外源給予緩釋型胰島素glargine對小鼠萊迪氏細胞類固醇生成作用之影響

Abstract

睪固酮 (Testosterone) 主要由雄性生殖系統中之睪丸萊迪氏細胞 (Leydig cell) 分泌，並受到下視丘-腦下垂體-性腺軸(Hypothalamic-pituitary-gonadal axis) 調控，當萊迪氏細胞接受排卵素 (Luteinizing hormone, LH) 的訊號時，便會啟動下游訊息傳遞路徑，增加類固醇生成。近年研究發現，有代謝疾病的男性，常發生血中睪固酮濃度較低的性腺功能低下現象，但血中LH濃度卻與健康男性沒有差異，暗示有其他可能因素影響睪固酮分泌。目前已知代謝疾病常伴隨著胰島素上升，然尚未有研究探討長期高胰島素對睪固酮分泌的影響，因此本研究目的在探討長期胰島素是否會影響睪丸萊迪氏細胞的睪固酮分泌。 為探討高胰島素對類固醇生成之影響，以皮下注射給予C57BL/6J小鼠緩釋型胰島素glargine，兩天一次為期八周。結果發現長期注射glargine使空腹血糖及胰島素顯著增加。而LH濃度沒有差異，但睪固酮卻顯著下降。分析睪丸內胰島素訊息傳遞及類固醇生成相關蛋白質表現，發現長期glargine的處理會使睪丸產生胰島素阻抗 (insulin resistance)，且類固醇生成相關酵素3β-HSD1則有顯著減少，顯示部分類固醇生成酵素受到抑制。而類固醇生成相關酵素之抑制因子，不論是DAX1的蛋白質表現量或AMPK的磷酸化程度皆沒有差異。為探討其可能機制，我們使用小鼠腫瘤細胞株MA-10以glargine處理48小時後，並以人類絨毛膜激性腺素 (Human chorionic gonadotropin, hCG)、毛喉素 (Forskolin) 及 8-Br-cAMP (Protein kinase A 促進劑) 刺激類固醇生成，結果發現glargine能抑制以毛喉素及hCG刺激下之類固醇生成，並降低StAR的蛋白質表現量，但CYP11A1的蛋白質表現量則沒有差異，然以8-Br-cAMP刺激則未觀察到類固醇生成下降。 綜合以上所述，長期處理glargine會導致萊迪氏細胞之類固醇生成下降，其可能透過降低部分類固醇生成相關蛋白質，如StAR及3β-HSD1的表現量，以及導致胰島素阻抗。然胰島素阻抗與glargine抑制萊迪氏細胞類固醇生成之關聯性，則需更多研究闡明。

Testosterone is mainly secreted by testicular Leydig cells and is regulated by hypothalamic-pituitary-gonadal axis in male. Once Leydig cell receives luteinizing hormone (LH), it will initiate downstream signal transduction and increase steroidogenesis. Recent studies have found that men with metabolic disorder often have low blood testosterone, one of the symptoms of hypogonadism. However, the blood LH level in these men are not different in comparison to healthy individuals. This indicates some other factors may affect testosterone secretion. Researches have shown that elevated insulin is common in men with metabolic diseases. However, no study has investigated whether long-term high blood insulin affects testosterone secretion. Hence, the aim of this study is to evaluate the effects of elevated insulin on testosterone secretion in testicular Leydig cells. To investigate the effects of high insulin on steroidogenesis, we subcutaneously injected slow-acting insulin, glargine, every 2 days for 8 weeks in C57BL/6J mice. We found that long-term injection of glargine leaded to increased fasting blood glucose and insulin levels. The testosterone concentration in glargine group was lower than control group, while the LH level was not different between two groups. We found that glargine treatment would cause testicular insulin resistance. Steroidogenic enzyme expression, 3β-HSD1, in glargine treated mice was significantly decreased in comparison to control group, implying the expressions of steroidogenic enzymes were partially inhibited. The expressions of steroidogenic suppressors, DAX1 and AMPK, were not different between two groups. To further investigate the possible mechanism, we used MA-10 mouse tumor Leydig cell line as in vitro model. MA-10 cells were treated with glargine for 48 hours and increased the steroid (progesterone) production after human chorionic gonadotropin (hCG), forskolin, and 8-Br-cAMP (Protein kinase A agonist) treatment. Results showed that glargine would inhibit the steroidogenesis of MA-10 cells induced by forskolin or hCG and decrease the expression of StAR, while the expression of CYP11A1 was not different. However, glargine treatment did not inhibit the steroid production of MA-10 cells in 8-Br-cAMP treated group. In conclusion, our results showed that long-term glargine would decrease steroidogenesis in Leydig cells, possibly is by means of decreasing the expressions of some steroidogenic proteins such as StAR and 3β-HSD1 and causing insulin resistance. However, the relationship between insulin resistance and glargine inhibited steroidogenesis on Leydig cells, needs more studies to verify.