Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Effects of oral exposure to arsenite on arsenic metabolism and transport in rat kidney.

Nephrotoxicity is within the recognized toxic effects of arsenic. In this study we assessed the effect of arsenite on the renal capacity to metabolize and handle arsenicals in rats exposed to drinking water with 0, 1, 5, or 10 ppm sodium arsenite for ten days. Arsenite treatment did not affect the gene expression of the main enzyme catalyzing methylation of arsenite, As3mt, while it reduced the expression of GSTO1 mRNA and protein. Arsenite decreased the expression of Aqp3, Mrp1, Mrp4, and Mdr1b (i.e., transporters and channels used by arsenic), but not that of Aqp7, Glut1, Mrp2, and Mdr1a. The protein abundance of AQP3 was also reduced by arsenite. Arsenite increased urinary NGAL and FABP3 and decreased Klotho plasma levels, without alteration of creatinine, which evidenced early tubular damage. Renal Klotho mRNA and protein expressions were also downregulated, which may exacerbate renal damage. No effect was observed in selected miRNAs putatively associated with renal injury. Plasma PTH and FGF23 were similar between groups, but arsenite decreased the renal expression of Fgfr1 mRNA. In conclusion, exposure to arsenite alters the gene expression of proteins involved in the cellular handling of arsenical species and elicits tubular damage.

Hepatic Synaptotagmin 1 is involved in the remodelling of liver plasma- membrane lipid composition and gene expression in male Apoe-deficient mice consuming a Western diet.

BACKGROUND AND AIMS: The molecular mechanisms by which the liver develops steatotic disease still remain unclear. Previous studies using nutritional and genetic models of hepatic steatosis in mice showed that liver synaptotagmin 1 (Syt1) expression was associated with lipid droplet area. Hepatic Syt1 overexpression was used as a tool to explore its effect on hepatic and plasma lipids. METHODS AND RESULTS: To find out a cause-effect, hepatic mouse Syt1 mRNA was cloned into a vector driving hepatocyte-specific expression and administered by hydrodynamic injection to male Apoe-deficient mice fed on a Western diet, the latter as a model of rapid spontaneous steatosis development. Hepatic microsomal, large vesicle, lysosomal and plasma membrane fractions were enriched in SYT1 protein following gene overexpression. In these conditions, very low density lipoprotein esterified cholesterol increased. Likewise, the transgene caused an alteration in lipid droplet surface and a positive correlation between Syt1 expression and hepatic total cholesterol content. A lipidomic approach evidenced a decrease in lysophosphatidylcholine, phosphatidylcholine and triglycerides in isolated plasma membrane fraction. Expressions of genes involved in biosynthesis of bile acids, fatty acid metabolism, lipoprotein dynamics and vesicular transport were modified by the increased SYT1 expression. CONCLUSIONS: These results indicate that this protein is involved in hepatic management of lipids and in the regulation of genes involved in lipid metabolism.

Diagnosis of genetic amyloidosis through the analysis of transthyretin gene mutation using high-resolution melting.

Transthyretin amyloidosis can be either the wild-type (ATTR-wt) or the hereditary form (ATTR-m) with autosomal dominant inheritance. ATTR seems to be an underdiagnosed disease, despite now being recognized as one of the most frequent causes of heart failure (HF) with preserved ejection fraction. The confirmation of diagnosis includes a genetic analysis as a critical step to distinguish between ATTR-wt and hereditary amyloidosis. The present study aimed to evaluate the potential application of High-Resolution Melting (HRM) analysis for identifying gene mutations in patients with suspected ATTR-m. We have adapted and validated the use of HRM for TTR mutations. We, therefore, sequenced the TTR gene and used HRM in a group of 134 patients suspected of suffering from amyloidosis. Seven patients were diagnosed with mutations in the TTR gene (p.Glu74Gln, heterozygous p.Val142Ile, and homozygous p.Val142Ile). HRM is capable of clearly detecting these TTR mutations, including the heterozygous and homozygous variants. The results show a 100% correlation between the HRM study and TTR sequencing. These results support future studies of applying HRM analysis as a diagnostic approach for ATTR-m, mainly for epidemiological studies.

Several phosphate transport processes are present in vascular smooth muscle cells.

We have studied inorganic phosphate (Pi) handling in rat aortic vascular smooth muscle cells (VSMC) using 32P-radiotracer assays. Our results have revealed a complex set of mechanisms consisting of 1) well-known PiT1/PiT2-mediated sodium-dependent Pi transport; 2) Slc20-unrelated sodium-dependent Pi transport that is sensitive to the stilbene derivatives 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) and 4-acetamido-4-isothiocyanostilbene-2,2-disulfonate (SITS); 3) a sodium-independent Pi uptake system that is competitively inhibited by sulfate, bicarbonate, and arsenate and is weakly inhibited by DIDS, SITS, and phosphonoformate; and 4) an exit pathway from the cell that is partially chloride dependent and unrelated to the known anion-exchangers expressed in VSMC. The inhibitions of sodium-independent Pi transport by sulfate and of sodium-dependent transport by SITS were studied in greater detail. The maximal inhibition by sulfate was similar to that of Pi itself, with a very high inhibition constant (212 mM). SITS only partially inhibited sodium-dependent Pi transport, but the Ki was very low (14 µM). Nevertheless, SITS and DIDS did not inhibit Pi transport in Xenopus laevis oocytes expressing PiT1 or PiT2. Both the sodium-dependent and sodium-independent transport systems were highly dependent on VSMC confluence and on the differentiation state, but they were not modified by incubating VSMC for 7 days with 2 mM Pi under nonprecipitating conditions. This work not only shows that the Pi handling by cells is highly complex but also that the transport systems are shared with other ions such as bicarbonate or sulfate.NEW & NOTEWORTHY In addition to the inorganic phosphate (Pi) transporters PiT1 and PiT2, rat vascular smooth muscle cells show a sodium-dependent Pi transport system that is inhibited by DIDS and SITS. A sodium-independent Pi uptake system of high affinity is also expressed, which is inhibited by sulfate, bicarbonate, and arsenate. The exit of excess Pi is through an exchange with extracellular chloride. Whereas the metabolic effects of the inhibitors, if any, cannot be discarded, kinetic analysis during initial velocity suggests competitive inhibition.

Substrates and inhibitors of phosphate transporters: from experimental tools to pathophysiological relevance.

The control of inorganic phosphate homeostasis is mediated through the activity of sodium-coupled Pi transporters located in the intestine, kidneys, and bone. To study these transporters in either the native tissue or after heterologous expression, it is very important to use specific inhibitors of the studied transporter, in order to know the corresponding relevance in the total Pi uptake and to differentiate from the activity of other transporters. Inhibitors are also necessary as drugs for treating Pi homeostasis disorders. Under normal physiological conditions, the renal and intestinal excretion of Pi matches dietary intestinal absorption, but when the number of non-functional nephrons increase in chronic kidney disease and end-stage renal disease, the excretion of surplus Pi is progressively impaired, thereby increasing the risk of hyperphosphatemia and Pi toxicity. When the compensatory mechanisms that increase Pi excretion fail, Pi toxicity can only be prevented by reducing the intestinal absorption of Pi through phosphate binders that reduced the free Pi concentration in the lumen, and inhibitors of intestinal Pi transporters and of the paracellular absorption route. Although many potentially interesting inhibitors have been reported to date, only a few are available for experimental purposes, and even fewer have been used in independent clinical trials. In this review, we summarize the different groups of compounds reported to date as inhibitors of Pi transport. To help understand and characterize the inhibition mechanisms, we also summarize the kinetic analysis approaches and screening methods that could be applied.

Identification and expression analysis of type II and type III Pi transporters in the opossum kidney cell line.

NEW FINDINGS: What is the central question of this study? The opossum kidney (OK) cell line is the main in vitro model of proximal tubular Pi transport, but it is incomplete because only the NaPiIIa Pi transporter has been identified. What is the main finding and its importance? We have cloned and characterized the Pi transporters NaPiIIc, PiT1 and PiT2 from OK cells and have analysed the relevance of the four transporters to Pi transport. All four transporters are involved in the upregulated Pi transport of cells incubated using a low-Pi medium, and only PiT1 is not involved in basal transport. ABSTRACT: The apical membrane of renal proximal tubular epithelial cells is the main controller of phosphate homeostasis, because it determines the rate of urinary Pi excretion. The opossum kidney (OK) cell line is a good model for studying this function, but only NaPiIIa (NaPi4) has been identified to date as a Pi transporter in this cell line. In this work, we have identified three additional Pi transporters that are present in OK cells: NaPiIIc, PiT1 and PiT2. All three sequences are similar to the corresponding orthologues, but PiT1 is missing the first transmembrane domain. Confluent cells exhibit characteristics of type II Pi transport, which increases with alkalinity and is inhibited by phosphonoformic acid (PFA), and they mainly express NaPiIIa and NaPiIIc, with a low abundance of PiT1 and PiT2. Proliferating cells show a higher expression of PiT1 and PiT2 and a low expression of NaPiIIa and NaPiIIc. Adaptation to a low Pi concentration for 24 h induces the expression of RNA from NaPiIIa and NaPiIIc, which is not prevented by actinomycin D. Small interfering RNA transfections revealed that PiT1 is not necessary for Pi transport, but it is necessary for adaptation to a low Pi , similar to NaPiIIa and PiT2. Our study reveals the complexity of the coordination between the four Pi transporters, the variability of RNA expression according to confluence and the heterogeneous correlation between Pi transport and RNA levels.

Identifying early pathogenic events during vascular calcification in uremic rats.

Vascular calcification in chronic kidney disease is a very complex process traditionally explained in multifactorial terms. Here we sought to clarify relevance of the diverse agents acting on vascular calcification in uremic rats and distinguish between initiating and complicating factors. After 5/6 nephrectomy, rats were fed a 1.2% phosphorus diet and analyzed at different time points. The earliest changes observed in the aortic wall were noticed 11 weeks after nephrectomy: increased Wnt inhibitor Dkk1 mRNA expression and tissue non-specific alkaline phosphatase (TNAP) expression and activity. First deposits of aortic calcium were observed after 12 weeks in areas of TNAP expression. Increased mRNA expressions of Runx2, BMP2, Pit1, Pit2, HOXA10, PHOSPHO1, Fetuin-A, ANKH, OPN, Klotho, cathepsin S, MMP2, and ENPP1 were also found after TNAP changes. Increased plasma concentrations of activin A and FGF23 were observed already at 11 weeks post-nephrectomy, while plasma PTH and phosphorus only increased after 20 weeks. Plasma pyrophosphate decreased after 20 weeks, but aortic pyrophosphate was not modified, nor was the aortic expression of MGP, Msx2, several carbonic anhydrases, osteoprotegerin, parathyroid hormone receptor-1, annexins II and V, and CD39. Thus, increased TNAP and Dkk1 expression in the aorta precedes initial calcium deposition, and this increase is only preceded by elevations in circulating FGF23 and activin A. The expression of other agents involved in vascular calcification only changes at later stages of chronic kidney disease, in a complex branching pattern that requires further clarification.

Intestinal phosphate absorption is mediated by multiple transport systems in rats.

Apical inorganic phosphate (Pi) transport in the small intestine seems to be mainly mediated by the sodium/Pi cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum for all three times. Alkaline pH inhibited Pi transport, despite the increasing concentration of [Formula: see text] (NaPi2b substrate), whereas acidity increased transport when the concentration of the PiT1/PiT2 substrate, [Formula: see text], was at its highest. The effect of Pi deprivation was maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 h of Pi deprivation in the duodenum, jejunum, and ileum, whereas PiT1 required 5-10 days in the duodenum and jejunum. Therefore, whereas transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and phosphonoformic acid precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression were also inconsistent when feeding was limited to 4 h daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries [Formula: see text] and is inhibited by phosphonoformate.NEW & NOTEWORTHY The combined effects of dietary inorganic phosphate (Pi) content, pH, and phosphonoformate inhibition suggest that the resulting apical Pi transport in the small intestine cannot be fully explained by the presence of NaPi2b, PiT1, or PiT2. We provide evidence of the presence of a new sodium-coupled Pi transporter that uses [Formula: see text] as the preferred substrate and is inhibited by phosphonoformate, and its expression correlates with Pi transport in all assayed conditions.

Cloning and expression of hepatic synaptotagmin 1 in mouse.

Mouse hepatic synaptotagmin 1 (SYT1) cDNA was cloned, characterized and compared to the brain one. The hepatic transcript was 1807 bp in length, smaller than the brain, and only encoded by 9 of 11 gene exons. In this regard, 5'-and 3'-untranslated regions were 66 and 476 bp, respectively; the open reading frame of 1266 bp codified for a protein of 421 amino acids, identical to the brain, with a predicted molecular mass of 47.4 kDa and highly conserved across different species. Immunoblotting of protein showed two isoforms of higher molecular masses than the theoretical prediction based on amino acid sequence suggesting posttranslational modifications. Subcellular distribution of protein isoforms corresponded to plasma membrane, lysosomes and microsomes and was identical between the brain and liver. Nonetheless, the highest molecular weight isoform was smaller in the liver, irrespective of subcellular location. Quantitative mRNA tissue distribution showed that it was widely expressed and that the highest values corresponded to the brain, followed by the liver, spleen, abdominal fat, intestine and skeletal muscle. These findings indicate tissue-specific splicing of the gene and posttranslational modification and the variation in expression in the different tissues might suggest a different requirement of SYT1 for the specific function in each organ.

Na+-independent phosphate transport in Caco2BBE cells.

Pi transport in epithelia has both Na(+)-dependent and Na(+)-independent components, but so far only Na(+)-dependent transporters have been characterized in detail and molecularly identified. Consequently, in the present study, we initiated the characterization and analysis of intestinal Na(+)-independent Pi transport using an in vitro model, Caco2BBE cells. Only Na(+)-independent Pi uptake was observed in these cells, and Pi uptake was dramatically increased when cells were incubated in high-Pi DMEM (4 mM) from 1 day to several days. No response to low-Pi medium was observed. The increased Pi transport was mainly caused by Vmax changes, and it was prevented by actinomycin D and cycloheximide. Pi transport in cells grown in 1 mM Pi (basal DMEM) decreased at pH > 7.5, and it was inhibited with proton ionophores. Pi transport in cells incubated with 4 mM Pi increased with alkaline pH, suggesting a preference for divalent phosphate. Pi uptake in cells in 1 mM Pi was completely inhibited only by Pi and partially inhibited by phosphonoformate, oxalate, DIDS, SITS, SO4 (2-), HCO3 (-), and arsenate. This inhibition pattern suggests that more than one Pi transporter is active in cells maintained with 1 mM Pi. Phosphate transport from cells maintained at 4 mM Pi was only partially inhibited by phosphonoformate, oxalate, and arsenate. Attempts to identify the responsible transporters showed that multifunctional anion exchangers of the Slc26 family as well as members of Slc17, Slc20, and Slc37 and the Pi exporter xenotropic and polytropic retrovirus receptor 1 are not involved.

Characterization of the cDNA and in vitro expression of the ram seminal plasma protein RSVP14.

In previous studies we have shown that seminal plasma (SP) proteins can prevent and repair cold-shock membrane damage to ram spermatozoa. Three proteins of approximately 14, 20 and 22kDa, mainly responsible for this protective ability, were identified in ram SP. They are exclusively synthesized in the seminal vesicles and, consequently, named RSVP14, RSVP20 and RSVP22. The aim of this study is to characterize and express the RSVP14 gene to provide new insights into the mechanisms through which SP proteins are able to protect spermatozoa. Additionally, a first approach has been made to the recombinant protein production. The cDNA sequence obtained encodes a 129 amino acid chain and presents a 25-amino acid signal peptide, one potential O-linked glycosylation site and seven phosphorylation sites on tyrosine, serine and threonine residues. The sequence contains two FN-2 domains, the signature characteristic of the bovine seminal plasma (BSP) protein family and related proteins of different species. More interestingly, it was shown that RSVP14 contains four disulphide bonds and a cholesterol recognition/interaction amino acid consensus (CRAC) domain, also found in BSP and similar proteins. Analysis of the relationships between RSVP14 and other mammalian SP proteins revealed a 76-85% identity, particularly with the BSP protein family. The recombinant protein was obtained in insect cell extracts and in Escherichia coli in which RSVP14 was detected in both the pellet and the supernatant. The results obtained corroborate the role of RSVP14 in capacitation and might explain its protective effect against cold-shock injury to the membranes of ram spermatozoa. Furthermore, the biochemical and functional similarities between RSVP14 and BSP proteins suggest that it might play a similar role in sperm functionality.

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.

In comparison with palm oil, dietary nut supplementation delays the progression of atherosclerotic lesions in female apoE-deficient mice.

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 hepatic Pon2 expression. 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.

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

Squalene is an abundant hydrocarbon present in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE-knock-out mice. To study its effects on microsomal proteins, 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.

Cystathionine ß-synthase deficiency causes infertility by impairing decidualization and gene expression networks in uterus implantation sites.

Hyperhomocysteinemia has been reported in human reproduction as a risk factor for early pregnancy loss, preeclampsia, and congenital birth defects like spina bifida. Female infertility was also observed in cystathionine beta synthase-deficient mice (Cbs-KO) as an animal model for severe hyperhomocysteinemia. The aim for the present research was to elucidate the time-point of pregnancy loss and to pinpoint gene and cellular changes involved in the underlying pathological mechanism. By mating 90-day-old wild-type and Cbs-KO female mice with their homologous male partners, we found that pregnancy loss in Cbs-KO occurred between the 8th and 12th gestation day during placenta formation. DNA microarrays were carried out on uterus from implantation and interimplantation samples obtained on day 8. The results allowed us to select genes potentially involved in embryo death; these were individually confirmed by RT-qPCR, and their expressions were also followed throughout pregnancy. We found that changes in expression of Calb1, Ttr, Expi, Inmt, Spink3, Rpgrip1, Krt15, Mt-4, Gzmc, Gzmb, Tdo2, and Afp were important for pregnancy success, since a different regulation in Cbs-KO mice was found. Also, differences in relationships among selected genes were observed, indicating a dysregulation of these genes in Cbs-KO females. In conclusion, our data provide more information on the gene expression cascade and its timely regulated process required for a successful pregnancy. In addition, we unveil new potential avenues to explore further investigations in pregnancy loss.

Proteomics and gene expression analyses of mitochondria from squalene-treated apoE-deficient mice identify short-chain specific acyl-CoA dehydrogenase changes associated with fatty liver amelioration.

Squalene, a hydrocarbon involved in cholesterol biosynthesis, is an abundant component in virgin olive oil. Previous studies showed that its administration decreased atherosclerosis and steatosis in male apoE knock-out mice. To study the effect of squalene on mitochondrial proteins in fatty liver, 1 g/kg/day of this isoprenoid was administered to those mice. After 10 weeks, hepatic fat was assessed and protein extracts from mitochondria enriched fractions from control and squalene-treated animals were analyzed by 2D-DIGE. Spots exhibiting significant differences were identified by MS analysis. Squalene administration modified the expression of eighteen proteins involved in different metabolic processes, 12 associated with hepatic fat content. Methionine adenosyltransferase I alpha (Mat1a) and short-chain specific acyl-CoA dehydrogenase (Acads) showed significant increased and decreased transcripts, respectively, consistent with their protein changes. These mRNAs were also studied in wild-type mice receiving squalene, where Mat1a was found increased and Acads decreased. However, this mRNA was significantly increased in the absence of apolipoprotein E. These results suggest that squalene action may be executed through a complex regulation of mitochondrial protein expression, including changes in Mat1a and Acads levels. Indeed, Mat1a is a target of squalene administration while Acads reflects the anti-steatotic properties of squalene.

Postprandial transcriptome associated with virgin olive oil intake in rat liver.

Liver has been proposed as a gatekeeper that regulates postprandial lipemia and a potential target for regulation by acute intake of virgin olive oil. To characterize the hepatic gene expression response to a fat gavage, male rats were fed a bolus of 5 ml of extra-virgin olive oil and the hepatic mRNA expression analyzed 4 hours later using DNA microarrays. To provide an initial screening of candidate genes, only twenty one with remarkably modified expression between both conditions (signal log2 ratio > 2.5 or < -2.5) were considered and confirmed by quantitative real time PCR. Those that presented biological significance were also analyzed 8 hours after the experimental approach. Hepatic A2m Slc13a5 and Nrep mRNA expressions were found significantly changed in both studied conditions and showed the highest significant associations with postprandial plasma triglycerides and lack of association with basal triglyceridemia. These results highlight new gene regulation in liver by postprandial triglyceridemia and will help to understand the complex human pathology providing the involvement of hepatic proteins and new strategies to cope with postprandial metabolism.

Nitric oxide involved in the IL-1ß-induced inhibition of fructose intestinal transport.

Interleukin-1ß (IL-1ß) is a pleiotropic cytokine produced by cells of the immune system and a large variety of other cell types including endothelial cells. It is released during inflammatory and infectious diseases, and possesses a wide spectrum of autocrine, paracrine and endocrine activities. The aim of this work was to examine the IL-1ß effect on D-fructose transport across rabbit jejunum and try to identify the mediators implicated in this process. A sepsis condition was induced for 90 min after intravenous (iv) administration of IL-1ß and body temperature was recorded. Studies on cellular intestinal integrity have not shown modifications of the epithelium and the basement membrane. D-fructose intestinal transport was studied in rabbit jejunum from control and treated animals and it was reduced in the latter ones. This cytokine decreased both the mucosal to serosal transepithelial flux and the transport across brush-border membrane vesicles of D-fructose. The inhibition was reversed by L-NAME (nitric oxide [NO] synthase inhibitor), but not by indomethacin (cyclooxygenase 1 and 2 inhibitor). Both inhibitors were administered iv 15 min before the IL-1ß. The protein levels of GLUT5 were not changed in all animal groups and those of mRNA were even increased. In summary, these findings indicate that IL-1ß, at the time assayed, induced a significant reduction in the relative intrinsic activity of GLUT5 and in this decrease are involved NO signalling pathways. In this way, blockage of D-fructose intestinal uptake by IL-1ß may be playing an essential role in the pathophysiology of septic shock.

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