Novel Interconnections in Lipid Metabolism Revealed by Overexpression of Sphingomyelin Synthase-1

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.

Keywords: ceramide; diacylglycerol; hepatocytes; lipid droplets; lipid metabolism; palmitic acid; phosphatidylcholine; phospholipid; sphingomyelin synthase; triacylglycerol.

FIGURE 1.

Characterization of SMS1 protein expression and activity in HepG2 cells. The HepG2-SMS1 cell line stably overexpresses the human V5-tagged 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. Hoechst 33258 (blue) and wheat germ agglutinin (WGA; red) were used for staining of the nuclei and Golgi. B, SM synthase activity measured in vitro. C–E, in situ labeling of SM in live cells using NBD-ceramide (C), [³H]palmitic acid (D), and BODIPY®-palmitic acid (E) as tracers. F, mass of SM measured by TLC separation of total lipid extract and quantification of inorganic phosphate. Mean values ± S.D. (error bars) are shown (n = 3 dishes/point). Results were confirmed in at least three independent experiments, and representative data are shown. *, p < 0.05; **, p < 0.01; ***, p < 0.001 according to Student's t test.

FIGURE 2.

Effect of SMS1 overexpression on SM and Hex-Cer levels and synthesis. A and B, quantification of sphingomyelin (A) and hexosylceramide (B) species in total lipid extracts of HepG2-SMS1 and control cells by mass spectrometry. C, NBD-ceramide incorporation into glucosylceramide in control and SMS1-overexpressing cells. D and E, effects of GCS inhibition using PDMP on the formation of NBD-glucosylceramide and NBD-SM. PDMP (25 μm) was added 1 h before the addition of NBD-ceramide, and cells were harvested at the indicated time points. Conversion of NBD-Cer to NBD-glucosylceramide or NBD-SM was quantified using HPLC. Data are shown as mean values ± S.D. (error bars) (n = 3 dishes/point). *, p < 0.05; **, p < 0.01; ***, p < 0.001 according to Student's t test. Results were confirmed in two independent experiments.

FIGURE 3.

Effect of palmitic acid on the levels of different sphingolipids. HepG2 cells were cultured for 18 h in the presence of either 1 mm palmitic acid delivered as a BSA complex (2:1, mol/mol) or 0.5 mm BSA as a vehicle control. Total lipid extracts were prepared, and ceramide (A), SM (B), and Hex-Cer (C) species were measured by mass spectrometry. Data are shown as mean values ± S.D. (error bars) (n = 3 dishes/point). *, p < 0.05 according to Student's t test. Results were confirmed in two independent experiments.

FIGURE 4.

SMS1 overexpression attenuates the ability of cells to accumulate TG. A–C, HepG2-SMS1 and HepG2-EV cells were incubated with 1 mm palmitic acid delivered as a BSA complex (2:1, mol/mol) or with vehicle control (0.5 mm BSA) for 18 h. A, formation of lipid droplets visualized by Oil Red-O staining. B and C, levels of TG (B) and total cholesterol (C) measured in total lipid extracts as described under “Experimental Procedures.” Mean values ± S.D. (error bars) are shown (n = 3 dishes/point). D–F, HepG2 cells transiently transfected with SMS1 or EV were incubated with 0.1 or 1 mm ³H-labeled palmitic acid for 18 h. The specific labeling in each case was kept at 50 mCi/mmol. Lipids were extracted and separated by TLC as described under “Experimental Procedures.” D, radioactivity from the bands corresponding to SM quantified by scintillation counting. E, representative scan for ³H-labeled TG at 1 mm palmitic acid. F, radioactivity associated with TG determined by scintillation counting. According to two-way analysis of variance, the main effects of palmitate treatment and SMS1 overexpression on TG were statistically significant. The interaction effect was not statistically significant. The results of Bonferroni post-test analyses are indicated (***, p < 0.001; **, p < 0.01; *, p < 0.05). Results were confirmed in at least four independent experiments.

FIGURE 5.

Effects of SMS1 overexpression on DGAT activity, TG secretion, and mitochondrial bioenergetics. HepG2 cells stably overexpressing the human SMS1 or EV controls. A, de novo TG biosynthesis assessed using BODIPY®-labeled palmitic acid as a tracer (8 μm for 18 h). Levels of BODIPY®-TG were quantified after TLC separation by scanning the plates using a Typhoon imager and normalizing the intensity of the TG bands to the intensity of the total lipid extract. B, TG levels in conditioned medium (18 h) were measured following extraction with Dole's reagent and separation on a TLC plate. C, OCRs of HepG2-SMS1 and HepG2-EV cells. A mitochondrial respiration assay was done using an XF96 extracellular flux analyzer (Seahorse Biosciences). The culture medium was serum-free and contained 10 mm glucose, 3 mm glutamine, and 1 mm pyruvate. Inhibitors (1.25 μm oligomycin, 1.0 μm FCCP, and 2.0 μm antimycin A or 2.0 μm rotenone) were injected at the indicated time points to block different components of the electron transport chain. D and E, DGAT activity (total (D) and overt and latent (E)) measured in permeabilized cells as described under “Experimental Procedures.” Latent activity was calculated as the difference between total and overt activities. Mean values ± S.D. (error bars) are shown (n = 3 dishes/point). F, effect of palmitic acid on DGAT activity. HepG2-EV or HepG2-SMS1 cells were cultured for 18 h in the presence of either 0.5 mm BSA as a vehicle control or 1 mm palmitic acid delivered as a BSA complex (2:1, mol/mol). Overt and latent DGAT activity was measured as described under “Experimental Procedures.” Results were confirmed in at least two independent experiments, and representative data are shown. ***, p < 0.001 (A) or as indicated (F) according to Student's t test. n = 3 dishes/point. Results were confirmed in at least two independent experiments.

FIGURE 6.

TG species distribution in SMS1 and control cells. HepG2 cells were cultured for 18 h in the presence of either 0.5 mm BSA as a vehicle control (upper panel) or 1 mm palmitic acid delivered as a BSA complex (2:1, mol/mol) (lower panel) for 18 h. TG fatty acid composition measured by mass spectrometry as described under “Experimental Procedures.” The graphs represent S-plots of OPLS-DA of TG fatty acid composition and indicate the major features that contributed to the group difference between HepG2-EV and HepG2-SMS1 in each case. Data are the average of three replicates.

FIGURE 7.

Effects of SMS1 overexpression and palmitic acid on the synthesis of major lipid classes. HepG2-SMS1 and EV control cells were supplemented with [³H]palmitic acid at low (0.1 mm) or high (1.0 mm) concentration for 18 h. The specific labeling in each case was kept at 50 mCi/mmol. Lipids were extracted and separated by TLC as described under “Experimental Procedures.” Radioactivity from the individual bands was quantified by scintillation counting. A, SM; B, TG; C, phosphatidylcholine; D, phosphatidylethanolamine; E, phosphatidylserine; F, phosphatidic acid; G, DG. According to two-way analysis of variance, a strong statistically significant main effect of palmitate treatment was detected for all lipids. The main effects of SMS1 overexpression on TG, PC, and DG were also statistically significant. A statistically significant interaction effect was seen for SM and TG. Results of Bonferroni post-test analyses are shown (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Results were confirmed in two independent experiments. Error bars, S.D.

FIGURE 8.

Effect of SMS1 overexpression on the de novo synthesis of PC. HepG2-SMS1 and EV control cells were cultured in complete growth medium supplemented with 0.3 μCi/well radiolabeled [¹⁴C]choline chloride for the indicated periods of time. Lipids were extracted and separated by TLC as described under “Experimental Procedures.” Radioactivity from the corresponding bands was quantified by scintillation counting. A, phosphatidylcholine; B, sphingomyelin. Data shown are the average ± S.D. (error bars), n = 3 (*, p < 0.05). Results were confirmed in two independent experiments.

FIGURE 9.

Proposed mechanism for the SMS1 regulation of TG synthesis. The figure illustrates the main pathways for glycerolipid and sphingolipid synthesis and their respective localization in the ER and Golgi apparatus. Chronic increases in SMS1 in the trans-Golgi generate a signal of enhanced utilization of PC, resulting in the stimulation of PC synthesis in the ER via CEPT1. As a result, the pool of DG substrate available for TG synthesis is diminished, causing a decline in TG synthesis. A change in the fatty acid composition of available DG substrate might also influence its metabolic conversion toward PC rather than TG synthesis due to different substrate preferences of CEPT1 and DGAT1 (see “Discussion”). Also shown are the two routes for utilization of palmitic acid in sphingolipid and glycerolipid synthesis. CERS, ceramide synthase; DGK1, diacylglycerol kinase 1; GlcCer, glucosylceramide; GPAT, glycero-3-phosphate acyltransferase; LPAT, lysophosphatidic acid acyltransferase; PA, phosphatidic acid; PAP, phosphatidic acid phosphatase.