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Introduction

Pancreatic β cells are key regulators of the process of glucose tolerance, and proper β cell function is essential for the maintenance of blood glucose homeostasis. Notably, defects in the mass and function of β cells are required for type 2 diabetes to develop (1). In the early phase of glucose metabolism abnormality, the levels of blood sugar are kept relatively stable through the compensatory function of islet β cells. When the limits of this compensatory function are exceeded, apoptosis of the pancreatic β cells increases (2). The mechanism may be associated with disorders of mitochondrial function, oxidative stress and elevated levels of proinflammatory cytokines (3). In vivo, estrogen treatment has been demonstrated to decrease β cell apoptosis and restore a certain degree of insulin secretion in mice (4). Estrogen is a steroid hormone that serves an important role in physiological processes by binding with intracellular receptors. However, estrogen receptors on the membranes of pancreatic β cells are not yet completely understood. The existence of a non-classical membrane estrogen receptor has been described by Nadal et al (5) and Ropero et al (6). Other researchers have suggested a possible role for G-protein coupled receptor 30 (GPR30) as an estrogen receptor involved in the effects of estrogen in the endocrine pancreas (7,8). In the review conducted by Ropero et al (9), a model for the roles of estrogen receptor β and GPR30 in the physiology of the endocrine pancreas was presented, and it was suggested that the GPR30 is expressed in mice and rat pancreatic β cells. GPR30 is a 17β-estradiol (17β-E2)-binding receptor, which is also known as the G protein-coupled estrogen receptor (GPER) (10). The 17β-E2-mediated activation of the GPER stimulates intracellular calcium mobilization and phosphoinositide 3-kinase (PI3K) activation (11). The functions and importance of the GPER in pancreatic function and glucose metabolism have been elucidated, which has revealed the therapeutic potential of GPER activity (12).

The PI3K/Akt pathway is normally activated by extracellular signals in physiological conditions, such as growth factors, cytokines and hormones. It has been demonstrated that G protein-coupled receptors directly stimulate the p110β and p110γ isoforms of PI3K via the βγ subunits of heterotrimeric G proteins, and activate the p110δ isoform of PI3K in β cells by an unknown mechanism (13–15). For this reason, it would be interesting to detect whether 17β-E2 activates the PI3K/Akt signaling pathway via the GPER in an insulin-secreting β-cell line (INS-1).

Autophagy is a biological process by which cytoplasmic macromolecules and unnecessary organelles are degraded in membranous vesicles, and is a widely present biological phenomenaonin eukaryotes (16,17). Autophagy serves an important role in various human diseases (18). It is becoming clear that the regulation of autophagic activity is associated with tumor formation and progression, and is also important to cancer therapy (19). In cancer cells, chemical inhibitors of autophagy increase the apoptosis induced by active-site mechanistic target of rapamycin (mTOR) inhibitors or dual PI3K/mTOR inhibitors, which suggests that the PI3K/Akt pathway has an important role in the process of autophagy (20,21). Autophagy of the mitochondria, also known as mitophagy, is important for mitochondrial quality control, and thus is essential in cellular energy provision, calcium homeostasis, redox signaling and apoptotic signaling (22,23). The health of the mitochondria is important, because mitochondria are essential to major cell metabolic pathways, and are a major cause of cell death (24). Mitochondrial dynamics, including fission and fusion, serve a key role in mitophagic signaling (25,26). 17β-E2 has been indicated to increase mitochondrial fusion, decrease fission processes and modify the normal development and function of mitochondria in MCF-7 breast cancer cells (27). According to the review of Zhang (28), it is possible to visualize mitophagy by the co-localization of mitochondrial proteins with lysosomal markers.

The aim of the present study was to investigate whether 17β-E2 regulates the PI3K/Akt pathway through the GPER and whether 17β-E2 inhibits mitophagy by activating the GPER/PI3K/Akt signaling pathway and thereby protects INS-1 cells.

Materials and methods

Reagents

17β-E2 was obtained from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). RPMI-1640 medium and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The selective GPER antagonist (G15) was bought from Tocris Bioscience (Minneapolis, MA, USA). The PI3K inhibitor (LY294002) and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Beyotime Institute of Biotechnology (Jiangsu, China). GPER antibody [sc-48524-R; polyclonal, rabbit anti-mouse, rat, human; western blotting (WB) 1:200; immunofluorescence (IF) 1:50], lysosomal-associated membrane protein 2 (LAMP2) antibody (sc-8100; polyclonal, goat anti mouse, rat and human; IF 1:50), the translocase of the mitochondrial outer membrane complex 20 (TOM20) antibody (sc-11415; polyclonal, rabbit anti mouse, rat and human; WB 1:200, IF 1:50), microtubule-associated protein-1 light chain 3 (LC3) antibody (sc-376404; monoclonal, mouse anti mouse and rat; WB 1:100), and the mitochondrial heat-shock protein 60 (Hsp60) antibody (sc-1052; polyclonal, goat anti mouse, rat and human; WB 1:200) were all obtained from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Total/phospho-Akt (t-Akt/p-Akt) polyclonal antibody (1:1,000; Ab8805/Ab8932) was obtained from Abcam (Cambridge, MA, USA). β-actin antibody (AF0003; monoclonal, mouse anti-human/mouse/rat; WB 1:1,000) was acquired from Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China). An immunofluorescence staining kit with Alexa Fluor 555-labeled donkey anti-rabbit immunoglobulin G (lgG) (P0179) was purchased from Beyotime Institute of Biotechnology. IFKine® Green conjugated donkey anti-goat IgG (A24231) was obtained from Abbkine Scientific Co., Ltd. (Redlands, CA, USA).

Cell culture

The rat insulin-secreting β-cell line (INS-1) was obtained from China Infrastructure of Cell Line Resources (Beijing, China) and maintained in RPMI-1640 medium supplemented with 10% FBS, L-glutamine, 50 mg/ml penicillin and 100 mg/ml streptomycin (Beyotime Institute of Biotechnology). The cells were grown in a humidified atmosphere containing 5% CO2 at 37°C. Prior to the experiment, the INS-1 cells were grown in Petri dishes in a serum-free medium for 24 h. The following day, the INS-1 cells were treated with different concentrations of 17β-E2 (0, 1, 10 and 100 nM), or with 100 nM 17β-E2 plus 15 µM G15 or 20 µM LY294002 for 24 h, respectively. For the combination treatments, the cells were pretreated with G15 or LY294002 for 30 min prior to treatment with 17β-E2.

IF analysis

The INS-1 cells were fixed in 4% paraformaldehyde buffered with 0.1 M phosphate (pH 7.3) for 30 min at room temperature and then washed with phosphate-buffered saline (PBS). The cells were permeabilized with 0.1% Triton X-100 for 30 min and washed with PBS. Following blocking with 5% bovine serum albumin (Gibco) in Tris-buffered saline with Tween-20 (TBST) for 30 min at room temperature, the INS-1 cells were incubated with specific primary antibodies targeting LAMP2, TOM20 and GPER overnight at 4°C. The next day, the INS-1 cells were washed with PBS and then incubated with donkey anti-goat IgG (1:1,000) or donkey anti-rabbit IgG (1:1,000) secondary antibody for 30 min at 37°C. Subsequently, the cells were stained with DAPI for 5 min at room temperature. After washing with PBS for 15 min, the stained cells were viewed using an Olympus FV1000 confocal laser-scanning microscope (Olympus Corporation, Tokyo, Japan) with peak emission wavelengths of 518 nm (green) and 565 nm (red).

Analysis using transmission electron microscopy (TEM)

The cells were treated and collected by trypsinization, fixed with 2.5% phosphate-buffered glutaraldehyde at room temperature for 4 h, and post-fixed in 1% phosphate-buffered osmium tetroxide at 4°C for 1.5 h. The cells were embedded using Epon 812 epoxy resin at room temperature overnight, sectioned, double stained with uranyl acetate and lead citrate at room temperature for 23 min, and analyzed using a JEM-1200EX transmission electron microscope (Jeol, Ltd., Tokyo, Japan).

Protein preparation and western blot analysis

The INS-1 cells were washed with cold PBS and harvested in radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology) containing protease inhibitors, including phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology) and phosphatase inhibitors (Nanjing KeyGen Biotech Co., Ltd., Nanjing, China). The cell lysates were incubated on ice for 30 min, and then collected and centrifuged at 12,000 × g for 10 min at 4°C. The supernatants were collected, mixed with 5X loading buffer and denatured by boiling for 10 min. The samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10% gel used) and transferred onto polyvinylidene fluoride membranes at 70 V for 1.5 h in a transfer buffer containing Tris (20 mM; bioWORLD, Dublin, OH, USA), 150 mM glycine (Beijing Solarbio Science and Technology Co., Ltd., Beijing, China) and 20% methanol (Liaoning Xinxing Chemical Group Co., Ltd., Liaoning, China). The protein determination of the samples was made using the Bicinchoninic Acid (BCA) method; 50 µg samples were loaded per lane. The membranes were incubated in non-fat dry milk for 120 min at room temperature, and were then washed thrice with TBST for 30 min. The membranes were incubated with primary antibodies in TBST overnight at 4°C. The membranes were then washed and incubated with horseradish peroxidase-conjugated anti-species secondary antibodies (1:1,000; A0208 and A0216; Beyotime Institute of Biotechnology) for 2 h at room temperature, and were washed thrice with TBST for 30 min. Proteins were visualized using the BeyoECL plus kit (Beyotime Institute of Biotechnology) and quantified using Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Statistical analysis

Data are expressed as the mean ± standard error of the mean, and the differences between the means were analyzed by one-way analysis of variance followed by a Dunnett post hoc test. P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using the Statistical Package for the Social Sciences (SPSS) 15.0 software package (SPSS, Inc., Chicago, IL, USA).

Results

GPERs are expressed in INS-1 cells

Since it has been shown GPERs are expressed in MIN6 cells (9), a mouse β-cell line, the present study aimed to detect the presence of GPERs in rat INS-1 cells. The presence of GPERs in the INS-1 cells was investigated using IF staining. As shown in Fig. 1, GPERs were present in the INS-1 cells. No GPER expression was detected in the negative controls lacking primary antibody (data not shown).

Figure 1 GPER is expressed in INS-1 cells. Immunofluorescence staining of INS-1 cells for GPER (red). Nuclei were stained with DAPI (blue). Results are representative of three different experiments. Scale bar, 10 µm. GPER, G protein-coupled estrogen receptor; INS-1, rat insulin-secreting β-cell line; DAPI, 4′,6-diamidino-2-phenylindole.

17β-E2 regulates the PI3K/Akt pathway via the GPER in INS-1 cells

17β-E2 (1, 10 and 100 nM) caused the expression levels of GPER protein to increase in an apparently dose-dependent manner, with a significant increase detected at a concentration of 100 nM (P<0.05; Fig. 2). In the 100 nM group, the expression level of GPER protein was increased 2.2-fold in comparison with the control. However, the stimulatory effect of 17β-E2 was eradicated by 15 µM G15, a GPER-specific antagonist (Fig. 2).

Figure 2 17β-E2 affects GPER expression in INS-1 cells. Serum-starved INS-1 cells were cultured with 17β-E2 at different concentrations (0, 1, 10 and 100 nM) with or without 15 µM G15 for 24 h. Prior to treatment with 17β-E2, the cells were pretreated with G15 for 30 min. Protein activation was measured by western blot analysis. Data are presented as the mean ± standard error of the mean based on triplicate determinations from a representative experiment. *P<0.05 vs. control (0 M group). 17β-E2, 17β-estradiol; GPER, G protein-coupled estrogen receptor; INS-1, rat insulin-secreting β-cell line.

The ability of 17β-E2 to regulate the PI3K/Akt pathway via the GPER in INS-1 cells was investigated via western blot analysis. The results demonstrated that 100 nM 17β-E2 significantly increased the protein levels of p-Akt in INS-1 cells (P<0.05; Fig. 3). However, the protein expression level of t-Akt remained unchanged. The effect of 17β-E2 on Akt activation in the INS-1 cells was blocked by the PI3K inhibitor LY294002 (20 µM) and the GPER inhibitor G15 (15 µM) (Fig. 3).

Figure 3 17β-E2 regulates the PI3K/Akt pathway via the GPER in INS-1 cells. Serum-starved INS-1 cells were cultured with 17β-E2 at concentration of 100 nM with or without 20 µM LY294002 or 15 µM G15 for 24 h. Prior to treatment with 17β-E2, the cells were exposed to LY294002 or G15 for 30 min. Protein activation was measured by western blot analysis. Data are presented as the mean ± standard error of the mean based on triplicate determinations from a representative experiment. *P<0.05 vs. control (untreated). 17β-E2, 17β-estradiol; PI3K, phosphoinositide 3-kinase; GPER, G protein-coupled estrogen receptor; INS-1, rat insulin-secreting β-cell line; p, phospho; t, total.

17β-E2 protects INS-1 cells from mitophagy via the GPER

Mitophagy can be visualized by the detection of mitophagosomes or autophagosomes using TEM or the co-localization of lysosomes and mitochondria with mitophagic proteins by IF staining. Whether 17β-E2 is involved in mitophagy in INS-1 cells was investigated using these methods in the present study.

Mitophagosomes were detected in the INS-1 cells by TEM, indicating that mitophagy occurs in INS-1 cells. Compared with the control group, fewer mitophagosomes were observed in the INS-1 cells treated with 17β-E2 (Fig. 4). Greater numbers of double-membrane vacuoles were observed in the INS-1 cells treated with 17β-E2 and G15 than that in the cells with 17β-E2 only.

Figure 4 17β-E2 protects INS-1 cells from mitophagy via the GPER. Transmission electron microscopy images indicating double membrane vacuoles in INS-1 cells treated with 100 nM 17β-E2 with or without the presence of 15 µM G15 for 24 h (original magnification, ×12,000). G15 was applied as a pretreatment for 30 min. Arrows indicate vacuoles. Results are representative of three different experiments. 17β-E2, 17β-estradiol; INS-1, rat insulin-secreting β-cell line; GPER, G protein-coupled estrogen receptor; N, nucleus.

In addition, the co-localization of TOM20 with LAMP2 was detected by IF staining. The results demonstrated a reduction in TOM20-positive granules and a co-localized reduction in LAMP2 expression in the INS-1 cells exposed to 17β-E2, indicating that the numbers of mitophagosomes or autophagosomes were decreased (Fig. 5). However, these effects of 17β-E2 were eliminated by the presence of G15 (Fig. 5).

Figure 5 17β-E2 protectes INS-1 cells from mitophagy via the GPER. Immunofluorescence staining of TOM20 (red) and LAMP2 (green), nuclear staining (DAPI, blue) and merged images. Serum-free INS-1 cells were cultured with 17β-E2 at concentration of 100 nM with or without G15 and LY294002 for 24 h. Prior to treatment with 17β-E2, the cells were exposed to G15 or LY294002 for 30 min. Results are representative of three different experiments. Scale bar, 10 µm. 17β-E2, 17β-estradiol; INS-1, rat insulin-secreting β-cell line; GPER, G protein-coupled estrogen receptor; TOM20, translocase of the mitochondrial outer membrane complex 20; LAMP2, lysosomal-associated membrane protein 2.

17β-E2 is involved in mitophagy through the GPER/PI3K/Akt pathway

Whether 17β-E2 participates in mitophagy through the PI3K/Akt pathway was then investigated. The results of IF staining revealed that there were increased numbers of LAMP2-positive granules with increased TOM20 expression in the INS-1 cells treated with 17β-E2 and LY294002 compared with the cells treated with 17β-E2 alone (Fig. 5).

17β-E2 regulates the expression of LC3, TOM20 and Hsp60 in INS-1 cells

The present study suggested that 17β-E2 may be involved in mitophagy in INS-1 cells, on the basis of the detection of mitophagosomes and autophagosomes by TEM and mitophagy-related protein co-location by IF. To further investigate this, the expression of LC3, TOM20 and Hsp60 was detected by western blot analysis. LC3-II is converted from LC3-I, and serves as a typical marker of completed autophagy (29). The exposure of INS-1 cells to 17β-E2 for 24 h resulted in the LC3-II protein levels being significantly reduced compared with those of the control group (Fig. 6). However, pretreatment with LY294002 or G15 attenuated the 17β-E2-induced reduction in LC3-II expression (Fig. 6). The 17β-E2 group also had significantly higher expression levels of TOM20 and Hsp60 compared with the control group (Fig. 6). However, the effect of 17β-E2 on these proteins was eradicated by pretreatment with LY294002 or G15 (Fig. 6).

Figure 6 17β-E2 regulates the expression of LC3, TOM20 and Hsp60 in INS-1 cells. Serum-free INS-1 cells were treated with 100 nM 17β-E2 with or without LY294002 (20 µM) or G15 (15 µM) for 24 h. Prior to exposure to 17β-E2, the cells were pretreated with LY294002 or G15 for 30 min. The protein expression was measured by western blot analysis. Data are presented as the mean ± standard error of the mean based on triplicate determinations from a representative experiment. *P<0.05 vs. control (untreated). 17β-E2, 17β-estradiol; LC3, microtubule-associated protein-1 light chain 3; GPER, G protein-coupled estrogen receptor; TOM20, translocase of the mitochondrial outer membrane complex 20; Hsp60, mitochondrial heat-shock protein 60; INS-1, rat insulin-secreting β-cell line.

Discussion

In the present study, the involvement of 17β-E2 in the process of mitophagy in INS-1 cells was investigated. The results indicated that 17β-E2 protects INS-1 cells from mitophagy, with this regulatory effect likely occurring through the GPER/PI3K/Akt pathway.

The PI3K/Akt signaling pathway is involved in almost every aspect of the physiological and pathological functions of cells, including growth, tumorigenesis, apoptosis and autophagy (30–32). The study conducted by Ropero et al (9) demonstrated that the INS-1 rat β-cell line expresses GPERs. The present study confirmed the expression of GPERs in INS-1 cells. It has been reported that E2 stimulates insulin secretion and induces glucagon secretion via the GPER in pancreatic islets (7). Therefore, the present study aimed to detect whether 17β-E2 regulates the PI3K/Akt signaling pathway via the GPER in INS-1 cells. Whether Akt phosphorylation levels are modulated by 17β-E2 via a GPER-dependent pathway was tested in the present study. The phosphorylation level of Akt was significantly increased following stimulation with 17β-E2 for 24 h, and the effect was blocked by pretreatment with the GPER and PI3K antagonists G15 and LY294002, respectively, for 30 min, indicating that the activation of the PI3K/Akt signaling pathway in INS-1 cells is regulated by 17β-E2 through the GPER. Additionally, 17β-E2 has been demonstrated to increase p-Akt levels via the GPER in cardiomyocytes, which may rescue the heart from pathological hypertrophy (33). The rapid phosphorylation of Akt by E2 has been shown to upregulate miR144 in SkBr3 breast cancer and HepG2 hepatocarcinoma cells, and this process has been indicated to occur in cancer-associated fibroblasts and cancer progression (34). Similar GPER-mediated modulatory effects of E2 on Akt phosphorylation have been demonstrated in endometrial cancer cells (35). However, E2 did not activate the PI3K/Akt pathway via the GPER in MCF-7 and MCF-10A cells (36). Therefore, the mechanism by which 17β-E2 activates the PI3K/Akt pathway remains unclear. To fully ascertain how 17β-E2 acts through the GPER, the use of a specific agonist of this receptor (such as G1) is planned in future research.

The association between 17β-E2 and mitophagy has become a topic of particular research interest. Mitochondria in pancreatic β cells are continuously recruited in fusion and fission processes (37). In an animal model of spinal cord injury, treatment with 17β-E2 significantly attenuated cell death (38). Furthermore, in a study conducted by Sastre-Serra et al (27), 17β-E2 increased mitochondrial fusion, decreased fission processes, and modified the normal development and functions of mitochondria in MCF-7 breast cancer cells through estrogen receptors. In the present study, 17β-E2 was demonstrated to protect INS-1 cells from mitophagy via the GPER. The presence of mitochondria in autophagosomes (mitophagosomes) was identified using TEM, which indicates that mitophagy occurs in INS-1 cells. TEM analysis demonstrated that there were few mitophagosomes and scarcely any autophagomes in the INS-1 cells treated with 17β-E2. However, the number of mitophagosomes and autophagomes was increased in the INS-1 cells cultured with 17β-E2 following pretreatment with G15. Therefore, it is suggested that the protective effect of 17β-E2 against mitophagy may occur through the activation of the GPER. This observation may be associated with inhibitory effect of 17β-E2 on the mitochondrial fission process, and may also indicate that the GPER serves an important role in this protective process.

It has been reported that PI3K/Akt signal transduction prevents cells from undergoing apoptosis (30,39). The PI3K/Akt pathway, the p38 mitogen-activated protein kinase signaling pathway, and reactive oxygen species participate in wogonoside-induced autophagy and apoptosis (40). Inactivation of the PI3K/Akt pathway prevents the translocation of damage-regulated autophagy modulator to the mitochondria and induces apoptosis in hepatocellular carcinoma cells by the mediation of mitophagy (41). Furthermore, inhibition of the PI3K/Akt/mTOR signaling pathway is accompanied by autophagy and mitophagy in human glioblastoma cells, glioblastoma stem cells and human prostate cancer (42,43). In the present study, the results indicated that 17β-E2 regulated mitophagy in INS-1 cells through the GPER/PI3K/Akt pathway. In addition, IF analysis of the INS-1 cells revealed that 17β-E2 reduced the number of LAMP2-labeled lysosomes that were co-localized with TOM20-labeled mitochondria in comparison with the cells treated with 17β-E2 following G15 or LY294002 pretreatment, which exhibited an increased presence of mitophagosomes, indicating that 17β-E2 alone reduced the formation of mitophagosomes. These results are consistent with the TEM results of the present study. Therefore, it is suggested that the PI3K/Akt pathway is also involved in the mechanistic pathway by which 17β-E2 exerts a protective effect on INS-1 cells via the GPER. Furthermore, the results of western blot analysis also supported this suggestion. As shown in Fig. 6, the expression of LC3-II was decreased and the expression of TOM20 and Hsp60 was increased in INS-1 cells exposed to 17β-E2, and this was partially consistent with the observations concerning the 17β-E2-induced suppression of mitophagy in INS-1 cells. mTOR is a downstream factor of the PI3K/Akt signaling pathway, which has an important role in the modulation of mitophagy (44–46). The inhibition of mTOR increases mitophagic activity through a ubiquitin-like-conjugating enzyme ATG3-dependent mechanism in natural killer cells (47). Therefore, it is hypothesized that 17β-E2 activates the PI3K/Akt signaling pathway by means of the GPER, which may subsequently inhibit mitophagy via the regulation of mTOR.

In conclusion, the results of the present study demonstrate that 17β-E2 protects INS-1 rat insulinoma cells from mitophagy via the GPER and acts through the PI3K/Akt signaling pathway. These results provide novel insights for understanding the pathophysiological functions of the GPER in pancreatic β cells.

Glossary

Abbreviations

Abbreviations:

Acknowledgments

The authors would like to thank the China Medical University Affiliated Hospital Laboratory Center for kindly providing the equipment. The present study was supported by the National Natural Science Foundation of China (grant nos. 81470998, 81071460 and 81271996).

Notes

[1] Competing interests

The authors declare that they have no competing interests.

References