Naringin activates AMPK resulting in altered expression of SREBPs

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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

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Naringin activates AMPK resulting in altered expression of

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SREBPs, PCSK9, and LDLR to reduce body weight

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in obese C57BL/6J mice

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

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China

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Biotransformation,Hunan Agricultural University, Changsha 410128, China

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§

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,

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Changsha 410128, China

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

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Correspondence to:

Hong-Bo Xiao

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College of Veterinary Medicine

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Hunan Agricultural University

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Furong District

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Changsha 410128 China

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Tel: 086-731-84673618

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Fax: 086-731-84635292

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E-mail: [email protected]

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ABSTRACT: Previous investigations have shown a molecular crosstalk among

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

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

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

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innovative pharmacologic objective for treating obesity. We scrutinized

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

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

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

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

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

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

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

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

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

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low-density

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

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

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

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

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

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INTRODUCTION

lipoprotein

cholesterol

level,

and

plasma

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PCSK9

the

production

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

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

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cholesterol synthetic gene expressions are modulated by the family of sterol

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

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

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

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

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

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

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

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

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

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

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

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

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suppress cleavage and transcriptional activation of SREBP, inhibit LDLR degradation

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

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

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pharmacologic objective for treating obesity.

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

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

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

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

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

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

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

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

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obesity by prohibiting oxidative stress.

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

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MATERIALS AND METHODS

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

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

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

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Biotechnology (USA).

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

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

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

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

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

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weekly. Animal Studies Subcommittee at Hunan Agricultural University approved all

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animal procedures.

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

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

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

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

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

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

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

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

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

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

The dose of naringin

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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GCTTCCAGAGAGGAGGCCAG 3’and, 5’GGAGCCATGGATTGCACATT 3’;

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

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AAGAT 3’ (31-35).

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

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

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

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polyvinylidene fluoride membrane, equal concentration of protein was separated

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

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

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p-AMPKαas reported method (35-38).

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

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

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

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

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

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

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

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

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

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

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

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Japan).

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

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

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means ± SD.

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RESULTS

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

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

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

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

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

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

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

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

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

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

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

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

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Analysis of Pearson’s correlation found LDLR expression correlated negatively

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

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

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

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

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

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

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fat weigh.

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DISCUSSION

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

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

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

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

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

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hypertriglyceridemia and hypercholesterolemia even when they are fed a low fat

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

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

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

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

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

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

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

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

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

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protogenic fungus that only grows on the heartwood of endemic Cinnamomum

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

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

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

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

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

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

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

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useful pharmacologic objective for regulating obesity.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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supposition that naringin could regulate the molecular crosstalk among AMPK and

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SREBPs, PCSK9, and LDLR to improve obesity.

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

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oxidative

stress.

To

start

with,

AMPK

suppresses oxidative

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

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

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

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

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

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

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naringin has obtained lots of attention because of its antioxidant pharmacological

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

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

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

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

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

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

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

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

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

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which in turn inhibit the obesity of mice.

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

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

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

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

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

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

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

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

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

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

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related to the relation between appetite and this molecular crosstalk.

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

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

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mice.

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ORCID

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

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Funding

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We thank Hunan Provincial Natural Science Foundation of China for financial

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support.

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ACKNOWLEDGMENT

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Project supported by Hunan Provincial Natural Science Foundation of China

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(14JJ2079).

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Legends for Figures

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Fig. 1. Naringin’s chemical structure.

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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)

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

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

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

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

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

517

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

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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)

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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

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

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

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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

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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