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Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

ABSTRACT: We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ?0.5%, whereas mice on lower-cholesterol diets developed only simple steatosis. Hepatic cholesterol crystals and crown-like structures also developed at a dietary cholesterol concentration ?0.5%. Crown-like structures consisted of activated Kupffer cells (KCs) staining positive for NLRP3 and activated caspase 1, which surrounded and processed cholesterol crystal-containing remnant LDs of dead hepatocytes. The KCs processed LDs at the center of crown-like structures in the extracellular space by lysosomal enzymes, ultimately transforming into lipid-laden foam cells. When HepG2 cells were exposed to LDL cholesterol, they developed cholesterol crystals in LD membranes, which caused activation of THP1 cells (macrophages) grown in coculture; upregulation of TNF-alpha, NLRP3, and interleukin 1beta (IL1?) mRNA; and secretion of IL-1beta. In conclusion, cholesterol crystals form on the LD membrane of hepatocytes and cause activation and cholesterol loading of KCs that surround and process these LDs by lysosomal enzymes.

SUBMITTER: Ioannou GN

PROVIDER: S-EPMC5456359 | biostudies-literature | 2017 Jun

REPOSITORIES: biostudies-literature

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Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

Ioannou George N GN Subramanian Savitha S Chait Alan A Haigh W Geoffrey WG Yeh Matthew M MM Farrell Geoffrey C GC Lee Sum P SP Savard Christopher C

Journal of lipid research 20170412 6

We recently reported that cholesterol crystals form in hepatocyte lipid droplets (LDs) in human and experimental nonalcoholic steatohepatitis. Herein, we assigned WT C57BL/6J mice to a high-fat (15%) diet for 6 months, supplemented with 0%, 0.25%, 0.5%, 0.75%, or 1% dietary cholesterol. Increasing dietary cholesterol led to cholesterol loading of the liver, but not of adipose tissue, resulting in fibrosing steatohepatitis at a dietary cholesterol concentration of ≥0.5%, whereas mice on lower-cho ...[more]

PMID: 28404639

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Project description:It is unclear what drives the development of fibrosing nonalcoholic steatohepatitis (NASH). We aimed to determine whether cholesterol crystallization within hepatocyte lipid droplets (LDs) distinguishes patients with fibrosing NASH from patients with isolated hepatic steatosis and to study pathways leading to cholesterol accumulation in hepatocyte LDs. Patients with fibrosing NASH (n = 16) were compared to patients with isolated steatosis (n = 14). Almost all patients with fibrosing NASH had free cholesterol staining by filipin (16/16) and cholesterol crystals (15/16) in hepatocyte LDs, mostly in association with the LD membrane, compared to only 3/14 with cholesterol crystals and 3/14 with faint filipin staining in patients with isolated steatosis (P < 0.05). We were unable to identify significant differences in the expression of genes in liver tissue related to cholesterol homeostasis or LD proteins between patients with fibrosing NASH and isolated steatosis. Human hepatoma cell line (HepG2) cells were supplemented with low-density lipoprotein (LDL)-cholesterol and oleic acid to develop large LDs, similar to those observed in patients with NASH. Fluorescent markers were used to track the uptake and intracellular trafficking of LDL-cholesterol. LDL-cholesterol was taken up by HepG2 cells and transported through the endosomal-lysosomal compartment directly to LDs, suggesting direct contact sites between late endosomes and LDs. Exposure of HepG2 cells to LDL-cholesterol resulted in a high concentration of cholesterol and cholesterol crystallization in LDs. Conclusion: Excess cholesterol is stored in the liver primarily within hepatocyte LDs where it can crystallize. Our findings are best explained by direct transport of cholesterol from late endosomes/lysosomes to LDs in hepatocytes. We found a strong association between the presence of LD cholesterol crystals and the development of fibrosing NASH in humans, suggesting a causal relationship.

Project description:Background & aimsInherited abnormalities in apolipoprotein E (ApoE) or low-density lipoprotein receptor (LDLR) function result in early onset cardiovascular disease and death. Currently, the only curative therapy available is liver transplantation. Hepatocyte transplantation is a potential alternative; however, physiological levels of hepatocyte engraftment and repopulation require transplanted cells to have a competitive proliferative advantage of over host hepatocytes. Herein, we aimed to test the efficacy and safety of a novel preparative regimen for hepatocyte transplantation.MethodsHerein, we used an ApoE-deficient mouse model to test the efficacy of a new regimen for hepatocyte transplantation. We used image-guided external-beam hepatic irradiation targeting the median and right lobes of the liver to enhance cell transplant engraftment. This was combined with administration of the hepatic mitogen GC-1, a thyroid hormone receptor-β agonist mimetic, which was used to promote repopulation.ResultsThe non-invasive preparative regimen of hepatic irradiation and GC-1 was well-tolerated in ApoE-/- mice. This regimen led to robust liver repopulation by transplanted hepatocytes, which was associated with significant reductions in serum cholesterol levels after transplantation. Additionally, in mice receiving this regimen, ApoE was detected in the circulation 4 weeks after treatment and did not induce an immunological response. Importantly, the normalization of serum cholesterol prevented the formation of atherosclerotic plaques in this model.ConclusionsSignificant hepatic repopulation and the cure of dyslipidemia in this model, using a novel and well-tolerated preparative regimen, demonstrate the clinical potential of applying this method to the treatment of inherited metabolic diseases of the liver.Lay summaryHepatocyte transplantation is a promising alternative to liver transplantation for the treatment of liver diseases. However, it is inefficient, as restricted growth of transplanted cells in the liver limits its therapeutic benefits. Preparative treatments improve the efficiency of this procedure, but no clinically-feasible options are currently available. In this study we develop a novel well-tolerated preparative treatment to improve growth of cells in the liver and then demonstrate that this treatment completely cures an inherited lipid disorder in a mouse model.

Dataset Information

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

Publications

Cholesterol crystallization within hepatocyte lipid droplets and its role in murine NASH.

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