Concentration-dependent effect of oleate on HepG2 steatosis and proliferation
We first compared the effect of BSA vs BSA-conjugated oleic acid on triacylglycerol accumulation in HepG2 cells (Fig. 1a, b). Treatment of HepG2 cells with oleate for 24 h induced concentration-dependent triacylglycerol accumulation compared with the control condition, with up to a 2.7-fold increase observed at 1 mmol/l oleate. To evaluate the effect of oleate on cell proliferation (Fig. 2a), fetal FCS and HGF were used as controls to increase and decrease HepG2 proliferation, respectively . Compared with the number of cells initially plated, all treatments except 1 mmol/l oleate and HGF led to a significantly increased cell number at the 72 h time point. When comparing the effect of BSA with that of BSA-conjugated oleate, divergent results were observed between 50 μmol/l and 1 mmol/l oleate. Indeed, exposure to 50 μmol/l oleate significantly increased the cell number after 72 h treatment, whereas 1 mmol/l oleate had no significant effect. We next analysed [3H]thymidine incorporation and the number of propidium iodine-positive cells after 72 h of treatment with BSA, 50 μmol/l or 1 mmol/l oleate, FCS or HGF. As seen in Fig. 2b, [3H]thymidine incorporation was increased by 50 μmol/l oleate and FCS but decreased by 1 mmol/l oleate and by HGF, albeit not significantly for the latter. Conversely, the number of propidium iodine-positive cells was unchanged by FCS or HGF but significantly decreased by 50 μmol/l and further decreased by 1 mmol/l oleate (Fig. 2c), indicating that increasing the oleate concentration indeed protects against cell death.
In summary, our results show that 50 μmol/l oleate increases HepG2 cell number by concomitantly increasing cell proliferation and decreasing cell death.
Oleate-mediated proliferation of HepG2 cells involves the regulation of cell cycle components
The differential effects on cell proliferation induced by the low vs high concentration of oleate prompted us to search for a differential modulation of known cell cycle regulators produced by these treatments. Notably, hyperphosphorylation of the cell-cycle regulator retinoblastoma protein (RB) and increased production of cyclin A and E have been implicated in HCC initiation and progression [22, 23]. As seen in Fig. 3a–d, a 24 h treatment with the low concentration of oleate increased the level of phosphorylated RB and the protein levels of cyclin A and E. In contrast, the high concentration of oleate decreased phosphorylated RB and had no significant effect on cyclin A and E protein levels. Similar results were observed with a 72 h treatment (data not shown). Furthermore, exposure to oleate had no effect on cyclin A and E transcript levels, which suggests a translational effect of oleate on their respective mRNAs (data not shown).
Thus, these early effects of oleate on cell-cycle components are likely to explain its proliferative action.
Oleate drives mTORC1-dependent HepG2 proliferation and induces resistance to rapamycin
We took advantage of the concentration-dependent effects of oleate treatment to search for the signalling pathways potentially involved in oleate-induced proliferation. We reasoned that the relevant pathway would be differentially regulated by the low vs high concentration of oleate. Among the potentially relevant candidate cascades, we examined the extracellular signal-regulated kinase (ERK)1/2, PKB and mTORC1 phosphorylation levels, and phosphatase and tensin homologue (PTEN) level by western blot analysis of HepG2 cells treated with 50 μmol/l or 1 mmol/l oleate (Fig. 4a–e). A 24 h treatment with the low and high oleate concentrations had a similar effect on ERK and PKB activation, i.e. it reduced ERK and increased PKB. Both concentrations were without effect on PTEN level. Interestingly, we observed a differential effect of oleate on markers of the activity of the mTORC1 pathway (Fig. 5a–d). Indeed, at a low concentration, oleate increased the phosphorylation of mTOR, that of its target 4E-BP1 and that of the ribosomal S6 protein, the downstream target of S6K. Importantly, the high concentration of oleate had the opposite effect on the phosphorylation state of these molecules. Similar results were observed with a 72 h treatment (data not shown).
To further illustrate the concentration-dependent effects of oleate on mTORC1 activation and cyclin A level, HepG2 cells were exposed for 72 h to different concentrations of oleate (Fig. 5e). Oleate increased mTORC1/4E-BP1 phosphorylation and cyclin A level at low concentrations with a maximal increase seen at 50 μmol/l oleate. Starting from 100 μmol/l, these effects decreased. Hence, the two concentrations chosen for our experiments seem to be appropriate because at 50 μmol/l the mTOR pathway is fully activated while at 1 mmol/l it is severely inhibited.
To confirm that mTORC1 activation supported oleate-induced proliferation of HepG2 cells, we analysed the effect of rapamycin on the proliferation induced by a 72 h treatment with 50 μmol/l oleate. Surprisingly, rapamycin treatment alone efficiently inhibited the proliferation of HepG2 cells and reduced the level of cyclin A, but was without effect in the presence of oleate (Fig. 6a, b). Hence, we reasoned that oleate rendered a component of the mTORC1 pathway insensitive to rapamycin. Indeed, it has been shown recently that cap-dependent translation could occur despite rapamycin treatment and S6K inhibition, and that rapamycin resistance may be specific to the mTORC1/4E-BPs arm of the mTORC1 pathway . As seen in Fig. 6c and d, in the presence of oleate, rapamycin reduced the phosphorylation of the ribosomal S6 protein to control levels, but not that of 4E-BP1.
Oleate increases cyclin A level and mTORC1 activity through PLD activity
The existence of an oleate-sensitive PLD that is only stimulated by low concentrations of oleate  and the notion that PA renders mTORC1 resistant to rapamycin prompted us to examine whether oleate-induced mTORC1 activation may involve activation of PLDs. As seen in Fig. 7a, PLD activity was increased in HepG2 cells treated for 72 h with 50 μmol/l, but it was decreased by 1 mmol/l oleate. Oleate had no effect on PLD1 or PLD2 mRNAs (data not shown). Moreover, inhibition of PLD-mediated PA production by 1-butanol abolished mTORC1 activation assessed by mTOR, S6 and 4E-BP1 phosphorylation (Fig. 7b–d). Furthermore, treatment with 1-butanol blunted the increase in cyclin A protein levels induced by 50 μmol/l oleate compared with the control condition (Fig. 7e).
Oleate activates mTORC1 and favours rapamycin resistance in SK-Hep1 hepatoma cells
To exclude the possibility that oleate’s main effects could be unique to HepG2 cells, we performed similar experiments on SK-Hep1 cells. Indeed, compared with the basal condition, 50 μmol/l oleate addition for 48 h robustly activated the mTORC1 pathway and increased the cyclin A level (Fig. 8a–e). However, l mmol/l oleate also induced a modest, albeit not significant, increase in mTOR phosphorylation and cyclin A level, and a significant increase in S6 and 4E-BP1 phosphorylation, compared with the basal condition. These differences in the effect of 1 mmol/l oleate between HepG2 and SK-Hep1 cells can be attributed to the low basal level of mTOR activation in the latter cell line.
To investigate whether oleate could also induce rapamycin resistance in this model, SK-Hep1 cells were treated concomitantly with 50 μmol/l oleate and 50 nmol/l rapamycin for 48 h (Fig. 9a–c). Interestingly, 4E-BP1 phosphorylation and cyclin A level were quite insensitive to rapamycin in the presence of oleate, whereas S6 phosphorylation was not. Overall, these findings are similar to the observations made in HepG2 cells.