Cristina Barranquero

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.

Postprandial Changes in High Density Lipoproteins in Rats Subjected to Gavage Administration of Virgin Olive Oil

Background and Aims The 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 Results To 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. Conclusion All 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.

Cysteinemia, rather than homocysteinemia, is associated with plasma apolipoprotein A-I levels in hyperhomocysteinemia: lipid metabolism in cystathionine β-synthase deficiency.

OBJECTIVE Genetic and dietary hyperhomocysteinemia has been found to decrease high density lipoproteins (HDL) and their apolipoprotein A1 (APOA1). To test the hypothesis that the presence of cysteine could normalize HDL levels in hyperhomocysteinemic cystathionine beta-synthase (Cbs)-deficient mice and that the inclusion of glycine would block this effect. METHODS Lipids and HDL cholesterol were studied in Cbs-deficient mice and wild-type animals fed a low-methionine diet supplemented with cysteine and glycine and in Cbs-deficient mice on the same diet supplemented only with cysteine. RESULTS Triglyceride and homocysteine levels were significantly decreased and increased, respectively in Cbs-deficient mice irrespective of treatment. However, plasma cholesterol, glucose and APOA1 were significantly decreased in homozygous Cbs-deficient mice when they received the cysteine and glycine-enriched beverage. This group of mice also showed decreased mRNA levels and increased hepatic content of APOA1 protein, the latter increase was observed in endothelial cells. A significant, inverse relationship was observed between plasma and hepatic APOA1 concentrations while a positive one was found between plasma levels of cysteine and APOA1. CONCLUSION These data suggest an altered hepatic management of APOA1 and that cysteine may be involved in the control of this apolipoprotein at this level. Overall these findings represent a new aspect of dietary regulation of HDL at the hepatic transendothelial transport.

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 squalenes 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.

Map3k8 Modulates Monocyte State and Atherogenesis in ApoE-/- Mice.

Objective— Map3k8 (Cot/Tpl2) activates the MKK1/2-ERK1/2, MAPK pathway downstream from interleukin-1R, tumor necrosis factor-&agr;R, NOD-2R (nucleotide-binding oligomerization domain-like 2R), adiponectinR, and Toll-like receptors. Map3k8 plays a key role in innate and adaptive immunity and influences inflammatory processes by modulating the functions of different cell types. However, its role in atherogenesis remains unknown. In this study, we analyzed the role of this kinase in this pathology. Approach and Results— We show here that Map3k8 deficiency results in smaller numbers of Ly6ChighCD11clow and Ly6ClowCD11chigh monocytes in ApoE−/− mice fed a high-fat diet (HFD). Map3k8−/−ApoE−/− monocytes displayed high rates of apoptosis and reduced amounts of Nr4a1, a transcription factor known to modulate apoptosis in Ly6ClowCD11chigh monocytes. Map3k8−/−ApoE−/− splenocytes and macrophages showed irregular patterns of cytokine and chemokine expression. Map3k8 deficiency altered cell adhesion and migration in vivo and decreased CCR2 expression, a determinant chemokine receptor for monocyte mobilization, on circulating Ly6ChighCD11clow monocytes. Map3k8−/−ApoE−/− mice fed an HFD showed decreased cellular infiltration in the atherosclerotic plaque, with low lipid content. Lesions had similar size after Map3k8+/+ApoE−/− bone marrow transplant into Map3k8−/−ApoE−/− and Map3k8+/+ApoE−/− mice fed an HFD, whereas smaller plaques were observed after the transplantation of bone marrow lacking both ApoE and Map3k8. Conclusions— Map3k8 decreases apoptosis of monocytes and enhances CCR2 expression on Ly6ChighCD11clow monocytes of ApoE−/− mice fed an HFD. These findings explain the smaller aortic lesions in ApoE−/− mice with Map3k8−/−ApoE−/− bone marrow cells fed an HFD, supporting further studies of Map3k8 as an antiatherosclerotic target.

Caloric restriction or telmisartan control dyslipidemia and nephropathy in obese diabetic Zücker rats

BackgroundThe obese Zücker diabetic fatty male rat (ZDF:Gmi™-fa) is an animal model of type II diabetes associated with obesity and related metabolic disturbances like dyslipidaemia and diabetic nephropathy. In addition, diabetic dyslipidaemia has been linked to vascular and glomerular damage too. Dietary fat restriction is a current strategy to tackle obesity and, telmisartan, as a renoprotective agent, may mediate cholesterol efflux by activating PPARγ. To test the hypothesis that both therapeutical alternatives may influence dyslipidaemia and nephropathy in the ZDF rat, we studied their effect on development of diabetes.MethodsMale Zücker Diabetic Fatty (ZDF) rats received a low-calorie diet, vehicle or telmisartan for 9 weeks. Blood samples were obtained for analyses of lipids and lipoproteins, LDL-oxidisability, HDL structural and functional properties. Urinalysis was carried out to estimate albumin loss. At the end of the experimental period, rats were sacrificed, liver extracted and APOA1 mRNA quantified.ResultsResults indicated that low-calorie diet and telmisartan can slower the onset of overt hyperglycaemia and renal damage assessed as albuminuria. Both interventions decreased the oxidative susceptibility of LDL and hepatic APOA1 mRNA expression but only dietary restriction lowered hyperlipidaemia.ConclusionEither a dietary or pharmacologic interventions with telmisartan have important beneficial effects in terms of LDL oxidative susceptibility and progression of albuminuria in obesity related type II diabetes.

Interleukin-1beta reduces galactose transport in intestinal epithelial cells in a NF-kB and protein kinase C-dependent manner.

Interleukins (IL), aside from their role in the regulation of the immune cascade, they have also been shown to modulate intestinal transport function. IL-1β is a potent inflammatory cytokine involved in many important cellular functions. The aim of this work was to study the in vitro effect of IL-1β on d-galactose transport across intestinal epithelia in rabbit jejunum and Caco-2 cells. The results showed that d-galactose intestinal absorption was diminished in IL-1β treated jejunum rabbits without affecting the Na(+), K(+)-ATPase activity. The presence of IL-1 cell-surface receptors was confirmed by addition to tissue of a specific IL-1 receptor antagonist (IL-1ra). The cytokine did not inhibit either the uptake of d-galactose nor modified the sodium-glucose transport (SGLT1) protein levels in the brush border membrane vesicles, suggesting an indirect IL effect. The IL-inhibition was significantly reversed in the presence of inhibitors of protein kinase C (PKC) and mitogen-activated protein kinases (MAPKs). The proteasome selective inhibitor completely abolished the IL-effect. Furthermore, the cytokine inhibition on galactose transport related to NF-kB activation was also confirmed in Caco-2 cells. In summary, the direct addition of IL-1β to intestinal epithelia inhibits d-galactose transport by a possible reduction in the SGLT1 activity. This event may be mediated by several transduction pathways activated during the inflammatory processes related to several protein kinases and nuclear factor, NF-kB. The IL-effect is independent of hormonal milieu and nervous stimuli.

Inhibitory effect of IL-1β on galactose intestinal absorption in rabbits.

Background/Aims: Recent studies from our laboratory have shown that nitric oxide is involved in the IL-1β-induced inhibition of D-fructose intestinal transport in rabbits. The aim of this work was to further the studies of IL-1β effect on D-galactose absorption in a septic state induced by intravenous administration of this cytokine. Methods: Galactose intestinal absorption was assessed employing three techniques: sugar uptake in jejunum everted rings, transepithelial flux in Ussing-type chambers and uptake assays in brush border membrane vesicles. The level of the Na+/D-glucose cotransporter (SGLT1) expression was analyzed by Western blot. Results: In sepsis condition the body temperature was increased and studies on cellular intestinal integrity have not shown modifications in the brush border membrane. However, D-galactose absorption across mucosa of jejunum was diminished in IL-1β treated rabbits. The levels of SGLT-1 were no significantly different in both animal groups (control and IL-1β treated), indicating that the cytokine could induce a reduction in the SGLT-1 functionality. The inhibition was significantly reversed by the activation of several PKC, PKA, MAPKs and nuclear factor (NF)-ĸB inhibitors administered 15 min before the IL-1β. Conclusion: The inhibitory effect of IL-1β on D-galactose absorption across mucosal side of enterocyte could be mediated by the activation of several kinases and nuclear factor (NF)-ĸB.

Involvement of Intracellular Signaling in the IL‐1β Inhibitory Effect on Fructose Intestinal Absorption

A variety of bacteria and their excreted/secreted products having direct effects on epithelial ion transport and permeability and the release of cytokines during bacterial infection may impact directly on epithelial function. Interleukin‐1β (IL‐1β) is a pleiotropic cytokine that affects the intestinal absorption of nutrients. The aim of this work was to study the intracellular signaling pathways involved in the inhibitory effect of IL‐1β on D‐fructose intestinal transport in rabbit jejunum and Caco‐2 cells. The results show that the cytokine inhibitory effect was completely reversed in presence of proteasome or PKC selective inhibitors in IL‐1β treated rabbits. In addition, the activation of PI3K abolished the IL‐1β effect. Likewise, these results were confirmed in Caco‐2 cells. In addition, p‐PI3K expression was increased by IL‐1β‐treatment whereas the expression of p‐PKCα was not significantly affected. In summary, the results suggest that IL‐1β could regulate the activation of pPKCα 73, pPI3K 55, and NF‐kB proteins. These events could exert an inhibitory effect on fructose intestinal absorption by a modification of GLUT5 insertion to brush‐border membrane and/or the functional transporter activity. This effect is independent of hormonal milieu and nervous stimuli. J. Cell. Physiol. 230: 896–902, 2015.