Data on plasma cortisol levels in nibbler fish Girella punctata reared under high-density conditions in either surface seawater or deep ocean water.

Deep ocean water (DOW) is the water obtained from depth of >200 m below the surface of Earth's oceans and is characterized by rich nutrients and cleanliness [1,2]. We have recently reported that DOW suppresses the high-density-induced increase of plasma cortisol levels (i.e., a stress marker) in Japanese flounder (Paralichthys olivaceus) [1]. The current study aimed to examine whether the cortisol-reducing effect of DOW was observed in other marine organisms as well by comparing the plasma cortisol levels of nibbler fish Girella punctata reared under high-density conditions between surface seawater (SSW) and DOW. The nibbler fish were caught from Tsukumo Bay of Noto Peninsula (Ishikawa Prefecture, Japan). The DOW was obtained from seawater 320 m below the Noto Bay surface at a facility (Aquas Noto, Ishikawa Prefecture, Japan), whereas SSW was obtained from Tsukumo Bay (Noto Peninsula, Ishikawa Prefecture). The dissolved oxygen was maintained at approximately 7 mg/L in DOW as well as in SSW. Before they were transferred to the high-density condition, nibbler fish were acclimated in SSW at 20°C for 1 week at a mean density of 100 g/62.5 L. To expose them to the high-density stress, each of fish was kept at a density of 10 kg/m3 in a single aquarium (60 × 25 × 30 cm) containing either SSW or DOW (n = 8). Subsequently, the fish were reared with SSW or DOW for 10 days at 20°C ± 1°C under a 12:12-h light-dark cycle. A heparin containing syringe was used to obtain the blood samples from the caudal vessels of the fish anesthetized with a 0.04% 2-phenoxyethanol (FUJIFILM Wako Pure Chemical Corporation). The blood sampling was performed on days 0, 5, and 10 after rearing in the small aquaria. The plasma samples were prepared from the collected blood by centrifuging it at 5200 × g for 5 min and the cortisol concentrations were determined using an enzyme-linked immunosorbent assay (ELISA) kit (Cosmo Bio Co. Ltd., Tokyo, Japan) from those samples. The plasma cortisol concentration of nibbler fish reared in SSW on day 10 was significantly higher than that on day 0, whereas those reared in DOW did not show significant difference on the respective days. The current data contributes to the generalization of the cortisol-reducing effect of DOW on fish, which has been proposed in Japanese flounder [1]. These data could be used for developing and designing experiments to analyze the mechanisms underlying the cortisol-reducing effects by using small fish such as zebrafish, a well-established animal model.

Kynurenine promotes Calcitonin secretion and reduces cortisol in the Japanese flounder Paralichthys olivaceus.

Deep ocean water (DOW) exerts positive effects on the growth of marine organisms, suggesting the presence of unknown component(s) that facilitate their aquaculture. We observed that DOW suppressed plasma cortisol (i.e., a stress marker) concentration in Japanese flounder (Paralichthys olivaceus) reared under high-density condition. RNA-sequencing analysis of flounder brains showed that when compared to surface seawater (SSW)-reared fish, DOW-reared fish had lower expression of hypothalamic (i.e., corticotropin-releasing hormone) and pituitary (i.e., proopiomelanocortin, including adrenocorticotropic hormone) hormone-encoding genes. Moreover, DOW-mediated regulation of gene expression was linked to decreased blood cortisol concentration in DOW-reared fish. Our results indicate that DOW activated osteoblasts in fish scales and facilitated the production of Calcitonin, a hypocalcemic hormone that acts as an analgesic. We then provide evidence that the Calcitonin produced is involved in the regulatory network of genes controlling cortisol secretion. In addition, the indole component kynurenine was identified as the component responsible for osteoblast activation in DOW. Furthermore, kynurenine increased plasma Calcitonin concentrations in flounders reared under high-density condition, while it decreased plasma cortisol concentration. Taken together, we propose that kynurenine in DOW exerts a cortisol-reducing effect in flounders by facilitating Calcitonin production by osteoblasts in the scales.

Melatonin is a potential drug for the prevention of bone loss during space flight.

Astronauts experience osteoporosis-like loss of bone mass because of microgravity conditions during space flight. To prevent bone loss, they need a riskless and antiresorptive drug. Melatonin is reported to suppress osteoclast function. However, no studies have examined the effects of melatonin on bone metabolism under microgravity conditions. We used goldfish scales as a bone model of coexisting osteoclasts and osteoblasts and demonstrated that mRNA expression level of acetylserotonin O-methyltransferase, an enzyme essential for melatonin synthesis, decreased significantly under microgravity. During space flight, microgravity stimulated osteoclastic activity and significantly increased gene expression for osteoclast differentiation and activation. Melatonin treatment significantly stimulated Calcitonin (an osteoclast-inhibiting hormone) mRNA expression and decreased the mRNA expression of receptor activator of nuclear factor κB ligand (a promoter of osteoclastogenesis), which coincided with suppressed gene expression levels for osteoclast functions. This is the first study to report the inhibitory effect of melatonin on osteoclastic activation by microgravity. We also observed a novel action pathway of melatonin on osteoclasts via an increase in CALCITONIN secretion. Melatonin could be the source of a potential novel drug to prevent bone loss during space flight.

α-Melanocyte-stimulating hormone promotes bone resorption resulting from increased osteoblastic and osteoclastic activities in goldfish.

We examined the effects of α-melanocyte-stimulating hormone (α-MSH) on bone metabolism using regenerating goldfish scales. Normally developed scales on the bodies of goldfish were removed to allow the regeneration of scales under anesthesia. Thereafter, the influence of α-MSH on the regeneration of goldfish scales was investigated in vivo. In brief, α-MSH was injected at a low dose (0.1 µg/g body weight) or a high dose (1 µg/g body weight) into goldfish every other day. Ten days after removing the scales, we collected regenerating scales and analyzed osteoblastic and osteoclastic activities as respective marker enzyme (alkaline phosphatase for osteoblasts, tartrate-resistant acid phosphatase for osteoclasts) activity in the regenerating scales as well as plasma calcium levels. At both doses, osteoblastic and osteoclastic activities in the regenerating scales increased significantly. Plasma calcium concentrations in the α-MSH-treated group (high doses) were significantly higher than those in the control group. Next, in vitro experiments were performed to confirm the results of in vivo experiments. In the cultured regenerating scales, osteoblastic and osteoclastic activities significantly increased with α-MSH (10-7 and 10-6 M) treatment. In addition, real-time PCR analysis indicated that osteoclastogenesis in α-MSH-treated scales was induced by the receptor activator of the NF-κB/receptor activator of the NF-κB ligand/osteoprotegerin pathway. Furthermore, we found that α-MSH receptors (melanocortin receptors 4 and 5) were detected in the regenerating scales. Thus, in teleosts, we are the first to demonstrate that α-MSH functions in bone metabolism and promotes bone resorption via melatonin receptors 4 and/or 5.

Sardine procalcitonin amino-terminal cleavage peptide has a different action from calcitonin and promotes osteoblastic activity in the scales of goldfish.

The nucleotide sequence of a sardine preprocalcitonin precursor has been determined from their ultimobranchial glands in the present study. From our analysis of this sequence, we found that sardine procalcitonin was composed of procalcitonin amino-terminal cleavage peptide (N-proCT) (53 amino acids), CT (32 amino acids), and procalcitonin carboxyl-terminal cleavage peptide (C-proCT) (18 amino acids). As compared with C-proCT, N-proCT has been highly conserved among teleosts, reptiles, and birds, which suggests that N-proCT has some bioactivities. Therefore, both sardine N-proCT and sardine CT were synthesized, and their bioactivities for osteoblasts and osteoclasts were examined using our assay system with goldfish scales that consisted of osteoblasts and osteoclasts. As a result, sardine N-proCT (10-7M) activated osteoblastic marker enzyme activity, while sardine CT did not change. On the other hand, sardine CT (10-9 to 10-7M) suppressed osteoclastic marker enzyme activity, although sardine N-proCT did not influence enzyme activity. Furthermore, the mRNA expressions of osteoblastic markers such as type 1 collagen and osteocalcin were also promoted by sardine N-proCT (10-7M) treatment; however, sardine CT did not influence their expressions. The osteoblastic effects of N-proCT lack agreement. In the present study, we can evaluate exactly the action for osteoblasts because our scale assay system is very sensitive and it is a co-culture system for osteoblasts and osteoclasts with calcified bone matrix. Both CT and N-proCT seem to influence osteoblasts and osteoclasts and promote bone formation by different actions in teleosts.

Calcitonin-typical suppression of osteoclastic activity by amphioxus calcitonin superfamily peptides and insights into the evolutionary conservation and diversity of their structures.

Calcitonin (CT) is a hormone that decreases serum calcium level by suppressing osteoclastic activity in the vertebrate bone. In vertebrates, the structure-function relationship of CTs has been studied extensively. We recently identified three CT superfamily peptides, Bf-CTFP1 to 3, and clarified the molecular and functional characteristics of their receptor and receptor activity-modifying protein in amphioxus, Branchiostoma floridae. However, the CT activity of Bf-CTFPs has yet to be investigated. In the present study, a functional analysis of Bf-CTFPs was performed using goldfish scales having both osteoclasts and osteoblasts. All Bf-CTFPs suppressed osteoclastic activity via a goldfish CT receptor. Although the primary amino acid sequences of the Bf-CTFPs showed low sequence similarity to vertebrate CTs, Bf-CTFP1 to 3 share three amino acids, Thr25, Thr27, and Pro32-NH2, that are required for receptor binding, with salmon CT. Moreover, homology model analysis revealed that the Bf-CTFPs form alpha-helical structures. The alpha-helical position and length of Bf-CTFP1 and 2 were conserved with those of a highly potent ligand, teleost CT. Interestingly, the composition of the alpha-helix of Bf-CTFP3 differed from those of teleost CT, despite that the action of Bf-CTFP3 on goldfish scales was the same as that of Bf-CTFP1 and 2. Collectively, the present study provides new insights into the structure-function relationship of CT and its functional evolution in chordates.