A total of 40 male AA broilers (1-day-old, 44.5 ± 3.4 g) were obtained from Jiangsu Jinghai Poultry Industry Group Co., Ltd. (Jiangsu Province Poultry Science Research Institute) for the study. The broilers were given ad libitum access to feed and water, and were fed a commercial mixed feed (purchased from New Hope Liuhe). The nutrient composition of the broiler basal diet was as follows: 18% crude protein, 9% crude ash, 5% crude fiber, 0.5% total phosphorus, 1.2% calcium, 0.8% sodium chloride, and 0.9% methionine. The lighting schedule followed a 16-h light/8-h dark cycle daily. For the first 3 days, the ambient temperature within the poultry house was maintained at 35 °C, gradually decreasing by 3 °C per week until reaching 23 °C.

At 38 days of age, the broilers were randomly assigned into two experimental groups: the control (CON) group (n = 20) and the CORT group (n = 20). The CORT group was subjected to chronic stress by receiving subcutaneous injections of CORT (Aladdin, USA) at a dosage of 4.0 mg/kg/day, divided into two doses administered daily for 7 consecutive days (2). The CON group received an equal volume of alcohol-saline solution as a vehicle control. The feed intake of the birds was recorded daily, and body weight was measured weekly throughout the experimental period. At 45 days of age, the broilers were randomly select 12 chickens from each group for subsequent analysis.

Anticoagulated blood samples were collected, placed on ice, and centrifuged at 2000 g for 15 min to separate plasma. Plasma levels of TG, glucose (GLU), total cholesterol (TCH), ALT and AST were measured using an automatic biochemical analyzer. The corresponding assay kits were purchased from Meikang Technology Co., Ltd. Hepatic TG levels were determined using a TG detection kit obtained from Beijing Pulilai Gene Technology Co., Ltd., following the manufacturer’s protocol.

Liver tissues were fixed in 4% paraformaldehyde and subsequently embedded in paraffin. Sections 5 μm in thickness were prepared and subjected to Hematoxylin and Eosin (H&E) staining and Oil Red staining. The stained sections were examined under an optical microscope, and images were captured for further histological analysis of tissue lesions.

Primary hepatocytes were isolated from 45-day-old AA broilers using the in situ two-step perfusion method with collagenase IV (22). Following a fasting period, broilers were anesthetized, and a catheter was inserted into the portal vein. The liver was fixed in situ, and stepwise perfusion was conducted until the liver became softened. The liver was then excised and transferred to a sterile dish, where blood vessels, fat, and connective tissues were carefully removed. The undigested peripheral portions of the liver were trimmed, and the remaining tissue was minced into small fragments. Hepatocytes were filtered through cell strainers, centrifuged at 50 × g for 3 min, and washed three times with DMEM.

The AML12 cell line was cultured in DMEM/F12 medium supplemented with 10% fetal bovine serum (NEWZERUM, New Zealand), 1% penicillin–streptomycin, and 1% ITS (Biotian, China) at 37 °C in a humidified atmosphere with 5% CO₂. When the cell density reached 60%, the cells were treated with 50 μM CORT (Aladdin, USA) and 100 μM palmitic acid (PA) for 24 h. The CON group was treated with an equal concentration of 100 μM PA alone.

Specific MFN1 and GR small-interfering RNA (siRNA) was synthesized by QingKe (Qingdao, China), and the sequences of the MFN1, GR siRNAs were in Supplementary Table S4. These siRNAs were transfected into AML12 using the JetPRIME® transfection reagent (jetPRIME®, Polyplus, China). Scrambled siRNA was used as the negative control (si-N.C.).

The coding sequence (CDS) of mouse MFN1 was retrieved from the NCBI database, and primers were designed using CE Design software. Using cDNA derived from mouse tissues as a template, the MFN1 sequence was cloned and ligated into the pcDNA3.1 vector to construct the MFN1 overexpression plasmid.

AML12 were seeded in 12-well plates at a density of 1 × 10^{^6} cells per well. Following the treatment as outlined in the experimental design, the cells were harvested by trypsinization and washed twice with PBS. Subsequently, the cells were incubated separately with MitoSOX^Red (MCE, China) and JC-1 (Biotian, China) working solution following the manufacturer’s protocols. After incubation, cells were centrifuged at 600 g for 5 min at room temperature, supernatant discarded, resuspended in 1 mL PBS, and analyzed by flow cytometry (BD FACSVerse, USA).

Total RNA was isolated from liver tissue and cells using Trizol (Tsingke, China) adhering to the manufacturer’s instructions. Subsequently, 1 μg of RNA was reverse transcribed to cDNA using the Reverse Transcription Master Kit (TransGen, China) according to standard protocol. Primers used in this study were designed using Primer Premier 5.0 software, based on primer sequences sourced from PubMed (Supplementary Table S1). qPCR was performed using the AQ601 kit (TransGen, China) with results normalized to PPIA, which served as the internal control for mRNA quantification. The relative gene expression was calculated using the (2^-∆∆Ct) method, with PPIA as the reference gene.

Total proteins from tissues and cells were extracted using the RIPA lysis buffer (1% Triton-100, 0.5% sodium deoxycholate, 150 mmol/L NaCl, 0.1% SDS, 50 mmol/L Tris, along with 1% protease inhibitor and 1% phosphatase inhibitor), and protein concentrations were determined using a BCA protein assay kit (TransGen, China). For analysis, 30 μg of tissue protein or 20 μg of cell protein was separated by SDS-PAGE and then transferred to nitrocellulose (NC) membranes. The membranes were blocked with 5% skim milk and incubated with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with secondary antibodies for 2 h at room temperature, and the proteins were detected with an ECL chemiluminescence kit (Biosharp, China) and and visualized using a protein imager (Tannon-5200, China). Band intensity was quantified using ImageJ software (Wayne Rasband, National Institutes of Health). The antibodies employed in this study are listed in Supplementary Table S3.

AML12 were seeded in 24-well plates at a density of 1 × 10^{^6} cells per well. Following the treatment as outlined in the experimental design, the cells were harvested by trypsinization and washed twice with PBS. Subsequently, the cells were incubated separately with BODIPY working solution (Beyotime, China), DAPI (Beyotime, China) and Nile Red staining solution (Solarbio, China) at 37 °C in the dark for 15 min. Then the stained cells were then washed three times with PBS and observed under a fluorescence microscope.

After crosslinking and lysing the treated cell samples, the lysate was subjected to sonication to obtain DNA fragments of approximately 300–500 bp. Each sample was divided into positive, negative, and input groups, with each group containing 99 μg of chromatin. For the positive group, an antibody against GR (Proteintech, China) was added and incubated overnight to form the antibody–antigen complex. Subsequently, Protein A/G agarose beads were added to both the positive and negative groups and incubated on a shaking platform at 4 °C for 2 h. The beads were then washed and eluted to isolate the DNA-protein-antibody complex. After reversing the crosslinking and removing RNA and proteins, purified DNA was extracted using the phenol/chloroform method. The released DNA was quantified using real-time PCR, and the enrichment of the target genes was analyzed. The list of primers used in the ChIP analysis is provided in the Supplementary Table S2.

The data obtained from this study were analyzed using appropriate statistical methods to ensure the reliability of the results. All data are presented as means ± standard error of the mean (SEM). Differences between groups were assessed using t-tests, with p-values less than 0.05 considered statistically significant and p < 0.01 indicating a highly significant difference. Data analysis was conducted using GraphPad Prism 8.0 software (GraphPad Software Inc., La Jolla, CA). All experiments were repeated at least three times to confirm the reproducibility of the results.

Continuous treatment with CORT for 7 days led to a significant reduction in body weight in broilers (Figure 1a) and pronounced liver enlargement (Figures 1b,c). Also, the liver can be clearly seen to be yellow in the CORT group (Figure 1d). Biochemical analyses indicated substantial increases in plasma TCH, GLU, ALT and AST levels, accompanied by marked elevations in TG (Figures 1e–i). Liver triglyceride levels were also significantly elevated (Figure 1k). HE staining demonstrated CORT treatment-induced hepatocyte swelling and prominent vacuolation of lipid droplets (Figure 1j). Additionally, Oil Red staining of liver sections revealed extensive lipid droplet deposition on the liver surface (Figure 1l). Mitochondria are crucial for the regulation of fatty acid metabolism and synthesis. Transmission Electron Microscopy (TEM) analysis of liver mitochondria (Figure 2a) revealed that CORT treatment resulted in significant mitochondrial fragmentation, along with a marked reduction in mitochondrial diameter (Figure 2c) and a substantial increase in mitochondrial numbers (Figure 2b). Notably, no significant changes were observed of the mitochondrial aspect (Figure 2d). Similar mitochondrial alterations were evident in hepatocytes, as shown by TEM analysis (Figure 2e). The results indicated a significant reduction in the diameter of primary hepatocyte mitochondria (Figure 2g) and an increase in their numbers (Figure 2f), with no notable changes in the mitochondrial aspect (Figure 2h). In summary, CORT treatment resulted in morphologically disordered mitochondria and liver damage, which contributing to the development of fatty liver.

Mitochondrial integrity is predominantly influenced by the dynamic balance between fusion and fission processes. Analysis of proteins and genes related to mitochondrial dynamics revealed a significant reduction in the mitochondrial fusion protein MFN1 at both the gene and protein levels in the liver. In contrast, other fusion proteins, including MFN2 and OPA1, as well as the fission protein Dynamin-related protein 1 (DRP1), showed no significant changes (Figures 3a–c). Similarly, in primary hepatocyte mitochondria, MFN1 levels were significantly diminished at both the gene and protein levels, while levels of fusion proteins MFN2 and OPA1 were reduced at the mRNA level. Notably, although protein levels of the fission protein DRP1 increased, its mRNA levels decreased (Figures 3d–f). These findings suggest that the mitochondrial fusion protein MFN1 plays a crucial role in the effects of CORT treatment.

To investigate the involvement of MFN1 in CORT-induced fatty liver, AML12 were treated with 100 μM CORT combined with 50 μM PA for 24 h for in vitro validation. Bodipy staining results demonstrated that CORT exacerbated lipid droplet accumulation in hepatocytes (Figure 4a), and TG levels in AML12 were significantly elevated (Figure 4b). TEM analysis of AML12 mitochondria (Figure 4c) indicated that CORT treatment led to mitochondrial swelling and rupture of cristae, differing from the in vivo observations. Specifically, the number of mitochondria was significantly reduced (Figure 4e), while their diameter was significantly elongated (Figure 4d). Both the shape of mitochondria were elongated, resulting in a significant decrease in mitochondrial index compared to the CON group (Figure 4f). Mitochondrial integrity is essential for normal function. Utilizing the MitoSOX^Red probe, we found that CORT treatment aggravated mitochondrial oxidative stress, leading to a significant increase in ROS levels (Figure 4g) and a marked decline in MMP (Figure 4h). Further analysis of mitochondrial dynamics-related proteins in AML12 revealed that CORT treatment significantly inhibited the expression of the MFN1 protein (Figures 4i,j). Collectively, these results suggest that CORT-induced dysregulation of MFN1 leads to mitochondrial functional impairment, subsequently promoting the progression of fatty liver.

Given that MFN1 is a crucial protein for maintaining mitochondrial membrane integrity, we investigated whether restoring its expression could enhance mitochondrial function and mitigate the onset of fatty liver. Functional validation was conducted through MFN1 overexpression. Nile Red staining of AML12 demonstrated that overexpression of MFN1 alleviated lipid droplet accumulation induced by CORT (Figure 5a) and significantly reduced TG levels (Figure 5b). TEM images of AML12 mitochondria indicated that overexpression MFN1 notably restored the normal morphology and cristae structure of mitochondria, although there were no significant changes in mitochondrial numbers (Figures 5c–f). Furthermore, overexpression of MFN1 significantly improved the elevation of ROS levels and the reduction in MMP induced by CORT (Figures 5g,h). Collectively, these findings suggest that MFN1 enhances mitochondrial function by restoring mitochondrial integrity, thereby alleviating the progression of fatty liver.

The functionality of glucocorticoids is depend on their receptors GR. As a transcriptional regulator, we explored the potential role of GR in CORT-induced fatty liver by modulating MFN1 expression. Heatmap showing the transcriptional factors related to glucocorticoid, only GR significantly suppressed in CORT group (Figure 6a). Given that GR function exhibits time dependence, we investigated the changes in GR expression at different time points during CORT treatment. The results indicated that GR protein expression was significantly inhibited after 6, 12, and 24 h of CORT exposure (Figures 6b,d). Additionally, since mitochondrial dynamics is a complex and dynamic process, we found that 6 and 12 h of CORT treatment had no significant effect on MFN1 expression; however, 24 h of CORT treatment resulted in a marked decrease in MFN1 protein levels (Figures 6c,e). Subsequently, we observed that knockdown of GR led to a significant decrease in MFN1 protein expression (Figures 6f,h), whereas knockdown of MFN1 did not affect GR expression (Figures 6g,i). This suggests that GR is involved in the upstream regulation of MFN1. To elucidate the mechanism underlying the changes in MFN1 expression in CORT-induced fatty liver, we utilized the JASPAR database to predict binding relationships between the mouse GR transcription factor and the MFN1 promoter (Figure 6j). The results indicated potential binding regions of mouse GR in the MFN1 promoter, specifically at MFN1 Fragment 1: −442 to −427 bp and MFN1 Fragment 2: −59 to −44 bp (Figure 6k). ChIP-PCR experiments revealed that the level of GR binding to the MFN1 promoter region in the CORT group was significantly higher than that in the CON group (Figure 6l). Therefore, GR regulates the expression of MFN1 by binding to its promoter region, thereby leading to the progression of CORT-induced fatty liver.