Tomoyo Kawakubo-Yasukochi

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.

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.

Osteocalcin and its endocrine functions

Bone has traditionally been regarded as a static structural organ that supports movement of the body and protects the internal organs. However, evidence has been accumulated in the past decade showing that bone also functions as an endocrine organ that regulates systemic glucose and energy metabolism. Osteocalcin, an osteoblast-specific secreted protein, acts as a hormone by stimulating insulin production and increasing energy expenditure and insulin sensitivity in target organs. Animal studies have shown that an increase in the circulating concentration of osteocalcin, including via exogenous application of the protein, prevents obesity and glucose intolerance. Moreover, a number of epidemiological analyses support the role of osteocalcin in the regulation of glucose and energy homeostasis in humans. Therefore, it has been suggested that osteocalcin could be a feasible preventive or therapeutic agent for metabolic disorders. In this review, we summarize the current knowledge regarding the endocrine functions of osteocalcin and its various modes of action.

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 β-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 β-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

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.

Differential Roles of Carboxylated and Uncarboxylated Osteocalcin in Prostate Cancer Growth

Serum levels of osteocalcin (OC), a bone matrix non-collagenous protein secreted by osteoblasts, are correlated with pathological bone remodeling such as the bone metastasis of cancer, as well as physiological bone turnover. The pathological roles in prostate cancer growth of the two existing types of serum OC, γ-carboxylated (GlaOC) and lower- (or un-) carboxylated (GluOC), have not yet been discriminatively examined. In the present study, we demonstrate that normal prostate epithelial cell growth was promoted by both types of OC, while growth of cancer cells in the prostate was accelerated by GlaOC but suppressed by GluOC. We suggest that OC regulates prostate cancer growth depending on the γ-carboxylation, in part by triggering reduced phosphorylation of receptor tyrosine kinases.

miR-200c-3p spreads invasive capacity in human oral squamous cell carcinoma microenvironment

Oral squamous cell carcinoma (OSCC) constitutes over 90% of all cancers in the oral cavity. The prognosis for patients with invasive OSCC is poor; therefore, it is important to understand the molecular mechanisms of invasion and subsequent metastasis not only to prevent cancer progression but also to detect new therapeutic targets against OSCC. Recently, extracellular vesicles—particularly exosomes—have been recognized as intercellular communicators in the tumor microenvironment. As exosomic cargo, deregulated microRNAs (miRNAs) can shape the surrounding microenvironment in a cancer‐dependent manner. Previous studies have shown inconsistent results regarding miR‐200c‐3p expression levels in OSCC cell lines, tissues, or serum—likely because of the heterogeneous characters of the specimen materials. For this reason, single‐cell clone analyses are necessary to effectively assess the role of exosome‐derived miRNAs on cells within the tumor microenvironment. The present study utilized integrated microarray profiling to compare exosome‐derived miRNA and exosome‐treated cell‐derived mRNA expression. Data were acquired from noninvasive SQUU‐A and highly invasive SQUU‐B tongue cancer cell clones derived from a single patient to determine candidate miRNAs that promote OSCC invasion. Matrigel invasion assays confirmed that hsa‐miR‐200c‐3p was a key pro‐invasion factor among six miRNA candidates. Consistently, silencing of the miR‐200c‐3p targets, CHD9 and WRN, significantly accelerated the invasive potential of SQUU‐A cells. Thus, our data indicate that miR‐200c‐3p in exosomes derived from a highly invasive OSCC line can induce a similar phenotype in non‐invasive counterparts.

Uncarboxylated osteocalcin induces antitumor immunity against mouse melanoma cell growth

Because of the poor response to chemotherapy and radiation therapy, new treatment approaches by immune-based therapy involving activated T cells are required for melanoma. We previously reported that the uncarboxylated form of osteocalcin (GluOC), derived from osteoblasts, potentially suppresses human prostate cancer cell proliferation by direct suppression of cell growth. However, the mechanisms in vivo have not been elucidated. In this study, we found that GluOC suppressed tumor growth of B16 mouse melanoma transplants in C57Bl/6N wild-type mice. Our data demonstrated that GluOC suppressed cell growth by downregulating phosphorylation levels of receptor tyrosine kinases and inducing apoptosis in vitro. Additionally, stimulation of primary mouse splenocytes with concanavalin A, a polyclonal T-cell mitogen, in the presence of GluOC increased T cell proliferation and their interferon-γ production. Taken together, we demonstrate that GluOC exerts multiple antitumor effects not only in vitro, but also in vivo through cellular immunostimulatory effects against B16 mouse melanoma cells.

In Vitro Anti-inflammatory Effects of the Phenylbutyric Acid Metabolite Phenylacetyl Glutamine

Sodium 4-phenylbutyrate (PBA), which exerts a wide range of anti-inflammatory effects, is rapidly cleared from the body (approximately 98%) by urinary excretion by 24 h after oral treatment in humans. PBA was almost entirely excreted to urine as phenylacetyl glutamine (PAGln). However, no data describe the potential anti-inflammatory effects of PAGln. The purpose of this study was to evaluate the anti-inflammatory effects of PAGln on mouse spleen cells and peritoneal cavity cells, and explore the potential mechanism underlying this effect. PAGln was added to mouse spleen cell cultures stimulated by concanavalin A, or mouse peritoneal cavity cell cultures stimulated by lipopolysaccharide. After 72 h of culture, levels of inflammatory cytokines in culture supernatants were measured using a sandwich enzyme-linked immunosorbent assay system, and levels of inflammatory proteins were assessed by Western blotting. PAGln significantly inhibited inflammatory cytokine (interferon-γ, interleukin-6, and tumor necrosis factor-α) production, decrease of cell number in the spleen cell, and suppressed the expression of inflammatory proteins (nuclear factor κB, and inducible nitric oxide synthase). These results suggest that PAGln possesses anti-inflammatory activity via inhibition of T cell activation and Toll-like receptor 4 signaling. This study of the anti-inflammatory mechanism of PAGln provides useful information about its potential for therapeutic applications.