Discovery of a novel regulator, 3β-sulfate-5-cholestenoic acid, of lipid metabolism and inflammation.

Mitochondrial oxysterols, cholestenoic acid (CA), 25-hydroxycholesterol (25HC), and 27-hydroxycholesterol (27HC), are potent regulators involved in many important biological events. This study aimed to investigate the metabolic pathways of these oxysterols and their roles between hepatocytes and macrophages. LC-MS/MS analysis showed a novel regulatory molecule, 3β-sulfate-5-cholestenoic acid (3SCA), following the addition of CA in media culturing hepatocytes. Further study showed that 3SCA could also be derived from 27HC. In comparison, 25HC was converted to 25HC3S, which mostly remained in the cells and nuclei. The functional study showed that 3SCA significantly downregulated the expression of genes involved in lipid metabolism in hepatocytes and suppressed gene expression of proinflammatory cytokines induced by lipopolysaccharide in human macrophages. Based on the results, we conclude that 3SCA acts as a secretory regulator for the regulation of lipid metabolism and inflammatory responses in hepatocytes and macrophages. These findings shed light on understanding the unique metabolic pathways of these oxysterols and their possible roles in liver tissues.NEW & NOTEWORTHY This study identifies a novel oxysterol metabolite, 3β-sulfate-5-cholestenoic acid (3SCA), secreted by hepatocytes, which regulates lipid metabolism and inflammatory responses in hepatocytes and macrophages. These findings reveal previously unknown metabolic pathways of mitochondrial oxysterols and their roles in the progression and recovery of metabolic dysfunction-associated steatotic liver disease (MASLD), offering novel insights into potential therapeutic targets.