The regulation of bone remodeling by an adipocyte-derived hormone implies that bone may exert a feedback control of energy homeostasis. To test this hypothesis we looked for genes expressed in osteoblasts, encoding signaling molecules and affecting energy metabolism. We show here that mice lacking the protein tyrosine phosphatase OST-PTP are hypoglycemic and are protected from obesity and glucose intolerance because of an increase in beta-cell proliferation, insulin secretion, and insulin sensitivity. In contrast, mice lacking the osteoblast-secreted molecule osteocalcin display decreased beta-cell proliferation, glucose intolerance, and insulin resistance. Removing one Osteocalcin allele from OST-PTP-deficient mice corrects their metabolic phenotype. Ex vivo, osteocalcin can stimulate CyclinD1 and Insulin expression in beta-cells and Adiponectin, an insulin-sensitizing adipokine, in adipocytes; in vivo osteocalcin can improve glucose tolerance. By revealing that the skeleton exerts an endocrine regulation of sugar homeostasis this study expands the biological importance of this organ and our understanding of energy metabolism.

The osteoblast-specific secreted molecule osteocalcin behaves as a hormone regulating glucose metabolism and fat mass in two mutant mouse strains. Here, we ask two questions: is the action of osteocalcin on ␤ cells and adipocytes elicited by the same concentrations of the molecule, and more importantly, does osteocalcin regulate energy metabolism in WT mice? Cell-based assays using isolated pancreatic islets, a ␤ cell line, and primary adipocytes showed that picomolar amounts of osteocalcin are sufficient to regulate the expression of the insulin genes and ␤ cell proliferation markers, whereas nanomolar amounts affect adiponectin and Pgc1␣ expression in white and brown adipocytes, respectively. In vivo the same difference exists in osteocalcin's ability to regulate glucose metabolism on the one hand and affect insulin sensitivity and fat mass on the other hand. Furthermore, we show that long-term treatment of WT mice with osteocalcin can significantly weaken the deleterious effect on body mass and glucose metabolism of gold thioglucose-induced hyperphagia and high-fat diet. These results establish in WT mice the importance of this novel molecular player in the regulation of glucose metabolism and fat mass and suggest that osteocalcin may be of value in the treatment of metabolic diseases.fat ͉ insulin ͉ diet-induced obesity ͉ diet-induced diabetes ͉ adiponectin W e recently showed that the uncarboxylated form of the osteoblast-specific secreted molecule osteocalcin functions as a hormone regulating glucose metabolism and fat mass (1). However, this unexpected role for osteocalcin had not previously been exclusively demonstrated in genetically modified animals such as the Osteocalcin Ϫ/Ϫ and Esp Ϫ/Ϫ mice (1, 2). The latter mouse model exhibits an osteocalcin gain of bioactivity. Data generated in these two animal models and cell-based assays showed that osteocalcin can increase ␤ cell proliferation, stimulate insulin expression and secretion by pancreatic ␤ cells, enhance energy expenditure, and increase expression of adiponectin, an insulin-sensitizing hormone produced by adipocytes (3, 4).The next critical question to answer is whether the functions of osteocalcin on energy metabolism extend to WT animals. Moreover, if it is the case, one needs to know whether identical or different concentrations of osteocalcin are required to affect glucose metabolism and fat mass. Answers to these questions are of critical importance for two reasons. First, if osteocalcin has an effect in WT mice it would firmly establish the notion that it is a physiologically important hormone; second, it would start addressing the therapeutic potential of this new player in the regulation of energy metabolism.Thus, we embarked on a systematic analysis of osteocalcin relevance in regulating energy metabolism in WT mice. We performed in vitro and in vivo assays to determine the doses of osteocalcin able to affect various aspects of energy metabolism and tested different doses of osteocalcin in WT mice fed either a normal diet or a diet favo...

Summary The synthesis of Type I collagen, the main component of the bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the osteoblast's energetic needs might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1 whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While Runx2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, Runx2 favors Glut1 expression, and this feed-forward regulation between Runx2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

The recent demonstration that osteoblasts have a role in controlling energy metabolism suggests that they express cell-specific regulatory genes involved in this process. Activating transcription factor 4 (ATF4) is a transcription factor that accumulates predominantly in osteoblasts, where it regulates virtually all functions linked to the maintenance of bone mass. Since Atf4 -/-mice have smaller fat pads than littermate controls, we investigated whether ATF4 also influences energy metabolism. Here, we have shown, through analysis of Atf4 -/-mice, that ATF4 inhibits insulin secretion and decreases insulin sensitivity in liver, fat, and muscle. Several lines of evidence indicated that this function of ATF4 occurred through its osteoblastic expression. First, insulin sensitivity is enhanced in the liver of Atf4 -/-mice, but not in cultured hepatocytes from these mice. Second, mice overexpressing ATF4 in osteoblasts only [termed here α1(I)Collagen-Atf4 mice] displayed a decrease in insulin secretion and were insulin insensitive. Third, the α1(I)Collagen-Atf4 transgene corrected the energy metabolism phenotype of Atf4 -/-mice. Fourth, and more definitely, mice lacking ATF4 only in osteoblasts presented the same metabolic abnormalities as Atf4 -/-mice. Molecularly, ATF4 favored expression in osteoblasts of Esp, which encodes a product that decreases the bioactivity of osteocalcin, an osteoblast-specific secreted molecule that enhances secretion of and sensitivity to insulin. These results provide a transcriptional basis to the observation that osteoblasts fulfill endocrine functions and identify ATF4 as a regulator of most functions of osteoblasts.

The osteoblast-secreted molecule osteocalcin favors insulin secretion, but how this function is regulated in vivo by extracellular signals is for now unknown. In this study, we show that leptin, which instead inhibits insulin secretion, partly uses the sympathetic nervous system to fulfill this function. Remarkably, for our purpose, an osteoblast-specific ablation of sympathetic signaling results in a leptin-dependent hyperinsulinemia. In osteoblasts, sympathetic tone stimulates expression of Esp, a gene inhibiting the activity of osteocalcin, which is an insulin secretagogue. Accordingly, Esp inactivation doubles hyperinsulinemia and delays glucose intolerance in ob/ob mice, whereas Osteocalcin inactivation halves their hyperinsulinemia. By showing that leptin inhibits insulin secretion by decreasing osteocalcin bioactivity, this study illustrates the importance of the relationship existing between fat and skeleton for the regulation of glucose homeostasis.

Global gene deletion studies in mice and humans have established the pivotal role of runt related transcription factor-2 (Runx2) in both intramembranous and endochondral ossification processes during skeletogenesis. In this study, we for the first time generated mice carrying a conditional Runx2 allele with exon 4, which encodes the Runt domain, flanked by loxP sites. These mice were crossed with a1(I)-collagen-Cre or a1(II)-collagen-Cre transgenic mice to obtain osteoblast-specific or chondrocyte-specific Runx2 deficient mice, respectively. As seen in Runx2 À/À mice, perinatal lethality was observed in a1(II)-Cre;Runx2 flox/flox mice, but this was not the case in animals in which a1(I)-collagen-Cre was used to delete Runx2. When using double-staining with Alizarin red for mineralized matrix and Alcian blue for cartilaginous matrix, we observed previously that mineralization was totally absent at embryonic day 15.5 (E15.5) throughout the body in Runx2 À/À mice, but was found in areas undergoing intramembranous ossification such as skull and clavicles in a1(II)-Cre;Runx2 flox/flox mice. In newborn a1(II)-Cre; Runx2 flox/flox mice, mineralization impairment was restricted to skeletal areas undergoing endochondral ossification including long bones and vertebrae. In contrast, no apparent skeletal abnormalities were seen in mutant embryo, newborn, and 3-week-old to 6-week old-mice in which Runx2 had been deleted with the a1(I)-collagen-Cre driver. These results suggest that Runx2 is absolutely required for endochondral ossification during embryonic and postnatal skeletogenesis, but that disrupting its expression in already committed osteoblasts as achieved here with the a1(I)-collagen-Cre driver does not affect overtly intramembranous and endochondral ossification. The Runx2 floxed allele established here is undoubtedly useful for investigating the role of Runx2 in particular cells.

In the originally published author list, we inadvertently misspelled Takashi Iezaki's surname. The author list has been corrected online.

The hypothesis that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is now gaining more support after the successful cloning of a number of genes coding for the signaling machinery required for this neurocrine at synapses in the brain. These include Glu receptors (signal detection), Glu transporters (signal termination) and vesicular Glu transporters (signal output through exocytotic release). Relatively little attention has been paid to the functional expression of these molecules required for Glu signaling in peripheral neuronal and non-neuronal tissues; however, recent molecular biological analyses show a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis -as an excitatory neurotransmitter in the central neurocrine system and an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, the possible Glu signaling methods are outlined in specific peripheral tissues including bone, testis, pancreas, and the adrenal, pituitary and pineal glands.Keywords: autocrine; glutamate; glutamate receptor; glutamate transporter; neurotransmitter; paracrine; vesicular glutamate transporter; peripheral tissues. Glutamate signaling moleculesGlutamate receptors L-Glutamate (Glu) is accepted as an excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Receptors for Glu (GluRs) are categorized into two major classes, metabotropic (mGluRs) and ionotropic (iGluRs) receptors, according to their differential intracellular signal transduction mechanisms and molecular homologies (Fig. 1) [1-3]. mGluRs are further divided into three distinct subtypes containing seven transmembrane domains, including group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8), in line with each receptor's exogenous agonists and intracellular second messengers [4,5]. The group I subtype stimulates formation of inositol 1,4,5-triphosphate and diacylglycerol, while both group II and III subtypes induce reduction of intracellular cyclic AMP (cAMP). On the basis of sequence homology and agonist preference, the latter iGluRs are classified into N-methyl-D-aspartate (NMDA), DL-a-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), and kainate (KA) receptors, which are associated with ion channels permeable to particular cations [6,7].NMDA receptor channels. These channels are highly permeable to Ca 2+ , with sensitivity to blockade by Mg 2+ in a voltage-dependent manner [8,9]. Functional NMDA receptor channels are comprised of heteromeric assemblies between the essential NR1 subunit and one of four different NR2 (A-D) subunits, in addition to one of two different NR3 (A-B) subunits. Expression of the NR2 subunit alone does not lead to composition of functional ion channels in any expression system, while coexpression of...