Characterization of oxidation of glutathione by cytochrome c

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+ level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

MicroRNA-506 modulates insulin resistance in human adipocytes by targeting S6K1 and altering the IRS1/PI3K/AKT insulin signaling pathway

The incidence of obesity has increased rapidly, becoming a worldwide public health issue that involves insulin resistance. A growing number of recent studies have demonstrated that microRNAs play a significant role in controlling the insulin signaling network. For example, miR-506-3p expression has been demonstrated to correlate with insulin sensitivity; however, the underlying mechanism remains unknown. In this study, we found that miR-506-3p enhanced glucose uptake by 2-deoxy-D-glucose uptake assays and regulated the protein expression of key genes involved in the PI3K/AKT insulin signaling pathway including IRS1, PI3K, AKT, and GlUT4. We next predicted ribosomal protein S6 kinase B1 (S6K1) to be a candidate target of miR-506-3p by bioinformatics analysis and confirmed using dual-luciferase assays that miR-506-3p regulated S6K1 expression by binding to its 3′-UTR. Moreover, modulating S6K1 expression counteracted the effects of miR-506-3p on glucose uptake and PI3K/AKT pathway activation. In conclusion, miR-506-3p altered IR in adipocytes by regulating S6K1-mediated PI3K/AKT pathway activation. Taken together, these findings provide novel insights and potential targets for IR therapy.