An ACE2-Alamandine Axis Modulates the Cardiac Performance of the Goldfish Carassius auratus via the NOS/NO System.

Alamandine is a peptide of the Renin Angiotensin System (RAS), either generated from Angiotensin A via the Angiotensin Converting Enzyme 2 (ACE2), or directly from Ang-(1-7). In mammals, it elicits cardioprotection via Mas-related G-protein-coupled receptor D (MrgD), and the NOS/NO system. In teleost fish, RAS is known to modulate heart performance. However, no information is available on the presence of a cardioactive ACE2/Alamandine axis. To fill this gap, we used the cyprinid teleost Carassius auratus (goldfish) for in silico and in vitro analyses. Via the NCBI Blast P suite we found that in cyprinids ace2 is phylogenetically detectable in a subcluster of proteins including ace2-like isoforms, and is correlated with a hypoxia-dependent pathway. By real-time PCR, Western Blotting, and HPLC, ACE2 and Alamandine were identified in goldfish heart and plasma, respectively. Both increased after chronic exposure to low O2 (2.6 mg O2 L-1). By using an ex-vivo working goldfish-heart preparation, we observed that in vitro administration of exogenous Alamandine dose-dependently stimulates myocardial contractility starting from 10-11 M. The effect that involved Mas-related receptors and PKA occurred via the NOS/NO system. This was shown by exposing the perfused heart to the NOS inhibitor L-NMMA (10-5 M) that abolished the cardiac effect of Alamandine and was supported by the increased expression of the phosphorylated NOS enzyme in the extract from goldfish heart exposed to 10-10 M Alamandine. Our data are the first to show that an ACE2/Alamandine axis is present in the goldfish C. auratus and, to elicit cardiac modulation, requires the obligatory involvement of the NOS/NO system.

Cytoskeletal Responses and Aif-1 Expression in Caco-2 Monolayers Exposed to Phorbol-12-Myristate-13-Acetate and Carnosine.

The dis(re)organization of the cytoskeletal actin in enterocytes mediates epithelial barrier dys(re)function, playing a key role in modulating epithelial monolayer's integrity and remodeling under transition from physiological to pathological states. Here, by fluorescence-based morphological and morphometric analyses, we detected differential responses of cytoskeletal actin in intestinal epithelial Caco-2 cell monolayers at two different stages of their spontaneous differentiation, i.e., undifferentiated cells at 7 days post-seeding (dps) and differentiated enterocyte-like cells at 21 dps, upon challenge in vitro with the inflammation-mimicking stimulus of phorbol-12-myristate-13-acetate (PMA). In addition, specific responses were found in the presence of the natural dipeptide carnosine detecting its potential counteraction against PMA-induced cytoskeletal alterations and remodeling in differentiated Caco-2 monolayers. In such an experimental context, by both immunocytochemistry and Western blot assays in Caco-2 monolayers, we identified the expression of the allograft inflammatory factor 1 (AIF-1) as protein functionally related to both inflammatory and cytoskeletal pathways. In 21 dps monolayers, particularly, we detected variations of its intracellular localization associated with the inflammatory stimulus and its mRNA/protein increase associated with the differentiated 21 dps enterocyte-like monolayer compared to the undifferentiated cells.

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.

Leptin receptor-deficient (knockout) zebrafish: Effects on nutrient acquisition.

In mammals, knockout of LEPR results in a hyperphagic, morbid obese, and diabetic phenotype, which supports that leptin plays an important role in the control of appetite and energy metabolism, and that its receptor, LEPR, mediates these effects. To date, little is known about the role(s) of lepr in teleost physiology. We investigated a zebrafish (Danio rerio) homozygous lepr knockout (lepr-/-) line generated by CRISPR/Cas9 in comparison to its wt counterpart with respect to nutrient acquisition, energy allocation, and metabolism. The metabolic characterization included oxygen consumption rate and morphometric parameters (yolk sac area, standard length, wet weight, and condition factor) as proxies for use and allocation of energy in developing (embryos, larvae, and juveniles) zebrafish and showed no particular differences between the two lines, in agreement with previous studies. One exception was found in oxygen consumption at 72 hpf, when zebrafish switch from embryonic to early larval stages and food-seeking behavior could be observed. In this case, the metabolic rate was significantly lower in lepr-/- than in wt. Both phenotypes showed similar responses, with respect to metabolic rate, to acute alterations (22 and 34 °C) in water temperature (measured in terms of Q10 and activation energy) compared to the standard (28 °C) rearing conditions. To assess lepr involvement in signaling the processing and handling of incoming nutrients when an exogenous meal is digested and absorbed, we conducted an in vivo analysis in lepr-/- and wt early (8 days post-fertilization) zebrafish larvae. The larvae were administered a bolus of protein hydrolysate (0%, 1%, 5%, and 15% lactalbumin) directly into the digestive tract lumen, and changes in the mRNA expression profile before and after (1 and 3 h) administration were quantified. The analysis showed transcriptional differences in the expressions of genes involved in the control of appetite and energy metabolism (cart, npy, agrp, and mc4r), sensing (casr, t1r1, t1r3, t1r2-1, t1r2-2, pept1a, and pept1b), and digestion (cck, pyy, try, ct, and amy), with more pronounced effects observed in the orexigenic than in the anorexigenic pathways, suggesting a role of lepr in their regulations. Differences in the mRNA levels of these genes in lepr-/-vs. wt larvae were also observed. Altogether, our analyses suggest an influence of lepr on physiological processes involved in nutrient acquisition, mainly control of food intake and digestion, during early development, whereas metabolism, energy allocation, and growth seem to be only slightly influenced.

Morpho-functional remodelling of the adult zebrafish (Danio rerio) heart in response to waterborne angiotensin II exposure.

Angiotensin II (AngII), the principal effector of the Renin-Angiotensin System, is a pluripotent humoral agent whose biological actions include short-term modulations and long-term adaptations. In fish, short-term cardio-tropic effects of AngII are documented, but information on the role of AngII in long-term cardiac remodelling is not fully understood. Here, we describe a direct approach to disclose long-term morpho-functional effects of AngII on the zebrafish heart. Adult fish exposed to waterborne teleost analogue AngII for 8 weeks showed enhanced heart weight and cardio-somatic index, coupled to myocardial structural changes (i.e. augmented compacta thickness and fibrosis), and increased heart rate. These findings were paralleled by an up-regulation of type-1 and type-2 AngII receptors expression, and by changes in the expression of GATA binding protein 4, nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 and superoxide dismutase 1 soluble mRNAs, as well as of cytochrome b-245 beta polypeptide protein, indicative of cardiac remodelling. Our results suggest that waterborne AngII can sustain and robustly affect the cardiac morpho-functional remodelling of adult zebrafish.

Correction to: The peptide transporter 1a of the zebrafish Danio rerio, an emerging model in nutrigenomics and nutrition research: molecular characterization, functional properties, and expression analysis.

[This corrects the article DOI: 10.1186/s12263-019-0657-3.].

The peptide transporter 1a of the zebrafish Danio rerio, an emerging model in nutrigenomics and nutrition research: molecular characterization, functional properties, and expression analysis.

BACKGROUND: Peptide transporter 1 (PepT1, alias Slc15a1) mediates the uptake of dietary di/tripeptides in all vertebrates. However, in teleost fish, more than one PepT1-type transporter might function, due to specific whole genome duplication event(s) that occurred during their evolution leading to a more complex paralogue gene repertoire than in higher vertebrates (tetrapods). RESULTS: Here, we describe a novel di/tripeptide transporter in the zebrafish (Danio rerio), i.e., the zebrafish peptide transporter 1a (PepT1a; also known as Solute carrier family 15 member a1, Slc15a1a), which is a paralogue (78% similarity, 62% identity at the amino acid level) of the previously described zebrafish peptide transporter 1b (PepT1b, alias PepT1; also known as Solute carrier family 15 member 1b, Slc15a1b). Also, we report a basic analysis of the pept1a (slc15a1a) mRNA expression levels in zebrafish adult tissues/organs and embryonic/early larval developmental stages. As assessed by expression in Xenopus laevis oocytes and two-electrode voltage clamp measurements, zebrafish PepT1a, as PepT1b, is electrogenic, Na+-independent, and pH-dependent and functions as a low-affinity system, with K 0.5 values for Gly-Gln at - 60 mV of 6.92 mmol/L at pH 7.6 and 0.24 mmol/L at pH 6.5 and at - 120 mV of 3.61 mmol/L at pH 7.6 and 0.45 mmol/L at pH 6.5. Zebrafish pept1a mRNA is highly expressed in the intestine and ovary of the adult fish, while its expression in early development undergoes a complex trend over time, with pept1a mRNA being detected 1 and 2 days post-fertilization (dpf), possibly due to its occurrence in the RNA maternal pool, decreasing at 3 dpf (~ 0.5-fold) and increasing above the 1-2 dpf levels at 4 to 7 dpf, with a peak (~ 7-fold) at 6 dpf. CONCLUSIONS: We show that the zebrafish PepT1a-type transporter is functional and co-expressed with pept1b (slc15a1b) in the adult fish intestine. Its expression is also confirmed during the early phases of development when the yolk syncytial layer is present and yolk protein resorption processes are active. While completing the missing information on PepT1-type transporters function in the zebrafish, these results open to future investigations on the similar/differential role(s) of PepT1a/PepT1b in zebrafish and teleost fish physiology.