ABSTRACT
Infertility represents a growing burden worldwide, with one in seven couples presenting difficulties conceiving. Among these, 10-15% of the men have idiopathic infertility that does not correlate with any defect in the classical sperm parameters measured. In the present study, we used a mouse model to investigate the effects of maternal undernutrition on fertility in male progeny. Our results indicate that mothers fed on a low-protein diet during gestation and lactation produce male offspring with normal sperm morphology, concentration, and motility but exhibiting an overall decrease of fertility when they reach adulthood. Particularly, in contrast to control, sperm from these offspring show a remarkable lower capacity to fertilize oocytes when copulation occurs early in the estrus cycle relative to ovulation, due to an altered sperm capacitation. Our data demonstrate for the first time that maternal nutritional stress can have long-term consequences on the reproductive health of male progeny by affecting sperm physiology, especially capacitation, with no observable impact on spermatogenesis and classical quantitative and qualitative sperm parameters. Moreover, our experimental model could be of major interest to study, explain, and ultimately treat certain categories of infertilities.
Subject(s)
Infertility, Male , Malnutrition , Adult , Animals , Female , Fertility , Humans , Infertility, Male/etiology , Lactation , Male , Malnutrition/complications , Mice , Pregnancy , Sperm Capacitation , Sperm Motility , Spermatozoa/physiologyABSTRACT
Insulin resistance (IR), currently called prediabetes (PD), affects more than half of the adult population worldwide. Type 2 diabetes (T2D), which often follows in the absence of treatment, affects more than 475 million people and represents 10-20% of the health budget in industrialized countries. A preventive public health policy is urgently needed in order to stop this constantly progressing epidemic. Indeed, early management of prediabetes does not only strongly reduce its evolution toward T2D but also strongly reduces the appearance of cardiovascular comorbidity as well as that of associated cancers. There is however currently no simple and reliable test available for the diagnosis or screening of prediabetes and it is generally estimated that 20-60% of diabetics are not diagnosed. We therefore developed an ELISA for the quantitative determination of serum Insulin-Regulated AminoPeptidase (IRAP). IRAP is associated with and translocated in a stoechiometric fashion to the plasma membrane together with GLUT4 in response to insulin in skeletal muscle and adipose tissue which are the two major glucose storage sites. Its extracellular domain (IRAPs) is subsequently cleaved and secreted in the blood stream. In T2D, IRAP translocation in response to insulin is strongly decreased. Our patented sandwich ELISA is highly sensitive (≥10.000-fold "normal" fasting concentrations) and specific, robust and very cost-effective. Dispersion of fasting plasma concentration values in a healthy population is very low (101.4 ± 15.9 µg/ml) as compared to those of insulin (21-181 pmol/l) and C-peptide (0.4-1.7 nmol/l). Results of pilot studies indicate a clear correlation between IRAPs levels and insulin sensitivity. We therefore think that plasma IRAPs may be a direct marker of insulin sensitivity and that the quantitative determination of its plasma levels should allow large-scale screening of populations at risk for PD and T2D, thereby allow the enforcement of a preventive health policy aiming at efficiently reducing this epidemic.
ABSTRACT
The genetic causes of oocyte meiotic deficiency (OMD), a form of primary infertility characterised by the production of immature oocytes, remain largely unexplored. Using whole exome sequencing, we found that 26% of a cohort of 23 subjects with OMD harboured the same homozygous nonsense pathogenic mutation in PATL2, a gene encoding a putative RNA-binding protein. Using Patl2 knockout mice, we confirmed that PATL2 deficiency disturbs oocyte maturation, since oocytes and zygotes exhibit morphological and developmental defects, respectively. PATL2's amphibian orthologue is involved in the regulation of oocyte mRNA as a partner of CPEB However, Patl2's expression profile throughout oocyte development in mice, alongside colocalisation experiments with Cpeb1, Msy2 and Ddx6 (three oocyte RNA regulators) suggest an original role for Patl2 in mammals. Accordingly, transcriptomic analysis of oocytes from WT and Patl2-/- animals demonstrated that in the absence of Patl2, expression levels of a select number of highly relevant genes involved in oocyte maturation and early embryonic development are deregulated. In conclusion, PATL2 is a novel actor of mammalian oocyte maturation whose invalidation causes OMD in humans.
Subject(s)
Codon, Nonsense , Exome Sequencing/methods , Gene Expression Profiling/methods , Infertility/genetics , Nuclear Proteins/physiology , Oocytes/metabolism , RNA-Binding Proteins/physiology , Adult , Animals , Cohort Studies , Female , Humans , Meiosis/genetics , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Nuclear Proteins/genetics , Oocytes/cytology , RNA-Binding Proteins/genetics , Young AdultABSTRACT
Spermatogenesis defects concern millions of men worldwide, yet the vast majority remains undiagnosed. Here we report men with primary infertility due to multiple morphological abnormalities of the sperm flagella with severe disorganization of the sperm axoneme, a microtubule-based structure highly conserved throughout evolution. Whole-exome sequencing was performed on 78 patients allowing the identification of 22 men with bi-allelic mutations in DNAH1 (n = 6), CFAP43 (n = 10), and CFAP44 (n = 6). CRISPR/Cas9 created homozygous CFAP43/44 male mice that were infertile and presented severe flagellar defects confirming the human genetic results. Immunoelectron and stimulated-emission-depletion microscopy performed on CFAP43 and CFAP44 orthologs in Trypanosoma brucei evidenced that both proteins are located between the doublet microtubules 5 and 6 and the paraflagellar rod. Overall, we demonstrate that CFAP43 and CFAP44 have a similar structure with a unique axonemal localization and are necessary to produce functional flagella in species ranging from Trypanosoma to human.
Subject(s)
Flagella/physiology , Infertility, Male/genetics , Microtubule Proteins/genetics , Mutation , Nuclear Proteins/genetics , Peptide Hydrolases/genetics , Spermatozoa/physiology , Trypanosoma/physiology , Adult , Animals , Axoneme , Clustered Regularly Interspaced Short Palindromic Repeats , Cohort Studies , Cytoskeletal Proteins , Fertility , Flagella/metabolism , Homozygote , Humans , Male , Mice , Mice, Knockout , Microscopy, Immunoelectron , Middle Aged , Sperm Motility , Spermatozoa/metabolism , Exome SequencingABSTRACT
Hyperglycemia associated with inflammation and oxidative stress is a major cause of vascular dysfunction and cardiovascular disease in diabetes. Recent data reports that a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), empagliflozin (Jardiance®), ameliorates glucotoxicity via excretion of excess glucose in urine (glucosuria) and significantly improves cardiovascular mortality in type 2 diabetes mellitus (T2DM). The overarching hypothesis is that hyperglycemia and glucotoxicity are upstream of all other complications seen in diabetes. The aim of this study was to investigate effects of empagliflozin on glucotoxicity, ß-cell function, inflammation, oxidative stress and endothelial dysfunction in Zucker diabetic fatty (ZDF) rats. Male ZDF rats were used as a model of T2DM (35 diabetic ZDF-Leprfa/fa and 16 ZDF-Lepr+/+ controls). Empagliflozin (10 and 30mg/kg/d) was administered via drinking water for 6 weeks. Treatment with empagliflozin restored glycemic control. Empagliflozin improved endothelial function (thoracic aorta) and reduced oxidative stress in the aorta and in blood of diabetic rats. Inflammation and glucotoxicity (AGE/RAGE signaling) were epigenetically prevented by SGLT2i treatment (ChIP). Linear regression analysis revealed a significant inverse correlation of endothelial function with HbA1c, whereas leukocyte-dependent oxidative burst and C-reactive protein (CRP) were positively correlated with HbA1c. Viability of hyperglycemic endothelial cells was pleiotropically improved by SGLT2i. Empagliflozin reduces glucotoxicity and thereby prevents the development of endothelial dysfunction, reduces oxidative stress and exhibits anti-inflammatory effects in ZDF rats, despite persisting hyperlipidemia and hyperinsulinemia. Our preclinical observations provide insights into the mechanisms by which empagliflozin reduces cardiovascular mortality in humans (EMPA-REG trial).
Subject(s)
Benzhydryl Compounds/therapeutic use , Diabetic Cardiomyopathies/drug therapy , Glucosides/therapeutic use , Hypoglycemic Agents/therapeutic use , Sodium-Glucose Transporter 2 Inhibitors , Animals , Aorta, Thoracic/drug effects , Aorta, Thoracic/metabolism , Benzhydryl Compounds/pharmacology , C-Reactive Protein/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Glucose/metabolism , Glucosides/pharmacology , Glycated Hemoglobin/metabolism , Hypoglycemic Agents/pharmacology , Insulin-Secreting Cells/drug effects , Insulin-Secreting Cells/metabolism , Male , Oxidative Stress , Rats , Rats, Zucker , Sodium-Glucose Transporter 2/metabolismABSTRACT
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.
Subject(s)
International Cooperation , Reactive Oxygen Species/metabolism , Animals , European Union , Humans , Molecular Biology/organization & administration , Molecular Biology/trends , Oxidation-Reduction , Reactive Oxygen Species/chemistry , Signal Transduction , Societies, ScientificABSTRACT
Azoospermia, characterized by the absence of spermatozoa in the ejaculate, is a common cause of male infertility with a poorly characterized etiology. Exome sequencing analysis of two azoospermic brothers allowed the identification of a homozygous splice mutation in SPINK2, encoding a serine protease inhibitor believed to target acrosin, the main sperm acrosomal protease. In accord with these findings, we observed that homozygous Spink2 KO male mice had azoospermia. Moreover, despite normal fertility, heterozygous male mice had a high rate of morphologically abnormal spermatozoa and a reduced sperm motility. Further analysis demonstrated that in the absence of Spink2, protease-induced stress initiates Golgi fragmentation and prevents acrosome biogenesis leading to spermatid differentiation arrest. We also observed a deleterious effect of acrosin overexpression in HEK cells, effect that was alleviated by SPINK2 coexpression confirming its role as acrosin inhibitor. These results demonstrate that SPINK2 is necessary to neutralize proteases during their cellular transit toward the acrosome and that its deficiency induces a pathological continuum ranging from oligoasthenoteratozoospermia in heterozygotes to azoospermia in homozygotes.
Subject(s)
Asthenozoospermia/genetics , Asthenozoospermia/physiopathology , Azoospermia/genetics , Azoospermia/physiopathology , Glycoproteins/deficiency , Serine Peptidase Inhibitors, Kazal Type/deficiency , Animals , Disease Models, Animal , Heterozygote , Homozygote , Male , Mice , Mice, KnockoutABSTRACT
The concept of oxidative stress (OS) that connects altered redox biology with various diseases was introduced 30 years ago and has generated intensive research over the past two decades. Whereas it is now commonly accepted that macromolecule oxidation in response to ROS is associated with a variety of pathologies, the emergence of NO as a key regulator of redox signalling has led to the discovery of the pathophysiological significance of reactive nitrogen species (RNS). RNS can elicit various modifications of macromolecules and lead to nitrative or nitro-OS. In order to investigate oxidative and nitro-OS in human and in live animal models, circulating biomarker assays have been developed. This article provides an overview of key biomarkers used to assess lipid peroxidation and NO/NO2 signalling, thereby stressing the necessity to analyse several OS biomarkers in relation to the overall (aerobic) metabolism and health condition of patients. In addition, the potential interest of heart rate variability as the non-invasive integrative biomarker of OS is discussed. 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.
Subject(s)
Nitric Oxide/metabolism , Animals , Biomarkers/metabolism , Humans , Oxidation-Reduction , Oxidative Stress , Reactive Nitrogen Species/metabolismABSTRACT
Phospholipase A2 (PLA2) activity has been shown to be involved in the sperm acrosome reaction (AR), but the molecular identity of PLA2 isoforms has remained elusive. Here, we have tested the role of two intracellular (iPLA2ß and cytosolic PLA2α) and one secreted (group X) PLA2s in spontaneous and progesterone (P4)-induced AR by using a set of specific inhibitors and knock-out mice. iPLA2ß is critical for spontaneous AR, whereas both iPLA2ß and group X secreted PLA2 are involved in P4-induced AR. Cytosolic PLA2α is dispensable in both types of AR. P4-induced AR spreads over 30 min in the mouse, and kinetic analyses suggest the presence of different sperm subpopulations, using distinct PLA2 pathways to achieve AR. At low P4 concentration (2 µm), sperm undergoing early AR (0-5 min post-P4) rely on iPLA2ß, whereas sperm undergoing late AR (20-30 min post-P4) rely on group X secreted PLA2. Moreover, the role of PLA2s in AR depends on P4 concentration, with the PLA2s being key actors at low physiological P4 concentrations (≤2 µm) but not at higher P4 concentrations (~10 µm).
Subject(s)
Acrosome Reaction/drug effects , Acrosome/enzymology , Exocytosis/drug effects , Group VI Phospholipases A2/metabolism , Group X Phospholipases A2/metabolism , Progesterone/pharmacology , Animals , Group VI Phospholipases A2/genetics , Group X Phospholipases A2/genetics , Male , Mice , Mice, Knockout , Progesterone/metabolismABSTRACT
Although protein nitration had already been reported in the late forties and found to specifically affect tyrosine residues 20 years later, it was not until the early nineties that this post-translational modification was reported to occur in vivo in mammalian cells. Over the years, this protein modification has increasingly proven to play a major role in a variety of physiological mechanisms through redox signaling and pathological conditions through nitro-oxidative stress, from protozoan parasites to humans. In this issue (Proteomics 2015, 15, 580-590), Kang et al. report the identification of the nitroproteome during mating in the yeast Saccharomyces cerevisiae and most interestingly on the changes in nitration induced by the mating signal α-factor of several of these proteins. The correlation of these modifications with the biological functions of these proteins strongly suggests a role for protein nitration in mating signal transduction in yeast, thereby confirming the conservation of this pathway throughout the evolution from unicellular eukaryotes to man. The ubiquity of protein tyrosine nitration, whose importance is now also recognized in plants, further highlights its significance as an essential signaling mechanism in eukaryotes.
Subject(s)
Nitrates/analysis , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Tyrosine/analysisABSTRACT
OBJECTIVE: In diabetes, vascular dysfunction is characterized by impaired endothelial function due to increased oxidative stress. Empagliflozin, as a selective sodium-glucose co-transporter 2 inhibitor (SGLT2i), offers a novel approach for the treatment of type 2 diabetes by enhancing urinary glucose excretion. The aim of the present study was to test whether treatment with empagliflozin improves endothelial dysfunction in type I diabetic rats via reduction of glucotoxicity and associated vascular oxidative stress. METHODS: Type I diabetes in Wistar rats was induced by an intravenous injection of streptozotocin (60 mg/kg). One week after injection empagliflozin (10 and 30 mg/kg/d) was administered via drinking water for 7 weeks. Vascular function was assessed by isometric tension recording, oxidative stress parameters by chemiluminescence and fluorescence techniques, protein expression by Western blot, mRNA expression by RT-PCR, and islet function by insulin ELISA in serum and immunohistochemical staining of pancreatic tissue. Advanced glycation end products (AGE) signaling was assessed by dot blot analysis and mRNA expression of the AGE-receptor (RAGE). RESULTS: Treatment with empagliflozin reduced blood glucose levels, normalized endothelial function (aortic rings) and reduced oxidative stress in aortic vessels (dihydroethidium staining) and in blood (phorbol ester/zymosan A-stimulated chemiluminescence) of diabetic rats. Additionally, the pro-inflammatory phenotype and glucotoxicity (AGE/RAGE signaling) in diabetic animals was reversed by SGLT2i therapy. CONCLUSIONS: Empagliflozin improves hyperglycemia and prevents the development of endothelial dysfunction, reduces oxidative stress and improves the metabolic situation in type 1 diabetic rats. These preclinical observations illustrate the therapeutic potential of this new class of antidiabetic drugs.
Subject(s)
Benzhydryl Compounds/pharmacology , Diabetes Complications/metabolism , Diabetes Mellitus, Experimental , Diabetic Angiopathies/metabolism , Glucosides/pharmacology , Oxidative Stress/drug effects , Sodium-Glucose Transporter 2 Inhibitors , Animals , Benzhydryl Compounds/administration & dosage , Blood Glucose/drug effects , Cytokines/genetics , Cytokines/metabolism , Diabetes Complications/drug therapy , Diabetic Angiopathies/drug therapy , Gene Expression , Glucose/metabolism , Glucosides/administration & dosage , Hemodynamics/drug effects , Inflammation Mediators/metabolism , Insulin/blood , Insulin/metabolism , Male , RNA, Messenger/genetics , Rats , Receptor for Advanced Glycation End Products , Receptors, Immunologic/metabolism , Signal Transduction , Streptozocin/adverse effectsABSTRACT
Mitochondria consume oxygen in the respiratory chain and convert redox energy into ATP. As a side process, they produce reactive oxygen species (ROS), whose physiological activities are still not understood. However, current analytical methods cannot be used to monitor mitochondrial ROS quantitatively and unambiguously. We have developed electrochemical biosensors based on peroxidase-redox polymer-modified electrodes, providing selective detection of H2O2 with nanomolar sensitivity, linear response over five concentration decades, and fast response time. The release of H2O2 by mitochondria was then monitored under phosphorylating or inhibited respiration conditions. We report the detection of two concomitant regimes of H2O2 release: large fluxes (hundreds of nM) under complexâ III inhibition, and bursts of a few nM immediately following mitochondria activation. These unprecedented bursts of H2O2 are assigned to the role of mitochondria as the hub of redox signaling in cells.
Subject(s)
Electrochemical Techniques , Hydrogen Peroxide/analysis , Mitochondria/metabolism , Biosensing Techniques , Carbon/chemistry , Electrodes , Horseradish Peroxidase/metabolism , Hydrogen Peroxide/metabolism , Oxidation-Reduction , Polymers/chemistry , Saccharomyces cerevisiae/metabolismABSTRACT
The reaction product of nitric oxide and superoxide, peroxynitrite, is a potent biological oxidant. The most important oxidative protein modifications described for peroxynitrite are cysteine-thiol oxidation and tyrosine nitration. We have previously demonstrated that intrinsic heme-thiolate (P450)-dependent enzymatic catalysis increases the nitration of tyrosine 430 in prostacyclin synthase and results in loss of activity which contributes to endothelial dysfunction. We here report the sensitive peroxynitrite-dependent nitration of an over-expressed and partially purified human prostacyclin synthase (3.3 µM) with an EC50 value of 5 µM. Microsomal thiols in these preparations effectively compete for peroxynitrite and block the nitration of other proteins up to 50 µM peroxynitrite. Purified, recombinant PGIS showed a half-maximal nitration by 10 µM 3-morpholino sydnonimine (Sin-1) which increased in the presence of bicarbonate, and was only marginally induced by freely diffusing NO2-radicals generated by a peroxidase/nitrite/hydrogen peroxide system. Based on these observations, we would like to emphasize that prostacyclin synthase is among the most efficiently and sensitively nitrated proteins investigated by us so far. In the second part of the study, we identified two classes of peroxynitrite scavengers, blocking either peroxynitrite anion-mediated thiol oxidations or phenol/tyrosine nitrations by free radical mechanisms. Dithiopurines and dithiopyrimidines were highly effective in inhibiting both reaction types which could make this class of compounds interesting therapeutic tools. In the present work, we highlighted the impact of experimental conditions on the outcome of peroxynitrite-mediated nitrations. The limitations identified in this work need to be considered in the assessment of experimental data involving peroxynitrite.
Subject(s)
Cytochrome P-450 Enzyme System/chemistry , Intramolecular Oxidoreductases/chemistry , Peroxynitrous Acid/chemistry , Protein Processing, Post-Translational , Sulfhydryl Compounds/chemistry , Tyrosine/analogs & derivatives , Animals , Cattle , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Humans , Intramolecular Oxidoreductases/genetics , Intramolecular Oxidoreductases/metabolism , Oxidation-Reduction , Peroxynitrous Acid/genetics , Peroxynitrous Acid/metabolism , Sf9 Cells , Spodoptera , Sulfhydryl Compounds/metabolism , Tyrosine/chemistry , Tyrosine/genetics , Tyrosine/metabolismABSTRACT
It is now demonstrated that mitochondria individually function differently because of specific energetic needs in cell compartments but also because of the genetic heterogeneity within the mitochondrial pool-network of a cell. Consequently, understanding mitochondrial functioning at the single organelle level is of high interest for biomedical research, therefore being a target for analyticians. In this context, we developed easy-to-build platforms of milli- to microwells for fluorescence microscopy of single isolated mitochondria. Poly(dimethylsiloxane) (PDMS) was determined to be an excellent material for mitochondrial deposition and observation of their NADH content. Because of NADH autofluorescence, the metabolic status of each mitochondrion was analyzed following addition of a respiratory substrate (stage 2), ethanol herein, and a respiratory inhibitor (stage 3), Antimycin A. Mean levels of mitochondrial NADH were increased by 32% and 62% under stages 2 and 3, respectively. Statistical studies of NADH value distributions evidenced different types of responses, at least three, to ethanol and Antimycin A within the mitochondrial population. In addition, we showed that mitochondrial ability to generate high levels of NADH, that is its metabolic performance, is not correlated either to the initial energetic state or to the respective size of each mitochondrion.
Subject(s)
Dimethylpolysiloxanes/chemistry , Microarray Analysis/methods , Mitochondria/metabolism , NAD/metabolism , Saccharomyces cerevisiae/cytology , Spectrometry, FluorescenceABSTRACT
BACKGROUND: The clinical significance of early transient hypoglycemia (ETH), a frequent event in preterm newborns, is a highly controversial issue. In experimental models, hypoglycemia has been reported to cause oxidative stress. Among the reactive species, early generated peroxynitrite is responsible for protein nitration and lipid peroxidation, a process referred to as nitrative stress. OBJECTIVES: The aim of the present study is to investigate whether ETH is associated with protein nitration in the preterm newborn. METHODS: Using a novel highly sensitive ELISA, we quantified plasma nitroalbumin (PNA) as a marker of peroxynitrite generation in 72 preterm newborns (28-36 weeks), among which 25 had a glycemia level of <2.5 mmol/l during the first hour of life (H1). RESULTS: PNA was significantly higher in ETH than in normoglycemic infants at H1 [median = 6.3 (3.8-8.8) vs. 3.4 ng/ml (2.1-5.1), p = 0.027] and at day 1 [median = 6.6 (5.6-15.3) vs. 3.9 ng/ml (2.3-4.6), p = 0.014]. PNA was inversely correlated with glycemia at H1 (r = -0.30, p = 0.01) and at day 1 (r = -0.63, p = 0.001). In ETH infants, lactatemia was inversely correlated with PNA. At day 1, PNA was higher in ETH infants treated by gavage than in those treated with intravenous dextrose [median = 8.9 ng/ml (7.1-10.4) vs. 4.4 ng/ml (2.6-5.7), p = 0.008]. CONCLUSIONS: These results indicate that ETH is associated with increased peroxynitrite generation resulting in systemic protein nitration in premature newborns. Treatment of ETH with intravenous dextrose is associated with lower PNA levels than gavage.
Subject(s)
Hypoglycemia/blood , Infant, Premature, Diseases/blood , Infant, Premature , Serum Albumin/analysis , Female , Fetal Blood/chemistry , Glucose/administration & dosage , Humans , Hypoglycemia/drug therapy , Infant, Newborn , Infant, Premature, Diseases/drug therapy , Male , Peroxynitrous Acid/metabolismABSTRACT
Angiotensin II (Ang II) plays a major role in the pathogenesis of insulin resistance and diabetes by inhibiting insulin's metabolic and potentiating its trophic effects. Whereas the precise mechanisms involved remain ill-defined, they appear to be associated with and dependent upon increased oxidative stress. We found Ang II to block insulin-dependent GLUT4 translocation in L6 myotubes in an NO- and O(2)(*-)-dependent fashion suggesting the involvement of peroxynitrite. This hypothesis was confirmed by the ability of Ang II to induce tyrosine nitration of the MAP kinases ERK1/2 and of protein kinase B/Akt (Akt). Tyrosine nitration of ERK1/2 was required for their phosphorylation on Thr and Tyr and their subsequent activation, whereas it completely inhibited Akt phosphorylation on Ser(473) and Thr(308) as well as its activity. The inhibitory effect of nitration on Akt activity was confirmed by the ability of SIN-1 to completely block GSK3alpha phosphorylation in vitro. Inhibition of nitric oxide synthase and NAD(P)Hoxidase and scavenging of free radicals with myricetin restored insulin-stimulated Akt phosphorylation and GLUT4 translocation in the presence of Ang II. Similar restoration was obtained by inhibiting the ERK activating kinase MEK, indicating that these kinases regulate Akt activation. We found a conserved nitration site of ERK1/2 to be located in their kinase domain on Tyr(156/139), close to their active site Asp(166/149), in agreement with a permissive function of nitration for their activation. Taken together, our data show that Ang II inhibits insulin-mediated GLUT4 translocation in this skeletal muscle model through at least two pathways: first through the transient activation of ERK1/2 which inhibit IRS-1/2 and second through a direct inhibitory nitration of Akt. These observations indicate that not only oxidative but also nitrative stress play a key role in the pathogenesis of insulin resistance. They underline the role of protein nitration as a major mechanism in the regulation of Ang II and insulin signaling pathways and more particularly as a key regulator of protein kinase activity.
Subject(s)
Angiotensin II/pharmacology , Glucose Transporter Type 4/metabolism , Insulin/pharmacology , Mitogen-Activated Protein Kinase 3/metabolism , Nitrates/metabolism , Proto-Oncogene Proteins c-akt/metabolism , Tyrosine/metabolism , Animals , Cells, Cultured , Glucose Transporter Type 4/antagonists & inhibitors , Humans , Insulin Receptor Substrate Proteins/metabolism , Muscle Fibers, Skeletal/metabolism , Muscle, Skeletal/cytology , Protein Transport , RatsABSTRACT
Reactive nitrogen species (RNS) have been shown to play a major role in the pathophysiology of hypoxic-ischemic cerebral injury. Using a novel sensitive ELISA allowing the quantification of nitrated albumin (nitroalbumin) in plasma, we tested the hypothesis that perinatal asphyxia increases nitrating RNS generation by verifying whether the concentration of one of its target proteins is correlated with the clinical outcome. We assayed nitroalbumin in 114 plasma samples collected during the first hour, at day 1, and at day 4 of life from 48 term newborns suffering from perinatal asphyxia and correlated this marker with neurological and systemic neonatal outcomes. Nitroalbumin levels at day 1, but not at days 0 and 4, were significantly increased in patients who developed moderate or severe encephalopathy compared to those who had a normal neurological evolution or developed mild encephalopathy (median: 14.4 ng/ml versus 7.3 ng/ml, respectively). In contrast, nitroalbumin concentration at day 1 was not associated with systemic complications. First-hour and fourth-day nitroalbumin concentrations did not differ with respect to the neonatal neurological course. At day 0, nitroalbumin levels also correlated with circulating leukocytes. We conclude that plasma nitroalbumin seems to be a specific marker of neurological injury after perinatal asphyxia and may serve as a secondary end-point in neuroprotective clinical trials.
Subject(s)
Albumins/chemistry , Albumins/metabolism , Asphyxia Neonatorum/metabolism , Hypoxia-Ischemia, Brain/metabolism , Oxidative Stress , Reactive Nitrogen Species/metabolism , Albumins/analysis , Biomarkers/blood , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Humans , Infant, NewbornABSTRACT
Angiotensin II (Ang II) induces a prominent and sustained nitration and activation of ERK1/2 in rat vascular smooth muscle cells, both mediated via AT1 receptor. Nitration and activation was also shown for recombinant non-activated extracellular signal-regulated kinase (ERK) and MEK. Nitration and phosphorylation of ERK1/2 by Ang II was significantly inhibited by NAD(P)H inhibitors and scavengers of oxygen and nitrogen reactive species and completely blocked by a selective inducible nitric-oxide synthase inhibitor. MEK inhibitor U0126 did not affect ERK nitration but completely blocked activation. These data indicate that Ang II nitrates and activates ERK1/2 via a reactive species-sensitive pathway.
Subject(s)
Angiotensin II/metabolism , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/enzymology , Nitrates/metabolism , Tyrosine/metabolism , Animals , Cells, Cultured , Enzyme Activation , Enzyme Inhibitors/metabolism , Free Radical Scavengers/metabolism , MAP Kinase Kinase 1/antagonists & inhibitors , MAP Kinase Kinase 1/metabolism , Mitogen-Activated Protein Kinase 1/metabolism , Mitogen-Activated Protein Kinase 3/metabolism , Molsidomine/analogs & derivatives , Molsidomine/metabolism , Myocytes, Smooth Muscle/cytology , NADPH Oxidases/antagonists & inhibitors , NADPH Oxidases/metabolism , Nitric Oxide Synthase/antagonists & inhibitors , Nitric Oxide Synthase/metabolism , Rats , Receptor, Angiotensin, Type 1/metabolismABSTRACT
The putative role of aluminium intake in young Bangladeshi children (1.5 to 4 years of age) with calcium-deficient rickets was evaluated in a non randomised controlled eight month trial. The effects of aluminium or stainless-steel cooking pots on bone metabolism were assessed by measuring blood calcium, phosphorus, alkaline phosphatase, parathyroid hormone, 1,25 dihydroxy vitamin D, aminoterminal propeptide of type 1 collagen (PINP), cross-linked carboxyterminal telopeptide of type 1 collagen (ICTP), aluminium and albumin, and by analysis of wrist radiographs. In both groups, blood alkaline phosphatase, 1,25 dihydroxy vitamin D and aluminium decreased significantly, white serum albumin increased (p < 0.01). These results suggest that the nutrition may well be of major importance, whereas the role of aluminium appears to be insignificant.
Subject(s)
Aluminum/metabolism , Calcium/metabolism , Cooking and Eating Utensils , Food , Rickets/therapy , Stainless Steel/chemistry , Bangladesh , Bone and Bones/metabolism , Child, Preschool , Cooking , Diet , Female , Humans , Infant , Male , Nutritional Physiological Phenomena , Rickets/metabolismABSTRACT
1. Heat stress (HS) is known to induce delayed preconditioning against myocardial infarction 24 h later, but the exact signalling pathway of this response remains to be elucidated. In previous studies, we have shown evidence for the implication of protein kinase C (PKC) and p38 mitogen-activated protein kinase (MAPK) in the HS-induced reduction in infarct size. Furthermore, in their phosphorylated state, small heat shock proteins (Hsp27) seem to confer cytoskeletal protection. In the present study, we sought to determine the effect of HS on the subcellular distribution of PKC isoforms and on Hsp27 phosphorylation. 2. Rats were subjected to either HS (42 degrees C for 15 min; HS group) or sham anaesthesia (sham group) before their hearts were excised. Myocardial tissue extracts obtained 20 min or 24 h after HS were processed for western blot analysis. 3. In the HS group, PKCepsilon translocated from the cytosolic to the particulate fraction (4426 +/- 128 vs 6258 +/- 316 arbitrary units; P = 0.002). Chelerythrine (5 mg/kg, i.p.), a PKC inhibitor, abolished this translocation. Western blot analysis of Hsp27 24 h after HS showed a marked increase in protein expression and phosphorylation in the particulate fraction. 4. In the present study, we have shown that HS induces the translocation of PKCepsilon from the cytosolic to the particulate fraction. Along with our previous observation that PKC is a trigger of HS-induced myocardial preconditioning, the results of the present study suggest an important role of the epsilon isoform of PKC in this cardioprotective mechanism. Furthermore, we have also demonstrated that the cytoprotective protein Hsp27 is phosphorylated following HS. Therefore, we can conclude that PKC and MAPK/Hsp27 are involved in the signalling pathway of HS-induced cardioprotection.