Nonalcoholic fatty liver disease (NAFLD) encompasses a spectrum of liver conditions, ranging from simple steatosis to severe non-alcoholic steatohepatitis (NASH), affecting a substantial portion of the global population. A significant proportion of NAFLD patients progress to hepatic fibrogenesis, potentially leading to cirrhosis and hepatocellular carcinoma. Hepatic stellate cells (HSCs) are the primary effectors of liver fibrosis. In a healthy liver, HSCs maintain a quiescent state, characterized by abundant lipid droplets (LDs) rich in retinol. However, during chronic liver injury, HSCs undergo activation, transforming into myofibroblast-like cells. This transformation involves the loss of LDs, increased α-smooth muscle actin (α-SMA) and α1 type I collagen synthesis, and extracellular matrix (ECM) imbalance, ultimately resulting in liver fibrosis. While significant progress has been made in understanding the genetic mechanisms driving HSC activation, effective therapeutic interventions remain limited. Therefore, strategies targeting HSC activation, inducing apoptosis, or necrosis of activated HSCs are crucial for developing novel therapeutic approaches. Perilipin 5 (PLIN5) is a LD-associated protein pivotal in lipid homeostasis in various mammalian cells. LDs are present in most cell types, including quiescent HSCs. Emerging evidence suggests a link between intracellular lipid content and HSC activation. PLIN5's roles in hepatocytes, heart, skeletal muscle, and islets have been established, encompassing lipid metabolism and insulin secretion. Previous studies hinted at PLIN5's potential in inhibiting HSC activation, but its role in the apoptosis of activated HSCs remains unclear. This study aimed to elucidate PLIN5's role in HSCs and its impact on HFD-induced NAFLD and liver fibrosis in vivo.

The literature extensively documents the pathogenesis of NAFLD and the crucial role of HSCs in liver fibrogenesis. Studies have highlighted the complex interplay between lipid metabolism, inflammation, and HSC activation. The importance of targeting HSCs for therapeutic intervention has been widely recognized. Research on PLIN5 has primarily focused on its function in lipid droplet metabolism and energy homeostasis in various tissues. However, knowledge regarding PLIN5’s function specifically in the liver, particularly concerning HSCs and NAFLD, remains limited. Previous studies suggested a potential inhibitory effect of PLIN5 on HSC activation, but this relationship required further investigation, especially its connection to HSC apoptosis and the molecular pathways involved. This study built upon previous research to comprehensively investigate the role of PLIN5 in HSC activation and its contribution to NAFLD pathogenesis.

The study employed both in vitro and in vivo approaches. In vitro experiments utilized Sprague-Dawley rats and C57BL/6J mice. HSCs were isolated from rat livers through pronase-collagenase perfusion and density gradient centrifugation. Activated HSCs were obtained through prolonged culturing or passaging. PLIN5 was overexpressed in activated HSCs using a lentiviral system. PLIN5 knockout mice were generated and fed a high-fat diet (HFD) for 20 weeks. Various assays were performed to measure TG, GSH, caspase-3 activity, ATP levels, and mitochondrial DNA copy number. Untargeted metabolomic analysis of mouse liver tissue was conducted using UPLC-MS/MS. Western blotting and qPCR were used to detect AMPK, mitochondrial function, cell proliferation, and apoptosis-related genes and proteins. In vivo experiments involved feeding PLIN5 knockout mice and wild-type C57BL/6J mice with either a normal diet or an HFD for 20 weeks. Liver tissues were subjected to histological examination (H&E, Sirius red, Masson's trichrome staining), and biochemical analyses were performed to assess lipid accumulation, fibrosis, and glucose homeostasis. Untargeted metabolomics was employed to identify metabolic changes associated with PLIN5 knockout. Statistical analysis involved one-way ANOVA with Tukey's post hoc test and Student's t-test. Multivariate statistical analysis (OPLS-DA) was used for metabolomics data analysis.

In vitro studies revealed that PLIN5 overexpression in activated HSCs significantly decreased mitochondrial ATP levels, inhibited cell proliferation, and increased cell apoptosis through AMPK activation. PLIN5 knockout in mice fed an HFD led to reduced liver fat deposition, decreased LD abundance and size, and attenuated liver fibrosis. Metabolomic analysis identified significant changes in glutamate and glutathione metabolism in PLIN5 knockout mice. In vivo experiments confirmed that PLIN5 knockout mice showed improved glucose homeostasis, reduced hepatic steatosis, and significantly less liver fibrosis compared to wild-type mice fed an HFD. Furthermore, PLIN5 knockout reduced the expression of pro-fibrotic markers (COL1A1 and α-SMA) and improved hepatic triglyceride content. The study also demonstrated that PLIN5 overexpression caused mitochondrial dysfunction in activated HSCs, characterized by decreased PGC-1α expression, reduced mtDNA copy number, and diminished citrate synthase activity, ultimately leading to AMPK activation, cell cycle arrest, and increased apoptosis. The addition of mitochondrial pyruvate (MP) reversed the effects of PLIN5 overexpression on HSCs, indicating the crucial role of mitochondrial metabolism in this process. In contrast, the caspase inhibitor Z-VAD-FMK only partially reversed PLIN5-induced apoptosis, suggesting that other mechanisms might be involved. The study also found that PLIN5 knockout mice exhibited upregulation of AMPK activity and downregulation of pro-apoptotic proteins, further supporting the protective role of PLIN5 deletion in HFD-induced liver injury.

The study's findings demonstrate a novel regulatory role for PLIN5 in HSCs and its impact on NAFLD progression. PLIN5 overexpression in HSCs promotes AMPK activation, leading to decreased mitochondrial function, reduced cell proliferation, and increased apoptosis. Conversely, PLIN5 deletion in vivo resulted in decreased liver steatosis, fibrosis, and improved glucose homeostasis. The observed metabolic changes, particularly in glutamate and glutathione metabolism, point to a complex interplay between PLIN5 and oxidative stress. The in vitro findings highlighting the role of mitochondrial dysfunction and AMPK activation are largely consistent with the in vivo observations, where PLIN5 knockout was associated with improved mitochondrial function and AMPK activation. However, some discrepancies between in vitro and in vivo results might stem from the fact that the in vivo model involves the whole organism, whereas in vitro experiments focused on isolated HSCs, ignoring the complex interactions among different cell types in the liver. The study also revealed that PLIN5 knockout mice showed improved hepatic GSH levels. Reduced GSH has been implicated in the development of apoptosis, indicating a potential protective mechanism for PLIN5 knockout against NAFLD through the reduction of oxidative stress. The results suggest that PLIN5 plays a significant role in regulating HSC activation and contributes to the progression of NAFLD.

This research reveals a critical role for PLIN5 in regulating HSC activation and NAFLD pathogenesis. PLIN5 overexpression in HSCs leads to mitochondrial dysfunction and apoptosis through AMPK activation, whereas PLIN5 deletion mitigates HFD-induced liver injury, steatosis, and fibrosis. Future research could focus on tissue-specific PLIN5 deletion in hepatocytes or HSCs to further clarify its effects and explore potential therapeutic strategies targeting PLIN5 for NAFLD treatment.

The study's limitations include the use of a whole-body PLIN5 knockout mouse model, which doesn't allow for isolating the effects of PLIN5 specifically in HSCs or hepatocytes. Future studies using conditional knockout mice targeting specific cell types would enhance the understanding of PLIN5's role in NAFLD. The in vitro study used oleic acid to induce LD formation, which might not completely replicate the in vivo condition. Further investigation is needed to fully elucidate the interactions between PLIN5 and other molecular pathways involved in NAFLD.