Background & Aims Type-2 diabetes and non-alcoholic steatohepatitis (NASH) are associated with insulin resistance and disordered cholesterol homeostasis. We investigated the basis for hepatic cholesterol accumulation with insulin resistance and its relevance to pathogenesis of NASH. Methods Alms1 mutant (foz/foz) and wild-type (WT) NOD.B10 mice were fed high-fat diets that contained varying percentages of cholesterol; hepatic lipid pools and pathways of cholesterol turnover were determined. Hepatocytes were exposed to insulin concentrations that circulate in diabetic foz/foz mice. Results Hepatic cholesterol accumulation was attributed to up-regulation of low density lipoprotein receptor (LDLR) via activation of sterol regulatory element binding protein-2 (SREBP-2), reduced biotransformation to bile acids, and suppression of canalicular pathways for cholesterol and bile acid excretion in bile. Exposing primary hepatocytes to concentrations of insulin that circulate in diabetic Alms1 mice replicated the increases in SREBP-2 and LDLR and suppression of bile salt export pump. Removing cholesterol from diet prevented hepatic accumulation of free cholesterol and NASH; increasing dietary cholesterol exacerbated hepatic accumulation of free cholesterol, hepatocyte injury or apoptosis, macrophage recruitment, and liver fibrosis. Conclusions In obese, diabetic mice, hyperinsulinemia alters nuclear transcriptional regulators of cholesterol homeostasis, leading to hepatic accumulation of free cholesterol; the resulting cytotoxicity mediates transition of steatosis to NASH.
The majority of patients with nonalcoholic fatty liver disease (NAFLD) have “simple steatosis,” which is defined by hepatic steatosis in the absence of substantial inflammation or fibrosis and is considered to be benign. However, 10%–30% of patients with NAFLD progress to fibrosing nonalcoholic steatohepatitis (NASH), which is characterized by varying degrees of hepatic inflammation and fibrosis, in addition to hepatic steatosis, and can lead to cirrhosis. The cause(s) of progression to fibrosing steatohepatitis are unclear. We aimed to test the relative contributions of dietary fat and dietary cholesterol and their interaction on the development of NASH. We assigned C57BL/6J mice to four diets for 30 weeks: control (4% fat and 0% cholesterol); high cholesterol (HC; 4% fat and 1% cholesterol); high fat (HF; 15% fat and 0% cholesterol); and high fat, high cholesterol (HFHC; 15% fat and 1% cholesterol). The HF and HC diets led to increased hepatic fat deposition with little inflammation and no fibrosis (i.e., simple hepatic steatosis). However, the HFHC diet led to significantly more profound hepatic steatosis, substantial inflammation, and perisinusoidal fibrosis (i.e., steatohepatitis), associated with adipose tissue inflammation and a reduction in plasma adiponectin levels. In addition, the HFHC diet led to other features of human NASH, including hypercholesterolemia and obesity. Hepatic and metabolic effects induced by dietary fat and cholesterol together were more than twice as great as the sum of the separate effects of each dietary component alone, demonstrating significant positive interaction. Conclusion Dietary fat and dietary cholesterol interact synergistically to induce the metabolic and hepatic features of NASH, whereas neither factor alone is sufficient to cause NASH in mice.
Gall-bladder epithelial cells (GBEC) are exposed to high concentrations of cholesterol in bile. Whereas cholesterol absorption by GBEC is established, the fate of this absorbed cholesterol is not known. The aim of this study was to determine whether ABCA1 (ATP-binding cassette transporter A1) mediates cholesterol efflux in GBEC. Polarized canine GBEC were cultured on porous membrane filters allowing separate access to apical (AP) and basolateral (BL) compartments. After AP loading of cells with model bile and [14C]cholesterol, cholesterol efflux was measured. Cholesterol loading together with 8-bromo-cAMP treatment, which increased ABCA1 expression, led to a significant increase in cholesterol efflux with apolipoprotein A-I (apoA-I) as the acceptor. Cholesterol efflux was observed predominantly into the BL compartment. Similar results were found for phospholipid efflux. Confocal immunofluorescence microscopy showed a predominantly BL ABCA1 localization. Interestingly, apoA-I added to either the AP or the BL compartments elicited BL lipid efflux with cAMP treatment. No paracellular or transcellular passage of 125I-apoA-I occurred. Ligands for the nuclear hormone receptors liver X receptor alpha (LXRalpha) and retinoid X receptor (RXR) elicited AP and BL cholesterol efflux, suggesting the involvement of both ABCA1- and non-ABCA1-mediated pathways. In summary, BL cholesterol/phospholipid efflux consistent with an ABCA1-mediated mechanism occurs in GBEC. This efflux pathway is stimulated by cAMP and by LXRalpha/RXR ligands, and in the case of the cAMP pathway appears to involve a role for biliary apoA-I.
Oxysterols are oxidation products of cholesterol that are generated by enzymatic reactions mediated by cytochrome P450 family enzymes or by non-enzymatic reactions involving reactive oxygen and nitrogen species. Oxysterols play various regulatory roles in normal cellular processes such as cholesterol homeostasis by acting as intermediates in cholesterol catabolism. Pathological effects of oxysterols have also been described, and various reports have implicated oxysterols in several disease states, including atherosclerosis, neurological disease, and cancer. Numerous studies show that oxysterols are associated with various types of cancer, including cancers of the colon, lung, skin, breast and bile ducts. The molecular mechanisms whereby oxysterols contribute to the initiation and progression of cancer are an area of active investigation. This review focuses on the current state of knowledge regarding the role of oxysterols in carcinogenesis. Mutagenicity of oxysterols has been described in both nuclear and mitochondrial DNA. Certain oxysterols such as cholesterol-epoxide and cholestanetriol have been shown to be mutagenic and genotoxic. Oxysterols possess pro-oxidative and pro-inflammatory properties that can contribute to carcinogenesis. Oxysterols can induce the production of inflammatory cytokines such as interleukin-8 and interleukin-1β. Certain oxysterols are also involved in the induction of cyclo-oxygenase-2 expression. Inflammatory effects can also be mediated through the activation of liver-X-receptor, a nuclear receptor for oxysterols. Thus, several distinct molecular mechanisms have been described showing that oxysterols contribute to the initiation and progression of cancers arising in various organ systems.
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.
A mechanistic model of mucous granule exocytosis by columnar epithelial cells must take into account the unique physical-chemical properties of mucin glycoproteins and the resultant mucus gel. In particular, any model must explain the intracellular packaging and the kinetics of release of these large, heavily charged species. We studied mucous granule exocytosis in gallbladder epithelium, a model system for mucus secretion by columnar epithelial cells. Mucous granules released mucus by merocrine exocytosis in mouse gallbladder epithelium when examined by transmission electron microscopy. Spherules of secreted mucus larger than intracellular granules were noted on scanning electron microscopy. Electron probe microanalysis demonstrated increased calcium concentrations within mucous granules. Immunofluorescence microscopic studies revealed intracellular colocalization of mucins and the cystic fibrosis transmembrane conductance regulator (CFTR). Confocal laser immunofluorescence microscopy confirmed colocalization. These observations suggest that calcium in mucous secretory granules provides cationic shielding to keep mucus tightly packed. The data also suggests CFTR chloride channels are present in granule membranes. These observations support a model in which influx of chloride ions into the granule disrupts cationic shielding, leading to rapid swelling, exocytosis and hydration of mucus. Such a model explains the physical-chemical mechanisms involved in mucous granule exocytosis.
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