Cichoric Acid Reverses Insulin Resistance and ... - ACS Publications

Subscriber access provided by UNIV OF NEBRASKA - LINCOLN

Article

Cichoric acid reverses insulin resistance and suppresses inflammatory responses in the glucosamine-induced HepG2 cells Di Zhu, Yutang Wang, Qingwei Du, Zhigang Liu, and Xuebo Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04533 • Publication Date (Web): 21 Nov 2015 Downloaded from http://pubs.acs.org on December 13, 2015

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 free 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 accessible to all readers and 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.

Journal of Agricultural and Food Chemistry 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 40

Journal of Agricultural and Food Chemistry

1

Cichoric Acid Reverses Insulin Resistance and Suppresses Inflammatory

2

Responses in the Glucosamine-induced HepG2 Cells

3

Di Zhu#†, Yutang Wang#†, Qingwei Du§, Zhigang Liu#, Xuebo Liu#*

4 5 #

6

Yangling 712100, China

7 8

College of Food Science and Engineering, Northwest A&F University,

§

Functional Food Engineering and Technology Research Center of Shaanxi Province, Xi’an 710054, China

9 10 11

†

Authors Di Zhu and Yutang Wang contributed equally to this work

12 13

* Correspondence to: Prof. Xuebo Liu, College of Food Science and Engineering,

14

Northwest A&F University, 28. Xi-nong Road, Yangling 712100, China.

15

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

16 17 18 19 20 21 22

ACS Paragon Plus Environment


23

ABSTRACT: Cichoric acid, a caffeic acid derivative found in Echinacea purpurea,

24

basil and chicory, has been reported to have bioactive effects, such as,

25

anti-inflammatory, anti-oxidant, and preventing insulin resistance. In this study, to

26

explore the effects of CA on regulating insulin resistance and chronic inflammatory

27

responses, the insulin resistance model was constructed by glucosamine in HepG2

28

cells. CA stimulated glucosamine-mediated glucose uptake by stimulating

29

translocation of the glucose transporter 2. Moreover, the production of reactive

30

oxygen, the expression of COX-2 and iNOS, and the mRNA levels of TNF-α and IL-6

31

were attenuated. Furthermore, CA was verified to promote glucosamine-mediated

32

glucose uptake and inhibited inflammation through PI3K/Akt, NF-κB and MAPK

33

signaling pathways in HepG2 cells. These results implied that CA could increase

34

glucose uptake, improve insulin resistance and attenuate glucosamine-induced

35

inflammation, suggesting that CA is a potential nature nutraceutical with anti-diabetic

36

properties and anti-inflammatory effects.

37

KEYWORDS: Cichoric acid, Insulin resistance, GLUT2, PI3K/Akt, Inflammatory

38

response

39 40 41 42 43 44


Page 2 of 40

Page 3 of 40


45

Introduction

46

Diabetes has gradually developed into a global disease with a serious impact on

47

human health.1 Accumulating evidences suggest insulin resistance is the common

48

basis of metabolic syndrome, such as type II diabetes, obesity, hypertension,

49

hyperlipidemia, and coronary heart disease.2 Liver is a target organ for insulin action

50

and plays an essential role in the systemic insulin resistance and inflammation.3.

51

Insulin resistance is known to play a critical role in further impair insulin signaling

52

and sensitize the liver to inflammatory injury, such as non-alcoholic fatty liver disease

53

(NAFLD) and non-alcoholic steatohepatitis (NASH),4 induced by a variety of stimuli5.

54

The latter progresses with hyperinsulinemia and inhibition of the insulin receptor

55

substrate (IRS).6 Our previous study also showed that dietary-induced hepatic insulin

56

resistance elicited serious fatty liver and inflammatory responses through inactivating

57

IRS-1/Akt pathway and stimulating MAPK and NF-κB pathway7.

58

Chronic inflammation is always accompanied by insulin resistance.8 Through the

59

mediation of pro-inflammatory cytokines, such as TNF-α and IL-6 mediation,

60

especially.9, 10 Studies have also indicated that TNF-α induced insulin resistance by

61

activating p38.11 JNK and p38 MAPK are important in the pathogenesis of insulin

62

resistance. In the obesity and insulin resistance patient, the p38 levels of the liver and

63

adipose tissue are reported to have been increased.12 JNK, a serine kinase, affects the

64

normal phosphorylation of IRS-1 tyrosine and hinders the insulin signal transduction,

65

leading to insulin resistance.13 The inflammatory responses induced by the activation

66

of NF-κB makes contribute to insulin resistance in liver.7 Thus, the key inflammatory



67

pathways, NF-κB and MAPKs, have important influence in the process of insulin

68

resistance.

69

Many plant extracts play positive roles in improving the insulin resistance, by

70

suppressing inflammation, such as salicylate14 and eriodictyol.15 Cichoric acid (CA),

71

which is a caffeic acid derivative, is a very important immunologically active

72

ingredient in Echinacea purpurea16, basil17 and chicory18. Echinacea is one of the

73

most popular natural health products (NHPs) in North America16, and the content of

74

CA in Echinacea purpurea roots was 16.80–24.30mg/g19. Studies have reported that

75

CA can enhance immune and anti-inflammatory properties20 and facilitate the glucose

76

uptake in L6 muscle cells21. These results suggest that CA is a potential nature

77

nutraceutical in improving insulin resistance. However, the possible mechanisms

78

underlying the effect of CA on restoring insulin resistance by suppressing

79

inflammation is rarely reported.

80

To simulate insulin resistance, glucosamine is a suitable inducer. Glucosamine

81

affects the regulation of fat accumulation and glucose absorption.22, 23The HepG2

82

cells which are derived from human embryonic liver tumor cells, retains many

83

biological characteristics of liver cells, with its surface high affinity expression of

84

insulin receptor to meet the standards required by a typical insulin receptor.24 This

85

study was designed to investigate the effect of CA against the development of insulin

86

resistance and inflammatory responses using HepG2 cells and elucidate the possible

87

mechanisms. To this end, we assessed the ability of CA on the glucose uptake and

88

insulin sensitivity and GLUT2 translocation via PI3K/Akt signaling, and the


Page 4 of 40

Page 5 of 40


89

intervention of CA on the inflammatory response via MAPKs and I-κB/NF-κB

90

signaling in the HepG2 cells with glucosamine-induced insulin resistance.

91

Materials and Methods

92

Reagents and antibodies

93

Cichoric acid (purity ≥ 98 %) was obtained from Chengdu Pufei De Biotech Co.,

94

Ltd (Chengdu, Sichuan, China), which was extracted from Echinacea purpurea.

95

RPMI 1640 medium and fetal bovine serum (FBS) were obtained from Thermo Fisher

96

Scientific. (Shanghai, China). MTT (purity ≥93%) was purchased from Wolsen

97

Biotechnology. Insulin and 2', 7'-dichlorofluorescin diacetate (DCFH-DA) were

98

purchased from Sigma Chemical Co. (St. Louis, MO, USA). All the inhibitors were

99

purchased from Selleck (Shanghai, China). Glucosamine (purity ≥ 99%) was provided

100

by Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). The Glucose

101

uptake analysis kit (E1010) from Applygen (Beijing, China). All other chemicals were

102

analytical grade.

103

The

primary antibodies

against COX-2,

iNOS,

GAPDH,

Lamin

B,

104

Na+/K+-ATPase α1, GLUT2 and horseradish peroxidase-conjugated secondary

105

antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

106

NF-κB p65, p-p44/42 MAPK (ERK1/2), p44/42 MAPK (ERK1/2), p-SAPK/JNK

107

(Thr183/Tyr185), SAPK/JNK, p-p38 MAPK, p38 MAPK, p-Akt and Akt were

108

purchased from Cell Signaling Technology Company (Shanghai, China). Alexa Fluor

109

488 conjugated Donkey anti-Goat IgG (H+L) was got from Invitrogen (Shanghai,

110

China).



111

Cell culture and treatment

112

HepG2 cells were obtained from the Fourth Military Medical University (Xi’an,

113

Shaanxi, China). After cells recovery, HepG2 cells were cultured at 37°C under a

114

humidified atmosphere containing 5% CO2 and 95% air in a RPMI 1640 medium

115

supplemented with 10% FBS and 1% penicillin-streptomycin. The logarithmically

116

growing cells were used in the follow experiment. CA, rosiglitazone (ROG) and

117

inhibitors were dissolved in DMSO, and glucosamine was dissolved in phosphate

118

buffer. In the control group, the medium was added the same amount of DMSO with

119

other groups, as control. Prior to drug treatment, the cells were starved for 2 h, using

120

serum-free medium.

121

Cell viability

122

The cell viability was detected by MTT. The HepG2 cells were digested to single

123

cell suspension with 0.25% trypsin and were adjusted concentration with RPMI-1640

124

medium to 1×106 cells/mL in 96-well cell culture plate, which were cultured at 37°C

125

with 5% (v/v) CO2 for 24 h. After treatments, the medium was replaced by MTT

126

solution (dissolved by serum-free DMEM) for 4 h at 37°C. Then formazan crystals

127

formed on the bottom of each plant. The culture solution were suck dry and 100 µL

128

DMSO was added to solute the crystals formed. The absorbance values were

129

measured at 570 nm using a micro plate reader (Bio-Rad Model 680, Bio-Rad

130

Laboratories Ltd., shanghai, China).

131

Glucose uptake

132

Insulin resistance model were optimized by the glucose uptake. After insulin


Page 6 of 40

Page 7 of 40


133

resistance model established, the medium was replaced with serum-free medium for 2

134

h. The control group, the glucosamine group, CA treatment group, and the positive

135

control ROG group were set up. With or without 100 nmol/L insulin, the medium was

136

replaced with serum-free medium (the control group and the glucosamine group), CA

137

(diluted to 0, 25, 50, 100 µmol/L with serum-free medium), or rosiglitazone (diluted

138

to 10 µmol/L with serum-free medium). And then, the medium supernatant was

139

collected respectively. In accordance with instructions of the glucose diagnostic kit,

140

the mediums were measured at 570 nm with enzyme standard instrument. According

141

to the optical density of each well and the standard curve of glucose, the glucose

142

content in the medium was calculated. The glucose content of the experimental

143

groups and the original RPMI-1640 mediums were measured. Glucose uptake was the

144

difference value between the original RPMI-1640 mediums and the medium

145

supernatant of the experimental group medium.

146

GLUT2 Immunofluorescence

147

On HepG2 cell membrane, GLUT2 was observed by immunofluorescence

148

staining. When cells grew to confluence on glass coverslips, the cells were washed 3

149

times with 1×PBS. Cells were fixed in freshly prepared 4% paraformaldehyde at room

150

temperature for 20 to 30 min. Then the cells were washed 3 times. 5% skim milk was

151

used to block at 37°C shaker for 30 minutes, and then discard the skim milk. The

152

primary antibodies anti-GLUT2 (diluted with 1×PBS) were added and the cells were

153

shook at 37°C for 2 h. After washed 3 times, cells were incubated with the Alexa

154

Fluor 488 conjugated Donkey anti-Goat IgG (H+L) (diluted with 1% PBS) at 37°C



155

shaker for 2 h without light. After washed 3 times, nuclei was stained with DAPI. It

156

was observed and photographed by an inverted fluorescence microscope (Olympus

157

IX71, Tokyo, Japan). Fluorescence excitation was obtained at 488 nm via

158

argon/krypton laser line for Alexa 488. The green and blue channels were assigned to

159

GLUT2 and the nuclei, respectively.

160

Measurement of NO assay

161

The NO content of the culture supernatant was detected by Griess method. First,

162

we configured A and B reagent of Griess. Reagent A was a mixed solution with 1.0%

163

anhydrous sulfanilic and 5% concentrated phosphoric acid (85%) and reagent B was

164

0.1% of N-(1-naphthyl)-ethylenediamine. In the 96-well plates, 50 uL of the culture

165

supernatant was added in per-well, and the reagent A was added in per-well for 10

166

min at 37°C. Then the Reagent B was added for another 10 min at 37°C. After the

167

solution mixed and shocked, the value of culture supernatant for per-well was

168

determined at OD 540 nm by enzyme standard instrument. Each sample was repeated

169

for 8 times. According to the standard curve produced by NaNO2 standard solution,

170

the concentration of NO was calculated, and was standardized by protein

171

concentration. The control group was set to be benchmark, and the corresponding

172

ratio was calculated for each group.

173

Detection of intracellular ROS production

174

After the treatments, cells were washed twice with PBS, 10 µM final

175

concentration of H2DCFDA solution was added to each well, which can diffuse

176

readily into the cells and be oxidized to DCF with intensity fluorescence by the


Page 8 of 40

Page 9 of 40


177

oxidant in cells. The cells were incubated at 37°C for 30 min. After the cells washed

178

twice, the generation of ROS was observed and took photographs under an inverted

179

fluorescence microscope. Cells were treated as above. After washing with PBS, the

180

cells were lysed immediately by the cell lysis buffer and the cell extract was collected.

181

The fluorescence was read in cell extract supernatant at OD 485/520 nm by

182

fluorescence micro plate reader, and was standardized by the protein concentration.

183

RNA isolation and PCR

184

The total mRNA was extracted from cultured cells using the mRNA extraction

185

kit. Used for reverse transcription, 1 µg of total mRNA were converted to first-strand

186

complementary DNA in 20 µL of the reaction using the cDNA synthesis kit. The

187

sequences for the PCR primers were as follows: Human β-actin: forward 5’-TGGATC

188

AGCAAGCAGGAGTA-3’, reverse 5’-TCGGCCACATTGTGAACTTT-3’; Human

189

TNF-α: forward 5’-GGCAGTCAGATCATCTTCTCGAA-3’, reverse 5’- TGAAGAG

190

GACCTGGGAGTAGATG-3’; Human IL-6: forward 5’- CTCAGCCCTGAGAAAG

191

GAGA-3’, reverse 5’-TTTTCTGCCAGTGCCTCTTT-3’. PCR reactions were

192

performed as follows: 10 min at 95°C; 40 cycles of 15 s at 95°C, 30 s at 55°C, and 45

193

s at 72°C; a final extension of 71 cycles of 30 s at 60°C. The PCR products were

194

resolved in a 1%-agarose gels.

195

Western Blot

196

The treated HepG2 cells were washed twice with PBS (pH 7.4) and lysed in

197

responsive lysis buffer, containing 1% PMSF and 20 mM NaF for 15 min. The cell

198

debris were scraped and collected into the 1.5 mL centrifuge tube. Centrifuge it for 10



199

min at 15000 r/min at 4°C, and collect the supernatant. Cell protein concentration was

200

measured by the BCA kit. SDS loading buffer (a quarter of the sample volume) was

201

added. The mix was placed in 95°C bath for 10 min to make protein denaturation, and

202

was stored at -20°C. Take the equal amount of each sample to SDS-PAGE

203

electrophoresis. The protein was concentrated by 80 V for 40 min, and separated by

204

120 V for 90 min. After constant 10 V semi-dry transfer for 25 min, the proteins on

205

the gel were transferred to PVDF membrane

206

semidry transfer apparatus (Bio-Rad, Shanghai, China). The membrane was placed in

207

5% nonfat milk to block, which was dissolved in TBST, at room temperature for 2 h.

208

Wash the membrane 4 times with TBST. The membrane was placed in diluted primary

209

antibody incubation overnight at 4°C. After washing for 4 times, membranes were

210

placed in diluted secondary antibody, and incubated with rocked at room temperature

211

for 2 h. Wash the membrane 4 times. The ECL emitting kit (Thermo Scientific,

212

Waltham, MA, USA) was uniformly added at 0.45 µm pore PVDF membrane, and

213

then Molecular Imager Chemidoc XRS System (Bio-Rad, Shanghai, China) and

214

Quantity one software was used to expose and gray semi-quantitative analysis.

215

Statistical analysis

(Millipore, Bedford, MA, USA) by a

216

All of the experiments were performed at least three independent experiments

217

per sample, and the data were presented as the means ± standard deviation (S.D.). The

218

data were analyzed by using the one-way analysis of variance25 followed by Tukey's

219

multiple-range test with the SPSS 19.0 system. Any p-values less than 0.05 were

220

considered to be statistically significant.


Page 10 of 40

Page 11 of 40


221

Results

222

Effect of CA on glucose uptake under the insulin resistance cell model induced by

223

glucosamine

224

Previous research demonstrated that glucosamine could induce insulin resistance

225

in several cell models.23,

26

226

reduced glucose uptake in dose-dependent and time-dependent manner in HepG2 cells,

227

which suggested that it was a potential model to stimulate insulin resistance on

228

hepatocytes. And this insulin resistance model was stable for 24 h after treated with

229

18 mM glucosamine for 18 h. The glucosamine and CA had no effects on cell

230

viability (Figure 1E).

As shown in Figure 1A and Figure 1B, glucosamine

231

To assess the effect of CA on reversing insulin resistance, glucosamine-induced

232

HepG2 cells were incubated with CA, in either absence or presence of insulin. In

233

Figure 1C, CA dramatically improved glucose uptake in a dose-dependent manner,

234

and CA further enhanced insulin-induced glucose uptake by 57.7%. In Figure 1D, CA

235

significantly reversed glucosamine-induced glucose uptake decreasing after 24 h

236

treatment. Rosiglitazone (ROG), an insulin sensitizer, was a positive control (10 µM).

237

In the following experiments, the insulin resistance model was constructed by 18 mM

238

glucosamine for 18 h, and the addition of CA was 100 µM for 24 h.

239

CA restored glucosamine-induced impairment of GLUT2 translocation through

240

activating PI3K/Akt pathway

241

To further explore whether CA improved glucose uptake by promoting the

242

translocation of GLUT2 from the cytoplasm to the membrane via PI3K/Akt pathway,



243

the expression of GLUT2 and the activation of Akt were detected. As shown in Figure

244

2A, by immunofluorescence staining, CA (100 µM) significantly improved GLUT2

245

translocation. In Figure 2B and 2C, the expression of GLUT2 on the cell membrane of

246

the glucosamine-induced cells was dramatically reduced by 73.0%, which led to

247

marked reduction of cell glucose uptake. CA (100 µM) or ROG (10 µM) substantially

248

elevated the decreased GLUT2 expression by 1.4-fold and 2.9-fold than control group,

249

respectively, in membrane fractions of glucosamine model. In contrast with the

250

insulin group, the treatment of insulin and CA (100 µM) was increased by 17.0%. As

251

downstream of insulin signaling pathway, the phosphorylation of Akt was detected.

252

As shown in Figure 2D and 2E, compared to glucosamine group, CA (100 µM) with

253

or without insulin increased the phosphorylation of Akt by 1.1-fold and 1.7-fold,

254

respectively.

255

Effects of CA on inflammatory responses induced by glucosamine

256

Insulin resistance has been linked to a low-grade chronic inflammatory

257

response.9, 10, 27 To assess the effects of CA on inflammation induced by glucosamine,

258

the pro-inflammatory cytokines and mediators were detected. As shown in Figure 3A,

259

the production of nitrite (NO stable final product) was significantly increased by 80.0%

260

in the glucosamine-induced HepG2 cells. CA (100 µM) dose-dependently attenuated

261

the glucosamine-induced release of nitrite by 10.1% in CA group. CA (100 µM)

262

significantly attenuated the increased mRNA levels of IL-6 and TNF-α in the

263

glucosamine-induced cells (Figure 3B).

264

As shown in Figure 3C and 3D, glucosamine increased the expression of iNOS


Page 12 of 40

Page 13 of 40


265

and COX-2 levels by 1.42 and 1.96 times than the control group, respectively. In

266

contrast with the glucosamine group, CA (100 µM) significantly reduced the

267

expression of iNOS and COX-2 by approximately 46.4% and 38.3%, respectively.

268

The inhibition of CA on the generation of ROS induced by glucosamine

269

Impairment of insulin signaling and hepatic insulin resistance has been attributed

270

to mitochondrial-generated ROS28. As shown in Figure 4A and 4B, glucosamine

271

markedly increased ROS levels by 16.2% compared with control group. As shown in

272

Figure 4C and 4D, the treatment of CA (100 µM) significantly decreased ROS level

273

by 37.8% compared with glucosamine group. NAC, ROS scavenger, and CA (100 µM)

274

had the synergistic effect on down-regulation expressions of iNOS and COX-2 by

275

54.6% and 33.6%, respectively. With the pretreatment of NAC, CA (100 µM)

276

synergistically promoted the glucose uptake by 25.9%, compared with the treatment

277

only with CA, as shown in Figure 4E.

278

Effect of CA on the inflammation and glucose uptake via suppressing MAPKs

279

pathway

280

Compared

to

control

group,

glucosamine

significantly

induced

the

281

phosphorylation of JNK and p38 by 59.4% and 56.8%, respectively. The expression

282

of the phosphorylation of JNK and p38 were markedly decreased by 30.8% and 47.2%

283

compared with CA (100 µM) treatment. However, CA has no significant effect on

284

phosphorylation of ERK1/2, as shown in Figure 5A and 5B. The ERK1/2 inhibitor

285

U0126, p38 inhibitor SB203580, and JNK inhibitor SP600125 were added, and the

286

expression of COX-2 and iNOS were substantially reduced in p38 and JNK inhibitors



287

treatment group (p

Cichoric Acid Reverses Insulin Resistance and ... - ACS Publications

Recommend Documents