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.

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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<0.001) and between apoA-II and atherosclerosis (r=0.68, P<0.001). These results indicate that trans-10, cis-12-CLA intake modifies HDL to form a proatherogenic apoA-II containing particle and promotes phenotypic changes compatible with metabolic syndrome. Cis-9, trans-11-CLA does not promote this detrimental effect.

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.

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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.