Myoglobin (Mb), an oxygen-binding heme protein highly expressed in heart and skeletal muscle, has been shown to undergo oxidative modifications on both an inter- and intramolecular level when exposed to hydrogen peroxide (H2O2) in vitro. Here, we show that exposure to H2O2 increases the peroxidase activity of Mb. Reaction of Mb with H2O2 causes covalent binding of heme to the Mb protein (Mb-X), corresponding to an increase in peroxidase activity when ascorbic acid is the reducing co-substrate. Treatment of H2O2-reacted Mb with ascorbic acid reverses the Mb-X crosslink. Reaction with H2O2 causes Mb to form dimers, trimers, and larger molecular weight Mb aggregates, and treatment with ascorbic acid regenerates Mb monomers. Reaction of Mb with H2O2 causes formation of dityrosine crosslinks, though the labile nature of the crosslinks broken by treatment with ascorbic acid suggests that the reversible aggregation of Mb is mediated by crosslinks other than dityrosine. Disappearance of a peptide containing a tryptophan residue when Mb is treated with H2O2 and the peptide’s reappearance after subsequent treatment with ascorbic acid suggest that tryptophan side chains might participate in the labile crosslinking. Taken together, these data suggest that while exposure to H2O2 causes Mb-X formation, increases Mb peroxidase activity, and causes Mb aggregation, these oxidative modifications are reversible by treatment with ascorbic acid. A caveat is that future studies should demonstrate that these and other in vitro findings regarding properties of Mb have relevance in the intracellular milieu, especially in regard to actual concentrations of metMb, H2O2, and ascorbate that would be found in vivo.
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 3β (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.
Ascorbate plays a key antioxidant role in protection of cells from damage by reactive oxygen species, which have been implicated in causing metabolic dysfunction such as diabetes. Thus, we hypothesized that ascorbate could aid in rescue from insulin resistance brought on by exposure to reactive oxygen species. In addition, we hypothesized that the peroxidase mimetic Fe(III)tetrakis(4‐benzoic acid)porphyrin (FeTBAP) could act synergistically with ascorbate in defense against oxidative stress. In order to measure the protective effect of ascorbate, we pretreated C2C12 myotubes with ascorbate. We then exposed the myotubes to glucose oxidase (GO), an enzyme that utilizes glucose to produce hydrogen peroxide, thus mimicking a state of oxidative stress prior to exposure to insulin. Data were quantified via western blot analysis monitoring phosphorylated AKT and total AKT. In order to assay the ability of FeTBAP in conjunction with ascorbate to protect against insulin resistance, C2C12 myotubes and isolated soleus were first pretreated with ascorbate and FeTBAP and then exposed to glucose oxidase followed by insulin. We found that ascorbate rescues insulin resistance brought on by exposure to reactive oxygen species. In addition, preliminary data from C2C12 myotubes and soleus illustrated a trend in which FeTBAP in combination with ascorbate protected against insulin resistance. Taken together, these data demonstrate a functional role of ascorbate in which the antioxidant on its own and in conjunction with FeTBAP rescues cells from peroxide induced 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 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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