Lightle S A, Oakley J I, Nikolova-Karakashian M N
Department of Physiology, University of Kentucky College of Medicine, Chandler Medical Center, MS 579, 800 Rose Street, Lexington, KY 40536, USA.
Mech Ageing Dev. 2000 Dec 1;120(1-3):111-25. doi: 10.1016/s0047-6374(00)00191-3.
Aging leads to a decreased ability of liver to metabolize drugs and increased expression and secretion of acute phase proteins, such as serum amyloid A (SAA), C-reactive protein (CRP), and alpha-1-acid glycoprotein (AGP). This phenomenon resembles some aspects of the acute phase response of host to inflammation; however, the molecular basis for the similarity is unclear. Ceramide and sphingosine are second messenger mediators of cellular responses to stress and inflammation. In liver, they play important role in mediating acute phase responses to IL1-beta. In this study, we use HPLC and thin layer chromatography to evaluate the effects of aging on steady-state levels of ceramide and sphingosine. We report that both lipids are elevated in liver of old (24 months) as compared to young (5 months) male Fisher 344 rats. To elucidate the mechanism(s) for ceramide elevation, we test the acidic (ASMase) and neutral sphingomyelinase (NSMase) in vitro using NBD-sphingomyelin as an exogenous substrate. SM synthase is also analyzed in vitro using NBD-ceramide and [3H]-dipalmitoylphosphatidylcholine (DPPC) as exogenous substrates. In accordance with the increases in the mass of ceramide, the activity of acid and neutral SMase is elevated in old animals. Michaelis-Menten analysis of NSMase implies that the apparent activation of this enzyme is caused by an increase in the Vmax of the enzyme. In contrast, SM synthase activity is lower in old animals as compared to young ones. These results show that aging is accompanied by an elevation in SM turnover and a decrease in its synthesis, resulting in accumulation of pro-inflammatory and growth inhibitory second messenger ceramide. Ceramidase, the only enzyme leading to sphingosine generation, is also measured in vitro using NBD-ceramide as a substrate and liver homogenate as an enzyme source. Its activity is higher in the old rats, as compared to young ones. The acid and neutral forms of the enzyme are affected the most, while the changes in the alkaline enzyme are not significant. The increases in the basal levels of ceramide and sphingosine in old animals may contribute to the onset of an inflammatory like state in liver during aging, exemplified by decreased P4502C11 mRNA expression and chronic induction of acute phase protein expression.
衰老导致肝脏代谢药物的能力下降，以及急性期蛋白如血清淀粉样蛋白A（SAA）、C反应蛋白（CRP）和α-1-酸性糖蛋白（AGP）的表达和分泌增加。这种现象类似于宿主对炎症的急性期反应的某些方面；然而，这种相似性的分子基础尚不清楚。神经酰胺和鞘氨醇是细胞对应激和炎症反应的第二信使介质。在肝脏中，它们在介导对IL1-β的急性期反应中起重要作用。在本研究中，我们使用高效液相色谱法和薄层色谱法评估衰老对神经酰胺和鞘氨醇稳态水平的影响。我们报告，与年轻（5个月）雄性Fisher 344大鼠相比，老年（24个月）大鼠肝脏中这两种脂质均升高。为了阐明神经酰胺升高的机制，我们使用NBD-鞘磷脂作为外源性底物在体外测试酸性（酸性鞘磷脂酶）和中性鞘磷脂酶（中性鞘磷脂酶）。还使用NBD-神经酰胺和[3H]-二棕榈酰磷脂酰胆碱（DPPC）作为外源性底物在体外分析鞘磷脂合成酶。与神经酰胺含量的增加一致，老年动物中酸性和中性鞘磷脂酶的活性升高。对中性鞘磷脂酶的米氏分析表明，该酶的明显激活是由酶的Vmax增加引起的。相反，与年轻动物相比，老年动物中鞘磷脂合成酶的活性较低。这些结果表明，衰老伴随着鞘磷脂周转率的升高及其合成的减少，导致促炎和生长抑制性第二信使神经酰胺的积累。还使用NBD-神经酰胺作为底物和肝脏匀浆作为酶源在体外测量唯一导致鞘氨醇生成的酶——神经酰胺酶。与年轻大鼠相比，老年大鼠中其活性更高。该酶的酸性和中性形式受影响最大，而碱性酶的变化不显著。老年动物中神经酰胺和鞘氨醇基础水平的升高可能导致衰老过程中肝脏出现类似炎症的状态，例如P4502C11 mRNA表达降低和急性期蛋白表达的慢性诱导。
