Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice

et al. (2013) Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice. PLoS ONE 8(2): e57375. doi:10.1371/journal.pone.0057375 Osteocalcin Induces Release of Glucagon-Like Peptide-1 and Thereby Stimulates Insulin Secretion in Mice Akiko Mizokami 0 Yu Yasutake 0 Jing Gao 0 Miho Matsuda 0 Ichiro Takahashi 0 Hiroshi Takeuchi 0 Masato Hirata 0 Nigel Irwin, University of Ulster, United Kingdom 0 1 Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, Kyushu University , Fukuoka , Japan , 2 Division of Orthodontics, Faculty of Dental Science, Kyushu University , Fukuoka , Japan , 3 Division of Applied Pharmacology, Kyushu Dental College , Kitakyushu , Japan The uncarboxylated form (ucOC), but not the c-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide-1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1. - Funding: This work was supported by KAKENHI from Japan Society for Promotion of Science to MH (24229009), JG (23792127), HT (24592805) and MM (24592804). AM is a Research Fellow (RPD program) of Japan Society for the Promotion of Science. The Uehara Memorial Foundation (to AM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Bone is one of the largest organs in the human body and undergoes remodeling both during childhood and throughout adulthood. Bone remodeling, characterized by repetitive bone resorption by osteoclasts and bone formation by osteoblasts, is tightly regulated at the local level by cytokines produced by bone cells as well as at the systemic level by hormones and neuropeptides [1,2]. Bone is also under the influence of hormones that regulate energy metabolism such as leptin, an adipocytederived hormone that regulates appetite and energy expenditure and which modulates postnatal bone acquisition through activation of several signaling pathways [3,4]. Another such hormone is insulin. Osteoblasts thus express functional insulin receptors, the stimulation of which in primary osteoblasts or osteoblast-like cell lines results in the up-regulation of bone anabolic markers including collagen synthesis, alkaline phosphatase production, and glucose uptake [57]. Bone is not merely a passive tissue that is subject to external influences, however. It is also an active endocrine organ that produces at least two hormones, fibroblast growth factor 23 [8] and osteocalcin [9]. Osteocalcin (OC) increases insulin production and sensitivity and thereby promotes glucose utilization and energy expenditure [9]. It also undergoes vitamin Kdependent carboxylation on three glutamic acid residues, which gives rise to GlaOC and confers a high affinity for the bone matrix. A small proportion of OC molecules remain uncarboxylated and are secreted into the circulation [10]. The acidic pH of the boneresorbing niche promotes the decarboxylation of GlaOC [11], and the resulting uncarboxylated osteocalcin (ucOC) is responsible for the stimulation of insulin secretion. In turn, insulin signaling in osteoblasts promotes bone formation by suppressing the expression of Twist2, an inhibitor of osteoblast development, and increases the expression of OC [12]. Furthermore, insulin signaling downregulates the expression of osteoprotegerin, an osteoblast-specific inhibitor of RANKL (receptor activator of nuclear factor-kB ligand), and thereby promotes bone resorption by osteoclasts, resulting in the release of active ucOC [11] and completing a feedforward loop. Incretin hormones also promote the secretion of insulin from pancreatic b cells in a glucose-dependent manner [13]. Glucagonlike peptide1 (GLP-1), one of the incretin hormones, is produced by enteroendocrine L cells of the small intestine and is secreted into the circulation in response to nutrient ingestion [14]. GLP-1 achieves its insulinotropic effect by binding to its specific receptor and thereby increasing the cytosolic concentrations of cAMP and Ca2+ in b cells [15]. It also stimulates b cell proliferation as well as protects the cells from apoptosis [13]. In the present study, we have therefore investigated whether, in addition to its direct effect on the pancreas, OC might increase GLP-1 secretion. Materials and Methods Cell culture STC-1 cells (kindly provided from Dr. G. Tsujimoto, Kyoto University) [16], a mouse enteroendocrine cell line were cultured under a humidified atmosphere of 5% CO2 at 37 uC in Dulbeccos modified Eagles medium supplemented with 15% horse serum, 5% fetal bovine serum, penicillin (100 U/ml), and streptomycin (0.1 mg/ml). The cells were routinely passaged at 80 to 90% confluence. RT-PCR analysis Total RNA (2 mg) isolated with an RNeasy Mini Kit (Qiagen, Valencia, CA) was subjected to RT-PCR (reverse-transcriptase polymerase chain reaction) analysis with the use of a ReverTra Ace kit (Toyobo, Osaka, Japan) and with the Gprc6a primers 59CCAGACGACCACAAATCCAG-39 (forward) and 59-GATTCATAACTCACCTGT-39 (reverse). Immunohistochemistry Mouse small intestine was dissected to the upper, middle, and lower thirds (as duodenum, jejunum, and ileum, respectively), fixed in 4% paraformaldehyde, dehydrated with a series of ethanol solutions, embedded in paraffin, and sectioned at a thickness of 6 mm. The sections were then depleted of paraffin and rehydrated with phosphate-buffered saline. Antigen retrieval was performed with an autoclave for 5 min at 121 uC in 10 mM sodium citrate buffer (pH 6.0), and nonspecific protein binding was blocked by incubation of the sections with 10% goat serum in phosphatebuffered saline. The sections were then incubated overnight at 4 uC with rabbit antibodies to Gprc6a (1:500 dilution, PAB16273; Abnova, Taipei, Taiwan) or with rabbit c-globulin (1:500 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) as a negati (...truncated)