4-Methylcatechol stimulates apoptosis and reduces insulin secretion by decreasing betacellulin and inhibin beta-A in INS-1 beta-cells

Abstract

Insulinoma INS-1 cell line is a pancreatic beta cell tumor which is characterized with high insulin content and secretion in response to increasing glucose levels. 4-Methylcatechol (4-MC) is a metabolite of quercetin, which is known as a potential drug for inhibition of tumorigenesis. The aim of this study was to determine the applying doses of 4-methylcatechol (4-MC) for triggening cell death and decreasing the cell function of rat insulinoma INS-1 beta cells. The rate of apoptosis and the amount of insulin in the cell and the secretions were determined by the ELISA method. Betacellulin (BTC) and inhibin beta-A amounts in both the cell and the glucose induced secretion were investigated by Western blotting. Furthermore, BTC, Inhibin beta-A, Ins1, Ins2, and GLUT2 gene expression levels were determined by the by the real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. We noted a significant decrease in cell viability, while an increase in apoptotic cell death by 4-MC treatment. It caused a decrease in the secretion of BTC, expressions of both BTC and inhibin beta-A. We showed a decrease in the expressions of Ins1 and GLUT2, while there is no alteration in the level of insulin protein. Insulin secretion levels increased in INS-1 cells given 4-MC by basal glucose concentration while they did not response to high concentration of glucose, which indicates that 4-MC disrupts the functionality of INS-1 cells. These results revealed that 4-MC induces apoptosis and decreases insulin secretion by reducing BTC and inhibin beta-A in insulinoma INS-1 cells. Thus, 4-MC may be offered as a potential molecule for treatment of insulinoma.

Keywords

4-Methylcatechol INS-1 cells apoptosis betacellulin inhibin beta-A insulin secretion

Introduction

Insulinoma is a tumor originating from the pancreatic beta cells, which are secreting insulin. Pancreatic beta cell tumor is a different form of the beta cell disease that is characterized by hyperinsulinemia and hypoglycemia.¹ There are many insulin-releasing cell lines obtained from various species. MIN6 and INS-1 cells are best at reflecting the physiological situation in beta cells when stimulated by glucose.²

Catechols induce various endobiotic and xenobiotic compounds and generate a large class of natural or synthetic origin substances. These molecules have a general catechol framework showing physiological, pharmacological, and toxicological properties.³ 4-Methylcatechol (4-MC), 4-nitrocatechol, 2,3-dihydroxynaphthalene, and other catechols are produced in the industry. Moreover, catechols are used as metal-ion-complex-forming agents and antioxidants.^3,4 Payton et al.⁵ showed that 4-MC inhibited the proliferation in melanoma cells and did not affect the proliferation in normal human epidermal melanocytes. 4-MC induces apoptosis in Sertoli and melanoma cells, and it also increases reactive oxygen species (ROS).^5,6 Interestingly, it has an important role in melanoma cell survival.⁵ However, the beneficial or cytotoxic effects and a relationship with apoptosis of 4-MC on insulinoma cells are unknown.

The proliferative function of betacellulin (BTC) in pancreatic beta cells is important to improve diabetes.⁷ Activin/inhibin beta regulates cell growth, differentiation, and programmed cell death in some cell types.^8
–10 There is no study about the role of BTC and inhibin beta-A in apoptosis induced by 4-MC in INS-1 cells. BTC level increases glucose-induced insulin secretion in mesenchymal stem cells in vitro and in vivo.¹¹ Activin A also stimulates insulin secretion in human pancreatic islets.¹² The relationship between BTC, activin A, and insulin secretion is unknown in INS-1 cells induced by 4-MC.

GLUT2 is functional for regulating insulin production in diabetic mice.¹³ However, it may not be required for regulation by glucose of insulin gene transcription.¹⁴ But, there is no information about the importance of 4-MC for GLUT2 and insulin secretion in insulinoma cells.

In this study, we have found that low-dose 4-MC protects cell viability, while at high doses it leads to death in insulinoma INS-1 cells. Based on the information above, this study aimed to determine the applying doses of 4-MC for triggering cell death and decreasing the cell function of insulinoma INS-1 beta cells.

Materials and methods

Cell culture

Rat insulinoma cell line INS-1 was a generous gift from Prof. Dr Claes B. Wollheim (University Medical Center, Geneva). INS-1 cells were cultured in a humidified atmosphere containing 5% carbon dioxide (CO₂) in Roswell Park Memorial Institute (RPMI) 1640 (invitrogen, Carlsbad, California, USA) containing 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (Sigma, St Louis, Missouri, USA), 50 μM 2-mercaptoethanol (Biorad, Hercules, California, USA), 1 mM sodium pyruvate (Sigma, USA), 5% fetal bovine serum (Gibco, USA), 100 IU/mL penicillin, and 100 μg/mL streptomycin (Gibco, USA). INS-1 cells were passed once a week by gentle trypsinization with 0.25% tripsin-EDTA (Sigma, USA), and cells between passages 2 and 9 were used for experiments.

MTT assay

We determined the toxic effects of 4-MC (Sigma, USA) at 50–1000 μM applying doses colorimetrically by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cells were seeded and 4-MC was given exogenously to cells for 24 h. MTT (0.5 mg/mL; Applichem, Darmstadt, Germany) assay was evaluated in 5% CO₂/95% humidified air at 37°C for 2 h and following the removal of the medium, isopropanol was added to solve formazan.¹⁵ The optical density was recorded with a spectrophotometer at 540 nm. Cell viability rate was calculated as % of control.

Measurement of apoptosis

We calculated the enrichment factor of apoptosis with Cell Death Detection ELISA Kit (Roche, Basel, Switzerland) at determined doses. We accepted the control group as 1 and calculated the enrichment factors of each group as fold increase according to control.

Western blotting

Proteins were separated by 7.5% gel SDS-PAGE and transferred to nitrocellulose membranes using a wet transfer method. The membranes were incubated for 1 h in the blocking solution (5% skim milk in TBST), then for primary antibody (for BTC; sc-5802, for inhibin beta-A; sc-50288) was applied. Then, membranes were incubated with the secondary antibody (anti-goat IgG, sc-2350, anti-rabbit IgG sc-2004) for 1 h and treated with luminol reagent (sc-2048). The protein bands were made visible with Gel Logic 1500 Imaging System Device. Data analysis was done with Kodak Carestream MI analysis program. Beta-actin was used to normalize cytoplasmic proteins, while Ponceau S was used for normalization of proteins in secretion samples.

Measurement of intracellular insulin levels

The cell lysates were analyzed fluorometrically by using an insulin analysis kit (Cisbio-62INSPEB, Codolet, France) according to the instructions of manufacturer.

Insulin secretion assay

The INS-1 cells were incubated for 2 h with basal glucose concentration (5.5 mM) in 5% CO₂/95% humidified air at 37°C after given 4-MC. Then, the cell secretion samples were taken and high-concentration glucose (15.5 mM) was applied for 2 h in 5% CO₂/95% humidified air at 37°C. The cell secretion samples were taken again and analyzed fluorometrically by using insulin analysis kit (Cisbio-62INSPEB) according to the instructions of manufacturer. The cells were lysed with tris-HCI/SDS and sonication. Total protein levels were calculated in supernatants by Qubit fluorimetric kit and instrument.

qRT-PCR

Trizol reagent method was applied for total ribonucleic acid (RNA) isolation. All samples containing 1 mg RNA were translated to complementary deoxyribonucleic acid by using SuperScript III First-Strand Kit (Invitrogen). The primer for inhibin beta-A was designed as follows: forward primer: TCATGCCAACTACTGCGAGG, reverse primer: ACAGTGAGGACCCGGACG, and probe: TGAGTGCCCGAGCCATATAGCAGGC. Primers of BTC, Ins1, Ins2, GLUT2, and Beta actin were bought from Applied Biosystems (Foster City, California, USA). Ct values were obtained according to manual protocol by using Applied Biosystems 7500 Fast Instrument. Expression of target genes were calculated with the Pfaffl method.¹⁶

Statistical analysis

Statistical analyses were performed with minimum triple data by analysis of variance (ANOVA) test and nonparametric Tukey’s test using GraphPad Prism 5 computer program (GraphPad Software, Inc., San Diego, California, USA). Results were reported as mean ± SEM. p values less than 0.05 were considered to be significant.

Results

4-MC has toxic effects on INS-1 cells at high doses

We examined the effects of 4-MC on the viability of INS-1 cells at 50–1000 μM (Figure 1a). We used relative nontoxic doses for following experiments. According to our results, when we administered 50 and 100 μM 4-MC, INS-1 cells were alive. Cell viability was below 50% for 450 µM. In addition, cells were alive at 150 and 200 µM doses; however, there were still 10–20% cell viability reduction. Therefore, low doses of 4-MC are moderate cytotoxic compared to high ones. Thus, we have chosen 250–450 μM doses of 4-MC for the experiments.

Figure 1.

(a) Cell viability % of INS-1 cells in different doses of 4-MC. (b) Apoptosis enrichment factor in INS-1 cells. ^a p < 0.001 versus control group; ^b p < 0.01 versus control group. 4-MC: 4-methylcatechol.

4-MC has apoptotic effects on INS-1 cells at moderate doses

We used the ELISA kit for the detection of apoptotic cell death and calculated enrichment factor (Figure 1b). According to the results, there was a significant difference between groups (p _ANOVA < 0.001). Apoptotic cell death level was statistically increased in the 300-, 350-, 400-, and 450-μM dose of 4-MC according to control group (p < 0.001; p < 0.01; p < 0.001; p < 0.001, respectively).

The decrease in the level and expression degree of BTC was consistent with apoptotic cell death induced by 4-MC

The BTC amount both in INS-1 cells and its secretion was measured by Western blotting (Figure 2a). A statistically significant result was obtained when the groups were compared between each other (p _ANOVA < 0.05). As a result of pairwise comparisons, a significant decrease was observed in the amount of intracellular BTC in the group that was administered 450-μM 4-MC compared to the control group (p < 0.05). Moreover, BTC levels in secretion samples were also statistically significant (p _ANOVA < 0.01). As a result of pairwise comparisons, the cell secretion in the groups that were administered 400- and 450-μM 4-MC decreased significantly when compared to the control group (p < 0.05; p < 0.01). Statistically significant results were obtained also in BTC expression of INS-1 cells when groups were compared between each other (p _ANOVA < 0.05). As a result of pairwise comparisons, the group that was administered 350-µM 4-MC decreased significantly in terms of BTC expression level when compared to the control group (p < 0.05).

Inhibin beta-A level was not changed, while its expression degree was decreased with apoptotic cell death induced by 4-MC

The cellular and secretion amounts of inhibitin beta-A was measured by Western blotting in INS-1 cells (Figure 2b). When the groups were compared between each other for intracellular or secretion levels of inhibin beta-A, no statistically significant result was obtained (p _ANOVA > 0.05). On the other hand, when inhibin beta-A expression levels in the groups were compared between each other, statistically significant results were obtained (p _ANOVA < 0.001). As a result of pairwise comparisons, inhibin expression levels decreased significantly in the groups that were administered 350-, 400-, and 450-µM 4-MC when compared to the control group (p < 0.01).

Figure 2.

(a) BTC amount assay in INS-1 cell lysate. ^a p < 0.05 versus control group. BTC amount assay in INS-1 cell secretion. ^a p < 0.05 versus control group; ^b p < 0.01 versus control group. BTC mRNA expression in INS-1 cells. ^a p < 0.05 versus control group. (b) Inhibin beta-A amount assay in INS-1 cell lysate. p > 0.05 versus control group. Inhibin beta-A amount assay in INS-1 cell secretion. p > 0.05 versus control group. Inhibin beta-A mRNA expression in INS-1 cells. ^a p < 0.01 versus control group. BTC: betacellulin.

4-MC suppresses Ins1 gene expression, while it has no effect on Ins2 gene expression and insulin protein content in INS-1 cells

Insulin levels in INS-1 cells were observed with insulin ELISA kit (Figure 3a). There was not any significant difference between all groups (p _ANOVA > 0.05). We observed a statistically significant alteration between groups in the levels of Ins1 mRNA (p _ANOVA < 0.05). Ins1 gene expression level was statistically decreased in the groups 4-MC treated at 350-, 400-, and 450-µM concentrations versus control group (p < 0.05). The difference in Ins2 mRNA expression levels was also not statistically significant between all groups (p _ANOVA > 0.05).

Figure 3.

(a) Insulin level in INS-1 cells. p > 0.05 for insulin level. Ins1 and Ins2 mRNA levels in INS-1 cells. ^a p < 0.05 for Ins1 versus control group. p > 0.05 for Ins2. (b) Insulin secretion level in INS-1 cells. ^a p < 0.01 versus control group given basal concentration glucose, ^b p < 0.01 versus control group given basal concentration glucose, and ^c p < 0.05 versus 350- and 400-μM 4-MC group given basal concentration glucose. (c) GLUT2 mRNA level in INS-1 cells. ^a p < 0.01 versus control group. 4-MC: 4-methylcatechol.

4-MC suppresses insulin secretion in response to high glucose concentration and GLUT2 gene expression in INS-1 cells

Insulin secretion levels, occurred in response to basal- and high glucose concentrations, were determined by using an insulin ELISA kit (Figure 3b). We observed a statistically significant difference between all groups (p _ANOVA < 0.01). Insulin secretion level was increased as a response to high glucose concentration versus basal glucose in control group (p < 0.01). We observed a significant increase in insulin secretion level response to basal glucose induction in 350- and 400 μM 4-MC-group given low-dose glucose (p < 0.05). There were no significant alteration between basal and high concentration glucose treatments (p _ANOVA > 0.05).

GLUT2 expression levels were shown by qRT-PCR method in INS-1 cells (Figure 3c). There was a statistically significant alteration between all groups (p _ANOVA < 0.01). GLUT2 expression levels significantly decreased in the 4MC-treated groups at 350-, 400-, and 450 µM concentrations according to the control group (p < 0.01).

Discussion

Insulinoma is originated from pancreatic beta cells in islets of Langerhans.^17,18 Although beta cell tumors are very small, they may cause death because of high levels of hormone production. Insulinomas may be benign or malign, and they are characterized by uncontrollable insulin secretion. Insulinomas secrete more proinsulin according to normal beta cells, thus they can be determined with increased ratio of proinsulin to insulin.^18
–20

4-MC is a phenolic substrate that is produced during the metabolism of quercetin found naturally in the combination of various fruits.²¹ 4-MC produces ROS as hydrogen peroxide and this situation shows that this compound has cytotoxic characteristic. Cell death was induced following 100–500 μM doses of 4-MC in mouse B16-F10 melanoma cells, mouse LL/2 Lewis lung carcinoma cells, rat C6 glioma cells, and rat PC1 pheochromocytoma cells.²² Conversely, Gezginci-Oktayoglu and Bolkent²³ have shown that apoptotic cell death decreases in hyperglycemic rat pancreatic beta cells pretreated with 10 μg/kg 4-MC in in vivo.

In this study, we determined that 4-MC has a preventive effect at lower doses on INS1 cells, while it leads to cell death at higher doses in vitro. We continued our experiments with higher doses of 4-MC that lead to cell death because the aim of this study was to stimulate the apoptotic cell death in INS-1 cell. Thus, the cells incubated with 250–450 μM 4-MC for 24 h and the induction of apoptotic cell death were investigated.

Apoptotic changes were shown by morphologic assessment occurred in 100-μM 4-MC-treated B16F10 mouse melanoma cells for 24 h. The increased caspase-3 activity was observed when the cells exposed to different concentrations of 4-MC for 6 h and the highest activity of caspase-3 observed by given 250-μM 4-MC.²² Interestingly, Gezginci-Oktayoglu and Bolkent^23,24 showed that apoptotic cell death decreased by 4-MC injection for 10 days in vivo, by both TUNEL method and by measurement of caspase 3-8 activities in pancreas tissue. The apoptotic effect of 4-MC in the beta cells is unknown under in vitro condition. We investigated the apoptotic changes in between 250 and 450 μM doses of 4-MC for 24 h in INS-1 cells. We determined apoptosis under above-mentioned conditions, especially at 450-μM 4-MC.

BTC has a mitogenic effect on INS-1 cells.²⁵ The positive immunoreactivity of BTC was found both in insulinoma and glucagonoma cells. The localization of BTC and its receptor play a role in physiologically important situations such as the growth and differentiation of islet cells.²⁶ Nevertheless, how the BTC in the cell is affected as a result of the administration of 4-MC was not studied.

In our study, we have not observed any alteration in the BTC expression while cell death occurs as a result of the decrease levels of intracellular and secreted BTC. It is well-known that BTC has an important role for cell survival. We have shown in our previous work that growth factor loss inhibits beta cell proliferation and activates apoptosis through increasing Act-A secretion or decreasing BTC secretion from beta cells.²⁷ Our results suggested that 4-MC causes decrease in BTC and this decrease triggered beta cell apoptosis.

BTC causes differentiation of the exocrine AR42J cells to cells that produce insulin together with Activin A.²⁸ Activin A inhibits mitogen-induced DNA synthesis in liver and induces the apoptosis of the hepatocytes in vivo and in vitro.^29
–31 There are many researches showing apoptotic effect of activin A.^32,33 Activins have important roles in cell growth, differentiation, and apoptosis.³⁴ It also regulates β-cell proliferation in rat and human islets.³² Activin A together with BTC increases β-cell mass of neonatal STZ-treated rats.³⁵ The apoptotic effect of activin A or inhibin beta-A on insulinoma cells is not known.

The expression level of inhibin beta-A may be decreased as a result of apoptotic cell death. It is well-known that inhibin beta-A also reduces cell proliferation and cell growth. No change was observed in the level of inhibin beta-A in both cellular and secretion samples, and no cell proliferation occurred at the given doses of 4-MC as a result of apoptotic cell death.

Insulin levels in pancreatic tissue of STZ-induced hyperglycemic rats were increased by pre-treatment of 4-MC for a relative long time.²³ This is the only study that gives idea about the effects of 4-MC on insulin level. We could not observe any change in insulin level of INS-1 cells after giving 4-MC. However, experimental results showed that especially, cell death at the highest dose of 4-MC was observed in many cells both biochemically and microscopically. Even though there were not a difference in endogenous insulin levels between experimental groups, we observed a decrease in cell number caused by 4-MC. For this reason, it may be suggested that the cells administered with 4-MC secrete more insulin than the control group. Insulin is important for the maintenance of beta cell survival.³⁶ It is possible that more releases of insulin are needed in order to continue the survival of the remained cells. On the other hand, the expression degree of Ins1 gene was decreased, and the expression degree of Ins2 was not changed. It is known that when one insulin gene is inactivated, the others are transcripted and protein level is elevated, at least partially, to compensate for the loss.³⁷ This information may explain the imbalance between the gene expression levels of Ins1 and Ins2.

GLUT2 has an important role in the release of glucose-induced insulin by functioning the glucose uptake into the cells.³⁸ GLUT2 expression was decreased in islets that do not respond to glucose in various diabetic animal models.^38,39 The effect of 4-MC on GLUT2 expression is not known in INS-1 cells. The reduction in the levels of the GLUT2 gene expression was paralleled by a decrease in insulin secretion occurred in response to rising glucose levels. Thus, we think that the decrease in the level of insulin secretion, which is expected to increase depending on the rising glucose levels, is related to the reduction in GLUT2 expression level, which plays an important role in the uptake of glucose.

Insulin secretion increased at 15.6-mM glucose according to 5.6-mM glucose in isolated primary beta cells. Insulin secretion is not changed in both groups given basal and high concentration of glucose in primary beta cell isolated from hyperglycemic rat.⁴⁰ In our study, insulin secretion induced when we give basal concentration of glucose after giving 4-MC to cells. High concentration of glucose may not have a stimulant effect. Insulin secretion in control group given high-concentration glucose increased according to control group given basal concentration glucose. Therefore, INS-1 cells are functional.

As a conclusion, insulinoma INS-1 cells undergo apoptosis, BTC and inhibin beta-A amount reduced as a result of apoptosis. The cells lose their insulin secretion function through decreasing GLUT2 expression by 24 h 4-MC treatment in a dose-dependent manner. Thus, 4-MC may be suggested as an important molecule for inducing pancreatic beta cell death. It could be developed as a drug for cancer therapy.

Footnotes

Acknowledgements

The authors would like to thank Prof. Dr Claes B. Wollheim (University Medical Center, Geneva) for providing kind gift of insulinoma INS-1 cell lines.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Research Fund of Istanbul University (project number: 14831 and BEK-2016-21753).

ORCID iD

A Karatug Kacar

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