Adipocyte-specific GPRC6A ablation promotes diet-induced obesity by inhibiting lipolysis.

The G protein-coupled receptor GPRC6A regulates various physiological processes in response to its interaction with multiple ligands, such as extracellular basic amino acids, divalent cations, testosterone, and the uncarboxylated form of osteocalcin (GluOC). Global ablation of GPRC6A increases the susceptibility of mice to diet-induced obesity and related metabolic disorders. However, given that GPRC6A is expressed in many tissues and responds to a variety of hormonal and nutritional signals, the cellular and molecular mechanisms underlying the development of metabolic disorders in conventional knockout mice have remained unclear. On the basis of our previous observation that long-term oral administration of GluOC markedly reduced adipocyte size and improved glucose tolerance in WT mice, we examined whether GPRC6A signaling in adipose tissue might be responsible for prevention of metabolic disorders. We thus generated adipocyte-specific GPRC6A knockout mice, and we found that these animals manifested increased adipose tissue weight, adipocyte hypertrophy, and adipose tissue inflammation when fed a high-fat and high-sucrose diet compared with control mice. These effects were associated with reduced lipolytic activity because of downregulation of lipolytic enzymes such as adipose triglyceride lipase and hormone-sensitive lipase in adipose tissue of the conditional knockout mice. Given that, among GPR6CA ligands tested, GluOC and ornithine increased the expression of adipose triglyceride lipase in cultured 3T3-L1 adipocytes in a manner dependent on GPRC6A, our results suggest that the constitutive activation of GPRC6A signaling in adipocytes by GluOC or ornithine plays a key role in adipose lipid handling and the prevention of obesity and related metabolic disorders.

Bone morphogenetic protein induces bone invasion of melanoma by epithelial-mesenchymal transition via the Smad1/5 signaling pathway.

Oral malignant melanoma, which frequently invades the hard palate or maxillary bone, is extremely rare and has a poor prognosis. Bone morphogenetic protein (BMP) is abundantly expressed in bone matrix and is highly expressed in malignant melanoma, inducing an aggressive phenotype. We examined the role of BMP signaling in the acquisition of an aggressive phenotype in melanoma cells in vitro and in vivo. In five cases, immunohistochemistry indicated the phosphorylation of Smad1/5 (p-Smad1/5) in the nuclei of melanoma cells. In the B16 mouse and A2058 human melanoma cell lines, BMP2, BMP4, or BMP7 induces morphological changes accompanied by the downregulation of E-cadherin, and the upregulation of N-cadherin and Snail, markers of epithelial-mesenchymal transition (EMT). BMP2 also stimulates cell invasion by increasing matrix metalloproteinase activity in B16 cells. These effects were canceled by the addition of LDN193189, a specific inhibitor of Smad1/5 signaling. In vivo, the injection of B16 cells expressing constitutively activated ALK3 enhanced zygoma destruction in comparison to empty B16 cells by increasing osteoclast numbers. These results suggest that the activation of BMP signaling induces EMT, thus driving the acquisition of an aggressive phenotype in malignant melanoma.

p130Cas induces bone invasion by oral squamous cell carcinoma by regulating tumor epithelial-mesenchymal transition and cell proliferation.

Bone invasion is a critical factor in determining the prognosis of oral squamous cell carcinoma (OSCC) patients. Transforming growth factor ß (TGF-ß) is abundantly expressed in the bone matrix and is involved in the acquisition of aggressiveness by tumors. TGF-ß is also important to cytoskeletal changes during tumor progression. In this study, we examined the relationship between TGF-ß signaling and cytoskeletal changes during bone invasion by OSCC. Immunohistochemical staining of OSCC samples from five patients showed the expression of p130Cas (Crk-associated substrate) in the cytoplasm and phosphorylated Smad3 expression in the nucleus in OSCC cells. TGF-ß1 induced the phosphorylation of Smad3 and p130Cas, as well as epithelial-mesenchymal transition (EMT) accompanied by the downregulation of the expression of E-cadherin, a marker of epithelial cells, and the upregulation of the expression of N-cadherin, or Snail, a marker of mesenchymal cells, in human HSC-2 cells and mouse squamous cell carcinome VII (SCCVII) cells. SB431542, a specific inhibitor of Smad2/3 signaling, abrogated the TGF-ß1-induced phosphorylation of p130Cas and morphological changes. Silencing p130Cas using an short hairpin RNA (shRNA) or small interfering RNA in SCCVII cells suppressed TGF-ß1-induced cell migration, invasion, EMT and matrix metalloproteinase-9 (MMP-9) production. Compared with control SCCVII cells, SCCVII cells with silenced p130Cas strongly suppressed zygomatic and mandibular destruction in vivo by reducing the number of osteoclasts, cell proliferation and MMP-9 production. Taken together, these results showed that the expression of TGF-ß/p130Cas might be a new target for the treatment of OSCC bone invasion.

Long-term oral administration of osteocalcin induces insulin resistance in male mice fed a high-fat, high-sucrose diet.

Uncarboxylated osteocalcin (GluOC), a bone-derived hormone, regulates energy metabolism by stimulating insulin secretion, pancreatic ß-cell proliferation, and adiponectin expression in adipocytes. Previously, we showed that long-term intermittent or daily oral administration of GluOC reduced the fasting blood glucose level, improved glucose tolerance, and increased the fasting serum insulin concentration as well as pancreatic ß-cell area in female mice fed a normal or high-fat, high-sucrose diet. We have now performed similar experiments with male mice and found that such GluOC administration induced glucose intolerance, insulin resistance, and adipocyte hypertrophy in those fed a high-fat, high-sucrose diet. In addition, GluOC increased the circulating concentration of testosterone and reduced that of adiponectin in such mice. These phenotypes were not observed in male mice fed a high-fat, high-sucrose diet after orchidectomy, but they were apparent in orchidectomized male mice or intact female mice that were fed such a diet and subjected to continuous testosterone supplementation. Our results thus reveal a sex difference in the effects of GluOC on glucose homeostasis. Given that oral administration of GluOC has been considered a potentially safe and convenient option for the treatment or prevention of metabolic disorders, this sex difference will need to be taken into account in further investigations.

Maternal oral administration of osteocalcin protects offspring from metabolic impairment in adulthood.

OBJECTIVE: Maternal diet during pregnancy has been found to influence the health of offspring. However, strategies for modulation of maternal energy metabolism without an adverse effect on the fetus have remained limited. It was recently shown that oral administration of uncarboxylated osteocalcin (GluOC) improves metabolic status in adult female mice. Whether maternal GluOC administration during gestation might improve the metabolic status of offspring was investigated. METHODS: Female C57BL/6 mice were fed a normal diet (ND) or high-fat, high-sucrose diet (HFS) and were given saline or GluOC by oral administration during pregnancy. The resulting offspring were in turn assigned to ND- or HFS-fed groups immediately after weaning, and their body weight, glucose metabolism, serum lipid parameters, and level of adipose tissue inflammation were subsequently assessed. RESULTS: Maternal HFS feeding during gestation had adverse effects on glucose and lipid parameters, body weight, and adipose tissue inflammation in female offspring fed the same diet, and these effects were attenuated by maternal oral GluOC administration. CONCLUSIONS: Maternal oral administration of GluOC protects HFS-fed female offspring from metabolic disorders induced by maternal obesity.

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 ß-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 ß-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.