Abstract
Food intake in fish and mammals is orchestrated by hypothalamic crosstalk between orexigenic (food intake stimulation) and anorexigenic (food intake inhibition) signals. Some of these signals are released by peripheral tissues that are associated with energy homeostasis or nutrient availability. During the fish larva stage, orexigenic stimulation plays a critical role in individual viability. The goal of this study was to assess the mRNA levels of the main neuropeptides involved in food intake regulation (npy, agrp, carppt, and pomc), in concert with the mRNA levels and peptide levels of ghrelin, under a fasting intervention at the larval stage in zebrafish (Danio rerio). Prior to the fasting intervention, the zebrafish larva cohort was reared for 20 days post fertilization (dpf) and then randomly divided into two groups of 20 individuals. One group was subjected to a fasting intervention for 5 days (fasted group), and the other group was fed normally (fed group); this experimental protocol was performed twice independently. At the end of the fasting period, individuals from each experimental group were divided into different analysis groups, for evaluations such as relative gene expression, immunohistochemistry, and liquid chromatography coupled to nano high-resolution mass spectrometry (nLC-HRMS) analyses. The relative expression levels of the following genes were assessed: neuropeptide Y (npy), agouti-related peptide (agrp), proopiomelanocortin (pomc), cocaine and amphetamine-regulated transcript (cartpt), ghrelin (ghrl), ghrelin O-acyltransferase (mboat4), growth hormone secretagogue receptor (ghsr), and glucokinase (gck). In the fasted group, significant upregulation of orexigenic peptides (npy – agrp) and ghsr was observed, which was associated with significant downregulation of gck. The anorexigenic peptides (pomc and cartpt) did not show any significant modulation between the groups, similar to mboat4. Contrary to what was expected, the relative mRNA upregulation of the orexigenic peptides observed in the fasted experimental group could not be associated with significant ghrelin modulation as assessed by three different approaches: qPCR (relative gene expression of ghrelin), nLC-HRMS (des-acyl-ghrelin levels), and immunohistochemistry (integrated optical density of prepropeptides in intestinal and hepatopancreas tissues). Our results demonstrate that zebrafish larvae at 25 dpf exhibit suitable modulation of the relative mRNA levels of orexigenic peptides (npy and agrp) in response to fasting intervention; nevertheless, ghrelin was not coregulated by fasting. Therefore, it can be suggested that ghrelin is not an essential peptide for an increase in appetite in the zebrafish larva stage. These results give rise to new questions about food intake regulation factors in the early stages of fish.
Highlights
Food intake in mammals and fish is regulated by the hypothalamus
These appetite-regulating neurons can be divided into two populations: neurons that coexpress neuropeptide Y (Npy) and agouti-related peptide, which are orexigenic neuropeptides, and neurons that coexpress proopiomelanocortin and the cocaine and amphetamine-regulated transcript, which are anorexigenic neuropeptides (Sobrino Crespo et al, 2014; Chu et al, 2015)
Significant differences were observed between the experimental groups for gck and ghsr (p = 0.001 and p = 0.0003, respectively)
Summary
Food intake in mammals and fish is regulated by the hypothalamus. In mammals, the key nuclei associated with the key appetite-regulating role are the arcuate nucleus neurons, which coordinate regulation in the central nervous system (CNS) by projecting their axons to other main hypothalamic and extrahypothalamic areas (Cansell et al, 2012). The hypothalamus of fish presents neuroanatomical differences from that of mammals (Biran et al, 2015), with the dorsal area of the periventricular and lateral hypothalamus being the region characterized by the same neuropeptides as the arcuate nucleus (Jeong et al, 2018) These appetite-regulating neurons can be divided into two populations: neurons that coexpress neuropeptide Y (Npy) and agouti-related peptide (agrp), which are orexigenic (food intake stimulatory) neuropeptides, and neurons that coexpress proopiomelanocortin (pomc) and the cocaine and amphetamine-regulated transcript (cart), which are anorexigenic (food intake inhibitory) neuropeptides (Sobrino Crespo et al, 2014; Chu et al, 2015). Food intake regulation is orchestrated by peripheral signals, such as peptides, nutrients, or nerve pathways, which inform the CNS about energy homeostasis and nutrient availability in the body (Anand and Brobeck, 1951; Gorissen et al, 2006; Moran and Ladenheim, 2016) Peripheral peptides exert their effects mainly by binding hypothalamic neuronal receptors. Food intake regulation shares important similarities with that in mammals (Volkoff and Peter, 2006; Volkoff et al, 2009); the lateral tuberal nucleus located in the ventral hypothalamus is the teleost homolog of the arcuate nucleus (Cerdá-Reverter et al, 2003), which is integrated by key appetite-regulating neuronal populations that coexpress the above neuropeptides (Volkoff, 2016; Rønnestad et al, 2017)
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