Application of Flow Cytometry Using Advanced Chromatin Analyses for Assessing Changes in Sperm Structure and DNA Integrity in a Porcine Model.

Chromatin status is critical for sperm fertility and reflects spermatogenic success. We tested a multivariate approach for studying pig sperm chromatin structure to capture its complexity with a set of quick and simple techniques, going beyond the usual assessment of DNA damage. Sperm doses from 36 boars (3 ejaculates/boar) were stored at 17 °C and analyzed on days 0 and 11. Analyses were: CASA (motility) and flow cytometry to assess sperm functionality and chromatin structure by SCSA (%DFI, DNA fragmentation; %HDS, chromatin maturity), monobromobimane (mBBr, tiol status/disulfide bridges between protamines), chromomycin A3 (CMA3, protamination), and 8-hydroxy-2'-deoxyguanosine (8-oxo-dG, DNA oxidative damage). Data were analyzed using linear models for the effects of boar and storage, correlations, and multivariate analysis as hierarchical clustering and principal component analysis (PCA). Storage reduced sperm quality parameters, mainly motility, with no critical oxidative stress increases, while chromatin status worsened slightly (%DFI and 8-oxo-dG increased while mBBr MFI-median fluorescence intensity-and disulfide bridge levels decreased). Boar significantly affected most chromatin variables except CMA3; storage also affected most variables except %HDS. At day 0, sperm chromatin variables clustered closely, except for CMA3, and %HDS and 8-oxo-dG correlated with many variables (notably, mBBr). After storage, the relation between %HDS and 8-oxo-dG remained, but correlations among other variables disappeared, and mBBr variables clustered separately. The PCA suggested a considerable influence of mBBr on sample variance, especially regarding storage, with SCSA and 8-oxo-dG affecting between-sample variability. Overall, CMA3 was the least informative, in contrast with results in other species. The combination of DNA fragmentation, DNA oxidation, chromatin compaction, and tiol status seems a good candidate for obtaining a complete picture of pig sperm nucleus status. It raises many questions for future molecular studies and deserves further research to establish its usefulness as a fertility predictor in multivariate models. The usefulness of CMA3 should be clarified.

Polyphenols and Triterpenes Combination in an In Vitro Model of Cardiac Damage: Protective Effects.

Olive products contain high levels of monounsaturated fatty acids as well as other minor components such as triterpenic alcohols and other pentacyclic triterpenes, which together form the main triterpenes of virgin olive oil. Olive fruits and leaves contain significant amounts of hydrophilic and lipophilic bioactives including flavones, phenolic acids and phenolic alcohols, amongst others. Several studies have shown the benefits of these substances on the cardiovascular system. Regardless, little is known about the specific combination of bioactive compounds in cardiovascular health. Thus, we aimed to test the combination of a triterpenes (TT70) and a polyphenols (HT60) olive oil bioactive extract in H9c2 cells under stress conditions: LPS and H2O2 stimulation. To evaluate the effectiveness of the combination, we measured cell viability, superoxide production and protein expression of caspase 3, eNOS, peNOS, TNF-α and Il-6. Overall, cells stimulated with LPS or H2O2 and co-incubated with the combination of triterpenes and polyphenols had increased cell survival, lower levels of superoxide anion, lower protein expression of eNOS and higher expression of peNOS, increased protein expression of SOD-1 and lower protein expression of TNF-α and Il-6. The specific combination of HT60+TT70 is of great interest for further study as a possible treatment for cardiovascular damage.

Molecular evolution of the switch for progesterone and spironolactone from mineralocorticoid receptor agonist to antagonist.

The mineralocorticoid receptor (MR) is highly conserved across vertebrate evolution. In terrestrial vertebrates, the MR mediates sodium homeostasis by aldosterone and also acts as a receptor for cortisol. Although the MR is present in fish, they lack aldosterone. The MR binds progesterone and spironolactone as antagonists in human MR but as agonists in zebrafish MR. We have defined the molecular basis of these divergent responses using MR chimeras between the zebrafish and human MR coupled with reciprocal site-directed mutagenesis and molecular dynamic (MD) simulation based on the crystal structures of the MR ligand-binding domain. Substitution of a leucine by threonine in helix 8 of the ligand-binding domain of the zebrafish MR confers the antagonist response. This leucine is conserved across fish species, whereas threonine (serine in rodents) is conserved in terrestrial vertebrate MR. MD identified an interaction of the leucine in helix 8 with a highly conserved leucine in helix 1 that stabilizes the agonist conformation including the interaction between helices 3 and 5, an interaction which has previously been characterized. This switch in the MR coincides with the evolution of terrestrial vertebrates and of aldosterone synthesis. It was perhaps mandatory if the appearance of aldosterone as a specific mediator of the homeostatic salt retention was to be tolerated. The conformational changes also provide insights into the structural basis of agonism versus antagonism in steroid receptors with potential implications for drug design in this important therapeutic target.

Proanthocyanidins Maintain Cardiac Ionic Homeostasis in Aldosterone-Induced Hypertension and Heart Failure.

Excess aldosterone promotes pathological remodeling of the heart and imbalance in cardiac ion homeostasis of sodium, potassium and calcium. Novel treatment with proanthocyanidins in aldosterone-treated rats has resulted in downregulation of cardiac SGK1, the main genomic aldosterone-induced intracellular mediator of ion handling. It therefore follows that proanthocyanidins could be modulating cardiac ion homeostasis in aldosterone-treated rats. Male Wistar rats received aldosterone (1 mg kg-1 day-1) +1% NaCl for three weeks. Half of the animals in each group were simultaneously treated with the proanthocyanidins-rich extract (80% w/w) (PRO80, 5 mg kg-1 day-1). PRO80 prevented cardiac hypertrophy and decreased calcium content. Expression of ion channels (ROMK, NHE1, NKA and NCX1) and calcium transient mediators (CAV1.2, pCaMKII and oxCaMKII) were reduced by PRO80 treatment in aldosterone-treated rats. To conclude, our data indicate that PRO80 may offer an alternative treatment to conventional MR-blockade in the prevention of aldosterone-induced cardiac pathology.

Supplementation with an insoluble fiber obtained from carob pod (Ceratonia siliqua L.) rich in polyphenols prevents dyslipidemia in rabbits through SIRT1/PGC-1α pathway.

PURPOSE: To investigate the mechanism implicated in the effect of an insoluble fiber (obtained from carob pod) rich in polyphenols (IFCP) in lipid metabolism in the liver. METHODS: Male New Zealand rabbits were fed with the following diets for 8 weeks: control diet (CT group), dyslipidemic diet supplemented with 0.5% cholesterol + 14% coconut oil (DL group) and dyslipidemic diet containing 0.5% cholesterol + 14% coconut oil plus 3% IFCP (DL + IFCP group). RESULTS: Dyslipidemic diet with IFCP was able to reduce development of mixed dyslipidemia, liver relative weight and collagen I protein expression compared to DL rabbits. Analyses of the main enzymes implicated in cholesterol and triglycerides metabolism revealed that IFCP increased hepatic concentration of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and cytochrome P450, family 7, subfamily a, polypeptide 1C (CYP7A1) (82.34, 114.42%, respectively) as well as protein expression of LDL receptor (42.48%) in DL rabbits. Importantly, IFCP also increased hepatic lipase (HL) levels (91.43%) and decreased glycerol phosphate acyltransferase (GPAT) and sterol regulatory element-binding protein 1C (SREBP1c) liver expression levels (20.38 and 41.20%, respectively). Finally, sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1α) hepatic expression increased in DL + IFCP group compared with DL (159.81 and 48.00%, respectively). CONCLUSIONS: These findings show that IFCP is able to abrogate the deleterious effects of hepatic dyslipidemia by modulating SIRT1 and PGC-1α pathways.

Carob pod insoluble fiber exerts anti-atherosclerotic effects in rabbits through sirtuin-1 and peroxisome proliferator-activated receptor-γ coactivator-1α.

The aim of this study was to evaluate the potential effects of an insoluble dietary fiber from carob pod (IFC) (1 g ⋅ kg(-1) ⋅ d(-1) in the diet) on alterations associated with atherosclerosis in rabbits with dyslipidemia. Male New Zealand rabbits (n = 30) were fed the following diets for 8 wk: 1) a control diet (SF412; Panlab) as a control group representing normal conditions; 2) a control supplemented with 0.5% cholesterol + 14% coconut oil (DL) (SF302; Panlab) for 8 wk as a dyslipidemic group; and 3) a control containing 0.5% cholesterol + 14% coconut oil plus IFC (1 g ⋅ kg(-1) ⋅ d(-1)) (DL+IFC) for 8 wk. IFC was administered in a pellet mixed with the DL diet. The DL-fed group developed mixed dyslipidemia and atherosclerotic lesions, which were associated with endothelial dysfunction, inflammation, and fibrosis. Furthermore, sirtuin-1 (SIRT1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) protein expression in the aorta were reduced to 77% and 63% of the control group, respectively (P < 0.05), in these rabbits. Administration of IFC to DL-fed rabbits reduced the size of the aortic lesion significantly (DL, 15.2% and DL+IFC, 2.6%) and normalized acetylcholine-induced relaxation (maximal response: control, 89.3%; DL, 61.6%; DL+IFC, 87.1%; P < 0.05) and endothelial nitric oxide synthase expression (DL, 52% and DL+IFC, 104% of the control group). IFC administration to DL-fed rabbits also reduced cluster of differentiation 36 (DL, 148% and DL+IFC, 104% of the control group; P < 0.05), plasminogen activator inhibitor-1 (DL, 141% and DL+IFC, 107% of the control group), tumor necrosis factor-α (DL, 166% and DL+IFC, 120% of the control group), vascular cell adhesion molecule-1 (DL, 153% and DL+IFC, 110% of the control group), transforming growth factor-ß (DL, 173% and DL+IFC, 99% of the control group), and collagen I (DL, 157% and DL+IFC, 112% of the control group) in the aorta. These effects were accompanied by an enhancement of SIRT1 and PGC-1α (160% and 121% of the control group, respectively; P < 0.05) vascular expression. In summary, we demonstrated for the first time, to our knowledge, that administration of IFC reduces the development of atherosclerosis in rabbits. This effect seems to be related to an improvement in endothelial function and a reduction of inflammation and fibrosis, most probably as a consequence of the reduction of serum concentrations of cholesterol and triglycerides. Increased expression of aortic SIRT1 and PGC-1α could play an important role in the observed effects of IFC in rabbits with dyslipidemia.

Changes in cardiac energy metabolic pathways in overweighed rats fed a high-fat diet.

BACKGROUND: Heart produces ATP through long-chain fatty acids beta oxidation. PURPOSE: To analyze whether in ventricular myocardium, high-fat diet may modify the expression of proteins associated with energy metabolism before myocardial function was affected. METHODS: Wistar Kyoto rats were divided into two groups: (a) rats fed standard diet (control; n = 6) and (b) rats fed high-fat diet (HFD; n = 6). Proteins from left ventricles were analyzed by two-dimensional electrophoresis, mass spectrometry and Western blotting. RESULTS: Rats fed with HFD showed higher body weight, insulin, glucose, leptin and total cholesterol plasma levels as compared with those fed with standard diet. However, myocardial functional parameters were not different between them. The protein expression of 3-ketoacyl-CoA thiolase, acyl-CoA hydrolase mitochondrial precursor and enoyl-CoA hydratase, three long-chain fatty acid ß-oxidation-related enzymes, and carnitine-O-palmitoyltransferase I was significantly higher in left ventricles from HFD rats. Protein expression of triosephosphate isomerase was higher in left ventricles from HFD rats than in those from control. Two α/ß-enolase isotypes and glyceraldehyde-3-phosphate isomerase were significantly increased in HFD rats as compared with control. Pyruvate and lactate contents were similar in HFD and control groups. Expression of proteins associated with Krebs cycle and mitochondrial oxidative phosphorylation was higher in HFD rats. CONCLUSIONS: Expression of proteins involved in left ventricle metabolic energy was enhanced before myocardial functionality was affected in rats fed with HFD. These findings may probably indicate higher cardiac energy requirement due to weight increase by HFD.

Spironolactone prevents alterations associated with cardiac hypertrophy produced by isoproterenol in rats: involvement of serum- and glucocorticoid-regulated kinase type 1.

Persistent ß-adrenergic receptor stimulation with isoproterenol is associated with cardiac hypertrophy as well as cardiac synthesis of angiotensin II. Serum- and glucocorticoid-regulated kinase type 1 (SGK-1) is a key mediator in structural, functional and molecular cardiac effects of aldosterone in rats. This study was designed to investigate the cardiac effects of the mineralocorticoid receptor antagonist spironolactone on the response to isoproterenol treatment in rats, as well as the involvement of the main mediator of cellular aldosterone action, SGK-1, in the heart. Male Wistar rats received isoproterenol (3 mg kg(-1) day(-1)) or vehicle for 15 days. Half of the animals in each group were simultaneously treated with spironolactone (200 mg kg(-1) day(-1)). Systolic and diastolic blood pressures were not significantly different among groups. Treatment with spironolactone normalized the increased left ventricular end-diastolic pressure observed in isoproterenol-treated rats. Isoproterenol treatment induced cardiac hypertrophy and increased collagen content, both of which were normalized by spironolactone treatment. The mRNA levels of transforming growth factor ß, connective tissue growth factor, matrix metalloprotease 2, matrix metalloprotease inhibitor 2, tumour necrosis factor α, interleukin 1ß, p22phox and xanthine dehydrogenase were increased (P < 0.05) in isoproterenol-treated rats, and this effect was prevented by spironolactone (P < 0.05). Spironolactone also reduced the elevated SGK-1 expression in isoproterenol-treated rats. The observed reduction of the principal mediator of aldosterone cellular actions, SGK-1, by spironolactone in hearts from isoproterenol-treated rats suggests a role of mineralocorticoids in the cardiac hypertrophy, fibrosis, inflammation, oxidation and diastolic dysfunction induced by isoproterenol treatment in rats.

A role for cardiotrophin-1 in myocardial remodeling induced by aldosterone.

Hyperaldosteronim is associated with left ventricular (LV) hypertrophy (LVH) and fibrosis. Cardiotrophin (CT)-1 is a cytokine that induces myocardial remodeling. We investigated whether CT-1 mediates aldosterone (Aldo)-induced myocardial remodeling in two experimental models. Wistar rats were treated with Aldo-salt (1 mg·kg(-1)·day(-1)) with or without spironolactone (200 mg·kg(-1)·day(-1)) for 3 wk. Wild-type (WT) and CT-1-null mice were infused with Aldo (1 mg·kg(-1)·day(-1)) for 3 wk. Hemodynamic parameters were analyzed. LVH, fibrosis, inflammation, and CT-1 expression were evaluated in both experimental models by histopathological analysis, RT-PCR, Western blot analysis, and ELISA. Hypertensive Aldo-treated rats exhibited increased LV end-diastolic pressure and -dP/dt compared with controls. The cardiac index, LV cross-sectional area and wall thickness, cardiomyocyte size, collagen deposition, and inflammation were increased in Aldo-salt-treated rats. Myocardial expression of molecular markers assessing LVH and fibrosis as well as CT-l levels were also augmented by Aldo-salt. Spironolactone treatment reversed all the above effects. CT-1 correlated positively with hemodynamic, histological, and molecular parameters showing myocardial remodeling. In WT and CT-1-null mice, Aldo infusion did not modify blood pressure. Whereas Aldo treatment induced LVH, fibrosis, and inflammation in WT mice, the mineralocorticoid did not provoke cardiac remodeling in CT-1-null mice. In conclusion, in experimental hyperaldosteronism, the increase in CT-1 expression was associated with parameters showing LVH and fibrosis. CT-1-null mice were resistant to Aldo-induced LVH and fibrosis. These data suggest a key role for CT-1 in cardiac remodeling induced by Aldo independent of changes in blood pressure levels.

Structural, functional, and molecular alterations produced by aldosterone plus salt in rat heart: association with enhanced serum and glucocorticoid-regulated kinase-1 expression.

We aimed to evaluate the structural, functional, inflammatory, and oxidative alterations, as well as serum and glucocorticoid-regulated kinase-1 (SGK-1) expression, produced in rat heart by aldosterone + salt administration. Fibrosis mediators such as connective tissue growth factor, matrix metalloproteinase 2, and tissue inhibitor of metalloproteinases 2 were also evaluated. Treatment with spironolactone was evaluated to prove mineralocorticoid mediation. Male Wistar rats received aldosterone (1 mg[middle dot]kg-1[middle dot]d-1) + 1% NaCl for 3 weeks. Half of the animals were treated with spironolactone (200 mg[middle dot]kg-1[middle dot]d-1). Systolic and diastolic blood pressures, left ventricle (LV) systolic pressure, and LV end-diastolic pressure were elevated (P < 0.05) in aldosterone + salt-treated rats. In aldosterone + salt-treated rats, -dP/dt decreased (P < 0.05), but +dP/dt was similar in all groups. Spironolactone normalized (P < 0.05) systolic blood pressure, diastolic blood pressure, LV systolic pressure, LV end-diastolic pressure, and -dP/dt. Relative heart weight, collagen content, messenger RNA expression of transforming growth factor beta, connective tissue growth factor, matrix metalloproteinase 2, tissue inhibitor of metalloproteinases 2, tumor necrosis factor alpha, interleukin-1[beta], p22phox, endothelial nitric oxide synhtase, and SGK-1 were increased (P < 0.05) in aldosterone + salt-treated rats, being reduced by spironolactone (P < 0.05). SGK-1 might be a key mediator in the structural, functional, and molecular cardiac alterations induced by aldosterone + salt in rats. All the observed changes and mediators are related with the activation of mineralocorticoid receptors.

A proteomic approach to determine changes in proteins involved in the myocardial metabolism in left ventricles of spontaneously hypertensive rats.

BACKGROUND: Different works have suggested that in the hypertrophied heart the energy metabolic pathway shifts to glycolysis. Our aim was to evaluate using proteomics the expression of proteins associated with different energetic metabolic pathways in hypertrophied left ventricles of spontaneously hypertensive rats (SHR). METHODS: 24-weeks-old SHR with stable hypertension and established left ventricle hypertrophy were used. Normotensive Wistar Kyoto rats were used as control. Proteins from left ventricles were analyzed by 2-dimensional electrophoresis and identified by comparison with a virtual rat heart proteomic map and mass spectrometry. RESULTS: Enoyl-CoA hydratase expression, an enzyme involved in fatty acid beta-oxidation, was reduced whereas the expression of other beta-oxidation enzymes, 3-ketoacyl-CoA thiolase and the mitochondrial precursor of acyl-CoA thioester hydrolase, was increased in the hypertrophied left ventricles. The expression of two enzymes involved in the first steps of glycolysis, fructose bisphosphate aldolase and triosephosphate isomerase, was reduced in the left ventricle of SHR. Pyruvate dehydrogenase expression, enzyme involved in glucose oxidation, was enhanced in the hypertrophied ventricles whereas proteins of the tricarboxylic acid cycle were not modified. Proteins involved in the mitochondrial oxidative phosphorylation were overexpressed whereas the alpha-subunit of the mitochondrial precursor of ATP synthase was downexpressed. CONCLUSIONS: Several proteins involved in the main energy metabolic pathways were up and downexpressed. Moreover, our results seem to suggest that probably neither fatty acid beta-oxidation nor glycolysis are the only sources for energy in the hypertrophied left ventricle.

Cardiac L-type calcium current is increased in a model of hyperaldosteronism in the rat.

Accumulating evidence supports the importance of aldosterone as an independent risk factor in the pathophysiology of cardiovascular disease. It has been postulated that aldosterone could contribute to ventricular arrhythmogeneity by modulation of cardiac ionic channels. The aim of this study was to analyse ex vivo the electrophysiological characteristics of the L-type cardiac calcium current (I(CaL)) in a model of hyperaldosteronism in the rat. Aldosterone was administered for 3 weeks, and cardiac collagen deposition and haemodynamic parameters were analysed. In addition, RT-PCR and patch-clamp techniques were applied to study cardiac L-type Ca(2+) channels in isolated cardiomyocytes. Administration of aldosterone induced maladaptive cardiac remodelling that was related to increased collagen deposition, diastolic dysfunction and cardiac hypertrophy. In addition, ventricular myocytes isolated from the aldosterone-treated group showed increased I(CaL) density and conductance and prolongation of the action potential duration. No changes in kinetics or in voltage dependence of activation and inactivation of I(CaL) were observed, but relative expression of Ca(V)1.2 mRNA levels was higher in cardiomyocytes isolated from the aldosterone-treated group. The present study demonstrates that aldosterone treatment induces myocardial fibrosis, cardiac hypertrophy, increase of I(CaL) density, upregulation of L-type Ca(2+) channels and prolongation of action potential duration. It could be proposed that aldosterone, through these mechanisms, might exert pro-arrhythmic effects in the pathological heart.

Aldosterone and the vascular system.

Aldosterone can act in different tissues exerting physiological and pathological effects. At the vascular level, aldosterone affects endothelial function since administration of aldosterone impaired endothelium-dependent relaxations. In addition, the administration of mineralocorticoid receptor antagonists ameliorate relaxation to acetylcholine in models of both hypertension and atherosclerosis and in patients with heart failure. A reduction in nitric oxide levels seems to be the main mechanism underlying this effect due to a reduction in its production as well as an increase in its degradation by reactive oxygen species. Aldosterone is a pro-inflammatory factor that can participate in the vascular inflammatory process associated with different pathologies including hypertension through activation of the NFkappaB system, which mediates the vascular production of different cytokines. This mineralocorticoid also participates in the vascular remodeling observed in hypertensive rats since the administration of eplerenone improved the media-to-lumen ratio in these animals. This effect seems to be due to an increase in extracellular matrix. In summary, aldosterone through mineralocorticoid receptors can participate in the vascular damage associated with different pathologies including hypertension through its prooxidant, pro-inflammatory and profibrotic effects that triggered endothelial dysfunction, an inflammatory process and vascular remodeling.

Mitochondrial oxidative stress and cardiac ageing. / Estrés oxidativo mitocondrial y envejecimiento cardíaco.

According with different international organizations, cardiovascular diseases are becoming the first cause of death in western countries. Although exposure to different risk factors, particularly those related to lifestyle, contribute to the etiopathogenesis of cardiac disorders, the increase in average lifespan and aging are considered major determinants of cardiac diseases events. Mitochondria and oxidative stress have been pointed out as relevant factors both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy and diabetic cardiomyopathy. During aging, cellular processes related with mitochondrial function, such as bioenergetics, apoptosis and inflammation are altered leading to cardiac dysfunction. Increasing our knowledge about the mitochondrial mechanisms related with the aging process, will provide new strategies in order to improve this process, particularly the cardiovascular ones.

Chronic Exercise Improves Mitochondrial Function and Insulin Sensitivity in Brown Adipose Tissue.

The aim of the present work was to study the consequences of chronic exercise training on factors involved in the regulation of mitochondrial remodeling and biogenesis, as well as the ability to produce energy and improve insulin sensitivity and glucose uptake in rat brown adipose tissue (BAT). Male Wistar rats were divided into two groups: (1) control group (C; n = 10) and (2) exercise-trained rats (ET; n = 10) for 8 weeks on a motor treadmill (five times per week for 50 min). Exercise training reduced body weight, plasma insulin, and oxidized LDL concentrations. Protein expression of ATP-independent metalloprotease (OMA1), short optic atrophy 1 (S-OPA1), and dynamin-related protein 1 (DRP1) in BAT increased in trained rats, and long optic atrophy 1 (L-OPA1) and mitofusin 1 (MFN1) expression decreased. BAT expression of nuclear respiratory factor type 1 (NRF1) and mitochondrial transcription factor A (TFAM), the main factors involved in mitochondrial biogenesis, was higher in trained rats compared to controls. Exercise training increased protein expression of sirtuin 1 (SIRT1), peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α) and AMP-activated protein kinase (pAMPK/AMPK ratio) in BAT. In addition, training increased carnitine palmitoyltransferase II (CPT II), mitochondrial F1 ATP synthase α-chain, mitochondrial malate dehydrogenase 2 (mMDH) and uncoupling protein (UCP) 1,2,3 expression in BAT. Moreover, exercise increased insulin receptor (IR) ratio (IRA/IRB ratio), IRA-insulin-like growth factor 1 receptor (IGF-1R) hybrids and p42/44 activation, and decreased IGF-1R expression and IR substrate 1 (p-IRS-1) (S307) indicating higher insulin sensitivity and favoring glucose uptake in BAT in response to chronic exercise training. In summary, the present study indicates that chronic exercise is able to improve the energetic profile of BAT in terms of increased mitochondrial function and insulin sensitivity.

Low Phytanic Acid-Concentrated DHA Prevents Cognitive Deficit and Regulates Alzheimer Disease Mediators in an ApoE-/- Mice Experimental Model.

Alzheimer's disease (AD) is the main cause of dementia and cognitive impairment. It has been associated with a significant diminution of omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) levels in the brain. Clinical trials with DHA as a treatment in neurological diseases have shown inconsistent results. Previously, we reported that the presence of phytanic acid (PhA) in standard DHA compositions could be blunting DHA's beneficial effects. Therefore, we aimed to analyze the effects of a low PhA-concentrated DHA and a standard PhA-concentrated DHA in Apolipoprotein E knockout (ApoE-/-) mice. Behavioral tests and protein expression of pro-inflammatory, pro-oxidant, antioxidant factors, and AD-related mediators were evaluated. Low PhA-concentrated DHA decreased Aß, ß-amyloid precursor protein (APP), p-tau, Ca2+/calmodulin-dependent protein kinase II (CAMKII), caspase 3, and catalase, and increased brain derived neurotrophic factor (BDNF) when compared to standard PhA-concentrated DHA. Low PhA-concentrated DHA decreased interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) protein expression in ApoE-/- mice when compared to standard PhA-concentrated DHA. No significant differences were found in p22phox, inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx), superoxide dismutase 1 (SOD-1), and tau protein expression. The positive actions of a low PhA-concentrated DHA were functionally reflected by improving the cognitive deficit in the AD experimental model. Therefore, reduction of PhA content in DHA compositions could highlight a novel pathway for the neurodegeneration processes related to AD.

Effects of Low Phytanic Acid-Concentrated DHA on Activated Microglial Cells: Comparison with a Standard Phytanic Acid-Concentrated DHA.

Docosahexaenoic acid (DHA, 22:6 n-3) is an essential omega-3 (ω-3) long chain polyunsaturated fatty acid of neuronal membranes involved in normal growth, development, and function. DHA has been proposed to reduce deleterious effects in neurodegenerative processes. Even though, some inconsistencies in findings from clinical and pre-clinical studies with DHA could be attributed to the presence of phytanic acid (PhA) in standard DHA treatments. Thus, the aim of our study was to analyze and compare the effects of a low PhA-concentrated DHA with a standard PhA-concentrated DHA under different neurotoxic conditions in BV-2 activated microglial cells. To this end, mouse microglial BV-2 cells were stimulated with either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2) and co-incubated with DHA 50 ppm of PhA (DHA (PhA:50)) or DHA 500 ppm of PhA (DHA (PhA:500)). Cell viability, superoxide anion (O2-) production, Interleukin 6 (L-6), cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), glutathione peroxidase (GtPx), glutathione reductase (GtRd), Caspase-3, and the brain-derived neurotrophic factor (BDNF) protein expression were explored. Low PhA-concentrated DHA protected against LPS or H2O2-induced cell viability reduction in BV-2 activated cells and O2- production reduction compared to DHA (PhA:500). Low PhA-concentrated DHA also decreased COX-2, IL-6, iNOS, GtPx, GtRd, and SOD-1 protein expression when compared to DHA (PhA:500). Furthermore, low PhA-concentrated DHA increased BDNF protein expression in comparison to DHA (PhA:500). The study provides data supporting the beneficial effect of low PhA-concentrated DHA in neurotoxic injury when compared to a standard PhA-concentrated DHA in activated microglia.

Molecular factors involved in the hypolipidemic- and insulin-sensitizing effects of a ginger (Zingiber officinale Roscoe) extract in rats fed a high-fat diet.

Hypolipidemic and hypoglycemic properties of ginger in animal models have been reported. However, information related to the mechanisms and factors involved in the metabolic effects of ginger at a hepatic level are limited. The aim of the present study was to investigate molecular factors involved in the hypoglycemic and hypolipidemic effects of a hydroethanolic ginger extract (GE) in the liver of rats fed a high-fat diet (HFD). The study was conducted in male Wistar rats divided into the following 3 groups: (i) Rats fed a standard diet (3.5% fat), the control group; (ii) rats fed an HFD (33.5% fat); and (iii) rats fed an HFD treated with GE (250 mg·kg-1·day-1) for 5 weeks (HFD+GE). Plasma levels of glucose, insulin, lipid profile, leptin, and adiponectin were measured. Liver expression of glycerol phosphate acyltransferase (GPAT), cholesterol 7 alpha-hydroxylase, peroxisome proliferator-activated receptors (PPAR), PPARα and PPARγ, glucose transporter 2 (GLUT-2), liver X receptor, sterol regulatory element-binding protein (SREBP1c), connective tissue growth factor (CTGF), and collagen I was measured. Data were analyzed using a 1-way ANOVA, followed by a Newman-Keuls test if differences were noted. The study showed that GE improved lipid profile and attenuated the increase of plasma levels of glucose, insulin, and leptin in HFD rats. This effect was associated with a higher liver expression of PPARα, PPARγ, and GLUT-2 and an enhancement of plasma adiponectin levels. Furthermore, GE reduced liver expression of GPAT, SREBP1c, CTGF, and collagen I. The results suggest that GE might be considered as an alternative therapeutic strategy in the management of overweight and hepatic and metabolic-related alterations.

Mitochondria and oxidative stress in heart aging.

As average lifespan of humans increases in western countries, cardiac diseases become the first cause of death. Aging is among the most important risk factors that increase susceptibility for developing cardiovascular diseases. The heart has very aerobic metabolism, and is highly dependent on mitochondrial function, since mitochondria generate more than 90 % of the intracellular ATP consumed by cardiomyocytes. In the last few decades, several investigations have supported the relevance of mitochondria and oxidative stress both in heart aging and in the development of cardiac diseases such as heart failure, cardiac hypertrophy, and diabetic cardiomyopathy. In the current review, we compile different studies corroborating this role. Increased mitochondria DNA instability, impaired bioenergetic efficiency, enhanced apoptosis, and inflammation processes are some of the events related to mitochondria that occur in aging heart, leading to reduced cellular survival and cardiac dysfunction. Knowing the mitochondrial mechanisms involved in the aging process will provide a better understanding of them and allow finding approaches to more efficiently improve this process.

Proanthocyanidins block aldosterone-dependent up-regulation of cardiac gamma ENaC and Nedd4-2 inactivation via SGK1.

Aldosterone plays a central role in the development of cardiac pathological states involving ion transport imbalances, especially sodium transport. We have previously demonstrated a cardioprotective effect of proanthocyanidins in aldosterone-treated rats. Our objective was to investigate for the first time the effect of proanthocyanidins on serum and glucocorticoid-regulated kinase 1 (SGK1), epithelial Na+ channel (γ-ENaC), neuronal precursor cells expressed developmentally down-regulated 4-2 (Nedd4-2) and phosphoNedd4-2 protein expression in the hearts of aldosterone-treated rats. Male Wistar rats received aldosterone (1mg kg-1day-1)+1% NaCl for 3weeks. Half of the animals in each group were simultaneously treated with the proanthocyanidins-rich extract (80% w/w) (PRO80, 5mg kg-1day-1). Hypertension and diastolic dysfunction induced by aldosterone were abolished by treatment with PRO80. Expression of fibrotic, inflammatory and oxidative mediators were increased by aldosterone-salt administration and blunted by PRO80. Antioxidant capacity was improved by PRO80. The up-regulated aldosterone mediator SGK1, ENaC and p-Nedd4-2/total Nedd4-2 ratio were blocked by PRO80. PRO80 blunted aldosterone-mineralocorticoid-mediated up-regulation of ENaC provides new mechanistic insight of the beneficial effect of proanthocyanidins preventing the cardiac alterations induced by aldosterone excess.