Figure 9.

ACSL1 interacts with lipid droplet tethering proteins when glucose is absent. Primary murine hepatocytes were isolated from C57BL/6J mice maintained on a standard chow diet. Cells were infected with either Ad-GFP or Ad-ACSL1–FLAG. Protein complexes were cross-linked, and FLAG-tagged proteins were isolated with FLAG affinity resin and eluted with FLAG peptide. A, CPT-1 was identified by immunoblot. B, primary murine hepatocytes were infected with Ad-GFP or Ad-ACSL1–FLAG. The media were replaced with fresh media containing 25 mm glucose and 1 mm pyruvate or media lacking glucose and pyruvate for 16 h before collection and FLAG-affinity chromatography. Interacting proteins were identified using antibodies directed against CPT-1, SNAP23, and VAMP4. Data are representative of three independent experiments. C, Hepa 1–6 cells were cultured for 3 h in DMEM containing either 0 mm glucose, 0 mm pyruvate, or 25 mm glucose and 1 mm pyruvate. Fresh media containing [³H]palmitate and 100 mm carnitine were added for 3 h. Oxidation was measured by counting [³H]H₂O released into the media (n = 4). Incorporation of palmitate was measured by extracting total lipid and counting ³H levels (n = 3). D, Hepa 1–6 cells were transfected with 0.5 μg of esiRNA targeting GFP or SNAP23 and cultured for 48 h in the presence of 5 mm glucose (n = 3) or 25 mm glucose (n = 2). [1-¹⁴C]Palmitic acid oxidation to ASM and CO₂ was measured. Data are presented as means ± S.E. E, Hepa 1–6 cells were transfected with 0.5 μg of esiRNA targeting GFP or SNAP23 and cultured for 48 h in the presence of 5 mm glucose (n = 2) or 25 mm glucose (n = 3). Cells were incubated with 100 μm [1-¹⁴C]oleate for 3 h (pulse) and then washed with 1% BSA in PBS before unlabeled chase media was added. [1-¹⁴C]Oleate oxidation to ASM and CO₂ was measured at 1, 3, and 6 h. White bars represent CO₂ production; colored bars represent ASM production. Data are presented as means ± S.E. F, SNAP23 expression levels in Hepa 1–6 transfected with either 0.5 μg of esiRNA targeting GFP or SNAP23, with β-actin as loading control.