Editor’s Note: In recent years, the global incidence of Non-Alcoholic Fatty Liver Disease (NAFLD) has been continuously rising, becoming the most common cause of chronic liver disease worldwide, with an increasing prevalence globally. In developed areas of China, the prevalence of NAFLD among adults is as high as 15%, making it the leading cause of chronic liver disease in the country. Currently, the treatment of NAFLD primarily focuses on improving metabolic syndrome, and there is a lack of widely clinically validated therapeutic drugs. However, with a deeper understanding of its pathogenesis in recent years, some new drugs still in animal testing and early clinical research stages have attracted attention. At the 32nd Annual Meeting of the Asia-Pacific Association for the Study of the Liver (APASL), the team led by Professor Ning Qin of Tongji Medical College, Huazhong University of Science and Technology, China, reported new research progress, providing new intervention strategies for the clinical treatment of NAFLD.
NAFLD Disease Burden and Harm
NAFLD is one of the major chronic liver diseases globally, with an overall prevalence of about 32.4% [1]. In China, the prevalence of NAFLD rose from 23.8% in the early 20th century to 32.9% in 2018, becoming the most common liver disease in the country [2].
The spectrum of NAFLD includes simple steatosis (NAFL), Non-Alcoholic Steatohepatitis (NASH), cirrhosis, and hepatocellular carcinoma (HCC). NAFL, characterized by more than 5% of the liver undergoing fatty degeneration without significant inflammation and hepatocyte damage, is the most common form of clinical NAFLD. 10%-25% of NAFLD patients may progress to the NASH stage, which involves liver inflammation and cell damage. Patients in the advanced stages have a significantly increased risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma [3]. The large population base, combined with an increasing incidence rate, makes NAFLD an urgent public health issue worldwide [4].
Despite some progress in elucidating the pathogenesis of NAFLD, identifying therapeutic targets, and advancing drug development, the exact mechanisms of NAFLD-related liver lipid accumulation and inflammatory damage are still unclear, greatly limiting the development of clinical drugs [5]. Therefore, a deeper analysis of the exact mechanisms and influencing factors of NAFLD lipid accumulation and inflammatory damage is a prerequisite for advancing the development of clinical drugs for NAFLD.
Autophagy’s Role in NAFLD Liver Lipid Accumulation and Inflammatory Damage
Autophagy, specifically macroautophagy, is a highly conserved metabolic process activated in cells under stress. It serves as an internal quality control system, degrading damaged cell components and providing energy for stressed cells, typically considered a cellular protective mechanism [6]. Extensive research has shown that autophagy dysfunction is a crucial component in the pathogenesis of NAFLD.
Within liver cells, autophagy directly participates in lipid metabolism by degrading lipid droplets [7], and indirectly by maintaining the normal function of organelles and proteases involved in lipid metabolism [8]. However, under the chronic lipid stimulation characteristic of NAFLD, liver cell autophagy becomes dysfunctional, which may be one of the intrinsic mechanisms for lipid accumulation in NAFLD liver cells.
On the other hand, autophagy is closely related to inflammation. It can inhibit inflammation by degrading intracellular inflammasomes and other key inflammatory pathway molecules, combat cell inflammatory damage through mechanisms like clearing inflammatory stimuli (bacteria, viruses, damaged organelles or proteins), inhibiting inflammatory phenotypes in immune cells, or suppressing STING activation [9]. Autophagy can also mediate the transmission of inflammatory signals between cells through secretory autophagy, exosomes, NETS, etc. [10]. In NAFLD liver, the relationship between autophagy and inflammation has been extensively researched, and improving autophagy is beneficial in combating liver inflammatory damage and fibrosis progression.
Published studies on NAFLD liver autophagy abnormalities involve aspects like upstream regulation of autophagy, autophagosome maturation, autophagosome-lysosome fusion, and lysosomal function, covering the entire autophagy pathway. Most research focuses on upstream regulation, with proteins like HIF-1α, Rubicon, MTOR, AMPK, TFEB, and ATG7 receiving widespread attention. However, there is still no proven clinical intervention, and studies on autophagosome maturation, fusion, and lysosomal function are relatively scarce.
NPC1 Mutation Induces Autophagy Abnormalities and Liver Steatosis
Cholesterol transport protein NPC1, a transmembrane protein on lysosomal and late endosomal membranes, transports cholesterol bound to lysobisphosphatidic acid (LBPA) on late endosomal membranes, aided by soluble cholesterol transport protein NPC2. NPC1 on late endosomal and lysosomal membranes regulates intracellular cholesterol and lipid redistribution, participating in the balance of cellular lipid metabolism. Genome-wide association studies in humans have confirmed the significance of common NPC1 variations in adult obesity and type 2 diabetes, with NPC1 heterozygous mutations increasing the risk of adult obesity.
Mutations in the NPC1 gene lead to a rare hereditary neurovisceral disease, Niemann-Pick disease (NPCD), characterized by hepatosplenomegaly and subsequent neurodegenerative changes. Hepatomegaly originates from intracellular cholesterol and lipid accumulation, a type of lysosomal storage disease (LSD). Interestingly, NPC1 mutant cells not only have lipid accumulation and inflammatory damage similar to NAFLD cells but also exhibit similar autophagy changes, indicated by significant increases in autophagy markers LC3II and autophagic degradation substrate P62, suggesting abnormalities in the downstream autophagy pathway (autophagosome-lysosome fusion or lysosomal degradation).
Furthermore, LBPA, an upstream molecule in NPC1 cholesterol transport, not only binds cholesterol and other lipids to increase the rate of cholesterol and lipid transport but also assists in protein sorting by the insulin-like growth factor 2 receptor (IGF-2R), participating in the sorting and transport of newly synthesized cellular proteins and likely involved in the sorting of lysosomal proteases, affecting lysosomal function. Moreover, stimulation with DOPG (a precursor of LBPA) or Thioperamide (proven to increase intracellular LBPA levels) significantly improves cell autophagy abnormalities and lipid accumulation induced by NPC1 mutation.
Autophagy Abnormalities in NAFLD Mouse and Cell Models, Reduced NPC1 and LBPA Levels
Based on the above background, it’s easy to link autophagy abnormalities in NAFLD liver with those in NPC1 mutant cells. To confirm this hypothesis, we established NAFLD mouse and human cell models by feeding mice a high-fat diet (HFD) for 16 weeks and stimulating THP1 or LO2 cell lines with palmitic acid (PA) in vitro. Consistent with most previous studies, autophagosome markers LC3II and autophagy substrate P62 significantly increased in the NAFLD group, while upstream regulatory proteins of autophagy such as P-AMPK, Beclin-1, ATG7, and PI3K showed no significant changes, indicating that the autophagy abnormality in NAFLD is likely located downstream in the autophagy pathway. Subsequent in vivo and in vitro stimulation of NAFLD mice and cell models with the upstream autophagy agonist rapamycin (RAPA) also confirmed that merely stimulating upstream autophagy does not significantly improve NAFLD-induced autophagy abnormalities and lipid accumulation.
Next, we examined the levels of NPC1 and LBPA in the livers of NAFLD mice. Immunofluorescence and Western results showed that NPC1 and LBPA levels were significantly reduced in the NAFLD group (Figure 1). Simultaneously, stimulation with LBPA antibodies and the NPCD cell model inducer U18666A in vitro induced increased levels of LC3II and P62 in cells, further confirming the possibility of this connection.
Figure 1. Significant Reduction in NPC1 Expression in NAFLD Mouse Liver
Targeting NPC1 or LBPA Improves NAFLD Cell Autophagy In Vitro
To further confirm the relationship between NAFLD liver’s deficiency in NPC1 or LBPA and autophagy abnormalities, we selected three intervention molecules targeting NPC1 or LBPA: Hydroxypropyl-β-cyclodextrin (HP-β-CD, which improves NPC1 function), DOPG (a precursor of LBPA, supplementing LBPA), and Thioperamide (stimulating an increase in cellular LBPA levels). In vitro, all three molecules could improve the autophagy abnormalities induced by NAFLD to some extent. However, repeated experiments showed that methods targeting LBPA had more pronounced effects (Figure 2).
Figure 2. Improvement of NAFLD Cell Autophagy In Vitro Through Targeting NPC1 or LBPA
Thioperamide Combined with RAPA Significantly Improves Lipid Accumulation in Mouse Liver Induced by HFD
Based on the results of in vitro experiments, we examined the in vivo effects of DOPG and Thioperamide by intraperitoneal injection. The results indicated that DOPG or Thioperamide alone could not significantly improve the hepatic steatosis induced by a high-fat diet (HFD) in mice, but they did increase the levels of LBPA in liver cells. However, the combination of the autophagy upstream agonist RAPA significantly improved hepatic steatosis in NAFLD mice, with the combination of Thioperamide and RAPA showing a more pronounced effect (Figure 3). Subsequent electron microscopy revealed that liver cells in HFD mice accumulated autophagosomes. Treatment with Thioperamide significantly reduced autophagic bodies, and the combination of Thioperamide and RAPA increased autophagosomes compared to Thioperamide alone. Additionally, special multi-lamellar body structures formed in the cells, and damaged organelles were observed entering single-layer vesicles for degradation.
These findings suggest that LBPA deficiency is involved in the formation of downstream autophagy abnormalities in NAFLD mouse liver. Targeting LBPA is a viable strategy to improve NAFLD by enhancing autophagy function. However, since the autophagy upstream in NAFLD liver is not activated, it is necessary to combine with autophagy upstream agonists to increase autophagic flux, thereby jointly improving the lipid deposition in NAFLD liver.
Figure 3. Thioperamide Combined with Rapamycin Significantly Improves Diet-Induced NAFLD in Mice
This figure demonstrates the effectiveness of combining Thioperamide with Rapamycin in significantly alleviating Non-Alcoholic Fatty Liver Disease (NAFLD) caused by a high-fat diet in mice. This combination therapy highlights the potential for targeted treatments in managing NAFLD by addressing specific mechanisms involved in the disease’s progression.
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TAG: APASL 2023, Voice of China, NAFLD
