Journal List > J Nutr Health > v.51(6) > 1111482

J Nutr Health. 2018 Dec;51(6):498-506. Korean.
Published online Dec 31, 2018.  https://doi.org/10.4163/jnh.2018.51.6.498
© 2018 The Korean Nutrition Society
Protective effect of lycopene against cytokine-induced β-cell apoptosis in INS-1 cells
Kyong Kim,1,** Se-Eun Jang,1,** Gong Deuk Bae,2 Hee-Sook Jun,2,3,4 and Yoon Sin Oh1
1Department of Food and Nutrition, Eulji University, Seongnam, Gyeonggi 13135, Korea.
2Lee Gil Ya Cancer and Diabetes Institute, Department of Molecular Medicine, Gachon University, Incheon 21999, Korea.
3College of Pharmacy, Gachon University, Incheon 21936, Korea.
4Gachon Gil Medical Center, Incheon 21565, Korea.

To whom correspondence should be addressed. tel: +82-31-740-7287, Email: ysoh@eulji.ac.kr

**These authors contributed equally to this article.

Received Aug 29, 2018; Revised Nov 06, 2018; Accepted Nov 20, 2018.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.


Abstract

Purpose

Lycopene, a carotenoid with anti-oxidant properties, occurs naturally in tomatoes and pink grapefruit. Although the beneficial effects of lycopene on various disorders have been established, little attention has been paid to the possible anti-diabetic effects of lycopene focusing on β-cells. Therefore, this study investigated the potential of lycopene to protect β-cells against apoptosis induced by a cytokine mixture.

Methods

For toxicity experiments, the cells were treated with 0.1 ~ 10 nM of lycopene, and the cell viability in INS-1 cells (a rat β-cell line) was measured using a MTT assay. To induce cytokine toxicity, the cells were treated with a cytokine mixture (20 ng/mL of TNFα+20 ng/mL of IL-1β) for 24 h, and the effects of lycopene (0.1 nM) on the cytokine toxicity were measured using the MTT assay. The expression levels of the apoptotic proteins were analyzed by Western blotting, and the level of intracellular reactive oxidative stress (ROS) was monitored using a DCFDA fluorescent probe. The intracellular ATP levels were determined using a luminescence kit, and mRNA expression of the genes coding for anti-oxidative stress response and mitochondrial function were analyzed by quantitative reverse-transcriptase PCR.

Results

Exposure of INS-1 cells to 0.1 nM of lycopene increased the cell viability significantly, and protected the cells from cytokine-induced death. Lycopene upregulated the mRNA and protein expression of B-cell lymphoma-2 (Bcl-2) and reduced the expression of the Bcl-2 associated X (Bax) protein. Lycopene inhibited apoptotic signaling via a reduction of the ROS, and this effect correlated with the upregulation of anti-oxidative stress response genes, such as GCLC, NQO1, and HO-1. Lycopene increased the mRNA expression of mitochondrial function-related genes and increased the cellular ATP level.

Conclusion

These results suggest that lycopene reduces the level of oxidative stress and improves the mitochondrial function, contributing to the prevention of cytokine-induced β-cell apoptosis. Therefore, lycopene could potentially serve as a preventive and therapeutic agent for the treatment of type 2 diabetes.

Keywords: lycopene; β-cell; cytokine; apoptosis; type 2 diabetes

Figures


Fig. 1
Effect of lycopene on cytokine mixture-induced cytotoxicity in INS-1 cells. (A) Effect of lycopene on proliferation of INS-1 cells. Cells were incubated in media containing various concentrations of lycopene (LY, 0.1, 1, and 10 nM) dissolved in THF for 24 and 48 h and cell viability was measured by MTT assay. (B) Cytokine mixture (CM, 20 ng/mL TNFα and 20 ng/mL IL-1β) was treated with or without 0.1 nM of LY and cell viability at 24 and 48 h was determined by MTT assay. Each values are means ± SEM from three independent experiments and normalized to percentage of control (CON, 24h). *p < 0.05 versus THF or cytokine mixture (CM).
Click for larger image


Fig. 2
Effect of lycopene on cytokine mixture-induced apoptosis in INS-1 cells. (A) Cells were treated with 0.1nM lycopene (LY) and Cytokine mixture (CM, 20 ng/mL TNFα and 20 ng/mL IL-1β) for 24 h. The cells were harvested and expression levels of β-cell lymphoma 2 (BcL-2) and Bcl-2-associated X protein (Bax) were measured by Western blot analysis. β-actin was used as the internal control. The bands were quantified by Image J software. (B) Cells were treated as described in (A) and cells were harvested after 12 h treatment. The mRNA levels of Bcl-2 and Bax were analyzed by quantitative RT-PCR. The mRNA levels were normalized with those of cyclophilin. The values represent the mean ± SEM from triplicate experiments. *p < 0.05 versus CM.
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Fig. 3
Effect of lycopene on cytokine mixture-induced ROS generation in INS-1 cells. (A) Cells were treated with 0.1 nM lycopene (LY) and Cytokine mixture (CM, 20 ng/mL TNFα and 20 ng/mL IL-1β) for 1 h. The cells were stained with 10 µM H2-DCFDA, and intracellular ROS generation was determined by DCF. (B) The cells were treated as described in Fig 2B, and mRNA expression levels of glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H dehydrogenase, quinone 1 (NQO1) and heme oxygenase 1 (HO-1) were analyzed by quantitative RT-PCR. The mRNA levels were normalized with those of cyclophilin. The values represent the mean ± SEM from triplicate experiments. *p < 0.05 versus CM.
Click for larger image


Fig. 4
Effect of lycopene on cytokine mixture-induced mitochondria dysfunction in INS-1 cells. (A) Cells were treated with 0.1nM lycopene (LY) and Cytokine mixture (CM, 20 ng/mL TNFα and 20 ng/mL IL-1β) for 24 h. Intracellular concentrations of Adenosine triphosphate (ATP) were determined using an ATP-dependent luminescent cell viability assay (B) Cells were treated as described in (A) and cells were harvested after 12 h treatment. The mRNA expression levels of transcription factor A, mitochondrial (TFAM) and peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α) were analyzed by quantitative RT-PCR. The mRNA levels were normalized with those of cyclophilin. The values represent the mean ± SEM from triplicate experiments. *p < 0.05 versus CM.
Click for larger image

Notes

This study was supported by a grant (NRF-2015R1D1A1A01058888; NRF-2018R1C1B6000998) from the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT & Future Planning.

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