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

Abstract

This research assessed the direct effects of insulin on nutrientsensing 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.

Integration of Nutrient Sensing in Fish Hypothalamus

“…In fish, several evidence suggests the involvement of Akt in the hypothalamic mechanisms related to food intake control. Treatment with nutrients activates this signal through increased phosphorylation status, as demonstrated in rainbow trout for oleate (Blanco et al, 2020), octanoate (Velasco et al, 2017b), glucose (Otero-Rodiño et al, 2017), and leucine (Comesaña et al, 2018a). This activation also occurred under anorectic conditions such as treatment with ceramide (Velasco et al, 2016b(Velasco et al, , 2017a, insulin (Blanco et al, 2020), leptin (Gong et al, 2016), or FFAR agonists (Velasco et al, 2020).…”

“…A role for hypothalamic AMPKα in food intake control is also supported by the increase observed in hypothalamus of food-deprived rainbow trout (Conde-Sieira et al, 2019). The involvement of AMPK is further supported by the decrease in phosphorylation status of AMPKα observed in rainbow trout hypothalamus under anorectic conditions like treatment with ceramide (Velasco et al, 2016b(Velasco et al, , 2017a, FFAR agonists (Velasco et al, 2020), or anorectic hormones like CCK (Velasco et al, 2019), GLP-1 (Velasco et al, 2019), or insulin (Blanco et al, 2020). Of the different isoforms of AMPKα, it seems that AMPKα2 is involved in feed intake regulation while AMPKα1 appears to modulate peripheral metabolism (Conde-Sieira et al, 2019).…”

“…This is comparable with that observed in the same species (Kamalam et al, 2012) or in Megalobrama amblycephala (Xu et al, 2017) when fed a carbohydrate-enriched diet. Also in rainbow trout, raising levels of nutrients like oleate (Velasco et al, 2016a(Velasco et al, , 2017bBlanco et al, 2020), octanoate (Velasco et al, 2017b), glucose (Otero-Rodiño et al, 2017), or β-hydroxybutyrate (Comesaña et al, 2019) also resulted in a decrease in phosphorylation status of AMPKα. The specificity of AMPKα response was supported by the disappearance of responses to oleate in the presence of the AMPKα inhibitor compound C (Velasco et al, 2017b).…”

“…In fish, mTOR was characterized in hypothalamus of several species including rainbow trout (Velasco et al, 2017b;Comesaña et al, 2018a;Blanco et al, 2020) and Japanese sea bass (Liang et al, 2019). In rainbow trout, mTOR levels and phosphorylation status responded with an increase to the presence of nutrients like oleate (Velasco et al, 2017b;Blanco et al, 2020), octanoate (Velasco et al, 2017b), glucose (Blanco et al, 2020), or leucine (Comesaña et al, 2018a,b). The presence of rapamycin blocked the response to fatty acids in hypothalamus (Velasco et al, 2017b) supporting the specificity of the response.…”

“…Additional studies relate mTOR to food intake regulation in fish. Thus, increased mTOR occurred under anorectic conditions like feeding a lipidenriched diet in rainbow trout (Librán-Pérez et al, 2015) and blunt snout bream (Dai et al, 2018) or different treatments in rainbow trout with PYY (Velasco et al, 2018), CCK (Velasco et al, 2019), insulin (Blanco et al, 2020), or ceramide (Velasco et al, 2017a). In other fish species, available information is limited.…”