Analyses of serum lipoprotein lipids in the present study using a mouse model of MASH-related fibrosis revealed that the levels of cholesterol in very-small LDL and very-large HDL and levels of both TGs and cholesterol in very-small HDL are associated with the severity of fibrosis. Levels of these lipoprotein subfractions increased in a manner dependent on fibrosis severity.
The concentrations of very-small LDL subfractions that showed dynamic changes in this study were identical to changes in levels of lipids known to increase with progression to simple steatosis and MASH in humans. These results suggest that the serum lipid profiles observed in the present study might be similar to those in humans and not specific only to mice. Previous reports of studies conducted in humans and animal models have mainly focused on analyses of lipid profiles during the phase of progression from steatosis to steatohepatitis, and few studies have focused on fibrosis progression. This is the first study to comprehensively examine the relationship between MASH-related fibrosis and serum lipid dynamics in lipoprotein subfractions.
LDL is composed of cholesterol, TGs, and phospholipids. In this study, LDL was classified into large, medium, small, and very-small LDL subfractions. sdLDL is readily oxidized and reportedly useful as a marker of coronary atherosclerosis [21–23]. In human clinical studies, sdLDL levels are reportedly higher in MASH patients than in patients with simple steatosis [18, 19]. The size of sdLDL particles (< 25.5 nm) in those studies corresponds to the middle, small, and very-small LDL subfractions of the present study. In line with a previous study, high concentrations of very-small LDL-C are associated with high NAS [19]. We also found a positive correlation between liver fibrosis progression and very-small LDL-C levels, which indicates that very-small LDL-C plays a role in liver fibrosis in MASH.
Serum levels of total HDL-C are known to be low in dyslipidemia in patients with MASLD [24]. HDL plays a role in the reverse cholesterol transport, transferring excess cholesterol from the periphery to the liver [25], and its dynamics may be affected by cholesterol in extrahepatic tissues. The formation of HDL initially involves ApoA1 from the liver and small intestine together with cholesterol and phospholipids as small HDL disks. The volume of these disk-shaped HDL particles increases via esterification of the surrounding cholesterol by lecithin-cholesterol acyltransferase to form spherical HDL [26]. In the present study, discoidal HDL was classified as very-small HDL, and spherical HDL particles were classified as very-large, large, medium, and small HDL. Our results showed no significant changes in serum total HDL-C concentrations with progression of liver fibrosis, but the subfraction levels of very-large and very-small HDL-C increased markedly with fibrosis progression. It is interesting to note that the HDL subfractions in which the lipid concentrations increased with liver fibrosis progression were those with the largest and smallest diameter. The levels of neither very-large HDL-C nor very-small HDL-C differed significantly when grouped based on NAS. This suggests that the lipids in these two lipoprotein subfractions that increased in level are fibrosis specific.
Cholic acid, a primary bile acid, is commonly used to promote absorption of lipids in diets high in fat and cholesterol, and it also decreases serum TG levels [27, 28]. As expected, TG levels in most lipoprotein subfractions decreased in the present study; however, it is noteworthy that only TG levels in very-small HDL increased with fibrosis. HDL-TG is associated with metabolic syndrome, diabetes, and obesity [29]. HDL3, which represents very-small HDL, is inherently anti-inflammatory, but TG-rich HDL3 is less anti-inflammatory than HDL with a lower TG content [30]. Therefore, it is possible that the increased levels of very-small HDL-TGs indicate a lack of anti-inflammatory activity by HDL3 in mice with severe fibrosis in the present study. These results suggest that focusing on both the quality and quantity of lipoproteins could aid in elucidating the mechanism of pathogenesis for conditions such as liver inflammation and fibrosis and facilitate the identification of new drug targets.
A limitation of this study must be considered. Mice were fed a cholesterol-added iHFC diet to induce fibrosis. No perfect diet-dependent increase in serum cholesterol was observed in our study, but an effect of dietary cholesterol on the increase in serum cholesterol cannot be completely excluded. Further studies will be necessary utilizing a diet-independent fibrosis induction model.
In summary, cholesterol levels in small LDL, large HDL, and very-small HDL as well as TG levels in very-small HDL were higher in model mice with advanced fibrosis than in those with mild fibrosis. In particular, the dynamic changes in lipids in the HDL subfraction were significantly related to progression of liver fibrosis, and these data could aid in elucidating the pathogenesis of liver fibrosis and facilitate the discovery of new markers.