Linking biochemical and individual-level effects of chlorpyrifos, triphenyl phosphate, and bisphenol A on sea urchin (Paracentrotus lividus) larvae.

The effects of three relevant organic pollutants: chlorpyrifos (CPF), a widely used insecticide, triphenyl phosphate (TPHP), employed as flame retardant and as plastic additive, and bisphenol A (BPA), used primarily as plastic additive, on sea urchin (Paracentrotus lividus) larvae, were investigated. Experiments consisted of exposing sea urchin fertilized eggs throughout their development to the 4-arm pluteus larval stage. The antioxidant enzymes glutathione reductase (GR) and catalase (CAT), the phase II detoxification enzyme glutathione S-transferase (GST), and the neurotransmitter catabolism enzyme acetylcholinesterase (AChE) were assessed in combination with responses at the individual level (larval growth). CPF was the most toxic compound with 10 and 50% effective concentrations (EC10 and EC50) values of 60 and 279 µg/l (0.17 and 0.80 µM), followed by TPHP with EC10 and EC50 values of 224 and 1213 µg/l (0.68 and 3.7 µM), and by BPA with EC10 and EC50 values of 885 and 1549 µg/l (3.9 and 6.8 µM). The toxicity of the three compounds was attributed to oxidative stress, to the modulation of the AChE response, and/or to the reduction of the detoxification efficacy. Increasing trends in CAT activity were observed for BPA and, to a lower extent, for CPF. GR activity showed a bell-shaped response in larvae exposed to CPF, whereas BPA caused an increasing trend in GR. GST also displayed a bell-shaped response to CPF exposure and a decreasing trend was observed for TPHP. An inhibition pattern in AChE activity was observed at increasing BPA concentrations. A potential role of the GST in the metabolism of CPF was proposed, but not for TPHP or BPA, and a significant increase of AChE activity associated with oxidative stress was observed in TPHP-exposed larvae. Among the biochemical responses, the GR activity was found to be a reliable biomarker of exposure for sea urchin early-life stages, providing a first sign of damage. These results show that the integration of responses at the biochemical level with fitness-related responses (e.g., growth) may help to improve knowledge about the impact of toxic substances on marine ecosystems.

In vitro insulin treatment reverses changes elicited by nutrients in cellular metabolic processes that regulate food intake in fish.

This research assessed the direct effects of insulin on nutrient-sensing mechanisms in the brain of rainbow trout (Oncorhynchus mykiss) using an in vitro approach. Cultured hypothalamus and hindbrain were exposed to 1 µmol l-1 insulin for 3 h, and signals involved in appetite regulation and nutrient-sensing mechanisms were measured. Additionally, the involvement of the phosphatidylinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway in the actions of insulin was studied by using the inhibitor wortmannin. Treatment with insulin alone did not elicit many changes in the appetite regulators and nutrient-sensing-related genes and enzymes tested in the hypothalamus and hindbrain. However, we found that, when insulin and nutrients were added together, insulin reversed most of the effects exerted by nutrients alone, suggesting that insulin changes responsiveness to nutrients at the central level. Effects reversed by insulin included expression levels of genes related to the sensing of both glucose (slc2a2, slc5a1, gck, pck1, pklr, g6pcb, gys1, tas1r3 and nr1h3 in the hindbrain, and slc2a2, pklr and pck1 in the hypothalamus) and fatty acid (cd36 in the hindbrain, and cd36 and acly in the hypothalamus). Nutrient-induced changes in the activity of Acly and Cpt-1 in the hindbrain and of Pepck, Acly, Fas and Hoad in the hypothalamus were also reversed by insulin. Most of the insulin effects disappeared in the presence of wortmannin, suggesting the PI3K/Akt pathway is a mediator of the effects of insulin reported here. This study adds new information to our knowledge of the mechanisms regulating nutrient sensing in fish.

Nesfatin-1 Regulates Feeding, Glucosensing and Lipid Metabolism in Rainbow Trout.

Nesfatin-1 is an 82 amino acid peptide that has been involved in a wide variety of physiological functions in both mammals and fish. This study aimed to elucidate the role of nesfatin-1 on rainbow trout food intake, and its putative effects on glucose and fatty acid sensing systems. Intracerebroventricular administration of 25 ng/g nesfatin-1 resulted in a significant inhibition of appetite, likely mediated by the activation of central POMC and CART. Nesfatin-1 stimulated the glucosensing machinery (changes in sglt1, g6pase, gsase, and gnat3 mRNA expression) in the hindbrain and hypothalamus. Central fatty acid sensing mechanisms were unaltered by nesfatin-1, but this peptide altered the expression of mRNAs encoding factors regulating lipid metabolism (fat/cd36, acly, mcd, fas, lpl, pparα, and pparγ), suggesting that nesfatin-1 promotes lipid accumulation in neurons. In the liver, intracerebroventricular nesfatin-1 treatment resulted in decreased capacity for glucose use and lipogenesis, and increased the potential of fatty acid oxidation. Altogether, the present results demonstrate that nesfatin-1 is involved in the homeostatic regulation of food intake and metabolism in fish.

Tissue-specific expression of ghrelinergic and NUCB2/nesfatin-1 systems in goldfish (Carassius auratus) is modulated by macronutrient composition of diets.

The macronutrient composition of diets is a very important factor in the regulation of body weight and metabolism. Several lines of research in mammals have shown that macronutrients differentially regulate metabolic hormones, including ghrelin and nesfatin-1 that have opposing effects on energy balance. This study aimed to determine whether macronutrients modulate the expression of ghrelin and the nucleobindin-2 (NUCB2) encoded nesfatin-1 in goldfish (Carassius auratus). Fish were fed once daily on control, high-carbohydrate, high-protein, high-fat and very high-fat diets for 7 (short-term) or 28 (long-term) days. The expression of preproghrelin, ghrelin O-acyl transferase (goat), growth hormone secretagogue receptor 1 (ghs-r1) and nucb2/nesfatin-1 mRNAs was quantified in the hypothalamus, pituitary, gut and liver. Short-term feeding with fat-enriched diets significantly increased nucb2 mRNA levels in hypothalamus and liver, preproghrelin, goat and ghs-r1 expression in pituitary, and ghs-r1 expression in gut. Fish fed on a high-protein diet exhibited a significant reduction in preproghrelin and ghs-r1 mRNAs in the liver. After long-term feeding, fish fed on high-carbohydrate and very high-fat diets had significantly increased preproghrelin, goat and ghs-r1 expression in pituitary. Feeding on a high-carbohydrate diet also upregulated goat and ghs-r1 transcripts in gut, while feeding on a high-fat diet elicited the same effect only for ghs-r1 in liver. Nucb2 expression increased in pituitary, while it decreased in gut after long-term feeding of a high-protein diet. Collectively, these results show for the first time in fish that macronutrients differentially regulate the expression of ghrelinergic and NUCB2/nesfatin-1 systems in central and peripheral tissues of goldfish.

In Situ Localization and Rhythmic Expression of Ghrelin and ghs-r1 Ghrelin Receptor in the Brain and Gastrointestinal Tract of Goldfish (Carassius auratus).

Ghrelin is a gut-brain peptide hormone, which binds to the growth hormone secretagogue receptor (GHS-R) to regulate a wide variety of biological processes in fish. Despite these prominent physiological roles, no studies have reported the anatomical distribution of preproghrelin transcripts using in situ hybridization in a non-mammalian vertebrate, and its mapping within the different encephalic areas remains unknown. Similarly, no information is available on the possible 24-h variations in the expression of preproghrelin and its receptor in any vertebrate species. The first aim of this study was to investigate the anatomical distribution of ghrelin and GHS-R1a ghrelin receptor subtype in brain and gastrointestinal tract of goldfish (Carassius auratus) using immunohistochemistry and in situ hybridization. Our second aim was to characterize possible daily variations of preproghrelin and ghs-r1 mRNA expression in central and peripheral tissues using real-time reverse transcription-quantitative PCR. Results show ghrelin expression and immunoreactivity in the gastrointestinal tract, with the most abundant signal observed in the mucosal epithelium. These are in agreement with previous findings on mucosal cells as the primary synthesizing site of ghrelin in goldfish. Ghrelin receptor was observed mainly in the hypothalamus with low expression in telencephalon, pineal and cerebellum, and in the same gastrointestinal areas as ghrelin. Daily rhythms in mRNA expression were found for preproghrelin and ghs-r1 in hypothalamus and pituitary with the acrophase occurring at nighttime. Preproghrelin, but not ghs-r1a, displayed a similar daily expression rhythm in the gastrointestinal tract with an amplitude 3-fold higher than the rest of tissues. Together, these results described for the first time in fish the mapping of preproghrelin and ghrelin receptor ghs-r1a in brain and gastrointestinal tract of goldfish, and provide the first evidence for a daily regulation of both genes expression in such locations, suggesting a possible connection between the ghrelinergic and circadian systems in teleosts.