Capsaicin ameliorates the redox imbalance and glucose metabolism

2 hours ago - Circadian rhythms are closely associated with metabolic homeostasis. Metabolic disorders can be alleviated by many bioactive components ...
0 downloads 0 Views 921KB Size
Subscriber access provided by Warwick University Library

Bioactive Constituents, Metabolites, and Functions

Capsaicin ameliorates the redox imbalance and glucose metabolism disorder in insulin-resistance model via circadian clock-related mechanisms MUWEN LU, Yaqi Lan, Jie Xiao, Mingyue Song, Chengyu Chen, Caowen Liang, Qingrong Huang, Yong Cao, and Chi-Tang Ho J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b04016 • Publication Date (Web): 18 Aug 2019 Downloaded from pubs.acs.org on August 20, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 25

Journal of Agricultural and Food Chemistry

1

Capsaicin ameliorates the redox imbalance and glucose metabolism disorder in

2

insulin-resistance model via circadian clock-related mechanisms

3 4

Muwen Lu†, Yaqi Lan†, Jie Xiao†, Mingyue Song†, Chengyu Chen‡, Caowen Liang†,

5

Qingrong Huang§, Yong Cao†,* and Chi-Tang Ho§,*

6 7



8

Food Science, South China Agricultural University, Guangzhou 510642, China

9



Guangdong Provincial Key Laboratory of Nutraceuticals and Functional Foods, College of

College of Natural Resources and Environment, South China Agricultural University,

10

Guangzhou 510642, China.

11

§

Department of Food Science, Rutgers University, New Brunswick, NJ 08901, USA

12 13 14 15

* To whom correspondence should be addressed. Tel: 848-932-5553 (CH). Fax: 732-932-6776;

16

Email: [email protected] (CH); [email protected] (YC)

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

17

ABSTRACT

18

Circadian rhythms are closely associated with metabolic homeostasis. Metabolic

19

disorders can be alleviated by many bioactive components through the controlling of clock

20

gene expressions. Capsaicin has been demonstrated with many beneficial effects including

21

anti-obesity and anti-insulin resistance activities, yet whether the rhythmic expression of

22

circadian clock genes are involved in the regulation of redox imbalance and glucose

23

metabolism disorder by capsaicin remains unclear. In this work, the insulin resistance was

24

induced in HepG2 cells by the treatment of glucosamine. Glucose uptake level, reactive oxygen

25

species (ROS), H2O2 production and mitochondrial membrane potential (MMP) were

26

measured with/without capsaicin co-treatment. The mRNA and protein expressions of core

27

circadian clock genes were evaluated by RT-qPCR and western blot analysis. Our study

28

revealed that circadian misalignment could be ameliorated by capsaicin. The glucosamine-

29

induced cellular redox imbalance and glucose metabolism disorder were ameliorated by

30

capsaicin in a Bmal1-dependent manner.

31

KEYWORDS: Capsaicin; circadian clock, insulin resistance, redox homeostasis

32 33

2

ACS Paragon Plus Environment

Page 2 of 25

Page 3 of 25

34

Journal of Agricultural and Food Chemistry

INTRODUCTION

35

Circadian rhythms are biological variables that oscillate cyclically with a period close to

36

24 h, allowing the organism to make adaptations to the constantly changing environment,

37

including light, temperature and nutrients.1 The central circadian clock is located in the

38

hypothalamus suprachiasmatic nucleus (SCN), which is composed of multiple circadian

39

oscillators operated by two interlocking transcription/translation feedback loop (TTFL).2 The

40

24-h rhythmic circadian gene expression regulated by TTFL is driven by four integral clock

41

proteins: two activators (CLOCK and BMAL1) and two inhibitors (PER and CRY), as well as

42

by other kinases and phosphatases.3 CLOCK and BMAL1 form CLOCK/BMAL1 heterodimer

43

and bind to the promoters of clock-controlled genes at E-boxes (5’-CACGTG-3’) in the nucleus,

44

inducing the transcription of other circadian genes such as Pers and Crys. As the protein

45

concentration of the PER and CRY accumulates, the polymers will be formed to inhibit the

46

transcription mediated by CLOCK/BMAL1 heterodimer.4

47

Metabolic disorders have become serious global health issues negatively affecting lives

48

of many people, which occur when the body's usual metabolic processes are disrupted.5,6

49

Insulin resistance, as a hallmark of the metabolic syndrome, occurs when cells are unable to

50

respond normally to the hormone insulin.7 Insulin resistance is intimately linked to a variety of

51

metabolic syndromes, such as hypertension, hyperlipidemia and atherosclerosis.8

52

Growing evidence indicates that circadian rhythm is closely related with metabolic

53

homeostasis and the disruption of circadian rhythms results in metabolic disorders.6,9 Therefore,

54

many metabolic systems may in turn affect the function of clock genes and circadian systems.

55

Various studies revealed that dietary bioactive component could regulate metabolic disorders

56

via the involvement of circadian clock genes.10-12 Qi, et al. reported that tea polyphenols could

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 25

57

alleviate metabolic syndrome and mitochondrial dysregulation in a circadian gene-dependent

58

manner.13 According to Sun, et al., resveratrol could attenuate the high-fat diet (HFD)-induced

59

circadian misalignment of lipid metabolism in male C57BL/6 mice.14 Cichoric acid, a

60

hydroxycinnamic acid occurs in a variety of plant species, could prevent free-fatty-acid-

61

induced disorders of lipid metabolism through the modulation of the circadian gene Bmal1

62

expressions in hepatocytes.15

63

Capsaicin, the main capsaicinoid in chili peppers with pungent and spicy flavor,16-18 has

64

many

65

cardioprotective,25 anti-oxidation26 and anti-obesity activities.27,28 Kang, et al. reported the

66

administration of capsaicin could reduce obesity-related metabolic disorders such as insulin

67

resistance and hepatic steatosis induced by HFD in male C57BL/6 mice.29 Later, they found

68

that capsaicin attenuated the metabolic dysregulation by enhancing expressions of adiponectin

69

and its receptor in obese diabetic KKAy mice that exhibits serious insulin resistance.30 Jeong,

70

et al. treated Sprague Dawley (SD) rats with capsaicin water suspension and revealed that

71

capsaicin could regulate the expression of circadian clock gene Per2.31 Therefore, capsaicin

72

may exert the preventative effect on insulin resistance by regulating the circadian clock gene

73

expressions. However, it remains unclear whether the rhythmic expressions of circadian clock

74

genes are involved in glucosamine-induced insulin resistance ameliorated by capsaicin in

75

human hepatocytes.

beneficial

effects

such

as

anti-inflammation,19

anti-cancer,20-23

analgesic,24

76

In this work, the protective effect of capsaicin on circadian disruption triggered by

77

glucosamine was investigated using HepG2 cell line. The glucosamine-induced oxidative

78

stress and mitochondrial dysfunction relieved by capsaicin were also evaluated in respect of

79

the core circadian clock gene expressions.

4

ACS Paragon Plus Environment

Page 5 of 25

80

Journal of Agricultural and Food Chemistry

MATERIALS AND METHODS

81

Materials

82

Capsaicin (purity ~99%) was purchased from Ji’an Shengda Fragrance Oils Company

83

(Ji’an, Jiangxi, China). Milli-Q water (18.3 MΩ) was used in all experiments.

84

Cell Culture and Viability Assay

85

HepG2 cell line was obtained from Collection of Cell Cultures of the Fourth Military

86

Medical University of the People's Liberation Army (Xi’an, Shaanxi, China). Modified

87

Roswell Park Memorial Institute (RPMI)-1640 medium was purchased from Thermo Fisher

88

Scientific (Waltham, MA, USA). HepG2 were cultured in RPMI medium supplemented with

89

10% fetal bovine serum (FBS), penicillin (100 kU/L) and streptomycin (100 mg/L). Cells were

90

maintained at 37 °C in a humidified atmosphere of 5% CO2.

91

The MTT assay was used to evaluate the viability of HepG2 cells. Cells were seeded at a

92

density of 2.0 × 104 cells/well in 96-well plates. After an overnight incubation, glucosamine

93

(20 mM) and capsaicin with different concentrations were added. At the end of each treatment,

94

MTT solution (Sigma, St. Louis, MO, USA) was added and incubated at 37 °C for 4 h.

95

Formazan crystal which displayed a purple color was detected by a by Bio-Rad iMark

96

microplate reader at 490 nm (Bio-Rad, Hercules, CA, USA). All experiments were performed

97

in triplicate.

98

Glucose Uptake

99

The glucose uptake study was performed according to the reported method with slight

100

modifications.32 HepG2 cells were treated with 20 mM glucosamine to induce insulin

101

resistance, and then co-treated with 50 μM capsaicin for 18 h after incubating with 100 nM

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

102

insulin. The supernatant was measured at wavelength of 540 nm by a glucose assay kit (Nanjing

103

Jiancheng Bioengineering Institute, Nanjing, China). The optical density of each sample was

104

measured and the glucose concentration was calculated. Six replicate wells were established

105

and all experiments were performed in triplicate.

106

Measurement of Reactive Oxygen Species (ROS) and H2O2 Level

107

Intracellular ROS levels were measured by using ROS assay kit (Beyotime Biotechnology,

108

Nanjing, China), which could be used to measure the hydroxyl, peroxyl, and other reactive

109

oxygen species activity within a cell. The cells were treated with 2´,7´-dichlorofluorescein-

110

diacetate (DCFDA), which could be converted to the fluorescent dichlorofluorescein (DCF) by

111

intracellular ROS. Fluorescence was read using an excitation wavelength at 495 nm and

112

emission wavelength at 529 nm in the plate reader Synergy H1 (BioTek, Winooski, VT, USA).

113

The extracellular H2O2 levels were detected by the Amplex Red hydrogen

114

peroxide/peroxidase assay kit (Invitrogen, Carlsbad, CA, USA). The Amplex Red reagent, in

115

combination with horseradish peroxidase (HRP), could react with H2O2 and produce a highly

116

fluorescent resorufin. Fluorescence intensity was measured by microplate reader using an

117

excitation wavelength at 490 nm and emission wavelength at 535 nm.

118

Measurement of Mitochondrial Membrane Potential (MMP)

119

MMP assay kit with JC-1 (Beyotime, Nanjing, China) was applied to measure the

120

mitochondrial membrane potential (MMP). After incubation overnight, cells were centrifuged,

121

resuspended in phosphate-buffered saline (PBS) and stained in cationic dye JC-1. Fluorescence

122

were detected by Synergy Neo2 hybrid multi-mode microplate reader (BioTek, Winooski, VT,

123

USA). The MMP levels was indicated by the intensity ratio of green/red fluorescence.

6

ACS Paragon Plus Environment

Page 6 of 25

Page 7 of 25

Journal of Agricultural and Food Chemistry

124

RNA Extraction and Real-time PCR

125

Total RNA was extracted from HepG2 cells by a RNA isolator (TaKaRa, Dalian, China),

126

and cDNA was synthesized using the Primescript RT reagent (TaKaRa). The relative mRNA

127

quantification was analyzed by RT-qPCR using a SYBR green I dye (TaKaRa) and the CFX96

128

Touch real-time PCR detection system (Bio-Rad). Gene-specific mouse primers used in this

129

study were summarized in Table 1. The relative transcript level of each target gene was

130

calculated according to the 2−Ct method for gene normalization to GAPDH.33

131

Western Blot Analysis

132

Cell lysates were prepared by solubilizing in SDS sample buffer. After proteins were

133

transferred onto a polyvinylidene difluoride (PVDF) membrane (Merck Millipore, Darmstadt,

134

Germany), they were stained for visualization and identified by immunodetection. Antibodies

135

including Anti-CLOCK (ab93804), Anti-BMAL1 (ab93806), Anti-CRY1 (ab54649), Anti-

136

CRY2 (ab155255), Anti-PER1 (ab136451) Anti-PER2 (ab179813) and Anti-GAPDH

137

(ab181602) were purchased from Abcam (Abcam, Cambridge, MA, USA). Quantitative

138

analysis of western blot was achieved by the Quantity One v4.6.2 (Bio-Rad).

139

Statistical Analysis

140

All of the data are expressed as means ± standard error. Variances between groups were

141

determined using one-way ANOVA by SPSS software. Significance level at p < 0.05, 0.01 ,

142

and 0.001 were considered statistically significant.

143

RESULTS AND DISCUSSION

144

Capsaicin Alleviated Glucosamine-impaired Glucose Uptake in HepG2 Cells

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

145

Different concentrations of capsaicin (0, 25, 50, 75, 100 μM, respectively) were applied

146

to HepG2 cells to examine the effects of capsaicin on cell viability during proliferation.

147

According to Figure 1A, the concentration of 50 μM was selected with 84.67% cell viability.

148

Studies revealed that insulin resistance could be induced by glucosamine through the

149

hexosamine biosynthesis pathway, causing insulin disturbances and glucose intolerance.34 In

150

this work, HepG2 cells were treated with glucosamine at concentration of 20 mM to induce

151

insulin resistance (Figure 1B).

152

Glucose uptake in cells treated with capsaicin and glucosamine (20 mM) is presented in

153

Figure 1C. The insulin-stimulated glucose uptake was reduced significantly from 100% to

154

33.94% after glucosamine treatment (p < 0.001) in HepG2 cells. After the capsaicin treatment

155

for 18 h, the glucose uptake increased effectively from 33.94% to 72.8% (p < 0.001), indicating

156

that capsaicin could alleviate the glucosamine-impaired glucose uptake. The glucose uptake

157

was further enhanced to 85.95% (p < 0.001) with the increase in capsaicin concentration,

158

suggesting that the glucosamine-induced insulin resistance could be alleviated by capsaicin in

159

a dose-dependent manner in HepG2 cells.

160

Regulation of Circadian Misalignment by Capsaicin in HepG2 Cells

161

Studies revealed that the disruption of circadian clock was closely related with insulin

162

resistance and obesity.5 Mi, et al. and Zhu, et al. both reported that glucosamine treatment

163

could induce insulin resistance in HepG2 cells,10,35 which resulted in the decrease in the

164

transcription level of Clock and Bmal1. In this work, circadian misalignment was induced by

165

the treatment of glucosamine in the insulin-resistance model. The expression levels of circadian

166

rhythm genes Clock, Bmal1, Per1, Per2, Cry1, and Cry2 in HepG2 cells were measured by

167

RT-qPCR, and results are presented in Figure 2 (A-F). Most of the oscillating genes in the

8

ACS Paragon Plus Environment

Page 8 of 25

Page 9 of 25

Journal of Agricultural and Food Chemistry

168

control group were expressed in a rhythmic pattern, such as Clock, Bmal1, Per2, Cry1 and

169

Cry2. However, after glucosamine treatment, the mRNA expressions of both activators (Clock

170

and Bmal1) and inhibitors (Per1) displayed relatively shallow oscillations, which were

171

reversed by capsaicin treatment effectively.

172

To further demonstrate the regulation effect of capsaicin in alleviating the circadian

173

misalignment triggered by glucosamine in insulin resistance models, the protein expressions

174

of core clock components in these groups were measured through western blot analysis.

175

According to Figure 3 (A-F), relative shallow oscillations were observed in the glucosamine-

176

treated group for the protein expressions of BMAL1, CRY1 and CRY2. The oscillatory

177

behavior was recovered by capsaicin co-treatment efficiently, which was in consistent with the

178

mRNA expression levels of clock genes. Therefore, capsaicin was proved to regulate the

179

glucosamine-induced circadian clock disruption at both RNA and protein level in insulin-

180

resistant HepG2 cell model.

181 182

Inhibitory Effects of Capsaicin on the Glucosamine-induced ROS Production and Mitochondria Dysfunction in HepG2 Cells

183

The pathophysiology of insulin resistance is complex and still incompletely understood,

184

yet it has been reported to be intricately linked to mitochondrial dysfunction and reactive

185

oxygen species (ROS) imbalance.36,37 The importance of ROS signaling and oxidative stress in

186

the development of insulin resistance has been implicated in many studies.38 An excessive

187

amount of ROS could damage cellular lipids, proteins, or DNA, leading to the inhibition of

188

signal transduction pathways and disruption of normal cellular functions. As shown in Figure

189

4 (A), the redox status in HepG2 cells was measured using DCFDA. Relative ROS level was

190

calculated and presented in Figure 4 (B). In comparation with the control, the relative ROS

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

191

level was increased from 100.00% to 326.98% for the glucosamine-treated group, showing that

192

glucosamine could induce the production of ROS in HepG2 cells and that the imbalance of

193

ROS may promote mitochondrial dysfunction. The ROS level was reduced to 141.10% after

194

the co-treatment of 50 M capsaicin, indicating that capsaicin had an inhibition effect on the

195

production of excess ROS caused by glucosamine.

196

During normal cellular metabolism, mitochondrial electron transport could result in the

197

formation of hydrogen peroxide (H2O2).39 It was reported that excess H2O2 emission could

198

inhibit the activities of specific mitochondrial enzymes and overall mitochondrial respiration,

199

leading to insulin resistance in both rodents and humans.40 According to Figure 4 (C), the H2O2

200

content in glucosamine-treated HepG2 cells was increased from 98.76% to 173.21% compared

201

to the control group, while it was reduced to 112.92% due to the existence of capsaicin,

202

indicating that capsaicin was effective in inhibiting the glucosamine-induced H2O2 emission.

203

The mitochondrial membrane potential (MMP, ΔΨm) was measured using JC-1 dye and

204

presented as the ratio of green/red using fluorescence microscopy, as was shown in Figure 4

205

(D). In healthy cells, ΔΨm was relatively high and JC-1 could aggregate with deep red

206

fluorescence. However, for unhealthy cells with low ΔΨm, JC-1 existed in a monomeric form

207

and exhibited green florescence. Therefore, the lower ratio of green/red fluorescence reflected

208

a higher polarization of the mitochondrial membrane. Compared with the control group, the

209

ratio of green/red fluorescence for glucosamine group was increased from 94.35% to 276.01%,

210

which was recovered to 105.44% by capsaicin co-treatment, demonstrating the alleviating

211

effect of capsaicin on glucosamine-induced mitochondria dysfunction in HepG2 cells.

212 213

Alleviation of Cellular Redox Imbalance by Capsaicin via Regulating the Circadian Clock Genes in mRNA Levels.

10

ACS Paragon Plus Environment

Page 10 of 25

Page 11 of 25

Journal of Agricultural and Food Chemistry

214

Compelling evidence suggested that many metabolic disorders could be prevented by

215

functional food ingredients via regulating circadian gene expressions in HepG2 cells.15,41 To

216

verify if the mRNA level of circadian clock genes could affect the modulation effect of

217

capsaicin on cellular redox homeostasis, small interfering RNA (siRNA) was used in HepG2

218

cells to decrease Bmal1 abundance. As shown in Figure 4 (A-B), by exposing to si-Bmal1, the

219

relative ROS level increased from 141.01% to 202.54%, indicating that the inhibition effect of

220

capsaicin on ROS production was weakened by the knockdown of Bmal1 gene. Same trend

221

could be observed for the H2O2 level and the mitochondrial membrane potential in HepG2 cells.

222

According to Figure 4 (C), the H2O2 concentration in si-Bmal1 group was increased from

223

112.92% to 143.61% compared with capsaicin group, showing a reduced effect in suppressing

224

the glucosamine-induced H2O2 emission. The green/red florescence ratio for the Bmal1-

225

knocked down group was also raised from 105.44% to 192.43%, reflecting a diminished

226

modulation effect on MMP by capsaicin in the presence of si-Bmal1 (Figure 4 D). Therefore,

227

the regulation effect of capsaicin on cellular redox homeostasis is dependent on the mRNA

228

level of the circadian clock gene Bmal1.

229 230

Capsaicin Ameliorated Glucose Metabolism Disorder in HepG2 Cells in a Bmal1Dependent Manner

231

To study the role of circadian clock gene Bmal1 in the regulation of glucose metabolic

232

disorder by capsaicin, Bmal1 was silenced using siRNAs and relative protein levels for core

233

circadian clock genes were measured. Based on the western blot analysis shown in Figure 5(A-

234

B), the protein level of BMAL1 was significantly reduced to 51.52% with si-RNA knockdown

235

in HepG2 cells. Meanwhile, the relative protein expressions of CLOCK, CRY2, PER1 and

236

PER2 were also suppressed compared with the control group, suggesting that Bmal1 as the

237

core clock gene could regulate the expression of other circadian genes involved in the

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

238

transcriptional feedback loop, which was in consistent with previous studies.42-44 As shown in

239

Figure 5(B), the treatment of glucosamine resulted in the down-regulation of protein levels of

240

circadian genes, such as BMAL1, CLOCK, CRY1 and PER2, which was reversed by capsaicin

241

co-treatment. However, the protein levels in capsaicin co-treated group failed to increase after

242

Bmal1 deletion, demonstrating that the modulation effect of capsaicin on expression of

243

circadian clock genes relied upon regular expression of Bmal1.

244

According to Figure 5 (C), the glucose uptake was measured under different conditions.

245

Compared with the control group, the glucose uptake in Bmal1- knockout group was reduced

246

to 77.90%, suggesting that si-Bmal1 impaired the glucose metabolism in HepG2 cells. The

247

glucosamine-induced glucose metabolic disorder was alleviated after capsaicin treatment with

248

glucose uptake improved from 64.59% to 85.64%. The amelioration effect of capsaicin on

249

glucose metabolic disorder was decreased after Bmal1 deletion with the glucose uptake level

250

of 66.11%, showing that capsaicin mitigated glucose metabolic dysfunction in HepG2 cells in

251

a Bmal1-dependent manner.

252

In summary, our study revealed that capsaicin could alleviate the circadian misalignment

253

and inhibit glucosamine-induced ROS production and mitochondria dysfunction in HepG2

254

cells. The glucose metabolism disorder was also relieved by capsaicin through regulating the

255

rhythmic expression of circadian clock gene Bmal1. These findings could provide novel

256

solutions in the prevention and treatment of obesity, insulin resistance as well as other

257

metabolic disorders through the modulation of clock genes.

258

Acknowledgements

259

This work was financially supported by the National Natural Science Foundation of China (No.

260

31901689).

12

ACS Paragon Plus Environment

Page 12 of 25

Page 13 of 25

Journal of Agricultural and Food Chemistry

261

Conflict of interest

262

The authors declare no competing financial interest.

263

Abbreviations:

264

BMAL1, brain and muscle arnt-like protein 1; CRY, cryptochrome. CAP, capsaicin; DCF,

265

dichlorofluorescein; DCFDA, 2’,7’-dichlorofluorescein-diacetate; FL, fluorescence; GAPDH,

266

glyceraldehyde 3-phosphate dehydrogenase; HFD, high-fat diet; HRP, horseradish peroxidase;

267

H2O2, hydrogen peroxide; JC-1, tetraethyl benzimidazolyl carbocyanine iodide; MMP (ΔΨm),

268

mitochondrial membrane potential; PER, Period circadian protein; ROS, reactive oxygen

269

species; PVDF, polyvinylidene difluoride; SCN, suprachiasmatic nucleus; SD rat, Sprague

270

Dawley rat; SDS, sodium dodecyl sulfate; SDS-PAGE, sodium dodecyl sulphate-

271

polyacrylamide

272

transcription/translation feedback loop.

gel

electrophoresis;

siRNA,

small

273

13

ACS Paragon Plus Environment

interfering

RNA;

TTFL,

Journal of Agricultural and Food Chemistry

274

Table 1. Primer sequences used for quantitative real-time PCR analysis. Forward primer

Reverse primer

GAPDH TCAAGAAGGTGGTGAAGCAGG TCAAAGGTGGAGGAGTGGGT Bmal1

ATGGGGCTGGATGAAGACAA

CTGTTGCCCTCTGGTCTACA

Clock

ACGACGAGAACTTGGCATTG

GGTGTTGAGGAAGGGTCTGA

Per1

AAGTCCGTCTTCTGCCGTAT

TATCCGGGGAGCTTCGTAAC

Per2

AGCCGGAGTTAGAGATGGTG

TCTGCTCCTCCTTCTGTGTG

Cry1

GTCTACATCCTGGACCCCTG

CTGGGAAACACATCTGCTGG

Cry2

GGGAGGAGAGACAGAAGCTC

AATAGGGAGAGGGGAGGTGT

275 276

14

ACS Paragon Plus Environment

Page 14 of 25

Page 15 of 25

Journal of Agricultural and Food Chemistry

277

Figure Captions

278

Figure 1. Capsaicin alleviated glucosamine-impaired glucose uptake in HepG2 cells. (A)

279

Relative viability of HepG2 cells treated with different concentrations of capsaicin measured

280

by MTT assay; (B) Relative cell viability treated with 50 μM capsaicin and co-treated with

281

/without 20 mM glucosamine; (C) Glucose uptake in groups treated with capsaicin (25, 50 μM)

282

and co-treated with/without glucosamine (20 mM). Data were presented as the mean value ±

283

SE (n≥ 6): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

284

Figure 2. Capsaicin regulated glucosamine-induced circadian misalignment in HepG2 cells.

285

(A-F) The mRNA expression levels of circadian rhythm genes Clock, Bmal1, Per1, Per2, Cry1,

286

and Cry2 in HepG2 cells measured by RT-qPCR and normalized to β-actin mRNA levels. Data

287

were presented as the mean value ± SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001

288

versus control group; (#) P < 0.05,( ##) P < 0.01 and (###) P < 0.001 versus glucosamine

289

group.

290

Figure 3. The effects of CAP on glucosamine-induced clock genes changes were determined

291

by western blots. Clock, Bmal1, Cry1, Cry2, Per1 and Per2 were detected in HepG2 cells, and

292

GAPDH was used as a loading control. (A)-(F) Densitometric analysis of the blots shown in

293

G. Data were presented as the mean value ± SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P

294

< 0.001 versus control group; (#) P < 0.05,( ##) P < 0.01 and (###) P < 0.001 versus

295

glucosamine group.

296

Figure 4. Capsaicin alleviated the imbalance in redox status induced by glucosamine in HepG2

297

cells. HepG2 cells were cultured with/without glucosamine and co-treated with capsaicin for

298

18 h. (A)-(B) The cellular oxidation status in different groups detected using DCFDA. (C)

299

Production of hydrogen peroxide (H2O2) measured by Amplex Red Hydrogen

300

Peroxide/Peroxidase Assay Kit. (D) The mitochondrial membrane potential reflected as the

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

301

ratio of green/red using fluorescence microscopy. Data were presented as the mean value ±

302

SE (n=3): (∗) P < 0.05, (∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

303

Figure 5.Capsaicin ameliorated glucose metabolism disorder induced by glucosamine via

304

modulating the protein expression of circadian clock genes. HepG2 cells were transfected with

305

si-Bmal1 for 48 h, and then cultured with/without glucosamine and co-treated with capsaicin

306

for 18 h. β-actin were used as a loading control. (A) Representative western blots of core

307

circadian clock genes after treatment with glucosamine and capsaicin in HepG2 cells. (B)

308

Densitometric analysis of the blots shown in A. (C) The glucose content in cells measured by

309

a glucose uptake analysis kit. Data were presented as the mean value ± SE (n= 3): (∗) P < 0.05,

310

(∗∗) P < 0.01, and (∗∗∗) P < 0.001 versus control group.

311 312 313

16

ACS Paragon Plus Environment

Page 16 of 25

Page 17 of 25

Journal of Agricultural and Food Chemistry

314

315 316

Figure 1.

317 318

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

319 320 321

Figure 2.

322

18

ACS Paragon Plus Environment

Page 18 of 25

Page 19 of 25

Journal of Agricultural and Food Chemistry

323

324

325 326

Figure 3.

327

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

328 329

330 331

Figure 4.

20

ACS Paragon Plus Environment

Page 20 of 25

Page 21 of 25

Journal of Agricultural and Food Chemistry

332 333

Figure 5.

334 335

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

336

References

337 338 339 340 341 342 343 344 345 346 347 348 349 350 351 352 353 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378 379 380

1. 2. 3. 4. 5. 6. 7. 8.

9. 10. 11. 12. 13. 14. 15. 16. 17.

Top, D.; Young, M. W. Coordination between differentially regulated circadian clocks generates rhythmic behavior. Cold Spring Harb. Perspect. Biol. 2018, 10, a033587.. Papazyan, R.; Zhang, Y.; Lazar, M. A. Genetic and epigenomic mechanisms of mammalian circadian transcription. Nat. Struct. Mol. Biol. 2016, 23, 1045-1052. Partch, C. L.; Green, C. B.; Takahashi, J. S. Molecular architecture of the mammalian circadian clock. Trends Cell Biol. 2014, 24, 90-99. Huang, N.; Chelliah, Y.; Shan, Y.; Taylor, C. A.; Yoo, S.-H.; Partch, C.; Green, C. B.; Zhang, H.; Takahashi, J. S. Crystal structure of the heterodimeric CLOCK:BMAL1 transcriptional activator complex. Sci. 2012, 337, 189-194. Shi, S. Q.; Ansari, T. S.; McGuinness, O. P.; Wasserman, D. H.; Johnson, C. H. Circadian disruption leads to insulin resistance and obesity. Curr. Biol. 2013, 23, 372-381. Mi, Y.; Qi, G.; Fan, R.; Ji, X.; Liu, Z.; Liu, X. EGCG ameliorates diet-induced metabolic syndrome associating with the circadian clock. BBA-Mol. Basis Disease 2017, 1863, 1575-1589. Kahn, B. B.; Flier, J. S. Obesity and insulin resistance. J. Clin. Invest. 2000, 106, 473481. Medina-Contreras, J. M. L.; Colado-Velazquez, J., 3rd; Gomez-Viquez, N. L.; MaillouxSalinas, P.; Perez-Torres, I.; Aranda-Fraustro, A.; Carvajal, K.; Bravo, G, Effects of topical capsaicin combined with moderate exercise on insulin resistance, body weight and oxidative stress in hypoestrogenic obese rats. Int. J. Obes. 2017, 41, 750758. Froy, O. Metabolism and circadian rhythms—Implications for obesity. Endocr. Rev. 2010, 31, 1-24. Mi, Y.; Qi, G.; Gao, Y.; Li, R.; Wang, Y.; Li, X.; Huang, S.; Liu, X. (-)-Epigallocatechin-3gallate ameliorates insulin resistance and mitochondrial dysfunction in HepG2 cells: involvement of Bmal1. Mol. Nutr. Food Res. 2017, 61, 1700440. Qi, G.; Mi, Y.; Liu, Z.; Fan, R.; Qiao, Q.; Sun, Y.; Ren, B.; Liu, X. Dietary tea polyphenols ameliorate metabolic syndrome and memory impairment via circadian clock related mechanisms. J. Funct. Foods 2017, 34, 168-180. Liu, F.; Zhang, X.; Zhao, B.; Tan, X.; Wang, L.; Liu, X. Role of food phytochemicals in the modulation of circadian clocks. J. Agric. Food Chem. 2019, 67, 8735-8739. Qi, G.; Mi, Y.; Fan, R.; Zhao, B.; Ren, B.; Liu, X. Tea polyphenols ameliorates neural redox imbalance and mitochondrial dysfunction via mechanisms linking the key circadian regular Bmal1. Food Chem. Toxicol. 2017, 110, 189–199. Sun, L.; Wang, Y.; Song, Y.; Cheng, X.-R.; Xia, S.; M. R. T. Rahman; Shi, Y.; Le, G. Resveratrol restores the circadian rhythmic disorder of lipid metabolism induced by high-fat diet in mice. Biochem. Biophys. Res. Commun. 2015, 458, 86-91. Guo, R.; Zhao, B.; Wang, Y.; Wu, D.; Wang, Y.; Yu, Y.; Yan, Y.; Zhang, W.; Liu, Z.; Liu, X. Cichoric acid prevents free-fatty-acid-induced lipid metabolism disorders via regulating Bmal1 in HepG2 cells. J. Agric. Food. Chem. 2018, 66, 9667-9678. Reyes-Escogido Mde, L.; Gonzalez-Mondragon, E. G.; Vazquez-Tzompantzi, E. Chemical and pharmacological aspects of capsaicin. Molecules 2011, 16, 1253-1270. Lu, M.; Ho, C. T.; Huang, Q. Extraction, bioavailability, and bioefficacy of capsaicinoids. J. Food Drug Anal. 2017, 25, 27-36.

22

ACS Paragon Plus Environment

Page 22 of 25

Page 23 of 25

381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 412 413 414 415 416 417 418 419 420 421 422 423 424 425 426 427

Journal of Agricultural and Food Chemistry

18. Lu, M.; Cao, Y.; Ho, C. T.; Huang, Q. Development of organogel-derived capsaicin nanoemulsion with improved bioaccessibility and reduced gastric mucosa irritation. J. Agric. Food. Chem. 2016, 64, 4735-4741. 19. Lee, E. J.; Jeon, M. S.; Kim, B. D.; Kim, J. H.; Kwon, Y. G.; Lee, H.; Lee, Y. S.; Yang, J. H.; Kim, T. Y. Capsiate inhibits ultraviolet B-induced skin inflammation by inhibiting Src family kinases and epidermal growth factor receptor signaling. Free Radic. Biol. Med. 2010, 48, 1133-1143. 20. Lu, H. F.; Chen, Y. L.; Yang, J. S.; Yang, Y. Y.; Liu, J. Y.; Hsu, S. C.; Lai, K. C.; Chung, J. G. Antitumor activity of capsaicin on human colon cancer cells in vitro and Colo 205 tumor xenografts in vivo. J. Agric. Food Chem. 2010, 58, 12999-3005. 21. Ziglioli, F.; Frattini, A.; Maestroni, U.; Dinale, F.; Ciuffreda, M.; Cortellini, P. Vanilloidmediated apoptosis in prostate cancer cells through a TRPV-1 dependent and a TRPV-1-independent mechanism. Acta Biomed. 2009, 80, 13-20. 22. Thoennissen, N. H.; O'Kelly, J.; Lu, D.; Iwanski, G. B.; La, D. T.; Abbassi, S.; Leiter, A.; Karlan, B.; Mehta, R.; Koeffler, H. P. Capsaicin causes cell-cycle arrest and apoptosis in ER-positive and -negative breast cancer cells by modulating the EGFR/HER-2 pathway. Oncogene 2010, 29, 285-296. 23. Huh, H. C.; Lee, S. Y.; Lee, S. K.; Park, N. H.; Han, I. S., Capsaicin induces apoptosis of cisplatin-resistant stomach cancer cells by causing degradation of cisplatininducible aurora-A protein. Nutr. Cancer 2011, 63, 1095-1103. 24. O'Neill, J.; Brock, C.; Olesen, A. E.; Andresen, T.; Nilsson, M.; Dickenson, A. H. Unravelling the mystery of capsaicin: a tool to understand and treat pain. Pharmacol. Rev. 2012, 64, 939-971. 25. Luo, X. J.; Peng, J.; Li, Y. J., Recent advances in the study on capsaicinoids and capsinoids. Eur. J. Pharmacol. 2011, 650, 1-7. 26. Chen, L.; Kang, Y. H., Anti-inflammatory and antioxidant activities of red pepper (Capsicum annuum L.) stalk extracts: Comparison of pericarp and placenta extracts. J. Funct. Foods 2013, 5, 1724-1731. 27. Yoshioka, M.; Imanaga, M.; Ueyama, H.; Yamane, M.; Kubo, Y.; Boivin, A.; St-Amand, J.; Tanaka, H.; Kiyonaga, A. Maximum tolerable dose of red pepper decreases fat intake independently of spicy sensation in the mouth. Br. J. Nutr. 2004, 91, 991995. 28. Lu, M.; Cao, Y.; Ho, C. T.; Huang, Q. The enhanced anti-obesity effect and reduced gastric mucosa irritation of capsaicin-loaded nanoemulsions. Food Funct. 2017, 8, 1803-1809. 29. Kang, J. H.; Goto, T.; Han, I. S.; Kawada, T.; Kim, Y. M.; Yu, R. Dietary capsaicin reduces obesity-induced insulin resistance and hepatic steatosis in obese mice fed a high-fat diet. Obesity (Silver Spring) 2010, 18, 780-787. 30. Kang, J.-H.; Tsuyoshi, G.; Le Ngoc, H.; Kim, H.-M.; Tu, T. H.; Noh, H.-J.; Kim, C.-S.; Choe, S.-Y.; Kawada, T.; Yoo, H.; Yu, R. Dietary capsaicin attenuates metabolic dysregulation in genetically obese diabetic mice. J. Med. Food 2011, 14, 310–315. 31. Jeong, K. Y.; Seong, J. Neonatal capsaicin treatment in rats affects TRPV1-related noxious heat sensation and circadian body temperature rhythm. J. Neurol. Sci. 2014, 341, 58-63. 32. Xie, W.; Wang, W.; Su, H.; Xing, D.; Pan, Y.; Du, L. Effect of ethanolic extracts of Ananas comosus L. leaves on insulin sensitivity in rats and HepG2. Comp. Biochem. Physiol. C. Toxicol. Pharmacol. 2006, 143, 429-435.

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

428 429 430 431 432 433 434 435 436 437 438 439 440 441 442 443 444 445 446 447 448 449 450 451 452 453 454 455 456 457 458 459 460 461

33. Livak, K. J.; Schmittgen, T. D. Analysis of relative gene expression data using realtime quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402-408. 34. Bailey, C. J.; Turner, S. L. Glucosamine-induced insulin resistance in L6 muscle cells. Diabetes Obes. Metab. 2004, 6, 293-8. 35. Zhu, D.; Wang, Y.; Du, Q.; Liu, Z.; Liu, X. Cichoric acid reverses insulin resistance and suppresses inflammatory responses in the glucosamine-induced HepG2 cells. J. Agric. Food Chem. 2015, 63, 10903-10913. 36. Barazzoni, R.; Gortan Cappellari, G.; Ragni, M.; Nisoli, E. Insulin resistance in obesity: an overview of fundamental alterations. Eating Weight Disorders : EWD 2018, 23, 149-157. 37. Fazakerley, D. J.; Minard, A. Y.; Krycer, J. R.; Thomas, K. C.; Stockli, J.; Harney, D. J.; Burchfield, J. G.; Maghzal, G. J.; Caldwell, S. T.; Hartley, R. C.; Stocker, R.; Murphy, M. P.; James, D. E. Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation. J. Biol. Chem. 2018, 293, 7315-7328. 38. McMurray, F.; Patten, D. A.; Harper, M. E. Reactive oxygen species and oxidative stress in obesity-recent findings and empirical approaches. Obesity (Silver Spring) 2016, 24, 2301-2310. 39. Nulton-Persson, A. C.; Szweda, L. I. Modulation of mitochondrial function by hydrogen peroxide. J. Biol. Chem. 2001, 276, 23357-23361. 40. Anderson, E. J.; Lustig, M. E.; Boyle, K. E.; Woodlief, T. L.; Kane, D. A.; Lin, C. T.; Price, J. W., 3rd; Kang, L.; Rabinovitch, P. S.; Szeto, H. H.; Houmard, J. A.; Cortright, R. N.; Wasserman, D. H.; Neufer, P. D. Mitochondrial H2O2 emission and cellular redox state link excess fat intake to insulin resistance in both rodents and humans. J. Clin. Invest. 2009, 119, 573-581. 41. Qi, G.; Wu, W.; Mi, Y.; Shi, R.; Sun, K.; Li, R.; Liu, X.; Liu, X. Tea polyphenols direct Bmal1-driven ameliorating of the redox imbalance and mitochondrial dysfunction in hepatocytes. Food Chem. Toxicol. 2018, 122, 181-193. 42. Buhr, E. D.; Takahashi, J. S. Molecular components of the mammalian circadian clock. Handb. Exp. Pharmacol. 2013, 217, 3-27. 43. Takahashi, J. S. Transcriptional architecture of the mammalian circadian clock. Nat. Rev. Genet. 2017, 18, 164-179. 44. Khapre, R. V.; Kondratova, A. A.; Susova, O.; Kondratov, R. V. Circadian clock protein BMAL1 regulates cellular senescence in vivo. Cell Cycle 2011, 10, 4162–4169.

462 463 464 465 466 467

24

ACS Paragon Plus Environment

Page 24 of 25

Page 25 of 25

Journal of Agricultural and Food Chemistry

468 469

Graphic Table of Contents

470

471

25

ACS Paragon Plus Environment