Investigation of Ras-related nuclear protein gene expression in obesity in relation with adipogenesis
Sinem Banu Demir1, Meliha Koldemir Gündüz1, Mehtap Çevik1, Çavlan Çiftçi2, Belgin Süsleyici1
1Marmara Üniversitesi, Fen Edebiyat Fakültesi, Biyoloji Bölümü, Moleküler Biyoloji Anabilim Dalı, İstanbul, Türkiye
2İstanbul Bilim Üniversitesi Tıp Fakültesi, Kardiyoloji Anabilim Dalı, İstanbul, Türkiye
Keywords: Adipocytes; gene expression; iCELLigence; obesity; Ras-related nuclear protein.
Abstract
Objectives: This study aims to investigate how the gene expression of Ras-related nuclear protein (RAN) will change as a response to the adipocytes in different stages of development, oxidative stress markers (stearic acid, linoleic acid, ethanol and hydrogen peroxide) to be applied in different dosages and also its effects on the proliferation ability of cells.
Materials and methods: In our study, to determine the role of the RAN gene in obesity, we differentiated mouse-derived 3T3-L1 fibroblast cells into preadipocytes and mature adipocytes by observing with iCELLigence system in real-time. These cells were exposed to cytotoxic markers at different concentrations and cell proliferations were monitored with iCELLgence system. The messenger RNA isolation from the obtained cells, complementary DNA synthesis from messenger RNA and the changes of RAN gene expression in response to cytotoxic markers obtained from the genetic material were identified by TaqMan real-time polimerase chain reaction.
Results: Implementation periods of oxidative stress markers were found to be IC50 value by using real-time cell monitoring, which was obtained by applying various concentrations. After 24 hours application of hydrogen peroxide (H2O2), IC50 value was found as 807 μM. While 24 hours application of 1 mM H2O2 had lethal effect on adipocytes 3T3-L1 cells, we did not observe any significant effect of H2O2 applications in the concentration range of 50-250 μM on proliferation of adipocytes. While RAN gene expression increased after four and five hours of exposure to 600 μM H2O2 application, it decreased after application of stearic acid, ethanol and 24 hours of H2O2. As a result of application of linoleic acid, RAN gene expression was completely silenced.
Conclusion: The observation of RAN gene expression having increased twice in adipocytes that were exposed to 600 μM H2O2 for four hours compared to control cells while five hours of exposure to the same dosage to increase nearly six times suggests the deterioration of glucose homeostasis in differentiated adipocyte cells. We think the fact of ethanol decreasing RAN gene expression contributes to the increase of insulin resistance.