Jamil H, Utal A K, Vance D E
Lipid and Lipoprotein Research Group, University of Alberta, Edmonton, Canada.
J Biol Chem. 1992 Jan 25;267(3):1752-60.
The mechanism by which glucagon and cAMP analogues inhibit phosphatidylcholine biosynthesis was investigated in rat hepatocytes. The studies were facilitated by preparation of an antibody to a synthetic peptide (D-F-V-A-H-D-D-I-P-Y-S-S-A) corresponding to residues 164-176 of CTP:phosphocholine cytidylyl-transferase. The antibody, which was purified by affinity chromatography, quantitatively immunoprecipitated cytidylyltransferase from rat liver cytosol. Various analogues of cAMP had no effect on the labeling of cytidylyltransferase with 32Pi in rat hepatocytes. Nor did the cAMP analogues have any effect on the distribution of cytidylyltransferase between cytosol and membranes. These results indicate that the supply of CDP-choline does not limit phosphatidylcholine biosynthesis in hepatocytes treated with cAMP analogues. A decreased supply of diacylglycerol was considered as an alternative mechanism for inhibition of phosphatidylcholine biosynthesis. An approximately 30% decrease in diacylglycerol concentration was observed in hepatocytes treated with the cAMP analogues or glucagon, compared with controls. A similar decrease of phosphatidylcholine biosynthesis was observed. The cAMP-mediated decrease in diacylglycerol levels and inhibition of phosphatidylcholine biosynthesis were reversed by addition of 0.5-1.5 mM oleic acid to the treated hepatocytes. A correlation coefficient of 0.93 was calculated between the levels of diacylglycerol and the rate of phosphatidylcholine biosynthesis. In another approach, the diacylglycerol levels were increased by an inhibitor of diacylglycerol lipase (U-57908) which also reversed the cAMP effects on diacylglycerol levels and phosphatidylcholine biosynthesis. We conclude that the cAMP-mediated inhibition of phosphatidylcholine biosynthesis was not due to an effect on the phosphorylation of cytidylyltransferase. Instead, phosphatidylcholine biosynthesis appears to be inhibited due to a decreased level of diacylglycerol, a substrate for CDP-choline: 1,2-diacylglycerol cholinephosphotransferase.
在大鼠肝细胞中研究了胰高血糖素和环磷酸腺苷（cAMP）类似物抑制磷脂酰胆碱生物合成的机制。通过制备针对与CTP：磷酸胆碱胞苷酰转移酶第164 - 176位残基对应的合成肽（D - F - V - A - H - D - D - I - P - Y - S - S - A）的抗体，促进了这些研究。通过亲和层析纯化的该抗体从大鼠肝脏胞质溶胶中定量免疫沉淀胞苷酰转移酶。各种cAMP类似物对大鼠肝细胞中胞苷酰转移酶用32Pi进行标记没有影响。cAMP类似物对胞苷酰转移酶在胞质溶胶和膜之间的分布也没有任何影响。这些结果表明，在经cAMP类似物处理的肝细胞中，CDP - 胆碱的供应并不限制磷脂酰胆碱的生物合成。二酰基甘油供应减少被认为是抑制磷脂酰胆碱生物合成的另一种机制。与对照相比，在用cAMP类似物或胰高血糖素处理的肝细胞中观察到二酰基甘油浓度降低约30%。观察到磷脂酰胆碱生物合成有类似程度的降低。向处理过的肝细胞中添加0.5 - 1.5 mM油酸可逆转cAMP介导的二酰基甘油水平降低和磷脂酰胆碱生物合成的抑制。计算出二酰基甘油水平与磷脂酰胆碱生物合成速率之间的相关系数为0.93。在另一种方法中，通过二酰基甘油脂肪酶抑制剂（U - 57908）提高二酰基甘油水平，这也逆转了cAMP对二酰基甘油水平和磷脂酰胆碱生物合成的影响。我们得出结论，cAMP介导的磷脂酰胆碱生物合成抑制不是由于对胞苷酰转移酶磷酸化的影响。相反，磷脂酰胆碱生物合成似乎是由于二酰基甘油水平降低而受到抑制，二酰基甘油是CDP - 胆碱：1,2 - 二酰基甘油胆碱磷酸转移酶的底物。
