Recent studies demonstrate widespread expression of ghrelin among tissues and have uncovered its pleiotropic nature. We have examined gene expression of ghrelin and its two receptor splice variants, growth hormone secretagogue receptors (GHS-R) 1a and 1b, in human bone biopsies and in the human pre-osteoblastic SV-HFO cell line during differentiation. Additionally, we examined proliferative effects of ghrelin and unacylated ghrelin (UAG) in differentiating and non-differentiating cells. We detected GHS-R1b mRNA in human bone and osteoblasts but not ghrelin's cognate receptor GHS-R1a, using two different real-time PCR assays and both total RNA and mRNA. In osteoblasts GHS-R1b mRNA expression remained low during the first 14 days of culture, but increased 300% in differentiating cells by day 21. Both human bone biopsies and osteoblasts expressed ghrelin mRNA, and osteoblasts were found to secrete ghrelin.Overall, ghrelin gene expression was greater in differentiating than non-differentiating osteoblasts, but was not increased during culture in either group. Ghrelin and UAG induced thymidine uptake dose-dependently, peaking at 1 and 10 nM respectively, at day 6 of culture in both non-differentiating and differentiating osteoblasts. The proliferative response to ghrelin and UAG declined with culture time and state of differentiation. The proliferative effects of ghrelin and UAG were suppressed by inhibitors of extracellular-signal-regulated kinase (ERK) and phosphoinositide-3 kinase, and both peptides rapidly induced ERK phosphorylation. Overall, our data suggest new roles for ghrelin and UAG in modulating human osteoblast proliferation via a novel signal transduction pathway.

Circadian rhythms are deeply rooted in the biology of virtually all organisms. The pervasive use of artificial lighting in modern society disrupts circadian rhythms and can be detrimental to our health. To investigate the relationship between disrupting circadian rhythmicity and disease, we exposed mice to continuous light (LL) for 24 weeks and measured several major health parameters. Long-term neuronal recordings revealed that 24 weeks of LL reduced rhythmicity in the central circadian pacemaker of the suprachiasmatic nucleus (SCN) by 70%. Strikingly, LL exposure also reduced skeletal muscle function (forelimb grip strength, wire hanging duration, and grid hanging duration), caused trabecular bone deterioration, and induced a transient pro-inflammatory state. After the mice were returned to a standard light-dark cycle, the SCN neurons rapidly recovered their normal high-amplitude rhythm, and the aforementioned health parameters returned to normal. These findings strongly suggest that a disrupted circadian rhythm reversibly induces detrimental effects on multiple biological processes.

The mutual interplay between energy homeostasis and bone metabolism is an important emerging concept. Ghrelin and leptin antagonize each other in regulating energy balance, but the role of this interaction in bone metabolism is unknown. Using ghrelin receptor and leptin-deficient mice, we show that ghrelin has dual effects on osteoclastogenesis, inhibiting osteoclast progenitors directly and stimulating osteoclastogenesis via a more potent systemic/central pathway. Using mice with combined ghrelin receptor and leptin deficiency, we find that this systemic osteoclastogenic activity is suppressed by leptin, thus balancing the two counterregulatory ghrelin pathways and leading to an unchanged bone structure. With aging, this osteoclastogenic ghrelin pathway is lost, unmasking the direct protective effect of ghrelin on bone structure. In conclusion, we identify a novel regulatory network linking orexigenic and anorectic metabolic factors with bone metabolism that is age dependent. There is now strong evidence that skeletal turnover is coupled with energy metabolism. The mechanism by which these processes are linked involves the adipose tissue-derived factor leptin (1, 2). Leptin acts as a signal from fat, by which bone can adapt to alterations in body weight and metabolism caused by changes in adiposity. Various routes of leptin action on bone have been described. Leptin can act directly on osteoblasts and osteoclasts to increase cortical bone mass (3), and leptin effects may also be mediated via the GH-IGF axis (4). In addition, leptin can act via neuro-endocrine circuits by both inhibiting bone resorption via stimulation of hypothalamic cocaine-and amphetamine-regulated transcript (CART) expression (5) and stimulating bone resorption and inhibiting bone formation via central circuits that stimulate sympathetic nervous system (SNS) function (5, 6). It has also been determined that there is a feed-forward regulation loop from osteoblasts involving osteocalcin that enhances pancreatic insulin production, modulates insulin sensitivity at peripheral sites, and increases energy expenditure (7). These processes enable interplay between the regulation of skeletal and energy metabolism and body weight.An important counterregulator of leptin action is the gut-derived peptide hormone ghrelin, interacting predominantly at hypothalamic agouti-related protein/neuropeptide Y neurons of the arcuate nucleus, where both ghrelin [GH secretagogue receptor (GHS-R)] and leptin receptors are expressed (8). Ghrelin modulates long-term energy homeostasis by stimulating weight gain (9) and has direct effects on the pancreas to suppress insulin secretion, together leading to worsened glucose homeostasis (e.g. Ref.

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Ghrelin receptor-deficient (Ghsr-/-) mice that lack acylated ghrelin (AG) signaling retain a metabolic response to unacylated ghrelin (UAG). Recently, we showed that Ghsr-deficiency affects bone metabolism. The aim of this study was to further establish the impact of AG and UAG on bone metabolism. We compared bone metabolism in Ghsr-/- (lacking only AG signaling) and ghrelin-deficient (Ghrl-/-; both AG and UAG deficient) male mice. Ghrl-/- mice had lower cortical bone mass, whereas Ghsr-/- mice had lower trabecular bone mass. This demonstrates bone compartment-specific effects of AG and a role for UAG in bone metabolism. Also, Ghrl-/- but not Ghsr-/- mice had increased bone formation rate and increased osteogenic stem cell numbers in their bone marrow. In ex vivo bone marrow cultures both AG and UAG inhibited osteoblast differentiation. This indicated that bone resorption must be increased in these mice. Accordingly, osteoclastogenesis rate was faster in bone marrow cultures from Ghsr-/- and Ghrl-/- mice, and osteoclast formation was inhibited by AG signaling and partially suppressed by UAG. In osteoblast cultures, AG markedly induced osteoprotegerin gene expression and both peptides reduced RANKL/osteoprotegerin ratio. These data describe unique cell-type specific effects of AG and UAG within a single tissue, supporting a tight and complex control of bone formation and resorption as well as a link between nutrition and bone metabolism. The balance between AG and UAG actions in the bone marrow may lead to bone compartmental-specific effects.

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Osteocytes are the predominant cells in bone, where they form a cellular network and display important functions in bone homeostasis, phosphate metabolism and mechanical transduction. Several proteins strongly expressed by osteocytes are involved in these processes, e.g., sclerostin, DMP-1, PHEX, FGF23 and MEPE, while others are upregulated during differentiation of osteoblasts into osteocytes, e.g., osteocalcin and E11. The receptor-type protein tyrosine phosphatase µ (RPTPμ) has been described to be expressed in cells which display a cellular network, e.g., endothelial and neuronal cells, and is implied in mechanotransduction. In a capillary outgrowth assay using metatarsals derived from RPTPμ-knock-out/LacZ knock-in mice, we observed that the capillary structures grown out of the metatarsals were stained blue, as expected. Surprisingly, cells within the metatarsal bone tissue were positive for LacZ activity as well, indicating that RPTPμ is also expressed by osteocytes. Subsequent histochemical analysis showed that within bone, RPTPμ is expressed exclusively in early-stage osteocytes. Analysis of bone marrow cell cultures revealed that osteocytes are present in the nodules and an enzymatic assay enabled the quantification of the amount of osteocytes. No apparent bone phenotype was observed when tibiae of RPTPμ-knock-out/LacZ knock-in mice were analyzed by μCT at several time points during aging, although a significant reduction in cortical bone was observed in RPTPμ-knock-out/LacZ knock-in mice at 20 weeks. Changes in trabecular bone were more subtle. Our data show that RPTPμ is a new marker for osteocytes.

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