensity ◽  
Embryonic Stem ◽  
Blastocyst Stage ◽  
Classification Model ◽  
Control Group ◽  
Compensatory Mechanism ◽  
Bovine Embryos ◽  
Acetyl Coa ◽  
Group 5

Intermediates of the energy metabolism (as acetyl Co-A) can donate their acetyl group to introduce acetylation in histones, establishing a relationship between metabolism and epigenetic control in somatic and embryonic stem cells. Embryos with different kinetics during the first cleavages also have alterations in epigenetic profile as well as in metabolism and energy substrate consumption during invitro culture. The aim of this work was to verify if and how this relation between metabolism and epigenetic parameters was also presented in invitro-produced bovine embryos. For that, we first characterised the pattern of H3K9ac, and the molecular pattern of enzymes involved with histone acetylation and acetyl-CoA production in female blastocysts derived from fast and slow cleavage embryos. To validate the results, we also produced bovine embryos cultured with an inhibitor of the pyruvate production, and consequently the acetyl Co-A generation to check if this could interfere in the H3K9ac pattern. For this, embryos were invitro produced following standard protocol and classified at 40 hours post-insemination as fast (4 or more cells) or slow (2 cells) and collected at the blastocyst stage. Blastocysts were immunostained to H3K9ac and the fluorescence intensity of each nucleus was quantified using ImageJ and analysed by Student's t-test. For transcript quantitation, RNAseq data were accessed from a previous report using the same kinetics classification model (Milazzotto et al. 2016Mol. Rep. Dev. 83, 324-336; https://doi.org/10.1002/mrd.22619) and analysed using limma-voom on Galaxy 3.38.3. To validate the results, bovine embryos were produced and cultured until Day 4 and then incubated until the blastocyst stage with different doses of iodoacetate (IA; 2 and 5mM) to reduce the intracellular levels of acetyl CoA. These blastocysts were also assessed by H3K9 acetylation. Slow blastocysts presented higher fluorescence intensity for H3K9ac than fast blastocysts (fast 13.33±0.37 AU vs. slow 38.14±1.17 AU; P&lt;0.0001). Despite the fact that there were no differences in transcripts related to this acetylation (ELP3 and HAT2), slow blastocysts presented higher levels of transcripts for PDHB1 and PDHA1, responsible for acetyl-CoA production (PDHB1: fast 11.6±0.2CPM vs. slow 13.1±0.2 counts per million; P&lt;0.01 PDHA1: fast 12.6±0.2 CPM vs. slow 13.2±0.3 CPM; P&lt;0.01). The reduction of acetyl-CoA in blastocysts induced by IA led to lower levels of H3K9ac in 1 and 2mM doses when compared with the control (control: 43.8±0.7 AU; 1 mM: 34.7±0.5 AU; 2 mM: 30.1±0.6 AU; P&lt;0.0001). Interestingly, H3K9ac levels were similar for 5mM IA and control group (5 mM: 41.2±1.4; P&gt;0.05), suggesting a compensatory mechanism in extreme cases to maintain the histone acetylation. As far as we know, this is the first work that describes a relation between metabolism and epigenetics in bovine embryos. Although the pattern of genes related to acetylation seems to be unaltered, changes in acetyl Co-A production pathway exert an influence on H3K9ac status. Grant support was provided by grant 2017/18384-0 and 2018/11668-6 from FAPESP and CAPES.

Overexpression and inhibition of 3-hydroxy-3-methylglutaryl-CoA synthase affect central metabolic pathways in tobacco

2020 ◽  
Author(s):  
Pan Liao ◽  
Shiu-Cheung Lung ◽  
Wai Lung Chan ◽  
Menglong Hu ◽  
Geoffrey Kwai-Wai Kong ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
Quantitative Proteomics ◽  
Tricarboxylic Acid Cycle ◽  
Specific Inhibitor ◽  
Primary Metabolism ◽  
Proteomics Analysis ◽  
Acetyl Coa ◽  
Cell Wall Modification ◽  
Mva Pathway ◽  
The Relationship

Abstract Little has been established on the relationship between the mevalonate (MVA) pathway and other metabolic pathways except for the sterol and glucosinolate biosynthesis pathways. In the MVA pathway, 3-hydroxy-3-methylglutaryl-CoA synthase (HMGS) catalyses the condensation of acetoacetyl-CoA and acetyl-CoA to form HMG-CoA. Our previous studies had shown that while the recombinant Brassica juncea HMGS1 (BjHMGS1) mutant S359A displayed 10-fold higher enzyme activity than wild-type (wt) BjHMGS1, transgenic tobacco overexpressing S359A (OE-S359A) exhibited higher sterol content, growth rate and seed yield than OE-wtBjHMGS1. Herein, untargeted proteomics and targeted metabolomics were employed to understand the phenotypic effects of HMGS overexpression in tobacco by examining which other metabolic pathways were affected. SWATH-MS quantitative proteomics analysis on OE-wtBjHMGS1 and OE-S359A identified the misregulation of proteins in primary metabolism and cell wall modification, while some proteins related to photosynthesis and the tricarboxylic acid cycle were upregulated in OE-S359A. Metabolomic analysis indicated corresponding changes in carbohydrate, amino acid and fatty acid contents in HMGS-OEs, and F-244, a specific inhibitor of HMGS, was applied successfully on tobacco to confirm these observations. Finally, the crystal structure of acetyl-CoA-liganded S359A revealed that improved activity of S359A likely resulted from a loss in hydrogen bonding between Ser359 and acyl-CoA which is evident in wtBjHMGS1. This work suggests that regulation of plant growth by HMGS can influence the central metabolic pathways. Furthermore, this study demonstrates that the application of the HMGS-specific inhibitor (F-244) in tobacco represents an effective approach for studying the HMGS/MVA pathway.

scholarly journals Identification and regulation of glycogen synthase kinase-3 during bovine embryo development

Reproduction ◽  
2010 ◽  
Vol 140 (1) ◽  
pp. 83-92 ◽  
Author(s):  
I M Aparicio ◽  
M Garcia-Herreros ◽  
T Fair ◽  
P Lonergan
Keyword(s):  
Embryo Development ◽  
Developmental Stages ◽  
Glycogen Synthase ◽  
Specific Inhibitor ◽  
Blastocyst Stage ◽  
Serine Phosphorylation ◽  
Glycogen Synthase Kinase ◽  
Bovine Embryo ◽  
Bovine Embryos ◽  
Cell Stage

The aim of this study was to examine the presence and regulation of glycogen synthase kinase-3α (GSK3A) and GSK-3β (GSK3B) in bovine embryos and their possible roles in embryo development. Our results show that GSK3A and GSK3B are present in bovine embryos at the two-cell stage to the hatched blastocyst stage. Bovine embryo development was associated with an increase in the phosphorylation of both isoforms, being statistically significant at blastocyst and hatched blastocyst stages, compared with earlier stages. Inhibition of GSK3 with CT99021 (3 μM) resulted in a significant increase in the percentage and quality of blastocysts, while inhibition of GSK3 with lithium chloride (LiCl; 20 mM) significantly reduced at the proportion of eight-cell embryos on day 3 and inhibited blastocyst formation. The use of LY294002 (10 μM), a specific inhibitor of phosphatidylinositol-3 kinase, also produced a significant decrease in embryo development. In addition, treatment with LiCl and LY294002 produced a significant decrease in the serine phosphorylation of both isoforms of GSK3. Finally, CT99021 and LiCl reduced the phosphorylation of β-catenin on Ser45 in two-cell embryos, while LY294002 increased it. Despite the fact that LiCl inhibited GSK3 activity, as demonstrated by β-catenin phosphorylation, its effects on the bovine embryo could be mediated through other signaling pathways leading finally to a decrease in the phosphorylation of GSK3 and a reduction in embryo development. Therefore, in conclusion, GSK3A/B serine phosphorylation was positively correlated with embryo development, indicating the importance of an accurate regulation of GSK3 activity during developmental stages to achieve normal bovine embryo development.

scholarly journals Biological differences between in vitro produced bovine embryos and parthenotes

Reproduction ◽  
2009 ◽  
Vol 137 (2) ◽  
pp. 285-295 ◽  
Author(s):  
Enrique Gómez ◽  
Alfonso Gutiérrez-Adán ◽  
Carmen Díez ◽  
Pablo Bermejo-Alvarez ◽  
Marta Muñoz ◽  
...  
Keyword(s):  
De Novo ◽  
Inner Cell Mass ◽  
Relative Proportion ◽  
Cell Mass ◽  
Blastocyst Stage ◽  
Bovine Embryos ◽  
Blastocyst Development ◽  
Cell Counts ◽  
Biological Differences

Parthenotes may represent an alternate ethical source of stem cells, once biological differences between parthenotes and embryos can be understood. In this study, we analyzed development, trophectoderm (TE) differentiation, apoptosis/necrosis, and ploidy in parthenotes andin vitroproduced bovine embryos. Subsequently, using real-time PCR, we analyzed the expression of genes expected to underlie the observed differences at the blastocyst stage.In vitromatured oocytes were either fertilized or activated with ionomycin +6-DMAP and cultured in simple medium. Parthenotes showed enhanced blastocyst development and diploidy and reduced TE cell counts. Apoptotic and necrotic indexes did not vary, but parthenotes evidenced a higher relative proportion of apoptotic cells between inner cell mass and TE. The pluripotence-relatedPOU5F1and the methylationDNMT3Agenes were downregulated in parthenotes. Among pregnancy recognition genes,TP-1was upregulated in parthenotes, whilePGRMC1andPLAC8did not change. Expression ofp66shcandBAX/BCL2ratio were higher, andp53lower, in parthenotes. Among metabolism genes,SLC2A1was downregulated, whileAKR1B1,PTGS2,H6PD, andTXNwere upregulated in parthenotes, andSLC2A5did not differ. Among genes involved in compaction/blastulation,GJA1was downregulated in parthenotes, but no differences were detected withinATP1A1andCDH1. Within parthenotes, the expression levels ofSLC2A1,TP-1, andH6PD, and possiblyAKR1B1, resemble patterns described in female embryos. The pro-apoptotic profile is more pronounced in parthenotes than in embryos, which may differ in their way to channel apoptotic stimuli, throughp66shcandp53respectively, and in their mechanisms to control pluripotency andde novomethylation.

Phosphorylated H2AX in parthenogenetically activated, in vitro fertilized and cloned bovine embryos

Zygote ◽  
2014 ◽  
Vol 23 (4) ◽  
pp. 485-493 ◽  
Author(s):  
A.F. Pereira ◽  
L.M. Melo ◽  
V.J.F. Freitas ◽  
D.F. Salamone
Keyword(s):  
Dna Damage ◽  
Developmental Stages ◽  
Blastocyst Stage ◽  
Preimplantation Embryos ◽  
Bovine Embryos ◽  
Dna Breaks ◽  
Embryo Production ◽  
Cell Stage ◽  
Γh2ax Foci

SummaryIn vitro embryo production methods induce DNA damage in the embryos. In response to these injuries, histone H2AX is phosphorylated (γH2AX) and forms foci at the sites of DNA breaks to recruit repair proteins. In this work, we quantified the DNA damage in bovine embryos undergoing parthenogenetic activation (PA), in vitro fertilization (IVF) or somatic cell nuclear transfer (SCNT) by measuring γH2AX accumulation at different developmental stages: 1-cell, 2-cell and blastocyst. At the 1-cell stage, IVF embryos exhibited a greater number of γH2AX foci (606.1 ± 103.2) and greater area of γH2AX staining (12923.6 ± 3214.1) than did PA and SCNT embryos. No differences at the 2-cell stage were observed among embryo types. Although PA, IVF and SCNT were associated with different blastocyst formation rates (31.1%, 19.7% and 8.3%, P < 0.05), no differences in the number of γH2AX foci or area were detected among the treatments. γH2AX is detected in bovine preimplantation embryos produced by PA, IVF and SCNT; the amount of DNA damage was comparable among those embryos developing to the blastocyst stage among different methods for in vitro embryo production. While IVF resulted in increased damage at the 1-cell embryo stage, no difference was observed between PA and SCNT embryos at any developmental stage. The decrease in the number of double-stranded breaks at the blastocyst stage seems to indicate that DNA repair mechanisms are functional during embryo development.

scholarly journals Identifying Sources of Hepatic Lipogenic Acetyl-CoA Using Stable Isotope Tracers and NMR

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
John G. Jones
Keyword(s):  
Nutrient Intake ◽  
De Novo ◽  
Acetyl Coa ◽  
Nutrient Sources ◽  
Isotope Tracers ◽  
Disease States ◽  
The Relationship ◽  
Insight Into ◽  
C Nmr

The role of hepatic de novo lipogenesis (DNL) in promoting fatty liver disease and hypertriglyceridemia during excessive nutrient intake is becoming firmly established. Certain nutrients such as fructose promote hepatic DNL activity and this has been at least partly attributed to their efficient conversion to the acetyl-CoA precursors of DNL. However, tracer studies indicate a paradoxically low level of fructose incorporation into lipids, which begs the question of what the actual lipogenic acetyl-CoA sources are under these and other conditions. Here, we describe novel approaches for measuring substrate contributions to lipogenic hepatic acetyl-CoA using 13C-tracers and 13C-NMR analysis of lipids and acetyl-CoA probes. We review and address aspects of hepatic intermediary fluxes and acetyl-CoA compartmentation that can confound the relationship between 13C-precursor substrate and lipogenic 13C-acetyl-CoA enrichments and demonstrate novel methodologies that can provide realistic estimates of 13C-enriched substrate contributions to DNL. The most striking realization is that the principal substrate contributors to lipogenic acetyl-CoA have yet to be identified, but they are probably not the so-called “lipogenic substrates” such as fructose. The proposed methods may improve our insight into the nutrient sources of DNL under various feeding and disease states.

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