Maintaining cholesterol homeostasis is crucial for sustaining human health and physiological function. Although the detrimental effects of chronic cholesterol overload on hepatic injury and fibrosis are well documented, the molecular mechanisms driving this pathology remain incompletely understood.

This study investigates the mechanistic role of chronic cholesterol overload in driving liver fibrosis and evaluates the therapeutic efficacy of honokiol as a targeted intervention.

High-cholesterol models induced by cholesterol and 25-hydroxycholesterol in human HepG2 cells or induced by cholesterol crystals in mouse bone marrow-derived macrophages were established. We also examined the effect of cholesterol on the livers of mice following a 20-week regimen of high-cholesterol diets.

Excess cholesterol interfered with normal cholesterol metabolism both in vitro and in vivo, and led to liver damage and fibrosis in vivo. Further research showed that cholesterol exposure triggered NLRP3 inflammasome activation and programmed cell death called pyroptosis; induced an increase in mitochondrial ROS and a disruption of intracellular redox homeostasis, followed by the opening of the mitochondrial permeability transition pore; and finally induced cellular DNA damage, resulting in the translocation of the double-stranded DNA fragment into the cytoplasm and the activation of the DNA-sensing adaptor STING. The activation of the NLRP3-cGAS-STING axis initiated the downstream cascade reaction and up-regulated the expression of pro-inflammatory cytokines, including IL-1β, TNF-α, and IFN-β, thus facilitating liver damage and fibrosis. Furthermore, honokiol, an active ingredient in Magnolia officinalis, could alleviate liver damage and fibrosis by blocking NLRP3 inflammasome activation, pyroptosis, and the cGAS-STING pathway.

Systematic evidence shows that cholesterol induces liver fibrosis through the activation of the NLRP3-cGAS-STING signaling axis and that honokiol demonstrates interventional efficacy in mitigating this process.

Excess cholesterol accumulation contributes to fibrogenesis in metabolic dysfunction-associated steatohepatitis (MASH), but how hepatic cholesterol metabolism becomes dysregulated in MASH is not completely understood. We show that human fibrotic MASH livers have decreased EH-domain-binding protein 1 (EHBP1), a genome-wide association study (GWAS) locus associated with low-density lipoprotein (LDL) cholesterol, and that EHBP1 loss- and gain-of-function increase and decrease MASH fibrosis in mice, respectively. Mechanistic studies reveal that EHBP1 promotes sortilin-mediated PCSK9 secretion, leading to LDL receptor (LDLR) degradation, decreased LDL uptake, and reduced TAZ, a fibrogenic effector. At a cellular level, EHBP1 deficiency affects the intracellular localization of retromer, a protein complex required for sortilin stabilization. Our therapeutic approach to stabilizing retromer is effective in mitigating MASH fibrosis. Moreover, we show that the tumor necrosis factor alpha (TNF-α)/peroxisome proliferator-activated receptor alpha (PPARα) pathway suppresses EHBP1 in MASH. These data not only provide mechanistic insights into the role of EHBP1 in cholesterol metabolism and MASH fibrosis but also elucidate an interplay between inflammation and EHBP1-mediated cholesterol metabolism.

Μetabolic dysfunction-associated steatotic liver disease (MASLD) comprises a heterogeneous condition in the presence of steatotic liver. There can be a hierarchy of metabolic risk factors contributing to the severity of metabolic dysfunction and, thereby, the associated risk of both liver and extrahepatic outcomes, but the precise ranking and combination of metabolic syndrome (MetS) traits that convey the highest risk of major adverse liver outcomes and extrahepatic disease complications remains uncertain. Insulin resistance, low-grade inflammation, atherogenic dyslipidaemia and hypertension are key to the mechanisms of liver and extrahepatic complications. The liver is pivotal in MetS progression as it regulates lipoprotein metabolism and secretes substances that affect insulin sensitivity and inflammation. MASLD affects the kidneys, heart and the vascular system, contributing to hypertension and oxidative stress. To address the global health burden of MASLD, intensified by obesity and type 2 diabetes mellitus epidemics, a holistic, multidisciplinary approach is essential. This approach should focus on both liver disease management and cardiometabolic risk factors. This Review examines the link between metabolic dysfunction and liver dysfunction and extrahepatic disease outcomes, the diverse mechanisms in MASLD due to metabolic dysfunction, and a comprehensive, personalized management model for patients with MASLD.

Hepatic deletion of methionine adenosyltransferase-1a (Mat1a) in mice reduces S-adenosylmethionine (SAMe), a key methyl donor essential for many biological processes, which promotes the development and progression of metabolic dysfunction-associated steatotic liver disease (MASLD). Hyperglycemia and reduced MAT1A expression, along with low SAMe levels, are common in MASLD patients. This study explores how Mat1a-knockout (KO) hepatocytes respond to prolonged high glucose conditions, focusing on glucose metabolism and lipid accumulation. Hepatocytes from methionine adenosyltransferase-1a-knockout (Mat1a-KO) mice were incubated in high glucose conditions overnight, allowing for analysis of key metabolic intermediates and gene expression related to glycolysis, gluconeogenesis, glyceroneogenesis, phospholipid synthesis, and very low density lipoprotein (VLDL) secretion. SAMe deficiency in Mat1a-KO hepatocytes led to reduced protein methyltransferase-1 activity, resulting in increased expression of glycolytic enzymes (glucokinase, phosphofructokinase, and pyruvate kinase) and decreased expression of gluconeogenic enzymes (phosphoenolpyruvate carboxykinase, fructose-1,6-bisphosphatase, and glucose-6-phosphatase). These alterations led to a reduction in dihydroxyacetone phosphate (DHAP), which subsequently inhibited mammalian target of rapamycin complex 1 (mTORC1) activity. This inhibition resulted in decreased phosphatidylcholine synthesis via the CDP-choline pathway and impaired VLDL secretion, ultimately causing lipid accumulation. Thus, under high glucose conditions, SAMe deficiency in hepatocytes depletes DHAP, inhibits mTORC1 activity, and promotes lipid buildup.

Lipid accumulation in the liver can negatively impact liver function and health, which is well-described for humans and other mammals, but relatively unexplored in Atlantic salmon. This study investigates the phenotypic, genetic, and transcriptomic variations related to individual differences in liver fat content within a group of slaughter-sized Atlantic salmon reared under the same conditions and fed the same feed. The objective was to increase the knowledge on liver fat deposition in farmed salmon and evaluate the potential for genetic improvement of this trait.

The study involved measuring liver fat content in a group of slaughter-sized Atlantic salmon. Genetic analysis included estimating heritability and conducting genome-wide association studies (GWAS) to identify quantitative trait loci (QTLs). Transcriptomic analysis was performed to link liver fat content to gene expression, focusing on genes involved in lipid metabolic processes.

There was a large variation in liver fat content, ranging from 3.6% to 18.8%, with frequent occurrences of high liver fat. Livers with higher levels of fat had higher proportions of the fatty acids 16:1 n-7, 18:2 n-6, and 18:1 n-9, and less of the long-chain omega-3 fatty acids. The heritability of liver fat was estimated at 0.38, and the genetic coefficient of variation was 20%, indicating substantial potential for selective breeding to reduce liver fat deposition in Atlantic salmon. Liver fat deposition appears to be a polygenic trait, with no large QTLs detected by GWAS. Gene expression analysis linked liver fat content to numerous genes involved in lipid metabolic processes, including key transcription factors such as LXR, SREBP1, and ChREBP.

The results indicated a connection between liver fat and increased cholesterol synthesis in Atlantic salmon, with potentially harmful free cholesterol accumulation. Further, the gene expression results linked liver fat accumulation to reduced peroxisomal β-oxidation, increased conversion of carbohydrates to lipids, altered phospholipid synthesis, and possibly increased de novo lipogenesis. It is undetermined whether these outcomes are due to high fat levels or if they are caused by underlying metabolic differences that result in higher liver fat levels in certain individuals. Nonetheless, the results provide new insights into the metabolic profile of livers in fish with inherent differences in liver fat content.

Hypercholesterolemia is often observed in individuals with type 2 diabetes. Cholesterol accumulation in subcellular compartments within islet β-cells can result in insulin secretory dysfunction, which is a key pathological feature of diabetes. Previously, we demonstrated that expression of the mitochondrial cholesterol transport protein, steroidogenic acute regulatory protein (StAR), is induced in islets under conditions of β-cell dysfunction. However, whether it contributes to mitochondrial cholesterol accumulation in β-cells and cholesterol-induced β-cell dysfunction has not been determined. Thus, we sought to examine the role of StAR in isolated mouse islets under conditions of excess exogenous cholesterol. Cholesterol treatment of islets upregulated StAR expression, which was associated with cholesterol accumulation in mitochondria, decreased mitochondrial membrane potential and impaired mitochondrial oxidative phosphorylation. Impaired insulin secretion and reduced islet insulin content were also observed in cholesterol-laden islets. To determine the impact of StAR overexpression in β-cells per se, a lentivirus was used to increase StAR expression in INS-1 cells. Under these conditions, StAR overexpression was sufficient to increase mitochondrial cholesterol content, impair mitochondrial oxidative phosphorylation, and reduce insulin secretion. These findings suggest that elevated cholesterol in diabetes may contribute to β-cell dysfunction via increases in StAR-mediated mitochondrial cholesterol transport and accumulation.

Liver x receptor alpha (LXRα) functions as an intracellular cholesterol sensor that regulates lipid metabolism at the transcriptional level in response to the direct binding of cholesterol derivatives. We have generated mice with a mutation in LXRα that reduces activity in response to endogenous cholesterol derived LXR ligands while still allowing transcriptional activation by synthetic agonists. The mutant LXRα functions as a dominant negative that shuts down cholesterol sensing. When fed a high fat, high cholesterol diet LXRα mutant mice rapidly develop pathologies associated with Metabolic Dysfunction-Associated Steatohepatitis (MASH) including ballooning hepatocytes, liver inflammation, and fibrosis. Strikingly LXRα mutant mice have decreased liver triglycerides but increased liver cholesterol. Therefore, elevated cholesterol in the liver may play a critical role in the development of MASH. Reengaging LXR signaling by treatment with synthetic agonist reverses MASH in LXRα mutant mice suggesting that LXRα normally functions to impede the development of liver disease.

The metabolic dysfunction-associated steatotic liver disease (MASLD) affects approximately 30 % of the global population. While excessive consumption of dietary fat induces steatosis, it does not develop fibrosis, indicating that additional factors are required as "second hits" for further progression of MASLD. Here, based on shear wave elastography, we compared the longitudinal patterns of fibrogenesis induced by different diets and show the crucial role of cholesterol accumulation in fibrosis progression.

Mice were fed chow, high-fat (HFD), high-fat high-cholesterol (HFHCD), choline-deficient, L-amino acid-defined high-fat (CDAHFD), or 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine diets over 12 weeks.

Mice fed with HFD gained significant amounts of body weight but did not show an increase in liver stiffness. In contrast, the addition of cholesterol in the same diet robustly induced liver stiffening starting from the first week, which was comparable to the CDAHFD-induced fibrosis model. Longitudinal tracking of liver stiffness revealed a two-step progression of fibrosis after prolonged feeding of HFHCD and CDAHFD, likely due to cellular cholesterol accumulation over a certain threshold after the transition point. Biochemical analyses suggested the critical role of both total and hepatic cholesterol accumulation in liver fibrosis development.

Collectively, our results underscore the significance of cholesterol in liver fibrosis development, also highlighting the benefit of monitoring liver stiffness to understand the pathogenesis of liver fibrosis.

Dietary cholesterol promotes metabolic dysfunction-associated steatohepatitis (MASH), with hepatic macrophages central to disease pathology. However, the mechanisms by which cholesterol-loaded macrophages influence MASH remain unclear.

In this study, mice were fed a cholesterol-rich choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD). Hepatic cholesterol levels, inflammatory markers, and pro-inflammatory macrophage polarization were assessed. In vitro studies examined the impact of cholesterol on macrophage polarization, identifying 7-dehydrocholesterol reductase (DHCR7) as a key cholesterol- and inflammation-responsive enzyme. DHCR7 expression in macrophages from MASH patients and model mice was evaluated. Functional studies involving in vitro knockdown and overexpression experiments, were complemented using myeloid-specific DHCR7 knockout mice. RNA sequencing was performed on liver tissues from wild-type and DHCR7 knockout mice to identify affected signaling pathways.

CDAHFD-fed mice exhibited local cholesterol accumulation and a pro-inflammatory macrophage phenotype in the liver. Cholesterol overload in vitro promoted M1 polarization and liver inflammation, reversible by simvastatin. DHCR7 expression, responded to cholesterol and polarization state, was downregulated in M1-polarized and hepatic macrophages from MASH patients and mice. DHCR7 suppression promoted pro-inflammatory phenotype, while its overexpression showed anti-inflammatory effects. Myeloid-specific DHCR7 deficiency in CDAHFD-fed mice worsened liver inflammation and pro-inflammatory macrophage infiltration. RNA sequencing identified the phosphoinositide 3-kinase (PI3K) pathway in DHCR7-regulated effects, with DHCR7-PI3K axis activation mitigating cholesterol-driven inflammation.

These findings unveil novel mechanistic insights into MASH pathogenesis, suggesting targeting macrophage-specific DHCR7 activation may offer a promising therapeutic strategy for MASH.

Liver damage from nonalcoholic steatohepatitis (NASH) presents a significant challenge to the health and productivity of ruminants. However, the regulatory mechanisms behind variations in NASH susceptibility remain unclear. The gut‒liver axis, particularly the enterohepatic circulation of bile acids (BAs), plays a crucial role in regulating the liver diseases. Since the ileum is the primary site for BAs reabsorption and return to the liver, we analysed the ileal metagenome and metabolome, liver and serum metabolome, and liver single-nuclei transcriptome of NASH-resistant and susceptible goats together with a mice validation model to explore how ileal microbial BAs metabolism affects liver metabolism and immunity, uncovering the key mechanisms behind varied NASH pathogenesis in dairy goats.

In NASH goats, increased total cholesterol (TC), triglyceride (TG), and primary BAs and decreased secondary BAs in the liver and serum promoted hepatic fat accumulation. Increased ileal Escherichia coli, Erysipelotrichaceae bacterium and Streptococcus pneumoniae as well as proinflammatory compounds damaged ileal histological morphology, and increased ileal permeability contributes to liver inflammation. In NASH-tolerance (NASH-T) goats, increased ursodeoxycholic acid (UDCA), isodeoxycholic acid (isoDCA) and isolithocholic acid (isoLCA) in the liver, serum and ileal contents were attributed to ileal secondary BAs-producing bacteria (Clostridium, Bifidobacterium and Lactobacillus) and key microbial genes encoding enzymes. Meanwhile, decreased T-helper 17 (TH17) cells and increased regulatory T (Treg) cells proportion were identified in both liver and ileum of NASH-T goats. To further validate whether these key BAs affected the progression of NASH by regulating the proliferation of TH17 and Treg cells, the oral administration of bacterial UDCA, isoDCA and isoLCA to a high-fat diet-induced NASH mouse model confirmed the amelioration of NASH through the TH17 cell differentiation/IL-17 signalling/PPAR signalling pathway by these bacterial secondary BAs.

This study revealed the roles of ileal microbiome and its secondary BAs in resilience and susceptibility to NASH by affecting the hepatic Treg and TH17 cells proportion in dairy goats. Bacterial UDCA, isoDCA and isoLCA were demonstrated to alleviate NASH and could be novel postbiotics to modulate and improve the liver health in ruminants. Video Abstract.

Background: The gut microbiota constitutes a complex microorganism community that harbors bacteria, viruses, fungi, protozoa, and archaea. The human gut bacterial microbiota has been extensively proven to participate in human metabolism, immunity, and nutrient absorption. Its imbalance, namely "dysbiosis", has been linked to disordered metabolism. Metabolic dysfunction-associated steatotic liver disease (MASLD) is one of the features of deranged human metabolism and is the leading cause of liver cirrhosis and hepatocellular carcinoma. Thus, there is a pathophysiological link between gut dysbiosis and MASLD. Aims and Methods: We aimed to review the literature data on the composition of the human bacterial gut microbiota and its dysbiosis in MASLD and describe the concept of the "gut-liver axis". Moreover, we reviewed the approaches for gut microbiota modulation in MASLD treatment. Results: There is consolidated evidence of particular gut dysbiosis associated with MASLD and its stages. The model explaining the relationship between gut microbiota and the liver has a bidirectional organization, explaining the physiopathology of MASLD. Oxidative stress is one of the keystones in the pathophysiology of MASLD and fibrosis generation. There is promising and consolidated evidence for the efficacy of pre- and probiotics in reversing gut dysbiosis in MASLD patients, with therapeutic effects. Few yet encouraging data on fecal microbiota transplantation (FMT) in MASLD are available in the literature. Conclusions: The gut dysbiosis characteristic of MASLD is a key target in its reversal and treatment via diet, pre/probiotics, and FMT treatment. Oxidative stress modulation remains a promising target for MASLD treatment, prevention, and reversal.

Non-alcoholic (now: metabolic) steatohepatitis (MASH) is the progressive inflammatory form of metabolic dysfunction-associated steatotic liver disease (MASLD), which often coexists and mutually interacts with type 2 diabetes (T2D), resulting in worse hepatic and cardiovascular outcomes. Understanding the intricate mechanisms of diabetes-related MASH progression is crucial for effective therapeutic strategies. This review delineates the multifaceted pathways involved in this interplay and explores potential therapeutic implications. The synergy between adipose tissue, gut microbiota, and hepatic alterations plays a pivotal role in disease progression. Adipose tissue dysfunction, particularly in the visceral depot, coupled with dysbiosis in the gut microbiota, exacerbates hepatic injury and insulin resistance. Hepatic lipid accumulation, oxidative stress, and endoplasmic reticulum stress further potentiate inflammation and fibrosis, contributing to disease severity. Dietary modification with weight reduction and exercise prove crucial in managing T2D-related MASH. Additionally, various well-known but also novel anti-hyperglycemic medications exhibit potential in reducing liver lipid content and, in some cases, improving MASH histology. Therapies targeting incretin receptors show promise in managing T2D-related MASH, while thyroid hormone receptor-β agonism has proven effective as a treatment of MASH and fibrosis.

Liver X receptor-α (LXRα) regulates cellular cholesterol abundance and potently activates hepatic lipogenesis. Here we show that at least 1 in 450 people in the UK Biobank carry functionally impaired mutations in LXRα, which is associated with biochemical evidence of hepatic dysfunction. On a western diet, male and female mice homozygous for a dominant negative mutation in LXRα have elevated liver cholesterol, diffuse cholesterol crystal accumulation and develop severe hepatitis and fibrosis, despite reduced liver triglyceride and no steatosis. This phenotype does not occur on low-cholesterol diets and can be prevented by hepatocyte-specific overexpression of LXRα. LXRα knockout mice exhibit a milder phenotype with regional variation in cholesterol crystal deposition and inflammation inversely correlating with steatosis. In summary, LXRα is necessary for the maintenance of hepatocyte health, likely due to regulation of cellular cholesterol content. The inverse association between steatosis and both inflammation and cholesterol crystallization may represent a protective action of hepatic lipogenesis in the context of excess hepatic cholesterol.

Hepatocellular carcinoma (HCC) is a malignant tumor that occurs in the liver, with a high degree of malignancy and relatively poor prognosis. Gypenoside L has inhibitory effects on liver cancer cells. However, its mechanism of action is still unclear. This study aims to investigate the inhibitory effects of gypenoside L on HCC in vitro and in vivo, and explore its potential mechanisms. The results showed that gypenoside L reduced the cholesterol and triglyceride content in HepG2 and Huh-7 cells, inhibited cell proliferation, invasion and metastasis, arrested cell cycle at G0/G1 phase, promoted cell apoptosis. Mechanistically, it targeted the transcription factor SREPB2 to inhibit the expression of HMGCS1 protein and inhibited the downstream proteins HMGCR and MVK, thereby regulating the mevalonate (MVA) pathway. Overexpression HMGCS1 led to significant alterations in the cholesterol metabolism pathway of HCC, which mediated HCC cell proliferation and conferred resistance to the therapeutic effect of gypenoside L. In vivo, gypenoside L effectively suppressed HCC growth in tumor-bearing mice by reducing cholesterol production, exhibiting favorable safety profiles and minimal toxic side effects. Gypenoside L modulated cholesterol homeostasis, enhanced expression of inflammatory factors by regulating MHC I pathway-related proteins to augment anticancer immune responses. Clinical samples from HCC patients also exhibited high expression levels of MVA pathway-related genes in tumor tissues. These findings highlight gypenoside L as a promising agent for targeting cholesterol metabolism in HCC while emphasizing the effectiveness of regulating the SREBP2-HMGCS1 axis as a therapeutic strategy.

Over the last 50 years, liver transplantation has evolved into a procedure routinely performed in many countries worldwide. Those able to access this therapy frequently experience a miraculous risk-benefit ratio, particularly if they face the imminently life-threatening disease. Over the decades, the success of liver transplantation, with dramatic improvements in early posttransplant survival, has aggressively driven demand. However, despite the emergence of living donors to augment deceased donors as a source of organs, supply has lagged far behind demand. As a result, rationing has been an unfortunate focus in recent decades. Recent shifts in the epidemiology of liver disease combined with transformative innovations in liver preservation suggest that the underlying premise of organ shortage may erode in the foreseeable future. The focus will sharpen on improving equitable access while mitigating constraints related to workforce training, infrastructure for organ recovery and rehabilitation, and their associated costs. Research efforts in liver preservation will undoubtedly blossom with the aim of optimizing both the timing and conditions of transplantation. Coupled with advances in genetic engineering, regenerative biology, and cellular therapies, the portfolio of innovation, both broad and deep, offers the promise that, in the future, liver transplantation will not only be broadly available to those in need but also represent a highly durable life-saving therapy.

Metabolic-associated steatotic liver disease (MASLD), the hepatic manifestation of metabolic syndrome, represents a growing global health concern. The intricate pathogenesis of MASLD, driven by genetic, metabolic, epigenetic, and environmental factors, leads to considerable clinical variability. Dysregulation of hepatic lipid metabolism, particularly cholesterol homeostasis, is a critical factor in the progression of MASLD and its more severe form, metabolic dysfunction-associated steatohepatitis (MASH). This review elucidates the multifaceted roles of cholesterol metabolism in MASLD, focusing on its absorption, transportation, biosynthesis, efflux, and conversion. We highlight recent advancements in understanding these processes and explore the therapeutic potential of natural products such as curcumin, berberine, and resveratrol in modulating cholesterol metabolism. By targeting key molecular pathways, these natural products offer promising strategies for MASLD management. Finally, this review also covers the clinical studies of natural products in MASLD, providing new insights for future research and clinical applications.

Excess cholesterol storage can induce the formation of cholesterol crystals in hepatocyte lipid droplets. Such crystals distinguish metabolic dysfunction associated steatohepatitis (MASH) from simple steatosis and may underlie its pathogenesis by causing cell damage that triggers liver inflammation. The mechanism linking cholesterol excess to its crystallization in lipid droplets is unclear. As cholesteryl esters localize to and accumulate in lipid droplets more readily than unesterified free cholesterol, we investigated whether cholesterol esterification by sterol O-acyltransferase (SOAT), also known as acyl co-A cholesterol acyltransferase (ACAT), is required for hepatocyte lipid droplet crystal formation.

Cholesterol crystals were measured in cholesterol loaded Hep3B hepatocytes, RAW264.7 macrophages, and mouse liver using polarizing light microscopy. We examined the effect of blocking SOAT activity on crystal formation and compared these results to features of cholesterol metabolism and the progression to intracellular crystal deposits.

Cholesterol loading of Hep3B cells caused robust levels of lipid droplet localized crystal formation in a dose- and time-dependent manner. Co-treatment with SOAT inhibitors and genetic ablation of SOAT1 blocked crystal formation. SOAT inhibitor also blocked crystal formation in low density lipoprotein (LDL) treated Hep3B cells, acetylated LDL treated RAW 264.7 macrophages, and in the liver of mice genetically predisposed to hepatic cholesterol overload and in mice with cholesterol enriched diet-induced MASH.

SOAT1-mediated esterification may underlie cholesterol crystals associated with MASH by concentrating it in lipid droplets. These findings imply that inhibiting hepatocyte SOAT1 may be able to alleviate cholesterol associated MASH. Moreover, that either a lipid droplet localized cholesteryl ester hydrolase is required for cholesterol crystal formation, or the crystals are composed of cholesteryl ester.

The purpose of this study was to examine the effect of pemafibrate in a murine model of non-alcoholic steatohepatitis (NASH).

Forty-two, 19-week-old, male, C57BL/6J mice were divided into three groups: a Control group (n = 14), a dextran sulfate sodium (DSS) group (n = 14), and a DSS + PEM group (n = 14). All mice were given a standard rodent diet for the first week, followed by a choline-deficient, high-fat diet (CDHF) for the next 12 weeks. The 22nd day after the animals arrived was taken as Day 1 of the experiment. The Control group continued the CDHF diet and MilliQ water. The DSS group continued the CDHF diet, but starting on Day 1, the group received 0.8 % DSS to drink for 7 consecutive days, followed by MilliQ water for 10 days; this was taken as one course, and it was repeated on the same schedule until autopsy. The DSS + PEM group received the CDHF diet with PEM 0.1 mg/kg/day. Their drinking water was the same as that of the DSS group. On Seven animals from each group were autopsied on each of Day 50 and Day 120, and histopathological and immunohistochemical examinations, as well as quantitative RNA and cytokine measurements, of autopsied mice were performed.

Pemafibrate improved hepatic steatosis (decreased steatosis area), improved liver inflammation enhanced by DSS (decreased aspartate transaminase and alanine aminotransferase), improved hepatic fibrosis promoted by DSS (decreased fibrotic areas and a marker of fibrosis), inhibited tumorigenesis, and decreased intestinal inflammation in the NASH model mice.

In a murine model of NASH, mixing PEM 0.1 mg/kg/day into the diet inhibited disease progression and tumor formation.

MicroRNAs (miRNAs) are promising therapeutic agents for non-alcoholic fatty liver disease (NAFLD). This study aimed to identify key genes/proteins involved in NAFLD pathogenesis and progression and to evaluate miRNAs influencing their expression.

Gene expression profiles from datasets GSE151158, GSE163211, GSE135251, GSE167523, GSE46300, and online databases were analyzed to identify significant NAFLD-related genes. Then, protein-protein interaction networks and module analysis identified hub genes/proteins, which were validated using real-time PCR in oleic acid-treated HepG2 cells. Functional enrichment analysis evaluated signaling pathways and biological processes. Gene-miRNA interaction networks identified miRNAs targeting critical NAFLD genes.

The most critical overexpressed hub genes/proteins included: TNF, VEGFA, TLR4, CYP2E1, ACE, SCD, FASN, SREBF2, and TGFB1 based on PPI network analysis, of which TNF, TLR4, SCD, FASN, SREBF2, and TGFB1 were up-regulated in oleic acid-treated HepG2 cells. Functional enrichment analysis for biological processes highlighted programmed necrotic cell death, lipid metabolic process response to reactive oxygen species, and inflammation. In the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the highest adjusted P-value signaling pathways encompassed AGE-RAGE in diabetic complications, TNF, and HIF-1 signaling pathways. In gene-miRNA network analysis, miR-16 and miR-124 were highlighted as the miRNAs exerting the most influence on important NAFLD-related genes.

In silico analyses identified NAFLD therapeutic targets and miRNA candidates to guide further experimental investigation.

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.

The global prevalence of nonalcoholic fatty liver disease (NAFLD) defined by the increased number of lipid droplets (LDs) in hepatocytes, have risen continuously in parallel with the obesity. LDs and related proteins are known to affect cellular metabolism and signaling. Seipin, one of the most important LD-related proteins, plays a critical role in LD biogenesis. Although the role of adipose tissue-specific Seipin silencing is known, hepatocyte-specific silencing upon cholesterol-mediated lipid accumulation has not been investigated. In our study, we investigated the effect of Seipin on endoplasmic reticulum (ER) stress and lipophagy in cholesterol accumulated mouse hepatocyte cells. In this direction, cholesterol accumulation was induced by cholesterol-containing liposome, while Seipin mRNA and protein levels were reduced by siRNA. Our findings show that cholesterol containing liposome administration in hepatocytes increases both Seipin protein and number of large LDs. However Seipin silencing reduced the increase of cholesterol mediated large LDs and Glucose-regulated protein 78 (GRP78) mRNA. Additionally, lysosome-LD colocalization increased only in cells treated with cholesterol containing liposome, while the siRNA against Seipin did not lead any significant difference. According to our findings, we hypothesize that Seipin silencing in hepatocytes reduced cholesterol mediated LD maturation as well as GRP78 levels, but not lipophagy.

Metabolic Dysfunction-Associated Steatotic Liver Disease (MASLD) constitutes the commonest cause of chronic liver disorder worldwide, whereby affecting around one third of the global population. This clinical condition may evolve into Metabolic Dysfunction-Associated Steatohepatitis (MASH), fibrosis, cirrhosis and hepatocellular carcinoma (HCC), in a predisposed subgroup of patients. The complex pathogenesis of MASLD is severely entangled with obesity, dyslipidemia and type 2 diabetes (T2D), so far so nutritional and lifestyle recommendations may be crucial in influencing the risk of HCC and modifying its prognosis. However, the causative association between HCC onset and the presence of metabolic comorbidities is not completely clarified. Therefore, the present review aimed to summarize the main literature findings that correlate the presence of inherited or acquired hyperlipidemia and metabolic risk factors with the increased predisposition towards liver cancer in MASLD patients. Here, we gathered the evidence underlining the relationship between circulating/hepatic lipids, cardiovascular events, metabolic comorbidities and hepatocarcinogenesis. In addition, we reported previous studies supporting the impact of triglyceride and/or cholesterol accumulation in generating aberrancies in the intracellular membranes of organelles, oxidative stress, ATP depletion and hepatocyte degeneration, influencing the risk of HCC and its response to therapeutic approaches. Finally, our pursuit was to emphasize the link between HCC and the presence of cardiometabolic abnormalities in our large cohort of histologically-characterized patients affected by MASLD (n=1538), of whom 86 had MASLD-HCC by including unpublished data.

Liver x receptor alpha (LXRα, Nr1h3) functions as an important intracellular cholesterol sensor that regulates fat and cholesterol metabolism at the transcriptional level in response to the direct binding of cholesterol derivatives. We have generated mice with a mutation in LXRα that reduces activity in response to endogenous cholesterol derived LXR ligands while still allowing transcriptional activation by synthetic agonists. The mutant LXRα functions as a dominant negative that shuts down cholesterol sensing. When fed a high fat, high cholesterol diet LXRα mutant mice rapidly develop pathologies associated with Metabolic Dysfunction-Associated Steatohepatitis (MASH) including ballooning hepatocytes, liver inflammation, and fibrosis. Strikingly LXRα mutant mice have decreased liver triglycerides but increased liver cholesterol. Therefore, MASH-like phenotypes can arise in the absence of large increases in triglycerides. Reengaging LXR signaling by treatment with synthetic agonist reverses MASH suggesting that LXRα normally functions to impede the development of liver disease.

Non-alcoholic steatohepatitis (NASH) is characterized by aberrant lipid metabolism in hepatocytes. We investigated the involvement of a histone H3K9 methyltransferase Suv39h2 in the pathogenesis of NASH.

NASH is induced by feeding the mice with a high-fat high-carbohydrate (HFHC) diet or a high-fat choline-deficient amino acid defined (HFD-CDAA) diet. The Suv39h2f/f mice were crossbred with the Alb-Cre mice to specifically delete Suv39h2 in hepatocytes.

Ablation of Suv39h2 in hepatocytes improved insulin sensitivity of the mice fed either the HFHC diet or the CDAA-HFD diet. Importantly, Suv39h2 deletion significantly ameliorated NAFLD as evidenced by reduced lipid accumulation, inflammation, and fibrosis in the liver. RNA-seq uncovered Vanin-1 (Vnn1) as a novel transcriptional target for Suv39h2. Mechanistically, Suv39h2 repressed Vnn1 transcription in hepatocytes exposed to free fatty acids. Consistently, Vanin-1 knockdown normalized lipid accumulation in Suv39h2-null hepatocytes. Importantly, a significant correlation between Suv39h2, Vanin-1, and hepatic triglyceride levels was identified in NASH patients.

Our study uncovers a novel mechanism whereby Suv39h2 may contribute to NASH pathogenesis and suggests that targeting the Suv39h2-Vanin-1 axis may yield novel therapeutic solutions against NASH.

Metabolic-associated steatotic liver disease (MASLD) is a cluster of pathological conditions primarily developed due to the accumulation of ectopic fat in the hepatocytes. During the severe form of the disease, i.e., metabolic-associated steatohepatitis (MASH), accumulated lipids promote lipotoxicity, resulting in cellular inflammation, oxidative stress, and hepatocellular ballooning. If left untreated, the advanced form of the disease progresses to fibrosis of the tissue, resulting in irreversible hepatic cirrhosis or the development of hepatocellular carcinoma. Although numerous mechanisms have been identified as significant contributors to the development and advancement of MASLD, altered lipid metabolism continues to stand out as a major factor contributing to the disease. This paper briefly discusses the dysregulation in lipid metabolism during various stages of MASLD.

Nonalcoholic steatohepatitis (NASH) is a chronic progressive liver disease that can progress to cirrhosis and hepatocellular carcinoma. The prevalence of NASH is increasing year by year. However, the etiology and progression of NASH, along with the processes leading to carcinogenesis, remain poorly understood. A range of animal models are used in research, but investigators have been unable to establish a model that results in tumorigenesis from a stable disease state. The present study aimed to create a stable, low-mortality model of NASH using abdominal ultrasonography (US) to assess NASH stage and diagnose liver tumors.

Thirty-four 19-week-old male C57BL/6J mice were fed a choline-deficient, high-fat (CDHF) diet. Twenty animals were given seven courses of 0.8 % dextran sulfate sodium (DSS) for 7 days followed by 10 days of MilliQ water (CDHF+DSS group). The remaining 14 animals drank only MilliQ water (CDHF group). All animals were weighed weekly and US was performed on Days 35 and 120. After necropsy, samples were taken for biochemical analysis and histopathological evaluation.

The CDHF+DSS group had significantly lower body weight on Days 35 and 120, and significantly higher liver/body weight (%) on Day 35 compared to the CDHF group. US on Days 35 and 120 revealed significantly shorter long intestine and higher colonic histological score in the CDHF+DSS group compared to the CDHF group. IL-1β and IL-6 levels in the large intestinal tissue were significantly higher in the CDHF+DSS group.

A stable, low-mortality model of NASH was created with a CDHF diet and intermittent 0.8 % DSS. Abdominal US can assess the degree of fatty degeneration and evaluate liver tumorigenesis without necropsy. This assessment procedure will reduce the number of mice killed unnecessarily during experiments, thereby contributing to animal welfare.

Non-alcoholic steatohepatitis (NASH) is a serious public health issue associated with the obesity pandemic. Obesity is the main risk factor for the non-alcoholic fatty liver disease (NAFLD), which progresses to NASH and then to end-stage liver disease. Currently, there are no specific pharmacotherapies of NAFLD/NASH approved by the FDA or other national regulatory bodies and the treatment includes lifestyle adjustment and medicines for improving lipid metabolism, enhancing sensitivity to insulin, balancing oxidation, and counteracting fibrosis. Accordingly, further basic research and development of new therapeutic approaches are greatly needed. Mesenchymal stem cells (MSCs) and MSC-derived extracellular vesicles prevent induced hepatocyte death in vitro and attenuate NASH symptoms in animal models of the disease. They interact with hepatocytes directly, but also target other liver cells, including Kupffer cells and macrophages recruited from the blood flow. This review provides an update on the pathogenesis of NAFLD/NASH and the key role of macrophages in the development of the disease. We examine in detail the mechanisms of the cross-talk between the MSCs and the macrophages, which are likely to be among the key targets of MSCs and their derivatives in the course of NAFLD/NASH cell therapy.

Accumulation of lipotoxic lipids, such as free cholesterol, induces hepatocyte death and subsequent inflammation and fibrosis in the pathogenesis of nonalcoholic steatohepatitis (NASH). However, the underlying mechanisms remain unclear. We have previously reported that hepatocyte death locally induces phenotypic changes in the macrophages surrounding the corpse and remnant lipids, thereby promoting liver fibrosis in a murine model of NASH. Here, we demonstrated that lysosomal cholesterol overload triggers lysosomal dysfunction and profibrotic activation of macrophages during the development of NASH. β-cyclodextrin polyrotaxane (βCD-PRX), a unique supramolecule, is designed to elicit free cholesterol from lysosomes. Treatment with βCD-PRX ameliorated cholesterol accumulation and profibrotic activation of macrophages surrounding dead hepatocytes with cholesterol crystals, thereby suppressing liver fibrosis in a NASH model, without affecting the hepatic cholesterol levels. In vitro experiments revealed that cholesterol-induced lysosomal stress triggered profibrotic activation in macrophages predisposed to the steatotic microenvironment. This study provides evidence that dysregulated cholesterol metabolism in macrophages would be a novel mechanism of NASH.

Nonalcoholic fatty liver disease (NAFLD, including nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH)) is a high-prevalence disorder, affecting about 1 billion people, which can evolve to more severe conditions like cirrhosis or hepatocellular carcinoma. NAFLD is often concomitant with conditions of the metabolic syndrome, such as central obesity and insulin-resistance, but a specific drug able to revert NAFL and prevent its evolution towards NASH is still lacking. With the liver being a key organ in metabolic processes, the potential therapeutic strategies are many, and range from directly targeting the lipid metabolism to the prevention of tissue inflammation. However, side effects have been reported for the drugs tested up to now. In this review, different approaches to the treatment of NAFLD are presented, including newer therapies and ongoing clinical trials. Particular focus is placed on the reverse cholesterol transport system and on the agonists for nuclear factors like PPAR and FXR, but also drugs initially developed for other conditions such as incretins and thyromimetics along with validated natural compounds that have anti-inflammatory potential. This work provides an overview of the different therapeutic strategies currently being tested for NAFLD, other than, or along with, the recommendation of weight loss.

Nonalcoholic steatohepatitis (NASH) can lead to cirrhosis and hepatocellular carcinoma in their advanced stages; however, there are currently no approved therapies. Here, we show that microRNA (miR)-33b in hepatocytes is critical for the development of NASH. miR-33b is located in the intron of sterol regulatory element-binding transcription factor 1 and is abundantly expressed in humans, but absent in rodents. miR-33b knock-in (KI) mice, which have a miR-33b sequence in the same intron of sterol regulatory element-binding transcription factor 1 as humans and express miR-33b similar to humans, exhibit NASH under high-fat diet feeding. This condition is ameliorated by hepatocyte-specific miR-33b deficiency but unaffected by macrophage-specific miR-33b deficiency. Anti-miR-33b oligonucleotide improves the phenotype of NASH in miR-33b KI mice fed a Gubra Amylin NASH diet, which induces miR-33b and worsens NASH more than a high-fat diet. Anti-miR-33b treatment reduces hepatic free cholesterol and triglyceride accumulation through up-regulation of the lipid metabolism-related target genes. Furthermore, it decreases the expression of fibrosis marker genes in cultured hepatic stellate cells. Thus, inhibition of miR-33b using nucleic acid medicine is a promising treatment for NASH.

Nonalcoholic fatty liver disease (NAFLD) is a range of pathologies arising from fat accumulation in the liver in the absence of excess alcohol use or other causes of liver disease. Its complications include cirrhosis and liver failure, hepatocellular carcinoma, and eventual death. NAFLD is the most common cause of liver disease globally and is estimated to affect nearly one-third of individuals in the United States. Despite knowledge that the incidence and prevalence of NAFLD are increasing, the pathophysiology of the disease and its progression to cirrhosis remain insufficiently understood. The molecular pathogenesis of NAFLD involves insulin resistance, inflammation, oxidative stress, and endoplasmic reticulum stress. Better insight into these molecular pathways would allow for therapies that target specific stages of NAFLD. Preclinical animal models have aided in defining these mechanisms and have served as platforms for screening and testing of potential therapeutic approaches. In this review, we will discuss the cellular and molecular mechanisms thought to contribute to NAFLD, with a focus on the role of animal models in elucidating these mechanisms and in developing therapies.

The rising prevalence of nonalcoholic fatty liver disease (NAFLD)-related cirrhosis highlights the need for a better understanding of the molecular mechanisms responsible for driving the transition of hepatic steatosis (fatty liver; NAFL) to steatohepatitis (NASH) and fibrosis/cirrhosis. Obesity-related insulin resistance (IR) is a well-known hallmark of early NAFLD progression, yet the mechanism linking aberrant insulin signaling to hepatocyte inflammation has remained unclear. Recently, as a function of more distinctly defining the regulation of mechanistic pathways, hepatocyte toxicity as mediated by hepatic free cholesterol and its metabolites has emerged as fundamental to the subsequent necroinflammation/fibrosis characteristics of NASH. More specifically, aberrant hepatocyte insulin signaling, as found with IR, leads to dysregulation in bile acid biosynthetic pathways with the subsequent intracellular accumulation of mitochondrial CYP27A1-derived cholesterol metabolites, (25R)26-hydroxycholesterol and 3β-Hydroxy-5-cholesten-(25R)26-oic acid, which appear to be responsible for driving hepatocyte toxicity. These findings bring forth a "two-hit" interpretation as to how NAFL progresses to NAFLD: abnormal hepatocyte insulin signaling, as occurs with IR, develops as a "first hit" that sequentially drives the accumulation of toxic CYP27A1-driven cholesterol metabolites as the "second hit". In the following review, we examine the mechanistic pathway by which mitochondria-derived cholesterol metabolites drive the development of NASH. Insights into mechanistic approaches for effective NASH intervention are provided.

Cholesterol is a crucial component of membrane bilayers by regulating their structural and functional properties. Cholesterol traffics to different cellular compartments including mitochondria, whose cholesterol content is low compared to other cell membranes. Despite the limited availability of cholesterol in the inner mitochondrial membrane (IMM), the metabolism of cholesterol in the IMM plays important physiological roles, acting as the precursor for the synthesis of steroid hormones and neurosteroids in steroidogenic tissues and specific neurons, respectively, or the synthesis of bile acids through an alternative pathway in the liver. Accumulation of cholesterol in mitochondria above physiological levels has a negative impact on mitochondrial function through several mechanisms, including the limitation of crucial antioxidant defenses, such as the glutathione redox cycle, increased generation of reactive oxygen species and consequent oxidative modification of cardiolipin, and defective assembly of respiratory supercomplexes. These adverse consequences of increased mitochondrial cholesterol trafficking trigger the onset of oxidative stress and cell death, and, ultimately, contribute to the development of diverse diseases, including metabolic liver diseases (i.e. fatty liver disease and liver cancer), as well as lysosomal disorders (i.e. Niemann-Pick type C disease) and neurodegenerative diseases (i.e. Alzheimer's disease). In this review, we summarize the metabolism and regulation of mitochondrial cholesterol and its potential impact on liver and neurodegenerative diseases.

Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is emerging as the most common cause of liver disease. For evaluation of therapies, there is a pressing need to identify non-invasive, mechanism-based biomarkers. A pro-fibrotic process relevant to human NASH involves a pathway in which a transcriptional regulator called TAZ (WWTR1) in hepatocytes induces the secretion of pro-fibrotic Indian hedgehog (IHH). We therefore reasoned that circulating IHH may be a useful mechanism-based marker to assess changes in NASH fibrosis.

Circulating IHH was assessed in wild-type and hepatocyte-TAZ-silenced NASH mice and in three separate cohorts of patients with mild-moderate NASH.

Circulating IHH was elevated in mice with diet-induced NASH compared with chow-fed mice or with NASH mice in which hepatocyte TAZ was silenced, which is an effective means to decrease NASH fibrosis. In patients with fatty liver disease with or without NASH, NASH fibrosis was associated with increased concentrations of circulating IHH.

The results of these analyses support further investigation to determine whether circulating IHH may be useful as a mechanism-based indicator of target engagement in anticipated future clinical trials testing NASH fibrosis therapies that block the IHH pathway.

Non-alcoholic steatohepatitis (NASH)-induced liver fibrosis is a common cause of liver disease. Circulating biomarkers that reflect liver fibrosis in NASH would be very useful to evaluate therapies. One mechanism of NASH fibrosis with potential as a therapeutic target involves a liver-secreted protein called Indian hedgehog (IHH). We report that circulating levels of IHH in experimental and human NASH associates with NASH and NASH-associated liver fibrosis, providing the premise for further investigation into using circulating IHH to evaluate anticipated future NASH therapies that block the IHH pathway in liver.

Patients with metabolic syndrome are often prescribed statins to prevent the development of cardiovascular disease. Conversely, data on their effects on non-alcoholic steatohepatitis (NASH) are lacking. We evaluated these effects by feeding APOE*3-Leiden mice a Western-type diet (WTD) with or without atorvastatin to induce NASH and hepatic fibrosis. Besides the well-known plasma cholesterol lowering (-30%) and anti-atherogenic effects (severe lesion size -48%), atorvastatin significantly reduced hepatic steatosis (-22%), the number of aggregated inflammatory cells in the liver (-80%) and hepatic fibrosis (-92%) compared to WTD-fed mice. Furthermore, atorvastatin-treated mice showed less immunohistochemically stained areas of inflammation markers. Atorvastatin prevented accumulation of free cholesterol in the form of cholesterol crystals (-78%). Cholesterol crystals are potent inducers of the NLRP3 inflammasome pathway and atorvastatin prevented its activation, which resulted in reduced expression of the pro-inflammatory cytokines interleukin (IL)-1β (-61%) and IL-18 (-26%). Transcriptome analysis confirmed strong reducing effects of atorvastatin on inflammatory mediators, including NLRP3, NFκB and TLR4. The present study demonstrates that atorvastatin reduces hepatic steatosis, inflammation and fibrosis and prevents cholesterol crystal formation, thereby precluding NLRP3 inflammasome activation. This may render atorvastatin treatment as an attractive approach to reduce NAFLD and prevent progression into NASH in dyslipidemic patients.

Hepatic cholesterol overload promotes steatohepatitis. Insufficient understanding of liver stress defense impedes therapy development. Here, we elucidate the role of stress defense transcription factors, nuclear factor erythroid 2 related factor-1 (NRF1) and -2 (NRF2), in counteracting cholesterol-linked liver stress. Using a diet that increases liver cholesterol storage, expression profiles and phenotypes of liver from mice with hepatocyte deficiency of NRF1, NRF2, or both are compared with controls, and chromatin immunoprecipitation sequencing is undertaken to identify target genes. Results show NRF1 and NRF2 co-regulate genes that eliminate cholesterol and mitigate inflammation and oxidative damage. Combined deficiency, but not deficiency of either alone, results in severe steatohepatitis, hepatic cholesterol overload and crystallization, altered bile acid metabolism, and decreased biliary cholesterol. Moreover, therapeutic effects of NRF2-activating drug bardoxolone require NRF1 and are supplemented by NRF1 overexpression. Thus, we discover complementary gene programming by NRF1 and NRF2 that counteract cholesterol-associated fatty liver disease progression.

Non-alcoholic fatty liver disease (NAFLD) is a global pandemic affecting 25% of the world's population and is a serious health and economic concern worldwide. NAFLD is mainly the result of unhealthy dietary habits combined with sedentary lifestyle, although some genetic contributions to NAFLD have been documented. NAFLD is characterized by the excessive accumulation of triglycerides (TGs) in hepatocytes and encompasses a spectrum of chronic liver abnormalities, ranging from simple steatosis (NAFL) to steatohepatitis (NASH), significant liver fibrosis, cirrhosis, and hepatocellular carcinoma. Although the molecular mechanisms that cause the progression of steatosis to severe liver damage are not fully understood, metabolic-dysfunction-associated fatty liver disease is strong evidence that mitochondrial dysfunction plays a significant role in the development and progression of NAFLD. Mitochondria are highly dynamic organelles that undergo functional and structural adaptations to meet the metabolic requirements of the cell. Alterations in nutrient availability or cellular energy needs can modify mitochondria formation through biogenesis or the opposite processes of fission and fusion and fragmentation. In NAFL, simple steatosis can be seen as an adaptive response to storing lipotoxic free fatty acids (FFAs) as inert TGs due to chronic perturbation in lipid metabolism and lipotoxic insults. However, when liver hepatocytes' adaptive mechanisms are overburdened, lipotoxicity occurs, contributing to reactive oxygen species (ROS) formation, mitochondrial dysfunction, and endoplasmic reticulum (ER) stress. Impaired mitochondrial fatty acid oxidation, reduction in mitochondrial quality, and disrupted mitochondrial function are associated with a decrease in the energy levels and impaired redox balance and negatively affect mitochondria hepatocyte tolerance towards damaging hits. However, the sequence of events underlying mitochondrial failure from steatosis to hepatocarcinoma is still yet to be fully clarified. This review provides an overview of our understanding of mitochondrial adaptation in initial NAFLD stages and highlights how hepatic mitochondrial dysfunction and heterogeneity contribute to disease pathophysiology progression, from steatosis to hepatocellular carcinoma. Improving our understanding of different aspects of hepatocytes' mitochondrial physiology in the context of disease development and progression is crucial to improving diagnosis, management, and therapy of NAFLD/NASH.