# L-Theanine Administration Modulates the Absorption of Dietary Nutrients and Expression of Transporters and Receptors in the Intestinal Mucosa of Rats

Authors: Qiongxian Yan; Haiou Tong; Shaoxun Tang; Zhiliang Tan; Xuefeng Han; Chuanshe Zhou
Journal: BioMed Research International
 (2017)

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## Abstract

L-theanine has various advantageous functions for human health; whether or not it could mediate the nutrients absorption is unknown yet. The effects of L-theanine on intestinal nutrients absorption were investigated using rats ingesting L-theanine solution (0, 50, 200, and 400 mg/kg body weight) per day for two weeks. The decline of insulin secretion and glucose concentration in the serum was observed by L-theanine. Urea and high-density lipoprotein were also reduced by 50 mg/kg L-theanine. Jejunal and ileac basic amino acids transportersSLC7a1 andSLC7a9, neutralSLC1a5 andSLC16a10, and acidicSLC1a1 expression were upregulated. The expression of intestinalSGLT3 andGLUT5 responsible for carbohydrates uptake andGPR120 andFABP2 associated with fatty acids transport were inhibited. These results indicated that L-theanine could inhibit the glucose uptake by downregulating the related gene expression in the small intestine of rats. Intestinal gene expression of transporters responding to amino acids absorption was stimulated by L-theanine administration.


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## Body

## 1. Introduction

L-theanine, as a non-protein-forming amino acid (AA), contributes to the umami taste and unique flavor of green tea. Its content in tea leaves is closely related to the quality and price of green tea [1, 2]. L-theanine is beneficial for remedying various nutritional and metabolic diseases in human, including providing antiobesity effects [3, 4], suppressing the body weight increases and fat accumulation [3, 5], and exerting antidiabetic effects [6, 7]. L-theanine is transported through the intestinal brush border membrane mainly via neutral AA systems B, A, ASC, N, and L, based on findings that L-theanine inhibited the absorption of glutamine and large neutral amino acids (AAs, leucine, and tryptophan) into organs [8–10]. Our knowledge data and previous findings also confirmed that most neutral AAs (threonine, valine, methionine, isoleucine, serine, alanine, tyrosine, and leucine) and certain basic AA (lysine) in the serum of L-theanine-administered rats were decreased [8, 11]. These researches indicated that L-theanine could competitively suppress the absorption of AAs.However, AAs absorption is dependent on the activities of AA transporters located in the brush border membrane of small intestine. Neutral AA transporters, solute carrier family 1, member 5(SLC1a5) and family 16, member 10(SLC16a10), are responsible for threonine, serine, alanine, cysteine, glutamine and phenylalanine, tyrosine, and tryptophan transporting, respectively. Basic AA transporters, solute carrier family 7, member 1(SLC7a1) and member 9(SLC7a9), are in charge of transporting arginine, lysine, histidine, alanine, serine, cysteine, threonine, asparagine, and glutamine. Acidic AA transporters solute carrier family 1, member 1(SLC1a1) and member 2(SLC1a2) transport glutamate and aspartate. It is reported that L-theanine competitively inhibited the uptake of glutamate substrate through solute carrier family 1, member 3(SLC1a3) andSLC1a2expressed in cancer cells [12, 13]. However, the expression pattern of glutamate transporter subtypes in tumor cells is different from normal cells. Therefore, it is necessary to investigate the efficacy of L-theanine on glutamate transporters in normal tissues. Whether or not the expression of different AA transport systems is mediated by L-theanine is unknown yet.Furthermore, it is reported that the fatty accumulation in mice was suppressed by the administration of green tea powder [4] and theanine was responsible for this suppressive effect [3]. Although serum glucose in rats was not changed, the insulin was reduced by oral theanine [14]. These literatures indicate that metabolism of lipid and insulin is regulated by L-theanine. In the enterocytes of rats, there are many transporters and receptors responses to sugar and fatty acids transport, including sodium dependent glucose transporters(SGLTs), glucose transporters, G-protein-coupled receptors, and fatty acid binding protein 2(FABP2) [15–21]. Whether these transporters and receptors involved in the regulation of L-theanine administration on absorption of glucose and lipid is unclear. Based on these questions, we measured the nutrient content in the blood and mRNA expression of related transporters and receptors in small intestine of rats after the intragastric administration of L-theanine for two weeks, aiming at figuring out the preliminary L-theanine-induced regulation mechanism in nutrients absorption in rats.


## 2. Material and Methods

### 2.1. Experimental Design

This experiment was conducted according to the animal care guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha city, Hunan province, China (number KYNEAAM-2013-0009). Sixty-four Sprague Dawley (SD) rats which are 3 weeks old weighing 74–92.2 g were used as experimental animals. The management of SD rats and L-theanine administration experiment was the same as Li et al. [22]. The animals were individually housed in plastic cages under laboratory conditions (25 ± 3°C, 70 ± 5% relative humidity, good ventilation, and a 12-h light-dark cycle) and had free access to food and pure water. After three days of adaptation, SD rats were randomly divided into four treatment groups. Each group contained eight male rats and eight female rats. During fasting (15:00–17:00 h), rats in the treatments received gastric intubation of four different doses of L-theanine (0, 50, 200, and 400 mg/kg body weight/day), respectively. L-theanine was freshly dissolved in 0.9% NaCl solution in advance before intubation every day. 1 mL of the L-theanine solution was daily administered to each rat for two weeks.


### 2.2. Blood and Tissue Samples Collection

At the end of the experiment, SD rats were fasted overnight and anesthetized by ether for 4 min, and then blood was collected from the jugular vein into tubes without anticoagulant. The blood samples were centrifuged at 3500 rpm for 15 min at 4°C, and then serum samples were collected and stored at −80°C until assay. The whole jejunum and ileum segments were collected and rinsed with ice-cold saline (0.9% NaCl wt/vol). Then the mucosae were carefully removed, quickly frozen in liquid nitrogen, and stored at −80°C prior to subsequent analyses.


### 2.3. Analysis of Serum

The glucose, total cholesterol, triglyceride (TG), urea, low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were determined by automatic biochemistry analyzer (Synchron Clinical System CX4 PRO, Beckman Coulter, USA) according to the instructions. Insulin was assayed by the ELISA kit purchased from Huamei Biotechnology Co., Ltd. (Wuhan, Hubei, China). Non-esterified fatty acids (NEFA) were measured by kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).


### 2.4. Real-Time Quantitative PCR

Total RNA was isolated from the mucosa of jejunum and ileum using the Trizol Reagent (Invitrogen, USA), andcDNA was synthesized using the Revert Aid First StrandcDNA synthesis kit (Applied Biosystems, Thermo Fisher Scientific, USA). For relative quantification of gene expression, the ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster, CA) was used. Primers were designed using the Primer 3 plus program, and sequences are listed in Table 1. The reaction system contained 5 μL SYBR® Premix Ex Taq™ (2x), 0.4 μL PCR forward primer (10 μM), 0.4 μL PCR reverse primer (10 μM), 0.2 μL ROX reference dye (50x), 1.0 μLcDNA, and 3 μL sterilized ddH2O. The thermal profile for all reactions was 30 s at 95°C, then 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. Each reaction was completed with a melting curve analysis to ensure the specificity of the reaction. All the samples were analyzed in duplicate, and the relative amount of each specific transcript was obtained after normalization against the endogenous control β-actin. The relative amounts of target genes were quantified according to the 2-ΔΔCT method [23].Table 1
Sequences of primers used for real-time quantitative PCR.
Gene
GenBank accession
Primer
Length (bp)
SLC7a1
NM_013111.3
Forward-CCTTCATCACTGGCTGGAACReverse-GGTTTGCCTATCAGCTCGTC
100
SLC1a5
NM_175758.3
Forward-GGAGAAATGGACTGGGTGTGReverse-CCAGCAAGAAGGCTCTGAAT
107
SLC1a2
NM_001035233.1
Forward-GGCAGTCATCTCCCTGTTGAReverse-AGACATTCATCCCGTCCTTG
101
SLC1a1
NM_013032.3
Forward-GGAGTCTTGGTTCGAGGACAReverse-GTGGCAGAATGACGAGCTTC
106
SLC16a10
NM_138831.1
Forward-TCACTGGTCATTCTGGGACAReverse-CCTAACAGCAAAGGGAGCAA
107
SLC7a9
NM_053929.1
Forward-ACCAAGTCAGGGGGTGAGTAReverse-AGATGATGGCGAAGGATGAG
115
SGLT1
NM_013033
Forward-GCCATCATCCTCTTCGCTATReverse-CGCTCTTCTGTGCTGTTACG
122
SGLT3
NM_001106383
Forward-GATGCTGGTGCTGAAACTGAReverse-CGCTGTTGAAGATGGAGGTC
101
GLUT2
NM_012879
Forward-GATTGCTCCAACCACACTCAReverse-CCTGATTGCCCAGAATGAAG
113
GLUT5
NM_031741
Forward-GGGCTCTTGGTCACACACAReverse-CGTCTTGTCTCTCGGCAACT
108
FATP
NM_053580.2
Forward-GGAAGGTTGCTGTGGTGTTCReverse-ATGGGAGCCAGAAGGGTAGA
120
GPR43
NM_001005877
Forward-AGGCTGTGGTGTTCAGTTCCReverse-GGGATTGCGGAGTAGTAGCA
113
GPR120
NM_001047088.1
Forward-AGACCACCGTTCTGGGACTReverse-GAAGAGGTTGAGCACCAAGC
119
FABP2
NM_013068.1
Forward-GAGGCCAAGCGGATCTTTAReverse-TGCATTATCAGCGAGATGGA
109
β-actin
NM_031144.3
Forward-TGTCACCAACTGGGACGATAReverse-GGGGTGTTGAAGGTCTCAAA
165


### 2.5. Statistical Analysis

Statistical analyses were conducted by one-way analysis of variance (ANOVA) using the Mixed Proc of SAS (version 8.2, SAS Institute, Cary, NC, USA). The main effect tested was the dose of L-theanine. When indicated by ANOVA, means were separated using least significant differences. Significance was declared atP < 0.05.


## 2.1. Experimental Design

This experiment was conducted according to the animal care guidelines of the Animal Care Committee, Institute of Subtropical Agriculture, the Chinese Academy of Sciences, Changsha city, Hunan province, China (number KYNEAAM-2013-0009). Sixty-four Sprague Dawley (SD) rats which are 3 weeks old weighing 74–92.2 g were used as experimental animals. The management of SD rats and L-theanine administration experiment was the same as Li et al. [22]. The animals were individually housed in plastic cages under laboratory conditions (25 ± 3°C, 70 ± 5% relative humidity, good ventilation, and a 12-h light-dark cycle) and had free access to food and pure water. After three days of adaptation, SD rats were randomly divided into four treatment groups. Each group contained eight male rats and eight female rats. During fasting (15:00–17:00 h), rats in the treatments received gastric intubation of four different doses of L-theanine (0, 50, 200, and 400 mg/kg body weight/day), respectively. L-theanine was freshly dissolved in 0.9% NaCl solution in advance before intubation every day. 1 mL of the L-theanine solution was daily administered to each rat for two weeks.


## 2.2. Blood and Tissue Samples Collection

At the end of the experiment, SD rats were fasted overnight and anesthetized by ether for 4 min, and then blood was collected from the jugular vein into tubes without anticoagulant. The blood samples were centrifuged at 3500 rpm for 15 min at 4°C, and then serum samples were collected and stored at −80°C until assay. The whole jejunum and ileum segments were collected and rinsed with ice-cold saline (0.9% NaCl wt/vol). Then the mucosae were carefully removed, quickly frozen in liquid nitrogen, and stored at −80°C prior to subsequent analyses.


## 2.3. Analysis of Serum

The glucose, total cholesterol, triglyceride (TG), urea, low-density lipoprotein cholesterol (LDL), and high-density lipoprotein cholesterol (HDL) were determined by automatic biochemistry analyzer (Synchron Clinical System CX4 PRO, Beckman Coulter, USA) according to the instructions. Insulin was assayed by the ELISA kit purchased from Huamei Biotechnology Co., Ltd. (Wuhan, Hubei, China). Non-esterified fatty acids (NEFA) were measured by kit produced by Nanjing Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China).


## 2.4. Real-Time Quantitative PCR

Total RNA was isolated from the mucosa of jejunum and ileum using the Trizol Reagent (Invitrogen, USA), andcDNA was synthesized using the Revert Aid First StrandcDNA synthesis kit (Applied Biosystems, Thermo Fisher Scientific, USA). For relative quantification of gene expression, the ABI Prism 7900 HT Fast Real-Time PCR System (Applied Biosystems, Foster, CA) was used. Primers were designed using the Primer 3 plus program, and sequences are listed in Table 1. The reaction system contained 5 μL SYBR® Premix Ex Taq™ (2x), 0.4 μL PCR forward primer (10 μM), 0.4 μL PCR reverse primer (10 μM), 0.2 μL ROX reference dye (50x), 1.0 μLcDNA, and 3 μL sterilized ddH2O. The thermal profile for all reactions was 30 s at 95°C, then 40 cycles of denaturation at 95°C for 5 s, and annealing at 60°C for 30 s. Each reaction was completed with a melting curve analysis to ensure the specificity of the reaction. All the samples were analyzed in duplicate, and the relative amount of each specific transcript was obtained after normalization against the endogenous control β-actin. The relative amounts of target genes were quantified according to the 2-ΔΔCT method [23].Table 1
Sequences of primers used for real-time quantitative PCR.
Gene
GenBank accession
Primer
Length (bp)
SLC7a1
NM_013111.3
Forward-CCTTCATCACTGGCTGGAACReverse-GGTTTGCCTATCAGCTCGTC
100
SLC1a5
NM_175758.3
Forward-GGAGAAATGGACTGGGTGTGReverse-CCAGCAAGAAGGCTCTGAAT
107
SLC1a2
NM_001035233.1
Forward-GGCAGTCATCTCCCTGTTGAReverse-AGACATTCATCCCGTCCTTG
101
SLC1a1
NM_013032.3
Forward-GGAGTCTTGGTTCGAGGACAReverse-GTGGCAGAATGACGAGCTTC
106
SLC16a10
NM_138831.1
Forward-TCACTGGTCATTCTGGGACAReverse-CCTAACAGCAAAGGGAGCAA
107
SLC7a9
NM_053929.1
Forward-ACCAAGTCAGGGGGTGAGTAReverse-AGATGATGGCGAAGGATGAG
115
SGLT1
NM_013033
Forward-GCCATCATCCTCTTCGCTATReverse-CGCTCTTCTGTGCTGTTACG
122
SGLT3
NM_001106383
Forward-GATGCTGGTGCTGAAACTGAReverse-CGCTGTTGAAGATGGAGGTC
101
GLUT2
NM_012879
Forward-GATTGCTCCAACCACACTCAReverse-CCTGATTGCCCAGAATGAAG
113
GLUT5
NM_031741
Forward-GGGCTCTTGGTCACACACAReverse-CGTCTTGTCTCTCGGCAACT
108
FATP
NM_053580.2
Forward-GGAAGGTTGCTGTGGTGTTCReverse-ATGGGAGCCAGAAGGGTAGA
120
GPR43
NM_001005877
Forward-AGGCTGTGGTGTTCAGTTCCReverse-GGGATTGCGGAGTAGTAGCA
113
GPR120
NM_001047088.1
Forward-AGACCACCGTTCTGGGACTReverse-GAAGAGGTTGAGCACCAAGC
119
FABP2
NM_013068.1
Forward-GAGGCCAAGCGGATCTTTAReverse-TGCATTATCAGCGAGATGGA
109
β-actin
NM_031144.3
Forward-TGTCACCAACTGGGACGATAReverse-GGGGTGTTGAAGGTCTCAAA
165


## 2.5. Statistical Analysis

Statistical analyses were conducted by one-way analysis of variance (ANOVA) using the Mixed Proc of SAS (version 8.2, SAS Institute, Cary, NC, USA). The main effect tested was the dose of L-theanine. When indicated by ANOVA, means were separated using least significant differences. Significance was declared atP < 0.05.


## 3. Results

As shown in Table2, glucose concentration was decreased by 400 mg/kg L-theanine administration compared to the control group (0 mg/kg L-theanine administration) (P< 0.05). Insulin concentration was linearly decreased by L-theanine administration (P< 0.001). There were no differences (P> 0.05) in the serum cholesterol, TG, NEFA, and LDL concentrations among the L-theanine treatments. Concentrations of urea and HDL were decreased by 50 mg/kg L-theanine treatment compared to the control group (P< 0.05).Table 2
Effects of L-theanine administration on average daily gain and biochemical parameters in serum of rats.
Item
Treatments (mg/kg BW⋅d)
P value
0
50
200
400
Linear
Quadratic
Average daily gain, g/d
5.24 ± 0.17b
6.01 ± 0.17a
6.15 ± 0.17a
5.98 ± 0.17a
0.038
<0.01
Glucose, mM
5.65 ± 0.29a
5.31 ± 0.29ab
5.74 ± 0.29a
4.77 ± 0.30b
NS
NS
Insulin, uIU/mL
43.2 ± 2.18a
41.7 ± 2.18a
26.3 ± 2.26b
19.0 ± 3.08c
<0.001
NS
Cholesterol, mM
2.23 ± 0.09
2.08 ± 0.09
2.13 ± 0.09
2.05 ± 0.09
NS
NS
Triglyceride, mM
1.23 ± 0.07
1.21 ± 0.07
1.02 ± 0.07
1.16 ± 0.07
NS
NS
NEFA, mM
1.27 ± 0.13
1.35 ± 0.10
1.45 ± 0.11
1.31 ± 0.10
NS
NS
Urea, mM
5.42 ± 0.16a
4.85 ± 0.16b
5.70 ± 0.16a
5.38 ± 0.17a
NS
NS
LDL, mM
0.339 ± 0.02
0.341 ± 0.02
0.354 ± 0.02
0.347 ± 0.02
NS
NS
HDL, mM
1.74 ± 0.06a
1.52 ± 0.06b
1.60 ± 0.06ab
1.60 ± 0.06ab
NS
NS
BW: body weight, NEFA: non-esterified fatty acids, LDL: low-density lipoprotein, HDL: high-density lipoprotein, and NS: not significant.a–cMeans within a row not bearing a common superscript letter differ (P<0.05). Data were reported as mean ± SE. Data of average daily gain were cited by Tong et al. (2016).Transcript levels of intestinal AA transporters in the intestine of rats are shown in Table3. Expression of acidic AA transporterSLC1a1 was upregulated in the jejunum and ileum (Quadratic,P< 0.001), while jejunalSLC1a2 transcript was linearly decreased (P< 0.001) with the increasing doses of L-theanine but increased by L-theanine treatments in the ileum (Quadratic,P< 0.05). Expression of neutral AA transporterSLC1a5 was increased by doses of L-theanine (jejunum, Linear,P< 0.001; ileum, Linear and Quadratic,P< 0.001); therein the maximal values both occurred in the 400 mg/kg L-theanine treatment. Another neutral AA transporterSLC16a10expression in the jejunum and ileum was upregulated by doses of L-theanine (Quadratic,P< 0.001). Basic AA transportersSLC7a1 (jejunum, Linear,P< 0.01; ileum, Linear and Quadratic,P< 0.001) andSLC7a9expression (jejunum, Quadratic,P< 0.001; ileum, Linear and Quadratic,P< 0.001) was increased with the increasing doses of L-theanine.Table 3
Effects of L-theanine administration on the transcript levels of AA transporters in rat intestine.
Item
Treatments (mg/kg BW⋅d)
P value
0
50
200
400
Linear
Quadratic
Jejunum
SLC1a1
1.00b
1.71 ± 0.43b
7.70 ± 0.50a
1.12 ± 0.55b
NS
<0.001
SLC1a2
1.00a
0.34 ± 0.06c
0.63 ± 0.07b
0.23 ± 0.07c
<0.001
NS
SLC1a5
1.00b
1.77 ± 0.43b
1.61 ± 0.38b
3.49 ± 0.41a
<0.001
NS
SLC16a10
1.00c
7.17 ± 0.84a
5.25 ± 0.79ab
3.83 ± 0.68b
NS
<0.001
SLC7a1
1.00c
8.40 ± 0.70a
3.93 ± 0.90b
8.88 ± 1.28a
0.0017
NS
SLC7a9
1.00c
6.40 ± 0.60b
10.9 ± 0.66a
1.70 ± 0.60c
NS
<0.001
Ileum
SLC1a1
1.00b
4.89 ± 0.35a
5.11 ± 0.39a
1.25 ± 0.39b
NS
<0.001
SLC1a2
1.00
1.47 ± 0.29
1.86 ± 0.24
1.02 ± 0.44
NS
0.01
SLC1a5
1.00b
1.45 ± 0.35b
0.94 ± 0.37b
4.45 ± 0.45a
<0.001
<0.001
SLC16a10
1.00c
8.27 ± 0.27b
13.1 ± 0.63a
1.52 ± 0.77c
NS
<0.001
SLC7a1
1.00b
1.71 ± 0.34b
1.18 ± 0.36b
4.62 ± 0.45a
<0.001
<0.001
SLC7a9
1.00b
1.93 ± 0.42b
7.21 ± 0.52a
2.09 ± 0.42b
<0.001
<0.001
BW: body weight,SLC1a1: solute carrier family 1, member 1, SLC1a2: solute carrier family 1, member 2, SLC1a5: solute carrier family 1, member 5, SLC16a10: solute carrier family 16, member 10, SLC7a1: solute carrier family 7, member 1, SLC7a9: solute carrier family 7, member 9, and NS: not significant. a–cMeans within a row not bearing a common superscript letter differ (P<0.05). Data were reported as mean ± SE.Gene expressions of glucose transporters and receptors in the intestine of rats are shown in Table4. Transcript level ofSGLT1 in the jejunum was stimulated (P< 0.05) by 400 mg/kg L-theanine compared to the 200 mg/kg L-theanine treatment, while in the ileum it was downregulated (Quadratic,P< 0.001); therein a minimum value appeared at the 200 mg/kg L-theanine group.SGLT3expression in the jejunum and ileum was decreased by L-theanine treatment (jejunum, Linear,P< 0.001; ileum, Linear and Quadratic,P< 0.001). Comparing with the 50 mg/kg L-theanine treatment, jejunalGLUT2 expression was suppressed (P< 0.05) by 200 mg/kg L-theanine. IleacGLUT2 expression was upregulated (P< 0.01) by 50 mg/kg L-theanine and then inhibited (P< 0.05) by high doses of L-theanine treatments compared to 50 mg/kg L-theanine. JejunalGLUT5 expression was inhibited (P< 0.05) by high doses of L-theanine treatment compared to the control group, while its expression in the ileum was linearly decreased (P< 0.01) by increasing doses of L-theanine.Table 4
Effects of L-theanine administration on the transcript levels of glucose transporters and receptors in rat intestine.
Item
Treatments (mg/kg BW⋅d)
P value
0
50
200
400
Linear
Quadratic
Jejunum
SGLT1
1.00ab
1.14 ± 0.20ab
0.80 ± 0.21b
1.65 ± 0.21a
NS
NS
SGLT3
1.00ab
1.06 ± 0.15a
0.53 ± 0.16b
0.03 ± 0.15c
<0.001
NS
GLUT2
1.00ab
1.44 ± 0.2a
0.77 ± 0.20b
1.12 ± 0.17ab
NS
NS
GLUT5
1.00a
0.16 ± 0.08c
0.72 ± 0.10b
0.68 ± 0.10b
NS
NS
Ileum
SGLT1
1.00a
0.60 ± 0.12b
0.17 ± 0.11c
1.10 ± 0.09a
NS
<0.001
SGLT3
1.00a
0.61 ± 0.07b
0.26 ± 0.06c
0.01 ± 0.005d
<0.001
<0.001
GLUT2
1.00c
3.07 ± 0.30a
1.31 ± 0.33bc
1.99 ± 0.30b
NS
NS
GLUT5
1.00a
0.58 ± 0.10b
0.53 ± 0.11b
0.46 ± 0.11b
0.003
NS
BW: body weight,SGLT1: sodium dependent glucose transporter 1, SGLT3: sodium dependent glucose transporter 3, GLUT2: glucose transporter protein, member 2, GLUT5: glucose transporter protein, member 5, and NS: not significant. a–dMeans within a row not bearing a common superscript letter differ (P < 0.05). Data were reported as mean ± SE.The mRNA abundance of the fatty acid transporters and receptors in the intestine of rats is shown in Table5. JejunalFATP expression was decreased by L-theanine treatments (Quadratic,P< 0.01), while its expression levels in the treatments of 50 and 400 mg/kg L-theanine were lower (P< 0.01) than that of control group and 200 mg/kg L-theanine treatment. IleacFATP expression was not affected by L-theanine treatments (P> 0.05). JejunalGPR43 expression was unchanged by L-theanine treatments (P> 0.05). However, its expression in the ileum of 50 mg/kg L-theanine treatment was decreased (P< 0.05) compared with control group and 200 mg/kg L-theanine treatment.GPR120 (jejunum, Linear and Quadratic,P< 0.001; ileum, Linear,P< 0.001) andFABP2(jejunum, Linear,P< 0.001; ileum, Linear and Quadratic,P< 0.001) expression levels were both suppressed by L-theanine treatments.Table 5
Effects of L-theanine administration on the transcript levels of fatty acid transporters and receptors in rat intestine.
Item
Treatments (mg/kg BW·d)
P value
0
50
200
400
Linear
Quadratic
Jejunum
FATP
1.00ab
0.27 ± 0.03b
1.86 ± 0.07a
0.40 ± 0.03b
NS
0.007
GPR43
1.00
0.79 ± 0.49
1.23 ± 0.57
0.86 ± 0.61
NS
NS
GPR120
1.00a
0.25 ± 0.03b
0.18 ± 0.03b
0.06 ± 0.03c
<0.001
<0.001
FABP2
1.00a
0.52 ± 0.08b
0.51 ± 0.08b
0.09 ± 0.08c
<0.001
NS
Ileum
FATP
1.00
1.52 ± 0.19
1.00 ± 0.19
1.76 ± 0.39
NS
NS
GPR43
1.00a
0.56 ± 0.13b
0.72 ± 0.14ab
1.02 ± 0.14a
NS
0.07
GPR120
1.00a
0.36 ± 0.11bc
0.64 ± 0.11b
0.21 ± 0.12c
<0.001
NS
FABP2
1.00a
0.38 ± 0.05b
0.16 ± 0.05c
0.08 ± 0.05c
<0.001
<0.001
BW: body weight, FATP: fatty acid transport protein, GPR43: G-protein-coupled receptor 43, GPR120: G-protein-coupled receptor 120, FABP2: fatty acid binding protein 2, and NS: not significant.a–cMeans within a row not bearing a common superscript letter differ (P < 0.05). Data were reported as mean ± SE.


## 4. Discussion

To the best of our knowledge, this experiment is a new attempt to investigate the link between serum nutrients and the expression of nutrient-associated transporters and receptors in the small intestine of L-theanine-administered rats. In this study, the declines of glucose, insulin, and urea in the serum were observed by L-theanine administration, indicating that L-theanine could inhibit the absorption of glucose, nitrogen, and secretion of insulin. Our results are partly in line with the data of Yamada et al. (2008) which observed reduced insulin level with unchanged glucose concentration in the serum of rats administrated by 4 g/kg oral L-theanine. These results are inconsistent with the findings of Zheng et al. (2004) which discovered that TG and NEFA levels in the serum of mice were decreased by 0.03% L-theanine administration. This discrepancy appears to be due to the dosage of L-theanine ingested, method of administration, and experimental period.The upregulating effects of L-theanine are reflected in the AA transporters expression at the mRNA level in small intestine in this study, exceptSLC1a2. This finding can partly explain the increased AAs concentrations in rat serum after L-theanine ingestion [11], including acidic acid (aspartic acid and glutamic acid), neutral acid (glutamine), and basic acid (histidine) (see Supplemental Table 1 in [11]; see Supplementary Material available online at https://doi.org/10.1155/2017/9747256), indicating that L-theanine promotes the AAs absorption in rat small intestine. The opposing effect of L-theanine on jejunalSLC1a2 expression was observed, reflecting that asparagine absorption in the jejunum might be blocked by L-theanine. Although direct evidences about the regulatory mechanism of AA transporters transcription by L-theanine are lacking, previous literatures showed that activating transcription factor 4 (ATF4) could transcriptionally upregulateSLC7a1 [24] and regulatory factor X proteins(RFXs) induced mRNA ofSLC1a1 [25]. After MatInspector online analysis [26], we find that there areATF4 binding sites in the promoter regions ofSLC7a1 (between nucleotides +10 and +18) andSLC7a9 (between nucleotides −155 and −146) genes andRXFs located inSLC1a1(between nucleotides −239 and −86) promoter sequence. Additionally, elements for E-box binding factors (EBOX) and cAMP-responsive element binding proteins(CREB) binding are identified in the promoter sequences ofSLC1a5,SLC7a1, andSLC7a9genes. Therefore, we speculated that L-theanine, as an amino acid, changedSLC1a1,SLC1a5,SLC7a1, andSLC7a9mRNA transcription via acting withATF4,RXF,EBOX, andCREB proteins.Glucose transporting from the intestinal lumen to the blood mainly depends on Na+-glucose cotransporterSGLT1, which absorbs glucose and galactose and the passive glucose transporterGLUT2, which acts as a glucose sensor [27–30].SGLT3 is also a glucose sensor in cholinergic neurons neighboring enterocytes and induces membrane currents upon Na+-glucose binding [27].GLUT5 is primarily in charge of fructose absorption into the cytosol. Although decreases inSGLT1andGLUT2 mRNA abundance in the intestine of rats receiving 200 mg/kg L-theanine, in which glucose absorption was declined, were not observed in this study, we found that intestinalSGLT3 and ileacGLUT5 transcripts in L-theanine-ingested rats were decreased in a dose-dependent manner. These results indicated that rats intestinalGLUT2 was less impressible thanGLUT5 to L-theanine administration at the transcriptional level, andSGLT3 andGLUT5genes rather thanSGLT1andGLUT2play a role in intestinal glucose absorption of L-theanine-ingested rats. It is reported that period circadian clock 1(PER1) exerted an indirect suppressive effect on ratSGLT1 promoter [31] and hepatic nuclear factors(HNF) regulatedSGLT1 andGLUT2 promoter activities [32, 33]. By analyzing [26], we also identifiedHNF-element located inSGLT3and peroxisome proliferator-activated receptor(PPARG) element encompassed inGLUT5 promoter regions. Therefore, we predicted that L-theanine may target transcription factors (PER1,HNF, andPPARG) and further inhibit the expression of glucose transporters mRNA.It is reported thatGPR43 binds short-chain fatty acids, whereasGPR120 responds to medium and long chain fatty acids [34, 35].FABP2 also displays high-affinity binding for long chain fatty acids and is believed to be involved with uptake and trafficking of lipids in the intestine [21]. In the present study,GPR120 andFABP2 transcripts in jejunum and ileum were decreased by L-theanine. JejunalFATP mRNA was also suppressed by 50 mg/kg and 400 mg/kg L-theanine. However, triglyceride and cholesterol contents in the serum of L-theanine-treated rats were not affected (Table 2). These results state that the intestinal uptake of dietary fatty acids might have been inhibited by L-theanine. Further research is needed to explore the regulatory mechanism of L-theanine on intestinal uptake of dietary lipids.In summary, L-theanine administration had decreased serum glucose probably by inhibiting intestinalSGLT3andGLUT5 mRNA expression in rats. Dietary fatty acids uptake might be suppressed by downregulatingGPR120 andFABP2 transcripts in the intestine of rats. Meanwhile, intestinal transporters responding to AAs absorption were upregulated by L-theanine administration. Our data provide theoretical basis for further investigation of L-theanine and nutrients interaction.



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*Source: 9747256-2017-07-24.xml*