Background

Metabolic dysfunction-associated steatotic liver disease (MASLD) is formerly known as nonalcoholic fatty liver disease (NAFLD) and is a major cause of chronic liver disease worldwide [1,2,3,4]. MASLD can develop as hepatic steatosis or severe conditions involving inflammation, such as metabolic dysfunction-associated steatohepatitis (MASH), liver fibrosis, cirrhosis, hepatocellular carcinoma (HCC), and liver failure [3, 4]. MASH is characterized by hepatic inflammation, hepatocyte ballooning, and intrahepatic fat accumulation [5, 6]. Patients with MASH are at a higher risk of liver cancer, liver failure, and cardiovascular disease than are healthy individuals [2, 5]. Key risk factors for MASH include diabetes, obesity, age, ethnicity, sex, and genetic polymorphisms, which can also affect MASH progression [7, 6B). Pathological analysis (Fig. 2) revealed that although both WD and HFC induced MASH, HFC induced a more severe condition—liver fibrosis. Therefore, we further compared the WD group (mild fibrosis) with the HFC group (severe fibrosis) to explore fibrosis-specific metabolites. A total of 17 metabolites were found to be associated with severe liver fibrosis (Fig. 6C). VLDLs and LDLs accounted for 35% and 47%, respectively, of the aforementioned metabolites; both VLDLs and LDLs were found to be predominant in the liver of mice with severe MASH (Fig. 6C). The 17 markers included the large VLDLs V1CH, V1PL, V2CH, V2TG, and V4CH; the small LDLs L5PN, L5CH, L5PL, and L5AB; and the HDL HDTG (Fig. 6C). Taken together, the results indicate VLDL and LDL are involved in the development of MASLD/MASH and induce severe fibrosis. These findings elucidate both the roles of VLDL and LDL as biomarkers of severe MASH and the pathophysiological changes that occur during the progression of MASH.

Fig. 6
figure 6

Expression levels of very low–density lipoprotein and low-density lipoprotein are strongly correlated with the severity of metabolic dysfunction-associated steatohepatitis/fibrosis. A Correlation between serological metabolites and collagen scores. Significant lipoproteins were selected on the basis of a p value of < 0.05 and a correlation coefficient of > 0.3. B Integrated analysis of significant metabolites and metabolites strongly associated with fibrosis. Blue indicates significant metabolites, whereas yellow indicates metabolites strongly correlated with fibrosis. C Shortened list of 17 significant metabolites identified from the comparison between HFC and WD groups (Table on the left). The pie chart presents the proportions of the 17 significant metabolites

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Upregulation of VLDLR expression in mouse models of diet-induced MASH

The liver is the most prominent contributor of lipoproteins because this organ is responsible for both the production and recycling of lipoproteins [47]. Lipoprotein receptors are crucial for systemic lipid metabolism. The expression of lipoprotein receptors, such as VLDLR, LDL receptor (LDLR), and HDL receptor (SR-B1), in normal organs has been studied in humans and mice [47, 48]. VLDL is believed to be produced only by the liver; VLDLR is expressed in the periphery of but not within the liver [49]. In our study, the expression of both LDL and VLDL was upregulated in mouse models of severe MASH, which prompted us to investigate the receptors of these lipoproteins in diseased liver tissues. The expression and distribution of VLDLR, LDLR, and SR-B1 were evaluated through immunohistochemical analysis (Fig. 7A) and was quantified using ImageJ (Fig. 7B). The expression of VLDLR was not similar between the diet- or feeding time–based groups, with the exception of the HCF group, which was fed for 32 weeks (Fig. 7B). After 16 weeks of feeding, the expression of LDLR was markedly downregulated in the experimental groups compared with that in the control group; nonetheless, the expression was gradually restored in the WD and HFC groups after 32 weeks of feeding (Fig. 7B). Notably, the expression of SR-B1 remained high and did not change with diet (Fig. 7B). In summary, the expression of VLDLR is considerably upregulated in severe liver fibrosis. The findings of increases in the levels of serological VLDL and LDL and the upregulation of VLDLR expression in the severe MASH of this study indicate a feedforward mechanism for lipid deposition.

Fig. 7
figure 7

Expression levels of lipoprotein receptors in mouse models of diet-induced MASH. A Results of immunohistochemical staining performed to measure the expression levels of very low–density lipoprotein receptor, low-density lipoprotein receptor, and high-density lipoprotein receptor (SR-B1) in the livers of mice with diet-induced MASH. The brown indicates receptor expression. B Receptor expression levels were quantified using ImageJ and analyzed using GraphPad Prism (version 8). The white, black, blue, and red dots indicate NC, HFD, WD, and HFC, respectively. Statistical significance: *p < 0.05, compared with NC. NC: normal chow; HFC: high-fat diet; WD: Western diet; HFC: high-fat, high-cholesterol; and MASH: metabolic dysfunction-associated steatohepatitis

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VLDLs serve as the biomarkers of liver fibrosis/cirrhosis in humans

The expression of large VLDLs and VLDLR are upregulated in mouse models of diet-induced MASH with severe fibrosis (Figs. 6C and 7). We analyzed the clinical specimens of a retrospective cohort of patients with liver fibrosis/cirrhosis to identify the correlation between serological metabolites and clinical features. The demographic characteristics of our cohort are summarized in Supplementary Table 5. On the basis of their METAVIR scores, the patients were stratified into mild and severe disease groups. A comparison of the metabolome and differential expression of relevant genes were performed. The results revealed significant increases in the levels of the following metabolites (very large VLDLs) in patients with severe fibrosis: XXL_VLDL_CE, XXL_VLDL_C, L_VLDL_CE, and L_VLDL_C (Fig. 8A). Human and experimental (mouse) MASH diseases were similar in terms of the upregulation of the expression of very large VLDLs. In summary, the severity of diet-induced MASH in mouse models can be evaluated to align with clinical diagnostic methods. Metabolomic profiling revealed a likely mechanism of VLDL recycling through VLDLR, which may be involved the pathogenesis of liver fibrosis/cirrhosis.

Fig. 8
figure 8

Very low–density lipoprotein and low-density lipoprotein as key biomarkers of fibrosis/cirrhosis in humans. A Significant metabolites identified through the differential gene expression analysis of a retrospective cohort of patients with fibrosis/cirrhosis. Log2 fold changes were calculated by comparing the FIB4 scores of patients with advanced disease (F3 or F4) with those of patients with no or mild fibrosis (F0–F2). Significant metabolites associated with cirrhosis. Log2 fold changes were determined by comparing patients with cirrhosis with those without cirrhosis. B Changes in lipoproteins during the progression of MASH/fibrosis in mice

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Discussion

In this study, three commonly employed experimental diet–induced MASH models were used to evaluate MASH severity by using clinically relevant diagnostic methods. The serological metabolites associated with MASH severity was identified. The roles of the lipoprotein–receptor axes in the pathogenesis of diet-induced MASH in mice were investigated.

Importance of mouse models of diet-induced MASH in clinical diagnosis

MASH is diagnosed on the basis of histopathological features, such as steatosis, hepatocyte ballooning, and lobular inflammation. Although fibrosis is not a histopathological feature of MASH, it can be used to predict the risk of mortality. The METAVIR scoring system is a commonly used tool for diagnosing fibrosis. The pathogenesis and progression of MASH is complex and involve cellular heterogeneity and alterations in the humoral matrix. Histopathological analysis is important in MASH diagnosis. Rodent models of experimental MASLD/MASH/fibrosis/cirrhosis can be established through diet, genetic modifications, toxin treatment, and a combination of different methods [11, 15, 50]. Few studies have been conducted to systemically compare diet-induced MASLD/MASH models, evaluate their importance in clinical diagnosis, and identify metabolite biomarkers. The present study was conducted to obtain valuable insight into various decompensated liver diseases, such as MASH, fibrosis, and cirrhosis. Inbred rodents are widely used for studying MASLD/MASH from a genetic perspective [51]. The present study was conducted using C57BL/6 mice, which are commonly used in transgenic animal studies.

Importance of noninvasive metabolomic tools in MASH diagnosis

The METAVIR scoring system is an invasive tool that involves liver biopsy, which involves the risks of major vein rupture and internal bleeding. Serological biomarkers, such as FIB4, can serve as noninvasive tools for disease diagnosis. However, the precision of clinical diagnosis performed on the basis of FIB4 is low (receiver operating characteristic curve score, approximately 70%) [52, 53]. In our study, high-throughput metabolomic profiling was performed with NMR spectroscopy; the results revealed a correlation between the phenotypic and metabolomic characteristics of mouse models of MASH. In addition, a novel biomarker of steatohepatitis was identified.

Regarding the relevance of experimental MASH to human MASH, elevated levels of VLDL, VLDL-cholesterol, and LDL-cholesterol can serve as the biomarkers of the progression of hepatic steatosis to MASH (Fig. 8A). High serum levels of total lipid and cholesterol (VLDL and LDL) are associated with intrahepatic cholesterol accumulation and hepatocyte injury in MASH [54, 55]. The similarity between experimental diet-induced MASH and human MASH in terms of lipoprotein metabolites indicated lipoprotein analysis may be valuable for clinical diagnosis. Therefore, the significance of our study lies in its identification of an association of large VLDLs and LDLs with the progression of MASH in animal models and patients (Fig. 8B).

Most studies conducted using animal models of MASH have explored lipid, glucose, and protein metabolites in the liver. Our findings reveal that the levels of lipoproteins increased with the severity of MASH. Through NMR spectroscopy–based metabolomic profiling, both VLDL and LDL were simultaneously explored in mice and humans. The consistency between the experimental MASH and human MASH in terms of metabolite biomarkers indicated that similarities were present in the pathophysiological alternations between the mouse and human, and that NMR spectroscopy–based metabolomic profiling can be valuable for research and clinical diagnosis.

Lipoproteins as the predominant biomarkers of diet-induced MASH in mice

Serum-insoluble lipids circulate in the bloodstream as lipoproteins, which are macromolecular complexes of free cholesterol, cholesterol esters, triglycerides, phospholipids, and apolipoproteins [56]. The liver is the primary site for the synthesis of LDL (16–30 nm) and VLDL (30–80 nm), which carry lipids and ApoB100 to tissue. An essential lipoprotein for the collection and transportation of extra serum cholesterol is HDL (8–16 nm; ApoA). Structurally, VLDL comprises a triglyceride-enriched core surrounded by a monolayer of phospholipids and incorporated proteins (e.g., ApoB-100), which facilitates the delivery and uptake of VLDL. Notably, most triglycerides incorporated into VLDL are derived from exogenous lipids and not synthesized through de novo lipogenesis [57, 58]. The impaired synthesis of ApoB-100 in patients with MASH may be associated with increased free fatty acid level, disrupted redox balance, hyperinsulinemia, and reduced gene expression, all of which hinder ApoB-100 synthesis and VLDL assembly, thus resulting in intrahepatic lipid accumulation [59]. Additionally, VLDL particles can be converted into LDL particles through hydrolyzation of triglycerides by LPL in the bloodstream [56].

In patients with MASLD, the expression of VLDL in the liver is upregulated, leading to increased levels of triglycerides. In addition, the clearance of LDL is reduced, which accelerates the development of atherosclerosis and cardiovascular disease. Excessive lipid storage in the liver promotes the secretion of VLDL and thus dyslipidemia [60]. Furthermore, an increase in the level of oxidized LDL occurs, which induces systemic inflammation [60].

The increase in the mean size of VLDL in patients with MASH and the reduction in the level of small VLDL in patients with liver fibrosis reflect changes in the number and state of hepatocytes resulting from such diseases [61]. Hepatocytes with increased levels of intracellular lipid can serve as the source of large VLDLs. MASH driven by insulin resistance and an increase in the intrahepatic lipid pool may increase the numbers of large VLDLs and the mean size of VLDLs [62,63,64].

Upregulation of VLDL/VLDLR expression indicates a positive feedforward mechanism for hepatic lipid accumulation

In adipose tissues, VLDLR is regulated by peroxisome proliferator–activated receptor [65]. Free cholesterol and fatty acid can promote stress response, inflammation, apoptosis, and fibrosis in the liver [66]. Large VLDLs induce the accumulation of triglycerides in macrophages and exhibit a higher affinity for VLDLR binding than do small VLDLs. Disease states may influence VLDL properties. Hepatic secretion of VLDL is impaired in patients with ApoB mutations, which often leads to fatty liver disease because of the excessive intrahepatic accumulation of fat. The extent of steatosis is associated with the size and number of VLDLs in the patient population [67]. Patients with hepatic steatosis and insulin resistance have increased levels of circulating ApoC-III, which is a strong inhibitor of LPL. After the LPL-mediated hydrolysis of triglycerides, lipoprotein remnants are removed through receptor-mediated pathways, primarily those operated in the liver. Obesity and insulin resistance contribute to reduced LDL clearance by reducing the activities of LDLR and LDLR-related protein 1, among others [68]. The intrahepatic accumulation of LDL due to reduced receptor-mediated uptake may directly inhibit LPL, resulting in a feedforward mechanism that drives lipid deposition and MASH development [69,70,71].

The dysregulation of lipid homeostasis in hepatocytes leads to the generation of toxic lipids that impair organelle functions, promoting inflammation, hepatocellular damage, and apoptosis [69]. In patients with MASH, the uptake of circulating lipids, particularly free fatty acids and lipoproteins, by the liver is higher [72]. Because the hepatic secretion of lipoprotein is higher in patients with MASLD, the deposition of fat in hepatocytes disrupts lipid homeostasis in these cells [72]. Further studies are required to evaluate the quantity and quality of changes in lipid metabolites during the pathogenesis of MASH.

Conclusions

Our findings provide key insight into the pathophysiology and serological metabolomics of experimental diet-induced MASH in relation to human MASH. The finding of an upregulation of lipoprotein expression indicates a feedforward mechanism underlies MASH development, and this mechanism may be targeted for the development of noninvasive diagnostic strategies.