2026 年 63 巻 論文ID: 2026002
In layer chicks, central administration of insulin and refeeding promote the phosphorylation of AKT and extracellular signal-regulated kinase (ERK) in the medulla oblongata. Broiler chicks consume more feed than layer chicks, suggesting distinct feeding regulation. This study aimed to clarify the involvement of medullary AKT and mitogen-activated kinases (MAPKs: ERK, c-Jun N-terminal kinase (JNK), and p38 MAPK) in regulating feeding of broiler chickens. The phosphorylation of AKT and p38 MAPK, but not that of ERK and JNK, was significantly higher in the medulla oblongata of broilers refed for 1 h after a 24-h fasting. Intracerebroventricular (ICV) administration of insulin significantly enhanced AKT phosphorylation in the medulla oblongata, but had no significant effect on the phosphorylation of MAPKs. Oral administration of glucose increased plasma glucose and the phosphorylation of AKT and p38 MAPK, but not ERK and JNK, in the medulla oblongata. ICV administration of the p38 MAPK activator anisomycin strongly induced the phosphorylation of p38 MAPK, but not JNK, without affecting feed intake. These findings suggest that medullary AKT contributes to insulin-induced suppression of feed intake in broiler chicks, while medullary p38 MAPK is phosphorylated in response to postprandial elevation of blood glucose, but is not involved in feeding regulation.
The hypothalamus and medulla oblongata play important roles in regulating energy intake and expenditure. Evidence from mammals has revealed that cellular signaling pathways in the hypothalamus and medulla oblongata regulate food intake[1,2,3,4,5]. For example, the hypothalamic AKT, mechanistic target of rapamycin, and Janus kinase/signal transducers and activators of transcription signaling pathways are activated by peripheral peptides, such as insulin and leptin, and nutrients, such as glucose and leucine, and their activation halts food intake[2,4]. Several studies in rodents have suggested that the central mitogen-activated protein kinase (MAPK) family is involved in controlling the energy balance. For example, extracellular signal-regulated kinase (ERK) signaling contributes to the regulation of food intake and glucose production in the dorsal vagal complex (DVC)[6,7,8]. Intracerebroventricular (ICV) administration of a c-Jun N-terminal kinase (JNK) inhibitor reduces food intake in wild-type mice[9]. Similarly, central JNK1 deficiency enhances the suppressive effects of insulin on food intake[10,11]. However, it remains unclear whether p38 MAPK, another member of the MAPK family, contributes to the control of food intake.
Modern broiler chickens consume more feed than layer chickens during the neonatal period[12]. Overconsumption of feed can lead to excessive accumulation of unwanted abdominal fat[13,14]. Although the causes of feed overconsumption in broiler chickens have not been completely clarified, differences in the regulation of feeding habits between broiler and layer chickens have been hypothesized. For example, although central administration of insulin suppresses food intake in both broiler[15,16] and layer[17,18,19] chicks, insulin resistance may occur in the central nervous system of broiler chicks[20]. Recent studies have shown that central injection of insulin and refeeding promote the phosphorylation of both AKT and the downstream molecule FOXO1 in the hypothalamus of layer chicks[19]; whereas only AKT, but not FOXO1, is phosphorylated in broiler chicks[16]. These findings raise the possibility that differences in insulin-induced signaling pathways contribute to hyperphagia in broiler chicks. Even though the phosphorylation of AKT and ERK is promoted by the central administration of insulin and refeeding in the medulla oblongata of layer chicks[21], it remains unclear whether medullary AKT and ERK respond to feeding conditions and insulin in broiler chicks. Similarly unknown is the contribution of other MAPKs.
In the present study, we investigated how feeding conditions and insulin injection affected AKT and MAPK signaling pathways in the medulla oblongata of broiler chickens, as well as the effect of p38 MAPK activator injections on feed intake.
All animal procedures were approved by the Institutional Animal Care and Use Committee and carried out according to Kobe University Animal Experimental Regulations (permission number: 2019-07-03-R1, 2023-05-02-R1). Newly hatched or1-day-old male broiler chicks (Ross 308) were purchased from local hatcheries (Yamamoto Co. Ltd., Kyoto, Japan) and maintained at 31 ± 2 °C in a room with an automatically controlled 23-h light/1-h dark cycle (23:00-24:00 dark). The birds were provided with free access to water and a commercial chicken starter diet (Nichiwa Sangyo Co., Ltd., Kobe, Japan).
Experimental design Effect of feeding conditions on phosphorylation of AKT and MAPKs in the medulla oblongata of broiler chicksFollowing a previous study on layer chicks[21], 7-day-old chicks were weighed and allocated to three groups based on their body weight (six birds per group). One group was fed ad libitum, and the other groups were deprived of feed for 24 h. Then, the ad libitum group and one fasting group (8-day old) were euthanized by decapitation, while the other fasting group was refed for 1 h before euthanasia. The dorsomedial region of the medulla oblongata was excised based on a stereotaxic atlas drawn by Kuenzel and Masson[22], immediately frozen in liquid nitrogen, and stored at -80 °C until use for western blotting.
Effect of central administration of insulin on phosphorylation of the medullary AKT and MAPKs in broiler chicksEight-day-old broiler chicks were divided based on body weight (eight birds per group). Porcine insulin was dissolved in saline solution containing 0.1% Evans blue. Either insulin (200 pmol/chick) or vehicle (control) was administered intracerebroventricularly in a volume of 10 µL after 3 h of fasting, as described by Davis et al.[23]. After 30 min, the chicks were euthanized by decapitation. Injections were confirmed by the presence of Evans blue dye in the lateral ventricle, as previously described[24]. The medulla oblongata was excised, immediately frozen in liquid nitrogen, and stored at -80 °C. Five samples were randomly selected for western blotting.
Effect of oral administration of glucose on phosphorylation of the medullary AKT and MAPKs in broiler chicksEight-day-old broiler chicks were divided according to body weight (six birds per group) and fasted for 24 h. Glucose dissolved in distilled water (400 mg/chick) or vehicle (control) was administered orally in a volume of 1 mL. Previously, oral administration of 200 mg glucose in a 0.5-mL volume successfully increased plasma glucose in 4-day-old chicks[25]. After 60 min, the chicks were euthanized by decapitation, and blood and medulla oblongata were collected. EDTA (1.67 mg/mL blood) was used as anticoagulant. The plasma was separated by centrifugation at 1,900 × g for 10 min at 4 °C. The excised medulla oblongata was frozen immediately in liquid nitrogen and stored at -80 °C for subsequent use in western blotting. Plasma glucose was measured using a commercial kit (LabAssayTM Glucose; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan).
Effect of central administration of anisomycin on phosphorylation of the medullary p38 MAPK and JNK in broiler chicksSeven-day-old broiler chicks were divided based on body weight (eight birds per group). Anisomycin (Cayman Chemical Co., Ann Arbor, MI, USA) was dissolved in saline containing 0.1% Evans blue and 5% dimethyl sulfoxide. Anisomycin (25 nmol/chick) or vehicle (control) was administered intracerebroventricularly as described above. After 30 min, the chicks were euthanized by decapitation. Injections were confirmed by the presence of Evans blue dye in the lateral ventricle, as previously described[24]. The medulla oblongata was excised, immediately frozen in liquid nitrogen, and stored at -80 °C. Five samples were randomly selected for western blotting.
Effect of central administration of anisomycin on feed intake in broiler chicks.Anisomycin (25 nmol/chick) or vehicle (control) was administered intracerebroventricularly to 8-day-old broiler chicks after 3 h of fasting as described above (ten birds per group). Feed intake was measured 30, 60, and 120 min after administration. At the end of the experiments, the chicks were euthanized to verify the success of the injection based on Evans blue staining[24]. Data from chicks without Evans blue dye in the lateral ventricle were omitted.
Western blot analysisWestern blot analysis was performed as previously described[21]. Briefly, frozen medulla samples were ultrasonicated in lysis buffer containing 150 mM NaCl, 10 mM tris(hydroxymethyl)aminomethane, 1 mM EDTA, 1 mM ethylene glycol bis(β-aminoethylether)-N,N,N’,N’-tetraacetic acid, 1% Triton X-100, 0.5% NP-40, 100 mM NaF, 23 mM sodium phosphate, 2 mM sodium orthovanadate, and protease inhibitor cocktail (Nacalai Tesque, Inc., Kyoto, Japan). Homogenates were centrifuged at 17,900 × g for 15 min at 4 °C, and supernatants were stored at -80 °C. The protein concentration was determined using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA). The lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blotting using HorizeBlot (ATTO Corp., Tokyo, Japan) according to the supplier’s recommendations. Bands were detected using Chemi-Lumi One Super (Nacalai Tesque, Inc.), visualized using Lumicube (Liponics Inc., Tokyo, Japan), and quantified using a CS Analyzer (ATTO Corp.). Anti-Akt (#9272), anti-phosphorylated Akt (Thr308) (#9275), anti-p44/42 MAPK (ERK1/2) (#4695), anti-phospho-p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (#9101), anti-p38 MAPK (#8690), anit-phospho-p38 MAPK (Thr180/Tyr182) (#4511), anti-SAPK/JNK (#9252), anti-phospho-SAPK/JNK (Thr183/Tyr185) (#9255), anti-β-actin (#8457), anti-α-tubulin (#2125), horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (#7074), and HRP-conjugated anti-mouse IgG (#7076) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Anti-β-actin and anti-α-tubulin antibodies were used as loading controls. After the detection of phospho-JNK, membranes were rinsed with RestoreTM Plus Western Blot Stripping Buffer (Thermo Fisher Scientific Inc., Rockford, IL, USA) and then reprobed with an anti-JNK antibody. The results are shown relative to the fasting and vehicle groups.
Statistical analysisData were analyzed using Dunnett’s test or t-test. All analyses were performed using Excel 2013 (Microsoft, Seattle, WA, USA) with the Statcel 3 add-in software (OMS, Tokyo, Japan).
Akin to a previous study on layer chicks[21] and compared to fasting birds, refeeding significantly increased the phosphorylation of medullary AKT and p38 MAPK in broiler chicks (Fig. 1). Instead, no significant differences were detected for ERK or JNK phosphorylation (Fig. 1). These results support the hypothesis that medullary AKT and p38 are physiologically involved in regulating feeding of broiler chicks.
Effect of feeding conditions on the phosphorylation of AKT and mitogen-activated protein kinases in the medulla oblongata of broiler chicks. Data are expressed as the mean ± SEM of six birds per group. The Tukey-Kramer method was used to analyze differences between groups; *P < 0.05 and **P < 0.01 with respect to the fasting group. Ad, ad libitum feeding; Fa, fasting; Re, refeeding.
In broiler chicks, plasma insulin increased after 24 h of fasting and subsequent refeeding for 1 h[16]. In layer chicks, phosphorylation of AKT and ERK increased in the medulla oblongata following the ICV administration of insulin[21]. This evidence suggests that insulin may promote phosphorylation of AKT and p38 MAPK in the medulla oblongata of broiler chicks. ICV administration of insulin significantly upregulated the phosphorylation of AKT, but not that of ERK, p38 MAPK, or JNK, in the medulla oblongata of broiler chicks (Fig. 2). Given that ICV insulin addition significantly decreased feed intake in broiler chicks[16], medullary AKT was likely involved in postprandial insulin-induced satiety in both broiler and layer chicks.
Effect of central administration of insulin on the phosphorylation of AKT and mitogen-activated protein kinases in the medulla oblongata of broiler chicks. Data are expressed as the mean ± SEM of five birds per group. The t-test was used to analyze differences between groups; *P < 0.05 with respect to the vehicle group. Ve, vehicle; Ins, insulin
Whereas refeeding and central injection of insulin increased the phosphorylation of both AKT and ERK in the medulla oblongata of layer chicks[21], the present study on broiler chicks shows that medullary ERK phosphorylation is not promoted by either treatment (Figs. 1 and 2). In rats, ERK is more sensitive to insulin than AKT in the DVC[6]. Moreover, insulin suppresses food intake via ERK but not AKT in the DVC[8], suggesting a more important role for ERK than AKT in this process. Therefore, unresponsive ERK in the medulla oblongata may explain excessive feed intake in broiler chicks as opposed to layer chicks.
Administration of anti-insulin serum decreases ERK phosphorylation in the skeletal muscles of broiler chickens[26]. We previously reported that the knockdown of insulin-like growth factor-1 (IGF-1) inhibited ERK phosphorylation in myotubes formed by the fusion of myoblasts derived from broiler embryos[27]. These findings suggest that ERK in the skeletal muscle of broiler chickens responds to insulin and IGF-1. Therefore, the unresponsiveness of medullary ERK in broiler chicks may be tissue-specific, and only future studies will clarify why it does not respond to nutritional conditions or insulin levels.
Preliminary results indicate that refeeding significantly increases plasma glucose compared to fasting (Supplementary Fig. 1). Therefore, glucose was administered orally to investigate the regulatory mechanisms of p38 MAPK phosphorylation in the medulla oblongata of broiler chicks. Oral administration of glucose significantly increased glycemia and the phosphorylation of p38 MAPK, contrasting with the results of insulin administration (Fig. 2). Instead, no significant changes were observed with respect to the levels of ERK and JNK (Fig. 3). Previous in vitro studies have shown that a high-glucose medium promotes p38 MAPK phosphorylation in rat pheochromocytoma cells[28] and rat dorsal root ganglia[29]. These findings suggest that the postprandial elevation of blood glucose, but not insulin, enhances p38 MAPK phosphorylation in the medulla oblongata of broiler chicks.
Effect of oral administration of glucose on the phosphorylation of AKT and mitogen-activated protein kinases in the medulla oblongata of broiler chicks. Data are expressed as the mean ± SEM of six birds per group. The t-test was used to analyze differences between groups; *P < 0.05 and **P < 0.01 with respect to the vehicle group. Ve, vehicle; Glc, glucose
The phosphorylation of AKT was also increased by the oral administration of glucose (Fig. 3). Although plasma insulin was not measured in the present study, oral administration of glucose augments plasma insulin in broilers[25]. Therefore, it is likely that increased AKT phosphorylation is due to glucose-induced elevation of plasma insulin. However, plasma glucose may also directly promote AKT phosphorylation in the medulla oblongata of broiler chicks and further studies are required to clarify whether blood glucose acts alone or in combination with blood insulin.
To investigate whether medullary p38 MAPK was involved in regulating feeding in broiler chicks, anisomycin was administered intracerebroventricularly. ICV addition of anisomycin significantly increased the phosphorylation of p38 MAPK, but not that of JNK (Fig. 4A). As no significant change was observed in feed intake (Fig. 4B), medullary p38 MAPK was not involved in regulating feeding of broiler chicks.
Effect of central administration of anisomycin on the phosphorylation of medullary p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK), along with feed intake in broiler chicks. Data are expressed as the mean ± SEM of five (A), nine (B, 0 nmol), and seven (B, 25 nmol) birds per group. The t-test was used to analyze differences between groups; *P < 0.05 with respect to the vehicle group. Ve, vehicle; An, anisomycin.
In fructose-induced hypertensive rats, the superoxide-mediated p38 MAPK signaling pathway impairs insulin signal transduction to promote insulin resistance in the nucleus tractus solitarius (NTS)[30]. Therefore, it is possible that the postprandial upregulation of p38 MAPK phosphorylation caused by elevated glycemia induces acute insulin resistance in the medulla oblongata of broiler chicks and attenuates satiety signaling.
Previous studies in rodents have suggested that the activation of central JNK signaling reduces food intake[9,10,11]. Here, no significant changes were observed in the phosphorylation of JNK (Figs. 1–3). Therefore, medullary JNK does not appear to be physiologically involved in feeding regulation in 1-week-old broiler chicks. However, as this is the sole study on the topic, investigation of older broilers and different strains will clarify the role of medullary JNK signaling in feeding regulation in chickens.
The DVC in the medulla oblongata includes several nuclei crucial for controlling food intake, including the area postrema (AP), NTS, and dorsal motor nucleus of the vagus[31]. Afferent vagal sensory neurons that sense a variety of gut parameters, such as gastric distension and the presence of nutrients in the gut, synapse in the AP and NTS[31]. The AP resides outside the blood-brain barrier and is positioned to sense blood-borne signals, including a variety of ingestion-related humoral signals from the gut[31]. The AP and NTS signal rostral brain structures, such as the hypothalamus and parabrachial nucleus, to control food intake[31], while receiving signals directly and indirectly from other brain regions, such as the hypothalamus[32,33,34]. Therefore, the phenomena observed in the present study may arise from afferent vagal neurons, blood-derived hormones and nutrients, and neurons projecting from other regions such as the hypothalamus.
In conclusion, the present study suggests that medullary AKT contributes to insulin-induced suppression of feed intake in broiler chicks and that medullary p38 MAPK is phosphorylated in response to postprandial elevation of blood glucose, but is not involved in feeding regulation. Although further studies are required, these findings provide new insights into candidate targets controlling feed intake in chickens, especially broilers.
This study was supported by JSPS KAKENHI (grant numbers 19K06353 and 23K05506).
Takaoki Saneyasu designed and conducted the experiments, analyzed the data, and wrote the manuscript; Miyu Kaihatsu, Kanami Nagata, Aika Kitayama, and Haruka Sakai conducted the experiments and analyzed the data; Hiroshi Kamisoyama designed the experiment and edited the manuscript; Junya Takegaki and Kazuhisa Honda revised the manuscript.
The authors declare no conflict of interest.
