Differentially Expressed Genes in Response to a Squalene-Supplemented Diet Are Accurate Discriminants of Porcine Non-Alcoholic Steatohepatitis.

Squalene is the major unsaponifiable component of virgin olive oil, the fat source of the Mediterranean diet. To evaluate its effect on the hepatic transcriptome, RNA sequencing was carried out in two groups of male Large White x Landrace pigs developing nonalcoholic steatohepatitis by feeding them a high fat/cholesterol/fructose and methionine and choline-deficient steatotic diet or the same diet with 0.5% squalene. Hepatic lipids, squalene content, steatosis, activity (ballooning + inflammation), and SAF (steatosis + activity + fibrosis) scores were analyzed. Pigs receiving the latter diet showed hepatic squalene accumulation and twelve significantly differentially expressed hepatic genes (log2 fold change < 1.5 or <1.5) correlating in a gene network. These pigs also had lower hepatic triglycerides and lipid droplet areas and higher cellular ballooning. Glutamyl aminopeptidase (ENPEP) was correlated with triglyceride content, while alpha-fetoprotein (AFP), neutralized E3 ubiquitin protein ligase 3 (NEURL3), 2'-5'-oligoadenylate synthase-like protein (OASL), and protein phosphatase 1 regulatory inhibitor subunit 1B (PPP1R1B) were correlated with activity reflecting inflammation and ballooning, and NEURL3 with the SAF score. AFP, ENPEP, and PPP1R1B exhibited a remarkably strong discriminant power compared to those pathological parameters in both experimental groups. Moreover, the expression of PPP1R1B, TMEM45B, AFP, and ENPEP followed the same pattern in vitro using human hepatoma (HEPG2) and mouse liver 12 (AML12) cell lines incubated with squalene, indicating a direct effect of squalene on these expressions. These findings suggest that squalene accumulated in the liver is able to modulate gene expression changes that may influence the progression of non-alcoholic steatohepatitis.

Squalene through Its Post-Squalene Metabolites Is a Modulator of Hepatic Transcriptome in Rabbits.

Squalene is a natural bioactive triterpene and an important intermediate in the biosynthesis of sterols. To assess the effect of this compound on the hepatic transcriptome, RNA-sequencing was carried out in two groups of male New Zealand rabbits fed either a diet enriched with 1% sunflower oil or the same diet with 0.5% squalene for 4 weeks. Hepatic lipids, lipid droplet area, squalene, and sterols were also monitored. The Squalene administration downregulated 9 transcripts and upregulated 13 transcripts. The gene ontology of transcripts fitted into the following main categories: transporter of proteins and sterols, lipid metabolism, lipogenesis, anti-inflammatory and anti-cancer properties. When the results were confirmed by RT-qPCR, rabbits receiving squalene displayed significant hepatic expression changes of LOC100344884 (PNPLA3), GCK, TFCP2L1, ASCL1, ACSS2, OST4, FAM91A1, MYH6, LRRC39, LOC108176846, GLT1D1 and TREH. A squalene-enriched diet increased hepatic levels of squalene, lanosterol, dihydrolanosterol, lathosterol, zymostenol and desmosterol. Strong correlations were found among specific sterols and some squalene-changed transcripts. Incubation of the murine AML12 hepatic cell line in the presence of lanosterol, dihydrolanosterol, zymostenol and desmosterol reproduced the observed changes in the expressions of Acss2, Fam91a1 and Pnpla3. In conclusion, these findings indicate that the squalene and post-squalene metabolites play important roles in hepatic transcriptional changes required to protect the liver against malfunction.

Characterization of novel mechanisms for steatosis from global protein hyperacetylation in ethanol-induced mouse hepatocytes.

Steatosis is the earliest and most common disease of the liver due to chronic ethanol consumption, and stems from alterations in the function of transcription factors related to lipid metabolism. Protein acetylation at the lysine residue (Kac) is known to have diverse functions in cell metabolism. Recent studies showed that ethanol exposure induces global protein hyperacetylation by reducing the deacetylase activities of SIRT1 and SIRT3. Although global acetylome analyses have revealed the involvement of a variety of lysine acetylation sites, the exact sites directly regulated by ethanol exposure are unknown. In this study, to elucidate the exact hyperacetylation sites that contribute to SIRT1 and SIRT3 downregulation, we identified and quantified a total of 1285 Kac sites and 686 Kac proteins in AML-12 cells after ethanol treatment (100 mM) for 3 days. All quantified Kac sites were divided into four quantiles: Q1 (0-15%), Q2 (15-50%), Q3 (50-85%), and Q4 (85-100%). Q4 had 192 Kac sites indicating ethanol-induced hyperacetylation. Using the Motif-x program, the [LXKL], [KH], and [KW] motifs were included in the Q4 category, where [KW] was a specific residue for SIRT3. We also performed gene ontology term and KEGG pathway enrichment analyses. Hyperacetylation sites were significantly enriched in biosynthetic processes and ATPase activities within the biological process and molecular function categories, respectively. In conclusion, ethanol regulates the acetylation of proteins in a variety of metabolic pathways mediated by SIRT1 and SIRT3. As a result, ethanol stimulates increased de novo fatty acid synthesis in hepatocytes.

Schisandrin B inhibits cell growth and induces cellular apoptosis and autophagy in mouse hepatocytes and macrophages: implications for its hepatotoxicity.

A number of drugs and herbal compounds have been documented to cause hepatoxicity. Schisandrin B (Sch B) is an active dibenzocyclooctadiene isolated from Schisandrae fructus, with a wide array of pharmacological activities. However, the potential hepatotoxicity of Sch B is a major safety concern, and the underlying mechanism for Sch B-induced liver toxic effects is not fully elucidated. In the present study, we aimed to investigate the liver toxic effects and the molecular mechanisms of Sch B in mouse liver and macrophage cells. The results have shown that Sch B exhibits potent grow inhibitory, proapoptotic, and proautophagic effects in AML-12 and RAW 264.7 cells. Sch B markedly arrested cells in G1 phase in both cell lines, accompanied by the down-regulation of cyclin dependent kinase 2 (CDK2) and cyclin D1 and up-regulation of p27 Kip1 and checkpoint kinase 1. Furthermore, Sch B markedly increased the apoptosis of AML-12 and RAW 264.7 cells with a decrease in the expression of B-cell lymphoma-extra-large and (Bcl-xl) B-cell lymphoma 2 (Bcl-2), but an increase in the expression of B-cell lymphoma 2-associated X protein (Bax). Sch B promoted the cleavage of caspase 3 and poly-adenosine diphosphate-ribose polymerase (PARP) in both cell lines. Additionally, Sch B significantly induced autophagy of AML-12 and RAW 264.7 cells. Sch B inhibited the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, as indicated by their altered phosphorylation, contributing to the proautophagic effect of Sch B. Taken together, our findings show that the inducing effects of Sch B on cell cycle arrest, apoptosis, and autophagy may contribute to its liver toxic effects, which might provide a clue for the investigation of the molecular toxic targets and underlying mechanisms for Sch B-induced hepatotoxicity in herbal consumers. More studies are warranted to fully delineate the underlying mechanisms, efficacy, and safety of Sch B for clinical use.

Retinoic acid regulates several genes in bile acid and lipid metabolism via upregulation of small heterodimer partner in hepatocytes.

Retinoic acid (RA) affects multiple aspects of development, embryogenesis and cell differentiation processes. The liver is a major organ that stores RA suggesting that retinoids play an important role in the function of hepatocytes. In our previous studies, we have demonstrated the involvement of small heterodimer partner (SHP) in RA-induced signaling in a non-transformed hepatic cell line AML 12. In the present study, we have identified several critical genes in lipid homeostasis (Apoa1, Apoa2 and ApoF) that are repressed by RA-treatment in a SHP dependent manner, in vitro and also in vivo with the use of the SHP null mice. In a similar manner, RA also represses several critical genes involved in bile acid metabolism (Cyp7a1, Cyp8b1, Mdr2, Bsep, Baat and Ntcp) via upregulation of SHP. Collectively our data suggest that SHP plays a major role in RA-induced potential changes in pathophysiology of metabolic disorders in the liver.