Thioredoxin domain containing 5 is involved in the hepatic storage of squalene into lipid droplets in a sex-specific way.

Hepatic thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family found associated with anti-steatotic properties of squalene and located in the endoplasmic reticulum and in lipid droplets. Considering that the latter are involved in hepatic squalene accumulation, the present research was aimed to investigate the role of TXNDC5 on hepatic squalene management in mice and in the AML12 hepatic cell line. Wild-type and TXNDC5-deficient (KO) mice were fed Western diets with or without 1% squalene supplementation for 6 weeks. In males, but not in females, absence of TXNDC5 blocked hepatic, but not duodenal, squalene accumulation. Hepatic lipid droplets were isolated and characterized using label-free LC-MS/MS analysis. TXNDC5 accumulated in this subcellular compartment of mice receiving squalene and was absent in TXNDC5-KO male mice. The latter mice were unable to store squalene in lipid droplets. CALR and APMAP were some of the proteins that responded to the squalene administration in all studied conditions. CALR and APMAP were positively associated with lipid droplets in the presence of squalene and they were decreased by the absence of TXNDC5. The increased squalene content was reproduced in vitro using AML12 cells incubated with squalene-loaded nanoparticles and this effect was not observed in an engineered cell line lacking TXNDC5. The phenomenon was also present when incubated in the presence of a squalene epoxidase inhibitor, suggesting a mechanism of squalene exocytosis involving CALR and APMAP. In conclusion, squalene accumulation in hepatic lipid droplets is sex-dependent on TXNDC5 that blocks its secretion.

Endoplasmic Reticulum Protein TXNDC5 Interacts with PRDX6 and HSPA9 to Regulate Glutathione Metabolism and Lipid Peroxidation in the Hepatic AML12 Cell Line.

Non-alcoholic fatty liver disease or steatosis is an accumulation of fat in the liver. Increased amounts of non-esterified fatty acids, calcium deficiency, or insulin resistance may disturb endoplasmic reticulum (ER) homeostasis, which leads to the abnormal accumulation of misfolded proteins, activating the unfolded protein response. The ER is the primary location site for chaperones like thioredoxin domain-containing 5 (TXNDC5). Glutathione participates in cellular oxidative stress, and its interaction with TXNDC5 in the ER may decrease the disulfide bonds of this protein. In addition, glutathione is utilized by glutathione peroxidases to inactivate oxidized lipids. To characterize proteins interacting with TXNDC5, immunoprecipitation and liquid chromatography-mass spectrometry were used. Lipid peroxidation, reduced glutathione, inducible phospholipase A2 (iPLA2) and hepatic transcriptome were assessed in the AML12 and TXNDC5-deficient AML12 cell lines. The results showed that HSPA9 and PRDX6 interact with TXNDC5 in AML12 cells. In addition, TXNDC5 deficiency reduced the protein levels of PRDX6 and HSPA9 in AML12. Moreover, lipid peroxidation, glutathione and iPLA2 activities were significantly decreased in TXNDC5-deficient cells, and to find the cause of the PRDX6 protein reduction, proteasome suppression revealed no considerable effect on it. Finally, hepatic transcripts connected to PRDX6 and HSPA9 indicated an increase in the Dnaja3, Mfn2 and Prdx5 and a decrease in Npm1, Oplah, Gstp3, Gstm6, Gstt1, Serpina1a, Serpina1b, Serpina3m, Hsp90aa1 and Rps14 mRNA levels in AML12 KO cells. In conclusion, the lipid peroxidation system and glutathione mechanism in AML12 cells may be disrupted by the absence of TXNDC5, a novel protein-protein interacting partner of PRDX6 and HSPA9.

Dietary proteins modulate high-density lipoprotein characteristics in a sex-specific way in Apoe-deficient mice.

OBJECTIVES: The type and amount of dietary protein have become a topic of renewed interest, considering their involvement in several diseases. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. In a previous study, we saw that compared with soybean protein, the consumption of avian proteins, depending on sex, resulted in similar or lower atherosclerosis with a higher paraoxonase 1 activity, an antioxidant enzyme carried by high-density lipoproteins (HDL). This suggests that under these conditions, the HDL lipoproteins may undergo important changes. The aim of this research was to study the influence of soybean, chicken, and turkey proteins on the characteristics of HDL. METHODS: Male and female Apoe-deficient mice were fed purified Western diets based on the AIN-93 diet, differing only in the protein source, for 12 wk. After this period, blood and liver samples were taken for analysis of HDL composition and hepatic expression of genes related to HDL metabolism (Abca1, Lcat, Pltp, Pon1, and Scarb1). Depending on sex, these genes define a different network of interactions. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, which can be due to larger very-low-density lipoproteins (VLDLs) calculated by molar ratio triacylglycerols/VLDL cholesterol and higher expression of Lcat. In contrast, in males, a higher ratio of paraoxonase1 to apolipoprotein A1 decreased the oxidative status of the different lipoproteins, and augmented Abca1 expression was observed. CONCLUSIONS: The source of protein has an effect on the development of atherosclerosis depending on sex by modifying HDL characteristics and the expression of genes involved in their properties.

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.

Squalene Loaded Nanoparticles Effectively Protect Hepatic AML12 Cell Lines against Oxidative and Endoplasmic Reticulum Stress in a TXNDC5-Dependent Way.

Virgin olive oil, the main source of fat in the Mediterranean diet, contains a substantial amount of squalene which possesses natural antioxidant properties. Due to its highly hydrophobic nature, its bioavailability is reduced. In order to increase its delivery and potentiate its actions, squalene has been loaded into PLGA nanoparticles (NPs). The characterization of the resulting nanoparticles was assessed by electron microscopy, dynamic light scattering, zeta potential and high-performance liquid chromatography. Reactive oxygen species (ROS) generation and cell viability assays were carried out in AML12 (alpha mouse liver cell line) and a TXNDC5-deficient AML12 cell line (KO), which was generated by CRISPR/cas9 technology. According to the results, squalene was successfully encapsulated in PLGA NPs, and had rapid and efficient cellular uptake at 30 µM squalene concentration. Squalene reduced ROS in AML12, whereas ROS levels increased in KO cells and improved cell viability in both when subjected to oxidative stress by significant induction of Gpx4. Squalene enhanced cell viability in ER-induced stress by decreasing Ern1 or Eif2ak3 expressions. In conclusion, TXNDC5 shows a crucial role in regulating ER-induced stress through different signaling pathways, and squalene protects mouse hepatocytes from oxidative and endoplasmic reticulum stresses by several molecular mechanisms depending on TXNDC5.

Thioredoxin Domain Containing 5 Suppression Elicits Serum Amyloid A-Containing High-Density Lipoproteins.

Thioredoxin domain containing 5 (TXNDC5) is a protein disulfide isomerase involved in several diseases related to oxidative stress, energy metabolism and cellular inflammation. In a previous manuscript, a negative association between fatty liver development and hepatic Txndc5 expression was observed. To study the role of TXNDC5 in the liver, we generated Txndc5-deficient mice. The absence of the protein caused an increased metabolic need to gain weight along with a bigger and fatter liver. RNAseq was performed to elucidate the putative mechanisms, showing a substantial liver overexpression of serum amyloid genes (Saa1, Saa2) with no changes in hepatic protein, but discrete plasma augmentation by the gene inactivation. Higher levels of malonyldialdehyde, apolipoprotein A1 and platelet activating factor-aryl esterase activity were also found in serum from Txndc5-deficient mice. However, no difference in the distribution of high-density lipoproteins (HDL)-mayor components and SAA was found between groups, and even the reactive oxygen species decreased in HDL coming from Txndc5-deficient mice. These results confirm the relation of this gene with hepatic steatosis and with a fasting metabolic derive remedying an acute phase response. Likewise, they pose a new role in modulating the nature of HDL particles, and SAA-containing HDL particles are not particularly oxidized.

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.

Dietary Avian Proteins Are Comparable to Soybean Proteins on the Atherosclerosis Development and Fatty Liver Disease in Apoe-Deficient Mice.

BACKGROUND AND AIM: The type and amount of dietary protein has become a topic of renewed interest in light of their involvement in metabolic diseases, atherosclerosis and thrombosis. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. The aim was to investigate the influence of chicken and turkey as sources of protein compared with that of soybean on atherosclerosis and fatty liver disease. METHODS AND RESULTS: To this purpose, male and female Apoe-deficient were fed purified Western diets differing in their protein sources for 12 weeks. After this period, blood, liver, aortic tree and heart base samples were taken for analyses of plasma lipids and atherosclerosis. Plasma triglycerides, non-esterified fatty acids, esterified cholesterol levels and radical oxygen species in lipoproteins changed depending on the diet and sex. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, as evidenced by the en face atherosclerosis analyses. The presence of macrophages and smooth muscle cells in plaques were not modified, and no changes were observed in hepatic lipid droplets in the studied groups either. Paraoxonase activity was higher in the group consuming turkey protein without sex differences, but only in females, it was significantly associated with aortic lesion areas. CONCLUSIONS: Compared to soybean protein, the consumption of avian proteins depending on sex resulted in similar or lower atherosclerosis development and comparable hepatic steatosis.

Current Insights into the Biological Action of Squalene.

Squalene is a triterpenic compound found in a large number of plants and other sources with a long tradition of research since it was first reported in 1926. Herein a systematic review of studies concerning squalene published in the last 8 years is presented. These studies have provided further support for its antioxidant, anti-inflammatory, and anti-atherosclerotic properties in vivo and in vitro. Moreover, an antineoplastic effect in nutrigenetic-type treatments, which depends on the failing metabolic pathway of tumors, has also been reported. The bioavailability of squalene in cell cultures, animal models, and in humans has been well established, and further progress has been made in regard to the intracellular transport of this lipophilic molecule. Squalene accumulates in the liver and decreases hepatic cholesterol and triglycerides, with these actions being exerted via a complex network of changes in gene expression at both transcriptional and post-transcriptional levels. Its presence in different biological fluids has also been studied. The combination of squalene with other bioactive compounds has been shown to enhance its pleiotropic properties and might lead to the formulation of functional foods and nutraceuticals to control oxidative stress and, therefore, numerous age-related diseases in human and veterinary medicine.