Transcriptome analyses of liver in newlyhatched chicks during the metabolic perturbation of fasting and re-feeding reveals THRSPA as the key lipogenic transcription factor

- Background: The fasting-refeeding perturbation has been used extensively to reveal specific genes and metabolic pathways that control energy metabolism in the chicken.
- The first few days after hatching pose the most critical period in the chicken’s terrestrial life.
- After consuming its first meal, the hatchling chick launches a predominant lipogenic drive in its major metabolic organ—the liver..
- We have developed functional genomic tools and gen- etic resources to gain a global view and more detailed understanding of genes and gene interaction networks that regulate important biological processes (e.g., growth, metabolism and development) in the chicken [1–4].
- Recently, we used the Affymetrix Chicken Genome Chip® to expand the repertoire of hepatic genes involved in the homeorhe- tic regulation of metabolism during the peri-hatch period [5].
- Our immediate interest in the present study was examination of global patterns of hepatic gene expres- sion in newly-hatched cockerels during a fasting- refeeding perturbation, given during the first 4 days (D0- D4) of terrestrial life.
- Additionally, the time from hatching to provision of feed can vary in the poultry industry, due to timing of the hatch and distance for transportation of hatchling chicks to the rearing houses.
- Previous studies of gene expression in regulatory and metabolic tissues during a bout of fasting and refeeding in the chicken have been implemented.
- A recent study investigating the impact of delayed feeding at hatch on gene expression patterns in liver and breast muscle revealed perturbations in developmental profiles of PPARG and CHREBP, which indicates a tran- sitional delay in the switch from lipid to carbohydrate metabolism in these tissues [11]..
- However, plasma NEFA levels in the D3REFED24h and D4REFED48h chicks were similar to their respective control fed groups (D3FED72h;.
- The majority of DEGs in the D1FED and D2REFED4h treatment groups were also highly expressed and clustered together in the condition (treatment) tree.
- Three additional clusters of lipogenic DEGs (Clus- ters 1, 2 and 6) have identical expression patterns, with high- est expression in the refed state.
- The microarray DEGs that mapped to known mamma- lian genes accrued in the Ingenuity Knowledge Base were considered as AR-DEGs by IPA.
- However, almost one-quarter of the chicken-specific DEGs determined from our microarray analysis were rejected by IPA due to the absence of a valid Entrez Gene ID accrued in the.
- Fewer AR-DEGs were found in the D0FAST4h vs.
- The greatest number of AR-DEGs was found in the D2FED48h vs.
- In contrast, Cluster A genes are down-regulated in the fully-fed (FED) and refed (REFED) groups on day 3(D3) and D4, whereas Cluster B genes are up-regulated after refeeding and in FED groups on D2, D3 and D4.
- The largest number of unique DEGs (750 AR-DEGs) was found in the D2FED48h vs.
- The largest number of unique genes (489 AR-DEGs) was found in the D2FAST48h vs..
- The top three upstream regulators identified by IPA in the D1FED24h vs.
- Among the most highly expressed up-regulated genes in the D1FED24h treatment were THRSPA, deiodinase 2 (DIO2), ME1 and PLIN2.
- Annotated lists of AR-DEGs found in the five contrasts are provided by multiple worksheets in Additional file 3.
- SERTAD2 has a direct action on both DIO2 and acyl-CoA oxidase 1 (ACOX1), which is the initial enzyme in the fatty acid β- oxidation pathway.
- Several additional genes were expressed higher in the D1FAST24h treatment, including PSAT1, CTH, LDHB, Table 1 IPA summary of liver transcriptomes in-hatchling chicks--D1FED24h vs.
- The top four up-regulated AR-DEGs in the D2FED48h vs..
- D2FAST48h contrast were THRSPA, ME1, SCD, and FADS2, while the highest expressed AR-DEGs in the D2FAST48h treatment were IGFBP2, adenylosuccinate lyase (ADSL), CYP3A7, and CYP4A22..
- expressed in the D2FED48h group and 22 AR-DEGs had greater expression in the D2FAST48h chicks.
- The import- ance of “Oxidation of Fatty Acids” was confirmed by the greater abundance of 24 AR-DEGs in D2FAST48h chicks, whereas only 4 AR-DEGs were higher in the D2FED48h treatment.
- Likewise, the IPA canonical pathway “LPS/IL1 Inhibition of RXR Function” had 18 AR-DEGs that were more abundant in the D2FAST48h group, compared to only 8 genes with higher expression in the D2FED48h chicks.
- Under the “LXR/RXR Activation” pathway, only 6 AR- DEGs were up-regulated, while 12 AR-DEGs were more abundant in the D2FAST48h treatment.
- The “Coagu- lation System” was more active in the D2FAST48h treat- ment (7 AR-DEGs) than in the D2FED48h condition (1 AR-DEG)..
- In this overview of lipid synthesis, AR-DEGs with red symbols are lipogenic genes, which are highly expressed in liver of D2FED48h chicks, whereas green symbols indicate greater abundance of lipolytic genes in the D2FAST48h treatment (Additional file 4).
- Most AR-DEGs were found in the cytoplasm (70 AR-DEGs, which are mainly metabolic enzymes, trans- porters, kinases and phosphatases) with fewer genes in the plasma membrane (15 AR-DEGs.
- The IPA summary of liver transcriptomes in the D2FAST48h vs.
- 4 A gene interaction network (Panel a) of lipogenic (green symbols) and lipolytic (red symbols) AR-DEGS found in the D1FED24h vs..
- Among the highest expressed genes in the D2FAST48h treatment were CYP4A22, UPP2, IGFBP2, and N-myc downstream regu- lated 1 (NDRG1), whereas THRSPA, hexokinase domain.
- containing 1 (HKDC), cytochrome P450 family 51 sub- family A member 1 (CYP51A1) and methylsterol mono- oxygenase 1 (MSMO1) were the most abundant AR- DEGs found in the D2REFED4h treatment..
- of NR3C1, and were highly expressed in the D2FAST48h treatment.
- Genes with red symbols are expressed higher in liver of D2FED48h cockerels, while green symbols indicate higher hepatic expression in the D2FAST48h treatment.
- A group of five upstream regulators control transcription of numerous metabolic enzymes, transporters, kinases and phosphatases in the cytoplasm, several transmembrane receptors, G-protein-coupled receptors, peptidases and enzymes in the plasma membrane, and even fewer growth factors, transporters and enzymes found in extracellular space.
- Ingenuity Pathway Analysis (IPA) was used for functional analysis of 641 “ Analysis Ready ” (AR)-DEGs found in the D2FAST48h vs.D2REFED4h contrast.
- The top up-regulated genes in this contrast in- cluded IGFBP2, ADSL, LDHB and mannose binding lectin 2 (MBL2), whereas THRSPA, ME1, SCD and galectin 2 (LGALS2) were the highest expressed AR- DEGs in the D3REFED24h treatment..
- or chemerin, an adipokine) and acetyl-CoA acyltransferase 2 (ACAA2), the enzyme that catalyzes the final reaction in the fatty acid β-oxidation pathway.
- 7b), 6 AR- DEGs were up-regulated, while 22 genes were down- regulated or expressed higher in the D3REFED24h treat- ment group.
- Another gene network identified by IPA in the D2FAST48h vs.
- Six direct targets of FOXO1 were up-regulated in the D2FAST48h vs.
- 6 This gene network, identified in the D2FAST48h vs.
- Actually, the green gene symbols indicate higher expression in liver of D2REFED4h cockerels, while red gene symbols indicate higher expression in the D2FAST48h treatment group.
- Ingenuity predicts inhibition of the concentration of lipid in the D2FAST48h vs.
- On the other hand, several cyto- plasmic genes expressed higher in the D4REFED48h treatment, are involved in lipid metabolism (SCD, INSIG1, ACAT2, HMGCR, FABP2, ELOVL5).
- Type 2 deiodinase (DIO2) controls generation of metabolically- active T3, which drives activity of THRSPA, the major lipogenic transcription factor in the chicken.
- Additional genes found in the plasma membrane (ADIPOR2, PLIN2, LPCAT3, FADS2) or extracellular space (LIPG, ANGPTL3, ANGPTL4) that support lipid metabolism are also expressed at higher levels in liver of D4REFED48h chicks..
- was centered on the interactions of four upstream regu- lators identified in the D2FAST48h vs.
- IPA predicted activation (orange lines and arrows) or inhibition (blue lines) of direct targets of SREBF2 identified in the D2FAST48h vs.
- As such, Ingenuity predicts that SREBF2 should be inhibited (blue symbol), which would lead to inhibition (blue arrows/edges) of 16 DEGs (green symbols) that control lipogenesis under the direction of the most highly-expressed gene in liver of fed or refed cockerels — the lipogenic transcription factor THRSPA.
- 8 A gene interaction network was identified in the D2FAST48h vs.
- The top 10 up-regulated and down-regulated AR-DEGs are presented along with their respective log2 ratio of treatment conditions, where positive numbers indicate higher expression in the D2FAST48h treatment and negative values indicate higher expression in the D4REFED48h treatment condition.
- Genes with red symbols are expressed higher in liver of D2FAST48h cockerels, while green symbols indicate higher hepatic expression in the D4REFED48h treatment.
- The expression of these metabolic genes was greatly depressed by fasting for 4, 24 or 48 h and re- stored after refeeding (4, 24 or 48 h), whereas only PPARG exhibited a progressive increase in hepatic ex- pression with age in the fed state.
- Three genes dem- onstrated a clear progressive increase in expression with age in the feed state (INSIG2, PPARA and UPP2)..
- FAT1 expression was higher in the fed state, depressed by fasting and restored with refeeding.
- Both FAT1 and GAPDH showed a pro- gressive increase with age in the fed state, both were de- pressed in the fasted state and recovered on D4 after being refed for 48 h.
- The only candidate that failed to show a positive correlation between microarray and qRT-PCR analysis was insulin induced gene 1 (INSIG1), which was not included in the Pearson’s Correlation Analysis.
- Unfortunately, two important lipid metabolism genes, FASN (RIGG07906 oligo) and PPARA (RIGG05780 oligo), appeared quite variable in the present microarray study and, there- fore, were not identified as DEGs by statistical ana- lysis, although qRT-PCR analysis did reveal them as DEGs..
- were expressed higher in liver of chicks under the D2FAST48h treatment, while 15 genes were expressed at higher levels in the D4REFED48h chicks.
- large group of lipogenic genes was depressed by fast- ing and quickly restored after refeeding, or remained high in the fully-fed state.
- Apparently, a handful (5–15) of key transcription factors regulates a larger number (100) of downstream genes involved in the synthesis or concen- tration of lipid in liver of newly-hatched chicks..
- Presently, THRSPA and DIO2 were the highest expressed hepatic genes in the D1FED24h vs.
- However, the exact mechanism(s) by which THRSPA affects the expression or activity of other transcription factors or downstream metabolic genes in the chicken remains unclear.
- Further investiga- tion will be required for elucidation of the exact mecha- nisms by which THRSPA promotes lipogenesis and for acknowledgement of THRSPA as the key lipogenic tran- scription factor regulating lipid metabolism in the chicken..
- The pair-wise contrasts made across 10 nutritional con- ditions proved useful in detecting the hierarchy of tran- scriptional control over responses of hundreds of downstream DEGs involved in the homeorhetic shift from lipolytic to lipogenic metabolism.
- In the present study, the adipokine RARRES2 was up- regulated by prolonged fasting (see Figs.
- And as emphasized in the transcriptional ana- lysis of liver from newly-hatched (D0) and fed D7 chicks, the hepatic lipogenic program is likely activated by both endocrine and nutrient signals once the chick consumes carbohydrate-rich feed and purges residual yolk lipids [10].
- Another hepatic gene that was highly expressed in the D4REFED48h chicks was DHCR24, an insulin induced gene that catalyzes the final step in cholesterol biosyn- thesis.
- DHCR24 regulation of DHCR24 expression and ultim- ately cholesterol synthesis, adds a new dimension to the complexity of transcriptional control over lipid metabol- ism in the chicken.
- Certainly, microRNAs are involved in the robust homeorhetic control of energy metabolism induced by fasting and refeeding in newly-hatched chicks.
- Presently, our gene interaction networks show THRSPA as the ultimate transcription factor controlling lipogenesis in the newly-hatched chicken.
- In the rat, PPARA also blocks THRSP ex- pression by interfering with THRB-T 3 binding to the multiple TREs in the THRSP promoter, rather than via a PPARA response element (PPAR-RE) [44].
- Our analyses of liver transcriptome during a fasting- refeeding perturbation immediately after hatching shows that THRSPA functions as the ultimate transcriptional regulator of lipogenesis and thermogenesis in the chicken.
- chicks in the fed or refed state.
- 1) were used in the following sections and described accordingly..
- Fertile eggs (Ross x Ross breed) were obtained from a commercial hatchery (Allen’s Hatchery, Seaford DE) and incubated in the Animal Facility, Department of Animal and Avian Sciences, University of Maryland (College Park, MD).
- After hatching, chicks in the T1, T3 and T5 groups (N = 15) were brooded with water freely available, but without access to feed (i.e., prolong-fasting) for 4, 24 and 48 h, respect- ively.
- Chicks in the T6, T8 and T10 groups (N = 15) were fasted for 48 h and subsequently re-fed for 4, 24 and 48 h, respectively, prior to tissue sampling.
- Chicks in the REFED groups (D2REFED4h or T6, D3REFED24h or T8 and D4REFED48h or T10) were fasted for 48 h and subsequently re-fed for 4, 24 and 48 h, respectively, prior to the time of tissue sampling.
- The coupling reaction took place in the dark for 1 hour at room temperature.
- The balanced-block hybridization design used for the 50 oligo arrays used in the present study is provided in Additional file 9..
- This minimum infor- mation about a microarray experiment (MIAME)-com- pliant dataset was deposited in the NCBI GEO database under accession number GSE9745..
- The DEGs accepted by IPA are considered as “Analysis Ready” (AR)-DEGs, if the gene is curated and annotated in the Ingenuity® Knowledge Base, which is mainly cu- rated from the mammalian biomedical literature and de- void of many avian-specific genes.
- In additional, a panel of four invariant genes (ATPCL, COX7A2L, GAPDH and PRL14) was included in the qRT-PCR ana- lysis for normalization of qRT-PCR expression.
- Additional file 4: Over-represented canonical and functional pathways identified by IPA in the D2FED48h vs D2FAST48h contrast.
- Additional file 5: Over-represented canonical and functional pathways identified by IPA in the D2FAST48h vs D3REFED24h contrast.
- Additional file 6: Over-represented canonical and functional pathways identified by IPA in the D2FAST48h vs D4REFED48h contrast.
- The funding agency provided funding for the research projects, but had no role in the design of the experiment, statistical analysis, interpretation of the data, nor writing of the manuscript.
- Transcriptional profiling of liver during the critical embryo-to- hatchling transition period in the chicken (Gallus gallus).
- Identification of microRNAs controlling hepatic mRNA levels for metabolic genes during the metabolic transition from embryonic to posthatch development in the chicken.
- Transcriptional and pathway analysis in the hypothalamus of newly hatched chicks during fasting and delayed feeding.
- Differential gene expression pattern in hypothalamus of chickens during fasting-induced metabolic reprogramming: functions of glucose and lipid metabolism in the feed intake of chickens.
- Location of a glucose-dependent response region in the rat S14 promoter.
- Sterol response element-binding protein 1c (SREBP1c) is involved in the polyunsaturated fatty acid suppression of hepatic S14 gene transcription

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