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GR-mediated transcriptional regulation of m6A metabolic genes contributes to diet-induced fatty liver in hens

Journal of Animal Science and Biotechnology volume 12, Article number: 117 (2021) Cite this article

Abstract

Background

Glucocorticoid receptor (GR) mediated corticosterone-induced fatty liver syndrome (FLS) in the chicken by transactivation of Fat mass and obesity associated gene (FTO), leading to demethylation of N6-methyladenosine (m6A) and post-transcriptional activation of lipogenic genes. Nutrition is considered the main cause of FLS in the modern poultry industry. Therefore, this study was aimed to investigate whether GR and m6A modification are involved in high-energy and low protein (HELP) diet-induced FLS in laying hens, and if true, what specific m6A sites of lipogenic genes are modified and how GR mediates m6A-dependent lipogenic gene activation in HELP diet-induced FLS in the chicken.

Results

Laying hens fed HELP diet exhibit excess (P < 0.05) lipid accumulation and lipogenic genes activation in the liver, which is associated with significantly increased (P < 0.05) GR expression that coincided with global m6A demethylation. Concurrently, the m6A demethylase FTO is upregulated (P < 0.05), whereas the m6A reader YTHDF2 is downregulated (P < 0.05) in the liver of FLS chickens. Further analysis identifies site-specific demethylation (P < 0.05) of m6A in the mRNA of lipogenic genes, including FASN, SREBP1 and SCD. Moreover, GR binding to the promoter of FTO gene is highly enriched (P < 0.05), while GR binding to the promoter of YTHDF2 gene is diminished (P < 0.05).

Conclusions

These results implicate a possible role of GR-mediated transcriptional regulation of m6A metabolic genes on m6A-depenent post-transcriptional activation of lipogenic genes and shed new light in the molecular mechanism of FLS etiology in the chicken.

Background

Fatty liver syndrome (FLS) is a metabolic disease mostly observed in laying hens, which is characterized by increased lipid accumulation in the liver [1]. FLS occurs at the rate between 4% and 20% in chickens kept in intensive systems, which may cause dramatic drop in egg production and increased mortality, leading to considerable economic losses [2, 3]. Several factors have been reported to contribute to the development of FLS, including genetics, environment, nutrition, toxic substances, and hormones [4, 5]. Among these, nutrition is considered the main cause of FLS in the modern poultry industry. Nutritionally over-fed laying hens are at risk for developing FLS [4], and a high-energy maize diet produces a higher incidence of FLS than a low-energy barley diet [6]. Also, high-energy low-protein (HELP) diet is used to establish a model of FLS in some previous publications [7,8,9].

FLS is caused primarily by an imbalance of hepatic energy influx and efflux. Glucocorticoids (GC) play an important role in hepatic metabolic homeostasis. Chronically elevated GC level is a common feature of fatty liver in humans and animal models [10, 11]. The actions of GC are primarily mediated by glucocorticoid receptor (GR) [12]. Over-activation of GR pathway leads to transcriptional up-regulation of lipogenic genes, causing hepatic steatosis [13]. In chickens, corticosterone (CORT) is the main active form of GC [14]. We have shown previously that excessive CORT administration causes FLS in chickens, which is characterized by excessive lipid accumulation in the liver [15,16,17]. GR is up-regulated in the liver of FLS chickens [15,16,17], yet it remains unknown how GR contributes to diet-induced FLS in the chicken.

Three families of protein components are involved in the dynamic m6A methylation. The methyltransferases complex formed by methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms’ tumor 1-associating protein (WTAP) [18,19,20] are “writers” to transfer methyl group to the adenosine of the consensus m6A motif in the target RNA. The two demethylases, termed as “erasers”, fat mass and obesity-associated protein (FTO) and α-ketoglutarate-dependent dioxygenase AlkB homolog 5 (ALKBH5) [21, 22], are responsible to remove the methyl group from the m6A. The “readers”, such as YTH-domain family 1–3 (YTHDF1–3), can recognize this methylation and regulate the RNA metabolism including stability or translation efficiency of the mRNA [23,24,25,26]. m6A modification plays a key role in lipid accumulation and energy metabolism [27, 28]. Recently, GR is reported to mediate corticosterone-induced fatty liver in the chicken by transactivation of FTO, leading to demethylation of m6A and post-transcriptional activation of lipogenic genes such as sterol regulatory element-binding protein-1 (SREBP1), fatty acid synthase (FASN) and stearoyl-CoA desaturase (SCD) [17]. However, it remains unclear what specific sites of lipogenic genes are demethylated and which m6A binding protein is involved in mediating m6A-dependent post-transcriptional activation of lipogenic genes.

Therefore, this study was aimed to investigate whether GR and m6A modification are involved in HELP diet-induced FLS in laying hens, and if yes, what specific m6A sites of lipogenic genes are modified and how GR mediates m6A-dependent lipogenic gene activation in HELP diet-induced FLS in the chicken.

Methods

Animals and treatment

Forty-eight Hy-Line Variety Brown laying hens (260 days of age, 1.69 ± 0.09 kg in body weight) were raised in the animal house of Nanjing Agricultural university, with the room temperature at approximately 24 °C, and the light regime of 16 L: 8D. Three hens were housed in each cage (60 cm × 46 cm × 44 cm) equipped with a nipple drinker. Hens were randomly divided into control (CON, twenty-four chickens in eight cages) and high-energy low protein diet (HELP, twenty-four chickens in eight cages) groups, fed control diet (2,610 kcal/kg metabolizable energy, 16.9% crude protein) and HELP diet (3,100 kcal/kg metabolizable energy, 12.1% crude protein), respectively, for 12 weeks. The ingredient and calculated composition of the diets used in the current study are presented in Table 1. Hens were subjected to feed restriction (110 g per hen per day) with free access to water throughout the experiment. After 12 weeks of dietary treatment, 1 hen from each cage was randomly selected and killed by rapid decapitation that is considered acceptable for euthanasia of birds according to American Veterinary Medical Association (AVMA) Guidelines for the Euthanasia of Animals: 2013 Edition. Liver samples were rapidly frozen in liquid nitrogen and kept at − 80 °C for further analysis. Among eight hens from HELP group, six hens were diagnosed as FLS according to the hepatic triglyceride (TG) content. So, the sample size was adjusted of both CON and HELP group to six. We conducted a statistical assessment with the replicate number by using G*Power 3.1.9.2 with power (1-β) set at 0.95 and α = 0.05. According to the TG content in the liver (CON = 21.62 ± 2.85 mg/g; HELP = 42.45 ± 7.62 mg/g), we have calculated the effective size d = 3.62. A sample size of 8 participants (4 per group) was needed. Therefore, 12 participants (6 per group) provide sufficient power to study the molecular mechanism underlying the diet-induced FLS in the present study.

Table 1 Ingredient and calculated composition of the diets (based on air-dried weight)
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Histological evaluation

To visualize the hepatic fat droplets, fresh frozen liver samples were embedded in optimal cutting temperature (OCT) compound and sliced into 8 μm sections. The frozen sections were stained with oil red O (Sigma Aldrich, Saint Louis, MO, USA) for 30 min, counter-stained with H&E for 30 s, then mounted in neutral resin. The slides were observed by using an optical light microscope (Olympus-BX53, Tokyo, Japan).

Determination of triglyceride content in liver

TG content in liver was measured by using TG assay kits (E1013, Applygen Technologies Inc., Beijing, China) following the manufacturer’s instructions. Briefly, 50 mg of frozen liver sample was homogenized in 1 mL of isopropanol manually in a glass homogenizer with 10 passes on ice, incubated at 4 °C for 10 min. The supernatants were collected and used to measure the hepatic TG contents following the instruction of the TG assay kit.

Determination of corticosterone content in plasma

CORT content in plasma was measured by using chicken CORT ELISA Kit (E-EL-0160c, Elabscience, TX, USA) following the manufacturer’s instructions.

Total RNA isolation and real-time PCR

Total RNA was isolated from liver sample (30 mg) using TRIzol Reagent (Invitrogen, Carlsbad, CA, USA) and reverse transcribed into cDNA by using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (R223–01, Vazyme, Nanjing, China). The coding sequences were used to design specific oligonucleotide primers (GenScript Biotech Co., Nanjing, China) for PCR (Table 2) with AceQ qPCR SYBR Green Master Mix (Q111–02, Vazyme, Nanjing, China) on the Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The relative mRNA abundance was calculated with the 2−ΔΔCt method using GAPDH as an internal reference.

Protein extraction and Western blotting analysis

Protein was extracted from 40 mg frozen liver sample as previously described [29]. The protein concentration was determined with a Pierce BCA Protein Assay Kit (Thermo Fisher, Waltham, MA, USA). Forty micrograms of protein were used for electrophoresis on the 10% SDS-PAGE gel. Western blot analysis for SREBP1 (AB12162, Abcam, Cambridge, MA, USA, diluted 1:500), SCD (sc-30081, Santa Cruz, CA, USA, diluted 1:200), GR (Custom made for chickens by Genecreate Biotech Co., Wuhan, China, diluted 1:1,000), METTL3 (AB98009, Abcam, diluted 1:1,000), METTL14 (AB98116, Abcam, diluted 1:1,000), FTO (AB77547, Abcam, diluted 1:1,000), YTHDF1 (17479–1-AP, Proteintech, Chicago, IL, USA, diluted 1:2,000), YTHDF2 (24744–1-AP, Proteintech, diluted 1:2,000) and YTHDF3 (25537–1-AP, Proteintech, diluted 1:1,000) were carried out according to the recommended protocols provided by the manufacturers, The density of each protein band was normalized by that of Tubulin α, the internal control. All antibodies were verified to work with chicken samples in previous publications [16, 17]. Images were captured by VersaDoc 4000MP system (Bio-Rad, Hercules, CA, USA) and the band density was analyzed with Quantity One software (Bio-Rad, USA).

RNA m6A dot blot assays

For m6A dot blot, 500 ng RNA sample was denatured at 95 °C for 5 min and transferred onto a Hybond-N+ membrane (GE Healthcare, Piscataway, NJ, USA). After UV crosslinking, the membrane was washed with TBST buffer, blocked with 5% non-fat milk, and incubated with anti-m6A antibody (AB151230, Abcam, diluted 1:1,000) overnight at 4 °C. Then, the membrane was incubated with secondary antibody at room temperature for 2 h. The signals were visualized by the chemiluminescence system (Bio-Rad, USA) and the dot density was analyzed with Quantity One software (Bio-Rad, USA), with staining of 0.02% methylene blue (in 0.3 mol/L sodium acetate, pH = 5.2) as loading control.

SELECT for detection of m6A

From the m6A-seq database obtained in a previous study on CORT-induced FLS chickens [17], sequences with m6A peaks were retrieved for lipogenic mRNAs including SREBP1, SCD and FASN, and subjected to specific m6A site analysis with SRAMP (http://www.cuilab.cn/sramp). One very high/high confidence m6A site was selected for each gene and verified by using a single-base elongation- and ligation-based qPCR amplification method (termed as “SELECT”). Briefly, 5 μg total RNA was incubated with 40 nmol/L Up Primer, 40 nmol/L Down Primer and 5 nmol/L dNTP in 17 μL 1× CutSmart buffer (50 mmol/L KAc, 20 mmol/L Tris-HAc, 10 mmol/L MgAc2, 100 μg/mL BSA) and annealed under the program as follows: 90 °C (1 min), 80 °C (1 min), 70 °C (1 min), 60 °C (1 min), 50 °C (1 min) and 40 °C (6 min). Next, 17 μL annealing products were incubated with a 3 μL of enzyme mixture containing 0.01 U Bst 2.0 DNA polymerase, 0.5 U SplintR ligase and 10 nmol ATP. The final 20 μL reaction mixture was incubated at 40 °C for 20 min, denatured at 80 °C for 20 min and kept at 4 °C. Quantitative PCR analysis was run under the following conditions: 95 °C, 5 min; (95 °C, 10 s; 60 °C, 45 s) for 40 cycles. The SELECT products of tested site were normalized to the RNA abundance of the mRNA transcript bearing this site. Primers used in SELECT assay are listed in the Table 3.

Chromatin immunoprecipitation (ChIP) assay

ChIP was carried out as previously described [30]. Briefly, 200 mg frozen liver samples were ground in liquid nitrogen and washed with PBS containing protease inhibitor cocktail (Roche, Basel, Switzerland). After cross-linking in 1% formaldehyde, the reaction was stopped with 2.5 mol/L glycine. The pellets were lysed and chromatin was sonicated to an average length of ∼ 300 bp and the protein-DNA complex was diluted in ChIP dilution buffer, incubated with 2 μg of GR antibody (sc-1004, Santa Cruz, California, USA) overnight at 4 °C. A negative control was included with normal IgG or no antibody. Protein G agarose beads (sc-2003, Santa Cruz, California, USA) were added to capture the immunoprecipitated chromatin complexes. Reverse cross-linking was performed at 65 °C for 5 h to release DNA fragments from the immunoprecipitated complex and DNA was purified. The putative GREs in FTO, YTHDF1 and YTHDF2 promoters were predicted using JASPAR 2020 (http://jaspar.genereg.net) [31]. Immunoprecipitated DNA was used as a template for real-time PCR. The primers used to amplify the sequences covering these putative GREs are listed in Table 2.

Table 2 Nucleotide sequences of primers
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Table 3 Nucleotide sequences of SELECT method
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Statistical analysis

Differences between two groups were analyzed by t-test using SPSS 20.0 software (SPSS Inc., Chicago, IL, USA). Data are expressed as means ± SEM. Pearson correlation analysis was performed for correlation analysis. The differences were considered statistically significant when P < 0.05.

Results

Lipogenic genes are activated in the liver of chickens fed HELP diet

Chickens fed HELP diet had significantly higher hepatic lipid accumulation compared with their control counterparts, as seen in Oil Red O staining (Fig. 1A) and hepatic TG (Fig. 1B) content (P < 0.05). Meanwhile, plasma CORT concentration was significantly elevated (P < 0.05) in HELP group (Fig. 1C). Moreover, hepatic expression of lipogenesis genes, such as SREBP1, FASN and SCD were significantly up-regulated (P < 0.05) at both mRNA (Fig. 1D) and protein (Fig. 1E) levels.

Fig. 1
figure 1

Lipogenic genes are activated in the liver of chickens fed HELP diet. A histological sections stained with oil-red, n = 3; B liver concentration of TG, n = 6; C the plasma corticosterone level, n = 6; D mRNA expression of SREBP1, FASN and SCD with qPCR, n = 6; E protein content of SREBP1 and SCD with western blot, n = 6. CON = Control; HELP = High-energy low-protein. Values are means ± SEM, *P < 0.05

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HELP diet increases hepatic GR expression and decreases global RNA m6A methylation

GR was significantly increased (P < 0.05), at both mRNA (Fig. 2A) and protein (Fig. 2B) levels, in the liver of chickens fed HELP diet. Meanwhile, HELP diet significantly decreased (P < 0.05) mRNA m6A levels (Fig. 2C) in the liver. Hepatic TG contents were negatively correlated to global m6A levels (r2 = − 0.7404, P < 0.01, Fig. 2D). No significant alterations were detected for the expression of RNA methyltransferases (METTL3 and METTL14) or the reader proteins (YTHDF1 and YTHDF3) (Figs. 2E, 3A-B, F-G), and no correlation found between hepatic TG contents and METTL3, METTL14, YTHDF1 and YTHDF3 protein content in liver (Fig. 3C-D, H, J). However, RNA demethylase FTO (Figs. 2E, 3A-B) was significantly increased (P < 0.05), and the reader protein YTHDF2 (Figs. 2E, 3F-G) was significantly decreased (P < 0.05) in the liver of chickens fed HELP diet. Moreover, hepatic TG contents are positively correlated to FTO protein content (r2 = 0.5916, P < 0.05, Fig. 3E) and negatively correlated to hepatic YTHDF2 protein content (r2 = − 0.856, P < 0.01, Fig. 3I) in FLS hens.

Fig. 2
figure 2

HELP diet increases hepatic GR expression and decreases global RNA m6A methylation. A and B protein content and mRNA expression of GR with western blot and qPCR, n = 6; C global RNA m6A level in the liver was detected by dot-blot, n = 6; D correlation analysis was performed between TG concertation and m6A modification; E mRNA expression of the RNA methyltransferases METTL3 and METTL14, the m6A demethylase FTO, and the reader proteins YTHDF1 and YTHDF3 were detected with qPCR, n = 6. CON = Control; HELP = High-energy low-protein. Values are means ± SEM, *P < 0.05

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Fig. 3
figure 3

HELP diet increases hepatic FTO and YTHDF2 protein content. A and B protein content of METTL3, METTL14, and FTO in the liver were detected by western blot, n = 6; C-E correlation analysis was performed between TG concertation and METTL3, METTL14, FTO, n = 6; F and G protein content of YTHDF1, YTHDF2 and YTHDF3 in the liver were detected by western blot, n = 6; H-J correlation analysis was performed between TG concertation and YTHDF1–3, n = 6. CON = Control; HELP = High-energy low-protein. Values are means ± SEM, *P < 0.05

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Levels of m6A on specific sites of lipogenic mRNAs are decreased in the liver of HELP diet-fed chickens

Specific m6A sites on 3’UTR of SREBP1, SCD and FASN mRNAs were selected for site-specific m6A quantification by SELECT method. Cycle threshold numbers were significantly increased (P < 0.05) at potential m6A site (X site, Fig. 4A, C and E), but not at the negative control site (N site, Fig. 3B, D and F), indicating decreased m6A modification on specific site of lipogenic mRNA 3’UTRs in the liver of HELP diet-fed chickens.

Fig. 4
figure 4

3’UTR of lipogenic mRNAs is m6A hypermethylated in the liver of HELP diet-fed chickens. A and B detection of m6A modification in SREBP1 3’UTR using SELECT, n = 6. X site was predicted by SRAMP and N site (non-modification site) was negative control. C and D detection of m6A modification in SCD 3’UTR using SELECT, n = 6. X site was predicted by SRAMP and N site (non-modification site) was negative control. E and F detection of m6A modification in FASN 3’UTR using SELECT, n = 6. X site was predicted by SRAMP and N site (non-modification site) was negative control. CON = Control; HELP = High-energy low-protein. Values are means ± SEM, *P < 0.05

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GR binding to the promoter of FTO and YTHDF2 genes is modulated in the liver of HELP diet-fed chickens

ChIP-PCR analysis revealed changes of GR binding on the promoter of FTO and YTHDF2 genes. Fragments containing putative GREs on the promoter of FTO (Fig. 5A), YTHDF2 (Fig. 5B) and YTHDF1 (Fig. 5C) were amplified after chromatin immunoprecipitation with GR antibody. GR binding to the fragment of FTO gene promoter (Fig. 5A) was significantly increased (P < 0.05), while that to the fragment 1 of YTHDF2 gene promoter (Fig. 5B) was significantly decreased (P < 0.05), in the liver of HELP diet-fed chickens. In contrast, GR binding to the fragment 2 of YTHDF2 or YTHDF1 gene promoter was not affected.

Fig. 5
figure 5

GR binding to the promoter of FTO and YTHDF2 genes is modulated in the liver of HELP diet-fed chickens. A schematic representation of GR transcription factor binding promoter of FTO (up) and ChIP-PCR assay was used to measure the binding of GR on FTO promoter in the liver of HELP diet-fed chickens (down, n = 3). B schematic representation of GR transcription factor binding promoter of YTHDF2 (up) and ChIP-PCR assay was used to measure the binding of GR on YTHDF2 promoter in the liver (down, n = 3). C schematic representation of GR transcription factor binding promoter of YTHDF1 (up) and ChIP-PCR assay was used to measure the binding of GR on YTHDF1 promoter in the liver (down, n = 3). CON = Control; HELP = High-energy low-protein. Values are means ± SEM, *P < 0.05

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Discussion

Accumulating evidences indicate that FTO-dependent RNA demethylation and nonalcoholic fatty liver disease are closely intertwined [32, 33]. FTO-dependent demethylation of m6A leads to an increase in lipogenic expression in hepatocytes through m6A modification [34, 35]. Previously, we used chronic administration of corticosterone (CORT) to establish an in vivo FLS model in the juvenile chickens, and to induce excessive lipid accumulation in primary chicken hepatocytes in vitro with combined treatment of oleic acid and dexamethasone (OA/DEX) [17]. In both in vivo and in vitro models, we found that GR-mediated transactivation of FTO and m6A demethylation contribute to lipogenic gene activation. Interestingly, HELP diet-induced FLS is also associated with global m6A demethylation and the activation of lipogenic genes in the liver of laying hens. It may not be totally unexpected, because all these fatty liver models, no matter how they are induced, share the same hormonal and biochemical status of elevated CORT and lipid concentration in the blood.

The major mechanism by which m6A exerts its effects is recruiting m6A-binding proteins [36]. m6A can be recognized by proteins that contain a YTH (YT521B homology) domain [37] or alternatively by eukaryotic initiation factor 3 (eIF3) [38, 39]. The functions of m6A-binding proteins are context-dependent, which means that different m6A-binding proteins bind m6A on different regions of mRNA to exert different functions in gene regulation [40]. Among three m6A-binding proteins determined in the present study, YTHDF2 was down-regulated at both mRNA and protein levels, indicating YTHDF2-mediated gene regulation. YTHDF2 binds transcripts carrying m6A in 3’UTR to induce mRNA degradation partially through recruiting the CCR4-NOT deadenylase complex [41]. YTHDF2 was reported to selectively recognize m6A sites in FASN mRNA, leading to increased FASN mRNA decay and decreased FASN protein content in HepG2 cell lines [42]. Previously, we conducted a m6A-seq analysis to elaborate the epitranscriptomic modification of m6A in the liver of CORT-induced FLS chickens [17]. From this published database, we selected some CORT-responsive m6A peaks in 3’UTR of the lipogenic transcripts, and identified specific HELP-responsive m6A sites on 3’UTR of lipogenic mRNAs, including SREBP1, SCD and FASN, with SELECT analysis. It is possible that the down-regulation of YTHDF2 impairs m6A-dependent lipogenic mRNA degradation, which leads to augmented lipogenesis and excessive lipid accumulation in the liver of FLS hens.

The molecular mechanism by which HELP-diet induces hepatic up-regulation of YTHDF2 in the chicken is unknown. YTHDF2 is negatively regulated by miR-145/miR-495/miR-493-3p at post-transcriptional level in hepatocellular carcinoma cells and prostate cancer cells [43,44,45]. Moreover, YTHDF2 can be SUMOylated in vivo and in vitro at the site of K571, which significantly increases its binding affinity with m6A-modified mRNAs [46]. HIF-2α is reported to transrepress YTHDF2 in hepatocellular carcinoma cells [47]. Based on the observation that YTHDF2 is down-regulated at both mRNA and protein level, we come up with a hypothesis that GR may directly transactivate YTHDF2. Silicon analysis using JASPAR online database identifies 2 putative GR binding sites for YTHDF2. These binding sites were then functionally validated using ChIP-PCR. Indeed, GR binding to YTHDF2 gene promoter is decreased in the liver of hens fed HELP diet. It remains a mystery how GR binding to the promoter of FTO and YTHDF2 genes is distinctively regulated, leading to FTO up-regulation and YTHDF2 down-regulation in HELP diet-induced FLS. Some unidentified co-factors must come into play to coordinate the down-stream effectors of GR action.

In this study, GR was upregulated at both mRNA and protein levels in the liver of hens fed HELP diet. The altered GR binding to the promoter of FTO and YTHDF2 genes indicate HELP diet-induced modulation in GR activation. GR can be activated via both ligand-dependent [48] and ligand-independent manners [49]. Previously, we reported CORT-dependent GR activation in the liver of CORT-treated chickens [16, 50]. In this study, chickens are not treated with CORT and the plasma CORT level was not determined. Therefore, we cannot draw a conclusion whether the altered GR expression and binding is dependent on CORT. Nevertheless, GR can be activated by cellular stressors through p38 MAPK-mediated phosphorylation of Ser134, which is a hormone-independent phosphorylation site on the human GR [51]. GR can also be activated by various stimuli in the absence of glucocorticoid ligands, such as elevated temperature, excessive inflammation, and cancer [52,53,54]. Therefore, both ligand-dependent and ligand-independent pathways are possible in HELP diet-induced alteration in hepatic GR activation.

Conclusions

The present results have shown that GR-mediated transcriptional regulation of FTO and YTHDF2 contributes to lipogenic gene activation by site-specific demethylation in HELP diet-induced chicken FLS. These findings add YTHDF2-mediated m6A modification as a new component of GR signaling in the regulation of fat metabolism in the liver and shed new light on developing effective therapeutic strategies in the prevention and treatment of HELP diet-induced chicken FLS.

Availability of data and materials

The datasets used and analyzed during the current study available from the corresponding author upon request.

Abbreviations

ALKBH5:

α-ketoglutarate-dependent dioxygenase AlkB homolog 5

ChIP:

Chromatin immunoprecipitation

CORT:

Corticosterone

DEX:

Dexamethasone

eIF3:

Eukaryotic initiation factor 3

FASN:

Fatty acid synthase

FLS:

Fatty liver syndrome

FTO:

Fat mass and obesity associated gene

GC:

Glucocorticoids

GR:

Glucocorticoid receptor

HELP:

High-energy low-protein

m6A:

N6-methyladenosine

METTL3:

Methyltransferase-like 3

METTL14:

Methyltransferase-like 14

OA:

Oleic acid

SCD:

Stearoyl-CoA desaturase

SELECT:

Single-base elongation- and ligation-based qPCR amplification method

SREBP1:

Sterol regulatory element-binding protein-1

TG:

Triglyceride

WTAP:

Wilms’ tumor 1-associating protein

YTHDF1–3:

YTH-domain family 1–3

References

  1. Wolford JH, Polin D. Lipid accumulation and hemorrhage in livers of laying chickens. A study on fatty liver-hemorrhagic syndrome (FLHS). Poult Sci. 1972;51(5):1707–13. https://doi.org/10.3382/ps.0511707.

    Article  PubMed  Google Scholar 

  2. Grimes TM. Causes of disease in two commercial flocks of laying hens. Aust Vet J. 1975;51(7):337–43. https://doi.org/10.1111/j.1751-0813.1975.tb15942.x.

    Article  PubMed  Google Scholar 

  3. Zhang Y, Liu Z, Liu R, Wang J, Zheng M, Li Q, et al. Alteration of hepatic gene expression along with the inherited phenotype of acquired fatty liver in chicken. Genes. 2018;9(4):199. https://doi.org/10.3390/genes9040199.

  4. Trott KA, Giannitti F, Rimoldi G, Hill A, Woods L, Barr B, et al. Fatty liver hemorrhagic syndrome in the backyard chicken: a retrospective histopathologic case series. Vet Pathol. 2014;51(4):787–95. https://doi.org/10.1177/0300985813503569.

    Article  PubMed  Google Scholar 

  5. Choi YI, Ahn HJ, Lee BK, Oh ST, An BK, Kang CW. Nutritional and hormonal induction of fatty liver syndrome and effects of dietary lipotropic factors in egg-type male chicks. Asian-Australas J Anim Sci. 2012;25(8):1145–52. https://doi.org/10.5713/ajas.2011.11418.

    Article  PubMed  PubMed Central  Google Scholar 

  6. Pearson AW, Curtis MJ, Butler EJ. Bacterial endotoxins and the pathogenesis of fatty liver--haemorrhagic syndrome in the laying hen. Res Vet Sci. 1981;31(2):259–61. https://doi.org/10.1016/S0034-5288(18)32507-4.

    Article  PubMed  Google Scholar 

  7. Jiang S, Cheng HW, Cui LY, Zhou ZL, Hou JF. Changes of blood parameters associated with bone remodeling following experimentally induced fatty liver disorder in laying hens. Poult Sci. 2013;92(6):1443–53. https://doi.org/10.3382/ps.2012-02800.

    Article  PubMed  Google Scholar 

  8. Rozenboim I, Mahato J, Cohen NA, Tirosh O. Low protein and high-energy diet: a possible natural cause of fatty liver hemorrhagic syndrome in caged white Leghorn laying hens. Poult Sci. 2016;95(3):612–21. https://doi.org/10.3382/ps/pev367.

    Article  PubMed  Google Scholar 

  9. Song Y, Ruan J, Luo J, Wang T, Yang F, Cao H, et al. Abnormal histopathology, fat percent and hepatic apolipoprotein a I and apolipoprotein B100 mRNA expression in fatty liver hemorrhagic syndrome and their improvement by soybean lecithin. Poult Sci. 2017;96(10):3559–63. https://doi.org/10.3382/ps/pex163.

    Article  PubMed  Google Scholar 

  10. Targher G, Bertolini L, Rodella S, Zoppini G, Zenari L, Falezza G. Associations between liver histology and cortisol secretion in subjects with nonalcoholic fatty liver disease. Clin Endocrinol. 2006;64(3):337–41. https://doi.org/10.1111/j.1365-2265.2006.02466.x.

    Article  Google Scholar 

  11. Vegiopoulos A, Herzig S. Glucocorticoids, metabolism and metabolic diseases. Mol Cell Endocrinol. 2007;275(1–2):43–61. https://doi.org/10.1016/j.mce.2007.05.015.

    Article  PubMed  Google Scholar 

  12. Hollenberg SM, Weinberger C, Ong ES, Cerelli G, Oro A, Lebo R, et al. Primary structure and expression of a functional human glucocorticoid receptor cDNA. Nature. 1985;318(6047):635–41. https://doi.org/10.1038/318635a0.

    Article  PubMed  PubMed Central  Google Scholar 

  13. Beaupere C, Liboz A, Fève B, Blondeau B, Guillemain G. Molecular mechanisms of glucocorticoid-induced insulin resistance. Int J Mol Sci. 2021;22(2):623. https://doi.org/10.3390/ijms22020623.

  14. Costantini D, Fanfani A, Dell'omo G. Effects of corticosteroids on oxidative damage and circulating carotenoids in captive adult kestrels (Falco tinnunculus). J Comp Physiol B. 2008;178(7):829–35. https://doi.org/10.1007/s00360-008-0270-z.

    Article  PubMed  Google Scholar 

  15. Hu Y, Sun Q, Liu J, Jia Y, Cai D, Idriss AA, et al. In ovo injection of betaine alleviates corticosterone-induced fatty liver in chickens through epigenetic modifications. Sci Rep. 2017;7(1):40251. https://doi.org/10.1038/srep40251.

    Article  PubMed  PubMed Central  Google Scholar 

  16. Hu Y, Sun Q, Hu Y, Hou Z, Zong Y, Omer NA, et al. Corticosterone-induced lipogenesis activation and lipophagy inhibition in chicken liver are alleviated by maternal betaine supplementation. J Nutr. 2018;148(3):316–25. https://doi.org/10.1093/jn/nxx073.

    Article  PubMed  Google Scholar 

  17. Hu Y, Feng Y, Zhang L, Jia Y, Cai D, Qian SB, et al. GR-mediated FTO transactivation induces lipid accumulation in hepatocytes via demethylation of m(6) a on lipogenic mRNAs. RNA Biol. 2020;17(7):930–42. https://doi.org/10.1080/15476286.2020.1736868.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Liu J, Yue Y, Han D, Wang X, Fu Y, Zhang L, et al. A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation. Nat Chem Biol. 2014;10(2):93–5. https://doi.org/10.1038/nchembio.1432.

    Article  PubMed  Google Scholar 

  19. Ping XL, Sun BF, Wang L, Xiao W, Yang X, Wang WJ, et al. Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Res. 2014;24(2):177–89. https://doi.org/10.1038/cr.2014.3.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Wang X, Feng J, Xue Y, Guan Z, Zhang D, Liu Z, et al. Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex. Nature. 2016;534(7608):575–8. https://doi.org/10.1038/nature18298.

    Article  PubMed  Google Scholar 

  21. Jia G, Fu Y, Zhao X, Dai Q, Zheng G, Yang Y, et al. N6-methyladenosine in nuclear RNA is a major substrate of the obesity-associated FTO. Nat Chem Biol. 2011;7(12):885–7. https://doi.org/10.1038/nchembio.687.

    Article  PubMed  PubMed Central  Google Scholar 

  22. Zheng G, Dahl JA, Niu Y, Fedorcsak P, Huang CM, Li CJ, et al. ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol Cell. 2013;49(1):18–29. https://doi.org/10.1016/j.molcel.2012.10.015.

    Article  PubMed  Google Scholar 

  23. Wang X, Lu Z, Gomez A, Hon GC, Yue Y, Han D, et al. N6-methyladenosine-dependent regulation of messenger RNA stability. Nature. 2014;505(7481):117–20. https://doi.org/10.1038/nature12730.

    Article  PubMed  Google Scholar 

  24. Wang X, Zhao BS, Roundtree IA, Lu Z, Han D, Ma H, et al. N(6)-methyladenosine modulates messenger RNA translation efficiency. Cell. 2015;161(6):1388–99. https://doi.org/10.1016/j.cell.2015.05.014.

    Article  PubMed  PubMed Central  Google Scholar 

  25. Shi H, Wang X, Lu Z, Zhao BS, Ma H, Hsu PJ, et al. YTHDF3 facilitates translation and decay of N(6)-methyladenosine-modified RNA. Cell Res. 2017;27(3):315–28. https://doi.org/10.1038/cr.2017.15.

    Article  PubMed  PubMed Central  Google Scholar 

  26. Liu N, Dai Q, Zheng G, He C, Parisien M, Pan T. N(6)-methyladenosine-dependent RNA structural switches regulate RNA-protein interactions. Nature. 2015;518(7540):560–4. https://doi.org/10.1038/nature14234.

    Article  PubMed  PubMed Central  Google Scholar 

  27. Wang X, Zhu L, Chen J, Wang Y. mRNA m6A methylation downregulates adipogenesis in porcine adipocytes. Biochem Biophys Res Commun. 2015;459(2):201–7. https://doi.org/10.1016/j.bbrc.2015.02.048.

    Article  PubMed  Google Scholar 

  28. Zhao X, Yang Y, Sun BF, Shi Y, Yang X, Xiao W, et al. FTO-dependent demethylation of N6-methyladenosine regulates mRNA splicing and is required for adipogenesis. Cell Res. 2014;24(12):1403–19. https://doi.org/10.1038/cr.2014.151.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Duan Y, Fu W, Wang S, Ni Y, Zhao R. Effects of tonic immobility (TI) and corticosterone (CORT) on energy status and protein metabolism in pectoralis major muscle of broiler chickens. Comp Biochem Physiol A Mol Integr Physiol. 2014;169:90–5. https://doi.org/10.1016/j.cbpa.2013.12.019.

    Article  PubMed  Google Scholar 

  30. Feng Y, Dong H, Sun B, Hu Y, Yang Y, Jia Y, et al. METTL3/METTL14 transactivation and m(6)A-dependent TGF-β1 translation in activated Kupffer cells. Cell Mol Gastroenterol Hepatol. 2021;12(3):839–56. https://doi.org/10.1016/j.jcmgh.2021.05.007.

    Article  PubMed  PubMed Central  Google Scholar 

  31. Fornes O, Castro-Mondragon JA, Khan A, van der Lee R, Zhang X, Richmond PA, et al. JASPAR 2020: update of the open-access database of transcription factor binding profiles. Nucleic Acids Res. 2020;48(D1):D87–92. https://doi.org/10.1093/nar/gkz1001.

    Article  PubMed  Google Scholar 

  32. Mizuno TM. Fat mass and obesity associated (FTO) gene and hepatic glucose and lipid metabolism. Nutrients. 2018;10(11). https://doi.org/10.3390/nu10111600.

  33. Severson TJ, Besur S, Bonkovsky HL. Genetic factors that affect nonalcoholic fatty liver disease: a systematic clinical review. World J Gastroenterol. 2016;22(29):6742–56. https://doi.org/10.3748/wjg.v22.i29.6742.

    Article  PubMed  PubMed Central  Google Scholar 

  34. Kang H, Zhang Z, Yu L, Li Y, Liang M, Zhou L. FTO reduces mitochondria and promotes hepatic fat accumulation through RNA demethylation. J Cell Biochem. 2018;119(7):5676–85. https://doi.org/10.1002/jcb.26746.

    Article  PubMed  Google Scholar 

  35. Bartosovic M, Molares HC, Gregorova P, Hrossova D, Kudla G, Vanacova S. N6-methyladenosine demethylase FTO targets pre-mRNAs and regulates alternative splicing and 3′-end processing. Nucleic Acids Res. 2017;45(19):11356–70. https://doi.org/10.1093/nar/gkx778.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Meyer KD, Jaffrey SR. Rethinking m(6) a readers, writers, and erasers. Annu Rev Cell Dev Biol. 2017;33(1):319–42. https://doi.org/10.1146/annurev-cellbio-100616-060758.

    Article  PubMed  PubMed Central  Google Scholar 

  37. Xu Y, Zhang W, Shen F, Yang X, Liu H, Dai S, et al. YTH domain proteins: a family of m(6) a readers in cancer progression. Front Oncol. 2021;11:629560. https://doi.org/10.3389/fonc.2021.629560.

    Article  PubMed  PubMed Central  Google Scholar 

  38. Meyer KD, Patil DP, Zhou J, Zinoviev A, Skabkin MA, Elemento O, et al. 5′ UTR m(6) a promotes cap-independent translation. Cell. 2015;163(4):999–1010. https://doi.org/10.1016/j.cell.2015.10.012.

    Article  PubMed  PubMed Central  Google Scholar 

  39. Zhou J, Wan J, Gao X, Zhang X, Jaffrey SR, Qian SB. Dynamic m(6) a mRNA methylation directs translational control of heat shock response. Nature. 2015;526(7574):591–4. https://doi.org/10.1038/nature15377.

    Article  PubMed  PubMed Central  Google Scholar 

  40. Shi H, Wei J, He C. Where, when, and how: context-dependent functions of RNA methylation writers, readers, and erasers. Mol Cell. 2019;74(4):640–50. https://doi.org/10.1016/j.molcel.2019.04.025.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Du H, Zhao Y, He J, Zhang Y, Xi H, Liu M, et al. YTHDF2 destabilizes m(6)A-containing RNA through direct recruitment of the CCR4-NOT deadenylase complex. Nat Commun. 2016;7(1):12626. https://doi.org/10.1038/ncomms12626.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Sun D, Zhao T, Zhang Q, Wu M, Zhang Z. Fat mass and obesity-associated protein regulates lipogenesis via m(6) a modification in fatty acid synthase mRNA. Cell Biol Int. 2021;45(2):334–44. https://doi.org/10.1002/cbin.11490.

    Article  PubMed  Google Scholar 

  43. Yang Z, Li J, Feng G, Gao S, Wang Y, Zhang S, et al. MicroRNA-145 modulates N(6)-methyladenosine levels by targeting the 3′-untranslated mRNA region of the N(6)-methyladenosine binding YTH domain family 2 protein. J Biol Chem. 2017;292(9):3614–23. https://doi.org/10.1074/jbc.M116.749689.

    Article  PubMed  PubMed Central  Google Scholar 

  44. Du C, Lv C, Feng Y, Yu S. Activation of the KDM5A/miRNA 495/YTHDF2/m6A-MOB3B axis facilitates prostate cancer progression. J Exp Clin Cancer Res. 2020;39(1):223. https://doi.org/10.1186/s13046-020-01735-3.

    Article  PubMed  PubMed Central  Google Scholar 

  45. Li J, Meng S, Xu M, Wang S, He L, Xu X, et al. Downregulation of N(6)-methyladenosine binding YTHDF2 protein mediated by miR-493-3p suppresses prostate cancer by elevating N(6)-methyladenosine levels. Oncotarget. 2018;9(3):3752–64. https://doi.org/10.18632/oncotarget.23365.

    Article  PubMed  Google Scholar 

  46. Hou G, Zhao X, Li L, Yang Q, Liu X, Huang C, et al. SUMOylation of YTHDF2 promotes mRNA degradation and cancer progression by increasing its binding affinity with m6A-modified mRNAs. Nucleic Acids Res. 2021;49(5):2859–77. https://doi.org/10.1093/nar/gkab065.

    Article  PubMed  PubMed Central  Google Scholar 

  47. Hou J, Zhang H, Liu J, Zhao Z, Wang J, Lu Z, et al. YTHDF2 reduction fuels inflammation and vascular abnormalization in hepatocellular carcinoma. Mol Cancer. 2019;18(1):163. https://doi.org/10.1186/s12943-019-1082-3.

    Article  PubMed  PubMed Central  Google Scholar 

  48. Newton R, Holden NS. Separating transrepression and transactivation: a distressing divorce for the glucocorticoid receptor? Mol Pharmacol. 2007;72(4):799–809. https://doi.org/10.1124/mol.107.038794.

    Article  PubMed  Google Scholar 

  49. Scheschowitsch K, Leite JA, Assreuy J. New insights in glucocorticoid receptor signaling-more than just a ligand-binding receptor. Front Endocrinol. 2017;8:16. https://doi.org/10.3389/fendo.2017.00016.

    Article  Google Scholar 

  50. Omer NA, Hu Y, Idriss AA, Abobaker H, Hou Z, Yang S, et al. Dietary betaine improves egg-laying rate in hens through hypomethylation and glucocorticoid receptor-mediated activation of hepatic lipogenesis-related genes. Poult Sci. 2020;99(6):3121–32. https://doi.org/10.1016/j.psj.2020.01.017.

    Article  PubMed  PubMed Central  Google Scholar 

  51. Galliher-Beckley AJ, Williams JG, Cidlowski JA. Ligand-independent phosphorylation of the glucocorticoid receptor integrates cellular stress pathways with nuclear receptor signaling. Mol Cell Biol. 2011;31(23):4663–75. https://doi.org/10.1128/mcb.05866-11.

    Article  PubMed  PubMed Central  Google Scholar 

  52. Sanchez ER. Heat shock induces translocation to the nucleus of the unliganded glucocorticoid receptor. J Biol Chem. 1992;267(1):17–20. https://doi.org/10.1016/S0021-9258(18)48448-7.

    Article  PubMed  Google Scholar 

  53. Verhoog NJ, Du Toit A, Avenant C, Hapgood JP. Glucocorticoid-independent repression of tumor necrosis factor (TNF) alpha-stimulated interleukin (IL)-6 expression by the glucocorticoid receptor: a potential mechanism for protection against an excessive inflammatory response. J Biol Chem. 2011;286(22):19297–310. https://doi.org/10.1074/jbc.M110.193672.

    Article  PubMed  PubMed Central  Google Scholar 

  54. Ritter HD, Antonova L, Mueller CR. The unliganded glucocorticoid receptor positively regulates the tumor suppressor gene BRCA1 through GABP beta. Mol Cancer Res. 2012;10(4):558–69. https://doi.org/10.1158/1541-7786.mcr-11-0423-t.

    Article  PubMed  Google Scholar 

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Acknowledgements

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Funding

This work was supported by the National Natural Science Foundation of China (31972638); the National Key Research and Development Program of China (2016YFD0500502); the Fundamental Research Funds for the Central Universities (KYZ201212), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and Jiangsu Collaborative Innovation Centre of Meat Production and Processing, Quality and Safety Control.

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Authors and Affiliations

  1. MOE Joint International Research Laboratory of Animal Health & Food Safety, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China

    Yue Feng, Yanlin Li, Wenduo Jiang, Yun Hu, Yimin Jia & Ruqian Zhao

  2. Key Laboratory of Animal Physiology & Biochemistry, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, P. R. China

    Yue Feng, Yanlin Li, Wenduo Jiang, Yun Hu, Yimin Jia & Ruqian Zhao

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  1. Yue Feng
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  2. Yanlin Li
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  3. Wenduo Jiang
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  4. Yun Hu
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  5. Yimin Jia
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  6. Ruqian Zhao
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Contributions

YF, performed the experiments, analyzed and interpreted the results, and drafted the manuscript. YL and WJ, performed the animal experiment, recorded and analyzed the phenotypic data and took the samples. YH and YJ, analyzed the data. RZ, contributed to experimental concepts and design, provided scientific direction, analyzed and interpreted the results, and finalized the manuscript. All authors read and approved the final manuscript.

Corresponding author

Correspondence to Ruqian Zhao.

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Ethics approval and consent to participate

The experimental protocol was approved by the Animal Ethics Committee of Nanjing Agricultural University. The sampling procedures complied with the “Guidelines on Ethical Treatment of Experimental Animals” (2006) No. 398 set by the Ministry of Science and Technology, China.

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Not applicable.

Competing interests

The authors declare that they have no competing interests.

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Feng, Y., Li, Y., Jiang, W. et al. GR-mediated transcriptional regulation of m6A metabolic genes contributes to diet-induced fatty liver in hens. J Animal Sci Biotechnol 12, 117 (2021). https://doi.org/10.1186/s40104-021-00642-7

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  • DOI: https://doi.org/10.1186/s40104-021-00642-7

Keywords

  • Chicken
  • Fatty liver syndrome
  • FTO
  • GR
  • Lipogenesis
  • m6A
  • YTHDF2

Journal of Animal Science and Biotechnology

ISSN: 2049-1891