Differential expression of two somatostatin receptor subtype 1 mRNAs in rainbow trout (Oncorhynchus mykiss)

Somatostatins (SSs) play important roles in the growth, development and metabolism of vertebrates. In this study, cDNAs for two unique somatostatin receptor variants were cloned and sequenced from rainbow trout. The two cDNAs, one consisting of 1755 bp and the other of 1743 bp, share 63.6% identity in nucleotide sequence and 94.1% identity in deduced amino acid sequence and presumably arose through gene duplication. Each cDNA encodes for a putative 371-amino acid somatostatin receptor (one designated sst1A and the other sst1B) containing seven transmembrane domains. Rainbow trout sst1A and sst1B have 64.4 and 65.5% similarity respectively with human sst1 and only 43-60% similarity with other subtypes. Trout sst1 mRNAs are differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues. Both sst1A and sst1B mRNAs were present in brain, stomach, liver, pancreas, upper and lower intestine, pyloric cecum, kidney and muscle, whereas only sst1B mRNA was present in the esophagus. sst1A mRNA was more abundant than sst1B in the optic tectum, whereas sst1B mRNA was more abundant than sst1A in liver. sst1A and sst1B mRNAs were equally abundant in pancreas. These findings contribute to the understanding of the evolution of the SS signaling system and provide insight into the mechanisms that regulate the expression of SS receptors.     

Effects of nutritional state, insulin, and glucagon on lipid mobilization in rainbow trout, Oncorhynchus mykiss.

The effects of nutritional state, insulin, and glucagon on lipid mobilization were determined in rainbow trout, Oncorhynchus mykiss. In nutritional state experiments, fish were either fed continuously (except 24 to 36 hr prior to experimentation) with commercial trout chow or fasted for 4 weeks. Lipase activity in liver tissue isolated from fasted fish and cultured for 5 hr was greater than that in tissue isolated from fed fish and cultured. The presence of glucose (5.55 mM) in the incubation medium accentuates lipolytic activity in both liver and adipose tissue. Hormone response was assessed both in vivo and in vitro. Salmon insulin was injected into anesthetized fish (fed continuously except 24 hr prior to injections) in 10 microliters of saline/g body weight; final hormone dose was 100 ng/g body weight. Tissue and plasma were sampled 1 and 3 hr after injection. Insulin resulted in depressed plasma FA concentration and reduced hepatic triacylglycerol lipase activity. In vitro effects of hormones were evaluated by incubating liver and adipose tissue pieces in Hanks-MEM. Glucagon (bovine/porcine) directly stimulated lipid breakdown in both liver and adipose tissue. These actions were manifested by enhanced FA and glycerol released into the culture medium and by elevated triacylglycerol lipase activity. Insulin (bovine) generally appeared antilipolytic as this agent inhibited glucagon-stimulated lipase activity and glucagon-stimulated FA release. Furthermore, insulin (in the presence of glucose) reduced net lipolysis, as indicated by glycerol release, compared to control cultures. These results indicate that nutritional state and glucose are important modulators of lipid mobilization and that glucagon and insulin act directly on lipid storage sites to coordinate lipolysis in rainbow trout.     

Glucagon and insulin response to dietary carbohydrate in rainbow trout (Oncorhynchus mykiss)

The effects of high carbohydrate (CH) diets on circulating levels of insulin and glucagon were studied in rainbow trout (Oncorhynchus mykiss). Fish (76.87 +/- 8.11 g) were fed for 60 days with three isocaloric diets: control (C) (12% CH), W (28% CH mainly from wheat), and S (28% CH from wheat plus gelatinized starch). After the diet treatment, trout fed the enriched CH diets showed a higher hepatosomatic index and liver glycogen content than controls. In addition, plasma glucose levels were also higher but, on the contrary, circulating insulin levels were greater in the control group. After the period of diet adaptation, fish were sampled 3, 6, 9, and 24 h after food administration. Glycemia levels correlated with the dietary carbohydrate content, and were minimum in controls (from 96.02 +/- 3.77 to 118.97 +/- 6.08 mg/100 ml), followed by W group (from 124.60 +/- 7.46 to 172.19 +/- 11.36 mg/100 ml) and maximum in the S group (from 133.51 +/- 9.36 to 217.88 +/- 13.36 mg/100 ml). Postprandial glucagon profiles showed an inverse relationship with glycemia, indicating that the ingestion of glucose inhibits glucagon secretion. There were no significant differences in postprandial insulin levels between groups except for W group, which presented lower levels of circulating insulin 9 h after feeding. These results indicate that in trout fed a CH diet glucose affects the secretion of glucagon more than insulin during the postprandial period.     

Environmental temperature increases plasma GH levels independently of nutritional status in rainbow trout (Oncorhynchus mykiss)

Like many poecilotherms, salmonids exhibit seasonal variations of growth rate in relation with seasonal temperatures and plasma GH level. However, temperature alters other parameters like food intake, which may directly modify the level of plasma GH. In order to determine whether temperature regulates plasma GH levels independently of nutritional status, fish were reared at 8, 12, or 16 degrees C and either fed ad libitum (fish with different food intake) to determine the global effect of temperature, or with the same ration (1.2%/body weight) to observe the temperature effect in fish with the same growth rate. Plasma insulin level was inversely proportional to the temperature (8, 12, and 16 degrees C) in fish fed ad libitum (12.1+/-0.3 ng/ml, 10.9+/-0.3 ng/ml, 9.5+/-0.4 ng/ml; P<0.001) and in restricted fish (14.0+/-0.3 ng/ml, 11.3+/-0.3 ng/ml, 10.0+/-0.2 ng/ml; P<0.0001), probably due to a prolonged nutrient absorption, and delayed recovery of basal insulin level at low temperature. Conversely, temperature did not affect plasma T3 level of fish fed ad libitum (2.5+/-0.2 ng/ml, 2.4+/-0.1 ng/ml, 2.5+/-0.1 ng/ml at 8, 12, and 16 degrees C) while fish fed with the same ration present less T3 at 16 degrees C than at 8 degrees C (1.83+/-0.1 ng/ml versus 1.2+/-0.1 ng/ml; P<0.001) throughout the experiment; these observations indicate that different plasma T3 levels reflect the different nutritional status of the fish. The levels of GH1 and GH2 mRNA, and GH1/GH2 ratio were not different for whatever the temperature or the nutritional status. Pituitary GH content, of fish fed ad libitum did not exhibit obvious differences at 8, 12, or 16 degrees C (254+/-9 ng/g bw, 237+/-18 ng/g bw, 236+/-18 ng/g bw), while fish fed with the same ration have higher pituitary GH contents at 16 degrees C than at 8 degrees C (401+/-30 ng/g bw versus 285+/-25 ng/g bw; P<0.0001). Interestingly, high temperature strongly increases plasma GH levels (2.5+/-0.3 ng/ml at 8 degrees C versus 4.8+/-0.6 ng/ml at 16 degrees C; P<0.0001) to the same extent in both experiments, since at a given temperature average plasma GH was similar between fish fed ad libitum or a restricted diet. Our results, demonstrate that temperature regulates plasma GH levels specifically but not pituitary GH content, nor the levels of GH1 and GH2 mRNA. In addition no differential regulation of both GH genes was evidenced whatever the temperature.     

Regulation of lipoprotein lipase activity in rainbow trout (Oncorhynchus mykiss) tissues

Lipoprotein lipase (LPL) is considered as a key enzyme in the lipid deposition and metabolism of many tissues. Information on LPL activity and its regulation in fish remains very scarce. In the present study, we have examined the nutritional regulation of LPL activity by conducting post-feeding and fasting experiments in rainbow trout (Oncorhynchus mykiss). As insulin plays an important role in the nutritional regulation of LPL activity in mammals, the effects of this hormone were tested in vivo by intraperitoneal administration. Moreover, we conducted in vitro studies using fat pads of rainbow trout to better clarify the direct role of insulin and tumor necrosis factor-alpha (TNFalpha) as possible regulators of LPL activity in rainbow trout. LPL activity in adipose tissue increased in response to feeding, 4h after ingestion of food, then decreasing to basal levels at 6h. No clear response was found in either red or white muscles, where LPL values were lower. Moreover, fasting produced a down-regulation of LPL activity in adipose tissue, concomitant with low levels of plasma insulin. While insulin administration stimulated LPL activity of adipose tissue 3h after injection, no response was observed in red or white muscles. Finally, in vitro studies using fat pads revealed that insulin significantly stimulated the proportion of LPL in active conformation at the extracellular level. On the other hand, TNFalpha did not greatly affect LPL activity using this in vitro model. These data indicate that LPL activity is regulated in a tissue-specific manner following food intake, and suggest that insulin is an important regulator of LPL activity in the adipose tissue of rainbow trout.     

Osmoregulatory actions of insulin-like growth factor-I in rainbow trout (Oncorhynchus mykiss)

The ability of insulin-like growth factor-I (IGF-I), insulin and GH to promote hypoosmoregulatory ability was examined in juvenile rainbow trout (Oncorhynchus mykiss). Following adaptation to 12 parts per thousand (p.p.t.) seawater for 5 days, fish were given a single injection of hormone or vehicle, then exposed to 29 p.p.t. for 24 h and examined for changes in plasma osmolarity, ions and glucose. Ovine GH (oGH; 0.2 micrograms/g) significantly improved the ability of rainbow trout to maintain plasma osmolarity and sodium levels following transfer to 29 p.p.t. seawater. Recombinant bovine IGF-I (0.01, 0.05 and 0.02 micrograms/g) also improved the hypoosmoregulatory ability of trout; the effect being dose-dependent and greater than that of oGH. Bovine insulin (0.01, 0.05 and 0.2 micrograms/g) had no statistically significant effect on plasma ions. The results indicate that IGF-I is a potential mediator of the action of GH in seawater adaptation of salmonids.     

Liver and brain glucocorticoid receptor in rainbow trout, Oncorhynchus mykiss: down-regulation by dexamethasone.

Total glucocorticoid binding sites were identified and quantitated in liver and brain of rainbow trout using an exchange method and [3H]dexamethasone as the ligand. Both tissues contained a predominantly cytosolic moiety that bound dexamethasone with high specificity. Binding was saturable, time dependent, and completely reversible. Scatchard analysis showed a linear relationship suggesting that receptors belong to a single class. Dexamethasone down-regulated both liver and brain receptors. Down-regulation was rapid (within hours) and dose dependent (ED50 = 1.5 mg/kg body weight). Dexamethasone-induced down-regulation was not a result of cytoplasm to nuclei translocation or due to increases in tissue concentrations of steroid. Dexamethasone administration resulted in a lowering of Bmax (82.3 +/- 2.5 to 20.6 +/- 10.5 fmol/mg protein) and an increase in Kd (15.6 +/- 0.2 to 44.3 +/- 5.0 nM) suggesting a conformational change in the receptor molecule as part of the mechanism. The brain and liver of the rainbow trout thus have glucocorticoid receptors similar to those described in the mammalian system. Further, these receptors are subjected to autologous regulation similar to their counterparts in other systems.     

Disposition of genistein in rainbow trout (Oncorhynchus mykiss) and siberian sturgeon (Acipenser baeri)

Genistein (G) is a xenoestrogen from soy present in fish diet. In vivo, a 50-fold difference in sensitivity to genistein on vitellogenin (VTG) synthesis was found when comparing trout and sturgeon. This difference was not linked to the estrogen receptor affinity nor to the sensitivity of induction of the VTG pathway. The study was performed to check if differences in the G disposition in the two species could explain their difference of sensitivity to G. A pharmacokinetic analysis of radiolabeled G was performed to determine its bioavailability and metabolism in both species. G was used at levels corresponding to fish farm exposure. G plasma levels after chronic ingestion were found to be 15.6 times higher in sturgeon than in trout. Sturgeon primarily produces sulfate conjugates after G ingestion whereas trout mainly produces glucuronides. Sturgeon was able to excrete orobol glucuronide in bile. An important first pass effect was suggested in both species. No accumulation of G or its metabolites was observed in the two species. Trout muscles accounted only for 0.14 of radioactivity 48 h post-ingestion similarly to sturgeon. Trout viscera accounted for 15% of the radioactivity 48 h post-ingestion. In sturgeon, 48 h post-ingestion, viscera accounted for 21.5% of the radioactivity. These rates decreased rapidly thereafter. The study partly explains the difference in sensitivity to G, previously recorded between the two species. In addition, it shows that human exposure to G through farmed fish consumption is negligible.     

Control of adipose tissue lipid metabolism by tumor necrosis factor-alpha in rainbow trout (Oncorhynchus mykiss)

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.     

Rainbow trout (Oncorhynchus mykiss) possess two insulin-encoding mRNAs that are differentially expressed

Insulin (INS) plays a critical role in the growth, development, and metabolism of vertebrates. In this study, two unique cDNAs that encode preproinsulin were isolated, cloned and sequenced from the endocrine pancreas (Brockmann body) of rainbow trout. One 592-bp cDNA (INS 1) encodes a 105-amino acid protein and the other 587-bp cDNA (INS 2) encodes a 107-amino acid protein. The sequences share 93% nucleotide identity and 91.4% deduced amino acid identity. Quantitative real-time PCR revealed that the two INS-encoding mRNAs were differentially expressed, both in terms of distribution among tissues as well as in terms of abundance within selected tissues of juvenile trout. Both INS 1 and INS 2 mRNAs were detected in pancreas, adipose tissue, pyloric cecum, and brain; however, only INS 1 mRNA was detected in upper and lower intestine and pituitary. In all cases where INS 1 and INS 2 were co-expressed, INS 1 was more abundant. INS 1 and INS 2 also were differentially expressed in various body regions (head, body, and tail) during embryonic development. Both INS 1 and INS 2 mRNAs were detected early in development (29 days post-fertilization), but their expression declined as development proceeded (through 90 days post-fertilization); in most cases, unlike the situation in juveniles, INS 2 mRNA was more abundant than INS 1 mRNA in embryos. These findings contribute to our understanding of the evolution, distribution, and function of INS.     

Mediation of humoral catecholamine secretion by the renin-angiotensin system in hypotensive rainbow trout (Oncorhynchus mykiss)

The individual contributions of, and potential interactions between, the renin-angiotensin system (RAS) and the humoral adrenergic stress response to blood pressure regulation were examined in rainbow trout. Intravenous injection of the smooth muscle relaxant, papaverine (10 mg/kg), elicited a transient decrease in dorsal aortic blood pressure (PDA) and systemic vascular resistance (RS), and significant increases in plasma angiotensin II (Ang II) and catecholamine concentrations. Blockade of alpha-adrenoceptors before papaverine treatment prevented PDA and RS recovery, had no effect on the increase in plasma catecholamines, and resulted in greater plasma Ang II concentrations. Administration of the angiotensin-converting enzyme inhibitor, lisinopril (10(-4) mol/kg), before papaverine treatment attenuated the increases in the plasma concentrations of Ang II, adrenaline, and noradrenaline by 90, 79, and 40%, respectively and also prevented PDA and RS recovery. By itself, lisinopril treatment caused a gradual and sustained decrease in PDA and RS, and reductions in basal plasma Ang II and adrenaline concentrations. Bolus injection of a catecholamine cocktail (4 nmol/kg noradrenaline plus 40 nmol/kg adrenaline) in the lisinopril+papaverine-treated trout, to supplement their circulating catecholamine concentrations and mimic those observed in fish treated only with papaverine, resulted in a temporary recovery in PDA and RS. These results indicate that the RAS and the acute humoral adrenergic response are both recruited during an acute hypotensive stress, and have important roles in the compensatory response to hypotension in rainbow trout. However, whereas the contribution of the RAS to PDA recovery is largely indirect and relies on an Ang II-mediated secretion of catecholamines, the contribution from the adrenergic system is direct and relies at least in part on plasma catecholamines.     

Recombinant prolactin receptor extracellular domain of rainbow trout (Oncorhynchus mykiss): subcloning, preparation, and characterization

The cDNA of the extracellular domain of rainbow trout (Oncorhynchus mykiss) prolactin receptor (trPRLR-ECD) was cloned in the prokaryotic expression vector pMON to enable its expression in Escherichia coli after induction with nalidixic acid. The bacterially expressed trPRLR-ECD protein, contained within the refractile body pellet, was solubilized in 4.5 M urea, refolded, and purified on a Q-Sepharose column, pH 8, by stepwise elution with NaCl. The bioactive monomeric 26-kDa fraction was eluted in 0.2 M NaCl, yielding 20 mg/2.5 L of induced culture. The purified protein was over 98% homogeneous, as shown by SDS-PAGE in the presence or absence of reducing agent and by chromatography on a Superdex column. Binding experiments using [125I]ovine placental lactogen (oPL) as a ligand revealed that human growth hormone (hGH), oPL, and ovine prolactin (oPRL) were the most effective competitors, with respective IC50 values of 1.32, 2.27, and 2.70 nM. Chicken (ch) PRL did not compete at all, and homologous trPRL was much less effective, with a corresponding IC50 value of 1826 nM. Gel-filtration was used to determine the stoichiometry of trPRLR-ECD's interaction with oPL, hGH, and oPRL. Only oPL yielded a 2:1 complex, whereas hGH and oPRL formed only 1:1 complexes, with excess trPRLR-ECD being seen at the initial 2:1 trPRLR-ECD:hGH or trPRLR-ECD:oPRL ratios. No studies were performed with chPRL because of its inability to compete with [125I]oPL or with trPRL because of its low affinity toward trPRLR-ECD. The present results agree with previous findings indicating, as in mammals, that homologous PRL interacts transiently with its receptor and suggest that transient homologous PRL-induced homodimerization of the receptor is sufficient to initiate a biological signal, despite the fact that, in classical binding experiments, only low specific binding can be detected.     

The acute effects of food and glucose challenge on plasma thyroxine and triiodothyronine levels in previously starved rainbow trout (Oncorhynchus mykiss)

The acute effects of a single meal on plasma L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3) levels were examined in rainbow trout starved for 3 days. Plasma T4 increased within 2 hr of food intake and remained elevated to 8 hr. Plasma T3 was not altered consistently. Feeding-induced elevations in plasma T4 were present only in trout weighing less than 250 g and if they consumed a ration exceeding 0.38% of body weight. Postprandial elevations in plasma glucose paralleled those in plasma T4, suggesting a possible relationship between glucose intake and food-induced alterations in plasma T4. In trout intraperitoneally (ip) injected 4 hr earlier with 0.7% NaCl containing 0.2 or 2.0 g/kg D-glucose, plasma T4 increased relative to that in saline-injected controls. In starved trout cannulated in the dorsal aorta to permit serial blood removal, ip injection of glucose (0.85 g/kg) increased plasma glucose at 1 hr and plasma T4 at 2 hr, but did not alter plasma T3. It is concluded that enhanced glucose availability associated with feeding starved trout contributes to the postprandial elevation in plasma T4.     

Differential expression of preprosomatostatin- and somatostatin receptor-encoding mRNAs in association with the growth hormone-insulin-like growth factor system during embryonic development of rainbow trout (Oncorhynchus mykiss)

Rainbow trout were used to evaluate the relationship between the somatostatin (SS) signaling and the growth hormone (GH)-insulin-like growth factor (IGF) systems during pre-hatch and post-hatch embryonic development. The expression of preprosomatostatins (PPSS), SS receptors (SSTR), GH receptors (GHR), IGF-1, IGF-2, and IGF type 1 receptors (IGFR1) was examined in various regions at the eyed-egg (29 days post-fertilization, dpf;), post-hatch (53dpf), swim-up (68dpf), and complete yolk-absorbed (90dpf) stages. In head, PPSSI mRNA abundance increased during development while that of PPSSII' decreased and that of PPSSII' remained unchanged. In body and tail, mRNA abundance of all PPSSs remained unchanged except that of PPSSII' which declined in the tail. SSTR expression increased as development progressed in all regions with the exception of SSTR1A mRNA which remained unchanged. mRNA levels of GHR1 declined in all regions of post-hatch embryos, whereas those of GHR2 remained unchanged. Expression of IGF-1 and IGF-2 in head and tail regions increased immediately after hatching, and then declined, whereas the expression of neither IGF changed during development in the body. The expression of IGFR1 mRNAs declined in all regions, reaching their lowest levels at 90dpf, with the exception of IGFR1A mRNA in the body which remained unchanged. The general decline in the expression of GH-IGF system components during development appears inversely related to a general increase in the expression of SS system elements, and suggests that these two systems interact to regulate the tissue expansion and tissue regression of embryogenesis.