Jo Rie, Shibata Hirotaka, Kurihara Isao, Yokota Kenichi, Kobayashi Sakiko, Murai-Takeda Ayano, Mitsuishi Yuko, Hayashi Takeshi, Nakamura Toshifumi, Itoh Hiroshi
Division of Endocrinology, Metabolism and Nephrology, Department of Internal Medicine, School of Medicine, Keio University, Tokyo, Japan.
Keiyu Hospital, Kanagawa, Japan.
Hypertens Res. 2023 Jan;46(1):19-31. doi: 10.1038/s41440-022-01036-6. Epub 2022 Oct 14.
This study investigated the mechanism underlying the beneficial effects of mineralocorticoid receptor (MR) antagonists in patients with resistant hypertension and diabetic nephropathy by examining post-translational modification of the MR by O-linked-N-acetylglucosamine (O-GlcNAc), which is strongly associated with type 2 diabetes. Coimmunoprecipitation assays in HEK293T cells showed that MR is a target of O-GlcNAc modification (O-GlcNAcylation). The expression levels and transcriptional activities of the receptor increased in parallel with its O-GlcNAcylation under high-glucose conditions. Liquid chromatography-tandem mass spectrometry revealed O-GlcNAcylation of the MR at amino acids 295-307. Point mutations in those residues decreased O-GlcNAcylation, and both the protein levels and transcriptional activities of MR. In db/db mouse kidneys, MR protein levels increased in parallel with overall O-GlcNAc levels of the tissue, accompanied by increased SGK1 mRNA levels. The administration of 6-diazo-5-oxo-L-norleucin, an inhibitor of O-GlcNAcylation, reduced tissue O-GlcNAc levels and MR protein levels in db/db mice. Thus, our study showed that O-GlcNAcylation of the MR directly increases protein levels and transcriptional activities of the receptor under high-glucose conditions in vitro and in vivo. These findings provide a novel mechanism of MR as a target for prevention of complications associated with diabetes mellitus.
本研究通过检测与2型糖尿病密切相关的O-连接N-乙酰葡糖胺（O-GlcNAc）对盐皮质激素受体（MR）的翻译后修饰，探讨了MR拮抗剂对顽固性高血压和糖尿病肾病患者有益作用的潜在机制。在HEK293T细胞中进行的免疫共沉淀试验表明，MR是O-GlcNAc修饰（O-GlcNAcylation）的靶点。在高糖条件下，受体的表达水平和转录活性与其O-GlcNAcylation水平平行升高。液相色谱-串联质谱分析显示，MR在氨基酸295-307处发生O-GlcNAcylation。这些残基的点突变降低了O-GlcNAcylation水平，以及MR的蛋白水平和转录活性。在db/db小鼠肾脏中，MR蛋白水平与组织的总体O-GlcNAc水平平行升高，同时伴有SGK1 mRNA水平升高。给予O-GlcNAcylation抑制剂6-重氮-5-氧代-L-正亮氨酸可降低db/db小鼠的组织O-GlcNAc水平和MR蛋白水平。因此，我们的研究表明，在体外和体内的高糖条件下，MR的O-GlcNAcylation直接增加了受体的蛋白水平和转录活性。这些发现为MR作为预防糖尿病相关并发症靶点提供了一种新机制。
