GPRC6A null mice exhibit osteopenia, feminization and metabolic syndrome

Abstract

Background: GPRC6A is a widely expressed orphan G-protein coupled receptor that senses extracellular amino acids, osteocalcin and divalent cations in vitro. The physiological functions of GPRC6A are unknown.

Methods/principal findings: In this study, we created and characterized the phenotype of GPRC6A(-/-) mice. We observed complex metabolic abnormalities in GPRC6A(-/-) mice involving multiple organ systems that express GPRC6A, including bone, kidney, testes, and liver. GPRC6A(-/-) mice exhibited hepatic steatosis, hyperglycemia, glucose intolerance, and insulin resistance. In addition, we observed high expression of GPRC6A in Leydig cells in the testis. Ablation of GPRC6A resulted in feminization of male GPRC6A(-/-) mice in association with decreased lean body mass, increased fat mass, increased circulating levels of estradiol, and reduced levels of testosterone. GPRC6A was also highly expressed in kidney proximal and distal tubules, and GPRC6A(-/-) mice exhibited increments in urine Ca/Cr and PO(4)/Cr ratios as well as low molecular weight proteinuria. Finally, GPRC6A(-/-) mice exhibited a decrease in bone mineral density (BMD) in association with impaired mineralization of bone.

Conclusions/significance: GPRC6A(-/-) mice have a metabolic syndrome characterized by defective osteoblast-mediated bone mineralization, abnormal renal handling of calcium and phosphorus, fatty liver, glucose intolerance and disordered steroidogenesis. These findings suggest the overall function of GPRC6A may be to coordinate the anabolic responses of multiple tissues through the sensing of extracellular amino acids, osteocalcin and divalent cations.

Figure 1. Generation of GPRC6A knockout mice.

(A) GPRC6A knockout targeting strategy. (B) Mouse genotyping by PCR shows the presence of exon 2 in wild-type (GPRC6A ^+/+) and its absence in homozygous GPRC6A knockout (GPRC6A ^−/−) mice. RT-PCR analysis (C) and Western Blot analysis (D) of GPRC6A expression in kidney from GPRC6A ^+/+ and GPRC6A ^−/− mice. RT-PCR condition, primer set and anti-mGPRC6A antibody are described in Materials and Methods.

Figure 2. Characterization of reproductive system of male GPRC6A ^−/− mice.

(A) Gross appearance of male GPRC6A ^−/− mice. The genito-anal distance is demarcated by arrows. (B) Comparison of the genito-anal distance in 16 week-old GPRC6A^+/+ and GPRC6A ^−/− male mice. (C) Gross appearance of testes of male GPRC6A^+/+ and GPRC6A ^−/− at age of 16 week-of-age. Upper panel shows testis and epididymis (magnification 10×) and lower panel shows dissected testis (magnification 20×) viewed under dissecting microscope. (D and E) Comparison of testicular weight (D) and seminal vesicle weight (E) in 16 week-old GPRC6A^+/+ and GPRC6A ^−/− mice. (F) H&E stained sections of testicular histology (upper panel; 200× magnification) and in-situ for GPRC6A in the testis (lower panel; 400× magnification) from GPRC6A^+/+ and GPRC6A ^−/− mice. The arrow heads depict sites of high GPRC6A expression in Leydig cells, and the arrows indicate that GPRC6A is also expressed in lower amounts in sertoli cells, spermatogonia and spermatids. (G) Mammary gland abnormalities in male GPRC6A ^−/− mice at 16 week-old. (H) Comparison of mammary fat pad mass in 16 week-old male GPRC6A^+/+ and GPRC6A ^−/− mice. Data represent the mean±SEM from 6 to 10 mice in each group. * indicates a significant difference from GPRC6A ^+/+ and GPRC6A ^−/− mice at p<0.05, respectively.

Figure 3. Possible mechanisms underlying altered testosterone/estrogen ratio in GPRC6A ^−/− male mice.

(A and B) RT-PCR and real-time RT-PCR analysis of androgen receptor (AR) expression. AR expression in testis (Te) and bone marrow (BM) was not different between GPRC6A^+/+ and GPRC6A ^−/− male mice. (C) Real time RT-PCR analysis of aromatase expression in testis. (D and E) Comparison of the aromatase protein expression in testis from GPRC6A^+/+ and GPRC6A ^−/− male mice. Small increments in aromatase protein expression were observed in GPRC6A ^−/− male mice by Western blot analysis (D) that was localized by immunhistochemistry (E) to Leydig cells (L, arrow head) and to a lesser degree in sertoli cells (SC), and spermatogonia (SG) (respectively indicated by arrows). (F and G) Real time RT-PCR analysis of Cyp17 and Sult1e1 expression in testis. (H and I) RT-PCR and real-time RT-PCR analysis of GnRH expression in brain.

Figure 4. GPRC6A deficiency is associated with a renal phenotype.

(A) Expression of GPRC6A messenger in kidney by in-situ hybridization showing localization of both proximal and distal tubular segments. (B-D) Kidney expression of NaPi IIa. Immunohistochemistry (B) demonstrates decreased Napi IIa protein expression and translocation to the brush border membrane in GPRC6A ^−/− mice. Loss of GPRC6A also resulted in decreased Napi IIa message expression by RT-PCR (C) and real-time RT-PCR analysis (D). Data represent the mean±SEM from 6 to 10 mice in each group. * indicates a significant difference from GPRC6A ^+/+ and GPRC6A ^−/− mice at p<0.05, respectively. (E and F) Low molecular weight proteinuria in GPRC6A ^−/− mice. Western-blot analysis using 4–12% SDS-Page gel and comasis blue staining identified an increase in urinary excretion of a low molecular weight protein in GPRC6A ^−/− mice (E) that was identified as β2-mcroglobulin by immunobloting with an anti-β2-mcroglobulin antibody (F). The arrow indicates β2-mcroglobulin.

Figure 5. GPRC6A deficiency is associated with a liver phenotype.

(A) Histological examination of liver from GPRC6A ^−/− male mice at age of 16 week old by H & E stained (left panel), Oil Red O stained (right panel). (B) Hepatic triglyceride levels in GPRC6A ^+/+ and GPRC6A ^−/− male mice at age of 16 week old. Liver triglyceride levels were expressed as mg/g liver tissue. (C and D) GTT (C) and ITT (D) in 3 month-old male GPRC6A ^+/+ and GPRC6A ^−/− mice. ITT data are presented as percentage of initial blood glucose concentration. Data represent the mean±SEM from more than 5 male mice in each group. * Significant difference from GPRC6A^+/+ and GPRC6A ^−/− mice at p<0.05.

Figure 6. Characterization of the bone phenotype of GPRC6A ^−/− mice.

(A–C) Comparison of the lean body mass (A), fat content (B) and bone mineral density of the femur analysis (C) by PIXImus^TM analysis in GPRC6A ^+/+ and GPRC6A ^−/− mice at ages ranging from 6 to 16 weeks. Data represent the mean±SEM from 6 to 10 mice in each group. * Significant difference from GPRC6A^+/+ and GPRC6A ^−/− mice at p<0.05. (D) Backscattered Scanning Electron Microscopy analysis of tibia cortical bone in 16-week-old GPRC6A^+/+ (upper panel) and GPRC6A ^−/− mice (lower panel). The arrows showed the diminished mineralization layer in the bone of GPRC6A ^−/− mice. (E) Toluidine blue-stained plastic sections of femur from 16-week-old GPRC6A^+/+ (upper panel) and GPRC6A ^−/− mice (lower panel). The arrows showed the unmineralized osteoid surfaces in the bone of GPRC6A ^−/− mice. (F) Plastic unstained sections of tibia cortical bone viewed under fluorescent light in 16-week-old GPRC6A^+/+ (upper panel) and GPRC6A ^−/− mice (lower panel) prelabeled with twice calcein (double label).