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Effects of Dietary Genistein on Plasma and Liver Lipids, Hepatic Gene Expression, and Plasma Metabolic Profiles of Hamsters with Diet-induced Hyperlipidemia Chaohua Tang, Kai Zhang, Qingyu Zhao, and Junmin Zhang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b01590 • Publication Date (Web): 27 Aug 2015 Downloaded from http://pubs.acs.org on September 1, 2015

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Journal of Agricultural and Food Chemistry

Effects of Dietary Genistein on Plasma and Liver Lipids, Hepatic Gene Expression, and Plasma Metabolic Profiles of Hamsters with Diet-induced Hyperlipidemia Chaohua Tang,† Kai Zhang,† Qingyu Zhao,† Junmin Zhang†,* †

Institute of Animal Science, Chinese Academy of Agricultural Sciences, No.2 Yuan Ming Yuan

West Road, Haidian District, 100193, Beijing, China. *

Correspondence:

Professor

Junmin

Zhang,

email

address:

86-010-62815537, tel.: 86-010-62815852. Short title: Dietary Genistein on Hyperlipidemia.

1

ACS Paragon Plus Environment

[email protected],

fax:


1

Abstract

2

Male hamsters were fed one of the following three diets (n = 15): normal fat diet (NFD), high fat

3

diet (HFD), or HFD + 2 g/kg genistein for 6 weeks, and the effects of dietary genistein on

4

hyperlipidemia were investigated using traditional and 1H NMR metabonomic approaches. At 6

5

weeks, compared with the hamsters in the NFD group, those in the HFD group had higher plasma

6

and liver lipids (P < 0.05). Hyperlipidemia was alleviated in the genistein group, with lower plasma

7

cholesterol (9.11 ± 0.40 vs. 12.4 ± 0.37 mmol/L), triglyceride (8.07 ± 1.08 vs. 14.7 ± 1.18 mmol/L),

8

LDL-cholesterol (2.69 ± 0.20 vs. 4.48 ± 0.27 mmol/L), malondialdehyde (7.77 ± 1.64 vs. 14.0 ±

9

1.15 µmol/L) and liver cholesterol (20.9 ± 1.01 vs. 29.9 ± 2.76 µmol/g) than those in the HFD

10

group (P < 0.05). Hepatic LDL receptor, estrogen receptors α and β mRNA expression in the

11

genistein group were significantly up-regulated, compared with the HFD group (P < 0.05). In the

12

1

13

differed in the three groups and the metabolic profile of the genistein group was shifted towards that

14

of the NFD group. These results extend our understanding of the beneficial effects of genistein on

15

hyperlipidemia.

16

Keywords: Genistein, Plasma lipid, Liver lipid, Hepatic gene expression, Plasma metabolic profile,

17

Hamster, Hyperlipidemia

H NMR metabonomic analysis, both the small and macromolecular plasma metabolite profiles

2


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Introduction

19

The elevation of blood lipids, hyperlipidemia, has a variety of etiologies. The clinically significant

20

consequences of hyperlipidemia include two life-threatening conditions, pancreatitis and

21

atherosclerosis. Atherosclerosis is responsible for a large proportion of cardiovascular diseases,

22

which are themselves responsible for up 10% of the worldwide disease burden.1 In 1999, the U.S.

23

Food and Drug Administration stated that soy protein, as part of a diet low in saturated fat and

24

cholesterol, may reduce the risk of coronary heart disease by lowering blood cholesterol levels.2

25

The soy isoflavones contained in soy protein may account for 60%–70% of the

26

hypocholesterolemic effects of soy protein.3

27

Soy isoflavones are reported to improve lipid metabolism in normocholesterolemic

28

premenopausal women and hyperlipidemic men.4, 5 Genistein (GSN), the principal soy isoflavone,

29

may modulate the estrogen receptors and estrogen-receptor-dependent gene expression and regulate

30

lipid metabolism through its biological similarity to estrogens.6 GSN acts through multiple

31

mechanisms, including the inhibition of cholesterol synthesis and its esterification, significantly

32

reducing the secretion by cells of apolipoprotein B, the primary lipoprotein of low-density

33

lipoprotein (LDL) particles.7 Both in vivo and in vitro studies have suggested that GSN lowers

34

cholesterol levels by increasing LDL receptor (LDL-R) activity.7, 8 Study has also suggested that

35

peroxisome proliferator-activated receptor α (PPARα) is activated by GSN in vitro,9 whereas it was

36

not affected by GSN in in vivo studies.10,

37

mitogen-activated protein kinase (MAPK) pathway in vitro.12 Taken together, these data indicate

38

that multiple mechanisms are responsible for the effects of GSN on lipid metabolism, and that these

39

require further investigation.

11

Another study reported that GSN affects the

40

Metabonomics, the high-throughput identification and quantification of small-molecule

41

metabolites in the metabolome, is a useful method with which to improve our understanding of the

42

dynamic biochemical compositions of living systems.13 Nuclear magnetic resonance (NMR) is one 3



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of two instruments (another is mass spectrometry) typically used for the acquisition of

44

metabonomic data, to characterize how metabolic profiles respond to bioactive components.14, 15

45

Metabonomic analyses also generate a more complete picture of the metabolic status of living

46

systems than do traditional approaches. One of the first studies to use metabonomic techniques in a

47

dietary intervention trial examined the effects of soy-derived proteins and soy isoflavones.

48

Significant differences were detected in the lipoprotein, amino acid, and carbohydrate profiles of

49

the treated subjects, which were related to the metabolic pathways responsible for osmolyte

50

fluctuation and energy metabolism. However, no previous study has investigated the changes in the

51

metabolic profiles induced by the ingestion of GSN in individuals with hyperlipidemia.

16

52

In this study, the hamster, an excellent animal model for studying hyperlipidemia,17 was used to

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determine the effects of GSN on high fat diet (HFD)-induced hyperlipidemia using both traditional

54

and 1H NMR metabonomic approaches. We also investigated the organ weights, the lipid levels in

55

the plasma and liver, the hepatic expression of genes related to lipid metabolism, and the plasma

56

metabolic profiles of the hamsters.

57

Materials and methods

58

Chemicals

59

GSN was purchased from Chengdu Must Bio-technology Co., Ltd (Chengdu, China). Oil-Red O

60

and 2-propanol were from Sigma–Aldrich (Shanghai, China). TRIzol Reagent was obtained from

61

Invitrogen (Carlsbad, CA, USA). Moloney murine leukemia virus reverse transcriptase and dNTPs

62

were purchased from TaKaRa Bio, Inc. (Dalian, China). EvaGreen® reagents were provided by

63

Biotium Inc. (Hayward, CA, USA). D2O was obtained from Cambridge Isotope Laboratories, Inc.

64

(Tewksbury, MA, USA) and sodium trimethylsilyl propionate from Merck KGaA (Darmstadt,

65

Germany).

66

Animal treatment and management

67

Forty-five male golden Syrian hamsters (9 weeks old) were purchased from Charles River 4


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Company (Vital River, Beijing, China). After acclimation for 1 week, the hamsters were randomly

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assigned to one of the following dietary groups (n = 15 each): normal fat diet (NFD) group, HFD

70

group, or HFD + 2 g/kg GSN group (equivalent to 76 mg/kg body weight). GSN (98%) was mixed

71

in with the feed. The dose of GSN used in this study was based on a former study in which the

72

serum and hepatic lipids of mice fed a HFD were not affected until the level of dietary GSN

73

supplementation reached 2 g/kg.10 No adverse effects occurred at the dose used based on a 90-day

74

feeding study which showed no toxicity in animals fed GSN at doses up to 63 mg/kg bodyweight/d

75

or a GSN/daidzein isoflavone mixture (2:1) at 100 mg/kg bodyweight/d.18 The present study was

76

conducted over 6 weeks and the hamsters were given access to feed and water ad libitum. Dietary

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consumption and bodyweight were measured every week and after 6 weeks. The NFD (protein 22

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g%, fat 5 g%, carbohydrate 54 g%) was formulated based on the hamsters’ nutritional requirements

79

and was produced by Beijing Keao Xieli Feed Co., Ltd (Beijing, China). The HFD (protein 20 g%,

80

fat 21 g%, carbohydrate 50 g%) was purchased from Research Diets Inc. (New Brunswick, NJ,

81

USA). The animals were housed in steel cages (five hamsters per cage) in an environmentally

82

controlled room (20–24 °C, 12 h alternating light:dark cycle). The study protocol was approved by

83

the Animal Care and Use Committee of the Chinese Academy of Agricultural Sciences.

84

Sample collection

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In order to meet the statistical requirements of metabonomics data (n≥6) and to reduce the errors

86

resulted from sampling time (were all the hamsters sampled, sampling would have taken a whole

87

day). After fasting for 12 h, six hamsters from each treatment group were killed under anesthesia

88

induced with pentobarbital. Blood was obtained from the orbital vein with heparin-treated glass

89

capillary tubes. Plasma was obtained after the blood was centrifuged (3000 rpm at 4 °C for 10 min),

90

and stored at −80 °C until analysis. The liver, heart, kidney, and abdominal and epididymal fat were

91

separated, weighed, and flash frozen in liquid nitrogen.

92

Plasma parameters, hepatic lipid determination, and Oil-Red O staining 5



93

The plasma total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL–C),

94

and LDL–cholesterol (LDL–C) were determined with an autoanalyzer (Hitachi 7060, Hitachi,

95

Chiyoda, Tokyo, Japan) with commercial kits (BioSino Bio-technology and Science Inc., Beijing,

96

China). The superoxide dismutase (SOD), malondialdehyde (MDA), and free fatty acids (FFA) in

97

the plasma, and hepatic TC and TG were measured with commercial kits (Jiancheng Biotechnology,

98

Nanjing, China). The same liver lobes were sampled from the hamsters in each group, immersed in

99

liquid nitrogen, and stored at −80 °C until frozen sectioning. The sections were rinsed with distilled

100

water, stained with 0.2% Oil-Red O and 60% 2-propanol for 10 min at 37 °C, and then rinsed again

101

with distilled water. Lipid accumulation in the liver was then observed under a microscope (Leica

102

DM4000B, Bensheim, Germany) and photographed.

103

Real-Time PCR

104

The expression of 13 genes involved in lipid metabolism in the liver was determined with

105

Real-Time PCR. Total RNA was extracted from the liver with TRIzol Reagent, and quantified

106

spectrophotometrically. Complementary DNA was synthesized with Moloney murine leukemia

107

virus reverse transcriptase, random hexamers, deoxyribonucleosides, RNase inhibitor, and 4 µg of

108

total RNA. The mRNA expression of the target genes was determined with Real-Time PCR using

109

EvaGreen® reagents in an MJ-Chromo 4 thermocycler (Bio-Rad, Hercules, CA, USA). Changes in

110

fluorescence during the PCR were calculated according to the manufacturer’s instructions. The

111

amplification of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA was used as the

112

internal control. The Real-Time PCR results were normalized as previously described.19 The

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gene-specific primers are listed in Table 1.

114

Plasma 1H NMR metabonomics

115

Before the measurements were made, the plasma samples (200 µL) were mixed with 300 µL of D2O

116

and 100 µL of sodium trimethylsilyl propionate D2O solution (1 mg/mL), and then centrifuged

117

(13,000 rpm at 4 °C for 10 min). The supernatant of each sample was transferred individually to a 5 6


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mm NMR tube.

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All NMR measurements were made on a Varian Inova 600 MHz spectrometer (Varian Inc.,

120

Palo Alto, CA, USA) equipped with a triple resonance inverse probe. A total of 64 transients were

121

collected into 32 K data points for each spectrum, with a spectral width of 8000 Hz and a recycle

122

delay of 2 s. The plasma was analyzed using the Carr–Purcell–Meiboom–Gill (CPMG) pulse

123

sequence

124

longitudinal-eddy-current (BPP-LED) pulse sequence to detect high-molecular-weight metabolites.

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Two 1H NMR spectra were acquired for each sample. The CPMG spectra were acquired using the

126

CPMG pulse sequence (-RD-90°-(τ-180°-τ)n-ACQ) with a total spin–spin relaxation delay (2nτ) of

127

320 ms and a delay Te of 400 µs. Irradiation of the water resonance was used during recycle delay.

128

The

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(-RD-90°-G1-180°-G1-90°-T-90°-G1-180°-G1-90°-τ-90°-ACQ) with a diffusion delay of 100 ms

130

and a delay Te of 5 ms, with water peak irradiation during recycle delay. The free induction decay

131

signal was zero filled. Line-broadening factors of 0.5 Hz (CPMG) and 3 Hz (BPP-LED) were added

132

and then Fourier transformed to the 1H NMR spectra. The 1H NMR spectra were manually

133

corrected for phase and baseline, with reference to the lactate doublet signal at 1.33 ppm. The

134

CPMG and BPP-LED spectra were binned into 0.01 ppm and 0.04 ppm integral regions,

135

respectively. To exclude the water signal, the region from 4.6–5.0 ppm was not included in the

136

statistical analysis. The spectra were normalized to remove any concentration effects.

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Statistical analysis

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Bodyweight, feed intake, organ weight, plasma parameters, hepatic lipid content, and the relative

139

expression of mRNAs are shown as mean values and their standard errors (SE). Student’s t test was

140

used to compare differences (NFD vs. HFD group, HFD vs. HFD + 2 g/kg GSN group). Differences

141

were considered significant at P < 0.05. The 1H NMR metabonomic data were examined with

142

multivariate analysis using the SIMCA-P+ software package (version 11; Umetrics, Umeå, Sweden),

to

detect

BPP-LED

low-molecular-weight

spectra

were

metabolites

acquired

using

the

7


and

with

BPP-LED

the

pulse

bipolar-pair

sequence


143

with the binned integral regions used as input. The plasma metabonomic data were analyzed after

144

centering was applied. A principal components analysis (PCA), an unsupervised pattern-recognition

145

technique, was used to analyze the datasets. The datasets were then explored further with a

146

supervised multivariate data analytical tool, orthogonal signal correction partial least squares

147

(OSC-PLS). The quality of each model was judged by its R2 and Q2 values. Permutation testing was

148

performed on each OSC-PLS model to assess its validity (with 20 permutations).

149

Results

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Feed intake, bodyweight, and organ weight

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The different dietary groups did not differ in their bodyweights at the end of the study (Table 2).

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The hamsters in the HFD group had lower average daily feed intake and heart weights (P < 0.05),

153

and higher liver, abdominal, and epididymal fat weights (P < 0.05) than the NFD group. Although

154

the average daily feed intake and the liver and epididymal fat weights of the HFD and HFD + 2

155

g/kg GSN groups did not differ significantly (P > 0.05), the hamsters fed the GSN diet had a

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significantly lower abdominal fat weight than those fed the HFD (P < 0.05).

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Plasma parameters and hepatic lipids

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Table 3 shows that the TC, TG, LDL–C, MDA, and FFA concentrations were all significantly

159

higher in the plasma of the HFD group than in that of the NFD group (P < 0.05). The TC, TG,

160

LDL–C, and MDA contents in the GSN-treated group were all significantly lower than those in the

161

HFD group (P < 0.05). The plasma FFA levels in the GSN-treated group were 29% lower than

162

those in the HFD group, although the difference was not significant.

163

The hepatic lipid contents in the HFD group were higher than those in the NFD group (29.9 vs.

164

12.2 µmol/g for TC, respectively, and 37.1 vs. 24.8 µmol/g for TG, respectively; P < 0.05). The

165

HFD + 2 g/kg GSN group had significantly lower hepatic TC (20.9 µmol/g; P < 0.05) and 16.6%

166

lower TG (31.0 µmol/g; P = 0.223) than the HFD group (Figure 1a). Oil-Red O staining showed

167

that the hepatic lipid droplets in the HFD + 2 g/kg GSN group were smaller than those in the HFD 8


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group (Figure 1b).

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Hepatic mRNA expression

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The mRNA expression of the lipid metabolism genes in the liver is shown in Figure 2. Some genes

171

related to LDL–C clearance, including those encoding the LDL-R and sterol regulatory

172

element-binding protein 2 (SREBP-2), were downregulated in the HFD group compared with the

173

NFD group (P < 0.05). LDL-R expression was significantly higher in the HFD + 2 g/kg GSN group

174

than in the HFD group (P < 0.05). The expression of cholesterol 7 α-hydroxylase (CYP7A1) and

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sterol 27-hydroxylase (CYP27A1), was lower in the HFD group than in the NFD group (P < 0.05),

176

and were unaffected by GSN treatment. The different diets significantly affected the expression of

177

the estrogen receptor (ER). The expression of both ERα and ERβ was downregulated in the HFD

178

group compared with the NFD group (P < 0.05), but was upregulated in the HFD + 2 g/kg GSN

179

group compared with the HFD group (P < 0.05).

180

The hepatic expression of ERK2, ERK5, and c-Jun N-terminal kinase (JNK), which are

181

involved in the MAPK pathway, was downregulated in the HFD group compared with the NFD

182

group (P < 0.05). However, there were no significant differences between the HFD and HFD + 2

183

g/kg GSN groups in ERK1, ERK2, or ERK5 mRNA expression. The transcription of both p38 and

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JNK almost returned to normal in the HFD + 2 g/kg GSN group relative to that in the HFD group,

185

but the change was not significant. The expression of the androgen receptor (AR) and PPARa were

186

downregulated in the HFD group (P < 0.05), but was unaffected by the GSN treatment.

187


188

As indicated by the CPMG score plots shown in Figures 3a and c, the plasma metabolic profiles of

189

the NFD and HFD groups were clearly separated in both the PCA and OSC-PLS analyses. However,

190

the HFD and HFD + 2 g/kg GSN groups could not be differentiated with PCA (Figure 3a), but

191

were separated with OSC-PLS (Figure 3c). In the OSC-PLS analysis, the NFD group was on the

192

right half of the ellipse and the HFD group was on the left half of the ellipse. The samples in the 9



193

HFD + 2 g/kg GSN group were differentiated from those in the HFD group and were shifted closer

194

to those in the NFD group. Validation of the OSC-PLS model with permutation testing resulted in a

195

Q2 intercept of (0.0, −0.27).

196

Figure 4 presents the results of BPP-LED analyzed with both PCA and OSC-PLS. In the PCA,

197

the HFD group was distributed between the lower (5/6) and upper (1/6) ellipse, the NFD group was

198

in the left ellipse, and the HFD + 2 g/kg GSN group was distributed between the right (5/6) and left

199

(1/6) ellipse (Figure 4a). The plasma BPP-LED results were also subjected to OSC-PLS, and three

200

clusters were apparent: the NFD, HFD, and HFD + 2 g/kg GSN groups. This result indicates that

201

metabolic changes occurred in the hamster plasma in response to the different dietary treatments

202

(Figure 4c). The metabolic profiles of the HFD + 2 g/kg GSN group differed from those of the

203

HFD group, and were closer to those of the NFD group. The OSC-PLS model was validated with

204

permutation testing, which had a Q2 intercept of (0.0, −0.27).

205

Table 4 shows the changes in the plasma metabolite concentrations according to CPMG and

206

BPP-LED. The concentrations of lactate and lipoprotein in the HFD group were lower and higher,

207

respectively, than in the NFD group. Very-LDL (VLDL; lipid, CH3), LDL and VLDL (lipid, CH2),

208

VLDL (lipid, CH2), lipid (CH2CH2CO), lipid (CH2C=C), lipid (CH2CO), and unsaturated fatty acids

209

(UFA; =CH) levels were higher in the HFD group than in the NFD group. Most of the abnormal

210

lipid metabolites, including VLDL (lipid, CH3), VLDL (lipid, CH2), lipid (CH2CH2CO), lipid

211

(CH2C=C), lipid (CH2CO), phosphatidylcholine, and UFA (=CH), were reversed by GSN

212

supplementation.

213

Discussion

214

Feed intake, bodyweight, organ weight, and plasma and hepatic lipid levels

215

There has been little agreement on the effects of GSN on the feed intake and bodyweights of animal

216

models. Some researchers have reported that GSN reduces feed intake or bodyweight, 20, 21 whereas

217

others have shown no effect of dietary GSN on feed intake and bodyweight.22 These discrepancies 10


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218

may be attributable to differences in the animal models used, HFD compositions, GSN doses, or the

219

durations of the experiments. It is unclear whether the changes in feed intake caused by GSN

220

influence the experimental results, because this factor is not accounted for in many experiments.

221

Feed intake was not affected by GSN in this study, so all the results were attributable to the

222

biological effects of GSN rather than to different patterns of food intake. Although 2 g/kg of dietary

223

GSN significantly reduced the liver weights of mice fed an HFD (18% fat),10 liver weight was not

224

significantly affected by the same concentration of GSN in the present study, which could be

225

explained by the differences in the HFD composition (21% fat in this study) and the animal species

226

used. GSN has previously been shown to affect adipose deposition,21 and we also observed

227

significantly less abdominal fat in the hamsters fed 2 g/kg GSN than in the HFD hamsters.

228

Plasma and hepatic lipid levels reflect the lipid metabolism in animals. At the end of 6 weeks,

229

the plasma and liver lipids in the HFD group were all significantly higher than those in the NFD

230

group, suggesting the induction of hyperlipidemia. This hyperlipidemia was alleviated in the 2 g/kg

231

GSN group, which had lower lipid levels in both the plasma and liver than did the HFD group.

232

Previous studies have shown that GSN reduces the plasma and hepatic lipids in animal models,

233

although the lowest observable levels have varied in different studies.10, 11, 23 One study found that 2

234

g/kg of dietary GSN reduced plasma TC and liver TG in mice fed a HFD.21 Another study found

235

that the serum and liver lipids of mice fed an HFD were not affected until GSN was increased to 2

236

g/kg.10 In this study, the lipids in both the plasma and livers of the 2 g/kg GSN group were lower

237

than those in the HFD group, which is consistent with previous studies.10, 21 We also found that

238

GSN exerted an antioxidant effect by reducing plasma MDA production, which is consistent with a

239

previous study in which lipid peroxidation was inhibited by GSN treatment.24 The biochemical

240

parameters of the plasma and liver demonstrated that 2 g/kg dietary GSN improved the lipid

241

metabolism of hamsters with diet-induced hyperlipidemia by reducing lipid circulation and

242

inhibiting lipid peroxidation. 11



243

Hepatic mRNA expression

244

Several mechanisms have been proposed to explain the antihyperlipidemic effects of GSN. One

245

study reported that of the 97 transcripts altered in the HFD-fed group, the expression of 84 were

246

normalized with GSN supplementation.21 Genes that are involved in cholesterol metabolism are

247

regulated by GSN,7 which was confirmed here by the observed increase in hepatic LDL receptor

248

mRNA expression, indicating an improvement in LDL–C clearance. Isoflavones have previously

249

been reported to increase the mature nuclear form of SREBP-2 in vitro.25 However, hepatic

250

SREBP-2 mRNA expression was not affected by GSN in the present study, consistent with another

251

study.11 CYP7A1 and CYP27A1 were upregulated in mice fed a soy protein diet plus 2%

252

cholesterol.26 We observed no effect of GSN on CYP7A1 or CYP27A1 expression in this study,

253

which warrants further investigation. Because GSN has a similar structure to estradiol, it is reported

254

to bind both ERα and ERβ,27 the two nuclear ERs upon which estradiol acts. The binding of

255

estradiol to the ERs inhibits lipogenesis primarily by reducing the activity of lipoprotein lipase, an

256

enzyme that regulates lipid uptake by adipocytes.6 Hepatic ERα and ERβ mRNA expression was

257

upregulated in the GSN group, indicating that the ERs were activated by GSN ingestion. This may

258

explain the primary mechanism underlying the hypolipidemic effects of GSN in this study, although

259

more evidence is required to demonstrate the molecular mechanism of GSN activity on

260

hyperlipidemia.

261

Increasing evidence has established that GSN acts not only through the ERs, but also via other

262

pathways. PPARα is important in the β-oxidation of fatty acids and is mainly expressed in tissues

263

such as the liver, where lipoprotein metabolism is crucial. GSN binds directly to and activates

264

PPARα in vitro, which may result in lipid catabolism and reduce blood lipids.6, 9 However, hepatic

265

PPARα mRNA expression was not affected by GSN supplementation in vivo.10, 11 We also found no

266

difference in hepatic PPARα mRNA expression in this study. Although GSN reportedly upregulates

267

the expression of ERK1/2 in vitro,12 we saw no significant effect of GSN treatment on any gene 12


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involved in the liver MAPK pathway. Therefore, the actions of GSN in vivo and in vitro may not be

269

consistent, so further studies of this discrepancy are required.

270


271

The aim of the 1H NMR metabonomic analysis was not to absolutely quantify the differences

272

between the groups, but to compare their relative differences. The distinct plasma metabolic profiles

273

of the HFD and NFD groups indicate that 1H NMR metabonomic data directly reflect the abnormal

274

metabolic profiles induced by HFD in hamsters, which recapitulates the results of previous studies.

275

28, 29

276

this is the first study to use an 1H NMR metabonomic analysis of plasma metabolites profiles to

277

identify the biological effects of GSN in hamsters fed an HFD. The restoration of the plasma

278

metabolic profiles in the 2 g/kg GSN group indicates that GSN improved hamster hyperlipidemia.

279

These results are consistent with the plasma and hepatic results that we obtained with traditional

280

approaches.

Although numerous studies have reported the beneficial effects of GSN on lipid metabolism,

281

A multivariate statistical analysis of the NMR spectra of the plasma samples revealed

282

differences in the intensities of the different spectral regions, which could be ascribed to differences

283

in lipoproteins, lactate, VLDL, LDL, phosphatidylcholine, UFA, or other lipids among the three

284

groups, as listed in Table 4. Lactate, an important intermediate of glycometabolism, was lower in

285

the HFD group than in the NFD group, consistent with a previous study,29 and the change was

286

reversed in the GSN group. A reduction in blood lactate implies that glycolysis is inhibited and the

287

energy consumption pattern may shifted to lipid oxidation in response to hyperlipidemia.29

288

Therefore, the reversion of lactate levels by GSN might contribute to the restoration of normal

289

energy consumption patterns. However, other NMR metabonomic studies have reported that blood

290

lactate is higher in hamsters fed an HFD. 30, 31 Therefore, further research is required. The BPP-LED

291

spectra were dominated by the signals from lipids, and GSN treatment restored the signal intensities

292

of VLDL, phosphorylcholine, UFA, and other lipids to those of the NFD group. VLDL and LDL, 13



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293

which are considered to be the major cholesterol-containing lipoproteins implicated in the

294

development of atherosclerosis, were restored to normal levels in the GSN group. LDL–C is mainly

295

cleared by the hepatic LDL-R pathway, in which ERs are critically involved.32 Therefore, in this

296

study, the reversion of VLDL and LDL in the GSN group observed with 1H NMR metabonomics

297

may be explained by the upregulated expression of hepatic ERα, ERβ, and the LDL receptor.

298

Polyunsaturated fatty acids, which have a high degree of unsaturation in the n-6 and n-3 series,

299

accelerate cell-mediated LDL peroxidation and therefore aggravate the atherosclerotic process.33

300

The restoration of the plasma lipid profiles after GSN supplementation suggests that hyperlipidemia

301

was globally ameliorated in these hamsters.

302

We have demonstrated that dietary 2 g/kg GSN ameliorated hyperlipidemia in hamsters by

303

reducing the weight of abdominal fat and the plasma and hepatic lipid contents. The improvement

304

in lipid metabolism was associated with the upregulated expression of the hepatic LDL receptor,

305

ERα, and ERβ mRNAs. 1H NMR and multivariate data analyses showed that both the abnormal

306

small and macromolecular metabolic plasma profiles in the HFD group of hamsters were shifted

307

towards normal profiles by GSN supplementation. Further research to absolutely quantify the

308

plasma lipid profiles in relation to GSN ingestion would be of great value.

309

Abbreviations used

310

GSN, genistein; NMR, nuclear magnetic resonance; NFD, normal fat diet; HFD, high fat diet;

311

HFD + 2 g/kg GSN, high fat diet + 2 g/kg genistein; ADFI, average daily feed intake; BW,

312

bodyweight; TC, total cholesterol; TG, triglyceride; HDL–C, high-density lipoprotein

313

(HDL)–cholesterol; LDL–C, low-density lipoprotein (LDL)–cholesterol; SOD, superoxide

314

dismutase; MDA, malondialdehyde; FFA, free fatty acids; GAPDH, glyceraldehyde phosphate

315

dehydrogenase; LDL-R, low-density lipoprotein (LDL)–receptor; SREBP-2, sterol regulatory

316

element-binding

317

27-hydroxylase; ER, estrogen receptor; ERK, extracellular regulated protein kinase; JNK, c-Jun

protein

2;

CYP7A1,

cholesterol

7

α-hydroxylase;

14


CYP27A1,

sterol

Page 15 of 29


318

N-terminal kinase; AR, androgen receptor; PPARα, peroxisome proliferator-activated receptor α;

319

MAPK, mitogen-activated protein kinase; CPMG, Carr–Purcell–Meiboom–Gill; BPP-LED,

320

bipolar-pair longitudinal-eddy current; PCA, principal components analysis; OSC-PLS, orthogonal

321

signal correction partial least squares.

322

Acknowledgment

323

We thank Bo Sun for her technical support during sample analysis.

324

Funding

325

This study was financially supported by the Agricultural Science and Technology Innovation

326

Program (ASTIP-IAS12) and the Special Basic Research Fund for Central Public Research

327

Institutes (2015ywf-zd-28).

328

Conflicts of interest

329

None.

15



330

References

331

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332

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423 424 425 426

19



Table 1 Oligonucleotide primers Primer

Sequence (5′-3′)

GAPDH-F

TGGCAAGTTCAAAGGCACAG

GAPDH-R

AACATACTCGGCGCCAGCATC

LDL-R-F

CTCCACTCTATCTCCAGCATTG

LDL-R-R

TTTCAGCCACCAAATTAACATC

ERα-F

ACCAAAGCCTCGGGAATG

ERα-R

GCGGCGTTGAACTCGTAG

ERβ-F

GGTGAAGGAGCTGCTGCTGA

ERβ-R

GGCTGGGCCAAGAAAATCC

SREBP-2-F

GGCTGGTTTGACTGGATGG

SREBP-2-R

CGGATAAGCAGGTTTGTAGGTT

CYP7A1-F

AGCAACTAAACAACCTGCCAGTACTA

CYP7A1-R

GTCCGGATATTCAAGGATGCA

CYP27A1-F

GGAGCAGTGGAAGGATCACC

CYP27A1-R

TGTTCAAAGCCTGGCGC

ERK1-F

TTGTATCATCAACATGAAGGCTCG

ERK1-R

TGAAGGTCAACATCCGGTCC

ERK2-F

GCCTTGCCCGTGTTGC

ERK2-R

AATACCCAGGATGTGATTCAGC

ERK5-F

AGAGTCGCCTGATGTCAACCT

ERK5-R

CGATGTCAGCGGGGTTCA

p38-F

CGAAAATGTGATTGGTCT

p38-R

CACGATGTTGTTCAGGTC

JNK-F

TCCAGCACCCATACATCAACG

JNK-R

CGCTCCTCCAAGTCCATTACC

AR-F

TCCGCTGGCTCTGACCG

AR-R

CAGCTGCACTCCCTCCATG

PPARα-F

CCTGTCTGTTGGGATGTCAC

PPARα-R

AGGTAGGCCTCGTGGATTCT

GAPDH, glyceraldehyde phosphate dehydrogenase; LDL-R, Low-density-lipoprotein receptor; ER, estrogen receptor; SREBP-2, sterol regulatory element-binding protein 2; CYP7A1, cholesterol 7 α-hydroxylase; CYP27A1, sterol 27-hydroxylase; ERK, extracellular regulated protein kinase; JNK, c-Jun N-terminal kinase; AR, androgen receptor; PPARα, peroxisome proliferator-activated receptor-alpha. 20


Page 20 of 29

Page 21 of 29


Table 2 Effects of 2 g/kg dietary genistein on bodyweight, feed intake, organ weight, and visceral fat weight in hamsters fed a high fat diet NFD Measures

HFD

HFD + 2 g/kg GSN

†

Mean

SE

Mean

SE

Mean

SE

Initial BW (g)

135

1.76

135

1.90

134

1.90

Final BW (g)

149

2.67

154

2.54

156

1.74

ADFI (g/d)

6.74**

0.01

5.69

0.21

5.81

0.10

ADFI and BW

Organ and visceral fat weight (g/100 g BW) Heart weight

0.32**

0.01

0.28

0.01

0.30

0.01

Liver weight

2.94***

0.12

4.54

0.15

4.32

0.11

Kidney weight

0.64

0.02

0.68

0.02

0.67

0.02

Abdominal fat

1.78*

0.17

2.82

0.34

2.05*

0.12

Epididymal fat

2.84*

0.16

3.45

0.19

3.19

0.13

NFD, normal fat diet; HFD, high fat diet; HFD + 2 g/kg GSN, high fat diet + 2 g/kg genistein; ADFI, average daily feed intake; BW, bodyweight. Mean values were significantly different from those of the HFD group: *P < 0.05, **P < 0.01, ***P < 0.001. †

Fifteen replicates were used for BW, three for ADFI, and six for organ weight and visceral fat weight per group.

21



Page 22 of 29

Table 3 Effects of 2 g/kg dietary genistein on plasma biochemical parameters in hamsters fed a high fat diet NFD

HFD

HFD + 2 g/kg GSN

†

Indices

Mean

SE

Mean

SE

Mean

SE

TC (mmol/L)

4.20***

0.16

12.4

0.37

9.11***

0.40

TG (mmol/L)

1.56***

0.17

14.7

1.18

8.07**

1.08

HDL-C (mmol/L)

0.26

0.01

0.26

0.01

0.29

0.02

LDL-C (mmol/L)

0.94***

0.10

4.48

0.27

2.69***

0.20

38.8

6.14

30.3

3.71

32.4

5.64

MDA (µmol/L)

7.94**

0.95

14.0

1.15

7.77*

1.64

FFA (µmol/L)

2767***

678

9337

843

6598

1255

SOD (U/mL)

NFD, normal fat diet; HFD, high fat diet; HFD + 2 g/kg GSN, high fat diet + 2 g/kg genistein; TC, total cholesterol; TG, triglycerides; HDL–C, high-density lipoprotein–cholesterol; LDL–C, low-density lipoprotein–cholesterol; SOD, superoxide dismutase; MDA, malondialdehyde; FFA, free fatty acid. Mean values were significantly different from those of the HFD group: *P < 0.05, **P < 0.01, ***P < 0.001. †

Six replicates were used per group.

22


Page 23 of 29


Table 4 Metabolic differences in plasma among the normal fat diet, high fat diet, and high fat diet + 2 g/kg genistein groups† NMR spectral region (ppm)

Assignment

HFD

HFD + 2 g/kg GSN

0.84–0.90

Lipoprotein

↑

↓

1.26–1.30

Lipoprotein

↑

↓

↓

↑

CPMG Spectra

1.32–1.34 Lactate 4.09–4.10 BPP-LED Spectra 0.82, 0.86

VLDL/LDL (lipid, CH3)

↓

↑

0.90

VLDL (lipid, CH3)

↑

↓

1.22

LDL (lipid, CH2)

↓

↑

1.26

LDL/VLDL (lipid, CH2)

↑

↑

1.30, 1.34

VLDL (lipid, CH2)

↑

↓

1.58

Lipid (CH2CH2CO)

↑

↓

2.02, 2.06

Lipid (CH2C=C)

↑

↓

2.22, 2.26

Lipid (CH2CO)

↑

↓

3.22

PCho

↓

↑

UFA (=CH)

↑

↓

NFD

HFD

5.3, 5.34

NFD, normal fat diet; HFD, high fat diet; HFD + 2 g/kg GSN, high fat diet + 2 g/kg genistein; VLDL, very low-density lipoprotein; LDL, low-density lipoprotein; Pcho, phosphatidylcholine; UFA, unsaturated fatty acids; CPMG, Carr–Purcell–Meiboom–Gill pulse sequence; BPP-LED, bipolar-pair longitudinal-eddy current. †

The symbols ↑ and ↓ indicate higher and lower concentrations in groups listed in the top compared with groups at the

bottom of the table.

23



Page 24 of 29

Figure Captions Figure 1 (a) Concentrations of total cholesterol (TC) and triglycerides (TG) in the liver. Values are presented as means (n = 6) and standard errors. Mean values were significantly different from those of the HFD group at *P < 0.05, ***P < 0.001. (b) Histology (Oil-Red O staining, ×400) of a representative liver section from each group. NFD, normal fat diet; HFD, high fat diet; HFD + 2 g/kg GSN, high fat diet + 2 g/kg genistein. Figure 2 Relative mRNA expression of genes related to lipid metabolism in the livers of the normal fat diet (NFD), high fat diet (HFD), and high fat diet + 2 g/kg genistein (HFD + 2 g/kg GSN) groups. The genes analyzed encoded the low-density lipoprotein receptor (LDL-R), sterol regulatory element-binding

protein

2

(SREBP-2),

cholesterol

7 α-hydroxylase

(CYP7A1),

sterol

27-hydroxylase (CYP27A1), estrogen receptor (ER), extracellular regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK), androgen receptor (AR), and peroxisome proliferator-activated receptor α (PPARα). GAPDH mRNA was amplified as the internal control. Mean values were significantly different from those of the HFD group at *P < 0.05, **P < 0.01, ***P < 0.001. Figure 3 Principal components analysis (PCA; a, b; R2X = 0.945, Q2 = 0.905) and orthogonal signal correction partial least squares (OSC-PLS; c, d; R2X = 0.891, R2Y = 0.728, Q2 = 0.62) scores plots and loading plots derived from the 1H CPMG NMR spectra of the plasma metabolic profiles of the normal fat diet (▲), high fat diet (□), and high fat diet + 2 g/kg GSN (◆) groups. Figure 4 Principal components analysis (PCA; a, b; R2X = 0.983, Q2 = 0.921) and orthogonal signal correction partial least squares (OSC-PLS; c, d; R2X = 0.99, R2Y = 0.724, Q2 = 0.7) scores plots and loading plots derived from the 1H BPP-LED NMR spectra of the plasma metabolic profiles of the normal fat diet (▲), high fat diet (□), and high fat diet + 2 g/kg genistein (◆) groups. 24


Page 25 of 29


(a)

(b)

NFD

HFD

HFD + 2 g/kg GSN

Figure 1.

25



Figure 2.

26


Page 26 of 29

Page 27 of 29


(a)

(b)

(c)

(d)

Figure 3.

27



(a)

(b)

(c)

(d)

Figure 4.

28


Page 28 of 29

Page 29 of 29


TOC Graphic(11.69*3.99cm )

Normal fat diet

Feed intake, organ weight

Plasma

High fat diet

Lipids contents Metabolic profiles

High fat diet +2 g/kg genistein

Liver

Lipids contents mRNA expression

29


Effects of Dietary Genistein on Plasma and Liver ... - ACS Publications

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