Elevated free cholesterol in a p62 overexpression model of non-alcoholic steatohepatitis

Abstract

AIM:To characterize how insulin-like growth factor 2 (IGF2 ) mRNA binding protein p62/IMP2-2 promotes steatohepatitis in the absence of dietary cholesterol. METHODS:Non-alcoholic steatohepatitis (NASH) was induced in wild-type mice and in mice overexpressing p62 specifically in the liver by feeding the mice a methionine and choline deficient (MCD) diet for either two or four weeks. As a control, animals were fed a methionine and choline supplemented diet. Serum triglycerides, cholesterol, glucose, aspartate aminotransferase and alanine transaminase were determined by standard analytical techniques. Hepatic gene expression was determined by real-time reverse transcription-polymerase chain reaction. Generation of reactive oxygen species in liver tissue was quantified as thiobarbituric acid reactive substances using a photometric assay and malondialdehyde as a standard. Tissue fatty acid profiles and cholesterol levels were analyzed by gas chromatographymass spectrometry after hydrolysis. Hepatocellular iron accumulation was determined by Prussian blue staining in paraffin-embedded formalin-fixed tissue. Filipin staining on frozen liver tissue was used to quantify hepatic free cholesterol levels. Additionally, nuclear localization of the nuclear factor kappa B (NF-κB) subunit p65 was examined in frozen tissues. RESULTS:Liver-specific overexpression of the insulin-like growth factor 2 mRNA binding protein 2-2 (IGF2BP2-2/IMP2-2/p62) induces steatosis with regular chow and amplifies NASH-induced fibrosis in the MCD mouse model. Activation of NF-κB and expression of NF-κB target genes suggested an increased inflammatory response in p62 transgenic animals. Analysis of hepatic lipid composition revealed an elevation of monounsaturated fatty acids as well as increased hepatic cholesterol. Moreover, serum cholesterol was significantly elevated in p62 transgenic mice. Dietary cholesterol represents a critical factor for the development of NASH from hepatic steatosis. Filipin staining revealed increased free cholesterol in p62 transgenic livers, which were not diet-derived. The mRNA levels of the rate-limiting enzyme for cholesterol synthesis 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase or HMGCR) were not significantly upregulated, potentially due to increased cholesterol biosynthesis via elevated sterol regulatory element binding transcription factor 2 (SREBF2 ) gene expression and increased iron deposition in transgenic animals. CONCLUSION:This study provides evidence that p62/IGF2BP2-2 drives the progression of NASH through elevation of hepatic iron deposition and increased production of hepatic free cholesterol.

Updates on Dietary Models of Nonalcoholic Fatty Liver Disease: Current Studies and Insights

“…Several studies have shown that an MCD diet can eventually induce hepatic fibrosis in WT mice 23,24,29,31,34–39 ; however, the time required for mice to develop fibrosis while on an MCD diet varies. In studies by Katsura et al and Simon et al, mice fed an MCD diet for 15 weeks or 2 weeks, respectively, developed fibrosis 35,38 . It is also unclear if longer feeding time on an MCD diet results in more severe fibrosis.…”

“…Several studies have shown that feeding WT mice an MCD diet for as few as 2 weeks and up to 15 weeks resulted in elevated liver enzymes, including aspartate transaminase (AST) and alanine transaminase (ALT) 23,24,31,35,38 . In the study by Itagaki et al WT mice were fed an MCD diet for 30 weeks total and developed increased levels of AST and ALT at only 2 weeks 31 .…”

“…Four studies showed that mice fed an MCD diet for as few as 2 weeks but up to 7 weeks developed no change or had decreased levels of serum triglycerides, cholesterol, and glucose 27,37,38,44 . In contrast, in a study by Kim et al mice that were fed an MCD diet for 4 weeks developed elevated serum triglycerides and total cholesterol levels 22 .…”

“…Although weight gain secondary to WD contributes to developing metabolic syndrome, which is associated with NAFLD in humans, the MCD diet mouse model has consistently been shown to induce weight loss 37,38,46 . Loss in body mass resulting from the MCD diet is thought to be secondary to increased metabolic activity 47 .…”

MCD diet-induced steatohepatitis is associated with alterations in asymmetric dimethylarginine (ADMA) and its transporters

“…Thus, mice or rats fed an MCD diet have frequently been used as animal models for NASH as they help clarify the pathogenesis of these disorders and underpin therapies for them [6]. Liver injury induced by an MCD diet is accompanied by elevated transaminases, protein cytokines, oxidative stress and changes in mitochondrial function [7].…”