María A. Navarro

Divergent mechanisms of cis9, trans11-and trans10, cis12-conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach

Conjugated linoleic acids (CLA) affect atherogenesis, but mechanisms are not well understood. We explored how two isomers of CLA, cis9, trans11‐CLA and trans10, cis12‐CLA, affected lipid and glucose metabolism, as well as hepatic protein expression, in apolipoprotein E knockout mice. After 12 wk of intervention, plasma triglyceride, NEFA, and glucose concentrations were significantly higher in the trans10, cis12‐CLA group, whereas plasma triglyceride, NEFA, glucose, and insulin concentrations were significantly lower in the cis9, trans 11‐CLA group, compared with control mice consuming linoleic acid. Proteomics identified significant up‐ or down‐regulation of 113 liver cytosolic proteins by either CLA isomer. Principal component analysis revealed that the treatment effect of cis9, trans11‐CLA was mainly explained by the up‐regulation of different posttranslational forms of heat shock protein 70 kD. In contrast, the treatment effect of trans10, cis12‐CLA was mainly explained by up‐regulation of key enzymes in the gluconeogenic, β‐oxidation, and ketogenesic pathways. Correlation analysis again emphasized the divergent effects of both CLA isomers on different pathways, but also revealed a linkage between insulin resistance and increased levels of hepatic serotransferrin. Thus, our systems biology approach provided novel insights into the mechanisms by which individual CLA isomers differentially affect pathways related to atherogenesis, such as insulin resistance and inflammation. Baukje De Roos, Garry Rucklidge, Martin Reid, Karen Ross, Gary Duncan, Maria A. Navarro, Jose M. Arbones‐Mainar, Mario A. Guzman‐Garcia, Jesus Osada, John Browne, Christine E. Loscher, Helen M. Roche Divergent mechanisms of cis9, trans11‐ and trans10, cis12‐conjugated linoleic acid affecting insulin resistance and inflammation in apolipoprotein E knockout mice: a proteomics approach. FASEB J. 19, 1–21 (2005)

Microarray analysis of hepatic gene expression identifies new genes involved in steatotic liver

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.

Efficacy of bioactive compounds from extra virgin olive oil to modulate atherosclerosis development.

As olive oil is the main source of calories in the Mediterranean diet, a great deal of research has been devoted to characterizing its role in atherosclerosis. Virgin olive oil is an oily matrix that contains hydrocarbons, mainly squalene; triterpenes such as uvaol, erythrodiol, oleanolic, and maslinic acid; phytosterols; and a wide range of phenolic compounds comprising simple phenols, flavonoids, secoiridoids, and lignans. In this review, we analyze the studies dealing with atherosclerosis and olive oil in several species. A protective role of virgin olive oil against atherosclerosis has been shown in ApoE-deficient mice and hamsters. In the former animal, sex, dose, and dietary cholesterol are modulators of the outcome. Contradictory findings have been reported for rabbits, a circumstance that could be due to the profusion of experimental designs, differing in terms of doses and animal strains, as well as sources of olive oils. This role has yet to be fully validated in humans. Minor components of olive oil have been shown to be involved in atherosclerosis protection. Nevertheless, evidence of the potential of isolated compounds or the right combination of them to achieve the antiatherosclerotic effect of virgin olive oil is inconclusive and will undoubtedly require further experimental support.

Intestinal d -Galactose Transport in an Endotoxemia Model in the Rabbit

Lipopolysaccharide (LPS) is an endotoxin causing sepsis. Studies from our laboratory revealed impaired intestinal absorption of l-leucine and d-fructose in LPS-treated rabbits. The aim of this study was to examine intestinal d-galactose transport following intravenous administration of LPS in the rabbit and to identify the cellular mechanisms driving this process. Endotoxin treatment diminished the buildup of d-galactose in intestinal tissue, the mucosal to serosal transepithelial flux of the sugar and its uptake by brush border membrane vesicles (BBMVs). Intracellular signaling pathways associated with protein kinase C (PKC), protein kinase A (PKA), p38 mitogen-activated protein kinase (p38MAPK), Jun N-terminal kinase (JNK), MAPK/extracellular signal-regulated kinases 1 and 2 (MEK1/2) and proteasome were found to be involved in this reduction in sugar uptake. Na+/glucose cotransporter 1 (SGLT1) protein levels in BBMVs were lower for LPS-treated animals than control animals. These findings indicate that LPS inhibits the intestinal absorption of d-galactose via a complex cellular mechanism that could involve posttranscriptional regulation of the SGLT1 transporter.

Selection of reference genes for gene expression studies in rats

Selection of the most stable reference gene is critical for a reliable interpretation of gene expression data using RT-PCR. In order so, 17 commonly used genes were analyzed in Wistar rat duodenum, jejunum, ileum and liver following a fat gavage and at two time periods. These reference genes were also tested in liver from Zucker (fa/fa) on a long-term dietary trial. Four strategies were used to select the most suitable reference gene for each tissue: ranking according to biological coefficient of variation and further validation by statistical comparison among groups, geNorm, NormFinder and BestKeeper programs. No agreement was observed among these approaches for a particular gene, nor a common gene for all tissues. Furthermore we demonstrated that normalising using an inadequate reference conveyed into false negative and positive results. The selection of genes provided by BestKeeper resulted in more reliable results than the other statistical packages. According to this program, Tbp, Ubc, Hprt and Rn18s were the best reference genes for duodenum, jejunum, ileum and liver, respectively following a fat gavage in Wistar rats and Rn18s for liver in another rat strain on a long-term dietary intervention. Therefore, BestKeeper is highly recommendable to select the most stable gene to be used as internal standard and the selection of a specific reference expression gene requires a validation for each tissue and experimental design.

Inhibitory effect of TNF-α on the intestinal absorption of galactose

Sepsis is a systemic response to infection in which toxins, such as bacterial lipopolysaccharide (LPS), stimulate the production of inflammatory mediators like the cytokine tumor necrosis factor alpha (TNF‐α). Previous studies from our laboratory have revealed that LPS inhibits the intestinal absorption of L‐leucine and D‐fructose in rabbit when it was intravenously administered, and that TNF‐α seems to mediate this effect on amino acid absorption. To extend this work, the present study was designed to evaluate the possible effect of TNF‐α on D‐galactose intestinal absorption, identify the intracellular mechanisms involved and establish whether this cytokine mediates possible LPS effects. Our findings indicate that TNF‐α decreases D‐galactose absorption both in rabbit intestinal tissue preparations and brush‐border membrane vesicles. Western blot analysis revealed reduced amounts of the Na+/glucose cotransporter (SGLT1) protein in the plasma membrane attributable to the cytokine. On the contrary, TNF‐α increased SGLT1 mRNA levels. Specific inhibitors of the secondary messengers PKC, PKA, the MAP kinases p38 MAP, JNK, MEK1/2 as well as the proteasome, diminished the TNF‐α‐evoked inhibitory effect. LPS inhibition of the uptake of the sugar was blocked by a TNF‐α antagonist. In conclusion, TNF‐α inhibits D‐galactose intestinal absorption by decreasing the number of SGLT1 molecules at the enterocyte plasma membrane through a mechanism in which several protein‐like kinases are involved. J. Cell. Biochem. 101: 99–111, 2007.

Understanding the role of dietary components on atherosclerosis using genetic engineered mouse models.

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.

Over-expression of Neuron-derived Orphan Receptor-1 (NOR-1) exacerbates neointimal hyperplasia after vascular injury

We have previously shown that NOR-1 (NR4A3) modulates the proliferation and survival of vascular cells in culture. However, in genetically modified animal models, somewhat conflicting results have been reported concerning the involvement of NOR-1 in neointimal formation after vascular injury. The aim of this study was to generate a transgenic mouse model over-expressing NOR-1 in smooth muscle cells (SMCs) and assess the consequence of a gain of function of this receptor on intimal hyperplasia after vascular injury. The transgene construct (SM22-NOR1) was prepared by ligating the full-length human NOR-1 cDNA (hNOR-1) and a mouse SM22α minimal promoter able to drive NOR-1 expression to SMC. Two founders were generated and two stable transgenic mouse lines (TgNOR-1) were established by backcrossing the transgene-carrying founders with C57BL/6J mice. Real-time PCR and immunohistochemistry confirmed that hNOR-1 was mainly targeted to vascular beds such as aorta and carotid arteries, and was similar in both transgenic lines. Vascular SMC from transgenic animals exhibit increased NOR-1 transcriptional activity (assessed by electrophoretic mobility shift assay and luciferase assays), increased mitogenic activity (determined by [(3)H]-thymidine incorporation; 1.58-fold induction, P < 0.001) and increased expression of embryonic smooth muscle myosin heavy chain (SMemb) than wild-type cells from control littermates. Using the carotid artery ligation model, we show that neointima formation was increased in transgenic versus wild-type mice (2.36-fold induction, P < 0.01). Our in vivo data support a role for NOR-1 in VSMC proliferation and vascular remodelling. This NOR-1 transgenic mouse could be a useful model to study fibroproliferative vascular diseases.

Lysyl oxidase (LOX) in vascular remodelling. Insight from a new animal model.

Lysyl oxidase (LOX) is an extracellular matrix-modifying enzyme that seems to play a critical role in vascular remodelling. However, the lack of viable LOX-deficient animal models has been an obstacle to deep in LOX biology. In this study we have developed a transgenic mouse model that over-expresses LOX in vascular smooth muscle cells (VSMC) to clarify whether LOX could regulate VSMC phenotype and vascular remodelling. The SM22α proximal promoter drove the expression of a transgene containing the human LOX cDNA. Two stable transgenic lines, phenotypically indistinguishable, were generated by conventional methods (TgLOX). Transgene expression followed the expected SMC-specific pattern. In TgLOX mice, real-time PCR and immunohistochemistry evidenced a strong expression of LOX in the media from aorta and carotid arteries, coincident with a higher proportion of mature collagen. VSMC isolated from TgLOX mice expressed high levels of LOX pro-enzyme, which was properly secreted and processed into mature and bioactive LOX. Interestingly, cell proliferation was significantly reduced in cells from TgLOX mice. Transgenic VSMC also exhibited low levels of Myh10 (marker of SMC phenotypic switching), PCNA (marker of cell proliferation) and MCP-1, and a weak activation of Akt and ERK1/2 in response to mitogenic stimuli. Accordingly, neointimal thickening induced by carotid artery ligation was attenuated in TgLOX mice that also displayed a reduction in PCNA and MCP-1 immunostaining. Our results give evidence that LOX plays a critical role in vascular remodelling. We have developed a new animal model to study the role of LOX in vascular biology.

Immunomodulatory properties of Beta-sitosterol in pig immune responses

The ability to control an immune response for the benefit and production efficiency of animals is the objective of immunomodulation in food-producing animals; substances that exert this control are called immunomodulators. A Spanish product (Inmunicín MAYMO®), based on food plant phytosterols, is being commercialized as complementary feed. The main component of this product is Beta-sitosterol (BSS). BSS and its glycoside (BSSG) have been shown to exhibit anti-inflammatory, anti-neoplasic, anti-pyretic and immune-modulating activity demonstrated by in vitro and in vivo experiments. The objective of the present study was to characterize the effect of BSS on the pig immune system using in vitro cell cultures first and to elucidate whether BSS possesses any in vivo activity in fattener pigs after vaccination with porcine reproductive and respiratory syndrome virus (PRRSV) modified life vaccine (MLV). Firstly, our in vitro results showed that BSS increased viable peripheral blood mononuclear cell (PBMC) numbers and it activated swine dendritic cells (DCs) in culture. Secondly, pigs treated with phytosterols prior to vaccination with PRRSV-MLV vaccine exhibited some changes in immunological parameters at different times post-vaccination, such as the proliferation ability of PBMC after phytohemaglutinin stimulation and increased apolipoprotein A1 plasma concentration which may contribute to enhance PRRSV vaccine response. In conclusion, the data in this report show that BSS can be considered an immunomodulator in pigs.