PNPLA3-I148M Variant Promotes the Progression of Liver Fibrosis by Inducing Mitochondrial Dysfunction

Abstract

Patatin-like phospholipase domain-containing 3 (PNPLA3) rs738409 polymorphism (I148M) is strongly associated with non-alcoholic steatohepatitis and advanced fibrosis; however, the underlying mechanisms remain largely unknown. In this study, we investigated the effect of PNPLA3-I148M on the activation of hepatic stellate cell line LX-2 and the progression of liver fibrosis. Immunofluorescence staining and enzyme-linked immunosorbent assay were used to detect lipid accumulation. The expression levels of fibrosis, cholesterol metabolism, and mitochondria-related markers were measured via real-time PCR or western blotting. Electron microscopy was applied to analyze the ultrastructure of the mitochondria. Mitochondrial respiration was measured by a Seahorse XFe96 analyzer. PNPLA3-I148M significantly promoted intracellular free cholesterol aggregation in LX-2 cells by decreasing cholesterol efflux protein (ABCG1) expression; it subsequently induced mitochondrial dysfunction characterized by attenuated ATP production and mitochondrial membrane potential, elevated ROS levels, caused mitochondrial structural damage, altered the oxygen consumption rate, and decreased the expression of mitochondrial-function-related proteins. Our results demonstrated for the first time that PNPLA3-I148M causes mitochondrial dysfunction of LX-2 cells through the accumulation of free cholesterol, thereby promoting the activation of LX-2 cells and the development of liver fibrosis.

Comparison of Wild-Type and High-risk PNPLA3 variants in a Human Biomimetic Liver Microphysiology System for Metabolic Dysfunction-associated Steatotic Liver Disease Precision Therapy

“…At the conclusion of the experimental time course, 96-well LAMPS were labeled with BODIPY 581/591 C11, a ratiometric fluorescent sensor for LPO, to determine the ratio of oxidized BODIPY (green) to reduced BODIPY (yellow) reflecting the overall LPO status within the model. Consistent with recent studies (76, 77), a significant increase in the ratio of Oxidized BODIPY/reduced BODIPY was observed in PNPLA3 GG variant 96-well plate LAMPS compared to the CC wild type in each media condition, demonstrating increased ROS production associated with the PNPLA3 GG variant (Fig S4A and S4B) .…”

“…Previous studies have shown that the increased steatosis sociated with MASLD progression impairs mitochondrial oxidative capacity resulting in the increased production of reactive oxygen species (ROS) (74, 75). Moreover, it has also recently been demonstrated that the PNPLA3 GG variant promotes the progression of MASLD by inducing mitochondrial dysfunction (76, 77). We next quantitatively assessed lipid peroxidation (LPO) in a 96-well plate format LAMPS (62) constructed with PNPLA3 GG variant or CC wild type hepatocytes and nonparenchymal cells (LSECs, LX-2 cells and THP-1 cells) that were maintained in NF, EMS, or LMS media for 5 days (Fig S4) .…”

In Vitro Lipid Overload Affects Cellular Proliferation, Apoptosis, and Senescence in a Time-Dependent Manner in HepG2 Hepatocytes and LX-2 Hepatic Stellate Cells

“…The pharmacologic inhibition of acetyl-coenzyme A carboxylase, which is involved in de novo lipogenesis, prevented HSC activation in a rodent model of nonalcoholic fatty liver disease [30]. This suggests that lipid challenge, fatty acid oxidation, de novo lipogenesis induction, and the presence of PNPLA3 polymorphisms involved in the steatotic liver might influence HSC activation and, thus, fibrosis progression [30][31][32].…”

Lipid droplets in steatotic liver disease

“…Genetic association studies have highlighted that some of the genetic factors which influence the individual's susceptibility to SLD are strongly associated with lipid droplet biogenesis and turnover (Table 1) [29,34,36,[39][40][41][42][43]. Specifically, certain genetic variants encoding lipid droplet proteins have been identified as instrumental in the onset and progression of SLD.…”