Data of intracellular insulin protein reduced by autophagy in INS-1E cells

Autophagy appears to be involved in maintaining normal intracellular insulin content by accelerating the insulin degradation rate in β-cells (Marsh et al., 2007) [1]. 2-deoxy-d-glucose (2-DG) is metabolized by hexokinase, and acts as an inhibitor of glycolysis. 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways (Aghaee et al., 2012) [2], and appears to be an ideal tool for studying autophagy. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β-cells (Tanemura et al., 2012) [3]. Here, we examined that 2-DG or rapamycin-induced autophagy may decrease the production of intracellular insulin in INS-1E insulinoma cells. Data showed that autophagy was increased by 2-DG or rapamycin by Western blotting and Immunofluorescence staining analyses. Also, intracellular insulin decreased by 2-DG or rapamycin. Furthermore, the autophagy inhibitors, bafilomycin A1 and/or 3-methyladenine, in the presence or absence of 2-DG or rapamycin increased intracellular insulin in INS-1E insulinoma cells.


a b s t r a c t
Autophagy appears to be involved in maintaining normal intracellular insulin content by accelerating the insulin degradation rate in β-cells (Marsh et al., 2007) [1]. 2-deoxy-d-glucose (2-DG) is metabolized by hexokinase, and acts as an inhibitor of glycolysis. 2-DG triggers glucose deprivation without altering other nutrients or metabolic pathways (Aghaee et al., 2012) [2], and appears to be an ideal tool for studying autophagy. Rapamycin induced upregulation of autophagy in both cultured isolated islets and pancreatic β-cells (Tanemura et al., 2012) [3]. Here, we examined that 2-DG or rapamycin-induced autophagy may decrease the production of intracellular insulin in INS-1E insulinoma cells. Data showed that autophagy was increased by 2-DG or rapamycin by Western blotting and Immunofluorescence staining analyses. Also, intracellular insulin decreased by 2-DG or rapamycin. Furthermore, the autophagy inhibitors, bafilomycin A1 and/or 3-methyladenine, in the presence or absence of 2-DG or rapamycin increased intracellular insulin in INS-1E insulinoma cells.
& Autophagy in INS-1E insulinoma cells were induced by treatment of 2-DG or rapamycin, and administration of bafilomycin A1 or 3-methyladenine inhibited autophagy, resulted in increase of intracellular insulin levels.

Experimental features
Autophagy showed Western blotting of LC3 II, Beclin 1, and insulin proteins and Immunofluorescence staining of LC3-positive granules or lysosomal-associated membrane protein 2 (Lamp2) under the conditions of 2-DG or rapamycin, and /or bafilomycin A1 or 3-methyladenine treatments.

Data source location
Wonju, Gangwon-do, Republic of Korea

Data accessibility
All data are provided with this article

Value of the data
The data provide 2-DG treatment increase autophagy and reduce intracellular insulin synthesis. The data show that rapamycin increase autophagy and subsequently result in a decrease in insulin production.
This Data could give a base for the detection of intracellular insulin in both cultured isolated islets and pancreatic β-cells by Western blotting analysis. The data support the development of intracellular insulin detection by drug-induced autophagy.

Data
The autophagy was increased by 2-DG or rapamycin treatment and a subsequent decrease in insulin production (Figs. 1 and 2). Immunofluorescence staining data showed that 2-DG or rapamycin increased LC3-positive granules or puncta were co-localized with the increased Lamp2, indicating that autophagosomes increase under conditions of 2-DG or rapamycin (Fig. 3). Subsequently, an increase in intracellular insulin was measured in INS-1E insulinoma cells following treatment with the autophagy inhibitors, bafilomycin A1 and/or 3-methyladenine, in the presence or absence of 2-DG ( Fig. 4A and B) or rapamycin ( Fig. 4C and D).

Immunof luorescence staining
We performed as described previously [5]. INS-1E cells grown on culture slides (BD Falcon Labware, REF 354108) were permeabilized and fixed in methanol at À20°C for 3 min. Cells were washed with phosphate-buffered saline (PBS), blocked with 10% bovine serum albumin (Sigma-Aldrich) in PBS for 10 min, and incubated with primary antibody in blocking buffer for 1 h at room temperature (RT). Cells were hybridized with secondary antibodies for 1 h at RT. The coverslips were mounted on glass slides using Vectashield mounting medium (Vector Labs Inc., Burlingame, CA, USA). Cells were viewed under a Leica TCS SP5 confocal microscope (Leica, Microsystems CMS GmbH, Wetzlar, Germany). The following primary antibodies were used: Lamp2 (Cell Signaling Technology) and LC3 (Cell Signaling Technology). The following secondary antibodies were used: Alexa 594 (red)-conjugated anti-rabbit IgG (Vector Laboratories Inc.) and fluorescein isothiocyanate (green)-labeled anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Cells stained with 4,6-diamidino-2-phenylindole (DAPI, Santa Cruz Biotechnology) for 10 min.

Statistical analysis
Significant differences were determined by ANOVA, followed by Tukey's test for multiple comparisons. Analysis was performed using the Prism Graph Pad v4.0 (Graph Pad Software Inc., San Diego, CA, USA). Values are expressed as means 7SD of at least three separate experiments, of which a representative experiment is depicted in the figures. P values o0.05 were considered statistically significant.