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Metformin inhibits the activation of melanocortin receptors 2 and 3 in vitro: A possible mechanism for its anti-androgenic and weight balancing effects in vivo?
The Journal of Steroid Biochemistry and Molecular Biology ( IF 2.7 ) Pub Date : 2020-04-28 , DOI: 10.1016/j.jsbmb.2020.105684 Shaheena Parween 1 , Silvia Rihs 1 , Christa E Flück 1
The Journal of Steroid Biochemistry and Molecular Biology ( IF 2.7 ) Pub Date : 2020-04-28 , DOI: 10.1016/j.jsbmb.2020.105684 Shaheena Parween 1 , Silvia Rihs 1 , Christa E Flück 1
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Metformin is recommended as one of the first-line drugs for the treatment of type 2 diabetes and the metabolic syndrome. In addition to its insulin sensitizing effects, it has been shown to attenuate androgen excess in women with polycystic ovary syndrome (PCOS) or congenital adrenal hyperplasia (CAH), as well as to ameliorate obesity. The mechanisms of metformin action seem manifold. Preclinical studies suggest that it inhibits the cellular stress response at the level of the mitochondrial OXPHOS system and through AMPK dependent and independent mechanisms. Recent studies have shown that metformin decreases ACTH secretion from pituitary and reduces ACTH-stimulated adrenal secretion. In this study we investigated its specific effect through the melanocortin receptor 2 (MC2R) on signaling targeting adrenal steroidogenesis. To assess this effect, we used mouse adrenal OS3 cells, which do not express the MC2R. Cells were transfected with the MC2R and stimulated by ACTH. Downstream cyclic AMP production was then assessed by a co-transfected cAMP-responsive vector producing luciferase that was measured by a dual luciferase assay. The amount of luciferase produced in this assay corresponds to the amount of receptor activation with varying amount of ACTH. The effect of metformin was then tested in this system. We found a significant inhibition of ACTH induced MC2R activation and signaling with 10 mM metformin. The ACTH concentration response curve (CRC) was half-log shifted and a ∼30 % reduction in maximum receptor response (Rmax) to ACTH in presence of metformin was observed. This effect was dose dependent with an IC50 of 4.2 mM. qRT-PCR analyses showed that metformin decreased ACTH induced MC2R expression. Metformin did not affect cell viability and basal cAMP levels. We also tested the effect of metformin on homologous melanocortin receptors (MCRs). No significant effect was found on MC1R and MC4R activity. However, a log shift of EC50 of ACTH stimulation on MC3R was observed with metformin treatment. Metformin also inhibited melanocortin stimulating hormone (αMSH) induced MC3R activity. In conclusion, we show that metformin acts on MC2R and MC3R signaling directly. The role of MC2R for steroidogenesis is well established. MC3R is involved in energy balance and seems to act as a rheostat when the metabolism is challenged. Our study may explain how metformin helps in weight loss and attenuates the excess response to ACTH in androgen excess disorders such as PCOS and CAH.
中文翻译：
二甲双胍在体外抑制黑皮质素受体2和3的激活：体内抗雄激素和体重平衡作用的可能机制是什么？
二甲双胍被推荐作为治疗2型糖尿病和代谢综合征的一线药物之一。除具有胰岛素增敏作用外，它还可以减轻患有多囊卵巢综合症（PCOS）或先天性肾上腺皮质增生（CAH）的女性的雄激素过多，并改善肥胖症。二甲双胍作用机制似乎多种多样。临床前研究表明，它在线粒体OXPHOS系统水平以及通过AMPK依赖和独立机制抑制细胞应激反应。最近的研究表明，二甲双胍可降低垂体的ACTH分泌并减少ACTH刺激的肾上腺分泌。在这项研究中，我们研究了其通过黑皮质素受体2（MC2R）对靶向肾上腺类固醇生成的信号的特异性作用。为了评估这种效果，我们使用了不表达MC2R的小鼠肾上腺OS3细胞。细胞用MC2R转染并被ACTH刺激。然后通过产生荧光素酶的共转染的cAMP-应答载体，通过双重荧光素酶测定法测量下游环状AMP的产生。在该测定中产生的萤光素酶的量对应于具有不同ACTH量的受体活化的量。然后在该系统中测试了二甲双胍的作用。我们发现10 mM二甲双胍对ACTH诱导的MC2R激活和信号传导具有显着抑制作用。ACTH浓度反应曲线（CRC）对数移动了一半，观察到二甲双胍存在时对ACTH的最大受体反应（Rmax）降低了约30％。这种作用是剂量依赖性的，IC50为4.2 mM。qRT-PCR分析表明二甲双胍降低了ACTH诱导的MC2R表达。二甲双胍不影响细胞生存力和基础cAMP水平。我们还测试了二甲双胍对同源黑皮质素受体（MCRs）的作用。没有发现对MC1R和MC4R活性有明显影响。但是，用二甲双胍治疗时，观察到了ACTH刺激对MC3R的EC50的对数漂移。二甲双胍还抑制黑皮质素刺激激素（αMSH）诱导的MC3R活性。总之，我们表明二甲双胍直接作用于MC2R和MC3R信号传导。众所周知，MC2R在类固醇生成中的作用。MC3R参与能量平衡，并且在新陈代谢受到挑战时似乎充当变阻器。我们的研究可能解释了二甲双胍如何帮助减轻体重并减轻雄激素过多症（例如PCOS和CAH）对ACTH的过度反应。
更新日期：2020-04-28
中文翻译：
二甲双胍在体外抑制黑皮质素受体2和3的激活：体内抗雄激素和体重平衡作用的可能机制是什么？
二甲双胍被推荐作为治疗2型糖尿病和代谢综合征的一线药物之一。除具有胰岛素增敏作用外，它还可以减轻患有多囊卵巢综合症（PCOS）或先天性肾上腺皮质增生（CAH）的女性的雄激素过多，并改善肥胖症。二甲双胍作用机制似乎多种多样。临床前研究表明，它在线粒体OXPHOS系统水平以及通过AMPK依赖和独立机制抑制细胞应激反应。最近的研究表明，二甲双胍可降低垂体的ACTH分泌并减少ACTH刺激的肾上腺分泌。在这项研究中，我们研究了其通过黑皮质素受体2（MC2R）对靶向肾上腺类固醇生成的信号的特异性作用。为了评估这种效果，我们使用了不表达MC2R的小鼠肾上腺OS3细胞。细胞用MC2R转染并被ACTH刺激。然后通过产生荧光素酶的共转染的cAMP-应答载体，通过双重荧光素酶测定法测量下游环状AMP的产生。在该测定中产生的萤光素酶的量对应于具有不同ACTH量的受体活化的量。然后在该系统中测试了二甲双胍的作用。我们发现10 mM二甲双胍对ACTH诱导的MC2R激活和信号传导具有显着抑制作用。ACTH浓度反应曲线（CRC）对数移动了一半，观察到二甲双胍存在时对ACTH的最大受体反应（Rmax）降低了约30％。这种作用是剂量依赖性的，IC50为4.2 mM。qRT-PCR分析表明二甲双胍降低了ACTH诱导的MC2R表达。二甲双胍不影响细胞生存力和基础cAMP水平。我们还测试了二甲双胍对同源黑皮质素受体（MCRs）的作用。没有发现对MC1R和MC4R活性有明显影响。但是，用二甲双胍治疗时，观察到了ACTH刺激对MC3R的EC50的对数漂移。二甲双胍还抑制黑皮质素刺激激素（αMSH）诱导的MC3R活性。总之，我们表明二甲双胍直接作用于MC2R和MC3R信号传导。众所周知，MC2R在类固醇生成中的作用。MC3R参与能量平衡，并且在新陈代谢受到挑战时似乎充当变阻器。我们的研究可能解释了二甲双胍如何帮助减轻体重并减轻雄激素过多症（例如PCOS和CAH）对ACTH的过度反应。
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