Amino Acid Carriers of the Solute Carrier Families 7 (SLC7) and 38 (SLC38) Are Involved in Leucine Sensing in the Brain of Atlantic Salmon (Salmo salar)

Sensing of amino acids in fish brain, especially branched-chain amino acids (BCAA) like leucine, is involved in regulation of feed intake through different mechanisms. However, there is limited information regarding the possible involvement of mechanisms dependent on amino acid carriers of the solute carrier families (SLC) known to be key regulators of intracellular leucine concentration, namely L-type amino acid transporter 1 (LAT1), and sodium-dependent neutral amino acid transporter 2 (SNAT2) and 9,(SNAT9), for which evidence of their participation is available in mammals. Comparative analysis amongst sequences revealed a complex pattern of paralogues in Atlantic salmon, for LAT1 (slc7a5aa, slc7a5ab, slc7a5ba, slc7a5bb, slc7a5ca, and slc7a5cb), SNAT2 (slc38a2a and slc38a2b) and SNAT9 (slc38a9). After establishing phylogenetic relationships of the different paralogues evaluated, samples of the selected brain areas were taken from Atlantic salmon to assess tissue distribution of transcripts. In an additional experiment, fish were fed two diets with different levels of leucine (high leucine: 35 g/kg vs. control leucine: 27.3 g/kg). The high leucine diet resulted in lower feed intake and increased mRNA abundance of specific paralogues of LAT1 (slc7a5aa, slc7a5ab, and slc7a5bb) and SNAT2 (slc38a2a and slc38a2b) though apparently not for SNAT9 in brain areas like hypothalamus and telencephalon involved in food intake regulation. The results obtained suggest a role for members of the SLC family in the anorectic effect of leucine and thus their involvement as additional amino acid sensing mechanism not characterised so far in fish regulation of feed intake.publishedVersio

Leucine did not stimulate growth and accretion in either stressed or unstressed Atlantic salmon

The aim of the current trial was to test whether leucine affected growth and accretion including test any effects on leucine upon stress. Quadruplicate tanks each containing 50 Atlantic salmon (Salmo salar) (mean start body weight of 524 ± 28 g) were fed diets containing 27.3, 30.0, 35.0 and 41.0 g leucine/kg diet for 74 days. Two tanks per dietary group were exposed to a stressor (5 min chasing) three days a week to test whether enriched leucine diet aid coping with chronic stress, while two tanks per dietary groups were left unstressed. The stressed fish consumed less feed and grew less than the unstressed fish, irrespective of diet. Leucine inclusion did not affect protein accretion, but leucine retention declined with increasing dietary leucine in both stressed and unstressed fish. No difference between the stressed or unstressed fish was present. Leucine did not affect relative liver size, but unstressed fish had slightly higher relative liver size compared with stressed fish (p = 0.05). Free leucine in the muscle and liver was not affected by dietary leucine, but unstressed fish had higher concentration of valine and isoleucine in the muscle compared with the stressed fish. Muscle of fish fed elevated leucine had lower mRNA expression of murf1 (p = 0.037) and higher expression of ppara (p = 0.012). Muscle of stressed fish had higher expression of the oxydative genes mnsod (p = 0.049) and catalase (p = 0.037) compared with the fish left unstressed, while in liver, there were no differences of expression of any of the genes tested. In conclusion, diets enriched in leucine had minor effects and neither protein accretion nor growth was affected in either stressed or unstressed fish.publishedVersio

The stress response in Atlantic salmon (Salmo salar L.): identification and functional characterization of the corticotropin-releasing factor (crf) paralogs

Corticotropin-Releasing Factor (CRF) is one of the main mediators of the Hypothalamic-Pituitary–Interrenal (HPI) axis to stress response. In Atlantic salmon, a comparative understanding of the crf1 paralogs role in the stress response is still incomplete. Our database searches have identified four crf1 genes in Atlantic salmon, named crf1a1, crf1a2, crf1b1 and crf1b2. Brain distribution analysis revealed that the four crf1 paralogs were widely distributed, and particularly abundant in the telencephalon, midbrain, and hypothalamus of Atlantic salmon post-smolts. To increase the knowledge on crf1-mediated response to stress, Atlantic salmon post-smolts were exposed to either repeated chasing, hypoxia or a combination of chasing and hypoxia for eight days, followed by a novel-acute stressor, confinement. Cortisol, glucose, lactate, and creatinine levels were used as markers for the stress response.publishedVersio

Impact of long-term fasting on the stomach-hypothalamus appetite regulating genes in Atlantic salmon postsmolts

Atlantic salmon will experience periods of fasting during its lifecycle. In nature, prolonged fasting periods occur owing to seasonal fluctuations in available feeds, migration or in relation to reproduction. In a culture setting, salmon is fasted mainly as part of planned operational handling prior to vaccination, delousing, transfer etc., and where fasting may last up to nine days. The mechanisms regulating the appetite during long-term fasting may vary among fish species. Here, we studied the impact of long-term fasting on neuro-endocrine regulation of appetite through the stomach-hypothalamic axis in Atlantic salmon post smolts (1.2 kg, ∼46 cm), reared in two experimental conditions (Fed and Fasted; triplicated tanks), and sampled after 4 weeks and 6 weeks of fasting. Fasted fish showed lower condition factor and hepatosomatic index at both sampling points compared to Fed group. In qPCR analysis, hypothalamic relative mRNA expression of agouti-related protein 1 (agrp1) was upregulated in fasted group at both sampling points. Among neuropeptide Y (npy) paralogs, only npya1 at 4 weeks was upregulated by fasting. As for cocaine- and amphetamine-regulated transcripts (cart), cart2a was elevated at 4 weeks, and cart2b at both 4 and 6 weeks in fasted group, while cart3a and cart4 showed no response to fasting. The pro-opiomelanocortin (pomc) a1, a2 and melanocortin-4 receptor (mc4r) a2 increased only after 6 weeks of fasting, while mc4rb1 did not respond to fasting. In stomach, 6 weeks of fasting resulted in a decrease of ghrelin1 (ghrl1), while expression of mboat4 was unaffected. The elevated levels of hypothalamic agrp1 and npya1 in fasted group support orexigenic roles for these neuropeptides. In addition, upregulation of cart2a, cart2b, pomca1 and pomca2 indicate that these play vital roles in appetite regulation and that fasting may halt and/or counteract hunger signals (agrp1 and npya1) to save energy from foraging search activities during catabolic conditions. Another possibility is that these neuropeptides play a role in fasting-induced stress. Based on the drop in mRNA expression of ghrl under catabolic conditions, we hypothesize that Ghrl might return as hunger signal once feed becomes available. We also propose that agrp1 is a potential appetite biomarker gene under feed deprived conditions.publishedVersio

Brain Distribution of 10 cart Transcripts and Their Response to 4 Days of Fasting in Atlantic Salmon (Salmo salar L.)

Cocaine- and amphetamine-regulated transcript (CART) has been known to be involved in feeding and energy balance in mammals, acting as an anorexigenic neuropeptide in hypothalamus. In Atlantic salmon, little is known about Cart brain localization and its function. In this study, in silico analysis revealed the existence of 10 cart paralogs, here named cart1a, 1b1, 1b2, 2a, 2b1, 2b2, 3a1, 3a2, 3b, and 4. The Atlantic salmon Cart sequences shared from 19 to 50% of identity with the human homolog and between 25 and 90% of sequence identity among paralogs, except for Cart4 which only shared 18–23% of identity. We further explored cart mRNA expressions in 8 brain regions (Olfactory Bulb-OB, Telencephalon-TEL, Midbrain-MB, Cerebellum-CE, Hypothalamus-HYP, Saccus vasculosus-SV, Pituitary-PT, and Brain Stem-BS) of Atlantic salmon smolt under 4 days of fasting and continuous fed conditions. The cart paralogs analyzed were widely distributed among the brain regions and OB, TEL, HYP, MB, and BS seemed to be the major sites of expression. The expression of cart1a and 1b showed quite similar pattern in MB, HYP, and BS. The expression of cart2a had the highest in MB followed by HYP and TEL. The cart3a transcript was widely distributed in rostrocaudal regions of brain except in OB and SV whereas cart3b was predominantly expressed in BS followed by MB. Expression of cart4 was high in HYP followed by TEL. With regards to effect of feeding status the Atlantic salmon cart2b, which is the most abundant among the paralogs, was upregulated after 4 days of fasting in OB, MB, and HYP compared to fed group. This may suggest an unexpected, but possible orexigenic role of cart2b in Atlantic salmon or a fasting induced stress effect. No other significant effect was observed. Collectively, the differential expressions of the cart paralogs in different brain regions suggest that they may have roles in regional integration of appetite signals and are possibly involved in regulating other brain functions in Atlantic salmon. The fact that salmon has 10 cart paralogs, while mammalians only one, opens interesting perspectives for comparative research on evolutionary adaptations of gene function in the control of appetite and energy homeostasis.publishedVersio

Effects of short term fasting on mRNA expression of ghrelin and the peptide transporters PepT1 and 2 in Atlantic salmon (Salmo salar)

Food intake is a vital process that supplies necessary energy and essential nutrients to the body. Information regarding luminal composition in the gastrointestinal tract (GIT) collected through mechanical and nutrient sensing mechanisms are generally conveyed, in both mammals and fish, to the hypothalamic neurocircuits. In this context, ghrelin, the only known hormone with an orexigenic action, and the intestinal peptide transporters 1 and 2, involved in absorption of dietary di- and tripeptides, exert important and also integrated roles for the nutrient uptake. Together, both are potentially involved in signaling pathways that control food intake originating from different segments of the GIT. However, little is known about the role of different paralogs and their response to fasting. Therefore, after 3 weeks of acclimatization, 12 Atlantic salmon (Salmo salar) post-smolt were fasted for 4 days to explore the gastrointestinal response in comparison with fed control (n = 12). The analysis covered morphometric (weight, length, condition factor, and wet content/weight fish %), molecular (gene expression variations), and correlation analyses. Such short-term fasting is a common and recommended practice used prior to any handling in commercial culture of the species. There were no statistical differences in length and weight but a significant lower condition factor in the fasted group. Transcriptional analysis along the gastrointestinal segments revealed a tendency of downregulation for both paralogous genes slc15a1a and slc15a1b and with significant lowered levels in the pyloric ceca for slc15a1a and in the pyloric ceca and midgut for slc15a1b. No differences were found for slc15a2a and slc15a2b (except a higher expression of the fasted group in the anterior midgut), supporting different roles for slc15 paralogs. This represents the first report on the effects of fasting on slc15a2 expressed in GIT in teleosts. Transcriptional analysis of ghrelin splicing variants (ghrl-1 and ghrl-2) showed no difference between treatments. However, correlation analysis showed that the mRNA expression for all genes (restricted to segment with the highest levels) were affected by the residual luminal content. Overall, the results show minimal effects of 4 days of induced fasting in Atlantic salmon, suggesting that more time is needed to initiate a large GIT response.publishedVersio

Daily rhythms of intestinal cholecystokinin and pancreatic proteases activity in Senegalese sole juveniles with diurnal and nocturnal feeding

The influence of diurnal and nocturnal feeding on daily rhythms of gut levels of cholecystokinin (CCK) and the activity of two key pancreatic proteases, trypsin and chymotrypsin, were examined in juveniles of Senegalese sole (Solea senegalensis), a species with nocturnal habits. Four feeding protocols were performed: P1) One morning meal; P2) Six meals during the light period; P3) Six meals during the dark period; and P4) 12 meals during 24 h. Daily activity patterns of both proteases were remarkably similar and showed a high correlation in all the experimental protocols. In P1, daily patterns of CCK and digestive enzymes showed a single maximum. In P2, CCK levels exhibited two peaks. Digestive enzymes activities showed slightly delayed peaks compared to CCK, although their daily fluctuations were not significant. In P3, intestinal CCK concentration exhibited two peaks at the end of light and dark periods, but only the second one was significant. The first maximum level of chymotrypsin activity occurred 4 h after the first CCK peak, while the second one coincided with the second CCK peak. Fluctuations of trypsin activity were not significant. In P4, CCK concentration showed three small peaks. Digestive enzymes daily fluctuations were not significant, although they showed an inverted trend with respect to CCK. The daily pattern of the gut CCK content in our study is in agreement with the anorexigenic function of this hormone. Our results support the existence of a negative feedback regulatory loop between CCK and pancreatic proteolytic enzymes in Senegalese sole juveniles.acceptedVersio

Exposure to cold temperatures differentially modulates neural plasticity and stress responses in post-smolt Atlantic salmon (Salmo salar)

The transfer success of farmed post-smolt Atlantic salmon (Salmo salar) to sea-cages rely on neural adaptions to promote stress resilience. As low temperatures impact physiology, this suggests that off-season transfer to cold waters may be challenging. To address this, post-smolts reared at 13 °C seawater were abruptly transferred to 10 °C, 7 °C, and 4 °C, then acclimated to these respective temperatures for 58-days followed by an acute challenge test (ACT) using confinement stress. Plasma and brain samples were collected after i) the abrupt temperature transfer at 1-h and 1-day, ii) 58-days of acclimation, and iii) 1-h post ACT. In tandem to measuring plasma cortisol levels, the expression of key genes involved in telencephalic regulation (crf, crfbp, mr, gr1, gr2 and hsd11b2) and neural plasticity (neurod, bdnf, pcna, and cfos) were analyzed. Post-smolts exposed to the 7 °C and 4 °C displayed the largest alteration in telencephalic functions, differentially regulating mr and gr1, to elevate the mr/g1 ratio for downregulating Gr1, proposing an elevated stress loads. After acclimation, these coincided with blunted stress responses capacities to ACTs for both cortisol and telencephalic neural activity (cfos), suggesting a continuation of challenges and reduced the capacity to mount a stress response. Concomitantly, these telencephalic alterations in CRs coincided with a differential modulation in neural plasticity, measured as elevated bdnf and neurod during the abrupt transfer period (acute) and after acclimation (prolonged), respectively, revealing neural responses are still robustly maintained to retain a degree of stress resilience. However, exposure of post-smolts to 4 °C clearly induced the most adverse and suppressive effects in telencephalic functions, cued by a suppression in pcna and stress response capacities, downregulation in the CRF system, and largest elevation in the mr/g1 ratio. Conversely, acclimating post-smolts to 7 °C elevated 11hsdb2 proposing a greater inhibition of cortisol action that may point to still adequate maintenance of CR and neural processes. Taken together, these findings show that cold temperatures alter key neural processes required for maintaining proper stress management, providing an alternative explanation for reductions in fish stress reactivity commonly observed with declining temperature. Therefore, exposing post-smolts at 13 °C to temperature reductions of 6 °C or greater should be avoided in aquaculture.publishedVersio

Anorectic role of high dietary leucine in farmed Atlantic salmon (Salmo salar L.): Effects on feed intake, growth, amino acid transporters and appetite-control neuropeptides.

Leucine has been identified to modulate feed intake and energy homeostasis in fish as in other vertebrates. Under allostatic conditions, energy expenditure may change, and adjustments to the processes that govern the energy homeostatic system may be necessary. We investigated the responsiveness of appetite-related neuropeptides involved in feed intake regulation in Atlantic salmon (Salmo salar) reared with high (35 g/kg leucine) or control (27.3 g/kg leucine) leucine-supplemented diets and/or under chronic stressor conditions (chasing) for eight weeks. We also analysed the response of amino acid transporters potentially involved in uptake of branched-chain amino acids (BCAA), including leucine, into areas of the brain where nutrient sensors may signal locally or to other areas involved in appetite control. At the end of the experiment, all fish were subjected to a novel-acute stressor (confinement). Our results show that fish fed with high leucine diet had a lower feed intake, growth, and hepatosomatic index (HSI) when compared to fish fed control leucine diet. In addition, increased mRNA expression of amino acid solute carrier (slc) genes in the diencephalon, and genes related to appetite control, such as proopiomelanocortin a1 (pomca1), in both the diencephalon and telencephalon, imply their involvement in leucine anorectic effect. Stress, as high leucine, reduced feed intake, growth and HSI of fish fed control or high leucine diet and antagonized the high leucine effect on the slc genes mRNA expression. An increase of neuropeptide y a1 (npya1) was observed both due to high dietary leucine and/or stress treatment which may represent a compensatory regulatory mechanism with the aim to reverse the decrease in feed intake. In summary, our results confirm an anorectic role of high dietary leucine via the activation of amino acid sensing mechanisms in the brain. Further, corticotropin-releasing hormone 1 b1 (crh1b1) and npya1 showed to play a role in the regulation of appetite in Atlantic salmon under stress conditions and/or high leucine levels.publishedVersio

Mitochondrial physiology

As the knowledge base and importance of mitochondrial physiology to evolution, health and disease expands, the necessity for harmonizing the terminology concerning mitochondrial respiratory states and rates has become increasingly apparent. The chemiosmotic theory establishes the mechanism of energy transformation and coupling in oxidative phosphorylation. The unifying concept of the protonmotive force provides the framework for developing a consistent theoretical foundation of mitochondrial physiology and bioenergetics. We follow the latest SI guidelines and those of the International Union of Pure and Applied Chemistry (IUPAC) on terminology in physical chemistry, extended by considerations of open systems and thermodynamics of irreversible processes. The concept-driven constructive terminology incorporates the meaning of each quantity and aligns concepts and symbols with the nomenclature of classical bioenergetics. We endeavour to provide a balanced view of mitochondrial respiratory control and a critical discussion on reporting data of mitochondrial respiration in terms of metabolic flows and fluxes. Uniform standards for evaluation of respiratory states and rates will ultimately contribute to reproducibility between laboratories and thus support the development of data repositories of mitochondrial respiratory function in species, tissues, and cells. Clarity of concept and consistency of nomenclature facilitate effective transdisciplinary communication, education, and ultimately further discovery