A Metalloporphyrin that Serves as a Peroxidase and Stimulates Insulin Signaling in Skeletal Muscle

Abstract

Hydrogen peroxide is a form of reactive oxygen species (ROS) that has been associated with metabolic dysfunction such as insulin resistance. We hypothesized that the metalloporphyrin Fe(III)tetrakis(4‐benzoic acid)porphyrin (FeTBAP), which is a peroxidase mimetic, would protect skeletal muscle cells from the damaging effects of peroxide. To determine whether FeTBAP could be a means to deliver peroxidase activity to living cells, we incubated C2C12 myotubes with FeTBAP. We then confirmed in SDS polyacrylamide gels that cell lysates contained FeTBAP's peroxidase activity by the appearance of blue bands corresponding to oxidized tetramethylbenzidine (TMB) when incubated in TMB and peroxide. In order to determine whether FeTBAP could lower peroxide levels in cells challenged with peroxide, we transfected C2C12 myoblasts with the cytosolic, ratiometric fluorescent peroxide sensor, HyPer3. Cells were then pretreated with FeTBAP and finally exposed to glucose oxidase (GO), an enzyme that utilizes glucose to produce hydrogen peroxide. Exposure to GO increased the Hyper3 fluorescence ratio, indicating higher cytosolic peroxide concentration. This increased intracellular peroxide was prevented in cells that were pretreated with FeTBAP. Carbonyl formation, a marker of oxidative protein modification, was also monitored in myotubes pretreated with FeTBAP and exposed to glucose oxidase. We found that there was an increase in carbonyl content in cells exposed to GO, while prior treatment with FeTBAP prevented an increase in carbonyl formation. To assess the ability of FeTBAP to protect myotubes from peroxide induced insulin resistance, cells were pretreated with FeTBAP followed by incubation in GO and then insulin. Exposure to GO decreased insulin‐stimulated phosphorylation of insulin receptor substrate 1 (IRS‐1, Y612). Prior treatment of cells to FeTBAP rescued myotubes from this peroxide induced insulin resistance. Next, we asked whether FeTBAP could impact insulin signaling in the absence of insulin. C2C12 myotubes and mouse soleus muscle were pretreated with FeTBAP followed by an incubation in insulin. We found that FeTBAP increases insulin signaling in myotubes and soleus to about the same level as insulin for phosphorylation of AKT (S473), IRS‐1 (Y612), and glycogen synthase kinase 3 beta (S9). Further, we found that FeTBAP stimulates glucose transport in myotubes. We conclude that FeTBAP can blunt peroxide induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting that it could be a lead compound for new approaches to prevention of insulin resistance.Support or Funding InformationThis project was supported by United States Public Health Service award R15DK102122 from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.