Stimulation of Akt Phosphorylation and Glucose Transport by Metalloporphyrins with Peroxynitrite Decomposition Catalytic Activity.

Iron porphyrin molecules such as hemin and iron(III) 4,4',4″,4‴-(porphine-5,10,15,20-tetrayl)tetrakis(benzoic acid) (FeTBAP) have previously been shown to influence insulin signaling and glucose metabolism. We undertook this study to determine whether a catalytic action of iron porphyrin compounds would be related to their stimulation of insulin signaling and glucose uptake in C2C12 myotubes. FeTBAP did not display nitrite reductase activity or alter protein S-nitrosylation in myotubes, eliminating this as a candidate mode by which FeTBAP could act. FeTBAP displayed peroxynitrite decomposition catalytic activity in vitro. Additionally, in myotubes FeTBAP decreased protein nitration. The peroxynitrite decomposition catalyst Fe(III)5,10,15,20-tetrakis(4-sulfonatophenyl)porphyrinato chloride (FeTPPS) also decreased protein nitration in myotubes, but the iron porphyrin Fe(III)tetrakis(1-methyl-4-pyridyl)porphyrin pentachlorideporphyrin pentachloride (FeTMPyP) did not. FeTBAP and FeTPPS, but not FeTMPyP, showed in vitro peroxidase activity. Further, FeTBAP and FeTPPs, but not FeTMPyP, increased Akt phosphorylation and stimulated glucose uptake in myotubes. These findings suggest that iron porphyrin compounds with both peroxynitrite decomposition activity and peroxidase activity can stimulate insulin signaling and glucose transport in skeletal muscle cells.

Reversible Oxidative Modifications in Myoglobin and Functional Implications.

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.

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.

Myoglobin as a versatile peroxidase: Implications for a more important role for vertebrate striated muscle in antioxidant defense.

Myoglobins (Mb) are ubiquitous proteins found in striated muscle of nearly all vertebrate taxa. Although their function is most commonly associated with facilitating oxygen storage and diffusion, Mb has also been implicated in cellular antioxidant defense. The oxidized (Fe3+) form of Mb (metMB) can react with hydrogen peroxide (H2O2) to produce ferrylMb. FerrylMb can be reduced back to metMb for another round of reaction with H2O2. In the present study, we have shown that horse skeletal muscle Mb displays peroxidase activity using 2,2'-azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) and 3,3',5,5'-tetramethylbenzidine (TMB) as reducing substrates, as well as the biologically-relevant substrates NADH/NADPH, ascorbate, caffeic acid, and resveratrol. We have also shown that ferrylMb can be reduced by both ethanol and acetaldehyde, which are known to accumulate in some vertebrate tissues under anaerobic conditions, such as anoxic goldfish and crucian carp, implying a potential mechanism for ethanol detoxification in striated muscle. We found that metMb peroxidase activity is pH-dependent, increasing as pH decreases from 7.4 to 6.1, which is biologically relevant to anaerobic vertebrate muscle when incurring intracellular lactic acidosis. Finally, we found that metMb reacts with hypochlorite in a heme-dependent fashion, indicating that Mb could play a role in hypochlorite detoxification. Taken together, these data suggest that Mb peroxidase activity might be an important antioxidant mechanism in vertebrate cardiac and skeletal muscle under a variety of physiological conditions, such as those that might occur in contracting skeletal muscle or during hypoxia.

Glucose-dependent trans-plasma membrane electron transport and p70S6k phosphorylation in skeletal muscle cells.

The reduction of extracellular oxidants by intracellular electrons is known as trans-plasma membrane electron transport (tPMET). The goal of this study was to characterize a role of tPMET in the sensing of glucose as a physiological signal. tPMET from C2C12 myotubes was monitored using a cell-impermeable extracellular electron acceptor, water-soluble tetrazolium salt-1 (WST-1). Superoxide dismutase in the incubation medium or exposure to an NADPH oxidase (NOX) isoform 1/4 inhibitor suppressed WST-1 reduction by 70%, suggesting a role of NOXs in tPMET. There was a positive correlation between medium glucose concentration and WST-1 reduction, suggesting that tPMET is a glucose-sensing process. WST-1 reduction was also decreased by an inhibitor of the pentose phosphate pathway, dehydroepiandrosterone. In contrast, glycolytic inhibitors, 3PO and sodium fluoride, did not affect WST-1 reduction. Thus, it appears that glucose uptake and processing in the pentose phosphate pathway drives NOX-dependent tPMET. Western blot analysis demonstrated that p70S6k phosphorylation is glucose-dependent, while the phosphorylation of AKT and MAPK did not differ in the presence or absence of glucose. Further, phosphorylation of p70S6k was dependent upon NOX enzymes. Finally, glucose was required for full stimulation of p70S6k by insulin, again in a fashion prevented by NOX inhibition. Taken together, the data suggest that muscle cells have a novel glucose-sensing mechanism dependent on NADPH production and NOX activity, culminating in increased p70S6k phosphorylation.

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage by extracellular oxidants. This process of transporting electrons from intracellular reductants to extracellular oxidants is not well defined. Here we present spectrophotometric assays by C2C12 myotubes to monitor tPMET utilizing the extracellular electron acceptors: water-soluble tetrazolium salt-1 (WST-1) and 2,6-dichlorophenolindophenol (DPIP or DCIP). Through reduction of these electron acceptors, we are able to monitor this process in a real-time analysis. With the addition of enzymes such as ascorbate oxidase (AO) and superoxide dismutase (SOD) to the assays, we can determine which portion of tPMET is due to ascorbate export or superoxide production, respectively. While WST-1 was shown to produce stable results with low background, DPIP was able to be re-oxidized after the addition of AO and SOD, which was demonstrated with spectrophotometric analysis. This method demonstrates a real-time, multi-well, quick spectrophotometric assay with advantages over other methods used to monitor tPMET, such as ferricyanide (FeCN) and ferricytochrome c reduction.

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.