Role of Cholesterol-Associated Steatohepatitis in the Development of NASH

Abstract

The rising prevalence of nonalcoholic fatty liver disease (NAFLD) and NAFLD-related cirrhosis in the United States and globally highlights the need to better understand the mechanisms causing progression of hepatic steatosis to fibrosing steatohepatitis and cirrhosis in a small proportion of patients with NAFLD. Accumulating evidence suggests that lipotoxicity mediated by hepatic free cholesterol (FC) overload is a mechanistic driver for necroinflammation and fibrosis, characteristic of nonalcoholic steatohepatitis (NASH), in many animal models and also in some patients with NASH. Diet, lifestyle, obesity, key genetic polymorphisms, and hyperinsulinemia secondary to insulin resistance are pivotal drivers leading to aberrant cholesterol signaling, which leads to accumulation of FC within hepatocytes. FC overload in hepatocytes can lead to ER stress, mitochondrial dysfunction, development of toxic oxysterols, and cholesterol crystallization in lipid droplets, which in turn lead to hepatocyte apoptosis, necrosis, or pyroptosis. Activation of Kupffer cells and hepatic stellate cells by hepatocyte signaling and cholesterol loading contributes to this inflammation and leads to hepatic fibrosis. Cholesterol accumulation in hepatocytes can be readily prevented or reversed by statins. Observational studies suggest that use of statins in NASH not only decreases the substantially increased cardiovascular risk, but may ameliorate liver pathology. Conclusion: Hepatic FC loading may result in cholesterol-associated steatohepatitis and play an important role in the development and progression of NASH. Statins appear to provide significant benefit in preventing progression to NASH and NASH-cirrhosis. Randomized controlled trials are needed to demonstrate whether statins or statin/ezetimibe combination can effectively reverse steatohepatitis and liver fibrosis in patients with NASH.

FIG. 1

Model of the CASH hypothesis. In the CASH model, hepatic cholesterol accumulation is the main driver of cellular derangement, causing NASH in a subset of patients, whereas dietary, genetic, and lifestyle co‐factors either lead to the accumulation of hepatic cholesterol (yellow arrows) or interact which hepatic cholesterol to promote CASH (blue arrows). Abbreviations: FFA, free fatty acids; HSD17B13, 17β hydroxysteroid dehydrogenase 13; LIPA, lysosomal acid lipase; and TM6SF2, transmembrane 6 superfamily member 2.

FIG. 2

Cholesterol trafficking through the hepatocyte. (A) Cholesterol uptake and synthesis. Dietary cholesterol is absorbed in the jejunal mucosa through NPC1L1, incorporated into chylomicrons (CMs), and reaches the liver in CM remnants. CM remnants are taken up by the liver through interaction of the apoE protein on the CM remnant and LDLR on hepatocytes, which also binds to circulating LDL particles through interaction with apoB‐100 on the LDL surface. After binding to the LDLR, the complex undergoes receptor‐mediated endocytosis, processing through the late endosome/lysosome compartment, and transport into the metabolically active pool of cholesterol in the cytosol through NPC1. CE taken from HDL particles are selectively transported into the cytosol through SR‐B1, followed by hydrolysis through nCEH to join the metabolically active pool of cholesterol in the cytosol. Cholesterol can also be taken up from bile through NPC1L1 on the canalicular membrane of hepatocytes, when cells are deprived of cholesterol. Finally, cholesterol can also be synthesized de novo through the HMGCoAR, which is tightly regulated by SREBP‐2, the principal transcriptional activator of HMGCoAR. (B) Cholesterol secretion and excretion. Transport of cholesterol out of the cell is performed primarily through members of a superfamily of ABC transporters that use ATP to transport lipids across membranes. ABCA1 is a transmembrane protein present on the basolateral plasma membrane of hepatocytes that removes lipids from the cell membrane to an extracellular acceptor apolipoprotein ApoA‐I. ABCA1 interacts with lipid‐free apoA‐1 to generate nascent HDL particles, promoting cholesterol efflux from the cell. On the canalicular membrane of hepatocytes, ABCG5 and ABCG8 form a heterodimer that functions to excrete sterols into the bile. Cholesterol may also be secreted into the circulation in the form of VLDL particles. Finally, cholesterol may be converted to bile acids and excreted into bile through BSEP, an ABC transporter (ABCB11) located on the canalicular membrane of hepatocytes. In the classical pathway, the rate‐limiting step for cholesterol conversion into bile acid is the microsomal cytochrome P450 CYP7A1, which results in 7‐hydroxycholesterol; however, alternative pathways include the mitochondrial CYP27A enzyme and 25‐hydroxylase enzyme, forming 27‐hydroxycholesterol or 25‐hydroxycholesterol, respectively. (C) Hepatocyte LD. The LD membrane consists of a monolayer of phospholipids, and FC and is covered with proteins, including perilipins. The interior of the LD consists of triglycerides and CEs. When the concentration of FC within the LD membrane exceeds the saturation threshold, FC can precipitate as cholesterol crystals in the periphery of the LD. Abbreviations: apoA‐1, apolipoprotein A‐1; apoB‐100, apolipoprotein B‐100; apoE, apolipoprotein E; BA, bile acid; CM, chylomicron; CoA, coenzyme A; NPC1, Niemann‐Pick type C1.

FIG. 3

Regulation of cholesterol homeostasis: The nuclear receptors SREBP‐2 (black arrows), FXR (orange arrows), and LXR (blue arrows) are intimately involved in regulating cholesterol metabolism in a number of different mechanisms. The SREBP‐2/Scap complex senses cholesterol content in the ER, and when cholesterol levels are low, SREPB‐2 disassociates with Scap, travels to the Golgi apparatus where it is cleaved, and then promotes transcription of genes involved in cholesterol synthesis and uptake. FXR senses bile acids and triggers the transcription of SR‐B1 and ABCG5/8, but inhibits the activity of CYP7A1, preventing further bile acid formation. LXR binds to oxysterols in the cell, and then, after combining with retinoid X receptor, up‐regulates ABCA1, CYP7A1, and ABCG5/8 transcription, but down‐regulates LDLR transcription. Abbreviations: BA, bile acid; and RXR, retinoid X receptor.

FIG. 4

Mechanisms of organelle dysfunction in cholesterol overload. (A) Overview of organelle cholesterol loading. Cholesterol entering the hepatocyte through LDL particles binds to the LDLR receptors and undergoes receptor‐mediated endocytosis. That cholesterol is then trafficked through the late endosome and lysosome, and ultimately is transferred to different cellular organelles. NPC1 mediates transfer of cholesterol to lipid droplets, where it is stored; however, FC can form cholesterol crystals within the LDs. StAR/MLN64 transfers cholesterol from the lysosome to the mitochondria (or StAR can transfer cholesterol from the LD to the mitochondria), where it is typically used for synthesis of steroidogenic signaling molecules; however, it can also be deposited into the mitochondrial membrane and interfere with the function of 2‐Oxo. NPC1/2 mediates transfer of cholesterol from the lysosome to the ER, where high cholesterol membrane content causes disruption of the calcium pump SERCA, decreasing the concentration of calcium in the endoplasmic reticulum lumen. FC in the cell can react with ROS through CYP450 enzymes and form oxysterols, which increases nuclear NF‐κB signaling. (B) ER stress. Excess cholesterol in the ER leads to dysfunction of SERCA, lowers the luminal calcium concentration (stimulating the UPR), activation of NLRP3 inflammasome, and pyroptosis. (C) Mitochondrial dysfunction. Cholesterol loading in the mitochondrial interferes with 2‐Oxo function, which depletes the mitochondrial glutathione pool, resulting in ROS generation, lipid peroxidation, release of cytochrome C, and trigger of apoptosis. Excessive ROS generation for cholesterol overload leads to the generation of toxic oxysterols, which triggers inflammatory signaling through NF‐κB. (D) LD cholesterol crystallization and activation of inflammatory cells. Excessive FC in hepatocyte LDs leads to the formation of cholesterol crystal in the periphery of the LDs. LD cholesterol deposition results in activation of the NLRP3 inflammasome, which results in release of IL‐1β, causing pyroptosis or necrosis. Processing of these cholesterol crystals by activated KC in crown‐like structures causes release of proinflammatory signaling molecules, specifically IL‐1B, IL‐18, TGF‐β, and MCP1, which recruits immune cells to the liver and transforms HSCs into myofibroblasts. Myofibroblasts elaborate collagen, which deposits in the liver and leads to fibrosis and cirrhosis. (E) The TAZ Pathway. FC accumulated on the plasma membrane gets internalized by ASTER B/C, which activates sAC. Elevations in cAMP levels results in phosphorylation of IP3R through PKA and causes release of Ca from the ER lumen. Elevated cytosolic Ca levels activates RhoA, which inhibits LATS1/2 through phosphorylation. LATS1/2 is unable to phosphorylate TAZ, and the dephosphorylated TAZ (active form) translocates to the nucleus to induce transcription of Ihh. Ihh is secreted out of the hepatocyte and is then able to induce profibrotic mRNA in HSCs, resulting in hepatic fibrosis. Abbreviations: AMP, adenosine monophosphate; ATP, adenosine triphosphate; Ca, calcium; cAMP, cyclic adenosine monophosphate; Cyt C, cytochrome C; IP3R, inositol 1,4,5‐trisphosphate receptor; GSH, glutathione; LATS 1/2, large tumor suppressor 1/2; MCP1, monocyte chemoattractant protein‐1; MLN64, metastatic lymph node 64 protein; NPC1, Niemann‐Pick type C1; PKA, protein kinase A; PO^4‐, phosphate; RhoA, ras homolog family member A; sAC, soluble adenylyl cyclase; and StAR, steroidogenic acute regulatory protein.