Fig. 7

Increased intracellular accumulation of neutral lipid and CEs as a result of esterification of cholesterol to fatty acyl-CoA.

(A) Oil Red O (ORO) staining of eGFP stable expressing (Ctrl), zebrafish saot1 stable expressing (Soat1), and zebrafish soat2 stable expressing (Soat2) HEK293 cells after incubation with low (15 μM oleic acids and 1 μg/mL cholesterol) or high (150 μM oleic acids and 10 μg/mL cholesterol) level of substrate at room temperature for 6 hrs. The expression of zebrafish Soat2 significantly increased the intracellular accumulation of neutral lipid indicated its enzymatic activity of cholesterol esterification, and this accumulation of neutral lipid is significantly increased with the increased levels of substrates. Note the significantly increased neutral lipid in WT-high compared to WT-low reflected the activity of endogenous human SOAT1 expressed by HEK293 cells. The letters above each column indicate the statistical groups, and the data sharing the same letters indicates no significant difference. (B) The cell lysate of HEK293 cells with zebrafish soat2 overexpression contained significantly higher CE content than either control group or soat1 overexpression group. Consistent with the ORO staining, the CE content in soat1 group was comparable to the control group. (C) RT-PCR confirmed the expression of both zebrafish soat1 and eGFP in Soat1-overexpressing HEK293 cells, zebrafish soat2 and eGFP in Soat2-overexpressing HEK293 cells and only eGFP in control group in which the HEK293 cells were transfect with empty vectors. (D) The HEK293 cells overexpressing zebrafish Soat2 (Soat2-DsRed) contained more and larger intracellular lipid droplets containing NBD-cholesterol as compared to the HEK293 cells overexpressing DsRed only (DsRed). (E) Avasimibe (AVA) significantly reduced the intracellular accumulation of neutral lipid, but the cells with zebrafish soat2 overexpression still showed a significantly more ORO staining than other AVA-treated cells. (F) Pyripyropene A (PPPA) could significantly reduce the intracellular accumulation of neutral lipid by inhibiting the enzymatic activity of both endogenous human SOAT1 and zebrafish Soat2 when compared to the vehicle control (DMSO). The letters above each column indicate the statistical groups, and the data sharing the same letters indicates no significant difference.