Upregulation of UCP2 by Adiponectin: The Involvement of Mitochondrial Superoxide and hnRNP K

Background: The adipocyte-derived hormone adiponectin elicits protective functions against fatty liver diseases and hepatic injuries at least in part by stimulating the expression of a mitochondrial inner membrane transporter, uncoupling protein 2 (UCP2). The present study was designed to investigate the cellular and molecular mechanisms underlying adiponectin-induced UCP2 expression. Methodology/Principal Findnigs: Mice were treated with adiponectin and/or different drug inhibitors. Parenchymal (PCs) and nonparenchymal (NPCs) cells were fractionated from the liver tissues for mitochondria isolation, Western blotting and quantitative PCR analysis. Mitochondrial superoxide production was monitored by MitoSOX staining and flow cytometry analysis. Compared to control mice, the expression of UCP2 was significantly lower in NPCs, but not PCs of adiponectin knockout mice (AKO). Both chronic and acute treatment with adiponectin selectively increased the mRNA and protein abundance of UCP2 in NPCs, especially in the enriched endothelial cell fractions. The transcription inhibitor actinomycin D could not block adiponectin-induced UCP2 expression, whereas the protein synthesis inhibitor cycloheximide inhibited the elevation of UCP2 protein but not its mRNA levels. Mitochondrial content of heterogeneous nuclear ribonucleoprotein K (hnRNP K), a nucleic acid binding protein involved in regulating mRNA transportation and stabilization, was significantly enhanced by adiponectin, which also evoked a transient elevation of mitochondrial superoxide. Rotenone, an inhibitor of mitochondrial respiratory complex I, abolished adiponectin-induced superoxide production, hnRNP K recruitment and UCP2 expression. Conclusions/Significance: Mitochondrial superoxide production stimulated by adiponectin serves as a trigger to initiate the translocation of hnRNP K, which in turn promotes UCP2 expressions in liver. © 2012 Zhou et al.published_or_final_versio

Membrane potential and delta pH dependency of reverse electron transport-associated hydrogen peroxide production in brain and heart mitochondria

Succinate-driven reverse electron transport (RET) is one of the main sources of mitochondrial reactive oxygen species (mtROS) in ischemia-reperfusion injury. RET is dependent on mitochondrial membrane potential (Δψm) and transmembrane pH difference (ΔpH), components of the proton motive force (pmf); a decrease in Δψm and/or ΔpH inhibits RET. In this study we aimed to determine which component of the pmf displays the more dominant effect on RET-provoked ROS generation in isolated guinea pig brain and heart mitochondria respiring on succinate or α-glycerophosphate (α-GP). Δψm was detected via safranin fluorescence and a TPP+ electrode, the rate of H2O2 formation was measured by Amplex UltraRed, the intramitochondrial pH (pHin) was assessed via BCECF fluorescence. Ionophores were used to dissect the effects of the two components of pmf. The K+/H+ exchanger, nigericin lowered pHin and ΔpH, followed by a compensatory increase in Δψm that led to an augmented H2O2 production. Valinomycin, a K+ ionophore, at low [K+] increased ΔpH and pHin, decreased Δψm, which resulted in a decline in H2O2 formation. It was concluded that Δψm is dominant over ∆pH in modulating the succinate- and α-GP-evoked RET. The elevation of extramitochondrial pH was accompanied by an enhanced H2O2 release and a decreased ∆pH. This phenomenon reveals that from the pH component not ∆pH, but rather absolute value of pH has higher impact on the rate of mtROS formation. Minor decrease of Δψm might be applied as a therapeutic strategy to attenuate RET-driven ROS generation in ischemia-reperfusion injury

4Pi microscopy reveals an impaired three-dimensional mitochondrial network of pancreatic islet beta-cells, an experimental model of type-2 diabetes.

Insulin production in pancreatic beta-cells is critically linked to mitochondrial oxidative phosphorylation. Increased ATP production triggered by blood glucose represents the beta-cells\u27 glucose sensor. Type-2 diabetes mellitus results from insulin resistance in peripheral tissues and impaired insulin secretion. Pathology of diabetic beta-cells might be reflected by the altered morphology of mitochondrial network. Its characterization is however hampered by the complexity and density of the three-dimensional (3D) mitochondrial tubular networks in these cell types. Conventional confocal microscopy does not provide sufficient axial resolution to reveal the required details; electron tomography reconstruction of these dense networks is still difficult and time consuming. However, mitochondrial network morphology in fixed cells can also be studied by 4Pi microscopy, a laser scanning microscopy technique which provides an approximately 7-fold improved axial resolution (approximately 100 nm) over conventional confocal microscopy. Here we present a quantitative study of these networks in insulinoma INS-1E cells and primary beta-cells in Langerhans islets. The former were a stably-transfected cell line while the latter were transfected with lentivirus, both expressing mitochondrial matrix targeted redox-sensitive GFP. The mitochondrial networks and their partial disintegration and fragmentation are revealed by carefully created iso-surface plots and their quantitative analysis. We demonstrate that beta-cells within the Langerhans islets from diabetic Goto Kakizaki rats exhibited a more disintegrated mitochondrial network compared to those from control Wistar rats and model insulinoma INS-1E cells. Standardization of these patterns may lead to development of morphological diagnostics for Langerhans islets, for the assessment of beta-cell condition, before their transplantations