Masato Hirata

Osteocalcin induces release of glucagon-like peptide-1 and thereby stimulates insulin secretion in mice.

The uncarboxylated form (ucOC), but not the γ-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.

Phospholipase C-related but Catalytically Inactive Protein Is Required for Insulin-induced Cell Surface Expression of γ-Aminobutyric Acid Type A Receptors

The γ-aminobutyric acid type A (GABAA) receptors play a pivotal role in fast synaptic inhibition in the central nervous system. One of the key factors for determining synaptic strength is the number of receptors on the postsynaptic membrane, which is maintained by the balance between cell surface insertion and endocytosis of the receptors. In this study, we investigated whether phospholipase C-related but catalytically inactive protein (PRIP) is involved in insulin-induced GABAA receptor insertion. Insulin potentiated the GABA-induced Cl− current (IGABA) by about 30% in wild-type neurons, but not in PRIP1 and PRIP2 double-knock-out (DKO) neurons, suggesting that PRIP is involved in insulin-induced potentiation. The phosphorylation level of the GABAA receptor β-subunit was increased by about 30% in the wild-type neurons but not in the mutant neurons, which were similar to the changes observed in IGABA. We also revealed that PRIP recruited active Akt to the GABAA receptors by forming a ternary complex under insulin stimulation. The disruption of the binding between PRIP and the GABAA receptor β-subunit by PRIP interference peptide attenuated the insulin potentiation of IGABA. Taken together, these results suggest that PRIP is involved in insulin-induced GABAA receptor insertion by recruiting active Akt to the receptor complex.

Oral administration of osteocalcin improves glucose utilization by stimulating glucagon-like peptide-1 secretion

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion and pancreatic β-cell proliferation. We previously showed that the effect of GluOC on insulin secretion is mediated largely by glucagon-like peptide-1 (GLP-1) secreted from the intestine in response to GluOC exposure. We have now examined the effect of oral administration of GluOC on glucose utilization as well as the fate of such administered GluOC in mice. Long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level and improved glucose tolerance in mice without affecting insulin sensitivity. It also increased the fasting serum insulin concentration as well as the β-cell area in the pancreas. A small proportion of orally administered GluOC reached the small intestine and remained there for at least 24h. GluOC also entered the general circulation, and the serum GLP-1 concentration was increased in association with the presence of GluOC in the intestine and systemic circulation. The putative GluOC receptor, GPRC6A was detected in intestinal cells, and was colocalized with GLP-1 in some of these cells. Our results suggest that orally administered GluOC improved glucose handling likely by acting from both the intestinal lumen and the general circulation, with this effect being mediated in part by stimulation of GLP-1 secretion. Oral administration of GluOC warrants further study as a safe and convenient option for the treatment or prevention of metabolic disorders.

Acceleration of bone regeneration by local application of lithium: Wnt signal-mediated osteoblastogenesis and Wnt signal-independent suppression of osteoclastogenesis.

Inhibition of glycogen synthase kinase (GSK)-3 and the consequent activation of the Wnt/β-catenin signaling pathway have been reported to increase bone volume. To develop a novel pharmacotherapy for injured bone, we investigated whether GSK-3 inhibitor was effective in promoting bone formation. In in vitro experiments, we examined the effects of GSK-3 inhibitors LiCl and SB216763 on osteoblastogenesis of mesenchymal progenitor C3H10T1/2 cells and osteoclastogenesis of osteoclast precursor RAW-D cells. Both inhibitors promoted osteoblast differentiation, assessed by alkaline phosphatase activity and calcium deposition, stimulating the Wnt/β-catenin signaling pathway and thereby inducing Runx2. On the other hand, the GSK-3 inhibitors suppressed osteoclast differentiation, assessed by tartrate-resistant acid phosphatase staining and number of nuclei in the cells, reducing NFATc1 expression independently of the Wnt/β-catenin signaling pathway. In subsequently performed in vivo studies, we examined the effect of locally administered Li2CO3 on the recovery from a partial defect made on the rat tibia. Computerized tomography and bone histomorphometry showed that Li2CO3 accelerated bone regeneration in defect lesion with increased lamellar bone ratio compared with the controls. These results suggested that local application of lithium (or other GSK-3 inhibitors) might effectively facilitate recovery from bone injury by promoting osteoblastogenesis and inhibiting osteoclastogenesis.

Signaling pathway for adiponectin expression in adipocytes by osteocalcin.

In addition to providing skeletal support, the bone is an endocrine organ that produces osteocalcin, whose uncarboxylated form (GluOC) increases insulin secretion either directly or indirectly by promoting incretin secretion. We have now investigated the signaling pathway by which GluOC increases expression of adiponectin in adipocytes. Activation of its putative receptor GPRC6A by GluOC induced the intracellular accumulation of cAMP and consequent activation of protein kinase A (PKA) in differentiated 3T3-L1 adipocytes. It also induced phosphorylation of CREB (cAMP response element binding protein), but this effect appeared to be mediated indirectly by extracellular signal-regulated kinase (ERK) rather than directly by PKA, given that it was attenuated by the ERK signaling inhibitor U0126. Activated PKA also induced activation of the tyrosine kinase Src, the small GTPase Rap1, an upstream of ERK and CREB phosphorylation. Activated CREB up-regulated the expression of peroxisome proliferator-activated receptor γ (PPARγ), which in turn led to induction of adiponectin expression. Finally, intermittent oral administration of GluOC in mice reduced the size of gonadal white adipocytes as well as increased the expression of PPARγ and adiponectin in these cells. Our results have thus revealed the signaling pathway by which GluOC induces adiponectin expression in adipocytes.

Involvement of phospholipase C-related inactive protein in the mouse reproductive system through the regulation of gonadotropin levels

Phospholipase C-related but catalytically inactive protein (comprising PRIP-1 and PRIP-2 [officially designated PLCL1 and PLCL2]) was first identified in our laboratory, but the biological functions have remained elusive. Therefore, we generated Plcl1 and Plcl2 double-knockout mice (Plcl1tm1Mh; Plcl2tm1Tta) to gain insight into the biological function. Double-knockout mice apparently grew normally and became fertile; however, during animal maintenance, we noticed that mutant couples exhibited decreased litter events and litter size, indicating dysfunction of the reproductive system. Cross-mating experiments to discriminate whether males or females were defective indicated that the cause appeared to be on the female side. Mutant female mice had an apparently smaller uterus by gross anatomical observation and had more estrous days during the cycles. Levels of serum luteinizing hormone (LH) and follicle-stimulating hormone were measured for 5–6 consecutive days and were significantly higher in the mutant, which was also confirmed by examining the secretion of LH from the explant culture of anterior pituitary glands of wild-type and double-knockout mice. These results suggest that through gonadotropin secretion, PRIP plays an important role in female reproduction.

Promotion of insulin-induced glucose uptake in C2C12 myotubes by osteocalcin.

A close relationship between the bone and systemic glucose metabolism has recently been the center of attention, since the uncarboxylated form of osteocalcin (GluOC), a bone-derived protein, but not the γ-carboxylated form, is involved in glucose metabolism. However, the analysis of GluOC effect using isolated organs and related cell lines are required to understand its roles in a whole systemic metabolic status. In the present study, we examined the effect of GluOC on cell lines derived from skeletal muscle to explore the mechanisms by which GluOC regulates glucose uptake. In the differentiated C2C12 myotubes, GluOC dose-dependently induced the phosphorylation of ERK without affecting intracellular cAMP and Ca(2+) levels. This effect was inhibited by U0126, an inhibitor of ERK kinase (MEK). Additionally, U73122, an inhibitor of phospholipase C tended to inhibit it as well. Furthermore, cell treatment with GluOC for a long period promoted insulin-induced Akt phosphorylation and glucose uptake in the myotubes, which was abolished by ERK signaling inhibition. These results indicate that GluOC does not triggered Akt phosphorylation and glucose uptake by itself but promotes insulin-induced glucose uptake in myotubes, probably by up-regulating Akt signaling through ERK activation.

Characterization of PXK as a Protein Involved in Epidermal Growth Factor Receptor Trafficking

ABSTRACT The phox homology (PX) domain is a phosphoinositide-binding module that typically binds phosphatidylinositol 3-phosphate. Out of 47 mammalian proteins containing PX domains, more than 30 are denoted sorting nexins and several of these have been implicated in internalization of cell surface proteins to the endosome, where phosphatidylinositol-3-phosphate is concentrated. Here we investigated a multimodular protein termed PXK, composed of a PX domain, a protein kinase-like domain, and a WASP homology 2 domain. We show that the PX domain of PXK localizes this protein to the endosomal membrane via binding to phosphatidylinositol 3-phosphate. PXK expression in COS7 cells accelerated the ligand-induced internalization and degradation of epidermal growth factor receptors by a mechanism requiring phosphatidylinositol 3-phosphate binding but not involving the WASP homology 2 domain. Conversely, depletion of PXK using RNA interference decreased the rate of epidermal growth factor receptor internalization and degradation. Ubiquitination of epidermal growth factor receptor by the ligand stimulation was enhanced in PXK-expressing cells. These results indicate that PXK plays a critical role in epidermal growth factor receptor trafficking through modulating ligand-induced ubiquitination of the receptor.

Phospholipase C-related but Catalytically Inactive Protein (PRIP) Modulates Synaptosomal-associated Protein 25 (SNAP-25) Phosphorylation and Exocytosis

Background: Phospho-modulation of SNARE function has not yet been shown. Results: Phospho-SNAP-25 was dephosphorylated by protein phosphatase-1, whose activity was regulated by PRIP, thus regulating exocytosis. Conclusion: Protein phosphatase-1, whose activity is regulated by PRIP, is the major phosphatase responsible for the dephosphorylation of SNAP-25. Significance: The results provide the first information regarding the phosphatases responsible for phospho-modulation of SNAP-25 and the regulation of exocytosis. Exocytosis is one of the most fundamental cellular events. The basic mechanism of the final step, membrane fusion, is mediated by the formation of the SNARE complex, which is modulated by the phosphorylation of proteins controlled by the concerted actions of protein kinases and phosphatases. We have previously shown that a protein phosphatase-1 (PP1) anchoring protein, phospholipase C-related but catalytically inactive protein (PRIP), has an inhibitory role in regulated exocytosis. The current study investigated the involvement of PRIP in the phospho-dependent modulation of exocytosis. Dephosphorylation of synaptosome-associated protein of 25 kDa (SNAP-25) was mainly catalyzed by PP1, and the process was modulated by wild-type PRIP but not by the mutant (F97A) lacking PP1 binding ability in in vitro studies. We then examined the role of PRIP in phospho-dependent regulation of exocytosis in cell-based studies using pheochromocytoma cell line PC12 cells, which secrete noradrenalin. Exogenous expression of PRIP accelerated the dephosphorylation process of phosphorylated SNAP-25 after forskolin or phorbol ester treatment of the cells. The phospho-states of SNAP-25 were correlated with noradrenalin secretion, which was enhanced by forskolin or phorbol ester treatment and modulated by PRIP expression in PC12 cells. Both SNAP-25 and PP1 were co-precipitated in anti-PRIP immunocomplex isolated from PC12 cells expressing PRIP. Collectively, together with our previous observation regarding the roles of PRIP in PP1 regulation, these results suggest that PRIP is involved in the regulation of the phospho-states of SNAP-25 by modulating the activity of PP1, thus regulating exocytosis.

Inhibition of BMP2-Induced Bone Formation by the p65 Subunit of NF-κB via an Interaction With Smad4

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11-770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65(-/-) MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.