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Perilipin 5 deletion protects against nonalcoholic fatty liver disease and hepatocellular carcinoma by modulating lipid metabolism and inflammatory responses

ABSTRACT: The molecular mechanisms underlying nonalcoholic fatty liver disease (NAFLD) transition to hepatocellular carcinoma (HCC) are incompletely understood. During NAFLD development, Perilipin 5 (PLIN5) can regulate lipid metabolism by suppressing lipolysis and preventing lipotoxicity. Other reports suggest that lack of PLIN5 decreases hepatic injury, indicating a protective role in NAFLD pathology. To better understand the role of PLIN5 in liver disease, we established mouse models of NAFLD and NAFLD-induced HCC, in which wild type and Plin5 null mice were exposed to a single dose of acetone or 7,12-dimethylbenz[a]anthracene (DMBA) in acetone, followed by a 30-week high-fat diet supplemented with glucose/fructose. In the NAFLD model, RNA-seq revealed significant changes in genes related to lipid metabolism and immune response. At the protein level, signaling pathways such as AMP-activated protein kinase (AMPK), signal transducer and activator of transcription 3 (STAT3), Janus kinase (JNK) and protein kinase B (AKT) were blunted in Plin5 deficient (Plin5-/-) mice compared to wild type mice (WT). In the NAFLD-HCC model, only WT mice developed hepatic tumors, while Plin5-/- mice were resistant to tumorigenesis. Furthermore, only 32 differentially expressed genes were identified that were associated with NAFLD progession in Plin5 null mice. Markers of mitochondrial function and immune response such as the peroxisome proliferator‐activated receptor-γ, coactivator 1‐α (PGC-1a) and pSTAT3 were decreased. Lipidomics analysis revealed differential levels of some sphingomyelins between WT and Plin5-/- mice. Interestingly, these changes were not detected in the HCC model, indicating a possible shift in the metabolism of sphingomelins during carcinogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE242852 | GEO | 2024/03/11

REPOSITORIES: GEO

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Project description:Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in tissues that are active in fatty acid oxidation such as the heart, oxidative muscle, the liver, and brown adipocytes. PLIN5 shares homology with PLIN1: it prevents lipolysis in basal state but promotes lipolysis upon the activation of protein kinase A (PKA). In vitro studies expressing exogenous PLIN5 have shown that PLIN5 is phosphorylated at serine 155 (S155) by PKA. In addition to the regulation of lipolysis, phosphorylation of PLIN5 at S155 has been shown to regulate expression of genes in lipid metabolism, mitochondria, inflammation, and autophagy by traveling to the nucleus and activating peroxisome proliferater-activated-receptor-gamma-coactivator-1-α (PGC1a). However, a role of PLIN5 phosphorylation at S155 has been primarily studied in vitro with exception of a few studies expressing exogenous wild type (WT) and phosphorylation resistant PLIN5 (S155A) in the heart or the liver of mice. Here, we aim to determine the extent of endogenous PLIN5 phosphorylation in vivo and its role in the regulation of lipid metabolism and gene expression in the liver using PLIN5 S155A knock in mice (S155A). First, we enriched LDs from the liver of fed and fasted C57BL6 WT mice to measure non-phosphorylated and S155 phosphorylated PLIN5 by LC-MS/MS. While peak area of non-phosphorylated PLIN5 was similar, S155 phosphorylation was increased in the liver from fasted mice compared with fed mice confirming that endogenous PLIN5 is phosphorylated at S155 when PKA is activated during fasting (3.0-fold increase, p<0.05). Then, we utilized Phos-tag gel as a simple method to assess the extent of PLIN5 phosphorylation. When the heart and the liver from WT and S155A mice were run on Phos-tag gel and immunoblotted for PLIN5, a phosphorylated band was significantly reduced in S155A tissues confirming that S155 is the dominant phosphorylation site in vivo. Next, we assessed phenotypes of S155A mice to gauge the necessity of S155 phosphorylation for metabolism and gene expression in the fasted liver. For the glucose tolerance test, 4-month-old male and female S155A mice showed no differences, but one year-old S115A males had impaired glucose tolerance. There was not a significant difference in weight or blood glucose in fasted young S155A females compared to wild type, but aged male S155A mice had a higher fasted blood glucose (p<0.05) without difference in weight. There was not a significant difference in serum insulin, triglycerides (TG), beta-hydroxybutyrate, or non-esterified fatty acids levels between WT and S155A mice in either males or females. The liver TG contents did not differ between WT and S155A mice of both genders. When qPCR tested the liver of fasted WT and S155A mice, Pgc1a target genes upregulated by fasting and proposed to be regulated by PLIN5 phosphorylation did not show striking difference in expression. Pgc1a, Ppara, Acot1, Pdk4, and Ascl1 expression was similar between WT and S155A liver of fasted mice. Cpt1a and G6pc showed trend of reduction in S155A liver but did not reach statistical significance (n=5 to 7). Inflammation markers such as IL1b and Ccl2 did not differ between WT and S155A liver of fasted mice either. We performed unbiased RNA sequencing of WT and S155A liver from fasted female mice to identify genes differentially regulated by phosphorylation of PLIN5 at S155. There were limited number of genes differentially expressed in S155A female livers with 3 genes < 0.05 for adjusted p value. Interestingly, RNA seq identified insulin receptor substrate 1 (Irs1) as a gene with significantly decreased expression in the liver of female S155A mice (Log2 fold change -0.74, adjusted p value 0.003). The difference was confirmed in qPCR of fasted liver for the female mice (p<0.05), but not for males. Interestingly, insulin receptor substrate 2 (Irs2) measured by qPCR was not different in the liver of female S155A mice but was reduced in male S155A mice (p<0.05). The reduction of IRS2 protein in the liver of S155A male mice was confirmed by Western blot. In conclusion, endogenous PLIN5 in the liver increases phosphorylation at S155 upon fasting in mice. However, the loss of S155 phosphorylation does not affect serum lipids or liver TG. Also, changes in the expression of PGC1a target genes were subtle in the liver of fasted S155A mice compared with WT mice indicating that the loss of PLIN5 S155 phosphorylation can be readily compensated by other mechanisms. Unexpectedly, IRS2 showed decreased expression in the liver of S155A male mice that may explain glucose intolerance in aged male mice. Thus, PLIN5 phosphorylation may impact the insulin signaling in the liver by supporting IRS2 expression in male mice.

Project description:Immunotherapy has opened hitherto unknown possibilities to treat cancer. Whereas some cancer types (e.g. melanoma) can be efficiently treated, others lack measurable positive effects (e.g. PDAC). Moreover, stratification of responders/non-responders is only possible in some cancer types (e.g. melanoma). Hepatocellular carcinoma (HCC) has a dismal prognosis, limited treatment options and survival benefit, and represents a potential cancer entity for successful immunotherapy. Here, we investigated NASH-triggered HCC in the context PD-1-targeted immunotherapy. Using flow cytometry, single cell RNA sequencing, immunohistochemistry and mass spectrometric analyses, we found a progressive increase of CD8+PD-1+ effector T-cells with a unique profile of exhaustion and activation markers rising with murine and human NASH severity. Notably, late-stage HCC treatment with PD-1-targeted immunotherapy enhanced hepatic carcinogenesis in mice. Dissecting potential mechanisms of action during tumor-initiation and -progression we analyzed the effects of PD-1-targeted immunotherapy at HCC initiation. PD-1-targeted immunotherapy induced a pro-tumorigenic environment, enhanced necro-inflammation and increased NAFLD-activation score (NAS), leading to increased liver cancer incidence, tumor number and nodule size. In contrast, anti-CD8 or anti-CD8/anti-NK1.1 treatment reduced NAS and abrogated the development of liver cancer, thus identifying CD8+PD-1+ T-cells as drivers of liver cancer in NASH-triggered HCC. Increased apoptotic signaling, STAT3 phosphorylation and hepatic proliferation were detected in intra-tumoral liver tissue upon PD-1-targeted immunotherapy. In line, PD-1-/- mice challenged with a NASH diet displayed early onset of hepatocarcinogenesis, corroborating the pro-tumorigenic role of absent or reduced PD-1. Mechanistically PD-1-targeted immunotherapy mainly affected hepatic abundance of CD8+PD-1+ T-cells, instead of altering the quality of Tox+CXCR6+ expressing CD8+PD-1+TNF+CD39+Gzmb+ T-cells found in NASH livers, leading to an aggressive, pro-tumorigenic liver environment. Single-cell mapping of human NASH-, borderline NASH- or unaffected livers corroborated our preclinical NASH results. Moreover, in human NASH livers a correlation of hepatic CD8+, PD-1+, TNF+ T-cells with fibrosis and NASH severity was observed. Accordingly, HCC patients with NASH etiology display a sharp increase in intra- and peri-tumoral CD8+ PD-1+ T-cells. In a cohort of 65 patients recruited across 6 centers in Germany and Austria, patients with NAFLD/NASH-driven HCC responded worse to PD-1-targeted immunotherapy by Nivolumab or Pembrolizumab compared to non-NAFLD patients. This resulted in significant reduced overall survival, in trends of faster disease progression and reduced progression free survival. Histological analysis of livers derived from HCC patients treated with PD-1-targeted immunotherapy displayed high levels of intra and peri-tumoral CD8+ PD-1+ T-cells and Ki67+ hepatocytes. Taken together, these data indicate that PD-1-targeted immunotherapy induces immune-related adverse effects in NAFLD/NASH-driven HCC through CD8+PD-1+ T-cells. Our data call for stratification of HCC patients subjected to PD-1-targeted immunotherapy, with NAFLD being a negative predictor.

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Perilipin 5 deletion protects against nonalcoholic fatty liver disease and hepatocellular carcinoma by modulating lipid metabolism and inflammatory responses

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