Naringin activates AMPK resulting in altered expression of SREBPs

Subscriber access provided by READING UNIV

Bioactive Constituents, Metabolites, and Functions

Naringin activates AMPK resulting in altered expression of SREBPs, PCSK9, and LDLR to reduce body weight in obese C57BL/6J mice GUO-GUANG SUI, hong-bo xiao, XIANG-YANG LU, and ZHI-LIANG SUN J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02696 • Publication Date (Web): 10 Aug 2018 Downloaded from http://pubs.acs.org on August 12, 2018

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 32

Journal of Agricultural and Food Chemistry

Naringin activates AMPK resulting in altered expression of

1

SREBPs, PCSK9, and LDLR to reduce body weight

2

3

in obese C57BL/6J mice

4

GUO-GUANG SUI†, HONG-BO XIAO†,*, XIANG-YANG LU ‡,§, ZHI-LIANG SUN¶

5

†

6

China

7

‡

8

Biotransformation，Hunan Agricultural University, Changsha 410128, China

9

§

College of Veterinary Medicine, Hunan Agricultural University, Changsha 410128,

Hunan Province University Key Laboratory for Agricultural Biochemistry and

Hunan Co-Innovation Center for Ultilization of Botanical Functional Ingredients,

10

Changsha 410128, China

11

¶

Hunan Engineering Research Center of Veterinary Drug, Changsha 410128, China

12 13

Correspondence to:

Hong-Bo Xiao

14

College of Veterinary Medicine

15

Hunan Agricultural University

16

Furong District

17

Changsha 410128 China

18

Tel: 086-731-84673618

19

Fax: 086-731-84635292

20

E-mail: [email protected]

21

1

ACS Paragon Plus Environment


Page 2 of 32

22

ABSTRACT: Previous investigations have shown a molecular crosstalk among

23

activated adenosine monophosphate-activated protein kinase (AMPK), proprotein

24

convertase subtilisin/kexin type 9 (PCSK9), sterol regulatory element-binding

25

proteins (SREBPs) and low-density lipoprotein receptor (LDLR) may be an

26

innovative pharmacologic objective for treating obesity. We scrutinized

27

beneficial effect of naringin, a flavanone-7-O-glycoside, on obesity and the

28

mechanisms in the present study. We arbitrarily divided fifty mice into five groups (n

29

=10): 25 or 50 or 100 mg/kg/day naringin-treated obese mice (gavage for 8 weeks),

30

untreated obese mice, and C57BL/6J control. After eight weeks, body weight was

31

51.8 ± 4.4 in untreated obese mice group while the weights were 41.4 ± 4.1, 34.6 ±

32

2.2, and 28.0 ± 2.3 in 25, 50,100 mg/kg naringin groups, respectively. Moreover,

33

naringin treatment significantly decreased plasma 8-isoprostane (an indicator of the

34

oxidative stress) level, fat weight, liver weight, hepatic total cholesterol concentration,

35

hepatic triglyceride concentration, plasma leptin level, plasma insulin content, plasma

36

low-density

37

concomitantly with down-regulated expression of SREBP-2, PCSK9, and SREBP-1,

38

up-regulated expression of p-AMPKα and LDLR. The present results suggest that

39

naringin activates AMPK resulting in altered expression of SREBPs, PCSK9, and

40

LDLR to reduce body weight of obese C57BL/6J mice.

41

KEYWORDS: Naringin; AMP-activated protein kinase; Obesity; C57BL/6J

42

INTRODUCTION

lipoprotein

cholesterol

level,

and

plasma

2


PCSK9

the

production

Page 3 of 32


43

It is generally acknowledged that obesity is caused by an excessive accumulation of

44

fat that can have detrimental effects on health. To begin with, fatty acid and

45

cholesterol synthetic gene expressions are modulated by the family of sterol

46

regulatory element-binding protein (SREBP) (1). The expression of SREBP is related

47

to obesity and lipid accumulation (2, 3). Then, it is detected that proprotein

48

convertase subtilisin/kexin type 9 (PCSK9) is a target gene of SREBPs at the

49

transcriptional level(1). PCSK9 is a kind of subtilisin-related serine endoproteases’

50

proteinase K subfamily. There is a link between PCSK9 and obesity(4, 5).

51

Furthermore, PCSK9 is a hepatic low-density lipoprotein receptor (LDLR) regulator.

52

In the liver, PCSK9 combines to the LDLR, the major mechanism of it involves in

53

binding at the surface of hepatocytes, coendocytosis and lysosomal degradation (6).

54

Liver-specific or global deactivation of PCSK9 gene in mice results in higher

55

abundance of LDLR protein on the surface of hepatocytes (7). Obesity is associated

56

with decreased LDLR expression (8, 9). Finally, adenosine monophosphate activated

57

protein kinase (AMPK) is a desirable curative aim of obesity(10). AMPK can

58

suppress cleavage and transcriptional activation of SREBP, inhibit LDLR degradation

59

by reducing PCSK9 expression(11). To sum up as it was previously stated, the

60

molecular crosstalk among AMPK and SREBPs, PCSK9, and LDLR may be a novel

61

pharmacologic objective for treating obesity.

62

As a flavanone-7-O-glycoside, naringin is naturally isolated from citrus fruits,

63

especially from grapefruit (12). Various pharmacological effects have been

64

detected in vitro or in animal studies (13-15). Naringin could improve the 3



65

obesity of high fat diet-fed rats (16), but the mechanism contributing to its protective

66

effect is not yet fully defined. AMPK, SREBPs, PCSK9, and LDLR is related to

67

oxidative stress (17-21). In addition, naringin has potent antioxidative capability

68

(22-25). On this basis, it could be inferred that naringin may regulate the molecular

69

crosstalk among AMPK and SREBPs, PCSK9, and LDLR to protect against the

70

obesity by prohibiting oxidative stress.

71 72

Therefore, we examined the useful effect of naringin on obesity and its mechanisms in obese C57BL/6J mice in the present study.

73 74

MATERIALS AND METHODS

75

Chemicals. Naringin (Fig. 1, purity: 98.0%) was got at Wuhan Xinxin Biotechnology

76

Co. Ltd (Wuhan, China). Some reagent was gained from Shanghai Sinopharm Chemical

77

Reagent Company Limited (China). Antibodies were purchased at Santa Cruz

78

Biotechnology (USA).

79 80

Experimental animals. Male C57BL/6J mouse was bought from Hunan

81

Agricultural University (Changsha, China). This study experiments was designed to

82

conform to the U.S. Department of Agriculture (USDA) regulations about care and

83

use of laboratory animals. Five mice were accommodated in a cage with unrestricted

84

access to water and fodder. Food consumption and body weight were examined

85

weekly. Animal Studies Subcommittee at Hunan Agricultural University approved all

86

animal procedures.

4


Page 4 of 32

Page 5 of 32


87 88

Experimental protocols. Fifty 9 and 10 weeks mice were divided arbitrarily into

89

five groups (n = 10): 25 or 50 or 100 mg/kg/day naringin-treated obese mice (gavage

90

for 8 weeks), untreated obese mice, and C57BL/6J control.

91

was previously shown (26, 27). Naringin was delivered in 0.9% saline as reported

92

previously (28). Only the vehicle was fed to untreated obese mice and C57BL/6J

93

control. C57BL/6J mice were nourished with high-fat diet (60% energy from fat, Test

94

Diets, IPS Product Supplies Ltd, UK) to develop chronic obesity mouse model

95

according to the diets used previously (29). Mice in the control group were fed a

96

normal fodder. Before euthanasia, mouse was starved overnight at the end of the

97

experiments. Specimens of plasma, epididymal fat, and liver were gained.

The dose of naringin

98 99

Determination of plasma 8-isoprostane level. According to the protocol provided

100

for evaluating free 8-isoprostane and esterified 8-isoprostane following formerly

101

reported method (30), plasma 8-isoprostane contents were determined in mice by

102

Caymen’s 8-isoprostane enzyme immunoassay (hemicals, Ann Arbor, MI, USA).

103 104

Analysis of mRNA expressions of SREBP-1, p-AMPKα, LDLR, PCSK9 and

105

SREBP-2. Real-time PCR was carried out to analyze p-AMPKα, SREBP-1, LDLR,

106

PCSK9 and SREBP-2 mRNA. Concisely, before reverse-transcribed, hepatic total

107

RNA was obtained with Invitrogen’s TRIzol reagent (Carlsbad, USA). Primer pairs

108

were utilized as follows: GAPDH: 5′-GTCCACCACCCTGTTGCTGTA-3′ 5


and


109

5′-GAGAATGGGAAGCTTGTCATC-3′; 36B4: 5’-ATCTGCTGCATCTGCTTGG-3’

110

and 5’-GCGACCTGGAAGTCCAACTAC-3’; LDLR: 5’-TGCGGTCCAGGGTCAT

111

CT-3’ and 5’-AGGCTGTGGGCTCCATAGG-3’; PCSK9: 5’-CCGACTGTGATGAC

112

CTCTGGA-3’ and 5’-TTGCAGCAGCTGGGAACTT-3’. SREBP-2: 5’ GCAGCAAC

113

GGGACCATTCT 3’ and 5’CCCCATGACTAAGTCCTTCAACT 3’; SREBP-1: 5’

114

GCTTCCAGAGAGGAGGCCAG 3’and, 5’GGAGCCATGGATTGCACATT 3’;

115

p-AMPKα: 5’ TGTTGTACAGGCAGCTGAGG 3’and 5’ AGAGGGCCGCAATAA

116

AAGAT 3’ (31-35).

117 118

Measurement of protein expressions of p-AMPKα, SREBP-1, LDLR, PCSK9

119

and SREBP-2. Dulbecco’s phosphate-buffered saline was cooled with ice. The

120

segregated tissue was homogenized in it. Before the separated protein transferred to

121

polyvinylidene fluoride membrane, equal concentration of protein was separated

122

using 12% SDS-PAGE. Western Blotting was carried out to measure protein contents

123

of mature SREBP-2 and SREBP-1 (mSREBP-2, mSREBP-1), PCSK9, LDLR, and

124

p-AMPKαas reported method (35-38).

125 126

Assessment of plasma PCSK9, leptin, insulin, HDL-C, and LDL-C. Plasma

127

PCSK9 concentration was assessed by using R&D Systems’ enzyme-linked

128

immunosorbent assay according as the demand of manufacturer. Insulin and leptin

129

levels were measured by Morinaga & Company’s ELISA (Japan). In light of

130

producer’s suggestions, plasma high or low density lipoprotein cholesterol (HDL-C or

131

LDL-C) concentrations were assessed by Hitachi’s Type 7170A automated analyzer 6


Page 6 of 32

Page 7 of 32

132


using bioMerieux’s kit (Lyon, France) according to the producer’s requirement.

133 134

Measurement of hepatic lipid content. Liver tissues were cut off and

135

homogenized in sodium chloride solution. Lipids were extracted with methanol and

136

chloroform (5mL, 1 : 2, vol/vol) (39). The chloroform layer was dried. Triglyceride

137

and total cholesterol ((TG and TC) were measured by Wako’s LabAssay kit (Osaka,

138

Japan).

139 140

Statistical analysis. SPSS 19.0 was employed. Datum was appraised using

141

Tukey's test and ANOVA. P≤ 0.05 was viewed as significant. Value was expressed as

142

means ± SD.

143 144

RESULTS

145

Plasma level of 8-isoprostane. Compared with normal mouse, 8-isoprostane levels

146

of obese mice were markedly elevated (P < 0.01). Compared obese mice, mice fed

147

with naringin (25 or 50 or 100 mg/kg) possessed lower 8-isoprostane (P < 0.05 or P
0.05).

167 168

Hepatic lipid concentrations. Hepatic lipid concentrations were given in Fig. 6.

169

Hepatic TC and TG were higher (P < 0.01) in obese mice than in the control group.

170

Naringin (25 or 50 or 100 mg/kg) treated animals had significantly decreased hepatic

171

TG and TC contents (P < 0.05 or P < 0.01).

172 173

Tssue weight, feed efficiency ratio, and body weight. There were marked

174

difference (P < 0.01) in liver weight, fat weight, feed efficiency, and body weight

175

when we collated obese mice with normal mice (Fig. 7). After treatment with naringin 8


Page 8 of 32

Page 9 of 32


176

(25 or 50 or 100 mg/kg), body weight, liver weigh, feed efficiency ratio, and fat

177

weight in obese mice were markedly diminished (P < 0.05 or P < 0.01).

178

Analysis of Pearson’s correlation found LDLR expression correlated negatively

179

with fat weigh (r=-0.795, P=0.006) and body weight (r=-0.811, P=0.005); PCSK9

180

expression correlated positively with fat weigh (r=0.802，P=0.003) and body weight

181

(r=0.768, P=0.004); SBRP expression was positively related to body weight (r=0.790,

182

P=0.001) and fat weigh (r=0.811, P=0.002). The stronger nonpositive correlation with

183

r=-0.738 and P=0.005 was achieved between AMPK expression and body weight. A

184

stronger correlation of r=-0.802; P=0.003 was detectable for AMPK expression and

185

fat weigh.

186 187

DISCUSSION

188

As is known to all, obesity is one of the most dangerous chronic diseases

189

characterized by an excess of body fat. AMPK, SREBPs, PCSK9, and LDLR are

190

closely associated with obesity. It has been demonstrated that elevated LDL-C plays a

191

major role in the obesity (4). Plasma LDLC is governed by its uptake into cells upon

192

binding to LDLR. In addition, LDLR−/− mice are obese displaying extreme

193

hypertriglyceridemia and hypercholesterolemia even when they are fed a low fat

194

unpurified diet (8). LDLR relative with 11 ligand-binding repeats (R11), also known

195

as SORL1 or SorLA, is a member of the LDLR family (40). Plasma soluble R11

196

(sLR11) reduces in overweight individuals (41). PCSK9 is a negative regulator of

197

LDLR. PCSK9 binds with the LDLR at hepatocytes’ surface, leading to elevated 9



198

plasma LDL-C (6). Moreover, obesity is related to elevated PCSK9 levels in young

199

women (5). Although there is no genetic evidence in mouse that overexpression of

200

PCSK9 leads to obesity, in spite of the hypercholesterolemia (42). However, it is

201

PCSK9 deficiency that seems to be associated with abnormal metabolism of adipose

202

tissue (43, 44). PCSK9 is a target gene of the SREBPs. Antrodia cinnamomea (a

203

protogenic fungus that only grows on the heartwood of endemic Cinnamomum

204

kanehirae Hayata) prevents obesity via regulating SREBP signaling (2). Furthermore,

205

increased expression of SREBP is relevant to the accumulation of obesity-driven lipid

206

in dairy cows’ liver during late gestation (3). Moreover, protein kinase is downstream

207

goal in inflammation-mediated obesity (45). It is interested that AMPK as a remedial

208

target has latent influence on obesity (46). AMPK is reported to alter the expression of

209

SREBP-2, PCSK9, and LDLR (47). It is clear from the above discussion that a

210

physical relation among activated AMPK and SREBPs, PCSK9, and LDLR may be a

211

useful pharmacologic objective for regulating obesity.

212

Naringin is a flavanoid obtained from grapefruit and other citrus fruits. Naringin

213

has a cholesterol-reducing effect. Naringin ameliorates TC, TG, and LDL-C in

214

subjects with moderate hypercholesterolemia (13), hyperlipidemic rabbits (14), rats

215

fed high-cholesterol and high-fat diet (15), and isoproterenol-stimulated myocardial

216

infarction wistar rats (48). In addition, after treatment with naringin, HDLC level is

217

increased in human while is not altered in rats (15). The similar results were got in the

218

present study. Besides, the present results substantiated previous finding that naringin

219

takes part in the improvement of lipogenesis as AMPK activator (49). In the present 10


Page 10 of 32

Page 11 of 32


220

study, naringin treatment also effectively decreased body weight, fat weight, liver

221

weight, hepatic TC and TG concentrations, plasma levels of LDLC, leptin, PCSK9

222

and insulin accompany with down-regulated expression of PCSK9, SREBP-2, and

223

SREBP-1, and up-regulated level of p-AMPKα and LDLR in obese mice. In addition,

224

the present finding confirmed previous report that there is an obvious dose-response

225

effect of naringin on body weight (16). Taken together, these discoveries uphold the

226

supposition that naringin could regulate the molecular crosstalk among AMPK and

227

SREBPs, PCSK9, and LDLR to improve obesity.

228

Interestingly, AMPK and SREBPs, PCSK9, and LDLR are associated with

229

oxidative

stress.

To

start

with,

AMPK

suppresses oxidative

230

c‐Myc‐positive melanoma cells (20). Oxidized low density lipoprotein (oxLDL)

231

increases the level of SREBP1 in Raw 264.7 cells (21). Hydrogen peroxide and

232

ox-LDL can induce SREBP2 in endothelial cells (17); next, PCSK9 expression is also

233

induced by oxLDL (18). In addition, the mitochondria of hypercholesterolemic LDLR

234

knockout (k/o) mice have reduced antioxidant ability (19). Finally, antioxidants

235

salicylate has the capacity to inhibit lectin-like oxidized LDLR-1 (50). In recent years,

236

naringin has obtained lots of attention because of its antioxidant pharmacological

237

effects. Naringin protects against oxidative stress in human adipose-derived

238

mesenchymal stem cells (51), experimentally induced inflammatory bowel disease in

239

rats (24), LPS-induced cardiac injury in mice (22), ferric nitrilotriacetate-stimulated

240

oxidative renal damage in rat (25), and hypercholesterolemic rats (23). We showed

241

that naringin treatment prohibited hepatic content of SREBP-1, PCSK9, and SREBP-2, 11


stress

in


242

elevated LDLR and p-AMPKα expression accompany with the reduced plasma

243

concentrations of 8-isoprostane in obese C57BL/6J mice. Because naringin possessing

244

antioxidant effect, we assumed that naringin could modulate the molecular crosstalk

245

among AMPK and SREBPs, LDLR, and PCSK9 by prohibiting lipid peroxidation,

246

which in turn inhibit the obesity of mice.

247

It is notable that the present results verified previous report that feed efficiency

248

ratio was obviously decreased in rodent after treatment with naringin (52). Naringin is

249

principally found in grapefruits. People employ grapefruit juice to lessen appetite for

250

weight loss and elevate taste sensation because the naringin in the juice motivates the

251

taste buds. Naringin is the chief bitter component of grapefruit juice; that is to say, it

252

is the ingredient that grants grapefruit its peculiar bitter flavor. Elevated taste acuity

253

for naringin is related to greater dislike for bitter compound (53). Therefore, the

254

feeding of naringin affects the appetite. This might be one significant mechanistic

255

view to the decrease in body weight of obese C57BL/6J mice after treatment with

256

naringin. However, lots of extra study is demanded to expound the mechanisms

257

related to the relation between appetite and this molecular crosstalk.

258

In summary, our study suggests that naringin activates AMPK resulting in altered

259

expression of SREBPs, PCSK9, and LDLR to reduce body weight of obese C57BL/6J

260

mice.

261 262

ORCID

263

Hong-Bo Xiao: 0000-0002-8941-2866 12


Page 12 of 32

Page 13 of 32


264

Funding

265

We thank Hunan Provincial Natural Science Foundation of China for financial

266

support.

267

ACKNOWLEDGMENT

268

Project supported by Hunan Provincial Natural Science Foundation of China

269

(14JJ2079).

270 271

REFERENCES

272

(1) Horton, J.D.; Goldstein, J.L.; Brown, M.S. SREBPs: activators of the complete

273

program of cholesterol and fatty acid synthesis in the liver. J. Clin. Invest. 2002,

274

109,1125-1131

275

(2) Peng, C.H.; Yang, M.Y.; Yang, Y.S.; Yu, C.C.; Wang, C.J. Antrodia cinnamomea

276

Prevents Obesity, Dyslipidemia, and the Derived Fatty Liver via Regulating

277

AMPK and SREBP Signaling. Am. J. Chin. Med. 2017, 45, 67-83.

278

(3) Prodanović, R.; Korićanac, G.; Vujanac, I.; Djordjević, A.; Pantelić, M.; Romić,

279

S.; Stanimirović, Z.; Kirovski, D. Obesity-driven prepartal hepatic lipid

280

accumulation in dairy cows is associated with increased CD36 and SREBP-1

281

expression. Res. Vet. Sci. 2016, 107, 16-19.

282

(4) Du, Y.; Li, S.; Cui, C.J.; Zhang, Y.; Yang, S.H.; Li, J.J. Leptin decreases the

283

expression of low-density lipoprotein receptor via PCSK9 pathway: linking

284

dyslipidemia with obesity. J. Transl. Med. 2016, 14, 276

285

(5) Levenson, A.E.; Shah, A.S.; Khoury, P.R.; Kimball, T.R.; Urbina, E.M.; de 13



Page 14 of 32

286

Ferranti, S.D.; Maahs, D.M.; Dolan, L.M.; Wadwa, R.P.; Biddinger, S.B.

287

Obesity and type 2 diabetes are associated with elevated PCSK9 levels in young

288

women. Pediatr. Diabetes. 2017, 18, 755-760.

289

(6) Duff, C.J.; Hooper, N.M. PCSK9: an emerging target for treatment of

290

hypercholesterolemia. Expert. Opin. Ther. Targets. 2011, 15, 157-168.

291

(7) Mbikay, M.; Mayne, J.; Chrétien, M. Proprotein convertases subtilisin/ kexin

292

type 9, an enzyme turned escort protein: hepatic and extra hepatic functions. J.

293

Diabetes. 2013, 5, 391-405.

294

(8)

Saraswathi, V., Morrow, J.D., Hasty, A.H. Dietary fish oil exerts hypolipidemic

295

effects in lean and insulin sensitizing effects in obese LDLR-/- mice. J.

296

Nutr. 2009, 139, 2380-2386.

297

(9)

Rodríguez-Arroyo,

G.; Paradisi,

I.; Vívenes-Lugo,

M.;

Castro-Guerra

298

D.; Rodríguez-Larralde Á. LEP, LDLR and APOA4 gene polymorphisms and

299

their relationship with the risk of overweight,obesity and chronic diseases in

300

adults of the State of Sucre, Venezuela. Biomedica. 2016, 36, 78-90.

301

(10) Anil, T.M.; Harish, C.; Lakshmi, MN.; Harsha, K.; Onkaramurthy, M.; Sathish

302

Kumar,

V.; Shree,

N.; Geetha,

V.; Balamurali,

303

AS.; Madhusudhan Reddy, B.; Govind, M.K.; Anup, M.O.; Moolemath,

304

Y.; Venkataranganna, M.V.; Jagannath, M.R.; Somesh, B.P. CNX-012-570,

305

a direct AMPK activator provides strong glycemic and lipid control along with

306

significant reduction in body weight; studies from both diet-induced obese

307

mice and db/db mice models. Cardiovasc. Diabetol. 2014, 13, 27. 14


G.V.; Gopala,

Page 15 of 32


308

(11) Li, Y.; Xu, S.; Mihaylova, M.M.; Zheng, B.; Hou, X.; Jiang, B.; Park, O.; Luo,

309

Z.; Lefai, E.; Shyy, J.Y.; Gao, B.; Wierzbicki, M.; Verbeuren, TJ.; Shaw,

310

R.J.; Cohen, R.A.; Zang, M. AMPK phosphorylates and inhibits SREBP

311

activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-

312

resistant mice. Cell. Metab. 2011, 13, 376-388.

313

(12) Han, Y.; Su, J.; Liu, X.; Zhao, Y.; Wang, C.; Li, X. Naringin alleviates early

314

brain injury after experimental subarachnoid hemorrhage by reducing oxidative

315

stress and inhibiting apoptosis. Brain. Res. Bull. 2016, 133, 42-50.

316

(13) Toth, P.P.; Patti, A.M.; Nikolic, D.; Giglio, R.V.; Castellino, G.; Biancucci,

317

T.; Geraci, F.; David, S.; Montalto, G.; Rizvi, A.; Rizzo, M. Bergamot Reduces

318

Plasma Lipids, Atherogenic Small Dense LDL, and Subclinical Atherosclerosis

319

in Subjects with Moderate Hypercholesterolemia: A 6 Months Prospective Study.

320

Front. Pharmacol. 2016, 6, 299.

321

(14) Xiao, Y.; Li, L.L.; Wang, Y.Y.; Guo, J.J.; Xu, W.P.; Wang, Y.Y.; Wang, Y.

322

Naringin administration inhibits platelet aggregation and release by reducing

323

blood cholesterol levels and the cytosolic free calcium concentration in

324

hyperlipidemic rabbits. Exp. Ther. Med. 2014, 8, 968-972

325

(15) Kim, S.Y.; Kim, H.J.; Lee, M.K.; Jeon, S.M.; Do, G.M.; Kwon, E.Y.; Cho,

326

Y.Y.; Kim,

D.J.; Jeong,

327

Naringin time-dependently

328

plasma cholesterol in rats fed high-fat and high-cholesterol diet. J. Med.

329

Food. 2006, 9, 582-586

K.S.; Park, lowers

Y.B.; Ha,

T.Y.; Choi,

hepatic cholesterol biosynthesis

15


M.S. and


330

(16)

Alam,

M.A.; Kauter,

K.; Brown,

L.

Naringin improves

Page 16 of 32

diet-induced

331

cardiovascular dysfunction and obesity in high carbohydrate, high fat diet-fed

332

rats. Nutrients. 2013, 5, 637-50.

333

(17) Chen, Z.; Wen, L.; Martin, M.; Hsu, C.Y.; Fang, L.; Lin, F.M.; Lin, T.Y.; Geary,

334

M.J.; Geary, G.G.; Zhao, Y.; Johnson, D.A.; Chen, J.W.; Lin, S.J.; Chien,

335

S.; Huang, H.D.; Miller, Y.I.; Huang, P.H.; Shyy, J.Y. Oxidative stress activates

336

endothelial innate immunity via sterol regulatory element bindingprotein 2

337

(SREBP2) transactivation of microRNA-92a. Circulation. 2015, 13, 805-814.

338

(18) Paim, B.A.; Velho, J.A.; Castilho, R.F.; Oliveira, H.C.; Vercesi, A.E. Oxidative

339

stress in hypercholesterolemic LDL (low-density lipoprotein) receptor knockout

340

mice is associated with low content of mitochondrial NADP-linked substrates

341

and is partially reversed by citrate replacement. Free. Radic. Biol. Med. 2008, 44,

342

444-451

343

(19) Tang, Z.; Jiang, L.; Peng, J.; Ren, Z.; Wei, D.; Wu, C.; Pan, L.; Jiang, Z.; Liu, L.

344

PCSK9 siRNA suppresses the inflammatory response induced by oxLDL

345

through inhibition of NF-κB activation in THP-1-derived macrophages. Int. J.

346

Mol. Med. 2012, 30, 931-938.

347

(20) Kfoury, A.; Armaro, M.; Collodet, C.; Sordet-Dessimoz, J.; Giner, MP.; Christen,

348

S.; Moco, S.; Leleu, M.; de Leval, L.; Koch, U.; Trumpp, A.; Sakamoto, K.;

349

Beermann, F.; Radtke, F. AMPK promotes survival of c-Myc-positive

350

melanoma cells by suppressing oxidative stress. EMBO. J. 2018, 37, e97673

351

(21) Wang, Y.G.; Yang, T.L.; Liraglutide reduces oxidized LDL-induced oxidative 16


Page 17 of 32


352

stress and

353

AMPK/SREBP1 pathway. J. Geriatr. Cardiol. 2015, 12, 410-416.

fatty

degeneration

in

Raw

264.7

cells

involving

the

354

(22) Xianchu, L.; Lan, P.Z.; Qiufang, L.; Yi, L.; Xiangcheng, R.; Wenqi, H.; Yang, D.

355

Naringin protects against lipopolysaccharide-induced cardiac injury in mice.

356

Environ. Toxicol. Pharmacol. 2016, 48, 1-6.

357

(23) Chtourou, Y.; Kamoun, Z.; Zarrouk, W.; Kebieche, M.; Kallel, C.; Gdoura,

358

R.; Fetoui, H. Naringenin ameliorates renal and platelet purinergic signalling

359

alterations in high-cholesterol fed rats through the suppression of ROS and

360

NF-κB signaling pathways. Food. Funct. 2016, 7, 183-193.

361

(24) Kumar, V.S.; Rajmane, A.R.; Adil, M.; Kandhare, A.D.; Ghosh, P.; Bodhankar,

362

S.L. Naringin ameliorates acetic acid induced colitis through modulation of

363

endogenous oxido-nitrosative balance and DNA damage in rats. J. Biomed.

364

Res. 2014, 28, 132-145.

365

(25) Singh., D., Chander., V., Chopra., K (2004) Protective effect of naringin, a

366

bioflavonoid on ferric nitrilotriacetate-induced oxidative renal damage in rat

367

kidney. Toxicology 201:1-8

368

(26) Aggarwal, A.; Gaur, V.; Kumar, A. Nitric oxide mechanism in the protective

369

effect of naringin against post-stroke depression (PSD) in mice. Life. Sci. 2010,

370

86, 928-935.

371

(27) Wang, L.; Zhang, Y.G.; Wang, X.M.; Ma, L.F.; Zhang, Y.M. Naringin protects

372

human

adipose-derived

373

peroxide-induced

mesenchymal

inhibition

of

stem

osteogenic

cells

against

differentiation.

17


hydrogen

Chem.

Biol.


374 375

Page 18 of 32

Interact. 2015, 242, 255-261. (28) Sun, X.; Li, F.; Ma, X. ; Ma, J.; Zhao, B.; Zhang, Y.; Li, Y.; Lv, J.; Meng, X. The

376

Effects of Combined Treatment with Naringin and Treadmill Exercise

377

Osteoporosis in Ovariectomized Rats. Sci. Rep. 2015, 5, 13009.

on

378

(29) Lawrence, C.B.; Brough, D.; Knight, E.M. Obese mice exhibit an altered

379

behavioural and inflammatory response to lipopolysaccharide. Dis. Model.

380

Mech. 2012, 5, 649-59.

381 382 383

(30) Joshua D, Lambert.; Mary J, Kennett.; Shengmin, Sang.; Kenneth R, Reuhl.; Jihyeung, Ju.; Chung, S.; Yang. Hepatotoxicity of High Oral Dose (-)-Epigallocatechin-3-Gallate in Mice. Food. Chem. Toxicol. 2010, 48, 409–416.

384

(31) Lagace, T.A.; Curtis, D.E.; Garuti, R.; McNutt, M.C.; Park, S.W.; Prather,

385

H.B.; Anderson, N.N.; Ho, Y.K.; Hammer, R.E.; Horton, J.D. Secreted PCSK9

386

decreases LDL receptors in hepatocytes and in livers of parabiotic mice. J. Clin.

387

Invest. 2006, 11, 2995-3005

388

(32) Feingold, K.R.; Moser, A.H.; Shigenaga, J.K.; Patzek, S.M.; Grunfeld, C.

389

Inflammation stimulates the expression of PCSK9. Biochem. Biophys. Res.

390

Commun. 2008, 374, 341-344.

391

(33) Yang, A.; Gyulay, G.; Mitchell, M.; White, E.; Trigatti, B.L.; Igdoura, S.A.

392

Hypomorphic

393

downregulation of VLDL production in mice. J. Lipid. Res. 2012, 53, 2573-2585.

394

(34) Le Lay, S.; Lefrère, I.; Trautwein, C.; Dugail, I.; Krief, S. Insulin and

395

sterol-regulatory element-binding protein-1c (SREBP-1C) regulation of gene

sialidase

expression

decreases

18


serum

cholesterol

by

Page 19 of 32


396

expression in 3T3-L1 adipocytes. Identification of CCAAT/enhancer-binding

397

protein beta as an SREBP-1C target. J. Biol. Chem. 2002, 277, 35625-34. Epub

398

2002 Jun 4.

399

(35) Horie, T.; Baba, O.; Kuwabara, Y.; Chujo, Y.; Watanabe, S.; Kinoshita,

400

M.; Horiguchi, M.; Nakamura, T.; Chonabayashi, K.; Hishizawa, M.; Hasegawa,

401

K.; Kume, N.; Yokode, M.; Kita, T.; Kimura, T.; Ono, K. MicroRNA-33

402

deficiency reduces the progression of atherosclerotic plaque in ApoE-/- mice. J.

403

Am. Heart. Assoc. 2012, 1, e003376.

404

(36) Jeong, H.J.; Lee, H.S.; Kim, K.S.;

Kim, Y.K.; Yoon, D.; Park, S.W.

405

Sterol-dependent regulation of proprotein convertase subtilisin/kexin type 9

406

expression by sterol-regulatory element binding protein-2. J. Lipid. Res. 2008, 49,

407

399-409

408

(37) Sirois, F.; Chrétien, M.; Mbikay, M. Comparing expression and activity

409

of PCSK9 in SPRET/EiJ and C57BL/6J mouse strains shows lack of correlation

410

with plasma cholesterol. Mol. Genet. Metab. Rep. 2016, 10, 11-17

411

(38) Jiang, D.; Wang, D.; Zhuang, X.; Wang, Z.; Ni, Y.; Chen, S.; Sun, F.

412

Berberine increases adipose triglyceride lipase in 3T3-L1 adipocytes through

413

the AMPK pathway. Lipids. Health. Dis. 2016, 15, 214.

414

(39) Folch, J.; Lees, M.; Sloane Stanley, G.H. A simple method for the isolation and

415

purification of total lipides from animal tissues. J. Biol. Chem. 1957, 226,

416

497-509.

417

(40) Klinger, S.C.; Glerup, S.; Raarup, M.K.; Mari, M.C.; Nyegaard, M.; Koster, 19



Page 20 of 32

418

G.; Prabakaran, T.; Nilsson, S.K.; Kjaergaard, M.M.; Bakke, O.; Nykjær, A.;

419

Olivecrona, G.; Petersen, C.M.; Nielsen, M.S. SorLA regulates the activity of

420

lipoprotein lipase by intracellular trafficking. J. Cell. Sci. 2011, 124, 1095-105.

421

(41)Berk,

K.A.; Vongpromek,

R.; Jiang,

M.; Schneider,

W.J.; Timman,

422

R.; Verhoeven, A.J.; Bujo, H.; Sijbrands, E.J.; Mulder, M.T.

423

soluble LDL receptor-relative LR11 decrease in overweight individuals with type

424

2 diabetes upon diet-induced weight loss. Atherosclerosis. 2015, 254, 67-72.

425

(42)Zaid

A.; Roubtsova

A.; Essalmani

R.; Marcinkiewicz

Levels of the

J.; Chamberland

426

A.; Hamelin J.; Tremblay M.; Jacques H.; Jin W.; Davignon J.; Seidah NG.; Prat A.

427

Proprotein convertase subtilisin/kexin type 9 (PCSK9): hepatocyte-specific

428

low-density lipoprotein receptor degradation and critical role in mouse liver

429

regeneration. Hepatology. 2008, 48, 646-54.

430

(43)Roubtsova

A.; Munkonda

MN.; Awan

Z.; Marcinkiewicz

J.; Chamberland

431

A.; Lazure C.; Cianflone K.; Seidah NG.; Prat A. Circulating proprotein

432

convertase subtilisin/kexin 9 (PCSK9) regulates VLDLR protein and triglyceride

433

accumulation in visceral adipose tissue. Arterioscler. Thromb. Vasc. Biol. 2011, 31,

434

785-791.

435

(44)Baragetti A.; Balzarotti

G.; Grigore

L.; Pellegatta

F.; Guerrini

U.; Pisano

436

G.; Fracanzani AL.; Fargion S.; Norata GD.; Catapano AL. PCSK9 deficiency

437

results in increased ectopic fat accumulation in experimental models and in

438

humans. Eur. J. Prev. Cardiol. 2017, 24, 1870-1877.

439

(45)Nandipati KC.; Subramanian S.; Agrawal DK. Protein kinases: mechanisms and 20


Page 21 of 32


440

downstream targets in inflammation-mediated obesity and insulin resistance. Mol.

441

Cell. Biochem. 2017, 426, 27-45.

442 443

(46)Guigas B.; Viollet B. Targeting AMPK: From Ancient Drugs to New Small-Molecule Activators. EXS. 2016, 107, 327-350.

444

(47)Lee KS.; Kwon YS.; Kim S.; Moon DS.; Kim HJ.; Nam KS. Regulatory

445

mechanism of mineral-balanced deep sea water on hypocholesterolemic effects in

446

HepG2 hepatic cells. Biomed. Pharmacother. 2017, 86, 405-413.

447

(48) Rajadurai, M.; Stanely Mainzen Prince, P. Preventive effect of naringin on lipids,

448

lipoproteins and lipid metabolic enzymes in isoproterenol-induced myocardial

449

infarction in Wistar rats. J. Biochem. Mol. Toxicol. 2006, 20, 191-197

450

(49)Pu P.; Gao DM.; Mohamed S.; Chen J.; Zhang J.; Zhou XY.; Zhou NJ.; Xie J.;

451

Jiang H. Naringin ameliorates metabolic syndrome by activating AMP-

452

activated protein kinase in mice fed a high-fat diet. Arch. Biochem. Biophys.

453

2012, 518, 61-70.

454

(50) Baltazar, M.T.; Dinis-Oliveira, R.J.; Duarte, J.A.; Bastos, M.L.; Carvalho, F.

455

Antioxidant properties and associated mechanisms of salicylates. Curr. Med.

456

Chem. 2011, 18, 3252–3264

457

(51) Wang, D.; Gao, K.; Li, X.; Shen, X.; Zhang, X.; Ma, C.; Qin, C.; Zhang, L.

458

Long-term naringin consumption reverses a glucose uptake defect and improves

459

cognitive deficits in a mouse model of Alzheimer's disease. Pharmacol. Biochem.

460

Behav. 2012, 102, 13-20

461

(52)Gorinstein,

S.; Leontowicz,

H.; Leontowicz,

M.; Krzeminski,

21


R.; Gralak,


462

M.; Delgado-Licon, E.; Martinez Ayala, AL.; Katrich, E.; Trakhtenberg, S.

463

Changes in plasma lipid and antioxidant activity in rats as a result of naringin

464

and red grapefruitsupplementation. J. Agric. Food. Chem. 2005, 53, 3223-3238.

465

(53)Drewnowski A.; Henderson SA.; Shore AB.Taste responses to naringin, a

466

flavonoid, and the acceptance of grapefruit juice are related to genetic sensitivity

467

to 6-n-propylthiouracil. Am J Clin Nutr. 1997, 66, 391-397.

468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 22


Page 22 of 32

Page 23 of 32


485

486 487 488 489 490 491 492 493 494 495 496 497 498 499 500 501 502 503 23



504

Legends for Figures

505

Fig. 1. Naringin’s chemical structure.

Page 24 of 32

506 507

Fig. 2. 8-isoprostane plasma level. Data are mean ± SD (n = 10). Naringin, naringin

508

at (L) 25 or (M) 50 or (H) 100 mg/kg. **, p < 0.01, vs. chow-fed. +, p < 0.05 and ++, p

509

< 0.01 vs. HF-fed. HF, high fat.

510 511

Fig. 3. SREBP-1, p-AMPKα, SREBP-2, PCSK9, and LDLR protein expression. (A)

512

Representative result of western blot. (B) Protein expression of p-AMPKα. (C)

513

Protein expression of SREBP-1. (D) Protein expression of SREBP-2. (E) Protein

514

expression of PCSK9. (F) Protein expression of LDLR. Data are mean ± SD (n = 10).

515

Naringin, naringin at (L) 25 or (M) 50 or (H) 100 mg/kg. +p < 0.05 or

516

HF-fed.**p < 0.01 vs. chow-fed. p-AMPKα, AMP-activated protein kinase (AMPK)

517

; mSREBP, mature sterol regulatory element-binding protein; PCSK9,

518

proprotein convertase subtilisin/kexin type 9; LDLR, low-density lipoprotein receptor;

519

HF, high fat.

++

p < 0.01 vs.

520 521

Fig. 4. SREBP-1, p-AMPKα, SREBP-2, PCSK9, and LDLR mRNA expression. (A)

522

p-AMPKα mRNA/GAPDH. (B) SREBP-1 mRNA/GAPDH. (C) SREBP-2 mRNA/

523

GAPDH. (D) PCSK9 mRNA/GAPDH. (E) LDLR mRNA/GAPDH. (F) p-AMPKα

524

mRNA/36B4. (G) SREBP-1 mRNA/36B4. (H) SREBP-2 mRNA/36B4. (I) PCSK9

525

mRNA/36B4. (J) LDLR mRNA/36B4. Data are mean ± SD (n = 10). Naringin, 24


Page 25 of 32


526

naringin at (L) 25 or (M) 50 or (H) 100 mg/kg. +p < 0.05 or ++p < 0.01 vs. HF-fed.**p

527

< 0.01 vs. chow-fed. HF, high fat.

528 529

Fig. 5. Plasma concentrations of PCSK9, LDLC, leptin and insulin. (A) Leptin

530

level.(B) Insulin level. (C) LDLC level. (D) PCSK9 level. (E) HDLC level. Data are

531

mean ± SD (n = 10). Naringin, naringin at (L) 25 or (M) 50 or (H) 100 mg/kg. +p

Naringin activates AMPK resulting in altered expression of SREBPs

Recommend Documents