Dietary squalene increases high density lipoprotein-cholesterol and paraoxonase 1 and decreases oxidative stress in mice

Background and Purpose: Squalene, the main hydrocarbon in the unsaponifiable fraction of virgin olive oil, is involved in cholesterol synthesis and it has been reported to own antiatherosclerotic and antiesteatosic effects. However, the squalene’s role on lipid plasma parameters and the influence of genotype on this effect need to be addressed. Experimental Approaches: Three male mouse models (wild-type, Apoa1- and Apoe- deficient) were fed chow semisynthetic diets enriched in squalene to provide a dose of 1 g/kg during 11 weeks. After this period, their plasma parameters and lipoprotein profiles were analyzed. Key Results: Squalene administration at a dose of 1 g/kg showed decreased reactive oxygen species in lipoprotein fractions independently of the animal background and caused an specific increase in high density lipoprotein (HDL)-cholesterol levels, accompanied by an increase in phosphatidylcholine and paraoxonase 1 and no changes in apolipoproteins A1 and A4 in wild-type mice. In these mice, the cholesterol increase was due to its esterified form and associated with an increased hepatic expression of Lcat. These effects were not observed in absence of apolipoprotein A1. The increases in HDL- paraoxonase 1 were translated into decreased plasma malondialdehyde levels depending on the presence of Apolipoprotein A1. Conclusions and Implications: Dietary squalene promotes changes in HDL- cholesterol and paraoxonase 1 and decreases reactive oxygen species in lipoproteins and plasma malondialdehyde levels, providing new benefits of its intake that might contribute to explain the properties of virgin olive oil, although the phenotype related to apolipoproteins A1 and E may be particularly relevant

Dietary erythrodiol modifies hepatic transcriptome in mice in a sex and dose-dependent way

Erythrodiol is a terpenic compound found in a large number of plants. To test the hypotheses that its long-term administration may influence hepatic transcriptome and this could be influenced by the presence of APOA1-containing high-density lipoproteins (HDL), Western diets containing 0.01% of erythrodiol (10 mg/kg dose) were provided to Apoe-and Apoa1-deficient mice. Hepatic RNA-sequencing was carried out in male Apoe-deficient mice fed purified Western diets differing in the erythrodiol content. The administration of this compound significantly up-regulated 68 and down-regulated 124 genes at the level of 2-fold change. These genes belonged to detoxification processes, protein metabolism and nucleic acid related metabolites. Gene expression changes of 21 selected transcripts were verified by RT-qPCR. Ccl19-ps2, Cyp2b10, Rbm14-rbm4, Sec61g, Tmem81, Prtn3, Amy2a5, Cyp2b9 and Mup1 showed significant changes by erythrodiol administration. When Cyp2b10, Dmbt1, Cyp2b13, Prtn3 and Cyp2b9 were analyzed in female Apoe-deficient mice, no change was observed. Likewise, no significant variation was observed in Apoa1-or in Apoe-deficient mice receiving doses ranging from 0.5 to 5 mg/kg erythrodiol. Our results give evidence that erythrodiol exerts a hepatic transcriptional role, but this is selective in terms of sex and requires a threshold dose. Furthermore, it requires an APOA1-containing HDL

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. © 2022 by the authors. Licensee MDPI, Basel, Switzerland

The Impact of Sensitive Research on the Researcher: Preparedness and Positionality

There is currently limited research exploring the impact of undertaking sensitive or challenging research on the researcher, although some textbooks explore researcher preparedness. This article presents a discussion of the findings from a research project which engaged with the seldom heard voices of researchers themselves. The aim was to explore researchers’ experiences of undertaking research on sensitive topics, or with marginalized groups, as this can expose researchers to emotionally disturbing situations throughout data collection and analysis, which can be psychologically challenging. Although ethical codes of practice include discussion around protection of both the researcher and the participant, in practice, the ethics approval process rarely considers the impact of the proposed research on the researcher. Their experiences are therefore seldom acknowledged or heard, resulting in potential distress for the researcher. Semistructured interviews were undertaken with social science researchers from a range of discipline backgrounds and at different points in their research careers (n = 10). This article explores two themes emerging from the data: preparedness and positionality. It considers what these themes mean in terms of supporting researchers who encounter challenging research data, and issues related to supporting researcher reflexivity and the requirements for institutional support offered to researchers will also be considered

Proteomics and gene expression analyses of squalene-supplemented mice identify microsomal thioredoxin domain-containing protein 5 changes associated with hepatic steatosis

Squalene is an abundant hydrocarbon present in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE-knock-out mice. To study its effects on microsomal proteins, 1 g/kg/day of squalene was administered to those mice. After 10 weeks, hepatic fat content was assessed and protein extracts of microsomal enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by peptide fingerprinting and MSMS analysis. Squalene administration modified the expression of thirty-one proteins involved in different metabolic functions and increased the levels of those involved in vesicle transport, protein folding and redox status. Only mRNA levels of 9 genes (Arg1, Atp5b, Cat, Hyou1, Nipsnap1, Pcca, Pcx, Pyroxd2, and Txndc5) paralleled these findings. No such mRNA changes were observed in wild-type mice receiving squalene. Thioredoxin domain-containing protein 5 (TXNDC5) protein and mRNA levels were significantly associated with hepatic fat content in apoE-ko mice. These results suggest that squalene action may be executed through a complex regulation of microsomal proteins, both at the mRNA and post-transcriptional levels and the presence of apoE may change the outcome. Txndc5 reflects the anti-steatotic properties of squalene and the sensitivity to lipid accumulation

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 re-newed 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 athero-sclerotic 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 aor-tic 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. © 2021 by the authors. Licensee MDPI, Basel, Switzerland

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

Experimental Tests and Preliminary Life Cycle Assessment

Funding Information: Thanks are also due to FCT for the project PTDC/BII-BIO/30884/2017. A.F.P. and M.M.M. thank FCT for funding through the Individual Call to Scientific Employment Stimulus (ref. 2020.01614.CEECIND/CP1596/CT0007 and CEEC-IND/02702/2017, respectively). O.D. is thankful for the Ph.D. grant supported by FCT (2021.05322.BD). Funding Information: This work was funded by Portuguese funds through Fundação para a Ciência e a Tecnologia (FCT/MCTES) in the framework of the projects UIDB/50006/2020, UIDP/50006/2020, and EXPL/BII-BIO/0436/2021 and also by BIP Proof 2022/2023 through UI-Transfer, a cofunding project by European Union through COMPETE 2020-Portugal 2020, CIQUP, Centro de Investigação em Química da Universidade do Porto (UIDB/00081/2020), and IMS, Institute of Molecular Sciences (LA/P/0056/2020). Publisher Copyright: © 2023 The Authors. Published by American Chemical Society.The control of the oxidative stability of biodiesel and blends of biodiesel with diesel is one of the major concerns of the biofuel industry. The oxidative degradation of biodiesel can be accelerated by several factors, and this is most critical in the so-called second generation biodiesel, which is produced from low-cost raw materials with lower environmental impacts. The addition of antioxidants is imperative to ensure the oxidative stability of biodiesel, and these are considered products of high commercial value. The antioxidants currently available on the market are from synthetic origin, so the existence/availability of alternative antioxidants of natural origin (less dependent on fossil sources) at a competitive price presents itself as a strong business opportunity. This work describes and characterizes a sustainable alternative to synthetic antioxidants used in the biodiesel market developed from extracts of vineyard pruning waste (VPW), which are naturally rich in phenolic compounds with antioxidant properties. A hydrothermal extraction process was applied as a more efficient and sustainable technology than the conventional one with the potential of the extracts as antioxidant additives in biodiesel evaluated in Rancitech equipment. The VPW extract showed comparable antioxidant activity as the commercial antioxidant butylated hydroxytoluene (BHT) typically used in biodiesel. The stability of the biodiesel is dependent from the amount of the extract added. Further, for the first time, the assessment of the environmental impacts of using natural extracts to control the oxidative stability of biodiesel in the production process is also discussed as a key factor of the process environmental sustainability.publishersversionpublishe

The calcium-Activated potassium channel KCa3.1 is an important modulator of hepatic injury

The calcium-Activated potassium channel KCa3.1 controls different cellular processes such as proliferation and volume homeostasis. We investigated the role of KCa3.1 in experimental and human liver fibrosis. KCa3.1 gene expression was investigated in healthy and injured human and rodent liver. Effect of genetic depletion and pharmacological inhibition of KCa3.1 was evaluated in mice during carbon tetrachloride induced hepatic fibrogenesis. Transcription, protein expression and localisation of KCa3.1 was analysed by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry. Hemodynamic effects of KCa3.1 inhibition were investigated in bile duct-ligated and carbon tetrachloride intoxicated rats. In vitro experiments were performed in rat hepatic stellate cells and hepatocytes. KCa3.1 expression was increased in rodent and human liver fibrosis and was predominantly observed in the hepatocytes. Inhibition of KCa3.1 aggravated liver fibrosis during carbon tetrachloride challenge but did not change hemodynamic parameters in portal hypertensive rats. In vitro, KCa3.1 inhibition leads to increased hepatocyte apoptosis and DNA damage, whereas proliferation of hepatic stellate cells was stimulated by KCa3.1 inhibition. Our data identifies KCa3.1 channels as important modulators in hepatocellular homeostasis. In contrast to previous studies in vitro and other tissues this channel appears to be anti-fibrotic and protective during liver injury