Betaine ameliorates impaired steroidogenesis and apoptosis in mice granulosa cells induced by high glucose concentration

ABSTRACT

Betaine is a bioactive peptide whose beneficial effects on diabetes complications have been considered, previously. The present study aimed to investigate the possible protective effects of betaine against hyperglycemia-induced steroidogenesis impairment and apoptosis in mice granulosa cells. Ovarian granulosa cells were isolated from C57/BL6 mice and cultured in steroidogenesis medium (SM) containing 30 ng/ml FSH and 0.5 µM testosterone. The cells were cultured in SM containing low (5 mM) or high (30 mM) glucose concentrations for 24 h in the presence or absence of betaine (5 mM). At the end of the experiment, estradiol and progesterone were measured by ELISA in the culture medium. Expression of apoptosis and steroidogenesis associated genes and caspase-3 activity were determined by qRT-PCR and colorimetric assays, respectively. Exposure of mice granulosa cells to high glucose concentration inhibited the steroidogenesis by decreasing estradiol and progesterone secretion and downregulation of steroidogenesis-related genes including 3βHSD, Cyp11a1, Cyp19a1, and StAR. Betaine treatment could ameliorate the steroidogenesis impairment at molecular and biochemical levels. High glucose concentration also enhanced apoptosis in mice granulosa cells that were characterized by elevation of caspase-3 activity, upregulation of bax gene and downregulation of bcl2 gene. Betaine treatment could attenuate the apoptotic-related changes induced by high glucose concentration in granulosa cells. According to the results of the present study, betaine could ameliorate the adverse effects of hyperglycemia on the physiological function of ovarian granulosa cells. The results highlight the potential role of betaine for the intervention of ovarian dysfunction in diabetic patients.

Abbreviations: AABA: Betaine-α-aminobutyric acid; AGEs: Advanced glycation end products; bax: bcl2 Associated X; bcl2: B-cell lymphoma 2; AMPK: AMP-activated protein kinase; BHMT: Betaine homocysteine methyltransferase; C/EBP: CCAAT-enhancer-binding proteins; Cyp11a1: Cholesterol side-chain cleavage cytochrome P450; Cyp19a1: Cytochrome P450 aromatase; DM: Diabetes mellitus; E2: Estradiol; ERS: Endoplasmic reticulum stress; GCs: Granulosa cells; GLUT: Glucose transporter; FSH: Follicle-stimulating hormone; 3βHSD: 3β-hydroxysteroid dehydrogenase; IL-1β: interleukin-1ß; LH: Luteinizing hormone; MDCK: Madin-Darby Canine Kidney cell; MT: Methionine synthase, MTT: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NLRP3: NLR Family Pyrin Domain Containing 3; NF-κB: Nuclear factor κB; P4: Progesterone; ROS: Reactive oxygen species; SGLT: Sodium dependent glucose transporter; SLC7A6: Solute Carrier Family 7 Member 6; StAR: Steroidogenic acute regulatory protein; STZ: Streptozotocin; Tumor necrosis factor α: TNF-α; TXNIP: Thioredoxin interacting protein.

KEYWORDS:

• Granulosa cells
• hyperglycemia
• apoptosis
• steroidogenesis
• betaine

Ethics approval

All protocols of the present study were approved by the research ethics committee of Shahid Chamran University of Ahvaz (EE/98.24.3.2.26576/scu.ac.ir).

Acknowledgments

This work was funded by a grant from Shahid Chamran University of Ahvaz Research Council (Grant No: SCU.VB98.231).

Disclosure statement

Authors declare that there is no conflict of interest.

Author contributions

All authors contributed to the study conception and design. MRT and MA designed the study. KAS performed the study and collected the data. MRT and MA analyzed the results. MRT and KAS drafted the article. MRT and MA revised the article critically for important intellectual content. All authors gave final approval of the version to be published. MRT is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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