Regulatory Role of Ghrelin Hormone in Fish Energy Metabolism and Feed Intake

The aquaculture sector has been striving to sustainably respond to the global demands for protein sources. However, it persistently faces challenges in terms of environmental, nutritional and feeding inputs. One strategy to combat some of these challenges is understanding the physiological and endocrine mechanisms in fish, and the utilization of endogenous and exogenous hormones.?

Ghrelin, a 28 amino acid acylated peptide hormone, is mainly secreted by the gut mucosa (Hatef et al., 2015; Sanchez-Breta?o et al., 2015; Abtahi et al., 2017). It is the only anabolic gastrointestinal hormone that has so far been identified (Lv et al., 2018). Some studies have demonstrated that this hormone is also widely found in the peripheral and central tissues particularly in the brain (Hatef et al., 2015; Sanchez-Breta?o et al., 2015; Blanco et al., 2017b).Ghrelin may have a participation in regulating energy balance? by enhancing food intake, utilization of carbohydrates and accumulation of body fats.

Majority of the studies investigating the role and mechanism of ghrelin in energy metabolism of fish were focused on model species belonging in the Cyprinidae family such as zebrafish (Danio rerio) and goldfish (Carassius auratus). To effectively utilize this hormone, it is empirical to investigate its relationship with its receptors, enzymes and transporters,? their abundance and anatomical distribution in various cells and tissues, modulation of nutrient source and how they influence the feed intake of fish.?

Ghrelin works with two other components of the ghrelinergic system, ghrelin O-acyl transferase (GOAT) enzyme and? growth hormone secretagogue receptor (GHS- R), to elicit its regulatory role not only in energy metabolism but also in reproduction and physiological processes. These roles establish ghrelin as a multifunctional hormone. GOAT enzyme enables the unique acylation of ghrelin in the third serine, this post translational process allows the binding of the hormone to its GHS receptor (Sanchez-Breta?o et al., 2015; Mani & Zigman 2017). One recent study by Hatef and colleagues (2015), sequenced the GOAT enzyme in zebrafish and found that it exhibited similar amino acid composition to that of other fish, and that the bioactive core of the sequences between fish and mammals showed a stronger conservation. Considerably, this suggests that scientific interventions in these species may be applicable to other vertebrates, for the least. They also confirmed the expression of GOAT mRNA and found that it is highly expressed in the gut, and followed by the ovary, brain and testis. Interestingly, in goldfish, the two variants of GOAT (goat V1 and goat v2) showed the highest mRNA expression in the gonads, secondarily by the anterior intestinal tract, with lesser but significant levels in brain and pituitary (Blanco et al., 2017a). This implies variation in tissue expression among fish species.?

For the GHS receptor, Sánchez-Breta?o et al (2015) performed the RT-qPCR analysis to evaluate the expression of ghs-r1a gene in goldfish. They found that the gene is widely expressed in the brain, from the telencephalon to the cerebellum, and that it is significantly abundant in specific hypothalamic nucleus. Notably, the area where the ghs-r1a is primarily expressed, is also where the other appetite regulating hormones are located (Sanchez-Breta?o et al., 2015). The role a GHS-R1a as a mediator of the effects of ghrelin on gene expression is elucidated when all inductions in expression is abolished by the use of the receptor antagonist [D-Lys3]-GHRP6 (Blanco et al., 2017).

Another way of elucidating the relationship of the enzyme and receptor associated with ghrelin is to confirm their localization in the tissues, and determine whether they colocalize with this hormone. Hatef et al (2015) determined the GOAT and ghrelin-like immunoreactivity in zebrafish J-loop, and established that GOAT immunopositive cells were scattered within the folds and throughout the apical regions of the villi. Ghrelin-like and GOAT were also found to colocalize in some cells. In goldfish, GOAT was present in intestinal mucosa and was also found to colocalize with ghrelin in some endocrine cells (Blanco et al., 2017a). GHS receptor on the other hand, was shown to extensively colocalize with ghrelin in a cell specific manner (Sanchez-Breta?o et al., 2015).

Aside from GOAT and GHS-R, another interesting concept to investigate is whether ghrelin modulates intestinal glucose transport in fish. Glucose, an ubiquitous source of energy, is transported by facilitative glucose carriers (GLUTs) and sodium/glucose co-transporters (SGLTs). Blanco et al (2017b) revealed that in goldfish, GOAT and GSH-R1a colocalize GLUT2, SGLT1 and/or SGLT2 in the intestine. The levels of these transporters in the intestine were found to be elevated by ghrelin hormone. Furthermore, an in vivo experiment demonstrated that glucose upregulates the ghrelinergic system in goldfish intestines (Blanco et al., 2017b) . In zebrafish, ghrelin, mediated by its G-protein coupled receptor, acts on carbohydrates through inhibition of insulin and stimulation of glucagon (Cruz et al., 2010). The study of Blanco et al (2017b) also investigated the possible transduction pathways and they found that all ghrelin-evoked expression inductions are blocked by PLC inhibitor rather than the PKA inhibitor. This implies that the PLC/PKC pathway participates in the transduction and will eventually modulate the expression on GLUT and SGLT transporters following the binding of ghrelin to its receptor.

It is also interesting to know the relationship between energy balance and circadian rhythm mediated by ghrelin. In a general aquaculture setting, fish are regularly fed two to three times per day based on their biomass. Strategic intervention in this feeding schedule employing the ghrelinergic system might facilitate the best utilization of feeds, bluntly speaking, determining the ‘right timing”. This concept entails determining whether the expression of ghrelin, together with its enzyme and receptor, is influenced by feeding time and method. Furthermore, would there be other factors that might influence their expression considering the differences in nutritional status and maturity of fish.?

The study of Hatef et al (2015) revealed that zebrafish fed at the regular feeding time exhibited a decrease or remained the same in the mRNA expression of? preproghrelin (a precursor of ghrelin) before the regular feeding time. Whereas, fish that were unfed at the regular time showed a significant increase not only in the ghrelin precursor but also in the GOAT mRNA expression. In goldfish, the study of Sanchez-Breta?o et al (2015) explored the pattern of expression of ghrelin and its GHS receptor. Their results suggest that the daily regulation of ghrelin-related genes is exerted only by bioactive peptide synthesis without affecting the receptor. Moreover, the expression pattern showed a nocturnal acrophase (the time period where the cycle peaks). These results imply that ghrelin is an endocrine signal involved in the integration of gastrointestinal signals by the circadian clocks (Delgado et al., 2017). This is further supported by the study of Blanco et al (2015), elucidating the significant differences in mRNA expression among time points which resulted in significant daily rhythms in selected tissues.Taken together, there is a rhythmical pattern of expression in the hypothalamus, pituitary and gastrointestinal tract of fish.

On the other hand, it is also worth knowing what suppresses the expression of ghrelin. In the feeding experiment of Hatef et al (2015), they discovered that GOAT mRNA expression in the gut and brain of unfed zebrafish was suppressed after an injection of acylated ghrelin.This suggests a possible feedback inhibition of GOAT. Similar to this, an in vitro experiment by Blanco et al (2017a) demonstrated the downregulation of GOAT gene and protein in goldfish intestine when administered with exogenous ghrelin. The short-term exposure of fish intestinal cells to varying ghrelin concentrations (0.1, 1 and 10 nM) significantly reduced the goat transcript levels. This supports that when an excess of acylated ghrelin is present in the system, GOAT exhibits a feedback inhibition mechanism.

Overall, ghrelinergic system plays an important regulatory role in fish by helping them thrive in varying conditions of their environment particularly on the availability of food.

References:

Abtahi, S., Mirza, A., Howell, E., & Currie, P. (n.d.). Ghrelin Enhances Food Intake and Carbohydrate Oxidation in a Nitric Oxide Dependent Manner. General Comparative Endocrinology, 250, 9-14. doi:10.1016/j.ygcen.2017.05.017

Akalu, Y., Molla, M. D., Dessie, G., & Ayelign, B. (2020). Physiological Effect of Ghrelin on Body Systems. International Journal of Endocrinology, 2020(1385138), 26. Volume 2020, Article ID 1385138, 26 pages https://doi.org/10.1155/2020/1385138

Blanco, A. M., Bertucci, J. I., Ramesh, N., Delgado, M. J., Valenciano, A. I., & Unniappan, S. (2017). Ghrelin Facilitates GLUT2-, SGLT1- and SGLT2-mediated Intestinal Glucose Transport in Goldfish (Carassius auratus). Scientific Reports, 7, 45024. DOI: 10.1038/srep45024

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Cruz, S. A., Tseng, Y.-C., Kaiya, H., & Hwang, P. P. (2010). Ghrelin affects carbohydrate-glycogen metabolism via insulin inhibition and glucagon stimulation in the zebrafish (Danio rerio) brain. Comp Biochem Physiol A Mol Integr Physiol, 156(2), 190-200. 10.1016/j.cbpa.2010.01.019

Delgado, M. J., Cerda-Reverter, J. M., & Soengas, J. L. (2017). Hypothalamic integration of metabolic, endocrine, and circadian signals in fish:Involvement in the control of food intake. Frontiers in Neuroscience, 11(354), 29. doi:10.3389/fnins.2017.00354

Gray, S. M., Laura, P., & Tong, J. (2019). Ghrelin regulation of glucose metabolism. Journal of Neuroendocrinology, 31(7), e12705. doi:10.1111/jne.12705.

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Lv, Y., Liang, T., Wang, G., & Li, Z. (2018). Ghrelin, a gastrointestinal hormone, regulates energy balance and lipid metabolism. Bioscience Reports, 38, 26. https://doi.org/10.1042/BSR20181061

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