2018 Volume 55 Issue 4 Pages 269-273
The physiological functions of insulin-like growth factor-binding proteins (IGFBPs) in mammals have been evaluated in several studies. However, the physiological roles of IGFBPs in chickens have not yet been elucidated. In this study, we examined the effects of short-term (6 h) fasting and refeeding on the mRNA levels of IGFBPs in chick liver and brain. Eighteen 8-day-old chicks were weighed and allocated to three groups on the basis of body weight, and subjected to ad libitum feeding, 6 h of fasting, or 6 h of fasting followed by 6 h of refeeding. After the chicks were euthanized by decapitation, the liver and brain were excised, and the brain was dissected into six segments (telencephalon, optic lobes, cerebellum, rostral part of the brainstem, middle part of the brainstem, and caudal part of the brainstem). IGFBP mRNA levels were determined by qRT-PCR. Fasting significantly increased the mRNA levels of IGFBP-1 and -2 in the chick liver, and these changes were reversed by 6 h of refeeding. The mRNA levels of IGFBP-3 in the middle part of the brainstem and IGFBP-5 in the optic lobes were decreased by 6 h of fasting and were not reversed after 6 h of refeeding. These findings suggest that IGFBP-1 and -2 in the liver, IGFBP-3 in the middle part of the brainstem, and IGFBP-5 in the optic lobes may play physiological roles in response to short-term changes in the nutritional status of chicks.
Insulin-like growth factor-binding proteins (IGFBPs) are thought to function as carrier proteins in circulation, and locally expressed IGFBPs can inhibit and/or potentiate IGF activities in mammals (Duan and Xu, 2005). In addition, some IGFBPs show IGF-independent biological effects. For example, IGFBP-3 and -5 have nuclear localization sequences, and the nuclear transport protein importin-β has been shown to mediate the translocation of both IGFBP-3 and -5 (Firth and Baxter, 2002). However, the physiological roles of IGFBPs in birds have not yet been fully investigated, although full-length cDNA sequences of IGFBP-1 to -5 are available in the GenBank database.
In genetically fat and lean chickens fasted for 48 h, among four circulating IGFBPs with molecular weights of 28, 34, 40, and 60 kDa, the 28-kDa, and to a lesser extent 34-kDa and 60-kDa IGFBPs were increased (Beccavin et al., 1999). In layer chickens, dietary protein restriction (Leili and Scanes, 1998), food deprivation (Leili et al., 1997), and dexamethasone (Leili and Scanes, 1998) affected the binding activity of plasma IGFBPs to IGF-1. Polymorphisms in IGFBP-2 (Lei et al., 2005; Li et al., 2006; Leng et al., 2009) and IGFBP-3 (Ou et al., 2009) may be related to growth and/or carcass traits. Kita et al. (2002) investigated the response of IGFBP-2 expression to variations in nutritional status in several tissues of layer chickens; they found that the mRNA level of IGFBP-2 in the liver was significantly increased after 2 days of food deprivation, and this increase was reversed after 6 h of refeeding. Thus, the expression of hepatic IGFBPs may influence the plasma level of IGFBPs, which in turn inhibit and/or potentiate IGF activities in chickens.
Recently, we found that central and peripheral administration of IGF-1 significantly suppressed food intake in chicks (Fujita et al., 2017). Additionally, we showed that the mRNA levels of IGF-1 in the liver were significantly increased upon refeeding and that the IGF-1 receptor is expressed throughout the brain (Fujita et al., 2017). Evidence demonstrates that IGF-1 crosses the blood-brain barrier (Reinhardt and Bondy, 1994; Armstrong et al., 2000; Nishijima et al., 2010). These findings suggest that hepatic IGF-1 functions as a satiety signalin the brain in chicks. The mRNA level of IGFBP-2 in the brain was significantly decreased after 2 days of food deprivation and this decrease was not reversed after 24 h of refeeding in layer chickens (Kita et al., 2002). Thus, it is possible that brain-expressed IGFBPs influence the function of circulating IGF-1 in the chicken brain. However, although the brainstem is proposed to contain the satiety center (Richards and Proszkowiec-Weglarz, 2007), the physiological-functions of IGFBPs in the brainstem in chicks have not been investigated.
In the present study, we examined the effects of short-term (6 h) fasting and refeeding on the mRNA levels of IGFBPs in chick liver and brain. Our findings suggest the physiological importance of IGFBP-1 and -2 in the liver, IGFBP-3 in the middle part of the brainstem, and IGFBP-5 in the optic lobes under the fasting and/or refeeding condition in chicks.
One-day-old male broiler chicks (ROSS 308) were purchased from a local hatchery (Ishii, Tokushima, Japan). They were given free access to water and a commercial-chick starter diet (Nippon Formula Feed, Kanagawa, Japan). This study was approved by the Institutional Animal Care and Use Committee and was carried out according to the Kobe University Animal Experimentation Regulation.
Eighteen 8-day-old chicks were weighed and divided in three groups on the basis of body weight, and they were euthanized by decapitation after 0 or 6 h of fasting, or 6 h of refeeding after 6 h of fasting. The whole brains were collected, preserved in RNAlater® tissue storage reagent (Sigma-Aldrich, St. Louis, MO, USA), and dissected into six segments (telencephalon, optic lobes, cerebellum, rostral part of the brainstem, middle part of the brainstem, and caudal part of the brainstem) as described previously (Aoki et al., 2017). Total RNA extraction and cDNA synthesis were conducted using Sepasol-RNA I (Nacalai Tesque, Kyoto, Japan) and ReverTra Ace® qPCR RT Master Mix with gDNA Remover (Toyobo, Osaka, Japan) as described previously (Honda et al., 2015a). cDNAs of chicken IGFBP -1, -2, -3, -4, and -5 (GenBank accession numbers: NM_01001294, NM_205359, NM_001101034, NM_204353, and XM_422069, respectively) were amplified with the following primers: IGFBP-1 sense, 5′-CCC AAC TGT AAC AAG AAT GGA TTT T-3′; IGFBP-1 antisense, 5′-CGG AAT CTC CAT CCA GTG AAG-3′; IGFBP-2 sense, 5′-AAT GGG CAG CGT GGA GAG T-3′; IGFBP-2 antisense, 5′-CTG GAT CAC CTT CCC ATG GA-3′; IGFBP-3 sense, 5′-ATC AGG CCA TCC CAA GCT T-3′; IGFBP-3 antisense, 5′-GAT GTG CTG TGG AGG CAA ATT-3′; IGFBP-4 sense, 5′-GAG CAC CCC AAC AAC AGC TT-3′; IGFBP-4 antisense, 5′-CCG TTG TTG ATG CGC TTT G-3′; IGFBP-5 sense, 5′-CAA GGC CGA ACG GGA AT-3′; IGFBP-5 antisense, 5′-TCC TCC GTC ATC TCC GAT GT-3′. cDNA of ribosomal protein S17 (GenBank accession number: NM_204217), as an internal standard, was amplified with previously reported primers (Honda et al., 2015b). mRNA levels were quantified in duplicate using an Applied Biosystems 7300 Real-Time PCR system and SYBR® Premix Ex Taq™ II (Tli RNaseH Plus) (Takara Bio, Shiga, Japan) according to the supplier's recommendations.
Data were analyzed with one-way analysis of variance and Fisher's protected least significant differences post-hoc test. All statistical analyses were performed using the commercial package StatView version 5 (SAS Institute, Cary, NC, USA).
As shown in Table 1, 6 h of fasting significantly increased the mRNA levels of IGFBP-1 and -2 in the chick liver, and these changes were reversed by 6 h of refeeding. The changes in IGFBP-2 are in agreement with the results of a study in 6-week-old layer chickens, in which hepatic IGFBP-2 mRNA expression was significantly increased after 2 days of food deprivation and this increase was reversed after 6 h of refeeding (Kita et al., 2002). Beccavin et al. (1999) reported that 48 h of fasting significantly increased plasma 28-kDa and 34-kDa IGFBPs in male lean chickens and suggested that these proteins are the equivalents of mammalian IGFBP-1 and -2, respectively. These findings suggest that the transcriptional regulation of hepatic IGFBP-1 and -2 may play a physiological role in response to a change in nutritional status induced by short-term fasting and refeeding in broiler chicks.
| Feeding | Fasting | Refeeding | |
|---|---|---|---|
| IGFBP-1 | 1.00±0.12a | 4.81±1.91b | 1.07±0.26a |
| IGFBP-2 | 1.00±0.14a | 2.72±0.78b | 0.58±0.05a |
| IGFBP-3 | 1.00±0.08 | 0.93±0.07 | 0.80±0.12 |
| IGFBP-4 | 1.00±0.20 | 0.64±0.07 | 0.59±0.05 |
| IGFBP-5 | 1.00±0.12 | 0.70±0.06 | 0.95±0.17 |
Values are means±SEM of six chicks in each group. Values with different letters are significantly different (P<0.05).
In mammals, the availability of blood IGFs to the receptors of target cells is limited by binding to IGFBPs. In chickens, the 28-kDa IGFBP (the equivalent of mammalian IGFBP-1) released by hepatoma cells inhibits exogenous IGF-1-stimulated amino acid uptake by the hepatoma cells (Duclos et al., 1998). Kita et al. (2002) reported that the response of IGFBP-2 gene expression to variations in nutritional status was rapid and differed in several tissues of layer chickens, which would help modulate the growth-promoting effect of circulating IGF-I through IGF-IGFBP complex formation. In the present study, the mRNA levels of hepatic IGFBP-1 and -2 were significantly increased by 6 h of fasting in broiler chicks. Therefore, it seems likely that the upregulation of hepatic IGFBP-1 and -2 expression may contribute to the suppression of IGF effects under the fasting condition in chickens.
Nagao et al. (2001) reported that intravascular administration of insulin reversed the fasting-induced increase in hepatic IGFBP-2 mRNA in layer chickens. Plasma insulin reportedly significantly decreases within 6 h of fasting in broiler chickens (Krestel-Rickert et al., 1986; Christensen et al., 2013; Saneyasu et al., 2017). Krestel-Rickert et al. (1986) reported that 4 h of fasting significantly decreased the plasma insulin level, and this decrease was returned to prefasted concentration within 15 min of feeding in broiler chickens. Bigot et al. (2003) reported that 30 min of refeeding significantly increased plasma insulin in 3-week-old broiler chickens. It is therefore likely that plasma insulin may be involved in the fasting- and refeeding-induced changes in IGFBP-2 mRNA in the liver in broiler chicks.
The mRNA levels of most IGFBPs throughout the brain were not changed by fasting and refeeding (Table 2). However, IGFBP-3 mRNA in the middle part of the brainstem was significantly decreased by 6 h of fasting, and this change was not reversed by 6 h of refeeding. The mRNA level of IGFBP-5 in the optic lobes was significantly decreased after 6 h of refeeding. In mammals, IGFBP-3 and -5 are associated with the cell surface and extracellular matrix and modulate IGF actions in the cells (Clemmons, 2001; Firth and Baxter, 2002). In addition, IGFBP-3 and -5 show IGF-independent biological effects (Firth and Baxter, 2002). The physiological importance of IGFBPs in the brain is not fully understood in both mammals and birds. However, it is possible that IGFBP-3 and -5 play important roles in the neurons of the middle part of the brainstem or optic lobes in response to fasting-induced changes in nutritional status in chickens.
| Feeding | Fasting | Refeeding | |
|---|---|---|---|
| Telencephalon | |||
| IGFBP-1 | 1.00±0.09 | 1.15±0.07 | 0.97±0.09 |
| IGFBP-2 | 1.00±0.04 | 1.11±0.10 | 0.95±0.05 |
| IGFBP-3 | 1.00±0.04 | 1.00±0.06 | 0.95±0.04 |
| IGFBP-4 | 1.00±0.06 | 0.90±0.09 | 1.10±0.07 |
| IGFBP-5 | 1.00±0.07 | 1.11±0.13 | 0.99±0.08 |
| Optic lobes | |||
| IGFBP-1 | 1.00±0.09 | 1.01±0.12 | 0.94±0.11 |
| IGFBP-2 | 1.00±0.07 | 0.92±0.09 | 0.89±0.14 |
| IGFBP-3 | 1.00±0.06 | 0.86±0.04 | 0.88±0.07 |
| IGFBP-4 | 1.00±0.14 | 0.94±0.11 | 0.91±0.13 |
| IGFBP-5 | 1.00±0.03a | 0.85±0.08ab | 0.68±0.08b |
| Cerebellum | |||
| IGFBP-1 | 1.00±0.13 | 1.22±0.05 | 1.05±0.21 |
| IGFBP-2 | 1.00±0.04 | 1.04±0.12 | 1.08±0.09 |
| IGFBP-3 | 1.00±0.12 | 0.97±0.07 | 1.12±0.10 |
| IGFBP-4 | 1.00±0.15 | 1.17±0.13 | 1.23±0.12 |
| IGFBP-5 | 1.00±0.11 | 1.26±0.07 | 1.00±0.14 |
| Rostralpart of the brainstem | |||
| IGFBP-1 | 1.00±0.06 | 1.05±0.10 | 0.81±0.07 |
| IGFBP-2 | 1.00±0.05 | 1.02±0.04 | 1.24±0.17 |
| IGFBP-3 | 1.00±0.04 | 0.94±0.03 | 0.95±0.04 |
| IGFBP-4 | 1.00±0.13 | 1.18±0.05 | 1.07±0.10 |
| IGFBP-5 | 1.00±0.04 | 0.98±0.07 | 1.01±0.08 |
| Middle part of the brainstem | |||
| IGFBP-1 | 1.00±0.08 | 1.02±0.06 | 0.89±0.10 |
| IGFBP-2 | 1.00±0.04 | 0.97±0.05 | 0.89±0.07 |
| IGFBP-3 | 1.00±0.04a | 0.84±0.02b | 0.84±0.02b |
| IGFBP-4 | 1.00±0.11 | 0.87±0.07 | 0.94±0.07 |
| IGFBP-5 | 1.00±0.07 | 0.78±0.05 | 0.80±0.07 |
| Caudalpart of the brainstem | |||
| IGFBP-1 | 1.00±0.09 | 1.05±0.03 | 0.84±0.09 |
| IGFBP-2 | 1.00±0.07 | 1.10±0.18 | 0.90±0.07 |
| IGFBP-3 | 1.00±0.04 | 0.93±0.03 | 0.87±0.04 |
| IGFBP-4 | 1.00±0.11 | 1.13±0.09 | 1.32±0.16 |
| IGFBP-5 | 1.00±0.13 | 0.98±0.07 | 0.94±0.06 |
Values are means±SEM of six chicks in each group. Values with different letters are significantly different (P<0.05).
We previously demonstrated that intravascular administration of IGF-1 significantly suppressed food intake in chicks (Fujita et al., 2017). The dorsal vagal complex (DVC) and arcuate nucleus (ARC) receive and integrate peripheral satiety signals in mammals (Williams and Elmquist, 2012). In chickens, the rostral and caudal parts of the brainstem include the DVC and in fundibular nucleus (the avian equivalent of mammalian ARC), respectively (Kuenzel and Masson, 1988). In addition, we have reported that the IGF-1 receptor is expressed in these parts of the brain in chicks (Fujita et al., 2017). The plasma IGF-1 concentration was significantly decreased by 48 h of fasting and reversed by 48 h of refeeding in layer chickens (Kita et al., 2002). The effects of short-term fasting on the plasma IGF-1 concentration in chickens had not been investigated. However, McMurtry et al. (1996) demonstrated that the half-life of free IGF-1 was 5.17±0.27 min in broiler chickens. We previously confirmed that hepatic IGF-1 mRNA levels were significantly decreased by 6 h of fasting in 8-day-old broiler chicks (Fujita et al., 2017) and by 4 h of fasting in 14-day-old broiler chicks (unpublished data). These findings suggest that the plasma IGF-1 concentration may be significantly decreased under the 6-h fasted condition, which in turn stimulates appetite in broiler chicks. On the other hand, the mRNA levels of IGFBPs in the rostral part and the caudal part of the brainstem were not influenced by 6 h of fasting and refeeding in chicks (Table 2). It is therefore possible that IGFBPs in these parts are not involved in appetite regulation, at least in the experimental conditions used in this study.
In summary, we examined the effects of fasting and refeeding on IGFBP expression in the liver and brain. Our findings suggest that IGFBP-1 and -2 in the liver, IGFBP-3 in the middle part of the brainstem, and IGFBP-5 in the optic lobes may play physiological roles in response to nutritional changes in broiler chicks.
