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The main pathological hallmark of diabetes is the loss of functional β-cells. Among several types of β-cell death in diabetes, the involvement of ferroptosis remains elusive. Therefore, we investigated the potential of diabetes-mimicking factors: high glucose (HG), proinflammatory cytokines, hydrogen peroxide (H2O2), or diabetogenic agent streptozotocin (STZ) to induce ferroptosis of β-cells in vitro. Furthermore, we tested the contribution of ferroptosis to injury of pancreatic islets in an STZ-induced in vivo diabetic model. All in vitro treatments increased loss of Rin-5F cells along with the accumulation of reactive oxygen species, lipid peroxides and iron, inactivation of NF-E2-related factor 2 (Nrf2), and decrease in glutathione peroxidase 4 expression and mitochondrial membrane potential (MMP). Ferrostatin 1 (Fer-1), ferroptosis inhibitor, diminished the above-stated effects and rescued cells from death in case of HG, STZ, and H2O2 treatments, while failed to increase MMP and to attenuate cell death after the cytokines’ treatment. Moreover, Fer-1 protected pancreatic islets from STZ-induced injury in diabetic in vivo model, since it decreased infiltration of macrophages and accumulation of lipid peroxides and increased the population of insulin-positive cells. Such results revealed differences between diabetogenic stimuli in determining the destiny of β-cells, emerging HG, H2O2, and STZ, but not cytokines, as contributing factors to ferroptosis and shed new light on an antidiabetic strategy based on Nrf2 activation. Thus, targeting ferroptosis in diabetes might be a promising new approach for preservation of the β-cell population. Our results obtained from in vivo study strongly justify this approach.

Aspergillus, Penicillium and Talaromyces species are frequently cited as contaminants of various substrata and are often associated with indoor environments. The main purpose of this study was to assess the potential pathogenicity of aerosol-derived fungi from Aspergillus, Penicillium and Talaromyces genera, isolated in the rooms for con?servation of cultural heritage artefacts, via estimating hemolytic activity. Hemolysis was detected in 20.58% of tested isolates at 37?C (11.76% partial and 8.82% complete) and 64.71% at 25?C (38.24% partial and 26.47% complete). The majority of isolates that caused ? hemolysis led to the significant oxidation of hemoglobin iron with methemoglobin content in blood agar medium, higher than 80%. Aspergillus melleus was the only tested fungi that caused formation of ferry hemoglobin after the incubation at 25?C. Obtained I values (index of activity for hemolytic exoenzymes) for ? hemolysis were in range of from 0.13 to 0.60 for 37?C, while for the temperature of 25?C values were in range of from 0.08 to 0.50. The same values for ? hemolysis were in range of from 0.03 to 0.08 (37?C), i.e. 0.06 to 0.49 (25?C). Monitoring of pathogenic airborne fungi in indoor environments and estimation of their virulence is essential for the adequate assessment of human health risks.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

IntroductionMetabolic syndrome arises from abnormal adipose function accompanied by insulin resistance. As early factors reflecting/impacting lipid storage dysfunction of adipose tissues, we sought to determine adipokine levels in subcutaneous and visceral adipose tissues (SAT and VAT).Material and methodsGene and protein expression of leptin, adiponectin, and resistin were analyzed in SAT and VAT of normal-weight and overweight/obese women, subclassified according to insulin resistance index, triglyceride, total-, LDL- and HDL-cholesterol levels into metabolically healthy and “at risk” groups.ResultsCompared with normal-weight women, obese women had higher serum leptin levels (PConclusionsLow adiponectin in both examined adipose tissues and inappropriate leptin expression levels in SAT appear to be important characteristics of obesity-related metabolic syndrome. Intriguingly, this adipokine dysregulation is primary seen in SAT, suggesting that endocrine dysfunction in this abdominal depot may be an early risk sign of metabolic syndrome.

Cell death plays an important role in diabetes-induced liver dysfunction. Ferroptosis is a newly defined regulated cell death caused by iron-dependent lipid peroxidation. Our previous studies have shown that high glucose and streptozotocin (STZ) cause β-cell death through ferroptosis and that ferrostatin-1 (Fer-1), an inhibitor of ferroptosis, improves β-cell viability, islet morphology, and function. This study was aimed to examine in vivo the involvement of ferroptosis in diabetes-related pathological changes in the liver. For this purpose, male C57BL/6 mice, in which diabetes was induced with STZ (40 mg/kg/5 consecutive days), were treated with Fer-1 (1 mg/kg, from day 1–21 day). It was found that in diabetic mice Fer-1 improved serum levels of ALT and triglycerides and decreased liver fibrosis, hepatocytes size, and binucleation. This improvement was due to the Fer-1-induced attenuation of ferroptotic events in the liver of diabetic mice, such as accumulation of pro-oxidative parameters (iron, lipofuscin, 4-HNE), decrease in expression level/activity of antioxidative defense-related molecules (GPX4, Nrf2, xCT, GSH, GCL, HO-1, SOD), and HMGB1 translocation from nucleus into cytosol. We concluded that ferroptosis contributes to diabetes-related pathological changes in the liver and that the targeting of ferroptosis represents a promising approach in the management of diabetes-induced liver injury.

Infertility is a significant global health problem that currently affects one of six couples in reproductive age. The quality of male reproductive cells dramatically decreased over the last years and almost every aspect of modern life additionally worsen sperm functional parameters that consequently markedly increase male infertility. This clearly points out the importance of finding a new approach to treat male infertility. Redox signaling mediated by reactive oxygen, nitrogen and sulfur species (ROS, RNS, and RSS respectively), has appeared important for sperm reproductive function. Present review summarizes the current knowledge of ROS, RNS, and RSS in male reproductive biology and identifies potential targets for development of novel pharmacological and therapeutic approaches for male infertility by targeted therapeutic modulation of redox signaling.

The main pathological hallmark of diabetes is the loss of functional

Haptoglobin (Hp) is a hemoglobin-binding protein that prevents free hemoglobin-induced tissue oxidative damage. In streptozotocin-induced diabetic rats, the initial elevation of Hp expression in the serum and liver tends to decrease with diabetes progression, contributing to increased oxidative stress. Glucose toxicity and diabetic complications are closely related to increased modification of nucleocytoplasmic proteins by O-linked-N-acetylglucosamine (O-GlcNAc). We examined the contribution of O-GlcNAcylation of NF-?B p65 to changes in liver Hp expression in diabetic rats. WGA-affinity chromatography revealed a progressive increase in O-GlcNAcylation in nuclear NF-?B p65 during eight weeks of diabetes. DNA-affinity chromatography followed by immunoblot analysis revealed that decreased Hp expression at 4 and 8 weeks of diabetes was accompanied by the absence of Hp gene hormone-responsive element (HRE) occupancy with NF-?B p65, low occupancy with glucocorticoid receptor (GR), and almost no changes in STAT3 occupancy compared to 2 weeks, when Hp expression was highest. Coimmunoprecipitation experiments indicate that these events were the result of impaired NF-?B p65/STAT3 and GR/STAT3 interactions. Results suggest that the attenuation of Hp expression associated with diabetes was at least in part the result of O-GlcNAcylation of NF-?B p65, which prevents the formation of an effective transcription initiation complex on the Hp gene promoter.

Protein expression/activity of antioxidative defense enzymes (AD) in seminal plasma of fertile men might be used as biomarkers of male fertility status. To test this concept, the present study examined the semen parameters of males among 14 normal idiopathic (normozoospermia) and 84 subnormal (teratozoospermia, oligoteratozoospermia, oligoasthenoteratozoospermia) infertile individuals\. We investigated levels of protein expression/activity of Cu, Zn superoxide dismutase (CuZnSOD), manganese superoxide dismutase (MnSOD), catalase and glutathione peroxidase (GSH-Px), their association with functional sperm parameters, as well as their potential to serve as biomarkers of specific sperm pathologies. Although the activity of CuZnSOD and protein expression of catalase were significantly correlated with several sperm parameters, underlying their potential role in etiology of various sperm abnormalities, investigation of their potential usefulness as a biomarker of semen quality showed that these AD enzymes could not distinguish subtle differences between various sperm pathologies. In contrast, GSH-Px activity was decreased in all groups with sperm pathologies and was a very good indicator of aberrations in functional sperm parameters, explaining up to 94.6% of infertility cases where functional sperm parameters were affected. Therefore, assessment of GSH-Px activity showed the potential to discriminate between infertile males with normal and subnormal semen characteristics and may prove useful in the evaluation of male (in)fertility. Abbreviations: AD: antioxidative defense; Cu, Zn SOD: copper, zinc superoxide dismutase; GSH-Px: glutathione peroxidase; MnSOD: manganese superoxide dismutase; NS: normospermia; OATS: oligoasthenoteratozoospermia; OTS: oligoteratozoospermia; ROC: receiver operating characteristic; ROS: reactive oxygen species; TS: teratozoospermia; WHO: world health organization

Ferroptosis is a recently described form of regulated cell death characterized by intracellular iron accumulation and severe lipid peroxidation due to an impaired cysteine-glutathione-glutathione peroxidase 4 antioxidant defence axis. One of the hallmarks of ferroptosis is a specific morphological phenotype characterized by extensive ultrastructural changes of mitochondria. Increasing evidence suggests that mitochondria play a significant role in the induction and execution of ferroptosis. The present review summarizes existing knowledge about the mitochondrial impact on ferroptosis in different pathological states, primarily cancer, cardiovascular diseases, and neurodegenerative diseases. Additionally, we highlight pathologies in which the ferroptosis/mitochondria relation remains to be investigated, where the process of ferroptosis has been confirmed (such as liver- and kidney-related pathologies) and those in which ferroptosis has not been studied yet, such as diabetes. We will bring attention to avenues that could be followed in future research, based on the use of mitochondria-targeted approaches as anti- and proferroptotic strategies and directed to the improvement of existing and the development of novel therapeutic strategies.

Background: The effect of insulin on the endocrine pancreas has been the subject of extensive study, but quantitative morphometric investigations of the exocrine pancreas are scarce. This study was therefore undertaken to investigate the effect of acute and chronic insulin administration (two doses, 0.4 IU and 4 IU) on the morphology of rat pancreas acini. Materials and methods: Semi-fine sections stained with methylene blue and basic fuchsine or haematoxylin and eosin-stained 5-micrometer thick paraffin sections were used for fractal and stereological analysis of exocrine acini. Acute insulin treatment, independent of applied doses increased fractal dimension in line with decreased lacunarity of pancreas acini. Chronic low dose insulin decreased fractal dimension and increased lacunarity of pancreas acini, but a high dose had the opposite effect. The volume densities (Vv) of cytoplasm, granules and nucleus are affected differently: acute low dose and high chronic dose significantly decreased granules Vv, and in line increased cytoplasmic Vv, whereas other examined structures showed slight changes without statistical significance. Results: The results obtained from this investigation indicate that insulin treatment induced structural remodelling of the exocrine pancreas suggesting a substantial role of insulin in its functioning. Conclusions: Additionally, we showed that fine architectural changes in acini could be detected by fractal analysis, suggesting this method as an alternative or addition to routine stereology.

Paper description: As a part of their microenvironment, extracellular hemoglobin has the potential to modify functional characteristics of satellite cells that act as myogenesis initiators. The effect of extracellular xenogeneic hemoglobin (porcine and bovine) was investigated on myoblast proliferation, migration and differentiation capacity, using C2C12 cells as a model. Extracellular hemoglobin modifies C2C12 cell proliferation and migration in a dose- and time-dependent manner and suppresses myogenic differentiation. These findings are important for treating muscle injuries in patients with hemoglobinemia-related diseases and conditions, especially when using satellite cells for cell therapy as the presence of extracellular hemoglobin can influence its outcome. Abstract: Functional characteristics of satellite cells (SCs) that act as myogenesis initiators and have emerged as a promising target for cell therapy, are dependent on their microenvironment. The aim of this study was to investigate the effect of cell-free hemoglobin, as a part of the microenvironment of SCs, on their functional characteristics. The C2C12 cell line served as the experimental model of the SC experimental model; hemoglobin isolated from porcine (PHb) and bovine (BHb) slaughterhouse blood served as the extracellular hemoglobin. The proliferation rate of C2C12 cells was assessed by the MTT test, migration capacity by the scratch assay, and myogenic differentiation capacity by histochemical staining and RT-PCR analysis of the expression of genes specific for myogenic lineage. The effect of hemoglobin on the proliferation and migration of C2C12 cells was dependent on its concentration and the animal species it was isolated from, but the effect of BHb was more prominent. Both PHb and BHb decreased the expression levels of myogenin and muscle specific creatine kinase at a 10 µM concentration. While PHb had no effect on the morphometric parameters of C2C12 myotubes, BHb modified the area and length of C2C12 myotubes cultivated in DMEM/2% horse serum and DMEM/10% fetal calf serum. While PHb and BHb had no effect on heme oxygenase 1 ( Hmox1 ) expression, they stimulated the expression of hypoxia-inducible factor 1-alpha ( Hif1α ) at a concentration of 10 µM. The mainly inhibitory effect of cell-free hemoglobin on myogenic differentiation suggests that it could be a relevant factor in the outcome of cell therapy of muscle injury. https://doi.org/10.2298/ABS200625032S Received: June 25, 2020; Revised: August 8, 2020; Accepted: August 10, 2020; Published online: August 25, 2020 How to cite this article: Stancic AZ, Drvenica IT, Bugarski BM, Ilic VL, Bugarski DS. Extracellular xenogeneic hemoglobin suppresses the capacity for C2C12 myoblast myogenic differentiation. Arch Biol Sci. 2020;72(3):379-91.

Introduction. Blood products, i.e. platelet rich plasma (PRP), leukocyte-poor plasma (PRP) and platelet poor plasma (PPP), have previously been used to improve muscle regeneration. In this study, six months' frozen-stored PPP of individuals who practiced different types of physical exercise was analysed; it could steer mouse C2C12 myoblast cells towards proliferation, migration and myogenic differentiation, and it could affect the morphology/shape of myotubes. Materials and Methods. PPP of male Olympic weightlifters, football players and professional folk dancers, aged 15-19, was collected 12 h post-training and stored for 6 months at -20°C. C2C12 cell proliferation was assessed by MTT test, motility by scratch assay, myogenic differentiation by myotube formation and gelatinase activity by gel-zymography. Results and Conclusions. PPP induced proliferation and migration of C2C12 cells. Proliferative capacity was as follows: weightlifters gt dancers gt football players; mean migratory capacity was: weightlifters = dancers gt football players. PPP induced formation of myotubes; significant inter-individual variations were detected: PPP from weightlifters induced formation of round myotubes, and PPP from football players and dancers induced formation of elongated myotubes. The mean myotube area was as follows: football players gt dancers gt weightlifters. PPP gelatinolytic activity was observed; it was negatively correlated with C2C12 myoblast proliferation. These results provide general but distinct evidence that PPP of individuals practicing certain types of exercise can specifically modify myoblast morphology/function. This is significant for explaining physiological responses and adaptations to exercise. In conclusion, longterm, frozen-stored PPP preserves its potential to modify myoblast morphology and function. Uvod. Krvna plazma obogaćena leukocitima, plazma sa niskim sadržajem leukocita i plazma sa niskim sadržajem trombocita (platelet poor plasma; PPP) su produkti krvi koji se koriste za stimulaciju regeneracije mišića. U ovom radu smo ispitivali da li zamrzavana PPP osoba koje se bave različitim tipovima fizičke aktivnosti, usmerava C2C12 myoblaste u pravcu povećane proliferacije, migracije i miogene diferencijacije, i da li utiče na morfologiju/izgled miotuba. Materijal i metode. PPP osoba muškog pola starih 15-19 godina je izolovana iz krvi dizača tegova, fudbalera i profesionalnih igrača folklora, 12 sati nakon treninga. Uzorci PPP su čuvani šest meseci na -20ºC. Uticaj PPP na proliferaciju C2C12 ćelija je analiziran MTT testom, na migraciju "scratch" testom, a uticaj na miogenu diferencijaciju je analiziran na osnovu sposobnosti PPP da indukuju formiranje miotuba. Želatinolitička aktivnost PPP je analizirana gel-zimografijom. Rezultati i zaključak. Uzorci PPP su indukovali proliferaciju i migraciju C2C12 ćelija, a kapacitet da stimulišu proliferaciju je bio: dizači tegova gt igrači gt fudbaleri. Kapacitet PPP da utiču na migraciju C2C12 ćelija je bio: dizači tegova = igrači gt fudbaleri. Svi uzorci PPP su indukovali formiranje miotuba, ali su zapažene značajne interindividualne varijacije. PPP dizača tegova su indukovali formiranje okruglih miotuba, dok su miotube formirane u prisustvu PPP igrača i fudbalera bile izdužene. Površina miotuba se, zavisno od tipa fizičke aktivnosti, menjala po sledećem rasporedu: fudbaleri gt igrači gt dizači tegova. Želatinolitička aktivnost PPP je nagativno korelirala sa proliferacijom C2C12 ćelija. Rezultati ove studije pokazuju da PPP osoba koje se bave određenim tipom fizičke aktivnosti mogu da na specifičan način modulišu morfologiju/funciju mioblasta. Ovaj rezultat je od značaja za objašnjnje fiziološkog odgovora i adaptacije na vežbanje. On pokazuje i da PPP nakon dugotrajnog zamrzavanja imaju očuvanu spospbnost modifikovanja morfologije i funkcije mioblasta.

We have tested in vitro effects of hemoglobin from bovine slaughterhouse blood (BHb) on stromal cells of mesodermal origin, with an aim to explore its use as a component of cell culture media. Human peripheral blood mesenchymal stromal cells (PB-MSCs) and three mouse cell lines (ATDC5, MC3T3-E1 and 3T3-L1) were employed to study BHb effects on their growth and migration. The cells multilineage differentiation capacity in the presence of BHb was evaluated after induced differentiation, by histochemical staining and by RT-PCR analysis of the expression of genes specific for chondrogenic, adipogenic and osteogenic lineages. The effects of BHb on the cell proliferation and motility were dependent on both, cell type and BHb concentration (0.1 μM, 1 μM and 10 μM). In the lowest concentration (0.1 µM) BHb showed the least prominent effect on the cell proliferation and migration. In this concentration BHb reduced the differentiation capacity of all tested cells and its effect was dependent of composition of induction medium and the culture period. Obtained data suggest that BHb has the potential to be used as a component of cell culture media through maintaining proliferation and reducing differentiation capacity of mesenchymal stromal cells.

The objective of our study was to develop controlled drug delivery system based on erythrocyte ghosts for amphiphilic compound sodium diclofenac considering the differences between erythrocytes derived from two readily available materials porcine slaughterhouse and outdated transfusion human blood. Starting erythrocytes, empty erythrocyte ghosts and diclofenac loaded ghosts were compared in terms of the encapsulation efficiency, drug releasing profiles, size distribution, surface charge, conductivity, surface roughness and morphology. The encapsulation of sodium diclofenac was performed by an osmosis based process gradual hemolysis. During this process sodium diclofenac exerted mild and delayed antihemolytic effect and increased potassium efflux in porcine but not in outdated human erythrocytes. FTIR spectra revealed lack of any membrane lipid disorder and chemical reaction with sodium diclofenac in encapsulated ghosts. Outdated human erythrocyte ghosts with detected nanoscale damages and reduced ability to shrink had encapsulation efficiency of only 8%. On the other hand, porcine erythrocyte ghosts had encapsulation efficiency of 37% and relatively slow drug release rate. More preserved structure and functional properties of porcine erythrocytes related to their superior encapsulation and release performances, define them as more appropriate for the usage in sodium diclofenac encapsulation process.

Insulin sensitivity and metabolic homeostasis depend on the capacity of adipose tissue to take up and utilize excess glucose and fatty acids. The key aspects that determine the fuel-buffering capacity of adipose tissue depend on the physiological levels of the small redox molecule, nitric oxide (NO). In addition to impairment of NO synthesis, excessive formation of the superoxide anion (О2•- ) in adipose tissue may be an important interfering factor diverting the signalling of NO and other reactive oxygen and nitrogen species in obesity, resulting in metabolic dysfunction of adipose tissue over time. Besides its role in relief from superoxide burst, enhanced NO signalling may be responsible for the therapeutic benefits of different superoxide dismutase mimetics, in obesity and experimental diabetes models. This review summarizes the role of NO in adipose tissue and highlights the effects of NO/О2•- ratio 'teetering' as a promising pharmacological target in the metabolic syndrome. Linked articles This article is part of a themed section on Redox Biology and Oxidative Stress in Health and Disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.12/issuetoc.

Slaughterhouse blood represents a valuable source of hemoglobin, which can be used in the production of heme-iron based supplements for the prevention/treatment of iron-deficiency anemia. In order to obtain a stable solid-state formulation, the effect of maltose addition (30 %) on the stability and storage of bovine and porcine hemoglobin in powders obtained by spray and freeze-drying (without maltose: Hb; with maltose: HbM) were investigated. Differential scanning calorimetry of spray- and freeze-dried powders indicated satisfying quality of the formulation prepared with maltose on dissolving back into solution. After two-year storage at room temperature (20?5?C) in solid forms, protected from moisture and light, rehydrated spray- and freeze-dried HbM were red, while Hb were brown. Dynamic light scattering showed the presence of native hemoglobin monomers in rehydrated spray- and freeze-dried HbM, but their agglomerates in Hb samples. UV?Vis spectrophotometry confirmed an absence of significant hemoglobin denaturation and methemoglobin formation in HbM freeze-dried powders. In spray-dried HbM, an increased level of methemoglobin was detected. The results confirmed the stabilizing effect of maltose, and suggested its use in the production of long-term stable solid-state formulations of hemoglobin, along with drying processes optimization.

Key points White to brown adipose tissue conversion and thermogenesis can be ignited by different conditions or agents and its sustainability over the long term is still unclear. Browning of rat retroperitoneal white adipose tissue (rpWAT) during cold acclimation involves two temporally apparent components: (1) a predominant non-selective browning of most adipocytes and an initial sharp but transient induction of uncoupling protein 1, peroxisome proliferator-activated receptor (PPAR) coactivator-1α, PPARγ and PPARα expression, and (2) the subsistence of relatively few thermogenically competent adipocytes after 45 days of cold acclimation. The different behaviours of two rpWAT beige/brown adipocyte subsets control temporal aspects of the browning process, and thus regulation of both components may influence body weight and the potential successfulness of anti-obesity therapies. Abstract Conversion of white into brown adipose tissue may have important implications in obesity resistance and treatment. Several browning agents or conditions ignite thermogenesis in white adipose tissue (WAT). To reveal the capacity of WAT to function in a brownish/burning mode over the long term, we investigated the progression of the rat retroperitoneal WAT (rpWAT) browning during 45 days of cold acclimation. During the early stages of cold acclimation, the majority of rpWAT adipocytes underwent multilocularization and thermogenic-profile induction, as demonstrated by the presence of a multitude of uncoupling protein 1 (UCP1)-immunopositive paucilocular adipocytes containing peroxisome proliferator-activated receptor (PPAR) coactivator-1α (PGC-1α) and PR domain-containing 16 (PRDM16) in their nuclei. After 45 days, all adipocytes remained PRDM16 immunopositive, but only a few multilocular adipocytes rich in mitochondria remained UCP1/PGC-1α immunopositive. Molecular evidence showed that thermogenic recruitment of rpWAT occurred following cold exposure, but returned to starting levels after cold acclimation. Compared with controls (22 ± 1°C), levels of UCP1 mRNA increased in parallel with PPARγ (PPARα from days 1 to 7 and PGC-1α on day 1). Transcriptional recruitment of rpWAT was followed by an increase in UCP1 protein content (from days 1 to 21). Results clearly showed that most of the adipocytes within rpWAT underwent transient brown-fat-like thermogenic recruitment upon stimulation, but only a minority of cells retained a brown adipose tissue-like phenotype after the attainment of cold acclimation. Therefore, browning of WAT is dependent on both maintaining the thermogenic response and retaining enough brown-like thermogenically competent adipocytes in the long-term. Both aspects of browning could be important for long-term energy homeostasis and body-weight regulation.

Infertility is one of the most serious medical issues that is dramatically rising worldwide. Extensive research dedicated to this problem clearly shows that infertility is a disease of modern society and that a nutrition has a great infl uence on the development of sterility. Thus, the impact of specifi c nutritional factors, i.e. diet pattern on both male and female fertility is included in this review. It is encouraging that modifi cation of nutritional habits can help couples to conceive spontaneously, or increase their chances of conception with in vitro fertilization (IVF) treatment.

Growing evidences suggest that statins exert several cholesterol-independent pleiotropic effects in diabetes, but there is no consensus whether they are positive or negative. To shed more light on this issue, we examined the effect of simvastatin on β-cell regeneration in a diabetic state. Diabetes was induced in male Mill Hill rats with a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were additionally separated into two subgroups: treated with simvastatin (5 mg/kg/day, i.g., 12 days) and control. Treatment of diabetic animals started after diabetes induction (glucose level ≥ 12 mmol/L). Our findings revealed that there is no increase in the area of insulin-immunopositive cells neither normalization of serum insulin level after simvastatin treatment of diabetic animals, although simvastatin increased nuclear immunopositivity for pancreas duodenum homeobox-1 (PDX-1) and proliferating cell nuclear antigen (PCNA). The data from this study suggest that 12-day treatment with simvastatin did not improve diabetes- induced disturbances in β-cell mass/function.

The biological activity of three previously synthesized 17 beta-carboxamide glucocorticoids (BG, BEG, and MPEA) was tested in vitro on mitogen stimulated and non-stimulated peripheral blood mononuclear cells (MNCs) and granulocytes from human healthy donors, and the results were compared to the conventional glucocorticoid dexamethasone. The tested 17 beta-carboxamide glucocorticoids did not induce decreases in MNC viability and proliferation, while modulation of reactive oxygen species (ROS) synthesis in granulocytes was dependent on the cell donor. The obtained results indicate the possibility of avoidance of strong lymphocyte suppression, which is generally recognized during administration of conventional glucocorticoids. Furthermore, the metabolism of the tested derivatives was predicted in silico. The predicted metabolites were synthesized and the in silico results were confirmed by in vitro evaluation of the metabolism of BG, BEG, and MPEA in human serum and in cultures of peripheral blood MNCs. The results of the biological activity and metabolism evaluation and of previous in vivo evaluations of biological activity indicate the soft drug nature of BG, BEG, and MPEA. In order to be fully considered as soft glucocorticoids, further investigations on the toxicity and activity of the formed metabolites are required.

The European Cooperation in Science and Technology (COST) provides an ideal framework to establish multi-disciplinary research networks. COST Action BM1203 (EU-ROS) represents a consortium of researchers from different disciplines who are dedicated to providing new insights and tools for better understanding redox biology and medicine and, in the long run, to finding new therapeutic strategies to target dysregulated redox processes in various diseases. This report highlights the major achievements of EU-ROS as well as research updates and new perspectives arising from its members. The EU-ROS consortium comprised more than 140 active members who worked together for four years on the topics briefly described below. The formation of reactive oxygen and nitrogen species (RONS) is an established hallmark of our aerobic environment and metabolism but RONS also act as messengers via redox regulation of essential cellular processes. The fact that many diseases have been found to be associated with oxidative stress established the theory of oxidative stress as a trigger of diseases that can be corrected by antioxidant therapy. However, while experimental studies support this thesis, clinical studies still generate controversial results, due to complex pathophysiology of oxidative stress in humans. For future improvement of antioxidant therapy and better understanding of redox-associated disease progression detailed knowledge on the sources and targets of RONS formation and discrimination of their detrimental or beneficial roles is required. In order to advance this important area of biology and medicine, highly synergistic approaches combining a variety of diverse and contrasting disciplines are needed., Graphical abstract fx1, Highlights • RONS are chemical mediators and a communication tool. • RONS and disturbed redox balance play a role in a broad range of diseases and aging. • Bacteria and toxins are important stimulators of cellular RONS formation. • Drugs should preserve beneficial redox signaling and inhibit detrimental RONS sources. • Redox drugs may target the origin, identity, location and time of RONS formation.

In the present study we hypothesized that myocardial adaptive phenotype in mammalian hibernation involves rearrangement of mitochondria bioenergetic pathways providing protective pattern in states of reduced metabolism and low temperature. European ground squirrels (Spermophilus citellus) were exposed to low temperature (4 ± 1 °C) and then divided into two groups: (1) animals that fell into torpor (hibernating group) and (2) animals that stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). Protein levels of selected components of the electron transport chain and ATP synthase in the heart increased after prolonged cold acclimation (mainly from day 7–21 of cold exposure) and during hibernation. Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) was also upregulated under both cold exposure and hibernating conditions. The phosphorylation state (Thr172) of 5′-AMP-activated protein kinase α increased early in cold exposure (at day 1 and 3) along with increased protein levels of phosphofructokinase and pyruvate dehydrogenase, whereas hypoxia inducible factor 1α protein levels showed no changes in response to cold exposure or hibernation. Hibernation also resulted in protein upregulation of three antioxidant defense enzymes (manganese and copper/zinc superoxide dismutases and glutathione peroxidase) and thioredoxin in the heart. Cold-exposed and hibernation-related phenotypes of the heart are characterized by improved molecular basis for mitochondrial energy-producing and antioxidant capacities that are achieved in a controlled manner. The recapitulation of such adaptive mechanisms found in hibernators could have broad application for myocardial protection from ishemia/reperfusion to improve hypothermic survival and cold preservation of hearts from non-hibernating species, including humans.

Introduction: Prevention represent the turning point in medicine, and if we have knowledge about it for long time, it isn't completely applicable, because of the fact that man kind aren't completely developed consciousness about influence of preventive measures on their health. We must learn people about preventive measures to live healthy, to feed themselves healthy, to haven't bad habits which disturb health, to avoid stress situations... The Aim of research: Establish in which extent are people on the area of general practice institution of town Sabac introduced about risk factors which leads to cerebrovascular diseases and what to do to avoid development of risk factors. Materials and methods: Research conducted in General practice institution of town Sabac in December 2011 on sample of 30 examinee. Persons are accidently selected. The data were obtained descriptive method and method of survey. The instrument used in this research is questionnaire. Results: Based on the research it provided information that examinee are mainly under the stress, that they suffer from hypertension, they have obesity, not properly take food, physically not active... but most of them normally goes to their medical examination and have wish to find out more about prevention of cerebrovascular diseases. Discussion: Nurses must trough their work seized more their users of their services and motivate them to have normally medical examination and take care of their health condition, to eliminate risk factors and cure diseases that they have already. Conclusion: Cerebrovascular diseases have huge social-medical importance. Excepting that cerebrovascular diseases are diseases with large-scale of death rate, equally is important fact that this neurological illness with large-scale disability and dramatically disorder life in family and patients surounding, and present material weight for social community. These facts highlights importance of prevention of cerebrovascular diseases.

Great progress has been made in our understanding of the browning process in white adipose tissue (WAT) in rodents. The recognition that i) adult humans have physiologically inducible brown adipose tissue (BAT) that may facilitate resistance to obesity and ii) that adult human BAT molecularly and functionally resembles beige adipose tissue in rodents, reignited optimism that obesity and obesity-related diabetes type 2 can be battled by controlling the browning of WAT. In this review the main cellular mechanisms and molecular mediators of browning of WAT in different physiological states are summarized. The relevance of browning of WAT in metabolic health is considered primarily through a modulation of biological role of fat tissue in overall metabolic homeostasis.

Infertility is a global problem that is on the rise, especially during the last decade. Currently, infertility affects approximately 10-15% of the population worldwide. The frequency and origin of different forms of infertility varies. It has been shown that reactive oxygen and nitrogen species (ROS and RNS) are involved in the aetiology of infertility, especially male infertility. Various strategies have been designed to remove or decrease the production of ROS and RNS in spermatozoa, in particular during in vitro fertilization. However, in recent years it has been shown that spermatozoa naturally produce a variety of ROS/RNS, including superoxide anion radical (O2 (⋅-)), hydrogen peroxide and NO. These reactive species, in particular NO, are essential in regulating sperm capacitation and the acrosome reaction, two processes that need to be acquired by sperm in order to achieve fertilization potential. In addition, it has recently been shown that mitochondrial function is positively correlated with human sperm fertilization potential and quality and that NO and NO precursors increase sperm motility by increasing energy production in mitochondria. We will review the new link between sperm NO-driven redox regulation and infertility herein. A special emphasis will be placed on the potential implementation of new redox-active substances that modulate the content of NO in spermatozoa to increase fertility and promote conception.

Objective. Metabolic homeostasis depends on adipocyte metabolic responses/processes, most of which are redox-regulated. Besides, visceral and subcutaneous adipose tissues (VAT and SAT, respectively) differ metabolically and in their contribution to metabolic complications, but their redox characteristics in humans are still unknown. To understand the molecular mechanisms of metabolic syndrome development, we analysed the redox characteristics of VAT and SAT in groups with various body weights and metabolic risks. Material and Methods. Fifty premenopausal women were classified according to body mass index into normal-weight and obese groups, and these groups were further sub-classified into metabolically healthy and metabolically obese ({''}at risk{''}) based on the homeostasis model assessment of insulin resistance (HOMA-IR) index and the triglyceride, total-, LDL- and HDL-cholesterol levels. Antioxidant components, NADPH oxidase protein and 4-hydroxynonenal (4-HNE) levels were analysed in VAT and SAT. Results. Compared with the SAT, the VAT showed a higher basal level of glutathione (GSH) and GSH-dependent enzyme activities. Compared with the metabolically healthy normal-weight controls, the obese groups of women showed lower GSH levels in both depots. However, in these groups, additional prooxidative changes (increased NADPH oxidase and 4-HNE and decreased levels of SOD and/or CAT) were observed only in VAT. Conclusions. Because of the critical role of thiol-redox homeostasis in lipogenesis, interdepot-differences in the GSH-dependent antioxidant part may be connected to the higher metabolic activity found in VAT. Analogously, the lower GSH levels that occur during obesity and the corresponding additional redox imbalance may be signs of VAT metabolic dysfunction that underlie the subsequent metabolic impairment. (C) 2014 Elsevier Inc. All rights reserved. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173054, 173055]

This study investigated the effect of Mn chelate supplementation on the activity of antioxidant enzymes: copper zincand manganese- superoxide dismutase (CuZnSOD and MnSOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR), glutathione S-transferase (GST) and the amount of glutathione (GSH) in the blood, liver, brain, heart and small intestine of the rat. Increased Mn intake of 34% above daily intake (0.851 ± 0.045 mg Mn / kg b.wt.) induced changes in the antioxidant defense in the tissues of the rat. Decreased activity of peroxides-converting enzymes CAT and GSHPx was found in all examined tissues (except GSH-Px activity in the liver). The amount of GSH was increased in plasma and liver, while it was decreased in heart and brain of supplemented animals. Treatment with Mn increases MnSOD activity in the liver, and CuZnSOD activity in the heart. The observed changes in antioxidant defense are closely connected with the newly established redox profile in the examined tissues after Mn supplementation.

Context: Brown adipose tissue (BAT) has the unique ability of generating heat due to the expression of mitochondrial uncoupling protein 1 (UCP1). A recent discovery regarding functional BAT in adult humans has increased interest in the molecular pathways of BAT development and functionality. An important role for estrogen in white adipose tissue was shown, but the possible role of estrogen in human fetal BAT (fBAT) is unclear. Objective: The objective of this study was to determine whether human fBAT expresses estrogen receptor alpha (ER alpha) and ER beta. In addition, we examined their localization as well as their correlation with crucial proteins involved in BAT differentiation, proliferation, mitochondriogenesis and thermogenesis including peroxisome proliferator-activated receptor gamma (PPAR gamma), proliferating cell nuclear antigen (PCNA), PPAR gamma-coactivator-1 alpha (PGC-1 alpha), and UCP1. Design: The fBAT was obtained from 4 human male fetuses aged 15, 17, 20, and 23 weeks gestation. ER alpha and ER beta expression was assessed using Western blotting, immunohistochemistry, and immunocytochemistry. Possible correlations with PPAR gamma, PCNA, PGC-1 alpha, and UCP1 were examined by double immunofluorescence. Results: Both ER alpha and ER beta were expressed in human fBAT, with ER alpha being dominant. Unlike ER beta, which was present only in mature brown adipocytes, we detected ER alpha in mature adipocytes, preadipocytes, mesenchymal and endothelial cells. In addition, double immunofluorescence supported the notion that differentiation in fBAT probably involves ER alpha. Immunocytochemical analysis revealed mitochondrial localization of both receptors. Conclusion: The expression of both ER alpha and ER beta in human fBAT suggests a role for estrogen in its development, primarily via ER alpha. In addition, our results indicate that fBAT mitochondria could be targeted by estrogens and pointed out the possible role of both ERs in mitochondriogenesis. Serbian Ministry of Education, Science, and Technological Development {[}173055, 173054]

Nitric oxide (NO) and vasoactive intestinal polypeptide (VIP) are important intestinal neurotransmitters that coexist in the gut enteric nervous system and play an important role in intestinal physiology (e.g., absorption, motility, fluid secretion and smooth muscle relaxation). It is also known that cold exposure alters several aspects of gastrointestinal physiology and induces hyperphagia to meet increased metabolic demands, but there are no data regarding NO and VIP involvement in intestinal response during acclimation to cold. The objective of this study was to determine the influence of long-term l-arginine supplementation on the expression of the three isoforms of nitric oxide synthase (NOS) and VIP in small intestine of rats acclimated to room temperature or cold. Animals (six per group) acclimated to room temperature (22 +/- A 1 A degrees C) and cold (4 +/- A 1 A degrees C), respectively, were treated with 2.25 \% l-arginine, a substrate for NOSs, or with 0.01 \% N (omega)-nitro-l-arginine methyl ester, an inhibitor of NOSs, for 45 days. The topographical distribution of VIP and NOSs expression in small intestine was studied by immunohistochemistry, and ImageJ software was used for semiquantitative densitometric analysis of their immunoexpression. Long-term dietary l-arginine supplementation increases VIP and NOSs immunoexpression at room temperature while at cold increases the endothelial NOS, inducible NOS and VIP but decrease neuronal NOS in rat small intestine. Our results demonstrate that long-term dietary l-arginine supplementation modulates NOSs and VIP immunoexpression in rat small intestine with respect to ambient temperature, pointing out the eNOS as a predominant NOS isoform with an immunoexpression pattern similar to VIP. Serbian Ministry of Education, Science and Technological Development {[}173055]

Hippocampal structural changes associated with diabetes-related cognitive impairments are well described, but their molecular background remained vague. We examined whether/how diabetes alters molecular basis of energy metabolism in hippocampus readily after diabetes onset, with special emphasis on its redox-sensitivity. To induce diabetes, adult Mill Hill hybrid hooded rats received a single alloxan dose (120 mg/kg). Both non-diabetic and diabetic groups were further divided in two subgroups receiving (i) or not (ii) superoxide dismutase (SOD) mimic, [Mn(II)(pyane)Cl2] for 7 days, i.p. Treatment of the diabetic animals started after blood glucose level ≥12 mM. Diabetes decreased protein levels of oxidative phosphorylation components: complex III and ATP synthase. In contrast, protein amounts of glyceraldehyde-3-phosphate dehydrogenase, pyruvate dehydrogenase, and hypoxia-inducible factor-1α - the key regulator of energy metabolism in stress conditions, were higher in diabetic animals. Treatment with SOD mimic restored/increased the levels of oxidative phosphorylation components and returned hypoxia-inducible factor-1α to control level, while diabetes-induced up-regulation of glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase, was additionally stimulated. To conclude, our results provide insight into the earliest molecular changes of energy-producing pathways in diabetes that may account for structural/functional disturbance of hippocampus, seen during disease progression. Also, data suggest [Mn(II)(pyane)Cl2] as potential therapeutic agent in cutting-edge approaches to threat this widespread metabolic disorder.

Considering the vital role of skeletal muscle in control of whole-body metabolism and the severity of long-term diabetic complications, we aimed to reveal the molecular pattern of early diabetes-related skeletal muscle phenotype in terms of energy metabolism, focusing on regulatory mechanisms, and the possibility to improve it using two redox modulators, L-arginine and superoxide dismutase (SOD) mimic. Alloxan-induced diabetic rats (120 mg/kg) were treated with L-arginine or the highly specific SOD mimic, M40403, for 7 days. As appropriate controls, non-diabetic rats received the same treatments. We found that L-arginine and M40403 restored diabetes-induced impairment of phospho-5′-AMP-activated protein kinase α (AMPKα) signaling by upregulating AMPKα protein itself and its downstream effectors, peroxisome proliferator-activated receptor-γ coactivator-1α and nuclear respiratory factor 1. Also, there was a restitution of the protein levels of oxidative phosphorylation components (complex I, complex II and complex IV) and mitofusin 2. Furthermore, L-arginine and M40403 induced translocation of glucose transporter 4 to the membrane and upregulation of protein of phosphofructokinase and acyl coenzyme A dehydrogenase, diminishing negative diabetic effects on limiting factors of glucose and lipid metabolism. Both treatments abolished diabetes-induced downregulation of sarcoplasmic reticulum calcium-ATPase proteins (SERCA 1 and 2). Similar effects of L-arginine and SOD mimic treatments suggest that disturbances in the superoxide/nitric oxide ratio may be responsible for skeletal muscle mitochondrial and metabolic impairment in early diabetes. Our results provide evidence that L-arginine and SOD mimics have potential in preventing and treating metabolic disturbances accompanying this widespread metabolic disease. Chemico-Biological Interactions (2017), 272: 188-196

The role of nitric oxide (NO) in cutaneous physiology/pathology became a growing research field since the discovery that almost all types of skin cells can synthetize this redox signaling molecule about 20 years ago. Now, it is evident that NO is an important player in skin physiological processes and in responses of cutaneous cells to external insults, while the impaired NO signaling has an important consequence in skin pathology. Skin disorders are common complications in diabetic conditions. Various metabolic/biochemical and immunological dysregulations in diabetic skin are tightly coupled with the disturbances in the redox state, primarily the ratio between NO and superoxide (O(cyrillic) 2 - ). This review describes possible therapeutic significance of different redox state modulators in the treatment of diabetic skin disorders. The focus is on those modulators that tightly control NO/O(cyrillic) 2 - ratio through the complex mechanisms affecting endogenous NO and O(cyrillic) 2 - producing and removing systems. The fact that classic antioxidants failed to show significant benefits in diabetes, emphasizes the importance of such redox mechanism-based and targeted approaches.

Setting the correct ratio of superoxide anion (O2•-) and nitric oxide (•NO) radicals seems to be crucial in restoring disrupted redox signaling in diabetic skin and improvement of •NO physiological action for prevention and treatment of skin injuries in diabetes. In this study we examined the effects of L-arginine and manganese(II)-pentaazamacrocyclic superoxide dismutase (SOD) mimic – M40403 in diabetic rat skin. Following induction of diabetes by alloxan (blood glucose level ≥12 mMol l −1) non-diabetic and diabetic male Mill Hill hybrid hooded rats were divided into three subgroups: (i) control, and receiving: (ii) L-arginine, (iii) M40403. Treatment of diabetic animals started after diabetes induction and lasted for 7 days. Compared to control, lower cutaneous immuno-expression of endothelial NO synthase (eNOS), heme oxygenase 1 (HO1), manganese SOD (MnSOD) and glutathione peroxidase (GSH-Px), in parallel with increased NFE2-related factor 2 (Nrf2) and nitrotyrosine levels characterized diabeti...

The present study describes utilization of two photon excitation fluorescence (2PE) microscopy for visualization of the hemoglobin in human and porcine erythrocytes and their empty membranes (i.e., ghosts). High-quality, label- and fixation-free visualization of hemoglobin was achieved at excitation wavelength 730 nm by detecting visible autofluorescence. Localization in the suspension and spatial distribution (i.e., mapping) of residual hemoglobin in erythrocyte ghosts has been resolved by 2PE. Prior to the 2PE mapping, the presence of residual hemoglobin in the bulk suspension of erythrocyte ghosts was confirmed by cyanmethemoglobin assay. 2PE analysis revealed that the distribution of hemoglobin in intact erythrocytes follows the cells’ shape. Two types of erythrocytes, human and porcine, characterized with discocyte and echinocyte morphology, respectively, showed significant differences in hemoglobin distribution. The 2PE images have revealed that despite an extensive washing out procedure after gradual hypotonic hemolysis, a certain amount of hemoglobin localized on the intracellular side always remains bound to the membrane and cannot be eliminated. The obtained results open the possibility to use 2PE microscopy to examine hemoglobin distribution in erythrocytes and estimate the purity level of erythrocyte ghosts in biotechnological processes.

Background/Aims: Study elucidates and compares the mitochondrial bioenergetic-related molecular basis of sevoflurane and propofol cardioprotection during aortic valve replacement surgery due to aortic valve stenosis. Methods: Twenty-two patients were prospectively randomized in two groups regarding the anesthetic regime: sevoflurane and propofol. Hemodynamic parameters, biomarkers of cardiac injury and brain natriuretic peptide (BNP) were measured preoperatively and postoperatively. In tissue samples, taken from the interventricular septum, key mitochondrial molecules were determined by Western blot, real time PCR, as well as confocal microscopy and immunohisto- and immunocyto-chemical analysis. Results: The protein levels of cytochrome c oxidase and ATP synthase were higher in sevoflurane than in propofol group. Nevertheless, cytochrome c protein content was higher in propofol than sevoflurane receiving patients. Propofol group also showed higher protein level of connexin 43 (Cx43) than sevoflurane group. Besides, immunogold analysis showed its mitochondrial localization. The mRNA level of mtDNA and uncoupling protein (UCP2) were higher in propofol than sevoflurane patients, as well. On the other hand, there were no significant differences between groups in hemodynamic assessment, intensive care unit length of stay, troponin I and BNP level. Conclusions: Our data indicate that sevoflurane and propofol lead to cardiac protection via different mitochondrially related molecular mechanisms. It appears that sevoflurane acts regulating cytochrome c oxidase and ATP synthase, while the effects of propofol occur through regulation of cytochrome c, Cx43, mtDNA transcription and UCP2. Copyright (C) 2012 S. Karger AG, Basel Ministry of Education and Science [173055, 173054]

Study elucidates molecular mechanisms underlying activation of catalase and glutathione peroxidase (GSH-Px) during cold-exposure by examining time-dependent changes in their mRNA, protein expression and activity, in interscapular brown adipose tissue (IBAT) of rats kept at room temperature (22 +/- 1 degrees C), or subjected to cold (4 +/- 1 degrees C) for 1, 3, 7, 12, 21 and 45 days. To examine involvement of nitric oxide (*NO) in catalase and GSH-Px regulation, rats were treated with L-arginine or N((1))-nitro-L-arginine-methyl ester (L-NAME). In all groups, cold increased catalase mRNA, protein expression and activity from day 3 to day 45 of acclimation. However, cold-acclimation increased GSH-Px mRNA content on day 3, protein content from day 1 to day 45 and activity no sooner than on day 45. At room temperature, 21- and 45-day L-arginine and L-NAME supplementation increased catalase and GSH-Px mRNA expression, but decreased their activity. Concomitantly, catalase protein content decreased, while GSH-Px protein expression increased compared to control. Data show that cold is dominant stimulus that determines IBAT catalase and GSH-Px responses, by setting tissue metabolic state. In thermogenic-active tissue, catalase is regulated on transcriptional level, while GSH-Px is regulated post-translationally. On the other side, it was shown that *NO affects catalase and GSH-Px only in thermogenic-inactive tissue. (C) 2011 Elsevier Ltd. All rights reserved. Ministry of Science and Technological Development of the Republic of Serbia [173055]; COST, Action [FA0602]

White to brown adipose tissue conversion and thermogenesis can be ignited by different conditions or agents and its sustainability over the long term is still unclear. Browning of rat retroperitoneal white adipose tissue (rpWAT) during cold acclimation involves two temporally apparent components: (1) a predominant non-selective browning of most adipocytes and an initial sharp but transient induction of uncoupling protein 1, peroxisome proliferator-activated receptor (PPAR) coactivator-1α, PPARγ and PPARα expression, and (2) the subsistence of relatively few thermogenically competent adipocytes after 45 days of cold acclimation. The different behaviours of two rpWAT beige/brown adipocyte subsets control temporal aspects of the browning process, and thus regulation of both components may influence body weight and the potential successfulness of anti-obesity therapies.Conversion of white into brown adipose tissue may have important implications in obesity resistance and treatment. Several browning agents or conditions ignite thermogenesis in white adipose tissue (WAT). To reveal the capacity of WAT to function in a brownish/burning mode over the long term, we investigated the progression of the rat retroperitoneal WAT (rpWAT) browning during 45 days of cold acclimation. During the early stages of cold acclimation, the majority of rpWAT adipocytes underwent multilocularization and thermogenic-profile induction, as demonstrated by the presence of a multitude of uncoupling protein 1 (UCP1)-immunopositive paucilocular adipocytes containing peroxisome proliferator-activated receptor (PPAR) coactivator-1α (PGC-1α) and PR domain-containing 16 (PRDM16) in their nuclei. After 45 days, all adipocytes remained PRDM16 immunopositive, but only a few multilocular adipocytes rich in mitochondria remained UCP1/PGC-1α immunopositive. Molecular evidence showed that thermogenic recruitment of rpWAT occurred following cold exposure, but returned to starting levels after cold acclimation. Compared with controls (22 ± 1 °C), levels of UCP1 mRNA increased in parallel with PPARγ (PPARα from days 1 to 7 and PGC-1α on day 1). Transcriptional recruitment of rpWAT was followed by an increase in UCP1 protein content (from days 1 to 21). Results clearly showed that most of the adipocytes within rpWAT underwent transient brown-fat-like thermogenic recruitment upon stimulation, but only a minority of cells retained a brown adipose tissue-like phenotype after the attainment of cold acclimation. Therefore, browning of WAT is dependent on both maintaining the thermogenic response and retaining enough brown-like thermogenically competent adipocytes in the long-term. Both aspects of browning could be important for long-term energy homeostasis and body-weight regulation.

Background. Semen analysis is the cornerstone in the evaluation of male (in) fertility. However, there are men with normal semen tests but with impaired fertilizing ability, as well as fertile men with poor sperm characteristics. Thus, there is rising interest to find novel parameters that will help to predict and define the functional capacity of spermatozoa. Methods. We examined whether there is a correlation between semen parameters (count, progressive motility, and morphology) and protein expression/activity of antioxidative defense enzymes in seminal plasma from 10 normospermic subjects. Results. Sperm progressive motility was in positive correlation with seminal plasma protein expression of both superoxide dismutase (SOD) isoforms (MnSOD and CuZnSOD) and catalase. Also, positive correlation was observed between sperm count and MnSOD protein expression, as well as between sperm morphology and protein expression of catalase in seminal plasma. In contrast, protein expression of glutathione peroxidase was not in correlation with any sperm parameter, while its activity negatively correlated with sperm morphology and motility. Conclusions. These data suggest that evaluation of protein expression of antioxidative defense enzymes in seminal plasma might be of importance in the evaluation of male fertility status and that could be used as an additional biomarker along with classic semen analysis in assessment of semen quality. Ministry of Education, Science and Technological Development of the Republic of Serbia {[}173054]; COST {[}BM1203]

Natural hypothermia, in addition to allowing energy saving in hostile conditions, has been associated with delayed aging and increased longevity. However, the molecular basis responsible for observed correlations between the use of daily torpor/hibernation and indices of rate of aging is hitherto unclear. Considering central role of mitochondria dysfunction in the ageing process, we examined several mechanisms that might be involved in mitochondrial protection against oxidative damage during euthermia-hypothermia (and vice versa) transition, in brown adipose tissue (BAT) of the European Ground Squirrel ( Spermophilus citellus ). Results showed that in hibernation increased protein expression of Mn superoxide dismutase coincides with decreased content of ATP synthase and uncoupling protein 1. This suggests that BAT mitochondria during hibernation are protected from oxidative injuries by suppressed oxidative capacity, as well as by upregulated antioxidant defense. Also, the data indicate that such molecular pattern of changes is initiated already during prehibernating period. Namely, in this period we observed accumulations of hypoxia-inducible factor-1α (HIF-1α) and nuclear factor (erythroid 2-related)-like 2, which are probably responsible for suppressed oxidative metabolism, i.e. increased antioxidant capacity, respectively. Increased expression of the mitofusin 1 and detection of the megamitochondria in the prehibernating period indicate intensive mitofusion process in the BAT. This may be another mechanism of protection of mitochondrial content/function during euthermia-hypothermia transition. The results of the study suggest mechanisms that might be associated with increased resistance of “hibernating” mitochondria to the oxidative damage. Also, the data showed that biochemistry responsible for redox balance within the cell involves integration of antioxidant response and transcription control of overall metabolism. Finally, the results go in favor of the previous reports that suggested HIF-1 as a negative modulator of aging.

Maintaining heart viability in states of unfavorable metabolite/oxygen supply has become a prime challenge in cardiac medicine. Mitochondria have been recognised as key actors in cardioprotection. Our goal was to reveal the myocardial protective phenotype related to mitochondrial bioenergetic pathways in the state of limited metabolite supply and low temperature using the physiological model of mammalian hibernation. European ground squirrels (Spermophilus citellus) were exposed to cold (4 ± 1 °C) and divided into two groups: (1) animals that fell into torpor (hibernating group) and (2) animals that stayed active and euthermic for 1, 3, 7, 12, or 21 days (cold-exposed group). We found increased protein levels of electron transport chain components and ATP synthase in late cold acclimation and in hibernation. Also, protein level of PGC-1α was upregulated. Phospho-AMPKα protein content was increased during early cold acclimation. HIF1α protein level was unchanged. Protein levels of manganese and copper-zinc superoxide dismutase and glutathione peroxidase were increased in hibernation. The hibernation related phenotype of the heart is characterised by controlled improvement of mitochondrial energy and antioxidative capacity. Its reproduction could have broad implications, from myocardial protection in ischaemia/reperfusion to the hypothermic survival and cold preservation of organs.

Redox homeostasis disturbance, mainly caused by increased O 2 •– level and/or decreased NO bioavailability, represents an important contributing factor in the (ethio)pathology of diabetes. We examined whether targeting of O 2 •– /NO ratio by two redox-modulating compounds, L-arginine (substrate for NO synthases) and Mn(II) pentaazamacrocyclic mimics of SOD, could improve diabetic state and associated impairment of energy metabolism. Multiple beneficial effects of L-arginine and SOD mimics in alloxan-induced diabetes were observed. First of all, acting directly on pancreas, L-arginine and SOD mimic induce β-cells regeneration. In skeletal muscle, those treatments restore diabetes-induced impairment in mitochondrial energy metabolism (OXPHOS) and glucose transport (GLUT4) by targeting AMPKα signaling. Similarly, SOD mimic improves energy metabolism in hippocampus of diabetic rats. L-arginine and SOD mimic stabilize diabetes-induced redox disbalance in diabetic skin acting on NO producing (NOS) and O 2 •– /H 2 O 2 removing (MnSOD and GPx) systems. The data suggest that fine settings of O 2 •– /NO ratio by L-arginine and SOD mimic could have beneficial implications for several pathological hallmarks of diabetes: impaired insulin synthesis and sensitivity as well as accompanying metabolic complications and speak in favor of therapeutic potential of these redox-active agents in diabetic conditions.

Global rise in male infertility give importance to find a new approach to increase fertility. Redox signaling emerged recently as important for spermatozoa functioning. Accordingly, we just showed great therapeutic potential of redox modulator SOD mimic M40403. Molecular mechanisms underlying beneficial effects of M40403 indicate NO involvement, but remain unsolved; we aimed to reveal this herein. Compared to the control, incubation of spermatozoa in Tyrode's medium for 3 h, decreased sperm motility, mitochondrial membrane potential (MMP), level of NO, expression of NO synthases (NOSs), and completely abolished nuclear localization of NF-E2-related nuclear factor 2 (Nrf2) that was followed by decrease in SODs, catalase and GPX expression. Treatment with M40403 decreased levels of O2•–, increased level of NO, restored expression of NOSs and MMP, and triggered marked nuclear translocation of Nrf2 followed by up-regulation of SODs, catalase and GPX and increase in sperm motility. In turn, M40403 + L-NAME treatment nullified all beneficial effects of M40403. These data reveal the redox based mechanisms of beneficial effects of M40403 SOD mimic in sperm cells, suggesting NO/Nrf2 signaling as a central pathway. Utilization of a redox modulator, M40403, as an inducer of Nrf2 is a promising pharmacological approach for the improvement of sperm fertilizing potential and beat infertility.

Clarification of molecular mechanisms underlying lipid buffering capacity of white adipose tissue (WAT) may aid in the improvement of therapeutic options for obesity and diabetes prevention. Results of our studies have shown that cold-induced regression of WAT involves transient increase of tissue lipid oxidation, uncoupling capacity, an occurrence of browning allsynchronized with increase in adiponectin and resistin synthesis and profound glutathione (GSH) depletion. This GSH decrease prompted us to examine if a decrement of GSH a priori recapitulates cold-induced favorable phenotype of WAT. To this end, room-temperature and cold-exposed rats were treated with L-buthionine-S,R-sulfoximine (BSO). Indeed, BSO treatment reduced rat body weight and lipid content in fat tissue on account of increased OXPHOS and uncoupling capacity of WAT in both, cold-exposed and room-temperature maintained rats. However, in comparison to control, BSO decreased expressions of adiponectin and resistin at room temperature and attenuated their adaptive increase on cold exposure. Decrement of GSH raise lipid burning phenotype in WAT, but it may interfere with endocrine role of WAT in overall metabolic homeostasis maintenance.

Duchenne-Becker muscular dystrophy (DBMD) might be caused by a widespread genetic defect in surface membranes, which could be expressed in membranes not pathologically involved in DBMD. This hypothesis was supported by a substantial amount of evidence of abnormalities in erythrocytes from patients with DBMD. Catalases are well studied enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. In previous years a lot of papers on oxidative status in DBMD are mostly confirmed increased activity of enzymes involved in the elimination of reactive oxygen species in order to protect the cells from damage, including superoxide dismutase, catalase and glutathione peroxidase. Immunohistochemistry and immunogold labeling were used to study erythrocytes from patients with Duchenne-Becker muscular dystrophy and from age-matched normal boys. There were significant differences in the catalase localization of erythrocytes from Duchenne patients when compared to controls. Hence, the internal catalase localization in the erythrocyte is atypical in DBMD, supporting the concept that a membrane and cytoskeletal defect involving multiple tissues is present in this disorder.

Mitochondria are key organelles maintaining cellular bioenergetics and integrity, and their regulation of {[}Ca2+](i) homeostasis has been investigated in many cell types. We investigated the short-term Ca-SANDOZ (R) treatment on brown adipocyte mitochondria, using imaging and molecular biology techniques. Two-month-old male Wistar rats were divided into two groups: Ca-SANDOZ (R) drinking or tap water (control) drinking for three days. Alizarin Red S staining showed increased Ca2+ level in the brown adipocytes of treated rats, and potassium pyroantimonate staining localized electron-dense regions in the cytoplasm, mitochondria and around lipid droplets. Ca-SANDOZ (R) decreased mitochondrial number, but increased their size and mitochondrial cristae volume. Transmission electron microscopy revealed numerous enlarged and fusioned-like mitochondria in the Ca-SANDOZ (R) treated group compared to the control, and megamitochondria in some brown adipocytes. The Ca2+ diet affected mitochondrial fusion as mitofusin 1 (MFN1) and mitofusin 2 (MFN2) were increased, and mitochondrial fission as dynamin related protein 1 (DRP1) was decreased. Confocal microscopy showed a higher colocalization rate between functional mitochondria and endoplasmic reticulum (ER). The level of uncoupling protein-1 (UCP1) was elevated, which was confirmed by immunohistochemistry and Western blot analysis. These results suggest that Ca-SANDOZ (R) stimulates mitochondrial fusion, increases mitochondrial-ER contacts and the thermogenic capacity of brown adipocytes. Serbian Ministry of Education, Science and Technological Development {[}173055, 173054]