Osteocalcin promotes β-cell proliferation during development and adulthood through Gprc6a

Abstract

Expanding β-cell mass through β-cell proliferation is considered a potential therapeutic approach to treat β-cell failure in diabetic patients. A necessary step toward achieving this goal is to identify signaling pathways that regulate β-cell proliferation in vivo. Here we show that osteocalcin, a bone-derived hormone, regulates β-cell replication in a cyclin D1-dependent manner by signaling through the Gprc6a receptor expressed in these cells. Accordingly, mice lacking Gprc6a in the β-cell lineage only are glucose intolerant due to an impaired ability to produce insulin. Remarkably, this regulation occurs during both the perinatal peak of β-cell proliferation and in adulthood. Hence, the loss of osteocalcin/Gprc6a signaling has a profound effect on β-cell mass accrual during late pancreas morphogenesis. This study extends the endocrine role of osteocalcin to the developmental period and establishes osteocalcin/Gprc6a signaling as a major regulator of β-cell endowment that can become a potential target for β-cell proliferative therapies.

Figure 1

Inactivation of Gprc6a in the β-cell lineage causes glucose intolerance and decreases insulin production. All data were obtained with 3-month-old Gprc6a fl/fl and Gprc6a_Pdx1^−/− mice, unless otherwise indicated. Quantitative PCR analysis of Gprc6a expression in indicated tissues and cells isolated from wild-type mice (A) or in islets isolated from control (Gprc6a fl/fl) and Gprc6a_Pdx1^−/− P15 mice (B). Results of GTT (C), GSIS (D), and ITT (E), with the right panels representing calculated areas under the curve for each test. Glucagon levels in plasma (F) and pancreas extracts (G). Serum insulin levels in ad libitum fed (H) or fasted (I) mice. Blood glucose levels in ad libitum fed (J) or fasted (K) mice. L: BW. M: Pancreas weight. Adip., adipocytes. *P ≤ 0.05; ***P ≤ 0.001.

Figure 2

Inactivation of Gprc6a in the β-cell lineage decreases β-cell proliferation and cyclin D1 expression. A: Quantification of the number of islets, proportional β-cell area, and β-cell mass, with representative images of whole sections immunolabeled with an anti-insulin antibody (red staining) shown for each genotype. Scale bars: 1 mm. B: Quantification of the percentage of insulin/BrdU-positive cells in pancreata from mice injected with BrdU (100 mg/kg) 16 h before being killed (n = 6 for each genotype). C: Western blot analysis of the levels of indicated factors in extracts from isolated islets. The left panel shows a representative image, and the histogram (right) shows quantification analysis of results obtained with three animals per genotype. Results are expressed relative to a 100% expression level in control extracts. D: Representative images of islets coimmunolabeled with anti-cyclin D1 (green) and anti-insulin (red) antibodies. Scale bars: 100 μm. E: Quantitative PCR analysis of Ccnd1 expression in islets isolated from Gprc6a fl/fl and Gprc6a_Pdx1^−/− mice (n = 3) and cultured in the presence of recombinant osteocalcin (Ocn) (10 ng/mL) or vehicle (Veh.) (n = 7–10 animals unless otherwise indicated). *P ≤ 0.05.

Figure 3

Gprc6a mediates osteocalcin regulation of β-cell proliferation. All data were obtained with 3-month-old mice unless otherwise indicated. A: Quantitative PCR analysis of Ins2 expression in islets isolated from Gprc6a fl/fl and Gprc6a_Pdx1^−/− mice (n = three) and cultured in presence of recombinant osteocalcin (Ocn) (1 ng/mL) or vehicle (Veh.). B: Insulin secretion from islets isolated from Gprc6a fl/fl and Gprc6a_Pdx1^−/− mice after treatment with recombinant osteocalcin (0.03 ng/mL) or vehicle in presence of 11.2 mmol/L glucose. Results were normalized to DNA content in each sample. C: BrdU incorporation in β-cells isolated from Gprc6a fl/fl and Gprc6a_Pdx1^−/− islets and cultured for 48 h in the presence of recombinant osteocalcin (0.03 ng/mL) or vehicle and of 100 µmol/L BrdU. Results were normalized to DNA content in each sample. D: Blood glucose levels in ad libitum fed (right histogram) or fasted (left histogram) mice of the indicated genotypes. E: GSIS test in mice of the indicated genotypes. The right histogram shows calculated areas under the curve. F: Quantification of the proportional β-cell area (left) and β-cell mass (right) in mice of indicated genotypes (?, fl, or wild-type allele). *P ≤ 0.05; **P ≤ 0.01.

Figure 4

Osteocalcin/Gprc6a signaling regulates proliferation of β-cells during development. Quantification of total (A) or active (B) osteocalcin (Ocn) levels in the serum of wild-type (WT) embryos and pups at indicated stages. For all stages, sera from at least six animals were individually analyzed except at E14.5, for which sera from four to five embryos were pooled in each sample. C: Femurs and pancreata of Osteocalcin-mCherry embryos at indicated stages were imaged under fluorescent light to detect Osteocalcin endogenous expression (dark background panels, red staining) or bright light (white background panels). D: Quantification of the percentage of insulin/BrdU-positive cells in pancreas from P10 pups injected with BrdU (100 mg/kg) 4 h before being killed (n = 3 or more for each genotype). E: Quantification of the proportional β-cell area in whole pancreata of E17.5 embryos of indicated genotypes. Representative images of whole sections immunolabeled with an anti-insulin antibody (red staining) are shown on the left. Scale bars: 1 mm. F: Quantification of the percentage of insulin/BrdU-positive cells in pancreas E17.5 embryos injected with BrdU (100 mg/kg) 4 h before being killed (n = 3 or more for each genotype). Representative images of islets coimmunolabeled with anti-BrdU (green) and anti-insulin (red) antibodies are shown on the left. Scale bars: 100 μm. *P ≤ 0.05.

Figure 5

Osteocalcin regulates cyclin D1 expression but does not affect endocrine cell differentiation during development. A and B: Representative images of islets on sections from Gprc6a fl/fl and Gprc6a_Pdx1^−/− E18.5 embryos coimmunolabeled with antibodies against indicated regulators of endocrine cell differentiation or markers of mature cell lineages (green) and with an anti-insulin antibody (red). Scale bars: 100 µm. C: Western blot analysis of the levels of indicated factors in extracts from isolated islets. The left panel shows representative images, and the right histogram shows quantification analysis of results obtained with three animals per genotype. Results are expressed relative to a 100% expression level in control extracts. D: Representative images of islets coimmunolabeled with anti-cyclin D1 (green) and anti-insulin (red) antibodies. Scale bars: 100 μm.