Abstract
This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin. HepG2 cells stably transfected with SMS1 (HepG2-SMS1) exhibit elevated enzyme activity in vitro and increased sphingomyelin content (mainly C22:0- and C24:0-sphingomyelin) but lower hexosylceramide (Hex-Cer) levels. HepG2-SMS1 cells have fewer triacylglycerols than controls but similar diacylglycerol acyltransferase activity, triacylglycerol secretion, and mitochondrial function. Treatment with 1 mm palmitate increases de novo ceramide synthesis in both cell lines to a similar degree, causing accumulation of C16:0-ceramide (and some C18:0-, C20:0-, and C22:0-ceramides) as well as C16:0- and C18:0-Hex-Cers. In these experiments, the palmitic acid is delivered as a complex with delipidated BSA (2:1, mol/mol) and does not induce significant lipotoxicity. Based on precursor labeling, the flux through SM synthase also increases, which is exacerbated in HepG2-SMS1 cells. In contrast, palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former. Furthermore, triacylglycerols from HepG2-SMS1 cells are enriched in polyunsaturated fatty acids, which is indicative of active remodeling. Together, these results delineate novel metabolic interactions between glycerolipids and sphingolipids.
Highlights
This study investigates the consequences of elevating sphingomyelin synthase 1 (SMS1) activity, which generates the main mammalian sphingolipid, sphingomyelin
Palmitate-induced lipid droplet formation is significantly reduced in HepG2-SMS1 cells. [14C]Choline and [3H]palmitate tracking shows that SMS1 overexpression apparently affects the partitioning of palmitate-enriched diacylglycerol between the phosphatidylcholine and triacylglycerol pathways, to the benefit of the former
Indirect immunofluorescence confirmed that SMS1 was overexpressed and that the protein co-localized with the Golgi marker wheat germ agglutinin (WGA) (Fig. 1A)
Summary
SMS1 (HepG2-SMS1), whereas HepG2-EV is the empty vector control cell line. A, expression and subcellular localization of the V5-tagged SMS1 protein (green) visualized by indirect immunofluorescence in permeabilized cells using antibody against the V5 tag. The direct inhibition of CEPT1 or the genetic deletion of phosphoethanolamine cytidylyltransferase, which catalyzes the formation of CDP-ethanolamine for PE synthesis, has been shown to stimulate the synthesis of TG by as much as 10-fold [8, 9]. The overexpression of DGAT1, on the other hand, has been shown to inhibit the synthesis of glycerophospholipids [10]. To directly test the relationship between SMS1 and glycerolipid synthesis, we stably overexpressed SMS1 in HepG2 cells. We show that HepG2-SMS1 cells exhibit an attenuated rate of TG synthesis, especially in the presence of excess palmitic acid. The chronic up-regulation of SMS1 activity appears to activate PC depletion-sensing mechanisms at the Golgi and to stimulate the Kennedy pathway of de novo PC synthesis, diverting DG precursors away from DGAT and TG synthesis
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