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Hepatic fat-specific protein 27 [cell death-inducing DNA fragmentation effector protein C (Cidec)/Fsp27] mRNA levels have been associated with hepatic lipid droplet extent under certain circumstances. To address its hepatic expression under different dietary conditions and in both sexes, apolipoprotein E (Apoe)-deficient mice were subjected to different experimental conditions for 11 wk to test the influence of cholesterol, Western diet, squalene, oleanolic acid, sex, and surgical castration on Cidec/Fsp27 mRNA expression. Dietary cholesterol increased hepatic Cidec/Fsp27β expression, an effect that was suppressed when cholesterol was combined with saturated fat as represented by Western diet feeding. Using the latter diet, neither oleanolic acid nor squalene modified its expression. Females showed lower levels of hepatic Cidec/Fsp27β expression than males when they were fed Western diets, a result that was translated into a lesser amount of CIDEC/FSP27 protein in lipid droplets and microsomes. This was also confirmed in low-density lipoprotein receptor (Ldlr)-deficient mice. Incubation with estradiol resulted in decreased Cidec/Fsp27β expression in AML12 cells. Whereas male surgical castration did not modify the expression, ovariectomized females did show increased levels compared with control females. Females also showed increased expression of peroxisome proliferator-activated receptor-γ coactivator 1-α (Pgc1a), suppressed by ovariectomy, and the values were significantly and inversely associated with those of Cidec/Fsp27β. When Pgc1a-deficient mice were used, the sex differences in Cidec/Fsp27β expression disappeared. Therefore, hepatic Cidec/Fsp27β expression has a complex regulation influenced by diet and sex hormonal milieu. The mRNA sex differences are controlled by Pgc1a., This research was supported by Ministerio de Economía y CompetitividadFondo Europeo de Desarrollo Regional (Grants SAF2015-63904-R, SAF2016-75441-R, and RTI2018-093864-B-I00), Fondo Social Europeo-Gobierno de Aragón (Grant B16_17R), and the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie Grant Agreement 721236-TREATMENT to M. Monsalve. Centro de Investigación Biomédica en Red Fisiopatología de la Obesidad y Nutrición (CB06/03/1012) is an initiative of Instituto de Salud Carlos III. L. V. Herrera-Marcos and S. Sancho-Knapik were recipients of Fondo Social Europeo-Gobierno de Aragón and Fundación Cuenca-Villoro (BE 203/2009) fellowships, respectively.

Squalene (SQ) is an intermediate hydrocarbon in the biosynthesis of phytosterols and terpenes in plants. It is widely used for applications such as skin moisturizers, vaccines, or in carriers for active lipophilic molecules. It has commonly been obtained from sharks, but restrictions on their use have created a need to find alternative sources. We present a review of studies concerning SQ in olive groves to characterize its content and to provide new aspects that may increase the circular economy of the olive tree. There is a large variation in SQ content in virgin olive oil due to cultivars and agronomic issues such as region, climate, types of soil, crop practices, and harvest date. Cultivars with the highest SQ content in their virgin olive oil were 'Nocellara de Belice', 'Drobnica', 'Souri', and 'Oblica'. An interaction between cultivar and aspects such as irrigation practices or agricultural season is frequently observed. Likewise, the production of high SQ content needs precise control of fruit maturation. Leaves represent an interesting source, if its extraction and yield compensate for the expenses of their disposal. Supercritical carbon dioxide extraction from olive oil deodorizer distillates offers an opportunity to obtain high-purity SQ from this derivative. Exploiting SQ obtained from olive groves for the pharmaceutical or cosmetic industries poses new challenges and opportunities to add value and recycle by-products. © 2019 Society of Chemical Industry.

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.

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.

Scope To investigate the effects of squalene, the main hydrocarbon present in extra virgin olive oil, on liver transcriptome in different animal models and to test the influence of sex on this action and its relationship with hepatic lipids. Methods and results To this purpose, male C57BL/6J Apoe-deficient mice are fed a purified Western diet with or without squalene during 11 weeks and hepatic squalene content is assessed, so are hepatic lipids and lipid droplets. Hepatic transcriptomic changes are studied and confirmed by RT-qPCR. Dietary characteristics and influence of squalene doses are tested in Apoe-deficient on purified chow diets with or without squalene. These diets are also given to Apoa1 and wild-type mice on C57BL/6J background and to C57BL/6J xOla129 Apoe-deficient mice. Squalene supplementation increases its hepatic content without differences among sexes and hormonal status. The Cyp2b10 and Cyp2c55 gene expressions are significantly up-regulated by the squalene intake in all models, with independence of sex, sexual hormones, dietary fat content, genetic background and dose, and in Apoe-deficient mice consuming extra-virgin olive oil. Conclusion Hepatic squalene increases the expression of these cytochromes and their changes in virgin olive oil diets may be due to their squalene content.

Ion channels have recently attracted attention as potential mediators of skin disease. Here, we explored the consequences of genetically encoded induction of the cell volume-regulating Ca2+-activated KCa3.1 channel (Kcnn4) for murine epidermal homeostasis. Doxycycline-treated mice harboring the KCa3.1+-transgene under the control of the reverse tetracycline-sensitive transactivator (rtTA) showed 800-fold channel overexpression above basal levels in the skin and solid KCa3.1-currents in keratinocytes. This overexpression resulted in epidermal spongiosis, progressive epidermal hyperplasia and hyperkeratosis, itch and ulcers. The condition was accompanied by production of the pro-proliferative and pro-inflammatory cytokines, IL-β1 (60-fold), IL-6 (33-fold), and TNFα (26-fold) in the skin. Treatment of mice with the KCa3.1-selective blocker, Senicapoc, significantly suppressed spongiosis and hyperplasia, as well as induction of IL-β1 (-88%) and IL-6 (-90%). In conclusion, KCa3.1-induction in the epidermis caused expression of pro-proliferative cytokines leading to spongiosis, hyperplasia and hyperkeratosis. This skin condition resembles pathological features of eczematous dermatitis and identifies KCa3.1 as a regulator of epidermal homeostasis and spongiosis, and as a potential therapeutic target, This work was supported by the Government of Aragón (DGA-METIC–consortium; B04_17R), FP7-PEOPLE-MC-CIG to RK, FIS-CB06/07/1036 to PG and RK, ERC-2014-StG-638284, DPI2016-75458-R, T39_17R to EP; FIS-PI16/02112 to ALGO; NHLBI-R01-HL080173, NCRRP20-RR018751 to HM. JLG received a DGA-PhD scholarship (C072/2014). BMB was supported by the NCATS (UL1 TR001860 and linked award TL1 TR001861)

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.

Aims: The North-to-South gradient in cardiovascular disease in Europe could be partially explained by the low prevalence of lipid-rich atheroma plaques (responsible for most culprit lesions) in Southern Europe. Consumption of certain foods delays atherosclerosis progression. In a mouse model of accelerated atherosclerosis we investigated whether the inclusion of walnuts within an atherogenic diet stabilizes advanced atheroma plaque.Methods: Apolipoprotein E-deficient mice (male, 10-week old) were randomized to receive either a control diet (9.6% of energy as fat, n=14), a palm oil-based high-fat diet (40% of energy as fat, n=15) or an isocaloric diet which fat was partially supplied by walnuts, in a dose equivalent to 30 g/d in humans (n=14). All diets contained 0.2% cholesterol. After 15 weeks of intervention, we evaluated changes in aortic atherosclerotic lesions (size and composition) and expression levels of markers for inflammation and oxidative stress. Results: There were no among-group differences in atherosclerotic size and extension, or oxidative stress. Compared to control diet, palm oil-diet induced plaque instability (higher lipid and necrosis, and lower collagen-to-lipid ratio). Walnut inclusion attenuated these features and decreased macrophage infiltration. Palm oil-based diet increased expression of chemokines, cytokines, inflammasome, and M1-macrophage marker compared to control diet. Such response was not observed in walnut group. Findings for walnut group could be explained by the differential aortic activation of NFkB (down-regulated) and Nrf2 (up-regulated). Conclusions: Isocaloric inclusion of walnuts in an unhealthy high-fat diet stabilizes advanced atheroma plaque. This contributes novel mechanistic evidence for the cardiovascular benefits of walnuts.

OBJECTIVES: The presence of lipid-rich, unstable atheroma plaques in the vascular tree is the harbinger of cardiovascular events. There is a low prevalence of unstable atheroma plaques in Mediterranean countries. This might explain in part the lower rates of cardiovascular disease in Southern Europe compared to Northern Europe and US. Consumption of certain foods paradigmatic of the Mediterranean Diet delays atherosclerosis progression. In a mouse model of accelerated atherosclerosis, we investigated whether the inclusion of walnuts within an atherogenic diet stabilizes advanced atheroma plaque. METHODS: Apolipoprotein E-deficient mice (male, 10-week old) were randomized to receive either regular chow (9.6% of energy as fat, n = 14); a palm oil-based high-fat diet (43% of energy as fat, n = 15); or an isocaloric high-fat diet in which palm oil was partially replaced by walnuts in a dose equivalent to 30 g/d in humans (n = 14). All diets contained 0.2% cholesterol. After 15 weeks of intervention, we evaluated changes in aortic atherosclerotic lesions (size and composition) and markers for inflammation, oxidative stress, necrosis and autophagy. RESULTS: There were no among-group differences in atherosclerotic size and extension, or oxidative stress. Compared to control diet, palm oil-diet induced plaque instability (higher lipid and necrosis, and lower collagen-to-lipid ratio). Walnut inclusion attenuated these features and decreased macrophage infiltration. Palm oil-based diet increased expression of chemokines, cytokines, inflammasome, and M1-macrophage marker compared to control diet. Such response was not observed in walnut group. Findings for walnut group could be explained by the differential aortic activation of NF-kB (down-regulated) and Nrf2 and autophagy (up-regulated). CONCLUSIONS: Isocaloric inclusion of walnuts within an unhealthy high-fat diet stabilizes advanced atheroma plaque. This contributes novel mechanistic evidence for the cardiovascular benefits of sustained walnut consumption. FUNDING SOURCES: Instituto de Salud Carlos III, Spain; Fondo de Investigación Sanitaria–Fondo Europeo de Desarrollo Regional, Spain. California Walnut Commission.

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.

Oleanolic acid is a triterpene widely distributed throughout the plant kingdom and present in virgin olive oil at a concentration of 57 mg/kg. To test the hypotheses that its long-term administration could modify hepatic gene expression in several animal models and that this could be influenced by the presence of APOA1-containing high-density lipoproteins (HDLs), diets including 0.01% oleanolic acid were provided to Apoe- and Apoa1-deficient mice and F344 rats. Hepatic transcriptome was analyzed in Apoe-deficient mice fed long-term semipurified Western diets differing in the oleanolic acid content. Gene expression changes, confirmed by reverse transcriptase quantitative polymerase chain reaction, were sought for their implication in hepatic steatosis. To establish the effect of oleanolic acid independently of diet and animal model, male rats were fed chow diet with or without oleanolic acid, and to test the influence of HDL, Apoa1-deficient mice consuming the latter diet were used. In Apoe-deficient mice, oleanolic acid intake increased hepatic area occupied by lipid droplets with no change in oxidative stress. Bmal1 and the other core component of the circadian clock, Clock, together with Elovl3, Tubb2a and Cldn1 expressions, were significantly increased, while Amy2a5, Usp2, Per3 and Thrsp were significantly decreased in mice receiving the compound. Bmal1 and Cldn1 expressions were positively associated with lipid droplets. Increased Clock and Bmal1 expressions were also observed in rats, but not in Apoa1-deficient mice. The core liver clock components Clock-Bmal1 are a target of oleanolic acid in two animal models independently of the diets provided, and this compound requires APOA1-HDL for its hepatic action.

Scientific reports 6, 28770 (2016). doi:10.1038/srep28770, Published by Nature Publishing Group, London

Background Electrical bioimpedance (BI) has been used to indirectly measure steatosis. This method has not yet been established in the clinics thus experimental studies are needed in big animals. We assessed BI to measure liver steatosis in porcine animals. Methods Twelve large-white × Landrace pigs weighing 35 kg were allocated to a study ( n = 9) and a control group ( n = 3). A special diet was used to promote steatosis among the study group: methionine deficient and choline-restricted diet that contains supplements of cholesterol, collate and excess of saturated fat. Control group animals were fed a normal diet. A new tetrapolar electrode model was used for BI measurement, which were performed during open laparotomy by inserting a probe into one of the lobes. Measurements were done in the third and fourth segments of the pig liver, placing the probe either on the surface or inserted into the parenchyma of the liver. Open biopsies were obtained at the end of the measurements. Histological samples were processed and stained with hematoxylin-eosin to estimate macrosteatosis. Pearson correlation coefficient between BI and percentage steatosis were calculated at different frequencies. Results After 4 months of the special diet all the animals in the study group developed steatosis (90% to 20%), whereas none of the control group was affected. Pearson correlation coefficients between BI and percentage of steatosis were significant (0.877–0.878) with the best correlations obtained with a probe placed on the fourth segment of the liver surface and the best frequency to perform the measurements being 50 and 75 kHz. Conclusions BI is an accurate, fast method for steatosis measurements, that is easier and cheaper than either open or needle biopsy.

The low incidence of cardiovascular disease in countries bordering the Mediterranean basin, where olive oil is the main source of dietary fat, has stimulated interest in the chemical composition of olive oil and in the production of other oils enriched with its minor components. This review summarizes what has been learned about the effects of different olive oil preparations on the development of atherosclerosis and about the prognostic value of associated plasma variables in the disease from experiments on genetically modified mice that spontaneously develop atherosclerosis. The limitations of this animal model associated with its morphological and physiological differences with humans are minimized by the similarity of the two genomes and by the potential for increased understanding attainable, given that the dietary interventions reported here would have taken 400 years to achieve in humans. As observed in traditional Mediterranean populations, it has been confirmed that extra virgin olive oil is beneficial when consumed judiciously and in a diet that is low in cholesterol due to the relative scarcity of animal products. Furthermore, the use of genomic techniques has led to the identification of new markers of response to olive oil. In conclusion, multidisciplinary research into extra virgin olive oil is expanding our knowledge of the substance's biological properties.

Double heterozygous mice lacking Apoa1 and Cbs genes show mild hyperhomocysteinemia in combination with hypoalphalipoproteinemia. This situation leads to a moderate hypertension associated with a dysregulation in nitric oxide metabolism. The aim of this study was to investigate the potential beneficial effects of statin treatment in these mice. After 4 weeks of simvastatin administration, plasma parameters; apolipoproteins A-I, A-II and A-IV; lipid profile; and blood pressure were assessed, Western blotting was performed in the aorta of these mice to measure endothelial nitric oxide synthase and caveolin-1 content. The high blood pressure level present in the double heterozygous group was corrected down to that of the wild-type group after simvastatin treatment (124 ± 7.7 vs. 109 ± 11.2 mmHg, p

Pig sphingomyelin synthase 1 (SMS1) cDNA was cloned, characterized and compared to the human ortholog. Porcine protein consists of 413 amino acids and displays a 97% sequence identity with human protein. A phylogenic tree of proteins reveals that porcine SMS1 is more closely related to bovine and rodent proteins than to human. Analysis of protein mass was higher than the theoretical prediction based on amino acid sequence suggesting a kind of posttranslational modification. Quantitative representation of tissue distribution obtained by real-time RT-PCR showed that it was widely expressed although important variations in levels were obtained among organs. Thus, the cardiovascular system, especially the heart, showed the highest value of all the tissues studied. Regional differences of expression were observed in the central nervous system and intestinal tract. Analysis of the hepatic mRNA and protein expressions of SMS1 following turpentine treatment revealed a progressive decrease in the former paralleled by a decrease in the protein concentration. These findings indicate the variation in expression in the different tissues might suggest a different requirement of Golgi sphingomyelin for the specific function in each organ and a regulation of the enzyme in response to turpentine-induced hepatic injury.

Folic acid is a vitamin that when used as a dietary supplementation can improve endothelial function. To assess the effect of folic acid on the development of atherosclerosis, male apolipoprotein E-deficient mice fed a standard chow diet received either water (control group) or an aqueous solution of folic acid that provided a dose of 75 microg/kg/day, for ten weeks. At the time of sacrifice, blood was drawn and the heart removed. The study measured plasma homocysteine, lipids, lipoproteins, low-density lipoprotein (LDL) oxidation, isoprostane, paraoxonase, and apolipoproteins, and aortic atherosclerotic areas. In folic acid-treated animals, total cholesterol, mainly carried in very low-density and low-density lipoproteins, increased significantly, and homocysteine, HDL cholesterol, paraoxonase, and triglyceride levels did not change significantly. Plasma isoprostane and apolipoprotein (apo) B levels decreased. The resistance of LDL to oxidization and plasma apoA-I and apoA-IV levels increased with a concomitant decrease in the area of atherosclerotic lesions. The administration of folic acid decreased atherosclerotic lesions independently of plasma homocysteine and cholesterol levels, but was associated with plasma levels of apolipoproteins A-I, A-IV and B, and decreased oxidative stress.

In human reproduction, hyperhomocysteinemia has been reported as a risk factor for early pregnancy loss and congenital birth defects. Hyperhomocysteinemia is also recognized as a cause of maternal obstetric complications such as preeclampsia. The role of plasma hyperhomocysteinemia in female fertility was examined using cystathionine beta synthase knockout (cbs KO) mice. Cbs KO females were infertile, showed alterations in the estrus cycle and an increased progesterone response during pseudo-pregnancy induction. Both cbs KO ovaries and ovulated oocytes showed no major morphological alterations. However, placental and uterine masses were decreased at day 18 of pregnancy and showed morphological abnormalities. In cbs-KO pregnant females, the number of uterine implantation sites was not decreased despite the low number of surviving embryos. Fertility was restored when cbs-deficient ovaries were transplanted to normal ovarectomized recipients. We detected an increased uterine expression of Grp78, a marker of endoplasmic reticulum stress, which was accompanied by the decreased levels of uterine cbs mRNA in both hyperhomocysteinemic heterozygous (fertile) and homozygous (non-fertile) females. Our results indicate that cbs -/- female infertility is a consequence of the uterine failure and demonstrate that uterine endoplasmic reticulum stress and cbs expression are not determinant of infertility, suggesting that uterine dysfunction is a consequence of either hyperhomocysteinemia or other factor(s) in the uterine environment of cbs -/- animals. In summary, these studies demonstrate the potential importance of homocysteine levels for uterine handling of embryos.

Hydroxytyrosol is a phenol found in olive oil. To verify the effect of hydroxytyrosol on the development of atherosclerosis, two groups of apo E deficient male mice on a standard chow diet were used: the control group receiving only water, and the second group an aqueous solution of hydroxytyrosol in order to provide a dose of 10 mg/kg/day to each mouse. This treatment was maintained for 10 weeks. At the moment of sacrifice, blood was drawn and heart removed. Plasma lipids, apolipoproteins and monocyte Mac-1 expression were assayed as well as aortic atherosclerotic areas in both groups. Data showed no significant changes in HDL cholesterol, paraoxonase, apolipoprotein B or triglyceride levels. However, hydroxytyrosol administration decreased apolipoprotein A-I and increased total cholesterol, atherosclerotic lesion areas and circulating monocytes expressing Mac-1. The latter was highly correlated with lesion areas (r = 0.65, P < 0.01). These results indicate that administration of hydroxytyrosol in low cholesterol diets increases atherosclerotic lesion associated with the degree of monocyte activation and remodelling of plasma lipoproteins. Our data supports the concept that phenolic-enriched products, out of the original matrix, could be not only non useful but also harmful. Our results suggest that the formulation of possible functional foods should approximate as much as possible the natural environment in which active molecules are found., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón., This research was supported by grants FEGA-FEOGA (CAO99-0014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón. R.C., S.A., M.A.N. and N.G. were recipient of DGA, FEGA-FEOGA and Fundación Cuenca Villoro fellowships.

Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote atherosclerosis in mice despite increasing blood concentrations of HDL cholesterol. This suggests that under these conditions, the HDL apolipoproteins (apo) produced are abnormal. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid (control), cis-9, trans-11-CLA or trans-10, cis-12-CLA (1.0% wt/wt) for 12 wk, and the effects on HDL metabolism and apoC-III levels recorded. Compared with the control and cis-9, trans-11-CLA mice, those fed the trans-10, cis-12-CLA diet had significantly higher HDL cholesterol concentrations, and had a higher incidence of hypertriglyceridemia and hepatic steatosis. Plasma apoA-I and paraoxonase concentrations were significantly lower in the trans-10, cis-12-CLA group than in the cis-9, trans-11-CLA group. These reductions were associated with decreased hepatic expression of these proteins and a shift toward lipid-poor apolipoprotein particles. The plasma apoA-II concentration increased with its corresponding mRNA concentration in the liver, and was preferentially bound to HDL in the trans-10, cis-12-CLA mice, thus explaining the increased HDL cholesterol concentrations in this group. Significant, positive associations were found between apoA-II and C-III (r=0.883, P

Apolipoprotein A-IV is a member of the apo A-I/C-III/A-IV gene cluster. In order to investigate its hypothetical coordinated regulation, an acute phase was induced in pigs by turpentine oil injection. The hepatic expression of the gene cluster as well as the plasma levels of apolipoproteins were monitored at different time periods. Furthermore, the involvement of the inflammatory mediators’ interleukins 1 and 6 and tumor necrosis factor in the regulation of this gene cluster was tested in cultured pig hepatocytes, incubated with those mediators and apo A-I/C-III/A-IV gene cluster expression at the mRNA level was measured. In response to turpentine oil-induced inflammation, a decreased hepatic apo A-IV mRNA expression was observed (independent of apo A-I and apo C-III mRNA) not correlating with the plasma protein levels. The distribution of plasma apo A-IV experienced a shift from HDL to larger particles. In contrast, the changes in apo A-I and apo C-III mRNA were reflected in their corresponding plasma levels. Addition of cytokines to cultured pig hepatocytes also decreased apo A-IV and apo A-I mRNA levels. All these results show that the down-regulation of apolipoprotein A-I and A-IV messages in the liver may be mediated by interleukin 6 and TNF-a. The well-known HDL decrease found in many different acute-phase responses also appears in the pig due to the decreased expression of apolipoprotein A-I and the enlargement of the apolipoprotein A-IV-containing HDL. 2005 Elsevier Ltd. All rights reserved.

La presente revision aborda el metabolismo lipoproteico comparado y la induccion de la aterosclerosis con sus controversias en varios modelos animales pertenecientes a un amplio espectro evolutivo que abarca desde los roedores (raton, conejo, rata, hamster, cobaya), las aves (paloma), los cetartiodactilos (cerdo) y los carnivoros (perro) hasta los primates (macacos, Rhesus, mono verde africano).

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.

Amyotrophic lateral sclerosis is a neurodegenerative disease associated with mutations in antioxidant enzyme Cu/Zn-superoxide dismutase 1. Albeit there is no treatment for this disease, new insights related to an exacerbated lipid metabolism have been reported. In connection with the hypermetabolic lipid status, the hypothesis whether nature of dietary fat might delay the progression of the disease was tested by using a transgenic mouse that overexpresses the human SOD1G93A variant. For this purpose, SOD1G93A mice were assigned randomly to one of the following three experimental groups: (1) a standard chow diet (control, n=21), (2) a chow diet enriched with 20% (w/w) extra virgin olive oil (EVOO, n=22) and (3) a chow diet containing 20% palm oil (palm, n=20). They received the diets for 8 weeks and the progression of the disease was assessed. On the standard chow diet, average plasma cholesterol levels were lower than those mice receiving the high-fat diets. Mice fed an EVOO diet showed a significant higher survival and better motor performance than control mice. EVOO group mice survived longer and showed better motor performance and larger muscle fiber area than animals receiving palm. Moreover, the EVOO-enriched diet improved the muscle status as shown by expression of myogenic factors (Myod1 and Myog) and autophagy markers (LC3 and Beclin1), as well as diminished endoplasmic reticulum (ER) stress through decreasing Atf6 and Grp78. Our results demonstrate that EVOO may be effective in increasing survival rate, improving motor coordination together with a potential amelioration of ER stress, autophagy and muscle damage.

Epidemiological studies have demonstrated the benefits of nut consumption on cardiovascular risk factors and CHD, attributed to their fatty acid profile, rich in unsaturated fatty acids, and also to other nutrients. The effect of nuts on atherosclerotic lesions was studied in female and male apoE-knockout mice fed a diet supplemented with 3 % (w/w) mixed nuts (mix: almonds, hazelnuts and walnuts in a proportion of 0·25:0·25:0·50, respectively), and compared with mice receiving an isoenergetic diet of similar fat content provided as palm oil. After 12 weeks, plasma lipid parameters and aortic lesions were measured. Males receiving nuts had lower plasma cholesterol than the palm oil group, and both sex groups had lower plasma non-HDL-cholesterol and lower content of reactive oxygen species in LDL than mice receiving the palm oil diet, the latter decrease being more pronounced in females than in males. Females consuming the nut diet showed a smaller aortic lesion area than those consuming palm oil, whereas no differences were observed in males. In females, hepatic paraoxonase 2 (Pon2) mRNA increased, and no change was observed in prenylcysteine oxidase 1 (Pcyox1) expression after the consumption of the nut-containing diet. In addition, aortic atherosclerotic lesions correlated directly with total plasma cholesterol and inversely with hepaticPon2expression. The results suggest that the beneficial effect of nut intake in female apoE-deficient mice may be attributed to reduced non-HDL-cholesterol levels and enhanced PON2 antioxidant activity.

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, 1g/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.

AIM Research suggests that sex may condition atherosclerosis development against different genetic backgrounds. This study addresses the hypothesis that this effect would be exerted by changes in the different apolipoproteins present in high-density lipoproteins. METHODS ApoE-deficient mice of both sexes with Ola 129 and C57BL/6J genetic backgrounds were fed a chow diet for 14 weeks. At the end of the dietary intervention, the development of atherosclerotic lesions, apolipoproteins, lipid metabolism, inflammation and paraoxonase were assessed. RESULTS Differences between atherosclerotic lesions in Ola 129 and C57BL/6J strains of apoE-deficient mice were sex-dependent and were only statistically significant in females. Plasma levels of HDL cholesterol and apolipoproteins related to these lipoparticles, such as apoA-I, apoA-II, apoA-IV, apoA-V and apoJ, were significantly different between these two strains and there were sex-related differences in some of these apolipoproteins. Hepatic steatosis was also related to the strain and was independent of sex. In females, changes in HDL cholesterol and apolipoproteins A-I and A-II were important determinants of atherosclerosis, while this was not the case in males. CONCLUSIONS Our results demonstrate that atherosclerosis-related differences between Ola129 and C57BL/6J genetic backgrounds in apoE-deficient mice are sex-dependent and that this finding is explained by the differences in HDL cholesterol and its apolipoprotein components, mainly apoA-I and A-II. Overall, our findings highlight the importance of taking sex into account in the analysis of atherosclerosis and lipid metabolism in animal models.

Since the hepatic mitogen, liver growth factor (LGF), improves vascular structure and function in a hypertensive rat model and exhibits antioxidant activity, it may play a role in the development of atherosclerosis.To test this hypothesis, 14 male and 11 female apolipoprotein E (apoE)-deficient mice with a C57BL/6J genetic background were injected intraperitoneally twice a week with 1.7 microg of LGF per mouse for ten weeks. Plasma carbohydrates, inflammatory and lipid parameters, apolipoproteins A-I and A-II and paraoxonase activity were assessed at the end of the experimental period. Histological and chemical analyses of the livers and quantification of aortic atherosclerotic lesions were also carried out.LGF administration changed neither plasma lipid nor inflammatory parameters. ApoA-I and arylesterase activity were not affected by LGF either, while apoA-II decreased significantly in males but not in females. Plasma apoA-II correlated positively with liver fat in males but negatively in females. Atherosclerotic area lesions in males receiving LGF were 25% lower than in control mice. Likewise, a significant reduction of fatty liver disease was also observed in males in association with decreased levels of insulin, leptin and resistin.These results indicate that administration of LGF modulates atherosclerotic lesions in a sex-dependent manner. This effect is independent of plasma cholesterol, triglycerides, IL-6, MCP-1 and TNF-alpha and is related to a remodelling of HDL particles characterised by a decrease in apoA-II induced by changes in hepatic mRNA expression. Hence, LGF administration could be used as a safe alternative to control fatty liver disease and atherosclerosis in males.

The hypothesis that the maslinic acid (MA) of olive oil (OO) dramatically influences hepatic gene expression was tested in mice. Two OOs only differing in the presence of MA were prepared. Using DNA microarrays, we analyzed hepatic gene expression in apolipoprotein E (apoE)-deficient mice with a C57BL/6J genetic background that were fed with isocaloric, isonitrogenous diets containing either 10% (w/w) OO or 10% MA-enriched OO. As an initial screening of potential candidate genes involved in a differential response, this study further considered only genes with remarkably modified expression (signal log2 ratio higher than1.5 or lower than −1.5). The nine genes fulfilling these prerequisites were confirmed by quantitative reverse transcriptase polymerase chain reaction and analyzed in C57BL/6J wild-type mice. Only Cyp2b9, Cyp2b13 and Dbp expressions appeared significantly increased, and Marco was significantly decreased in apoE-deficient mice receiving the MA-enriched diet. Dbp was up-regulated to an extent depending on the genetic background of the mice and negatively associated with the expression of Marco, a gene strongly up-regulated by the absence of apoE. These expression changes could be used as markers of the intake of the MA-enriched OO and are influenced by genetic background generated by the absence or the presence of apoE. Overall, these results (a) indicate that MA in virgin OO is highly active in controlling hepatic gene expression and (b) highlight the important interaction between the response to MA and the presence of apoE. They also confirm that virgin OO cannot be simplistically classified as monounsaturated fatty-enriched oil without paying attention to its active minor components., This research was supported by grants from FEDER-CICYT (SAF2007-60173, AGL2005-00572 and AGL2008-02285/ALI), Redes DGA (B-69) and FISS de Investigación Cooperativa (C03-01 and G03-140) and by Fundación Española del Corazón and Instituto Aragonés de Ciencias de la Salud. S.A. and N.G. were recipients of DGA and Fundación Cuenca Villoro fellowships.

Trans-10, cis-12-conjugated linoleic acid (CLA)-enriched diets promote fatty liver in mice, while cis-9, trans-11-CLA ameliorates this effect, suggesting regulation of multiple genes. To test this hypothesis, apoE-deficient mice were fed a Western-type diet enriched with linoleic acid isomers, and their hepatic gene expression was analyzed with DNA microarrays. To provide an initial screening of candidate genes, only 12 with remarkably modified expression between both CLA isomers were considered and confirmed by quantitative RT-PCR. Additionally mRNA expression of 15 genes involved in lipid metabolism was also studied. Ten genes (Fsp27, Aqp4, Cd36, Ly6d, Scd1, Hsd3b5, Syt1, Cyp7b1, and Tff3) showed significant associations among their expressions and the degree of hepatic steatosis. Their involvement was also analyzed in other models of steatosis. In hyperhomocysteinemic mice lacking Cbs gene, only Fsp27, Cd36, Scd1, Syt1, and Hsd3b5 hepatic expressions were associated with steatosis. In apoE-deficient mice consuming olive-enriched diet displaying reduction of the fatty liver, only Fsp27 and Syt1 expressions were found associated. Using this strategy, we have shown that expression of these genes is highly associated with hepatic steatosis in a genetic disease such as Cbs deficiency and in two common situations such as Western diets containing CLA isomers or a Mediterranean-type diet. Conclusion: The results highlight new processes involved in lipid handling in liver and will help to understand the complex human pathology providing new proteins and new strategies to cope with hepatic steatosis.

Double heterozygous mice lacking one allele of Cbs and Apoa1 develop hyperhomocysteinemia and hypoalphalipoproteinemia together with moderate hypertension. To study the influence of the genetic background into this specific phenotype, four groups of male mice were established: control and double heterozygous groups in C57BL/6J and in C57BL/6J x 129 backgrounds, respectively. Nitric oxide levels, systolic blood pressure, plasma lipid parameters, arylesterase activity and aorta histology were analyzed as well as oligonucleotide array hybridization of liver RNA. Results demonstrated that double heterozygous mice in C57BL/6J substrate had a milder phenotype showing lower increase in blood pressure compared to double heterozygous group in hybrid background. The severity of the phenotype in the latter group was associated with lower nitric oxide and arylesterase activity levels, and hyperplasia of the vascular media layer. Hepatic profiling of both genetic substrates showed profound differences in expression of contractile proteins that could explain these pathological findings. In summary, the phenotypic presentation of hypertension is associated with multiple processes from vascular bedside to liver as evidenced by nitric oxide production or paraoxonase levels.

BACKGROUND: Squalene is an intermediate of cholesterol biosynthesis which can be obtained from the diet where it is abundant, for example, in olive oil. The effect of this isoprenoid on the development of atherosclerosis was investigated on apoE-knockout mice. METHODS AND RESULTS: Two groups of animals, separated according to sex, were fed on standard chow diet: the control group receiving only vehicle and the second group an aqueous solution of squalene to provide a dose of 1g/kg/day in male and female mice. This treatment was maintained for 10 weeks. At the end of this period, plasma lipid parameters, oxidative stress markers and hepatic fat were measured as well as cross-sectional lesion area of aortic root in both groups. Data showed that in males squalene feeding reduced atherosclerotic lesion area independently of plasma lipids and activation of circulating monocytes. In contrast, squalene intake did not decrease lesion area in females, despite reducing plasma cholesterol and triglycerides, isoprostane and percentage of Mac-1 expressing white cells. In males, atherosclerotic lesion area was positively and significantly associated with hepatic fat content and the plasma triglycerides were also strongly associated with liver weight. CONCLUSIONS: These results indicate that administration of squalene modulates lesion development in a gender specific manner, and that accumulation of hepatic fat by liver is highly correlated with lesion progression in males. Hence, squalene administration could be used as a safe alternative to correct hepatic steatosis and atherosclerosis particularly in males.

BACKGROUND Hyperhomocysteinemia and hypoalphalipoproteinemia are two well-reported risk factors for cardiovascular disease. The effects of the synergistic combination of these two factors on vascular function need to be investigated. METHODS AND RESULTS Four groups of male mice were used: a control wild-type group; a group of mice heterozygous for cystathionine beta-synthase deficiency; a group of mice heterozygous for apolipoprotein A-I deficiency; and, finally, a group of double heterozygous mice, with both cystathionine beta-synthase and apolipoprotein A-I deficiency. To characterize the resulting phenotype, several parameters including plasma apolipoproteins, lipid profiles, homocysteine, blood pressure and aortic protein were analyzed. As expected, our results indicate that double heterozygous mice are a model of mild hypoalphalipoproteinemia and hyperhomocysteinemia. Further, the additive combination of both risk factors resulted in a significant increase in blood pressure compared with control animals (136 +/- 8.0 versus 126 +/- 7.5 mm Hg, P < 0.01) that was not present in single heterozygous mice. The increase in blood pressure was associated with decreased plasma nitric oxide levels, left ventricle hypertrophy and was independent of low-density lipoprotein (LDL) cholesterol, para-oxonase activity and kidney histological changes. Concomitant decreases in levels of apolipoprotein A-IV (APOA-IV) and caveolin-1 content were also found in the double heterozygous group. CONCLUSIONS Our findings suggest an additive adverse effect of hypoalphalipoproteinemia and hyperhomocysteinemia on endothelial function to generate clinical hypertension and cardiac muscle hypertrophy mediated by dysregulation in nitric oxide metabolism.

The hypothesis that the unsaponifiable fraction of olive oil dramatically influences hepatic gene expression was tested in mice. Two olive oils, obtained from the same olive cultivar but by different technological procedures, were characterized to show that they differed mainly in terms of the composition/quantity of this unsaponifiable fraction. Using DNA microarrays, hepatic gene expression was analysed in apoE-deficient mice fed one of two isoenergetic, isonitrogenous diets containing either 10% (w/w) olive oil or unsaponifiable fraction-enriched olive oil. To provide an initial screening of potential candidate genes involved in a differential response, only genes with remarkably modified expression (signal log2 ratio ≥3 or < -3) were further considered. The eleven genes fulfilling these prerequisites were confirmed by quantitative RT-PCR, and then analysed in apoE-deficient mice with a C57BL/6J genetic background. Orosomucoid and serum amyloid A2 were upregulated (to variable extents depending on the genetic background) in the absence of hepatic steatosis and inflammation. Fabp5 and Mt2 were also strongly upregulated. Several proteases were highly suppressed by the unsaponifiable-enriched olive diet, independent of the genetic background. The findings indicate that change in the expression of these genes is a good marker of the intake of the unsaponifiable fraction of olive oil. The results highlight the important biological effects of the unsaponifiable fraction of olive oil. The term 'monounsaturated fatty acid-enriched oil' no longer appears appropriate for describing all the oils to which it is currently applied since it does not adequately reflect that they have different biological effects. © The Authors 2007., This research was supported by grants FEGA-FEOGA (CAO99-014), Ministerio de Educación y Ciencia, CICYT (SAF2004-08 173-C03-02 and AGL2005-00 572), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes FISS de investigación cooperativa C03-01 and G03-140 and by the Fundación Española del Corazón.

To test the hypothesis that extra virgin olive oils from different cultivars added to Western diets might behave differently than palm oil in the development of atherosclerosis, apoE-deficient mice were fed diets containing different cultivars of olive oil for 10weeks. Female mice were assigned randomly to one of the following five groups: (1–4) fed chow diets supplemented with 0.15% (w/w) cholesterol and 20% (w/w) extra virgin olive oil from the Arbequina, Picual, Cornicabra, or Empeltre cultivars, and (5) fed a chow diet supplemented with 0.15% cholesterol and 20% palm oil. Compared to diets containing palm oil, a Western diet supplemented with one of several varieties of extra virgin olive oil decreased atherosclerosis lesions, reduced plaque size, and decreased macrophage recruitment. Unexpectedly, total plasma paraoxonase activity, apoA-I, plasma triglycerides, and cholesterol played minor roles in the regulation of differential aortic lesion development. Extra virgin olive oil induced a cholesterol-poor, apoA-IV-enriched lipoparticle that has enhanced arylesterase and antioxidant activities, which is closely associated with reductions in atherosclerotic lesions. Given the anti-atherogenic properties of extra virgin olive oil evident in animal models fed a Western diet, clinical trials are needed to establish whether these oils are a safe and effective means of treating atherosclerosis.

Oils enriched in monounsaturated fatty acids do not seem to behave similarly in protecting against the development of atherosclerosis in animal models, which has been attributed to the presence of soluble phenolic compounds. To test the relevance of other components of oils in the prevention of atherosclerosis, two olive oils from the same cultivar devoid of soluble phenolic compounds were prepared using different procedures (pressure or centrifugation), characterized and fed to apolipoprotein E-deficient mice as 10% (w/w) of their diet. The 2 olive oils had similar levels of monounsaturated fatty acids and squalene, but they differed in their content of linoleic, phytosterols, tocopherols, triterpenes and waxes, which were particularly enriched in the test olive oil obtained by centrifugation. In mice that received a diet enriched in the olive oil derived through centrifugation, the progression of atherosclerosis was delayed compared to the mice that received standard olive oil. That effect was associated with decreases in plasma triglycerides, total and non-high-density lipoprotein cholesterol and isoprostane 8-iso-prostaglandin F2α. Our results clearly indicate that the preparation of olive oil is crucial in determining its antiatherosclerotic effect, which extends beyond the presence of phenolic compounds. The test olive oil exerted its antiatherosclerotic effects by modifying plasma lipids and oxidative stress, and it might be a good candidate to replace other fats in functional foods., This research was supported by grants FEGA-FEOGA (CAO99-014), CICYT (SAF2004-08173-C03-02 and AGL2002-00495), Junta de Andalucía (CAO01-002), FISS 01/0202, Redes DGA (A-26) and FISS de investigación cooperativa C03-01 and G03-140 and by Fundación Española del Corazón.

The generation by genetic engineering of two murine models to investigate atherosclerosis, such as the apoE- and LDLr- deficient mice, is providing an extraordinaire knowledge of the effect of different nutrients on this complex disease. The present revision provides a comprehensive overview of the advances in this field that point to a remarkable complexity. While some controversies over puzzling results could be explained invoking potential nutrient interactions or different food sources of nutrients, it also appears that other factors such as sex, genetic background or immunological status are emerging as generators of differential responses to nutrients during the atherosclerotic process.

Research suggests that conjugated linoleic acid (CLA) may inhibit atherosclerosis, but there are contradictory results in different animal models fed heterogeneous mixtures of CLA isomers. This study addressed the hypothesis that the individual CLA isomers may exert different atherogenic properties. ApoE −/− mice were fed isocaloric, isonitrogenous westernized diets containing 0.15% cholesterol and enriched with 1% (w/w) cis -9, trans -11-CLA (c9,t11-CLA), trans -10, cis -12-CLA (t10,c12-CLA) or linoleic acid (control diet) for 12 weeks. At the end of the dietary intervention, the effects of CLA isomers on the development of atherosclerotic vascular lesions, lipid metabolism, inflammation and oxidative stress were assessed. The t10,c12-CLA diet had a profound pro-atherogenic effect, whereas c9,t11-CLA impeded the development of atherosclerosis. En face aortic lesion assessment showed more dorsal and lumbar extensions presenting atherosclerotic foci after the t10,c12-CLA diet. Furthermore, animals fed t10,c12-CLA had pronounced hyperlipidemia, higher 8- iso -prostaglandin F 2α levels, higher vulnerable atherosclerotic plaque with a lower smooth muscle and fibre contents and higher macrophage content and activation, assayed as plasma chitotriosidase compared to the control or c9,t11-CLA dietary groups. Plasma chitotriosidase activity was more closely associated with the extent of the plaque than with MOMA staining or than monocyte chemoattractant protein-1 levels. Our results demonstrate that CLA isomers differentially modulate the development of atherosclerosis, c9,t11-CLA impedes, whereas t10,c12-CLA promotes atherosclerosis. These opposing effects may be ascribed to divergent effects on lipid, oxidative, inflammatory and fibro muscular components of this pathology. Plasma chitotriosidase is a better indicator of dietary fat interventions that alter plaque monocyte activity in this murine model.

Background and aimsThe present study was designed to verify the influence of acute fat loading on high density lipoprotein (HDL) composition, and the involvement of liver and different segments of small intestine in the changes observed.Methods and resultsTo address these issues, rats were administered a bolus of 5-ml of extra-virgin olive oil and sacrificed 4 and 8 hours after feeding. In these animals, lipoproteins were analyzed and gene expressions of apolipoprotein and HDL enzymes were assessed in duodenum, jejunum, ileum and liver. Using this experimental design, total plasma and HDL phospholipids increased at the 8-hour-time-point due to increased sphingomyelin content. An increase in apolipoprotein A4 was also observed mainly in lipid-poor HDL. Increased expression of intestinal Apoa1, Apoa4 and Sgms1 mRNA was accompanied by hepatic decreases in the first two genes in liver. Hepatic expression of Abcg1, Apoa1bp, Apoa2, Apoe, Ptlp, Pon1 and Scarb1 decreased significantly following fat gavage, while no changes were observed for Abca1, Lcat or Pla2g7. Significant associations were also noted for hepatic expression of apolipoproteins and Pon1. Manipulation of postprandial triglycerides using an inhibitor of microsomal transfer protein -CP-346086- or of lipoprotein lipase -tyloxapol- did not influence hepatic expression of Apoa1 or Apoa4 mRNA.ConclusionAll these data indicate that dietary fat modifies the phospholipid composition of rat HDL, suggesting a mechanism of down-regulation of hepatic HDL when intestine is the main source of those particles and a coordinated regulation of hepatic components of these lipoproteins at the mRNA level, independently of plasma postprandial triglycerides.

The expression of Synaptotagmin 1 (Syt1) has been found to be associated with the lipid droplets in liver. Here, we studied the expression of Syt1 in Apoe-deficient mice receiving cholesterol, Western diet, squalene, and oleanolic acid. We also studied the influence of sex and impact of surgical castration. Dietary cholesterol increased hepatic Syt1 expression, an effect that was enhanced when cholesterol was combined with saturated fat present in a Western diet. This potentiation was modified by the administration of 10 mg/kg oleanolic acid or 1 g/kg squalene. Females fed chow or Western diet showed higher levels of hepatic Syt1 expression as compared to male mice on the same diet. Surgical castration of males did not modify the Syt1 expression; however, ovariectomy led to decreased levels. The data show that hepatic Syt1 expression is influenced by diet and hormonal milieu.