Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

<p>Abstract</p> <p>Background</p> <p>Accumulated evidence suggests that hydrogen peroxide (H₂O₂) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H₂O₂in neuronal insulin receptor activation and the origin of insulin-induced H₂O₂in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H₂O₂is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H₂O₂production, thus playing an integral role in insulin receptor autophosphorylation in neurons.</p> <p>Results</p> <p>Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H₂O₂release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H₂O₂scavenger, inhibited both insulin-stimulated H₂O₂release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H₂O₂production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H₂O₂release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN.</p> <p>Conclusion</p> <p>Results of the present study suggest that insulin-induced H₂O₂is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H₂O₂production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.</p

Comparative analysis of cytosolic and mitochondrial ATP synthesis in embryonic and postnatal hippocampal neuronal cultures

ATP in neurons is commonly believed to be synthesized mostly by mitochondria via oxidative phosphorylation. Neuronal mitochondria have been studied primarily in culture, i.e., in neurons isolated either from embryos or from neonatal pups. Although it is generally assumed that both embryonic and postnatal cultured neurons derive their ATP from mitochondrial oxidative phosphorylation, this has never been tested experimentally. We expressed the FRET-based ATP sensor AT1.03 in cultured hippocampal neurons isolated either from E17 to E18 rat embryos or from P1 to P2 rat pups and monitored [ATP]c simultaneously with mitochondrial membrane potential ΔΨm; TMRM) and NAD(P)H autofluorescence. In embryonic neurons, transient glucose deprivation induced a near-complete decrease in [ATP]c, which was partially reversible and was accelerated by inhibition of glycolysis with 2-deoxyglucose. In the absence of glucose, pyruvate did not cause any significant increase in [ATP]c in 84% of embryonic neurons, and inhibition of mitochondrial ATP synthase with oligomycin failed to decrease [ATP]c. Moreover, ΔΨm was significantly reduced by oligomycin, indicating that mitochondria acted as consumers rather than producers of ATP in embryonic neurons. In sharp contrast, in postnatal neurons pyruvate added during glucose deprivation significantly increased [ATP]c (by 54 ± 8%), whereas oligomycin induced a sharp decline in [ATP]c and increased ΔΨm. These signs of oxidative phosphorylation were observed in all tested P1-P2 neurons. Measurement of ΔΨm with the potential-sensitive probe JC-1 revealed that neuronal mitochondrial membrane potential was significantly reduced in embryonic cultures compared to the postnatal ones, possibly due to increased proton permeability of inner mitochondrial membrane. We conclude that, in embryonic, but not postnatal neuronal cultures, ATP synthesis is predominantly glycolytic and the oxidative phosphorylation-mediated synthesis of ATP by mitochondrial F1Fo-ATPase is insignificant. © 2013 Surin, Khiroug, Gorbacheva, Khodorov, Pinelis and Khiroug.Peer reviewe

Single fluorescent protein-based Ca2+ sensors with increased dynamic range

<p>Abstract</p> <p>Background</p> <p>Genetically encoded sensors developed on the basis of green fluorescent protein (GFP)-like proteins are becoming more and more popular instruments for monitoring cellular analytes and enzyme activities in living cells and transgenic organisms. In particular, a number of Ca²⁺sensors have been developed, either based on FRET (Fluorescence Resonance Energy Transfer) changes between two GFP-mutants or on the change in fluorescence intensity of a single circularly permuted fluorescent protein (cpFP).</p> <p>Results</p> <p>Here we report significant progress on the development of the latter type of Ca²⁺sensors. Derived from the knowledge of previously reported cpFP-based sensors, we generated a set of cpFP-based indicators with different spectral properties and fluorescent responses to changes in Ca²⁺concentration. Two variants, named Case12 and Case16, were characterized by particular high brightness and superior dynamic range, up to 12-fold and 16.5-fold increase in green fluorescence between Ca²⁺-free and Ca²⁺-saturated forms. We demonstrated the high potential of these sensors on various examples, including monitoring of Ca²⁺response to a prolonged glutamate treatment in cortical neurons.</p> <p>Conclusion</p> <p>We believe that expanded dynamic range, high brightness and relatively high pH-stability should make Case12 and Case16 popular research tools both in scientific studies and high throughput screening assays.</p

Pharmacogenetics and individualized approach to the therapy of bronchial asthma

Bronchial asthma is a chronic inflammatory disease of the airways. Ineffective treatment can significantly reduce the quality and duration of life of the patients. The article presents a review of current research devoted to the study of genetic determinism of the response to the treatment with inhaled corticosteroids, β2 -agonists of short-effect and antagonists of leukotriene receptors in patients with bronchial asthma. The contribution of genetic factors to the variability of therapeutic response in patients in each class of these antiasthmatic drugs is discussed in this article.Data describing Gly16 allele participation in phenotype formation with poor bronchial asthma course and decreased effectiveness of β2 -agonists therapy and inhaled glucocorticosteroids are also presented. The association of Gly16 genotype gene of β2 -adrenergic receptor with the decreased effect of broncholith therapy of β2 -adrenomimetric receptor of short effect has been determined in this study. It was shown that ALOX5 gene promotor polymorphism is linked with variations of response to antileukotriene drugs. Thus, it can be concluded that multiform gene variants can change the bronchial asthma patients’ response to the conducted therapy and the genetic information can be used to determine the probable prognoses of individuals’ reactions to definite anti-asthmatic remedies. The authors consider the pharmacogenetic test to help to identify the patients that are torpid to the treatment

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons-1

<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons"</p><p>http://www.biomedcentral.com/1471-2202/8/84</p><p>BMC Neuroscience 2007;8():84-84.</p><p>Published online 8 Oct 2007</p><p>PMCID:PMC2089077.</p><p></p>CCP (0.5 μmol/L). HOrelease from cultures for 1 min was measured as described in Materials and Methods. Results were normalized by cell density. Columns represent the means ± SD of HOvalues obtained from five to nine cultures. Dotted line represents a detection limit of the assay (7 nmol/L). B: CGN cultures were pre-incubated for 30 min in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L) for 20 min. Malonate (2 mmol/l) or FCCP (0.5 μmol/L) were added to cultures 5 min before the insulin exposure. Autophosphorylation of insulin receptor was measured as described in Materials and Methods. In each experiment, amount of phosphorylated insulin receptor β-subunit (pYpY-IR) was normalized to total amount of insulin receptor β-subunit and expressed as a percentage of the response produced to 100 nmol/L insulin. Columns represent the means ± SD of pYpY-IR values obtained from four to nine culture dishes. *P < 0.05 vs. control.P < 0.05 vs. insulin

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons-3

<p><b>Copyright information:</b></p><p>Taken from "Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons"</p><p>http://www.biomedcentral.com/1471-2202/8/84</p><p>BMC Neuroscience 2007;8():84-84.</p><p>Published online 8 Oct 2007</p><p>PMCID:PMC2089077.</p><p></p>/L). HOrelease from cultures for 1 min was measured as described in Materials and Methods. Results were normalized by cell density. Columns represent the means ± SD of HOvalues obtained from five to nine cultures. Dotted line represents a detection limit of the assay (7 nmol/L). B: CGN cultures were pre-incubated for 30 min in the absence or presence of N-acetylcysteine (5 mmol/l) in Hepes-buffered salt solution and then exposed to insulin (100 nmol/L) for 20 min. Autophosphorylation of insulin receptor was measured as described in Materials and Methods. In each experiment, amount of phosphorylated insulin receptor β-subunit (pYpY-IR) was normalized to total amount of insulin receptor β-subunit and expressed as a percentage of the response produced to 100 nmol/L insulin. Columns represent the means ± SD of pYpY-IR values obtained from four to nine culture dishes. *P < 0.05 vs. control.P < 0.05 vs. insulin