Influence of nitrogen levels on nutrient transporters and regulators of

Subscriber access provided by LUNDS UNIV

Agricultural and Environmental Chemistry

Influence of nitrogen levels on nutrient transporters and regulators of protein synthesis in small intestinal enterocytes of piglets Zhimei Tian, Xianyong Ma, Dun Deng, Yiyan Cui, and Weidong Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06712 • Publication Date (Web): 20 Feb 2019 Downloaded from http://pubs.acs.org on February 21, 2019

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 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 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.

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 38

Journal of Agricultural and Food Chemistry

1

Influence of nitrogen levels on nutrient transporters and regulators of protein synthesis in small

2

intestinal enterocytes of piglets

3

Zhimei Tian†, ‡, #, §, Xianyong Ma,†, ‡, #, §, Dun Deng†, ‡, #, §, Yiyan Cui†, ‡, #, §, Weidong Chen

,†, ‡, #,

4

§

5

(†Institute of Animal Science, Guangdong Academy of Agricultural Sciences, ‡The Key

6

Laboratory of Animal Nutrition and Feed Science (South China) of Ministry of Agriculture, #State

7

Key Laboratory of Livestock and Poultry Breeding, §Guangdong Engineering Technology

8

Research Center of animal Meat quality and Safety Control and Evaluation, Guangzhou 510640,

9

China)



Corresponding author: Xianyong Ma, Email, [email protected], 86-020-61368896.



Corresponding author: Weidong Chen, Email, [email protected], 86-020-61368838.

This research was funded by Guangdong international science and technology cooperation project (2014A050503049), Guangdong Modern Agro-industry Technology Research System (2016LM1080, 2017LM1080, 2018LM1080), National Basic Research Program of China (2013CB127301), Operating Project of Guangdong Key Laboratory of Animal Breeding and Nutrition (2017B030314044).

ACS Paragon Plus Environment


11

Abstract:

12

To investigate effects of dietary nitrogen level on nutrient absorption and utilization in small

13

intestinal enterocyte of piglets , weaned piglets were fed for 10 d with diets containing 20%, 17%,

14

or 14% crude protein (CP) with supplementation to meet requirements for essential amino acids in

15

vivo, and IPEC-1 cells were cultured with different nitrogen levels (NL) in culture medium (70%,

16

85%, 100%) in vitro by mono-cultured and co-cultured of IPEC-1 and GES-1 cells. The results

17

showed that: (1) In animal trail, decreased dietary CP reduced transcript abundance of nutrient

18

transporters like CAT1, PepT1, GLUT2 and SGLT-1 in jejunal mucosa (0.09 ± 0.03, P < 0.0001;

19

0.40 ± 0.04, P = 0.0087; 0.20 ± 0.07, P = 0.0003; 0.35± 0.02, P = 0.0001 ), but 17% CP diet did not

20

affect jejunal protein synthesis; (2) The transcript abundance of nutrient transporters displayed the

21

similarly effective tendency in jejunal mucosa and co-cultured IPEC-1 rather than mono-cultured

22

IPEC-1; (3) Decreased nitrogen levels reduced expressive abundance of PI3K, Class 3 PI3K, TSC2

23

and 4E-BP1 in mono-cultured IPEC-1, but 85% nitrogen level did not affect expressive abundance

24

of PI3K, TSC2, mTORC1, 4E-BP1 and S6K1 in co-cultured IPEC-1 . In general, decreased 3% CP

25

or 15% nitrogen level reduced relative transcript expression of nutrient transporters, but did not

26

affected protein synthesis in jejunal mucosa and co-cultured IPEC-1. Therefore, decreased 3%

27

dietary CP increased utilized and synthetic efficient of nitrogen resource in small intestine, and was

28

benefit for saving the dietary nitrogen resource.

29

Key words:

crude protein, transporter, protein synthesis, small intestine, IPEC-1 cells

30 31

Abbreviations

32

CP : Crude protein; EAA: Essential amino acid; NHE3: Sodium/hydrogen exchanger 3; ASTC2:


Page 2 of 38

Page 3 of 38


33

Alanine serine cysteine threonine transporters 2; B0+AT: Glycoprotein-associated amino acid

34

transporter; EAAT3: Glutamate and aspartate transporter 3; CAT1: Cationic AA transporters 1;

35

4F2hc: 4F2 cell-surface antigen heavy chain; Y+LAT1: y+L amino acid transporter-1; PepT1:

36

Peptide transporter 1; APOA1: Apolipoprotein A1; FATP-1c: Fatty acid transport protein 1c;

37

GLUT2: Glucose transporter 2; SGLT-1: Sodium-glucose co-transporter-1; mTORC1: Mammalian

38

target of rapamycin complex 1; S6K1: S6 kinase 1; 4E-BP1: Initiation factor 4E binding protein 1;

39

TSC2: Tuberous sclerosis complex 2; PI3K: Phosphatidylinositol 3 kinase.

40 41

Introduction

42

In swine production, reducing dietary crude protein (CP) is often regarded as a nutritional and

43

economic strategy to decrease N excretion and improve gastrointestinal health of weaned piglets1.

44

These studies suggested that low CP diets, supplemented with essential amino acids (EAA),

45

maintained performance and improved gastrointestinal health. Some works indicated that diets with

46

low CP decreased feed efficiency, thereby impairing growth performance of pigs2-3. Our previous

47

study also revealed that reducing dietary CP changed expression of gastrointestinal enzymes,

48

consistent with increasing intestinal digestion and absorption, but did not impairing growth

49

performance of piglets4-5. The influence of low-protein diets on growth performance and intestinal

50

health is likely associated with the capacity for absorption and utilization of nutrients6.

51

Dietary protein provides important nutrients and plays a physiological function in regulating

52

protein, carbohydrate and lipid metabolism in pigs. Amino acid availability affects many cell

53

functions including modulating expression of nutrient transporters and regulating cell signaling

54

pathways7. Low-protein diets affected utilization of free AA and expression of genes transporting



55

AA in skeletal muscle of pigs8. The intestine is the main digestive and absorptive organ in animals,

56

and the jejunum is the key segment for intestinal digestion and absorption6. The absorption of

57

nutrients is mediated by transporters regulating uptake and efflux of nutrients through the mucosal

58

cells in response to nutrient level. ATPase and the sodium/hydrogen exchanger (NHE3) are

59

responsible for the absorption of nutrients including fatty acids, AA and glucose9-10. AA transporters

60

and the intestinal peptide transporter are identified as seven systems and play important transport

61

activities and functions on FAA and small peptide11. Apolipoprotein A1 (APOA1) and fatty acid

62

transport protein (FATP-1c) is involved in plasma triglyceride metabolism by activating lipoprotein

63

lipase and mediates transport of aliphatic acids respectively12. The facilitated glucose transporter

64

GLUT2 and the sodium-glucose co-transporter SGLT-1 mediates glucose transport in intestinal

65

epithelial cells13. In addition, AA affects both nutrient transport and protein synthesis by activating

66

the mammalian target of rapamycin complex 1 (mTORC1and its downstream targets S6 kinase 1

67

(S6K1) and the initiation factor 4E binding protein (4E-BP1)14-15. Tuberous sclerosis complex 2

68

(TSC2) regulates the amino acid-sensing mTOR pathway through its subcellular location and

69

activity and mediates phosphatidylinositol 3 kinase (PI3K) signaling cascade16-17. Therefore, we

70

implied that decreased nitrogen level maybe affected nutrient metabolism by regulating the

71

expression of transporters and protein synthesis by mediating mTOR, TSC2 and PI3K signal

72

pathway.

73

The present study investigated the possible mechanisms by which low protein diets might

74

affect intestinal absorption of AA by analyzing transport, utilization of FAA and protein synthesis

75

in vivo and in vitro. The impact of reduced dietary protein on the expression of selected nutrient

76

transporters and protein synthesis in jejunum of weaned piglets was examined and the results


Page 4 of 38

Page 5 of 38


77

obtained in vivo were also assessed using intestinal porcine epithelial cells (IPEC-1) in two in vitro

78

culture systems.

79

Materials and Methods

80

Chemicals

81

All reagents of components in DMEM/F12 were purchased from Sigma (Merck, New Jersey,

82

USA) and suitable for cell culture. Insulin–transferrin–selenium (Lot: 41400045, Gibco) and EGF

83

(Lot: PHG0313, Gibco) were purchased from Thermo Fisher (Waltham, MA, USA).

84

Sulfosalicylic acid (Lot: S7422) and MTT (Lot: M2128) were purchased from Sigma (Merck, New

85

Jersey, USA).

86

Animals and diet

87

Eighteen cross-bred (Duroc×Landrace×Yorkshire) piglets with average initial body weight

88

9.57 ± 0.64 kg were weaned at 28 d of age and were randomly assigned to 3 treatment groups and

89

allowed a 7-d adaptation period. Diets with 20% CP (NCP), 17% CP (LCP), and 14% CP (VLCP)

90

was based on NRC (2012) recommendation as shown Table 1 and was supplemented with Lys, Met,

91

Thr, and Trp to meet the requirements of weaned piglets18. Piglets were housed individually in cages

92

and given ad libitum access to water and diets throughout the 10-d experimental period (d 35 to 45

93

of age).

94

Tissue sample collection

95

Animals were anaesthetized and slaughtered with intravenous injection of sodium

96

pentobarbital. The jejunum was immediately dissected and emptied of contents. Segment of mid-

97

jejunum was cut longitudinally and washed with ice-cold phosphate buffered saline (PBS) then

98

placed on ice. The jejunal mucosa was scraped and collected with glass slides, and then frozen in



99

liquid nitrogen and held at -80oC.

100

Cell Culture Conditions

101

Two established cell lines were used: porcine IPEC-1 was gift from Dr G Wu (Texas A&M

102

University) and a transformed human gastric epithelial cell line (GES-1), obtained from Dr X Wang

103

(South China Agricultural University). The control culture medium was same to Dulbecco's

104

Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12) (Gibco, Life Technologies,

105

Gaithersburg, MD) and this served as the normal nitrogen level (NNL) medium. In animal

106

experiment, diets were decreased 3% and 6% crude protein levels from 20% CP balanced with Lys,

107

Met, Thr, and Trp. Based on animal experimental design, nitrogen levels were regarded DMEM-

108

F12 medium as 100% nitrogen, and decreased the contents of amino acids components down to 85%

109

(LNL) and 70% nitrogen levels (VLNL). The total nitrogen of DMEM-F12 medium was calculated

110

and multiplied respectively by 0.85 and 0.70, then minus the nitrogen content of Lys, Met, Thr, Trp

111

and calculated other amino acids ratios. The components of amino acids were respectively added as

112

0.83 and 0.66 proportion balanced Lys, Met, Thr, Trp. and other non-AA components. In final, the

113

total nitrogen levels of amino acids were respectively 85% and 70% of DMEM-F12 medium. (as

114

shown in table 2). IPEC-1 and GES-1 cells were grown in NNL, LNL and VLNL media

115

supplemented with 10% FBS, 0.05 μg/ml epidermal growth factor (EGF) (Gibco) and 100 U/ml

116

penicillin and 100 μg/ml streptomycin (Sigma, St Louis, MO) at 37°C under 5% CO2 in air; media

117

were replaced every 2 d.

118

6-well transwell plates (Corning, Corning, NY) with NNL until confluency, then with differentiation

119

media (VLNL, LNL or NNL supplemented with 1% insulin–transferrin–selenium (ITS) and 0.05

120

μg/ml EGF) for 15 d. Protocol 2: IPEC-1 and GES-1 cells were respectively seeded and co-cultured

Protocol 1: IPEC-1 cells were seeded and cultured in the top chamber of


Page 6 of 38

Page 7 of 38


121

in the top and bottom chambers of transwell plates with NNL until confluency occurred, then

122

differentiated, as described above.

123

Free amino acids consumed from conditioned media

124

Concentrations of free AA (FAA) in media after cell culture were analyzed on an L-8900

125

automatic amino acid analyzer (Hitachi, Tokyo, Japan). Media were aspirated and clarified by

126

centrifugation for 15 min at 1,000 g at 4°C. Supernatants (400 μl) were deproteinized by adding

127

20% (W/V) sulfosalicylic acid (1.2 ml), vortexing vigorously and re-centrifuging for 15 min at

128

12,000 g at 4°C. Supernatants were passed through a 0.22 μm filter and FAA were measured. The

129

measured values were subtracted from FAA concentrations in the fresh corresponding medium to

130

determine FAA consumed19.

131

Cell viability

132

The number of live cells was measured by the MTT assay. Both IPEC-1 and GES-1 cells in

133

NNL, LNL and VLNL media were separately seeded with 6,000 cells per well in 96-well plates and

134

cultured at 37°C under 5% CO2 for 24, 48 and 72 h. A stock solution (5 mg/ml) of MTT was added

135

(10 µl) to each well, and culture continued for 4 h. Media was aspirated from each well and replaced

136

with 100 µl DMSO and the plate was shaken for 15 min before reading absorbance at 570 nm using

137

a plate reader. Values for LNL and VLNL were expressed as fractions of those for NNL.

138

Relative quantification of gene transcripts

139

Total RNA was extracted from jejunal mucosa (100 mg) or cells cultured in 6-well plates with

140

TRIzol reagent (TaKaRa, Otsu, Japan) and dissolved in RNase-free water. RNA (1 µg) was reverse-

141

transcribed following the manufacturer’s protocol (TaKaRa, Otsu, Japan).

142

The primers (Table 3) designed with Primer Premier 5.0 (Applied Biosystems, Carlsbad, CA)



143

and the specificity of primers was verified by sequencing products and blasting sequences against

144

NCBI database. Real-time PCR (Bio-Rad System) was performed according to our in-house

145

protocol. The housekeeping gene -actin was used to normalize data of each sample as an internal

146

control for efficiency of reverse transcription. Data were quantified using the comparative cycle

147

threshold (Ct) method and the relative expression values for each gene were calculated using the

148

2−ΔΔCt method with efficiency correction and using 1 control sample as calibrator.

149

Western-blotting

150

Total proteins of jejunal mucosa and IPEC-1 cells were extracted using protein extraction

151

reagents (Thermo Fisher, MA, USA) and 40µg proteins were separated by SDS-PAGE. Then the

152

proteins were electro-transferred onto the PVDF membrane (Millipore, MA, USA) at 180mA for

153

90min, and then incubated with primary antibodies at 4°C overnight after blocking with 5% BSA

154

in TBS at room temperature for 30min. All primary antibodies were fit to detecting porcine proteins

155

expression and the information as follows : β-actin (4970, 1:500, CST), p-PI3K (4228, 1:250, CST),

156

p-Class 3 PI3K (4263, 1:250, CST),TSC2 (23402, 1:200, CST), p-4E-BP1 (2855, 1:500, CST), p-

157

S6K1 (97596, 1:500, CST) and p-mTORC1 (5536, 1:500, CST). The HRP-conjugated secondary

158

antibodies were diluted at 1: 1000 and incubated for 2 hours at room temperature. Then the

159

membrane developed the blots using Amersham Imager 600 system (GE Healthcare, UK, USA).

160

Statistical analysis

161

The effects of treatments, dietary CP level in vivo or medium nitrogen level in vitro, were

162

assessed by one-way analysis of variance (ANOVA) with Tukey’s post hoc test to compare the means.

163

Data are summarized as means ± standard deviation (SD) of relative transcript abundance (n = 6),

164

live cells (n = 12) and FAA concentration (n = 6). Statistical analyses were performed and the data


Page 8 of 38

Page 9 of 38


165

were analyzed using Prism 6 (GraphPad Software, Inc. San Diego, CA). Statistical significance was

166

set at P < 0.05, P < 0.01, and P < 0.001.

167

Results:

1681. 1. Effect of dietary crude protein on relative transcript abundance of genes for nutrient transporters 169

in jejunum of piglets.

170

With the exception for EAAT3, which increased in abundance with VLCP and LCP diets (Fig.

171

1), expression of other transporters changed in a similar but reverse fashion: reducing dietary CP to

172

17% or 14% lead to decreased transcript abundance. The decreases in CAT1, PepT1, GLUT2, SGLT-

173

1 and ATPase transcripts were significant (P < 0.05), as was the increase in EAAT3 (P < 0.05).

174

2. Effect of nitrogen levels in medium on expression of nutrient transporters in IPEC-1 cells cultured

175

alone.

176

To explore the jejunal mucosal results further, the effects of reducing nitrogen levels in culture

177

medium on small intestinal epithelial cells were examined. In IPEC-1 cells cultured alone (Protocol

178

1, Fig. 2), nitrogen level in the medium significantly affected the expression of AA transporters

179

EAAT3 and 4F2hc (P < 0.05) but not that of ASTC2, B0+AT, CAT1 and y+LAT1 (P > 0.05).

180

Transcripts of 4F2hc were more abundant in cells grown in LNL (P < 0.05) than in cells cultured

181

in NNL or VLNL. Transcripts of AA transporters ASTC2, B0+AT, CAT1 and y+LAT1 were not

182

significantly affected by the different media. The expression of EAAT3 decreased with increased

183

nitrogen levels (P < 0.05). Transcripts of the small peptide transporter PepT1 were unaffected by

184

nitrogen level in the media (P > 0.05). The relative expression of APOA1, FATP-1C, ATPase and

185

NHE3 was lowest in IPEC-1 cells grown in LNL medium, but this medium resulted in highest

186

abundance of transcripts of the glucose transporters, GLUT2 and SGLT-1; there were no effects of



187

medium on expression of FATP-1C and GLUT2.

188

3. Effect on transcripts for nutrient transporters of nitrogen levels in media when IPEC-1 were co-

189

cultured with GES-1 cells

190

The expression of jejunal nutrient transporters affected by dietary CP were not mimicked in

191

IPEC-1 cells cultured alone (Protocol 1) in media of different levels of nitrogen. The co-culture

192

system (Protocol 2) of IPEC-1 and gastric epithelial cells (GES-1) was postulated to better simulate

193

the physiological environment of the gastrointestinal tract in vitro. Consistent with that hypothesis,

194

the effects of media with reduced nitrogen on transporter expression in the IPEC-1 cells were

195

remarkably similar (Fig. 3) to the effects of CP levels on jejunal mucosa in vivo. In the co-cultured

196

IPEC-1 cells, decreased nitrogen levels reduced significantly the relative expression of AA

197

transporters (B0+AT, and y+LAT1) and the small peptide transporter (PepT1) (P < 0.05), while the

198

transcript abundance of EAAT3 was increased with decreased nitrogen levels (P < 0.05), The

199

expression of 4F2hc and CAT1 was not affected by the different media. Relative expression of the

200

lipid transporters (APOA1, FATP-1C), glucose transporter (GLUT2), ATPase and NHE3 had similar

201

changes to the AA transporters with highest expression in NNL medium (P < 0.05); SGLT-1

202

transcripts were unaffected by nitrogen level.

203

4. Effect of nitrogen levels on viability and morphology of IPEC-1 and GES-1 cells.

204

The viability of IPEC-1 cells was unaffected by nitrogen levels (Fig. 4a) in the culture media

205

at any stage tested (P > 0.05). In contrast, the number of live GES-1 cells (Fig. 4b) was affected

206

immediately and thereafter with lowest survival in VLNL medium (P < 0.05); there were no

207

significant differences between LNL and NNL. There was no obvious effect of nitrogen levels in

208

the media on the morphology of IPEC-1 or GES-1 cells grown in either protocol, but IPEC-1 cells


Page 10 of 38

Page 11 of 38


209

displayed different morphology when cultured alone from that observed when they were co-cultured

210

with the GES-1 cells (Fig. 4c and 4d). The IPEC-1 cells were larger and had a more obviously

211

differentiated morphology in the co-culture system.

212

5. Amino acid consumption by mono-cultured and co-cultured IPEC-1.

213

The different morphologies and gene expression of nutrient transporters in the two culture

214

protocols for the IPEC-1 cells raised the possibility that their utilization of AA might have been

215

differentially affected by nitrogen levels in the media. Amino acid utilization by IPEC-1 in the two

216

protocols was assessed by measuring concentrations remaining in conditioned media after the last

217

2-d period of culture, relative to concentrations provided in the fresh media. With the exception for

218

Val, the greatest consumption of AA by mono-cultured IPEC-1 cells (Table 4 and Fig. 7) occurred

219

in the LNL medium (P < 0.05); for most AA, their consumption was not different between NNL

220

and LNL. In the case of Val, consumption directly followed the reduction in total nitrogen level in

221

the medium. There was net addition of some AA, most notably Ala, to the conditioned media but

222

no significant differences between media of different nitrogen levels. When considered in toto, the

223

net consumption of AA (Fig. 5) was greatest when IPEC-1 cells were grown in LNL medium (P
0.05).

239

In co-cultured IPEC-1 cells and jejunal mucosa, the abundance of Class I PI3K, Class III PI3K,

240

TSC2, and S6K1 transcript and translate showed very similar changes, all increasing as the medium

241

nitrogen level or dietary CP increased. There was lower expression of Class I PI3K, TSC2, and

242

S6K1 transcripts and translate in VLNL (VLCP) than that in LNL (LCP) and NNL (NCP) media (P

243

< 0.05) while transcripts and translate abundance of Class Ⅲ PI3K was higher in NNL (NCP) than

244

that in VLNL (VLCP) and LNL (LCP) media (P < 0.05). With an increase of nitrogen level or

245

dietary CP, the expression of mTORC1 increased between VLNL (VLCP) and NNL (NCP) media

246

(P < 0.05), with intermediate level of expression in co-cultured IPEC-1 cells and jejunal mucosa in

247

LNL (LCP) medium.

248

Discussion

249

Research investigation of decreasing dietary protein while maximizing AA utilization is still

250

urgent needed for precision nutrition to minimize the environmental impact of animal production.

251

Within the gastrointestinal tract (GIT) of monogastric animals, most absorption occurs across the

252

epithelium of the small intestine with the proximal jejunum being the major site of AA and peptide


Page 12 of 38

Page 13 of 38


253

absorption20. Our results showed that the relative expression of nutrient transporter genes was

254

decreased with reduced dietary CP, suggesting that reducing dietary CP might impair absorption of

255

AA, fatty acids and glucose in jejunum of weaned piglets of 9 to 11 kg BW. This finding is similar

256

with that of Wu et al.18 for AA transporters in older pigs of 11 to 20 kg BW. Among the transporter

257

transcripts, only those of the glutamate transporter EAAT3 showed increased abundance when

258

dietary CP was reduced. One possible reason is that Glutamate is catabolized by jejunal mucosa21.

259

However, Li et al.8 demonstrated that reducing dietary protein enhanced skeletal muscle absorption

260

of AA by up-regulating gene expression of AA transporters. It suggested that there were

261

organizational differences on nutrient transport in response to dietary CP.

262

In order to explore these in vivo results, we used cell experiment in vitro to verify the results

263

in vivo. The effects of media with reduced nitrogen level on gene expression in mono-cultured

264

IPEC-1 bore no resemblance to results found in jejunal mucosa (compare Figs 1 and 2).

265

imitate physiological functions, many co-cultured models of different cell lines have been

266

established, depending upon experimental objectives22-24. Recognizing that the environment

267

provided by the luminal digesta within the small intestine in part reflects its earlier transit of the

268

stomach, the IPEC-1 cells employed here incorporated co-culture with a transformed gastric cell

269

line, GES-1. When IPEC-1 cells were co-cultured with GES-1 cells, morphological evidence

270

indicated that the cells displayed larger size and more complete differentiation (Fig 4c and 4d) and

271

the effect of reduced nitrogen in the medium on gene expression related to nutrient transport more

272

closely mimicked the jejunal situation in vivo with diets of varying CP content. Antunes et al.,25 also

273

reported that co-cultured cells had increased cell size and thickness of membranes. Our previous

274

report clearly documented that the diets used here had differential effects on the gastric and enteric


To better


275

expression in vivo of several genes related to digestion and absorption of nutrients, and receptors

276

releasing hormones4. The results suggested that some secretory function of the gastric epithelial

277

cells affected the morphology of intestinal epithelial cells rather than their proliferation and survival,

278

along with altered expression of genes related to nutrient transport26.

279

The intracellular availability of AA is coordinated with AA transporters located in the cellular

280

membrane, which in turn may be affected by concentration of FAA27. Consumption of FAA from

281

the media showed that mono-cultured IPEC-1 cells most efficiently used the FAA of the LNL

282

medium compared to either VLNL or NNL media. The result of FAA consumption was in accord

283

with the relative abundance of 4F2hc, which suggested that 4F2hc was the most importance

284

transporter for promoting utilization and transport of the multimeric transporter of neutral branched

285

chain and aromatic AA compared to the other transporters in mono-cultured IPEC-1. The effects of

286

nitrogen levels on expression of intracellular fatty acid transporters was opposite to that of glucose

287

and AA transporter 4F2hc. In the co-cultured system, VLNL medium impaired the utilization of Ser

288

and Leu by IPEC-1 compared to LNL or NNL media and the net release of Glu and Ala was greater

289

in LNL medium. In general, the results on utilization of FAA was consistent with the expression of

290

amino acids transporters, which suggested that nitrogen level affected amino acids utilization and

291

transport by controlling expression of FAA transporters in two cultured systems. Taken together,

292

the results indicated that the presence of gastric cells affected AA utilization by the intestinal

293

epithelial cells. Decreased nitrogen levels in the medium reduced the expression of small peptide

294

transporters, fatty acid transporter, glucose transporter and AA transporters with the exception for

295

EAAT3, accounting for the reduction in AA utilization. The discrepancy of effects on EAAT3 may

296

be due to its dual function as both an AA transporter and sensor28. Similar changes in the expression


Page 14 of 38

Page 15 of 38


297

of ATPase and NHE3 in co-cultured IPEC-1 cells suggest that expression of their genes was

298

associated with the absorption of nutrients including fatty acid, AA and glucose by providing energy

299

and homeostasis. The results revealed that the co-cultured system reliably modeled the in vivo

300

behavior of jejunal epithelium and decreased medium nitrogen did not affect cell growth, meanwhile

301

was benefit for saving and promoting utilization of nitrogen resource.

302

Apart from their obvious role in providing substrates for protein synthesis, dietary protein or

303

FAA also serve as signaling molecules to regulate protein synthesis29. In co-cultured IPEC-1 and

304

jejunal mucosa, decreased 15% nitrogen level or 3% CP did not affect transcript and translate

305

abundance of the key target protein 4E-BP1 and S6K1, which suggested that properly decreased

306

nitrogen level did not impair protein synthesis of enterocyte. Decreased 15% nitrogen level or 3%

307

CP increased synthetic efficiency of protein, thereby controlling cell proliferation and animal

308

growth by feedback mechanism. mTORC1 enhances cellular protein synthesis by stimulating S6K1

309

activity and releasing 4E-BP from the complex of eukaryotic initiation factor (eIF) 4E and 4E-BP.

310

Effects of different nitrogen level (CP) media on expression of mTORC1 seemed to relate to AA

311

consumption and expression of S6K1 in IPEC-1 cells in either culture system, suggesting that AA

312

influence S6K1 rather than 4E-BP1 by regulating mTORC1. While nitrogen levels determined 4E-

313

BP transcript abundance in mono-cultured IPEC-1, they affected the expression of S6K1 in co-

314

cultured IPEC-1 and jejunal mucosa. The research pointed out that the complex of 4E-BP1 and

315

S6K1 may be controlled upstream by TSC230 and AA can regulate mTORC1 activity by additional

316

mechanisms, independent of TSC231-32. The transcript and translate abundance of TSC2 was similar

317

to that of S6K1 in co-cultured IPEC-1 and jejunal mucosa, but was similar to that of 4E-BP1 in

318

mono-cultured IPEC-1; there might be distinct mechanisms through which AA affected TSC2 and



319

possibly protein synthesis in the two culture systems. Reducing nitrogen levels in the medium

320

decreased class I and class III PI3K transcripts in similar fashion to those of TSC2. The research

321

reported that amino acids influence mTOR signaling through class III rather than class I PI3K33. In

322

the co-cultured IPEC-1 cells, it appeared that AA influenced S6K1 expression via class I and class

323

III PI3K controlling mTORC1 and by class I PI3K regulating TSC2. Recent research indicates that

324

both mTORC1 and phosphorylates S6K1 to enhance protein synthesis and has negative-feedback

325

effects rendering the IR-PI3K-Akt signaling axis refractory to insulin with S6K134-35. Therefore, it

326

can be deduced that the signaling pathway for AA regulating protein synthesis in the IPEC-1 cells

327

was associated with that of insulin. In both culture systems, decreasing nitrogen levels likely

328

regulated cellular protein synthesis through distinct signaling pathways. In mono-cultured IPEC-1,

329

nitrogen levels affected protein synthesis by PI3K (class 1 and class 3) and TSC2 signal pathway

330

regulating 4E-BP and by mTORC1 regulating S6K1. However, nitrogen levels affected protein

331

synthesis by PI3K (class 1 and class 3), TSC2 and mTORC1 mediating S6K1 rather than 4E-BP1

332

in co-cultured IPEC-1 and jejunal mucosa.

333

The study revealed that the co-cultured IPEC-1 cells had similar physiologic properties and

334

responses to nitrogen levels as did the jejunal mucosa of piglets. Meanwhile the study suggested

335

that decreased CP diets or nitrogen level media balanced with required amounts of Lys, Met, Thr,

336

and Trp reduced the relative expression of intestinal nutrient transporters in piglets, but 17% CP or

337

85% NL did not affect the protein synthesis in small intestine. Therefore, 17% CP diet or 85% NL

338

enhanced synthetic efficiency of protein and did not impair growth performance or proliferation.

339

Declarations

340

There is no conflict of interests in the present study. All authors consent to participate and


Page 16 of 38

Page 17 of 38


341

publish this article. Xianyong Ma and Weidong Chen carried out the design of the experiment;

342

Zhimei Tian participated in measure and analysis of the animal and cell sample and drafted the

343

manuscript. Dun Deng and Yiyan Cui participated in the sample collection and data analysis.

344

Acknowledgements

345

We gratefully acknowledge the helpful suggestions on presentation made by W. B. Currie

346

(Cornell University, Ithaca, NY). This research was supported by Guangdong international science

347

and technology cooperation project (2014A050503049), Guangdong Modern Agro-industry

348

Technology Research System (2017LM1080, 2018LM1080), National Basic Research 973 Program

349

of China (2013CB127301), Operating Project of Guangdong Key Laboratory of Animal Breeding

350

and Nutrition (2017B030314044).

351



References:

352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394

1. Nyachoti, C. M.; Omogbenigun, F. O.; Rademacher, M.; Blank, G., Performance responses and indicators of gastrointestinal health in early-weaned pigs fed low-protein amino acid-supplemented diets. J Anim Sci 2006, 84, (1), 125-134. 2. Wellock, I. J.; Fortomaris, P. D.; Houdijk, J. G.; Kyriazakis, I., Effects of dietary protein supply, weaning age and experimental enterotoxigenic Escherichia coli infection on newly weaned pigs: performance. Animal 2008, 2, (6), 825-833. 3. Wellock, I. J.; Fortomaris, P. D.; Houdijk, J. G. M.; Kyriazakis, I., Effect of weaning age, protein nutrition and enterotoxigenic Escherichia coli challenge on the health of newly weaned piglets. Livestock Science 2007, 108, 102-105. 4. Tian, Z. M.; Ma, X. Y.; Yang, X. F.; Fan, Q. L.; Xiong, Y. X.; Qiu, Y. Q.; Wang, L.; Wen, X. L.; Jiang, Z. Y., Influence of low protein diets on gene expression of digestive enzymes and hormone secretion in the gastrointestinal tract of young weaned piglets. J Zhejiang Univ Sci B 2016, 17, (10), 742751. 5. Ma, X.; Tian, Z.; Deng, D.; Cui, Y.; Qiu, Y., Effect of Dietary Protein Level on the Expression of Proteins in the Gastrointestinal Tract of Young Pigs. J Agric Food Chem 2018, 66, (17), 4364-4372. 6. Bergen, W. G.; Wu, G., Intestinal nitrogen recycling and utilization in health and disease. J Nutr 2009, 139, (5), 821-825. 7. Bergen, W. G.; Wu, G., Intestinal nitrogen recycling and utilization in health and disease. J Nutr 2009, 139, (5), 821-825. 8. Li, Y. H.; Li, F. N.; Wu, L.; Liu, Y. Y.; Wei, H. K.; Li, T. J.; Tan, B. E.; Kong, X. F.; Wu, F.; Duan, Y. H.; Oladele, O. A.; Yin, Y. L., Reduced dietary protein level influences the free amino acid and gene expression profiles of selected amino acid transceptors in skeletal muscle of growing pigs. J Anim Physiol Anim Nutr (Berl) 2017, 101, (1), 96-104. 9. Zhao, H.; Shiue, H.; Palkon, S.; Wang, Y.; Cullinan, P.; Burkhardt, J. K.; Musch, M. W.; Chang, E. B.; Turner, J. R., Ezrin regulates NHE3 translocation and activation after Na+-glucose cotransport. Proc Natl Acad Sci U S A 2004, 101, (25), 9485-9490. 10. Anderson, C. M.; Grenade, D. S.; Boll, M.; Foltz, M.; Wake, K. A.; Kennedy, D. J.; Munck, L. K.; Miyauchi, S.; Taylor, P. M.; Campbell, F. C.; Munck, B. G.; Daniel, H.; Ganapathy, V.; Thwaites, D. T., H+/amino acid transporter 1 (PAT1) is the imino acid carrier: An intestinal nutrient/drug transporter in human and rat. Gastroenterology 2004, 127, (5), 1410-1422. 11. Broer, S.; Broer, A., Amino acid homeostasis and signalling in mammalian cells and organisms. Biochem J 2017, 474, (12), 1935-1963. 12. Shu, G.; Zhu, X. T.; Wang, X. Q.; Song, Y. Z.; Bin, Y. F.; Zhang, Y. L.; Gao, P.; Jiang, Q. Y., Identification and gene expression of porcine fatty acid transport protein 1 isoforms. J Anim Physiol Anim Nutr (Berl) 2009, 93, (4), 439-446. 13. Zhang, S.; Yang, Q.; Ren, M.; Qiao, S.; He, P.; Li, D.; Zeng, X., Effects of isoleucine on glucose uptake through the enhancement of muscular membrane concentrations of GLUT1 and GLUT4 and intestinal membrane concentrations of Na+/glucose co-transporter 1 (SGLT-1) and GLUT2. Br J Nutr 2016, 116, (4), 593-602. 14. Rasmussen, B. B.; Richter, E. A., The balancing act between the cellular processes of protein synthesis and breakdown: exercise as a model to understand the molecular mechanisms regulating muscle mass. J Appl Physiol (1985) 2009, 106, (4), 1365-1366.


Page 18 of 38

Page 19 of 38

395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427 428 429 430 431 432 433 434 435 436


15. Kim, D. H.; Sarbassov, D. D.; Ali, S. M.; King, J. E.; Latek, R. R.; Erdjument-Bromage, H.; Tempst, P.; Sabatini, D. M., mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 2002, 110, (2), 163-175. 16. Demetriades, C.; Doumpas, N.; Teleman, A. A., Regulation of TORC1 in response to amino acid starvation via lysosomal recruitment of TSC2. Cell 2014, 156, (4), 786-799. 17. Zhang, H.; Cicchetti, G.; Onda, H.; Koon, H. B.; Asrican, K.; Bajraszewski, N.; Vazquez, F.; Carpenter, C. L.; Kwiatkowski, D. J., Loss of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through downregulation of PDGFR. J Clin Invest 2003, 112, (8), 1223-1233. 18. Wu, L.; He, L. Q.; Cui, Z. J.; Liu, G.; Yao, K.; Wu, F.; Li, J.; Li, T. J., Effects of reducing dietary protein on the expression of nutrition sensing genes (amino acid transporters) in weaned piglets. J Zhejiang Univ Sci B 2015, 16, (6), 496-502. 19.

GAO, H. L.; CONG, W.; OUYANG, F., Amino acids metabolism of vero cells in batch culture.

The Chinese Journal of Process Engineering 2001(02):176-179. 20. Silk, D. B.; Grimble, G. K.; Rees, R. G., Protein digestion and amino acid and peptide absorption. Proc Nutr Soc 1985, 44, (1), 63-72. 21. Lin, M.; Zhang, B.; Yu, C.; Li, J.; Zhang, L.; Sun, H.; Gao, F.; Zhou, G., L-Glutamate supplementation improves small intestinal architecture and enhances the expressions of jejunal mucosa amino acid receptors and transporters in weaning piglets. PLoS One 2014, 9, (11), e111950. 22. Arnold, J. T.; Kaufman, D. G.; Seppala, M.; Lessey, B. A., Endometrial stromal cells regulate epithelial cell growth in vitro: a new co-culture model. Hum Reprod 2001, 16, (5), 836-845. 23. Viney, M. E.; Bullock, A. J.; Day, M. J.; MacNeil, S., Co-culture of intestinal epithelial and stromal cells in 3D collagen-based environments. Regen Med 2009, 4, (3), 397-406. 24. Ishimoto, Y.; Satsu, H.; Totsuka, M.; Shimizu, M., IEX-1 suppresses apoptotic damage in human intestinal epithelial Caco-2 cells induced by co-culturing with macrophage-like THP-1 cells. Biosci Rep 2011, 31, (5), 345-351. 25. Antunes, F.; Andrade, F.; Araujo, F.; Ferreira, D.; Sarmento, B., Establishment of a triple co-culture in vitro cell models to study intestinal absorption of peptide drugs. Eur J Pharm Biopharm 2013, 83, (3), 427-435. 26. Broer, S., Amino acid transport across mammalian intestinal and renal epithelia. Physiol Rev 2008, 88, (1), 249-286. 27. Gilbert, E. R.; Li, H.; Emmerson, D. A.; Webb, K. J.; Wong, E. A., Dietary protein quality and feed restriction influence abundance of nutrient transporter mRNA in the small intestine of broiler chicks. J Nutr 2008, 138, (2), 262-271. 28. Hyde, R.; Taylor, P. M.; Hundal, H. S., Amino acid transporters: roles in amino acid sensing and signalling in animal cells. Biochem J 2003, 373, (Pt 1), 1-18. 29. Miyazaki, M.; Esser, K. A., Cellular mechanisms regulating protein synthesis and skeletal muscle hypertrophy in animals. J Appl Physiol (1985) 2009, 106, (4), 1367-1373. 30. Inoki, K.; Zhu, T.; Guan, K. L., TSC2 mediates cellular energy response to control cell growth and survival. Cell 2003, 115, (5), 577-590. 31. Hahn-Windgassen, A.; Nogueira, V.; Chen, C. C.; Skeen, J. E.; Sonenberg, N.; Hay, N., Akt activates the mammalian target of rapamycin by regulating cellular ATP level and AMPK activity. J Biol Chem 2005, 280, (37), 32081-32089.



437 438 439 440 441 442 443 444 445 446 447 448

32. Smith, E. M.; Finn, S. G.; Tee, A. R.; Browne, G. J.; Proud, C. G., The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. J Biol Chem 2005, 280, (19), 18717-18727. 33. Nobukuni, T.; Joaquin, M.; Roccio, M.; Dann, S. G.; Kim, S. Y.; Gulati, P.; Byfield, M. P.; Backer, J. M.; Natt, F.; Bos, J. L.; Zwartkruis, F. J.; Thomas, G., Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci U S A 2005, 102, (40), 14238-14243. 34. Shimobayashi, M.; Hall, M. N., Multiple amino acid sensing inputs to mTORC1. Cell Res 2016, 26, (1), 7-20. 35. Chauvin, C.; Koka, V.; Nouschi, A.; Mieulet, V.; Hoareau-Aveilla, C.; Dreazen, A.; Cagnard, N.; Carpentier, W.; Kiss, T.; Meyuhas, O.; Pende, M., Ribosomal protein S6 kinase activity controls the ribosome biogenesis transcriptional program. Oncogene 2014, 33, (4), 474-483.

449 450


Page 20 of 38

Page 21 of 38

451 452 453 454 455 456 457 458 459


Figure legends Fig.1. Relative expression of genes of nutrient transporters in jejunal mucosa of piglets as affected by dietary crude protein Fig.2. Relative expression of genes of nutrient transporters in response to varying medium nitrogen levels in mono-cultured IPEC-1 cells Fig.3. Relative expression of genes of nutrient transporters in IPEC-1 cells when co-cultured with GES-1 cells in response to nitrogen levels in the media Fig.4. Cell viability and morphology when grown with media containing three nitrogen levels in mono-cultured and co-cultured systems

460

(a-b) Influence of nitrogen levels on proliferation of IPEC-1 (a) and GES-1 (b); (c)

461

Morphology of IPEC-1 cultured alone with three nitrogen levels (left); Morphology of IPEC-1 co-

462

cultured with GES-1 with three nitrogen levels (Right) (Bar: 50 µm).

463 464 465 466

Fig.5. Total net removal of AA by mono-cultured IPEC-1 cells in media of different nitrogen levels Fig.6. Total net removal of AA by IPEC-1 cells co-cultured with GES-1 cells in media of different nitrogen levels

467

Fig. 7. Heatmap of Free amino acid consumption in two cultured system.

468

Fig.8. Transcript and translate abundance of genes related to protein synthesis signaling

469

pathway in IPEC-1 cells mono-cultured or co-cultured in media of three nitrogen levels

470

(a) Relative transcript and translate expression of genes in mono-cultured IPEC-1 cells; (b)

471

Relative transcript and translate expression of genes in co-cultured IPEC-1 cells; (c) Relative

472

transcript and translate expression of genes in jejunal mucosa.



473

Page 22 of 38

Table 1. Composition and nutrient levels of diets Dietary CP (%)‡ Component 14% CP

17% CP

20% CP

Corn

71.8

66.5

63.7

Soybean meal

13.4

18.8

19.8

Whey powder

4.40

4.30

4.30

Fish meal

1.50

4.00

9.00

Soybean oil

4.10

2.60

0.80

Lys

0.88

0.62

0.38

Met

0.27

0.19

0.10

Thr

0.33

0.21

0.09

Trp

0.08

0.04

0.01

Calcium hydrophosphate

1.15

0.74

0.00

Limestone

0.79

0.70

0.52

Salt

0.30

0.30

0.30

†1% premix compound

1.00

1.00

1.00

Total

100

100

100

Digestible energy (MJ/kg)

14.6

14.6

14.6

Crude protein

14.0

17.0

20.0

Total calcium

0.70

0.71

0.69

Total phosphorus

0.53

0.55

0.57

Arg

0.75

0.91

1.09

His

0.34

0.40

0.46

Ile

0.49

0.60

0.70

Leu

1.15

1.32

1.49

Lys

1.23

1.23

1.23

Met+Cys

0.68

0.68

0.68

Phe

0.59

0.69

0.80

Thr

0.73

0.73

0.73


Page 23 of 38


Trp

0.20

0.20

0.20

Val

0.53

0.65

0.77

Analyzed nutrient levels Arg

0.71

0.93

1.09

His

0.30

0.37

0.44

Ile

0.46

0.60

0.71

Leu

1.11

1.32

1.52

Lys

1.26

1.25

1.26

Met+Cys

0.63

0.65

0.62

Phe

0.56

0.70

0.81

Thr

0.76

0.75

0.76

Trp

0.20

0.20

0.20

Val

0.54

0.64

0.72

EAA/NEAA

0.90

0.80

0.70

474

†Premix provided these amounts of vitamins and minerals per kilogram on an as-fed basis: vitamin

475

A, 10 800 IU; vitamin D3, 4000 IU; vitamin E, 40 IU; vitamin K3, 4 mg; vitamin B1, 6 mg; vitamin

476

B2, 12 mg; vitamin B6, 6 mg; vitamin B12, 0.05 mg; biotin, 0.2 mg; folic acid, 2 mg; niacin, 50 mg;

477

D-calcium pantothenate, 25 mg; Fe, 100 mg

478

40 mg as manganese oxide; Zn, 100 mg as zinc oxide; I, 0.5 mg as potassium iodide; and Se, 0.3

479

mg as sodium selenite.

480

‡All values are expressed in percent (%), except for digestible energy.

as ferrous sulfate; Cu, 150 mg as copper sulfate; Mn,



482

Page 24 of 38

Table 2. Composition and nutrient levels of media Nitrogen Levels (mg/L)† Component NNL

LNL

VLNL

Glycine

18.8

15.5

12.3

L-Alanine

4.45

3.68

2.92

L-Arginine hydrochloride

148

122

96.7

L-Asparagine-H2O

7.50

6.21

4.92

L-Aspartic acid

6.65

5.50

4.36

L-Cysteine hydrochloride-H2O

17.6

14.5

11.5

L-Cystine 2HCl

31.3

25.9

20.5

L-Glutamic Acid

7.35

6.08

4.82

L-Glutamine

365

302

239

L-Histidine hydrochloride-H2O

31.5

26.1

20.6

L-Isoleucine

54.8

45.1

35.7

L-Leucine

59.1

48.9

38.7

L-Lysine hydrochloride

91.3

91.3

91.3

L-Methionine

17.2

17.2

17.2

L-Phenylalanine

35.5

29.4

23.3

L-Proline

17.3

14.3

11.3

L-Serine

26.3

21.7

17.2

L-Threonine

53.5

53.5

53.5

L-Tryptophan

9.02

9.02

9.02

L-Tyrosine disodium salt dihydrate

55.8

46.2

36.6

L-Valine

52.6

43.8

34.7

Total nitrogen of Amino acids (mg)

179

152

125

0.0035

0.0035

0.0035

8.98

8.98

8.98

Amino Acids

Non-Amino acids Biotin Choline chloride


Page 25 of 38


D-Calcium pantothenate

2.24

2.24

2.24

Folic Acid

2.65

2.65

2.65

Niacinamide

2.02

2.02

2.02

Pyridoxine hydrochloride

2.03

2.03

2.03

Riboflavin

0.219

0.219

0.219

Thiamine hydrochloride

2.17

2.17

2.17

Vitamin B12

0.68

0.68

0.68

i-Inositol

12.6

12.6

12.6

Calcium Chloride (CaCl2) (anhyd.)

117

117

117

0.0013

0.0013

0.0013

Ferric Nitrate (Fe(NO3)3"9H2O)

0.05

0.05

0.05

Ferric sulfate (FeSO4-7H2O)

0.417

0.417

0.417

Magnesium Chloride (anhydrous)

28.6

28.6

28.6

Magnesium Sulfate (MgSO4) (anhyd.)

48.8

48.8

48.8

Potassium Chloride (KCl)

312

312

312

Sodium Chloride (NaCl)

6996

6996

6996

Sodium Phosphate dibasic (Na2HPO4) anhydrous

71.0

71.0

71.0

Sodium Phosphate monobasic (NaH2PO4-H2O)

62.5

62.5

62.5

Zinc sulfate (ZnSO4-7H2O)

0.432

0.432

0.432

D-Glucose (Dextrose)

3151

3151

3151

HEPES

3575

3575

3575

Hypoxanthine Na

2.39

2.39

2.39

Linoleic Acid

0.042

0.042

0.042

Lipoic Acid

0.105

0.105

0.105

Phenol Red

8.1

8.1

8.1

Putrescine 2HCl

0.081

0.081

0.081

Sodium Pyruvate

55

55

55

0.365

0.365

0.365

Cupric sulfate (CuSO4-5H2O)

Thymidine 483



484

Page 26 of 38

Table 3. Primers used for real-time PCR analysis Size Gene

Primer sequence (5'→3')

TM Accession No.

(bp)

(°C)

Transporter genes ASTC2

F: GATGGAGGATGTGGGGATGCT

129

XM_003355984.4

57

134

XM_021093176.1

57

176

NM_001164649.1

59

174

XM_003353809.4

58

138

XM_021065165.1

60

400

XM_013978228.2

60

99

NM_214347.1

58

116

NM_214398.1

59

222

XM_021076151.1

55

273

NM_001097417.1

60

144

XM_021072101.1

58

186

XM_021091182.1

60

100

XM_021077063.1

59

R: TAGGGGTTTTTGCGAGTGAAG B0+AT

F: CTTGTCCCTGTTCCTGGTGTT R: TCTGAGCCCATCCGAACTTAT

EAAT3

F: ATCCACTCCATTGTTATTCTGC R: CTCTTGTCCACCTGGTTCTTCT

4F2hc

F: GAGGTGAGACGGCACAGAG R: CTCGAACCCACCAAGGAC

CAT1

F: CTGGTACACCATGTTCGGCT R: GCTGTCATGGCCTTCCTCTT

Y+LAT1

F: TTCTCTTACTCGGGCTGGGA R: GCGCCATGAGACCATTGAAC

PepT1

F: TTATCCCGCCAGTACCCAGA R: CAGACTTCGACCACAACGGA

APOA1

F: GATTTTGCCACCGTGTATGT R: TCCCAGTTGTCCAGGAGTTT

FATP-1c

F: GGTTGGTGCTTGTGGCTT R: ATCTTCTTGCTGGTGGCG

GLUT2

F: TTGTCACAGGCATTCTTGTTAGTCA R: TTCACTTGATGCTTCTTCCCTTTC

SGLT-1

F: TCATCATCGTCCTGGTCGTCTC R: CTTCTGGGGCTTCTTGAATGTC

ATPase

F: AGCAGTTATGTGGGGACGAAATGT R: AGAGCCAGGGAAGCGAGTGTGT

NHE3

F: ACCACCCTCATCGTCATCTTCT


Page 27 of 38


R: GCTCTCGCTGTTCACTCCTCTT Genes involved in protein synthesis mTORC1

F: AGCCCATAAGAAAACGGGGA

246

XM_003127584.6

60

158

XM_021076847

59

220

XM_021093598.1

59

146

XM_003354670.4

59

218

NM_001244225.1

59

144

XM_021067294.1

55

R: AAAGGACACCAGCCGATGTA Class I PI3K

F: TGAGTCGGGTGCTGGAACTG R: CGTCCCTAACCGATTCCGAC

Class III I3K

F: ACGGCAGGCAGATGATGAGGA R: AGGAGGGGAAGACACTGGAGG

TSC2

F: GACAAGCACCGCTGTGACAAGAAG R: TCGTAGTCCAGGGGCGTGATGA

4E-BP1

F: TCTTCAGCACCACCCCAGGA R: TGACTCTTCACCGCCCGC

S6K1

F: AATACGACAGCCGAACTCCG R: TCACACATCCCCTTCCCACC

485

Note: F: forward; R: reverse; TM: melting temperature, used as the annealing temperature

486



487

Page 28 of 38

Table 4. Free amino acid consumption1 from media by IPEC-1 cells cultured alone Amino acid (mmoles/L)

Nitrogen level in medium (NL) VLNL

LNL

NNL

P

Decreased Gly

0.026±0.011a

-0.05±0.01b

-0.006±0.002ab

0.018

Val

-0.191±0.001c

-0.082±0.001b

-0.01±0.011a

0.001

Cys

0.003±0.002a

-0.054±0.001b

0.009±0.006a

0.002

Met

-0.005±0.002a

-0.038±0.003b

-0.003±0.005a

0.009

Ile

0.023±0.007a

-0.167±0.009b

-0.036±0.035a

0.017

Leu

0.008±0.015a

-0.198±0.001b

-0.054±0.001ab

0.032

Tyr

0.022±0.002a

-0.089±0.001b

-0.013±0.011a

0.002

Phe

0.019±0.003a

-0.091±0.004b

-0.013±0.009a

0.002

Lys

-0.013±0.021a

-0.231±0.005b

-0.031±0.017a

0.004

His

0.015±0.002a

-0.042±0.002b

-0.004±0.009a

0.011

Arg

0.066±0.019a

-0.294±0.013b

-0.045±0.020a

0.002

Pro

0.053±0.011a

-0.071±0.030b

0.014±0.013ab

0.045

Ala

0.311±0.105

0.202±0.006

0.237±0.086

0.645

Cysthi

0.002±0.001

0.002±0.001

0.004±0.002

0.465

5Hyl

0.009±0.002

0.004±0.004

0.018±0.018

0.686

Orn

0.017±0.011

0.008±0.005

0.019±0.013

0.721

Increased

488

Note: values are measured concentrations in fresh media minus those in conditioned media

489


Page 29 of 38

490


Table 5. Free amino acid consumption1 from media by IPEC-1 cells co-cultured with GES-1 cells Nitrogen level in medium

Amino acid (mmoles/L)

VLNL

LNL

NNL

P

Decreased Thr

-0.075±0.011

-0.077±0.008

-0.098±0.018

0.469

Ser

-0.069±0.006a

-0.087±0.001ab

-0.110±0.011b

0.021

Gly

-0.054±0.005

-0.033±0.009

-0.029±0.007

0.112

Val

-0.1195±0.007

-0.116±0.013

-0.102±0.019

0.654

Cys

-0.032±0.002

-0.030±0.003

-0.029±0.009

0.937

Met

-0.019±0.002

-0.0195±0.001

-0.027±0.006

0.324

Ile

-0.062±0.007

-0.080±0.004

-0.107±0.018

0.080

Leu

-0.073±0.007a

-0.100±0.004ab

-0.134±0.021b

0.040

Tyr

-0.028±0.004

-0.039±0.003

-0.053±0.011

0.116

Phe

-0.035±0.004

-0.042±0.002

-0.057±0.009

0.107

Lys

-0.105±0.013

-0.110±0.009

-0.134±0.024

0.462

His

-0.021±0.005

-0.015±0.001

-0.033±0.007

0.096

Arg

-0.132±0.012

-0.152±0.009

-0.203±0.034

0.133

Pro

-0.025±0.004

-0.012±0.0003

-0.003±0.003

0.007

Glu

0.019±0.005b

0.050±0.008a

0.061±0.003a

0.007

Ala

0.092±0.004c

0.141±0.006a

0.117±0.005b

0.002

Orn

0.017±0.001

0.020±0.001

0.021±0.002

0.115

Increased

491 492 493 494



495

Page 30 of 38

Fig. 1

mRNA expression

2.0

14% CP 17% CP 20% CP

aa

1.5

a

b

a

a

a

a

1.0

b 0.5

bb b

bb

b

b

b

b

497


NH E3

se AT Pa

LT -1 SG

Pe pT 1 AP O A1 FA TP -1 c G LU T2

1

CA T1

3

4F 2h c

Y+ LA T

496

EA AT

AS TC

2 B0 +A T

0.0

Page 31 of 38

498


Fig. 2 70% Nitrogen level 85% Nitrogen level 100% Nitrogen level

mRNA expression

5

a

4 3

a

2

b

1

a

a

b b b

b

b b

b

a a a a b b

0 A

C ST

2 B

A 0+

T

A EA

T3

c 2h F 4

C

T1 A

LA Y+

T1

1 pT e P

A

PO

A

1

c -1 P T FA

499 500


G

LU

T2 SG

-1 LT A

a TP

se N

E3 H


501

Page 32 of 38

Fig. 3

mRNA expression

3

70% Nitrogen level 85% Nitrogen level 100% Nitrogen level

a aa

a

2

a

1

a

b

bb

bb

a

ab bb

bb

a b

ab b

a bb

a

bb

502 503


HE 3 N

se TP a A

LT -1 SG

Pe pT 1 A PO C 1 FA TP -1 c G LU T2

y

+L AT 1

AT 1 C

4F 2h c

T

T3 EA A

0+ A B

A

ST C

2

0

Page 33 of 38


504

Fig. 4

505

a

b

70% nitrogen level 85% nitrogen level 100% nitrogen level

1.5

1.5

508 509

Cell activity

507

Cell activity

506 1.0

0.5

1.0

a a b

a b

ab

b

a a

b

a a

0.5

0.0

0.0 0h

h 24

h 48

h 72

0h

Mono-cultured IPEC-1

c 70% NL

85% NL

100% NL


h 24

h 48

h 72

Co-cultured IPEC-1


Fig. 5

Nitrogen difference (uM)

510

511

4

a

a

2

70% nitrogen level 85% nitrogen level 100% nitrogen level

0 -2 -4

b

512


Page 34 of 38

Page 35 of 38

Fig. 6

Nitrogen difference (uM)

513


0

70% Nitrogen level 85% Nitrogen level 100% Nitrogen level

-5

-10

-15

514 515



516

Fig. 7

517 518


Page 36 of 38

Page 37 of 38

524

a

1.5 1.0

a

bb

bb

bb

0.5

a

a

a

ab

p-TSC2 p-4EB-P1

bb

p-S6K1

0.0 1 TO RC

3 Cl as s

p-mTORC1 Actin

m

PI 3K

525

p-PI3K p-Class 3 PI3K

b

S6 K1

523

2.0

4E -B P1

522

70% Nitrogen level 70%NL 85%NL 100%NL 85% Nitrogen level 100% Nitrogen level

TS C2

521

a

PI 3K

520

Fig. 8

mRNA expression

519


526 527

b 70% Nitrogen level 85% Nitrogen level 100% Nitrogen level

mRNA expression

2.0

85%NL

100%NL p-PI3K

a

1.5

a a

1.0 0.5

70%NL

a

a

a a

p-TSC2

a

bb

b

p-Class 3 PI3K

ab

b

p-4EB-P1

b

b

p-S6K1

0.0 P

K I3 C

ss la

3

P

K I3

C TS

2

P -B 4E

1 S

6K

1

R TO

C

p-mTORC1

1

Actin

m

528 529

c

mRNA expression

2.0

a

1.5

a a

a

a

a

bb

b

14% CP 17% CP 20% CP

a

a

1.0

14% CP 17% CP 20% CP

b ab

b

b

0.5

p-PI3K p-Class 3 PI3K p-TSC2 p-4EB-P1 p-S6K1

1 m

TO R

C

1 S6 K

4E -B P1

2 TS C

p-mTORC1 Actin

C

la ss

3

PI 3K

PI 3K

0.0

530



531 532

TOC graphic of this article


Page 38 of 38

Influence of nitrogen levels on nutrient transporters and regulators of

Recommend Documents