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

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

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons-2

<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> insulin (5 nmol/L) and CS (50 μmol/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 (100 nmol/L). P < 0.05 vs. insulin (5 nmol/L)

Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons-0

<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

Anti-Cancer Potential of Transiently Transfected HER2-Specific Human Mixed CAR-T and NK Cell Populations in Experimental Models: Initial Studies on Fucosylated Chondroitin Sulfate Usage for Safer Treatment

Human epidermal growth factor receptor 2 (HER2) is overexpressed in numerous cancer cell types. Therapeutic antibodies and chimeric antigen receptors (CARs) against HER2 were developed to treat human tumors. The major limitation of anti-HER2 CAR-T lymphocyte therapy is attributable to the low HER2 expression in a wide range of normal tissues. Thus, side effects are caused by CAR lymphocyte “on-target off-tumor” reactions. We aimed to develop safer HER2-targeting CAR-based therapy. CAR constructs against HER2 tumor-associated antigen (TAA) for transient expression were delivered into target T and natural killer (NK) cells by an effective and safe non-viral transfection method via nucleofection, excluding the risk of mutations associated with viral transduction. Different in vitro end-point and real-time assays of the CAR lymphocyte antitumor cytotoxicity and in vivo human HER2-positive tumor xenograft mice model proved potent cytotoxic activity of the generated CAR-T-NK cells. Our data suggest transient expression of anti-HER2 CARs in plasmid vectors by human lymphocytes as a safer treatment for HER2-positive human cancers. We also conducted preliminary investigations to elucidate if fucosylated chondroitin sulfate may be used as a possible agent to decrease excessive cytokine production without negative impact on the CAR lymphocyte antitumor effect