Î²-cryptoxanthin induces UCP-1 expression via RAR pathway

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Bioactive Constituents, Metabolites, and Functions

#-cryptoxanthin induces UCP-1 expression via RAR pathway in adipose tissue Hideyuki Hara, Haruya Takahashi, Shinsuke Mohri, Hiroki Murakami, Satoko Kawarasaki, Mari Iwase, Minoru Sugiura, Nobuyuki Takahashi, Tsuyoshi Goto, and Teruo Kawada J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b01930 • Publication Date (Web): 02 Sep 2019 Downloaded from pubs.acs.org on September 2, 2019

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Journal of Agricultural and Food Chemistry

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β-cryptoxanthin induces UCP-1 expression via RAR pathway in adipose tissue

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Hideyuki Hara†, Haruya Takahashi†, Shinsuke Mohri†, Hiroki Murakami†, Satoko

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Kawarasaki†, Mari Iwase†, Nobuyuki Takahashi†,‡, Minoru Sugiura#,

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Tsuyoshi Goto†,‡, *, and Teruo Kawada†,‡

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†Laboratory

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School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan

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‡Research

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# Department

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and Fisheries, Shimizu, Shizuoka 424-0292, Japan

of Molecular Function of Food, Division of Food Science and Biotechnology, Graduate

Unit for Physiological Chemistry, Kyoto University, Kyoto 606-8501, Japan of Citriculture, National Institute of Fruit Tree Sciences, Ministry of Agriculture, Forestry

10 11

*Denotes corresponding author: Tsuyoshi Goto, Ph.D., Laboratory of Molecular Function of Food, Division of Food Science and Biotechnology,

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Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan, FAX: +81-774-38-3753, E-mail: [email protected]

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This work was supported in part by Japan Society for the Promotion of Science (JSPS) Grants-in-Aid for

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Scientific Research (Grant Number 16K14927, 16K07734, 16H02551, and 18K14420).

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Key word: -cryptoxanthin, carotenoid, adipocytes, thermogenesis, RAR, UCP1

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1 ACS Paragon Plus Environment


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Abbreviations: atRA, all-trans retinoic acid; BAT, brown adipose tissue; CA, β-carotene; CX,

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-cryptoxanthin; HFD, high fat diet; IBMX, 3-isobutyl-1-methylxanthin; IWAT, inguinal white

21

adipose tissue; NEFA, non-esterified fatty acid; PVDF, polyvinylidene difluoride; RAR, retinoic

22

acid receptor; RARE, retinoic acid response element; TG, triacylglycerol; UCP, uncoupling

23

protein; WAT, white adipose tissue.


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ABSTRACT

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While -cryptoxanthin is hypothesized to have a preventive effect on lifestyle-related

27

diseases, its underlying mechanisms are unknown. We investigated the effect of

28

-cryptoxanthin on energy metabolism in adipose tissues and its underlying mechanism.

29

C57BL/6J mice were fed a high fat diet (HFD) (60% kcal fat) containing 0% or 0.05%

30

-cryptoxanthin for 12 weeks. -cryptoxanthin treatment was found to reduce body fat

31

gain and plasma glucose level, while increasing energy expenditure. The expression of

32

uncoupling protein (UCP) 1 was elevated in adipose tissues in the treatment group.

33

Furthermore, the in vivo assays showed that the Ucp1 mRNA expression was higher in

34

the -cryptoxanthin treatment group, an effect that disappeared upon co-treatment with

35

a retinoic acid receptor (RAR) antagonist. In conclusion, we report that -cryptoxanthin

36

reduces body fat and body weight gain, and that -cryptoxanthin increases the

37

expression of UCP1 via the RAR pathway.

38



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INTRODUCTION

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In obesity, adipocytes increase the secretion of some malignant cytokines, leading to

42

insulin resistance, and a decrease in adiponectin production; this enhances insulin

43

sensitivity

44

exercise. Increase in the incidence of obesity is a serious problem in developed

45

countries. Preventing obesity and obesity-related diseases is, therefore, an important

46

focus area of ongoing research. Recently, the improvement of obesity has focused on

47

specific food ingredients.

1-3.

Obesity is mainly caused by an excessive caloric intake and a lack of

48

-cryptoxanthin (chemical structure shown in Fig. 1A) is a kind of xanthophyll,

49

found in mandarin oranges and is also one of the main carotenoids present in human

50

serum

51

amount of mandarin orange intake, and people living in areas where the mandarin

52

orange is popular tend to have high -cryptoxanthin serum concentration

53

Interestingly, some epidemiologic studies have found that there is an inverse correlation

54

between serum concentration of -cryptoxanthin and the advance of some

55

obesity-induced lifestyle-related diseases

56

that of other carotenoids, including -carotene, -carotene, and zeaxanthin

57

-cryptoxanthin seems to have a role in preventing lifestyle-related diseases. However,

4-5.

Previous studies have reported that its serum concentration reflects the

9-10.

6-8.

This inverse correlation is stronger than 11-12.

Thus,


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58 59


the underlying mechanism of this action is not yet clear. Previous studied reported that -cryptoxanthin was stored in the tissues after 13.

60

absorption into the intestine, one of which was adipose tissue

61

types of fat-storage tissues, white and brown adipose tissue (WAT and BAT,

62

respectively). WAT deposits excess energy as triglycerides. On the other hand, brown

63

adipocytes dissipate energy through thermogenesis

64

temperature by producing heat, mainly via shivering. Humans and other small

65

mammals can increase their energy expenditure and produce heat in BAT of the

66

interscapular region with the help of uncoupling protein (UCP) 1. UCP1 is a protein

67

that is expressed in the inner membrane of the mitochondria and contributes to

68

thermogenesis in BAT

69

UCP1-expressing adipocytes (termed ‘brown-like adipocytes’) appear in WAT and

70

contribute to thermogenesis in the same way as BAT 19-20. Recent studies have reported

71

that there is a negative correlation between body mass index and the amount of active

72

BAT in humans

73

UCP1 in WAT are expected to prevent obesity and type-2 diabetes.

21.

16-18.

14-15.

Mammals have two

We maintain our body

After cold exposure or -adrenergic agonist treatment,

Therefore, strategies that could increase the expression levels of

74

All-trans retinoic acid (atRA), which is a metabolites of -cryptoxanthin is a kind of

75

vitamin A that affects metabolism in various embryonic and adult tissues and plays a



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22-23.

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role in processes such as cell differentiation, proliferation, and apoptosis

77

actions of atRA are mediated mainly by the retinoic acid receptor (RAR), which is a

78

member of the ligand-activated nuclear hormone receptor superfamily

79

studies found that the Ucp1 gene in mice and humans has RAR-responsive elements in

80

its enhancer region 26-27. Treatment of cultured rodent adipocytes with atRA upregulated

81

UCP1 expression and enhanced oxidative metabolism

82

reported that atRA treatment in mice upregulates UCP1 expression in both WAT and

83

BAT

84

rodent adipocytes

85

UCP1 expression in the same way as atRA.

86 87

30-31.

28-29.

22-25.

These

Previous

Furthermore, it has been

Other studies found that -cryptoxanthin has the ability to bind RAR in 32,

which led us to hypothesize that -cryptoxanthin upregulates

In this report, we investigated the effect of -cryptoxanthin on UCP1 expression using both animal and cell experiments.

88 89

MATERIALS AND METHODS

90

Chemicals and animals. In order to determine the effect of -cryptoxanthin on the

91

energy metabolism, a feeding test was performed. -cryptoxanthin and -carotene were

92

purchased from Wako (Osaka, Japan). Six-week-old C57BL/6J male mice were

93

purchased from LSG Corporation (Tokyo, Japan). All mice were housed in separated


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cages at 23 ± 1 °C, under 12 h light/dark cycle. High fat diet (HFD), comprising 60%

95

fat (kcal%) was purchased from Research Diet (MO, USA). The mice were divided

96

into two groups (n = 8), differing only in the type of diet. After a week of

97

acclimatization period, treatment mice were provided with HFD containing 0.05%

98

-cryptoxanthin for 12 weeks. The control group was pair-fed on HFD according to the

99

0.05% -cryptoxanthin group, for equating the energy intake. The animal care

100

procedures and methods were approved by the Animal Care Committee of Kyoto

101

University (approval code: 27-62). To perform the GTT assay (8 weeks), mice were

102

fasted for 5 h before obtaining a blood sample from the tail vein (0 min point).

103

Subsequently, mice were orally administered a glucose solution (2 mg glucose /g body

104

weight), then blood samples were collected at different time points. To measure the

105

effect of -cryptoxanthin intake on blood parameters, the mice were fasted for 5 h

106

before blood collection. The plasma concentrations of glucose, triacylglycerol (TG),

107

insulin, leptin, c-peptide, and Non-esterified Fatty Acid (NEFA) were determined

108

using Wako Glucose C-test kit, Wako Triacylglycerol E-test kit (Wako Pure

109

Chemicals, Osaka, Japan), Morinaga Ultrasensitive Mouse Insulin Assay Kit,

110

Morinaga Ultrasensitive Mouse Leptin Assay Kit, Morinaga Ultrasensitive Mouse

111

C-peptide Assay Kit (Morinaga Institute of Biological Science, Yokohama, Japan),



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and LabAssay™ NEFA kit (Wako Pure Chemicals), respectively. All the kits were

113

used according to manufacturer’s instructions. To measure the effect of

114

-cryptoxanthin intake on energy expenditure increase, the oxygen consumption was

115

measured using an indirect calorimetric system (Oxymax Equal Flow 8

116

Chamber/Small Subject System; Columbus Instruments, Columbus, OH, USA). Mice

117

were allowed to acclimatize in the individual metabolic cages for 2 hours prior to the

118

experiment. The oxygen consumption data for each metabolic cage were collected

119

every 9 min, with room air as a reference, and measured for 20 hours. The locomotor

120

activity was measured using an Actimo-S system (Bio Research Centre, Nagoya,

121

Japan).

122 123

RNA preparation and quantification of gene expression. To measure the effect of

124

-cryptoxanthin intake on mRNA expression related to lipid metabolism in adipose

125

tissues, total RNA was extracted from inguinal WAT and interscapular BAT using

126

Sepasol-RNA I Super reagent (Nacalai Tesque), in accordance with the

127

manufacturer’s protocol. Total RNA was reverse-transcribed using M-MLV reverse

128

transcriptase (Promega Corporation, Fitchburg, WI, USA). To quantify the mRNA

129

expression, real-time PCR was performed using a Light cycler system (Roche


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Diagnostics, Mannheim, Germany) and SYBR Green (TOYOBO CO., LTD., Osaka,

131

Japan). The oligonucleotide primer sets of genes were designed as follows: mouse Ucp1

132

(Fwd:

133

5′-AGCCGGCTGAGATCTTGTTT-3′),

134

5′-CTGTTAGGCCTCAACACCGAAC-3′;

135

5′-CTGTCATGGCTAGGCGGTACAT-3′),

136

5′-CCCTGCCATTGTTAAGACC-3′; Rev: 5′-TGCTGCTGTTCCTGTTTTC-3′), mouse

137

Cidea

138

5′-TACTACCCGGTGTCCATTTCT-3′),

139

5′-CCAAATCTCCACGGTCTGTT-3′;

140

mouse

141

5′-GCATTGATCCAGGAATTTCCA-3′), and mouse 36B4 as an internal control (Fwd:

142

5′-TCCTTCTTCCAGGCTTTGGG-3′; Rev: 5′-GACACCCTCCAGAAAGCGAG-3′).

143

All data indicating mRNA expression levels are presented as a ratio relative to a control

144

in each experiment.

5′-CAAAGTCCGCCTTCAGATCC-3′;

(Fwd:

Rar

mouse

Rev: Cpt1b

Rev: mouse

Pgc1a

5′-ATCACAACTGGCCTGGTTACG-3′;

(Fwd:

mouse Rev:

(Fwd:

Cycs

(Fwd:

Rev: (Fwd:

5′-GTCTGCCCTTTCTCCCTTCT-3′),

5′-ACAGATCTCCGCAGCATCAG-3′;

Rev:

145 146

Protein extraction for western blotting. To measure the effect of -cryptoxanthin

147

intake on protein expression related to lipid metabolism in adipose tissues, we 9 ACS Paragon Plus Environment


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extracted proteins from adipose tissue and quantified amount of expression by

149

western blotting. Inguinal WAT mitochondria were prepared as described by Cannon

150

and Lindberg

151

sucrose solution containing protease inhibitors. The homogenates were centrifuged at

152

8,500 × g for 10 min at 4 °C. After removing the fat layer and supernatant, the pellets

153

were resuspended in the sucrose solution and centrifuged at 800 × g for 10 min to

154

remove the nuclei and cell debris. The supernatants were then centrifuged at

155

8,500 × g for 10 min. The final pellets were resuspended in a small volume of the

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sucrose solution. In the case of interscapular BAT, tissues were minced and

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homogenized in lysis buffer (25 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton

158

X-100, 1mM EDTA, 0.5% deoxycholate, and 0.1% SDS) containing protease inhibitors.

159

The homogenates were centrifuged at 15,000 × g for 30 min at 4 °C, and the

160

supernatant was used for the assay.

33.

In brief, the tissues were minced and homogenized in 300 mM

161 162

Western blotting. The protein concentration was measured using a protein assay kit

163

(Bio-Rad laboratories, Hercules, CA, USA). Protein samples were diluted with

164

Laemmli SDS-PAGE sample buffer and boiled for 5 min in the presence of

165

2-mercaptoethanol. The samples were separated using 12.5% SDS-PAGE and


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166

transferred to the same polyvinylidene difluoride (PVDF) membranes (GE Healthcare,

167

Little Chalfont, UK). The membranes were blocked with PBS (containing 5%

168

skimmed milk and 0.1% tween-20) for 1 h. After blocking, the membranes were

169

incubated in a rabbit anti-UCP1 antibody (Sigma) overnight at 4 °C. The membranes

170

were treated with HRP-conjugated anti-rabbit IgG (Santa Cruz Biotechnology, San

171

Antonio, TX, USA) for 2 h at room temperature. Protein bands were visualized by

172

ELC chemiluminescence detection system (Millipore, Bedford, MA, USA).

173 174

Histological analysis. To observe the effect of -cryptoxanthin intake on the shape

175

and size of adipose tissues, we performed a histological analysis. The interscapular

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BAT and inguinal WAT were fixed in 4% paraformaldehyde and stored at 4 °C until

177

use. The fixed samples were embedded in paraffin. Each sample were cut into 7-μm

178

slices and stained with hematoxylin/eosin.

179 180

Extraction of urine catecholamines and catecholamine assays. To clarify the

181

effect of -cryptoxanthin intake on sympathetic activation, we measured the levels of

182

catecholamines in the urine. Urine samples were collected for 48 h in metabolic cages.

183

The urine was collected in 1 mL of 6 M HCl solution for collection and storage. The



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34.

184

samples were purified via activated alumina, as previously described

185

Catecholamines in the urine extract samples were assayed by high-performance

186

liquid chromatography (HPLC) with electrochemical detection. The detector

187

potential was set at 700 mV, maintained across a glassy carbon working electrode.

188

Methanol-buffer (10:90, v/v), comprising 50 mM potassium phosphate buffer (pH

189

3.5), 10 μM EDTA·2Na, and 100 mg/L sodium 1-octanesulphonate, was used as the

190

mobile phase at a flow rate of 1 mL/min.

191 192

Luciferase assay. To measure the effect of -cryptoxanthin on retinoic acid response

193

element and UCP1 promoter activity in adipose cells, we used a luciferase assay

194

system. IWAT immortalized cells were kindly provided by Dr. Shingo Kajimura from

195

University of California. IWAT immortalized cells were transfected with pGL3 retinoic

196

acid response element (RARE)-luc or UCP1-pro-tk luc (a reporter plasmid) and pGL

197

4.74 (an internal control for normalizing the transfection efficiency) using

198

Lipofectamine (Invitrogen Corp.), according to manufacturer's instruction. Luciferase

199

activity was assayed using the dual luciferase system (Promega, MO, U.S.A), according

200

to the manufacturer's protocol.

201


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IWAT immortalized cells and C3H10T1/2 cell culture. To measure the effect of

203

-cryptoxanthin on UCP1 related gene expression, we used cultured adipose cells.

204

Undifferentiated IWAT immortalized cells were cultured in a growth medium

205

comprising DMEM, supplemented with 10% (v/v) fetal bovine serum, 100 units/mL

206

penicillin, and 100 µg/mL streptomycin, at 37 °C under 5% CO2. At 95% confluence,

207

the cells were incubated in a differentiation medium containing 2 μg/mL

208

dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine (IBMX), 5 μg/mL insulin, 1 nM

209

T3, and 0.125 μM indomethacin for 2 days. On the 2nd day, the medium was replaced

210

with a fresh growth medium containing 5 μg/mL insulin and 1 nM T3. -cryptoxanthin

211

and -carotene were dissolved in THF containing 0.1% tween 20 and added to growth

212

media, along with LE540, which was dissolved in DMSO. C3H10T1/2 cells were

213

cultured, maintained, and differentiated using a previously described method 35. Briefly,

214

undifferentiated C3H10T1/2 cells were cultured in a growth medium comprising

215

DMEM, supplemented with 10% (v/v) fetal bovine serum, 100 units/mL penicillin, and

216

100 µg/mL streptomycin, at 37 °C under 5% CO2. Two days after reaching confluence,

217

the cells were cultured in a differentiation medium containing 0.25 μM dexamethasone,

218

1 µM troglitazone, 10 μg/mL insulin, and 0.5 mM IBMX and incubated for 48 h. On the

219

2nd day, the medium was replaced with a fresh regular growth medium containing



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insulin (5 μg/mL).

221 222

Analysis of atRA metabolism using HPLC and LC-MS. In the HPLC analysis,

223

C3H10T1/2 cells (day 8) were treated with 10 µM atRA or β-cryptoxanthin for 8 h. Cell

224

associated atRA was collected and homogenized using the extract buffer (2M

225

hydroxylamine: methanol = 1: 2). After the addition of 5M KOH and hexane, 4N NaOH,

226

saturated NaCl solution and hexane were added to aqueous layer and fractionated. The

227

organic layer was centrifuged (13,200 rpm, 20 min). The organic buffer was removed

228

using a rotary evaporator. The residue was dissolved in HPLC buffer B (acetonitrile:

229

methanol = 75: 25, including 5% THF and 0.035% acetic acid). HPLC analysis was

230

performed using a Hitachi HPLC system (Hitachi High-Technologies, Tokyo, Japan).

231

An aliquot of the extracted sample was injected into an 5C18-AR- II column (6.0 × 150

232

mm; Nacalai Tesque, Kyoto, Japan), with a column temperature of 45°C.

233

buffer A (water including 5% THF and 0.05% acetic acid) and B were used. The buffer

234

gradient consisted of 30 to 75% B for 0 to 3 min and 75.0% to 100% B for 3 to 8 min, at

235

a flow rate of 1900 µL/min. The PDA detector was set at 356 nm. The LC-MS analysis

236

and sample extracts were prepared as described in a previous study, with some

237

modifications

36.

HPLC

Briefly, LC-MS was performed using a HPLC system coupled to an


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LTQ Orbitrap XL-MS system (Thermo Fisher Scientific, CA, USA). These data were

239

acquired using X-Calibur (Thermo Fisher Scientific) and Compound Discoverer 2.1

240

(Thermo Fisher Scientific).

241 242

Statistical analysis. The data are presented as mean ± standard error of mean.

243

Statistical differences between the groups were assessed using one-way analysis of

244

variance (ANOVA). Differences between groups were compared using the Student’s

245

t-test (for two groups) and ANOVA, followed by Tukey’s or Tukey-Kramer method.

246

Values of p < 0.05 were considered statistically significant.

247 248

RESULTS

249

Effect of -cryptoxanthin on body weight and energy expenditure. To evaluate the

250

effect of -cryptoxanthin on the metabolism of high fat diet (HFD)-fed mice, C57BL/6J

251

mice were fed HFD with or without 0.05% -cryptoxanthin for 12 weeks. As seen in

252

Table.1, -cryptoxanthin feeding did not affect energy intake. Although -cryptoxanthin

253

did not affect the plasma TG and NEFA level (Table.1), the body weight gain and

254

plasma glucose level were significantly reduced in the treatment group (Fig. 2A, B). On

255

the other hand, β-cryptoxanthin had no effect on the GTT data (Fig. 2C, D). In



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particular, -cryptoxanthin intake significantly reduced inguinal and epididymal WAT

257

and interscapular BAT weight gain after 12 weeks (Table.1).

258

We next investigated the effect of -cryptoxanthin on energy metabolism. We

259

measured the mice oxygen consumption and rectal temperature after 8 and 10 weeks of

260

feeding, respectively. -cryptoxanthin feeding significantly increased the oxygen

261

consumption from 2100 to 2300 hours (Fig. 2E, F). Locomotor activity was not affected

262

by -cryptoxanthin feeding (Fig. 2G). Furthermore, -cryptoxanthin feeding

263

significantly increased the rectal temperature (Fig. 2H).

264 265

Effect of -cryptoxanthin on thermogenesis-associated protein expression. To

266

elucidate the mechanism of energy expenditure elevation by -cryptoxanthin feeding,

267

we assessed the thermogenesis-associated protein expression levels in adipose tissue.

268

Interestingly, -cryptoxanthin feeding increased the UCP1 expression levels in inguinal

269

WAT (Fig. 3A). The mRNA expression of Ucp1 also increased in inguinal WAT upon

270

-cryptoxanthin treatment (Fig. 3C). On the other hand, the UCP1 expression level and

271

Ucp1 mRNA expression in interscapular BAT were not increased (Fig. 3B, D).

272

Histological analysis of adipose tissues showed that -cryptoxanthin reduced the size

273

of adipocytes in inguinal WAT (Fig. 4A). On the other hand, -cryptoxanthin did not 16 ACS Paragon Plus Environment

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reduce the size of adipocytes in interscapular BAT (Fig. 4B). These results suggest that

275

-cryptoxanthin feeding increases the UCP1 expression level in inguinal WAT, thus,

276

effectively reducing the size of adipocytes.

277 278

Effect of -cryptoxanthin on catecholamine excretion and RAR activation. Some

279

studies have reported that UCP1 expression was elevated mainly upon sympathetic

280

nerve activation

281

activation, we measured the amount of catecholamine excretion. Three weeks after

282

-cryptoxanthin feeding, we took urine samples from mice and measured noradrenaline

283

and adrenaline levels using HPLC. We did not find a significant difference between the

284

control and -cryptoxanthin treatment groups (Fig. 5A). In addition, we measured the

285

peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc1,

286

Carnitine Palmitoyltransferase 1B (Cpt1b), Cell death activator A (Cidea), and

287

cytochrome c somatic (Cycs) mRNA expression levels, which are regulated by

288

sympathetic nerve activation in inguinal WAT as browning and mitochondrial

289

biogenesis markers. Again, there was no significant difference in these levels between

290

the control and -cryptoxanthin feeding groups (Fig. 5B). In conclusion, the effect of

37-38.

To elucidate the effect of -cryptoxanthin on sympathetic nerve



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291

-cryptoxanthin on UCP1 upregulation was found to not be caused by sympathetic

292

nerve activation.

293

Previous studies have reported that retinoic acid, one of -cryptoxanthin’s 28.

294

metabolites, has an ability to increase UCP1 expression levels by RAR activation

295

Furthermore, it was also reported that -cryptoxanthin activated RAR

296

we hypothesized that UCP1 upregulation by -cryptoxanthin feeding was caused by

297

RAR activation. To investigate this, we examined the effect of -cryptoxanthin on Rar

298

mRNA expression in inguinal WAT, which is an RAR activation marker

299

observed that -cryptoxanthin contributes to increase in the mRNA expression level of

300

Rar (Fig. 5C). This result suggests that the upregulation of UCP1 expression by

301

-cryptoxanthin feeding was caused by RAR activation.

32, 39.

Therefore,

40-41.

We

302 303

Effect of -cryptoxanthin on RAR activity and Ucp1 mRNA expression. Based on

304

the results of the feeding experiment, we hypothesized that UCP1 upregulation by

305

-cryptoxanthin feeding was caused by RAR activation. We used luciferase reporter

306

assay to clarify the mechanism of UCP1 upregulation by -cryptoxanthin. We used

307

IWAT immortalized cells transfected with pGL3-RARE-luciferase plasmid to measure

308

the ability of -cryptoxanthin to activate RAR. Upon treating the cells with 10 µM 18 ACS Paragon Plus Environment

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-cryptoxanthin for 8 hours, we observed a -cryptoxanthin-mediated increase in

310

luciferase activity (Fig. 6A). We also used IWAT immortalized cells transfected with

311

UCP1-pro-luciferase plasmid and treated the cells with 1 or 10 µM -cryptoxanthin for

312

8 hours. As expected, -cryptoxanthin increased the luciferase activity in these cells as

313

well (Fig. 6B).

314

Next, we investigated the underlying mechanism of this effect in adipocytes. For this,

315

we used IWAT immortalized cells. On day 8 of adipocyte differentiation induction, we

316

treated

317

that -cryptoxanthin had the ability to upregulate both Ucp1 and Rar (RAR activation

318

marker) mRNA in adipocytes. We co-treated these adipocytes with 10 µM

319

-cryptoxanthin and 0, 0.8, 2, or 5 µM LE540 (RAR antagonist) for 8 hours to measure

320

the RAR activation dependency of UCP1 upregulation. We found that LE540 blocked

321

the upregulation of Ucp1, and that Rar mRNA expression was induced

322

by -cryptoxanthin (Fig. 7A, B). We also confirmed the same observations in

323

C3H10T1/2 cells (Supplemental figure 1 A). Together, these data suggest that

324

-cryptoxanthin upregulates Ucp1 mRNA expression in adipocytes via RAR pathway.

325 326

the

cells

with

10

µM

-cryptoxanthin

for

8

hours.

We

found

-carotene (chemical structure shown in Fig. 1B) is one of the most common carotenoids, with a structure similar to that of -cryptoxanthin

42,

although 19

ACS Paragon Plus Environment


Page 20 of 51

327

-cryptoxanthin has a hydroxyl group that is not present in -carotene. When we treated

328

IWAT immortalized cells with 10 µM -cryptoxanthin or -carotene for 8 hours on day

329

8 of differentiation induction to adipocyte, we found that -cryptoxanthin was able to

330

upregulate Ucp1 and Rar mRNA expression stronger than -carotene (Fig. 7C, D). We

331

also confirmed the same observations in C3H10T1/2 cells (Supplemental figure 1 B).

332

This indicates that the hydroxyl group is important for upregulating Ucp1 mRNA

333

expression level in adipocytes.

334 335

DISCUSSION

336

In this study, we demonstrate for the first time that -cryptoxanthin has an effect of

337

suppressing body weight gain, via a mechanism that is partly contributed by the UCP1

338

and RAR pathways. The in vivo assays showed that body weight gain and blood glucose

339

concentration were significantly decreased upon -cryptoxanthin feeding in mice (Fig.

340

2A, B). Furthermore, -cryptoxanthin feeding enhanced their oxygen consumption

341

and rectal temperature (Fig. 2E, H). Thus, -cryptoxanthin feeding decreases body

342

weight gain and fat accumulation by increasing energy expenditure, suggesting that

343

-cryptoxanthin feeding enhances thermogenesis. On the other hand, our data showed

344

that -cryptoxanthin increased the energy expenditure in only 2 hours prior to the


Page 21 of 51


345

experiment (dark period), suggesting that the effect of -cryptoxanthin on

346

thermogenesis is temporary and definite. We showed that β-cryptoxanthin has no effect

347

on the GTT data (Fig. 2C, D). Although the level of plasma glucose at the end point of

348

the animal experiment were found to decrease (Fig. 2B), the plasma insulin levels had a

349

tendency to decrease (Table 1). These data suggested that the effect of -cryptoxanthin

350

on the glucose metabolism is also definite.

351

In this study, the mice were fed a high fat diet (HFD) containing 0.05%

352

-cryptoxanthin. If this condition were to be extrapolated to humans, the amount of

353

-cryptoxanthin would be approximately 300 mg/day. However, the -cryptoxanthin

354

content found in mandarin oranges is small amount, which is not comparable to the

355

circulating levels in human. Therefore, to have an effect on the human metabolism,

356

-cryptoxanthin would most likely need to be administered via dietary supplementation.

357

Thermogenesis mediated by UCP1 plays an important role in regulating energy 16-18, 43.

358

expenditure. UCP1 is a major determinant of BAT thermogenic activity

359

Previous studies have reported that there is a negative correlation between the level of

360

UCP1 expression in adipose tissue and body weight

361

thermogenesis via the upregulation of UCP1 expression is one of the mechanisms

362

underlying the effect of -cryptoxanthin. Our data indicate that the UCP1 expression

21.

We hypothesize that



Page 22 of 51

363

level in not interscapular BAT (classical brown adipocytes) but inguinal WAT

364

(beige/brite adipocytes) increased upon -cryptoxanthin treatment (Fig. 3). The

365

previous study has been reported that the molecular signatures of adult human brown

366

adipocytes resemble those of mouse beige adipocytes rather than mouse classical brown

367

adipocytes

368

inguinal WAT is important to explain the thermogenesis effect.

369

44.

Therefore, we believe that the UCP1 protein expression level in

UCP1 is upregulated mainly by 3-adrenoceptor stimulation

37.

However,

370

-cryptoxanthin feeding did not increase the amount of catecholamine excretion and did

371

not affect the mRNA expression of 3-adrenoceptor in adipose tissue, which is a

372

3-adrenoceptor stimulation marker 45 (Fig. 5A,B). These results showed that the UCP1

373

upregulation by -cryptoxanthin feeding was unrelated to 3-adrenoceptor stimulation.

374

It has been recently reported that -cryptoxanthin suppresses the differentiation of

375

adipocytes through RAR activation

39,

and that RAR activation by atRA in adipose

376

tissue upregulates UCP1 expression

28.

Based on these reports, we hypothesized that

377

-cryptoxanthin feeding upregulates UCP1 expression via RAR activation in adipose

378

tissue and contributes to a decrease in body weight gain. We measured the Rar mRNA

379

levels in mice adipose tissue, as a RAR activation marker

380

-cryptoxanthin feeding significantly upregulated the Rar mRNA expression (Fig. 5C).

40-41,

and found that


Page 23 of 51


381

To evaluate the effect of -cryptoxanthin on thermogenesis, we measured the oxygen

382

consumption in mice. Although the total oxygen consumption in dark and light sessions

383

did not differ between the two groups, -cryptoxanthin fed mice had significantly

384

elevated oxygen consumption from 2100 to 2300 hours (Fig. 2E, F). It has been

385

reported that UCP1 has a significant contribution to diet-induced thermogenesis 17. The

386

timeframe of 2100 to 2300 hours marks the beginning of dark session. It seems that

387

elevation of UCP1 expression by -cryptoxanthin feeding enhanced the diet-induced

388

thermogenesis, which, in turn, contributed to the increased oxygen consumption from 9

389

P.M. to 11P.M., which marks an active period for mice, including activities such as

390

eating.

391

In vitro assays showed that -cryptoxanthin upregulated the Ucp1 expression in white

392

adipocytes via RAR activation. -cryptoxanthin treatment increased the luciferase

393

activity in cells transfected with RARE luc and UCP1 luc plasmid (Fig. 6). Furthermore,

394

RAR antagonist co-treatment completely blocked the effect of -cryptoxanthin on Ucp1

395

upregulation, in both IWAT immortalized cells and C3H10T1/2 cells (Fig. 7A and

396

Supplemental figure1A). Based on these findings, we suggest that -cryptoxanthin

397

contributes to upregulation of UCP1 via RAR activation in white adipocytes. In

398

conjunction with the in vivo assay result that -cryptoxanthin increased oxygen



Page 24 of 51

399

consumption, our findings suggest that UCP1 upregulation in adipose tissue is one of

400

the mechanisms underlying the effect of -cryptoxanthin. A previous study indicated

401

that RARE exists in both mice and human UCP1 promoter

402

that -cryptoxanthin upregulates UCP1 expression in humans in the same way as mice.

403

It has been reported that -cryptoxanthin is partly metabolized to atRA, a strong RAR

404

agonist

405

including liver and white adipocytes 46, 49-50. In the in vitro assay, cells were treated with

406

-cryptoxanthin for only 8 hours. Therefore, even a small amount of -cryptoxanthin

407

has the potential to metabolize to other metabolites, for example, atRA. On the other

408

hand, in the in vivo assay, mice were fed -cryptoxanthin for 12 weeks. -cryptoxanthin

409

is absorbed through small intestine and deposited mainly in liver and white adipose

410

tissues

411

other metabolites. Thus, it is likely that -cryptoxanthin and/or its metabolites together

412

activate RAR 46, 48, and that the atRA metabolized from -cryptoxanthin may contribute

413

to RAR activation. To investigate this possibility, we performed the experiment using a

414

cell culture treated with -cryptoxanthin or atRA and demonstrated that the levels of

415

atRA did not increase after -cryptoxanthin treatment (Supplemental figure 2A).

416

Furthermore, the results of LC-MS analysis on mice adipose tissue and plasma

46-48,

42 51-53.

26-28.

Therefore, it is likely

by beta-carotene dioxygenase 2 (BCDO2) enzyme in some organs,

Twelve weeks is a long enough period to metabolize -cryptoxanthin to


Page 25 of 51


417

suggested that -cryptoxanthin has little effect on atRA metabolism (Supplemental

418

figure 2B). These results suggested that the effect of atRA metabolized from

419

-cryptoxanthin on RAR activation is not taken into consideration in this study.

420

Some epidemiologic studies have reported that -cryptoxanthin concentration in 9-12.

421

serum is inversely associated with contraction of some lifestyle-related diseases

422

addition, the negative correlation between serum -cryptoxanthin concentration and the

423

incidence rate of lifestyle-related diseases is stronger than the correlation observed for

424

other carotenoids, including -carotene, -carotene, and zeaxanthin 11. Previous studies

425

have also reported that -cryptoxanthin results in stronger activation of RAR than other

426

carotenoids 32. We hypothesized that -cryptoxanthin causes a stronger upregulation of

427

UCP1 expression than the other carotenoids. -carotene is the most common carotenoid

428

in our food and its concentration in plasma is similar to -cryptoxanthin 42. Hence, we

429

compared -cryptoxanthin and -carotene in terms of Ucp1 mRNA upregulation

430

activity. The results showed that -cryptoxanthin has a stronger activity in terms of

431

Ucp1 and Rar upregulation than -carotene (Fig. 7C, D).

432

Some previous reports have elucidated the activity of -cryptoxanthin

4, 54-56.

In

We

433

previously found that -cryptoxanthin has PPAR antagonist activity in mice

434

(unpublished data), and other groups have reported the positive effect of



Page 26 of 51

435

-cryptoxanthin on osteoporosis and liver inflammation

436

-cryptoxanthin upregulated the UCP1 expression in inguinal WAT via partly the

437

RAR pathway, thereby increasing the energy expenditure and decreasing the body

438

weight gain and fat accumulation. Based on these results, -cryptoxanthin-mediated

439

thermogenesis via UCP1 and RAR pathway partly enhances the energy expenditure

440

and reduces the accumulation of fat. In conclusion, -cryptoxanthin feeding can

441

induce UCP1 expression in beige adipocytes via the RAR pathway and may partly

442

contribute to the enhancement of the metabolism.

57-58.

In our present study,

443 444 445 446

Acknowledgements The authors thank Ms. S. Shinoto, Ms. M. Sakai, Ms. R. Yoshii, and Mr. M. Komori for secretarial and technical support.

447 448

Author contributions for this study were as follows: H.H, H.T, S.M performed

449

experimental work and analyzed data, H.H, H.T, S.M, H.M, S.K, M.I, M.S, N.T, T.G,

450

and T.K conceptualized and designed the experiments. All authors contributed to

451

interpretation of the data and editing of the manuscript. H.H and H.T wrote the

452

manuscript. T.G. and T.K supervised this work.


Page 27 of 51


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Li, D.; Xiao, Y.; Zhang, Z.; Liu, C., Light-induced oxidation and isomerization

Amengual, J.; Gouranton, E.; van Helden, Y. G.; Hessel, S.; Ribot, J.; Kramer,

Lobo, G. P.; Isken, A.; Hoff, S.; Babino, D.; von Lintig, J., BCDO2 acts as a

Burri, B. J., Beta-cryptoxanthin as a source of vitamin A. Journal of the

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Dhuique-Mayer, C.; Borel, P.; Reboul, E.; Caporiccio, B.; Besancon, P.; Amiot,

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M. J., Beta-cryptoxanthin from citrus juices: assessment of bioaccessibility using an in

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vitro digestion/Caco-2 cell culture model. The British journal of nutrition 2007, 97 (5),

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883-90.

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54.

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G.,

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inflammation in mouse Sertoli cells. Reproductive toxicology 2016, 60, 148-55.

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The carotenoid beta-cryptoxanthin stimulates the repair of DNA oxidation damage in

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addition to acting as an antioxidant in human cells. Carcinogenesis 2009, 30 (2),

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308-14.

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S.; Thompson, H. J., Plasma xanthophyll carotenoids correlate inversely with indices of

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oxidative DNA damage and lipid peroxidation. Cancer epidemiology, biomarkers &

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prevention : a publication of the American Association for Cancer Research,

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cosponsored by the American Society of Preventive Oncology 2000, 9 (4), 421-5.

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Nagashimada, M.; Kaneko, S.; Naito, S.; Ota, T., beta-Cryptoxanthin alleviates

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against

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diet-induced nonalcoholic steatohepatitis by suppressing inflammatory gene expression

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in mice. PloS one 2014, 9 (5), e98294.

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in Osteoporosis. J. Heal. Sci. 2008, 54 (4), 356-369.

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655


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Table 1. Effects of β-cryptoxanthin on body weight, food intake, tissue weight, and

658

plasma parameter in HFD-fed C57BL/6J mice. 　

　

control

　

CX

Body weight gain (Δg)

21.49±0.87

18.43±0.51*

Energy intake (kcal/day)

15.51±0.19

15.29±0.32

2.98±0.03

2.94±0.06

Inguinal WAT (g)

1.18±0.063

0.99±0.045*

Epididymal WAT (g)

2.21±0.11

1.89±0.088*

interscapular BAT (g)

0.15±0.0042

0.13±0.066*

Gastrocnemius muscle (g)

0.28±0.0064

0.27±0.0085

Liver (g)

1.22±0.025

1.13±0.030*

Kidney (g)

0.36±0.0089

0.35±0.0070

TG (mg/dL)

55.9±4.57

51.8±3.97

NEFA (mEq/L)

0.64±0.032

0.56±0.032

Insulin (ng/mL)

2.02±0.12

1.70±0.14

Leptin (ng/mL)

49.0±3.66

39.8±2.44

Food intake (g/day)

Tissue weight

Plasma parameter



Page 40 of 51

659

Data represent the means ± SEM (n = 7–8). Statistical comparisons were made with the

660

control group; *p < 0.05 was considered significant. CX: -cryptoxanthin group.

661


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663

FIGURE LEGENDS

664

Figure 1. Chemical structure of (A) -cryptoxanthin and (B) -carotene.

665 666

Figure 2. Effect of β-cryptoxanthin on metabolism in HFD-fed C57BL/6J mice. (A)

667

Body weight, (B) plasma glucose concentration, (C, D) glucose tolerance test (GTT), (E,

668

F) oxygen consumption, (G) locomotor activity, and (H) rectal temperature data of mice

669

treated with or without β-cryptoxanthin. Data represent the means ± SEM (n = 7–8,

670

except for locomotor activity measurement; n = 4 for locomotor activity measurement).

671

Statistical comparisons were made with the control group; *p < 0.05 was considered

672

significant. CX: -cryptoxanthin group

673 674

Figure 3. Effect of β-cryptoxanthin on UCP1 expression in HFD-fed C57BL/6J mice.

675

Western blotting images (left) and quantification data (right) of UCP-1 in (A) inguinal

676

WAT or (B) interscapular BAT. The mRNA expression level of Ucp-1 in (C) inguinal

677

WAT or (D) interscapular BAT. Data represent the means ± SEM (n = 7–8). Statistical

678

comparisons were made with the control group; **p < 0.01 was considered significant.

679 680

Figure 4. Effect of β-cryptoxanthin intake for 12 weeks on adipocytes size in HFD-fed



Page 42 of 51

681

C57BL/6J mice. Hematoxylin Eosin stained images (upper figure), cell population

682

(bottom left figure) and cell diameter (bottom right figure) of (A) inguinal WAT and (B)

683

interscapular BAT. Statistical comparisons were made with the control group; **p

Î²-cryptoxanthin induces UCP-1 expression via RAR pathway

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