Mitochondrial H2O2 as an enable signal for triggering autophosphorylation of insulin receptor in neurons.

BACKGROUND: Insulin receptors are widely distributed in the brain, where they play roles in synaptic function, memory formation, and neuroprotection. Autophosphorylation of the receptor in response to insulin stimulation is a critical step in receptor activation. In neurons, insulin stimulation leads to a rise in mitochondrial H2O2 production, which plays a role in receptor autophosphorylation. However, the kinetic characteristics of the H2O2 signal and its functional relationships with the insulin receptor during the autophosphorylation process in neurons remain unexplored to date. RESULTS: Experiments were carried out in culture of rat cerebellar granule neurons. Kinetic study showed that the insulin-induced H2O2 signal precedes receptor autophosphorylation and represents a single spike with a peak at 5-10 s and duration of less than 30 s. Mitochondrial complexes II and, to a lesser extent, I are involved in generation of the H2O2 signal. The mechanism by which insulin triggers the H2O2 signal involves modulation of succinate dehydrogenase activity. Insulin dose-response for receptor autophosphorylation is well described by hyperbolic function (Hill coefficient, nH, of 1.1±0.1; R2=0.99). N-acetylcysteine (NAC), a scavenger of H2O2, dose-dependently inhibited receptor autophosphorylation. The observed dose response is highly sigmoidal (Hill coefficient, nH, of 8.0±2.3; R2=0.97), signifying that insulin receptor autophosphorylation is highly ultrasensitive to the H2O2 signal. These results suggest that autophosphorylation occurred as a gradual response to increasing insulin concentrations, only if the H2O2 signal exceeded a certain threshold. Both insulin-stimulated receptor autophosphorylation and H2O2 generation were inhibited by pertussis toxin, suggesting that a pertussis toxin-sensitive G protein may link the insulin receptor to the H2O2-generating system in neurons during the autophosphorylation process. CONCLUSIONS: In this study, we demonstrated for the first time that the receptor autophosphorylation occurs only if mitochondrial H2O2 signal exceeds a certain threshold. This finding provides novel insights into the mechanisms underlying neuronal response to insulin. The neuronal insulin receptor is activated if two conditions are met: 1) insulin binds to the receptor, and 2) the H2O2 signal surpasses a certain threshold, thus, enabling receptor autophosphorylation in all-or-nothing manner. Although the physiological rationale for this control remains to be determined, we propose that malfunction of mitochondrial H2O2 signaling may lead to the development of cerebral insulin resistance.

Phosphono analogues of 2-oxoglutarate protect cerebellar granule neurons upon glutamate excitotoxicity.

Glutamate excitotoxicity is an important contributor to neuronal loss. Glutamate-induced Ca(2+) deregulation and accompanying mitochondrial depolarization are closely associated with the onset of apoptotic and necrotic neuronal death. We investigated the role in these phenomena of 2-oxoglutarate dehydrogenase (OGDH), the enzyme participating in mitochondrial degradation of glutamate. To achieve this goal, we used specific effectors of cellular OGDH, succinyl phosphonate and its phosphonoethyl ether. Preincubation of cerebellar granule neurons with these phosphono analogues of 2-oxoglutarate was shown to protect the cells from glutamate-induced Ca(2+) deregulation and irreversible mitochondrial depolarization, followed simultaneously by fluorescence of fura-2FF and rhodamine 123, respectively. The protection was characterized by delay in onset and decreased propagation of Ca(2+) deregulation and by reversibility of the associated mitochondrial depolarization. Compared to its phosphonoethyl ether, succinyl phosphonate exhibited both higher affinity to OGDH in vitro and better protection from Ca(2+) deregulation in situ, supporting the assumption that neuroprotection by phosphonates involves their interaction with cellular OGDH. Preincubation of cerebellar granule neurons with succinyl phosphonate decreased neuronal death after excitotoxic action of glutamate. Thus, specific inhibitors of OGDH alleviate glutamate-induced calcium deregulation, mitochondrial depolarization, and neuronal death.

Synthetic regulators of the 2-oxoglutarate oxidative decarboxylation alleviate the glutamate excitotoxicity in cerebellar granule neurons.

Impairment of the 2-oxoglutarate oxidative decarboxylation by the 2-oxoglutarate dehydrogenase complex (OGDHC) is associated with the glutamate accumulation, ROS production and neuropathologies. We hypothesized that correct function of OGDHC under metabolic stress is essential to overcome the glutamate excitotoxic action on neurons. We show that synthetic phosphono analogs of 2-oxoglutarate, succinyl phosphonate and its phosphono ethyl ester, improve the catalysis by brain OGDHC through inhibiting the side reaction of irreversible inactivation of its first component, 2-oxoglutarate dehydrogenase. Under the substrate and cofactor saturation, the component and complex undergo the inactivation during catalysis with the apparent rate constant 0.2 min(-1). The inactivation rate is reduced by 90% and 60% in the presence of 50 microM succinyl phosphonate and its phosphono ethyl ester, correspondingly. In cultured cerebellar granule neurons exposed to excitotoxic glutamate, the phosphonates (100 microM) protect from the irreversible impairment of mitochondrial function and delayed calcium deregulation. The deregulation amplitude is decreased by succinyl phosphonate and its phosphono ethyl ester by 50% and 30%, correspondingly. Thus, succinyl phosphonate is more potent than its phosphono ethyl ester in protecting both the isolated brain OGDHC from inactivation and cultured neurons from the glutamate-induced calcium deregulation. The correlation of the relative efficiency of the phosphonates in vitro and in situ indicates that their cellular effects are due to targeting OGDHC, which is in accord with independent studies. We conclude that the compounds preserving the 2-oxoglutarate dehydrogenase activity are of neuroprotective value upon metabolic disbalance induced by glutamate excess.

Dicholine salt of succinic acid, a neuronal insulin sensitizer, ameliorates cognitive deficits in rodent models of normal aging, chronic cerebral hypoperfusion, and beta-amyloid peptide-(25-35)-induced amnesia.

BACKGROUND: Accumulated evidence suggests that insulin resistance and impairments in cerebral insulin receptor signaling may contribute to age-related cognitive deficits and Alzheimer's disease. The enhancement of insulin receptor signaling is, therefore, a promising strategy for the treatment of age-related cognitive disorders. The mitochondrial respiratory chain, being involved in insulin-stimulated H2O2 production, has been identified recently as a potential target for the enhancement of insulin signaling. The aim of the present study is to examine: (1) whether a specific respiratory substrate, dicholine salt of succinic acid (CS), can enhance insulin-stimulated insulin receptor autophosphorylation in neurons, and (2) whether CS can ameliorate cognitive deficits of various origins in animal models. RESULTS: In a primary culture of cerebellar granule neurons, CS significantly enhanced insulin-stimulated insulin receptor autophosphorylation. In animal models, CS significantly ameliorated cognitive deficits, when administered intraperitoneally for 7 days. In 16-month-old middle-aged C57Bl/6 mice (a model of normal aging), CS enhanced spatial learning in the Morris water maze, spontaneous locomotor activity, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to the age-matched control (saline). In rats with chronic cerebral hypoperfusion, CS enhanced spatial learning, passive avoidance performance, and increased brain N-acetylaspartate/creatine levels, as compared to control rats (saline). In rats with beta-amyloid peptide-(25-35)-induced amnesia, CS enhanced passive avoidance performance and increased activity of brain choline acetyltransferase, as compared to control rats (saline). In all used models, CS effects lasted beyond the seven-day treatment period and were found to be significant about two weeks following the treatment. CONCLUSION: The results of the present study suggest that dicholine salt of succinic acid, a novel neuronal insulin sensitizer, ameliorates cognitive deficits and neuronal dysfunctions in animal models relevant to age-related cognitive impairments, vascular dementia, and Alzheimer's disease.

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

BACKGROUND: 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 H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. RESULTS: Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release 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. CONCLUSION: Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons.

Ischemic and hemorrhagic disturbances in cerebral circulation alter contractile responses of the rat middle cerebral artery.

In our study, we examined middle cerebral artery (MCA) contractile responses in two animal models. After hemorrhagic disturbances in rats of Krushinsky-Molodkina strain (KMRs) a decrease in contractile responses to serotonin (5-HT) was observed. During incomplete global cerebral ischemia, MCAs had increased responsiveness to endothelin-1 (ET-1), but reduced responsiveness to serotonin. These findings suggest that cerebral circulation disorders alter cerebrovascular function possibly leading to secondary disturbances in brain circulation.