- Results: We performed transcriptome and proteome profiling on chicken livers at five embryonic stages (E7, E12, E14, E17 and E21) between the fat and lean chicken lines. - At each stage and 169 differentially expressed genes were found by the digital gene expression, respectively, which were significantly enriched in the metabolic, PPAR signaling and fatty acid metabolism pathways. - Quantitative proteomics analysis found 20 differentially expressed proteins related to lipid metabolism, PPAR signaling, fat digestion and absorption, and oxidative phosphorylation pathways.. - Combined analysis showed that genes and proteins related to lipid transport (intestinal fatty acid-binding protein, nucleoside diphosphate kinase, and apolipoprotein A-I), lipid clearance (heat shock protein beta-1) and energy metabolism (NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10 and succinate dehydrogenase flavoprotein subunit) were significantly differentially expressed between the two lines.. - Conclusions: For hepatic lipid metabolism at embryonic stages, molecular differences related to lipid transport, lipid clearance and energy metabolism exist between the fat and lean chicken lines, which might contribute to the striking differences of abdominal fat deposition at post-hatch stages.. - Unlike in mammals, chicken lipogenesis is very limited in the adipose tissue [6], and more than 70% of de novo fatty acid synthesis takes place in the liver instead [7].. - Fatty acids synthesized in the liver are incorporated into triacylglycerols, and secreted as very low-density lipo- protein (VLDL). - Thus, accumulation of triacylglycerols in adipocytes is closely related to lipid metabolism in the liver [9].. - In previous studies, using adipose and liver tissues from the fat and lean chick- ens at various post-hatch stages (1, 4 and 7 weeks of age), we have found a number of differentially expressed genes (DEGs) and proteins (DEPs) related to lipid metabolism by the microarray and proteo- mics methods, such as peroxisome proliferator- activated receptor gamma (PPAR γ), liver basic fatty acids binding protein (LBFABP), LPL, adipocyte fatty acid-binding protein (AFABP), apolipoprotein A-I (ApoA-I), and long-chain acyl-coenzyme A dehydro- genase (ACADL) [10–13].. - From the fat and lean chicken lines at generation 14, which had 4.5-fold difference of AFP at 7 weeks of age [14], we collected hepatic tissues from embryos at five important developmental stages (E7, E12, E14, E17 and E21). - To identify genes differentially expressed (false dis- covery rate ≤ 0.001 and fold changes ≥2) and poten- tially involved in hepatic lipid metabolism, we compared the gene expression profiles between the fat and lean chicken lines. - DEGs at E7 were enriched in the GO term, the ubiquitin-protein ligase activity. - 2 Differentially expressed genes (DEGs) identified for hepatic tissues in chicken embryos. - a Number of DEGs at five developmental stages between the fat and lean lines. - Up and Down, up- and down-regulated DEGs in the fat line. - up- and down-regulated in the fat line. - We compared protein profiles between the fat and lean lines at each time point to find key proteins related to lipid metabolism. - We found 14 (protein spots protein spots protein spots protein spots 31, 57) and 1 (protein spot 40) differentially expressed protein spots (P <. - Among these differentially expressed protein spots, 3 protein spots were common to E7 and E12, and the protein spot 40 was common to E7 and E21. - At E7, differentially expressed protein spots and were up-regulated and down-regulated in the fat line, respectively. - 7) and E14 (protein spots signal intensities of these differentially expressed protein spots in the lean line was obviously stronger than those in the fat line. - At E17, the differen- tially expressed protein spot 31 was down-regulated, whereas spot 57 was up-regulated in the fat line. - At E21, differentially expressed protein spot 40 had higher expres- sion abundance in the fat line.. - A total of 26 differentially expressed protein spots were found at the five embryonic stages between the fat and lean lines, and after removing the 4 protein spots that repeated multiple times, 22 differentially expressed protein spots were identified by MALDI-TOF-MS. - Significantly enriched GO terms in the biological process, cellular component and molecular function (top five GO terms) categories, respectively (P <. - These DEPs were mainly involved in the PPAR signaling path- way, citrate cycle (TCA cycle), fat digestion and absorp- tion, oxidative phosphorylation, aminoacyl-tRNA biosynthesis and MAPK signaling pathways.. - Integrated analysis on transcriptome and proteome data For the 14 DEPs identified by the comparative proteo- mics, 2 of them, alpha-fetoprotein and OVAL, had no data in the digital gene expression experiment. - However, only were three genes, lysyl-tRNA synthetase (KARS), tyrosyl-tRNA synthetase (YARS) and intestinal fatty acid-binding protein (FABP2), validated to be sig- nificantly differentially expressed at E7 and E17, respect- ively (Fig. - We selected these two proteins, based on the facts that ApoA-I antibody is prepared in the lab and ready-to-use, FABP2 antibody is commercially available and the anti- genic epitope of the commercial FABP2 antibody is rela- tively conserved (72% similarity to human). - Subgroup 1 included 2 proteins, in which YARS and FABP2 were significantly higher in the fat birds at E7 and E17. - In Group II, 9 DEPs were not significantly differentially expressed at the transcriptional levels, which could also be classified into 2 subgroups. - a 12 DEGs identified by digital gene expression. - Y-axis, log2 (fold change of gene expression levels of the fat line vs. - and development, we previously found genes and mo- lecular pathways important for hepatic lipid metabolism (PPAR γ, LBFABP, ApoA-I, AFABP and glycol- metabolism) in the liver and adipose tissues at 1, 4 and 7 weeks of age by microarray and proteomics analyses [10–13]. - In the current study, we compared transcriptome and proteome profiling on hep- atic tissues sampled from 5 different embryonic stages between the two lines. - At E12, 5 differentially expressed protein spots were identified. - x-axis, differentially expressed protein spots. - Table 2 Features of the 20 differentially expressed proteins identified by MALDI-TOF-MS Stage Protein. - Inconsistency be- tween mRNA and protein expression levels probably comes from a variety of factors involved in the regula- tion of mRNA and protein abundances, such as (post-) transcriptional and (post-) translational regulation, pro- tein modification, protein-protein interaction, and other regulatory mechanisms as well [16]. - Proteins related to lipid transport, FABP2, NDK and ApoA-I, were found to be differentially expressed be- tween the two lines in the present study. - KARS, YARS and FABP2 were significantly differentially expressed. - Table 2 Features of the 20 differentially expressed proteins identified by MALDI-TOF-MS (Continued) Stage Protein. - which were also validated to be significantly differentially expressed between the two lines at E17 by western blot and qRT-PCR. - FABP2 is involved in lipid metabolism, especially in the uptake, intracellular metabolism and transport of long chain fatty acids [17, 18]. - The higher expression level of FABP2 in the fat birds may imply that the fat birds have stronger capacity of lipid transport.. - In the present study, mass spectrometry results showed NDK had two isoforms (protein spots 40 and 58). - The abundance of protein spot 40 in the fat line was significantly higher than that in the lean line at E7 and E21, and the transcriptional level of NDK was higher in the lean line compared to the fat line at E7 and E21.. - However, for protein spot 58 at E7, its mRNA and pro- tein abundance was down-regulated in the fat birds. - NDK catalyzes phosphoryl transfer from a nucleoside triphosphate to a nucleoside diphosphate, and functions in the metabolic pathway [21]. - How- ever, no transcriptional difference for ApoA-I in the embryonic liver was found between the fat and lean broilers at E17, suggesting the ApoA-I may be regulated at the translational level. - Thus, we speculate that ApoA-I can influence embryonic liver lipid metabolism, and con- tribute to the striking differences of abdominal fat de- position between the fat and lean chicken lines.. - HSPB1 could be related to lipid clearance, and protein levels were significantly higher in the lean birds at E7 and E12 as revealed by quantitative proteomics. - For ApoA-I, the lean line were significantly higher than the fat line (a, b). - In contrast, for FABP2, the fat line had strikingly higher protein level than the lean line (a, b). - We previously found that HSP27 protein was down-regulated in the abdominal adipose tissue of fat birds [12, 13], and here we found that HSP27 was down-regulated in the liver tissue of fat Table 3 Joint analyses on differentially expressed genes and proteins in qRT-PCR and comparative proteomics. - The protein and transcriptional abun- dance of SULT was all higher in the lean line at E7, whereas the protein level of SULT was opposite to its transcriptional level at E12. - The different ex- pression of SULT between the two lines at E7 and E12 showed that SULT may participate in hepatic lipid me- tabolism via PPARγ in the chicken. - Both transcriptional and protein expression levels of TXNDC5 was higher in the fat chickens at E7. - Moreover, we found also that proteins in the oxidative stress pathway were differentially expressed. - The protein abun- dance of SDHA was higher in the fat line, whereas the protein level was opposite to its transcriptional level at E7. - The mRNA and protein levels of NDUFB10 were higher in the lean lines at E7. - NDUFB10 is a subunit of the NADH dehydrogenase (ubiquinone) complex, located in the mitochondrial. - The different expression of SDHA and NDUFB10 indicated that the fat birds and lean birds had differences in regard to energy metabolism.. - We found two Aminoacyl-tRNA synthetases (KARS and YARS), which are important for amino acid synthe- sis, were differentially expressed between the fat and lean birds in liver tissue at E7. - Both the protein and mRNA expression levels of YARS were significantly higher in the fat birds at E7. - The protein level of KARS was significantly higher in the fat birds at E7 and the mRNA level was significantly higher in the lean birds at E7. - But the functions of the two proteins in chicken hepatic lipid metabolism are not very clear.. - ITPA had higher protein and mRNA abundances in the lean broilers at E7. - The protein abun- dance of CORO1C was significantly higher in the lean broilers at E14, whereas transcriptional abundance was higher in the fat broilers. - As mentioned above, we found that genes/proteins related to lipid transport and energy metabolism were differentially expressed in the embryonic liver between the fat and lean lines. - Molecular differences related to lipid transport, lipid clearance and energy metabolism exist for hepatic lipid metabolism at embryonic stages between the fat and lean chicken lines, which might contribute to the striking differences of abdominal fat deposition at post-hatch stages.. - The fertilized chicken eggs were chosen from the 14th generation of Northeast Agricultural University broiler lines divergently selected for high and low abdominal fat content (NEAUHLF), 200 each for the fat and lean lines, respectively. - The fertilized chicken eggs were all hatched in the same conditions at Northeast Agricultural Univer- sity hatchery.. - Unambiguous tags mapped to one gene were annotated and novel transcripts were discovered by comparing to known transcripts in the database. - Differentially expressed genes were identified according to the methods described previously [51], and false discovery rate was used for multiple testing correction. - Details of the experimental design were briefly described as follows.. - The DeCyder BVA module was used to performing comparative cross-gel statistical analysis of all spots, permitting the detection of differentially expressed spots between experimental groups (t-test, P <. - qRT-PCR. - DEGs identified in liver of E7 embryos between the fat and lean lines. - DEGs identified in liver of E12 embryos between the fat and lean lines. - DEGs identified in liver of E14 embryos between the fat and lean lines. - DEGs identified in liver of E17 embryos between the fat and lean lines. - DEGs identified in liver of E21 embryos between the fat and lean lines. - DEGs: Differentially expressed genes;. - DEPs: Differentially expressed proteins. - The funding bodies had no role in the specific design of the current study and collection, analysis and interpretation of data and in writing of the manuscript.. - CYW and BHC participated in the analysis of data. - ZQD participated in the analysis of data and helped write the manuscript. - Evolution of the modern broiler and feed efficiency. - In vitro hepatic lipogenesis in the hen and chick. - Microarray analysis of genes differentially expressed in the liver of lean and fat chickens. - Identification of differentially expressed proteins in adipose tissue of divergently selected broilers. - Intestinal fatty acid binding protein may favor differential apical fatty acid binding in the intestine. - Characterization of the chicken apolipoprotein A-I gene 5 ′ -flanking region. - Heat shock factors and the control of the stress response.. - TXNDC5 gene polymorphism contributes to increased risk of hepatocellular carcinoma in the Korean male population. - The quaternary structure of the saccharomyces cerevisiae succinate dehydrogenase. - Lipid metabolism in the chick embryo. - Biochemistry of the avian embryo. - The significance of digital gene expression profiles.
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