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Estradiol Prevents High Glucose-Induced β-cell Apoptosis by Decreased BTG2 Expression

Scientific Reportsvolume 8, Article number: 12256 (2018) | Download Citation

Abstract

Hyperglycemia stimulates several pathways to induce pancreatic β-cell apoptosis. In our previous study by mRNA analysis, we demonstrated that B-cell translocation gene 2 (BTG2) expression was up-regulated in INS-1 cells cultured under high glucose conditions, but this effect was reversed by estrogen. In the present study, we demonstrated that BTG2 mRNA and protein expressions in both INS-1 cells and mouse pancreatic islets increased under high glucose conditions compared to those cultured under basal glucose conditions, while in the presence of estrogen, the BTG2 mRNA and protein expressions decreased. SiRNA-BTG2 significantly reduced cell apoptosis, cleaved-caspase 3, and Bax, compared to the siRNA-control in INS-1 cultured under high glucose conditions. We further demonstrated that BTG2 promoter activity was activated under high glucose conditions whereas estrogen significantly reduced it. The effects of estrogen on BTG2 expression were inhibited by estrogen receptor inhibitors. Also, under high glucose conditions, p53 and Bax mRNA and protein expressions increased, but they decreased in the presence of estrogen. Again, the effect of estrogen on p53 and Bax expression was inhibited by estrogen receptor inhibitors. Taken together, this study demonstrates that estrogen reduces pancreatic β-cell apoptosis under high glucose conditions via suppression of BTG2, p53, and Bax expressions.

Introduction

Hyperglycemia induces pancreatic β-cell apoptosis through several pathways, including glyceraldehyde autoxidation, protein kinase C (PKC) activation, glycation, sorbitol metabolism, hexosamine pathway and oxidative phosphorylation1,2,3,4. However, it is possible that a novel pathway is still undiscovered. This study explored whether a possible novel pathway of high-glucose-increased pancreatic β-cell apoptosis, our preliminary work suggested that high glucose up-regulated of B-cell translocation gene 2 (BTG2) mRNA expression when compared to basal glucose.

BTG2 is also known as pheochromocytoma cell 3 (PC3) in the rat and tetradecanoyl phorbol acetate-inducible sequence 21 (TIS21) in the mouse5. BTG2 belongs to the BTG/Tob gene family6. BTG2 is known to have both physiological and pathological processes7. BTG2 is an antiproliferative (ARPO) tumor suppressor protein which is involved in cellular function, cell cycle progression, cell migration, cellular growth and differentiation, and apoptosis7. BTG2 roles are different in tumor cells. For example, a high level of BTG2 is associated with a poor prognosis in bladder cancer patients8. Cisplatin up-regulated BTG2-attenuated prostate cancer cell proliferation9. BTG2 inhibits cell invasion and proliferation in gastric cancer10. In normal cells, BTG2 mediates hepatic gluconeogenesis via induction of CREB in liver cells11. In the pancreatic β-cell line, GLP-1 positively increased BTG2 expression which up-regulated PDX-1 to increase insulin secretion12. BTG2 expression is induced by growth factors, DNA damage, and cytotoxic and genotoxic stress through a p53-dependent or p53-independent mechanism13,14. However, high-glucose-regulated BTG2 expression has not been studied. Also, the role of BTG2 in pancreatic β-cells apoptosis is still unknown.

Estrogen has been shown to prevent diabetes by increased glucose metabolism15 and antioxidants16. For example, in estrogen-deficient animal models, estrogen receptor α-deficient (α ERKO) or aromatase-deficient (ArKO−/−) mice, insulin resistance and abnormal metabolism are developed, which are risk factors for diabetes16,17. Estrogen replacement in estrogen-deficient animal models protects pancreatic β-cell apoptosis against streptozotocin18. Estrogens protected pancreatic β-cell from oxidative stress-induced apoptosis16,19,20 and gluco-lipotoxicity21 in mouse and human islets and protect survival of human islets transplanted in diabetic mice in vivo22. A previous study from our group demonstrated that estrogen protects against high glucose-induced pancreatic β-cell apoptosis via reduction of endoplasmic reticulum (ER) stress and oxidative stress23. The effect of estrogen on regulated BTG2 expression is still unrevealed in pancreatic β-cells.

Our preliminary results indicated that high glucose up-regulated BTG2 mRNA expression and estradiol suppressed BTG2 mRNA expression. Therefore, we hypothesized that estradiol protects pancreatic β-cell apoptosis against glucotoxicity via BTG2 suppression. This study aimed to examine whether or not estradiol suppresses BTG2 expression to prevent high-glucose-induced pancreatic β-cell apoptosis.

Materials and Methods

Animals

The animal experimentation protocol was approved by the Institutional Animal Care and Use Committee, Faculty of Medicine, Siriraj Hospital, Mahidol University (Approval No: SI-ACUP 002/2553). Male ICR outbred 8–12 week old mice were purchased from the National Laboratory Animal Center, Mahidol University, Bangkok, Thailand. The mice were kept in a 12-h light/dark cycle environment at 25° ± 2 °C and 60% humidity. They were housed 5–6 per cage with a wooden chip bedding, and were provided chow pellet ad libitum (Perfect Companion Group Co., Ltd., Bangkok, Thailand).


INS-1 cell culture

INS-1 cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in humidified air containing 5% CO2, and the culture media were changed every 2 days.


Mouse pancreatic islet isolation and culture

Pancreatic islets were isolated by collagenase digestion using the modified method of Lacy & and Kostianovsky24 and Gotoh25. Briefly, pancreases were infused with collagenase-P and digested at 37 °C. The islets were separated by using a histopaque gradient and manually picked under a stereomicroscope. Isolated islets were cultured in an RPMI 1640 medium supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in 5% CO2. The culture medium was changed every 2 days. All methods were carried out in accordance with ACUC guidelines.


Cleaved-caspase 3 activity assay

INS-1 cells were cultured either in normal or high glucose RPMI 1640 media, with or without 10–8 M 17 β-estradiol, in a 96-well plate for 72 h. The cleaved-caspase 3 activity was measured using a Caspase-Glo assay kit (Promega, USA). The assay was performed following the manufacturer’s protocol. Briefly, Caspase-Glo 3/7 Reagent was added to the cell culture plate, which was subsequently shaken gently at 300–500 rpm for 30 seconds. The plate was then incubated at room temperature for 30 minutes. The luminescence of each sample was measured in a plate-reading luminometer.


RNA isolation and reverse transcriptase-polymerase chain reaction

The total RNA was extracted from INS-1 cells or mouse pancreatic islets by using the High Pure RNA Isolation Kit (Roche Diagnostics Corporation, USA) and following the manufacturer’s instructions. The concentration of total RNA was measured with a ND-1000 Spectrophotometer (Nanodrop, USA). First-strand complementary DNA (cDNA) was generated from 0.5–1 μg of total RNA using the SuperScript III Reverse Transcriptase (RT) and Random Hexamer Primer (Invitrogen, USA) according to the manufacturer’s instructions. Primers were synthesized by Sigma-Aldrich (Sigma-Aldrich, USA). The rat primers for real-time PCR were as follows. The BTG2 forward primer was 5′-GGT TGG AGA AAA TCG GGA AAC-3′, and the reverse primer was 5′-GCC TTC TGA GAA GCC CTC ATC C-3′26. The Bax forward primer was 5′-CCA GGA CGC ATC CAC CAA GAA GC-3′, and the reverse primer was 5′-TGC CAC ACG GAA GAA GAC CTC TCG-3′27. The β-Actin forward primer was 5′-ATG AAG TGT GAC GTT GAC ATC GTC-3′, and the reverse primer was 5′-CCT AGA AGC ATT TGC GGT GCA CGA TG-3′. The real-time PCR for mouse primers were as follows. The BTG2 forward primer was 5′-GGT TGG AGA AAA TTG GGA AAC-3′, and the reverse primer was 5′-GCC TTC TAA GAA GCC CTC ATC-3′. The real-time PCR was performed to amplify specific DNA sequences with the Brilliant II SYBR Green QPCR Master Mix (Agilent Technologies, USA). The reactions were carried out on the Mx3005P instrument (Stratagene, USA). The quantity of gene expression was calculated by the 2−∆∆Ct method and was presented as fold changes, compared to those of the control.


Small interference RNA (siRNA) transfection

Transfection of siRNA directed against BTG2 mRNA (Dharmacon, USA) was performed using Lipofectamine 2000 (Invitrogen, USA), as detailed by the manufacturer. INS-1 cells were seeded into a 6-well plate for 24 h before transfection. The double-stranded siRNAs were transfected. After 6 h, the medium was changed to complete the culture medium. As a control, the cells were treated with siRNA-Control (Dharmacon, USA) under identical conditions. Twenty-four h after the siRNA transfection, the cells were treated with 11.1 mM or 40 mM glucose for 72 h. They were then harvested, and the BTG2, cleaved caspase-3 and Bax were determined using Western blotting. As for the cell lysate preparation, apoptotic and adhered cells were extracted with an RIPA buffer. The lysate was subjected to 15% SDS-PAGE, and the protein expression of BTG2, cleaved caspase-3 and Bax were determined by immunoblotting. BTG2 was detected by the anti-BTG2 antibody (Santa Cruz Biotechnology, USA), the anti-cleaved caspase-3 antibody (Cell Signalling, USA), rabbit polyclonal anti-Bax (Santa Cruz Biotechnology, USA), or the anti-β-actin antibody (Santa Cruz Biotechnology, USA) as an internal control. The membrane was probed with horseradish peroxidase-conjugated antibody. The immunoreactive proteins were visualized by SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific, Rockford, IL, USA), and were detected by using a G:BOX chemiluminescence imaging system (Syngene, Frederick, MD, USA).


Western blotting analysis

The total protein of INS-1 cells and mouse pancreatic islets were extracted by using a RIPA buffer. Nuclei proteins were extracted from the cells by using the Nuclear and Cytoplasmic Extraction Reagent Kit (Pierce, USA). The protein concentrations were then determined by a micro BCA assay. The total protein was separated on a 4–12% (wt/vol) SDS-PAGE. After that, the protein was transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, USA). The membrane was blocked with 5% skimmed milk before being incubated overnight at 4 °C with one of the following primary antibodies: rabbit polyclonal anti-BTG2 (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-p53 (Santa Cruz Biotechnology, USA), rabbit polyclonal anti-Bax (Santa Cruz Biotechnology, USA), or mouse monoclonal anti-β-Actin (Santa Cruz Biotechnology, USA). After washing, the membrane was incubated with one of the following secondary antibodies: horseradish peroxidase-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, USA), or horseradish peroxidase-conjugated anti-mouse IgG (Santa Cruz Biotechnology, USA), at room temperature. The protein bands were detected with an enhanced chemiluminescence system (Pierce Biotechnologies, USA) and exposed on x-ray films. The band intensities of proteins were quantified by using ImageJ v 1.43 software. All Western blot results were shown in supplement data.


Promoter assay

The INS-1 BTG2 promoter (−43 to −1802) was amplified from INS-1 genomic DNA by PCR using Pfu DNA polymerase (Stratagene, La Jolla, CA, USA). The PCR products of the BTG2 promoter were confirmed by automated DNA sequencing before being separately subcloned into pGL3 reporter vectors to generate INS-1 BTG2 promoter-firefly luciferase reporter plasmids.

The INS-1 cells were transfected with 1 µg luciferase reporter plasmid, pGL3-basic, or pGL3-Btg2 gene promoter together with an internal control renilla luciferase plasmid, pRL-SV40. After transfection and culturing for 24 h, the culture medium was changed into a basal glucose-containing medium or a high glucose-containing medium, with or without 10−8 M estrogen, before being cultured for 72 h. The firefly luciferase activity was normalized by the internal control renilla luciferase activity. The dual-luciferase reporter assay was performed according to the manufacturer’s instructions (Promega Corp., Fitchburg, WI, USA). The experiments were performed in six-plicate and on three independent occasions.


Statistical Analysis

Data were analyzed by using SPSS Statistics for Windows, version 17 (SPSS Inc., Chicago, Ill., USA) and expressed as mean ± standard error of mean (S.E.M). The differences between the groups of results were determined by one-way ANOVA, followed by Tukey’s post hoc test. A P-value less than 0.05 was considered to be statistically significant.

Results

Estradiol increased pancreatic β-cell viability after culture under high glucose conditions

To examine whether estradiol increased pancreatic β-cell viability under high glucose conditions, INS-1 cells were cultured under different conditions before measuring the apoptotic cell death by the cleaved-caspase 3 activity. INS-1 cells cultured in normal glucose were used as a control, and 10−8 M 17-β estradiol did not change the cleaved-caspase 3 activity compared to that of the control. As expected, the cleaved-caspase 3 significantly increased in INS-1 cells cultured in a high glucose medium compared to that of the control. In contrast, INS-1 cells cultured in a high glucose medium with 10−8 M 17-β estradiol significantly reduced the cleaved-caspase 3, suggesting that estrogen increased viable cells when the cells were cultured in high glucose (Fig. 1A).

Figure 1
Figure 1

Cell viabilities were measured by cleaved-caspase 3 activity. (A) The cell viability in INS-1 cells was determined using cleaved-caspase 3 activity. (B) Fold change of BTG2 mRNA normalized to β-actin mRNA at 48 h. (C) A representative Western blot analysis of BTG2 and β-actin from INS-1. The bar graph below is BTG2 protein level normalized to β-actin protein. The data is presented as mean ± S.D. of 3 independent experiments. The data IS presented as mean ± S.D. of 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, compared to the high-glucose-treated group.

High glucose conditions increased BTG2 expression in pancreatic β-cells, and effect reversed by estradiol

To identify the signaling pathway of estradiol that decreased pancreatic β-cell death against the high (40 mM) glucose medium, a signaling RT² Profiler PCR Array was performed. The preliminary results suggested that the BTG2 mRNA expression was higher in the high glucose medium than in the normal glucose medium (data not shown). To confirm the RT² Profiler PCR Array results, a conventional real-time PCR was performed for the samples from the experimental conditions. INS-1 cells cultured in the high glucose medium had a significantly increased BTG2 mRNA expression compared to those cultured in normal glucose. The presence of estradiol in the high glucose medium significantly reduced the BTG2 mRNA expression (Fig. 1B). The BTG2 protein expression corresponded with the BTG2 mRNA expression (Fig. 1C).

To examine the effects of high glucose and estrogen on BTG2 mRNA and protein expression, mouse pancreatic islets were cultured under experimental conditions, and real-time PCR and Western blot analyses were performed. The BTG2 mRNA and protein expressions were significantly upregulated by the high glucose. Estrogen significantly reduced the BTG2 mRNA and protein expressions compared to those cultured in high glucose alone (Fig. 2A). Thus, a high glucose medium increased BTG2 mRNA expression in both INS-1 and islets, whereas estradiol reversed BTG2 mRNA and protein expressions in both INS-1 cells and islets in high glucose conditions (Fig. 2B).

Figure 2
Figure 2

Effect of estrogen on BTG2 mRNA and protein expression. (A) Fold change of BTG2 mRNA normalized to β-actin mRNA at 10 days from mouse pancreatic islets. (B) A representative Western blot analysis of BTG2 and β-actin from mouse pancreatic islets. The bar graph below is BTG2 protein level normalized to β-actin protein. The data is presented as mean ± S.D. of 3 independent experiments. *P < 0.05, **P < 0.01 compared to the high-glucose-treated group.

BTG2 knockdown rescued pancreatic β-cells apoptosis from high-glucose conditions

To investigate the role of BTG2 in protecting pancreatic β-cells apoptosis, BTG2 silencing was performed in INS-1 cells cultured in basal and high glucose media (Fig. 3A–C). After SiRNA-BTG2 knockdown, cellular apoptosis was determined by the detection of cleaved-caspase 3 and Bax using Western blotting analysis. SiRNA-BTG2 diminished the BTG2 protein expression in INS-1 cells cultured in basal and high-glucose media, and cleaved-caspase 3 and Bax were significantly decreased in INS-1 cells with SiRNA-BTG2 knockdown cultured in a high glucose medium. These findings were similar to the results for cells cultured in a basal glucose medium with mock treatment, siRNA-control and siRNA BTG2, whereas INS-1 cells cultured in a high glucose medium with mock treatment and siRNA-control showed markedly increased cleaved-caspase 3, BTG2 and Bax protein levels compared with those cultured in a basal glucose medium. To confirm these results, SiRNA-BTG2 knockdown was performed and cell apoptosis was assessed by Annexin V/PI staining. SiRNA BTG2 significantly decreased cell apoptosis when compared to siRNA-control. These results suggest that BTG2 silencing protects against high-glucose-induced pancreatic β-cell apoptosis.

Figure 3
Figure 3

Effect of BTG2 knockdown on levels of cleaved-caspase 3, Bax and cell apoptosis. INS-1 cells cultured under basal and high glucose media with mock treatment, siRNA-control, and siRNA-BTG2. Cleaved-caspase 3 and BTG2 were detected by Western blot analysis. (A) The image shows a representative Western blot of cleaved-caspase3, BTG2, and β-actin from INS-1 cells. The bar graph below shows cleaved-caspase 3 protein levels normalized to β-actin protein. The results are presented as mean ± S.D. of 4 independent experiments. (B) The image shows a representative Western blot of Bax, BTG2, and β-actin from INS-1 cells. The bar graph below shows Bax protein levels normalized to β-actin protein. The results are presented as mean ± S.D. of 4 independent experiments. (C) The cell viability in knock down BTG2 was determined using Annexin V/PI assay. The bar graph below shows percentage of early apoptosis. The results are presented as mean ± S.D. of 3 independent experiments. (D) Effect of estrogen in transcription of Btg2 promoter activity. The experiments were performed in 3 independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared to the high-glucose-treated group.

Estradiol regulated BTG2 promoter activity

In a breast cancer study, it was demonstrated that estradiol suppressed the BTG2 promoter in MCF-7 and Hela cells28. To examine whether estrogen regulated BTG2 mRNA expression, the INS-1 BTG2 promoter (−43 to −1802) was cloned into pGL3 reporter vector. INS-1 cells in high glucose conditions significantly increased BTG2 promoter activity compared to those cultured under basal glucose conditions. Estradiol in high glucose condition significantly reduced BTG2 promoter activity compared to the high glucose condition alone (Fig. 3D). The presence or absence of estradiol under the basal glucose conditions did not change BTG2 promoter activity. This result confirms that high glucose condition induces BTG2 promoter activity, but the addition of estradiol into INS-1 cells cultured under high glucose conditions decreases BTG2 promoter activity.

Estradiol decreased BTG2 and Bax mRNA and protein expressions through both nuclear and membrane estrogen receptors

Our previous results showed that high glucose conditions induced BTG2 mRNA and protein expressions, while the presence of estradiol under high glucose conditions reversed this effect. To investigate whether estradiol decreased BTG2 mRNA and protein expressions via the nuclear or membrane estrogen receptor, nuclear or membrane estrogen receptor inhibitors were added under the experimental conditions. ICI 182,780 and 4-HT, a nuclear estrogen receptor inhibitor and a nuclear estrogen alpha receptor inhibitor, respectively, did not abolish the effect of estradiol in reducing the BTG2 mRNA and protein expressions. Also, G15, a membrane estrogen receptor inhibitor, did not by itself diminish the effect of estradiol. Only in the presence of both ICI 182,780 and G15 was the effect of estradiol diminished, with no difference in BTG2 mRNA and protein expressions evident compared with those for the high glucose conditions (Fig. 4A,B).

Figure 4
Figure 4

Effect of estrogen on BTG2 and Bax mRNA and protein expressions from INS-1 cells cultured under basal and high glucose conditions. (A) Fold change of BTG2 mRNA normalized to β-actin mRNA at 48 h with or without nuclear and membrane estrogen receptor inhibitors. (B) Fold change of Bax mRNA normalized to β-actin mRNA at 48 h. (C) A representative Western blot analysis of BTG2 and β-actin from INS-1 cell cultured. The bar graph below is BTG2 protein level normalized to β-actin protein. (D) A representative Western blot analysis of Bax and β-actin from INS-1 cell cultured at 72 h. The bar graph below is Bax protein level normalized to β-actin protein. The data are presented as mean ± S.D. of 3 independent experiments. NS is non-significant. **P < 0.01, ***P < 0.001 compared to the high-glucose-treated group.

It has been proposed that BTG2 induces apoptosis via activated Bax29. To correlate BTG2 expression and pancreatic β-cell apoptosis, Bax mRNA and protein expressions were measured by RT-PCR and Western blot analyses. High glucose conditions significantly increased Bax mRNA and protein expressions compared to basal glucose conditions. INS-1 cells cultured with estradiol and a high glucose medium significantly reduced Bax mRNA and protein expressions compared to those cultured in a high glucose medium alone. To examine whether Bax mRNA and protein expressions responded in a similar manner to BTG2 in the presence of nuclear and/or membrane estrogen receptor inhibitors, ICI 182,780, 4-HT and G15 were added under the experimental conditions. Comparable with BTG2 expression, Bax mRNA and protein expression induction under the high glucose conditions were decreased by estradiol. The effect of estradiol in the high glucose conditions was attenuated by a combination of ICI 182,780 and G15 (Fig. 4C,D).

Estradiol reduced p53 protein expression

BTG2 expression uses either a p53-dependent or a p53-independent pathway in prostate carcinoma cells9. To examine whether BTG2 expression is associated with p53, INS-1 cells cultured under experimental conditions were assessed for nuclear p53 expression by Western blot analysis. High glucose conditions significantly increased the p53 protein expression in the nucleus compared to that under basal glucose conditions. However, estradiol significantly reduced the p53 protein expression in the nucleus compared to the high glucose conditions alone. Neither the nuclear estrogen receptor inhibitor nor the membrane estrogen receptor inhibitor reversed the estradiol effect when co-cultured in a high glucose medium. In the presence of both, the nuclear and membrane estrogen receptor inhibitors attenuated the effects of estradiol on the nuclear p53 expression under high glucose conditions (Fig. 5A).

Figure 5
Figure 5

Effect of estrogen on p53 and Bax protein expressions under normal and high glucose. (A) Fold change of p53 normalized to β-actin protein from INS-1 cells with or without nuclear and membrane estrogen receptor inhibitors. (B) Above picture is a representative Western blot analysis of p53 and β-actin. The bar graph below is p53 protein level normalized to β-actin protein. (C) Above picture is a representative Western blot analysis of Bax and β-actin. The bar graph below is Bax protein level normalized to β-actin protein. All data are presented as mean ± S.D. of 3 independent experiments. NS is not significant. **P < 0.01, ***P < 0.001 compared to the high-glucose-treated group.

Estrogen reduced p53 and Bax protein expressions in mouse pancreatic islets

To confirm the effects of high glucose and estradiol that were observed in INS-1 cells, mouse pancreatic islets were isolated and cultured under basal and high glucose conditions with or without estradiol for 72 h. As observed in the INS-1 cells, the high glucose conditions induced p53 and Bax protein expressions in mouse pancreatic islets compared to those cultured in the basal glucose medium. Estradiol with high glucose significantly reduced the p53 and Bax protein expressions, compared to those cultured in high glucose alone (Fig. 5B,C).

Discussion

Hyperglycemia is a stressful condition that produces both oxidative and ER stress4,30,31. Both types of stress cause DNA damage32,33, which activates early growth response genes34. BTG2 is one of the early growth response genes35. BTG2 has different effects, depending on the cell type7. In our preliminary study by mRNA analysis using the RT2 PCR array, the results showed that high glucose conditions increased BTG2 mRNA expression, and estradiol reversed the effect of the high glucose. BTG2 seemed to correlate with high-glucose-induced cell death. This hypothesis was tested by this study, which aimed to demonstrate the association of the BTG2 level and high-glucose-inducing cell death. The results of this study showed that the high glucose conditions increased cell death and up-regulated the BTG2 mRNA and protein expressions. The fold of the BTG2 mRNA expression with conventional real-time PCR was lower than with the RT2 PCR array. This might be due to the better optimized conditions of the commercial RT2 PCR array than conventional real-time PCR. Although the specificity of the primers was different, the pattern of BTG2 mRNA induction was similar. The up-regulation of BTG2 was found in both rat pancreatic β-cell line (INS-1 cells) and mouse pancreatic islets. BTG2 is known as an immediate early gene which responds to stress36. High glucose levels produced cellular stress in the form of oxidative and endoplasmic reticulum stress4,30,31. Thus, the cellular stress produced by high glucose likely stimulated BTG2 expression. Also, BTG2 has been proposed as a protein involved in the programed cell death of PC1237. On the contrary, BTG2 up-regulated by GLP-1 in pancreatic β-cells is associated with increased PDX-1 and insulin secretion12. Another beneficial role of BTG2 has been reported that BTG2 is a co-activator to up-regulating antioxidant38. This role is supported our study that estrogen has a trend to increase BTG2 in basal glucose. Estrogen might up-regulate antioxidant via increase BTG2. It is known that BTG2 plays a role in both physiological and pathological processes7. Our knockdown BTG2 experiment indicated that high-glucose-induced BTG2 is a pathological process, whereas the up-regulation of BTG2 by GLP-1 is a role of BTG2 in physiological processes.

This study also demonstrated that estradiol protected pancreatic β-cell apoptosis against high glucose via decreased BTG2 mRNA and protein expressions. Again, this finding was found in both rat pancreatic β-cell lines (INS-1 cells) and mouse pancreatic islets. A previous study suggested that estrogen reduced BTG2 transcription in breast cancer cells28. That study also suggested that the estrogen receptor can interact with other transcription factors, including AP-1, Sp1, p53 and NF-kB, which are contained in the BTG2 promoter. Furthermore, they performed ChIP-on-chip analysis and found that the ERα binding site was present around −2000 upstream of the BTG2 start site. To examine this possibility in pancreatic β-cells in this present study, the INS-1 BTG2 promoter was cloned to perform a promoter assay. The promoter assay confirmed that high glucose increased BTG2 promoter activity, while estrogen significantly decreased BTG2 promoter activity. The promoter assay results support our previous findings. In breast cancer cells, it was demonstrated that the estrogen receptor alpha plays a role in the reduction of BTG2 promoter activity28. To investigate this observation, 4 HT (the estrogen receptor alpha inhibitor), ICI 182,780 (the nuclear receptor inhibitor), and G15 (the G-protein coupled estrogen receptor inhibitor) were added to the culture experiments. Estradiol effect on BTG2 expression was ameliorated in the presence of both the nuclear and G-protein coupled estrogen receptor inhibitors. This suggests that estrogen exerts its effect through both the nuclear and G-protein coupled estrogen receptors. In parallel with our previous study, it has been shown that estrogen decreases ER stress and cell apoptosis via the nuclear and membrane estrogen receptors23,39. It is worth mentioning that estrogen has been known to protect pancreatic β-cell apoptosis against toxic substances through multiple pathways16,40,41.

BTG2 is known to induce cell apoptosis via increased Bax29. Our result confirmed that Bax mRNA and protein expressions were altered in response to the BTG2 expression. The activated Bax bound together to form a homodimer and then inserted pores on the mitochondrial membrane and released cytochrome C42. The released cytochrome C triggers the mitochondrial-induced apoptosis pathway43. This result provided a mechanism for BTG2-induced pancreatic β-cell apoptosis through Bax. It is known that BTG2 expression can be induced through a p53-dependent or a p53-independent mechanism13,14. This study further showed that the nuclear p53 level is increased. Normally, p53 is inactivated in the cytoplasm compartment. When p53 is activated, the activated p53 moves into the nucleus44. The activated p53 acts as a transcription factor to activate the expression of apoptotic genes such as Bax45. p53-induced cell apoptosis was also found in cardiac myocyte cultured under high glucose conditions46. Our results showed that high glucose conditions increased the p53, BTG2 and Bax in the INS-1 cells and mouse isolated pancreatic islets. Knockdown BTG2 significantly decreased Bax in high glucose condition. Thus, it is likely that the high glucose condition increased pancreatic β-cell apoptosis through the p53-BTG2-Bax pathway. Furthermore, our results showed that estradiol directly suppressed BTG2 promoter activity. Estradiol might separately suppress both BTG2 and p53 expression. Estrogen-reduced p53 signaling has been observed in other cells. In breast cancer, induction of p53 increases cell apoptosis, whereas estrogen promotes breast cancer cell proliferation by a decreased p53 pathway47. Estrogen-protected ischemia reperfusion induced cardiomyocytes apoptosis by suppression of the p53 pathway48. Estrogen prevented mesangial cells apoptosis through inhibition of p53 expression49. It is possible that estrogen suppressed BTG2 expression through reduced p53. However, our results also showed that estradiol directly suppressed transcriptional activation of the BTG2 promoter by the luciferase promoter assay. Thus, our results suggest that estrogen might suppress both the p53 and BTG2 promoters.

In summary, our results show that high glucose conditions induce BTG2, p53 and Bax expressions, which are associated with increased pancreatic β-cell apoptosis (Fig. 6). Estradiol can suppress the BTG2 promoter under high glucose conditions. The protective effect of estradiol against high-glucose-induced cell death through the reduction of BTG2, p53 and Bax expressions is diminished by inhibition of both the nuclear and the membrane estrogen receptors. However, the detailed molecular mechanisms on how estrogen suppresses p53 and BTG2 require further investigation.

Figure 6
Figure 6

A proposed model of estrogen reduced BTG2 expression. High glucose stimulated BTG2 and/or p53 expression. Then, BTG2 and p53 induced apoptosis via Bax. P53 also activated BTG2. Estrdiol prevents high-glucose-induced apoptosis via suppressing BTG2 promoter.

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Acknowledgements

This work was supported by a Mahidol University Grant (to SK), a Siriraj Graduate Thesis Scholarship (to SKa, KS), and a TRF Research Grant (to JS). SK was supported by a Chalermphrakiat Grant from the Faculty of Medicine, Siriraj Hospital, Mahidol University. MJ and PY were supported by Chalermphrakiat Grants from the Faculty of Medicine, Siriraj Hospital, Mahidol University, and the Thailand Research Fund (Grant no. IRG5980006). We thank Malika Churintaraphan and Samarn Onreabroi for their technical assistance.

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Affiliations

  1. Department of Physiology, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand

    • Suwattanee Kooptiwut
    • , Suchada Kaewin
    • , Namoiy Semprasert
    •  & Kanchana Suksri
  2. Division of Molecular Medicine, Research Department, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand

    • Jatuporn Sujjitjoon
    • , Mutita Junking
    •  & Pa-thai Yenchitsomanus

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Contributions

S.K. and P.Y. were responsible for the study concept and design, and S.Ka., N.S., K.S., J.S., M.J. and S.K. for the acquisition of data. S.Ka., J.S., M.J., P.Y., K.S. and S.K. were responsible for the analysis and interpretation of experimental data. S.K. drafted the manuscript. P.Y. and S.K. critically revised the manuscript for important intellectual content. S.K. was responsible for the study supervision. All authors approved the final version of the manuscript.

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The authors declare no competing interests.

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Correspondence to Suwattanee Kooptiwut.

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https://doi.org/10.1038/s41598-018-30698-x

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