2026 年 63 巻 論文ID: 2026004
When facing dehydration, birds use body fat and proteins as a source of endogenous water. Recently, we found that osmotic stress triggered protein catabolism in skeletal muscles of chicks. In the present study, we investigated whether osmotic stress also affected fat metabolism. Twelve 21-day-old male chicks were allocated to two groups based on body weight. The chicks received either saline (0.15 M NaCl) or hypertonic saline (1.5 M NaCl) intraperitoneally (5 mL/kg body weight). After 1 h of feed and water deprivation, the chicks were euthanized by decapitation. Blood, breast muscle (pectoralis major), abdominal adipose tissue, liver, and kidney samples were collected, followed by analysis of plasma components and gene expression. Hypertonic saline significantly increased the plasma levels of non-esterified fatty acids (NEFA); adipose triglyceride lipase (ATGL) mRNA in adipose tissue; pyruvate dehydrogenase kinase 4 (PDK4) mRNA in breast muscle, adipose tissue, and liver; phosphoenolpyruvate carboxykinase 1 mRNA in the liver; and atrogin-1 mRNA in breast muscle. It also increased the plasma content of the hormone vasotocin. Subsequent intravascular administration of vasotocin significantly elevated plasma NEFA, but not ATGL or PDK4 mRNA in adipose tissue. Collectively, our findings suggest that osmotic stress alters nutrient metabolism in the peripheral tissues of chicks and that vasotocin may be partly involved in fatty acid mobilization.
Physiological indicators of thirst in chickens[1,2,3,4] are important criteria of animal welfare in the poultry industry[5,6]. Increased blood osmolality is probably the strongest homeostatic signal for thirst in mammals[7], and osmotic stress can be compensated for by the intake of water and its conservation by the kidneys[8]. In chicks, water deprivation elevates blood osmolality[9], and the kidneys play a key role in water conservation[10]. Body fat and protein are additional sources of endogenous water in birds facing dehydration[11,12,13]. Considering that water scarcity negatively influences chicken growth[14,15], understanding the regulatory mechanism of osmotic balance in domestic fowl is important not only for animal welfare but also for efficient poultry production.
The aquaporin (AQP) family plays a critical role in water conservation via urine concentration in the kidneys[10]. This role is supported by increased mRNA levels of AQP1 and 2 in the kidneys of 7-day-old layer chicks subjected to intraperitoneal administration of hypertonic saline[16]. We recently observed that the same treatment significantly increased the mRNA levels of renal AQP1 and breast muscle atrogin-1 in 8-day-old layer chicks[17]. Atrogin-1, a regulatory enzyme of the ubiquitin-proteasome system, is a marker of accelerated proteolysis in chicken skeletal muscle[18,19,20]. In mammals, muscle atrophy triggers protein degradation by both the ubiquitin-proteasome system and autophagy, with the former playing a prominent role in degrading myofibrillar components[21]. Therefore, osmotic stress likely induces both anti-diuresis and muscle proteolysis in chicks.
In contrast, the effect of osmotic stress on lipid metabolism in chickens remains unclear. Intraperitoneal administration of hypertonic saline significantly increased plasma corticosterone and vasotocin in 3-week-old broiler chicks[22]. A similar increase in plasma non-esterified fatty acids (NEFA) was observed upon glucocorticoid addition in chicks[23,24], and intravascular administration of vasotocin in pigeons[25]. The main determinant of plasma NEFA levels is adipose tissue lipolysis[26]. In mice, this process is induced by vasopressin, a mammalian equivalent of vasotocin[27]. Collectively, these findings suggest that hypertonic saline administration induces lipolysis in chicks.
In the present study, we examined the effect of intraperitoneal hypertonic saline administration on plasma NEFA and corticosterone, as well as on gene expression in chicken breast muscle, adipose tissue, liver, and kidney. We also examined the impact of intravascular vasotocin administration on plasma NEFA and gene expression in adipose tissue. Our findings demonstrate that osmotic stress induces transcriptional changes in nutrient metabolism-related genes in these tissues.
The percentage of abdominal fat in layer and broiler chicks at 21 days of age is 1.02 ± 0.02%[28] and 1.25 ± 0.27%[29], respectively. Given that similar values are reported also at 28 days of age[28,29], it is likely that both layer and broiler chicks accumulate body fat up to 21 days of age. Because broiler chicks grow extremely quickly and exhibit hyperphagia, they have been deemed unsuitable for basic research in avian physiology. Instead, the regulatory mechanisms of body fluid balance have been studied in layer chicks[9,16,17,22,30,31]. Therefore, 21-day-old layer chicks were used in the present study.
One-day-old male chicks (White Leghorn) were purchased from local hatcheries (Japan Layer K.K., Gifu, Japan). They were provided free access to water and a commercial chick starter diet (Nichiwa Sangyo Co., Ltd., Kobe, Japan). The chicks were reared in electrically heated cages under a 23-h/1-h light/dark cycle. The temperature was maintained at 31 ± 2 °C during the first 7 days, and then reduced gradually to 25 ± 2 °C by day 21. This study was approved by the Institutional Animal Care and Use Committee and was conducted in accordance with Kobe University Animal Experimentation Regulation (2024–10-01-R1).
Experiment 1. Intraperitoneal administration of hypertonic saline in chicksIntraperitoneal administration of 3 M NaCl (5 mL/kg body weight) to 7-day-old chicks[16] or 8-day-old chicks[17] significantly elevated plasma sodium and renal AQP1 mRNA levels 1 h after administration. However, a lower dose of NaCl was also reported to stimulate water intake in chickens. Intraperitoneal administration of 2.5 M NaCl (2 mL/kg body weight) significantly increased water intake in 6-week-old broiler chickens[32]; whereas intravascular administration of 1.0–2.0 M NaCl (2.5 mL/kg body weight) did so in 12–18-week-old layer chickens[30]. Therefore, it is possible that 3 M NaCl is too high to evaluate the physiological response of chicks to osmotic stress, and hypertonic saline of 1.5 M NaCl (5 mL/kg body weight) was administered to 21-day-old chicks in the present study.
The chicks were provided free access to food and water before administration. Twelve 21-day-old chicks were weighed and allocated to two groups based on body weight; the groups received saline (0.15 M NaCl) or hypertonic saline (1.5 M NaCl) intraperitoneally (5 mL/kg body weight). After 1 h of feed and water deprivation, the chicks were euthanized by decapitation. The blood was collected in tubes containing EDTA (1.25 mg/mL of blood), and the plasma was separated by centrifugation at 1,910 × g for 10 min at 4 °C. A centrally located sample of abdominal adipose tissue was excised and placed immediately on dry ice for real-time PCR analysis. Similarly, centrally excised breast muscle and liver samples were preserved in RNAlater® (Sigma-Aldrich, St. Louis, MO, USA).
Experiment 2. Intravascular administration of vasotocin in chicksTwelve 21-day-old chicks were weighed and allocated to two groups based on body weight; the groups received 0 or 0.25 µg/mL vasotocin via the wing vein (5 mL/kg body weight). After 1 h of feed and water deprivation, the chicks were euthanized, blood and abdominal adipose tissue were collected, and plasma and abdominal adipose tissue were prepared as described in Experiment 1.
Plasma component analysisNEFA and corticosterone levels were measured using commercial kits (LabAssay NEFA, Wako Pure Chemical Industries, Ltd., Osaka, Japan; Corticosterone ELISA Kit, AssayPro LLC, St. Charles, MO, USA).
Real-time PCR analysisTotal RNA was extracted from breast muscle, kidney, liver, and adipose tissue using Sepasol-RNA I Super G (Nacalai Tesque, Inc., Kyoto, Japan). First-strand cDNA was synthesized from total RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Ltd., Osaka, Japan). Each mRNA was quantified with TB Green Premix Ex Taq II (Tli RNase H Plus; TaKaRa Bio Inc., Otsu, Japan) according to the supplier’s recommendations using the Thermo QuantStudio 1 Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA). Primer sequences are listed in Table 1. The mRNA levels of target genes, including adipose triglyceride lipase (ATGL), AQP1–3, atrogin-1, carnitinepalmitoyltransferase 1A (CPT1A), insulin-like growth factor-1 (IGF-1), lipoprotein lipase (LPL), phosphoenolpyruvate carboxykinase (PCK), and pyruvate dehydrogenase kinase 4 (PDK4), were normalized to those of ribosomal protein S17 (RPS17).
| Gene | Nucletotide sequence | GenBank Accession Number | |
| ATGL | Sense | 5’-GCT GAT CCA GGC CTG TGT CT-3’ | NM_001113291 |
| Antisense | 5’-TGG AGG TAT CAG CCC ACA GTA GA-3’ | ||
| LPL | Sense | 5’-GAC AGC TTG GCA CAG TGC AA-3’ | NM_205282 |
| Antisense | 5’-CAC CCA TGG ATC ACC ACA AA-3’ | ||
| PDK4 | Sense | 5’-AGT CTG CTT CCA AAC ATT ACC AAA C-3’ | NM_001199909 |
| Antisense | 5’-CAG TCT GCT TTG GAC CTT TAC TTG-3’ | ||
| Atrogin-1 | Sense | 5’-CAC CTT GGG AGA AGC CTT CAA-3’ | NM_001030956 |
| Antisense | 5’-CCG GGA GTC CAG GAT AGC A-3’ | ||
| CPT1A | Sense | 5’-GCC CTG ATG CCT TCA TTC AA-3’ | NM_001012898 |
| Antisense | 5’-ATT TTC CCA TGT CTC GGT AGT GA-3’ | ||
| PCK1 | Sense | 5’-GCC TTC ACT CGG TTG GAT GT-3’ | NM_205471 |
| Antisense | 5’-CGA TCC GGG AGA TGA GCA AT-3’ | ||
| IGF-1 | Sense | 5’-GCT GCC GGC CCA GAA -3’ | NM_001004384 |
| Antisense | 5’-ACG AAC TGA AGA GCA TCA ACC A -3’ | ||
| AQP1 | Sense | 5’-GGG ACA TCT CCT TGC AAT TGA TTA C-3’ | NM_001039453 |
| Antisense | 5’-TGA TTG GGC CAA CCC AGA AGA-3’ | ||
| AQP2 | Sense | 5’-GGC CAT CAA CAA GCT GCA TAA-3’ | NM_001292072 |
| Antisense | 5’-GAC CCC ATG TTG TCC TCC CT-3’ | ||
| AQP3 | Sense | 5’-TTT GGG CTC TAC CAT GAT GCC-3’ | XM_424500 |
| Antisense | 5’-TGT TGA GAT GGG TAG GTG GC-3’ | ||
| RPS17 | Sense | 5’-GCG GGT GAT CAT CGA GAA GT-3’ | NM_204217 |
| Antisense | 5’-GCG CTT GTT GGT GTG GAA GT-3’ |
ATGL, adipose triglyceride lipase; AQP, aquqporin; CPT, carnitinepalmitoyltransferase; IGF, insulin-like growth factor; LPL, lipoprotein lipase; PCK, phosphoenolpyruvate carboxykinase; PDK, pyruvate dehydrogenase kinase; RPS, ribosomal protein
Data were analyzed using Student’s t-test. Differences were considered statistically significant at P < 0.05.
In this study, we aimed to determine whether osmotic stress affected fat metabolism in chicks. As shown in Fig. 1, plasma NEFA and ATGL mRNA levels were elevated in adipose tissue of chicks subjected to hypertonic saline administration (Fig. 1B). ATGL is the rate-limiting enzyme in adipose tissue lipolysis[33]. These results suggest that osmotic stress induces lipolysis in chicks. In addition, intraperitoneal administration of hypertonic saline had a significant impact on the expression of various genes in adipose tissue, liver, and breast muscle samples. Thus, osmotic stress may affect nutrient metabolism in peripheral tissues.
Effect of intraperitoneal administration of hypertonic saline on plasma components and gene expression in adipose tissue, liver, breast muscle, and kidney of chicks. Data are expressed as the mean ± SEM of six replicates per group. **P < 0.01 with respect to the saline group.
Akin to ATGL mRNA, PDK4 mRNA was also significantly higher in adipose tissue of chicks treated with hypertonic saline (Fig. 1B). We previously showed that glucagon, the primary lipolytic hormone in chickens[34], significantly stimulated PDK4 expression in chicken primary adipocytes[35]. Glucocorticoids exert a similar effect on ATGL mRNA in adipose tissue[23] and plasma NEFA in chickens[23,24]. Therefore, it is possible that glucagon and glucocorticoids boosted plasma NEFA in chicks given hypertonic saline. Although no significant change was observed in plasma corticosterone 1 h after administration (Fig. 1A), we cannot exclude that corticosterone levels were transiently elevated and reversed within 1 h. Time-course analysis of plasma glucagon and corticosterone after administration of hypertonic saline in chicks is necessary to provide a better understanding of the underlying transcriptional changes in adipose tissue.
Inactivation of the pyruvate dehydrogenase complex by upregulation of PDK4 can divert glucose catabolism to fatty acid utilization in skeletal muscles, adipose tissue, liver, and heart[36]. In the present study, hypertonic saline markedly increased the mRNA levels of PDK4 in the liver and breast muscle of chicks (Fig. 1C and D), suggesting that hypertonic saline induces a metabolic switch from glucose to fatty acids in both tissues. However, under the present experimental conditions, no significant changes in CPT1A mRNA were detected in the breast muscle or liver. CPT1 plays a pivotal role in mitochondrial beta-oxidation and its activity can be regulated at both post-translational and transcriptional level[37]. Further studies are required to clarify whether hypertonic saline induces fatty acid oxidation in the breast muscle and liver of chicks.
Significant increases in plasma NEFA and the mRNA levels of ATGL and PDK4 induced by hypertonic saline suggest that the energy source shifts from carbohydrates to fatty acids. While the reason for this change is unclear; metabolic water from body fat is an important source of endogenous water in birds facing dehydration[11,12,13]. Lipids yield 1.89-fold more metabolic water per gram of wet tissue than glycogen in birds[11]. Therefore, hypertonic saline administration may have stimulated fatty acid utilization to produce metabolic water under the present experimental conditions.
Hepatic mRNA levels of PCK1, a critical enzyme in gluconeogenesis[34], were significantly increased by hypertonic saline (Fig. 1C). Tinker et al. reported that 29% and 10% of hepatic PCK activity was present in the cytosolic fraction in day-old chicks and 6-week-old chicks, respectively[38]. Therefore, hypertonic saline likely induces gluconeogenesis in the liver. In newborn dogs, IGF-1 suppressed PCK1 expression in the liver[39]. In the present study, hypertonic saline significantly decreased hepatic IGF-1 mRNA levels in chicks (Fig. 1C). Because glucagon and glucocorticoids are gluconeogenic hormones in chickens[40,41], they may be involved in the hypertonic saline-induced upregulation of PCK1 in the liver.
Atrogin-1 mRNA in breast muscle and NEFA in plasma were significantly increased following hypertonic saline treatment (Fig. 1A and D). In previous studies, atrogin-1 mRNA levels in chicken embryonic myotubes were decreased by IGF-1[42,43], and circulating IGF-1 was released mainly from the liver[34]. Notably, IGF-1 has anti-lipolytic effects on chicken adipose tissue in vitro[44,45]. Therefore, downregulation of hepatic IGF-1 by hypertonic saline may decrease circulating IGF-1 levels, inducing transcriptional changes in breast muscle and adipose tissue of chicks.
Breast muscle IGF-1 was downregulated by hypertonic saline treatment (P = 0.088) (Fig. 1D). Circulating IGF-1 promotes skeletal muscle growth in chickens[34]. In addition, IGF-1 acts in an autocrine/paracrine manner to stimulate muscle hypertrophy in mammals[46]. These findings suggest that osmotic stress suppresses muscle growth by downregulating IGF-1 in the liver and skeletal muscles of chicks.
Renal mRNA levels of AQPs were also analyzed in this study. Although various transcriptional changes were observed in other tissues, hypertonic saline (1.5 M NaCl) did not affect renal mRNA levels of AQP1, 2, or 3 (Fig. 1E). Antidiuresis did not appear to play an important role in the response to osmotic stress under present experimental conditions.
All four vasotocin receptors are highly expressed in brain regions, including the pituitary, but not in the peripheral tissues of chickens[47], suggesting that the major target site of vasotocin is the brain. However, in pigeons, vasotocin induced free fatty acid release from adipose tissue in vitro[48]. Saito et al. reported that intraperitoneal administration of hypertonic saline significantly elevated plasma vasotocin levels in chicks within 5 min of administration[31]. These findings support the hypothesis that vasotocin is involved in hypertonic saline-induced changes in chicken adipose tissue (Fig. 1). Therefore, we examined whether vasotocin affected plasma NEFA and gene expression in adipose tissue of chicks 1 h after intravascular administration. Vasotocin increased significantly plasma NEFA, but not gene expression in adipose tissue (Fig. 2). Several regulatory mechanisms, including post-translational regulation of adipocyte lipolysis, have been discovered in mammals[49]. Thus, vasotocin likely elevates plasma NEFA through post-translational regulation of lipolysis in chicken adipose tissue.
Effect of intravascular administration of vasotocin on plasma non-esterified fatty acids (NEFA) and gene expression in abdominal adipose tissue of chicks. Data are expressed as the mean ± SEM of six replicates for each group. *P < 0.05 with respect to the saline group.
Several studies have demonstrated various effects of hypertonic saline within 1 h of administration in chickens[16,17,22,30,31], prompting us to do the same in the present study. However, intraperitoneal hypertonic saline administration has been shown to increase renal AQP2 mRNA levels in chicks not only 1 h, but also as late as 3 h after administration[16]. Therefore, the effects of hypertonic saline could appear or be magnified only at a later time. Further studies are required to examine the long-term effects of osmotic stress on chicks.
In conclusion, hypertonic saline administration induces various transcriptional changes in the peripheral tissues of chicks. Our findings suggest that osmotic stress affects primarily nutrient metabolism, but understanding osmoregulation across different body parts will require additional studies.
This study was supported by JSPS KAKENHI (grant number 24K01907).
Kaoruko Murata, Nnamdi Godswill Dialoke, Yuhui Zhang, and Kazuhisa Honda performed the experiments and analyzed the data; Kazuhisa Honda designed the experiments; Kaoruko Murata, Nnamdi Godswill Dialoke, and Kazuhisa Honda wrote the manuscript; Junya Takegaki and Takaoki Saneyasu revised the manuscript.
The authors declare no conflict of interest.
