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The prevalence of metabolic disorders characterized by chronic inflammation has been on a sharp rise for decades. As such, tools that address metabolic and inflammatory dysregulation are of great importance. Plant stanols are well-known for reducing intestinal cholesterol absorption and may also have direct anti-inflammatory effects. In this study, our aim was to investigate to what extent the benefits of dietary plant stanol supplementation depend on dietary cholesterol intake in an experimental mouse model for cholesterol-induced metabolic inflammation. Here, we used Ldlr−/− mice transplanted with Npc1nih-derived bone marrow, featuring feature bone marrow-derived immune cells characterized by chronic inflammation induced by lysosomal lipid accumulation. Npc1nih- and Npc1wt-transplanted mice were placed on either a high fat, high cholesterol (HFC) or on a chow diet low in cholesterol, with or without 2% plant stanols supplementation. At the end of the study, the metabolic and inflammatory status of the mice was analyzed. Plant stanol supplementation to the HFC diet reduced liver cholesterol levels and improved lipid metabolism and liver inflammation, particularly in Npc1nih-tp mice. In contrast, plant stanol supplementation to the chow diet did not significantly improve the aforementioned parameters, though similar reductive trends to those in the HFC diet setting were observed regarding liver cholesterol accumulation and liver inflammatory markers. The effects of dietary plant stanol supplementation on dietary cholesterol-induced inflammation are largely dependent on dietary cholesterol intake. Future research should verify whether other models of metabolic inflammation exhibit similar stanol-related effects on inflammation.

Dietary and lifestyle changes are leading to an increased occurrence of non-alcoholic fatty liver disease (NAFLD). Using a hyperlipidemic murine model for non-alcoholic steatohepatitis (NASH), we have previously demonstrated that the lysosomal protease cathepsin D (CTSD) is involved with lipid dysregulation and inflammation. However, despite identifying CTSD as a major player in NAFLD pathogenesis, the specific role of extracellular CTSD in NAFLD has not yet been investigated. Given that inhibition of intracellular CTSD is highly unfavorable due to its fundamental physiological function, we here investigated the impact of a highly specific and potent small-molecule inhibitor of extracellular CTSD (CTD-002) in the context of NAFLD. Treatment of bone marrow-derived macrophages with CTD-002, and incubation of hepatic HepG2 cells with a conditioned medium derived from CTD-002-treated macrophages, resulted in reduced levels of inflammation and improved cholesterol metabolism. Treatment with CTD-002 improved hepatic steatosis in high fat diet-fed rats. Additionally, plasma levels of insulin and hepatic transaminases were significantly reduced upon CTD-002 administration. Collectively, our findings demonstrate for the first time that modulation of extracellular CTSD can serve as a novel therapeutic modality for NAFLD.

Recently, the importance of lysosomes in the context of the metabolic syndrome has received increased attention. Increased lysosomal cholesterol storage and cholesterol crystallization inside macrophages have been linked to several metabolic diseases, such as atherosclerosis and non-alcoholic fatty liver disease (NAFLD). Two-hydroxypropyl-beta-cyclodextrin (HP-B-CD) is able to redirect lysosomal cholesterol to the cytoplasm in Niemann-Pick type C1 disease, a lysosomal storage disorder. We hypothesize that HP-B-CD ameliorates liver cholesterol and intracellular cholesterol levels inside Kupffer cells (KCs). Hyperlipidemic low-density lipoprotein receptor knockout (Ldlr(-/-)) mice were given weekly, subcutaneous injections with HP-B-CD or control PBS. In contrast to control injections, hyperlipidemic mice treated with HP-B-CD demonstrated a shift in intracellular cholesterol distribution towards cytoplasmic cholesteryl ester (CE) storage and a decrease in cholesterol crystallization inside KCs. Compared to untreated hyperlipidemic mice, the foamy KC appearance and liver cholesterol remained similar upon HP-B-CD administration, while hepatic campesterol and 7alpha-hydroxycholesterol levels were back increased. Thus, HP-B-CD could be a useful tool to improve intracellular cholesterol levels in the context of the metabolic syndrome, possibly through modulation of phyto- and oxysterols, and should be tested in the future. Additionally, these data underline the existence of a shared etiology between lysosomal storage diseases and NAFLD.