Thyroid hormone stimulates hepatic IGF-I mRNA expression in a bony fish,the tilapia Oreochromis mossambicus,in vitro and in vivo

To gain more knowledge about the physiological regulation of hepatic insulin-like growth factor-I (IGF-I) production in bony fish, we examined the potential influence of thyroid hormone (T3, 3,5,3'-triiodothyronine) on the expression of IGF-I in the liver of the tilapia Oreochromis mossambicus, using in vitro and in vivo methods. The in vitro experiments were performed using a recently established primary hepatocyte cell culture and IGF-I expression was determined by means of semiquantitative RT-PCR. T3 (100 nM) significantly enhanced the synthesis rate of IGF-I mRNA in short (>8h) and long (>42h) time courses. The stimulating effect of T3 was detected already after 1h. After 4h, the IGF-I mRNA expression was more than 150% of the starting amount. In long time courses, after 6h the IGF-I mRNA value was about 170% of that in untreated cells and at the end of the experiment, it was still three times higher than in the control. In addition, the increase in IGF-I mRNA expression evoked by T3 (1 nM to 1 microM) was dose-dependent. In the in vivo approach, 10 individuals of tilapia received 4 daily intraperitoneal injections of T3 (6 microg/g body weight). IGF-I mRNA was assessed using dot blot technique with a tilapia specific IGF-I cRNA probe. The T3 treatment led to an increase of the IGF-I mRNA level up to 45% in the liver compared to the untreated animals. In conclusion, our results show that T3 directly stimulates the hepatic production of IGF-I in the tilapia in vitro and in vivo and indicate that in tilapia liver regulatory mechanisms seem to exist, as they are discussed for mammals.     

A quantitative real-time RT-PCR assay for salmon IGF-I mRNA, and its application in the study of GH regulation of IGF-I gene expression in primary culture of salmon hepatocytes

The hormone insulin-like growth factor-I (IGF-I) regulates vertebrate growth. The liver produces most circulating IGF-I, under the control of pituitary growth hormone (GH) and nutritional status. To study the regulation of liver IGF-I production in salmon, we established a primary hepatocyte culture system and developed a TaqMan quantitative real-time RT-PCR assay for salmon IGF-I gene expression. A portion of the coho salmon acidic ribosomal phosphoprotein P0 (ARP) cDNA was sequenced for use as a reference gene. A systematic bias across the 96 well PCR plate was discovered in an initial IGF-I assay, which was corrected when the assay was redesigned. IGF-I mRNA levels measured with the validated assay correlated well with levels measured with an RNase protection assay, and were highest in liver compared with other tissues. We examined the time course of hepatocyte IGF-I gene expression over 48 h in culture, the response to a range of GH concentrations in hepatocytes from fed and fasted fish, and potential effects of variation in IGF-I in the medium. IGF-I gene expression decreased over time in culture in hepatocytes in plain medium, and in cells treated with 5 nM GH with or without a combination of metabolic hormones (1 microM insulin, 100 nM triiodothyronine, and 0.1 nM dexamethasone). GH stimulated IGF-I gene expression at all time points. In cells treated with GH plus metabolic hormones, IGF-I gene expression was intermediate between the controls and GH alone. Increasing concentrations of GH resulted in biphasic IGF-I gene expression response curves in cells from fed and fasted fish, with the threshold for stimulation from 0.5 to 2.5 nM GH, maximal response from 5 to 50 nM, and a reduced response at 500 nM. Medium IGF-I (5 nM) did not affect basal or GH stimulated IGF-I gene expression. This study shows that primary hepatocyte culture and the TaqMan IGF-I assay can be used to study the regulation of hepatic IGF-I gene expression in salmon, and provides the first evidence of a biphasic response to GH concentration in fish hepatocyte culture.     

IGF-I is distinctly located in the bony fish pituitary as revealed for Oreochromis niloticus, the Nile tilapia, using real-time RT-PCR, in situ hybridisation and immunohistochemistry

In bony fish, IGF-I released from the liver under the control of pituitary GH is the main endocrine regulator of growth, maintenance and development, and the amount of circulating IGF-I regulates synthesis and release of GH. In mammals and amphibia, evidence indicates that anterior pituitary endocrine cells also contain IGF-I. However, only preliminary and conflicting data exist on IGF-I gene expression in bony fish pituitary. Thus, we investigated the presence of IGF-I in the tilapia (Oreochromis niloticus) pituitary by quantitative real-time RT-PCR, in situ hybridisation and immunohistochemistry. The absolute amount of IGF-I mRNA in the whole pituitary (7.4+/-3.3 x 10(-3)pg/microg total RNA) was 1000-times lower than in liver (7.5+/-3.1 pg/microg total RNA). IGF-I peptide occurred in both neuro- and adenohypophysis but IGF-I gene expression was mainly restricted to the adenohypophysis. In the neurohypophysis, only few cells, probably pituicytes, contained IGF-I mRNA whereas IGF-I peptide was found also in numerous axons in the pars nervosa. In the adenohypophysis, both IGF-I mRNA and peptide were present in the majority of ACTH cells in all individuals investigated. In alpha-MSH cells, only IGF-I mRNA but no IGF-I peptide was detected likely suggesting an immediate release of IGF-I after synthesis. IGF-I mRNA and peptide were further observed in GH cells but their presence showed pronounced inter-individual differences likely due to the physiological, e.g., nutritional, status of the individual. IGF-I released from the GH cells may serve as auto/paracrine mediator of a negative feedback mechanism in addition to liver-derived endocrine IGF-I. Generally, the constitutive synthesis of IGF-I in ACTH cells and the varying content in GH and alpha-MSH cells suggest particular roles for IGF-I. Local IGF-I may regulate synthesis and release of pituitary hormones in an autocrine and/or paracrine manner as well as prevent apoptosis and stimulate proliferation of endocrine cells.     

Involvement of cyclic adenosine monophosphate in the stimulation of gonadotropin secretion from the pituitary of the teleost fish, tilapia.

The present study examines the involvement of cAMP in the transduction of the short-term effect of gonadotropin-releasing hormone (GnRH) on gonadotropin release in the teleost fish, tilapia. A 5 min pulse of dibutyryl cyclic AMP (dbcAMP; 0.03-3 mM) or forskolin (0.1-10 microM) resulted in dose-dependent surges in tilapia gonadotropin (taGTH) secretion from the perifused pituitary. The initial increase in taGTH in response to dbcAMP (3 mM) occurred within 6 min. The concentration of cAMP in the effluent medium increased about 20-fold after a pulse of [D-Ala6,Pro9-NEt]-luteinizing hormone-releasing hormone (LHRH) (GnRHa; 100 nM). To rule out the possibility that the observed effects were due to stimulation by endogenous GnRH release from intact nerve terminals present in the fragments, further experiments were performed in primary cultures of dispersed pituitary cells. Exposure (30 min) of the cells to forskolin (0.01-1.0 microM) resulted in a dose-dependent increase in taGTH release similar to that achieved by GnRHa (1 pM to 10 nM). Also 8-bromo cAMP (0.01-1.0 mM) evoked a dose-related increase in taGTH release. A 3-fold increase in the release occurred in the presence of isobutylmethylxanthine (IBMX) (0.2 mM), similar to that obtained by GnRHa (1.0 nM) in the absence of IBMX. However, when combined, the increase in taGTH release was 16-fold. Moreover, exposure of the cultured cells to GnRHa (0.1 or 10 nM, 60 min) resulted in a dose-related elevation of intracellular cAMP levels and taGTH release.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of epidermal growth factor on insulin-like growth factor-I (IGF-I) and IGF-binding protein synthesis by adult rat hepatocytes.

Growth hormone has been established as a primary regulator of IGF-I gene expression in adults, not only in liver but also in many extrahepatic tissues. We considered the possibility that IGF-I production by adult rat liver could also be stimulated by epidermal growth factor (EGF), a peptide known to be involved in liver regeneration. Chromatographic analysis performed after acid treatment of conditioned media revealed the presence of both immunoreactive (IR) IGF-I and IGF binding protein (IGFBP). Both IR IGF-I and IGFBP were present in the conditioned medium of adult rat hepatocytes in basal conditions. The stimulation of IGF-I and IGFBP secretion by EGF appears to be dose-dependent with a significant increment already evident at 5 nM. That EGF stimulates secretion is supported by the finding that IGF-I and IGFBP-1 mRNA levels are increased after EGF supplementation. We conclude that adult rat hepatocytes spontaneously produce IGF-I and IGFBP, and that EGF is able to increase their synthesis and secretion. This non-growth hormone-dependent regulation of IGF-I and IGFBP-1 production by adult rat hepatocytes in culture indicates an important autocrine/paracrine role for IGF-I, particularly during liver regeneration after extensive organ mass loss.     

Quantification of common carp (Cyprinus carpio) IGF-I and IGF-II mRNA by real-time PCR: differential regulation of expression by GH

IGFs are potent mitogens for many different cell types and play important roles in growth and development. A multitude of regulatory factors modulate the expression of IGFs. In some teleosts, liver IGF-I expression has been demonstrated to depend on the presence of GH. However, the GH dependence of IGF-II expression in teleosts is controversial. Moreover, most IGF expression studies in bony fish have been focused on the liver, and information on extrahepatic tIssues are conflicting and inconsistent. This is partly due to the fact that the traditional methods of mRNA measurement such as Northern blot and RT-PCR are not sensitive enough to detect changes in IGF levels in extrahepatic tIssues because of the low levels of IGFs in these tIssues. In addition, there have been few studies on the IGF system of non-salmonid teleosts. Our laboratory has thus begun such studies on a local tropical fast-growing fish, the common carp (Cyprinus carpio). In this study, real-time quantitative PCR assays were developed for the accurate measurement of IGF-I and IGF-II mRNA levels in common carp tIssues. This quantitative method was based on the measurement of a fluorescent labeled probe, which was cleaved by Taq polymerase during PCR by the 5'-->3' nuclease activity. The signal generated was directly proportional to the starting copy number of the target molecules in the sample. Hence, it was possible to detect and quantify the mRNA levels of both IGF-I and IGF-II reliably in very small amounts of tIssues obtained from juvenile common carp. Using these assays, the expression pattern of IGF-I and IGF-II in various common carp tIssues was studied, and their differential response to GH stimulation was also investigated.     

Differential expression of IGF-I mRNA and peptide in the male and female gonad during early development of a bony fish, the tilapia Oreochromis niloticus

Insulin-like growth factor I (IGF-I) plays a key role in the complex system that regulates bony fish growth, differentiation, and reproduction. The major source of circulating IGF-I is liver, but IGF-I-producing cells also occur in other organs, including the gonads. Because no data are available on the potential production sites of IGF-I in gonad development, developmental stages of monosex breedings of male and female tilapia from 0 day postfertilization (DPF) to 90 DPF were investigated for the production sites of IGF-I at the peptide (immunohistochemistry) and mRNA (in situ hybridization) level. IGF-I mRNA first appeared in somatic cells of the male and female gonad anlage at 7 DPF followed by IGF-I peptide around 9-10 DPF. Gonad anlagen were detected from 7 DPF. Starting at 7 DPF, IGF-I peptide but no IGF-I mRNA was observed in male and female primordial germ cells (PGCs) provided that IGF-I mRNA was not under the detection level, this observation may suggest that IGF-I originates from the somatic cells and is transferred to the PGCs or is of maternal origin. While in female germ cells IGF-I mRNA and peptide appeared at 29 DPF, in male germ cells both were detected as late as at 51-53 DPF. It is assumed that the production of IGF-I in the germ cells is linked to the onset of meiosis that in tilapia ovary starts at around 28 DPF and in testes at around 52-53 DPF. In adult testis, IGF-I mRNA and peptide occurred in the majority of spermatogonia and spermatocytes as well as in Leydig cells, the latter indicating a role of IGF-I in the synthesis of male sex steroids. In adult ovary, IGF-I mRNA and IGF-I peptide were always present in small and previtellogenic oocytes but only IGF-I peptide infrequently occurred in oocytes at the later stages. IGF-I expression appeared in numerous granulosa and some theca cells of follicles at the lipid stage and persisted in follicles with mature oocytes. The results suggest a crucial role of local IGF-I in the formation, differentiation and function of tilapia gonads.     

Epidermal growth factor stimulates glycogen synthesis in fetal rat hepatocytes: comparison with the glycogenic effects of insulin-like growth factor I and insulin

The effects of epidermal growth factor (EGF) on glycogen metabolism and the binding of [125I]iodo-EGF to receptors in fetal rat hepatocytes have been examined. The actions of EGF have been compared with those of insulin-like growth factor I (IGF-I) and insulin. EGF (0.1-45 nM) stimulated dose-dependent increases in [14C]glucose incorporation into glycogen (8.8-31.1%, P less than 0.01) and total cellular glycogen content (5.6-21.4%, P less than 0.05). The concentration of EGF causing half-maximal stimulation of glycogen synthesis was 2 ng/ml, and maximal stimulation occurred at 1 h of incubation. EGF had no effect on the uptake of the nonmetabolizable monosaccharide [14C]O-methyl-D-glucose, suggesting that the glycogenic effect of EGF was not mediated through stimulation of glucose transport. Although IGF-I (1-100 nM) and insulin (14 nM to 10 microM) also stimulated glycogen synthesis in fetal liver, the maximal effects of these hormones occurred at 2 h incubation, and the dose-response curves of IGF-I and insulin were not parallel to that of EGF. In addition, the maximal glycogenic effect of EGF was only 40% that of insulin or IGF-I, and the effects of EGF and insulin on [14C]glucose incorporation were additive. These findings suggest that EGF stimulates glycogen synthesis through a mechanism distinct from that of IGF-I or insulin. The binding of [125I]iodo-EGF to fetal hepatocytes was specific, saturable, and time- and temperature-dependent. Maximal specific binding occurred at 1 h of incubation at 37 C or at 24 h of incubation at 4 C. Unlabeled EGF (0.05-250 ng/ml) caused a dose-dependent inhibition of the binding of [125I]iodo-EGF to fetal hepatocytes, with half-maximal displacement of [125I]iodo-EGF by 1.7 ng unlabeled EGF/ml. The specific binding of [125I] iodo-EGF was not inhibited by high concentrations of insulin or IGF-I, suggesting that the differences in the mechanisms by which EGF, insulin, and IGF-I stimulate glycogenesis may be explained in part by differences in the binding of these hormones to fetal liver receptors. In addition to having mitogenic effects in fetal tissue, EGF or other EGF-like growth factors may have acute effects on fetal hepatic intermediary metabolism and may contribute to the accumulation of liver glycogen in the mammalian fetus during late gestation.     

Insulin-like growth factor I regulates growth hormone secretion and messenger ribonucleic acid levels in human pituitary tumor cells

GH secretion and mRNA levels were measured in cultured human GH adenoma cells incubated in serum-free medium for up to 48 h. A human recombinant insulin-like growth factor I (IGF-I) analog, Thr-59-IGF-I (6.5 nM), inhibited basal GH secretion by up to 60% in tumor cell cultures. The 30-50% stimulation of GH secretion by GH-releasing hormone (GHRH) was prevented by simultaneous exposure of the cells to IGF-I (6.5 nM). Gel electrophoresis of total RNA derived from GH cell adenoma tissue, followed by transfer and hybridization with 32P-labeled human GH cDNA, revealed a distinct mRNA species of about 1.0 kilobases. Using cytoplasmic dot blot hybridization, IGF-I inhibited the levels of human GH mRNA sequences in these cells and also prevented the GHRH-induced stimulation of GH mRNA. A monoclonal antibody to the type I IGF-I receptor (alpha IR3) prevented the inhibitory effects of IGF-I on basal and GHRH-stimulated GH secretion. This antibody also prevented the IGF-I-induced suppression of GH mRNA sequences. PRL secretion in these cells was not altered by IGF-I. Furthermore, relative levels of beta-actin mRNA were unaltered by IGF-I. Thus, IGF-I suppresses basal and GHRH-stimulated GH secretion and GH mRNA levels in pituitary adenoma cells, indicating that IGF-I acts selectively on the somatotroph to directly regulate GH gene expression.     

Differential regulation of Igf1 and Igf2 mRNA levels in tilapia hepatocytes: effects of insulin and cortisol on GH sensitivity

Igf1 and Igf2 stimulate growth and development of vertebrates. In mammals, liver-derived endocrine Igf1 mediates the growth promoting effects of GH during postnatal life, whereas Igf2 stimulates placental and fetal growth and is not regulated by GH. Insulin enhances Igf1 production by the mammalian liver directly, and by increasing hepatocyte sensitivity to GH. We examined the regulation of igf1 and igf2 mRNA levels by GH, insulin, and cortisol, and the effects of insulin and cortisol on GH sensitivity in primary cultured hepatocytes of tilapia, a cichlid teleost. GH increased mRNA levels of both igf1 and igf2 in a concentration-related and biphasic manner over the physiological range, with a greater effect on igf2 mRNA level. Insulin increased basal igf2 mRNA level, and strongly increased GH-stimulated igf2 mRNA level, but slightly reduced basal igf1 mRNA level and did not affect GH-stimulated igf1 mRNA level. Cortisol inhibited GH stimulation of igf1, but increased GH stimulation of igf2 mRNA level. The synergistic effect of insulin and GH on igf2 mRNA level was confirmed in vivo. These results indicate that insulin and cortisol differentially modulate the response of igf1 and igf2 mRNA to GH in tilapia hepatocytes, and suggest that the regulation of liver Igf2 production differs between fish and mammals. Regulation of liver Igf2 production in fish appears to be similar to regulation of liver Igf1 production in mammals.     

Ethinylestradiol differentially interferes with IGF-I in liver and extrahepatic sites during development of male and female bony fish

Growth and sexual development are closely interlinked in fish; however, no reports exist on potential effects of estrogen on the GH/IGF-I-axis in developing fish. We investigate whether estrogen exposure during early development affects growth and the IGF-I system, both at the systemic and tissue level. Tilapia were fed from 10 to 40 days post fertilization (DPF) with 17alpha-ethinylestradiol (EE(2)). At 50, 75, 90, and 165 DPF, length, weight, sex ratio, serum IGF-I (RIA), pituitary GH mRNA and IGF-I, and estrogen receptor alpha (ERalpha) mRNA in liver, gonads, brain, and gills (real-time PCR) were determined and the results correlated to those of in situ hybridization for IGF-I. Developmental exposure to EE(2) had persistent effects on sex ratio and growth. Serum IGF-I, hepatic IGF-I mRNA, and the number of IGF-I mRNA-containing hepatocytes were significantly decreased at 75 DPF, while liver ERalpha mRNA was significantly induced. At 75 DPF, a transient decline of IGF-I mRNA and a largely reduced number of IGF-I mRNA-containing neurons were observed in the female brain. In both sexes, pituitary GH mRNA was significantly suppressed. A transient downregulation of IGF-I mRNA occurred in ovaries (75 DPF) and testes (90 DPF). In agreement, in situ hybridization revealed less IGF-I mRNA signals in granulosa and germ cells. Our results show for the first time that developmental estrogen treatment impairs GH/IGF-I expression in fish, and that the effects persist. These long-lasting effects both seem to be exerted indirectly via inhibition of pituitary GH and directly by suppression of local IGF-I in organ-specific cells.     

Effects of ethanol on insulin-like growth factor-Ⅰ system in primary cultured rat hepatocytes： Implications of JNK1/2 and alcoholdehydrogenase

AIM： To evaluate the effects of ethanol on the insulin- like growth factor-Ⅰ （IGF-Ⅰ） system involved in c-Jun N-terminal kinase （JNK1/2） and alcoholdehydrogenase （ADH） activity in primary cultured rat hepatocytes. METHODS： Hepatocytes isolated from male Sprague-Dawley rats were incubated with various concentrations of ethanol for different durations of time. The cells were pretreated with SP600125 （10 μmol/L） and 4-MP （200 μmol/L）, and then treated with ethanol （200 mmol/L）. We then measured IGF-Ⅰ secretion, IGF-Ⅰ mRNA expression, cell viability and JNK1/2 activity by radioimmunoassay, RT-PCR, MTT assay and Western blot, respectively （n = 6）. RESULTS： Ethanol induced the activity of phospho （p）-JNK1/2, reaching a maximum at 60 min and then decreasing at 180 min. The effects of ethanol on the IGF-Ⅰ system were increased at 60 min （secretion： 7.11 ± 0.59 ng/mg protein vs 4.91 ± 0.51 ng/mg, mRNA expression： 150.2% ± 10.2% vs 101.5% ± 11.3%, P = 0.045） and then decreased at 180 min （secretion： 3.89 ± 0.25 ng/mg vs 5.4 ± 0.54 ng/mg protein; mRNA expression： 41.5% ± 10.4% vs 84.7% ± 12.1%, P = 0.04）, however cell viability was decreased in a dose- and time-dependent manner. SP600125 blocked the ethanol-induced changes （at 60 min）. Additionally, 4-methylpyrazole prevented the ethanol-induced decreases in the IGF-Ⅰ system, cell viability and p-JNK1/2 activity （at 180 min）. CONCLUSION： This study suggests that ethanol- induced p-JNK1/2 activation is associated with the IGF-Ⅰ system and cell viability in hepatocytes. Furthermore, alcohol dehydrogenase is involved in the relationship between ethanol-induced inactivation of p-JNK1/2 and the changes of the IGF-Ⅰ system and cell viability.     

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.     

Quantitative aspects of the effects of insulin, epidermal growth factor and dexamethasone on DNA synthesis in cultured adult rat hepatocytes

Epidermal growth factor (EGF) and insulin in combination have previously been shown to initiate S-phase in primary cultures of adult rat hepatocytes. We here describe the detailed time course and dose-dependency of the effects of EGF and insulin on DNA synthesis in cultured hepatocytes. The DNA synthesis was assessed either biochemically or autoradiographically with a fairly good correlation between the two methods. DNA synthesis started 24-30 h after plating of the cells and peaked at approximately 70 h. Up to 70% of the cells entered DNA synthesis during this period. EGF and insulin acted synergistically on the DNA synthesis. Dexamethasone raised the DNA synthesis slightly, maximal effect occurred at concentrations above 2.5 nM and this agent was routinely used in the experiments with EGF and insulin. In the presence of 0.4 microM insulin from the time of plating, EGF dose-dependently increased the DNA synthesis with maximal effect at 5-15 nM. When added in combination with 1.7 nM EGF, insulin enhanced the DNA synthesis over the concentration range from 0.1 to 3 nM. These studies show that primary cultures of hepatocytes are useful in assessing the quantitative aspects of the interactions between the growth stimulating effects of hormones.