Abstract
The N-myc downstream-regulated gene 2 (NDRG2) is involved in cell differentiation and apoptosis, but its function in the pancreas remains to be established. Herein we examine the expression and function of NDRG2 in the endocrine pancreas. NDRG2 immunoreactivity was localized mainly in the cytoplasm of pancreatic β cells. When β-TC3 cells were exposed chronically to high levels of free fatty acid (FFA), cell viability was impaired, and Akt and NDRG2 phosphorylation were reduced. NDRG2 is a potential substrate of protein kinase Akt. Overexpression of constitutively active Akt enhanced NDRG2 phosphorylation and abolished the apoptosis induced by FFA in β-TC3 cells, whereas NDRG2 knock-down attenuated Akt-mediated protection of β cells against fatty acid-triggered apoptosis. Collectively, these data indicate that NDRG2 acts as a key molecule in pancreatic β cells and is involved in the Akt-mediated protection of β cells against lipotoxicity.
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Acknowledgments
This work was supported by grants from the National Natural Science Foundation of China (nos. 30600314, 30670303, 30830054, 30801309 and 30170465). We thank members of our laboratory for helpful discussions.
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L. Shen, X. Liu and W. Hou contributed equally to this work.
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Supplemental Figure 1. Fluorescence microscopy shows the infection efficiency in β-TC3 cells infected with different MOI of Ad-EGFP for 48 h (JPG 399 kb)
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Supplemental Figure 2. Fluorescence microscopy shows the transfection efficiency in β-TC3 cells transfected with fluorescent dye-labeled siRNAs for 12 h (JPG 196 kb)
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Supplemental Figure 3. Immunocytochemical staining of apoptotic INS-1, β-TC3 and β-TC6 cells using NDRG2 antibody and Cy3-conjugated secondary antibody shows the localization of NDRG2 (red). DAPI staining (blue) reveals the position and morphology of the cell nuclei. Magnification 400x (JPG 318 kb)
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Supplemental Figure 4. Western blotting shows the expression and phosphorylation of Akt in β-TC3 cells chronically exposed to different high palmitic acid concentrations. Cells were incubated with either 1% BSA or the indicated concentration of palmitic acid complexed to 1% BSA for 72 h, and cell lysates separated by SDS-PAGE and subjected to Western blotting prior to detection with antibodies against Akt, phospho-Akt (Ser473) and β-actin. A histogram of the quantitative analysis of Western blot results shows a significant decrease of relative density of phospho-Akt (Ser473) compared to Akt in β-TC3 cells exposed to 0.5 mM palmitic acid complexed to 1% BSA (*P < 0.05 compared to the control) (JPG 216 kb)
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Supplemental Figure 5. The half-life of NDRG2 protein in β-TC3 cells treated with cycloheximide (CHX) for 2 h, 4 h, 8 h or 24 h was assessed by Western blotting (JPG 136 kb)
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Supplemental Figure 6. The cells were infected with Ad-myristoylated Aktα for 48 h and transfected with 60 nM NDRG2 siRNA or control siRNA and then incubated with 1 mM palmitic acid complexed to 1% BSA for 72 h prior to detection of apoptotic cells by flow cytometry (JPG 306 kb)
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Supplemental Figure 7. β-TC3 cells were incubated with 1 mM palmitic acid complexed to 1% BSA for the indicated times and GSIS was measured by ELISA assay (JPG 175 kb)
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Shen, L., Liu, X., Hou, W. et al. NDRG2 is highly expressed in pancreatic β cells and involved in protection against lipotoxicity. Cell. Mol. Life Sci. 67, 1371–1381 (2010). https://doi.org/10.1007/s00018-010-0258-1
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DOI: https://doi.org/10.1007/s00018-010-0258-1