Regulation of Dehydroepiandrosterone(DHEA) on cAMP/PKA Signalling System and cAMP-response Element Binding Protein (CREB) in Cultured Primary Chicken Hepatocytes
Abstract：Considerable research efforts have been expended to study the factors that are associated with fat deposition in poultry production. Dehydroepiandrosterone (DHEA, 3b-hydroxy-5-androsterone-17-one) is a steroidal compound that is secreted by the mammalian adrenal cortex gland. It is known to be a fat-reducing agent by activation of the steroid hormone receptors. In the present study, cultured primary chicken hepatocytes exposed to DHEA (0, 0.01, 0.1, 1.0 10 and 100 μmol/L, respectively) dissolved in medium were left for 20 min for cAMP assay by RIA(radioimmunoassay) kit. We found that the levels of cAMP were significantly higher in 0.1~100 μmol/L DHEA groups(P<0.05), especially a maximum accumulation in 0.1 μmol/L DHEA group that compared with the control group(P<0.01). Chicken hepatocytes pre-incubated with 0.25 mmol/L IBMX(3-isobutyl-1-methylxanthine) or vehicle for 5 min, followed by the addition of 0.1 μmol/L DHEA, 20 μmol/L forskolin or vehicle for 20 min at 37℃ were tested for adenylate cyclase (AC) activity. Cell lysates isolated from hepatocytes exposed to 0.1 μmol/L DHEA, 0.25 mmol/L IBMX or vehicle were tested for phosphodiesterase (PDE) and cAMP-dependent kinase A (PKA) activity. Additionally, chicken hepatocytes cultured in media as described above were exposed to 0.1 μmol/L DHEA, 20 μmol/L forskolin or vehicle for 1 h at 37℃. The cells were also exposed to 10 μmol/L H89(PKA- selective inhibitor), 2 μmol/L PKA inhibitor peptide (PKI) or vehicle for 30 min at 37℃. Then, 0.1 μmol/L DHEA was added, the cells were cultured for another 1 h, and scraped for the measurement of CREB phosphorylation levels. The results showed that, no significant differences were found in AC activity in both the DHEA and control groups(P>0.05). As expected, a full stimulation was achieved at 20 μmol/L forskolin(P<0.05), a powerful agonist of AC activity. In contrast, 0.1 μmol/L DHEA markedly suppressed PDE activity at 20 min as compared with the control group(P<0.05). The inhibitor IBMX was also potent in suppressing PDE activity during the incubation period(P<0.05). Cells incubated with 0.1 μmol/L DHEA for 20 min exhibited a significant increase in PKA activity(P<0.05). 0.1 μmol/L DHEA treatment lead to a significant increase in CREB phosphorylation(P<0.05). No significant differences was observed in hepatocytes pretreated with PKA inhibitors(P>0.05). The results suggest that DHEA can activate the cAMP/PKA signaling pathway and regulate lipid metabolism by enhancing CREB phosphorylation level, and provide a better understanding for improvements proposed for DHEA in decrease of body fat in broiler chickens.
