A clear link between ADR

A clear link between α2-ADR stimulation and decreased respiration in β-cells is established and consistent with proteomics data. Current discoveries in islets have identified physical interactions between ADR-coupled G proteins and cellular components such as insulin vesicles and ion channels (Zhao et al., 2010b, Zhao et al., 2010c, Straub and Sharp, 2012). For example, Gαo reduces insulin vesicle docking and in our data, is increased following ADR stimulation (Zhao et al., 2010a). Moreover, studies in other cell types have demonstrated that G proteins, including Gαi, are present in mitochondria providing framework for changes observed in oxidative phosphorylation (Lyssand and Bajjalieh, 2007, Beninca et al., 2014). We believe these studies support activation of α2-ADR may cause changes in protein abundance through novel down-stream targets of G protein signaling. Rearrangements in metabolic proteins after epinephrine exposure in Min6 cells support a direct role in β-cell oxidative metabolism. Given the evidence that G proteins can act directly on mitochondria and that many G protein functions are unknown, we hypothesize that proteins involved in oxidative phosphorylation are downstream targets for α2-ADR signaling. We found epinephrine exposure altered the abundance of every complex in the ETC. Complex I had increased expression in an essential subunit and mixed expression changes in the accessory catalytic subunits (Su et al., 2012). Cytochrome c subunits of complex III and complex IV were also increased, inconsistent with physiological assessments. However, succinate dehydrogenase (complex II) and several essential subunits to ATP synthase were decreased with epinephrine. Change in complex II abundance was confirmed by immunoblot. While complex II does not promote ATP synthesis directly through contribution to the proton gradient, it is the only ETC enzyme involved in both respiration and the citric dna stain cycle (Rutter et al., 2010). When we evaluated the mitochondrial function of Min6 cells after epinephrine exposure there was a decrease in cellular reducing capacity and ATP concentration, consistent with lower levels of succinate dehydrogenase (complex II). However, MMP was increased in cells following acute epinephrine exposure, which might reflect a decrease in proton carriers coincident with reduced cytochrome c oxidase activity (electron transfer to molecular oxygen) even though some Complex IV subunits are upregulated. Although ATP synthase subunit Atp5a was not different by either measure, downregulation of Atp5o, Atp5jp, and Atp5h in the proteomics analysis indicate reductions in the synthesis of ATP consistent with the decreased intracellular ATP concentration following epinephrine exposure. Additional mechanisms not tested in the present study may include decreased production/mobilization and translocation of ADP into the mitochondrial matrix (Chance and Williams, 1955, Gnaiger, 2001). Together, the upregulation of subunits for Complexes I and III and decrease in ATP concentration through inhibition in ATP synthase can explain the increase in MMP, but additional work is required to determine the overall regulation of mitochondrial respiration by adrenergic stimulation. Differentially expressed proteins expanded beyond oxidative phosphorylation and included protein turnover and DNA replication. We anticipated the acute response to stress (CA) would consist of signals that suppress cell growth, which reflects a balance between the production of new proteins and protein breakdown. Proteins annotated to ribosomal pathways reflected increased and decreased expression, yet the 18 subunits of the proteasome were increased, however subunits Psma1 and Psmb1 were not different when evaluated by immunoblot. The proteomic results suggest increased protein breakdown through up-regulation of the proteasome and changes in normal protein translation (ribosomes). Although we could not confirm changes in Psma1 and Psmb1 by immunoblot, the proteasome was previously implicated in islets following chronic ADR stimulation (Kelly et al., 2017) and in diabetic islets (Bugliani et al., 2013).