C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor B (NPR-B) are physiological potent positive regulators of endochondral bone growth; therefore, the CNP/NPR-B signaling pathway is one of the most promising therapeutic targets for treating growth failure and dwarfism. In this article, we summarized the pharmacological properties of a novel CNP analog peptide ASB20123 as a therapeutic agent for short stature. ASB20123, one of the CNP/ghrelin chimeric peptides, is composed of CNP(1-22) and human ghrelin(12-28, E17D). Compared to CNP(1-22), ASB20123 showed similar agonist activity for NPR-B and improved biokinetics with a longer plasma half-life in rats. In addition, the distribution of ASB20123 to the cartilage was higher than that of CNP(1-22) after single subcutaneous (sc) injection to mice. These results suggested that the C-terminal part of ghrelin, which has clusters of basic amino acid residues and a BX7B motif, might contribute to the retention of ASB20123 in the extracellular matrix of the growth plate. Multiple sc doses of ASB20123 potently stimulated skeletal growth in rats in a dose-dependent manner, and sc infusion was more effective than bolus injection at the same dose. Our data indicated that high plasma levels of ASB20123 would not necessarily be required for bone growth acceleration. Thus, pharmaceutical formulation approaches for sustained-release dosage forms to allow chronic exposure to ASB20123 might be suitable to ensure drug effectiveness and safety.

Insulin and leptin intracellular signaling pathways converge and act synergistically on the hypothalamic phosphatidylinositol-3-OH kinase/3-phosphoinositide-dependent protein kinase 1 (PDK1). However, little is known about whether PDK1 in agouti-related peptide (AGRP) neurons contributes to energy homeostasis. We generated AGRP neuron-specific PDK1 knockout (AGRPPdk1(-/-)) mice and mice with selective expression of transactivation-defective Foxo1 (Δ256Foxo1(AGRP)Pdk1(-/-)). The AGRPPdk1(-/-) mice showed reductions in food intake, body length, and body weight. The Δ256Foxo1(AGRP)Pdk1(-/-) mice showed increased body weight, food intake, and reduced locomotor activity. After four weeks of calorie-restricted feeding, oxygen consumption and locomotor activity were elevated in AGRPPdk1(-/-) mice and reduced in Δ256Foxo1(AGRP)Pdk1(-/-) mice. In vitro, ghrelin-induced changes in [Ca(2+)](i) and inhibition of ghrelin by leptin were significantly attenuated in AGRPPdk1(-/-) neurons compared to control neurons. However, ghrelin-induced [Ca(2+)](i) changes and leptin inhibition were restored in Δ256Foxo1(AGRP)Pdk1(-/-) mice. These results suggested that PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms.

Neuromedin U (NMU) is a neuropeptide that is expressed in the gastrointestinal tract and central nervous system. NMU interacts with two G protein-coupled receptors, NMU-R1 and NMU-R2. Whereas NMU-R2 localizes predominantly to nerve cells, NMU-R1 is expressed in peripheral tissues including lymphocytes and monocytes, suggesting a role of NMU in immunoregulation. However, the functions of NMU in peripheral tissues have not been clarified. In this study, using NMU-deficient mice, we first demonstrated that NMU plays an important role in mast cell-mediated inflammation. Complete Freund's adjuvant-induced mast cell degranulation as well as edema and neutrophil infiltration, which occurred weakly in mast cell-deficient WBB6F(1)-W/W(v) mice, did not occur in NMU-deficient mice. Moreover, intraplantar injection of NMU into paws induced early inflammatory responses such as mast cell degranulation, vasodilation, and plasma extravasation in WT mice but not in WBB6F(1)-W/W(v) mice. NMU-R1 was highly expressed in primary mast cells, and NMU induced Ca(2+) mobilization and degranulation in peritoneal mast cells. These data indicate that NMU promotes mast cell-mediated inflammation; therefore, NMU receptor antagonists could be a novel target for pharmacological inhibition of mast cell-mediated inflammatory diseases.

To investigate the precise role of Notch/Rbp-j signaling in the pancreas, we inactivated Rbp-j by crossing Rbp-j floxed mice with Pdx.cre or Rip.cre transgenic mice. The loss of Rbp-j at the initial stage of pancreatic development induced accelerated alpha and PP cell differentiation and a concomitant decrease in the number of Neurogenin3 (Ngn3)-positive cells at E11.5. Then at E15, elongated tubular structures expressing ductal cell markers were evident; however, differentiation of acinar and all types of endocrine cells were reduced. During later embryonic stages, compensatory acinar cell differentiation was observed. The resultant mice exhibited insulin-deficient diabetes with both endocrine and exocrine pancreatic hypoplasia. In contrast, the loss of Rbp-j specifically in beta cells did not affect beta cell number and function. Thus, our analyses indicate that Notch/Rbp-j signaling prevents premature differentiation of pancreatic progenitor cells into endocrine and ductal cells during early development of the pancreas.

P450 oxidoreductase (POR) catalyzes electron transfer to microsomal P450 enzymes. Its deficiency causes Antley-Bixler syndrome (ABS), and about half the patients with ABS have ambiguous genitalia and/or impaired steroidogenesis. POR mRNA expression is up-regulated when mesenchymal stem cells (MSCs) differentiate into steroidogenic cells, suggesting that the regulation of POR gene expression is important for steroidogenesis. In this context we examined the regulation of POR expression in ovarian granulosa cells by gonadotropins, and its possible role in steroidogenesis.

Here, we describe cDNA cloning and purification of the ghrelin gene sequences and ghrelin peptides from the Japanese true mole, Mogera imaizumii. The gene spans >2.9kbp, has four exons and three introns, and shares structural similarity with those of terrestrial animals. Mature mole ghrelin peptide was predicted to be 28 amino acids long (GSSFLSPEHQKVQQRKESKKPPSKPQPR) and processed from a prepropeptide of 116 amino acids. To further elucidate molecular characteristics, we purified ghrelin peptides from mole stomach. By mass spectrometry, we found that the mole ghrelin peptides had higher ratios of the odd-number fatty acids (C9 and C11 as much as C8) attached to the third serine residue than other vertebrate ghrelin. Truncated forms of ghrelins such as [1-27], [1-19], [1-16] and [1-15], and that lacked the 14th glutamine residue (des-Gln14 ghrelin) were produced in the stomach. Marked expression of ghrelin mRNA in lung was observed as in stomach and brain. Phylogenetic analysis indicated that the branch of M. imaizumii has slightly higher dN/dS ratios (the nucleotide substitution rates at non-synonymous and synonymous sites) than did other eulipotyphlans. Peptide length was positively correlated with human ghrelin receptor activation, whereas the length of fatty-acyl chains showed no obvious functional correlation. The basal higher luciferase activities of the 5'-proximal promoter region of mole ghrelin were detected in ghrelin-negative C2C12 cells and hypoxic culture conditions impaired transcriptional activity. These results indicated that moles have acquired diverse species of ghrelin probably through distinctive fatty acid metabolism because of their food preferences. The results provide a gateway to understanding ghrelin metabolism in fossorial animals.

The growth hormone secretagogue-receptor (GHS-R) was discovered in humans and pigs in 1996. The endogenous ligand, ghrelin, was discovered 3 years later, in 1999, and our understanding of the physiological significance of the ghrelin system in vertebrates has grown steadily since then. Although the ghrelin system in non-mammalian vertebrates is a subject of great interest, protein sequence data for the receptor in non-mammalian vertebrates has been limited until recently, and related biological information has not been well organized. In this review, we summarize current information related to the ghrelin receptor in non-mammalian vertebrates.

Highly relevant mouse models of human neuroblastoma (NB) are needed to evaluate new therapeutic strategies against NB. In this study, we characterized transgenic mice with bilateral adrenal tumors. On the basis of information from the tumoral gene expression profiles, we examined the antitumor effects of unencapsulated and liposomal doxorubicin (DXR), alone and in combination with gefitinib, on adrenal NB. We showed that intravenous injection of unencapsulated or liposomal DXR alone inhibited tumor growth in a dose-dependent manner, as assessed by magnetic resonance imaging (MRI). However, liposomal DXR did not exhibit greater antitumor effect than unencapsulated DXR. Immunohistochemical analysis revealed that the adrenal tumor vasculature with abundant pericyte coverage was a less leaky structure for liposomes. Combination therapy with unencapsulated or liposomal DXR plus gefitinib strongly suppressed tumor growth and delayed tumor regrowth than treatment with unencapsulated or liposomal DXR alone, even at a lower dose of DXR. Dynamic contrast-enhanced MRI analysis revealed that gefitinib treatment increased blood flow in the tumor, indicating that gefitinib treatment changes the tumor vascular environment in a manner that may increase the antitumor effect of DXR. In conclusion, the combination of gefitinib and DXR induces growth inhibition of adrenal NBs in transgenic mice. These findings will provide helpful insights into new treatments for NB.

An abnormal eating behavior is often associated with diabetes mellitus in individuals. In the present study, we investigated the mechanisms underlying the relationship among uncontrolled diabetes, food intake, and the production of ghrelin, an orexigenic hormone, in spontaneous diabetic Torii (SDT) rats. Male SDT rats and age-matched control Sprague-Dawley (SD) rats were housed from 8 to 38 weeks of age. Body weight and daily food intake were measured weekly, whereas blood and whole stomach samples were obtained at the age of 8, 25, and 38 weeks in both SDT and SD rats. The SDT rats at both 25 and 38 weeks of age demonstrated significantly lower body weights despite almost doubled food consumption compared with the SD rats of the same age. The SDT rats showed overt hyperglycemia at 25 and 38 weeks of age with concomitant hypoinsulinemia. The plasma active ghrelin levels and the ratio to total ghrelin levels of SDT rats at 38 weeks of age were significantly higher than those of SD rats of the same age. Stomach ghrelin and ghrelin O-acyltransferase messenger RNA expression levels were higher in SDT rats than in SD rats after the induction of diabetes, with a concomitant decrease of stomach ghrelin-immunopositive cell numbers in SDT rats at 38 weeks of age. The SDT rats with uncontrolled hyperglycemia show hyperphagia with a concomitant increase of plasma active ghrelin concentration. This report is the first to clarify the relevance of ghrelin to hyperphagia in diabetic state over an extended period.

Ghrelin (GRLN) is now known to be an appetite-stimulating and growth hormone (GH)-releasing peptide that is predominantly synthesized and secreted from the stomachs of various vertebrate species from fish to mammals. Here, we report a GRLN-like peptide (GRLN-LP) in a cartilaginous fish, the red stingray, Dasyatis akajei.

There are many orphan G protein-coupled receptors (GPCRs), for which ligands have not yet been identified, in both vertebrates and invertebrates, such as Drosophila melanogaster. Identification of their cognate ligands is critical for understanding the function and regulation of such GPCRs. Indeed, the discovery of bioactive peptides that bind GPCRs has enhanced our understanding of mechanisms underlying many physiological processes. Here, we identified an endogenous ligand of the Drosophila orphan GPCR, CG34381. The purified ligand is a peptide comprised of 28 amino acids with three intrachain disulfide bonds. The preprotein is coded for by gene CG14871. We designated the cysteine-rich peptide "trissin" (it means for triple S-S bonds) and characterized the structure of intrachain disulfide bonds formation in a synthetic trissin peptide. Because the expression of trissin and its receptor is reported to predominantly localize to the brain and thoracicoabdominal ganglion, trissin is expected to behave as a neuropeptide. The discovery of trissin provides an important lead to aid our understanding of cysteine-rich peptides and their functional interaction with GPCRs.

Ghrelin is an important regulator of energy--and glucose homeostasis. The octanoylation at Ser(3) is essential for ghrelin's biological effects but the mechanisms involved in the octanoylation are unknown. We investigated whether the gustatory G-protein, α-gustducin, and the free fatty acid receptors GPR40 and GPR120 are involved in the fatty acid sensing mechanisms of the ghrelin cell.

We have identified cDNA encoding a functional growth hormone secretagogue-receptor 1a (GHS-R1a, ghrelin receptor) in two species of anuran amphibian, the bullfrog (Rana catesbeiana), and the Japanese tree frog (Hyla japonica). Deduced receptor protein for bullfrog and Japanese tree frog (tree frog) was comprised of 374- and 371-amino acids, respectively. The two receptors shared 86% identity, and are grouped to the clade of the tetrapod homologs by phylogenetic analysis. In functional analyses, ghrelin and GHS-R1a agonists increased intracellular Ca(2+) concentration in GHS-R1a-transfected-HEK293 cell, but ligand selectivity of ghrelin with Ser(3) and Thr(3) was not observed between the two receptors. Bullfrog GHS-R1a mRNA was mainly expressed in the brain, stomach, and testis. In the brain, the gene expression was detected in the diencephalon and mesencephalon, but not in the pituitary. Tree frog GHS-R1a mRNA was predominantly expressed in the gastrointestinal tract and ovary, but not detected in the pituitary. In bullfrog stomach but not the brain, GHS-R1a mRNA expression increased after 10 days of fasting. For tree frog, GHS-R1a mRNA expression was increased in the brain, stomach and ventral skin by 10 days of fasting, and in the stomach and ventral skin by a dehydration treatment. Intracerebroventricular injection of ghrelin in dehydrated tree frog did not affect water absorption from the ventral skin. These results suggest that ghrelin is involved in energy homeostasis and possibly in osmoregulation in frogs.

In animal models, ghrelin production is suppressed by LPS administration. To elucidate the detailed molecular mechanisms involved in the phenomenon, we investigated the effects of LPS and LPS-inducible cytokines, including TNF-α, IL-1β, and IL-6, on the expression of ghrelin in the ghrelin-producing cell line MGN3-1. These cells expressed IL-1R, and IL-1β significantly suppressed ghrelin mRNA levels. The suppressive effects of IL-1β were attenuated by knockdown of IKKβ, suggesting the involvement of the NF-κB pathway. These results suggested that IL-1β is a major regulator of ghrelin expression during inflammatory processes.

Endocannabinoids acting on the cannabinoid-1 receptor (CB1R) or ghrelin acting on its receptor (GHS-R1A) both promote alcohol-seeking behavior, but an interaction between the two signaling systems has not been explored. Here, we report that the peripheral CB1R inverse agonist JD5037 reduces ethanol drinking in wild-type mice but not in mice lacking CB1R, ghrelin peptide or GHS-R1A. JD5037 treatment of alcohol-drinking mice inhibits the formation of biologically active octanoyl-ghrelin without affecting its inactive precursor desacyl-ghrelin. In ghrelin-producing stomach cells, JD5037 reduced the level of the substrate octanoyl-carnitine generated from palmitoyl-carnitine by increasing fatty acid β-oxidation. Blocking gastric vagal afferents abrogated the ability of either CB1R or GHS-R1A blockade to reduce ethanol drinking. We conclude that blocking CB1R in ghrelin-producing cells reduces alcohol drinking by inhibiting the formation of active ghrelin and its signaling via gastric vagal afferents. Thus, peripheral CB1R blockade may have therapeutic potential in the treatment of alcoholism.

Some chemotherapeutic agents have been reported to promote lung metastasis. However, there have been no reports regarding chemotherapy-induced liver metastasis. We hypothesized that chemotherapy might also enhance liver metastasis. The present study aimed to create a chemotherapy-enhanced liver metastasis mouse model and investigate its mechanism.

Two novel peptides, neuromedin U precursor-related peptide (NURP) and neuromedin S precursor-related peptide (NSRP), are produced from neuromedin U (NMU) and neuromedin S (NMS) precursors, respectively, as these precursors have multiple consensus sequences for proteolytic processing. Our group has shown previously that one of these two novel peptides, NURP, stimulates body temperature and locomotor activity, but not food intake. However, the physiological function of the other peptide, NSRP, has remained unclear. Therefore, the aim of this study was to characterize differences in the regions of the rat brain targeted by the NMU/NMS peptide family, including NURP and NSRP, and their physiological functions. First, we explored the regions of c-Fos expression after intracerebroventricular (i.c.v.) injection of NURP and NSRP and found that these were fewer than after i.c.v. injection of NMU and NMS in the hypothalamus, possibly because NURP and NSRP cannot activate NMU/NMS receptors. In the ventral subiculum, which is one region of the hippocampus, c-Fos expression was evident only after i.c.v. injection of NURP. We also examined the effects of NSRP on food intake, body temperature and locomotor activity. Like NURP, NSRP increased both body temperature and locomotor activity, but not food intake, indicating that NSRP is also a functional peptide. However, these effects of NSRP were distinctly weaker than those of NURP. These findings suggest differences in the affinity of NURP and/or NSRP for specific receptors, or in their respective biological activities.

Autosomal dominant hypocalcemia (ADH) is caused by gain-of-function mutations of the calcium sensing receptor (CaSR). It is characterized by hypercalciuria in spite of hypocalcemia. Vitamin D deficiency increases calcium reabsorption in the distal tubules of the kidneys, resulting in hypocalciuria.

Beige adipocytes are thermogenic adipocytes with developmental and anatomical properties distinct from those of classical brown adipocytes. Recent studies have revealed several key molecular regulators of beige adipocyte development. CD105, also called endoglin, is a membrane protein composed of TGF-β receptor complex. It regulates TGF-β-family signal transduction and vascular formation in vivo. We report here that CD105 maintains the thermogenic gene program of beige adipocytes by regulating Smad2 signaling. Cd105-/- adipocyte precursors showed augmented Smad2 activation and decreased expression of thermogenic genes such as Ucp1 and Prdm16-which encodes a transcriptional regulatory protein for thermogenesis-after adipogenic differentiation. Smad2 signaling augmentation by the constitutively active form of Smad2 decreased the expression of thermogenic genes in beige adipocytes. Loss of thermogenic activity in Cd105-/- beige adipocytes was rescued by Prdm16 expression. These data reveal a novel function of CD105 in beige adipocytes: maintaining their thermogenic program by regulating Smad2 signaling.

Nesfatin-1 is secreted, meal-responsive anorexigenic peptide encoded in the precursor nucleobindin-2 [NUCB2]. Circulating nesfatin-1 increases post-prandially, but the dietary components that modulate NUCB2/nesfatin-1 remain unknown. We hypothesized that carbohydrate, fat and protein differentially regulate tissue specific expression of nesfatin-1. NUCB2, prohormone convertases and nesfatin-1 were detected in mouse stomach ghrelinoma [MGN3-1] cells. NUCB2 mRNA and protein were also detected in mouse liver, and small and large intestines. MGN3-1 cells were treated with glucose, fatty acids or amino acids. Male C57BL/6 mice were chronically fed high fat, high carbohydrate and high protein diets for 17 weeks. Quantitative PCR and nesfatin-1 assays were used to determine nesfatin-1 at mRNA and protein levels. Glucose stimulated NUCB2 mRNA expression in MGN3-1 cells. L-Tryptophan also increased NUCB2 mRNA expression and ghrelin mRNA expression, and nesfatin-1 secretion. Oleic acid inhibited NUCB2 mRNA expression, while ghrelin mRNA expression and secretion was enhanced. NUCB2 mRNA expression was significantly lower in the liver of mice fed a high protein diet compared to mice fed other diets. Chronic intake of high fat diet caused a significant reduction in NUCB2 mRNA in the stomach, while high protein and high fat diet caused similar suppression of NUCB2 mRNA in the large intestine. No differences in serum nesfatin-1 levels were found in mice at 7 a.m, at the commencement of the light phase. High carbohydrate diet fed mice showed significantly elevated nesfatin-1 levels at 1 p.m. Serum nesfatin-1 was significantly lower in mice fed high fat, protein or carbohydrate compared to the controls at 7 p.m, just prior to the dark phase. Mice that received a bolus of high fat had significantly elevated nesfatin-1/NUCB2 at all time points tested post-gavage, compared to control mice and mice fed other diets. Our results for the first time indicate that nesfatin-1 is modulated by nutrients.