Endocrine Abstracts

Introduction: GPRC6A is a widely expressed seven-transmembrane G-protein coupled receptor mediating L-α-amino acids signaling (mainly, arginine, lysine, and ornithine), which has been proposed as receptor for osteocalcin. By interacting with GPRC6A, in β-cells, osteocalcin is thought to modulate the glucose-induced insulin secretion. However, GPRC6A functions have been studied only in rodents or in rodents-derived cells.

Aim of this study was to characterize the GPRC6A expression in different culture conditions in 1.1B4 cells, a human model of pancreatic β-cells.

Methods: 1.1B4 cells were cultured in RPMI-1640 medium supplemented with 10% FBS. To evaluate GPRC6A expression cells were seeded at different densities, from 2000 to 40 000/cm² and harvested every day over 7 days.

At the end of the experiments total RNA was extracted, reverse transcribed to cDNA and amplified through PCR by means of GPRC6A specific primers. In parallel GPRC6A protein expression in cell lysates was evaluated by western blotting.

Results: Our results show that at low cell densities (2000–5000/cm²) GPRC6A expression was kept very low over the culture period. Instead, at higher cell densities (>10 000/cm²) the expression level is increased over time parallel to the cell growth, and it remained stable from day 4 up to day 7. Western blotting results, however, differed from gene expression data since from day 3 the protein level remained stable.

Conclusions: GPRC6A expression in 1.1B4 human pancreatic β-cells is very unstable and it is strikingly dependent on seeding cell density and days on cultures.