Cholesterol-containing lipid crystals can directly stiffen the rat steatotic liver before fibrosis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is characterized by liver steatosis with cardiometabolic risk factors like dyslipidemia. Patients may progress from steatosis alone to complications such as fibrosis, end-stage liver disease, and hepatocellular carcinoma. The cause of progression is unclear. We previously showed that liver stiffening can drive fibrosis. However, the mechanical contributions of hepatic lipid and especially cholesterol accumulation are not known. We used rat dietary models to investigate how lipid accumulation affects liver mechanics. Liver stiffness was measured using rheology and magnetic resonance elastography, and associations between stiffness and lipid droplets (LDs) or cholesterol-containing lipid crystals were measured by microindentation-visualization. Polarized light, confocal reflection, and cryo-electron microscopy were employed to assess crystal abundance and structure. LDs and crystals extracted from livers were embedded in fibrous tissue mimics to isolate mechanical effects away from inflammation or fibrosis. Methyl-β-cyclodextrin perfusion was performed to assess whether cholesterol depletion reduced crystal abundance and tissue stiffness. Increased hepatic cholesterol storage led to the formation of cholesterol-containing lipid crystals in the liver. Steatotic livers with crystals stiffened before fibrosis while steatotic livers without crystals did not stiffen or fibrose. Lipid crystals stiffened tissue mimics while LDs did not, suggesting that crystals directly cause stiffening. Cholesterol depletion reduced crystal abundance and reverted tissue stiffness to near controls without changing inflammation, suggesting key roles for cholesterol in tissue stiffening. Lipid crystals cause profibrogenic liver stiffening, connecting high dietary cholesterol to MASLD progression, and may be a target for new diagnostic tools and therapeutics for progressive MASLD.