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.     

Short-term responses of coronary circulation to cortisol and estrogen in trout (Oncorhynchus mykiss)

This study was designed to investigate the short-term effects of cortisol and 17-beta-estradiol on the intact coronary tree of rainbow trout (Oncorhynchus mykiss). A non-working, isolated, and perfused heart preparation was used. The coronary pressure was monitored together with the coronary flow in order to calculate the coronary resistance. The drug effects were expressed as percent change in coronary resistance. At concentrations higher than 10(-5) mol l(-1), cortisol elicited a significant vasoconstriction (p<0.001) within 10 min of perfusion. The simultaneous administration of cortisol and adenosine (both at 10(-4) mol l(-1)) induced a significant reduction (p<0.001) of the coronary tree response elicited by each drug alone. The perfusion of the intact coronary trout system with 20 ng ml(-1) of 17-beta-estradiol elicited a significant vasodilative response (p<0.001) within 5-15 min of perfusion. This vasodilation did not involve nitric oxide, because no significant effect of Nomega-nitro-L-arginine (L-NA, a nitric oxide synthase inhibitor) in presence of the estrogen was observed. 17-beta-estradiol was also able to reduce the vasoconstriction induced by 10(-3) mol l(-1) acetylcholine. From these results it is possible to suggest that the steroid hormones, cortisol and 17-beta-estradiol, expound their action on the trout coronary tree through a non-genomic mechanism.     

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.     

Actions of growth hormone on carbohydrate metabolism and osmoregulation of rainbow trout (Oncorhynchus mykiss)

Rainbow trout Oncorhynchus mykiss were injected intraperitoneally with slow-release implants of vegetable oil alone or containing ovine growth hormone (oGH) (2 and 5 microgg(-1) body weight), and sampled after 5 days to assess the simultaneous effects of GH on both osmoregulation and carbohydrate metabolism. An enhanced hypoosmoregulatory capacity of oGH-implanted fish is suggested by the increase observed in gill Na+,K+-ATPase activity, and the decrease observed in plasma ion concentration (Na+ and Cl-) and osmolality. GH treatment also elicited increased plasma glucose levels and metabolic changes in liver, gills, kidney, and brain. Major metabolic changes elicited by GH treatment included (1) decreased glycolytic potential and capacity for exporting glucose in liver, (2) enhanced glycogenolytic potential and capacity for use of exogenous glucose in gills and kidney, as well as increased glycolytic capacity in the later tissue, and (3) enhanced glycogenolytic and glycolytic capacities in brain. These metabolic changes elicited by GH treatment support a role for GH in the control of carbohydrate metabolism in salmonids that could be related either to the metabolic changes occurring during osmotic acclimation in nature (a process in which changes in GH levels and carbohydrate metabolism have both been reported) or to metabolic changes associated with growth.     

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.     

Growth hormone and insulin-like growth factor-1 differentially stimulate the expression of preprosomatostatin mRNAs in the Brockmann bodies of rainbow trout, Oncorhynchus mykiss

We previously characterized three cDNAs obtained from the endocrine pancreas (Brockmann body) of rainbow trout that encode for distinct preprosomatostatin (PPSS) molecules: PPSS I containing somatostain-14 (SS-14) at its C-terminus and two separate PPSS IIs, PPSS II' and PPSS II', containing [Tyr7,Gly10]-SS-14 at their C-termini. In this study, we examined the control of PPSS I, PPSS II', and PPSS II' mRNA expression by growth hormone (GH) and insulin-like growth factor-1 (IGF-1). Rainbow trout implanted with GH for 21 days displayed elevated pancreatic expression of all PPSS mRNAs compared to control animals. Growth hormone directly stimulated the expression of all pancreatic PPSS mRNAs in vitro in a dose-dependent manner; however, GH was a more potent stimulator of PPSS II' expression than of PPSS I or PPSS II' expression. Insulin-like growth factor-1 also directly stimulated the expression of PPSS mRNAs in a dose-dependent manner in Brockmann bodies incubated in vitro; IGF-1 was a more potent stimulator of PPSS I and PPSS II' expression than of PPSS II' expression. These results indicate that the expression of PPSS mRNAs in the Brockmann body of trout is differentially regulated by GH and IGF-1 and suggest that SS mediate the feedback regulation of GH and IGF-1.     

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.     

Two different messenger RNAs for salmon gonadotropin-releasing hormone are expressed in rainbow trout (Oncorhynchus mykiss) brain.

Two different precursor genes encoding the decapeptide salmon GnRH (sGnRH) are present in most salmonid species. In rainbow trout, a precedent Southern blot study revealed the existence of two different sGnRH genes and, recently, two different genes and their complementary DNAs that encode the identical peptide sGnRH were isolated from ovary and testis. Our study confirms the existence of two different mRNAs encoding sGnRH (sGnRH mRNA-I and sGnRH mRNA-II) in the brain of rainbow trout and, for the first time, full-length complementary DNA sequences are given. Central and peripheral distributions of the two messengers are described and seem to indicate different regulation of their expression. sGnRH mRNA-I is found essentially in the olfactory bulbs and telencephalon, whereas sGnRH mRNA-II is more widely expressed in the brain. Our observations allow speculation on the respective roles of two genes encoding the same decapeptide.     

Effects of environmental temperature on IGF1, IGF2, and IGF type I receptor expression in rainbow trout (Oncorhynchus mykiss)

Recently, we have demonstrated in rainbow trout that environmental temperature may, independently of nutritional status, directly stimulate plasma growth hormone (GH) that is recognised as being an insulin-like growth factor (IGF) system regulator. The aim of this study was to determine whether temperature may directly regulate the IGF system or indirectly regulate it through plasma GH or nutritional status. For this purpose, rainbow trout were reared at 8, 12, or 16 degrees C and fed either ad libitum (similar nutritional status) to evidence the global effect of temperature, or with the same ration (1.2% body weight/day), to determine the temperature effect in fish with the same growth rate. Endocrine and autocrine/paracrine regulations of the IGF system were determined by measuring plasma IGF1 and IGF2, liver and muscle IGF1 and IGF2 mRNA as well as IGFRIa, IGFRIb mRNA, and the quantity of IGF type I receptor in muscle. Our results show that neither rearing temperature nor the nutritional status of fish affected the expression of both IGF receptor genes in muscle. Nevertheless, the quantity of IGF type I receptor determined by a binding study, appeared to be inversely proportional (P<0.05) to the rearing temperature without any relationship with nutritional status, suggesting a direct effect of temperature on its turnover. After 2 weeks of treatment, the levels of IGF1 mRNA in muscle at 8 degrees C were 2-fold higher (P<0.05) than at 16 degrees C in both ad libitum and restricted feed fish, whereas after 6 weeks, this difference was no longer observed. In both experiments, the levels of plasma IGF2 were 10-fold higher than the levels of plasma IGF1 (mean 105+/-3.0 versus 13.5+/-0.6 ng/ml), and plasma levels were correlated with their respective mRNA liver concentrations (r2=0.14 and 0.25, respectively; P<0.01). In the ad libitum feeding experiment, plasma and mRNA levels of IGF1 were related to the rearing temperature (P<0.05), while for IGF2 no effect was seen. In contrast, in the restricted feeding experiment, plasma and IGF2 mRNA levels were inversely proportional to the rearing temperature (P<0.0001) while plasma IGF1 was unaltered. Levels of plasma IGF1 were related to the growth rate in both experiments, while levels of plasma IGF2 appeared to be associated with the nutritional status of the fish. Our results suggest that the autocrine/paracrine expression of IGF1 and IGF2 in muscle is not a key regulator of the growth promoting effect of temperature. Conversely, temperature seems to promote growth through IGF1 secretion by the liver following GH stimulation, and impairment of nutritional status would prevent the IGF1 stimulation by temperature. In addition, the growth-promoting effect of temperature did not affect plasma IGF2, which appeared to be more related to the metabolic status of the fish.     

Transforming growth factor beta inhibits steroidogenic acute regulatory (StAR) protein expression in human ovarian thecal cells

In this study, we investigated the effects of TGFβ1 on steroidogensis and expression of the steroidogenic acute regulatory (StAR) protein which regulates an important early step in the steroidogenic pathway. We utilized a human ovarian thecal like tumor (HOTT) cell model and investigated the effects of activin-A, inhibin-A, or TGFβ1 in the presence of forskolin and the effect of dibutyryl cyclic AMP (dbcAMP) on steroid accumulation in the culture medium. Cells were also treated with different concentration of TGFβ1 in the presence of forskolin, combined steroid production was measured at the end of 48 h and after 3 h incubation with 22R-hydroxycholesterol. In the presence of TGFβ1 there was a dose-dependent inhibition of androstenedione production. Inhibition in combined steroid production was apparent at the highest concentration of TGFβ1 tested. In the presence of 22R-hydroxycholesterol, combined steroid production was significantly inhibited at lower concentrations. TGFβ1 inhibited StAR protein expression in a concentration dependent manner. There was also a similar inhibition in StAR mRNA. These results suggest that the effect of TGFβ1 on steroid production and possibly follicular development may be in part due to its effects on StAR expression.     

Corticotropin-releasing factor and neuropeptide Y mRNA levels are modified by glucocorticoids in rainbow trout, Oncorhynchus mykiss

The primary stress response involves neuronal activation that ultimately leads to the release of glucocorticoids. Circulating glucocorticoids are thought to influence their own synthesis and release through a negative feedback mechanism that inhibits the activity of the hypothalamic and pituitary components of the stress axis. This study was designed to address the hypothesis that glucocorticoids modify corticotropin-releasing factor (CRF) and neuropeptide Y (NPY) mRNA levels in the rainbow trout (Oncorhynchus mykiss) brain. Cortisol implantation significantly reduced CRF1 and NPY mRNA levels in fish exposed to an isolation stress. In contrast, cortisol implantation did not prevent the stress-induced elevation of CRF1 and NPY mRNA levels during confinement. Treatment with the glucocorticoid receptor antagonist RU-486 reduced CRF1 mRNA levels in both isolated and confined fish, but had no effect on NPY mRNA. Although the cytochrome P450 inhibitor metyrapone reduced ACTH-induced cortisol secretion in vitro, plasma cortisol levels were elevated in isolated trout treated with metyrapone. Nevertheless, metyrapone implantation increased CRF1 and NPY mRNA levels in confined fish. Together, these results implicate cortisol as a modulator of CRF and NPY mRNA levels in the preoptic area of the trout brain, but that cortisol is only one such regulating mechanism.