Lipidomic signatures discriminate subtle hepatic changes in the progression of porcine nonalcoholic steatohepatitis.

Recently, the development of nonalcoholic steatohepatitis (NASH) in common strains of pigs has been achieved using a diet high in saturated fat, fructose, cholesterol, and cholate and deficient in choline and methionine. The aim of the present work was to characterize the hepatic and plasma lipidomic changes that accompany the progression of NASH and its reversal by switching pigs back to a chow diet. One month of this extreme steatotic diet was sufficient to induce porcine NASH. The lipidomic platform using liquid chromatography-mass spectrometry analyzed 467 lipid species. Seven hepatic phospholipids [PC(30:0), PC(32:0), PC(33:0), PC(33:1), PC(34:0), PC(34:3) and PC(36:2)] significantly discriminated the time of dietary exposure, and PC(30:0), PC(33:0), PC(33:1) and PC(34:0) showed rapid adaptation in the reversion period. Three transcripts (CS, MAT1A, and SPP1) showed significant changes associated with hepatic triglycerides and PC(33:0). Plasma lipidomics revealed that these species [FA 16:0, FA 18:0, LPC(17:1), PA(40:5), PC(37:1), TG(45:0), TG(47:2) and TG(51:0)] were able to discriminate the time of dietary exposure. Among them, FA 16:0, FA 18:0, LPC(17:1) and PA(40:5) changed the trend in the reversion phase. Plasma LDL-cholesterol and IL12P40 were good parameters to study the progression of NASH, but their capacity was surpassed by hepatic [PC(33:0), PC(33:1), and PC(34:0)] or plasma lipid [FA 16:0, FA 18:0, and LPC(17:1)] species. Taken together, these lipid species can be used as biomarkers of metabolic changes in the progression and regression of NASH in this model. The lipid changes suggest that the development of NASH also affects peripheral lipid metabolism.NEW & NOTEWORTHY A NASH stage was obtained in crossbred pigs. Hepatic [PC(33:0), PC(33:1) and PC(34:0)] or plasma [FA 16:0, FA 18:0 and LPC(17:1)] species were sensitive parameters to detect subtle changes in development and regression of nonalcoholic steatohepatitis (NASH). These findings may delineate the liquid biopsy to detect subtle changes in progression or in treatments. Furthermore, phospholipid changes according to the insult-inducing NASH may play an important role in accepting or rejecting fatty livers in transplantation.

TXNDC5 Plays a Crucial Role in Regulating Endoplasmic Reticulum Activity through Different ER Stress Signaling Pathways in Hepatic Cells.

The pathogenesis of non-alcoholic fatty liver disease (NAFLD) is influenced by a number of variables, including endoplasmic reticulum stress (ER). Thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family and acts as an endoplasmic reticulum (ER) chaperone. Nevertheless, the function of TXNDC5 in hepatocytes under ER stress remains largely uncharacterized. In order to identify the role of TXNDC5 in hepatic wild-type (WT) and TXNDC5-deficient (KO) AML12 cell lines, tunicamycin, palmitic acid, and thapsigargin were employed as stressors. Cell viability, mRNA, protein levels, and mRNA splicing were then assayed. The protein expression results of prominent ER stress markers indicated that the ERN1 and EIF2AK3 proteins were downregulated, while the HSPA5 protein was upregulated. Furthermore, the ATF6 protein demonstrated no significant alterations in the absence of TXNDC5 at the protein level. The knockout of TXNDC5 has been demonstrated to increase cellular ROS production and its activity is required to maintain normal mitochondrial function during tunicamycin-induced ER stress. Tunicamycin has been observed to disrupt the protein levels of HSPA5, ERN1, and EIF2AK3 in TXNDC5-deficient cells. However, palmitic acid has been observed to disrupt the protein levels of ATF6, HSPA5, and EIF2AK3. In conclusion, TXNDC5 can selectively activate distinct ER stress pathways via HSPA5, contingent on the origin of ER stress. Conversely, the absence of TXNDC5 can disrupt the EIF2AK3 cascade.

Pgc1a is responsible for the sex differences in hepatic Cidec/Fsp27ß mRNA expression in hepatic steatosis of mice fed a Western diet.

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/Fsp27ß 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/Fsp27ß 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/Fsp27ß 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/Fsp27ß. When Pgc1a-deficient mice were used, the sex differences in Cidec/Fsp27ß expression disappeared. Therefore, hepatic Cidec/Fsp27ß expression has a complex regulation influenced by diet and sex hormonal milieu. The mRNA sex differences are controlled by Pgc1a.

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-αR, 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.

Involvement of intracellular signaling in the IL-1ß 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-1ß (IL-1ß) 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-1ß 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-1ß treated rabbits. In addition, the activation of PI3K abolished the IL-1ß effect. Likewise, these results were confirmed in Caco-2 cells. In addition, p-PI3K expression was increased by IL-1ß-treatment whereas the expression of p-PKCα was not significantly affected. In summary, the results suggest that IL-1ß 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.

Thioredoxin domain containing 5 is involved in the hepatic storage of squalene into lipid droplets in a sex-specific way.

Hepatic thioredoxin domain-containing 5 (TXNDC5) is a member of the protein disulfide isomerase family found associated with anti-steatotic properties of squalene and located in the endoplasmic reticulum and in lipid droplets. Considering that the latter are involved in hepatic squalene accumulation, the present research was aimed to investigate the role of TXNDC5 on hepatic squalene management in mice and in the AML12 hepatic cell line. Wild-type and TXNDC5-deficient (KO) mice were fed Western diets with or without 1% squalene supplementation for 6 weeks. In males, but not in females, absence of TXNDC5 blocked hepatic, but not duodenal, squalene accumulation. Hepatic lipid droplets were isolated and characterized using label-free LC-MS/MS analysis. TXNDC5 accumulated in this subcellular compartment of mice receiving squalene and was absent in TXNDC5-KO male mice. The latter mice were unable to store squalene in lipid droplets. CALR and APMAP were some of the proteins that responded to the squalene administration in all studied conditions. CALR and APMAP were positively associated with lipid droplets in the presence of squalene and they were decreased by the absence of TXNDC5. The increased squalene content was reproduced in vitro using AML12 cells incubated with squalene-loaded nanoparticles and this effect was not observed in an engineered cell line lacking TXNDC5. The phenomenon was also present when incubated in the presence of a squalene epoxidase inhibitor, suggesting a mechanism of squalene exocytosis involving CALR and APMAP. In conclusion, squalene accumulation in hepatic lipid droplets is sex-dependent on TXNDC5 that blocks its secretion.

Effect of virgin olive oil as spreadable preparation on atherosclerosis compared to dairy butter in Apoe-deficient mice.

Olive oil is the main source of lipid energy in the Mediterranean diet and there is strong evidence of its health benefits. The effect of extra virgin olive oil (EVOO) in the form of a preparation of spreadable virgin olive oil (S-VO) on the progression of atheroma plaques was investigated in Apoe-deficient mice, a model of accelerated atherosclerosis. METHODS: Two isocaloric Western purified diets containing 20% fat, either as S-VO or as dairy butter, were used to feed 28 males and 16 females of two-month-old Apoe-deficient mice for 12 weeks. S-VO was prepared by blending more than 75% virgin olive oil with other vegetal natural fat to obtain a solid fat. Plasma total cholesterol, triglycerides and HDL cholesterol were measured. Hepatic lipid droplets were analyzed. Areas of atherosclerotic aortic lesions were quantified in cross-sectional images of the proximal aorta and en face analysis of the whole aorta. RESULTS: Total plasma cholesterol was increased in mice on the butter-supplemented diet in both female and male mice compared to S-VO, and the ratio of TC/HDL-cholesterol was significantly lower in S-VO than in the butter diet, although only in males, and no differences in plasma triglycerides were observed. No significant differences in hepatic lipid droplets were observed between diets in either sex. Aortic lesion areas were significantly higher in mice consuming the butter versus the S-VO diet in both sexes. CONCLUSION: Extra virgin olive oil prepared in spreadable form maintained the delay in atheroma plaque progression compared to butter.

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.

Dietary proteins modulate high-density lipoprotein characteristics in a sex-specific way in Apoe-deficient mice.

OBJECTIVES: The type and amount of dietary protein have become a topic of renewed interest, considering their involvement in several diseases. However, little attention has been devoted to the effect of avian proteins despite their wide human consumption. In a previous study, we saw that compared with soybean protein, the consumption of avian proteins, depending on sex, resulted in similar or lower atherosclerosis with a higher paraoxonase 1 activity, an antioxidant enzyme carried by high-density lipoproteins (HDL). This suggests that under these conditions, the HDL lipoproteins may undergo important changes. The aim of this research was to study the influence of soybean, chicken, and turkey proteins on the characteristics of HDL. METHODS: Male and female Apoe-deficient mice were fed purified Western diets based on the AIN-93 diet, differing only in the protein source, for 12 wk. After this period, blood and liver samples were taken for analysis of HDL composition and hepatic expression of genes related to HDL metabolism (Abca1, Lcat, Pltp, Pon1, and Scarb1). Depending on sex, these genes define a different network of interactions. Females consuming the turkey protein-containing diet showed decreased atherosclerotic foci, which can be due to larger very-low-density lipoproteins (VLDLs) calculated by molar ratio triacylglycerols/VLDL cholesterol and higher expression of Lcat. In contrast, in males, a higher ratio of paraoxonase1 to apolipoprotein A1 decreased the oxidative status of the different lipoproteins, and augmented Abca1 expression was observed. CONCLUSIONS: The source of protein has an effect on the development of atherosclerosis depending on sex by modifying HDL characteristics and the expression of genes involved in their properties.

Dietary squalene supplementation decreases triglyceride species and modifies phospholipid lipidomic profile in the liver of a porcine model of non-alcoholic steatohepatitis.

Squalene is a key minor component of virgin olive oil, the main source of fat in the Mediterranean diet, and had shown to improve the liver metabolism in rabbits and mice. The present research was carried out to find out whether this effect was conserved in a porcine model of hepatic steatohepatitis and to search for the lipidomic changes involved. The current study revealed that a 0.5% squalene supplementation to a steatotic diet for a month led to hepatic accumulation of squalene and decreased triglyceride content as well as area of hepatic lipid droplets without influencing cholesterol content or fiber areas. However, ballooning score was increased and associated with the hepatic squalene content. Of forty hepatic transcripts related to lipid metabolism and hepatic steatosis, only citrate synthase and a non-coding RNA showed decreased expressions. The hepatic lipidome, assessed by liquid chromatography-mass spectrometry in a platform able to analyze 467 lipids, revealed that squalene supplementation increased ceramide, Cer(36:2), and phosphatidylcholine (PC[32:0], PC[33:0] and PC[34:0]) species and decreased cardiolipin, CL(69:5), and triglyceride (TG[54:2], TG[55:0] and TG[55:2]) species. Plasma levels of interleukin 12p40 increased in pigs receiving the squalene diet. The latter also modified plasma lipidome by increasing TG(58:12) and decreasing non-esterified fatty acid (FA 14:0, FA 16:1 and FA 18:0) species without changes in total NEFA levels. Together this shows that squalene-induced changes in hepatic and plasma lipidomic profiles, non-coding RNA and anti-inflammatory interleukin are suggestive of an alleviation of the disease despite the increase in the ballooning score.

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

BACKGROUND/AIMS: Recent studies from our laboratory have shown that nitric oxide is involved in the IL-1ß-induced inhibition of D-fructose intestinal transport in rabbits. The aim of this work was to further the studies of IL-1ß 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-1ß treated rabbits. The levels of SGLT-1 were no significantly different in both animal groups (control and IL-1ß 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-1ß. CONCLUSION: The inhibitory effect of IL-1ß on D-galactose absorption across mucosal side of enterocyte could be mediated by the activation of several kinases and nuclear factor (NF)-ĸB.

Thioredoxin Domain Containing 5 Suppression Elicits Serum Amyloid A-Containing High-Density Lipoproteins.

Thioredoxin domain containing 5 (TXNDC5) is a protein disulfide isomerase involved in several diseases related to oxidative stress, energy metabolism and cellular inflammation. In a previous manuscript, a negative association between fatty liver development and hepatic Txndc5 expression was observed. To study the role of TXNDC5 in the liver, we generated Txndc5-deficient mice. The absence of the protein caused an increased metabolic need to gain weight along with a bigger and fatter liver. RNAseq was performed to elucidate the putative mechanisms, showing a substantial liver overexpression of serum amyloid genes (Saa1, Saa2) with no changes in hepatic protein, but discrete plasma augmentation by the gene inactivation. Higher levels of malonyldialdehyde, apolipoprotein A1 and platelet activating factor-aryl esterase activity were also found in serum from Txndc5-deficient mice. However, no difference in the distribution of high-density lipoproteins (HDL)-mayor components and SAA was found between groups, and even the reactive oxygen species decreased in HDL coming from Txndc5-deficient mice. These results confirm the relation of this gene with hepatic steatosis and with a fasting metabolic derive remedying an acute phase response. Likewise, they pose a new role in modulating the nature of HDL particles, and SAA-containing HDL particles are not particularly oxidized.

Pulsed electric field increases the extraction yield of extra virgin olive oil without loss of its biological properties.

Introduction: Pulsed electric field (PEF) has been used for improving extraction of extra virgin olive oil (EVOO). However, the biological changes induced by the consumption of pulsed electric field-obtained extra virgin olive oil (PEFEVOO) have not been studied yet. Materials and methods: EVOO oils from Empeltre variety were prepared by standard (STD) cold pressure method involving crushing of the olives, malaxation and decanting and by this procedure including an additional step of PEF treatment. Chemical analyses of EVOO oils were done. Male and female Apoe-deficient mice received diets differing in both EVOOs for 12 weeks, and their plasma, aortas and livers were analyzed. Results: PEF application resulted in a 17% increase in the oil yield and minimal changes in chemical composition regarding phytosterols, phenolic compounds and microRNA. Only in females mice consuming PEF EVOO, a decreased plasma total cholesterol was observed, without significant changes in atherosclerosis and liver steatosis. Conclusion: PEF technology applied to EVOO extraction maintains the EVOO quality and improves the oil yield. The equivalent biological effects in atherosclerosis and fatty liver disease of PEF-obtained EVOO further support its safe use as a food.

Hepatic galectin-3 is associated with lipid droplet area in non-alcoholic steatohepatitis in a new swine model.

Non-alcoholic fatty liver disease (NAFLD) is currently a growing epidemic disease that can lead to cirrhosis and hepatic cancer when it evolves into non-alcoholic steatohepatitis (NASH), a gap not well understood. To characterize this disease, pigs, considered to be one of the most similar to human experimental animal models, were used. To date, all swine-based settings have been carried out using rare predisposed breeds or long-term experiments. Herein, we fully describe a new experimental swine model for initial and reversible NASH using cross-bred animals fed on a high saturated fat, fructose, cholesterol, cholate, choline and methionine-deficient diet. To gain insight into the hepatic transcriptome that undergoes steatosis and steatohepatitis, we used RNA sequencing. This process significantly up-regulated 976 and down-regulated 209 genes mainly involved in cellular processes. Gene expression changes of 22 selected transcripts were verified by RT-qPCR. Lipid droplet area was positively associated with CD68, GPNMB, LGALS3, SLC51B and SPP1, and negatively with SQLE expressions. When these genes were tested in a second experiment of NASH reversion, LGALS3, SLC51B and SPP1 significantly decreased their expression. However, only LGALS3 was associated with lipid droplet areas. Our results suggest a role for LGALS3 in the transition of NAFLD to NASH.

Dietary squalene modifies plasma lipoproteins and hepatic cholesterol metabolism in rabbits.

To evaluate the effects of squalene, the main unsaponifiable component of virgin olive oil, on lipid metabolism, two groups of male New Zealand rabbits were fed a 1% sunflower oil-enriched regular diet or the same diet containing 0.5% squalene for 4 weeks. Plasma triglycerides, total- and HDL-cholesterol and their lipoproteins were assayed. Analyses of hepatic lipid droplets, triglycerides, total- and non-esterified cholesterol, squalene, protein and gene expression, and cholesterol precursors were carried out. In the jejunum, the squalene content and mRNA and protein APOB expressions were measured. Finally, we studied the effect of cholesterol precursors in AML12 cells. Squalene administration significantly increased plasma total cholesterol, mainly carried as non-esterified cholesterol in IDL and large LDL, and corresponded to an increased number of APOB100-containing particles without accumulation of triglycerides and decreased reactive oxygen species. Despite no significant changes in the APOB content in the jejunum, the latter displayed increased APOB mRNA and squalene levels. Increases in the amounts of non-esterified cholesterol, squalene, lanosterol, dihydrolanosterol, lathosterol, cholestanol, zymostenol, desmosterol and caspase 1 were also observed in the liver. Incubation of AML12 cells in the presence of lanosterol increased caspase 1. In conclusion, squalene administration in rabbits increases the number of modified APOB-containing lipoproteins, and hepatic cholesterol biosynthesis is linked to caspase 1 probably through lanosterol.

Dietary Avian Proteins Are Comparable to Soybean Proteins on the Atherosclerosis Development and Fatty Liver Disease in Apoe-Deficient Mice.

BACKGROUND AND AIM: The type and amount of dietary protein has become a topic of renewed 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 atherosclerotic 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 aortic 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.

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.

LPS-squalene interaction on D-galactose intestinal absorption.

The dynamic and complex interactions between enteric pathogens and the intestinal epithelium often lead to disturbances in the intestinal barrier, altered fluid, electrolyte, and nutrient transport and can produce an inflammatory response. Lipopolysaccharide (LPS) is a complex polymer forming part of the outer membrane of Gram-negative bacteria. On the other hand, squalene is a triterpene present in high levels in the extra-virgin olive oil that has beneficial effects against several diseases and it has also anti-oxidant and anti-inflammatory properties. The aim of this work was to study whether the squalene could eliminate the LPS effect on D-galactose intestinal absorption in rabbits and Caco-2 cells. The results have shown that squalene reduced the effects of LPS on sugar absorption. High LPS doses increased D-galactose uptake through via paracellular but also decreased the active sugar transport because the SGLT1 levels were diminished. However, the endotoxin effect on the paracellular way seemed to be more important than on the transcellular route. At the same time, an increased in RELM-ß expression was observed. This event could be related to inflammation and cause a decrease in SGLT1 levels. In addition, MLCK protein is also increased by LPS which could lead to an increase in sugar transport through tight junctions. At low doses, the LPS could inhibit SGLT1 intrinsic activity. Bioinformatic studies by docking confirm the interaction between LPS-squalene as well as occur through MLCK and SGLT-1 proteins.

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

BACKGROUND: The 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. METHODS: Male 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. RESULTS: Results 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. CONCLUSION: Either 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.

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 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.