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.     

Time course of the GH/IGF axis response to fasting and increased ration in chinook salmon (Oncorhynchus tshawytscha)

Body growth in vertebrates is chiefly regulated by the GH/IGF axis. Pituitary growth hormone (GH) stimulates liver insulin-like growth factor-I (IGF-I) production. During fasting, plasma IGF-I levels decline due to the development of liver GH resistance, while GH levels generally increase. In mammals, decreased insulin during fasting is thought to cause liver GH resistance. However, the sequence of events in the GH/IGF axis response to fasting is not well characterized, especially in non-mammalian vertebrates. We assessed the time course of the GH/IGF axis response to fasting and increased ration in chinook salmon. Fish were placed on Fasting, Increased, or Control rations, and sampled daily for 4 days and at more widely spaced intervals through 29 days. Plasma IGF-I, GH, insulin, and 41 kDa IGF binding protein (putative salmon IGFBP-3), and liver IGF-I gene expression were measured. Control and Increased ration fish did not differ strongly. Plasma IGF-I and 41 kDa IGFBP were significantly lower in Fasted versus Control fish from day 4 onward, and liver IGF-I gene expression was significantly lower from day 6 onward. Liver IGF-I gene expression and plasma IGF-I levels were correlated. Plasma insulin was lower in Fasted fish from day 6 onward. There was a trend toward increased GH in Fasted fish on days 1-2, and GH was significantly increased Fasted fish from day 3 onward. Fasted GH first increased (days 1-3) to a plateau of 10-20 ng/ml (days 4-12) and then increased dramatically (days 15-29), suggesting that the GH response to fasting had three phases. The early increase in GH, followed by the decrease in plasma IGF-I after 4 days, suggests that GH resistance developed within 4 days.     

Free and protein-bound insulin-like growth factor-I (IGF-I) and IGF-binding proteins in plasma of coho salmon, Oncorhynchus kisutch.

Total and free insulin-like growth factor-I (IGF-I) levels were quantified in plasma from growth hormone (GH)-treated and fasted coho salmon. Total IGF-I was measured by radioimmunoassay after acid-ethanol extraction and free IGF-I was separated from protein-bound IGF-I using ultrafiltration by centrifugation. Total and free IGF-I increased in plasma after GH treatment and decreased after fasting. The level of free IGF-I, however, was maintained at approximately 0.3% in both experiments. Unsaturated binding activity in plasma for IGF-I was assessed by incubation with (125)I-recombinant salmon IGF-I ((125)I-sIGF-I). Although there was no difference in binding activity between GH-treated and control fish, fasted fish showed higher binding activity than did fed fish, suggesting induction of unsaturated binding protein by fasting. IGF-binding protein (IGFBP) bands were observed in plasma of coho salmon by Western ligand blotting using (125)I-sIGF-I. A low-molecular-weight (22 kDa) band was clear in fasted fish but not detectable in fed fish. The IGFBP band, which has molecular weight similar to that of human IGFBP-3 (41 kDa), was more intense in GH-treated fish than in controls. The molecular distribution of IGF-I in plasma was examined by gel filtration under neutral conditions. Most IGF-I was eluted around 40 kDa. This result suggests that the major form of bound IGF-I in the circulation of coho salmon may be in a 40-kDa binary complex rather than in a 150-kDa ternary complex, as in mammals.     

Effects of fasting on growth hormone, growth hormone receptor, and insulin-like growth factor-I axis in seawater-acclimated tilapia, Oreochromis mossambicus

Effects of fasting on the growth hormone (GH)--growth hormone receptor (GHR)-insulin-like growth factor-I (IGF-I) axis were characterized in seawater-acclimated tilapia (Oreochromis mossambicus). Fasting for 4 weeks resulted in significant reductions in body weight and specific growth rate. Plasma GH and pituitary GH mRNA levels were significantly elevated in fasted fish, whereas significant reductions were observed in plasma IGF-I and hepatic IGF-I mRNA levels. There was a significant negative correlation between plasma levels of GH and IGF-I in the fasted fish. No effect of fasting was observed on hepatic GHR mRNA levels. Plasma glucose levels were reduced significantly in fasted fish. The fact that fasting elicited increases in GH and decreases in IGF-I production without affecting GHR expression indicates a possible development of GH resistance.     

Resolving the growth-promoting and metabolic effects of growth hormone: Differential regulation of GH-IGF-I system components

Growth hormone regulates numerous processes in vertebrates including growth promotion and lipid mobilization. During periods of food deprivation, growth is arrested yet lipid depletion is promoted. In this study, we used rainbow trout on different nutritional regimens to examine the regulation of growth hormone (GH)-insulin-like growth factor-I (IGF-I) system elements in order to resolve the growth-promoting and lipid catabolic actions of GH. Fish fasted for 2 or 6 weeks displayed significantly reduced growth compared to their fed counterparts despite elevated plasma GH, while refeeding for 2 weeks following 4 weeks of fasting partially restored growth and lowered plasma GH. Fish fasted for 6 weeks also exhausted their mesenteric adipose tissue reserves. Sensitivity to GH in the liver was reduced in fasting fish as evidenced by reduced expression of GH receptor type 1 (GHR 1) and GHR 2 mRNAs and by reduced (125)I-GH binding capacity. Expression of GHR 1 and GHR 2 mRNAs also was reduced in the gill of fasted fish. In adipose tissue, however, sensitivity to GH, as indicated by GHR 1 expression and by (125)I-GH binding capacity, increased after 6 weeks of fasting in concert with the observed lipid depletion. Fasting-associated growth retardation was accompanied by reduced expression of total IGF-I mRNA in the liver, adipose and gill, and by reduced plasma levels of IGF-I. Sensitivity to IGF-I was reduced in the gill of fasted fish as indicated by reduced expression of type 1 IGF-I receptor (IGFR 1A and IGFR 1B) mRNAs. By contrast, fasting did not affect expression of IGFR 1 mRNAs or (125)I-IGF-I binding in skeletal muscle and increased expression of IGFR 1 mRNAs and (125)I-IGF-I binding in cardiac muscle. These results indicate that nutritional state differentially regulates GH-IGF-I system components in a tissue-specific manner and that such alterations disable the growth-promoting actions of GH and promote the lipid-mobilizing actions of the hormone.     

Response of the somatotropic axis of juvenile coho salmon to alterations in plane of nutrition with an analysis of the relationships among growth rate and circulating IGF-I and 41 kDa IGFBP

The effect of different feeding levels on plasma levels of insulin-like growth factor-I (IGF-I), 41 kDa insulin-like growth factor binding protein (41 kDa IGFBP), and growth hormone (GH) were assessed in post-smolt coho salmon. Fish were fed at either stable (1 and 2% body weight/day) or varying (1-0.5-1%, 2-0.5-2% body weight/day) feeding rates and plasma was sampled from 10 fish/treatment at 2-3 week intervals over five dates from June to September, resulting in a total of 200 samples. Fish fed at higher rates grew faster and had higher plasma IGF-I and 41 kDa IGFBP levels. Plasma GH levels were variable but generally showed an inverse relationship to feeding rate. Both plasma IGF-I and 41 kDa IGFBP increased seasonally, average IGF-I levels doubled from June to September, regardless of feeding rate. On any one date both IGF-I and 41 kDa IGFBP were highly related to growth rate with regression coefficients ranging from 0.36 to 0.68 (IGF-I) and from 0.33 to 0.70 (41 kDa IGFBP). No relationship was found between IGF-I:41 kDa IGFBP ratio and individual growth rate. Overall, both feeding rate and date were important in explaining variation in IGF-I and 41 kDa IGFBP levels.     

Changes in mRNA expression of grouper (Epinephelus coioides) growth hormone and insulin-like growth factor I in response to nutritional status

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) are key links to nutritional condition and growth regulation in teleost. To understand the endocrine mechanism of growth regulation in grouper, we cloned the cDNAs for grouper GH and IGF-I and examined their mRNA expression during different nutritional status. Grouper GH cDNA is 936 base pairs (bp) long excluding the poly-A tail. It contained untranslated regions of 85 and 231bp in the 5'- and 3'-ends, respectively. It has an open reading frame of 612bp coding for a signal peptide of 17 amino acids (aa) and a mature hormone of 187aa residues. Based on the aa sequence of the mature hormone, grouper GH shows higher sequence identity (>76%) to GHs of perciforms than to GHs of cyprinids and salmonids (53-69%). Grouper preproIGF-I cDNA consisted of 558bp, which codes for 186aa. This is composed of 44aa for the signal peptide, 68aa for the mature peptide comprising B, C, A, and D domains, and 74aa for the E domain. Mature grouper IGF-I shows very high sequence identity to IGF-I of teleost fishes (84-97%) compared to advanced groups of vertebrates such as chicken, pig, and human (80%). Using DNA primers specific for grouper GH and IGF-I, the changes in mRNA levels of pituitary GH and hepatic IGF-I in response to starvation and refeeding were examined by a semi-quantitative RT-PCR. Significant elevation of GH mRNA level was observed after 2 weeks of food deprivation, and increased further after 3 and 4 weeks of starvation. GH mRNA level in fed-controls did not change significantly during the same period. Hepatic IGF-I mRNA level decreased significantly starting after 1 week of starvation until the 4th week. There was no significant change in IGF-I mRNA levels in fed-controls. One week of refeeding can restore the GH and IGF-I mRNA back to its normal levels. Deprivation of food for 1-4 weeks also resulted in cessation of growth and decrease in condition factor.     

mRNA expression patterns for GH, PRL, SL, IGF-I and IGF-II during altered feeding status in rabbitfish, Siganus guttatus

Feeding time is a major synchronizer of many physiological rhythms in many organisms. Alteration in the nutritional status, specifically fasting, also affects the secretion rhythms of growth hormone (GH) and insulin-like growth factor-I (IGF-I). In this study, we investigated whether the expression patterns for the mRNAs of GH, prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor I and II (IGF-I and IGF-II) in the liver of juvenile rabbitfish (Siganus guttatus) follow a rhythm according to feeding time and whether these hormone rhythms changes with starvation. Hormone mRNA levels were determined by real time PCR. The daily expression pattern for the mRNAs of GH, PRL and SL was not altered whether food was given in the morning (10:00 h) or in the afternoon (15:00 h). The daily GH mRNA expression pattern, however, was affected when food was not available for 3 days. In contrast, the daily expression pattern for IGF-I mRNA reaches its peak at roughly 5-6h after feeding. This pattern, however, was not observed with IGF-II mRNA. During 15-day starvation, GH mRNA levels in starved fish were significantly higher than the control fish starting on the 9th day of starvation until day 15. The levels returned to normal after re-feeding. In contrast to GH, PRL mRNA levels in starved fish were significantly lower than the control group starting on the 6th day of starvation until 3 days after re-feeding. SL mRNA levels were not significantly different between the control and starved group at anytime during the experiment. Both IGF-I and IGF-II mRNA levels in starved group were significantly higher than the control fish on the 3rd and 6th day of starvation. mRNA levels of both IGF-I and II in the starved fish decreased starting on the 9th day of starvation. While IGF-I mRNA levels in the starved group continued to decrease as starvation progressed, IGF-II mRNA levels were not significantly different from the control during the rest of the starvation period. The results indicate that aside from GH and IGF-I, PRL and IGF-II are likewise involved in starvation in rabbitfish.     

The effect of temperature, stress, and cortisol on plasma IGF-I and IGFBPs in sunshine bass

The mechanisms through which stress and cortisol regulate insulin like growth factor-I (IGF-I) and insulin like growth factor binding proteins (IGFBPs) were studied in sunshine bass, by measuring plasma IGF-I and IGFBPs in fish maintained at 5, 10, 15, 20, 25, or 30 degrees C, fish subjected to an acute 15 min confinement stress at 25 and 30 degrees C, and fish fed 100 mg cortisol/kg feed. Plasma IGF-I concentrations were higher at 25 and 30 degrees C than at 20 degrees C and below. A 15 min confinement stress resulted in a decrease in IGF-I 2h post-confinement. Plasma concentrations of IGFBP with molecular weights of 24, 28, and 33 kDa were similar for fish acclimated to different temperatures, except for 5 degrees C where a 33-kDa IGFBP was significantly reduced. After a 15 min low-water stress at 25 degrees C, a 33-kDa IGFBP was reduced and IGFBPs with molecular weights of 24 and 28 kDa were increased at 2 and 6h, respectively. A 15 min low-water stress at 30 degrees C, resulted in no change in levels of a 33-kDa IGFBP over the 6-h recovery period. However, levels of a 24- and 28-kDa IGFBP were significantly increased at 2 and 6h, respectively. A single feeding with 100 mg cortisol/kg feed increased plasma cortisol but did affect plasma concentrations of IGF-I or any of the three IGFBPs. Acute stress appears to result in a decrease in IGF-I, but the mechanism of the decrease does not appear to be caused by cortisol released during the stress.     

Endocrine effects of growth hormone overexpression in transgenic coho salmon

Non-transgenic (wild-type) coho salmon (Oncorhynchus kisutch), growth hormone (GH) transgenic salmon (with highly elevated growth rates), and GH transgenic salmon pair fed a non-transgenic ration level (and thus growing at the non-transgenic rate) were examined for plasma hormone concentrations, and liver, muscle, hypothalamus, telencephalon, and pituitary mRNA levels. GH transgenic salmon exhibited increased plasma GH levels, and enhanced liver, muscle and hypothalamic GH mRNA levels. Insulin-like growth factor-I (IGF-I) in plasma, and growth hormone receptor (GHR) and IGF-I mRNA levels in liver and muscle, were higher in fully fed transgenic than non-transgenic fish. GHR mRNA levels in transgenic fish were unaffected by ration-restriction, whereas plasma GH was increased and plasma IGF-I and liver IGF-I mRNA were decreased to wild-type levels. These data reveal that strong nutritional modulation of IGF-I production remains even in the presence of constitutive ectopic GH expression in these transgenic fish. Liver GHR membrane protein levels were not different from controls, whereas, in muscle, GHR levels were elevated approximately 5-fold in transgenic fish. Paracrine stimulation of IGF-I by ectopic GH production in non-pituitary tissues is suggested by increased basal cartilage sulphation observed in the transgenic salmon. Levels of mRNA for growth hormone-releasing hormone (GHRH) and cholecystokinin (CCK) did not differ between groups. Despite its role in appetite stimulation, neuropeptide Y (NPY) mRNA was not found to be elevated in transgenic groups.     

The role of the IGF-I system for vitellogenesis in maturing female sterlet, Acipenser ruthenus Linnaeus, 1758

Transition from previtellogeneic to vitellogenic oocyte growth is a critical phase for folliculogenesis in sturgeon and may often be postponed for several years. Recent findings on the involvement of insulin-like growth factor I (IGF-I) in cell differentiation processes of oocyte follicle and ovarian steroidogenesis of teleosts in vitro led to the hypothesis that paracrine IGF-I could function as a potential trigger in vivo. For the first time, IGF-I and its corresponding receptor (IGF-IR) were identified in a non-teleostean fish. Real-time PCR assays for IGF-I and IGF-IR mRNA were established, normalising mRNA expression of the target genes to beta-microglobulin (beta2m). We clearly show that expression of IGF-I in the gonad is a substantial source for IGF-I-mediated effects in follicles compared to liver, brain, muscle and adipose tissue. Among these tissues, IGF-IR mRNA was highest in the gonad. With regard to different cohorts of coexisting follicles, highest expression of IGF-I and IGF-IR were met in developing follicles, indicating that IGF-I functions as an intraovarian modulator of follicle faith. Comparing previtellogenic follicles in females that matured within two years with non-maturing females f the same age, revealed an increases of 2.3-fold for IGF-I and 2.8-fold for IGF-IR mRNA expression in maturing females. These findings implicate an important role of paracrine IGF-I in early vitellogenesis and identify it as candidate vitellogenesis inducing factor (VIF), determining the faith of the follicle.     

Effect of GH and IGF-I treatment on reproduction,growth, and plasma hormone concentrations in domestic nutria (Myocastor coypus)

The role of GH and IGF-I in the control of reproduction, growth, and hormone secretion in domestic nutria was examined. In the first series of experiments, we studied the effects of single and multiple (daily for 20 days) injections of recombinant hGH (15 microg/animal) on plasma triiodothyronine (T3), thyroxine (T4), and progesterone (P) concentrations, as well as on the duration of pregnancy (time between start of mating and birth of pups), number of pups born, and body weight of adult females and their newborn pups. In the second series of experiments, the effects of single and multiple (daily for 28 days) injections of recombinant hIGF-I (1 microg/animal) on plasma IGF-I, IGFBP-3, T3, T4 concentrations, the duration of pregnancy, and number of offspring delivered were assessed. It was found that either single or multiple GH treatment resulted in significant increase in plasma T3, T4, but not P concentration. Furthermore, it significantly increased the body weight of adults and newborn pups. No influence of GH on the duration of pregnancy and the number of offspring was observed. IGF-I treatment caused an increase in plasma IGF-I concentration, a reduction in plasma IGFBP-3, T3, and T4 concentrations, and a shorter duration of pregnancy but did not alter the number of pups delivered. Our observations suggest that GH and IGF-I may be involved in the control of hormone secretion, growth, and reproduction in domestic nutria. Reproductive processes are controlled by IGF-I rather than by GH, whilst GH may be involved in the stimulation of prenatal and postnatal growth. The differential effects of these substances on thyroid hormones and reproductive parameters suggest that the actions of GH on these processes are probably not mediated by IGF-I.     

Developmental changes in growth hormone, insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in plasma of young growing pigs.

The relationship between plasma concentrations of normally secreted GH and insulin-like growth factor-I (IGF-I) was investigated in pigs after weaning. Frequent blood sampling for between 12 and 24 h showed that plasma GH was pulsatile in pigs of 10, 20 and 35 kg liveweight. Pulses were brief in duration, low in amplitude and variable in frequency. Basal and average daily plasma concentrations of GH changed significantly with development, increasing by about 50% between 10 and 20 kg liveweight. Concentrations of IGF-I in plasma showed little or no evidence of diurnal periodicity and were not increased by GH pulses. Average daily concentrations of both IGF-I and IGF-II in plasma progressively increased between 10 and 35 kg liveweight, as did the total desaturated IGF-binding protein (IGFBP) activity of plasma. A strong positive correlation was observed between the total concentration of IGFs (IGF-I plus IGF-II) in the circulation and plasma IGFBP activity. The developmental rise in IGFBP activity of plasma was associated with increased labelling with 125I-labelled human IGF-II in ligand blots of binding proteins of apparent molecular masses greater than 200, 50, 43 and 29 kDa. One class of binding proteins of 34.5 kDa decreased with development. This study of young growing pigs shows that normally secreted endogenous GH exerts no significant immediate control over plasma IGF-I concentrations, and that plasma levels of IGF-I and IGF-II increase with maturation in this species.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the growth hormone/insulin-like growth factor axis by treatment with 17 alpha-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.     

Tissue-specific regulation of the growth hormone/insulin-like growth factor axis during fasting and re-feeding: Importance of muscle expression of IGF-I and IGF-II mRNA in the tilapia

The effects of prolonged nutrient restriction (fasting) and subsequent restoration (re-feeding) on the growth hormone (GH)/insulin-like growth factor (IGF) axis were investigated in the tilapia (Oreochromis mossambicus). Mean weight and specific growth rate declined within 1 week in fasted fish, and remained lower than controls throughout 4 weeks of fasting. Plasma levels of IGF-I were lower than fed controls during 4 weeks of fasting, suggesting a significant catabolic state. Following re-feeding, fasted fish gained weight continuously, but did not attain the weight of fed controls at 8 weeks after re-feeding. Specific growth rate increased above the continuously-fed controls during the first 6 weeks of re-feeding, clearly indicating a compensatory response. Plasma IGF-I levels increased after 1 week of re-feeding and levels were not otherwise different from fed controls. Plasma GH levels were unaffected by either fasting or re-feeding. No consistent effect of fasting or re-feeding was observed on liver expression of GH receptor (GH-R), somatolactin (SL) receptor (SL-R), IGF-I or IGF-II. In contrast, muscle expression of GH-R increased markedly during 4 weeks of fasting, and then declined below control levels upon re-feeding for weeks 1 and 2. Similarly, muscle expression of SL-R increased after 4 weeks of fasting, and reduced below control levels after 1 and 2 weeks of re-feeding. On the other hand, muscle expression of IGF-I was strongly reduced throughout the fasting period, and levels recovered 2 weeks after re-feeding. Muscle expression of IGF-II was not affected by fasting, but was reduced after 1 and 2 weeks of re-feeding. These results indicate that GH/IGF axis, particularly muscle expression of GH-R, SL-R and IGF-I and -II, is sensitive to nutritional status in the tilapia.     

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 (150 kDa) 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 50 kDa 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.     

Production and characterization of recombinantly derived peptides and antibodies for accurate determinations of somatolactin, growth hormone and insulin-like growth factor-I in European sea bass (Dicentrarchus labrax)

A specific radioimmunoassay (RIA) for European sea bass (Dicentrarchus labrax) growth hormone (GH) was developed and validated. For this purpose, a stable source of GH was produced by means of recombinant DNA technology in a bacteria system. The identity of the purified protein (ion exchange chromatography) was demonstrated by Western blot and a specific GH antiserum was raised in rabbit. In Western blot and RIA system, this antiserum recognized specifically native and recombinant GH, and it did not cross-react with fish prolactin (PRL) and somatolactin (SL). In a similar way, a specific polyclonal antiserum against the now available recombinant European sea bass SL was raised and used in the RIA system to a sensitivity of 0.3 ng/ml (90% of binding of tracer). Further, European sea bass insulin-like growth factor-I (IGF-I) was cloned and sequenced, and its high degree of identity with IGF-I peptides of barramundi, tuna, and sparid fish allowed the use of a commercial IGF-I RIA based on barramundi IGF-I antiserum. These assay tools assisted for the first time accurate determinations of SL and GH-IGF-I axis activity in a fish species of the Moronidae family. Data values were compared to those found with gilthead sea bream (Sparus aurata), which is currently used as a Mediterranean fish model for growth endocrinology studies. As a characteristic feature, the average concentration year round of circulating GH in growing mature males of European sea bass was higher than in gilthead sea bream. By contrast, the average concentration of circulating SL was lower. Concerning to circulating concentration of IGF-I, the measured plasma values for a given growth rate were also lower in European sea bass. These findings are discussed on the basis of a different energy status that might allowed a reduced but more continuous growth in European sea bass.     

Daily expression patterns for mRNAs of GH, PRL, SL, IGF-I and IGF-II in juvenile rabbitfish, Siganus guttatus, during 24-h light and dark cycles

Most animals respond to changes in the external environment in a rhythmic fashion. In teleost fishes, daily rhythms are observed in plasma concentrations of some hormones but it is not clear whether these rhythms are exogenous or are entrained by predictable cues. We investigated whether the expression patterns for the mRNAs of growth hormone (GH), prolactin (PRL) and somatolactin (SL) in the pituitary gland, and insulin-like growth factor-I and II (IGF-I and IGF-II) in the liver, follow a daily rhythm when juvenile rabbitfish (Siganus guttatus) are reared under a normal 24-h light and dark cycle (LD), and when they are exposed to either continuous light (LL) or darkness (DD). Hormone mRNA levels were determined by real time PCR. Under LD conditions, GH mRNA expression in the pituitary was significantly lower during the light phase than during the dark phase suggesting a diurnal rhythm of expression. The rhythm disappeared when fish were exposed to LL or DD conditions. PRL mRNA expression pattern was irregular in all 3 conditions. Very low levels of SL mRNA were observed during the mid day under LD conditions. The expression pattern of SL mRNA became irregular under LL and DD conditions. No pattern could be observed in the expression profile of IGF-I and II mRNA in the liver during LD and LL conditions but a single peak in mRNA level was observed under DD conditions in both IGF-I and II. The results indicate that except for GH, the daily expression pattern for the mRNAs of the hormones examined do not seem to follow a rhythm according to light and dark cycles.     

Administration of insulin-like growth factor-I, but not growth hormone, increases maternal weight gain in late pregnancy without affecting fetal or placental growth.

During late pregnancy in the rat, circulating levels of insulin-like growth factor-I (IGF-I) and some IGF-binding proteins (IGFBP) decline. The aim of the present study was to determine the relationship of GH to circulating IGF and IGFBP in the late-pregnant rat and to examine the effects on maternal, fetal and placental growth of preventing the decline in serum IGF and IGFBP concentrations. During the first 9 days of pregnancy, IGF-I concentrations increased from 340 to 500 micrograms/l. Recombinant human (rh) GH at 2.4 mg/kg per day and rhIGF-I at 1.4 mg/kg per day were infused into pregnant rats via osmotic mini pumps during the second half of pregnancy. After pump implantation on day 11 of pregnancy, only IGF-I infusion significantly increased circulating IGF-I. A maximum IGF-I concentration of 907 micrograms/l was measured on day 14 during treatment with IGF-I, after which the serum concentration decreased to 510 micrograms/l by day 20 of pregnancy. The serum IGFBPs were examined using a Western ligand blot technique. Infusion of neither GH nor IGF-I returned the IGFBPs to non-pregnant levels. Administration of IGF-I slightly increased IGFBP-3 and a smaller 32 kDa IGFBP at days 17 and 20 of pregnancy. Neither fetal nor placental weight was significantly different between treatment groups. However, administration of IGF-I significantly increased maternal weight gain during the 10-day treatment period.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.