Register      Login
Reproduction, Fertility and Development Reproduction, Fertility and Development Society
Vertebrate reproductive science and technology
RESEARCH ARTICLE

Betaine ameliorates high glucose-induced oxidative stress in granulosa cells

Mohammad Hussein Abnosi https://orcid.org/0000-0002-1485-8847 A * , Mohammad Reza Tabandeh B and Fatmeh Mosavi-aroo A
+ Author Affiliations
- Author Affiliations

A Biology Department, Faculty of Sciences, Arak University, Arak, Iran.

B Department of Biochemistry and Molecular Biology, Faculty of Veterinary Medicine, Shahid Chamran University, Ahwaz, Iran.

* Correspondence to: m-abnosi@araku.ac.ir

Handling Editor: Geraldine Hartshorne

Reproduction, Fertility and Development 35(6) 395-405 https://doi.org/10.1071/RD22247
Published online: 22 March 2023

© 2023 The Author(s) (or their employer(s)). Published by CSIRO Publishing

Abstract

Context: In diabetes, abnormalities of granulosa cells (GCs) and steroidogenesis are associated with hyperglycaemia-induced oxidative stress. Betaine has beneficial effect in experimental model of diabetes by reducing oxidative stress, inflammation, and apoptosis.

Aims: In this study we investigate the effects of betaine to prevent oxidative stress in GCs induced by high glucose and improve steroidogenesis.

Methods: Primary GCs, isolated from ovarian follicles of C57BL/6 mice were cultured in 5 mM (control) and 30 mM (hyperglycaemia) of glucose and in presence of 5 mM of betaine for 24 h. Then antioxidant enzymes, malondialdehyde, oestradiol and progesterone were measured. In addition, the expression of Nrf2 and NF-κB, antioxidant enzymes (Sod1, Gpx and Cat) were analysed by qRT-PCR assay.

Key results: We observed significant (P < 0.001) up-regulation of NF-κB and down-regulation of Nrf2 due to high concentration of glucose. Also significant (P < 0.001) down-regulation of related antioxidant genes (Cat, Sod1 and GPx) and activity reduction of these enzymes as well as significant (P < 0.001) elevation of malondialdehyde was observed. In addition, betaine treatment compensated the drastic effect of high glucose induced oxidative stress via down-regulating the expression of NF-κB and up-regulating the expression of Nrf2, Cat, Sod1 and GPx. It was also shown that betaine in the presence of FSH significantly (P < 0.001) restored the oestradiol and progesterone level.

Conclusion: Betaine compensated the antioxidant stress in mouse GCs under hyperglycaemic condition via regulation of Nrf2/NF-κB at transcription level.

Implications: As betaine is a natural product and no side effect has been reported to today, we suggest more research needs to be carried out especially on patients whom suffer from diabetes to find the probability of using betaine as a therapeutic agent.

Keywords: betaine, cell culture, follicle stimulating hormone, gene expression, granulosa cells, progesterone, steroid hormone, stress.


References

Agarwal, A, Aponte-Mellado, A, Premkumar, BJ, Shaman, A, and Gupta, S (2012). The effects of oxidative stress on female reproduction: a review. Reproductive Biology and Endocrinology 10, 49.
The effects of oxidative stress on female reproduction: a review.Crossref | GoogleScholarGoogle Scholar |

Ahmed, SMU, Luo, L, Namani, A, Wang, XJ, and Tang, X (2017). Nrf2 signaling pathway: pivotal roles in inflammation. Biochimica et Biophysica Acta (BBA) - Molecular Basis of Disease 1863, 585–597.
Nrf2 signaling pathway: pivotal roles in inflammation.Crossref | GoogleScholarGoogle Scholar |

Akman, L, Erbas, O, Akdemir, A, Yavasoglu, A, Taskiran, D, and Kazandi, M (2015). Levetiracetam ameliorates ovarian function in streptozotocin-induced diabetic rats. Gynecological Endocrinology 31, 657–662.
Levetiracetam ameliorates ovarian function in streptozotocin-induced diabetic rats.Crossref | GoogleScholarGoogle Scholar |

Alirezaei, M, Niknam, P, and Jelodar, G (2012). Betaine elevates ovarian antioxidant enzyme activities and demonstrates methyl donor effect in non-pregnant rats. International Journal of Peptide Research and Therapeutics 18, 281–290.
Betaine elevates ovarian antioxidant enzyme activities and demonstrates methyl donor effect in non-pregnant rats.Crossref | GoogleScholarGoogle Scholar |

Asadi, N, Kheradmand, A, and Gholami, MR (2019). Biochemical and histological evaluation of protective effect of betaine in experimental varicocele using animal model. International Journal of Peptide Research and Therapeutics 25, 719–726.
Biochemical and histological evaluation of protective effect of betaine in experimental varicocele using animal model.Crossref | GoogleScholarGoogle Scholar |

Ashry, M, Folger, JK, Rajput, SK, Baroni, J, and Smith, GW (2022). FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling. Domestic Animal Endocrinology 78, 106678.
FSH stimulated bovine granulosa cell steroidogenesis involves both canonical and noncanonical WNT signaling.Crossref | GoogleScholarGoogle Scholar |

Asmat, U, Abad, K, and Ismail, K (2016). Diabetes mellitus and oxidative stress – a concise review. Saudi Pharmaceutical Journal 24, 547–553.
Diabetes mellitus and oxidative stress – a concise review.Crossref | GoogleScholarGoogle Scholar |

Balkan, J, Öztezcan, S, Küçük, M, Çevikbaş, U, Koçak-Toker, N, and Uysal, M (2004). The effect of betaine treatment on triglyceride levels and oxidative stress in the liver of ethanol-treated guinea pigs. Experimental and Toxicologic Pathology 55, 505–509.
The effect of betaine treatment on triglyceride levels and oxidative stress in the liver of ethanol-treated guinea pigs.Crossref | GoogleScholarGoogle Scholar |

Bernhisel, MA, Holman, JF, Haney, AF, and Schomberg, DW (1987). Estrogen and progesterone production by granulosa cell monolayers derived from in vitro fertilization procedures: lack of evidence for modulation by androgen. The Journal of Clinical Endocrinology & Metabolism 64, 1251–1256.
Estrogen and progesterone production by granulosa cell monolayers derived from in vitro fertilization procedures: lack of evidence for modulation by androgen.Crossref | GoogleScholarGoogle Scholar |

Bradford, MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248–254.
A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Crossref | GoogleScholarGoogle Scholar |

Canty, TG, Boyle, EM, Farr, A, Morgan, EN, Verrier, ED, and Pohlman, TH (1999). Oxidative stress induces NF-κB nuclear translocation without degradation of IκBα. Circulation 100, II-361–Ii-364.
Oxidative stress induces NF-κB nuclear translocation without degradation of IκBα.Crossref | GoogleScholarGoogle Scholar |

Chabrolle, C, JeanPierre, E, Tosca, L, Ramé, C, and Dupont, J (2008). Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells. Reproductive Biology and Endocrinology 6, 11.
Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells.Crossref | GoogleScholarGoogle Scholar |

Chang, AS, Dale, AN, and Moley, KH (2005). Maternal diabetes adversely affects preovulatory oocyte maturation, development, and granulosa cell apoptosis. Endocrinology 146, 2445–2453.
Maternal diabetes adversely affects preovulatory oocyte maturation, development, and granulosa cell apoptosis.Crossref | GoogleScholarGoogle Scholar |

Chowdhury, I, Thomas, K, and Thompson, WE (2016). Prohibitin (PHB) roles in granulosa cell physiology. Cell and Tissue Research 363, 19–29.
Prohibitin (PHB) roles in granulosa cell physiology.Crossref | GoogleScholarGoogle Scholar |

Colton, SA, Humpherson, PG, Leese, HJ, and Downs, SM (2003). Physiological changes in oocyte-cumulus cell complexes from diabetic mice that potentially influence meiotic regulation. Biology of Reproduction 69, 761–770.
Physiological changes in oocyte-cumulus cell complexes from diabetic mice that potentially influence meiotic regulation.Crossref | GoogleScholarGoogle Scholar |

Corbett, HE, Dubé, CD, Slow, S, Lever, M, Trasler, JM, and Baltz, JM (2014). Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter. Biology of Reproduction 90, 81.
Uptake of betaine into mouse cumulus-oocyte complexes via the SLC7A6 isoform of y+L transporter.Crossref | GoogleScholarGoogle Scholar |

Craig, SAS (2004). Betaine in human nutrition. The American Journal of Clinical Nutrition 80, 539–549.
Betaine in human nutrition.Crossref | GoogleScholarGoogle Scholar |

Devine, PJ, Perreault, SD, and Luderer, U (2012). Roles of reactive oxygen species and antioxidants in ovarian toxicity. Biology of Reproduction 86, 27.
Roles of reactive oxygen species and antioxidants in ovarian toxicity.Crossref | GoogleScholarGoogle Scholar |

Fan, C-Y, Wang, M-X, Ge, C-X, Wang, X, Li, J-M, and Kong, L-D (2014). Betaine supplementation protects against high-fructose-induced renal injury in rats. The Journal of Nutritional Biochemistry 25, 353–362.
Betaine supplementation protects against high-fructose-induced renal injury in rats.Crossref | GoogleScholarGoogle Scholar |

Fiorentino, VT, Prioletta, A, Zuo, P, and Folli, F (2013). Hyperglycemia-induced oxidative stress and its role in diabetes mellitus related cardiovascular diseases. Current Pharmaceutical Design 19, 5695–5703.
Hyperglycemia-induced oxidative stress and its role in diabetes mellitus related cardiovascular diseases.Crossref | GoogleScholarGoogle Scholar |

Ganesan, B, Buddhan, S, Anandan, R, Sivakumar, R, and AnbinEzhilan, R (2010). Antioxidant defense of betaine against isoprenaline-induced myocardial infarction in rats. Molecular Biology Reports 37, 1319–1327.
Antioxidant defense of betaine against isoprenaline-induced myocardial infarction in rats.Crossref | GoogleScholarGoogle Scholar |

Ganesh Yerra, V, Negi, G, Sharma, SS, and Kumar, A (2013). Potential therapeutic effects of the simultaneous targeting of the Nrf2 and NF-κB pathways in diabetic neuropathy. Redox Biology 1, 394–397.
Potential therapeutic effects of the simultaneous targeting of the Nrf2 and NF-κB pathways in diabetic neuropathy.Crossref | GoogleScholarGoogle Scholar |

Gargouri, M, Magné, C, and El Feki, A (2016). Hyperglycemia, oxidative stress, liver damage and dysfunction in alloxan-induced diabetic rat are prevented by Spirulina supplementation. Nutrition Research 36, 1255–1268.
Hyperglycemia, oxidative stress, liver damage and dysfunction in alloxan-induced diabetic rat are prevented by Spirulina supplementation.Crossref | GoogleScholarGoogle Scholar |

Go, EK, Jung, KJ, Kim, JY, Yu, BP, and Chung, HY (2005). Betaine suppresses proinflammatory signaling during aging: the involvement of nuclear factor-κB via nuclear factor-inducing kinase/IκB kinase and mitogen-activated protein kinases. The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60, 1252–1264.
Betaine suppresses proinflammatory signaling during aging: the involvement of nuclear factor-κB via nuclear factor-inducing kinase/IκB kinase and mitogen-activated protein kinases.Crossref | GoogleScholarGoogle Scholar |

Hagar, H, Husain, S, Fadda, LM, Attia, NM, Attia, MMA, and Ali, HM (2019). Inhibition of NF-κB and the oxidative stress-dependent caspase-3 apoptotic pathway by betaine supplementation attenuates hepatic injury mediated by cisplatin in rats. Pharmacological Reports 71, 1025–1033.
Inhibition of NF-κB and the oxidative stress-dependent caspase-3 apoptotic pathway by betaine supplementation attenuates hepatic injury mediated by cisplatin in rats.Crossref | GoogleScholarGoogle Scholar |

Kang, B, Wang, X, Xu, Q, Wu, Y, Si, X, and Jiang, D (2018). Effect of 3-nitropropionic acid inducing oxidative stress and apoptosis of granulosa cells in geese. Bioscience Reports 38, BSR20180274.
Effect of 3-nitropropionic acid inducing oxidative stress and apoptosis of granulosa cells in geese.Crossref | GoogleScholarGoogle Scholar |

Khadrawy, O, Gebremedhn, S, Salilew-Wondim, D, Taqi, MO, Neuhoff, C, Tholen, E, Hoelker, M, Schellander, K, and Tesfaye, D (2019). Endogenous and exogenous modulation of Nrf2 mediated oxidative stress response in bovine granulosa cells: potential implication for ovarian function. International Journal of Molecular Sciences 20, 1635.
Endogenous and exogenous modulation of Nrf2 mediated oxidative stress response in bovine granulosa cells: potential implication for ovarian function.Crossref | GoogleScholarGoogle Scholar |

Khodayar, MJ, Kalantari, H, Khorsandi, L, Rashno, M, and Zeidooni, L (2020). Upregulation of Nrf2-related cytoprotective genes expression by acetaminophen-induced acute hepatotoxicity in mice and the protective role of betaine. Human & Experimental Toxicology 39, 948–959.
Upregulation of Nrf2-related cytoprotective genes expression by acetaminophen-induced acute hepatotoxicity in mice and the protective role of betaine.Crossref | GoogleScholarGoogle Scholar |

King, GL, and Loeken, MR (2004). Hyperglycemia-induced oxidative stress in diabetic complications. Histochemistry and Cell Biology 122, 333–338.
Hyperglycemia-induced oxidative stress in diabetic complications.Crossref | GoogleScholarGoogle Scholar |

Kinyua, AW, Doan, KV, Yang, DJ, Huynh, MKQ, Choi, Y-H, Shin, DM, and Kim, KW (2018). Insulin regulates adrenal steroidogenesis by stabilizing SF-1 activity. Scientific Reports 8, 5025.
Insulin regulates adrenal steroidogenesis by stabilizing SF-1 activity.Crossref | GoogleScholarGoogle Scholar |

Kono, Y (1978). Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase. Archives of Biochemistry and Biophysics 186, 189–195.
Generation of superoxide radical during autoxidation of hydroxylamine and an assay for superoxide dismutase.Crossref | GoogleScholarGoogle Scholar |

Koroliuk, MA, Ivanova, LI, Maorova, IG, and Tokarev, VE (1988). A method of determining catalase activity. Laboratornoe Delo 1, 16–19.

Lai, Q, Xiang, W, Li, Q, Zhang, H, Li, Y, Zhu, G, Xiong, C, and Jin, L (2018). Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome. Frontiers of Medicine 12, 518–524.
Oxidative stress in granulosa cells contributes to poor oocyte quality and IVF-ET outcomes in women with polycystic ovary syndrome.Crossref | GoogleScholarGoogle Scholar |

Lei, L, Ge, J, Zhao, H, Wang, X, and Yang, L (2019). Role of endoplasmic reticulum stress in lipopolysaccharide-inhibited mouse granulosa cell estradiol production. Journal of Reproduction and Development 65, 459–465.
Role of endoplasmic reticulum stress in lipopolysaccharide-inhibited mouse granulosa cell estradiol production.Crossref | GoogleScholarGoogle Scholar |

Li, C, Wang, Y, Li, L, Han, Z, Mao, S, and Wang, G (2019). Betaine protects against heat exposure–induced oxidative stress and apoptosis in bovine mammary epithelial cells via regulation of ROS production. Cell Stress and Chaperones 24, 453–460.
Betaine protects against heat exposure–induced oxidative stress and apoptosis in bovine mammary epithelial cells via regulation of ROS production.Crossref | GoogleScholarGoogle Scholar |

Liddell, JR (2017). Interplay between Nrf2 and NF-κB in neuroinflammatory diseases. Journal of Clinical & Cellular Immunology 8, 489.
Interplay between Nrf2 and NF-κB in neuroinflammatory diseases.Crossref | GoogleScholarGoogle Scholar |

Ma, Q (2013). Role of Nrf2 in oxidative stress and toxicity. Annual Review of Pharmacology and Toxicology 53, 401–426.
Role of Nrf2 in oxidative stress and toxicity.Crossref | GoogleScholarGoogle Scholar |

Margolin, Y, Aten, RF, and Behrman, HR (1990). Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells. Endocrinology 127, 245–250.
Antigonadotropic and antisteroidogenic actions of peroxide in rat granulosa cells.Crossref | GoogleScholarGoogle Scholar |

Nayki, U, Onk, D, Balci, G, Nayki, C, Onk, A, and Gunay, M (2016). The effects of diabetes mellitus on ovarian injury and reserve: an experimental study. Gynecologic and Obstetric Investigation 81, 424–429.
The effects of diabetes mellitus on ovarian injury and reserve: an experimental study.Crossref | GoogleScholarGoogle Scholar |

Nna, VU, Bakar, ABA, Ahmad, A, and Mohamed, M (2019). Down-regulation of steroidogenesis-related genes and its accompanying fertility decline in streptozotocin-induced diabetic male rats: ameliorative effect of metformin. Andrology 7, 110–123.
Down-regulation of steroidogenesis-related genes and its accompanying fertility decline in streptozotocin-induced diabetic male rats: ameliorative effect of metformin.Crossref | GoogleScholarGoogle Scholar |

Ortega, I, Wong, DH, Villanueva, JA, Cress, AB, Sokalska, A, Stanley, SD, and Duleba, AJ (2012). Effects of resveratrol on growth and function of rat ovarian granulosa cells. Fertility and Sterility 98, 1563–1573.
Effects of resveratrol on growth and function of rat ovarian granulosa cells.Crossref | GoogleScholarGoogle Scholar |

Ramalho-Santos, J, Amaral, S, and Oliveira, PJ (2008). Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species. Current Diabetes Reviews 4, 46–54.
Diabetes and the impairment of reproductive function: possible role of mitochondria and reactive oxygen species.Crossref | GoogleScholarGoogle Scholar |

Ryu, JS, Kim, TK, Chung, JY, and Lee, GM (2000). Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant Chinese hamster ovary cell cultures differs among cell lines. Biotechnology and Bioengineering 70, 167–175.
Osmoprotective effect of glycine betaine on foreign protein production in hyperosmotic recombinant Chinese hamster ovary cell cultures differs among cell lines.Crossref | GoogleScholarGoogle Scholar |

Saha, S, Buttari, B, Panieri, E, Profumo, E, and Saso, L (2020). An overview of Nrf2 signaling pathway and its role in inflammation. Molecules 25, 5474–5505.
An overview of Nrf2 signaling pathway and its role in inflammation.Crossref | GoogleScholarGoogle Scholar |

Samie, KA, Tabandeh, MR, and Afrough, M (2020). Betaine ameliorates impaired steroidogenesis and apoptosis in mice granulosa cells induced by high glucose concentration. Systems Biology in Reproductive Medicine 66, 400–409.
Betaine ameliorates impaired steroidogenesis and apoptosis in mice granulosa cells induced by high glucose concentration.Crossref | GoogleScholarGoogle Scholar |

Sharma, R, Buras, E, Terashima, T, Serrano, F, Massaad, CA, Hu, L, Bitner, B, Inoue, T, Chan, L, and Pautler, RG (2010). Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice. PLoS ONE 5, e13463.
Hyperglycemia induces oxidative stress and impairs axonal transport rates in mice.Crossref | GoogleScholarGoogle Scholar |

Sohel, MMH, Amin, A, Prastowo, S, Linares-Otoya, L, Hoelker, M, Schellander, K, and Tesfaye, D (2018). Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway. Cell and Tissue Research 374, 629–641.
Sulforaphane protects granulosa cells against oxidative stress via activation of NRF2-ARE pathway.Crossref | GoogleScholarGoogle Scholar |

Tohyama, M, Hirata, S, Kai, K, Tsunoda, M, Hashimoto, M, Goto, H, Iwashita, T, and Hirai, T (2006). Effect of addition of betaine in the medium of in-vitro production of bovine blastocysts. Animal Science Journal (Japan) 77, 479–483.

Tosca, L, Froment, P, Solnais, P, Ferré, P, Foufelle, F, and Dupont, J (2005). Adenosine 5′-monophosphate-activated protein kinase regulates progesterone secretion in rat granulosa cells. Endocrinology 146, 4500–4513.
Adenosine 5′-monophosphate-activated protein kinase regulates progesterone secretion in rat granulosa cells.Crossref | GoogleScholarGoogle Scholar |

Uchiyama, M, and Mihara, M (1978). Determination of malonaldehyde precursor in tissues by thiobarbituric acid test. Analytical Biochemistry 86, 271–278.
Determination of malonaldehyde precursor in tissues by thiobarbituric acid test.Crossref | GoogleScholarGoogle Scholar |

Wang, Q, Chi, MM, Schedl, T, and Moley, KH (2012). An intercellular pathway for glucose transport into mouse oocytes. American Journal of Physiology-Endocrinology and Metabolism 302, E1511–E1518.
An intercellular pathway for glucose transport into mouse oocytes.Crossref | GoogleScholarGoogle Scholar |

Wardyn, JD, Ponsford, AH, and Sanderson, CM (2015). Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways. Biochemical Society Transactions 43, 621–626.
Dissecting molecular cross-talk between Nrf2 and NF-κB response pathways.Crossref | GoogleScholarGoogle Scholar |

Wu, Y, Li, Y, Liao, X, Wang, Z, Li, R, Zou, S, Jiang, T, Zheng, B, Duan, P, and Xiao, J (2017). Diabetes induces abnormal ovarian function via triggering apoptosis of granulosa cells and suppressing ovarian angiogenesis. International Journal of Biological Sciences 13, 1297–1308.
Diabetes induces abnormal ovarian function via triggering apoptosis of granulosa cells and suppressing ovarian angiogenesis.Crossref | GoogleScholarGoogle Scholar |

Xu, B, Hu, Y, Luo, Y-T, Xu, H, Qi, S-H, Zhang, Y-Y, Zhao, D, and Yuan, D-Z (2019). Changes in expression of follicular glucose transporters may be involved in ovarian function impairment during diabetic hyperglycemia. Annals of Clinical & Laboratory Science 49, 785–793.

Yagisawa, M, Doi, Y, Uenohara, T, Toda, M, Shigematsu, N, and Nakata, R (2006). Betaine supplementation suppresses plasma homocysteine level elevation induced by folate deficiency in rats. Nutrition Research 26, 266–270.
Betaine supplementation suppresses plasma homocysteine level elevation induced by folate deficiency in rats.Crossref | GoogleScholarGoogle Scholar |

Zhang, M, Zhang, H, Li, H, Lai, F, Li, X, Tang, Y, Min, T, and Wu, H (2016). Antioxidant mechanism of betaine without free radical scavenging ability. Journal of Agricultural and Food Chemistry 64, 7921–7930.
Antioxidant mechanism of betaine without free radical scavenging ability.Crossref | GoogleScholarGoogle Scholar |

Zhao, G, He, F, Wu, C, Li, P, Li, N, Deng, J, Zhu, G, Ren, W, and Peng, Y (2018). Betaine in inflammation: mechanistic aspects and applications. Frontiers in Immunology 9, 1070.
Betaine in inflammation: mechanistic aspects and applications.Crossref | GoogleScholarGoogle Scholar |