Figure 7.

Pulse-chase analyses of MCFA-TG formation in WT primary hepatocytes, incubated with alkyne-palmitate and chased with decanoic acid upon inhibitory treatment. After isolation, 7.5 × 10⁴ primary mouse hepatocytes were plated in 24-well plates per well. (A) Cells were pre-incubated with either vehicle (con) or 50 μM of etomoxir (Eto), 3 μM of DGAT1-inhibitor (D1i) and a combination of both inhibitors (Eto + D1i). (B) Cells were preincubated with either vehicle (con) or 3 μM of DGAT1-inhibitor (D1i), 15 μM of DGAT2 inhibitor (D2i) or 50 μM of teglicar (Tegli). Cells were then incubated with 100 μM of FA 17:0;Y for 2 min and chased with 100 μM of decanoic acid (FA 10:0) for either 0, 1, 2, 5 or 10 min in the presence of the respective inhibitor. Lipids were extracted, and alkyne-labelled species were identified and quantified by multiplexed click-MS analysis as described in the Materials and Methods section. Absolute amounts of the lipid species within the lipid class of MCFA-TG;Y (Σ of a43:1, a43:0, a45:2, a45:1) are shown in pmol, and statistical evaluation of changes upon inhibitory treatment in comparison to the respective negative control (con) are shown for the last chase time (t = 10 min). The data represent mean ± SD for n = 3 biological replicates. ∗∗∗p ≤ 0.0002, ∗∗∗∗p ≤ 0.0001.