Molecular cloning and tissue distribution of cholecystokinin-1 receptor (CCK-1R) in yellowtail Seriola quinqueradiata and its response to feeding and in vitro CCK treatment.

In vertebrates, the peptide cholecystokinin (CCK) is one of the most important neuroregulatory digestive hormones. CCK acts via CCK receptors that are classified into two subtypes, CCK-1 receptor (CCK-1R; formally CCK-A) and CCK-2 receptor (formally CCK-B). In particular, the CCK-1R is involved in digestion and is regulated by CCK. However, very little information is known about CCK-1R in fish. Therefore, we performed molecular cloning of CCK-1R cDNA from the digestive tract of yellowtail Seriola quinqueradiata. Phylogenetic tree analysis showed a high sequence identity between the cloned yellowtail CCK receptor cDNA and CCK-1R, which belongs to the CCK-1R cluster. Furthermore, the expression of yellowtail CCK receptor mRNA was observed in gallbladder, pyloric caeca, and intestines, similarly to CCK-1R mRNA expression in mammals, suggesting that the cloned cDNA is of CCK-1R from yellowtail. In in vivo experiments, the CCK-1R mRNA levels increased in the gallbladder and pyloric caeca after feeding, whereas in vitro, mRNA levels of CCK-1R and digestive enzymes in cultured pyloric caeca increased by the addition of CCK. These results suggest that CCK-1R plays an important role in digestion stimulated by CCK in yellowtail.

Molecular characterization, tissue distribution patterns and nutritional regulation of IGFBP-1, -2, -3 and -5 in yellowtail, Seriola quinqueradiata.

Insulin-like growth factor-binding proteins (IGFBPs) play a vital role in regulating the biological activities of IGFs. In this study, we cloned and determined full-length cDNA sequences of yellowtail IGFBP-1, -2, -3 and -5. Their tissue distribution was determined by real-time quantitative RT-PCR, which revealed that IGFBP-1, -2, -3 and -5 are widely distributed in yellowtail tissues. In yellowtail, both IGFBP-1 and -2 are highly expressed in the liver, IGFBP-3 is predominantly expressed in the heart and skin, with the lowest expression in the liver, and IGFBP-5 is highly expressed in the liver and kidneys. The widespread tissue expression of the yellowtail IGFBPs suggests that they may act in an autocrine and/or paracrine manner in the regulation of IGF activity. The effects of nutritional deprivation on yellowtail IGFBPs and IGF-I were also examined. During a 15-day starvation period, significant elevation was observed in hepatic yellowtail IGFBP-1. Refeeding restored its level to that of the control. No significant change was observed in the hepatic yellowtail IGFBP-2 mRNA levels in starved fish compared with control fish during the starvation period. Interestingly, during the early period of food deprivation, a significant increase was observed in hepatic yellowtail IGFBP-3 and -5 mRNA levels, concomitant to the significant elevation in hepatic IGF-I mRNA from day 3 to day 9. The unexpected increase in growth stimulatory IGFBPs and IGF-I during nutritional deprivation may represent a species-specific response to changes in nutritional condition.

Nutrient control of release of pancreatic enzymes in yellowtail (Seriola quinqueradiata): involvement of CCK and PY in the regulatory loop.

Cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptides are key regulators of pancreatic enzyme secretion in vertebrates. CCK stimulates enzyme secretion whereas peptide Y (PY), a NPY-related peptide, plays an antagonistic role to that of CCK. In fish, very little is known about how different nutrients affect the synthesis of CCK and PY in the digestive tract, and the mechanism by which CCK and PY actually regulate digestive enzyme secretion is not well understood. In order to determine how different nutrients stimulate the synthesis of CCK and PY in yellowtail (Seriola quinqueradiata), CCK and PY mRNA levels in the digestive tract were measured after oral administration of a single bolus of either phosphate-buffered saline (PBS: control), starch (carbohydrate), casein (protein), oleic acid (fatty acid) or tri-olein (triglyceride). In addition, in order to confirm the synthesis and secretion of digestive enzymes, the mRNA levels and enzymatic activities of three digestive enzymes (lipase, trypsin and amylase) were also analyzed. Casein, oleic acid and tri-olein increased the synthesis of lipase, trypsin and amylase, while starch and PBS did not affect the activity of any of these enzymes. CCK mRNA levels rose, while PY mRNA levels were reduced in fish administered casein, oleic acid and tri-olein. These results suggest that in yellowtail, CCK and PY maintain antagonistic control of pancreatic enzyme secretion after intake of protein and/or fat.

Changes in cholecystokinin and peptide Y gene expression with feeding in yellowtail (Seriola quinqueradiata): relation to pancreatic exocrine regulation.

In fish, the regulation of digestive enzyme secretion by hormonal control such as cholecystokinin (CCK) and neuropeptide Y (NPY)-related peptide is not well understood. To investigate the roles of fish CCK and peptide Y (PY) in digestive enzyme secretion, mRNA levels of CCK and PY, pyloric caeca enzyme activities and mRNA levels of pancreatic digestive enzymes (lipase, trypsin and amylase) were measured at pre- and post-prandial stages in yellowtail. Pyloric caeca were sampled at 0, 0.5, 1.5, 3, 6, 12, 24 and 48 h after feeding. The mRNA levels of trypsin and amylase increased after feeding, suggesting that transcription was induced by feed ingestion. Digestive enzyme activities decreased in exocrine pancreas after feeding, suggesting the stored enzyme was secreted from pancreas post-prandially. mRNA levels for CCK displayed a time-dependent increase, peaking between 1.5 and 3 h after-feeding followed by a rapid decrease 3 to 6 h after feeding. The mRNA expression pattern of PY was inverse to the pattern of CCK, decreasing until 1.5 h after feeding and then rising to initial levels by 12 h after feeding. These results suggest that CCK and PY work antagonistically in the exocrine pancreas of yellowtail.

Effects of exogenous cholecystokinin and gastrin on the secretion of trypsin and chymotrypsin from yellowtail (Seriola quinqueradiata) isolated pyloric caeca.

The humoral control of secretion of the proteolytic enzymes trypsin and chymotrypsin was studied in yellowtail (Seriola quinqueradiata). In vitro trials were performed to investigate the effects of cholecystokinin (CCK) and two commercially available gastrin peptides. Isolated preparations of pyloric caeca/pancreas release trypsin and chymotrypsin when incubated with cholecystokinin (CCK) at 10 microM and gastrin I (G1) at 50 microM after 15 min of incubation. On the other hand, G1 at 10 microM and gastrin-related peptide (G2) did not enhance trypsin and chymotrypsin secretion. The studies concerning the CCK effects at different incubation temperatures have shown that trypsin and chymotrypsin secretion at 25 degrees C was stimulated by CCK after 15 min, while at 10, 15 and 20 degrees C the stimulatory effects of CCK were observed only after 30 min of incubation. The CCK effects were increased at higher incubation temperatures and longer incubation periods.

Cholecystokinin and peptide Y in yellowtail (Seriola quinqueradiata): molecular cloning, real-time quantitative RT-PCR, and response to feeding and fasting.

In fish, the peptide hormones cholecystokinin (CCK) and peptide Y (PY) may be involved in pancreatic exocrine secretion, as found with mammalian CCK and peptide YY (PYY); CCK stimulates, whereas PYY inhibits, pancreatic exocrine secretion in mammals. However, there is very little information on these hormones in fish; in particular, the function of PY is still unknown. Therefore, as a first step for understanding the role of CCK and PY in regulating pancreatic exocrine in fish, the cDNAs of CCK and PY were cloned from the digestive tract of yellowtail (Seriola quinqueradiata). The peptide sequence of yellowtail CCK-8, DYLGWMDF, is identical to sequences found in several teleosts. The mature form of yellowtail PY consists of 36 amino acids and has high identity to other fish PYs (88.9-97.2%). Real-time quantitative RT-PCR assays were developed to measure yellowtail CCK and PY mRNA levels. CCK mRNA levels were extremely high in the brain and, among the digestive organs, high concentrations were found in the pyloric caeca and anterior intestine. PY mRNA levels were low in the brain and highest in the anterior intestine. In fasting experiments, mRNA levels of CCK and PY in the anterior intestine showed an antagonistic change after fasting; CCK decreased whereas PY increased. These data suggest that CCK and PY in yellowtail may relate to digestion including, enzyme secretion.