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.

Dietary squalene modifies plasma lipoproteins and hepatic cholesterol metabolism in rabbits.

To evaluate the effects of squalene, the main unsaponifiable component of virgin olive oil, on lipid metabolism, two groups of male New Zealand rabbits were fed a 1% sunflower oil-enriched regular diet or the same diet containing 0.5% squalene for 4 weeks. Plasma triglycerides, total- and HDL-cholesterol and their lipoproteins were assayed. Analyses of hepatic lipid droplets, triglycerides, total- and non-esterified cholesterol, squalene, protein and gene expression, and cholesterol precursors were carried out. In the jejunum, the squalene content and mRNA and protein APOB expressions were measured. Finally, we studied the effect of cholesterol precursors in AML12 cells. Squalene administration significantly increased plasma total cholesterol, mainly carried as non-esterified cholesterol in IDL and large LDL, and corresponded to an increased number of APOB100-containing particles without accumulation of triglycerides and decreased reactive oxygen species. Despite no significant changes in the APOB content in the jejunum, the latter displayed increased APOB mRNA and squalene levels. Increases in the amounts of non-esterified cholesterol, squalene, lanosterol, dihydrolanosterol, lathosterol, cholestanol, zymostenol, desmosterol and caspase 1 were also observed in the liver. Incubation of AML12 cells in the presence of lanosterol increased caspase 1. In conclusion, squalene administration in rabbits increases the number of modified APOB-containing lipoproteins, and hepatic cholesterol biosynthesis is linked to caspase 1 probably through lanosterol.

Hepatic subcellular distribution of squalene changes according to the experimental setting.

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies.

Hepatic subcellular distribution of squalene changes according to the experimental setting

Squalene is the main unsaponifiable component of virgin olive oil, the main source of dietary fat in Mediterranean diet, traditionally associated with a less frequency of cardiovascular diseases. In this study, two experimental approaches were used. In the first, New Zealand rabbits fed for 4 weeks with a chow diet enriched in 1% sunflower oil for the control group, and in 1% of sunflower oil and 0.5% squalene for the squalene group. In the second, APOE KO mice received either Western diet or Western diet enriched in 0.5% squalene for 11 weeks. In both studies, liver samples were obtained and analyzed for their squalene content by gas chromatography-mass spectrometry. Hepatic distribution of squalene was also characterized in isolated subcellular organelles. Our results show that dietary squalene accumulates in the liver and a differential distribution according to studied model. In this regard, rabbits accumulated in cytoplasm within small size vesicles, whose size was not big enough to be considered lipid droplets, rough endoplasmic reticulum, and nuclear and plasma membranes. On the contrary, mice accumulated in large lipid droplets, and smooth reticulum fractions in addition to nuclear and plasma membranes. These results show that the squalene cellular localization may change according to experimental setting and be a starting point to characterize the mechanisms involved in the protective action of dietary squalene in several pathologies