Agouti overexpression in a transgenic model regulates integrity, permeability and electrogenic amino acid transport in zebrafish intestine

Overexpression of asip1 in transgenic zebrafish disrupts dorsoventral pigment pattern in addition to increasing food intake levels and linear growth. A higher feed intake is unnecessary in transgenic fish to enable larger and heavier growth. A plausible explanation may rely on the enhanced feeding efficiency mediated by improved nutrient absorption in transgenic animals. To test this hypothesis, wide scope transcriptomic techniques were used to elucidate the potential pathways involved in the enhanced nutrient absorption and intestinal epithelium permeability/integrity. In addition, the electrogenic capacity for amino acid transport was analysed. Transcriptomic analysis reveal that amino acid, monocarboxylates, ionic and vitamin transmembrane transporters were substantially modified. Enrichment analysis also revealed an inhibition of intestinal lipid metabolism and down-regulation of KEGG pathways related to membrane integrity suggesting augmented intestinal laxity that may enhance paracellular transport. Electrophysiological experiments carried out in Ussing chambers show that asip1 overexpression decrease membraned tissue resistance (Rt), indicating a modification of the intestinal barrier function in ASIP1 transgenic animals. Similarly, paracellular permeability was higher in transgenic zebrafish. Both the decrease in Rt and the increase in permeability point to an ASIP1-dependent decrease in the tissue barrier function. Electrogenic amino acid transport was also enhanced in transgenic animals providing strong indication that ASIP1 fish can extract more amino acids from their diet at similar feeding levels. Both transcriptomic and electrophysiological results suggest that asip1-overexpressing zebrafish display improved nutrient absorption and by extension a higher feed efficiency which explains enhanced growth in the absence of augmented food intake. The enhanced growth of ASIP1 zebrafish potentially mediated by improved nutrient uptake and feed efficiency suggests that the melanocortin system, specifically asip1 overexpression, is a potential target for the development of genetically engineered fish displaying improved performance and no differential lipid accumulation.info:eu-repo/semantics/publishedVersio

Insights into the Function and Evolution of Taste 1 Receptor Gene Family in the Carnivore Fish Gilthead Seabream (Sparus aurata)

A plethora of molecular and functional studies in tetrapods has led to the discovery of multiple taste 1 receptor (T1R) genes encoding G-protein coupled receptors (GPCRs) responsible for sweet (T1R2 + T1R3) and umami (T1R1 + T1R3) taste. In fish, the T1R gene family repertoires greatly expanded because of several T1R2 gene duplications, and recent studies have shown T1R2 functional divergence from canonical mammalian sweet taste perceptions, putatively as an adaptive mechanism to develop distinct feeding strategies in highly diverse aquatic habitats. We addressed this question in the carnivore fish gilthead seabream (Sparus aurata), a model species of aquaculture interest, and found that the saT1R gene repertoire consists of eight members including saT1R1, saT1R3 and six saT1R2a-f gene duplicates, adding further evidence to the evolutionary complexity of fishT1Rs families. To analyze saT1R taste functions, we first developed a stable gene reporter system based on Ca2+-dependent calcineurin/NFAT signaling to examine specifically in vitro the responses of a subset of saT1R heterodimers to L-amino acids (L-AAs) and sweet ligands. We show that although differentially tuned in sensitivity and magnitude of responses, saT1R1/R3, saT1R2a/R3 and saT1R2b/R3 may equally serve to transduce amino acid taste sensations. Furthermore, we present preliminary information on the potential involvement of the Gi protein alpha subunits saGαi1 and saGαi2 in taste signal transduction

Molecular characterization of taste receptors in the seabream

Trabajo presentado en el 11º Congreso de la Asociación Ibérica de Endocrinología Comparada (AIEC), celebrado en Vigo (España), del 13 al 15 de julio de 2017Peer reviewe

Taste sensing and gut feeling: Possible involvement of taste receptor type 1 family in seabream Sparus aurata.

Trabajo presentado en Aquaculture Europe 2019, celebrado en Berlín (Alemania), del 7 al 10 de octubre de 2019Taste perception of sweet and umami stimuli in vertebrates is largely controlled by a family of G-protein-coupled receptors, the taste receptor type 1 (T1R) family, with three members that combine in heterodimers to form the taste receptors. The mammalian T1R1/T1R3 receptor responds to umami compounds such as amino acids, whereas T1R2/T1R3 is activated by sweet substances. In mammals, it has been well established that taste sensing in the oral cavity (taste buds) not only guides the consumption of foods, but the presence of taste receptors in specialized cells of the gastrointestinal tract (enteroendocrine cells) can also regulate digestive, absorptive and metabolic functions through gut sensing mechanisms (Alpers, 2010; Depoortere, 2014). In fish, however, very little information is available, particularly in species of aquaculture interest. We have previously cloned the full-length cDNA of five T1R genes in Sparus aurata (sa), including the specific heterodimer subunit of the umami taste (saT1R1), three novel sweet taste duplicate genes (saT1R2x, saT1R2y, saT1R2z) and the saT1R3 gene common to both umami and sweet taste heterodimers. The objective of this study was to further characterize their functional properties and patterns of expression in both larval and adult seabream tissues

Regulación sensorial de la ingesta en peces: aspectos moleculares y comportamentales

Trabajo presentado en el XVIII Congreso nacional de acuicultura, celebrado en Cádiz (España) del 21 al 24 de noviembre de 2022

Taste beyond oral perception in seabream sparus aurata: new evidence for functional gut chemical sensing in fish

Resumen del trabajo presentado en Aquaculture Europe 2022, celebrado en Rimini (Italia) del 27 al 30 de septiembre de 2022.[Introduction]: Taste receptors (TRs) are classically associated to the perception of gustatory sensations in oral tissues. However, many TRs are also found in different body tissues including the gastrointestinal tract (GIT), where they perform important roles (Finger and Kinnamon, 2011). In the GIT, the presence of TRs in specialized cells (enteroendocrine cells: EECs) regulates digestive, absorptive and metabolic functions through gut sensing mechanisms (Alpers, 2010). Functional actions of TRs are associated to their co-expression with gut peptides, including ghrelin (GHR), cholecystokinin (CCK), glucagon-like peptide-1 (GLP-1) or peptide YY (PYY), to cite a few. Once a TR is activated, an intracellular signaling cascade is initiated, terminating with peptide secretion in the EEC basolateral side, which can signal neighboring (paracrine action) or remote (endocrine action) cells, or activate afferent neurons (neural action). The taste receptor type 1 (T1R) family is one of the TR types that is commonly expressed in EECs, acting as heterodimeric sensors of nutrients in the mammalian gut: T1R1/ T1R3 responding to umami compounds such as amino acids, and T1R2/T1R3 to sweet substances. We previously showed the expression of the Sparus aurata (sa) T1R gene repertoire in oropharyngeal, GIT and brain tissues. However, this does not necessarily imply a conserved role in gut sensing. Furthermore, expression in GIT was observed in early larval stages but no expression was found in oropharyngeal tissues up to 12 days post-hatching (dph), which was puzzling. The present study aimed to provide direct evidence for mRNAs co-expression of saT1R genes (mostly t1r3, as the common element of both receptors) with EEC-type peptides such as ghr, cck, pyy and proglucagon (pg), to establish a morphological link indicating possible roles of T1R in gut nutrient-sensing mechanisms and in the regulation of fish digestive processes. A second objective was to extend the period of investigation during larval ontogeny to establish the temporal pattern of expression in GIT and oral tissues for the entire saT1R gene repertoire in relation to first-feeding

Exploring the potential for an evolutionarily conserved role of the taste 1 receptor gene family in gut sensing mechanisms of fish

In this study, we investigated the transcriptional spatio-temporal dynamics of the taste 1 receptor (T1R) gene family repertoire in seabream (Sparus aurata [sa]), during larval ontogeny and in adult tissues. In early larval development, saT1R expression arises heterochronously, i.e. the extraoral taste-related perception in the gastrointestinal tract (GIT) anticipates first exogenous feeding (at 9 days post hatching [dph]), followed by the buccal/intraoral perception from 14 dph onwards, supporting the hypothesis that the early onset of the molecular machinery underlying saT1R expression in the GIT is not induced by food but rather genetically hardwired. During adulthood, we characterized the expression patterns of saT1R within specific tissues (n = 4) distributed in oropharingeal, GIT and brain regions substantiating their functional versatility as chemosensory signaling players to a variety of biological functions beyond oral taste sensation. Further, we provided for the first time direct evidences in fish for mRNA co-expression of a subset of saT1R genes (mostly saT1R3, i.e. the common subunit of the heterodimeric T1R complexes for the detection of “sweet” and “umami” substances), with the selected gut peptides ghrelin (ghr), cholecystokinin (cck), hormone peptide yy (pyy) and proglucagon (pg). Each peptide defines the enteroendocrine cells (ECCs) identity, and establishes on morphological basis, a direct link for T1R chemosensing in the regulation of fish digestive processes. Finally, we analyzed the spatial gene expression patterns of 2 taste signaling components functionally homologous to the mammalian G(i)α subunit gustducin, namely saG(i)α1 and saG(i)α2, and demonstrated their co-localization with the saT1R3 in EECs, thus validating their direct involvement in taste-like transduction mechanisms of the fish GIT. In conclusion, data provide new insights in the evolutionary conservation of gut sensing in fish suggesting a conserved role for nutrient sensors modulating entero-endocrine secretion.This research was funded by LUCTA SA. Some aspects were covered by the National Research Agency (AEI, Spain) (grant number: PID2019-103969RB-C33) to Jose M. Cerda-Reverter.Peer reviewedPeer reviewedPeer reviewe

Dietary discrimination of positive and negative organoleptic cues in gilthead seabream (Sparus aurata) in relation to its physiological state

Trabajo presentado en el XX International symposium on fish nutrition and feeding towards precision fish nutrition and feeding, celebrado en Sorrento (Italia) del 05 al 09 de junio de 2022

The ontogeny and brain distribution dynamics of the appetite regulators NPY, CART and pOX in larval Atlantic cod (Gadus morhua L.)

Similar to many marine teleost species, Atlantic cod undergo remarkable physiological changes during the early life stages with concurrent and profound changes in feeding biology and ecology. In contrast to the digestive system, very little is known about the ontogeny and the localization of the centers that control appetite and feed ingestion in the developing brain of fish. We examined the expression patterns of three appetite regulating factors (orexigenic: neuropeptide Y, NPY; prepro-orexin, pOX and anorexigenic: cocaine- and amphetamine-regulated transcript, CART) in discrete brain regions of developing Atlantic cod using chromogenic and double fluorescent in situ hybridization. Differential temporal and spatial expression patterns for each appetite regulator were found from first feeding (4 days post hatch; dph) to juvenile stage (76 dph). Neurons expressing NPY mRNA were detected in the telencephalon (highest expression), diencephalon, and optic tectum from 4 dph onward. CART mRNA expression had a wider distribution along the anterior-posterior brain axis, including both telencephalon and diencephalon from 4 dph. From 46 dph, CART transcripts were also detected in the olfactory bulb, region of the nucleus of medial longitudinal fascicle, optic tectum and midbrain tegmentum. At 4 and 20 dph, pOX mRNA expression was exclusively found in the preoptic region, but extended to the hypothalamus at 46 and 76 dph. Co-expression of both CART and pOX genes were also observed in several hypothalamic neurons throughout larval development. Our results show that both orexigenic and anorexigenic factors are present in the telencephalon, diencephalon and mesencephalon in cod larvae. The telencephalon mostly contains key factors of hunger control (NPY), while the diencephalon, and particularly the hypothalamus may have a more complex role in modulating the multifunctional control of appetite in this species. As the larvae develop, the overall progression in temporal and spatial complexity of NPY, CART and pOX mRNAs expression might be correlated to the maturation of appetite control regulation. These observations suggest that teleost larvae continue to develop the regulatory networks underlying appetite control after onset of exogenous feeding