Cichoric Acid Prevents Free-Fatty-Acid-Induced Lipid Metabolism

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Bioactive Constituents, Metabolites, and Functions

Cichoric Acid Prevents Free Fatty Acids Induced Lipid Metabolism Disorders via Regulating Bmal1 in HepG2 Cells Rui Guo, Beita Zhao, Yijie Wang, Dandan Wu, Yutang Wang, Yafan Yu, Yuchen Yan, Wentong Zhang, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02147 • Publication Date (Web): 23 Jul 2018 Downloaded from http://pubs.acs.org on July 24, 2018

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Journal of Agricultural and Food Chemistry

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Cichoric Acid Prevents Free Fatty Acids Induced Lipid

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Metabolism Disorders via Regulating Bmal1 in HepG2 Cells

3

Rui Guo, Beita Zhao, Yijie Wang, Dandan Wu, Yutang Wang, Yafan Yu, Yuchen Yan,

4

Wentong Zhang, Zhigang Liu*, Xuebo Liu*

5

Laboratory of Functional Chemistry and Nutrition of Food, College of Food Science

6

and Engineering, Northwest A&F University, Yangling, China

7

* Corresponding authors:

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Prof. Xuebo Liu, College of Food Science and Engineering, Northwest A&F

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University, Xinong Road 22, Yangling 712100, China. Tel: +862987092492; Fax:

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+86-029-87092325; E-mail: [email protected]

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Dr. Zhigang Liu, College of Food Science and Engineering, Northwest A&F

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University, Xinong Road 22, Yangling 712100, China. Tel: +862987092817; Fax:

13

+86-029-87092817; E-mail: [email protected]

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ACS Paragon Plus Environment


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ABSTRACT

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Cichoric acid (CA), a polyphenol component from Echinacea

16

purpurea, exhibits preventive effects on liver lipid metabolism disorders

17

in obesity. This research aimed to determine the role of circadian rhythm

18

signaling during the process of CA attenuated lipid accumulation in

19

hepatocytes. In the current study, CA treatments improved cell

20

morphology changes and hepatic lipid levels, which were triggered by

21

free fatty acids (2:1, oleate: palmitate) in a dose dependent way. Besides,

22

CA (200 µM) regulated the circadian rhythm expressions of clock genes

23

and the relatively shallow daily oscillations. Moreover, silencing Bmal1

24

significantly blocked p-Akt/Akt pathway to 80.1% ± 1.5% and

25

p-GSK3β/GSK3β pathway to 64.7% ± 2.8% (p < 0.05), and elevated the

26

expressions of FAS and ACC to 122.4% ± 5.6% and 114.9% ± 1.7% in

27

protein levels (p < 0.05), 166.5% ± 18.5% and 131.4% ± 5.5% in mRNA

28

levels additionally (p < 0.05). Therefore, our results demonstrated that

29

CA has a Bmal1 resistance to lipid accumulation by enhancing the

30

Akt/GSK3β signaling pathways and modulating the downstream

31

expressions related to lipid metabolism, which indicated that CA might be

32

served as a natural and promising non-alcoholic fatty liver diseases

33

(NAFLD) modulator.

34 35

Keywords: Cichoric acid / Circadian rhythm / BMAL1 / lipid metabolism / Nonalcoholic fatty liver disease 2


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3



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1 Introduction

38

Circadian rhythm is an internal 24 hours oscillation of various

39

physiological processes and behaviors. It is synchronized with daylight

40

and dark cycles in a wide range of species 1. Circadian locomotor output

41

cycles kaput (CLOCK) and brain and muscle arnt-like protein 1 (BMAL1)

42

are two core circadian transcription factors that form heterodimers. They

43

combine with E-box elements in the gene promoters, including two major

44

oscillators (Per and Cry), and induce their expressions 2. Circadian

45

rhythm regulates a large amount of fundamental metabolic pathways and

46

multiple metabolites, and controls nearly 10-20% transcriptome and

47

proteome, which includes a large number of metabolic genes and proteins

48

in the peripheral tissues

49

which chronic circadian rhythm disruption caused by irregular lifestyle or

50

high-fat diet-induced obesity increases the risk of metabolic diseases or

51

exacerbates pathological states 5. Lipid metabolism disorders in liver lead

52

to the pathogenesis of non-alcoholic fatty liver diseases (NAFLD), which

53

could further develop into non-alcoholic steatohepatitis. NAFLD is a very

54

common chronic liver disease among people’s daily life, which is in

55

connection with the uptake of free fatty acid (FFA) as well as increased

56

delivery in the liver. The causes may be due to excessive dietary intake

57

and the synthesis of triglycerides (TG), the failure of extremely low

58

density lipoprotein synthesis, and TG output, or FFA accumulation due to

3, 4

. Epidemiological studies have illustrated

4


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6, 7

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the oxidative damage of the mitochondrial β-oxidation in liver

.

60

Hepatic steatosis is associated with several genetic variants of clock

61

genes 8. Knocking down Dec1, which is a critical regulator of circadian

62

rhythm, could inhibit Clock-Bmal1 activity and the accumulation of

63

hepatic triglycerides induced by the increased expression of Srebp1c 9.

64

Related haplotypes and Clock gene variants are associated with

65

susceptibility to NAFLD in humans

66

literature showed that NAFLD was correlated with Bmal1 mutation,

67

therefore, in this research we provided a potential mechanism that

68

BMAL1 is involved in the treatment of CA on lipid metabolism disorder.

10

. Nevertheless, there is no direct

69

CLOCK/BMAL1 binds to the SIRT1 promoter and regulates hepatic

70

insulin sensitivity 5. At the same time, BMAL1 plays a very important

71

role on regulating adipose differentiation and lipogenesis in mature

72

adipocytes 11. And also, the compensatory growth of β-cell caused by the

73

increased insulin related to diet-induced obesity are controlled by

74

BMAL1

75

fatty acid synthesis and β-oxidation pathways

76

disorders caused by high fat diet may be the target of NAFLD.

77

12

. Evidence showed that core circadian genes are involved in 13

. Therefore, circadian

Multiple researches have showed that many phenolic compounds 14-18

78

have remarkable effects on lipid metabolism in liver

79

(CA) is a vital active ingredient in basil, Echinacea purpurea, and chicory,

80

which is also a derivative of caffeic acid 19. Ample evidence has shown 5


. Cichoric acid


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that CA induced apoptosis in 3T3-L1 preadipocytes through PI3K/Akt

82

signal pathway

83

related proteins and enhanced the antioxidant defense system by

84

diminishing liver injury, inflammation and insulin resistance, efficiently

85

inhibited the obesity mice by high-fat diet

86

glucose homeostasis by reducing the expression of ACC (the major

87

regulatory enzyme of fatty acid biosynthesis) in glucosamine-induced

88

HepG2 cell, which indicated its anti-obesity. However, the specific

89

mechanisms of CA regulating lipid metabolism involved in the clock

90

genes is still unclear.

20

. CA also regulated the secretions of fat generation

21

. CA regulated hepatic

91

Current work has elucidated that CA treatments can protect HepG2

92

cells in high fatty acid condition by (i) investigating the effects of CA on

93

FFA-induced morphology changes, mitochondrial dysfunction, and

94

hepatic lipid profile; (ii) determining the effects of CA on circadian

95

misalignment and the relatively shallow daily oscillations of the clock

96

genes; (iii) characterizing the effects of CA defeated lipid metabolism

97

disorders via Akt/GSK3β pathways in a Bmal1-dependent efficacy, and

98

(iv) uncovering the important role of BMAL1 on lipid drop accumulation.

99

In

summary,

it

provides

new

insights

into

regulating

100

lipogenesis/adipogenesis by CA, which is relevant to circadian clock.

101

2 Methods and Materials

102

2.1 Chemicals 6


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RPMI-1640 medium was purchased from Hyclone Company (the

104

United States). Fetal bovine serum was purchased from Thermo Fisher

105

Scientific (Shanghai, China). Horse serum was purchased from Hyclone

106

Company (the United States). Penicillin-streptomycin was purchased

107

from Beyotime (Shanghai, China). Oleic acid was purchased from

108

Sigma-Aldrich (St. Louis, MO, the United States). Palmitic acid was

109

purchased from Sigma-Aldrich (St. Louis, MO, the United States).

110

Bovine aerum albumin was purchased from Sigma (the United States).

111

Cichoric Acid (purity ≥98%), extracted from Echinacea purpurea, was

112

purchased from Pufei De Biotech Company (Chengdu, Sichuan, China).

113

3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide was

114

purchased from Wolsen Biotechnology

115

2′,7′-Dichlorodi-hydrofluorescein

116

Beyotime Institute of Biotechnology (Jiangsu, China). PrimeScriptTM RT

117

Master Mix reverse transcription kit was purchased from TaKaRa (Dalian,

118

China). SYBR green PCR kit was purchased from TaKaRa (Dalian,

119

China). CLOCK (ab93804) and BMAL1 (ab93806) antibodies, were

120

purchased from Abcam (Abcam, Cambridge, MA). PPARγ (H-100),

121

PGC-1α (H-300), SREBP-1 (C20), β-actin (SC-47778) and GAPDH

122

(SC-25778) antibodies, were purchased from Santa Cruz Biotechnology

123

(Santa Cruz, CA, the United States). SIRT1 (1F3) (8469), AKT (4060),

124

p-AKT (ser473) (8242), GSK3β (D5C5Z), p-GSK3β (Ser9) (9336), FAS

diacetate

(Xi'an,

was

7


Shaanxi, China). purchased

from


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(C20G5) and ACC antibodies, were purchased from Cell Signaling

126

Technology Company (Shanghai, China).

127

2.2 Culture and treatments of HepG2 cells

128

HepG2 cells were provided by the Fourth Military Medical

129

University (Shaanxi, China) and cultured in DMEM medium (Hyclone

130

Company, the United States) added with 10% fetal bovine serum

131

(Thermo

132

penicillin-streptomycin at 37 degree centigrade, and supplemented 5%

133

carbon dioxide in a humid environment. Oleic acid (OA) (Sigma-Aldrich,

134

St. Louis, MO, the United States) and palmitic acid (PA) (Sigma-Aldrich,

135

St. Louis, MO, the United States) were conjugated to bovine serum

136

albumin (BSA) (Sigma, the United States) to dissolve in the culture

137

media evenly. 100 µM FFA (OA:PA = 2:1) was made as previously

138

described 22. CA (Pufei De Biotech Company Chengdu, Sichuan, China)

139

was dissolved in DMSO, and the cells were co-treated with various

140

concentrations of CA (0, 50, 100, 200 µM) for 24 hours. In the control

141

group, the same amount of DMSO and 0.2 % BSA were added. Before

142

drug treatments, the cells were starved for 2 hours with serum-free

143

medium.

144

2.3 Synchronization of HepG2 by serum shock

Fisher

Scientific,

Shanghai,

China)

and

100

µg/mL

145

HepG2 cells were serum-shocked (50% horse serum (Hyclone

146

Company, the United States)) for 2 hours and treated with FFA (100 µM) 8


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and CA (200 µM) for 24 hours. Then, the mRNA and protein analysis

148

were performed at the interval of 8 hours between 28 hours’ time points

149

to 52 hours’ time points.

150

2.4 The transfection of HepG2 cells

151

During transfection, the si-Control, si-Bmal1 or si-Clock plasmids

152

were transfected into cells cultured in 6-well plates using Lipofectamine

153

2000 (Invitrogen). The primer sequences were used: si-Control, forward,

154

5′-

155

AACCAUGUAGUUGAGGUCATT

156

GGCACAUCGUGUUAUGAAUTT

157

AUUCAUAACACGAUGUGCCTT

158

GCUGCAGUAACUACAUUCATT

159

UGAAUGUAGUUACUGCAGCTT -3′. Following transfection for 48

160

hours, the cells were incubated with FFA (100 µM) and CA (200 µM) for

161

24 hours and then collected for further analysis.

162

2.5 Cell viability

UGACCUCAACUACAUGGUUTT -3′;

-3′, si-Bmal1, -3′,

-3′;

reverse,

5′-

forward,

reverse,

si-Clock, -3′,

5′-

forward,

reverse,

5′-

5′5′-

163

HepG2 cells were seeded in 96-well plate with a density of 1×106

164

cells/mL, and cultured for about 24 hours. After treatments, the medium

165

was

166

3-(4,5-dimethy-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT)

167

(Wolsen Biotechnology Xi'an, Shaanxi, China) solution for 4 hours. After

168

the insoluble formazan crystals being dissolved by DMSO, the

replaced

by

9


0.5

mg/mL


169

absorbance were measured with a microplate reader (Bio-Rad, Hercules,

170

CA) at 560 nm.

171

2.6 Analyses of HepG2 cells lipid levels

172

The HepG2 cells' harvest, treatments and detection were performed 23

173

as described previously

. The reduced glutathione (GSH, A006-2),

174

superoxide dismutase (SOD, A001-3), malondialdehyde (MDA, A003-4),

175

HDL cholesterol (HDL-C, A112-1), LDL cholesterol (LDLC, A113-1),

176

total cholesterol (TC, A111-1), triglycerides (TG, A110-1) activity levels

177

were measured with enzymatic assay kits (Nanjing Jiancheng

178

Bioengineering Institute, Nanjing, China). The amount of protein was

179

measured by bicinchoninic acid kit (Thermo Fisher, the United States).

180

2.7 JC-1, DCF and Oil red O staining

181

Mitochondrial membrane potential was determined using JC-1 dye.,

182

after treatment, the cells were treated with 5 g/mL JC-1 for 30 minutes at

183

37 degree centigrade, rinsed two times with PBS, and then observed by a

184

fluorescence microscope (Olympus IX71, Tokyo, Japan).

185

As described in the previous research

24

, cells were rinsed by PBS

186

three times, then incubated in serum free medium added with DCF dyes

187

(Beyotime Institute of Biotechnology, Jiangsu, China) for 60 minutes in

188

37 degree centigrade. The cell oxidation state was observed by a

189

fluorescence microscope.

190

The method of oil red O staining is as described previously 25. 0.2 % 10


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of oil red O in isopropyl alcohol was filtered. After incubation, cells were

192

fixed with 3.7% paraformaldehyde in PBS for 2 minutes, then staining

193

with oil red O for 30 minutes. Stained TGs were pictured by an inverted

194

fluorescence microscope. After being dissolved by isopropyl alcohol, TG

195

content was normalized for total protein content and measured by a

196

microplate reader (Bio-Rad, Hercules, CA) at 490 nm.

197

2.8 RNA Isolation and Real Tine-qPCR

198

As mentioned above, the total mRNA was extracted by the

199

extraction kit (TaKaRa, MiniBEST Universal RNA Extraction Kit, Dalian,

200

China) 26. After evaluation of RNA purity, the PrimeScriptTM RT Master

201

Mix reverse transcription kit (TaKaRa, Dalian, China) was used to

202

reverse RNA into cDNA and the SYBR green PCR kit (TaKaRa, Dalian,

203

China) and the CFX96TM real-time system (Bio Rad, CA) to quantify the

204

expression. As following show of Table 1 is the gene specific human

205

primers

206

expression, which were normalized to β-actin.

207

2.9 Western Blots and Antibodies

27-29

. The method of 2-△△Ct was used to calculate the relative gene

208

The treated HepG2 cells were dissolved in the SDS sample buffer.

209

SDS-PAGE analysis and western blots detection were carried out through

210

previous studies from our laboratory

211

PGC-1α, SREBP-1, SIRT1, AKT, p-AKT (ser473), GSK3β, p-GSKβ

212

(Ser9), FAS and ACC were used as primary antibodies, and the dilution

30, 31

. CLOCK, BMAL1, PPARγ,

11



213

rates were 1:1000. β-actin and GAPDH were used as reference proteins,

214

and the dilution rates were 1:5000. Quantity One 4.6.2 software was used

215

to performed densitometric analysis of western blots.

216

2.10 Statistical Analysis

217

Each sample need to take at least three independent experiments and

218

all of the data need to presented as the means ± SD. The unidirectional

219

factorial analysis of variance (ANOVA) by Graphpad Prism 6 software

220

was used to analyze the significant difference between the control and the

221

treatment samples. When p value is less than 0.05, the data is considered

222

statistically significant and marked with different superscripts.

223

3 Results

224

3.1 Effects of CA on FFA induced morphology changes, lipid droplets

225

formation, mitochondrial function and oxidized status in HepG2

226

cells.

227

To explore the possible regulatory mechanisms, HepG2 cells were

228

stimulated with 100 µM FFA and various concentrations of CA (0, 50,

229

100, 200 µM) for 24 hours, after which, the morphology was observed by

230

an optical microscope. The morphology of cells changed obviously in

231

FFA group when compared with the control group, such as decreased cell

232

adherence, reduced cell number and cell shrinkage. Nevertheless, these

233

changes were improved by CA treatments in a notable dose-dependent

234

pattern (Fig. 1A and B), and high concentration treatments of CA (100 12


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µM and 200 µM) successfully restored HepG2 cell viability by 10.5%

236

and 27.0% (p < 0.05), compared to FFA group. Previous experiments

237

showed that 200 µM CA had no effect on cell morphology and viability

238

(Fig. S1) 32. Oil Red O staining indicated that treatment with CA (50, 100,

239

and

240

accumulation. Lower concentrations treatments of CA (50 µΜ and 100

241

µM) had no significant inhibition (Fig. 1A and C), however, higher

242

concentration treatments of CA (200 µM) remarkably decreased lipid

243

accumulation to 38.4%. 200 µM CA co-treatments showed a significant

244

increase by 16.6% in mitochondrial membrane potential compared to FFA

245

group by using JC-1 staining (Fig. 1A and D).

200

µM)

dose-dependently

suppressed

intracellular

lipid

246

200 µM CA co-treatments also showed a 49.1% decrease in ROS

247

concentration compared to FFA group by using DCF staining (Fig. 1A

248

and E). Consistently, Glutathione (GSH) is one of the endogenous

249

antioxidants, which is capable for preventing the damage to cell

250

components by reactive oxygen species 33. Superoxide dismutase (SOD)

251

is an enzyme that protects the tissue from the harmful effects of

252

superoxide radicals 34. Malondialdehyde (MDA) is a marker for oxidative

253

stress. As showed in Fig. 1F-H, cellular oxidized status (GSH, MDA,

254

SOD) were recovered by 200 µM CA co-treatments compared to FFA

255

group significantly. However, it's worth noting that SOD activities was

256

elevated in FFA group which may presented more superoxide was 13



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30, 35

257

generated, that was opposite to previous studies

. The underlying

258

mechanism might be the high level of oxygen free radicals induced by

259

FFA, made HepG2 cells enhanced the antioxidant capacity inductively,

260

that enhanced the level of SOD.

261

3.2 Effects of CA on FFA induced lipids levels of HepG2 cells.

262

TG, TC, LDL-C and HDL-C level were closely related to lipid

263

metabolism, which could reflect the liver function. As presented in Fig. 2,

264

FFA substantially increased cellular TG, TC, and LDL-C when

265

comparing with control group, however, CA treatments markedly

266

diminished TG, TC and LDL-C levels in a dose-dependent way.

267

Interestingly, HDL-C level was markedly decreased after FFA treatment,

268

while it has no significant change after CA treatment.

269

3.3 Effects of CA on FFA induced circadian misalignment under high

270

fatty acid conditions.

271

Recent studies indicated that HFD feeding impairs hepatic molecular 36

272

circadian rhythm in mice

273

hepatocytes steatosis and circadian rhythm, HepG2 cells were treated

274

with FFA (OA: PA =2:1) to induce lipid accumulation as described

275

previously

276

200 µM CA administration, were 64.9% and 75.1% higher than FFA

277

group (Fig. 4A).

278

37

. To determine the relationship between

. The protein level of CLOCK and BMAL1 recovered by

To determine whether CA exposure could improve the mRNA 14


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279

oscillation of clock genes of synchronized HepG2 cells in FFA, after 2

280

hours of serum shock, HepG2 cells were continuously exposed to 200 µM

281

CA for 24 hours. As exposed in Fig. 3A-G, strong rhythmic expressions

282

of Bmal1, Clock, Per1, Per2, Cry2 and Reverbα were observed in control

283

group. The mRNA levels of Clock and Bmal1 were similar to each other

284

in their cyclic oscillations, and the two genes both reached the highest

285

level at ZT36 to ZT44 (Fig. 3A and B). Conversely, Per1 and Per2, as the

286

negative regulator of circadian rhythm, which was conversed to the peak

287

of Clock/Bmal1 (Fig. 3C and D). CA treatments (200 µM) also recovered

288

Cry1/Cry 2 and Reverbα in Fig. 3E, F and G. As expected, the daily

289

expressions of Clock, Bmal1, Per2 and Cry2 were disordered, and CA

290

efficiently reversed the relatively shallow daily oscillations of the clock

291

genes triggered by FFA, rising their peak ratios of the mRNA levels.

292

These data indicated that the downregulating of clock genes was

293

improved by CA under the condition of hepatic lipid accumulation.

294

3.4 Effects of CA on FFA induced hepatic lipids metabolism

295

imbalance via mediating Akt/GSK3β pathways.

296

To

evaluate

whether

CA supplementation

prevented

lipid

297

metabolism disordering effectively in HepG2 cells, the activation of Akt,

298

GSK3β and relative proteins were determined. 200 µM CA co-treatments

299

dramatically reduced the lipogenesis protein expression of FAS, ACC and

300

SREBP-1, decreased 60.8%, 34.0% and 41.6% respectively compared to 15



301

FFA group (Fig. 4A). The Akt/GSK3β signaling pathway plays an

302

essential role in lipid metabolism. Akt regulates adipogenesis via the

303

phosphorylation and inactivation of substrates, such as GSK3β, which

304

directly regulates PPARα 35. 200 µM CA group showed a significant 89.7%

305

increase in the phosphorylation of Akt, simultaneously, a significant 72.0%

306

increase in the p-GSK3β/GSK3β (Fig. 4B). While, Apoa4, which

307

enhanced TG secretion from the liver and inhibit glucose production 38,

308

was not affected, neither did PPARγ or SIRT1 (Fig. S3 and S5).

309

3.5 Effects of CA on FFA induced lipid metabolism disorder by

310

regulating the circadian clock in protein levels.

311

To further study whether CA can maintain liver lipid homeostasis

312

through circadian clock, HepG2 cells were transfected with si-Bmal1 or

313

si-Clock for 48 hours. BMAL1 and CLOCK were notably inhibited to

314

35.3% and 37.4% in protein levels (Fig. 5A). SIRT1 primarily activates

315

PGC-1α, a key coactivator for PPARα signaling, reducing the expression

316

of fatty acid mitochondria β-oxidation genes in the liver of fasted mice 39,

317 318 319

40

. After deacetylating by SIRT1, LXRα is beneficial for inhibiting

intestinal cholesterol uptake and promoting reverse cholesterol transport 41

. As presented in Fig. 5B, SIRT1 and PGC-1α controlled fatty acid

320

β-oxidation, and CA co-treatments significantly recovered the decrease

321

induced by FFA. However, both si-Bmal1 and si-Clock suppressed the

322

improvement of PGC-1α and SIRT1 by CA. Additionally, silencing 16


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Bmal1 prevented the recovery of FAS and ACC. Neither si-Bmal1 nor

324

si-Clock played a part in PPARγ (Fig. S4). While, the phosphorylation of

325

AKT and GSK3β were markedly impaired by silencing Bmal1, silencing

326

Clock had no effect (Fig. 5C). Consistently, silencing Bmal1 blunted the

327

lipogenesis and fatty acid β-oxidation in the presence of CA.

328

3.6 Effects of CA on FFA induced lipid metabolism disorder by

329

regulating the circadian clock in mRNA levels.

330

Bmal1 or Clock also participated in the program which CA restored

331

lipid metabolism misalignment induced by FFA in mRNA levels. Bmal1

332

and Clock were remarkably repressed to 34.3% and 44.3% in mRNA

333

levels. (Fig. 6A). Pparα which promots the lipolysis of triglycerides into

334

free fatty acids. As showed in Fig. 6B, si-Bmal1 statistically significantly

335

down-regulated CA-induced increase of Pparα expression by 39.4% (p

Cichoric Acid Prevents Free-Fatty-Acid-Induced Lipid Metabolism

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