脱氢表雄酮(DHEA)对鸡胚原代肝细胞cAMP/PKA信号通路和cAMP反应元件结合蛋白(CREB)的影响与调节  

Regulation of Dehydroepiandrosterone(DHEA) on cAMP/PKA Signalling System and cAMP-response Element Binding Protein(CREB) in Cultured Primary Chicken Hepatocytes

作　　者：唐雪[1,2] 马海田[2] 邹思湘[2] 

机构地区：[1]江南大学食品学院,食品科学与技术国家重点实验室,无锡214122 [2]南京农业大学,农业部动物生理生化重点开放实验室,南京210095

出　　处：《农业生物技术学报》2012年第6期649-656,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(No.30600439)

摘　　要：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20min后收集细胞,放射免疫测定法(RIA)检测胞内cAMP水平。结果发现,0.1～100μmol/L DHEA孵育肝细胞均可显著提高胞内cAMP水平(P<0.05),其中0.1μmol/L DHEA效果最为显著(P<0.01)。0.1μmol/L DHEA孵育肝细胞20min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,0.1μmol/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)。0.1μmol/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。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-reducingagent 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 0.25μmol/L IBMX （3-isobutyl- 1-methylxanthine） or vehicle for 5 min, followed by the addition of 0.1μmol/L DHEA, 20 μmol/L forskolin or vehicle for 20 min at 37°C were tested for adenylate cyclase （AC） activity. Cell lysates isolated from hepatocytes exposed to 0.1 μmol/L DHEA, 0.25 μmol/L IBMX or vehicle were tested forphosphodiesterase （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, 20μmol/L forskolin or vehicle for 1 h at 37°C. The cells were also exposed to 10 μmol/L H89 （PKAselective inhibitor）, 2 μmol/L PKA inhibitor peptide （PKI） or vehicle for 30 min at 37°C. 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 20μmol/L forskolin （P〈0.05）, a powerful agonist of AC activity. In contrast, 0.1μmol/L DHEA markedly suppressed PDE activity at 20 min as compared with the contro

关 键 词：脱氢表雄酮(DHEA) 鸡胚原代肝细胞 肝脏脂肪代谢 cAMP/PKA信号通路 cAMP反应元件结合蛋白(CREB) 

分 类 号：Q426[生物学—神经生物学]