Circulating insulin-like growth factor binding proteins in fish: Their identities and physiological regulation.

Insulin-like growth factor binding proteins (IGFBPs) play crucial roles in regulating the availability of IGFs to receptors and prolong the half-lives of IGFs. There are six IGFBPs present in the mammalian circulation with IGFBP-3 being most abundant. In mammals IGFBP-3 is the major carrier of circulating IGFs, facilitated by forming a ternary complex with IGF and an acid-labile subunit (ALS). IGFBP-1 is generally inhibitory to IGF action by preventing it from interacting with its receptors. In teleosts, the third-round of vertebrate whole genome duplication created paralogs of each IGFBP, except IGFBP-4. In the fish circulation, three major IGFBPs are typically detected at molecular ranges of 20-25, 28-32 and 40-50kDa. However, their identities are not well established. Three major circulating IGFBPs in Chinook salmon have been identified through protein purification and cDNA cloning. Salmon 28- and 22-kDa IGFBPs are co-orthologs of IGFBP-1, termed IGFBP-1a and -1b, respectively. They are induced under catabolic conditions such as stress and fasting but their responses are somewhat different, with IGFBP-1b being the most sensitive of the two. Cortisol stimulates production and secretion of these IGFBP-1 subtypes while, unlike in mammals, insulin may not be a primary suppressor. Salmon 41-kDa IGFBP, a major carrier of IGF-I, is not IGFBP-3, as might be expected extrapolating from mammals, but is in fact IGFBP-2b. Salmon IGFBP-2b levels in plasma are high when fish are fed, and GH treatment increases its circulating levels similar to mammalian IGFBP-3. These findings suggest that salmon IGFBP-2b acquired the role and regulation similar to mammalian IGFBP-3. Multiple replications of fish IGFBPs offer a unique opportunity to investigate molecular evolution of IGFBPs.

Federal seafood safety response to the Deepwater Horizon oil spill.

Following the 2010 Deepwater Horizon oil spill, petroleum-related compounds and chemical dispersants were detected in the waters of the Gulf of Mexico. As a result, there was concern about the risk to human health through consumption of contaminated seafood in the region. Federal and Gulf Coast State agencies worked together on a sampling plan and analytical protocols to determine whether seafood was safe to eat and acceptable for sale in the marketplace. Sensory and chemical methods were used to measure polycyclic aromatic hydrocarbons (PAHs) and dispersant in >8,000 seafood specimens collected in federal waters of the Gulf. Overall, individual PAHs and the dispersant component dioctyl sodium sulfosuccinate were found in low concentrations or below the limits of quantitation. When detected, the concentrations were at least two orders of magnitude lower than the level of concern for human health risk. Once an area closed to fishing was free of visibly floating oil and all sensory and chemical results for the seafood species within an area met the criteria for reopening, that area was eligible to be reopened. On April 19, 2011 the area around the wellhead was the last area in federal waters to be reopened nearly 1 y after the spill began. However, as of November 9, 2011, some state waters off the Louisiana coast (Barataria Bay and the Delta region) remain closed to fishing.

Circulating salmon 28- and 22-kDa insulin-like growth factor binding proteins (IGFBPs) are co-orthologs of IGFBP-1.

Circulating insulin-like growth factor binding proteins (IGFBPs) play pivotal roles in stabilizing IGFs and regulating their availability to target tissues. In the teleost circulation, three major IGFBPs are typically detected by ligand blotting with molecular masses around 20-25, 28-32 and 40-45kDa. However, their identity is poorly established and often confused. We previously identified salmon 22- and 41-kDa forms as IGFBP-1 and -2b, respectively. In the present study, we cloned the cDNA of 28-kDa IGFBP from Chinook salmon (Oncorhynchus tshawytscha) as well as rainbow trout (Oncorhynchus mykiss) based on the partial N-terminal amino acid sequence of purified protein and identified it as an ortholog of IGFBP-1. Structural and phylogenetic analyses revealed that the 28-kDa IGFBP is more closely related to human IGFBP-1 and zebrafish IGFBP-1a than the previously identified salmon IGFBP-1 (i.e. 22-kDa IGFBP). We thus named salmon 28- and 22-kDa forms as IGFBP-1a and -1b, respectively. Salmon IGFBP-1a contains a potential PEST region involved in rapid protein turnover and phosphorylation sites typically found in mammalian IGFBP-1, although the PEST and phosphorylation scores are not as high as those of human IGFBP-1. There was a striking difference in tissue distribution patterns between subtypes; Salmon igfbp-1a was expressed in a variety of tissues while igfbp-1b was almost exclusively expressed in the liver, suggesting that IGFBP-1a has more local actions. Direct seawater exposure (osmotic stress) of Chinook salmon parr caused increases in both IGFBP-1s in plasma, while IGFBP-1b appeared to be more sensitive. The presence of two co-orthologs of IGFBP-1 in the circulation in salmon, and most likely in other teleosts, provides a good opportunity to investigate subfunction partitioning of duplicated IGFBP-1 during postnatal growth.

Circulating salmon 41-kDa insulin-like growth factor binding protein (IGFBP) is not IGFBP-3 but an IGFBP-2 subtype.

In vertebrates, most circulating insulin-like growth factor (IGF) is bound to multiple forms of IGF-binding proteins (IGFBPs) that differ both structurally and functionally. In mammals, the largest reservoir of IGF in the circulation comes from a large (150kDa) ternary complex comprised of IGF bound to IGFBP-3, which is bound to an acid label subunit (ALS), and this variant of IGFBP is regulated by growth hormone (GH) and feed intake. Although multiple variants of IGFBPs ranging from 20 to 50kDa have been found in fishes, no ternary complex is present and it has been assumed that the majority of circulating IGF is bound to fish IGFBP-3. Consistent with this assumption is previous work in salmon showing the presence of a 41-kDa IGFBP that is stimulated by GH, decreases with fasting and increases with feeding. However, the hypothesis that the salmon 41-kDa IGFBP is structurally homologous to mammalian IGFBP-3 has not been directly tested. To address this issue, we cloned cDNAs for several Chinook salmon IGFBPs, and found that the cDNA sequence of the 41-kDa IGFBP is most similar to that of mammalian IGFBP-2 and dissimilar to IGFBP-3. We found an additional IGFBP (termed IGFBP-2a) with high homology to mammalian IGFBP-2. These results demonstrate that salmon 41-kDa IGFBP is not IGFBP-3, but a paralog of IGFBP-2 (termed IGFBP-2b). Salmon IGFBP-2s are also unique in terms of having potential N-glycosylation sites and splice variants. Additional research on non-mammalian IGFBPs is needed to fully understand the molecular/functional evolution of the IGFBP family and the significance of the ternary complex in vertebrates.

The pituitary-thyroid axis during the parr-smolt transformation of Coho salmon, Oncorhynchus kisutch: quantification of TSH ß mRNA, TSH, and thyroid hormones.

The objective of this investigation was to quantify pituitary thyroid stimulating hormone (TSH) ß mRNA, pituitary and plasma TSH and plasma thyroid hormone levels during the parr-smolt transformation of Coho salmon that occurs in spring from February to May. The status of the pituitary-thyroid axis was assessed using an RNase protection assay for pituitary TSH ß mRNA and radioimmunoassays for salmon pituitary and plasma TSH and thyroid hormones. TSH ß mRNA was highest during late winter (February) (4.9 pg/µg DNA) and gradually declined during spring (2.3 pg/µg DNA). In contrast, pituitary and plasma TSH levels showed a small, but statistically non-significant change during smoltification. Despite minimal change in plasma TSH levels, characteristically large increases in plasma T4 (January-3.3 ng/ml to April-10.2 ng/ml) and significant, but modest increases in plasma T3 (February-2.4 ng/ml to April-5.8 ng/ml) were observed. Regression analysis showed a significant positive relationship between plasma T4 and T3 and negative relationship between plasma T3 and pituitary TSH ß mRNA. However, all other relations were not significant. These data suggest a significant role for peripheral regulation (i.e. T4-T3 conversion, change in tissue sensitivity, hormone degradation rate) as well as evidence of central regulation via negative feedback at the level of the pituitary gland in regulation of thyroid activity in salmon. Furthermore, the increased thyroid sensitivity to TSH (shown previously), in the face of relatively constant plasma TSH levels, may be the major factor responsible for the increased thyroid activity observed during smoltification.

Postprandial changes in plasma growth hormone, insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins in coho salmon fasted for varying periods.

We examined postprandial changes in circulating growth hormone (GH), insulin, insulin-like growth factor (IGF)-I, and IGF-binding proteins (IGFBPs) in yearling coho salmon under different feeding regimes. Fish were initially fasted for 1 day, 1 wk, or 3 wk. Fasted fish were then fed, and blood was collected at 4-h intervals over 26 h. After the various periods of fasting, basal levels of insulin were relatively constant, whereas those of IGF-I, IGFBPs and GH changed in proportion to the duration of the fast. A single meal caused a rapid, large increase in the circulating insulin levels, but the degree of the increase was influenced by the fasting period. IGF-I showed a moderate increase 2 h after the meal but only in the regularly fed fish. Plasma levels of 41-kDa IGFBP were increased in all groups within 6 h after the single meal. The fasting period did not influence the response of 41-kDa IGFBP to the meal. IGFBP-1 and GH decreased after the meal to the same extent among groups regardless of the fasting period. The present study shows that insulin and IGF-I respond differently to long (weeks)- and short (hours)-term nutritional changes in salmon; insulin maintains its basal level but changes acutely in response to food intake, whereas IGF-I adjusts its basal levels to the long-term nutritional status and is less responsive to acute nutritional input. IGFBPs maintain their sensitivity to food intake, even after prolonged fasting, suggesting their critical role in the nutritional regulation of salmon growth.

Measurement of circulating salmon IGF binding protein-1: assay development, response to feeding ration and temperature, and relation to growth parameters.

Fish plasma/serum contains multiple IGF binding proteins (IGFBPs), although their identity and physiological regulation are poorly understood. In salmon plasma, at least three IGFBPs with molecular masses of 22, 28 and 41 kDa are detected by Western ligand blotting. The 22 kDa IGFBP has recently been identified as a homolog of mammalian IGFBP-1. In the present study, an RIA for salmon IGFBP-1 was established and regulation of IGFBP-1 by food intake and temperature, and changes in IGFBP-1 during smoltification, were examined. Purified IGFBP-1 from serum was used for as a standard, for tracer preparation and for antiserum production. Cross-linking (125)I-labelled IGFBP-1 with salmon IGF-I eliminated interference by IGFs. The RIA had little cross-reactivity with salmon 28 and 41 kDa IGFBPs (< 0.5%) and measured IGFBP-1 levels as low as 0.1 ng/ml. Fasted fish had significantly higher IGFBP-1 levels than fed fish (21.6 +/- 4.6 vs 3.0 +/- 2.2 ng/ml). Plasma IGFBP-1 was measured in individually tagged 1-year-old coho salmon reared for 10 weeks under four different feeding regimes as follows: high constant (2% body weight/day), medium constant (1% body weight/day), high variable (2% to 0.5% body weight/day) and medium variable (1% to 0.5% body weight/day). Fish fed with the high ration had lower IGFBP-1 levels than those fed with the medium ration. Circulating IGFBP-1 increased following a drop in feeding ration to 0.5% and returned to the basal levels when feeding ration was increased. Another group of coho salmon were reared for 9 weeks under different water temperatures (11 or 7 degrees C) and feeding rations (1.75, 1 or 0.5% body weight/day). Circulating IGFBP-1 levels were separated by temperature during the first 4 weeks; a combined effect of temperature and feeding ration was seen in week 7; only feeding ration influenced IGFBP-1 level thereafter. These results indicate that IGFBP-1 is responsive to moderate nutritional and temperature changes. There was a clear trend that circulating IGFBP-1 levels were negatively correlated with body weight, condition factor (body weight/body length(3) x 100), growth rates and circulating 41 kDa IGFBP levels but not IGF-I levels. During parr-smolt transformation of coho salmon, IGFBP-1 levels showed a transient peak in late April, which was opposite to the changes in condition factor. Together, these findings suggest that salmon IGFBP-1 is inhibitory to IGF action. In addition, IGFBP-1 responds to moderate changes in dietary ration and temperature, and shows a significant negative relationship to condition factor.

Growth hormone and insulin-like growth factors in fish: where we are and where to go.

This communication summarizes viewpoints, discussion, perspectives, and questions, put forward at a workshop on "Growth hormone and insulin-like growth factors in fish" held on September 7th, 2004, at the 5th International Symposium on Fish Endocrinology in Castellon, Spain.

Identification of the salmon somatolactin receptor, a new member of the cytokine receptor family.

Somatolactin (SL) is a pituitary hormone of the GH/prolactin (PRL) family that so far has been found only in fish. Compared with GH and PRL, the primary structure of SL is highly conserved among divergent fish species, suggesting it has an important function and a discriminating receptor that constrains structural change. However, SL functions are poorly understood, and receptors for SL have not yet been identified. During cloning of GH receptor cDNA from salmon, we found a variant with relatively high (38-58%) sequence identity to vertebrate GH receptors and low (28-33%) identity to PRL receptors; however, the recombinant protein encoding the extracellular domain showed only weak binding of GH. Ligand binding of the recombinant extracellular domain for this receptor confirmed that the cDNA encoded a specific receptor for SL. The SL receptor (SLR) has common features of a GH receptor including FGEFS motif, six cysteine residues in the extracellular domain, a single transmembrane region, and Box 1 and 2 regions in the intracellular domain. These structural characteristics place the SLR in the cytokine receptor type I homodimeric group, which includes receptors for GH, PRL, erythropoietin, thrombopoietin, granulocyte-colony stimulating factor, and leptin. Transcripts for SLR were found in 11 tissues with highest levels in liver and fat, supporting the notion that a major function of SL is regulation of lipid metabolism. Cloning SLR cDNA opens the way for discovery of new SL functions and target tissues in fish, and perhaps novel members of this receptor family in other vertebrates.

Androgen effects on plasma GH, IGF-I, and 41-kDa IGFBP in coho salmon (Oncorhynchus kisutch).

Among many species of salmonids, fast growing fish mature earlier than slow growing fish, and maturing males grow faster than non-maturing ones. To study the potential endocrine basis for this reciprocal relationship we examined the in vivo effects of the androgens, testosterone (T) and 11-ketotestosterone (11-KT), on plasma growth hormone (GH), insulin-like growth factor-I (IGF-I) and 41-kDa IGF binding protein (41-kDa IGFBP) (putative IGFBP-3) in coho salmon, Oncorhynchus kisutch. Immature male and female, two-year old fish (avg. wt. 31.7 +/- 0.63 g) were injected with coconut oil containing T or 11-KT at a dose of 0.1, 0.25, or 1 microg/g body weight. Blood samples were taken 1 and 2 weeks postinjection, and analyzed by immunoassay for T, 11-KT, GH, IGF-I, and 41-kDa IGFBP. Steroid treatments elevated the plasma T and 11-KT levels to physiological ranges typical of maturing fish. Plasma IGF-I and 41-kDa IGFBP levels increased in response to both T and 11-KT in a significant and dose-dependent manner after 1 and 2 weeks, but GH levels were not altered. These data suggest that during reproductive maturation, in addition to the previously demonstrated effects of the IGFs on steroidogenesis, the gonadal steroids may in turn play a significant role in regulating IGF-I and its binding proteins in fish. The interaction between the reproductive and growth axes may provide a regulatory mechanism for bringing about the dimorphic growth patterns observed between maturing and non-maturing salmonids and other species of fish.

Salmon growth hormone receptor: molecular cloning, ligand specificity, and response to fasting.

To better understand the role of growth hormone in regulating fish growth, the cDNA of growth hormone receptor (GHR) was cloned from the liver of masu salmon (Oncorhynchus masou) and characterized. The masu salmon GHR (msGHR) sequence revealed common features of a GHR, including a (Y/F)GEFS motif in the extracellular domain, a single transmembrane region, and Box 1 and Box 2 in the intracellular domain. However, the amino acid sequence identity was low (49%) compared to GHRs of other vertebrates including seven teleosts, and the putative msGHR protein lacked one pair of cysteine residues in the extracellular domain. To verify the identity of the msGHR, the recombinant protein of the extracellular domain was expressed with a histidine tag protein (His-msGHR-ECD), refolded and purified for analysis of its ligand specificity. In competition experiments, the specific binding between His-msGHR-ECD and radioiodine-labeled salmon GH was displaced completely by only salmon GH, and not by salmon prolactin or somatolactin. A real-time RT-PCR assay was used to measure salmon GHR mRNA in the liver of fed and fasted coho salmon (Oncorhynchus kisutch). The levels of hepatic GHR mRNA were lower in fasted fish compared to fed fish after 3 weeks, suggesting that GHR gene expression is reduced following a long-term fast. These results confirm the identity of the salmon GHR based on ligand specificity and response to fasting.

Disparate regulation of insulin-like growth factor-binding proteins in a primitive, ictalurid, teleost (Ictalurus punctatus).

Vertebrate growth is principally controlled by growth hormone (GH) and, its intermediary, insulin-like growth factor-I (IGF-I). The actions of IGF-I are modulated by high-affinity binding proteins called insulin-like growth factor binding-proteins (IGFBPs). Channel catfish exhibit atypical responses (increased percentage body fat and reduced percentage protein) to GH treatment, despite GH-dependent IGF-I production. Among possible explanations for this atypical response to GH treatment is an unusual regulation of blood IGFBPs. In this species, there has been one report of a single 33-kDa plasma binding protein. To examine the occurrence and regulation of plasma IGFBPs in this species, two strains of channel catfish (Norris and USDA-103) were treated with weekly injections of recombinant bovine GH at different temperatures (21 degrees C versus 26 degrees C). In a separate experiment involving catfish of a different strain, endogenous GH levels were altered via injection of the GH secretagogue, bGHRH(1-29)-amide, and held in fresh water or transferred to brackish water (12 ppt). Following these treatments, the type and regulation of plasma IGFBPs in these catfish strains were examined by Western ligand blotting. We have identified five IGFBPs (19, 35, 44, 47, and >80 kDa) in catfish plasma that are differentially altered by experimental treatment and genetic lineage. Levels of the 19-kDa IGFBP were elevated in catfish of Norris and USDA-103 strains that were exposed to a higher environmental temperature (26 degrees C versus 21 degrees C), but was not seen in those animals used for the GH secretagogue/salinity study. In most vertebrates, treatment with GH increases levels of plasma IGFBP-3 (approximately 40-50 kDa). In the USDA-103 and Norris catfish strains, bGH injection reduced plasma levels of the 44- and 47-kDa IGFBPs. Similarly, elevations in plasma GH levels in GH secretagogue-treated and brackish water-adapted catfish resulted in reductions of the 44- and 47-kDa IGFBPs as well as a reduction in presence of a 35-kDa IGFBP that was not detected in the Norris or USDA-103 strains. Reduced levels of the 35, 44, and 47 kDa IGFBPs, seen in the plasma of the GH secretagogue-treated and brackish water-adapted animals, suggests that the atypical response of channel catfish to GH treatment is not attributed to the use of heterologous (bovine) GH. This negative response of the 35-47 kDa IGFBPs to GH has not been reported in any teleost or vertebrate (healthy) and may be partly responsible for the atypical physiological responses of channel catfish to GH treatment.

Purification of a 41-kDa insulin-like growth factor binding protein from serum of chinook salmon, Oncorhynchus tshawytscha.

In salmon, at least three insulin-like growth factor binding proteins (IGFBPs) with molecular masses of 41, 28, and 22kDa exist in serum. The 41-kDa IGFBP is up-regulated by growth hormone treatment and down-regulated by fasting, suggesting that it is a homolog of IGFBP-3. We purified the 41-kDa IGFBP from chinook salmon serum by IGF-I affinity chromatography followed by reversed-phase high pressure liquid chromatography. Purified IGFBP appeared as doublet bands on electrophoresis and was N-glycosylated. Analysis of partial N-terminal amino acid sequence revealed that salmon 41-kDa IGFBP has the cysteine rich domain conserved among IGFBP family. In a binding assay using 125I-salmon IGF-I, purified 41-kDa IGFBP specifically bound salmon IGF-I, human IGF-I and human IGF-II, but neither Long R(3)IGF-I nor salmon insulin, showing that binding characteristics of the salmon IGFBP are similar to those of mammalian IGFBPs. Although the partial amino acid sequence of 41-kDa IGFBP showed highest homologies with zebrafish and seabream IGFBP-2, the highly conserved nature of the N-terminus makes it impossible to identify the type of IGFBP from partial sequence data. However, based on physiological responses, molecular weight and type of glycosylation, the 41-kDa IGFBP is most similar to mammalian IGFBP-3.

Central administration of corticotropin-releasing hormone stimulates locomotor activity in juvenile chinook salmon (Oncorhynchus tshawytscha).

This study evaluated the effect of corticotropin-releasing hormone (CRH) on locomotor activity, habitat choice, and social behavior in juvenile spring chinook salmon (Oncorhynchus tshawytscha). An intracerebroventricular (ICV) injection of CRH caused a dose-dependent increase in locomotor activity. The stimulatory effect of exogenous CRH on locomotor activity lasted for at least 24 h. Injection (ICV) of a peptide antagonist of CRH, alpha-helical CRH(9-41) (ahCRH), prevented the increase in locomotor activity when administered concurrently with CRH. Furthermore, fish administered the antagonist alone had significantly lower locomotor activity levels compared to saline-injected control fish. The effects of CRH are often dependent on the social context. However, no evidence was found that the presence of conspecifics during the testing procedure affected locomotor activity following ICV injections of CRH. Similarly, ICV injections of CRH or ahCRH did not have a significant effect on the mean time spent in contact with a conspecific. However, the position of fish in the tank was affected by the treatments. ICV injections of CRH significantly increased the amount of time that fish spent near the center of the tank. Furthermore, ICV injections of ahCRH significantly increased the mean time taken for fish to find cover in the tank. The effect of CRH and ahCRH on locomotor activity was not related to changes in plasma cortisol or thyroxine. These results support the hypothesis that endogenous CRH within the central nervous system is involved in the stimulation of locomotor activity in fish. Furthermore, CRH may also alter habitat choice in a novel environment.