Figure 3.
13C-Oleate Tracing Reveals a Critical Buffering Role for TG-Resident Unsaturated FAs
(A) Effect of SCDi on total TG abundances as measured by LC-MS.
(B) Effect of oleate pre-loading with or without DGAT shRNA on subsequent A498 cell survival (by Annexin-PI) during serum limitation and SCD inhibition.
(C) Schematic of the experimental workflow. DGAT2 knockout cells were serum-starved for 24 hr and then loaded for 24 hr with 10 μM [U13C]-oleate (C18:1) ± DGAT1 inhibitor (T863, 2 μM). The medium was then replaced and the tracer removed, and cells were subjected to a 48-hr washout.
(D) TG labeling patterns after 24-hr loading with [U13C]-oleate with or without DGATi, where numbers of mono-unsaturated FA (MUFA) and FA carbons are indicated. 1×, 2×, and 3× indicate whether TGs have one, two, or three oleates (includes [13C18]-20:1) conjugated to their glycerol backbones.
(E) BODIPY and DAPI staining directly after [U13C]-oleate loading with or without DGATi.
(F) Labeling patterns as assessed by incorporation of the 13C label in 18:1 and 20:1 FAs in TG, DG, PC, and PE species.
(G) Model of the metabolic mechanism by which TGs alleviate the saturation of certain lipid classes (e.g., PCs) under conditions of unsaturated lipid deprivation by releasing stored oleate.
Data are means of triplicate wells confirmed in independent experiments (A, B, and D) or means of three independent experiments each conducted in triplicate (F); error bars represent SD. Statistical significance by t test or ANOVA, as appropriate. ∗p < 0.05, ∗∗p < 0.05, and ∗∗∗p < 0.005. See also Figure S3.