Our study reveals that in both in vivo and in vitro models of NASH, the cholesterol synthesis pathway is activated, accompanied by hepatic FC deposition and concurrent inflammation and fibrosis, while MST1 expression is reduced. MST1 deficiency promotes SREBP2 nuclear translocation, upregulates the expression of key cholesterol synthesis genes, enhances hepatic FC accumulation, and exacerbates NASH-related liver damage, inflammation, and fibrosis. Our findings indicate that MST1 regulates SREBP2 through the phosphorylation of AMPKα at the Thr172 site. MST1 overexpression upregulates AMPKα Thr172 phosphorylation, inhibits SREBP2 nuclear translocation, downregulates cholesterol synthesis gene expression, reduces hepatic FC deposition, and ameliorates NASH-related liver inflammation and fibrosis.
NASH, as a progressive stage of NAFLD, has been widely associated with hepatic accumulation of toxic lipid-free cholesterol (FC). Lipidomic analyses in human liver tissues indicate significantly elevated FC levels in NASH patients compared to NAFL individuals.32 In NAFLD patients, the co-occurrence of NASH and fibrosis parallels the hepatic aggregation of FC. Experimental induction of hepatic FC accumulation accelerates liver inflammation and fibrosis, while correcting excessive hepatic FC load can alleviate the severity of NASH.33 In NASH patients and NASH mouse models induced by high fat and high cholesterol (HFHC) diets, cholesterol crystals formed in the LDs of steatotic hepatocytes can activate Kupffer cells,34 KCs secrete proinflammatory cytokines, such as TNF-α, IL-1β, and IL-6, which recruit neutrophils and circulating monocytes through the interaction of CCL2 and CCR2. Monocytes differentiate into proinflammatory macrophages, further amplifying liver inflammation, while stimulating hepatic stellate cells through TGF-β, promoting their transformation into activated myofibroblasts, thereby promoting fibrosis.35, 36 In the study of NASH model mice induced by a high-fat and high cholesterol diet, we also observed a correlation consistent with the above research results, further clarifying the correlation between liver FC overload and NASH inflammatory injury.
As an important site for cholesterol metabolism, the liver is known to have many disruptions in liver cholesterol metabolism throughout the progression of NAFLD, which can lead to liver cholesterol deposition. Hyperinsulinemia and inflammation lead to the loss of SCAP/SREBP2 inhibition, causing excessive cholesterol accumulation in the liver of mice.37–39 NAFLD/NASH patients exhibit increased nuclear SREBP2, HMGCR mRNA, HMGCR protein, and phosphorylation levels alongside decreased LDLR, ABCA1, ABCG1, and ABCG5 mRNA levels. This imbalance results in heightened cholesterol synthesis and reduced excretion, contributing to increased hepatic cholesterol accumulation.32,33,37–40 In our findings from the NASH model mice, serum TC, LDL, hepatic TC, and FC levels were notably elevated. However, SREBP2 activity remained unimpeded, leading to increased nuclear SREBP2 expression and enhanced transcriptional expression of cholesterol synthesis genes HMGCR and HMGCS1. These results align with the previously mentioned disruptions in cholesterol synthesis metabolism.
As a core kinase in the Hippo signaling pathway, MST1 has been associated with hepatic lipid metabolism disorders. Significant lipid metabolism disturbances are observed in MST1 gene knockout mice, believed to be directly linked to decreased liver Sirt1 expression, enhanced ubiquitination degradation pathways, and increased expression of SREBP-1c.16 However, studies investigating its correlation with hepatic cholesterol metabolism are scarce. Existing studies have prominently highlighted the interplay between core components of the Hippo pathway, such as LATS1/2, Yes-associated protein (YAP), and transcriptional coactivator with PDZ-binding motif (TAZ), with hepatic cholesterol metabolism. Research by Xiaobo Wang et al. demonstrates that excessive hepatic cholesterol induces downstream calcium signaling, triggering RhoA activity and inhibiting LATS1/2, leading to increased protein levels of TAZ and YAP at the transcriptional level. Elevated TAZ in liver cells can induce NASH fibrosis.41,42 Zhiping Shu and colleagues confirmed YAP as a crucial coactivator of SREBP-1c and SREBP-2, controlling the expression of FAS and HMGCR genes in hepatocytes. Overexpression of LATS1 inhibits YAP dephosphorylation and nuclear translocation, improving systemic insulin resistance and hepatic steatosis in diabetic mice.43 Yael Aylon's research demonstrates that LATS2 binds to the endoplasmic reticulum-linked precursors of SREBP-1 and SREBP-2, inhibiting their processing and subsequently dampening the nuclear transcriptional activity of SREBPs.44 These studies collectively suggest a close association between the core component MST1 within the Hippo signaling pathway and hepatic lipid metabolism, cholesterol metabolism, as well as the occurrence and progression of NASH.
Building upon our previous work,20 we propose the potential of MST1 to regulate hepatic cholesterol synthesis metabolism through AMPK. Our study elucidates the interaction between MST1 and AMPK, providing further validation that MST1 can phosphorylate the Thr172 site of AMPK. Treatment of MST1 knockout mice with AICAR confirmed AMPK as a substrate of MST1 and demonstrated its ability to reverse hepatic FC overload and liver damage caused by MST1 deficiency. Additional mechanistic investigations revealed that AMPK regulates the nuclear activation of SREBP2 and, at the transcriptional level, modulates the expression of SREBP2 and its downstream targets HMGCR and HMGCS1. These findings are consistent with prior research by other investigators.31,45–47 By inducing MST1 overexpression in in vivo and in vitro models, we have elucidated that MST1 phosphorylates AMPKα at Thr172, leading to a cascade of changes in the AMPK/SREBP2 signaling pathway. This modulation affects SREBP2 nuclear translocation and alters the expression of SREBP2 and downstream target genes HMGCR and HMGCS1. The reduction in cholesterol synthesis alleviates hepatic free cholesterol load, resulting in an improvement in the degree of NASH-related liver inflammation and fibrosis.
In conclusion, our study demonstrates that MST1 can alleviate hepatic free cholesterol load and thus mitigate the progression of NASH through the regulation of AMPK/SREBP2 signaling. This highlights MST1 as a promising target for the treatment of NAFLD/NASH.