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The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

Yu C, Cui S, Zong C, Gao W, Xu T, Gao P, Chen J, Qin D, Guan Q, Liu Y, Fu Y, Li X, Wang X - J. Biol. Chem. (2015)

Bottom Line: This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice.NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation.In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as "positive and negative regulation."

View Article: PubMed Central - PubMed

Affiliation: From the The Department of Cell Biology, Shandong University School of Medicine, Jinan, China, 250012.

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Analysis of the effect of TG or PA on NR4A1 expression in mouse islets. Mouse islets were isolated from C57BL/6J mice, and total RNA of mouse islets was prepared using an RNeasy Mini Kit. A–D, relative mRNA levels of CHOP and NR4A1 in response to 0.5 μm TG (A and B) or 0.4 mm PA (C and D) at various time points were determined by qPCR. E, determination of the induced NR4A1 expression in pancreatic β-cells upon TG or PA treatment. Mouse islets were treated with 0.5 μm TG for 6 h, and double immunofluorescence staining was performed with anti-insulin and anti-NR4A1 antibodies from different species. The top panel is an islet treated with DMSO as a control, and the lower panel is an islet treated with TG. Blue represents DAPI, green represents NR4A1, red represents insulin, and MERGE of the three colors. The histograms indicate relative fluorescence intensity (=total red densitometry value/islet surface area). The data show the means of three independent experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus 0 h or DMSO control.
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Figure 2: Analysis of the effect of TG or PA on NR4A1 expression in mouse islets. Mouse islets were isolated from C57BL/6J mice, and total RNA of mouse islets was prepared using an RNeasy Mini Kit. A–D, relative mRNA levels of CHOP and NR4A1 in response to 0.5 μm TG (A and B) or 0.4 mm PA (C and D) at various time points were determined by qPCR. E, determination of the induced NR4A1 expression in pancreatic β-cells upon TG or PA treatment. Mouse islets were treated with 0.5 μm TG for 6 h, and double immunofluorescence staining was performed with anti-insulin and anti-NR4A1 antibodies from different species. The top panel is an islet treated with DMSO as a control, and the lower panel is an islet treated with TG. Blue represents DAPI, green represents NR4A1, red represents insulin, and MERGE of the three colors. The histograms indicate relative fluorescence intensity (=total red densitometry value/islet surface area). The data show the means of three independent experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus 0 h or DMSO control.

Mentions: To investigate whether ER stress-inducing reagents increase NR4A1 expression in mouse islets, we isolated islet cells from 16-week-old C57BL/6J mice. The isolated mice islets were treated with TG (0.5 μm) or PA (0.4 mm) for different time points. We found that TG and PA treatment induced a time-dependent rise in mRNA for sXBP1, Bip (immunoglobulin-binding protein) (data not shown), and CHOP (Fig. 2, A and C), which are markers for ER stress. As expected, TG or PA treatment also increased the NR4A1 mRNA level in a time-dependent manner in purified mouse islets (Fig. 2, B and D). TG- or PA-induced NR4A1 mRNA expression peaked at earlier time points than that of CHOP (Fig. 2, A–D). Olivier Briand (26) previously also showed that PA induces NR4A1 expression in mouse islet cells. To confirm if the increased NR4A1 expression induced by TG and PA in mouse islets occurred in β-cells, we did double immunofluorescence staining with specific antibodies against NR4A1 and Insulin. The images (Fig. 2E) showed that TG treatment increase NR4A1 expression (green), and it was mostly co-localized with insulin (red) in islets. These results confirmed that the two ER stress inducers truly induce NR4A1 expression in β-cells of mouse islets.


The Orphan Nuclear Receptor NR4A1 Protects Pancreatic β-Cells from Endoplasmic Reticulum (ER) Stress-mediated Apoptosis.

Yu C, Cui S, Zong C, Gao W, Xu T, Gao P, Chen J, Qin D, Guan Q, Liu Y, Fu Y, Li X, Wang X - J. Biol. Chem. (2015)

Analysis of the effect of TG or PA on NR4A1 expression in mouse islets. Mouse islets were isolated from C57BL/6J mice, and total RNA of mouse islets was prepared using an RNeasy Mini Kit. A–D, relative mRNA levels of CHOP and NR4A1 in response to 0.5 μm TG (A and B) or 0.4 mm PA (C and D) at various time points were determined by qPCR. E, determination of the induced NR4A1 expression in pancreatic β-cells upon TG or PA treatment. Mouse islets were treated with 0.5 μm TG for 6 h, and double immunofluorescence staining was performed with anti-insulin and anti-NR4A1 antibodies from different species. The top panel is an islet treated with DMSO as a control, and the lower panel is an islet treated with TG. Blue represents DAPI, green represents NR4A1, red represents insulin, and MERGE of the three colors. The histograms indicate relative fluorescence intensity (=total red densitometry value/islet surface area). The data show the means of three independent experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus 0 h or DMSO control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 2: Analysis of the effect of TG or PA on NR4A1 expression in mouse islets. Mouse islets were isolated from C57BL/6J mice, and total RNA of mouse islets was prepared using an RNeasy Mini Kit. A–D, relative mRNA levels of CHOP and NR4A1 in response to 0.5 μm TG (A and B) or 0.4 mm PA (C and D) at various time points were determined by qPCR. E, determination of the induced NR4A1 expression in pancreatic β-cells upon TG or PA treatment. Mouse islets were treated with 0.5 μm TG for 6 h, and double immunofluorescence staining was performed with anti-insulin and anti-NR4A1 antibodies from different species. The top panel is an islet treated with DMSO as a control, and the lower panel is an islet treated with TG. Blue represents DAPI, green represents NR4A1, red represents insulin, and MERGE of the three colors. The histograms indicate relative fluorescence intensity (=total red densitometry value/islet surface area). The data show the means of three independent experiments, *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus 0 h or DMSO control.
Mentions: To investigate whether ER stress-inducing reagents increase NR4A1 expression in mouse islets, we isolated islet cells from 16-week-old C57BL/6J mice. The isolated mice islets were treated with TG (0.5 μm) or PA (0.4 mm) for different time points. We found that TG and PA treatment induced a time-dependent rise in mRNA for sXBP1, Bip (immunoglobulin-binding protein) (data not shown), and CHOP (Fig. 2, A and C), which are markers for ER stress. As expected, TG or PA treatment also increased the NR4A1 mRNA level in a time-dependent manner in purified mouse islets (Fig. 2, B and D). TG- or PA-induced NR4A1 mRNA expression peaked at earlier time points than that of CHOP (Fig. 2, A–D). Olivier Briand (26) previously also showed that PA induces NR4A1 expression in mouse islet cells. To confirm if the increased NR4A1 expression induced by TG and PA in mouse islets occurred in β-cells, we did double immunofluorescence staining with specific antibodies against NR4A1 and Insulin. The images (Fig. 2E) showed that TG treatment increase NR4A1 expression (green), and it was mostly co-localized with insulin (red) in islets. These results confirmed that the two ER stress inducers truly induce NR4A1 expression in β-cells of mouse islets.

Bottom Line: This conclusion was further confirmed by experiments exploiting siRNA to knockdown NR4A1 expression in MIN6 cells or exploiting NR4A1 knock-out mice.NR4A1 overexpression in MIN6 cells or mouse islets resulted in Survivin up-regulation.In conclusion, NR4A1 protects pancreatic β-cells against ER stress-mediated apoptosis by up-regulating Survivin expression and down-regulating CHOP expression, which we termed as "positive and negative regulation."

View Article: PubMed Central - PubMed

Affiliation: From the The Department of Cell Biology, Shandong University School of Medicine, Jinan, China, 250012.

Show MeSH
Related in: MedlinePlus