Addition of very-low density lipoproteins (VLDL), fatty acids and cholesterol to lipid-reduced (LR) growth conditions reverses the increased activation of the lipogenic pathway.

Gene expression of FASN, ACLY, HMGCR and ACSS2 was analyzed by qPCR in T24 cells were cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL (a), different fatty acid mixtures (b) and different concentrations cholesterol (c). VLDL was added at a concentration of 607 µg triglycerides/ml serum (corresponding to the concentration triglycerides in normal FBS). Fatty acid (FA) mixtures were as follows, FA Mix 1: 20 µM linoleic (18∶2), 20 µM α-linolenic (18∶3), 5 µM arachidonic (20∶4), 5 µM docosahexaenoic acid (22∶6), FA Mix 2: 10 µM 18∶2, 15 µM 18∶3, 10 µM 20∶4, 15 µM 22∶6 and FA Mix 3: 20 µM 18∶2, 20 µM 18∶3, 5 µM 20∶4, 5 µM 22∶6, 30 µM oleic acid, 30 µM palmitic acid. Different cholesterol (Ch) concentrations are as indicated in the figures (25 µM, 50 µM or 100 µM). Data are normalized to 18S and represented as mean ± S.D. (triplicate per experiment and n = 3). Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. #Significantly different (#p≤0,05; ##p≤0,01; ###p≤0,001; ####p≤0,0001 ) from LR control. (d, e, f)14C-incorporation into cellular lipids was determined in T24 cells, cultured for 48 hours in normal (N) or LR growth conditions in the presence or absence of VLDL (d), different fatty acid mixtures (e) and different concentrations cholesterol (f) as mentioned in (a, b and c). During the last 4 hours 14C-acetate was added and the incorporation of radioactivity in cellular lipids was normalized to sample DNA content. Representative experiment is shown, experiment was repeated two times. Significance was determined by one-way ANOVA followed by Tukey's multiple comparison test. *Significantly different (*p≤0,05; **p≤0,01; ***p≤0,001; ****p≤0,0001) from normal medium control. #Significantly different (#p≤0,05; ##p≤0,01; ###p≤0,001; ####p≤0,0001) from LR control.