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Fasting Induces Gene Expression of Insulin-like Growth Factor-binding Proteins in Skeletal Muscles of Chicks
2025 年 62 巻 論文ID: 2025022
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2025 年 62 巻 論文ID: 2025022
In mammals, evidence suggests that insulin-like growth factor-binding proteins (IGFBPs) affect skeletal muscle growth in an autocrine and paracrine manner. In the present study, fasting induced significant transcriptional changes in IGFBP genes in the skeletal muscles of layer and broiler chickens. Twelve hours of fasting significantly increased mRNA levels of IGFBP-1 in the biceps femoris (BF; largest skeletal muscle in the thigh) of both chicken types. mRNA levels of IGFBP-2 in both the pectoralis major (PM; breast muscle) and the BF significantly increased in layer chicks and tended to increase in broiler chicks. Fasting significantly decreased mRNA levels of IGFBP-3 in the BF and PM of both chicken type. mRNA levels of IGFBP-4 and -5 differed responses in the PM and BF of layer and broiler chicks. mRNA levels of most IGFBP genes were not affected by insulin-like growth factor-1 (IGF-1) in chicken embryonic myotubes, suggesting that skeletal muscle IGFBPs were transcriptionally regulated in an IGF-1-independent manner. Overall, these findings suggested that IGFBP-1, -2, and -3, which were expressed in skeletal muscles, played conserved roles in layer and broiler chicks.
Chickens are regarded as a model avian species in many aspects[1,2,3]. Furthermore, two major commercial chickens, broilers and layers, have been established due to long-term genetic selection and show differences in muscle development[4]. For example, the breast muscle yield of broilers is approximately 1.5-fold higher than that of layer chicken[5]. Lee et al.[6] have reported greater number and size of muscle bundles in the breast and leg muscles of broilers than in those of layer chickens. Therefore, comparing the responsiveness of broiler and layer chickens to dietary nutrients may provide valuable insights into the avian physiology of commercial chickens.
Breast and thigh muscles account for a large portion of the body mass of chickens[7]. The pectoralis major (PM; breast muscle) and biceps femoris (BF; largest skeletal muscle in the thigh) exhibit distinct muscle properties. For instance, dietary supplementation with L-arginine suppresses protein synthesis in the PM of broiler chicks, but not in the BF[8]. Another study has shown that satellite cells derived from the PM and BF of 5-week-old broiler chickens have different levels of responsiveness to serum mitogens[9]. Hu et al. have demonstrated a difference in insulin sensitivity between the PM and BF in 7-day-old broiler chicks[10]. Thus, a comparison of the PM and BF in chickens may provide new insights into the specificity of skeletal muscle physiology in chickens, which, in turn, will benefit the poultry industry.
Insulin-like growth factor-binding proteins (IGFBPs) function as carrier proteins for insulin-like growth factor-1 (IGF-1) in the bloodstream in vertebrates[11]. Most circulating IGF-1 binds IGFBPs that regulate IGF activity in mammals[12]. In addition, in vivo and in vitro studies have shown that locally expressed IGFBPs affect skeletal muscle development in mammals. For instance, the IGFBP-2 antibody inhibits C2C12 myoblast differentiation[13] and reduction of IGFBP-3 secretion using antisense oligonucleotides inhibits human myoblast differentiation[14]. Positive effects of IGFBP-4 have also been reported during muscle hypertrophy[15], and knockdown of IGFBP-5 impairs myogenic differentiation and inhibits myotube formation in C2C12 cells[16]. In chickens, the microRNA miR-34b-5p may repress proliferation and promote the differentiation of chicken myoblasts by targeting IGFBP-2[17]. IGFBP-3 knockdown prevents myotube formation in chicken myoblasts, suggesting that IGFBP-3 promotes the differentiation of chicken myoblasts into skeletal muscle[18]. Moreover, transcriptional regulation of IGFBPs in skeletal muscles by feeding status has been reported in fish[19,20,21,22] and mammals[23,24]. These findings suggest that skeletal muscle IGFBPs play important physiological roles in vertebrate muscle growth.
In chickens, IGF-1 is the primary hormone that promotes skeletal muscle growth. For instance, continuous infusion of IGF-1 for 2 weeks significantly increases the growth rate of broiler chicks[25] and treatment with IGF-1 promotes the proliferation of chicken embryonic myoblasts[26]. Previous in vitro studies suggest that treatment with IGF-1 suppresses proteolytic pathways in chicken myotubes[27,28]. Subcutaneously administered IGF-1 affects IGFBP-3 expression in the gastrocnemius of rats[29] and intraperitoneal administration of IGF-1 increases IGFBP-1 expression in the skeletal muscle of rockfish[22]. In addition, subcutaneous IGF-I administration shows different effects on mRNA levels of muscle ring finger 1 between the gastrocnemius and soleus in rats[30] and on the proportion of Type I muscle fibers between the extensor digitorum longus and soleus in mice[31]. Therefore, it is possible that IGF-1 has different effects on IGFBP expression in different skeletal muscle types. Although the effects of IGF-1 on the gene expression of skeletal muscle IGFBPs have not been investigated in chickens, IGF-1 regulates IGFBP-2 gene expression in the ovarian granulosa cells of chicken follicles[10] and ovarian cells[32]. Hence, IGF-1 may regulate the gene expression of IGFBPs in chicken skeletal muscles.
The present study was conducted to characterize the gene expression of IGFBPs in response to fasting in the skeletal muscles of chicks and to determine the influence of IGF-1 on chicken embryonic myotubes. Our findings suggested that several IGFBPs play physiological roles in chicken skeletal muscle growth.
This study was approved by the Institutional Animal Care and Use Committee (2021-11-02) and performed according to the Kobe University Animal Experimentation Regulations. One-day-old male layer (White leghorn) and broiler (Ross 308) chicks were purchased from local hatcheries (Japan Layer K. K., Gifu, Japan and Yamamoto Co., Ltd., Kyoto, Japan, respectively). Chicks were reared in electrically heated battery cages. The temperature was kept at 31 ± 2°C during the first 7 days, and then reduced gradually according to age until reaching 25 ± 2°C at 21 days. Chicks were given free access to water and a commercial chick starter diet (NICHIWA SANGYO Co., Ltd., Kobe, Japan) under a 23-h/1-h light/dark cycle–a light schedule commonly used in the poultry industry. The commercial chick starter diet met the nutritional requirements of both broiler and layer chicks.
Sixteen 21-one-day-old male layer or broiler chicks were weighed and allocated to two groups based on body weight (eight birds in each group). At 21 days of age, the chicks in the feeding group were euthanized by decapitation by a skilled person after 0 h of fasting. Chicks in the fasting group were euthanized by decapitation after 12 h of fasting. The central pieces of the PM and BF were excised and preserved in RNAlater® tissue storage reagent (Sigma-Aldrich Co., St. Louis, MO, USA). Muscles were stored at -80 °C prior to total RNA extraction for gene expression analysis.
Myoblast culture and in vitro treatmentTranscriptional changes may not be induced by a short incubation time, whereas a long incubation time may induce not only a direct effect of IGF-1, but also a secondary effect of IGF-1-induced nutritional changes in primary cultures of chicken embryonic myotubes (CEM). There is evidence that IGF-1, insulin, and amino acids induce transcriptional changes in CEM after 3 h of incubation in vitro[28]. Therefore, a 3 h incubation time was chosen to examine the effect of IGF-1 on mRNA levels of IGF-related genes. Fertilized layer and broiler eggs were purchased from a commercial source and incubated at 37.5 °C under 60%–70% relative humidity with rotation every 1 h until embryonic myoblasts were isolated from the excised breast or thigh muscles of 14-day-old embryos. After washing with phosphate-buffered saline (PBS), muscle samples were minced using surgical scissors and digested with Hank’s balanced salt solution (+) (Nacalai Tesque, Inc., Kyoto, Japan) containing 0.2% collagenase (Worthington Biochemical Corp., Lakewood, NJ, USA) for 20 min at 37 °C. Cells were collected by centrifugation and resuspended in a growth medium consisting of Dulbecco’s modified Eagle’s medium (DMEM, Nacalai Tesque, Inc.) supplemented with 15% FetalClone III (GE Healthcare Bio-Sciences AB, Uppsala, Sweden), 1× non-essential amino acid solution (Nacalai Tesque, Inc.), and 1× gentamicin/amphotericin solution (Life Technologies, Carlsbad, CA, USA). The cell suspension was filtered using a cell strainer to remove tissue debris and then transferred to an uncoated flask to allow fibroblast attachement. After 1 h, the unattached cells were transferred to another uncoated flask, and this procedure was repeated three times. The cell suspension containing the unattached cells was collected and centrifuged at 1200 × g for 5 min, and the resulting cell pellet was resuspended. Myoblasts were counted and seeded on to collagen I-coated 12-well plates (Iwaki, Tokyo, Japan) at a density of 1 × 105 cells/well. Myoblasts were incubated in the growth medium described above at 37 °C and 5% CO2 in humidified air until myotube formation. On the 5th day, myotubes were treated with serum-free medium with or without 200 ng/mL recombinant human IGF-1 (Novus Biologicals, LLC, CO, USA) for 3 h.
Real-time PCRmRNA expression was measured using real-time reverse transcription polymerase chain reaction (RT-PCR) as previously described[26]. Briefly, total RNA from the PM and BF muscles or CEM cultures after washing with PBS was extracted using Sepazol-RNA I (Nacalai Tesque, Inc.). First strand cDNA was synthesized from total RNA using Rever Tra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo Co. Ltd, Osaka, Japan). mRNA levels were quantified for each primer using TB Green Premix Ex Taq II (Tli RNase H Plus; Takara Bio Inc., Otsu, Japan) according to the supplier’s recommendations and based on a relative standard curve using the Thermo Scientific Piko Real Real-Time PCR System (Thermo Fisher Scientific Oy, Vantaa, Finland). Primer sequences are listed in Table 1. mRNA levels of the target genes were normalized to those of the ribosomal protein S17.
| Gene | Nucletotide sequence | GenBank Accession Number | |
| IGFBP-1 | Sense | 5’-GCC AGG ACA AAT CCA TCC CT-3’ | NM_001001294 |
| Antisense | 5’-GCT CCT TCT GAC AAG GTC CC-3’ | ||
| IGFBP-2 | Sense | 5’-AAT GGG CAG CGT GGA GAG T-3’ | NM_205359 |
| Antisense | 5’-CTG GAT CAC CTT CCC ATG GA-3’ | ||
| IGFBP-3 | Sense | 5’-ATC AGG CCA TCC CAA GCT T-3’ | NM_001101034 |
| Antisense | 5’-GAT GTG CTG TGG AGG CAA ATT-3’ | ||
| IGFBP-4 | Sense | 5’-GAG CAC CCC AAC AAC AGC TT-3’ | NM_204353 |
| Antisense | 5’-CCG TTG TTG ATG CGC TTT G-3’ | ||
| IGFBP-5 | Sense | 5’-CAA GGC CGA ACG GGA AT-3’ | XM_422069 |
| Antisense | 5’-TCC TCC GTC ATC TCC GAT GT-3’ | ||
| IGF-1 | Sense | 5’-GCT GCC GGC CCA GAA -3’ | NM_001004384 |
| Antisense | 5’-ACG AAC TGA AGA GCA TCA ACC A -3’ | ||
| IGF1R | Sense | 5’- GGA GAA TTT CAT GGG TCT GAT TG-3’ | NM_205032 |
| Antisense | 5’- CAT GGG AAT GGC GAA TCT TC-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’ | ||
| RPS17 | Sense | 5’-GCG GGT GAT CAT CGA GAA GT-3’ | NM_204217 |
| Antisense | 5’-GCG CTT GTT GGT GTG GAA GT-3’ |
IGFBP, insulin-like growth factor-binding protein; IGF-1, insulin-like growth factor-1; IGF1R, insulin-
like growth factor-1 receptor; RPS, ribosomal protein
Data have been analyzed using the Student’s t-test in Excel 2013 (Microsoft, Redmond, WA, USA).
All differences were considered significant at P < 0.05.
In the PM and BF of layer chicks (Fig. 1), mRNA levels of IGFBP-2 were significantly increased after 12 h of fasting, whereas IGFBP-3 mRNA levels significantly decreased. mRNA levels of IGFBP-1, -4, and -5 significantly increased with fasting in the BF, but not in the PM. In contrast, mRNA levels of IGF-1 significantly decreased with fasting in the PM, but not in the BF. mRNA levels of insulin-like growth factor-1 receptor (IGF-1R) significantly increased with fasting in both muscles.
Effects of fasting on mRNA levels of insulin-like growth factor-related genes in the pectoralis major and biceps femoris in layer chicks. Data are represented as means ± standard error of the mean (S.E.M.) of eight chicks in each group and are expressed as a percentage of the mean in the feeding group. * and ** indicate statistical differences (P < 0.05, and P < 0.01, respectively) between the feeding and fasting group in each muscle.
In the PM and BF of boiler chicks (Fig. 2), fasting significantly decreased mRNA levels of IGFBP-3, similar to those in layer chicks. mRNA levels of IGFBP-2 slightly increased after fasting in the PM (P = 0.060) and BF (P = 0.068). Fasting significantly decreased IGFBP-5 mRNA levels in both muscles. Fasting significantly decreased IGFBP-4 and increased IGFBP-1 mRNA levels in the BF. Fasting significantly decreased IGF-1 mRNA levels in the PM. mRNA levels of IGF-1R significantly increased in both muscles with fasting. These results, combined with those from layer chicks, suggested that fasting-induced changes in IGFBP-2 and -3 were conserved in chicken skeletal muscles.
Effects of fasting on mRNA levels of insulin-like growth factor-related genes in the pectoralis major and biceps femoris in broiler chicks. Data are represented as means ± standard error of the mean (S.E.M.) of eight chicks in each group and are expressed as a percentage of the mean in the feeding group. †, *, and ** indicate statistical differences (P < 0.10, P < 0.05, and P < 0.01, respectively) between the feeding and fasting group in each muscle.
Next, the effects of IGF-1 treatment on IGF-related gene expression in CEM cells were examined. Similar to previous studies (Nakashima et al., 2017; Nakashima and Ishida, 2018), mRNA levels of atrogin-1 significantly decreased with IGF-1 treatment in layer (Fig. 3) and broiler (Fig. 4) CEMs, suggesting that IGF-1 signaling induces gene expression under fasting conditions. mRNA levels of IGFBP-3 in the PM-derived CEMs significantly decreased with IGF-1 treatment (Fig. 3). However, most IGFBP genes were not affected by IGF-1. mRNA levels of IGF-1R in broiler BF-derived CEMs significantly decreased following IGF-1 treatment (Fig. 4). mRNA levels of IGF-1R in layer PM-derived CEMs slightly decreased with IGF-1 treatment (Fig. 3; P = 0.053).
Effects of insulin-like growth factor-1 on mRNA levels of insulin-like growth factor-related genes in chicken embryonic myotubes derived from pectoralis major and biceps femoris of layer embryos. Data are represented as means ± standard error of the mean (S.E.M.) of five–six wells in each group and are expressed as a percentage of the mean in the control group. †, *, and ** indicate statistical differences (P < 0.10, P < 0.05, and P < 0.01, respectively) between the control and treatment groups.
Effects of insulin-like growth factor-1 on the mRNA levels of insulin-like growth factor-related genes in chicken embryonic myotubes derived from pectoralis major and biceps femoris of broiler embryos. Data are represented as means ± standard error of the mean (S.E.M.) of six wells in each group and are expressed as a percentage of the mean in the control group. * and ** indicate statistical differences (P < 0.05, and P < 0.01, respectively) between the control and treatment groups.
IGFBP-1 expression was significantly upregulated by fasting in the BF of layer and boiler chicks. There is evidence that mRNA levels of IGFBP-1 in the PM from 1 to 3 weeks of age are dramatically decreased compared to embryonic ages in both layer and broiler chicks[18]. However, in the present study, mRNA levels of IGFBP-1 in layer and broiler BF muscles under fasting conditions were 3.79- and 3.89-fold higher than those under ad libitum feeding conditions. Although the autocrine and paracrine effects of IGFBP-1 on skeletal muscle development remain unclear, IGFBP-1 inhibits IGF-1-induced DNA synthesis and impairs cell growth in chicken embryonic fibroblasts[33]. Burch et al. have reported that the addition of a 25 kDa amniotic IGFBP, later known as IGFBP-1, to the culture medium inhibits both basal and IGF-1-stimulated growth of cartilage in vitro[34]. Thus, skeletal muscle IGFBP-1 may suppress the role of IGF-1 in the growth of chicken BF muscles under fasting conditions.
Fasting significantly upregulated IGFBP-2 expression in the PM and BF of layer chicks. In addition, IGFBP-2 mRNA levels in the PM and BF of broiler chicks slightly increased with fasting. Jia et al. have shown that skeletal muscle IGFBP-2 mRNA levels are indirectly related to the body weight of chicks and levels are higher in slow-growing chickens than those in fast-growing chickens[35]. IGFBP-2 suppresses myotube formation by inhibiting the expression of myogenesis-related genes, such as myogenic differentiation 1 and myosin heavy chains, in cultured chicken primary myoblasts[17]. Thus, IGFBP-2 may suppress skeletal muscle growth in chicks under fasting conditions.
Fasting significantly decreased mRNA levels of IGFBP-3 in the PM and BF of both chicken types. Safian et al. have reported that fasting increases IGFBP-3 mRNA levels in the skeletal muscles of flounder[20]. These findings suggest that the role of IGFBP-3 in skeletal muscle is conserved between fish and birds. There is evidence that downregulation of IGFBP-3 in human[14] and chicken myoblasts[18] suppresses myoblast differentiation in vitro, suggesting that the role of IGFBP-3 in myoblasts is conserved between mammals and birds. These findings support the hypothesis that IGFBP-3 promotes muscle growth in birds under feeding conditions. Thus, the physiological role of skeletal muscle IGFBP-3 in chickens requires further clarification (e.g., the effects of IGFBP-3 knockdown on protein metabolism in CEMs).
The physiological roles of IGFBP-4 and -5 have not yet been elucidated in vertebrates. However, overexpression of IGFBP-4 in skeletal muscles is accompanied by increased IGF-1 gene expression and skeletal muscle hypertrophy in clenbuterol-treated rats[15]. Ren et al.[16] have reported that IGFBP-5 knockdown suppresses muscle cell differentiation in C2C12 cells. Additionally, mRNA levels of IGFBP-4 and -5 in breast muscles of layer and broiler chicks significantly decrease after hatching[18]. These findings suggested that IGFBP-4 and -5 were involved in chicken muscle development at the embryonic stage. In the present study, fasting altered mRNA levels of IGFBP-4 and -5 in broiler and layer chicks and the responses differed between chick types. Further studies are required to clarify the role of IGFBPs in chicks after hatching.
Fasting significantly affected the gene expression of many IGFBPs in chicken skeletal muscles. However, IGF-1 did not affect most IGFBPs in CEMs in vitro (Figs. 3 and 4). Only IGFBP-3 was observed in layer PM-derived CEMs, but mRNA levels of IGFBP-3 decreased with fasting in the PM and BF in vivo. Furthermore, the plasma fasting decreased plasma IGF-1 concentration in chickens[36,37]. Taken together, these findings suggested that chick skeletal muscle IGFBPs were transcriptionally regulated by feeding conditions in an IGF-1-independent manner. Intravascular administration of insulin suppresses fasting-induced IGFBP-2 expression in the liver and gizzard of 6-week-old layer chicks[38]. IGFBP-1 gene expression may be downregulated by insulin and upregulated by glucocorticoid in human osteoblasts[39] and rat hepatocytes[40,41]. Ernst and White have reported that insulin downregulates IGFBP-2 mRNA levels in C2C12 myotubes[42]. Additionally, glucose inhibits IGFBP-1 secretion from Hep G2 cells[43]. In chickens, fasting decreases plasma insulin[44,45] and glucose levels[44,45,46,47], and elevates plasma corticosterone and non-esterified fatty acid levels[48,49,50,51]. These findings suggest that changes in plasma hormone and nutrient levels affect IGFBP mRNA levels in chicken skeletal muscle. Further studies are required to examine the effects of insulin, glucocorticoids, and/or other nutrient treatments on IGF-related gene expression in CEM cells.
mRNA levels of IGF-1R significantly increased with fasting in all muscles and chicks examined in this study. A mammalian study has proposed that the negative feedback circuit in the IGF-1 signaling pathway is activated by the suppression of IGF-1R expression in skeletal muscles[52]. Plasma IGF-1 concentration decreases during fasting in chickens[36,37]. In the present study, mRNA levels of IGF-1R significantly decreased with IGF-1 treatment in broiler BF-derived CEMs. mRNA levels of IGF-1R in layer PM-derived CEMs slightly decreased following IGF-1 treatment (P = 0.053). Although the effects were not consistent between CEMs, these findings suggest that the negative feedback circuit of IGF-1 signaling accompanied by downregulation of IGF-1R gene expression in skeletal muscles has been partly conserved between mammals and chickens.
Fasting decreased IGF-1 mRNA levels only in the PM of both chick types. IGF-1 is the primary hormone that promotes skeletal muscle growth in chicks. In chicks, IGF-1 mRNA levels in the PM are significantly higher than those in other skeletal muscles, including the BF[53]. Although the autocrine and paracrine effects of IGF-1 on skeletal muscle growth remain unclear, IGF-1 mRNA levels in the PM may be upregulated to maintain muscle mass under ad libitum feeding conditions. Further studies are required to fully elucidate the evolution of breast muscle development in chickens.
Because primer pairs differed between IGFBP genes, it was difficult to compare the abundance of each IGFBP mRNA in the PM and BF. In addition, there was no evidence of a comparison of mRNA levels between IGFBP genes in chicken skeletal muscles. However, Guo et al.[18] have reported that the expression levels of all IGF system members, including IGFBPs, sharply decreased after hatching in layer and broiler chickens, indicating that the IGF system plays a much greater role in the embryonic stage and early period after hatching. The findings in the present study suggest that skeletal muscle IGFBPs may also function in growing chickens, although the mRNA levels may be lower than those at embryonic age. Therefore, the physiological importance and major role of each IGFBP in the skeletal muscles of growing chickens needs to be investigated in future studies.
There is no evidence showing the circadian rhythm of IGFBP gene expression in mammals and birds, but significant daily rhythmic mRNA expression of IGF-1 and IGFBP-2 has been reported in goldfish skeletal muscle under a 12-h/12-h light/dark cycle[54]. There is evidence that clock gene deficiencies in mice result in downregulation of IGF-1 production in skeletal muscles under a 12-h/12-h light/dark cycle[55]. However, in the present study, chicks were maintained under a 23-h/1-h light/dark cycle (23:00–24:00 dark) and fasted from 22:00 to 10:00. Therefore, the circadian rhythm would only have a small impact, if any, under these experimental conditions.
The present study showed that several IGFBP genes in chicken skeletal muscle were transcriptionally altered in response to feeding status. For example, mRNA levels of IGFBP-1 significantly increased in the BF, and mRNA levels of IGFBP-2 and -3 in the skeletal muscle of chicks appeared to be altered by fasting. Overall, these findings suggested that these IGFBPs play physiological roles in chicken skeletal muscle in response to feed intake.
Asmaa S. El-Far received a full scholarship [PD. 77] from the Ministry of Higher Education of the Arab Republic of Egypt. This study was supported by JSPS KAKENHI (grant number: 21K05892).
Asmaa S. El-Far and Kazuhisa Honda designed the experiments; Asmaa S. El-Far, Haruki Osada, and Yumi Sakanashi conducted the experiments and analyzed the data; Asmaa S. El-Far drafted the manuscript; Takaoki Saneyasu and Kazuhisa Honda supervised the experiments and edited the manuscript.
The authors declare no conflict of interests.
