Dietary cholesterol stiffens the steatotic liver

Introduction:: Hepatic steatosis is the accumulation of fat in the liver, which can progress to fibrosis and cirrhosis. The appearance of intracellular lipid aggregates precede other pathological changes in fatty liver diseases, and we have shown that simple fat accumulation, or steatosis, softens liver tissue in the absence of inflammation, fibrosis, and perfusion. However, fibrosis in the liver and other organs is often driven by stiffening. Non-alcoholic fatty liver disease (NAFLD) correlates significantly with high cholesterol and cholesterol can have modifying effects such as ordering of lipid aggregates in cells, leading us to hypothesize that the mechanical contribution of intrahepatic fat may be dependent on changes in the composition and structure of lipid aggregates.

We investigate the effects of dietary cholesterol on the whole organ and microenvironmental mechanics of fatty liver. Using rodent dietary models, we examine whether dietary cholesterol leads to altered mechanics in the steatotic liver at the macro or meso scale. We find that increased dietary cholesterol, unlike a simple high fat diet, results in a stiffer-than-normal steatotic liver. We further find that cholesterol in the lipid aggregates in the livers of high fat, high cholesterol-fed animals forms crystals. These results provide a potential cause for variability of progression in NAFLD, and may help inform the diagnosis and treatment of fatty liver diseases in their earliest phases.

Materials and Methods:: We fed rats either a high fat (HF), or high fat high cholesterol (HFHC) diet to investigate the effects of cholesterol on the fatty liver. All experiments were performed at 3, 6, and 9 weeks after the start of the selected diet. Lipid content, inflammation, and fibrosis were quantified by staining with BODIPY 493/503, anti-myeloperoxidase, and picrosirius red. Birefringent lipid crystals within the liver were visualized using polarized light microscopy.

Ex vivo parallel plate rheometry was used to measure whole liver mechanics without confounding from fluid pressure. Microindentation with modifications to demarcate measured regions for histological analysis was performed as described previously [1]. Briefly, demarcation with DAPI within a parafilm ring after the microindenter probe was fully indented results in a DAPI ring around the indented region. Liver samples were then stained with Oil Red O and visualized by whole mount epifluoresence microscopy, permitting quantification of lipid content within the indented regions. Confocal reflectance microscopy was performed on liver tissue samples to assess for reflectance indicative of changes in the structure of lipid aggregates.

Results, Conclusions, and Discussions:: We found that livers from HFHC rats were significantly stiffer than those from rats fed either HF or normal diets by shear rheometry, despite significant fat accumulation in both HF and HFHC. Staining for markers of fibrosis showed that stiffening precedes fibrosis. HFHC fatty rat livers contained crystalline aggregates exhibiting birefringence and reflectance under polarized light microscopy and confocal reflectance microscopy; these were not found in HF fatty or normal livers. Microindentation showed that local stiffness was increased in sub-millimeter regions with increased crystalline content.

The finding that a HFHC diet induced liver stiffening while a HF diet alone did not is potentially of great importance in NAFLD, in which progression of the disease is highly variable. The stiffening caused by the addition of dietary cholesterol could be a key contributor to disease progression, as tissue stiffening is a well-established source of profibrotic stimuli in other models of liver injury. Dietary cholesterol-induced liver stiffening may thus have implications in preventative care and developing therapeutics for NAFLD and other fatty liver diseases.

The birefringence observed in fatty livers with added dietary cholesterol is suggestive of a change towards greater order in the structure of lipid aggregates in the liver. Published work in cell lines shows that increasing the amount of cholesterol or cholesteryl esters causes intracellular lipid droplets to become more ordered and gain crystalline properties. These birefringent crystals may be responsible for the increase in cell and tissue stiffness we observe in HFHC livers.

In conclusion, we show that dietary cholesterol causes stiffening of the fatty liver at the macro and meso scales and that this stiffening is associated with the ordering of lipid aggregates into more crystalline structures. These findings may open new avenues into novel diagnostics, preventative care, and therapeutics for diseases of the fatty liver and of other tissues where cholesterol can accumulate.

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References (Optional): : [1] Li, D, Janmey, PA, Wells, RG. Local fat content determines global and local stiffness in livers with simple steatosis. FASEB BioAdvances. 2023; 00: 1- 11. doi:10.1096/fba.2022-00134