Alleviation of body weight loss by dietary methionine is independent of insulin-like growth factor-I in protein-starved young chickens
ABSTRACT
It is well known that in protein-starved chickens, small amounts of amino acid supplement, especially methionine, reduces nitrogen excretion and thereby improves nitrogen balance. On the other hand, excess intake of methionine causes growth depression and the growth-depressive effect of excess methionine can be alleviated by consumption of dietary glycine. Insulin-like growth factor-I (IGF-I) is one of various growth-promoting factors relating to the efficiency of animal production and is known to be very sensitive to changes in nutritional status. In the present study, the interactive effect of glycine on nitrogen sparing effect of methionine in protein-starved chickens was examined. In addition, the relation of IGF-I and its specific binding protein to the nitrogen sparing effect of supplemented methionine was also investigated. Two-days refeeding of methionine supplemented to protein-free diet could promptly alleviate body weight loss in protein-starved chickens, and the alleviation of body weight loss by methionine was not improved by glycine supplements. Moreover, such acute alleviation of body weight loss by dietary methionine was independent of the change in plasma IGF-I concentration.
INTRODUCTION
There are 20 amino acids in body proteins, and all are physiologically essential. However, nutritionally these amino acids can be divided into two categories: those that animals cannot synthesize at all or not enough for metabolic requirements (essential) and those that can be synthesized from other amino acids (nonessential). It has been well recognized that methionine is one of the essential amino acids for both avian and mammalian species. The supplementation of methionine in a protein-free diet had a nitrogen-sparing effect leading to the reduction of urinary nitrogen excretion in rats (Yoshida & Moritoki 1974; Yokogoshi & Yoshida 1976; Yokogoshi et al. 1977), dogs (Allison et al. 1947) and pigs (Lubaszewska et al. 1973). In protein-starved chicks, small amounts of amino acid supplement, especially methionine alone or in combination with arginine, also reduced nitrogen excretion and thereby improved nitrogen balance (Muramatsu & Okumura 1979a,b,c, 1980a,b; Okumura & Muramatsu 1978a,b). On the other hand, it was reported that methionine was one of the most toxic amino acids and excess intake of methionine caused growth depression with serious tissue damage (Harper et al. 1970). The growth-depressive effect of dietary excess methionine could be alleviated by consumption of dietary glycine and serine in rats (Benevenga & Harper 1970), and similar effect of glycine was also found in chicks (Hafez et al. 1978). The alleviating effect of glycine to the disadvantage of dietary excess methionine may be applied to the nitrogen-sparing effect of methionine in protein-starved chickens.
Insulin-like growth factor-I (IGF-I) is one of the various growth-promoting factors relating to the efficiency of animal production. IGF-I has been characterized and shown to be 70 amino acid polypeptides (Ballard et al. 1990). IGF-I plays a role in the postnatal growth of numerous mammalian species. In chickens, hypophysectomy results in a reduction in both growth rate and plasma IGF-I concentration, and sex-linked dwarf chickens, which lack a functional growth hormone receptor, and also have significantly reduced levels of plasma IGF-I (Huybrechts et al. 1985; Tanaka et al. 1996), suggesting that IGF-I also contributes to postnatal growth in avian species. Furthermore, it was also found that lowered plasma IGF-I concentration caused by food-deprivation remained low until 24 h after refeeding a commercial diet and recovered to the level of fed controls after 48 h of refeeding (Kita et al. 2002). In most circumstances, IGF-I makes a complex with specific, high-affinity IGF-binding proteins (IGFBPs). So far, six different IGFBPs have been found in mammals and their genes have also been cloned (Shimasaki & Ling 1991). IGFBPs are thought to prolong the half-life of circulating IGFs (Cohen & Nissley 1976) and also act as modulators of IGF activity (Elgin et al. 1987; Rutanen et al. 1988). Several types of IGFBPs have also been found in blood of avian species and Schoen et al. (1995) have cloned and characterized a complementary DNA (cDNA) of chicken IGFBP-2 which is the most abundant IGFBP in the circulation of chickens. Gene expression and plasma level of IGFBP-2 responded promptly to the alteration in nutritional status in chickens. Previously, we revealed that hepatic and gizzard IGFBP-2 gene expression was increased by fasting and decreased by refeeding (Kita et al. 2002). In this study, we also indicated that the change in tissue IGFBP-2 gene expression was modulated by dietary protein quality, including the level of limiting amino acids. However, the relation of IGF-I and IGFBP-2 to nitrogen sparing action of dietary methionine in chicks receiving a protein-free diet has not been clarified.
In the present study therefore the change in plasma IGF-I concentration and tissue IGFBP-2 gene expression was examined in protein-starved young chickens receiving methionine alone. In addition, the interactive effect of dietary methionine and glycine on nitrogen-sparing action was investigated.
MATERIALS AND METHODS
Animals and diets
One hundred single-comb White Leghorn male chicks from a local hatchery (GHEN Corporation Ltd, Gifu, Japan) were fed a commercial chick mash diet (crude protein 215 g/kg, metabolizable energy 12.1 kJ/g; Toyohashi Shiryou Ltd, Toyohashi, Japan) from hatching until 39 days of age in electrically heated brooders. At this age, 25 birds of uniform body weight were selected and divided evenly into five experimental groups of five birds each. The birds were placed in individual stainless steel metabolism cages. Continuous illumination was provided. All birds in five experimental groups were initially fed a protein-free diet ad libitum for 2 days. Then chicks in five experimental groups were given: (i) crude protein (CP) 20% diet; (ii) a protein-free diet; (iii) a protein-free diet supplemented with methionine; (iv) a protein-free diet supplemented with glycine; and (v) a protein-free diet supplemented with both methionine and glycine. The composition of experimental diets is shown in Table 1, and all experimental diets were given ad libitum for 2 days. At the end of the experiment, the birds were anesthetized by diethyl ether and blood was collected by heart puncture. Plasma was separated immediately and stored at −20°C. Then the liver and gizzard were removed and frozen immediately in liquid nitrogen and stored at −80°C. Animal care was in compliance with applicable guidelines from the Nagoya University Policy on Animal Care and Use.
| Ingredients (g/kg) | CP 20% | Protein-free | Protein-free + glycine | Protein-free + methionine | Protein-free + methionine + glycine |
|---|---|---|---|---|---|
| Isolated soybean protein† | 239.0 | – | – | – | – |
| L-methionine | 2.9 | – | – | 3.0 | 3.0 |
| L-threonine | 1.2 | – | – | – | – |
| Glycine | 4.2 | – | 7.0 | – | 7.0 |
| Cornstarch | 505.4 | 752.7 | 745.7 | 749.7 | 742.7 |
| Cellulose | 154.3 | 154.3 | 154.3 | 154.3 | 154.3 |
| Maize oil | 30.0 | 30.0 | 30.0 | 30.0 | 30.0 |
| Mineral mixture‡ | 58.5 | 58.5 | 58.5 | 58.5 | 58.5 |
| Vitamin mixture‡ | 2.0 | 2.0 | 2.0 | 2.0 | 2.0 |
| Choline chloride | 1.5 | 1.5 | 1.5 | 1.5 | 1.5 |
| Inositaol | 1.0 | 1.0 | 1.0 | 1.0 | 1.0 |
- † 84% crude protein.
- ‡ Kita et al. (1996).
Chemical analysis
Plasma IGF-I concentration was measured by radioimmunoassay according to the method described by Ballard et al. (1990) modified by Kita et al. (1996). Polyclonal antiserum raised in rabbits against human IGF-I was generously gifted by Dr P. C. Owens (Department of Obstetrics and Gynecology, Adelaide University, Adelaide, SA, Australia). IGFBP-2 messenger RNA (mRNA) level was quantified using Northern blot analysis described by Schoen et al. (1995). Briefly, total RNA was extracted from tissue samples, and to detect tissue IGFBP-2, Northern hybridization was conducted. In the present study, to certify that equal amounts of total RNA samples were loaded onto agarose gels for electrophoresis, chicken ribosomal protein S17 mRNA was measured. Chicken ribosomal protein S17 cDNA was generously gifted by Dr B. Trueb (Laboratorium fur Biochemie I, ETH Zentrum, Zurich, Switzerland). The intensities of chicken IGFBP-2 and ribosomal protein S17 bands were measured using a bio-imaging analysis system (BAS 2000; Fuji Photo Film, Co. Ltd, Tokyo, Japan).
Statistical analysis
Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's multiple range test using the General Linear Model Procedures (GLM; SAS/STAT Version 6, SAS Institute, Cary, NC, USA). Two-way ANOVA was also performed to test main and interactive effects of methionine and glycine supplementation in protein-free diets. Differences between means were considered to be significant at P < 0.05.
RESULTS
Influence of dietary supplementation with methionine and/or glycine to a protein-free diet on body weight change, feed intake and plasma IGF-I concentration of protein-starved young chickens is shown in Table 2. During 2 days of protein starvation before feeding various experimental diets, the average body weight loss of protein-starved chickens was −37.0 g ± 9.1 g (SD). Refeeding with CP 20% diet to protein-starved chickens produced 31.2 g gain in body weight. When protein-starved chickens were kept on feeding the protein-free diet for a further 2 days, an additional 22.0 g of body weight was lost. The body weight loss of chickens given the protein-free diet supplemented with methionine was significantly less than that of protein-starved chickens. No effect of glycine supplementation to the protein-free diet was observed on body weight change. There were no significant differences among any dietary treatments in feed intake. Plasma IGF-I concentration of protein-starved chickens followed by refeeding with CP 20% diet was significantly higher than those of chickens given the protein-free diets supplemented with amino acids. Dietary supplementation with methionine and/or glycine to a protein-free diet had no effect on plasma IGF-I concentration compared to that in protein-starved chickens. Although gene expression of ribosomal protein S17 was detected, which confirmed that RNA samples were successfully loaded on agarose gel electrophoresis, IGFBP-2 mRNA was not detected in RNA samples extracted from liver and gizzard of chickens in all dietary treatments (data not shown).
| Refeeding diets | Body weight change (g/2 days) | Feed intake (g/2 days) | Plasma IGF-I concentration (ng/mL) |
|---|---|---|---|
| Crude protein 20% | 31.2 ± 5.7a | 112.5 ± 5.7 | 27.3 ± 4.0a |
| Protein-free | −22.0 ± 4.4c | 83.2 ± 16.9 | 19.1 ± 2.1ab |
| Protein-free + methionine | −9.8 ± 3.8b | 130.4 ± 11.5 | 15.1 ± 2.8b† |
| Protein-free + glycine | −24.6 ± 4.8c | 115.8 ± 11.3 | 12.6 ± 1.5b |
| Protein-free + methionine + glycine | −12.5 ± 1.7b† | 104.1 ± 13.5 | 16.7 ± 3.0ab† |
| Analysis of variance (probability) | |||
| methionine | 0.009 | 0.206 | 0.973 |
| glycine | 0.525 | 0.820 | 0.315 |
| Methionine × glycine | 0.990 | 0.044 | 0.102 |
- abc Means (±SE) in the same column with different superscript letters were significantly different (n = 5, P < 0.05).
- † †One missing value.
DISCUSSION
In the present study, first, the nitrogen-sparing effect of dietary methionine and/or glycine in protein-starved young chickens was examined. During 2 days of protein starvation before feeding various experimental diets, chickens lost 37.0 g body weight, and successive feeding of the protein-free diet for a further 2 days, an additional 22.0 g of body weight was lost. However, supplementation with methionine to the protein-free diet alleviated body weight loss by 6 g/day, which was in good agreement with the nitrogen-sparing effect of dietary methionine extensively studied in several mammalian species (Allison et al. 1947; Lubaszewska et al. 1973; Yoshida & Moritoki 1974; Yokogoshi & Yoshida 1976; Yokogoshi et al. 1977) and chickens (Okumura & Muramatsu 1978a,b; Muramatsu & Okumura 1979a,b,c, 1980a,b). On the other hand, body weight loss of chickens given the protein-free diet supplemented with glycine alone was similar to that of birds given the protein-free diet with no supplementation. This result suggested that supplementation of dietary glycine alone to protein-free diet had no effect on body weight change in protein-starved chickens. As reported previously that glycine had the effect of alleviating growth depression caused by excess dietary methionine intake (Benevenga & Harper 1970; Toue et al. 2006), in the present study, the interactive effect of glycine to enhance the nitrogen-sparing effect of methionine in protein-starved chickens was expected. However, as shown in Table 2, body weight loss of chickens fed the protein-free diet supplemented with both methionine and glycine was similar to that of chickens given the protein-free diet supplemented with only methionine. These results suggest that the effect of glycine on methionine toxicity might be available when dietary amino acids, except for methionine and glycine, were fully sufficient to nutrient requirements.
In our previous studies, plasma IGF-I concentrations of chickens under severe nutritional conditions like protein starvation and feed deprivation, were halved compared to those of birds fed normal diets sufficient for nutritional requirements (Kita et al. 1996, 2002; Kita & Okumura 1999). As it was also found that lowered plasma IGF-I concentration caused by food deprivation remained low until 24 h after refeeding a commercial diet, and recovered to the level of fed controls after 48 h of refeeding (Kita et al. 2002), in the present study, plasma IGF-I concentration was measured after 2 days after refeeding with experimental diets. As we had expected, plasma IGF-I concentration of protein-starved chickens refed CP 20% diet for 2 days showed the highest value of all (Table 2). As shown in Table 2, the supplementation with methionine could successfully alleviate growth depression by protein starvation. However, regardless of the supplementation with methionine and/or glycine, plasma IGF-I concentration of protein-starved chickens was not affected by amino acid supplementation. These results suggest that the acute effect of supplemented methionine to spare body nitrogen of protein-starved chickens seems to be independent from the change in plasma IGF-I concentrations.
In most circumstances, IGF-I makes a complex with specific, high-affinity IGF-binding proteins (IGFBPs). So far, six different IGFBPs have been found in mammals and their genes have also been cloned (Shimasaki & Ling 1991). IGFBPs are thought to prolong the half-life of circulating IGFs (Cohen & Nissley 1976) and also act as modulators of IGF activity (Elgin et al. 1987; Rutanen et al. 1988). Previously, we revealed that IGFBP-2 gene expression in the gizzard and liver was increased by fasting and decreased by refeeding (Kita et al. 2002). However, in the present study no influence of feeding a protein-free diet and the supplementation of methionine and/or glycine to the protein-free diet on tissue IGFBP-2 gene expression was observed (data not shown). The depression of IGFBP-2 gene expression enhanced by refeeding was highly associated with the elevation in plasma insulin concentration stimulated by refeeding (Nagao et al. 2001), and it was suggested that little change in plasma insulin level might be brought about by protein starvation and the supplementation with methionine and/or glycine to the protein-free diet.
From these observations, we conclude that 2 days refeeding of methionine could promptly spare body nitrogen in protein-starved chickens, and the nitrogen-sparing effect of methionine was not improved by glycine supplements. Moreover, such acute alleviation of body weight loss by methionine seemed to be independent from the change in plasma IGF-I concentrations.
ACKNOWLEDGMENTS
Financial support was provided by a grant-in-aid (Number 19380149) for Scientific Research (B) from The Japan Society for the Promotion of Science (JSPS). In this study, iodination for RIA were carried out in the Radioisotope Center of Nagoya University, Japan. The authors thank the Radioisotope Center for support in this study.
