Evaluation of the effect of ACTH1-39 on primary cultured chicken hepatocytes

To evaluate the effect of ACTH_1-39 on chicken liver, the chicken hepatocytes were prepared and maintained as a monolayer culture. Briefly, one-month-old male chicken hepatocytes were isolated by perfusion of a liver with Ca²⁺-free KRB buffer to remove blood cells, and being digested with 0.25% collagenase-I for 10 min. Hepatocytes were obtained following filtering and washing using M199 medium. Then the hepatocytes were plated in a Corning CellBIND 48-well microplate (Corning, NY, cat. no.3338) with M199 medium containing 100 U/ml penicillin and 100 mg/ml streptomycin (Gibco), 5 μg/ml bovine insulin (Sigma), and 5% FBS at 37°C in a 5% CO₂ atmosphere. To adhere to the wall, the cells were incubated on the 48-well plate for 4 h.

Glucose production from primary cultured chicken hepatocytes was measured as previously described (Collins et al., 2006). Briefly, cells were washed three times with warm phosphate-buffered saline (PBS) to remove glucose, followed by treatment with 100 nM (or different concentration gradients) chicken ATCH_1-39 for 30 min or 60 min in glucose-free medium containing gluconeogenic substrates (20 mM sodium lactate and 2 mM sodium pyruvate). Glucose concentration was determined with a glucose assay kit (Applygen, Beijing, China, catalog no. E1011). Briefly, the 5 µl medium was added into 195 µl reagent and incubated for 20 min at 37°C. Absorbance in 550 nm was measured in a standard glucose solution.

For lipid quantification by Oil Red O staining, after treatment with ACTH_1-39 for 24 h, primary cultured chicken hepatocytes were washed with PBS and then fixed with 4% paraformaldehyde for 30 min. After PBS washing, the Oil Red O working solution was added to stain for 30 min. The stained lipids were then visualized by light microscopy after washing in PBS. To quantify the lipid content, the Oil Red O stained in the cells was extracted with isopropanol by measuring the OD value at 540 nm.

For measurement of triglyceride content, after treated with ACTH_1-39 for 24 h, hepatocytes were harvested and triglyceride (TG) content was analyzed using a commercial triglyceride content assay kit (Applygen, Beijing, China, catalog no. E1013). Results were normalized to the protein content of each sample, as determined using a BCA assay kit (Beyotime Institute of Biotechnology, China, catalog no. P0010).

For quantitative real-time PCR, primary cultured chicken hepatocytes were treated with various concentrations of ACTH_1-39 (or other composition) at 37°C for 6 h. The relative amount of mRNA was calculated using the comparative Ct method. Chicken β-actin gene was used as the reference gene. Amplification of specific transcripts was confirmed by analyzing the melting curve profile performed at the end of each run and by determining the size of the PCR products using agarose electrophoresis and ethidium bromide staining.