Larry G. Riley

Effects of fasting on growth hormone/insulin-like growth factor I axis in the tilapia, Oreochromis mossambicus

Effects of fasting on the growth hormone (GH)-insulin-like growth factor I (IGF-I) axis were examined in the tilapia (Oreochromis mossambicus) acclimated to fresh water. Fasting for 2 weeks resulted in significant reductions in body weight, specific growth rate and hepatosomatic index in both males and females. Significant reductions in specific growth rates were observed after 1 and 2 weeks in both sexes, although the decrease in body weight was not significant in the female. A significant reduction was also seen in the condition factor of females after 2 weeks. No change was seen in the gonadosomatic index in either sex. Two weeks of fasting also produced a significant reduction in plasma IGF-I but not in plasma GH, prolactin (PRL(188)) or cortisol. Significant reductions in the hepatic IGF-I mRNA were seen in both sexes. On the other hand, a significant increase was observed in cortisol receptor mRNA in the female liver. Plasma IGF-I levels were correlated significantly with specific growth rate, condition factor and hepatosomatic index, indicating that plasma IGF-I is a good indicator of growth in the tilapia. No change was seen in plasma glucose or osmolality after 2 weeks of fasting. During fasting, tilapia appears to convert metabolic energy from growth to basal metabolism including maintenance of ion and water balance.

Identification of tilapia ghrelin and its effects on growth hormone and prolactin release in the tilapia, Oreochromis mossambicus.

We have identified ghrelin and cDNA encoding precursor protein from the stomach of a euryhaline tilapia, Oreochromis mossambicus. The sequence of 20-amino acid tilapia ghrelin is GSSFLSPSQKPQNKVKSSRI. The third serine residue was modified by n-decanoic acid. The carboxyl-terminal end of the peptide possessed an amide structure. RT-PCR analysis revealed high levels of gene expression in the stomach and low levels in the brain, kidney and gill. Tilapia ghrelin stimulated growth hormone (GH) and prolactin (PRL) release from the organ-cultured tilapia pituitary at a dose of 10 nM. Thus, a novel regulatory mechanism of GH secretion by gastric ghrelin seems to be conserved in the tilapia. Stimulation of PRL release by homologous ghrelin has been reported in human, bullfrog and eel, and suggests the presence of growth hormone secretagogue receptor not only on somatotrophs but also on PRL cells of the tilapia pituitary.

Rat Ghrelin Stimulates Growth Hormone and Prolactin Release in the Tilapia, Oreochromis mossambicus

Abstract Recently, ghrelin (Ghr), a new peptide which specifically stimulates growth hormone (GH) release from the pituitary, was identified in the rat and human stomach. Ghrelin has been shown to stimulate GH release by acting through a growth hormone secretagogue (GHS) receptor in the rat. The present study describes the in vitro effect of rat Ghr on the release of GH and two forms of prolactin (PRL177 and PRL188) in the tilapia, Oreochromis mossambicus. Rat Ghr stimulated the release of GH in a dose-related manner after 8 and 24 hr of incubation. Rat Ghr also significantly stimulated the release of PRL177 and PRL188 in a dose-related manner after 24 hr. Rat Ghr had no effect on the pituitary content of GH or PRL188, but significantly increased PRL177 content. These results show for the first time that rat Ghr significantly stimulates GH and PRL release in teleosts, and suggest that Ghr and a GHS receptor are present in fish.

Effects of environmental osmolality on release of prolactin, growth hormone and ACTH from the tilapia pituitary

Prolactin (PRL) plays a central role in freshwater (FW) adaptation in teleost fish. Evidence now suggests that growth hormone (GH) acts in the seawater (SW) adaptation in at least some euryhaline fish. Reflecting its important role in FW adaptation, plasma levels of PRL(188) and PRL(177) are higher in tilapia (Oreochromis mossambicus) adapted to FW than in those adapted to SW. A transient but significant increase in plasma GH was observed 6h after transfer from FW to SW. Elevated plasma PRL levels were seen in association with reductions in plasma osmolality after blood withdrawal in FW fish whereas no significant change was seen in plasma GH levels. When pituitaries from FW tilapia were incubated for 7 days, secretion of both PRLs was significantly greater in hyposmotic medium than in hyperosmotic medium for the first 24h. Secretion of GH from the same pituitary was relatively low during this period compared with PRL secretion. No consistent effect of medium osmolality on GH release was seen for the first day, but its cumulative release was increased significantly in hyperosmotic medium after 2 days and thereafter. On the other hand, ACTH release was extremely low compared with the secretion of PRLs and GH and there was no consistent effect of medium osmolality. These results indicate that PRL release from the tilapia pituitary is stimulated both in vivo and in vitro as extracellular osmolality is reduced, whereas the secretion of GH increases temporarily when osmolality is increased. ACTH seems to be relatively insensitive to the changes in environmental osmolality.

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17α-methyltestosterone and seawater rearing in the tilapia, Oreochromis mossambicus

Effects of 17 alpha-methyltestosterone (MT) treatment and environmental salinity on the growth hormone (GH)/insulin-like growth factor (IGF) axis were examined in the euryhaline tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to 1 of 4 groups: FW, MT treatment in FW, SW, and MT treatment in seawater (SW). After 147 days, FW controls had the lowest levels of GH mRNA followed by FW fish treated with MT and SW control fish. Seawater fish fed with a diet containing MT, which grew the fastest, had significantly higher levels of GH mRNA than all the other groups. A significant correlation was observed between GH mRNA and the size of the individual fish. By contrast, plasma GH levels did not vary significantly among the groups. Pituitary GH mRNA levels, plasma IGF-I levels, and fish size varied in a correlated pattern, i.e., SW+MT>FW+MT=SW control>FW control. The tilapia pituitary produces two prolactins (PRLs), PRL(177) and PRL(188). Prolactin(177), but not PRL(188), exhibits growth-promoting actions in FW tilapia. Pituitary mRNA levels of both PRLs were significantly higher in fish reared in FW than those reared in SW. Treatment with MT significantly increased mRNA levels of both PRLs in FW, but had no effect on SW fish. No correlation was seen between plasma PRL levels and growth or between PRL mRNA levels and growth. These results indicate that SW rearing and MT treatment stimulate the GH/IGF-I axis, and suggest that pituitary GH mRNA at this stage of development is a better indicator of growth than plasma levels of GH and IGF-I.

Effects of transfer from seawater to fresh water on the growth hormone/insulin-like growth factor-I axis and prolactin in the Tilapia, Oreochromis mossambicus.

The effect of freshwater (FW) transfer on growth and on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis was examined in the tilapia, Oreochromis mossambicus. Tilapia were raised in seawater (SW) for 5 months and then transferred to FW for an additional 40 days. The growth rate of the fish transferred to FW was significantly reduced compared with the growth rate of fish that remained in SW. Plasma levels of GH were significantly elevated in FW-transferred fish, as were plasma IGF-I levels. Pituitary GH and liver IGF-I mRNA levels, on the other hand, were significantly reduced in the fish transferred to FW. There was a significant correlation between body mass and mRNA levels of GH and IGF-I, but not with plasma levels of GH and IGF-I. Fish transferred to FW had significantly higher prolactin (PRL)(177) levels than the SW control fish, although there was no difference in plasma PRL(188) levels. Consistent with the hyperosmoregulatory effects of PRL, mRNA levels of both PRL(177) and PRL(188) were significantly higher in FW-transferred fish than in the fish in SW. These results suggest that transferring tilapia from SW to FW activates the GH/IGF-I axis, but growth is still inhibited, possibly due to the greater metabolic cost of osmoregulation in FW than in SW.

Plasma ghrelin and growth hormone regulation in response to metabolic state in hybrid striped bass: Effects of feeding, ghrelin and insulin-like growth factor-I on in vivo and in vitro GH secretion

The regulation of growth hormone (GH) secretion by ghrelin during variable metabolic states is poorly understood. We examined plasma GH and ghrelin in hybrid striped bass (HSB) undergoing seasonally-based feeding and temperature manipulations. Fasting for 21 days (d) at 24 degrees C resulted in catabolism and up-regulation of plasma GH and ghrelin relative to fed controls. Continued fasting during cold-banking (14 degrees C, 90 d) resulted in a further 43-fold increase in ghrelin while GH remained elevated. A subsequent 19 day refeeding period at 24 degrees C elicited hyperphagic and compensatory growth responses, accompanied by declines in ghrelin and GH. We then tested the role of ghrelin in stimulating GH release in vivo and in vitro. Intraperitoneal injections of ghrelin resulted in dose-dependent increases in plasma GH after 6 hours (h). Ghrelin also increased GH release from HSB pituitaries during 6h incubations. Lastly, we assessed how metabolic state, ghrelin and insulin-like growth factor-I (IGF-I) affect in vitro pituitary GH release. Spontaneous GH release was 5.2-fold higher from pituitaries of fasted compared with fed animals. Ghrelin was equally effective in stimulating GH release from pituitaries of fed and starved animals, while it was ineffective in enhancing GH release from pituitaries of starved (21 d) then refed (4d) HSB. Incubation with IGF-I inhibited GH release regardless of metabolic state. These studies are the first to show that seasonally-based periods of feed deprivation and low temperature yield sustained increases in GH secretion that are likely mediated, at least partially, through elevated ghrelin, reduced IGF-I negative feedback and fasting-induced spontaneous GH release.

Effects of environmental salinity and 17α-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17alpha-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3-5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.

Absence of Effects of Short-Term Fasting on Plasma Ghrelin and Brain Expression of Ghrelin Receptors in the Tilapia, Oreochromis mossambicus

Abstract Ghrelin is an important endocrine peptide that links the gastrointestinal system and brain in the regulation of food intake and energy expenditure. In human, rat, and goldfish plasma levels of ghrelin and GH are elevated in fasted animals, suggesting that ghrelin is an orexigenic signal and a driving force behind the elevated plasma levels of GH during fasting. Ghrelin’s orexigenic action is mediated by the ghrelin receptor (GHS-R1a and GHS-R1b) which is localized on neuropeptide Y (NPY) neurons in the brain. Studies were undertaken to investigate the effect of short-term fasting on plasma ghrelin and brain expression of GHS-R1a, GHS-R1b, and NPY in the tilapia. Fasting for 7 days had no effect on plasma ghrelin concentrations, whereas significant increases in plasma levels of GH were observed on day 3. Fasting significantly reduced plasma levels of IGF-I on days 3 and 7, and of glucose on days 3, 5, and 7. Brain expression of ghrelin and GHS-R1b were significantly elevated in fasted fish on day 3, but were significantly reduced on day 5. This reduction was likely due to a significant increase in the expression in the fed controls on day 5 compared to day 0. No change was detected in the expression of GHS-R1a or NPY in the brain. These results indicate that ghrelin is not acting as a hunger signal in short-term fasted tilapia and is not responsible for the elevated levels of plasma GH.

Identification and genomic sequence of a ghrelin receptor (GHS-R)-like receptor in the Mozambique tilapia, Oreochromis mossambicus.

The growth hormone secretagogue-receptor (GHS-R) is an endogenous receptor for the gut hormone ghrelin (GRLN). Two isoforms of GHS-R have been identified in several animals: functional GHS-R1a and a splice variant of unknown function, GHS-R1b. Here we report identification of a GHS-R-like receptor (GHSR-LR) in the Mozambique tilapia, Oreochromis mossambicus. The cDNA is 1584 bp in length and encodes a 384-amino acid GHS-R1a ortholog. The amino acid sequence of tilapia GHS-R1a is 54, 60, 80 and 89% identical to that of rat, chicken, pufferfish, and seabream GHS-R1a, respectively. Genomic PCR revealed that the tilapia GHS-R gene is composed of two exons separated by a single intron. In addition, a GHS-R1b ortholog, which is generated by alternative splicing of the GHS-R gene and contains part of the intron, was identified and predicted to be a 298-amino acid protein. Functional analyses of tilapia GHS-R1a were conducted using mammalian HEK 293 and CHO cells, but the expected increase in intracellular calcium ions by tilapia or rat GRLN was not observed. We found that the GHS-R1a ortholog is expressed in greater quantities than the GHS-R1b ortholog in all tissues assayed. Further studies are required to conclude that our identified protein is the GHS-R for tilapia, although the gene structure and amino acid sequence showed high similarities to other GHS-R genes; thus, we designated this protein GHSR-LR.