GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-^KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-^KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-^KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-^KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.

GPRC6A 被提议用于调节小鼠的能量代谢，但在人类中，第三细胞内环 (ICL3) 中的 KGKY 多态性被提议导致细胞内滞留和功能丧失。为了在体内测试这种人类多态性的生理重要性，我们通过使用 CRISPR/Cas9（成簇规律间隔的短回文重复序列-CRISPR 相关蛋白 9）系统替换 GPRC6A 小鼠基因 ICL3 中的 RKLP 序列，对小鼠进行靶向基因组人源化使用独特的人类 KGKY 序列来创建Gprc6a- ^{KGKY 敲入}小鼠。人类 KGKY 序列的敲入导致基础血糖水平降低和循环血清胰岛素和 FGF-21 浓度增加。Gprc6a- ^{KGKY 敲入}尽管胰岛素敏感性受损和丙酮酸介导的糖异生作用增强，但小鼠表现出葡萄糖耐量改善。Gprc6a ^{-KGKY 敲入}小鼠的肝脏转录组分析确定了葡萄糖、糖原和脂肪代谢途径的改变。因此，人类特有的GPRC6A- ^KGKY变体似乎是一种功能获得性多态性，可正向调节小鼠的能量代谢。