Hydrogen Peroxide Causes Iron Dysregulation in C2C12 Skeletal Muscle Cells

Abstract

Diabetes is becoming an increasingly prevalent issue in the United States. Diabetes and insulin resistance is associated with increased levels of circulating iron and signs of iron dysregulation. Unbound iron in cells leads to increased amounts of free radicals causing damage, including insulin resistance. Ongoing research in our lab has shown that insulin resistant rats displayed iron dysregulation in liver tissue, marked by hepatic iron accumulation, with a decreased ability to store the excess iron in ferritin storage proteins. The purpose of this study is to develop a cell model of iron dysregulation to replicate findings in previous studies in order to identify the mechanism by which this iron dysregulation occurs. Using C2C12 mouse skeletal muscle cells, we investigated the effects of hydrogen peroxide on iron regulation, and its role in the development of iron dysregulation. C2C12 cells were treated with 200 μM hydrogen peroxide for 12 hours. Following treatment with hydrogen peroxide, cells were harvested for protein and iron analysis. Protein levels of transferrin receptor (TfR) and ferritin heavy chain (FHC) were measured using western blot analysis. Total iron was measured using a total iron colorimetric assay. Hydrogen peroxide treatment decreased TfR protein levels by 24±16% (p=.03). We then developed a cellular model to reflect the stress caused by elevated circulating iron levels by treating C2C12 cells with varying concentrations (0–100 μM) of FeCl3 for 24 hours. Iron treatment resulted in an increase in total cellular iron in a dose‐dependent manner. As expected, exposure to elevated iron caused a reduction in TfR expression and an increase in FHC in a dose‐dependent manner. Using this high iron model with a 24‐hour treatment of 10 μM FeCl3, we added 200 μM hydrogen peroxide during the last 12 hours of iron treatment to impose conditions of elevated oxidative stress. The combination of iron and hydrogen peroxide resulted in an increase in TfR by 46±9% (p=.05) and a decrease in FHC by 49±13% (p=0.018), showing a complete reversal compared to hydrogen peroxide treatment alone. Total iron increased by 69±6.7% (p=0.0005). Our results show that treatment with hydrogen peroxide in the presence of iron causes an accumulation of iron and a decrease in ferritin expression, potentially leading to an increase of free iron and the production of reactive oxygen species. This represents a condition of iron dysregulation and increased oxidative stress that can contribute to insulin resistance as well as other cellular pathologies.Support or Funding InformationSupported by BYU Mentoring Enhancement GrantThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.