PHYSIOLOGY, ENDOCRINOLOGY, AND REPRODUCTION Research Note The spleen accumulates advanced glycation end products in the chicken: Tissue comparison made with rat K. Kita1 Faculty of Agriculture, Iwate University, Iwate 020-8550, Japan study, therefore, the radioactive AGE prepared by reacting 14C-glucose and amino acids were intravenously administrated, and comparison of tissue accumulation of 14C-labeled AGE was made between chickens and rats. At 30 min after administration, tissues (18–20) were collected, and the radioactivity incorporated into tissues was determined. High levels of radioactivity per gram of tissue in the liver and kidney were observed in both rats and chickens. In chickens but not rats, a large amount of 14C-labeled AGE incorporated into 1 g of spleen was observed, and the specific accumulation of AGE in the avian spleen might have a particular role in immune response in avian species.
Key words: advanced glycation end product, tissue accumulation, spleen, chicken, rat 2014 Poultry Science 93:429–433 http://dx.doi.org/10.3382/ps.2013-03576 al., 2008). It was also reported that Nε-carboxymethyllysine (CML), which is known to be one type of noncross-linking AGE, was detected in skeletal muscles of diabetic rats (Snow et al., 2006; Snow and Thompson, 2009). Compared with protein-bound AGE originated from structural proteins, the information about tissue accumulation of free AGE in the blood has been limited. The intravenous administration of 3H-labeled pentosidine revealed that radioactive pentosidine rapidly accumulated in the kidney, was filtered by renal glomeruli, and was excreted in the urine of rats (Miyata et al., 1998). Recently, it was reported that, in rats, intravenously administrated 18F-labeled CML was quickly distributed via the blood and rapidly excreted through the kidneys within 20 min after injection (Xu et al., 2013). Hyperglycemia is commonly observed in avian species, and the preeminent traits of this class are as follows: 1) high blood glucose concentrations typically 2 to 3 times higher than human, which should accelerate amino-carbonyl reaction and generate high concentration of AGE; 2) an elevated basal body temperature (about 3°C higher than mammals), which should contribute to the nonenzymatic attachment of glucose to proteins and amino acids (Klandorf et al., 1995; Iqbal et al., 1999b). In avian species, pentosidine was de-
INTRODUCTION Glycation, or Maillard reaction, starts from a nonenzymatic amino-carbonyl reaction binding carbonyl group of reducing sugars to an amino group of proteins and amino acids (Maillard, 1912). Glycation leads to formation of a Schiff base followed by rearrangement into stable Amadori products. Amadori products undergo further complex reactions to form advanced glycation end products (AGE). The acceleration of Maillard reaction during hyperglycemia increases the production and accumulation of AGE, which is implicated in the gradual development of diabetic complications in diabetes mellitus (Brownlee, 2001; Singh et al., 2001). Tissue accumulation of AGE originated from structural proteins has been widely examined in various tissues of diabetic rats. Chronic hyperglycemia caused a significant increase in the concentration of pentosidine, which is one of representative AGE formed by nonenzymatic glycation of lysine and arginine residues, mainly in the aorta and skin of rats (Mikulíková et ©2014 Poultry Science Association Inc. Received August 21, 2013. Accepted October 13, 2013. 1 Corresponding author: [email protected]
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ABSTRACT Glycation starts from nonenzymatic amino-carbonyl reaction that binds carbonyl group of reducing sugars to the amino group of amino acids. Glycation leads to further complex reactions to form advanced glycation end products (AGE). Because AGE are implicated in the gradual development of diabetic complications, tissue accumulation of AGE has been widely examined in various tissues of rats. Avian species are known to have high body temperature and blood glucose concentration compared with mammals. Although these characteristics enabled chickens to be used as experimental models for diabetes mellitus, the information of AGE accumulation in various tissues of chickens has not been limited so far. In the present
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tected in collagen, which is the main structural protein of connective tissue of skin and tendon in broiler hens (Iqbal et al., 1997, 1999a, 2000). Glucose can bind to not only structural proteins but also to secreted protein. Although glycated albumin was detected in the serum of chickens (Klandorf et al., 1995), little is known about tissue accumulation of free glycated proteins and AGE existing in the circulation. In the present study, therefore, the radioactive AGE formed from 14C-glucose and amino acids were intravenously administrated to rats and chickens, and tissue accumulation of AGE was compared between chickens and rats.
Preparation of Radioactive AGE The radioactive AGE were prepared by reacting amino acids with radioactive glucose. Initially, 50 mg each of 20 amino acids (alanine, arginine monohydrochloride, asparagine monohydrate, aspartic acid, cysteine hydrochloride, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine monohydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine) was added in 50 mL of water. Then, 3.6 g of glucose (2 M of final concentration) was dissolved in 10 mL of amino acid suspension, and 1.85 MBq of radioactive glucose containing U-14C-D-glucose (7.4 MBq/mL, Amersham Japan, Tokyo, Japan) was added. Subsequently, the mixture solution was incubated at 200°C for 3 h. After incubation, free radioactive and nonradioactive glucose unbound to amino acids was removed by Sephadex G-10 (Amersham Japan) column chromatography. The mobile phase was water and the flow rate was 1.45 mL/min. The drops for 1 min each were collected in sampling tubes, and 60 tubes were sampled in total. Glucose concentration in collected fractions was measured by a commercial kit (Glucose C-II Test Wako, Wako Pure Chemical Industries Inc., Osaka, Japan). The radioactivity in collected 14C-labeled AGE samples was determined by using a liquid scintillation counter (Aloka Co., Ltd., Tokyo, Japan). The 14C-labeled AGE solution excluding free glucose was used for intravenous administration to rats and chickens.
Birds and Experimental Procedures In the rat experiment, 7 male Wistar rats (6 wk old) were purchased from a local supplier (Japan SLC Inc., Hamamatsu, Japan). They were kept in plastic cages, in a temperature-controlled room at 24 ± 2°C, with artificial light from 0700 to 1900 h. All rats were fed a stock diet (Labo MR, Nihon Nosan Co. Ltd., Yokohama, Japan) with free access to water until radioactive AGE administration. To investigate the accumulation of 14C-AGE in various tissues, 7,900 Bq/kg of BW of 14C-labeled AGE was injected intravenously via tail vein under light anesthesia with diethyl ether. Body
Measurement of Radioactivity Incorporated into Tissues After thaw of frozen samples, approximately 0.4 g of tissues was weighed accurately and homogenized in 0.5 mL of 0.5% (wt/vol) NaOH/0.1% (vol/vol) Triton-X-100 solution. A part of tissue homogenate was weighed accurately, mixed with 0.5 mL of ACS-II scintillator cocktail (Amersham Japan), and its radioactivity was measured using a liquid scintillation counter. Animal experiments were conducted at Nagoya University, and animal care was in compliance with applicable guidelines from the Nagoya University Policy on Animal Care and Use.
Statistical Analysis Data were analyzed by one-way ANOVA to assess the significance of the effects of difference in tissues. Then, Duncan’s multiple range test was performed to compare between all pairs of means. All statistical analyses were performed using the GLM procedures (SAS/ STAT version 6, SAS Institute Inc., Cary, NC). Differences between means were considered to be significant at P < 0.05.
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MATERIALS AND METHODS
weight of rats was 181.0 ± 1.6 (SE) g. At 30 min after injection, rats were anesthetized with diethyl ether, and blood was collected by heart puncture. After rats were killed by deep anesthesia with diethyl ether, cerebrum, cerebellum, eyes, heart, thymus, lung, stomach, duodenum, jejunum, left cecum, rectum, liver, pancreas, kidney, spleen, testis, left gastrocnemius muscle, and a small part of skin were removed. All tissues were rinsed in ice-cold Dulbecco’s phosphate buffered saline, blotted, weighed, and frozen in liquid N2. Frozen samples were stored at −30°C until analysis. In the chicken experiment, 30 male Single-Comb White Leghorn chicks (1-d-old) were obtained from a local hatchery (Ghen Co. Ltd., Gifu, Japan). Chicks were maintained on a commercial chick mash diet (CP 207 g/kg, ME 12.1 kJ/g, Toyohashi Feed Mills Co., Ltd., Toyohashi, Japan). Chicks were housed in an electrically heated brooder (28 ± 2°C) with continuous illumination. At 12 d of age, 8 birds of uniform BW [120.3 ± 1.5 (SE) g] were selected and 11,500 Bq/kg of BW of 14C-labeled AGE was injected via wing vein of each bird. At 30 min after injection, chicks were anesthetized by diethyl ether, and then blood was collected by heart puncture. After neck dislocation, cerebrum, mesocephalon, cerebellum, left eye, heart, left lung, proventriculus, gizzard, duodenum, jejunum, colon, rectum, liver, pancreas, left kidney, spleen, testis, left major breast muscle, left minor breast muscle, and a small part of breast skin were removed. All tissues were rinsed in ice-cold Dulbecco’s phosphate buffered saline, blotted, weighed, and frozen in liquid nitrogen. Frozen samples were stored at −30°C until analysis.
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RESULTS The weight of various tissues of rats and chickens is shown in Table 1. In rats, liver weight was the heaviest of all. The second heaviest tissue was jejunum. There
Table 1. Tissue weight of chickens and rats1 Tissue Large brain Mid brain Small brain Eye Heart Thymus Lung Proventriculus Gizzard Stomach Duodenum Jejunum Colon Rectum Liver Pancreas Kidney Spleen Testis Gastrocnemius muscle Major breast muscle Minor breast muscle
Chicken2 0.73 0.22 0.16 0.70 0.99 0.51 1.10 5.20 0.83 3.24 0.58 0.34 4.55 0.81 0.16 0.66 0.06 3.28 0.93
0.010fg 0.011ij 0.011ij 0.034fg 0.043de — ± 0.058h ± 0.018d ± 0.139a — ± 0.064ef ± 0.123c ± 0.023gh ± 0.039i ± 0.098b ± 0.032df ± 0.008ij ± 0.047fgh ± 0.006j — ± 0.079c ± 0.033de ± ± ± ± ±
Rat3 1.13 ± 0.022d — 0.22 ± 0.005gh 0.12 ± 0.009i 0.68 ± 0.016efg 0.63 ± 0.045fg 0.95 ± 0.032de — — 1.13 ± 0.016d 0.51 ± 0.046g 4.81 ± 0.147b 0.73 ± 0.068efg 0.90 ± 0.172def 8.86 ± 0.236a 0.96 ± 0.047de 1.68 ± 0.074c 0.43 ± 0.018gh 1.71 ± 0.137c 1.07 ± 0.143d — —
a–jMeans not sharing a common superscript in the same column are significantly different (P < 0.05). 1Means ± SE. 2n = 8 of chickens. 3n = 7 of rats.
were no significant difference in tissue weight between kidney and testis, and they were the third heaviest tissues in rats. In chickens, gizzard weight was the heaviest in all tissues. The second heaviest tissue was liver. The following heavy tissues were large breast muscle and jejunum, and the weights of both tissues were not significantly different. The radioactivity per gram of tissue of rats administrated intravenously with 14C-labeled AGE is shown in Figure 1. The radioactivity per unit tissue weight in the liver and kidney was the highest compared with other tissues. There were no significant differences in all other tissues. The radioactivity per gram of tissue of chickens is represented in Figure 2. Similarly to rats, the highest radioactivity per gram of tissue was detected in the liver. The second highest incorporation of 14C-labeled AGE into 1 g of spleen was measured in chickens, which was not observed in the rat experiment. The third highest radioactivity per gram of tissue was measured in the kidney. Tissues following after kidney were rectum and testis.
DISCUSSION In the present study, to compare chickens with rats in tissue accumulation of AGE administrated intravenously, the radioactive AGE were prepared from amino acids and 14C-glucose. After glycation, free radioactive and nonradioactive glucose unbound to amino acids was successfully removed by using Sephadex G-10 column chromatography, which was confirmed by the follow-
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Figure 1. Incorporation of 14C-advanced glycation end products (AGE) in grams of tissue of rats administrated with radioactive AGE via tail vein. Bars represent means ± SE (n = 7). Bars not sharing a common letter (a,b) are significantly different (P < 0.05).
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ing determination of glucose concentration in collected fractions including 14C-labeled AGE (data not shown). Glycation is one of nonenzymatic chemical reactions taken place spontaneously in vivo, and this reaction forms AGE that have been known to be important factors associated with the onset of diabetic complications. In comparison with mammals, the information about tissue accumulation of free AGE existing in the blood has been limited regardless of the advantage in avian species to be experimental models for diabetes. In the present study, therefore, the tissue accumulation of 14C-labeled AGE injected intravenously was compared between chickens and rats, and it was revealed that the apparent major site of AGE accumulation was liver in both species (Figures 1 and 2). The highest accumulation of 14C-labeled AGE in the liver seems to be plausible because liver is the largest and second largest visceral organs of rats and chickens, respectively (Table 1). Meanwhile, hepatic exposure to high levels of AGE augmented hepatic fibrosis associated with upregulation of receptors for AGE (RAGE; Goodwin et al., 2013), suggesting that free AGE in the circulation were bound to RAGE and incorporated into hepatic cytoplasm. Furthermore, to understand hepatic clearance pathways for AGE, the site of AGE binding in the liver of rats was investigated using in vivo radioautography techniques (Youssef et al., 1998). After injection of 125Ilabeled AGE into the abdominal aorta, the radioautography revealed that binding was localized primarily in Küpffer cells (Naito et al., 2004). It is known that Küpffer cells are the largest population of tissue macrophages and have a peculiar functional characteristic to clear various substances in the blood. These results
suggested that, in chickens as well as rats, AGE existing in the circulation could be readily incorporated into the liver and might be cleared from circulation by RAGE and phagocytosis of Küpffer cells. When rats were intravenously administrated 3H-labeled pentosidine, radioactive pentosidine rapidly accumulated in the kidney and was excreted in the urine (Miyata et al., 1998). Recently, 18F-labeled CML was also intravenously administrated into rats (Xu et al., 2013). In this report, 18F-labeled CML was quickly distributed via the blood and rapidly excreted through the kidneys within 20 min after injection. As represented in Figure 1, in rats, a large amount of 14C-labeled AGE was incorporated in 1 g of kidney rats within 30 min after administration, and there was no significant difference between liver and kidney. Similarly to rats, the high level of radioactivity was also detected in 1 g of liver and kidney of chickens (Figure 2). As 3Hlabeled pentosidine administrated intravenously to rats was rapidly accumulated in the kidney and excreted in the urine (Miyata et al., 1998), a large amount of 14Clabeled AGE administrated intravenously to chickens would be excreted in the urine similarly to rats. The chicken is one of hyperglycemic animals in which high blood glucose concentrations are typically 2 to 3 times higher than human, suggesting that class aves could easily form AGE compared with mammals (Klandorf et al., 1995; Iqbal et al., 1999b). As shown in Figure 2, it was very interesting that a considerable level of radioactivity was observed in 1 g of spleen of chickens compared with rats. The value for radioactivity was almost one-half of that in 1 g of liver and significantly higher than that in 1 g of kidney. This is
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Figure 2. Incorporation of 14C-advanced glycation end products (AGE) in grams of tissue of chickens administrated with radioactive AGE via wing vein. Bars represent means ± SE (n = 8). Bars not sharing a common letter (a–h) are significantly different (P < 0.05).
RESEARCH NOTE
ACKNOWLEDGMENTS Financial support was provided by a Grant-in-Aid (Number 17658122) for Exploratory Research from The Ministry of Education, Science Sports and Culture, Japan.
REFERENCES Brett, J., A. M. Schmidt, S. D. Yan, Y. S. Zou, E. Weidman, D. Pinsky, R. Nowygrod, M. Neeper, C. Przysiecki, A. Shaw, A. Migheli, and D. Stern. 1993. Survey of the distribution of a newly characterized receptor for advanced glycation end products in tissues. Am. J. Pathol. 143:1699–1712. Brownlee, M. 2001. Biochemistry and molecular cell biology of diabetic complications. Nature 414:813–820. Goodwin, M., C. Herath, Z. Jia, C. Leung, M. T. Coughlan, J. Forbes, and P. Angus. 2013. Advanced glycation end products augment experimental hepatic fibrosis. J. Gastroenterol. Hepatol. 28:369–376. Iqbal, M., P. B. Kenney, N. H. Al-Humadi, and H. Klandorf. 2000. Relationship between mechanical properties and pentosidine in
tendon: Effects of age, diet restriction, and aminoguanidine in broiler breeder hens. Poult. Sci. 79:1338–1344. Iqbal, M., P. B. Kenney, and H. Klandorf. 1999a. Age-related changes in meat tenderness and tissue pentosidine: Effect of diet restriction and aminoguanidine in broiler breeder hens. Poult. Sci. 78:1328–1333. Iqbal, M., L. L. Probert, N. H. Alhumadi, and H. Klandorf. 1999b. Protein glycosylation and advanced glycosylated endproducts (AGEs) accumulation: An avian solution? J. Gerontol. A Biol. Sci. 54:B171–B176. Iqbal, M., L. L. Probert, and H. Klandorf. 1997. Effect of dietary aminoguanidine on tissue pentosidine and reproductive performance in broiler breeder hens. Poult. Sci. 76:1574–1579. Klandorf, H., S. B. Holt, J. A. McGowan, Y. Pinchasov, D. Deyette, and R. A. Peterson. 1995. Hyperglycemia and non-enzymatic glycation of serum and tissue proteins in chickens. Comp. Biochem. Physiol. C 110:215–220. Lee, K. B., D. J. Brooks, and J. O. Thomas. 1998. Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3′-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli. Gene 225:97–105. Lum, H. K., K. D. Lee, and G. Yu. 2000. The chicken genome contains no HMG1 retropseudogenes but a functional HMG1 gene with long introns. Biochim. Biophys. Acta 1493:64–72. Maillard, L. C. 1912. Action of amino acids on sugars. Formation of melanoidins in a methodical way. Compte-Rendu de l’Académie des Sciences 154:66–68. Mikulíková, K., A. Eckhardt, J. Kunes, J. Zicha, and I. Miksík. 2008. Advanced glycation end-product pentosidine accumulates in various tissues of rats with high fructose intake. Physiol. Res. 57:89–94. Miyata, T., Y. Ueda, K. Horie, M. Nangaku, S. Tanaka, C. van Ypersele de Strihou, and K. Kurokawa. 1998. Renal catabolism of advanced glycation end products: The fate of pentosidine. Kidney Int. 53:416–422. Naito, M., G. Hasegawa, Y. Ebe, and T. Yamamoto. 2004. Differentiation and function of Kupffer cells. Med. Electron Microsc. 37:16–28. Neeper, M., A. M. Schmidt, J. Brett, S. D. Yan, F. Wang, Y. C. Pan, K. Elliston, D. Stem, and A. Shaw. 1992. Cloning and expression of RAGE: A cell surface receptor for advanced glycosylation endproducts of proteins. J. Biol. Chem. 267:14998–15004. Schmidt, A. M., S. D. Yan, J. Brett, R. Mora, R. Nowygrod, and D. Stern. 1993. Regulation of human mononuclear phagocyte migration by cell surface-binding proteins for advanced glycation end products. J. Clin. Invest. 91:2155–2168. Singh, R., A. Barden, T. Mori, and L. Beilin. 2001. Advanced glycation end-products: A review. Diabetologia 44:129–146. Snow, L. M., C. B. Lynner, E. M. Nielsen, H. S. Neu, and L. V. Thompson. 2006. Advanced glycation end product in diabetic rat skeletal muscle in vivo. Pathobiology 73:244–251. Snow, L. M., and L. V. Thompson. 2009. Influence of insulin and muscle fiber type in nepsilon-(carboxymethyl)-lysine accumulation in soleus muscle of rats with streptozotocin-induced diabetes mellitus. Pathobiology 76:227–234. Tan, J. K. H., and H. C. O’Neill. 2007. Dendritic cell development in the context of the spleen microenvironment. Stem Cells 25:2139–2145. Xu, H., Z. Wang, Y. Wang, S. Hu, and N. Liu. 2013. Biodistribution and elimination study of fluorine-18 labeled Nε-carboxymethyllysine following intragastric and intravenous administration. PLoS ONE 8:e57897. Youssef, S., T. Soulis, and M. E. Cooper. 1998. Hepatic advanced glycation end product binding is increased in experimental diabetes. Cell. Mol. Biol. 44:1095–1100. Zhu, X. M., Y. M. Yao, H. P. Liang, S. Xu, N. Dong, Y. Yu, and Z. Y. Sheng. 2009. The effect of high mobility group box-1 protein on splenic dendritic cell maturation in rats. J. Interferon Cytokine Res. 29:677–686.
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the first finding that avian spleen is the specific organ having a higher ability to incorporate AGE than rats. Although the physiological function of AGE in avian species has not been clarified so far, avian spleen might have the special role for metabolism and catabolism of AGE. To date, several types of RAGE have been cloned and identified (Neeper et al., 1992; Brett et al., 1993; Schmidt et al., 1993), and high mobility group box1 (HMGB1) protein was known to be one of RAGE that appeared in rat spleen. This protein had a potential immunostimulatory signal inducing the maturation and differentiation of splenic dendritic cells (Zhu et al., 2009). Dendritic cells were reported as antigen-presenting cells capable of activating naive T cells and initiating adaptive immunity (Tan and O’Neill, 2007). In chickens, the genomic sequence of chicken HMGB1 has already been identified from chicken lymphocyte cDNA library (Lee et al., 1998; Lum et al., 2000). These results suggested that the specific accumulation of AGE in avian spleen might have a particular role in immune response in avian species. This issue will be elucidated in the future. This work compares tissue accumulation of free AGE administrated intravenously between rats and chickens. In both rats and chickens, the highest radioactivity was determined in the liver. In chickens, the second highest radioactivity was detected in the gizzard because this tissue was avian specific and the heaviest organ in all tissues. The high levels of radioactivity per gram of tissue in the liver and kidney were observed in both rats and chickens. In avian species, the spleen was capable of accumulating a large amount of AGE as well as kidney. In conclusion, exogenous AGE administrated intravenously into the circulation mainly accumulate in the liver and kidney of both rats and chickens, and a considerable amount of AGE can accumulate in the spleen of chickens but not rats.
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Key words: advanced glycation end product, tissue accumulation, spleen, chicken, rat 2014 Poultry Science 93:429–433 http://dx.doi.org/10.3382/ps.2013-03576 al., 2008). It was also reported that Nε-carboxymethyllysine (CML), which is known to be one type of noncross-linking AGE, was detected in skeletal muscles of diabetic rats (Snow et al., 2006; Snow and Thompson, 2009). Compared with protein-bound AGE originated from structural proteins, the information about tissue accumulation of free AGE in the blood has been limited. The intravenous administration of 3H-labeled pentosidine revealed that radioactive pentosidine rapidly accumulated in the kidney, was filtered by renal glomeruli, and was excreted in the urine of rats (Miyata et al., 1998). Recently, it was reported that, in rats, intravenously administrated 18F-labeled CML was quickly distributed via the blood and rapidly excreted through the kidneys within 20 min after injection (Xu et al., 2013). Hyperglycemia is commonly observed in avian species, and the preeminent traits of this class are as follows: 1) high blood glucose concentrations typically 2 to 3 times higher than human, which should accelerate amino-carbonyl reaction and generate high concentration of AGE; 2) an elevated basal body temperature (about 3°C higher than mammals), which should contribute to the nonenzymatic attachment of glucose to proteins and amino acids (Klandorf et al., 1995; Iqbal et al., 1999b). In avian species, pentosidine was de-
INTRODUCTION Glycation, or Maillard reaction, starts from a nonenzymatic amino-carbonyl reaction binding carbonyl group of reducing sugars to an amino group of proteins and amino acids (Maillard, 1912). Glycation leads to formation of a Schiff base followed by rearrangement into stable Amadori products. Amadori products undergo further complex reactions to form advanced glycation end products (AGE). The acceleration of Maillard reaction during hyperglycemia increases the production and accumulation of AGE, which is implicated in the gradual development of diabetic complications in diabetes mellitus (Brownlee, 2001; Singh et al., 2001). Tissue accumulation of AGE originated from structural proteins has been widely examined in various tissues of diabetic rats. Chronic hyperglycemia caused a significant increase in the concentration of pentosidine, which is one of representative AGE formed by nonenzymatic glycation of lysine and arginine residues, mainly in the aorta and skin of rats (Mikulíková et ©2014 Poultry Science Association Inc. Received August 21, 2013. Accepted October 13, 2013. 1 Corresponding author: [email protected]
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ABSTRACT Glycation starts from nonenzymatic amino-carbonyl reaction that binds carbonyl group of reducing sugars to the amino group of amino acids. Glycation leads to further complex reactions to form advanced glycation end products (AGE). Because AGE are implicated in the gradual development of diabetic complications, tissue accumulation of AGE has been widely examined in various tissues of rats. Avian species are known to have high body temperature and blood glucose concentration compared with mammals. Although these characteristics enabled chickens to be used as experimental models for diabetes mellitus, the information of AGE accumulation in various tissues of chickens has not been limited so far. In the present
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tected in collagen, which is the main structural protein of connective tissue of skin and tendon in broiler hens (Iqbal et al., 1997, 1999a, 2000). Glucose can bind to not only structural proteins but also to secreted protein. Although glycated albumin was detected in the serum of chickens (Klandorf et al., 1995), little is known about tissue accumulation of free glycated proteins and AGE existing in the circulation. In the present study, therefore, the radioactive AGE formed from 14C-glucose and amino acids were intravenously administrated to rats and chickens, and tissue accumulation of AGE was compared between chickens and rats.
Preparation of Radioactive AGE The radioactive AGE were prepared by reacting amino acids with radioactive glucose. Initially, 50 mg each of 20 amino acids (alanine, arginine monohydrochloride, asparagine monohydrate, aspartic acid, cysteine hydrochloride, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine monohydrochloride, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine) was added in 50 mL of water. Then, 3.6 g of glucose (2 M of final concentration) was dissolved in 10 mL of amino acid suspension, and 1.85 MBq of radioactive glucose containing U-14C-D-glucose (7.4 MBq/mL, Amersham Japan, Tokyo, Japan) was added. Subsequently, the mixture solution was incubated at 200°C for 3 h. After incubation, free radioactive and nonradioactive glucose unbound to amino acids was removed by Sephadex G-10 (Amersham Japan) column chromatography. The mobile phase was water and the flow rate was 1.45 mL/min. The drops for 1 min each were collected in sampling tubes, and 60 tubes were sampled in total. Glucose concentration in collected fractions was measured by a commercial kit (Glucose C-II Test Wako, Wako Pure Chemical Industries Inc., Osaka, Japan). The radioactivity in collected 14C-labeled AGE samples was determined by using a liquid scintillation counter (Aloka Co., Ltd., Tokyo, Japan). The 14C-labeled AGE solution excluding free glucose was used for intravenous administration to rats and chickens.
Birds and Experimental Procedures In the rat experiment, 7 male Wistar rats (6 wk old) were purchased from a local supplier (Japan SLC Inc., Hamamatsu, Japan). They were kept in plastic cages, in a temperature-controlled room at 24 ± 2°C, with artificial light from 0700 to 1900 h. All rats were fed a stock diet (Labo MR, Nihon Nosan Co. Ltd., Yokohama, Japan) with free access to water until radioactive AGE administration. To investigate the accumulation of 14C-AGE in various tissues, 7,900 Bq/kg of BW of 14C-labeled AGE was injected intravenously via tail vein under light anesthesia with diethyl ether. Body
Measurement of Radioactivity Incorporated into Tissues After thaw of frozen samples, approximately 0.4 g of tissues was weighed accurately and homogenized in 0.5 mL of 0.5% (wt/vol) NaOH/0.1% (vol/vol) Triton-X-100 solution. A part of tissue homogenate was weighed accurately, mixed with 0.5 mL of ACS-II scintillator cocktail (Amersham Japan), and its radioactivity was measured using a liquid scintillation counter. Animal experiments were conducted at Nagoya University, and animal care was in compliance with applicable guidelines from the Nagoya University Policy on Animal Care and Use.
Statistical Analysis Data were analyzed by one-way ANOVA to assess the significance of the effects of difference in tissues. Then, Duncan’s multiple range test was performed to compare between all pairs of means. All statistical analyses were performed using the GLM procedures (SAS/ STAT version 6, SAS Institute Inc., Cary, NC). Differences between means were considered to be significant at P < 0.05.
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MATERIALS AND METHODS
weight of rats was 181.0 ± 1.6 (SE) g. At 30 min after injection, rats were anesthetized with diethyl ether, and blood was collected by heart puncture. After rats were killed by deep anesthesia with diethyl ether, cerebrum, cerebellum, eyes, heart, thymus, lung, stomach, duodenum, jejunum, left cecum, rectum, liver, pancreas, kidney, spleen, testis, left gastrocnemius muscle, and a small part of skin were removed. All tissues were rinsed in ice-cold Dulbecco’s phosphate buffered saline, blotted, weighed, and frozen in liquid N2. Frozen samples were stored at −30°C until analysis. In the chicken experiment, 30 male Single-Comb White Leghorn chicks (1-d-old) were obtained from a local hatchery (Ghen Co. Ltd., Gifu, Japan). Chicks were maintained on a commercial chick mash diet (CP 207 g/kg, ME 12.1 kJ/g, Toyohashi Feed Mills Co., Ltd., Toyohashi, Japan). Chicks were housed in an electrically heated brooder (28 ± 2°C) with continuous illumination. At 12 d of age, 8 birds of uniform BW [120.3 ± 1.5 (SE) g] were selected and 11,500 Bq/kg of BW of 14C-labeled AGE was injected via wing vein of each bird. At 30 min after injection, chicks were anesthetized by diethyl ether, and then blood was collected by heart puncture. After neck dislocation, cerebrum, mesocephalon, cerebellum, left eye, heart, left lung, proventriculus, gizzard, duodenum, jejunum, colon, rectum, liver, pancreas, left kidney, spleen, testis, left major breast muscle, left minor breast muscle, and a small part of breast skin were removed. All tissues were rinsed in ice-cold Dulbecco’s phosphate buffered saline, blotted, weighed, and frozen in liquid nitrogen. Frozen samples were stored at −30°C until analysis.
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RESULTS The weight of various tissues of rats and chickens is shown in Table 1. In rats, liver weight was the heaviest of all. The second heaviest tissue was jejunum. There
Table 1. Tissue weight of chickens and rats1 Tissue Large brain Mid brain Small brain Eye Heart Thymus Lung Proventriculus Gizzard Stomach Duodenum Jejunum Colon Rectum Liver Pancreas Kidney Spleen Testis Gastrocnemius muscle Major breast muscle Minor breast muscle
Chicken2 0.73 0.22 0.16 0.70 0.99 0.51 1.10 5.20 0.83 3.24 0.58 0.34 4.55 0.81 0.16 0.66 0.06 3.28 0.93
0.010fg 0.011ij 0.011ij 0.034fg 0.043de — ± 0.058h ± 0.018d ± 0.139a — ± 0.064ef ± 0.123c ± 0.023gh ± 0.039i ± 0.098b ± 0.032df ± 0.008ij ± 0.047fgh ± 0.006j — ± 0.079c ± 0.033de ± ± ± ± ±
Rat3 1.13 ± 0.022d — 0.22 ± 0.005gh 0.12 ± 0.009i 0.68 ± 0.016efg 0.63 ± 0.045fg 0.95 ± 0.032de — — 1.13 ± 0.016d 0.51 ± 0.046g 4.81 ± 0.147b 0.73 ± 0.068efg 0.90 ± 0.172def 8.86 ± 0.236a 0.96 ± 0.047de 1.68 ± 0.074c 0.43 ± 0.018gh 1.71 ± 0.137c 1.07 ± 0.143d — —
a–jMeans not sharing a common superscript in the same column are significantly different (P < 0.05). 1Means ± SE. 2n = 8 of chickens. 3n = 7 of rats.
were no significant difference in tissue weight between kidney and testis, and they were the third heaviest tissues in rats. In chickens, gizzard weight was the heaviest in all tissues. The second heaviest tissue was liver. The following heavy tissues were large breast muscle and jejunum, and the weights of both tissues were not significantly different. The radioactivity per gram of tissue of rats administrated intravenously with 14C-labeled AGE is shown in Figure 1. The radioactivity per unit tissue weight in the liver and kidney was the highest compared with other tissues. There were no significant differences in all other tissues. The radioactivity per gram of tissue of chickens is represented in Figure 2. Similarly to rats, the highest radioactivity per gram of tissue was detected in the liver. The second highest incorporation of 14C-labeled AGE into 1 g of spleen was measured in chickens, which was not observed in the rat experiment. The third highest radioactivity per gram of tissue was measured in the kidney. Tissues following after kidney were rectum and testis.
DISCUSSION In the present study, to compare chickens with rats in tissue accumulation of AGE administrated intravenously, the radioactive AGE were prepared from amino acids and 14C-glucose. After glycation, free radioactive and nonradioactive glucose unbound to amino acids was successfully removed by using Sephadex G-10 column chromatography, which was confirmed by the follow-
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Figure 1. Incorporation of 14C-advanced glycation end products (AGE) in grams of tissue of rats administrated with radioactive AGE via tail vein. Bars represent means ± SE (n = 7). Bars not sharing a common letter (a,b) are significantly different (P < 0.05).
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ing determination of glucose concentration in collected fractions including 14C-labeled AGE (data not shown). Glycation is one of nonenzymatic chemical reactions taken place spontaneously in vivo, and this reaction forms AGE that have been known to be important factors associated with the onset of diabetic complications. In comparison with mammals, the information about tissue accumulation of free AGE existing in the blood has been limited regardless of the advantage in avian species to be experimental models for diabetes. In the present study, therefore, the tissue accumulation of 14C-labeled AGE injected intravenously was compared between chickens and rats, and it was revealed that the apparent major site of AGE accumulation was liver in both species (Figures 1 and 2). The highest accumulation of 14C-labeled AGE in the liver seems to be plausible because liver is the largest and second largest visceral organs of rats and chickens, respectively (Table 1). Meanwhile, hepatic exposure to high levels of AGE augmented hepatic fibrosis associated with upregulation of receptors for AGE (RAGE; Goodwin et al., 2013), suggesting that free AGE in the circulation were bound to RAGE and incorporated into hepatic cytoplasm. Furthermore, to understand hepatic clearance pathways for AGE, the site of AGE binding in the liver of rats was investigated using in vivo radioautography techniques (Youssef et al., 1998). After injection of 125Ilabeled AGE into the abdominal aorta, the radioautography revealed that binding was localized primarily in Küpffer cells (Naito et al., 2004). It is known that Küpffer cells are the largest population of tissue macrophages and have a peculiar functional characteristic to clear various substances in the blood. These results
suggested that, in chickens as well as rats, AGE existing in the circulation could be readily incorporated into the liver and might be cleared from circulation by RAGE and phagocytosis of Küpffer cells. When rats were intravenously administrated 3H-labeled pentosidine, radioactive pentosidine rapidly accumulated in the kidney and was excreted in the urine (Miyata et al., 1998). Recently, 18F-labeled CML was also intravenously administrated into rats (Xu et al., 2013). In this report, 18F-labeled CML was quickly distributed via the blood and rapidly excreted through the kidneys within 20 min after injection. As represented in Figure 1, in rats, a large amount of 14C-labeled AGE was incorporated in 1 g of kidney rats within 30 min after administration, and there was no significant difference between liver and kidney. Similarly to rats, the high level of radioactivity was also detected in 1 g of liver and kidney of chickens (Figure 2). As 3Hlabeled pentosidine administrated intravenously to rats was rapidly accumulated in the kidney and excreted in the urine (Miyata et al., 1998), a large amount of 14Clabeled AGE administrated intravenously to chickens would be excreted in the urine similarly to rats. The chicken is one of hyperglycemic animals in which high blood glucose concentrations are typically 2 to 3 times higher than human, suggesting that class aves could easily form AGE compared with mammals (Klandorf et al., 1995; Iqbal et al., 1999b). As shown in Figure 2, it was very interesting that a considerable level of radioactivity was observed in 1 g of spleen of chickens compared with rats. The value for radioactivity was almost one-half of that in 1 g of liver and significantly higher than that in 1 g of kidney. This is
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Figure 2. Incorporation of 14C-advanced glycation end products (AGE) in grams of tissue of chickens administrated with radioactive AGE via wing vein. Bars represent means ± SE (n = 8). Bars not sharing a common letter (a–h) are significantly different (P < 0.05).
RESEARCH NOTE
ACKNOWLEDGMENTS Financial support was provided by a Grant-in-Aid (Number 17658122) for Exploratory Research from The Ministry of Education, Science Sports and Culture, Japan.
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the first finding that avian spleen is the specific organ having a higher ability to incorporate AGE than rats. Although the physiological function of AGE in avian species has not been clarified so far, avian spleen might have the special role for metabolism and catabolism of AGE. To date, several types of RAGE have been cloned and identified (Neeper et al., 1992; Brett et al., 1993; Schmidt et al., 1993), and high mobility group box1 (HMGB1) protein was known to be one of RAGE that appeared in rat spleen. This protein had a potential immunostimulatory signal inducing the maturation and differentiation of splenic dendritic cells (Zhu et al., 2009). Dendritic cells were reported as antigen-presenting cells capable of activating naive T cells and initiating adaptive immunity (Tan and O’Neill, 2007). In chickens, the genomic sequence of chicken HMGB1 has already been identified from chicken lymphocyte cDNA library (Lee et al., 1998; Lum et al., 2000). These results suggested that the specific accumulation of AGE in avian spleen might have a particular role in immune response in avian species. This issue will be elucidated in the future. This work compares tissue accumulation of free AGE administrated intravenously between rats and chickens. In both rats and chickens, the highest radioactivity was determined in the liver. In chickens, the second highest radioactivity was detected in the gizzard because this tissue was avian specific and the heaviest organ in all tissues. The high levels of radioactivity per gram of tissue in the liver and kidney were observed in both rats and chickens. In avian species, the spleen was capable of accumulating a large amount of AGE as well as kidney. In conclusion, exogenous AGE administrated intravenously into the circulation mainly accumulate in the liver and kidney of both rats and chickens, and a considerable amount of AGE can accumulate in the spleen of chickens but not rats.
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