Liver lipid droplet cholesterol content is a key determinant of metabolic dysfunction-associated steatohepatitis.

Unlabelled: Metabolic dysfunction-associated steatohepatitis (MASH) represents a progressive form of steatotic liver disease which increases the risk for fibrosis and advanced liver disease. The accumulation of discrete species of bioactive lipids has been postulated to activate signaling pathways that promote inflammation and fibrosis. However, the key pathogenic lipid species is a matter of debate. We explored candidates using various dietary, molecular, and genetic models. Mice fed a choline-deficient L-amino acid-defined high-fat diet (CDAHFD) developed steatohepatitis and manifested early markers of liver fibrosis associated with increased cholesterol content in liver lipid droplets within 5 days without any changes in total liver cholesterol content. Treating mice with antisense oligonucleotides (ASOs) against or treatment with bempedoic acid or atorvastatin decreased liver lipid droplet cholesterol content and prevented CDAHFD-induced MASH and the fibrotic response. All these salutary effects were abrogated with dietary cholesterol supplementation. Analysis of human liver samples demonstrated that cholesterol in liver lipid droplets was increased in humans with MASH and liver fibrosis and was higher in PNPLA3 I148M (variants rs738409) than in HSD17B13 variants (rs72613567). Together, these data identify cholesterol in liver lipid droplets as a critical mediator of MASH and demonstrate that COASY knockdown and bempedoic acid are novel therapeutic approaches to reduce liver lipid droplet cholesterol content and thereby prevent the development of MASH and liver fibrosis.

Significance Statement: Metabolic dysfunction-associated steatohepatitis (MASH) is a progressive liver disease linked to fibrosis. The role of specific lipid species in its pathogenesis remains debated. Using dietary, molecular, and genetic models, we found that mice on a choline-deficient, high-fat diet (CDAHFD) developed steatohepatitis and early fibrosis, marked by increased cholesterol in liver lipid droplets within five days. Targeting COASY with antisense oligonucleotides or treating with bempedoic acid or atorvastatin reduced lipid droplet cholesterol and prevented MASH. However, dietary cholesterol supplementation negated these effects. Human liver samples confirmed elevated lipid droplet cholesterol in MASH and fibrosis, especially in PNPLA3 I148M carriers. These findings highlight cholesterol reduction as a potential MASH therapy.