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Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells.

Shi X, Deng H, Dai Z, Xu Y, Xiong X, Ma P, Cheng J - Oxid Med Cell Longev (2015)

Bottom Line: At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress.Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2.Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China.

ABSTRACT
Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.

No MeSH data available.


Related in: MedlinePlus

Nr2e1 silencing affects oxidative stress and antioxidant enzyme levels. (a) Comparison of intracellular levels of ROS between control (MIN6-shc) and Nr2e1 silenced (MIN6-sh2 and MIN6-sh3) cells. Cells were cultured in complete medium with or without 0.5 mM palmitate for 24 h and then the fluorescence intensity of DHE was measured using flow cytometry. Data represent means ± SD of three observations; #P ≤ 0.05 compared to cells in normal conditions. ∗P ≤ 0.05 compared to MIN6-shc cells. (b) SOD and GSH-Px activity decreased in MIN6-sh2 and MIN6-sh3 cells. (c) GSH content decreased in MIN6-sh2 and MIN6-sh3 cells. (d) The relative mRNA expressions of Sod1, Gpx, Gclc, and Gclm were downregulated by Nr2e1 silencing. Data represent means ± SD of three observations; ∗P ≤ 0.05; all comparisons with MIN6-shc cells.
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fig6: Nr2e1 silencing affects oxidative stress and antioxidant enzyme levels. (a) Comparison of intracellular levels of ROS between control (MIN6-shc) and Nr2e1 silenced (MIN6-sh2 and MIN6-sh3) cells. Cells were cultured in complete medium with or without 0.5 mM palmitate for 24 h and then the fluorescence intensity of DHE was measured using flow cytometry. Data represent means ± SD of three observations; #P ≤ 0.05 compared to cells in normal conditions. ∗P ≤ 0.05 compared to MIN6-shc cells. (b) SOD and GSH-Px activity decreased in MIN6-sh2 and MIN6-sh3 cells. (c) GSH content decreased in MIN6-sh2 and MIN6-sh3 cells. (d) The relative mRNA expressions of Sod1, Gpx, Gclc, and Gclm were downregulated by Nr2e1 silencing. Data represent means ± SD of three observations; ∗P ≤ 0.05; all comparisons with MIN6-shc cells.

Mentions: Because Nr2e1 knockdown MIN6 cells exhibit increased sensitivity to lipotoxicity, we investigated the underlying mechanism that contributes to beta cell survival. It is well known that oxidative stress is a mechanism whereby lipotoxicity exerts harmful effects to damage beta cells, so we next investigated the levels of oxidative stress in MIN6-shc, MIN6-sh2, and MIN6-sh3 cells. Flow cytometry analysis using a ROS fluorescent probe (DHE) demonstrated an increase in ROS upon incubation of MIN6 cells with palmitate for 24 hours. Notably, this effect of palmitate was enhanced in MIN6-sh2 and MIN6-sh3 cells (Figure 6(a)). Because of low antioxidant enzyme production, beta cells have limited ability to counter ROS production. To determine if the promotion in oxidative stress in MIN6-sh2 and MIN6-sh3 cells resulted from decreases in antioxidant enzyme levels, we measure the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); results showed that Nr2e1 silencing inhibited the activity of GSH-Px and SOD activity and downregulated intracellular GSH content in MIN6 cells (Figures 6(b) and 6(c)). In addition, the mRNA expressions of Sod1, Gpx, and the GCL subunits Gclc and Gclm were measured. Significant changes were confirmed in the mRNA expressions of Sod1 and Gpx, which was perfectly consistent with that in their activities (Figure 6(d)). The levels of Gclc and Gclm mRNA were also substantially attenuated by Nr2e1 silencing (Figure 6(d)). These results indicate that Nr2e1 deficiency induced oxidative stress via ROS overproduction followed by disorder in the oxidant/antioxidant system.


Nr2e1 Deficiency Augments Palmitate-Induced Oxidative Stress in Beta Cells.

Shi X, Deng H, Dai Z, Xu Y, Xiong X, Ma P, Cheng J - Oxid Med Cell Longev (2015)

Nr2e1 silencing affects oxidative stress and antioxidant enzyme levels. (a) Comparison of intracellular levels of ROS between control (MIN6-shc) and Nr2e1 silenced (MIN6-sh2 and MIN6-sh3) cells. Cells were cultured in complete medium with or without 0.5 mM palmitate for 24 h and then the fluorescence intensity of DHE was measured using flow cytometry. Data represent means ± SD of three observations; #P ≤ 0.05 compared to cells in normal conditions. ∗P ≤ 0.05 compared to MIN6-shc cells. (b) SOD and GSH-Px activity decreased in MIN6-sh2 and MIN6-sh3 cells. (c) GSH content decreased in MIN6-sh2 and MIN6-sh3 cells. (d) The relative mRNA expressions of Sod1, Gpx, Gclc, and Gclm were downregulated by Nr2e1 silencing. Data represent means ± SD of three observations; ∗P ≤ 0.05; all comparisons with MIN6-shc cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig6: Nr2e1 silencing affects oxidative stress and antioxidant enzyme levels. (a) Comparison of intracellular levels of ROS between control (MIN6-shc) and Nr2e1 silenced (MIN6-sh2 and MIN6-sh3) cells. Cells were cultured in complete medium with or without 0.5 mM palmitate for 24 h and then the fluorescence intensity of DHE was measured using flow cytometry. Data represent means ± SD of three observations; #P ≤ 0.05 compared to cells in normal conditions. ∗P ≤ 0.05 compared to MIN6-shc cells. (b) SOD and GSH-Px activity decreased in MIN6-sh2 and MIN6-sh3 cells. (c) GSH content decreased in MIN6-sh2 and MIN6-sh3 cells. (d) The relative mRNA expressions of Sod1, Gpx, Gclc, and Gclm were downregulated by Nr2e1 silencing. Data represent means ± SD of three observations; ∗P ≤ 0.05; all comparisons with MIN6-shc cells.
Mentions: Because Nr2e1 knockdown MIN6 cells exhibit increased sensitivity to lipotoxicity, we investigated the underlying mechanism that contributes to beta cell survival. It is well known that oxidative stress is a mechanism whereby lipotoxicity exerts harmful effects to damage beta cells, so we next investigated the levels of oxidative stress in MIN6-shc, MIN6-sh2, and MIN6-sh3 cells. Flow cytometry analysis using a ROS fluorescent probe (DHE) demonstrated an increase in ROS upon incubation of MIN6 cells with palmitate for 24 hours. Notably, this effect of palmitate was enhanced in MIN6-sh2 and MIN6-sh3 cells (Figure 6(a)). Because of low antioxidant enzyme production, beta cells have limited ability to counter ROS production. To determine if the promotion in oxidative stress in MIN6-sh2 and MIN6-sh3 cells resulted from decreases in antioxidant enzyme levels, we measure the activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); results showed that Nr2e1 silencing inhibited the activity of GSH-Px and SOD activity and downregulated intracellular GSH content in MIN6 cells (Figures 6(b) and 6(c)). In addition, the mRNA expressions of Sod1, Gpx, and the GCL subunits Gclc and Gclm were measured. Significant changes were confirmed in the mRNA expressions of Sod1 and Gpx, which was perfectly consistent with that in their activities (Figure 6(d)). The levels of Gclc and Gclm mRNA were also substantially attenuated by Nr2e1 silencing (Figure 6(d)). These results indicate that Nr2e1 deficiency induced oxidative stress via ROS overproduction followed by disorder in the oxidant/antioxidant system.

Bottom Line: At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress.Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2.Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.

View Article: PubMed Central - PubMed

Affiliation: Department of Endocrinology, Zhongnan Hospital of Wuhan University, Wuhan, Hubei 430071, China.

ABSTRACT
Nuclear receptor subfamily 2 group E member 1 (Nr2e1) has been regarded as an essential regulator of the growth of neural stem cells. However, its function elsewhere is unknown. In the present study, we generated Nr2e1 knockdown MIN6 cells and studied whether Nr2e1 knockdown affected basal beta cell functions such as proliferation, cell death, and insulin secretion. We showed that knockdown of Nr2e1 in MIN6 cells resulted in increased sensitivity to lipotoxicity, decreased proliferation, a partial G0/G1 cell-cycle arrest, and higher rates of apoptosis. Moreover, Nr2e1 deficiency exaggerates palmitate-induced impairment in insulin secretion. At the molecular level, Nr2e1 deficiency augments palmitate-induced oxidative stress. Nr2e1 deficiency also resulted in decreases in antioxidant enzymes and expression level of Nrf2. Together, this study indicated a potential protective effect of Nr2e1 on beta cells, which may serve as a target for the development of novel therapies for diabetes.

No MeSH data available.


Related in: MedlinePlus