Abstract-Inhibition of glycogen synthase kinase (GSK)-3 reduces ischemia/reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to explore possible molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK inhibitors before ischemia significantly improved recovery of function. To assess the effect of GSK inhibitors on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSK inhibitors and were treated with uncoupler or cyanide or were made anoxic. GSK inhibition slowed ATP consumption under these conditions, which could be attributable to inhibition of ATP entry into the mitochondria through the voltage-dependent anion channel (VDAC) and/or adenine nucleotide transporter (ANT) or to inhibition of the F 1 F 0 -ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer but not the inner mitochondrial membrane, and we found that GSK inhibitors slow AMP production similar to their effect on ATP consumption. This suggests that GSK inhibitors are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSK inhibition had no effect on ATP consumption or AMP production. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not caused by opening of the mitochondrial permeability transition pore. Because GSK is a kinase, we assessed whether protein phosphorylation might be involved. Therefore, we performed Western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSK inhibitors. Liquid chromatographic-mass spectrometric analysis revealed 1 of these proteins to be VDAC2. Taken together, we found that GSK-mediated signaling modulates transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest that GSK regulates VDAC and that VDAC may be an important regulatory site in ischemia/reperfusion injury. (Circ Res. 2008;103:983-991.)

We investigated the effect of diazoxide on neuronal survival in primary cultures of rat cortical neurons against oxygenglucose deprivation (OGD). Diazoxide pre-treatment induced delayed pre-conditioning and almost entirely attenuated the OGD-induced neuronal death. Diazoxide inhibited succinate dehydrogenase and induced mitochondrial depolarization, free radical production and protein kinase C activation. The putative mitochondrial ATP-sensitive potassium channel blocker 5-hydroxydecanoate abolished the protective effect of diazoxide while the non-selective K ATP channel blocker glibenclamide did not. The non-selective K ATP channel openers nicorandil and cromakalim did not improve viability. Superoxide dismutase mimetic, M40401, or protein kinase C inhibitor, chelerythrine, prevented the neuroprotective effect of diazoxide. Diazoxide did not increase reduced glutathione and manganese-superoxide dismutase levels but we found significantly higher reduced glutathione levels in diazoxidepre-conditioned neurons after OGD. In pre-conditioned neurons free radical production was reduced upon glutamate stimulation. The succinate dehydrogenase inhibitor 3-nitropropionic acid also induced pre-conditioning and free radical production in neurons. Here, we provide the first evidence that diazoxide induces delayed pre-conditioning in neurons via acute generation of superoxide anion and activation of protein kinases and subsequent attenuation of oxidant stress following OGD. The succinate dehydrogenase-inhibiting effect of diazoxide is more likely to be involved in this neuroprotection than the opening of mitochondrial ATP-sensitive potassium channels.

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AimTo examine individual variability between perceived physical features and hormones of pubertal maturation in 9–10-year-old children as a function of sociodemographic characteristics.MethodsCross-sectional metrics of puberty were utilized from the baseline assessment of the Adolescent Brain Cognitive Development (ABCD) Study—a multi-site sample of 9–10 year-olds (n = 11,875)—and included perceived physical features via the pubertal development scale (PDS) and child salivary hormone levels (dehydroepiandrosterone and testosterone in all, and estradiol in females). Multi-level models examined the relationships among sociodemographic measures, physical features, and hormone levels. A group factor analysis (GFA) was implemented to extract latent variables of pubertal maturation that integrated both measures of perceived physical features and hormone levels.ResultsPDS summary scores indicated more males (70%) than females (31%) were prepubertal. Perceived physical features and hormone levels were significantly associated with child’s weight status and income, such that more mature scores were observed among children that were overweight/obese or from households with low-income. Results from the GFA identified two latent factors that described individual differences in pubertal maturation among both females and males, with factor 1 driven by higher hormone levels, and factor 2 driven by perceived physical maturation. The correspondence between latent factor 1 scores (hormones) and latent factor 2 scores (perceived physical maturation) revealed synchronous and asynchronous relationships between hormones and concomitant physical features in this large young adolescent sample.ConclusionsSociodemographic measures were associated with both objective hormone and self-report physical measures of pubertal maturation in a large, diverse sample of 9–10 year-olds. The latent variables of pubertal maturation described a complex interplay between perceived physical changes and hormone levels that hallmark sexual maturation, which future studies can examine in relation to trajectories of brain maturation, risk/resilience to substance use, and other mental health outcomes.

. MitoKATP opener, diazoxide, reduces neuronal damage after middle cerebral artery occlusion in the rat. Am

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