Eicosapentaenoic and Docosahexaenoic Acid Supplementation Increases HDL Content in n-3 Fatty Acids and Improves Endothelial Function in Hypertriglyceridemic Patients.

High-density lipoproteins (HDLs) are known to enhance vascular function through different mechanisms, including the delivery of functional lipids to endothelial cells. Therefore, we hypothesized that omega-3 (n-3) eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content of HDLs would improve the beneficial vascular effects of these lipoproteins. To explore this hypothesis, we performed a placebo-controlled crossover clinical trial in 18 hypertriglyceridemic patients without clinical symptoms of coronary heart disease who received highly purified EPA 460 mg and DHA 380 mg, twice a day for 5 weeks or placebo. After 5 weeks of treatment, patients followed a 4-week washout period before crossover. HDLs were isolated using sequential ultracentrifugation for characterization and determination of fatty acid content. Our results showed that n-3 supplementation induced a significant decrease in body mass index, waist circumference as well as triglycerides and HDL-triglyceride plasma concentrations, whilst HDL-cholesterol and HDL-phospholipids significantly increased. On the other hand, HDL, EPA, and DHA content increased by 131% and 62%, respectively, whereas 3 omega-6 fatty acids significantly decreased in HDL structures. In addition, the EPA-to-arachidonic acid (AA) ratio increased more than twice within HDLs suggesting an improvement in their anti-inflammatory properties. All HDL-fatty acid modifications did not affect the size distribution or the stability of these lipoproteins and were concomitant with a significant increase in endothelial function assessed using a flow-mediated dilatation test (FMD) after n-3 supplementation. However, endothelial function was not improved in vitro using a model of rat aortic rings co-incubated with HDLs before or after treatment with n-3. These results suggest a beneficial effect of n-3 on endothelial function through a mechanism independent of HDL composition. In conclusion, we demonstrated that EPA and DHA supplementation for 5 weeks improved vascular function in hypertriglyceridemic patients, and induced enrichment of HDLs with EPA and DHA to the detriment of some n-6 fatty acids. The significant increase in the EPA-to-AA ratio in HDLs is indicative of a more anti-inflammatory profile of these lipoproteins.

Association between rs662 (A > G) and rs854560 (A > T) polymorphisms in PON1 gene and the susceptibility for psoriasis in mestizo population of Western Mexico.

Psoriasis is a chronic, autoimmune skin disease. In psoriasis, PON1 activity is diminished and peroxidation biomarkers are elevated. The most studied PON1 polymorphisms are rs662 (A > G) and rs854560 (A > T), which have been associated with the antioxidant activity of PON1, risk of cardiovascular diseases and psoriasis development. The aim of this study, was to determine the association of rs662 (A > G) and rs854560 (A > T) PON1 polymorphisms with psoriasis susceptibility in Western Mexico population. In this case-control study, we included 104 psoriasis patients and 124 control subjects. The genotyping of polymorphisms rs662 (A > G) and rs854560 (A > T) of PON1 was carried out by PCR-RFLPs. The lipid profiles were quantified by enzymatic colorimetric method, and PON1 activity was determined by spectrophotometry. The lipid profile levels, except HDL-C and atherogenic index, were higher in patients vs. controls. Patients presented lower paraoxonase and arylesterase activity. The G allele of rs662 (A > G) is associated with risk for psoriasis, while the T allele of rs854560 (A > T) is associated with low susceptibility to psoriasis. The AG haplotype was more frequent within the patient group (p < 0.05). The AA and AG genotypes of rs662 (A > G) and TT and AA genotypes of rs854560 (A > T) are associated with lower PONase and ARE activity in patients vs. controls. Patients with the G allele of rs662 (G > A) and T alleles of rs854560 (A > T) show significant differences in the lipid levels in comparison to controls. These results suggest that carriers of G allele of rs662 (A > G) present a greater susceptibility to psoriasis.

Trp Fluorescence Redshift during HDL Apolipoprotein Denaturation Is Increased in Patients with Coronary Syndrome in Acute Phase: A New Assay to Evaluate HDL Stability.

High-density lipoproteins' (HDL) stability is a determinant of their residence times in plasma and consequently an important parameter that influences the beneficial properties of these lipoproteins. Since there are no accessible procedures for this purpose, here, we describe the methodological conditions to assess the stability of the HDL based on the redshift of the fluorescence spectrum of tryptophans contained in the structure of HDL-apolipoproteins during incubation with urea 8M. Along the HDL denaturation kinetics, the main variations of fluorescence were observed at the wavelengths of 330, 344, and 365 nm at room temperature. Therefore, HDL denaturation was estimated using the tryptophan (Trp)-ratio of fluorescence intensity (rfi) at such wavelengths. By setting 100% of the measurable denaturation at 26 h, HDL reached 50% after 8 h of incubation with urea. Then, for further analyses we determined the percentage of HDL denaturation at 8 h as an estimation of the stability of these lipoproteins. To explore the potential usefulness of this test, we analyzed the stability of HDL isolated from the plasma of 24 patients diagnosed with acute coronary syndrome (ACS). These HDL presented significantly higher percentages of denaturation (64.9% (58.7-78.4)) than HDLs of healthy individuals (23.3% (20.3-27.0)). These results indicate that HDL in ACS are less stable than in control subjects. Moreover, the percentage of denaturation of HDL correlated with body mass index and aspartate transaminase plasma activity. Furthermore, apo-I, HDL-cholesterol, HDL-triglycerides, and apo A-I-to-triglycerides ratio correlated with the percentage of HDL denaturation, suggesting that the lipoprotein composition is a main determinant of HDL stability. Finally, the percentage of HDL denaturation is the parameter that predicted the presence of ACS as determined by a machine learning procedure and logistic regression analysis. In conclusion, we established the methodological conditions to assess the stability of HDL by a fluorescence-based method that merits exploration in prospective studies for evaluating the coronary artery disease risk.

Atorvastatin and Fenofibrate Increase the Content of Unsaturated Acyl Chains in HDL and Modify In Vivo Kinetics of HDL-Cholesteryl Esters in New Zealand White Rabbits.

Previous studies demonstrated modifications of high-density lipoproteins (HDL) structure and apolipoprotein (apo) A-I catabolism induced by the atorvastatin and fenofibrate combination. However, it remains unknown whether such structural and metabolic changes of HDL were related to an improvement of the HDL-cholesteryl esters (HDL-CE) metabolism. Therefore, we determined the structure of HDL and performed kinetic studies of HDL-CE radiolabeled with tritium in rabbits treated with atorvastatin, fenofibrate, and a combination of both drugs. The atorvastatin and fenofibrate combination increased the HDL size and the cholesterol and phospholipid plasma concentrations of the largest HDL subclasses. Moreover, the relative amount of unsaturated fatty acids contained in HDL increased, in detriment of saturated fatty acids as determined by gas chromatography-mass spectrometry. The transfers of cholesteryl esters (CE) from HDL to very low-density lipoproteins/low-density lipoproteins (VLDL/LDL) and vice versa were enhanced with atorvastatin, alone or in combination. Moreover, the direct elimination of CE from plasma via VLDL/LDL decreased with fenofibrate, whereas the direct elimination of CE via HDL augmented with the combination treatment. Taken together, the rise of unsaturated fatty acid content and the size increase of HDL, suggest that atorvastatin and fenofibrate induce more fluid HDL particles, which in turn favor an enhanced CE exchange between HDL and VLDL/LDL. Our results contribute to a better understanding of the relationship between the structure and function of HDL during the use of anti-dyslipidemic drugs.

Atorvastatin and Fenofibrate Exert Opposite Effects on the Vascularization and Characteristics of Visceral Adipose Tissue in New Zealand White Rabbits.

Statins may precipitate the onset of type 2 diabetes (T2D) in high-risk patients. In contrast, only the subset of individuals with insulin resistance and/or diabetes receives cardiovascular benefits with fibrates. In this context, previous observations from our laboratory suggested that atorvastatin induced an increase in visceral adipose tissue (VAT), whereas fenofibrate had the opposite effects in rabbits. Therefore, we determined the mass, morphology, and vascularization of VAT in New Zealand white rabbits (n = 6/group) that received 0.33 or 2.6 mg/kg/d of atorvastatin or fenofibrate, respectively, during 2 months. As expected, the cholesterol from the atorvastatin group was lower after treatment, while triglycerides decreased in the fenofibrate group. The mass of VAT from the fenofibrate group was 46% lower compared to the controls, meanwhile atorvastatin was associated with a larger diameter of adipocytes (+65%) than that of the control and fenofibrate groups. Fibroblast growth factor 2 (FGF2) gene expression was lower in the fenofibrate group than in the control group (-54%). By contrast, vascular endothelial growth factor A (VEGF-A) gene expression in fenofibrate-treated rabbits was 110% higher than in the control group. In agreement with the gene expression, the marker of angiogenesis platelet endothelial cell adhesion molecule 1 was slightly but significantly higher (+10%) in rabbits treated with fenofibrate than in controls, as determined by immunohistochemistry. These results suggest that fenofibrate is associated with a favorable remodeling of VAT, that is, reduced mass and increased vascularization in normolipemic rabbits; in contrast, atorvastatin induced a nonfavorable remodeling of VAT. These results may be related to the cardiovascular benefits of fenofibrate and the increased risk of T2D in high-risk patients induced by atorvastatin.

Hyperuricemia is Associated with Increased Apo AI Fractional Catabolic Rates and Dysfunctional HDL in New Zealand Rabbits.

The potential cause-effect relationship between uric acid plasma concentrations and HDL functionality remains elusive. Therefore, this study aimed to explore the effect of oxonic acid (OA)-induced hyperuricemia on the HDL size distribution, lipid content of HDL subclasses, and apo AI turnover, as well as HDL functionality in New Zealand white rabbits. Experimental animals received OA 750 mg/kg/day by oral gavage during 21 days. The HDL-apo AI fractional catabolic rate (FCR) was determined by exogenous labeling with 125I, and HDL subclasses were determined by sequential ultracentrifugation and PAGE. Paraoxonase-1 activity (PON-1) and the effect of HDL on relaxation of aorta rings in vitro were determined as an indication of HDL functionality. Oxonic acid induced a sixfold increase of uricemia (0.84 ± 0.06 vs. 5.24 ± 0.12 mg/dL, P < 0.001), and significant decreases of triglycerides and phospholipids of HDL subclasses, whereas HDL size distribution and HDL-cholesterol remained unchanged. In addition, HDL-apo AI FCR was significantly higher in hyperuricemic rabbits than in the control group (0.03697 ± 0.0038 vs. 0.02605 ± 0.0017 h-1 respectively, P < 0.05). Such structural and metabolic changes were associated with lower levels of PON-1 activities and deleterious effects of HDL particles on endothelium-mediated vasodilation. In conclusion, hyperuricemia is associated with structural and metabolic modifications of HDL that result in impaired functionality of these lipoproteins. Our data strongly suggest that uric acid per se exerts deleterious effects on HDL that contribute to increase the risk of atherosclerosis.

Increased HDL Size and Enhanced Apo A-I Catabolic Rates Are Associated With Doxorubicin-Induced Proteinuria in New Zealand White Rabbits.

The catabolism and structure of high-density lipoproteins (HDL) may be the determining factor of their atheroprotective properties. To better understand the role of the kidney in HDL catabolism, here we characterized HDL subclasses and the catabolic rates of apo A-I in a rabbit model of proteinuria. Proteinuria was induced by intravenous administration of doxorubicin in New Zealand white rabbits (n = 10). HDL size and HDL subclass lipids were assessed by electrophoresis of the isolated lipoproteins. The catabolic rate of HDL-apo A-I was evaluated by exogenous radiolabelling with iodine-131. Doxorubicin induced significant proteinuria after 4 weeks (4.47 ± 0.55 vs. 0.30 ± 0.02 g/L of protein in urine, P < 0.001) associated with increased uremia, creatininemia, and cardiotoxicity. Large HDL2b augmented significantly during proteinuria, whereas small HDL3b and HDL3c decreased compared to basal conditions. HDL2b, HDL2a, and HDL3a subclasses were enriched with triacylglycerols in proteinuric animals as determined by the triacylglycerol-to-phospholipid ratio; the cholesterol content in HDL subclasses remained unchanged. The fractional catabolic rate (FCR) of [(131)I]-apo A-I in the proteinuric rabbits was faster (FCR = 0.036 h(-1)) compared to control rabbits group (FCR = 0.026 h(-1), P < 0.05). Apo E increased and apo A-I decreased in HDL, whereas PON-1 activity increased in proteinuric rabbits. Proteinuria was associated with an increased number of large HDL2b particles and a decreased number of small HDL3b and 3c. Proteinuria was also connected to an alteration in HDL subclass lipids, apolipoprotein content of HDL, high paraoxonase-1 activity, and a rise in the fractional catabolic rate of the [(131)I]-apo A-I.

Atorvastatin and fenofibrate combination induces the predominance of the large HDL subclasses and increased apo AI fractional catabolic rates in New Zealand white rabbits with exogenous hypercholesterolemia.

The anti-atherogenic properties of high-density lipoproteins (HDLs) may be related to their structure and metabolism. The HDL physicochemical characteristics that determine their plasma clearance during treatment with statins and fibrates are not well understood. In this study, we analyzed HDL-apo AI fractional catabolic rates (FCRs), size distributions, and the lipid composition of the HDL subclasses in New Zealand white rabbits with exogenous dyslipidemia that received low doses of atorvastatin and fenofibrate. Hypercholesterolemia decreased only partially with the combination of both drugs. HDL size distribution shifted toward larger particles among the groups of rabbits that received atorvastatin, fenofibrate, or their combination, compared with both the control group and the dyslipidemic group. The HDL subclasses were significantly rich in cholesterol in each of the groups compared with controls. The structural changes noted in the HDL subclasses were not associated with impaired plasma paraoxonase-1 (PON1) activity. The groups receiving monotherapy and the drug combination group were all associated with a higher apo AI FCR value compared with both the dyslipidemic rabbits and the control group. In conclusion, the combination of atorvastatin and fenofibrate induced a more favorable HDL subclass profile than did the individual use of these drugs. Similarly, the apo AI FCR values were augmented in every group receiving drug treatment (either monotherapy or combination therapy) in the setting of hypercholesterolemia. The anti-atherogenic properties of HDLs, excluding their capacity to bind PON1, may be enhanced by the structural and metabolic modifications induced by the combination of atorvastatin and fenofibrate.

Insulin-like growth factor I (IGF-I) regulates endocrine activity of the embryonic testis in the mouse.

Insulin-like growth factor I (IGF-I) is important for gonadal and reproductive functions in mammals, although the physiological role of this growth factor during gonadal development in rodents remains largely unknown. Here, we examined the steady-state levels of IGF-I mRNA by the reverse transcriptase polymerase chain reaction (RT-PCR). IGF-I protein expression was also detected by Western blot. The effect of IGF-I as promoter of 17alpha-hydroxylase/C17-20 lyase and 17beta-hydroxysteroid dehydrogenase enzyme activity in vitro was evaluated by radioimmunoassay. Onset of IGF-I gene expression was on day E10 (urogenital ridge stage). IGF-I mRNA expression was markedly reduced on days E12 and E13 (testicular differentiation stage). IGF-I transcripts increased on day E14 and their transcription levels were maintained throughout the stages analyzed. Several IGF-I protein bands of 31-100 kDa were observed. Culture experiments demonstrated that 17alpha-hydroxyprogesterone and testosterone (T) secretion levels increased in the presence of IGF-I on days E11-E17. Additive effects of IGF-I plus (Bu)2cAMP were also seen during testicular development. It is proposed that IGF-I regulates the expression of key steroidogenic enzymes important for endocrine activity of the testis during prenatal development leading to establishment of the male phenotype and fertility.

Connexin43 is expressed in mouse fetal ovary.

Developmental studies have shown that connexin43 (Cx43) is expressed in the ovary from the first day of life and throughout the rest of postnatal development. In both mouse embryonic ovaries and testes, target-directed deletion of Cx43 gene induces a significant decrease in germinal cells, but the exact mechanism determining this reduction remains unknown. Moreover, recently we found that Cx43 is abundantly expressed in mouse testes from the earliest stages of its fetal development. In the present work we investigate whether Cx43 transcript and protein are expressed in mouse embryonic ovaries. Total RNA was analyzed with specific Cx43 oligonucleotides in RT-PCR studies. A Cx43 PCR product was detected in ovaries at 16.5 and 18.5 days postcoitum (dpc). Bands of 43-45 kDa, characteristic of Cx43, were detected in immunoblots of total homogenates of ovaries at 14.5 and 18.5 dpc. Cell type-specific expression of Cx43 was investigated using double-labeled sections incubated with specific antibodies against Cx43 and the enzyme 3beta-hydroxysteroid dehydrogenase (3betaHSD) or a germ cell nuclear antigen (GCNA1), which are cell markers of steroidogenic and germinal cells, respectively. At 18.5 dpc, Cx43 was found in conglomerates of 3betaHSD-positive cells. Cx43 was also localized at homocellular junctions between parenchyma pregranulosa cells, and at heterocellular junctions between pregranulosa and germinal cells. At these two latter localizations, Cx43 was traced back to 12.5 dpc. In conclusion, this study demonstrates for the first time that from the earliest stages of embryonic ovary development, Cx43 is expressed in principal cell types involved in control of female fertility. These data suggest that the gap junctions formed with Cx43 between somatic and germinal cells may be necessary for prenatal expansion of germinal cells at initial stages of fetal gonadal development.

Early endothelial nitrosylation and increased abdominal adiposity in Wistar rats after long-term consumption of food fried in canola oil.

OBJECTIVE: The aim of this study was to establish whether the long-term consumption of reused canola oil contributes to the development of dyslipidemia, obesity, and endothelial function. METHODS: Canola oil was used for one frying cycle (1 FC) of corn flour dough or reused 10 times (10 FC). Rats received chow diet (control) or supplemented with 7% raw oil (RO), 1 FC or 10 FC oil (n = 10 per group). Food consumption, blood pressure (BP), and body weight plasma glucose, plasma lipids were monitored. Vascular reactivity was analyzed using aorta rings stimulated with phenylephrine and acetylcholine. Nitrotyrosine presence in aorta rings was analyzed by immunohistochemistry. RESULTS: After 10 wk of follow-up, visceral adipose tissue was significantly more abundant in 1 FC (7.4 ± 0.6 g) and 10 FC (8.8 ± 0.7 g) than the RO (5.0 ± 0.2 g; P = 0.05 versus 10 FC group) or control group (2.6 ± 0.3 g; P = 0.05 versus all groups). Despite similar plasma cholesterol, triglycerides, and BP among groups, a significantly reduced acetylcholine-induced vascular relaxation was observed in the three groups receiving the oil-supplemented diet (47.2% ± 3.6%, 27.2% ± 7.7%, and 25.9% ± 7.6% of relaxation, for the RO, 1 FC, and 10 FC, respectively; P < 0.05 for all versus 62.4% ± 9.7% of the control group). Endothelial dysfunction was concomitant with the presence of nitrotyrosine residues at a higher extent in the groups that received heated oils compared with the RO group. CONCLUSION: High canola oil intake over 10 wk was associated with increased adipose tissue and early endothelial dysfunction probably induced by peroxinitrite formation. Such deleterious effects were significantly potentiated when the consumed oil had been used repeatedly for frying.

Shift of high-density lipoprotein size distribution toward large particles in patients with proteinuria.

BACKGROUND: The potential atheroprotective role of the different HDL subclasses may depend on the metabolic factors that affect their plasma concentrations. The kidney is supposed to be one of the main catabolic sites for these lipoproteins. However, little is known about the impact of proteinuria on HDL size distribution and HDL structure. The aim of this study is to establish the influence of proteinuria on HDL size distribution and cholesterol plasma concentration of HDL subclasses. METHODS: Forty patients within a range of proteinuria from 0.2 to 10.0 g/g estimated by the urinary protein-to-creatinine ratio and 40 healthy controls were enrolled in the study. HDL subclasses were separated by sequential ultracentrifugation followed by a polyacrylamide gradient electrophoresis; gels were stained enzymatically for cholesterol and with Coomasie blue for proteins. HDL size distribution and plasma concentration of the five HDL subclasses were calculated by optical densitometry. RESULTS: When determined by protein, large HDL2b and HDL2a relative proportions were higher in patients than in control subjects, whereas the contrary was observed for small HDL3b and 3c. Consistently, HDL3a, 3b, and 3c were negatively correlated with proteinuria when data were adjusted by age, gender, body mass index, and blood pressure. Size distribution followed a different pattern when determined by cholesterol, suggesting an abnormal lipid composition that was further supported by a protein-to-cholesterol ratio significantly higher in most of the HDL subclasses in proteinuric patients than in the control group. Moreover, proteinuria statistically explains the HDL2b and HDL3c cholesterol plasma concentrations. CONCLUSIONS: Proteinuria is associated with a shift of HDL size distribution towards large particles and cholesterol-poor HDL subclasses. These results support the idea of a selective loss by the kidney of small HDL in patients with proteinuria; whether these abnormalities reflect an impaired reverse cholesterol transport and an increased risk of coronary heart disease remains to be elucidated.