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
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
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]
429
430
Kita
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.
MATERIALS AND METHODS
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
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.
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.
431
RESEARCH NOTE
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).
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-
432
Kita
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).
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
RESEARCH NOTE
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.
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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