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.