The Effects of Palmitic Acid on Nitric Oxide Production by Rat Skeletal Muscle: Mechanism via Superoxide and iNOS Activation

Background: Increased plasma concentrations of free fatty acids (FFA) can lead to insulin resistance in skeletal muscle, impaired effects on mitochondrial function, including uncoupling of oxidative phosphorylation and decrease of endogenous antioxidant defenses. Nitric oxide (NO) is a highly diffusible gas that presents a half-life of 5-10 seconds and is involved in several physiological and pathological conditions. The effects of palmitic acid on nitric oxide (NO) production by rat skeletal muscle cells and the possible mechanism involved were investigated. Methods: Primary cultured rat skeletal muscle cells were treated with palmitic acid and NO production was assessed by nitrite measurement (Griess method) and 4,5-diaminofluorescein diacetate (DAF-2-DA) assay. Nuclear factor-kappa B (NF-kappa B) activation was evaluated by electrophoretic mobility shift assay and iNOS protein content by western blotting. Results: Palmitic acid treatment increased nitric oxide production. This effect was abolished by treatment with NOS inhibitors, L-nitro-arginine (LNA) and L-nitro-arginine methyl esther (L-NAME). NF-kappa B activation and iNOS content were increased due to palmitic acid treatment. The participation of superoxide on nitric oxide production was investigated by incubating the cells with DAF-2-DA in the presence or absence of palmitic acid, a superoxide generator system (X-XO), a mixture of NOS inhibitors and SOD-PEG (superoxide dismutase linked to polyethylene glycol). Palmitic acid and X-XO system increased NO production and this effect was abolished when cells were treated with NOS inhibitors and also with SOD-PEG. Conclusions: In summary, palmitic acid stimulates NO production in cultured skeletal muscle cells through production of superoxide, nuclear factor-kappa B activation and increase of iNOS protein content. Copyright (C) 2012 S. Karger AG, Basel

Effects of discontinuous Percoll gradient centrifugation on the quality of bovine spermatozoa evaluated with computer-assisted semen analysis and fluorescent probes association

The objective of this study was to evaluate the quality of bovine frozen-thawed sperm cells after Percoll gradient centrifugation. Frozen semen doses were obtained from six bulls of different breeds, including three taurine and three Zebu animals. Four ejaculates per bull were evaluated before and after discontinuous Percoll gradient centrifugation. Sperm motility was assessed by computer-assisted semen analysis and the integrity of the plasma and acrosomal membranes, as well as mitochondrial function, were evaluated using a combination of fluorescent probes propidium iodide, fluorescein isothiocyanate-conjugated Pisum sativum agglutinin and 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolcarbocyanine iodide. The procedure of Percoll gradient centrifugation increased the percentage of total and progressive sperm motility, beat frequency, rectilinear motility, linearity and rapidly moving cells. In addition, the percentage of cells with intact plasma membrane and mitochondrial membrane potential was increased in post-centrifugation samples. However, the percentage of sperm cells with intact acrosomal membrane was markedly reduced. The method used selected the motile cells with intact plasma membrane and higher mitochondrial functionality in frozen-thawed bull semen, but processing, centrifugation and/or the Percoll medium caused damage to the acrosomal membrane.

Transcriptional profiling of Saccharomyces cerevisiae exposed to propolis

Abstract Background Propolis is a natural product of plant resins collected by honeybees (Apis mellifera) from various plant sources. Our previous studies indicated that propolis sensitivity is dependent on the mitochondrial function and that vacuolar acidification and autophagy are important for yeast cell death caused by propolis. Here, we extended our understanding of propolis-mediated cell death in the yeast Saccharomyces cerevisiae by applying systems biology tools to analyze the transcriptional profiling of cells exposed to propolis. Methods We have used transcriptional profiling of S. cerevisiae exposed to propolis. We validated our findings by using real-time PCR of selected genes. Systems biology tools (physical protein-protein interaction [PPPI] network) were applied to analyse the propolis-induced transcriptional bevavior, aiming to identify which pathways are modulated by propolis in S. cerevisiae and potentially influencing cell death. Results We were able to observe 1,339 genes modulated in at least one time point when compared to the reference time (propolis untreated samples) (t-test, p-value 0.01). Enrichment analysis performed by Gene Ontology (GO) Term finder tool showed enrichment for several biological categories among the genes up-regulated in the microarray hybridization such as transport and transmembrane transport and response to stress. Real-time RT-PCR analysis of selected genes showed by our microarray hybridization approach was capable of providing information about S. cerevisiae gene expression modulation with a considerably high level of confidence. Finally, a physical protein-protein (PPPI) network design and global topological analysis stressed the importance of these pathways in response of S. cerevisiae to propolis and were correlated with the transcriptional data obtained thorough the microarray analysis. Conclusions In summary, our data indicate that propolis is largely affecting several pathways in the eukaryotic cell. However, the most prominent pathways are related to oxidative stress, mitochondrial electron transport chain, vacuolar acidification, regulation of macroautophagy associated with protein target to vacuole, cellular response to starvation, and negative regulation of transcription from RNA polymerase II promoter. Our work emphasizes again the importance of S. cerevisiae as a model system to understand at molecular level the mechanism whereby propolis causes cell death in this organism at the concentration herein tested. Our study is the first one that investigates systematically by using functional genomics how propolis influences and modulates the mRNA abundance of an organism and may stimulate further work on the propolis-mediated cell death mechanisms in fungi.

Heparin and penicillamine–hypotaurine–epinephrine (PHE) solution during bovine in vitro fertilization procedures impair the quality of spermatozoa but improve normal oocyte fecundation and early embryonic development

The presence of heparin and a mixture of penicillamine, hypotaurine, and epinephrine (PHE) solution in the in vitro fertilization (IVF) media seem to be a prerequisite when bovine spermatozoa are capacitated in vitro, in order to stimulate sperm motility and acrosome reaction. The present study was designed to determine the effect of the addition of heparin and PHE during IVF on the quality and penetrability of spermatozoa into bovine oocytes and on subsequent embryo development. Sperm quality, evaluated by the integrity of plasma and acrosomal membranes and mitochondrial function, was diminished (P<0.05) in the presence of heparin and PHE. Oocyte penetration and normal pronuclear formation rates, as well as the percentage of zygotes presenting more than two pronuclei, was higher (P<0.05) in the presence of heparin and PHE. No differences were observed in cleavage rates between treatment and control (P>0.05). However, the developmental rate to the blastocyst stage was increased in the presence of heparin and PHE (P>0.05). The quality of embryos that reached the blastocyst stage was evaluated by counting the inner cell mass (ICM) and trophectoderm (TE) cell numbers and total number of cells; the percentage of ICM and TE cells was unaffected (P>0.05) in the presence of heparin and PHE (P<0.05). In conclusion, this study demonstrated that while the supplementation of IVF media with heparin and PHE solution impairs spermatozoa quality, it plays an important role in sperm capacitation, improving pronuclear formation, and early embryonic development

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.

Ruolo delle alterazioni mitocondriali indotte dall'ipossia nella patogenesi della malattia di alzheimer

Le alterazioni della funzionalità mitocondriale detengono un ruolo cruciale nella patogenesi della malattia di Alzheimer (AD), sostenendo il processo neurodegenerativo attraverso meccanismi quali la riduzione della disponibilità energetica e la iperproduzione di ROS. Alle numerose ipotesi di patogenesi dell’AD, si è recentemente affiancata la cosiddetta ipotesi vascolare. Nei soggetti AD è stata riscontrata una significativa riduzione della disponibilità di ossigeno a livello neuronale (ipossia neuronale). Da numerosi studi è poi emerso che l’ipossia gioca un ruolo fondamentale nello sviluppo dell’AD contribuendo a più vie patogenetiche contemporaneamente. Tuttavia, non sono stati ancora chiariti tutti i meccanismi attraverso cui l’ipossia esplica la sua azione di danno. Lo scopo di questo studio è stato quello di contribuire a chiarire il ruolo patologico dell’ipossia nell’AD, analizzando principalmente le alterazioni della funzionalità mitocondriale indotte dalla riduzione della disponibilità di ossigeno. Nella prima fase dello studio cellule PC12 sono state coltivate in presenza di β-amiloide e ipossia. In questo modello abbiamo osservato un potenziamento dei fenomeni di deplezione dell’ATP e di generazione delle ROS indotti dalla Aβ quando anche l’ipossia era presente come fonte di danno cellulare, ipotizzando per i due fattori un effetto congiunto di tipo additivo. Nella seconda fase abbiamo esposto all’ipossia fibroblasti prelevati da pazienti AD portatori di mutazioni a carico dei geni APP e PSEN. La presenza di mutazioni predisponenti ad un fenotipo AD era in grado di determinare un danno bioenergetico e ossidativo. Le alterazioni bioenergetiche riscontrate in normossia risultavano ulteriormente potenziate quando i fibroblasti erano coltivati in ipossia, mentre lo stato di stress ossidativo veniva evidenziato solo in condizioni ipossiche. Sulla base dei risultati finora conseguiti si può ipotizzare che uno dei meccanismi attraverso cui l’ipossia esplica la sua azione di danno nella AD, possa essere dovuto alla capacità di potenziare ulteriormente le alterazioni della funzionalità mitocondriale.

Statin therapy and the expression of genes that regulate calcium homeostasis and membrane repair in skeletal muscle

In skeletal muscle of patients with clinically diagnosed statin-associated myopathy, discrete signs of structural damage predominantly localize to the T-tubular region and are suggestive of a calcium leak. The impact of statins on skeletal muscle of non-myopathic patients is not known. We analyzed the expression of selected genes implicated in the molecular regulation of calcium and membrane repair, in lipid homeostasis, myocyte remodeling and mitochondrial function. Microscopic and gene expression analyses were performed using validated TaqMan custom arrays on skeletal muscle biopsies of 72 age-matched subjects who were receiving statin therapy (n = 38), who had discontinued therapy due to statin-associated myopathy (n = 14), and who had never undergone statin treatment (n = 20). In skeletal muscle, obtained from statin-treated, non-myopathic patients, statins caused extensive changes in the expression of genes of the calcium regulatory and the membrane repair machinery, whereas the expression of genes responsible for mitochondrial function or myocyte remodeling was unaffected. Discontinuation of treatment due to myopathic symptoms led to a normalization of gene expression levels, the genes encoding the ryanodine receptor 3, calpain 3, and dystrophin being the most notable exceptions. Hence, even in clinically asymptomatic (non-myopathic) patients, statin therapy leads to an upregulation in the expression of genes that are concerned with skeletal muscle regulation and membrane repair.

A role for low hepatic copper concentrations in nonalcoholic Fatty liver disease

Copper has a role in antioxidant defense, lipid peroxidation, and mitochondrial function, and copper deficiency has been linked to atherogenic dyslipidemia. We aimed to investigate the potential role of copper availability in the pathogenesis of nonalcoholic fatty liver disease (NAFLD).

S100A1 gene therapy for heart failure: a novel strategy on the verge of clinical trials

Representing the common endpoint of various cardiovascular disorders, heart failure (HF) shows a dramatically growing prevalence. As currently available therapeutic strategies are not capable of terminating the progress of the disease, HF is still associated with a poor clinical prognosis. Among the underlying molecular mechanisms, the loss of cardiomyocyte Ca(2+) cycling integrity plays a key role in the pathophysiological development and progression of the disease. The cardiomyocyte EF-hand Ca(2+) sensor protein S100A1 emerged as a regulator both of sarcoplasmic reticulum (SR), sarcomere and mitochondrial function implicating a significant role in cardiac physiology and dysfunction. In this review, we aim to recapitulate the translation of S100A1-based investigation from first clinical observations over basic research experiments back to a near-clinical setting on the verge of clinical trials today. We also address needs for further developments towards "second-generation" gene therapy and discuss the therapeutic potential of S100A1 gene therapy for HF as a promising novel strategy for future cardiologists. This article is part of a Special Section entitled "Special Section: Cardiovascular Gene Therapy".

Mechanisms of hepatocellular toxicity associated with dronedarone--a comparison to amiodarone

Dronedarone is a new antiarrhythmic drug with an amiodarone-like benzofuran structure. Shortly after its introduction, dronedarone became implicated in causing severe liver injury. Amiodarone is a well-known mitochondrial toxicant. The aim of our study was to investigate mechanisms of hepatotoxicity of dronedarone in vitro and to compare them with amiodarone. We used isolated rat liver mitochondria, primary human hepatocytes, and the human hepatoma cell line HepG2, which were exposed acutely or up to 24h. After exposure of primary hepatocytes or HepG2 cells for 24h, dronedarone and amiodarone caused cytotoxicity and apoptosis starting at 20 and 50 µM, respectively. The cellular ATP content started to decrease at 20 µM for both drugs, suggesting mitochondrial toxicity. Inhibition of the respiratory chain required concentrations of ~10 µM and was caused by an impairment of complexes I and II for both drugs. In parallel, mitochondrial accumulation of reactive oxygen species (ROS) was observed. In isolated rat liver mitochondria, acute treatment with dronedarone decreased the mitochondrial membrane potential, inhibited complex I, and uncoupled the respiratory chain. Furthermore, in acutely treated rat liver mitochondria and in HepG2 cells exposed for 24h, dronedarone started to inhibit mitochondrial β-oxidation at 10 µM and amiodarone at 20 µM. Similar to amiodarone, dronedarone is an uncoupler and an inhibitor of the mitochondrial respiratory chain and of β-oxidation both acutely and after exposure for 24h. Inhibition of mitochondrial function leads to accumulation of ROS and fatty acids, eventually leading to apoptosis and/or necrosis of hepatocytes. Mitochondrial toxicity may be an explanation for hepatotoxicity of dronedarone in vivo.

Gene expression in skeletal muscle of coronary artery disease patients after concentric and eccentric endurance training

Low-intensity concentric (CET) and eccentric (EET) endurance-type training induce specific structural adaptations in skeletal muscle. We evaluated to which extent steady-state adaptations in transcript levels are involved in the compensatory alterations of muscle mitochondria and myofibrils with CET versus EET at a matched metabolic exercise intensity of medicated, stable coronary patients (CAD). Biopsies were obtained from vastus lateralis muscle before and after 8 weeks of CET (n=6) or EET (n=6). Transcript levels for factors involved in mitochondrial biogenesis (PGC-1alpha, Tfam), mitochondrial function (COX-1, COX-4), control of contractile phenotype (MyHC I, IIa, IIx) as well as mechanical stress marker (IGF-I) were quantified using an reverse-transcriptase polymerase chain reaction approach. After 8 weeks of EET, a reduction of the COX-4 mRNA level by 41% and a tendency for a drop in Tfam transcript concentration (-33%, P=0.06) was noted. This down-regulation corresponded to a drop in total mitochondrial volume density. MyHC-IIa transcript levels were specifically decreased after EET, and MyHC-I mRNA showed a trend towards a reduction (P=0.08). Total fiber cross-sectional area was not altered. After CET and EET, the IGF-I mRNA level was significantly increased. The PGC-1alpha significantly correlated with Tfam, and both PGC-1alpha and Tfam significantly correlated with COX-1 and COX-4 mRNAs. Post-hoc analysis identified significant interactions between the concurrent medication and muscular transcript levels as well as fiber size. Our findings support the concept that specific transcriptional adaptations mediate the divergent mitochondrial response of muscle cells to endurance training under different load condition and indicate a mismatch of processes related to muscle hypertrophy in medicated CAD patients.

Influence of inspired oxygen on glucose-lactate dynamics after subdural hematoma in the rat

The mechanisms causing brain damage after acute subdural hematoma (SDH) are poorly understood. A decrease in cerebral blood flow develops immediately after the hematoma forms, thus reducing cerebral oxygenation. This in turn may activate mitochondrial failure and tissue damage leading to ionic imbalance and possibly to cellular breakdown. The purpose of this study was to test whether a simple therapeutic measure, namely increased fraction of inspired oxygen (FiO2 100), and hence increased arterial and brain tissue oxygen tension, can influence brain glucose and lactate dynamics acutely after subdural hematoma in the rat. Twenty-five male Sprague-Dawley anesthetized rats were studied before, during and after induction of the SDH in two separate groups. The Oxygen group (n = 10) was ventilated with 100% oxygen immediately after induction of the SDH. The Air group (n = 10) was ventilated during the entire study with 21% oxygen. Brain microdialysate samples were analyzed for glucose and lactate. All rats were monitored with femoral arterial blood pressure catheters, arterial blood gas analysis, arterial glucose, lactate and end tidal CO2 (EtCO2). Five male Sprague-Dawley rats were sham operated to measure the effect of oxygen challenge on glucose-lactate dynamics without injury. Arterial oxygen tension in the Oxygen group was 371 +/- 30 mmHg and was associated with significantly greater increase in dialysate lactate in the first 30 min after induction of SDH. Dialysate glucose initially dropped in both groups, after SDH, but then reverted significantly faster to values above baseline in the Oxygen group. Changes in ventilatory parameters had no significant effect on dialysate glucose and lactate parameters in the sham group. Extracellular dialysate lactate and glucose are influenced by administration of 100% O2 after SDH. Dialysate glucose normalizes significantly quicker upon 100% oxygen ventilation. We hypothesize that increased neural tissue oxygen tension, in presence of reduced regional CBF, and possibly compromised mitochondrial function, after acute SDH results in upregulation of rate-limiting enzyme systems responsible for both glycolytic and aerobic metabolism. Similar changes have been seen in severe human head injury, and suggest that a simple therapeutic measure, such as early ventilation with 100% O2, may improve cerebral energy metabolism, early after SDH. Further studies to measure the generation of adenosine triphosphate (ATP) are needed to validate the hypothesis.

Arginine vasopressin in vasodilatory shock: effects on metabolism and beyond

PURPOSE OF REVIEW: To describe the effects of arginine vasopressin other than its vasoconstrictive and antidiuretic potential in vasodilatory shock. RECENT FINDINGS: Arginine vasopressin influences substrate metabolism by stimulation of hepatic glucose release, gluconeogenesis, ureogenesis and fatty acid esterification. Although arginine vasopressin is a secretagogue of different hormones, only prolactin increases during arginine vasopressin therapy. Plasmatic and cellular coagulation are affected by arginine vasopressin, resulting in thrombocyte aggregation. Therefore, platelet count typically decreases following arginine vasopressin infusion in critically ill patients. In addition, arginine vasopressin reduces bile flow and may increase bilirubin concentrations. Despite its potential to decrease serum sodium, no change in electrolytes was observed in critically ill patients receiving arginine vasopressin. Although arginine vasopressin is an endogenous antipyretic, body temperature is not decreased by central venous arginine vasopressin infusion. In addition, arginine vasopressin modulates immune function through V1 receptors. Compared with norepinephrine, arginine vasopressin may have protective effects on endothelial function. Net arginine vasopressin effects on gastrointestinal motility seem to be inhibitory and are dose dependent. SUMMARY: Except for its antidiuretic and vasoconstrictive actions, the effects of arginine vasopressin in patients with vasodilatory shock have so far only been partially examined. Potential influences of arginine vasopressin on metabolism and immune, liver and mitochondrial function remain to be assessed in future studies.

The Pyruvate decarboxylase1 gene of Arabidopsis is required during anoxia but not other environmental stresses

Ethanolic fermentation is classically associated with flooding tolerance when plant cells switch from respiration to anaerobic fermentation. However, recent studies have suggested that fermentation also has important functions in the presence of oxygen, mainly in germinating pollen and during abiotic stress. Pyruvate decarboxylase (PDC), which catalyzes the first step in this pathway, is thought to be the main regulatory enzyme. Here, we characterize the PDC gene family in Arabidopsis. PDC is encoded by four closely related genes. By using real-time quantitative polymerase chain reaction, we determined the expression levels of each individual gene in different tissues, under normal growth conditions, and when the plants were subjected to anoxia or other environmental stress conditions. We show that PDC1 is the only gene induced under oxygen limitation among the PDC1 gene family and that a pdc1 null mutant is comprised in anoxia tolerance but not other environmental stresses. We also characterize the expression of the aldehyde dehydrogenase (ALDH) gene family. None of the three genes is induced by anoxia but ALDH2B7 reacts strongly to ABA application and dehydration, suggesting that ALDH may play a role in aerobic detoxification of acetaldehyde. We discuss the possible role of ethanolic fermentation as a robust back-up energy production pathway under adverse conditions when mitochondrial function is disturbed.

MR Spectroscopy and Spectroscopic Imaging for Evaluation of Skeletal Muscle Metabolism: Basics and Applications in Metabolic Diseases

Magnetic resonance spectroscopy (MRS) and spectroscopic imaging (MRSI) provide metabolic information on the musculoskeletal system, thus helping to understand the biochemical and pathophysiological nature of numerous diseases. In particular, MRS has been used to study the energy metabolism of muscular tissue since the very beginning of magnetic resonance examinations in humans when small-bore magnets for studies of the limbs became available. Even more than in other organs, the observation of non-proton-nuclei was important in muscle tissue. Spatial localization was less demanding in these studies, however, high temporal resolution was necessary to follow metabolism during exercise and recovery. The observation of high-energy phosphates during and after the application of workload gives insight into oxidative phosphorylation, a process that takes place in the mitochondria and characterizes impaired mitochondrial function. New applications in insulin-resistant patients followed the development of volume-selective 1H-MRS in whole-body magnets. Nowadays, multinuclear MRS and MRSI of the musculoskeletal system provide several windows to vital biochemical pathways noninvasively. It is shown how MRS and MRSI have been used in numerous diseases to characterize an involvement of the muscular metabolism.