Department of Physiology, University of Kentucky College of Medicine, Lexington, KY 40536, USA.
Markey Cancer Center, Redox Metabolism Shared Resource Facility, University of Kentucky, Lexington, KY 40536, USA.
Cells. 2021 May 21;10(6):1278. doi: 10.3390/cells10061278.
Ceramide and diacylglycerol (DAG) are bioactive lipids and mediate many cellular signaling pathways. Sphingomyelin synthase (SMS) is the single metabolic link between the two, while SMS2 is the only SMS form located at the plasma membrane. SMS2 functions were investigated in HepG2 cell lines stably expressing SMS2. SMS2 overexpression did not alter sphingomyelin (SM), phosphatidylcholine (PC), or ceramide levels. DAG content increased by approx. 40% and led to downregulation of DAG-dependent protein kinase C (PKC). SMS2 overexpression also induced senescence, characterized by positivity for β-galactosidase activity and heterochromatin foci. HepG2-SMS2 cells exhibited protruded mitochondria and suppressed mitochondrial respiration rates. ATP production and the abundance of Complex V were substantially lower in HepG2-SMS2 cells as compared to controls. SMS2 overexpression was associated with inflammasome activation based on increases in IL-1β and nlpr3 mRNA levels. HepG2-SMS2 cells exhibited lipid droplet accumulation, constitutive activation of AMPK based on elevated Thr phosphorylation, increased AMPK abundance, and insensitivity to insulin suppression of AMPK. Thus, our results show that SMS2 regulates DAG homeostasis and signaling in hepatocytes and also provide proof of principle for the concept that offset in bioactive lipids' production at the plasma membrane can drive the senescence program in association with steatosis and, seemingly, by cell-autonomous mechanisms.
神经酰胺和二酰基甘油（DAG）是生物活性脂质，介导许多细胞信号通路。神经鞘氨醇合酶（SMS）是两者之间的唯一代谢联系，而 SMS2 是唯一位于质膜的 SMS 形式。在稳定表达 SMS2 的 HepG2 细胞系中研究了 SMS2 的功能。SMS2 过表达不会改变神经鞘磷脂（SM）、磷脂酰胆碱（PC）或神经酰胺水平。DAG 含量增加约 40%，导致 DAG 依赖性蛋白激酶 C（PKC）下调。SMS2 过表达还诱导衰老，其特征为β-半乳糖苷酶活性和异染色质焦点阳性。HepG2-SMS2 细胞表现出伸出的线粒体和抑制的线粒体呼吸速率。与对照相比，HepG2-SMS2 细胞中的 ATP 产生和复合物 V 的丰度明显降低。基于 IL-1β 和 nlpr3 mRNA 水平的增加，SMS2 过表达与炎症小体激活相关。HepG2-SMS2 细胞表现出脂滴积累，基于 Thr 磷酸化升高的 AMPK 组成性激活，AMPK 丰度增加，以及对胰岛素抑制 AMPK 的不敏感。因此，我们的结果表明，SMS2 调节肝细胞中 DAG 的动态平衡和信号转导，并为生物活性脂质在质膜中产生的偏移可以驱动与脂肪变性相关的衰老程序的概念提供原理证明，并且似乎通过细胞自主机制。
