Hormone-sensitive lipase deficiency affects the expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis.

Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL+/+, HSL+/- and HSL-/- mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL-/- testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRß, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL+/- mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRα (Nr1h3) and LXRß (Nr1h2) decreased in testis from HSL-/- compared with HSL+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.

Quantitative lipidomic analysis of plasma and plasma lipoproteins using MALDI-TOF mass spectrometry.

Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins.

The antioxidant butylated hydroxyanisole potentiates the toxic effects of propylparaben in cultured mammalian cells.

Butylated hydroxyanisole and propylparaben are phenolic preservatives commonly used in food, pharmaceutical and personal care products. Both chemicals have been subjected to extensive toxicological studies, due to the growing concern regarding their possible impacts on environmental and human health. However, the cytotoxicity and underlying mechanisms of co-exposure to these compounds have not been explored. In this study, a set of relevant cytotoxicity endpoints including cell viability and proliferation, oxidative stress, DNA damage and gene expression changes were analyzed to assess whether the antioxidant butylated hydroxyanisole could prevent the pro-oxidant effects caused by propylparaben in Vero cells. We demonstrated that binary mixtures of both chemicals induce greater cytotoxic effects than those reported after single exposureto each compound. Simultaneous treatment with butylated hydroxyanisole and propylparaben caused G0/G1 cell cycle arrest as a result of enhanced generation of oxidative stress and DNA double strand breaks. DNA microarray analysis revealed that a cross-talk between transforming growth factor beta (TGFß) and ataxia-telangiectasia mutated kinase (ATM) pathways regulates the response of Vero cells to the tested compounds in binary mixture. Our findings indicate that butylated hydroxyanisole potentiates the pro-oxidant effects of propylparaben in cultured mammalian cells and provide useful information for their safety assessment.

Quantitative profile of lipid classes in blood by normal phase chromatography with evaporative light scattering detector: application in the detection of lipid class abnormalities in liver cirrhosis.

BACKGROUND: The lack of analytical methods specific for each lipid class, particularly for phospholipids and sphyngolipids, makes necessary their separation by preparative techniques before quantification. LC-MS would be the election method but for daily work in the clinical laboratory this is not feasible for different reasons, both economic and time consuming. In the present work, we have optimized an HPLC method to quantify lipid classes in plasma and erythrocytes and applied it to samples from patients with cirrhosis. METHODS: Lipid classes were analyzed by normal phase liquid chromatography with evaporative light scattering detection. We employed a quaternary solvent system to separate twelve lipid classes in 15 min. RESULTS: Interday, intraday and recovery for quantification of lipid classes in plasma were excellent with our methodology. The total plasma lipid content of cirrhotic patients vs control subjects was decreased with diminished CE (81±33 vs 160±17 mg/dL) and PC (37±16 vs 60±19 mg/dL). The composition of erythrocytes showed a decrease in acidic phospholipids: PE, PI and PS. CONCLUSION: Present methodology provides a reliable quantification of lipid classes in blood. The lipid profile of cirrhotics showed alterations in the PC/PE plasma ratio and in the phospholipid content of erythrocytes, which might reflect alterations in hepatocyte and erythrocyte membrane integrity.

HSL-knockout mouse testis exhibits class B scavenger receptor upregulation and disrupted lipid raft microdomains.

There is a tight relationship between fertility and changes in cholesterol metabolism during spermatogenesis. In the testis, class B scavenger receptors (SR-B) SR-BI, SR-BII, and LIMP II mediate the selective uptake of cholesterol esters from HDL, which are hydrolyzed to unesterified cholesterol by hormone-sensitive lipase (HSL). HSL is critical because HSL knockout (KO) male mice are sterile. The aim of the present work was to determine the effects of the lack of HSL in testis on the expression of SR-B, lipid raft composition, and related cell signaling pathways. HSL-KO mouse testis presented altered spermatogenesis associated with decreased sperm counts, sperm motility, and infertility. In wild-type (WT) testis, HSL is expressed in elongated spermatids; SR-BI, in Leydig cells and spermatids; SR-BII, in spermatocytes and spermatids but not in Leydig cells; and LIMP II, in Sertoli and Leydig cells. HSL knockout male mice have increased expression of class B scavenger receptors, disrupted caveolin-1 localization in lipid raft plasma membrane microdomains, and activated phospho-ERK, phospho-AKT, and phospho-SRC in the testis, suggesting that class B scavenger receptors are involved in cholesterol ester uptake for steroidogenesis and spermatogenesis in the testis.

The apolipoprotein A5 (APOA5) gene predisposes Caucasian children to elevated triglycerides and vitamin E (Four Provinces Study).

OBJECTIVE: Apolipoprotein A-V plays an important role in lipid metabolism regulation, particularly modulating triglyceride levels, as has been shown by many association studies in adults. The aim of this study was to analyse the effect of APOA5 on lipid profiles and fat-soluble vitamins (due to its strong relationship with triglyceride metabolism) in children. METHODS: We determined polymorphisms -1131T>C and S19W in the APOA5 gene in 964 6-8-year-old participants of the 4P study and analysed the influence of the APOA5 gene on plasma lipid levels (total cholesterol, LDL cholesterol, HDL cholesterol and triglycerides), apolipolipoproteins (apo A-I and apo B) and fat-soluble antioxidant vitamin (α-tocopherol, γ-tocopherol, lycopene, α-carotene, ß-carotene and retinol) levels. RESULTS: The allele frequencies of both polymorphisms were comparable to those described in adult Caucasian populations (0.08 and 0.07 for -1131T>C and S19W, respectively). Boys carrying the -1131C allele have a 12% increase in circulating triglyceride levels (p=0.016) and a 7% decrease in HDL phospholipid levels (p=0.016). Linked to its effect on triglycerides, boys with the -1131C allele also have a 5% increase in plasma α-tocopherol levels (p=0.032). This effect was not observed in female participants. Boys carrying the rare allele for the S19W polymorphism have a 4% increase in circulating cholesterol levels (p=0.045), whereas girls have a 9% increase in circulating triglyceride levels (p=0.029). Linked to its effect on triglycerides, female carriers of the rare allele for S19W also have a 6% increase in α-tocopherol levels (p=0.009). CONCLUSION: In children, the effect of APOA5 gene variants on triglyceride levels is related to gender, and because of the strong relationship between lipid metabolism and fat-soluble antioxidant vitamins, it also involves a significant elevation in α-tocopherol concentrations.

Hormone-sensitive lipase expression and IHC localization in the rat ovary, oviduct, and uterus.

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.

Induction of the endoplasmic reticulum stress protein GADD153/CHOP by capsaicin in prostate PC-3 cells: a microarray study.

The effect of capsaicin, main pungent ingredient of hot chilli peppers, in the gene expression profile of human prostate PC-3 cancer cells has been analyzed using a microarray approach. We identified 10 genes that were down-regulated and five genes that were induced upon capsaicin treatment. The data obtained from microarray analysis were then validated using quantitative real-time PCR assays and Western blot analysis. The most remarkable change was the up-regulation of GADD153/CHOP, an endoplasmic reticulum stress-regulated gene. Activation of GADD153/CHOP protein was corroborated by immunofluorescence and Western blot. We then tested the contribution of GADD153/CHOP to protection against capsaicin-induced cell death using RNA interference. Blockage of GADD153/CHOP expression by small interfering RNA, significantly reduced capsaicin-induced cell death in PC-3 cells. Taken together, these results suggested that capsaicin induces the antiproliferative effect through a mechanism facilitated by ER stress in prostate PC-3 cells.

Liquid chromatographic method for the simultaneous determination of different lipid-soluble antioxidants in human plasma and low-density lipoproteins.

We describe a reverse phase HPLC method, employing a simple methanol:water gradient as mobile phase, for the determination of several lipophilic antioxidants, such as retinol, gamma-tocopherol, alpha-tocopherol, lycopene, alpha-carotene and beta-carotene among others, using UV detection. Additionally, this method allows the simultaneous separation of probucol, an hypocholesterolemic drug with antioxidant properties. Retinol acetate and alpha-tocopherol acetate were added to samples as internal standards. A NovaPack ODS C18, 150 x 3.9 mm, 0.4 microm column was used and the flow rate was set constant at 1m/min, which allowed the separation of all the desired antioxidants in a total run time of 35 min. A photodiode array detector was used because of its advantages to study the purity of the peaks, however, any programmable multiwavelength UV/VIS detector could be employed given the good resolution of the peaks. The analytical recoveries of the studied compounds were > 96% and the detection limits were: retinol 0.050 microg/ml, gamma-tocopherol 0.137 microg/ml, alpha-tocopherol 0.906 microg/ml, lycopene 0.022 microg/ml, alpha-carotene 0.008 microg/ml, beta-carotene 0.015 microg/ml and probucol 1.503 microg/ml. The intra- and inter-assay coefficients of variation were calculated by using two human plasma samples with different levels of lipophilic antioxidants. The simplicity, rapidity and economy, make this method suitable for the routine measurement of plasma and low-density lipoproteins antioxidants, and may also be used in large scale epidemiological studies. The method has been used to measure antioxidants in samples from patients undergoing treatment with probucol, showing there is a good correlation between the probucol content in LDL and that in total plasma.

Influence of birth weight on the apo E genetic determinants of plasma lipid levels in children.

To evaluate the influence of birth weight on apolipoprotein (apo) E genetic determinants of plasma lipids levels in prepubertal children we studied 933 healthy children (491 males and 442 females) 6 to 8 years old (mean age of 6.7 y), whose weight was recorded at birth. Plasma lipid and apolipoprotein concentrations and apo E genotypes were determined. We observed a greater effect of the apo E polymorphism on total cholesterol (TC), LDL-cholesterol (LDL-C) and especially apo B levels in children with birth weight in the lower tertile compared with those with birth weights in higher tertiles. Taking the epsilon3 allele homozygosity as reference, in boys with birth weights in the low tertile the overall lowering effect of the epsilon2 allele on TC, LDL-C and apo B was greater (10.5% (p < 0.01), 20.2% (p < 0.01) and 18.8% (p < 0.01), respectively) than in those in the highest tertile (5.6% on TC, 10.3% on LDL-C and 12.6% (p < 0.01) on apo B). A similar trend in this effect between tertiles of birth weight was also observed in girls. For both sexes, linear regression analysis demonstrates a positive and significant interaction between birth weight and epsilon2, which may explain the fact that the decrease in TC, LDL-C and apo B associated with the epsilon2 allele is more marked the lower the birth weight. Taking into account the prevalence of apo E polymorphism, and that appears to be the main genetic factor affecting plasma lipids, the interaction of apo E genotype and birth weight could be an important determinant of TC, LDL-C and apo B levels, and, as a consequence, of atherosclerosis.

[Metabolic factors in school children population associated with adult cardiovascular mortality. Four provinces' study]. / Factores metabólicos en la población escolar asociados a mortalidad cardiovascular en los adultos. Estudio Cuatro Provincias.

BACKGROUND: Environmental factors in early stages of life may contribute to adult cardiovascular disease. We have examined certain anthropometric and biochemical variables in children aged 6-7 years from four Spanish provinces with high differences in mortality due to ischemic heart disease (IHD). PATIENTS AND METHOD: We performed a cross-sectional survey of 1,255 children (50.3% males) attending schools in Cádiz and Murcia provinces with a relatively high IHD mortality and Madrid and Orense provinces with a relatively low IHD mortality. Weight, body mass index (BMI) and prevalence of obesity were analyzed, and plasma glucose and lipid levels were measured by standardized methods. RESULTS: Compared with children in the two low-IHD-mortality provinces, those in the two high-IHD-mortality provinces had greater weight (p < 0.05) and BMI (p < 0.01) and higher prevalence of obesity (p < 0.01). Moreover, they had significantly higher (p < 0.01) plasma glucose, triglyceride and apo A-I levels. CONCLUSIONS: The higher prevalence of overweight and obesity, along with higher plasma glucose and triglyceride levels, in provinces with high coronary artery disease mortality indicates that children from these provinces are metabolically different from those in provinces with low coronary artery disease mortality. These alterations may thus contribute to the different IHD mortality in adulthood.

Factores metabólicos en la población escolar asociados a mortalidad cardiovascular en los adultos. Estudio Cuatro Provincias / Metabolic factors in school children population associated with adult cardiovascular mortality. Four Provinces Study

FUNDAMENTO: Existen evidencias sobre la contribución de factores ambientales en etapas tempranas de la vida a la aparición de la enfermedad cardiovascular en la edad adulta. Por ello, en el presente trabajo hemos examinado variables antropométricas y bioquímicas en niños de 6-7 años de cuatro provincias españolas con una gran variación en la mortalidad por cardiopatía isquémica (CI). PACIENTES Y MÉTODO: Estudio transversal en 1.255 niños en edad escolar (50,3 por ciento varones) de Cádiz y Murcia, provincias con alta mortalidad por CI, y de Madrid y Orense, provincias con baja mortalidad por CI, en los que se han analizado peso, índice de masa corporal (IMC) y prevalencia de obesidad, y se han determinado los valores plasmáticos de glucosa y el perfil lipídico mediante métodos estandarizados. RESULTADOS: Los niños de las provincias con mayor mortalidad tienen mayor peso (p < 0,05), IMC (p < 0,01) y prevalencia de obesidad (p < 0,01). Además, presentan valores plasmáticos significativamente más altos (p < 0,01) de glucosa, triglicéridos y apo A-I. CONCLUSIONES: La mayor prevalencia de sobrepeso y obesidad, junto con los elevados valores de glucosa y triglicéridos asociados a las provincias de alta mortalidad, indica que los niños de estas provincias son metabólicamente distintos de los de provincias de baja mortalidad. Estas alteraciones podrían contribuir a la distinta mortalidad cardiovascular en la edad adulta. (AU)