An overview of the cholesterol metabolism and its proinflammatory role in the development of MASLD

Abstract

Cholesterol is an essential lipid molecule in mammalian cells. It is not only involved in the formation of cell membranes but also serves as a raw material for the synthesis of bile acids, vitamin D, and steroid hormones. Additionally, it acts as a covalent modifier of proteins and plays a crucial role in numerous life processes. Generally, the metabolic processes of cholesterol absorption, synthesis, conversion, and efflux are strictly regulated. Excessive accumulation of cholesterol in the body is a risk factor for metabolic diseases such as cardiovascular disease, type 2 diabetes, and metabolic dysfunction–associated steatotic liver disease (MASLD). In this review, we first provide an overview of the discovery of cholesterol and the fundamental process of cholesterol metabolism. We then summarize the relationship between dietary cholesterol intake and the risk of developing MASLD, and also the animal models of MASLD specifically established with a cholesterol-containing diet. In the end, the role of cholesterol-induced inflammation in the initiation and development of MASLD is discussed.

INTRODUCTION

Cholesterol, also known as cholesterine, is a vital cyclopentane polyhydrophene derivative found in eukaryotic cells. It is typically lipid-soluble, meaning that it is insoluble in water but easily soluble in organic solvents. In 1769, François Poulletier de la Salle discovered and isolated cholesterol from the alcohol-soluble part of human gallstones.¹ However, it was not until 1815 when Michel Eugène Chevreul, known as the father of lipid chemistry, rediscovered this compound and named it cholesterine, which means solid bile in Greek (the chole for bile and the stereos for solid).^2,3 Nowadays, the frequently used word cholesterol consists of chole (bile) and stereos (solid), followed by the chemical suffix -ol indicating alcohol. Cholesterol has been studied for centuries due to its association with diseases. Scientists have made unremitting efforts to explore the chemical composition, structure, physiological function, and metabolism of cholesterol and have made many significant breakthroughs so far (Figure 1A, B); for example, more than 10 Nobel Prize works are directly or indirectly related to cholesterol and lipids (Figure 1C).

FIGURE 1.

Timeline of important events and promising discoveries in cholesterol research. (A) Timeline of advances in the research history of cholesterol. (B) The number of publications published each year using the following search query in a January 2024 search of PubMed: “cholesterol” or “cholesterol and fatty liver.” (C) Nobel Prizes in the research field of cholesterol and lipids. Abbreviations: CAD, coronary artery disease; FDA, Food and Drug Administration; FH, familial hypercholesterolemia; HMG, 3-hydroxy-3-methylglutaryl-CoA; SREBP, sterol regulatory element-binding protein.

Cholesterol is a compound that is commonly found in animal tissues, with the highest concentrations found in the brain and nerve tissues. It is also present in significant amounts in the liver, kidneys, and skin. Cholesterol is composed of a hydrophobic tail and a hydrophilic hydroxyl head. It has the basic structure of a cyclopentane polyhydrophenanthrene. The unique structure of cholesterol gives it a range of important physiological functions. First, it is integrated into the phospholipid bilayer membrane to form the lipid rafts and thus becomes a crucial component of the cell membrane, regulating membrane fluidity and phase transition.^4–6 Second, cholesterol can also serve as the synthetic precursor for bioactive molecules such as bile acids, steroid hormones, and lipid-soluble vitamins.^7,8 Third, cholesterol plays a role in important signal transduction pathways through protein modification. Studies have found that cholesterol can modify proteins in the Hedgehog signaling pathway and thus plays an important role in embryonic development and cell proliferation.^9,10 In addition, cholesterol is essential for the formation of synapses and myelin sheaths in the nervous system. Moreover, cholesterol can also interact with many sterol transport proteins to promote cholesterol transport and regulate its subcellular distribution.^11,12 Overall, cholesterol is essential for the survival and functioning of the cells and the body. However, there is ample evidence from clinical, laboratory, and epidemiological studies that excessive cholesterol can lead to a variety of severe diseases, including atherosclerosis, gallstones, metabolic dysfunction–associated steatotic liver disease (MASLD), cardiovascular disease, type 2 diabetes, cancer, and Alzheimer disease.^5,13–15 In addition, abnormal cholesterol transport within cells can also result in neurodegenerative disorders, such as Niemann-Pick type C (NPC).^16,17

Since the discovery of the association between cholesterol and arteriosclerosis, it has been recognized that cholesterol’s pathogenicity is linked to the inflammatory response it triggers.¹⁸ The activation of macrophages induced by cholesterol is a hallmark of inflammation, and this activation exacerbates diseases associated with chronic metabolic inflammation, such as atherosclerosis, metabolic dysfunction–associated steatohepatitis (MASH), and obesity.¹⁹ For that matter, chronic tissue inflammation has emerged as a key feature of cholesterol accumulation. Therefore, in the event of a disease, macrophage activation serves as the primary driving force that initiates chronic inflammation, while other types of immune cells participate in the subsequent inflammation and progression of the disease.

In this review, we examine recent advances and the underlying mechanisms of cholesterol and related inflammation in MASLD. First, we provide an overview of the fundamental process of cholesterol metabolism and its involvement in the development of MASLD, along with an introduction of the cholesterol-induced animal model used to study MASLD. Second, we also summarize the findings related to liver inflammation and MASLD, in which we particularly focus on the functions of different inflammatory cells, such as macrophages, CD4+ T cells, CD8+ T cells, in the development of cholesterol-induced MASLD.

CHOLESTEROL METABOLISM

Due to the significance and potential harm of cholesterol, the body requires a strict and precise regulatory mechanism to maintain the balance of cholesterol metabolism. At the organism level, the body pool of cholesterol is maintained in the balance between input and output. Input includes dietary sources and de novo synthesis, while output includes fecal excretion in the form of biliary cholesterol and bile acids (Figure 2). Generally, the human body needs about 1 g of cholesterol per day, of which about 600–900 mg comes from endogenous biosynthesis and about 300–500 mg is absorbed from diet.²⁰ The contribution of endogenous biosynthesis versus dietary absorption has been estimated as a ratio of 7:3.²¹ The mechanism for maintaining cholesterol homeostasis is very complex. In general, it can be regulated in three ways: through de novo synthesis, exogenous absorption and transportation, and transformation and efflux.²²

FIGURE 2.

Cholesterol homeostasis in human and mouse. The body pool of cholesterol in adult male human (without parentheses) and adult male mouse (with parentheses) are maintained by a dynamic balance between cholesterol input and output. Abbreviation: BW, body weight.

De novo synthesis

Cholesterol is the only sterol that is biosynthesized by animals. All living cells have the ability to synthesize cholesterol internally. However, the majority of biosynthesis takes place in the liver (about 50%) and intestine (about 24%).²⁰ At the organelle level, cholesterol synthesis occurs in the endoplasmic reticulum (ER), where the 3-hydroxy-3-methylglutaryl CoA reductase (HMGCR) and squalene monooxygenase are rate-limiting enzymes.²² HMGCR is a glycoprotein localized in the ER that catalyzes the reduction of 3-hydroxy-3-methylglutaryl-CoA to mevalonate. Downstream of HMGCR, squalene monooxygenase, encoded by SQLE, plays a role in converting nonsteroidal intermediate squalene to 2,3-oxidosqualene.^22,23

The synthesis of cholesterol requires several components, including acetyl-CoA, ATP, oxygen, and NADPH. This process involves >20 enzymes and nearly 30 enzymatic reactions, which begin with acetyl-CoA and can be divided into 3 stages^22,24,25 (Figure 3). The first stage involves the synthesis of isopentenyl pyrophosphate from acetyl-CoA, which is called the mevalonate pathway. The second stage involves the transformation of isopentenyl pyrophosphate into squalene. The third stage involves the conversion of squalene into cholesterol, which is a complex process and undergoes >20 steps.

FIGURE 3.

An overview of cholesterol biosynthesis pathway. The dark colors represent a schematic representation of the three stages of cholesterol biosynthesis. Abbreviations: FDPS, farnesyl diphosphate synthase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; MDD, mevalonate diphosphate decarboxylase; PMK, phosphomevalonate kinase; MK, mevalonate kinase; OSC, oxidosqualene cyclase; SQS, squalene synthase.

The regulation of cholesterol biosynthesis in the liver involves 2 primary pathways: transcriptional regulation, which is mediated by the family of transcription factors known as sterol regulatory element-binding proteins, and post-transcriptional negative feedback regulation, which occurs through degradation of HMGCR. The relevant details have been well summarized in the published review articles.^22,24

Exogenous absorption and transportation

In addition to endogenous biosynthesis, cholesterol can also be obtained through absorption in the intestine. Cholesterol in the intestine primarily originates from food and bile. Some animal-derived foods, such as organ meats, shrimp, dairy products, and egg yolks, are known to be high in cholesterol. Over 85% of the cholesterol in these foods is present in the nonesterified form, while the remaining amount exists as cholesterol esters.²⁶ Cholesterol esters need to undergo enzymatic hydrolysis to become free cholesterol in the digestive tract before they can be absorbed in the small intestine. Since cholesterol is highly insoluble in water, it must first combine with bile acids to form negatively charged mixed micelles. These micelles can then diffuse to the surface of enterocytes, allowing for absorption.²⁶

NPC1L1, a 13-transmembrane protein that is highly expressed in the small intestine, was identified as the protein that can specifically and efficiently facilitate cholesterol absorption by Altmann et al.²⁷ The N-terminal of the protein is situated outside the cell membrane, facing the intestinal lumen, while the C-terminal is located inside the cell. The 3–7 transmembrane segments constitute the sterol sensing domain.²⁸ In humans and other primates, NPC1L1 is also expressed on the canalicular membrane of the hepatocyte, where it is responsible for reabsorbing cholesterol that is secreted into the bile by the hepatocyte.²⁹

To understand the mechanisms of NPC1L1 mediate cholesterol absorption, the Baoliang Song team conducted a series of significant studies. They found that NPC1L1 has 2 functional domains, one of which has a conserved YVNxxF (where x stands for any amino acid) endocytosis signal at the C-terminus. When the cholesterol content on the plasma membrane is low, the YVNxxF of the NPC1L1 protein binds to the plasma membrane and cannot initiate endocytosis. When cholesterol flows in the intestinal lumen, the N-terminus domain of the NPC1L1 protein can specifically bind to cholesterol.³⁰ At the same time, the proteins Flotillin-1 and Flotillin-2, which are constituents of lipid rafts on the plasma membrane, bind to NPC1L1 and help NPC1L1 to create a microdomain rich in cholesterol around it.³¹ Local high cholesterol (HC) causes conformational changes in the NPC1L1 protein, leading to the dissociation of the C-terminal YVNxxF sequence from the plasma membrane. The exposed cytoplasmic YVNxxF endocytic signal is recognized by the adaptor protein Numb, which further recruits clathrin/AP2 to assemble and form an endocytic complex, initiating vesicular endocytosis.^32,33

Although it has been suggested that endocytosis mediated by NPC1L1 may be the primary pathway for cholesterol entry into the cell, the mechanism by which cholesterol is transported from the cell membrane to the ER after entry has remained a longstanding question. A recent study³⁴ by Peter Tontonoz team shows that when cholesterol is transported to the cell membrane by NPC1L1, elevated cholesterol levels recruit Aster proteins (Aster-B and Aster-C) to the cell membrane. These proteins then transport cholesterol from the cell membrane to the ER using a nonvesicular transport pathway for further processing (Figure 4).

FIGURE 4.

Cholesterol and lipoprotein metabolism. Abbreviations: ABCA1, ATP-binding cassette transporter A1; ABCG5/G8, ATP-binding cassette transporter G5/G8; ACAT1/2, acetyl-coA acetyltransferase ½; ASBT, apical sodium-dependent bile acid transporter; BA, bile acid; BSEP, bile salt export pump; CE, cholesterol ester; CM, chylomicrons; CMr, chylomicron remnants; CYP27A1, cytochrome P450 27A1; CYP7A1, cytochrome P450 7A1; FC, free cholesterol; HMGCR, 3-hydroxy-3-methylglutaryl-CoA reductase; LD, lipid droplets; LDLR, Low density lipoprotein receptor; LPL, lipoprotein lipase; LRP1, low-density lipoprotein receptor-related protein-1; MTTP, microsomal triglyceride transfer protein; NPC1L1, Niemann-pick C1 Like 1; SM, squalene monooxygenase; SR-B1, scavenger receptor B1.

In addition to NPC1L1, CD36 has also been demonstrated to play a direct role in cholesterol uptake in the small intestine. In 2006, Nauli et al³⁵ discovered that Cd36 knockout mice retained more cholesterol in the intestinal lumen and exhibited a 50% reduction in cholesterol output into the lymph. They speculated that CD36 may mediate cholesterol uptake in the proximal intestine. In 2007, by using a micellar solution of ¹⁴C labeled cholesterol, Nassir et al³⁶ found that cholesterol uptake was reduced by more than 60% in enterocytes isolated from the proximal small intestines of Cd36 knockout mice compared to wild type mice. This suggests that CD36 plays a role in cholesterol absorption in the proximal intestine.

After cholesterol is transported into the enterocytes, the majority of it is re-esterified. The only known cholesterol esterase is acyl-CoA:cholesterol acyltransferase (ACAT), which catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acids. ACAT protein is located in the ER. ACAT1 is expressed in various tissues and cells, while ACAT2 is specifically expressed in the small intestine and liver cells. Inside the enterocytes, when cholesterol is transported to the ER, ACAT2 catalyzes the formation of cholesterol to cholesterol ester (CE).³⁷ Under the action of apolipoproteins B (ApoB) and microsomal triglyceride transfer protein (MTTP), these CEs are assembled together with triglycerides, phospholipids, and a small amount of free cholesterol to form chylomicrons (Figure 4). The chylomicrons are then secreted into the circulatory system through the basement membrane of enterocytes.³⁸

Cholesterol can be absorbed and utilized by the relevant tissues once it enters the circulatory system. In hepatic tissue, cholesterol in chylomicrons is absorbed by hepatocytes through the low-density lipoprotein receptor-related protein pathway, while the absorption of cholesterol in LDL and HDL primarily relies on 2 pathways: the endocytosis pathway facilitated by the LDL receptor and the selective uptake pathway facilitated by the scavenger receptor class B I (SR-BI)^39–41 (Figure 4). In addition, numerous studies have demonstrated that CD36, a class B scavenger receptor, and scavenger receptor A (SR-A) can facilitate the uptake of exogenous cholesterol in specific cell types, such as macrophages or KCs.^42–46

Low-density lipoprotein receptor recognizes lipoproteins containing ApoB100 or apolipoproteins E. LDL in the blood binds to the ligand-binding domain of the extracellular domain of LDLR, which leads to the exposure of the NPxY endocytic motif (where x stands for any amino acid) in the intracellular region.^47,48 This motif then binds to the endocytic adapters, autosomal recessive hypercholesterolemia, and disabled homolog 2 directly, recruiting clathrin and AP2 to form endocytic vesicles to initiate the endocytosis.^49–52 Once endocytic vesicles are transported to lysosomes, LDL is degraded and separated from the LDLR. LDLR subsequently returns to the surface of the cell membrane for reuse, while CE is hydrolyzed into free cholesterol. The free cholesterol is then transferred to the ER through lysosome-peroxisome-ER membrane contact for re-esterification^22,53 or transported to other organelles, such as mitochondria and cell membranes.⁵⁴ Downregulation of LDLR expression makes lipids to accumulate in the blood, leading to hypercholesterolemia. The endocytosis of LDL and the feedback regulation of endogenous cholesterol synthesis in hepatocytes mediated by LDLR are key factors in the regulation of LDL levels in the blood.⁵³

SR-BI is a multifunctional membrane protein receptor that is expressed in the liver, intestinal tract, adrenal, gonad, and vascular cells (such as macrophages and endothelial cells).^55,56 Acton et al⁴⁰ demonstrated that SR-BI mediates the uptake of cholesterol esters from HDL by hepatocytes with high affinity. In contrast to the endocytosis of LDL cholesterol mediated by the LDL receptor, hepatic SR-BI selectively uptakes CE from circulating HDL without degrading the entire lipoprotein particle. First, the HDL binds to the extracellular loop domain of SR-BI using the amphiphilic α-helix domain. After binding, SR-BI promotes the transfer of CE from lipoprotein particles into the cell. Finally, the CE-depleted HDL is released into the blood.^55,57–59 Papale et al⁶⁰ discovered that the hydrophobicity of N-terminal extracellular regions encompassing V67, L140/L142, V164, or V221 of SR-BI is critical for the uptake of HDL cholesterol in an evolutionarily conserved manner. Although the mechanism by which SR-BI promotes the transfer of CE into the cell is not completely clear, a well-accepted model suggests that HDL particles form hydrophobic channels with the extracellular domain of the cell membrane SR-BI, subsequently, CE diffuses in a concentration gradient manner.^59–61

In the small intestine and liver, the ATP-binding cassette transporter A1 (ABCA1) is responsible for transporting intracellular cholesterol and phospholipids to apoA-I to form nascent HDL. Once formed, through the action of lecithin:cholesterol acyltransferase, cholesterol ester transfer protein, and phospholipid transfer protein, the nascent HDL acquires additional cholesterol from extrahepatic tissues and matures into HDL.⁶² This mature HDL is more readily absorbed by the liver via SR-BI.

Transformation and efflux

Lacking certain enzymes capable of breaking down the steroid nucleus, animal cells are limited in their ability to degrade cholesterol into carbon dioxide and water in a step-by-step manner. Instead, cholesterol is converted into other compounds through oxidation and reduction. Excess cholesterol can be converted to CE by ACAT1/2 and stored in lipid droplets. It can also generate oxysterols through enzymatic or nonenzymatic conversion. It can be converted into bile acids, vitamin D, and steroid hormones.⁶³ In addition, excess cholesterol in enterocytes can be directly excreted into the intestinal lumen, while in hepatocytes, it can be excreted into the bile. Moreover, it can be excreted into HDL in both enterocytes and hepatocytes (Figure 4) and then participate in the reverse cholesterol transport to the liver.^41,64

In the liver, free cholesterol can be metabolized either directly into the bile duct or by converting it into the more water-soluble bile acids. The body excretes about 1.2 g of cholesterol per day, half of which is in the form of bile acids. Cholesterol 7α-hydroxylase is a cytochrome P450 enzyme localized in the ER of hepatocytes, while sterol 27-hydroxylase (CYP27A1) is expressed in mitochondria. During bile acid synthesis, these 2 enzymes perform the initial and rate-limiting step in the classical pathway and the hydroxylation of cholesterol in the alternative pathway, respectively.^65,66 The expression and activity of these enzymes are inhibited by feedback from the final product bile acids. Meanwhile, they are stimulated by the substrate cholesterol. In humans, dietary cholesterol induces bile acid synthesis through the CYP27A1-mediated alternative pathway.⁶⁷ In mouse and rat models, a HC diet stimulates the transcription of the cholesterol 7α-hydroxylase gene, leading to increased bile acid synthesis.^68–70 Furthermore, cholesterol can also regulate bile acid metabolism by altering gut microbiota. For example, research has shown that a HC diet can significantly increase the abundance of Bacteroides, Clostridium, and Lactobacillus, consequently raising the levels of hydrophobic unconjugated bile acids cholic acid, deoxycholic acid, muricholic acid and chenodeoxycholic acid in mice.⁷¹

Secreted into the blood as a major component of lipoproteins is an important pathway for hepatic cholesterol to entry into the bloodstream. Endogenously synthesized and exogenously obtained cholesterol can finally be packaged by the liver into VLDL and then transported into the bloodstream in the form of secretory vesicles. This process requires the participation of other proteins, such as ApoB100 and MTTP (Figure 4). Among them, MTTP plays a crucial role in the assembly and secretion of VLDL in the liver. Deficiency of MTTP leads to beta-lipoproteinemia, a rare genetic disease characterized by reduced levels of ApoB-containing lipoproteins in plasma and symptoms of fatty liver.³⁸ Inhibition of MTTP expression is currently being used to treat homozygous familial hypercholesterolemia, which helps reduce levels of ApoB-containing lipoproteins, such as LDL-C, in these patients.^72,73

The efflux of cholesterol in the form of free cholesterol into the blood and bile is another essential mechanism for maintaining hepatic cholesterol homeostasis. ABCA1 in hepatocytes and ABCG1 in macrophages are key proteins that mediate the export of cholesterol from the liver. Patients with Tangier disease have plasma HDL levels that are <5% of normal due to the absence of ABCA1 in vivo, and their triglyceride levels are significantly elevated.⁷⁴ Liver-specific ABCA1 knockout mice exhibit a lipoprotein phenotype similar to Tangier disease subjects, with only 20% of normal plasma HDL levels.⁷⁵ Evidence has revealed that hepatic ABCA1 is located on the basolateral surface and is associated with endocytic vesicles.⁷⁶ In addition, ABCG5 and ABCG8, which are exclusively expressed in hepatocytes and enterocytes, also belong to the ABC transporter superfamily, and they combine to form heterodimers that mediate the efflux of intracellular cholesterol into the intrahepatic bile duct or into the intestinal lumen in hepatocytes and enterocytes, respectively (Figure 4). Silencing of the ABCG5/G8 gene in mice results in a decrease in biliary cholesterol and an increase in plasma and hepatic cholesterol levels.⁷⁷ However, the specific mechanism by which ABCG5 and ABCG8 recognize and export cholesterol has not been clearly clarified.⁷⁸

The liver X receptors (LXRs), namely LXRα and LXRβ, are transcription factors that regulate the efflux of cholesterol. They function as whole-body cholesterol sensors and regulate the homeostasis of cholesterol metabolism by controlling cholesterol export, bile acid production, fatty acid synthesis, and the expression of lipid transporters.⁷⁹ They can activate the expression of genes involved in cholesterol efflux, including ABCA1, ABCG5/G8, and cholesterol 7α-hydroxylase. When the level of cholesterol becomes too high in intestinal cells, LXRs upregulate ABCG5/G8, which are located at the brush border membrane of intestinal cells. This upregulation facilitates the removal of excess cholesterol into the intestinal lumen.⁸⁰ The mechanisms by which the LXR regulates cholesterol homeostasis have been well summarized in previously published review articles.^81,82

DIETARY CHOLESTEROL INTAKE AND HUMAN MASLD

The histological spectrum of liver damage, ranging from steatosis to steatohepatitis, can occur in individuals who do not consume excessive amounts of alcohol (<30 g/d in men and <20 g/d in women). This condition was previously termed NAFLD.⁸³ NAFLD is a cluster of conditions associated with metabolic dysfunction. To describe these conditions more accurately, a new terminology referred to as metabolic dysfunction–associated steatotic liver disease (MASLD) was introduced to replace NAFLD.⁸⁴ Since the 1980s, the prevalence of MASLD has increased dramatically worldwide, making it a significant health concern for the global population in the 21st century. According to recent reports, MASLD affects more than 30% of adults worldwide.⁸⁵ MASLD is a progressive disease that encompasses a range of related disorders, such as simple steatosis, MASH, liver fibrosis, and potentially cirrhosis and HCC.^86,87 Generally, simple steatosis is defined as the presence of cytoplasmic lipid droplets in >5% of hepatocytes.^86,87 The further development of simple steatosis results in MASH, which is characterized by the development of necroinflammation and hepatocyte damage and progresses to later stages.⁸³ The development of MASLD is influenced by various factors, including diet, metabolism, genetics, and gut microbes. Among these factors, diet is considered an important causative factor.^88–91

In the past decades, rapid economic growth, globalization, and urbanization have resulted in a significant shift in the dietary habits of people worldwide. Specifically, there has been an increase in the consumption of meats and eggs, while the intake of fruits, vegetables, and whole grains has decreased.⁹² For example, in China, from 1992 to 2012, the proportion of energy provided by fat in the diet of Chinese residents increased from 16.0% to 32.3%, the intake of red meat increased from 42.2 g/d to 64.4 g/d, while the intake of eggs increased from 13.5 to 23.5 g/day.⁹³ Meats and eggs, in addition to being rich in protein, are also high in cholesterol and are major contributors to dietary cholesterol.⁹⁴ It has been demonstrated that the yolk of a 65 g egg contains 237 mg of cholesterol,⁹² 100 g of cooked mutton contains 130 mg of cholesterol.⁹⁵ Additionally, organ meats, such as beef liver and lamb liver, have a more higher cholesterol content. For instance, 100 g beef liver contains 271 mg of cholesterol, while 100 g lamb liver contains 433 mg of cholesterol.⁹⁵ Despite the nutritional benefits of more consumption of meats and eggs, there are concerns about their high content of cholesterol and their impact on metabolic disorders. It has long been believed that excessive intake of HC diets, such as eggs, red meats, and processed meats, is linked to a higher risk of developing type 2 diabetes,^96,97 cardiovascular disease, and cancer.^98–100 However, increased cholesterol intake also appears to be one of the main causes of the rising prevalence of MASLD.¹⁰¹ Investigation of the dietary records found that cholesterol intake was significantly higher in patients with MASLD, including patients with MASH, than in healthy controls.^102,103 For example, based on a study of 73 subjects, Allard et al¹⁰⁴ found that compared to those with minimal findings who consume 269.5 mg of cholesterol per day, patients with simple steatosis consume 290.8 mg of cholesterol per day, while patients with MASH consume 357.9 mg of cholesterol per day. Interestingly, patients with nonobese MASLD consumed more cholesterol than patients with obese MASLD.¹⁰²

In addition to the small sample size (<100 subjects) studies mentioned above, some large-scale population–based studies have found that excessive intake of these HC diets increases the risk of MASLD in the population. For eggs, a case-control study conducted in Iran by Mokhtari et al¹⁰⁵ included 169 patients with MASLD and 782 controls. The study found that individuals who consumed two to 3 eggs per week had a 3.71 times higher risk of developing MASLD compared to those who consumed <2 eggs per week. According to a population-based study conducted in the United States, which included 14,369 participants and oversampled certain subgroups such as Blacks, Mexican Americans, and individuals with lower socioeconomic status, Mazidi et al¹⁰⁶ found subjects with higher egg intake have a greater chance of developing MASLD. Specifically, the study revealed that subjects in the highest tertile of egg consumption had a 6% higher likelihood of developing MASLD. For meats, Hashemian et al¹⁰⁷ conducted a Golestan cohort study with 50,045 participants in Iran and found that individuals who had a high consumption of total red meat and organ meat were at increased risk of MASLD. Zelber-Sagi et al¹⁰⁸ conducted a cross-sectional study in Israel with 789 individuals and found that consuming high amounts of red and/or processed meat is associated with both MASLD and insulin resistance (IR). Noureddin et al¹⁰⁹ performed a multiethnic cohort study with over 215,000 participants from various ethnic backgrounds, including African Americans, Native Hawaiians, Japanese Americans, Latinos, and whites in the United States. They discovered that consuming higher amounts of red meat, processed red meat, and cholesterol increases the risk of MASLD. Kim et al¹¹⁰ conducted a nationwide, prospective cohort study that included 77,795 participants in the United States. The study had a 20-year long-term follow-up period. The researchers found that consuming red meat, both unprocessed and processed, was significantly associated with an increased risk of developing MASLD.

CHOLESTEROL-INDUCED ANIMAL MODELS OF MASLD

Although these population-based studies have shown an association between excessive intake of a HC diet and MASLD, the underlying mechanism is still unclear. Therefore, appropriate animal models are needed for further research to elucidate the pathophysiological mechanisms of MASLD. To date, the role of cholesterol in the development of MASLD has been established in numerous animal models (Table 1). Here, we present a brief overview of the most frequently used models, with a particular focus on the impact of dietary cholesterol as one of the key determinants in the progression of MASLD. The advantages and disadvantages of these animal models are summarized in Figure 5.

TABLE 1.

Impact of dietary cholesterol in animal models of MASLD

		Diet compositions
Types	Animal models	Fat	Cholesterol	Cholic acid	Experimental time	Phenotypes	References
Mice	C57BL/6J mice	7.5%	1.25%	0.5%	6, 12, or 24 wk	1. Dyslipidemia, hepatic steatosis, and MASH 2. nonobese	111
	—	60%	1.25%	0.5%	—	1. Dyslipidemia, hepatic steatosis, MASH, and fibrosis 2. nonobese	—
	C57BL/6J mice	16%	1.25%	0.5%	3 wk	1. Hepatic steatosis, hypercholesterolemia 2. TLR4-induced hepatitis	112
	C57BL/6J mice	15%	1.5%	0.1%	25 or 55 wk	1. Hypercholesterolemia, hepatic steatosis, fibrosis, and tumor formation 2. nonobese 3. Increased plasma MCP-1 and hepatic PDGF	113
	C57BL/6J mice	4%	1.0%	0	30 wk	1. Hypercholesterolemia, hepatic steatosis, with little inflammation and no fibrosis 2. nonobese	114
	—	15%	1.0%	0	—	1. Hypercholesterolemia, hepatic steatosis, MASH, and fibrosis 2. Adipose tissue inflammation 3. Obesity	—
	C57BL/6J mice	43% kcal	0.75%	0	20 wk	1. Hepatic steatosis, MASH, and fibrosis 2. Overweight and IR 3. Macrophage inflammatory response and hepatocyte apoptosis	115
	C57BL/6J mice	45% kcal	0.2%	0	16 wk	1. Increased features of MetS, MASH, and fibrosis 2. Obesity 3. Increased circulating leptin and IL-6 levels	116
	C57BL/6J mice	23%	0.19%	0	26 wk	1. MASH-HCC aggravated by dietary cholesterol 2. Obesity	117
	C57BL/6 mice	43.7% kcal	0.013%	0	14 months	1. Fatty liver, MASH, fibrosis and HCC 2. Obesity and IR	118
		43.7% kcal	0.203%	0		1. Fatty liver 2. Obesity and IR
Rats	SD rats	40% kcal	1.25%	0.5%	7 wk	1. Liver steatosis 2. Without peripheral adiposity gain	119
	SD rats	28.75%	1.25%	2.0%	9 wk	1. Hypercholesterolemia, increased MASH and liver fibrosis in a cholesterol dose-dependent manner. 2. nonobese	120
		28.75%	2.5%	2.0%	—	—	—
	SD rats	30%	2.0%	0.2%	20 wk	1. Hepatic steatosis, inflammation, and fibrosis 2. Obesity and IR 3. Increased LPS and TNF-α secretions	121
	Wistar rats	5%	1.0%	0.3%	12 wk	1. Hypercholesterolemia, hepatic steatosis, and fibrosis 2. Obesity	122
	Wistar rats	39%	1%	0.25%	16 wk	1. Hepatic steatosis, hepatocyte ballooning, and MASH 2. Increased expression of proinflammatory and pro-fibrogenic genes 3. nonobese	123
	SHRSP5/Dmcr rats	35.3% kcal	5%	2%	2, 8, and 14 wk	1. Hypercholesterolemia, hepatic steatosis, MASH, and fibrosis 2. nonobese 3. Increased focal or spotty necrosis with lymphocyte infiltrations	124
Hamsters	Golden Syrian hamsters	25.6% kcal	0.5%	0	5 wk	1. Hyperlipidemia, hyperglycemia and hepatic steatosis 2. Obesity	125
	Golden Syrian hamsters	23.0%	1%	0	12 wk	1. Hypercholesterolemia, hepatic steatosis, MASH, and fibrosis 2. Accumulation of unesterified cholesterol in KCs	126
Guinea pigs	Guinea pigs	10.0%	0.33%	0	2, 4, or 6 mo	1. Hepatic lipid deposition 2. nonobese	127
	Guinea pigs	20.0%	0.35%	0	16 or 25 wk	1. Hepatic steatosis, MASH, and fibrosis 2. Enhanced lipogenesis, decreased fatty acid oxidation, and failed exportation of lipids	128
Rabbits	New Zealand rabbits	—	1%	0	7 wk	1. Hepatic lipid deposition, sinusoidal fibrosis 2. Cholesterol crystals were found in hepatocytes, endothelial cells, and KCs 3. nonobese	129
	New Zealand rabbits	—	0.3%	0	24, 34 wk	1. Cholesterolemia, hepatic steatosis, centrolobular liver fibrosis 2. Fat-storing cells gradually lose lipid droplets during fibrosis formation	130
	Japanese white rabbits	—	1%	0	8, 12 wk	1. Hypercholesterolemia, hepatic steatosis, MASH, and fibrosis 2. nonobese and non-IR	131
	Japanese white rabbits	12%	0.75%	0	9 mo	1. Hypercholesterolemia, hepatic steatosis, MASH, and advanced liver fibrosis 2. non-IR	132
Pigs	Ossabaw pigs	46% kcal	2%	0.7%	24 wk	1. Hyperlipidemia, metabolic syndrome, significant microvesicular steatosis and fatty KCs 2. Obesity	133
	Wuzhishan pigs and Tibetan pigs	15%	1.5%	0	24 wk	1. Hypercholesterolemia, hepatic steatosis and inflammation 2. Obesity	134
	Microminipigs	12%	5%	0.7%	12 wk	1. Hyperlipidemia, atherosclerosis, hepatic steatosis 2. Obesity	135
	Domestic pigs	27.2%	1%	0	10 wk	1. Hypercholesterolemia, atherosclerosis, hepatic steatosis and systemic inflammation 2. nonobese	136
Tree shrews	Tree shrews	20%	1.25%	0.5%	3, 6, and 10 wk	1. Hypercholesterolemia, hepatic steatosis, MASH, and fibrosis 2. nonobese 3. The liver takes up LDL-c via the lipoprotein lipase	137

Abbreviations: IR, insulin resistance; LPS, lipopolysaccharides; MASH, metabolic dysfunction–associated steatohepatitis; MASLD, metabolic dysfunction–associated steatotic liver; MCP-1, monocyte chemoattractant protein-1; MetS, metabolic syndrome; SD, Sprague-Dawley; SHRSP5/Dmcr, stroke-prone spontaneously hypertensive rat.

FIGURE 5.

Animal models of NAFLD induced by cholesterol-containing diet. The inner circle indicates the different animal models, where green indicates rodent models and blue indicates nonrodent models. The outer circle shows the characteristics of the relevant animal model. Abbreviations: MASLD, metabolic dysfunction–associated steatotic liver disease.

Rodent models

Mice

In terms of models for HC, cholesterol is typically used alone or in combination with other ingredients, and the cholesterol content generally ranges from 0.1% to 2%. The atherogenic (Ath) diet, which contains normal fat content, a relatively high dose of cholesterol (1%–1.25% by weight), and cholic acid (0.5% by weight), has initially been used to study atherosclerosis.^138,139 However, mice receiving this diet also developed steatohepatitis. When mice were treated with a combination of 1.25% cholesterol and 0.5% cholic acid, liver steatosis could be observed after 6 weeks, and MASH appeared at 24 weeks.¹¹¹ Mechanistically, toll-like receptor 4 was considered to be partly responsible for the development of this Ath diet-induced steatohepatitis, as TLR deletion mice showed a significant improvement in liver injury enzymes and leukocyte infiltration.¹¹² Sumiyoshi et al¹¹³ fed mice with a HC diet, which consists of 1.5% cholesterol and 0.1% cholic acid, after 55 weeks treatment, they found the mice displayed hypercholesterolemia, hepatic steatosis, fibrosis, and tumor formation in the form of focal nodular hyperplasia, they concluded that the liver fibrosis and focal nodular hyperplasia may be partly due to an increase of plasma monocyte chemoattractant protein-1 and liver PDGF expression after a long term of HC–diet treatment. Although the Ath diet can replicate the pathophysiological findings of MASH, the mice fed this diet did not show systemic IR. Only with the addition of a 60% HF component (cocoa butter), it can induce hepatic IR and further accelerate the progression from MASH to fibrosis.¹¹¹ This suggests that these mouse models can be used to study nonobese MASLD in humans.

To establish the obese MASLD model, obesogenic diets, which are a combination of high fat (HF) and HC, have been widely used. Savard et al¹¹⁴ discovered that diets containing either 15% fat (by weight, HF) or 1% cholesterol (by weight, HC) resulted in obesity and liver steatosis but not MASH and liver fibrosis, in a mouse model after 30 weeks of feeding. However, the combination of HF and HC led to significant body weight gain and severe liver steatosis, MASH, and liver fibrosis, suggesting that dietary fat and cholesterol have a synergistic effect in promoting the development of MASH. Henkel et al¹¹⁵ treated mice for 20 weeks with a diet containing 43% fat (percentage of kcal) and 0.75% cholesterol (by weight). They found that, in contrast to cholesterol-free HF diets, this diet not only induced the development of MASH and liver fibrosis but also caused overweight and IR. These effects closely resemble many clinical features observed in patients with metabolic syndrome and MASH. Similarly, Mells et al¹¹⁶ fed mice a diet containing 45% fat (percentage of kcal) and 0.2% cholesterol (by weight). After 16 weeks, the mice exhibited significant features of metabolic syndrome, MASH, and liver fibrosis. In another study, a high-fat and high-cholesterol (HFHC) diet containing 23% fat (by weight) and 0.19% cholesterol (by weight) was found to induce obesity and significant MASH-related HCC after 26 weeks of treatment in diethylnitrosamine-treated mice. However, a parallel HF noncholesterol diet containing 23% fat and 0% cholesterol only induces obesity and simple steatosis.¹¹⁷ Moreover, HFHC diet consisting of 43.7% fat (percentage of kcal) and 0.203% cholesterol (by weight)-induced obesity and a sequential progression of steatosis, MASH, fibrosis and eventually HCC in mice after 14 months of induction. This progression was accompanied by IR. On the other hand, a HF and low-cholesterol diet, consisting of 43.7% fat (percentage of kcal) and 0.013% cholesterol (by weight), only induced obesity and hepatic steatosis.¹¹⁸ These data suggest that a HF diet can only induce obesity and simple hepatic steatosis, while the addition of cholesterol to a HF diet can lead to the formation of obese MASLD, including MASH, and also suggest that dietary cholesterol is an essential factor for the development of the entire spectrum of MASLD.

To overcome the time-consuming limitations of inducing an animal model of MASH, a new mice model has been established. Mice fed a diet high in fat (60% percentage of kilocalories), cholesterol (1.25% by weight), and cholic acid (0.5% by weight), along with 2% hydroxypropyl-β-cyclodextrin in their drinking water, rapidly developed significant features of MASH and hepatic IR within 3 weeks. This time-saving model has the potential to be used for rapidly detecting the effects of novel drugs targeting MASH.^140,141

Rats

Similar to mice, rats are also frequently used in MASLD modeling. In Sprague-Dawley (SD) rats, an Ath diet containing 40% fat (percentage of kilocalories), 0.5% cholic acid, and 1.25% cholesterol-induced significant liver steatosis characterized by hepatic triglyceride and cholesterol accumulation compared with control diet-fed rats after 7 weeks of treatment.¹¹⁹ Also, in SD rats fed an HF diet containing 30% palm oil (by weight) and 2% cholic acid, or HFHC diet containing 28.75% palm oil (by weight), 2% cholic acid, and either 1.25% or 2.5% cholesterol for 9 weeks, Ichimura and colleagues¹²⁰ found that rats fed the HFHC diets displayed MASH and liver fibrosis in a dose-dependent manner. Additionally, 40% of rats progressed to liver cirrhosis when treated with the HFHC diet containing 2.5% cholesterol. In contrast, rats fed the HF diet developed mild hepatic steatosis and liver inflammation without fibrosis.¹²⁰ In another study conducted by the same group, using the same HFHC diets, it was found that all the rats exhibited a liver cirrhosis phenotype after 18 weeks of treatment.¹⁴² Maciejewska et al¹²¹ fed SD rats an HFHC diet consisting of 30% fat (percentage of kcal), 0.2% cholic acid, and 2% cholesterol. After 2, 4, 8, 12, 16, and 20 weeks of dietary exposure, they observed that the rats developed hepatic steatosis and inflammation at 4 weeks and liver fibrosis at 12 weeks, suggesting that dietary cholesterol could gradually induce the development of MASLD in SD rats.

For Wistar rats, when fed a diet supplemented with 1% cholesterol and 0.3% cholate for 12 weeks, the rats exhibited hypercholesterolemia starting from 3 weeks and hepatic fibrosis starting from 9 weeks.¹²² Furthermore, an HFHC diet (65% kcal from fat, 1% cholesterol, 0.25% cholate) induced significant hepatic steatosis, hepatocyte ballooning, and inflammation in Wistar rats after 16 weeks treatment.¹²³ The Wistar rat models induced by a HC diet or by a HF and HC diet have been widely used to test the protective effects of drugs,¹⁴³ natural compounds,¹⁴⁴ and certain foods^145–147 on MASLD.

The stroke-prone spontaneously hypertensive rat (SHRSP5/Dmcr), formerly known as the arteriolipidosis-prone rat, is the fifth generation of the original SHRSP rat, which originated from the normotensive Wistar-Kyoto rat in Japan.^148,149 In 2012, Nakajima lab treated the SHRSP5/Dmcr rats with an HFHC diet containing 35.3% fat (percentage of kcal), 5% cholesterol, and 2% cholic acid for 2, 8, and 14 weeks. Their aim was to develop a model of arteriosclerosis. Incidentally, hepatic steatosis was observed at 2 weeks, multilobular necrosis and liver fibrosis were observed at 8 weeks. Thus, the SHRSP5/Dmcr rats were proven to be an appropriate model for studying the development of MASLD induced by cholesterol.¹²⁴ At the same time, they found that the TNFα and p50/p65 molecular signals (NFκB pathway) appeared to be major factors in promoting the progression of the disease.¹⁵⁰ Furthermore, in this fibrotic steatohepatitis rat model, it was found that multilobular necrosis was caused by the suppression of caspase activity. Meanwhile, the researchers identified serum K18Asp396 levels, which is a neoepitope produced when keratin 18 is cleaved by caspases, as a potential biomarker for hepatocyte necrosis.¹⁵¹ Moreover, Nakajima lab also found that the HFHC diet caused ER stress and disrupted the autophagy process, which was caused by the accumulation of unfolded proteins and the prevention of fusion between the autophagosome and lysosome receptively. Consequently, these 2 events promoted the development of MASH and fibrosis in the livers of SHRSP5/Dmcr rats.¹⁵² In another study, Horai et al¹⁵³ once again demonstrated that a high-cholesterol diet, similar to the one used in Nakajima lab, can induce the entire course of MASLD in a time-dependent manner in SHRSP5/Dmcr rats. However, similar to the commonly used Ath diet-induced mice models, this HFHC diet-induced SHRSP5/Dmcr rat is a nonobese MASLD model characterized by the absence of apparent hyperglycemia, IR, and obesity.¹²⁴

Hamsters

Hamsters have higher plasma LDL levels and lower HDL levels compared to mice and rats. Additionally, their hepatocytes excrete only apolipoprotein B-100, which closely mimics human lipoprotein metabolism.^154,155 Therefore, hamsters have been used as an animal model to study diet-induced atherosclerosis since the 1980s.¹⁵⁶ Nowadays, they are increasingly becoming a choice of animal model for studying lipid metabolism and MASLD. In 2011, Bhathena et al¹²⁵ fed Golden Syrian hamsters a HF (25.6% kcal from fat) and HC (0.5%) diet for 5 weeks; they found that the hamsters developed hepatic steatosis accompanied by hyperlipidemia, hyperglycemia, and obesity. This suggested that hamsters fed with a HF and HC diet may be a good animal model to study diet-induced MASLD complicated by other metabolic syndromes. In 2016, Lin et al¹²⁶ conducted a study where they fed Golden Syrian hamsters and C57BL/6 mice the same HF and HC diet (11.5% coconut oil, 11.5% corn oil, and 1% cholesterol). After 12 weeks, they observed significant hepatic fat deposition in both models. Interestingly, there were notable differences between the 2 models. Hamsters also showed severe hepatocellular hypertrophy, inflammation, and bridging fibrosis. Additionally, they had significantly elevated plasma LDL cholesterol levels and an accumulation of unesterified cholesterol in KCs. But in mice, only mild hepatic inflammation was observed, accompanied by a slight increase in plasma LDL cholesterol levels, and no accumulation of unesterified cholesterol in KCs. Therefore, this study demonstrated that dietary cholesterol-induced increases in plasma LDL cholesterol and accumulation of cholesterol in KCs are important factors in promoting the development of MASLD.

Guinea pigs

Guinea pigs have lipoprotein profiles comparable to those of humans, with LDL being the predominant form of circulating cholesterol. As early as 1951, Cook and Thomson¹⁵⁷ found that compared to a HF diet (16.3% olive oil, by weight), a HF and HC diet (16.3% olive oil and 1.6% cholesterol, by weight) could induce weight loss and fatty liver phenotypes in guinea pigs. Next, in 1977 and 1980, researchers discovered that after feeding the guinea pigs a diet containing 10% cottonseed oil (by weight) and 1% cholesterol, a significant accumulation of neutral lipids in the liver was observed within 1 week.¹⁵⁸ This phenotype was accompanied by increased activity of hepatic lysosomal enzymes, such as β-glucuronidase, β-acetyl-glucosaminidase, and cathepsin D.¹⁵⁹ Later studies showed that a diet containing 10% fat (by weight) and 0.33% cholesterol could induce guinea pigs to develop enlarged livers with elevated markers of liver damage and hepatic lipid deposition at 24 weeks,¹²⁷ while a 20% fat (by weight) and 0.35% cholesterol diet-induced MASH in guinea pigs at 25 weeks.¹²⁸ Taken together, these studies have shown that the guinea pig model induced by a HC diet is characterized by dyslipidemia and hepatic steatosis but not accompanied by obesity. Therefore, this supports the use of the guinea pig model to study MASLD in nonobese individuals.

Nonrodent models

Rabbits

Cholesterol-fed rabbits have often been used as an experimental model for atherosclerosis due to their hypercholesterolemia.¹⁶⁰ However, studies have found that these rabbits also frequently exhibit the phenotype of MASLD. Wanless et al¹²⁹ fed New Zealand rabbits a diet containing 1% cholesterol for 7 weeks. They observed liver steatosis, as evidenced by the heavy loading of lipid droplets, in all 12 cholesterol-induced rabbits. Additionally, mild sinusoidal liver fibrosis was observed in 2 out of the 12 rabbits. Mechanistically, they speculated that bile salts and other cholesterol oxidation products may play an important role. Buyssens et al¹³⁰ induced New Zealand rabbits with a 2% cholesterol diet for 8 weeks and a 0.3% cholesterol diet for 24 and 34 weeks. They observed hepatic steatosis in all rabbits at these time points. However, they only observed pronounced centrolobular liver fibrosis in rabbits induced with 0.3% cholesterol for 24 weeks and 34 weeks. Kainuma et al¹³¹ found that a 1% cholesterol diet could induce hepatic steatosis, liver inflammation, and liver fibrosis in Japanese white rabbits after 12 weeks of treatment. Nevertheless, it is important to note that these animals were nonobese and non-IR. Ogawa et al¹³² treated Japanese white rabbits with a diet supplemented with 12% corn oil (by weight) and 0.75% cholesterol. After 9 months of induction, they successfully created a rabbit model of MASH with advanced liver fibrosis (almost cirrhosis), but without IR. These studies show that either a HC diet or a HF and HC diet can induce a nonobese rabbit model of MASLD. Therefore, it is important to consider the role of the HF content in the diet in this process. In fact, Wang et al¹⁶¹ found that there was no significant difference in the induction of liver steatosis between the HC diet and the HFHC diet. Although more data are needed to confirm this conclusion, their preliminary data suggests that HC is the key factor in inducing MASLD, and HF cannot accelerate the formation of MASLD in the rabbit model.

In the above experiments, adult rabbits (at least 10 weeks old) were widely used. However, the longer prepubertal stage also allowed for the use of young rabbits (4–6 wk old) to create a pediatric MASLD model through the administration of a HC diet.¹⁶²

Pigs

The general anatomy and certain aspects of lipid metabolism, including cholesterol, in pigs are more similar to the human condition compared to rodent models. Therefore, pigs serve as an excellent model for studying metabolic syndrome, and there are various breeds to choose from. In Ossabaw pigs, it has been found that an atherogenic diet (46% kcal from fat and 2% cholesterol and 0.7% cholate by weight) feeding caused metabolic syndrome, significant microvesicular steatosis, and fatty KCs with obesity at 24 weeks.¹³³ In Wuzhishan pigs and Tibetan pigs, both indigenous to China, the treatment with a HF and HC diet consisting of 15% shortening oil (by weight) and 1.5% cholesterol for 24 weeks caused hepatic steatosis and inflammation, along with obesity, hypertension, severe hypercholesterolemia, and hyperinsulinemia.¹³⁴ In a study conducted on Microminipigs, a novel breed of swine, it was discovered that a diet high in fat and cholesterol (12% lard, 5% cholesterol, and 0.7% sodium cholate) resulted in histopathological fatty changes and the accumulation of lipid droplets in hepatocytes. Additionally, the pigs experienced a significant increase in body weight over a period of 3 months.¹³⁵

Unlike these pigs that all developed obese MASLD, some pig breeds exhibited nonobese MASLD. Koopmans et al¹³⁶ demonstrated that Domestic (Landrace × Yorkshire, D-line) pigs, when fed a HF and HC diet (27.2% fat and 1% cholesterol by weight) for 10 weeks, exhibited significant deposition of liver lipids, atherosclerosis, hypercholesterolemia, and systemic inflammation, with no change in body weight. These findings suggest that genetic backgrounds, or breed differences, have a significant impact on MASLD in pigs.

Tree shrews

Tree shrews (Tupaia belangeri chinensis) are considered to have a close affinity to primates.¹⁶³ Due to this advantage, tree shrews have been used as animal models to study a variety of human diseases, including depression, breast cancer, and viral hepatitis.¹⁶⁴ Our previous studies have shown that tree shrews induced by a high-calorie diet can also be used as an animal model to study nonobese MASLD.¹⁶⁵ We found that a HF and HC diet (20% fat, 1.25% cholesterol, and 0.5% sodium cholate by weight) induced significant hepatic steatosis in the tree shrew model at 3 weeks, steatohepatitis at 6 weeks, and liver fibrosis at 10 weeks.¹³⁷ In contrast, it takes at least 30 weeks to replicate these courses in a mouse model induced with HF and HC.¹¹⁴ Mechanistically, we found that under the stimulation of dietary cholesterol, the liver takes up LDL-c in the circulatory system through the lipoprotein lipase pathway instead of the traditional LDLR pathway, resulting in a significant accumulation of lipids in the liver.¹³⁷ Our data suggest that the tree shrew model, induced by a HF and HC diet, mimics most of the progression of MASLD within 10 weeks. Furthermore, our findings indicate that lipoprotein lipase plays a crucial role in the development of MASLD, offering a novel perspective for studying this condition in nonobese individuals.

Most of the cholesterol-containing diet-induced animal models were associated with inflammation but without obesity and insulin resistance. Only a few were obese and insulin-resistant, which may be attributed to the synergistic effect of cholesterol and other components of diet. For example, a study showed that cholesterol in combination with hydrogenated vegetable oil induces phenotypes such as elevated fasting blood glucose, impaired glucose tolerance, and impaired insulin tolerance in mice.¹¹⁶ In another study, the authors found that dietary cholesterol in a soybean oil diet caused insulin resistance and affected hepatic insulin signaling. It significantly reduced insulin receptor substrate 2 expression and glycogen content in the mice liver.¹¹⁵ Since IRS-2 is the main pathway for insulin-dependent regulation of hepatic glucose metabolism and directly mediates hepatic glycogen synthesis, it suggests that cholesterol affects hepatic glucose metabolism by downregulating IRS-2 in this case. Taken together, these data suggest that inflammation may play a more significant role than impaired glucose metabolism in cholesterol-induced MASLD models.

CHOLESTEROL-INDUCED INFLAMMATION PROMOTES THE DEVELOPMENT OF MASLD

Although the links between cholesterol and inflammation are most clearly demonstrated in the case of atherosclerosis in which the macrophages are the decisive factor, similar mechanisms may also play a role in other metabolic disorders such as obesity or MASLD. For example, in MASLD, the buildup of cholesterol in KCs leads to liver inflammation, thereby promoting the progression of MASH in both mice and humans.¹⁶⁶ In addition to the KCs, there are also many other immune cells in the liver, including dendritic cells (DCs), neutrophils, and lymphocytes, such as CD4+ T cells, CD8+ T cells, natural killer (NK) cells, and NK T cells, which can be further classified as innate or adaptive immune cells.¹⁶⁷ Understanding the relationship between these immune cells and cholesterol metabolism helps to define how dietary cholesterol contribute to injury during the development of MASLD.

Innate immune cells and MASLD

The innate immune system acts as the first line of defense and involves the activation and recruitment of innate immune cells, including macrophages, DCs, neutrophils, NK cells, and NK T cells.¹⁶⁸ As well known, dietary cholesterol-induced activation of macrophages was initially thought to be the key factor in the development of atherosclerosis. However, its impact on MASLD, particularly MASH, has garnered increasing attention and concern. The liver macrophages in homeostasis comprise KCs, which reside within the liver and function in many aspects, such as pathogen scavenging and cholesterol metabolism regulation.¹⁶⁹ The association between cholesterol and KCs in the development of MASH has been well studied both in human patients and in animal models induced by the HFHC diet. For example, Ni et al¹⁷⁰ reported that infiltration of F4/80+ macrophages as well as activation of KCs with increased expression of proinflammatory cytokines, including Tnf, Il6, and Il1b were observed in the HFHC diet-induced MASH model. In terms of mechanisms, studies discovered that cholesterol crystals, formed by free cholesterol and found in lipid droplets within hepatocytes, activated the NOD-, LRR-, and pyrin domain-containing 3 inflammasome signaling pathway and triggered the activation and aggregation of KCs. This led to the formation of hepatic crown-like structures around necrotic hepatocytes (Figure 6). These structures subsequently contribute to the advancement of simple steatosis to MASH.^171,172 Meanwhile, in both human and mouse models, free cholesterol also deposits in the activated KCs to form foam cells that resemble the appearance of macrophages in atherosclerotic lesions.¹⁷¹ In these foam cells, NLRP3 is activated, and the NLRP3 inflammasome serves as the mechanistic link between KCs exposure to cholesterol and MASH^173,174 (Figure 6). Studies also showed that the activation of NLRP3 can lead to severe liver inflammation and fibrosis, even in the absence of cholesterol induction.^175,176 Furthermore, Li et al¹⁷⁷ found that a diet consisting of 21% fat and 0.15% cholesterol activated the NLRP3 inflammasome by inducing the galectin-3 and TLR4, thereby promoting MASH in mice. Corresponding with this, inhibition of NLRP3 inflammasome improved liver inflammation and fibrosis in atherogenic diet-induced MASH model.¹⁷⁸ However, a recent study from Ioannou et al¹⁷⁹ demonstrated that neither genetic deletion nor pharmacologic inhibition of NLRP3 inflammasome improved HF and HC diet–induced MSAH in mice. Taken together, these data suggest that the role of NLRP3 is complex, and further validation is needed to determine its true involvement in cholesterol-containing diet-induced MASH.

FIGURE 6.

Role of cholesterol crystal in the development of MASH. Abbreviations: aHSC, activated-hepatic stellate cell; EC, endothelial cell; hCLS, hepatic crown-like structures; NLRP3, NOD-, LRR-, and pyrin domain-containing 3.

In addition to free cholesterol deposition, the KCs also acquire cholesterol in the form of modified lipoproteins, such as oxidized LDL, through CD36 and scavenger receptor A. Studies by Bieghs et al^44,46 showed that deletion of Cd36 and macrophage scavenger receptor 1 decreased lysosomal cholesterol levels within KCs and attenuated liver inflammation. Consistently, they found that strategies targeting the reduction of oxidized LDL content in the entire liver and lysosomal cholesterol levels in KCs of mice treated with an HFHC diet had a beneficial effect on hepatic inflammation.^180,181 Moreover, Govaere et al¹⁶⁶ found that macrophage scavenger receptor 1 expression was associated with the formation of hepatic foam macrophages and correlated with the degree of MASLD in both human patients with MASLD and HFHC diet–treated mice. The deletion of macrophage scavenger receptor 1 protected mice against HFHC diet-induced MASLD, resulting in fewer hepatic foamy macrophages, reduced hepatic inflammation, improved dyslipidemia, and altered hepatic lipid metabolism. Together, these observations provide insight into the importance of cholesterol-activated KCs in participating in the underlying mechanism of MASH.

Beyond KCs, the DCs, neutrophils, NK cells, and NK T cells are also important components of the liver’s innate immune system.¹⁶⁷ They contribute to inflammation by producing cytokines, chemokines, eicosanoids, nitric oxide, and ROS.¹⁶⁸ Many studies have reported that these cells play an important role in the development of MASLD.^182–185 However, the animal models used in these studies were either induced with an methionine- and choline-deficient diet^182,184,185 or with a HF diet.¹⁸³ Few studies have focused on the mechanism of these cells in the development of MASLD under HC diet treatment. Even from the perspective of cholesterol metabolism, only a few studies have reported on the mechanism of NK T cells in the development of MASLD-HCC while neglecting the other cell types. For instance, Tang et al¹⁸⁶ discovered that cholesterol accumulation in hepatocytes, driven by sterol regulatory element-binding protein-2, induced lipid peroxide accumulation and resulted in deficient cytotoxicity in NK T cells. This deficiency selectively suppressed the antitumor immune surveillance of NK T cells and subsequently promoted the development of MASLD-HCC in both the diet-associated MASLD-HCC mice model and patients with MASLD-HCC.

Adaptive immune cells

The adaptive immune system acts as the second line of defense by antigenic specificity, immunologic memory, and diversity, thus providing long-term protection. T cells represent the cellular component of the adaptive immune system and consist of multiple differentially active subsets. Conventional T cells can be further classified into CD4+ T cells and CD8+ T cells.¹⁸⁷

CD4+ T cells and MASLD

CD4+ T cells, including T helper 1 (Th1), Th2, Th17, Th22, and regulatory T cells,¹⁸⁷ play a pivotal role in hepatocellular injury, autoimmunity, and the dysregulation of CD4+ T cell function is emerging as a critical pathological factor in the development of MASLD. These roles have been well summarized in previous reviews,^187–189 and the effects of the main CD4+ T cells in the development of MASLD are summarized in Figure 7. However, in these published studies, the animal models were established either with HF diet¹⁹⁰ or with an methionine- and choline-deficient diet.¹⁹¹ Only a few studies used a HF and HC diet (15% fat and 2% cholesterol by weight) to explore the function of Th17/regulatory T cells in MASLD.¹⁹² Thus, to our knowledge, the function of CD4+ T cells in HF and HC diet–induced MASLD has not been thoroughly investigated.

FIGURE 7.

Overview of the different cell types and characteristics of CD4+ T cells in the pathophysiology of MASLD. Abbreviations: AHR, aryl hydrocarbon receptor; CCR, C-C motif chemokine receptor; CTLA4, cytotoxic T-lymphocyte-associated protein 4; CXCR, C-X-C motif chemokine receptor; FoxP3, forkhead box P3; GATA-3, GATA binding protein 3; IFN-γ, interferon-gamma; RA, retinoic acid; ROR-γt, retinoic acid-related orphan receptor γ-t; STAT, signal transducer and activator of transcription; T-bet, T-box expressed in T cells; Th1, T helper 1; Th17, T helper 17; Th2, T helper 2; Th22 T helper 22; Treg, regulatory T cell.

CD8+ T cells and MASLD

The role of CD8+ T cells in promoting the progression of MASLD has been investigated in mouse model. For instance, a study found that the number of CD8+ T cells increased, and the activated CD8+ cells, along with NK T cells, activated the NF-κB pathway in hepatocytes. This activation promoted steatosis and the transition from MASH to liver cancer in choline-deficient HF diet–induced mice.¹⁹³ In line with this, depletion of CD8+ T cells improves MASH in mice fed a HF, high-carbohydrate diet.¹⁹⁴ In addition to these studies in models induced by cholesterol-free diets, researchers also explored the function of CD8+ T cells under the treatment of a cholesterol-containing diet. Breuer et al¹⁹⁵ established an obese and hyperlipidemic MASH mice model using a western diet that contains 42% of kcal fat and 0.15% cholesterol. They discovered that mice in this model exhibited a significant elevation and activation of hepatic CD8+ T cells, which is consistent with the phenotype observed in patients with obese MASLD. Mechanistically, they concluded that under the conditions of obesity and hyperlipidemia, hepatic CD8+ T cells secrete IL-10 to regulate hepatic inflammation and induce HSC activation. To the activation of hepatic CD8+ T cells, studies have reported that cholesterol could activate CD8+ T cells through antigen-presenting cells, such as DCs and macrophages.^171,196 Similarly, Breuer et al¹⁹⁵ found that the intracellular accumulation of cholesterol firstly activated DCs and KCs and then contributed to the CD8+ T cells activation in the obese and hyperlipidemic MASH model.

Therapeutic targets based on cholesterol metabolism and inflammation

Given that cholesterol and inflammation play crucial roles in the development of MASLD, strategies focusing on cholesterol metabolism and inflammation may help alleviate disease. In fact, there are already some lipid-lowering drugs being used clinically for managing cholesterol metabolism. Statins, for example, can reduce cholesterol synthesis by inhibiting the activity of HMGCR, thereby alleviating MASLD.¹⁹⁷ Ezetimibe is also effective in relieving liver steatosis because it inhibits NPC1L1, reducing cholesterol absorption in the intestine and liver.¹⁹⁸

In recent years, new drugs targeting inflammation have been discovered for the treatment of MASLD. Nuclear receptor farnesoid X receptor agonists can improve glucose and lipid metabolism and also have anti-inflammatory and antifibrotic effects, making them suitable for treating MASH.¹⁹⁹ Obeticholic acid, a synthetically modified analog of chenodeoxycholic acid, is the prototype of this class of agents and has been shown to significantly improve MASH and fibrosis in clinical trials.^200,201 In addition to obeticholic acid, several other nonbile acid farnesoid X receptor agonists, such as EDP-305, cilofexor, nidufexor, and tropifexor, have similar potential, and their therapeutic effects are currently under evaluation.¹⁹⁹ In addition, a phase 2 study demonstrated that the FGF-19 analog, NGM282, also enhances farnesoid X receptor activity, thereby improving MASH.^202,203 CCR2/5 plays a crucial role in innate immunity by recruiting monocytes. Inhibition of CCR2/5 with cenicriviroc has been found to reduce MASH and fibrosis in a clinical study, and the drug is currently in a phase 3 clinical trial.^199,204 Other anti-inflammatory drugs, such as hepatic macrophage polarization (Annexin A5), NLRP3 inhibitor (sulforaphen), TLR4 antagonist (sparstolonin B), anti-TNF-α drugs (thalidomide and infliximab), ERK antagonist (ravoxertinib), also show potential therapeutic effects on MASLD and are currently undergoing preclinical studies.²⁰⁵

CONCLUSIONS AND PERSPECTIVES

As one of the most important lipid molecules in mammalian cells, cholesterol not only constitutes a vital component of cell membranes but also serves as a precursor for the synthesis of bile acids and hormones. It plays an important biological function and is closely related to our life and health. With the continuous advancement of research, our understanding of cholesterol metabolism has become increasingly in-depth. For example, the NPC1L1-mediated endocytosis pathway, Aster-mediated intracellular nonvesicular transport, sterol regulatory element-binding protein-LDLR pathway, and HMGCR degradation pathway have been found to play important roles in the absorption and synthesis of cholesterol. The regulation of cholesterol metabolism is rigorous and complex. Dysregulation of cholesterol metabolism can lead to the development of various diseases, including cancer, type 2 diabetes, cardiovascular diseases, and MASLD. As far as MASLD is concerned, numerous studies have found that excessive intake and accumulation of cholesterol can promote the onset and development of MASLD. To study this, several animal models have been established using cholesterol-containing diets. Evidences showed that cholesterol-induced inflammatory responses, mediated by various types of immune cells, are key factors in these conditions.

Although significant progress has been made in the study of cholesterol and MASLD, there are still several important questions and directions that need to be explored at various physiological and pathological levels. First, it is important to investigate whether there are gut microbes that can transform and degrade dietary cholesterol, thus reducing its absorption and potentially attenuating cholesterol-induced MASLD. Second, regarding the mode of action of cholesterol, is it cholesterol itself or cholesterol derivatives that enter the liver and contribute to the development of MASLD? If cholesterol derivatives play an important role, who catalyzes the production of these derivatives? Third, in terms of the pathogenesis of cholesterol-induced hepatic inflammation, the main focus has been on the connection between cholesterol and innate immune cells, particularly macrophages. However, the relationship between cholesterol and adaptive immune cells, such as CD4+ and CD8+ T cells, has not been thoroughly investigated. Therefore, further studies are needed to gain a better understanding of the role of CD4+ and CD8+ T cells in relation to the progression of cholesterol-induced MASLD. It will also be interesting to study whether cholesterol acts as an antigen to cause liver inflammation through the adaptive immune system. Finally, regarding the role of cholesterol in promoting the development of MASLD, while there has been significant progress in understanding its impact on later stages such as MASH, fibrosis, and MASLD-HCC, the molecular mechanisms through which cholesterol promotes the initial simple steatosis are still unclear.