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In order to determine the cortisol response after an immune challenge in the gilthead seabream (Sparus aurata), a cortisol receptor (GR) was cloned, sequenced and its expression determined after lipopolysaccharide (LPS) treatment. To clone the gilthead seabream GR (sbGR), consecutive PCR amplifications and screening of a pituitary cDNA library were performed. We obtained a clone of 4586 bp encoding a 784aa protein. Northern blot analysis from head kidney, heart and intestine revealed that the full length sbGR mRNA was approximately 6.5 Kb. A LPS treatment, used as an acute stress model, was employed to characterise the expression of sbGR and some selected genes involved in the immune response (IL-1beta, TNF-alpha, Mx protein, cathepsin D and PPAR-gamma). All genes were expressed in all tissues examined and responses were tissue and time dependent revealing differential gene expression profiles after LPS administration. Furthermore, analysis of plasma cortisol levels after LPS injection, showed an acute response to inflammatory stress with a significant increase two and six h after injection, recovering to basal levels 12 h post-stress in all LPS concentrations tested.

Tumor necrosis factor-alpha (TNF alpha) is a cytokine with multiple biological functions which, in mammals, has been shown to modulate muscle and adipose tissue metabolism. In fish, TNF alpha has been identified in several species. However, few studies have examined the role of TNF alpha in fish outside the immune system. In this study, we assessed the effects of human recombinant TNF alpha and conditioned media from rainbow trout lipopolysaccharide (LPS)-stimulated macrophages (LPS-MCM) on lipolysis in isolated rainbow trout adipocytes. Furthermore, we studied the effects of an LPS injection in vivo on lipid metabolism. In our study, human recombinant TNF alpha stimulated lipolysis in trout adipocytes in a time- and dose-dependent manner. Similarly, LPS-MCM stimulated lipolysis in trout adipocytes when compared with control conditioned medium. Experiments using specific inhibitors of the MAP kinase pathway showed that p44/42 and p38 are partially involved in the lipolytic effects of TNF alpha. On the other hand, adipocytes from LPS-injected rainbow trout showed higher basal lipolysis than adipocytes from control fish after 24 h, while this effect was not seen at 72 h. Furthermore, lipoprotein lipase (LPL) activity in adipose tissue of LPS-injected fish was lower than in the controls at 24 h. These data suggest that TNF alpha plays an important role in the control of lipid metabolism in rainbow trout by stimulating lipolysis in vitro and in vivo and by down-regulating LPL activity of adipose tissue in vivo.

Insulin and IGF-I binding has been studied in brown trout (Salmo trutta) wheat germ agglutinin semipurified receptors from embryos (organogenesis), larvae (yolk sac), juveniles (2.98 +/- 0.21 g bw) and adults (111.6 +/- 6.92 and 522 +/- 53 g bw). Embryos and larvae were sampled at 5 and 12 weeks after fertilization (December 1999 and February 2000) and juvenile and adults were taken simultaneously (July 1999) and under the same feeding conditions to minimize potential nutritional and seasonal effects. Insulin receptor number was maximal at 12 weeks (144 fmol/mg glycoprotein) and progressively decreased in subsequent samplings. No alterations in affinity were detected (K(d) range, 0.21-0.32 nM) and changes in number of receptor paralleled changes in total specific binding. IGF-I receptor number was highest at 5 weeks (1044 fmol/mg) and was significantly higher than values for insulin in all samplings. The affinity of IGF-I receptor did not change (K(d) range, 0.11-0.18 nM) but was consistently higher than that for the insulin receptor. A more rapid decrease of IGF-I binding and receptor number was found with age. However, the ratio of insulin/IGF-I binding established in 12-week-old larvae (0.18 +/- 0.01) was thereafter maintained at very similar values in juveniles and adults (0.15-0.17). Tyrosine kinase activity (TKA) for insulin receptors ranged between 136 and 183% and there were no significant changes with age. For the IGF-I receptor, TKA ranged from 174 to 281% and was significantly higher in 5-week-old larvae coincident with the highest levels of receptor number and declined gradually in parallel with binding levels. In conclusion, the greater abundance of IGF-I receptors during embryonic and larval development is maintained throughout juvenile and adult stages. This would suggest a key role for IGF-I in the growth and metabolism of trout muscle., (Copyright 2001 Academic Press.)

To determine whether fish have an insulin-like growth factor II/mannose 6-phosphate (IGF-II/M6-P) receptor similar to that of mammals, we have performed binding, cross-linking, and immunoprecipitation experiments with wheat-germ-agglutinin- and mannose 6-phosphate (M6-P)-affinity-purified receptor preparations from fish embryos. In both receptor preparations, IGF-II binding was specific, because labeled IGF-II could only be completely displaced by cold IGF-II but not by IGF-I or insulin. Labeled IGF-II bound to a protein with a molecular mass of approximately 250 kDa, which could be immunoprecipitated with an antibody against the rat IGF-II receptor. IGF-II stimulated tyrosine kinase activity in wheat germ agglutinin preparations and was more potent than insulin or IGF-I, but neither peptide stimulated tyrosine kinase activity in M6-P preparations. Two fish cell lines (CHSE-214 and EPC) were used to confirm the IGF-II binding data obtained in the receptor preparations, revealing the presence of highly specific IGF-II binding and the absence of insulin binding. Furthermore, a decrease of the IGF-I receptors on the cell surface did not alter IGF-II binding in EPC cells. In conclusion, we have detected the presence of IGF-II/M6-P receptors in fish embryos that are similar in structure and specificity for their ligand to those found in mammals.

The regulation of ovarian steroidogenesis in vitro by coho salmon FSH and LH was investigated in intact coho salmon follicles and isolated follicular layers at various stages of oocyte maturation, from late vitellogenesis until the completion of germinal vesicle breakdown (GVBD). In granulosa layers from all stages, LH, but not FSH, stimulated 17alpha,20beta-dihydroxy-4-pregnen-3-one (17, 20beta-P) production. In theca-interstitial layers from all stages, FSH and LH stimulated steroid production, LH being more potent than FSH. The basal steroid output of intact follicles was significantly lower than that of isolated follicular layers, and their response to FSH and LH also differed. First, the intact follicles produced 17alpha-hydroxyprogesterone in response to FSH during the central germinal vesicle stage while theca-interstitial layers did not. Second, estradiol-17beta production was not inhibited by LH during final oocyte maturation in intact follicles, as observed for granulosa layers. Our results indicate that LH is the determining factor regulating the production of the maturation-inducing steroid, 17,20beta-P, and the induction of GVBD in the salmonid ovary. In summary, we have provided evidence for maturation-associated changes in the effects of FSH and LH in the salmonid ovary, which further supports the hypothesis that FSH and LH have distinct functions in the teleost ovary.

In fish, insulin is believed to act on adipose tissue to promote lipid accumulation, but a direct role for insulin in fish adipose tissue lipogenesis has yet to be demonstrated. To investigate the role of insulin and insulin-like growth factor I (IGF-I) in fish adipose tissue function, we have investigated the presence and the regulation of insulin and IGF-I receptors in adipose tissue of brown trout (Salmo trutta). Receptors for insulin and IGF-I were detected in trout adipose tissue, with IGF-I receptors being more abundant (two- to tenfold) and having a higher affinity (twofold) than insulin receptors. In contrast to the situation in mammals, arginine treatment, which elevates the levels of insulin and IGF-I in plasma, increased the number of insulin receptors 1.7-fold and the number of IGF-I receptors 2.3-fold. When plasma levels of insulin and IGF-I were decreased by fasting, insulin receptor numbers fell 3.6-fold and IGF-I receptor numbers fell 2.2-fold. These results demonstrate for the first time the presence of specific insulin and IGF-I receptors in adipose tissue of ectothermic vertebrates and suggest that adipose tissue may be a target for the actions of insulin and IGF-I in fish.

In this study we report that the use of a heterologous radioimmunoassay (RIA) is valid for the detection of insulin-like growth factor-I (IGF-I) levels in plasma of a variety of fish species. Parallelism between standard curves and plasma dilutions were observed and the standard curve obtained with mammalian IGF-I presented the same characteristics as that obtained with coho salmon recombinant IGF-I. The RIA was biologically validated since total plasma IGF-I values were significantly modified by different experimental conditions. Hyperinsulinemia induced either by arginine or insulin injection was accompanied by increases in IGF-I plasma levels in brown trout (Salmo trutta). In contrast, parallel decreases in insulin and IGF-I circulating levels were observed after 45 days of fasting and 20 days after a single streptozotocin injection. Administration of arginine in fasted fish led to a relative increase in insulin and IGF-I plasma concentrations, while arginine injection in fish previously treated with streptozotocin increased IGF-I levels only. The above data suggest that insulin, together with other factors, may act to increase the levels of IGF-I in plasma.

Insulin and glucagon, two of the most studied pancreatic hormones bind to specific membrane receptors to exert their biological actions. Insulin-like growth factors IGF-I and IGF-II are structurally related to insulin, although they are expressed ubiquitously. The biological functions of the IGFs are mediated by different transmembrane receptors, which includes the insulin, IGF-I and IGF-II receptors. The interaction of insulin, insulin related peptides and glucagon with the corresponding receptors has been studied extensively in mammals and continues to be so. At the same time, research on ectothermic animals has made enormous progress in the recent years. This paper summarizes current knowledge on insulin, IGF-I and glucagon receptors, from a comparative point of view with special attention to non-mammalian vertebrates. The review covers adult and mostly typical target tissues, and with very few exceptions, developmental aspects are not considered. Binding characteristics, tissue distribution and structure of insulin and IGF-I receptors will be considered first, because both ligands and receptors are structurally related and have overlapping functions. These sections will be followed by similar distribution of information on glucagon receptors. Readers interested in either structure or functions of insulin, IGFs and glucagon in nonmammalian vertebrates are referred to other reviews (Mommsen TP, Plisetskaya EM. Insulin in fishes and agnathans: history, structure and metabolic regulation. Rev Aquat Sci 1991;4:225-259; Mommsen TP, Plisetskaya EM. Metabolic and endocrine functions of glucagon-like peptides: evolutionary and biochemical perspectives. Fish Physiol Biochem 1993;11:429-438; Duguay SJ, Mommsen TP. Molecular aspects of pancreatic peptides. In: Sherwood NM, Hew CL, editors, Fish Physiology. vol 13. 1994:225-271; Plisetskaya EM, Mommsen TP. Glucagon and glucagon-like peptides in fishes. Int Rev Citol 1996;168:187-257.).

The major regulator of lipolysis in white adipocytes and brown adipocytes is cAMP and the actions of cAMP are mediated by protein kinase A (PKA). Multiple subunits of PKA, including RII beta, R1 alpha, C alpha, and C beta 1, are expressed in fat cells but the major holoenzyme assembled under normal conditions contains RII beta and C alpha. Targeted disruption of the RII beta gene in mice revealed that both white and brown adipocytes are capable of compensating by increasing the level of RI alpha. Nevertheless, the mice display a lean phenotype, have an elevated metabolic rate due to activation and induction of uncoupling protein in brown fat, and are resistant to diet-induced obesity and insulin resistance. Although the metabolic disturbances in white and brown fat tissue may explain most of the phenotypic changes, the loss of neuronal expression of RII beta may also contribute to the alterations in energy balance. Specific neuronal defects have been characterized that prevent the normal changes in gene expression seen with drugs that act through the dopaminergic pathway. The RII beta mutant mouse provides an interesting model of obesity resistance and demonstrates that chronic changes in the PKA signaling system can lead to stable alterations in energy storage and utilization.

In teleosts, ovarian steroidogenesis is under the control of two gonadotropic hormones, GTH I and GTH II, that are structurally and functionally homologous to FSH and LH. The intracellular mechanisms by which GTH I and GTH II stimulate steroidogenesis in the teleost ovary are not well understood. The purpose of this study was to investigate the involvement of the cAMP/protein kinase A (PKA) and protein kinase C (PKC)/Ca2+ signaling pathways in the steroidogenic actions of GTH I and GTH II in the ovary of the brook trout (Salvelinus fontinalis). The cAMP/PKA pathway mediated the actions of GTH I before germinal vesicle breakdown (preGVBD) and GTH II after GVBD and before ovulation (preOV). Experimental increases in intracellular cAMP concentration mimicked the steroidogenic effects of GTH I and GTH II, and an antagonistic analog of cAMP partially blocked them. In addition, GTH I and GTH II stimulated the production of cAMP in preGVBD and preOV follicles, respectively. Activation of the PKC/Ca2+ pathway by a phorbol ester or a Ca2+ ionophore blocked the GTH I- and GTH II-induced steroid production, whereas inhibition of PKC by specific inhibitors potentiated the effects of GTH I. These results suggest that the cAMP/PKA signaling pathway mediates the stimulatory effects of GTH I and GTH II on steroidogenesis, and they also suggest the additional involvement of the PKC/Ca2+ signaling pathway in modulating the actions of gonadotropins in brook trout ovarian follicles.

In this study, receptors for insulin and insulin-like growth factor I (IGF-I) in isolated theca-interstitial layers and granulosa cells of the coho salmon preovulatory ovary were characterized, and the effects of IGF-I on ovarian steroidogenesis were examined. Specific receptors for insulin and IGF-I were found in granulosa and theca-interstitial layers. In both follicular layers, IGF-I receptors were greater in number and higher in affinity than insulin receptors. The effects of IGF-I on in vitro production of testosterone (T) and 17 alpha-hydroxyprogesterone (17OH-P) by theca-interstitial layers and of 17 beta-estradiol (E2) and 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-P) by granulosa cell layers were evaluated during the preovulatory period. Both human and salmon recombinant IGF-I inhibited the basal and GTH II-stimulated T and 17OH-P production by theca-interstitial layers throughout the preovulatory period. In contrast, IGF-I stimulated the production of both E2 and 17,20 beta-P by granulosa cell layers prior to germinal vesicle breakdown (GVBD) but only stimulated the production of 17,20 beta-P by granulosa cell layers after GVBD. The inhibitory effects of IGF-I on steroid production by the theca-interstitial layer and the opposite stimulatory effects on steroid production by the granulosa cell layer, coupled by the presence of specific IGF-I receptors in both follicular layers, suggest that IGF-I may play a role in the regulation of steroidogenesis in the preovulatory coho salmon ovary.

Cyclic AMP is an important second messenger in the coordinated regulation of cellular metabolism. Its effects are mediated by cAMP-dependent protein kinase (PKA), which is assembled from two regulatory (R) and two catalytic (C) subunits. In mice there are four R genes (encoding RI alpha, RI beta, RII alpha, and RII beta) and two C gene (encoding C alpha and C beta), expressed in tissue-specific patterns. The RII beta isoform is abundant in brown and white adipose tissue and brain, with limited expression elsewhere. To elucidate its functions, we generated RII beta knockout mice. Here we report that mutants appear healthy but have markedly diminished white adipose tissue despite normal food intake. They are protected against developing diet-induced obesity and fatty livers. Mutant brown adipose tissue exhibits a compensatory increase in RI alpha, which almost entirely replaces lost RII beta, generating an isoform switch. The holoenzyme from mutant adipose tissue binds cAMP more avidly and is more easily activated than wild-type enzyme. This causes induction of uncoupling protein and elevations of metabolic rate and body temperature, contributing to the lean phenotype. Our results demonstrate a role for the RII beta holoenzyme in regulating energy balance and adiposity.

The ability of coho salmon gonadotropins GTH I and GTH II to stimulate testicular production of 11-ketotestosterone (11-KT) and 17 alpha,20 beta-dihydroxy-4- pregnen-3-one (17,20 beta-P) in vitro, as well as the involvement of cyclic AMP (cAMP) as a mediator of the actions of GTH I and GTH II, were investigated in maturing male coho salmon. Testicular tissue was incubated in the presence or absence of the test substances at 15 degrees. Both GTH I and GTH II stimulated the in vitro production of 11-KT and 17,20 beta-P in a concentration- and time-dependent manner. In testicular tissue at stage IV of spermatogenesis, GTH I and GTH II stimulated the in vitro production of steroids with similar potency. The steroidogenic activity of GTH I and GTH II appeared to be mediated, at least in part, by cAMP as indicated by the ability of dibutyryl cAMP and forskolin to mimic the steroidogenic actions of GTHs, and the similar ability of GTH I and GTH II to increase intracellular cAMP levels in the testicular tissue. The calcium ionophore A23187 stimulated basal but not GTH-stimulated production of steroids in vitro. In addition, differential effects of a protein kinase C inhibitor, H-7, on 11-KT and 17,20 beta-P production were found. Basal and GTH-stimulated production of 11-KT were inhibited by H-7; whereas basal and GTH-stimulated production of 17,20 beta-P were stimulated by H-7. These results suggest that protein kinase C may also be involved in the steroidogenic actions of GTH I and GTH II.

Somatolactin (SL), a newly discovered pituitary hormone of the teleost pars intermedia, is structurally similar to prolactin and growth hormone. The function(s) of SL are not yet established, although evidence suggests that it may play a role in reproduction. Possible steroidogenic activity of coho salmon SL was tested and compared with gonadotropin I (GTH I) in incubations of ovarian follicles or testicular fragments. SL stimulated production of 11-ketotestosterone and testosterone by testicular fragments, and production of estradiol by ovarian follicles in a concentration-dependent manner. However, the steroidogenic activity of SL was considerably less than that of GTH I. These results suggest that SL may play a role in regulation of gonadal function in salmon.