A peroxidase mimetic protects skeletal muscle cells from peroxide challenge and stimulates insulin signaling.

Reactive oxygen species such as hydrogen peroxide have been implicated in causing metabolic dysfunction such as insulin resistance. Heme groups, either by themselves or when incorporated into proteins, have been shown to scavenge peroxide and demonstrate protective effects in various cell types. Thus, we hypothesized that a metalloporphyrin similar in structure to heme, Fe(III)tetrakis(4-benzoic acid)porphyrin (FeTBAP), would be a peroxidase mimetic that could defend cells against oxidative stress. After demonstrating that FeTBAP has peroxidase activity with reduced nicotinamide adenine dinucleotide phosphate (NADPH) and NADH as reducing substrates, we determined that FeTBAP partially rescued C2C12 myotubes from peroxide-induced insulin resistance as measured by phosphorylation of AKT (S473) and insulin receptor substrate 1 (IRS-1, Y612). Furthermore, we found that FeTBAP stimulates insulin signaling in myotubes and mouse soleus skeletal muscle to about the same level as insulin for phosphorylation of AKT, IRS-1, and glycogen synthase kinase 3ß (S9). We found that FeTBAP lowers intracellular peroxide levels and protects against carbonyl formation in myotubes exposed to peroxide. Additionally, we found that FeTBAP stimulates glucose transport in myotubes and skeletal muscle to about the same level as insulin. We conclude that a peroxidase mimetic can blunt peroxide-induced insulin resistance and also stimulate insulin signaling and glucose transport, suggesting a possible role of peroxidase activity in regulation of insulin signaling.

Trans-Plasma Membrane Electron Transport and Ascorbate Efflux by Skeletal Muscle.

Trans-plasma membrane electron transport (tPMET) and the antioxidant roles of ascorbate reportedly play a role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as insulin resistance. Skeletal muscle comprises the largest whole-body organ fraction suggesting a potential role of tPMET and ascorbate export as a major source of extracellular antioxidant. We hypothesized that skeletal muscle is capable of tPMET and ascorbate efflux. To measure these processes, we assayed the ability of cultured muscle cells, satellite cells, and isolated extensor digitorum longus (EDL) and soleus (SOL) to reduce two extracellular electron acceptors, water soluble tetrazolium salt 1 (WST-1), and dichlorophenolindophenol (DPIP). Ascorbate oxidase (AO) was utilized to determine which portion of WST-1 reduction was dependent on ascorbate efflux. We found that muscle cells can reduce extracellular electron acceptors. In C2C12 myotubes and satellite cells, a substantial portion of this reduction was dependent on ascorbate. In myotubes, glucose transporter 1 (GLUT1) inhibitors along with a pan-GLUT inhibitor suppressed tPMET and ascorbate efflux, while a GLUT4 inhibitor had no effect. The adenosine 5'-monophosphate (AMP)-activated protein kinase activator 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) suppressed both tPMET and ascorbate efflux by myotubes, while insulin had no effect. Taken together, our data suggest that muscle cells are capable of tPMET and ascorbate efflux supported by GLUT1, thus illustrating a model in which resting muscle exports electrons and antioxidant to the extracellular environment.