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  • br Materials and methods br

    2023-11-30


    Materials and methods
    Results and discussion First, we examined whether hypothalamic Akt-mediated signaling pathways are regulated postprandially in chickens. Refeeding significantly (P < 0.05) increased the levels of pAkt (Thr308) and pS6, and the phosphorylation of Akt (Thr308) and FOXO1 (Fig. 1). With the corresponding activation of Akt/mTOR and inactivation of FOXO1 in the hypothalami, plasma NHS-12-Biotin concentrations significantly increased in chicks after 1 h of refeeding (958 ± 245% of 24 h-fasting group; P < 0.05). These results suggest that hypothalamic Akt/mTOR signaling and FOXO1 were activated/inactivated postprandially, possibly in response to the elevation of blood insulin levels in chicks. Leptin, as well as insulin, suppresses food intake and induces PI3 K and mTOR activation in the central nervous system of mammals [[3], [8], [28]]. In contrast, ICV injection of a synthetic partial leptin peptide of chicken does not affect food intake in chicks [29], and the leptin activity in chicken serum is undetectable [30]. These findings suggest that leptin is not involved in feeding regulation in chicks, and raise the possibility that insulin, and not leptin, is a major regulator of the hypothalamic Akt-mediated signaling pathways in chicks. Therefore, we next examined whether insulin regulates the Akt-mediated signaling pathways in chicken hypothalami. In the preliminary study, an ICV administration of 50 pmol of insulin significantly decreased 1 h of cumulative food intake in 3 h-fasted chicks (vehicle, 3.16 ± 0.27 g; insulin, 1.38 ± 0.18 g; P < 0.05). Therefore, we injected the same dose of insulin in experiment 2 and observed a significant (P < 0.05) increase in the phosphorylation of Akt, FOXO1, and S6 in chicken hypothalami (Fig. 2). These findings demonstrated that, similar to in mammals [6], a sufficient dose of insulin to inhibit food intake activates the hypothalamic Akt/mTOR signaling pathway and inactivates FOXO1 in chicks. Finally, we examined whether the inhibition of Akt-mediated signaling affects food intake in chicks. Central injections of LY294002 or rapamycin significantly (P < 0.05) enhanced food intake in fasted chicks (Fig. 3). In a separate study, ICV injection of 4 nmol LY294002 or 25 μg rapamycin significantly prevented 50 pmol insulin-induced increases in the hypothalamic levels of pAkt (Thr308) or pS6, respectively (supplementary figure), indicating that LY294002 and rapamycin inhibit PI3 K and mTOR, respectively, in chicken hypothalami. Although we did not measure the plasma insulin concentration in experiment 3, it is expected to have increased after refeeding, because we confirmed in the preliminary study that 4 h of fasting significantly decreased plasma insulin concentrations in chicks (52.6 ± 6.33% of 0 h-fasting; P < 0.05), and a previous study on chicks reported that 30–60 min of refeeding after 24 h-fasting dramatically increased the plasma insulin concentration [18]. Furthermore, refeeding and central administration of insulin activated Akt-mediated signaling pathways (Fig. 1, Fig. 2). Therefore, all these findings suggest that, similar to in mammals [[3], [7]], hypothalamic PI3 K and mTOR play important roles in feeding regulation in chicks, and that the increase in blood insulin levels appears to suppress food intake via the hypothalamic Akt-mediated signaling pathways in chicks. Further in vivo studies, e.g. using an insulin receptor antagonist, are required to clarify whether hypothalamic Akt-mediated signaling is physiologically involved in the insulin-induced suppression of food intake in chicks. Akt is a well-documented Ser and Thr kinase [[31], [32]]. To become fully active in mammals, Akt requires phosphorylation on two conserved residues, Thr308 and Ser473 [31]. Phosphorylation of Thr308 is required for Akt activity and is catalyzed by 3-phosphoinositide-dependent kinase 1 [31]. The activity of Akt phosphorylated at Thr308 may be sufficient for a subset of its physiological roles [31]. However, maximal activation of Akt requires its additional phosphorylation on Ser473, which is catalyzed by mTORC2 [31]. Phosphorylation of Ser473 alone is not sufficient to promote Akt activity, but boosts it by up to tenfold [31]. In this study, ICV administration of insulin significantly increased Akt phosphorylation on Thr308 and Ser473 in chicken hypothalami (Fig. 2). In contrast, refeeding did not significantly increase the phosphorylation of Akt (Ser473) (Fig. 1). Insulin can cross the blood-brain barrier and reach the brain and cerebrospinal fluid (CSF) [33]. Transport of insulin from plasma to CSF decreases during fasting [33]. Therefore, 1 h of refeeding might fail to completely recover the transport ability; as such, the hypothalamic insulin level might be insufficient to fully activate Akt. However, FOXO1 phosphorylation and the pS6 level significantly increased after 1 h of refeeding. It is therefore likely that although hypothalamic Akt activity may not be completely reversed by 1 h of refeeding in chicks, the levels attained are sufficient to phosphorylate downstream targets.