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 RlA(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-l-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 n,mol/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 (f<0.05), a powerful agonist of AC activity. In contrast, 0.1 (Amol/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.%控制肉鸡脂肪过多沉积是肉鸡生产中亟待解决的问题.脱氢表雄酮(dehydroepiandrosterone,DHEA)是人体分泌最为丰富的肾上腺类固醇激素,可经由类固醇激素受体介导发挥生理功能,降低机体生脂能力.本研究以鸡胚原代肝细胞为研究对象,选用含DHEA终浓度为0(对照)、0.01、0.1、1.0、10和100μmol/L的培养液孵育肝细胞20 min后收集细胞,放射免疫测定法(RIA)检测胞内cAMP水平.结果发现,0.1～100 μmol/L DHEA孵育肝细胞均可显著提高胞内cAMP水平(P＜0.05),其中0.1 μmnol/L DHEA效果最为显著(P＜0.01).0.1 μmol/L DHEA孵育肝细胞20 min或1h后收集细胞,RIA法分别检测胞内腺苷酸环化酶(adenylate cyclase,AC)和磷酸二酯酶(phosphodiesterase,PDE)活性,(γ-32P)ATP掺入法测定cAMP依赖性蛋白激酶A(protein kinase A,PKA)活性,Western blot法测定cAMP反应元件结合蛋白(cAMP-response element binding protein,CREB)磷酸化水平.结果显示,0.1 μmol/L DHEA孵育肝细胞20min可显著降低胞内PDE活性(P＜0.05),提高PKA活性(P＜0.05),但对AC活性无显著性影响(P＜0.05).0.1 μmol/L DHEA处理肝细胞1h可显著提高CREB蛋白磷酸化水平(P＜0.05),且这种效应可被PKA抑制剂阻断(P＞0.05).本研究结果提示,DHEA调控肉鸡肝脏脂肪代谢,降低脂肪沉积的机制可能与激活胞内cAMP/PKA信号系统,活化转录因子CREB有关.
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