Accepted Manuscript

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Type of manuscript: Regular Article

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Article Title

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Effects of dietary carbohydrate and energy intake on LAT1 protein expression in rat

5skeletal muscle

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7

Authors

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Aki Yokogawa1*, Keigo Tanaka1*, Ikuru Miura1, 2, Shinya Watanabe1, 3, Yusuke Iwao1,

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Daisuke Takakura¹, Tongzhu Zhou1, Kohei Kido1, 2, 4, Kentaro Kawanaka1, 2**1011

Affiliations and mailing address12

1. Faculty of Sports and Health Sciences, Fukuoka University, 8-19-1 Nanakuma,

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Jonan-ku, Fukuoka 814-0180, Japan.

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2. Institute for Physical Activity, Fukuoka University, 8-19-1 Nanakuma, Jonan-ku,

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Fukuoka 814-0180, Japan.

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3. Department of Nutrition and Integrative Physiology, University of Utah, 250 1850 E,

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Salt Lake City, Utah, 84112, USA

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4. Health and Medical Research Institute, National Institute of Advanced Industrial

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Science and Technology, 2217-14 Hayashi-cho, Takamatsu, Kanagawa 761-0301,

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Japan

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*These two authors equally contributed to this work.

25**Corresponding Author

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Kentaro Kawanaka

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Faculty of Sports and Health Science,

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Fukuoka University,29

8-19-1 Nanakuma, Jonan-ku,

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Fukuoka, 814-0180, Japan.

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E-mail: kawanaka@fukuoka-u.ac.jp

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Phone no: +81-92-871-6631

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Fax no: +81-92-865-6029

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Number of tables and figures

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Tables: 2, Figures: 437

Running Title: Diet and skeletal muscle LAT1 protein expression

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42

Abstract

43

Glucose has been reported to reduce the expression of L-type amino acid transporter 1

44(LAT1) protein in C2C12 myocytes. We therefore hypothesized that increased dietary45

carbohydrate and energy intake would reduce LAT1 protein expression in rodent

46skeletal muscle. Here, we tested this hypothesis. In Experiment 1 to examine the effects

47

of dietary carbohydrate intake, male Sprague Dawley (SD) rats were divided into low-48

carbohydrate (low-CHO) and high-carbohydrate (high-CHO) diet groups. Each group49

was fed a low-CHO (20% carbohydrate) or high-CHO (70% carbohydrate) diet,50

respectively. Total energy intakes of both groups were matched by pair feeding. In

51

Experiment 2 to examine the effects of dietary energy intake, rats were divided into

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low-Energy diet (fed 68% of ad libitum energy intake) and high-Energy diet (ad53libitum) groups. After 7 days of dietary manipulation, the lower leg muscles on one side54

were percutaneously stimulated and subjected to one acute bout of resistance exercise.

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The contralateral leg muscle served as an internal control. We collected gastrocnemius

56

muscle 6 h after contraction. In both Experiments 1 and 2, when results were analyzed

57

by two-way analysis of variance, no main effect of diet on LAT1 protein concentration

58

was observed. There was also no main effect of resistance exercise, or no interaction

59

between diet and exercise. These results do not support our hypothesis that increased60

dietary carbohydrate and energy intake reduce LAT1 protein expression in rodent

61skeletal muscle. Furthermore, diet may not affect the effects of resistance exercise on

62

LAT1 protein expression.

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Keywords64

L-type amino acid transporter 1, leucine, gastrocnemius muscle, dietary carbohydrate

65

ratio, total energy intake

66

標題

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糖質ならびにエネルギー摂取量がラット骨格筋の LAT1 タンパク質発現に及ぼ

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す影響

69

著者名

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横川晶 1,田中景梧 1, 三浦征 1,2,渡邉真也 1,3, 岩尾悠介 1, 髙倉大輔 1, 周桐竹 1,

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木戸康平 1,2,4, 川中健太郎 1,2

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所属

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1. 福岡大学スポーツ健康科学部、日本、福岡

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2. 福岡大学身体活動研究所、日本、福岡

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3. ユタ大学栄養統合生理学教室、米国、ユタ州、ソルトレイクシティ

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4. 産業技術総合研究所健康医療研究部門、日本、高松

77

概要

78

グルコースは、C2C12 筋細胞における L 型アミノ酸トランスポーター1

79（LAT1）タンパク質の発現を低下させることが報告されている。そこで我々

80は、食事からの糖質とエネルギーの摂取量が増加すれば、ラット骨格筋におけ

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る LAT1 タンパク質発現が減少するという仮説を立てて検証した。実験 1 で82

は、糖質摂取が LAT1 タンパク質発現に与える影響について調べるために、

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SD 系雄性ラットを低糖質食群と高糖質食群に振り分けた。各群にはそれぞれ

84

低糖質食（糖質 20％）または高糖質食（糖質 70％）を与えた。両群の総エネ

85

ルギー摂取量はペアフィーディング法で一致させた。実験 2 においては、エネ

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ルギー摂取量の違いが LAT1 タンパク質発現に与える影響について調べるた

87

め、ラットを低エネルギー食群（自由摂取エネルギー量の 68％を給餌）と、高88

エネルギー食群（自由摂取）に分けた。7 日間の食事介入後、片側の下腿筋を89

経皮的に刺激し、1 回の急性レジスタンス運動を行った。対側の下腿筋はコン90

トロール脚として使用した。筋収縮の 6 時間後に腓腹筋を摘出した。実験 1 と91

2 の結果を二元配置の分散分析で解析したところ、LAT1 タンパク質濃度に対92する食餌の主効果ならびに、レジスタンス運動の主効果は見られず、交互作用

93も見られなかった。これらの結果は、食餌からの糖質およびエネルギーの摂取

94

量増加は、骨格筋における LAT1 タンパク質発現を減少させるという我々の仮95説を支持するものではなかった。さらに、食餌はレジスタンス運動が LAT1 タ

96

ンパク質発現量に与える影響には関与しない可能性がある。

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Introduction

101

Being the main components of lean body mass and the protein reservoir in the human102

body, skeletal muscles not only control locomotion but they are fundamental for103

breathing, eating, energy expenditure, as well as for glucose, amino acids, and lipids

104

homeostasis and for maintaining a high quality of life1). Loss of muscle mass and105function has been linked to the risk of metabolic diseases, poor quality of life, increased

106

morbidity, all-cause mortality, and frailty2,3,4). Muscle mass is regulated by the balance107

between muscle protein synthesis and protein breakdown rates. Therefore, muscle

108

hypertrophy could theoretically be caused by increased muscle protein synthesis,

109

decreased degradation, or both. However, among these factors, an increase in protein

110synthesis may be required to increase skeletal muscle mass5,6).

111

The essential amino acids, in particular leucine, stimulate muscle protein

112synthesis, which is generally dependent on their delivery to intracellular sensors and113

effector molecules associated with the mTORC1 activity 7,8,9,10,11). The influx of114

essential amino acids into skeletal muscle is mediated by amino acid transporters

115

ubiquitously expressed in the plasma membrane of many cell types. The L-type amino

116acid transporter 1 (LAT1) is the most highly expressed large neutral amino acid117

transporter in skeletal muscle 12), although others such as LAT2, LAT3, and LAT4 may

118

be present and able to transport essential amino acids 13,14). Thus, LAT1 expression

119levels in skeletal muscle may directly contribute to muscle protein synthesis via amino

120acid transport and regulate muscle mass.

121

LAT1 primarily transports leucine into the sarcoplasma while co-transporting122

glutamine out of the cell, whereas the sodium-coupled neutral amino acid transporter 2

123(SNAT2) co-transports both sodium and glutamate into the sarcoplasma 15). These two

1

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transport proteins work in tandem such that SNAT2 brings in glutamine for LAT1 to

125pump back out of the cell while concomitantly transporting leucine into the cell.

126

Amino acid transporter expression in skeletal muscle may be dynamic and127

acutely responsive to dietary amino acids intake and resistance exercise. For example,

128

amino acid ingestion stimulates an increase in LAT1 and SNAT2 gene expression, an

129event which is followed by increases in protein expression 3 h post-ingestion 16). These

130

changes have also been reported in response to resistance exercise, whereby overnight131

resistance-braked running wheel exercise increases LAT1 protein expression mouse132

skeletal muscle 17). Gene expression of LAT1 and SNAT2 has also been reported to133

increase in human muscle at 6 hours after resistance exercise, followed by increased

134

protein expression over a 24-hour recovery period 18).

135

On the other hand, it was reported that in retinal capillary endothelial cells, LAT1

136

gene expression decreases with increasing glucose concentration in the medium 19). The

137

same result was reported in C2C12 myocytes 20). Thus, glucose down-regulates LAT1

138

protein expression. However, it is unclear whether dietary carbohydrate intake affects

139

LAT1 protein expression in rodent skeletal muscle, and whether increased dietary140

carbohydrate suppresses the exercise effect on LAT1.

141

In the Experiment 1 of the present study, we examined the effect of dietary142

carbohydrate ratio on LAT1 protein expression in rat skeletal muscle under the same143

energy intake. We also examined whether the effect of resistance exercise on muscle

144

LAT1 protein expression is affected by dietary carbohydrate ratio. In addition, it might145

be possible that energy intake, rather than dietary carbohydrate intake per se, affects the146

expression of LAT1 protein. Therefore, in the Experiment 2 of this study, we also

147

examined the effect of dietary energy intake.

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148

Materials and Methods

149

Experiment 1 protocol. This experiment examined the effect of the amount of150

carbohydrate intake on LAT1 expression in skeletal muscle under conditions of equal151

energy intake. 10-week-old Sprague Dawley (SD) rats, purchased from Kyudo company

152(Saga, Japan). The rats were housed under 12h light/dark cycle at 22.0 ± 2.0℃ and 50%

153± 10% humidity. After preliminary housing for 1 week, they were randomly divided

154

into two groups: fed a low-carbohydrate diet (Protein : Fat : Carbohydrate balance = 2 :155

6 : 2) group (low-CHO) and fed a high-carbohydrate diet (Protein : Fat : Carbohydrate156

balance = 2 : 1 : 7) group (high-CHO) and fed each diet for 7 days. Carbohydrate ratios

157

for low- and high-CHO diets followed previous studies 21). The diet composition is158

shown in Table 1. The experimental period, energy intake was standardized for both159

dietary conditions by pair-feeding. Both groups were allowed to drink freely, and160

amount of food intake was measured daily.

161
162

Experiment 2 protocol. This experiment examined the effect of the amount of energy163

intake on LAT1 expression in skeletal muscle. 9-week-old male SD rat, purchased from

164

Kyudo company. The rats were housed under the same conditions as in Experiment 1.

165

After preliminary housing for 1 week, they were randomly divided into two groups: a166

low-Energy group and high-Energy group and fed a high-fat diet (Protein : Fat :167

Carbohydrate balance = 2 : 6 : 2) for 7 days. The diet composition is shown in Table 1.

168

The low-Energy group was pair-fed with rats fed a high-carbohydrate diet (Protein :169

Fat : Carbohydrate balance = 2 : 1 : 7) at the same week of age to standardize energy

170

intake, while the high-Energy group was fed ad libitum. Both groups were allowed to171

drink freely, and amount of food intake was measured daily. When rats are fed ad

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172

libitum on a high-fat diet, their energy intake is well known to exceed their energy

173

requirements. Our approach therefore resulted in overfeeding in the high-Energy group,

174

whereas the low-Energy groups rats consumed an amount equivalent to their energy

175

requirements. It allowed us to examine the effects of overfeeding, even though both

176

group rats ate the same type of chow. The study was approved by the Animal Care and177

Use Committee of Fukuoka University and was conducted (No. 2113104).

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179

Resistance exercise. In both Experiment 1 and 2, after dietary intervention, all rats

180

underwent muscle contraction using transcutaneous electrical stimulation under181

anesthesia (4%–5% sevoflurane). Electrical stimulation was applied to the right leg

182(exercise leg), and the left leg was used as a control (non-exercise leg). Electrical183

stimulation was set at a frequency of 100Hz and a voltage of 30V, and muscle

184

contractions of 3 seconds were repeated 10 times. This was considered as one set, and a185

total of 5 sets with a 3-minute interval in between. This acute resistance exercise

186

protocol has been reported to increase the rate of muscle protein synthesis 22). It has also

187

been reported that continuous application of this protocol induces muscle hypertrophy

188

22)

. After resistance exercise, rats were monitored until they awoke from anesthesia.

189
190

Sampling. After 6 h of resistance exercise, the rats were weighed, and their191

gastrocnemius muscles were collected under anesthesia (4%–5% sevoflurane). Blood

192

samples were also collected from aorta. EDTA-treated blood samples were centrifuged193

at 1000g for 10 min to collect plasma. All the samples were stored at -80℃ until used

194

for analysis.

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Measurement of plasma glucose level.

197

Plasma glucose levels was measured using a glucose CⅡ-test (Fujifilm, Tokyo, Japan).

198

The concentration of glucose in the plasma was measured according to the standard

199

protocol of the kit. Absorbances were measured using a GloMax Discover System

200(Promega Corporation, WI, USA).

201
202

Western blotting. Frozen gastrocnemius samples were homogenized in RIPA buffer

203(Fujifilm, Tokyo) with protease inhibitor (Roche, Basel, Switzerland) and phosphatase204

inhibitor (Roche), and the total protein concentration of each was measured using a

205

BCA protein assay kit (Thermo Fisher Scientific, MA, USA). Protein lysate was mixed

206

with Laemmli sample loading buffer (BioRad, CA, USA) and heated at 90℃ for 10

207

minutes. The aliquots containing equal amounts of protein were separated by SDS-

208

PAGE and the proteins were transferred to PVDF membranes. The membranes were

209

blocked using 3% skim milk in TBS-Tween 20 for 60 minutes at room temperature and210

then incubated with LAT1 antibody (SantaCruz #sc-374232, TX, USA) for overnight at

211

4℃. The membrane was then incubated with the secondary antibody (Vector

212

Laboratories, CA, USA) for 90 min at room temperature. The LAT1 protein was

213

visualized using Immnobilo Forte Western HRP (Millipore, MA, USA). An Amersham

214

imager 600 (GE Healthcare Life Science, MA, USA) was used for imaging, and the215

images were quantified by image Lab software (BioRad). Membranes were stained with

216

Ponceau-S (Sigma-Aldrich, MO, USA) to confirm equal amounts of protein loading.

217
218

Statistical analysis. Data are expressed as mean ± standard error (SEM). GraphPad

219

prism 10 (GraphPad, CA, USA) was used for the statistical analysis. Two-way ANOVA

5

220

was used for comparisons between the four groups, and an unpaired t-test was used for221

comparisons between the two groups. P < 0.05 was considered to represent statistical222

significance.

223
224

Results

225

Experiment 1. The amount of carbohydrate intake does not affect LAT1 expression in226

skeletal muscle under conditions of the same energy intake. The total energy, protein,227

fat, carbohydrate intake, body weight, and plasma glucose are shown in Table 2. There

228

were no changes in total energy intake and body weight of the high-CHO group and229

low-CHO group during the 7-day dietary intervention period, confirming that pair-230

feeding was performed appropriately. As we assumed, the high-CHO group consumed

231

approximately 3.5 times as much carbohydrate as the low-CHO group. There was no232

change in plasma glucose between low-CHO and high-CHO groups.

233

The protein expression level of LAT 1 in gastrocnemius are shown in Figure234

1. No main effect of diet on the LAT1 protein expression was observed in

235

gastrocnemius. Equally, there was no main effect of resistance exercise, nor were their236

interaction observed. These results suggest that dietary carbohydrate ratio does not237

affect LAT1 protein expression under conditions of the same energy intake.

238

The change in LAT1 protein expression level due to resistance exercise was239

calculated for each animal by subtracting the value of the contralateral control leg

240

muscle from the value of the exercised leg muscle (Figure 2). Resistance exercise

241

increased LAT1 protein expression by 51% and 22% in the low- and high-CHO groups,242

respectively. Although this value was not statistically significant, it tended to be higher

243

in the low-CHO group compared to the high-CHO group (p = 0.12, effect size=0.91).

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244
245

Experiment 2. The amount of energy intake does not affect LAT1 expression in skeletal

246

muscle under conditions of the same type of chow. The total energy, protein, fat,247

carbohydrate intake, body weight was shown in Table 3. As we assumed, total energy

248

intake were increased in the high-Energy group compared to the low-Energy group249

during the 7-day dietary intervention period. The high-Energy group consumed an

250

average of approximately 27 kcal/day more energy than the low-Energy group.

251

The protein expression level of LAT 1 in gastrocnemius are shown in Figure252

3. No main effect of diet on the LAT1 protein expression was observed in

253

gastrocnemius. Equally, there was no main effect of resistance exercise, nor were their254

interaction observed. These results suggest that the amount of energy intake does not

255

affect LAT1 expression level in skeletal muscle.

256

As, in the Experiment 1, the change in LAT1 protein expression level due to257

resistance exercise was calculated (Figure 4). Resistance exercise increased LAT1

258

protein expression by 12% and 43% in the low- and high-Energy groups, respectively.

259

However, no significant differences were observed in the change in LAT1 protein

260

expression due to resistance exercise between the low- and high-Energy group (p=0.35,

261

effect size=0.62).

262
263
264

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266

Discussion
In C2C12 myocytes, high concentration of glucose in the culture medium was267

reported to reduce the LAT1 mRNA expression 20) . Findings from this previous study

268

suggest the possibility that increased muscle glucose uptake decreases LAT1 gene269

expression. In the experiment 1 of our present study, plasma glucose levels were not270

significantly different between low-CHO and high-CHO group rats. Even though high271

carbohydrate intake may cause a temporary increase in blood glucose levels, increased272

insulin secretion from the pancreas increases muscle glucose uptake and blood glucose

273

levels falls to normal levels. Thus, blood glucose level may remain constant regardless274

of dietary carbohydrate intake. Therefore, the absence of differences in plasma glucose

275

level does not necessarily mean that muscle glucose uptake is the same between groups.

276

In other words, glucose uptake may be higher in the muscles of high-CHO rats than in277

the muscles of low-CHO rats. In support of this idea, higher muscle glycogen levels

278

were observed in the high-CHO group rats than in the low-CHO group rats in another279

experiment conducted under the same dietary conditions as the present study

280(Kawanaka et al. unpublished data). However, in the experiment 1 of our present study,281

no effect of dietary carbohydrate ratio on LAT1 protein expression was observed,282

suggesting that increased glucose uptake may not reduce LAT1 protein expression in283

skeletal muscle. Furthermore, in the experiment 1, LAT1 protein expression tended to284

be higher in contralateral control leg of high-CHO rats than in low-CHO rats. Since

285

there is a possibility of type 2 error, we cannot completely rule out the possibility that286

increased dietary carbohydrate intake increases muscle LAT1 expression.

287

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288

In the experiment 1 of our present study, the two groups, i.e., the high-CHO

289

and low-CHO diet groups, were compared by pair feeding to ensure the same energy

290

intake. Taking this into account, previous study showing that high glucose in the culture291

medium reduce LAT1 mRNA expression in myocytes 20) suggest another possibility

292that the addition of energy, rather than increased glucose uptake, reduces LAT1 gene293

expression. However, this possibility is also unlikely. This is because, in the experiment294

2 of our present study, LAT1 protein expression in the high-Energy group was not295

different from the low-Energy group, despite higher energy intake.

296

The reason for the discrepancy between the previous study and the present

297

study is unclear. However, the effects of in vivo dietary manipulation on animal skeletal

298

muscle may be very different from the effects of media manipulation on C2C12 cells. In

299

C2C12 myocytes, pharmacological AMPK activation by AICAR increases LAT1

300mRNA levels 20). Thus, AMPK may up-regulate LAT1 gene expression. On the other301

hand, high concentrations of glucose in the medium decrease AMPK activity in C2C12302

cells 20). Thus, glucose may down-regulate LAT1 gene expression via inactivation of

303

AMPK. Although glucose concentration in the culture medium affected AMPK activity

304

in C2C12 cells, dietary carbohydrate and energy intake may not have affected AMPK

305

activity in rat skeletal muscle. And this may be the reason why we could not observe306

changes in LAT1 protein expression. AMPK activity was not measured in this study.

307

Therefore, further studies measuring AMPK activity are required to examine this308

possibility.

309

In the previous study, LAT1 mRNA expression was highest in C2C12310

myocytes cultured in 0 mM glucose medium, and low concentrations of glucose (1.4311mM) reduced LAT1 mRNA expression to its lowest level. Further increases in glucose

9

312

concentration had no further effect. Thus, the effect of carbohydrate on LAT1 gene313

expression might be maximal at very small amounts of glucose. Therefore, we cannot

314

rule out the possibility that even very small amounts of carbohydrate intake may reduce315

the LAT1 protein expression in skeletal muscle compared to no carbohydrate intake.

316

The energy percentage of carbohydrate in our low-CHO diet is 20%, which is higher

317

than the 5% energy percentage of carbohydrate in very low carbohydrate diet such as318

ketogenic diet. We cannot rule out the possibility that our low-CHO diet reduced the319

expression of LAT1 protein compared to the carbohydrate-free or ketogenic diet. This is320

a limitation of our study and requires further investigation.

321

In the Experiment 2, the high-Energy group rats were fed ad libitum on a322

high-fat diet (Protein : Fat : Carbohydrate balance =2 : 6 : 2). When rats were fed high-323

fat-diet, their energy intake is well known to exceed their energy requirements. Our

324

approach therefore resulted in overfeeding in the high-Energy group. On the other hand,325

energy intake of the low-Energy group rats was matched by pair feeding to rats fed a

326

high-carbohydrate diet (Protein : Fat : Carbohydrate balance = 2 : 1 : 7), even though

327

they were on a high-fat diet. Thus, the energy intake of the low-Energy group rats was

328

adjusted to their energy requirement level. Muscle LAT1 protein expression was not329

different between low- and high-Energy group rats, suggesting that overfeeding does330

not have any effect on muscle LAT1 protein expression. However, the effects of331

decreased energy intake below energy requirement, i.e., caloric restriction, were332

unclear. This requires further investigation.

333

In the present study, we applied an in situ electrical stimulation-induced

334

resistance exercise model in which current was applied to the leg muscles on one side to335

induce involuntary muscle contraction. Because the contralateral leg muscle was used as

10

336

a control, the increase in LAT1 protein due to exercise could be calculated for each337

animal by subtracting the value of the contralateral control leg muscle from the value of338

the exercised leg muscle. And although this value was not statistically significant, it

339

tended to be higher in the low-CHO group than in the high-CHO group. In other words,

340

the higher the dietary carbohydrate ratio, the lower the exercise effect on LAT1 protein

341

expression may be. Acute resistance exercise has been reported to increase LAT1

342

protein expression in skeletal muscle 17, 18). The exercise-induced increase in LAT1

343

expression presumably increases leucine entry into muscle cells and promotes muscle

344

protein synthesis. This may be one mechanism by which exercise induces muscle

345

hypertrophy. If a high-CHO diet exacerbates exercise-induced increase in LAT1

346

protein, then a diet with a high carbohydrate ratio might adversely affect the effects of347

exercise in promoting muscle protein synthesis and muscle mass. However, this idea

348

warrants caution in the interpretation. This is because when the results of Experiment 1

349

were analyzed by two-way analysis of variance, there was no main effect of resistance

350

exercise or no interaction between dietary carbohydrate ratio and exercise. Future351

studies will be needed to determine the effect of dietary carbohydrate ratio on exercise352

effect on LAT1 protein expression.

353

In conclusion, during the 7-day dietary manipulation period, changing the354

dietary carbohydrate ratio under the same energy intake did not significantly affect the355

expression of LAT1 protein in the rat gastrocnemius muscle. In addition, changing

356

dietary energy intake did not affect muscle LAT1 protein expression. These results do357

not support our hypothesis that increased dietary carbohydrate or energy intake

358

decreases LAT1 protein expression in skeletal muscle.

359

11

360

Acknowledgements

361This research was funded by a JSPS Grant-in-Aid Scientific Research Grant (22H03490

362

to Kentaro Kawanaka). We are grateful to the Center for Experimental Animals at

363

Fukuoka University for their support. We also thank the staff and graduate students of364

the Institute for Physical Activity at Fukuoka University for their support and dedication365

in conducting this study.

366
367
368

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449

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Authors’ contribution statement

451

A. Yokogawa, K. Tanaka, I. Miura, S. Watanabe, K. Kido, and K. Kawanaka conceived452

and designed the study; A. Yokogawa, K. Tanaka, I. Miura, Y. Iwao, S. Watanabe, D.

453

Takakura, K. Kido, and K. Kawanaka performed the experiments; A. Yokogawa, T.

454

Tanaka, I. Miura, and K. Kawanaka analyzed the data; all authors interpreted the455

results; T. Tanaka and I. Miura prepared the figures; I. Miura and K. Kawanaka drafted456

the manuscript; all authors approved the final version of the manuscript.

457
458

Conflict of interest

459

The author(s) declare that there are no conflicts of interest.

460
461
462

17

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464

Table 1. The composition of food used in this study
High-Carbohydrate

Low-Carbohydrate(high-fat diet)

gm%

kcal%

gm%

kcal%

Protein

19.2

20

26

20

Carbohydrate

67.3

70

26

20

Fat

4.3

10

35

60

Total
kcal/gm

100
3.85

100
5.24

gm

kcal

gm

kcal

Casein, 30 Mesh

200

800

200

800

L-Cystine

3

12

3

12

Corn Starch

506.2

2024.80

0

Maltodextrin 10

125

500

125

500

Sucrose

68.8

275.2

68.8

275

Cellulose, BW200

50

0

50

0

Soybean Oil

25

225

25

225

Lard

20

180

245

2205

10

0

10

0

13

0

13

0

5.5

0

5.5

0

16.5

0

16.5

0

10

40

10

40

Choline Bitartrate

2

0

2

0

FD&C Yellow Dye
#5

0.04

0

FD&C Blue Dye #1

0.01

0

0.05

0

1055.054057

773.854057

Mineral Mix S
10026
DiCalcium
Phosphate
Calcium Carbonate
Potassium Citrate,
1 H2O
Vitamin Mix
V10001

Total

465
18

Table 2. The effect of dietary carbohydrate ratio on body weight, total energy intake,total protein intake, total fat intake, total carbohydrate intake
high-CHO
low-CHO
Body weight (g)416.3 ± 4.8
409.7 ± 4.3
Total energy intake (kcal/week)
530.0 ± 13.3
530.0 ± 13.3
Total protein intake (kcal/week)
106.0 ± 2.7
106.0 ± 2.7
Total fat intake (kcal/week)
53.0 ± 1.3
318.0 ± 8.0 *Total carbohydrate intake (kcal/week)
371.0 ± 9.3
106.0 ± 2.7 *Plasma glucose (mg/dl)
125.1 ± 6.8
124.9 ± 8.8

466
467

Values are means ± SEM (n = 9/group). *p＜0.05 vs. high-CHO
Table 3. The effect of energy surplus intake on body weight, total energy intake,total protein intake, total fat intake, total carbohydrate intake
low-Energy
high-Energy
Body weight (g)394.5 ± 6.7
425.3 ± 6.8 *Total energy intake (kcal/week)
607.1 ± 10.1
799.5 ± 16.1 *Total protein intake (kcal/week)
121.4 ± 2.0
159.9 ± 3.2 *Total fat intake (kcal/week)
364.3 ± 6.0
479.7 ± 9.7 *Total carbohydrate intake (kcal/week)
121.4 ± 2.0
159.9 ± 3.2 *468

Values are means ± SEM (n = 6/group). *p＜0.05 vs. low-Energy

469
470

19

471

Figure472

Experiment 1.

473
474
475
476

477
478

Figure 1

479
480

20

481
482
483
484
485

486
487

Figure 2

488
489

21

490

Experiment 2

491
492

493
494

Figure 3

495
496

22

497
498
499
500
501
502

503
504

Figure 4

505
506

23

507

Figure Legends

508

Figure 1. Effect of carbohydrate intake on LAT1 protein expression.

509

Con, control (non-exercise leg); Ex, exercise (exercise leg); AU, arbitrary units. Values

510

are expressed as mean ± SEM (n = 7-9/group). Two-way ANOVA was used for

511

statistical processing.

512
513

Figure 2. Effect of carbohydrate intake on the rate of change in LAT1 protein

514

expression by resistance exercise.

515

AU, arbitrary units. Values are expressed as mean ± SEM (n = 7-9/group).

516
517

Figure 3. Effect of different energy intake on LAT1 protein expression.

518

Con, control (non-exercise leg); Ex, exercise (exercise leg); low-Ene, low-Energy; high-519

Ene, high-Energy; AU, arbitrary units. Values are expressed as mean ± SEM (n =520

6/group). Two-way ANOVA was used for statistical processing.

521
522

Figure 4. Effect of different energy intake on the rate of change in LAT1 protein

523

expression by resistance exercise.

524

AU, arbitrary units. Values are expressed as mean ± SEM (n = 6/group).

52524