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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 is a widely expressed G-protein coupled receptor that regulates energy metabolism. Global deletion of Gprc6a in mice is reported to result in a metabolic syndrome-like phenotype and conditional deletion of Gprc6a in pancreatic β-cell and skeletal muscle respectively impair insulin secretion and glucose uptake. In the current study, we explore the hepatic functions of GPRC6A by conditionally deleting Gprc6a in hepatocytes by cross breeding Alb-Cre and Gprc6a flox/flox mice to obtain Gprc6a Liver-cko mice. Gprc6a Liver-cko mice on a normal diet showed excessive hepatic fat accumulation and glycogen depletion. These mice also exhibit impaired glucose and pyruvate tolerance, but normal insulin sensitivity. Decreased circulating FGF-21 levels and FGF-21 message expression in the liver were found in Gprc6a Liver-cko mice. Hepatic transcriptome analysis identified alterations in multiple pathways regulating glucose, fat and glycogen metabolism in Gprc6a Liver-cko mice. Taken together, our studies suggest that GPRC6A directly regulates hepatic metabolism as well as regulates the production and release of FGF-21 to control systemic energy homeostasis. GPRC6A's unique regulation of β-cell, skeletal muscle and hepatic function may represent a new therapeutic target for treating disordered energy metabolism metabolic syndrome and type 2 diabetes.

The skeleton is an endocrine organ that regulates energy metabolism through the release of the osteoblast-derived hormone, osteocalcin (Ocn), and phosphate and vitamin D homeostasis through the secretion by osteoblasts and osteocytes of the novel hormone, FGF23 Ocn activates a widely expressed G-protein coupled receptor, GPRC6A, to regulate insulin secretion by pancreatic β-cells, testosterone secretion by testicular Leydig cells, fatty acid metabolism in the liver, and insulin sensitivity of muscle and fat, as well as other functions. FGF23 targets a limited number of tissues, including kidney, parathyroid gland, choroid plexus, and pituitary gland that co-express FGF receptors and α-Klotho complexes. Ectodomain shedding and secretion of a soluble form of Klotho also is purported to act as an anti-ageing hormone. Further elucidation of these novel endocrine networks is likely to lead to new appreciation of the cooperation between various organ systems to regulate phosphate, vitamin D, and energy metabolism.

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. [ABSTRACT FROM AUTHOR]