Subscriber access provided by UNIVERSITY OF KENTUCKY
Article
Effects of alpha-ketoglutarate on glutamine metabolism in piglet enterocytes in vivo and in vitro Liuqin He, Huan Li, Niu Huang, Junquan Tian, Zhiqiang Liu, xihong zhou, kang yao, tiejun li, and Yulong Yin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00433 • Publication Date (Web): 22 Mar 2016 Downloaded from http://pubs.acs.org on March 26, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 27
Journal of Agricultural and Food Chemistry
1
Effects of alpha-ketoglutarate on glutamine metabolism in piglet enterocytes in vivo and in
2
vitro
3
Liuqin He,†,‡ Huan Li,§ Niu Huang,§ Junquan Tian,†,‡ Zhiqiang Liu,† Xihong Zhou,†, * Kang
4
Yao,†,§, * Tiejun Li, †,#, * and Yulong Yin†
5
†
6
Engineering Research Center of Healthy Livestock, Scientific Observing and Experimental
7
Station of Animal Nutrition and Feed Science in South-Central, Ministry of Agriculture, Institute
8
of Subtropical Agriculture, Chinese Academy of Sciences, Changsha, Hunan , 410125, China
9
‡
10
§
11
410128, China
12
#
Key Laboratory of Agro-ecological Processes in Subtropical Region, Hunan Provincial
University of the Chinese Academy of Sciences, Beijing, 10008, China College of Animal Science and Technology, Hunan Agricultural University, Changsha, Hunan,
Hunan Co-Innovation Center of Animal Production Safety, Changsha, Hunan, 410128, China
13
14
15
16
17
18
19
20
21
22
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
23
ABSTRACT
24
Alpha-ketoglutarate (AKG) plays a vital part in the tricarboxylic acid cycle and is a key
25
intermediate in the oxidation of L-glutamine (Gln). The study was to evaluate effects of AKG on
26
Gln metabolism in vivo and in vitro. A total of twenty-one piglets were weaned at 28 days with a
27
mean body weight (BW) of 6.0 ± 0.2 kg, and randomly divided into 3 groups: corn soybean meal
28
based diet (CON group); the basal diet with 1% alpha-ketoglutarate (AKG treatment group); and
29
the basal diet with 1% L-glutamine (GLN treatment group). Intestinal porcine epithelial cells-1
30
(IPEC-1) was incubated to investigate effects of 0.5, 2, and 3 mM AKG addition on Gln
31
metabolism. Our results showed that there were no differences (P > 0.05) among the 3 treatments
32
in initial BW, final BW and average daily feed intake. However, average daily gain (P = 0.013)
33
and the ratio of gain : feed (P = 0.041) of AKG group were greater than the other two groups. In
34
comparison with the CON group, the AKG and GLN groups exhibited an improvement in villus
35
length, mucosal thickness, and crypt depth in the jejunum of piglets. Serum concentrations of Asp,
36
Glu, Val, Ile, Tyr, Phe, Lys, and Arg in the piglets fed the 1% AKG or Gln diet were lower than
37
those in the CON group. Compared with CON group, the mRNA expression of jejunal and ileal
38
amino acid (AA) transporters in the AKG and GLN groups were significantly increased (P < 0.05).
39
Additionally, the in vitro study showed that the addition of 0.5, 2, and 3 mM of AKG
40
dose-dependently decreased (P < 0.05) the net utilization of Gln and formulation of ammonia, Glu,
41
Ala and Asp by IPEC-1. In conclusion, dietary AKG supplementation, as a replacement for Gln,
42
could improve Gln metabolism in piglet enterocytes and enhance the utilization of AA.
43
KEY WORDS: alpha-ketoglutarate; glutamine; amino acids; piglet; enterocytes
44
45
46
47
ACS Paragon Plus Environment
Page 2 of 27
Page 3 of 27
Journal of Agricultural and Food Chemistry
48
INTRODUCTION
49
L-glutamine (Gln),as a predominant amino acid (AA) in the body, contributes more than 50%
50
of the total intracellular free α-AA pool in skeletal muscle and blood.1 Compelling evidences show
51
that Gln plays a key role in the intestinal health by serving as a crucial metabolic fuel for all fast
52
dividing cells,2, 3 and as a precursor of glutathione,4 pyrimidines and purines.5 Thus, Gln promotes
53
the synthesis of protein and inhibits protein catabolism in enterocytes.5,
54
glutamate (Glu), is readily converted to alpha-ketoglutarate (AKG).7 Two other AA that relate
55
closely to the tricarboxylic acid (TCA) cycle are alanine (Ala), which is derived from pyruvate,
56
and
57
metabolite/precursor of Gln is AKG, which plays a pivotal role in intermediate metabolism, and
58
enables the redistribution of nitrogen towards anabolic or catabolic pathways.9 Notably, AKG
59
serves as an intermediate in the TCA cycle, and the oxidation of Gln to CO2 and water requires the
60
formation of AKG.10 Therefore, AKG is considered to be one of the crucial molecules in
61
interorgan nitrogen transport, protein metabolism, as well as regulation of gene expression and
62
cellular redox state. 11, 12
aspartate (Asp)
is
derived
from oxaloacetate.8
However,
6
Briefly, Gln, via
the most
important
63
Emerging evidences showed beneficial effects of AKG in animal nutrition, particularly with
64
regulating AA transporters gene expression13 and the mammalian target of rapamycin signaling
65
pathway in the pig intestine.14 Notably, AKG may have a sparing effect on Glu and Gln in cells by
66
serving as a fuel source in growing pigs.15 Furthermore, AKG has been applied to be a gut nutrient
67
and a potential inhibitor of Gln catabolism.16 And through the synthesis of Gln, polyamines, and
68
arginine (Arg),3 AKG play a major role in the growth and development of small intestine.17 In the
69
practical application, although Gln is currently used as a new feed additive to enhance nitrogen
70
metabolism and reduce metabolic stress in animal production,18, 19 AKG is more inexpensive,
71
soluble and stable than Gln, thus it would potentially reduce feed cost and increase the efficiency
72
of utilization. To date, the mechanisms responsible for the action of AKG as Gln replacement on
73
intestinal Gln metabolism in piglets remains unknown. Therefore, the present study was to
74
determine the effects of AKG on intestinal Gln metabolism in vivo and in vitro.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
75
76
MATERIALS AND METHODS
77
Animals and Experimental Design
78
The animal experiments were approved by the Institutional Animal Care and Use Committee
79
of the Institute of Subtropical Agriculture, Chinese Academy of Sciences (2013020). Twenty-one
80
piglets with a mean body weight (BW) of 6.0 ± 0.2 kg were weaned at 28 days, and randomly
81
assigned into three treatments based on weaning weight (7 piglets/ treatment): one group was fed a
82
corn soybean meal based diet (CON group); another was fed the basal diet plus 1%
83
alpha-ketoglutarate
84
China; purity ≥ 99.2%) (AKG treatment group); and the third was fed the basal diet plus 1%
85
L-glutamine6 (Wuhan Yuancheng Gongchuang Technology co., LTD, Wuhan, Hubei, China;
86
purity ≥ 99.5%) (GLN treatment group). The composition and nutrient levels of all diets met the
87
nutrient specifications for 5 to 10 kg BW pig according to the NRC-recommended requirements
88
(NRC, 2012) and showed in Table 1. After 7 days of adaption, piglets were fed their respective
89
diets 3 times per day at 8:00, 13:00 and 18:00. During the experiment, piglets were housed
90
individually and given free access to water. Average daily weight gain (ADG) and feed intake
91
(ADFI) were calculated. The duration of whole experiment was four weeks.
13
(Wuhan Yuancheng Gongchuang Technology co., LTD, Wuhan, Hubei,
92
Sample Collection and Analytical Methods
93
After the whole feeding period, blood samples (10mL) were taken from jugular vein, then
94
pigs were anaesthetised with sodium pentobarbital intravenously (50mg/kg BW) and bled by
95
exsanguination. Tissue samples from jejunum and ileum, were collected (after being cleaned with
96
ice-cold phosphate-buffered saline), immediately frozen in liquid nitrogen, and stored at -80°C.
97
And one jejunum and ileum segment were fixed in 10% neutral buffered formalin for examination
98
of intestinal morphology. Blood samples were centrifuged at 3,000 ×g for 10 min at 4°C, and then
99
stored at -20°C for analysis of free AA by an automatic amino acid analyzer (L-8900; Hitachi
100
Global Inc., Hitachi, Japan).
ACS Paragon Plus Environment
Page 4 of 27
Page 5 of 27
Journal of Agricultural and Food Chemistry
101
Intestinal histomorphology
102
Paraffin sections (approximately 5 mm) of jejunum and ileum samples were stained with
103
hematoxylin and eosin, and villus length and crypt depth were measured using a light microscope
104
with a computer-assisted morphometric system (BioScan Optimetric, BioScan Inc., Edmonds, WA,
105
USA). Villus length, mucosal thickness, and crypt depth were defined as previous study did. 20
106 107
Relative quantification of mRNA expression of AA transporters by Real-time Quantitative RT-PCR
108
The mRNA expression of solute carrier family 1, member 1 (EAAC1), solute carrier family 7,
109
member 9 (b0+), solute carrier family 7, member 7 (y+ LAT1), and solute carrier family 1, member
110
5 (ASCT2) in the jejunum and ileum were analyzed by real-time quantitative RT-PCR as
111
previous study described.21 The primer were shown in Table 2. The relative gene expression was
112
expressed as a ratio of the target gene to the control gene using the formula 2 - ( ∆∆Ct), where ∆∆ Ct
113
= (Ct Target - Ct GAPDH )treatment - (Ct Target - Ct GAPDH)control.
114
Culture of intestinal epithelial cells
115
Intestinal porcine epithelial cells-1 (IPEC-1) were given by the lab of the Department of
116
Animal Science, Texas A&M University as a gift and cultured as described previously.22 Briefly,
117
the cells were grown in uncoated plastic culture flasks (100mm2) in Dulbecco’s modified
118
Eagle’s-F12 Ham medium(DMEM-F12). Confluent cells were trypsinized and seeded with
119
approximately 1×104 cells per well (6-well cell culture plates) and maintained at 37°C with 5%
120
CO2. After incubation for 16 h, the cells were cultured in a medium containing 0.5, 2, and 3 mM
121
of AKG respectively for 3 days. This medium contained physiological concentrations of AA
122
found in pig plasma.22 There were seven independent replicates AKG dose. The medium was
123
changed every day. After 3 days of culture, cells (5×106 /mL) were cultured 3 h with 2mM of
124
L-[U-14C] Gln. Briefly, after 3 h culture, medium and cells was collected for the analysis of
125
ammonia, Glu, Ala, and Asp using High Performance Liquid Chromatography (Waters 2695;
126
Waters Inc., MA, USA). Additionally, 14C-labled Gln was measured as described by Yao. 10
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
127
Statistical analysis
128
Data were analyzed by the one-way analysis of variance and a mixed procedure (PROC
129
MIXED) using SAS software version 9.2 (SAS Institute Inc., Cary, NC). Additionally, dietary
130
treatment is considered as fixed effect and animal as randomized factor. Data were presented as
131
Least Squares Means ± SEM. Mean values were considered to be significantly different when P
0.05) among the three treatment groups in initial BW, final BW and
138
ADFI. However, ADG and gain: feed (G/F) of AKG group were greater than the other groups (P >
139
0.05), and there was difference (P < 0.05) between AKG and CON groups.
140
Intestinal histomorphology
141
The results of intestinal morphology is summarized in Figure 1. In comparison with the CON
142
group, the AKG and GLN groups exhibited an increase in villus length, mucosal thickness and
143
crypt depth in the jejunum of piglets. And there was difference (P < 0.05) in villus length among
144
the three treatment groups. However, in ileum, there were no differences (P > 0.05) in villus
145
length, mucosal thickness and crypt depth among the three treatment groups.
146
Free AA concentration in serum
147
Table 4 shows the effect of AKG and Gln on serum contents of free AA in weaned piglets.
148
There were no differences(P > 0.05) in the contents of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg
149
between the AKG and the GLN groups, however, these AA concentrations differed from the CON
150
group (P < 0.05), and the value in the CON group was the greatest. Notably, the content of Thr in
ACS Paragon Plus Environment
Page 6 of 27
Page 7 of 27
Journal of Agricultural and Food Chemistry
151
the AKG group was the highest among the three groups, Thr concentration was (P < 0.05) higher
152
in both the AKG and GLN groups than the CON group.
153
The expression of AA transporters mRNA abundance.
154
Data on the mRNA abundance of AA transporters in jejunum and in ileum are shown in
155
Figure 2 (A and B). In the jejunum (Figure 2A), there were differences (P < 0.01) in the mRNA
156
expression of ASCT2, b0+, y+LAT1, and EAAC1 among the three groups, and the mRNA
157
abundance of ASCT2, b0+, and y+LAT1 in the GLN group was the highest, followed by the AKG
158
and CON groups, in descending order. However, the mRNA abundance of EAAC1 in the AKG
159
group was the highest. Furthermore, in the ileum (Figure 2B), remarkable differences (P < 0.05) in
160
the mRNA abundance of ASCT2, b0+, y+LAT1, and EAAC1 were also detected among the three
161
groups. And the expression of b0+, y+LAT1, and EAAC1 was much greater in the GLN group than
162
that in the AKG group, the opposite was observed for ASCT2. Additionally, the mRNA
163
abundance of ASCT2, b0+, and y+LAT1 of the AKG group in jejunum was greater than those in
164
ileum.
165
Effects of AKG on Gln catabolism and its metabolites production in IPEC-1 cells
166
As shown in Figure 3 (A, B, C, D, and E), the different concentration of AKG affected the
167
utilization of Gln and the production of ammonia, Glu, Ala, and Asp in IPEC-1 cells. Interestingly,
168
the increased contents of AKG from 0.5 to 3 mM decreased (P < 0.05) the net utilization of Gln
169
and inhibited (P < 0.05) the production of ammonia, Glu, Ala, and Asp. However, no differences
170
(P > 0.05) in the contents of Gln, ammonia, Glu, Ala, and Asp were determined between 2 and 3
171
mM of AKG groups in IPEC-1.
172
173
DISCUSSION
174
Alpha-ketoglutarate, as a replacement for Gln, could improve the growth performance of
175
piglets. This may be explained by the reasons that as an intermediate of the TCA cycle, AKG is
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
176
essential for the oxidation of fatty acids, AA, and glucose,12 thus produce enough energy for
177
intestinal cell growth and proliferation.23 Furthermore, as a precursor for the synthesis of Gln and
178
Glu in multiple tissues,24 AKG bridges carbohydrate and nitrogen metabolism for both
179
conservation of AA and ammonia detoxification.12 As an important fuel for all rapidly dividing
180
cells, Gln improves the synthesis of protein and inhibits protein catabolism in enterocytes.24
181
However, as a feed additive, Gln is easy to be decomposed25, 26 and did not improve the growth
182
performance as remarkably as AKG did. Therefore, compared with the GLN treatment, AKG
183
could maintain the unremarkable growth performance of piglets under lower ADFI condition.
184
Additionally, since G/F affects the economic return in pork production, our work has important
185
implications for the sustainability of the swine industry.
186
The present study showed an increase in villus length, mucosal thickness and crypt depth in
187
the jejunum of AKG and Gln-supplemented piglets. This result suggests a net improvement of the
188
intestinal health in the AKG and GLN groups. That is because Gln serves as a critical oxidative
189
substrate for the intestinal mucosa and a precursor of vital molecules.27-29 Furthermore, as a
190
precursor of Gln and Glu, AKG metabolism via the TCA cycle generates reduced coenzymes used
191
by the mitochondria for ATP synthesis.12, 30 And the enterocytes consume a large amount of ATP,
192
which is required for nutrient absorption and intestine health maintenance.31, 32 In our current
193
study, our results showed that Gln and AKG supplementation beneficially improved the intestinal
194
morphology in the jejunum of weaned piglets. These findings indicate that dietary AKG
195
supplementation may be also an important factor for the maintenance of intestinal health as well
196
as Gln.
197
Amino acids are key regulators of intestinal health and metabolic pathways that regulate
198
nutrient utilization.33 In the current study, compared with the CON group, the serum
199
concentrations of Asp, Glu, Val, Ile, Tyr, Phe, Lys, and Arg in piglets fed the 1% AKG or Gln diet
200
were decreased, while the opposite was observed for Thr. These results suggest that dietary AKG
ACS Paragon Plus Environment
Page 8 of 27
Page 9 of 27
Journal of Agricultural and Food Chemistry
201
or Gln supplementation could improve the utilization rate of those AA related with AKG and Gln
202
metabolism in serum and other tissues. Notably, the important function of Thr as an essential AA
203
is to stimulate protein synthesis and AA abundance, and may be regulate feed intake to some
204
degree,34 which may also explain why ADFI in the AKG group and GLN group was higher than
205
the CON group. Moreover, unlike with branched-chain AA,35 whose catabolism is initiated in
206
extra-hepatic organs and cells, the degradation of Lys, Phe, and Tyr occurs primarily in the liver.9
207
It is possible that AKG and Gln enhance the utilization of these AA for tissue protein synthesis or
208
promote their oxidation in the liver.36 Tracer studies will be required to examine these
209
possibilities.
210
The absorption of AA requires many transporter systems that differ with respect to their
211
substrate specificity and driving force.20 It has been reported that some genes (such as ASCT2,
212
EAAC1, bo+, y+LAT1) that are involved in the control of growth or AA metabolism are regulated
213
by AA availability.21, 37 An interesting finding of this study is that AKG and Gln increased the
214
mRNA expression of ASCT2, EAAC1, b0+, and y+LAT1 in the intestine of weaned piglets. Of
215
note, except the expression of EAAC1 in jejunum and ASCT2 in ileum, the expression of other
216
AA transporters in the GLN treatment group was higher than that in the AKG treatment group,
217
thus the results was consistent with the concentrations of AA in serum. To some extent, AA can
218
play a vital role in the control of AA transporters expression by a vast, complex regulatory system,
219
which may also affect the energy balance and endocrine system.32 This result can also be
220
explained by the extensive catabolism of both Gln and Glu in pig enterocytes.38 Additionally,
221
AKG as a precursor of Gln and Glu may have a potent ‘sparing’ effect on endogenous Gln pools.17
222
Interestingly, in our data we found that the addition of AKG could affect Gln catabolism and
223
the production of its metabolites in IPEC-1. The addition of 0.5, 2, and 3 mM of AKG
224
dose-dependently decreased the utilization of Gln and the formulation of Ala, Asp, Glu and
225
ammonia. Based on the chemical equilibrium of AKG dehydrogenase,39 AKG may inhibit Glu,
226
Asp, Ala and ammonia transaminases in cells, thereby inhibiting the catabolism of these AA and
227
Gln.31, 40 Alternatively, AKG may also inhibit cellular Gln transport,41 by affecting the expression
228
of phosphate-dependent glutaminase or directly inhibiting the catalytic activity of glutaminase.10,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
229
42
230
and AKG is an important intermediate in the oxidation of Gln, which is largely converted to AKG
231
and catabolized to produce ATP in the intestine.12 Therefore, all of these changes can contribute to
232
and decrease in Gln catabolism and its metabolites production. In summary, these interesting
233
findings may have important implications for the application of AKG as Gln replacement in
234
livestock.
Furthermore, to our knowledge, degradation of Gln produces Ala, Asp, Glu, ammonia and CO2,
235
236
ABBREVIATIONS USED
237
AKG, alpha-ketoglutarate; Gln, glutamine; AA, amino acid; Glu, glutamate; Ala, alanine; Asp,
238
aspartate; BW, body weight; ADFI, average daily feed intake; ADG, average daily gain; G/F,
239
gain : feed; IPEC-1, intestinal porcine epithelial cells-1; EAAC1, solute carrier family 1, member
240
1; b0+, solute carrier family 7, member 9; y+ LAT1, solute carrier family 7, member 7; ASCT2,
241
solute carrier family 1, member 5.
242
CORRESPONDING AUTHOR
243
*(K.Y.) Fax: +86 84615285.E-mail:
[email protected] or (T.L.) Fax: +86 84615285.E-mail:
244
[email protected] or (X.Z.) Fax: +86 84615285. E-mail:
[email protected] 245
FUNDING
246
This work was supported by National Basic Research Program of China (2013CB127301 and
247
2013CB127306), Chinese Academy of Sciences “Hundred Talent" award for Kang Yao, National
248
Natural Science Fundation Project (31472106 and 31472107), National Science and Technology
249
Support Project (2013BAD21B04).
250
ACKNOWLEDGMENTS
ACS Paragon Plus Environment
Page 10 of 27
Page 11 of 27
Journal of Agricultural and Food Chemistry
251
All of the authors declare no conflicts of interest. Thanks for supporting of Changsha Lvye
252
Biotechnology Limited Company Academician Expert Workstation, Guangdong Wangda Group
253
Academician Workstation for Clean Feed Technology Research and Development in swine.
254 255 256
REFERENCES 1.
Nordgren, A.; Karlsson, T.; Wiklund, L. Glutamine concentration and tissue exchange
257
with intravenously administered alpha-ketoglutaric acid and ammonium: A dose-response study in
258
the pig. Nutrition. 2002, 18, 496-504.
259
2.
Palmieri, E. M.; Spera, I.; Menga, A.; Infantino, V.; Iacobazzi, V.; Castegna, A.
260
Glutamine synthetase desensitizes differentiated adipocytes to proinflammatory stimuli by raising
261
intracellular glutamine levels. Febs Lett. 2014, 588, 4807-4814.
262
3.
Fillmann, H.; Kretzmann, N. A.; San-Miguel, B.; Llesuy, S.; Marroni, N.;
263
Gonzalez-Gallego, J.; Tunon, M. J. Glutamine inhibits over-expression of pro-inflammatory genes
264
and down-regulates the nuclear factor kappaB pathway in an experimental model of colitis in the
265
rat. Toxicology. 2007, 236, 217-226.
266
4.
Chellamuthu, V. R.; Ermilova, E.; Lapina, T.; Luddecke, J.; Minaeva, E.; Herrmann, C.;
267
Hartmann, M. D.; Forchhammer, K. A Widespread Glutamine-Sensing Mechanism in the Plant
268
Kingdom. Cell. 2014, 159, 1188-1199.
269
5.
Caballero-Solares, A.; Viegas, I.; Salgado, M. C.; Siles, A. M.; Saez, A.; Meton, I.;
270
Baanante, I. V.; Fernandez, F. Diets supplemented with glutamate or glutamine improve protein
271
retention and modulate gene expression of key enzymes of hepatic metabolism in gilthead
272
seabream (Sparus aurata) juveniles. Aquaculture. 2015, 444, 79-87.
273
6.
Zhong, X.; Li, W.; Huang, X. X.; Wang, Y. X.; Zhang, L. L.; Zhou, Y. M.; Hussain, A.;
274
Wang, T. Effects of glutamine supplementation on the immune status in weaning piglets with
275
intrauterine growth retardation. Arch. Anim Nutr. 2012, 66, 347-356.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
276
7.
Cooper, A. J. L.; Kuhara, T. alpha-Ketoglutaramate: an overlooked metabolite of
277
glutamine and a biomarker for hepatic encephalopathy and inborn errors of the urea cycle. Metab
278
Brain Dis. 2014, 29, 991-1006.
279 280 281
8.
Neu, J.; Shenoy, V.; Chakrabarti, R. Glutamine nutrition and metabolism: Where do we
go from here? Faseb J. 1996, 10, 829-837. 9.
282
1-17.
283
10.
Wu, G. Y. Amino acids: metabolism, functions, and nutrition. Amino Acids. 2009, 37,
Yao, K.; Yin, Y. L.; Li, X. L.; Xi, P. B.; Wang, J. J.; Lei, J.; Hou, Y. Q.; Wu, G. Y.
284
Alpha-ketoglutarate inhibits glutamine degradation and enhances protein synthesis in intestinal
285
porcine epithelial cells. Amino Acids. 2012, 42, 2491-2500.
286
11.
Filip, R.; Wdowiak, L.; Harrison, A. P.; Pierzynowski, S. G. Dietary supplementation
287
with phytohemagglutinin in combination with alpha-ketoglutarate limits the excretion of nitrogen
288
via urinary tract. Ann. Agr. Env Med. 2008, 15, 309-315.
289
12.
He, L. Q.; Xu, Z. Q.; Yao, K.; Wu, G. A.; Yin, Y. L.; Nyachoti, C. M.; Kim, S. W. The
290
Physiological Basis and Nutritional Function of Alpha-ketoglutarate. Curr Protein Pept Sc. 2015,
291
16, 576-581.
292
13.
Hou, Y. Q.; Wang, L.; Ding, B. Y.; Liu, Y. L.; Zhu, H. L.; Liu, J. A.; Li, Y. T.; Wu, X.;
293
Yin, Y. L.; Wu, G. Y. Dietary alpha-ketoglutarate supplementation ameliorates intestinal injury in
294
lipopolysaccharide-challenged piglets. Amino Acids. 2010, 39, 555-564.
295 296 297
14.
Rhoads, J. M.; Wu, G. Y. Glutamine, arginine, and leucine signaling in the intestine.
Amino Acids. 2009, 37, 111-122. 15.
Junghans, P.; Derno, M.; Pierzynowski, S.; Hennig, U.; Rudolph, P. E.; Souffrant, W. B.
298
Intraduodenal infusion of alpha-ketoglutarate decreases whole body energy expenditure in
299
growing pigs. Clin Nutr. 2006, 25, 489-496.
300
16.
Winkler, S.; Holzenbein, T.; Karner, J.; Roth, E. Kinetics of organ specific metabolism
301
of a bolus injection into the jejunum of glutamine, alpha-ketoglutarate, ornithine and ornithine -
302
alpha-ketoglutarate. Clin Nutr. 1993, 12, 56-7.
ACS Paragon Plus Environment
Page 12 of 27
Page 13 of 27
Journal of Agricultural and Food Chemistry
303
17.
Coudray-Lucas, C.; Lasnier, E.; Renaud, A.; Ziegler, F.; Settembre, P.; Cynober, L. A.;
304
Ekindjian, O. G. Is alpha-ketoisocaproyl-glutamine a suitable glutamine precursor to sustain
305
fibroblast growth? Clin Nutr. 1999, 18, 29-33.
306
18.
Haynes, T. E.; Li, P.; Li, X. L.; Shimotori, K.; Sato, H.; Flynn, N. E.; Wang, J. J.; Knabe,
307
D. A.; Wu, G. Y. l-Glutamine or l-alanyl-l-glutamine prevents oxidant- or endotoxin-induced
308
death of neonatal enterocytes. Amino Acids. 2009, 37, 131-142.
309 310 311
19.
Pierzynowski, S. G.; Sjodin, A. Perspectives of glutamine and its derivatives as feed
additives for farm animals. J. Anim. Feed Sci. 1998, 7, 79-91. 20.
He, L. Q.; Yang, H. S.; Hou, Y. Q.; Li, T. J.; Fang, J.; Zhou, X. H.; Yin, Y. L.; Wu, L.;
312
Nyachoti, M.; Wu, G. Y. Effects of dietary l-lysine intake on the intestinal mucosa and expression
313
of CAT genes in weaned piglets. Amino Acids. 2013, 45, 383-391.
314
21.
He, L.; Wu, L.; Xu, Z.; Li, T.; Yao, K.; Cui, Z.; Yin, Y.; Wu, G. Low-protein diets
315
affect ileal amino acid digestibility and gene expression of digestive enzymes in growing and
316
finishing pigs. Amino Acids. 2016, 48, 21-30.
317
22.
Tan, B.; Yin, Y. L.; Kong, X. F.; Li, P.; Li, X. L.; Gao, H. J.; Li, X. G.; Huang, R. L.;
318
Wu, G. Y. l-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal
319
cells. Amino Acids. 2010, 38, 1227-1235.
320
23.
Kristensen, N. B.; Jungvid, H.; Fernandez, J. A.; Pierzynowski, S. G. Absorption and
321
metabolism of alpha-ketoglutarate in growing pigs. J. Anim Physiol. Anim Nutr (Berl). 2002, 86,
322
239-245.
323
24.
Wang, L.; Hou, Y. Q.; Yi, D.; Li, Y. T.; Ding, B. Y.; Zhu, H. L.; Liu, J.; Xiao, H.; Wu,
324
G. Y. Dietary supplementation with glutamate precursor alpha-ketoglutarate attenuates
325
lipopolysaccharide-induced liver injury in young pigs. Amino Acids. 2015, 47, 1309-1318.
326 327 328
25.
Contineanu, I.; Neacsu, A.; Perisanu, S. T. The standard enthalpies of formation of
L-asparagine and L-alpha-glutamine. Thermochim Acta. 2010, 497, 96-100. 26.
Ingraham, L.; Li, M. S.; Renfro, J. L.; Parker, S.; Vapurcuyan, A.; Hanna, I.; Pelis, R. M.
329
A Plasma Concentration of alpha-Ketoglutarate Influences the Kinetic Interaction of Ligands with
330
Organic Anion Transporter 1. Mol Pharmacol. 2014, 86, 86-95.
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
331
27.
Wang, B.; Wu, Z.; Ji, Y.; Sun, K.; Dai, Z.; Wu, G. l-Glutamine Enhances Tight Junction
332
Integrity by Activating CaMK Kinase 2-AMP-Activated Protein Kinase Signaling in Intestinal
333
Porcine Epithelial Cells. J. Nutr. 2016, 146, 501-8.
334
28.
Yu, H.; Gao, Q.; Dong, S.; Lan, Y.; Ye, Z.; Wen, B. Regulation of dietary glutamine on
335
the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus
336
japonicus (Selenka). Fish Shellfish Immun. 2016, 50, 56-65.
337
29.
Coutinho, F.; Castro, C.; Rufino-Palomares, E.; Ordonez-Grande, B.; Gallardo, M. A.;
338
Oliva-Teles, A.; Peres, H. Dietary glutamine supplementation effects on amino acid metabolism,
339
intestinal nutrient absorption capacity and antioxidant response of gilthead sea bream (Sparus
340
aurata) juveniles. Comp Biochem. Phys. A. 2016, 191, 9-17.
341
30.
Lin, M.; Zhang, B.; Yu, C.; Li, J.; Zhang, L.; Sun, H.; Gao, F.; Zhou, G. L-Glutamate
342
supplementation improves small intestinal architecture and enhances the expressions of jejunal
343
mucosa amino acid receptors and transporters in weaning piglets. Plos One. 2014, 9, e111950.
344
31.
Lambert, B. D.; Filip, R.; Stoll, B.; Junghans, P.; Derno, M.; Hennig, U.; Souffrant, W.
345
B.; Pierzynowski, S.; Burrin, D. G. First-pass metabolism limits the intestinal absorption of enteral
346
alpha-ketoglutarate in young pigs. J. Nutr. 2006, 136, 2779-2784.
347
32.
Wu, G. Y. Intestinal mucosal amino acid catabolism. J. Nutr. 1998, 128, 1249-1252.
348
33.
Jobgen, W. S.; Fried, S. K.; Fu, W. J.; Meininger, C. J.; Wu, G. Y. Regulatory role for
349
the arginine-nitric oxide pathway in metabolism of energy substrates. J. Nutr Biochem. 2006, 17,
350
571-588.
351
34.
352 353
Dworkin, J. Ser/Thr phosphorylation as a regulatory mechanism in bacteria. Curr Opin.
Microbiol. 2015, 24, 47-52. 35.
Morales, A.; Arce, N.; Cota, M.; Buenabad, L.; Avelar, E.; Htoo, J. K.; Cervantes, M.
354
Effect of dietary excess of branched-chain amino acids on performance and serum concentrations
355
of amino acids in growing pigs. J. Anim Physiol. Anim Nutr (Berl). 2016, 100, 39-45.
356
36.
Chaudhry, K. K.; Shukla, P. K.; Mir, H.; Manda, B.; Gangwar, R.; Yadav, N.;
357
McMullen, M.; Nagy, L. E.; Rao, R. Glutamine supplementation attenuates ethanol-induced
358
disruption of apical junctional complexes in colonic epithelium and ameliorates gut barrier
359
dysfunction and fatty liver in mice. J. Nutr Biochem. 2016, 27, 16-26.
ACS Paragon Plus Environment
Page 14 of 27
Page 15 of 27
Journal of Agricultural and Food Chemistry
360
37.
Wu, L.; He, L. Q.; Cui, Z. J.; Liu, G.; Yao, K.; Wu, F.; Li, J.; Li, T. J. Effects of
361
reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in
362
weaned piglets. J. Zhejiang Univ. Sci. B. 2015, 16, 496-502.
363 364 365
38.
Reeds, P. J.; Burrin, D. G.; Stoll, B.; Jahoor, F. Intestinal glutamate metabolism. J. Nutr.
2000, 130, 978s-982s. 39.
Jr, W.; Shpun, S.; Dantzler, W. H.; Wright, S. H. Effect of alpha-ketoglutarate on
366
organic anion transport in single rabbit renal proximal tubules. Am J. Physiol-Renal. 1998, 274,
367
F165-F174.
368
40.
Miles, E. D.; McBride, B. W.; Jia, Y.; Liao, S. F.; Boling, J. A.; Bridges, P. J.;
369
Matthews, J. C. Glutamine synthetase and alanine transaminase expression are decreased in livers
370
of aged vs. young beef cows and GS can be upregulated by 17beta-estradiol implants. J. Anim Sci.
371
2015, 93, 4500-9.
372
41.
Tardito, S.; Oudin, A.; Ahmed, S. U.; Fack, F.; Keunen, O.; Zheng, L.; Miletic, H.;
373
Sakariassen, P. O.; Weinstock, A.; Wagner, A.; Lindsay, S. L.; Hock, A. K.; Barnett, S. C.;
374
Ruppin, E.; Morkve, S. H.; Lund-Johansen, M.; Chalmers, A. J.; Bjerkvig, R.; Niclou, S. P.;
375
Gottlieb, E. Glutamine synthetase activity fuels nucleotide biosynthesis and supports growth of
376
glutamine-restricted glioblastoma. Nat Cell Biol. 2015, 17, 1556-68.
377
42.
Szeliga, M.; Cwikla, J.; Obara-Michlewska, M.; Cichocki, A.; Albrecht, J. Glutaminases
378
in slowly proliferating gastroenteropancreatic neuroendocrine neoplasms/tumors (GEP-NETs):
379
Selective overexpression of mRNA coding for the KGA isoform. Exp. Mol Pathol. 2015, 100,
380
74-78.
381 382 383 384 385 386 387 388
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
389
FIGURES CAPTIONS
390
Figure 1. Effects of alpha-ketoglutarate and glutamine on the histomorphology of small intestine
391
of weaned piglets. CON group, corn soybean meal based diet; AKG group, the basal diet
392
containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln
393
supplementation. a,b Values with different letters are significantly different (P < 0.05).
394
Figure 2. Effects of alpha-ketoglutarate and glutamine on the mRNA abundance of AA
395
transporters in jejunum (A) and in ileum (B) of weaned piglets. CON group, corn soybean meal
396
based diet; AKG group, the basal diet containing 1.0% AKG supplementation; GLN group, the
397
basal diet containing 1.0% Gln supplementation. a,b,c Values with different letters are significantly
398
different (P < 0.05).
399
Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal
400
porcine epithelial cells-1. a,b Values with different letters are significantly different (P < 0.05). n=7.
401
A: The net utilization of glutamine (Gln) in intestinal porcine epithelial cells-1 (IPEC-1) cultured
402
for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM of alpha-ketoglutarate (AKG).
403
Negative data of glutamine denote the net utilization of Gln. B: The production concentration of
404
glutamate (Glu) in IPEC-1 cultured for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM
405
of AKG. C: The production concentration of alanine (Ala) in IPEC-1 cultured for 3 h in the
406
presence of 2 mM of Gln plus 0.5, 2, and 3 mM of AKG. D: The production concentration of
407
aspartate (Asp) in IPEC-1 cultured for 3 h in the presence of 2 mM of Gln plus 0.5, 2, and 3 mM
408
of AKG. E: The production concentration of ammonia in IPEC-1 cultured for 3 h in the presence
409
of 2 mM of Gln plus 0.5, 2, and 3 mM of AKG.
410
411
412
413
ACS Paragon Plus Environment
Page 16 of 27
Page 17 of 27
Journal of Agricultural and Food Chemistry
414
Table 1. Feedstuff ingredients and nutrient composition of basal diet Item
Concentration (%)
Feed Ingredient Corn
57.34
Soybean meal
25.62
Rice bran
2.00
Fish meal
5.00
Dried whey
5.00
Soy oil
0.88
CaHPO4
0.88
Vitamin-mineral premix 1
1.00
Limestone
0.80
ZnO Acidifier
0.30 0.30
NaCl
0.30
L-Lysine·HCl
0.17
Choline chloride
0.20
Mould
0.10
inhibitor
DL-Methionine
0.11
Total
100.00
Nutrition Composition Digestible energy (MJ/kg)
14.22
Crude protein
20.00
Lys Met
1.30 0.30
Met+Cys
0.65
Thr
1.05
Trp
0.25
Ca
0.80
P
0.69
Available phosphorus
0.45
NaCl
0.46
415
1
416
40 IU; vitamin K3, 4 mg; vitamin B1, 6 mg; vitamin B2, 12 mg; vitamin B6, 6 mg; vitamin B12,
Supplied per kilogram of finished feed: vitamin A, 10,800 IU; vitamin D3, 4,000 IU; vitamin E,
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
417
0.05 mg; biotin, 0.2 mg; folic acid, 2 mg; niacin, 50 mg; D-Calcium pantothenate, 25 mg; Fe, 100
418
mg as ferrous sulfate; Cu, 150 mg as copper sulphate; Mn, 40 mg as manganese oxide; Zn, 100
419
mg as zinc oxide; I, 0.5 mg as potassium iodide; and Se, 0.3 mg as sodium selenite.
420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444
ACS Paragon Plus Environment
Page 18 of 27
Page 19 of 27
Journal of Agricultural and Food Chemistry
445
Table 2. Primers used for quantitative reverse transcription-PCR
Gene
Accession No.
Primers F:5′-AAGGAGTAAGAGCCCCTGGA-3′
GAPDH
NM_001206359 R:5′-TCTGGGATGGAAACTGGAA-3′ F:5′-GGCACCGCACTCTACGAAGCA-3′
EAAC1
NM_001164649 R:5′-GCCCACGGCACTTAGCACGA-3′ F:5′-GATTGTGGAGATGGAGGATGTGG-3′
ASCT2
XM_003355984 R:5′-TGCGAGTGAAGAGGAAGTAGATGA-3′ F:5′-GAACCCAAGACCACAAATC-3′
b0
+
NM_001110171 R: 5′-CCCAGTGTCGCAAGAAT-3′ F:5′-TTTGTTATGCGGAACTGG-3′
+
y LAT1
NM_001110421 R:5′-AAAGGTGATGGCAATGAC-3′
446
447
448
449
450
451
452
453
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
454
Page 20 of 27
Table 3. Effects of alpha-ketoglutarate and glutamine on growth performance of piglets1
Diet Treatment2 Item
P-value CON group
AKG group
GLN group
Initial body weight , kg
5.92±0.25
5.96±0.20
6.20±0.21
0.642
Final body weight, kg
13.87±1.16
15.65±1.33
14.24±0.72
0.467
Average daily feed intake, g/d
529.34±32.5
556.42±61.4
594.51±43.3
0.372
Average daily gain, g/d
333.02±44.2b
405.21±25.3 a
380.03±17.3a
0.013
Gain: feed, g/g
0.60±0.00b
0.77±0.02a
0.64±0.05a
0.041
455
1
456
differ (P < 0.05).
457
2
458
containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln
459
supplementation.
Values are LSMean ± SEM, n=7.
a,b
Values in the same row with different superscript letters
Dietary treatments: CON group, corn soybean meal based diet; AKG group, the basal diet
460
461
462
463
464
465
466
467
468
ACS Paragon Plus Environment
Page 21 of 27
Journal of Agricultural and Food Chemistry
469
Table 4. Effects of alpha-ketoglutarate and glutamine on serum concentrations of amino acids in
470
piglets (µg /mL)1
Diet Treatment2
Item
P-value
CON group
AKG group
GLN group
Ile
25.45±1.89a
20.45±1.98ab
18.89±1.34b
0.042
Leu
39.54±2.93
33.24±3.08
29.81±2.28
0.155
Lys
a
58.33±8.87
b
27.46±7.53
16.95±1.73
b
0.015
Met
15.85±1.55
15.57±0.75
15.04±0.42
0.711
Phe
a
22.48±1.88
b
15.77±1.51
13.88±0.92
b
0.013
Thr
58.81±12.6b
90.44±3.29a
60.31±3.65b
0.022
Val
a
47.59±2.68
b
Ala Arg
47.75±8.78
Asp
20.8±0.53a
35.48±4.45
b
31.8±1.73
0.041
77.29±6.81
79.63±10.25
54.53±2.66
0.057
a
ab
35.52±5.27
27.08±1.10
b
0.024
18.46±0.74ab
15.77±0.80b
0.009
Glu
124.7±10.4
a
b
89.98±7.29
84.35±6.52
b
0.018
Gly
79.88±4.84
94.71±9.02
84.97±3.30
0.650
His
14.72±1.99
13.31±1.23
10.21±0.73
0.337
Pro
38.45±4.41
36.22±3.28
28.49±1.38
0.510
Ser
20.56±2.15
20.03±1.73
18.33±1.25
0.865
Tyr
23.66±2.6a
18.04±1.48b
15.72±0.88b
0.046
471
1
472
differ (P < 0.05).
473
2
474
containing 1.0% AKG supplementation; GLN group, the basal diet containing 1.0% Gln
475
supplementation.
Values are LSMean ± SEM, n=7.
a,b
Values in the same row with different superscript letters
Dietary treatments: CON group, corn soybean meal based diet; AKG group, the basal diet
476
477
478
479
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 1. Effects of alpha-ketoglutarate and glutamine on the histomorphology of small intestine of weaned piglets. 48x34mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 22 of 27
Page 23 of 27
Journal of Agricultural and Food Chemistry
Figure 2. Effects of alpha-ketoglutarate and glutamine on the mRNA abundance of AA transporters in jejunum (A) and in ileum (B) of weaned piglets. 99x50mm (300 x 300 DPI)
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 . 44x39mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 24 of 27
Page 25 of 27
Journal of Agricultural and Food Chemistry
Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 87x43mm (300 x 300 DPI)
ACS Paragon Plus Environment
Journal of Agricultural and Food Chemistry
Figure 3. Effects of alpha-ketoglutarate and glutamine catabolism and its metabolites in intestinal porcine epithelial cells-1 87x43mm (300 x 300 DPI)
ACS Paragon Plus Environment
Page 26 of 27
Page 27 of 27
Journal of Agricultural and Food Chemistry
TOC 77x63mm (300 x 300 DPI)
ACS Paragon Plus Environment