Methimazole-induced hypothyroidism disrupts testicular glycogen regulation without affecting insulin expression or signaling in rats

Thyroid hormones (TH) are essential regulators of energy metabolism and reproductive function. Hypothyroidism is a highly prevalent endocrine disorder and a risk factor for male infertility; however, its effects on the molecular pathways regulating testicular energy metabolism remains poorly understood. This study investigated the impact of hypothyroidism on insulin expression, insulin signaling, and glycogen regulation in the testis. Male Wistar rats were divided into control (CT) and hypothyroid (Hypo) groups, with hypothyroidism induced by methimazole (0.03%) in the drinking water for 21 days. Testicular mRNA expression of insulin and glucose transporters were evaluated by qPCR, while proteins related to insulin signaling, glucose metabolism, and glycophagy were analyzed by Western blot. Testicular insulin content was assessed by immunohistochemistry. Hypothyroidism reduced testosterone levels, decreased testicular mass, and impaired sperm parameters. It increased insulin receptor total protein levels and phosphorylation, without changes in the phospho/total ratio, while IRS-1, p-IRS1, AKT, p-AKT, and PTP-1B remained unchanged. Slc2a1 expression was upregulated and Slc2a3 downregulated, indicating adaptative changes in glucose uptake. Protein levels of AMPK, p-AMPK, GSK-3β, and p-GSK-3β remained unchanged. Increased total and phosphorylated GYS levels were observed without changes in the phospho/total ratio, suggesting potential modulation of glycogen metabolism. Although expression of the glycogen-binding protein STBD1 was increased, reduced GABARAPL1 levels suggest impaired glycophagy, consistent with unchanged testicular glycogen content observed in the hypothyroid group. Furthermore, testicular insulin expression remained unaffected. These findings suggest that hypothyroidism-induced testicular dysfunction is not primarily driven by local insulin resistance but may involve impaired glycogen mobilization via glycophagy. • Hypothyroidism increases testicular insulin receptor expression and phosphorylation • Downstream insulin signaling (IRS-1/AKT) is preserved in hypothyroid testes • Hypothyroidism alters glucose transporter expression, suggesting adaptive glucose uptake • Increased GYS phosphorylation suggests impaired glycogen synthesis in the testis • Reduced GABARAPL1 indicates defective glycophagy-mediated glycogen mobilization