## 4 Discussion  
The blood levels of 7 KCh are associated with cardiovascular disease events [4; 5; 9]. As the enzymes required for 7 KCh catabolism are expressed in the liver, 7 KCh catabolism in other tissues like the heart and any intervention measures to reduce its toxicity remain to  
Figure 5: 7 KCh alters intracellular levels of TGs and CEs in HL-1 cells. HL-1 cells were untreated (Con) or treated with indicated concentrations (10 or 20 uM) of 7 KCh for 24 h and harvested for LC-MS/MS-based quantification of TGs (**A**) and CEs (**B**). The relative abundance (Rel. Abundance) of each metabolite per 10\({}^{5}\) cells is shown. Data are mean \(\pm\) SD of three experiments. *\(p<0.05\), **\(p<0.01\), ***\(p<0.001\). be investigated. This study integrated metabolomic and transcriptomic data to study the 7 KCh-induced alteration of metabolic pathways in cardiac cells and represents the first one to delineate how an oxysterol induces lipid metabolic reprogramming and enhances CEs accumulation in cardiac cells. The enhanced production of cholesteryl esters is probably associated with SREBP- and PPAR\(\alpha\)-mediated upregulated expression of downstream genes, such as _Scat_ genes, in 7 KCh-treated cells. Decreased expression of genes involved in fatty acyl-CoA biosynthesis suggests a reduction in fatty acid synthesis. As the fatty acids synthesis declined, fatty acids required for esterification of 7 KCh were supplied through the catalytic action of phospholipase A2 (PLA2) in cardiomyocytes. The lysophospholipids were markedly higher in 7 KCh-treated cells than untreated cells, which is consistent with the enhanced expression of PLA2G12A (phospholipase A2, group XIIA). The proposed scheme is shown in Figure 6.  
Figure 6: A schematic diagram illustrating 7 KCh-induced remodeling of lipid metabolism in HL-1 cells. **(A)** 7 KCh inhibits TG biosynthesis and activates PLA2 to release fatty acid (FA) from phospholipids (e.g., PC). This provides FA for the esterification of Chol (yellow oval) to form CE (brown oval). 7 KCh inhibits cleavage and activation of SREBP. Decreased expression of _Srebf2_ and _Insig1_ genes may also reduce the transcriptional activity of SREBP. Inhibition of SREBP-mediated transcription reduces the expression of genes involved in the MVA pathway and biosynthesis. Additionally, 7 KCh may activate cell stress response that involves ATF4 and PERK signaling pathways (not shown here). Metabolites marked in blue (or red) represent those metabolites whose levels are lowered (or elevated) in 7 KCh-treated cells versus control cells. 7-Ketocholesterol, 7 KCh; cholesterol, Chol; cholesterol ester, CE; triglyceride, TG; phosphatidylinositol, PC; lysophosphatidylcholine, lysoC; glycerol-3-phosphate G3P; lysophosphatidate, LPA; phosphatidate, PA; diacylglycerol, DAG; fatty acid, FA; sterol regulatory element-binding transcription factor, SREBP; SREBP chaperone, SCAP; insulin-induced gene protein, Insig; site-1 protease, S1P; site-2 protease, S2P. **(B)** A significant difference in the expression of genes involved in lipid synthesis between 7 KCh-treated cells and control cells is shown. The fold change (\(\log_{2}\left(\frac{7\text{KCh}}{\text{Con}}\right)\)) is indicative of \(\log_{2}\left(\frac{\text{Expression units}}{\text{ in (FFKM) of the gene in 7 KCh-treated cells}}\right)\) for the specified gene (adjusted \(p\) value \(<0.05\), \(n=3\)). The accession number and descriptions of these genes are given in Supplementary Table S1. The genes are categorized according to their functions in cholesterol metabolism. It is noted that the _Insig_ gene (_Insig1_), _SREBF_ gene (_Srebf2_), and cholesterol influx-associated gene low density lipoprotein receptor (_Ldlr_) are downregulated in response to 7 KCh. The metabolism of 7 KCh is noteworthy. The extrahepatic metabolism of 7 KCh is mainly through its esterification to fatty acids by cytosolic SOAT and possibly through its subsequent selective efflux to HDL [8].