Molecular and pharmacological characterization of the melanocortin type 1 receptor in the sea bass.

Melanocortin 1 receptor (MC1R) plays a key role in the physiology of the vertebrate pigment system. Point mutations producing hyperactive or inactive receptors result in darkening or paling effects, respectively. We report the molecular and pharmacological characterization, as well as the tissue expression pattern, of the sea bass Mc1r. Similar to other MC1Rs, the sea bass gene is highly polymorphic and nine DNA polymorphisms, seven of them involving an amino acid substitution, were detected. SbMc1r is mainly expressed in the testis, fat and liver with moderate levels in the ventral and dorsal skin. The sea bass receptor was activated by all the melanocortins tested, with ACTH showing the lowest efficiency. The acetylation level of the MSH isoforms seems to be critical for the effectively of the agonist. Agouti-related protein (AGRP) drastically inhibited the basal activity of the receptor in vitro, as an inverse agonist does, but only in the presence of phosphodiesterase inhibitors. This observation suggests that sbMc1r is constitutively activated and inversely regulated by AGRP, which is expressed in the skin of different fish species.

Characterization of the sea bass melanocortin 5 receptor: a putative role in hepatic lipid metabolism.

The melanocortin 5 receptor (MC5R) plays a key role in the regulation of exocrine secretion in mammalian species. This receptor has also been characterized in some fish species but its function is unknown. We report the molecular and pharmacological characterization, as well as the tissue expression pattern, of sea bass MC5R. Cloning of five active alleles showing different levels of sensitivity to endogenous melanocortin and one non-functional allele demonstrate the allelic complexity of the MC5R locus. The sea bass receptor was activated by all the melanocortins tested, with ACTH and desacetyl-MSH and beta-MSH showing the lowest efficiency. The acetylation of the MSH isoforms seems to be critical for the effectiveness of the agonist. Agouti-related protein had no effect on basal or agonist-stimulated activation of the receptor. SbMC5R was mainly expressed in the brain but lower expression levels were found in several peripheral tissues, including liver. Progressive fasting did not induce up- or downregulation of hypothalamic MC5R expression, suggesting that central MC5R is not involved in the regulation of food intake in the sea bass. MTII, a sbMC5R agonist, stimulated hepatic lipolysis in vitro, measured as free fatty acid release into the culture medium after melanocortin agonist exposure of liver fragments, suggesting that MC5R is involved in the regulation of hepatic lipid metabolism. Taken together, the data suggest that different allelic combinations may confer differential sensitivity to endogenous melanocortin in tissues where MC5R is expressed and, by extension, in hepatic lipid metabolism.

Endogenous modification of macronutrient selection pattern in sea bass (Dicentrarchus labrax, L.).

The use of a single diet with a well defined composition to feed fish throughout their life cycle is an oversimplification that probably does not respond to their metabolic requirements. For example, the seasonal reproduction that characterizes most fish species demands changes in nutritional requirements. Bearing this in mind, the macronutrient selection pattern was studied from January to August in twelve individually housed sea bass exposed to a constant photoperiod (12L:12D h) and temperature (23+/-0.5 degrees C). The endogenous "seasonal" effect on food and energy intake regulation and macronutrient selection was determined, using protein (P), carbohydrate (CH), and fat (F) packaged separately into gelatine capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Energy intake changed monthly, the highest values being recorded in May and June and the lowest values in March and April. The preliminary results illustrated "seasonal" changes in the sea bass macronutrient selection pattern with, which showed a predominantly proteinic selection during April (53% P, 21% CH, 25% F) and lipidic in July (35% P, 19% CH, 42% F); the increase in fat selection from May to July being statistically significant. This is the first evidence supporting the existence of an endogenous rhythm in the "seasonal" energy regulation and macronutrient selection in fish through post-ingestive mechanisms and probably involving chemosensory detection in the gut and/or post-absorptive mechanisms, although the exact mechanisms involved have yet to be clarified.

Role of cholecystokinin and its antagonist proglumide on macronutrient selection in European sea bass Dicentrarchus labrax, L.

Teleost fish are able to adjust their energy intake when fed on pure macronutrient sources, although the exact mechanisms regulating macronutrient selection remain unknown. Since cholecystokinin (CCK) has been reported to modify macronutrient selection patterns in mammals, we explored the effect of CCK administered orally to European sea bass on the selection of separately encapsulated macronutrients. CCK doses of 0.05, 0.15 and 0.25 mg/kg BW administered in gelatine capsules for 5 consecutive days produced a significant inhibition of total food intake (21, 28 and 51%, respectively) at highest doses, evenly reducing the quantity of all the macronutrients ingested and, without affecting their relative proportions in the diet. Oral administration of proglumide, a non-specific CCK receptor antagonist, at doses of 5, 15 and 25 mg/kg BW, induced a quantitative total food intake increase of 2, 18 and 44%, respectively, and an increase of 52% in CH and 43% in P quantity ingested at highest dose. Co-administration of proglumide (25 mg/kg BW) and CCK (0.25 mg/kg BW) in a single preload capsule blocked the effects observed with CCK alone. In conclusion, orally administered CCK induced an anorexigenic effect on both total food and single macronutrient intake, an effect that is counteracted by the CCK antagonist proglumide.

Effect of restricted feeding schedule on seasonal shifting of daily demand-feeding pattern and food anticipatory activity in European sea bass (Dicentrarchus labrax L.).

The effect of restricted feeding schedule was investigated on the seasonal shifting of daily demand-feeding pattern and food anticipatory activity in European sea bass (Dicentrarchus labrax) held under natural environmental conditions in an outdoor laboratory. To that end, demand-feeding behavior was continuously monitored for approximately one year in four groups of 15 fish each exposed to natural fluctuations of water temperature (from 13.2 degrees C to 27.4 degrees C) and photophase (from 9.5 h to 14.5 h of light). When the animals were subjected to a time-restricted feeding schedule, the demand-feeding rhythm rapidly synchronized to the three periods of food availability: the first meal (FM) from 08:00 to 09:00 h, the second meal (SM) from 16:00 to 17:00 h, and the third meal (TM) from 00:00 to 01:00 h. The occurrence of demand-feeding activity into the three periods of food availability displayed a double seasonal shift: fish that self-fed mostly during the daytime periods of feeding availability (FM and SM) in summer and autumn changed to nocturnal feeding (TM) from December to April, returning to diurnal preferences in April. Food-demands appeared to be predominantly associated with feed availability, reaching its maximum levels during the hours of reward. In addition, feeding anticipatory activity (FAA) was observed. A relationship was detected between the duration of FAA and feeding-time, with shortest FAA (30-60 min) when mealtime occurred just after sunrise (FM) or sunset (TM). These findings demonstrate the ability of sea bass to self-feed under time-restricted schedules, and show a seasonal-phase inversion in demand-feeding activity in spite of the restrictions in their feeding availability. Sea bass can use external signals as reference to anticipate the time of feed availability. This information may be useful for designing new feeding strategies for European sea bass fish farming.

Influence of nutrient preload on encapsulated macronutrient selection in European sea bass.

The sea bass is a teleost that is able to regulate its energy intake by selecting from pure macronutrient sources, although the regulatory mechanisms involved in this selection are unknown. Nutrient preloads are known to reduce food intake and modify macronutrient selection patterns in mammals, but no information is available on its effects in fish. The aim of the present work was to determine the effect of orally administered macronutrient preloads of protein (P), fat (F) or carbohydrate (CH) on the subsequent macronutrient selection, using for the purpose feed consisting of CH, P or F packaged separately in gelatin capsules. The macronutrient preloads left the total food intake unaltered, but caused differential changes in the pattern of macronutrient selection. The CH preload increased the selection of CH (39%) and decreased that of P (20%), independently of the fish's previous nutritional preferences. The F preload induced an F increase (32%) and a P decrease (18%) in P-preferring fish, but not in F-preferring fish in which the macronutrient selection pattern remained unaffected. The P preload stimulated F selection by 42% in P-preferring fish, but left the macronutrient selection pattern unchanged in F-preferring fish. In conclusion, oral macronutrient preloads affected the pattern of macronutrient selection in fish, acting by post-ingestive mechanisms. The effect was influenced by the fish's previous nutritional preference and/or status, which could depend on its metabolic capacity.

Macronutrient selection through post-ingestive signals in sharpsnout seabream fed gelatine capsules and challenged with protein dilution.

Sharpsnout seabream ability for macronutrient self-selection was studied using gelatine capsules containing pure macronutrients. In particular, the existence of non-oropharyngeal chemosensory pathways involved on protein (P), fat (F) and carbohydrate (CH) selection, as well as sharpsnout seabream response to dietary protein dilution were investigated. In a sequence of experimental phases, sharpsnout seabream were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. The animals composed a diet containing 62.7% P, 21.3% CH and 16.0% F, in terms of macronutrient percentage intake, and this selection pattern was maintained throughout all experimental phases. In a second experiment, individually kept sharpsnout seabream were challenged with protein dilution. After protein capsules were diluted (from 91.9% P to 56.3% P) with cellulose, the animals increased their protein intake to compensate for dilution in such a way that their energy intake was not significantly modified (17.4 kJ/100 g BW vs. 17.6 kJ/100 g BW after dilution). These results show that sharpsnout seabream feeding on encapsulated diets are able to select and maintain a particular diet composition, as well as sustain their energy intake, without using the diet's oropharyngeal chemosensory properties. Moreover, they were also able to maintain their protein intake after dilution, which highlights the importance of this macronutrient in this omnivorous species.

Oral serotonin administration affects the quantity and the quality of macronutrients selection in European sea bass Dicentrarchus labrax L.

Teleost fish are able to regulate their energy intake selecting from pure macronutrients sources, but the regulatory mechanisms involved in macronutrients selection remain unknown. Serotonin (5-HT) reduces food intake in mammals and fish and modifies the macronutrients selection pattern in mammals; however, no information is available about its role on macronutrients selection in fish. The aim was to determine the effect of orally administered 5-HT (0.1, 0.5 and 2.5 mg kg BW(-)(1)) into gelatine capsules on the subsequent macronutrient selection of sea bass, using for this purpose gelatine capsules including carbohydrates, protein, or lipids separately. The voluntary ingested 5-HT was released into the plasma of fish, reaching a level two times greater than the controls, 45 min after the ingestion of a capsule containing 2.5 mg kg BW(-1) of 5-HT. The indoleamine, at doses of 0.1, 0.5 and 2.5 mg kg BW(-1), produced a reduction in total food intake of 31%, 49% and 37%, respectively, compared to the baseline, modifying the macronutrient selection pattern. The percentage of fat selected was significantly reduced whereas the percentage of protein significantly increased after administration of highest dose, but no changes were observed in the proportion of carbohydrate for any 5-HT doses. In conclusion, oral administration of 5-HT affected both amount of food intake and pattern of macronutrients selected. This is the first evidence supporting a role of 5-HT as a neurohumoral mediator involved in macronutrients selection in fish.

Effects of salinity on food intake and macronutrient selection in European sea bass.

Salinity is one of the most relevant environmental parameters in regards to fish physiology, modifying food intake and growth performance in many fish species; however, its possible effects on macronutrient selection are still unknown. The aim of this study was to determine the effects of three salinity levels (25 per thousand, 7 per thousand, and 0 per thousand) on total food intake and encapsulated macronutrient selection in a euryhaline teleost, European sea bass. A total of 40 fish (five per tank) with an average body weight of 52.4 +/- 7.1 g were used. Lowering the salinity level from 25 per thousand to 7 per thousand and 0 per thousand reduced food intake by 27% and 42%, respectively. Regarding macronutrient selection, these salinity changes significantly decreased the percentage of CH intake by 31% and 27%, while increasing that of P by 30% and 25%, respectively. Fat selection remained unaltered, with an average value of 22% for all tested salinities. Specific growth rate (SGR) and feed conversion efficiency (FCE) were affected by macronutrient selection pattern, which in turn was salinity-dependent. These results indicate a strong influence of salinity on European sea bass food intake and macronutrient selection.

Fish macronutrient selection through post-ingestive signals: effect of selective macronutrient deprivation.

Recent reports describe teleosts as being able to regulate energy intake by selecting from pure macronutrient sources, although the regulatory mechanisms involved in this selection remain unknown. The aim of the present work was to determine the effect of selective macronutrient deprivation on energy regulation and macronutrient selection, using for this purpose carbohydrate (CH), protein (P), and fat (F) packaged separately into gelatin capsules, a method that prevents the diet chemosensory properties at oropharyngeal level from interfering with macronutrient selection. Twenty-four individually housed sea bass (Dicentrarchus labrax) were subjected to two experiments: (a) two-macronutrient deprivation, and (b) one-macronutrient deprivation. In two-macronutrient deprivation, fish were fed sequentially with P, CH, or F, and in one-macronutrient deprivation, they were fed sequentially with two separately packaged macronutrients (P and CH, CH and F, or P and F). There was a rapid reduction of macronutrient intake in two-macronutrient deprivation, reaching 80% inhibition after 4, 5, and 7 days of P, CH, and F intake, respectively. In one-macronutrient deprivation, the energy intake was significantly reduced during selective F deprivation, but not with P or CH deprivation. Although the fish were being fed with only two macronutrients, the relative proportions of these macronutrients in each selective deprivation phase were the same as the baseline. These results show that in deprivation studies fish need at least F plus one other macronutrient to regulate their energy intake, and that their macronutrient selection is stable even when one is absent. In summary, fish seem to regulate energy and macronutrient selection through post-ingestive mechanisms probably involving chemosensory detection in the gut, and/or post-absorptive mechanisms.

Oral administration of melatonin reduces food intake and modifies macronutrient selection in European sea bass (Dicentrarchus labrax, L.).

Seasonal and circadian changes have been observed in dietary selection and feeding behavior of fish. It is known that the light-dark cycle is the principal mediator of the production of pineal-derived melatonin in fish, and also that the digestive tract synthesizes and secretes melatonin and indeed is the principal extrapineal source of this secretor product, suggesting that melatonin could be involved in the processes of feeding and/or digestion. Fish are capable of regulating their energy intake from separate sources of macronutrients, but the mechanisms of this selection process are unknown. In the present paper, we explored the effect of melatonin administered orally to European sea bass on their selection of encapsulated macronutrients. Melatonin doses of 0.1, 0.5, and 2.5 mg/kg body weight were administered in gelatin capsules. The voluntarily ingested melatonin was absorbed into the fish's plasma, 45 min after the administration reaching a level depending on the dose that was up to 26 times greater than the controls with the highest dose of melatonin. The indole produced a dose-dependent inhibition of total food intake of 9, 26, and 34%, respectively, and also modified the pattern of macronutrient selection. Carbohydrate intake was significantly reduced (by 17, 33, and 42% for the three doses, respectively), but the observed reductions in fat and protein intake were not statistically significant. On the contrary, fat percentage significantly increased for the highest dose, but no changes were observed in the protein or carbohydrate percentages for any of the melatonin doses. In conclusion, orally administered melatonin affected both the amount of food consumed and the pattern of macronutrients selected. This is the first evidence for the existence of neurohumoral mediators involved in the selection of macronutrients in fish.

Macronutrient selection through postingestive signals in sea bass fed on gelatine capsules.

The role of the food orosensory properties on protein (P), fat (F) and carbohydrate (CH) self-selection was investigated in fish fed gelatine capsules containing pure macronutrients. A total of 40 sea bass (39.6+/-6.2 g initial body weight) distributed in eight 75-l tanks were used. In a sequence of experimental phases, sea bass were fed a pelleted complete diet, an encapsulated complete diet or a combination of separately encapsulated pure macronutrients. In order to induce associative learning, capsules containing a given macronutrient were paired with a particular colour. Our results demonstrate that fish are able to regulate food intake, so as to balance their energy intake, when they are fed a complete encapsulated diet and, therefore, without using the orosensory properties of the diet. Moreover, sea bass learn to discriminate and select among colour-coded, pure macronutrient capsules to compose a complete and balanced diet using colour as the only external cue. The composition of selected diet was 55% P, 23% CH and 22% F in terms of macronutrient percentage. The diet orosensory properties do not seem to be necessary to regulate macronutrient intake either, suggesting that an associative learning between capsule colour and content can be established through monitoring of macronutrient intake by postingestive and/or postabsorptive mechanisms. These results provide the first insight into energy and macronutrient self-selection by fish fed on gelatine capsules containing separate macronutrients.