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.     

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.     

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.     

Developmental regulation of insulin-like growth factor-I and growth hormone receptor gene expression.

During development, the insulin-like growth factor I (IGF-I) gene is expressed in a tissue specific manner; however, the molecular mechanisms governing its developmental regulation remain poorly defined. To examine the hypothesis that expression of the growth hormone (GH) receptor accounts, in part, for the tissue specific expression of the IGF-I gene during development, the developmental regulation of IGF-I and GH receptor gene expression in rat tissues was examined. The level of IGF-I and GH receptor mRNA was quantified in RNA prepared from rats between day 17 of gestation (E17) and 17 months of age (17M) using an RNase protection assay. Developmental regulation of IGF-I gene expression was tissue specific with four different patterns of expression seen. In liver, IGF-I mRNA levels increased markedly between E17 and postnatal day 45 (P45) and declined thereafter. In contrast, in brain, skeletal muscle and testis, IGF-I mRNA levels decreased between P5 and 4M but were relatively unchanged thereafter. In heart and kidney, a small increase in IGF-I mRNA levels was observed between the early postnatal period and 4 months, whereas in lung, minimal changes were observed during development. The changes in GH receptor mRNA levels were, in general, coordinate with the changes in IGF-I mRNA levels, except in skeletal muscle. Interestingly, quantification of GH receptor levels by Western blot analysis in skeletal muscle demonstrated changes coordinate with IGF-I mRNA levels. The levels of the proteins which mediate GH receptor signaling (STAT1, -3, and -5, and JAK2) were quantified by Western blot analysis. These proteins also are expressed in a tissue specific manner during development. In some cases, the pattern of expression was coordinate with IGF-I gene expression, whereas in others it was discordant. To further define molecular mechanisms for the developmental regulation of IGF-I gene expression, protein binding to IGFI-FP1, a protein binding site that is in the major promoter of the rat IGF-I gene and is important for basal promoter activity in vitro, was examined. Gel shift analyses using a 34-base pair oligonucleotide that contained IGFI-FP1 did not demonstrate changes in protein binding that paralleled those in IGF-I gene expression, suggesting that protein binding to IGFI-FP1 does not contribute to the developmental regulation of IGF-I gene expression, at least in brain and liver. In summary, the present studies demonstrate coordinate expression of the IGF-I gene and GH receptor during development and suggest that GH receptor expression contributes to the tissue specific expression of the IGF-I gene during development.     

A peak in gh-receptor expression is associated with growth activation in Atlantic salmon vertebrae, while upregulation of igf-I receptor expression is related to increased bone density

Growth hormone (GH) and insulin-like growth factor-I (IGF-I) play major roles in the endocrine regulation of fish growth, but their interdependency and mode of action has not been well elucidated. The GH-IGF-I system is essential for normal vertebral growth in mouse, but this has not been studied in fish. To study the interplay between GH, IGF-I, and their receptors, postsmolt Atlantic salmon were studied during spring growth (January-June 2003). From January to June, fish were sampled regularly for plasma and vertebral bone. The vertebra was collected from the same anterior-posterior position. The growth hormone receptor (ghr) (There is no determined nomenclature of salmon genes but we stick to the nomenclature which is consequent for zebrafish, where all gene names are named with small letters and in italic.) expression in the vertebrae peaked in the end of February coinciding with high levels of plasma GH and IGF-I, and an increase of vertebral growth rate. From April to June, plasma IGF-I levels decreased together with ghr expression in the vertebrae, while plasma GH did not decrease. In May and June, expression of the igf-I receptor (igf-Ir) increased 4- to 5-fold, which coincided with an increase in bone density. The changes seen in gene expression of the IGF-I and GH receptors suggest that these hormones are involved in vertebral growth and bone density.     

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.     

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.     

Dynamic transcriptional regulation of autocrine/paracrine igfbp1, 2, 3, 4, 5, and 6 in the skeletal muscle of the fine flounder during different nutritional statuses

The IGF-binding proteins (IGFBPs) play a dual role in the regulation of the activity and bioavailability of IGFs in different tissues. Diverse evidence has shown that IGFBPs can inhibit and/or potentiate IGF actions. In this study, igfbp1, 2, 3, 4, 5, and 6 were isolated in the fine flounder, a flat fish species that shows slow growth and inherent Gh resistance in muscle. Subsequently, the expression of all igfbps was assessed in the skeletal muscle of flounder that underwent different nutritional statuses. igfbp1 was not expressed in muscle during any of the nutritional conditions, whereas igfbp3 and igfbp5 were the lowest and the highest igfbps expressed respectively. A dynamic expression pattern was found in all the igfbps expressed in skeletal muscle, which depended on the nutritional status and sampling period. During the fasting period, igfbp2, 4, and 5 were downregulated, whereas igfbp3 was upregulated during part of the fasting period. The restoration of food modulated the expression of the igfbps dynamically, showing significant changes during both the long- and short-term refeeding. igfbp3 and igfbp6 were downregulated during short-term refeeding, whereas igfbp5 was upregulated, and igfbp2 and igfbp4 remained stable. During long-term refeeding, the expression of igfbp2, 4, 5, and 6 increased, while igfbp3 remained unchanged. In conclusion, this study shows for the first time the isolation of all igfbps in a single fish species, in addition to describing a dynamic nutritional and time-dependent response in the expression of igfbps in the skeletal muscle of a nonmammalian species.     

Nutrition-regulated lipolysis in rainbow trout (Oncorhynchus mykiss) is associated with alterations in the ERK, PI3K-Akt, JAK-STAT, and PKC signaling pathways

Previous studies have shown that food deprivation, which occurs naturally in the life cycle of many species of fish, results in cessation of growth and catabolism of stored energy reserves, including lipids. In this study, we used rainbow trout (Oncorhynchus mykiss) to identify the cellular mechanisms involved with this metabolic shift. Fish were placed on one of five dietary regimes--fed continuously for 2 or 4 weeks, fasted continuously for 2 or 4 weeks, or fasted 2 weeks then refed 2 weeks--and the effects on organismal growth and lipid catabolism and on the activation state of signaling elements (e.g., Akt, ERK, JAK-STAT, PKC) in selected tissues were measured. Fasting for either 2 or 4 weeks significantly retarded growth in terms of body weight, body length, and body condition; refeeding restored growth such that body length and body condition were similar to measures seen in continuously fed fish. Fasting activated lipid catabolism by stimulating the mRNA expression and catalytic activity of hormone-sensitive lipase (HSL). Two HSL-encoding mRNAs have been characterized, and the expression of both forms of mRNA in 2- and 4-week fasted fish were significantly elevated over levels in fed fish in all tissues. In adipose tissue, liver, and white muscle, HSL activity was significantly elevated in 2- and 4-week fasted fish compared to fed animals; whereas in red muscle, HSL activity was significantly elevated compared to fed fish after 4 weeks of fasting. Refeeding reversed both fasting-associated HSL mRNA expression and HSL activity. Fasting resulted in the deactivation of Akt, JAK2, and STAT5 in adipose tissue, liver, and red and white muscle. By contrast, fasting activated ERK and PKC in all tissues measured. Refeeding reversed fasting-associated alterations in the activation state of all signal elements. These findings suggest that deactivation of Akt and JAK-STAT in conjunction with activation of ERK and PKC underlie fasting-associated growth retardation and lipolysis.     

The effects of recombinant bovine somatotropin (rbST) on tissue IGF-I, IGF-I receptor, and GH mRNA levels in rainbow trout, Oncorhynchus mykiss

Numerous studies demonstrated that rbST increased growth rates in several fish species, and several species exhibit GH production in tissues other than the pituitary. The role of tissue GH and IGF-I in regulating fish growth is poorly understood. Therefore an experiment was conducted to examine the effects of rbST treatment on tissue GH, IGF-I, and IGF-I receptor-A (rA) expression in rainbow trout. Rainbow trout (550 +/- 10 g) received either intra-peritoneal injections of rbST (120 microg/g body weight) or vehicle on days 0 and 21, and tissue samples were collected on days 0, 0.5, 1, 3, 7, and 28 (n = 6/day/trt). Total RNA was isolated and assayed for steady-state levels of IGF-I, IGF-IrA, and GH mRNA using quantitative RT-PCR. Insulin-like growth factor-I mRNA levels increased in liver, gill, gonad, muscle, brain, and intestine in response to rbST treatment (P < 0.10). Liver IGF-I mRNA increased (P < 0.01) 0.5 day after treatment and remained elevated throughout the trial. Intestine IGF-I mRNA increased (P < 0.05) in treated fish from day 1 to day 3, then decreased to day 7 and increased again at day 28, and remained elevated above control levels throughout the trial. Gill IGF-I mRNA levels increased (P < 0.05) 1 day after treatment and remained elevated throughout the trial. Heart IGF-IrA mRNA levels decreased (P < 0.05) while gonad GH mRNA levels increased (P < 0.10) following rbST treatment. These results demonstrate that rbST treatment increased IGF-I mRNA levels in extra-hepatic tissues, and decreased heart IGF-IrA and increased gonad GH mRNA levels. Because the primary source for endocrine IGF-I is liver, the increased IGF-I mRNA reported in extra-hepatic tissues may indicate local paracrine/autocrine actions for IGF-I for local physiological functions.     

Dual mode of cortisol action on GH/IGF-I/IGF binding proteins in the tilapia,Oreochromis mossambicus

Glucocorticoids are known to impede somatic growth in a wide range of vertebrates. In order to clarify the mechanisms through which they may act in an advanced teleost fish, we examined the effects of cortisol administration on the growth hormone (GH)/insulin-like growth factor-I (IGF-I)/IGF-binding protein (IGFBP) system in the tilapia (Oreochromis mossambicus). In a short-term experiment, fish were injected intraperitoneally with cortisol (2 or 10 microg/g), and killed at 2, 4, 8 and 24 h after the injection. In a longer-term experiment, fish were killed 24 and 48 h after cortisol injection (2, 10 and 50 microg/g). Cortisol at doses of 2 and 10 microg/g significantly increased IGFBPs of four different sizes (24, 28, 30, and 32 kDa) in the plasma within 2 h without altering plasma levels of IGF-I or GH. On the other hand, cortisol at doses of 10 and 50 microg/g significantly reduced plasma IGF-I levels after 24 and 48 h. IGF-I mRNA levels in the liver were also significantly reduced by cortisol at doses of 10 and 50 microg/g after 48 h, suggesting that a decrease in plasma IGF-I levels is mediated through the attenuation of IGF-I gene expression in the liver. In contrast, no significant change was observed in plasma or pituitary contents of GH at any time point examined, which would appear to indicate that cortisol reduces IGF sensitivity to GH (GH-resistance). These results clearly indicate that cortisol induces a rapid increase in plasma IGFBPs and a more delayed decrease in IGF-I production. The dual mode of cortisol action may contribute to the inhibitory influence of cortisol on somatic growth in teleosts.     

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.     

Growth hormone regulates the level of insulin-like growth factor-I mRNA in rat skeletal muscle

Levels of mRNA for the insulin-like growth factor-I (IGF-I) in rat heart and skeletal muscle and its dependence on GH were investigated using a solution hybridization assay. Levels of IGF-I mRNA decreased following hypophysectomy, and replacement therapy with human GH (hGH) normalized heart and skeletal muscle levels. The stimulatory effect of hGH was dose-dependent, the lowest effective dose being 100 micrograms. A significant increase of IGF-I mRNA was observed 60 min after s.c. administration of 100 micrograms hGH and the maximum increase was apparent 6-12 h after hGH injection. Administration of 200 micrograms IGF-I or 11 micrograms insulin did not significantly change levels of IGF-I mRNA. The results show that GH regulates the level of IGF-I mRNA in rat heart and skeletal muscle and give further support to the hypothesis that locally produced IGF-I might be a local mediator for the direct stimulatory effect of GH on the growth and development of heart and skeletal muscle.