Optimal Dietary Ferulic Acid for Suppressing the Obesity-Related

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Bioactive Constituents, Metabolites, and Functions

Optimal dietary ferulic acid for suppressing the obesityrelated disorders in leptin-deficient obese C57BL/6J-ob/ob mice Weiwei Wang, Yiou Pan, Li Wang, Hang Zhou, Ge Song, Yongwei Wang, Jianxue Liu, and Aike Li J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 25 Mar 2019 Downloaded from http://pubs.acs.org on March 25, 2019

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Journal of Agricultural and Food Chemistry 1

Optimal dietary ferulic acid for suppressing the obesity-related disorders in leptin-deficient obese C57BL/6J-ob/ob mice Weiwei Wang1, Yiou Pan1,2, Li Wang1, Hang Zhou1, Ge Song1, Yongwei Wang1, Jianxue Liu2, Aike Li1* 1Academy 2Henan

*To

of State Administration of Grain, Beijing, P. R. China 100037

University of Science and Technology, Luoyang, P. R. China 471023

whom correspondence should be addressed. E-mail: [email protected]

Tel. +86-010-56542666; Fax. +86-010-56542666

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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Abstract

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Ferulic acid (FA) is a major polyphenolic compound and has been shown to

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improve the glucose and lipid homeostasis in high-fat diet-induced obese mice. Here,

4

we found the optimal level of dietary FA to ameliorate obesity and obesity-correlated

5

disorders, and identified the responses of gut microbiota to dietary FA in genetic

6

leptin-deficient obese (ob/ob) mice. The ob/ob mice exhibited persistent higher body

7

weights, feed efficiency, white adipose tissue weights and hepatic lipid accumulation,

8

compared with those of the WT mice. However, 0.5% dietary FA suppressed these

9

symptoms in ob/ob mice. The diversity of gut microbiota and the total abundance of

10

obesity- and anti-obesity-related genera were not influenced after FA intervention in

11

ob/ob mice. These data suggest that sufficient intake of FA (0.5%) could be useful for

12

treating obesity or obesity-related diseases, and this weight-control effect is possibly

13

not correlated with the gut-brain axis.

14

Keywords: ferulic acid, obesity, hyperglycemia, gutmicrobiota, ob/ob mice


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Introduction

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Ferulic acid (FA) is a hydroxycinnamic acid, an abundant polyphenolic

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compound found in vegetables, fruits and grain, particularly rich in grain bran. Higher

18

level of FA (255-362 mg kg-1 grain) is found in brown rice, compared with that in

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milled rice (61-84 mg kg-1 grain)1. FA content is 452-731 mg kg-1 grain in

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whole-wheat flour2, of which almost 90% exists in the wheat bran. The level of FA is

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5300-5400 mg kg-1 grain in fine wheat bran. Corn has the highest content of FA in all

22

fractions among all grains3. The FA content is 232-1788 mg kg-1 grain in corn flours

23

and 1740 mg kg-1 grain in corn (dehulled kernels)3-5. What’s more, FA is of low

24

toxicity after oral administration, with the acute LD50 equal to 3200 mg kg-1 body

25

weight (BW) in mice6, and the LD50 equal to 2445 and 2113 mg kg-1 BW in male and

26

female rats, respectively7.

27

FA has been widely applied to prevent reactive oxygen species (ROS)-related

28

diseases, such as cancer, cardiovascular diseases and diabetes mellitus8. FA inhibits

29

proliferation of Caco-2 colon cancer cells by up-regulating genes related with

30

centrosome assembly and the gene for the structural maintenance of chromosome

31

protein SMC1L19. It can reduce systolic blood pressure; improve the structure and

32

function of the heart and blood vessel in hypertensive rats10. Furthermore, numerous

33

studies have shown that FA plays a beneficial role in treating diabetes through

34

regulating apoptosis and pro-inflammatory cytokines and alleviating insulin

35

resistant11.

36

Obesity, characterized by excess adipose deposition and impaired lipid and



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glucose metabolism, has been becoming a worldwide epidemic, which has high risk to

38

cause type II diabetes, cardiovascular diseases and cancers12, 13. Growing evidences

39

have demonstrated that the gut microbiota plays a key role in the development of

40

obesity, obesity-related type II diabetes and insulin resistance14-16. Most of studies

41

about the effects of phenolic compounds on gut microbiota have centered on their

42

antimicrobial activity and their potential prebiotic features17. Previous reports have

43

shown that green tea polyphenols suppressed the host body weight and hyperlipemia

44

in obese through changing of composition and diversity of colonic microbiota18.

45

However, limited information is available about whether and how the polyphenols

46

influence gut microbiota in obese human or animals. Recently, a study has shown that

47

FA inhibits the glucose and lipid metabolic dysequilibrium in high-fat diet-induced

48

obese mice19. Dietary FA can increase fecal lipid excretion and lipogenic enzyme

49

activities in high fat diet-fed mice20. And the lipid profiles can be ameliorated and the

50

lipid peroxidation can be restrained by supplementary intake of FA while rats suffered

51

with nicotine-induced toxicity21. However, the effects of dietary FA on lipid

52

deposition and metabolism in genetically obese mice remain unclear.

53

Likewise, it is seldom known about the optimal level of dietary FA to alleviate

54

changes in obese mice. The objective of the present study was to identify the optimal

55

level of dietary FA to ameliorate obesity and obesity-related disorders, and to identify

56

the responses of gut microbiota to dietary FA in leptin-deficient C57BL/6J-ob/ob

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obese mice.

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Materials and Methods


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Ethics statement. The study was carried out in accordance with the Animal Ethics

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Committee Guidelines (Registration number: 2015M03) of Academy of State

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Administration of Grain.

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Animals and diets. Male C57BL/6J-ob/ob mice (32.01 ± 0.27 g) and their counterpart

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wild-type (WT) C57BL/6J mice controls (21.65 ± 0.16 g) were purchased at 5 wks of

64

age from Beijing HFK Bioscience Co., Ltd (Beijing, China). They were housed in

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individual ventilated cages (2 mice per cage) in a temperature (23 ± 2 ºC) and

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humidity (50% relative humidity) maintained room with a 12-h light-dark cycle. After

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an acclimation period (1 week), the mice were divided into one of 6 dietary groups (n

68

= 8) on the basis of their body weights: two groups (WT and ob/ob control groups)

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fed the AIN-93M22 basal diet (Supplementary Table S1; prepared by TROPHIC,

70

Nantong, China), and four ob/ob groups fed the basal diets supplemented with FA (the

71

content of FA in diets was 0.05%, 0.125%, 0.25% and 0.5%, w/w, respectively) for 9

72

wks. FA (≥ 99% pure) was purchased from Aladdin (Shanghai, China). WT Mice and

73

ob/ob control mice were given free access to food throughout the entire study,

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whereas mice in the other four groups were pair-fed the amount of food consumed by

75

the ob/ob control mice. The food consumption and body weight were measured

76

weekly.

77

Preparation of serum and tissue samples. After food deprivation for 4 h, the mice

78

were sacrificed by using carbon dioxide inhalation at the end of the experiment.

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Serum were obtained by centrifuging whole blood at 2,000 × g for 15 min at 4ºC.

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Liver and white adipose tissue (WAT) were obtained and weighed.



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Determination of serum and hepatic lipids. Serum total triglyceride (TG), total

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cholesterol (TC), low-density lipoprotein cholesterol (LDL), and high-density

83

lipoprotein cholesterol (HDL) were measured using a biochemistry analyzer (Roche

84

Modular, Roche, USA). Livers were homogenized in distilled solution and the hepatic

85

TG and TC were determined by the assay kit (Nanjing Jiancheng Bioengineering Ins.,

86

Nanjing, China). The histopathological characteristics of mice livers were analyzed as

87

described in Park et al. (2011)23.

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Analysis of Fatty acid composition of WAT. The fatty acid composition of WAT was

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measured by the method of Pugo-Gunsam et al.24 with some modifications. In brief,

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triacylglycerols of WAT were extracted by petroleumether and were hydrolyzed to

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fatty acids by 0.5 mol/L NaOH in methanol. Fatty acids were transmethylated using

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BF3 in methanol (12-15%). Fatty acid methyl esters were then extracted in isooctane

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and used for GC analysis.

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DNA extraction and 16S rRNA gene sequencing. The cecal contents (n = 6) of WT

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and ob/ob mice fed 0 and 0.5% FA were collected and were immediately frozen and

96

stored at -80ºC for analysis. Genomic DNA was extracted using a modified

97

cetyltrimethylammonium bromide (CTAB) DNA extraction protocol25. The V3 and

98

V4 hypervariable regions of 16S rRNA genes were amplified by PCR (98 ºC for 1

99

min→30 cycles at 98 ºC for 10 s, 50 ºC for 30 s, 72 ºC for 30s→72 ºC for 5 min),

100

using

primer

(341F

5’-CCTAYGGGRBGCASCAG-3’

101

GGACTACNNGGGTATCTAAT-3’) with the barcode. The amplified products were

102

extracted from 2% agarose gels in Tris-acetate-EDTA (TAE) buffer and purified by


and

806R

5’-

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GeneJET Gel Extraction Kit (Thermo Fisher Scientific, Waltham, Massachusetts,

104

USA). Amplicon library was generated using Ion Plus Fragment Library Kit (Thermo

105

Fisher Scientific) following manufacturer's protocol. Quality of the library was

106

assessed on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific), and equal amount

107

of amplicon from each sample was pooled together. Then the library was single-end

108

sequenced on an Ion S5 XL platform (Thermo Fisher Scientific).

109

Statistical analysis. Values are expressed as means ± SEM. SAS Statistics (SAS

110

Institute Inc., NC, USA) was employed to analyze data of growth performance, tissue

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weights, blood and hepatic lipid profiles, fatty acid composition of WAT were

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analyzed by one-way ANOVA. Significant differences among group means were

113

determined by the Student-Newman-Keuls comparison test26. Single-end reads of 16S

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rRNA genes were identified under unique barcode in the individual read and truncated

115

by cutting off the barcode and primer sequence. Low-quality reads were filtered in

116

accordance with the Cutadapt (version V1.9.1) quality-control process. An

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Operational Taxonomic Unit (OTU) was defined as sequences with ≥ 97% similarity

118

according to UPARSE (version V7.0.1001). The phylogenetic affiliation of each OUT

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was analyzed by SILVA 16S rRNA database (SSUrRNA), and the distances between

120

phylotypes were calculated using MUSCLE (version 3.8.31). The alpha-diversity

121

(Chao 1 and PD_whole_tree) indices were calculated using Qiimesoftware package

122

(version 1.9.1) and the curves for them were composed by R packages (version

123

2.15.3). Principal coordinate analysis (PCoA) was performed based on weighted

124

UniFrac distance-metrics analysis by R programming language. A linear discriminant



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analysis (LDA) effect size pipeline (LEfSe) was used to determine differentially

126

abundant taxa (biomarkers) with LDA score higher than 2. ANOSIM analysis was

127

performed using R software. Comparisons of microbial abundance in different

128

experimental groups were performed using Tukey or Wilcox test with R software.

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Spearman’s correlation analysis was conducted to illustrate the correlations between

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gut microbe and metabolic parameters. Probability values ≤ 0.05 were considered as

131

significant.

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Results

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Response of weight gain and WAT accumulation to FA supplementation in ob/ob mice

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The BW of WT mice increased slightly during the experimental period. At week

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9, the BW of WT control mice was 27.21 ± 1.02 g. And ob/ob mice exhibited

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persistent higher (P < 0.01) body weights compared with that of their counterpart WT

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control mice (data not shown). Starting from week 4, the BW of ob/ob mice fed the

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0.5% FA diet was lower (P < 0.05) than that of ob/ob control mice (Figure 1). 0.5%

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FA also lowered the average BW gain and feed efficiency during the experimental

140

period compared with those of ob/ob control mice (Table 1). However, no significant

141

difference was observed in the BW of ob/ob mice fed 0.05%, 0.125%, 0.25% FA diet

142

or control diet (P > 0.05).

143

The WAT weights of ob/ob mice were significantly higher than that of WT mice,

144

while those were lowered when ob/obmice were fed FA (Table 1; P < 0.05).

145

However, no significant distinction was observed in the WAT weights among ob/ob

146

mice fed 0.05%, 0.125%, 0.25% or 0.5% FA diet (P> 0.05).


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Response of hepatic lipid accumulation to FA supplementation in ob/ob mice

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The liver weights of ob/ob mice were significantly higher than those in WT mice

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(Table 1; P < 0.05). A tendency was observed that 0.05%, 0.25% or 0.5% FA could

150

lower the liver weights of ob/ob control group. Contents of TG and TC in livers of

151

ob/ob control mice were significantly higher (P < 0.05) than those in WT control

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mice. However, dietary FA alleviated this metabolic variation (Figure 2). Moreover,

153

ob/ob mice fed 0.25% or 0.5% FA restored the contents of TG and TC in liver to the

154

normal level as the WT mice have. The similar results were shown as

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histopathological consequence in Figure 3. Numerous lipid droplets were clearly

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observed in ob/ob mice fed control diet (Figure 3B) as compared to those in WT mice.

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On the contrary, smaller and fewer lipid droplets were observed in the livers of

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FA-fed ob/ob mice (Figure 3C-F) compared with those of the ob/ob control mice.

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Especially, the lipid droplets looked smaller in ob/ob mice fed the 0.25% or 0.5% FA

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diet (Figure 3E, F) than those in ob/ob control mice.

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Response of blood lipid accumulation to FA supplementation in ob/ob mice

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As compared to WT control mice, the ob/ob mice exhibited higher contents of TC,

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TG, HDL, LDL and HDL/LDL ratio in serum (Table 2, P< 0.05). However, the

164

concentrations of TC, TG and LDL were lower (P< 0.05), while the concentration of

165

HDL and HDL/LDL ratio was higher (P< 0.05) in ob/ob mice fed 0.25% FA diet than

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those in ob/ob control mice.

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Response of the contents of obesity-related monounsaturated fatty acids in WAT to FA

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supplementation in obese ob/ob mice



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The contents of palmitic acid (C16:0), palmitoleic acid (C16:1), stearic acid

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(C18:0), and oleic acid (C18:1) in WAT were higher in ob/ob mice than those in WT

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mice (Table 3, P < 0.05). However, the amount of C16:1, C18:1 and the fatty acid

172

desaturation index (C16:1/C16:0 and C18:1/C18:0 ratios) in ob/ob mice fed 0.25% or

173

0.5% FA were significantly less (P < 0.05) compared with those in ob/ob control

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mice.

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Response of the structure and composition of gut microbiota to FA supplementation in

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obese ob/ob mice

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To identify the responses of gut microbiota to FA supplementation in obese ob/ob

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mice, the structure and composition of gut microbiota was determined using six

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replicates per group either for WT, FA 0 and FA 0.5 group. The Chao 1 curves

180

(Figure 4A) approached the saturation plateau, indicating most of the OTUs of our

181

samples were captured in our research. However, no significant difference was

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identified (P > 0.05) for the Chao 1 (Figure 4B) and PD_whole_tree (Figure 4C)

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indices among all the three groups. PCoA on all taxonomic levels (Figure 4D)

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showed that WT mice and ob/ob mice had a radically different microbiota profile,

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whereas ob/ob mice fed 0.5% FA and ob/ob control mice had a similar microbiota

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profile.

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The compositions of gut microbiota were quite dissimilar among mice in WT, FA

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0 and FA 0.5 groups. At the phylum level (Figure 5A), the gut microbial composition

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of all the mice was taken the lead by the phyla Bacteroidetes, Proteobacteria and

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Firmicutes. WT mice had significantly higher Bacteroidetes than that of either ob/ob


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mice fed basal diet or 0.5% FA diet (WT vs OB vs FA: 0.42 ± 0.05 vs 0.30 ± 0.03 vs

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0.30 ± 0.03, P< 0.05). Interestingly, ob/ob mice fed 0.5% FA had significantly higher

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Proteobacteria (FA vs WT: 0.34 ± 0.02 vs 0.18 ± 0.06, P < 0.05) and lower

194

Firmicutes (FA vs WT: 0.20 ± 0.02 vs 0.32 ± 0.04, P < 0.05) than those of WT mice.

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However, there was no difference of Proteobacteria and Firmicutes between ob/ob

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mice fed 0.5% FA and ob/ob control mice (P > 0.05). At the family level (Figure 5B),

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an obvious decrease in Muribaculaceae (belonging to the phylum Bacteroidetes) was

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perceived in the mice of FA 0.5 group and WT group compared with the mice in FA 0

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group (FA vs WT vs OB: 0.050 ± 0.004 vs 0.054 ± 0.007 vs 0.076 ± 0.008, P < 0.05).

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Using LEfSe algorithm, we identified 54 OTUs differed in abundance between WT

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mice and ob/ob control mice (Figure 6A), with 30 OTUs specialized for WT mice,

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and 24 OTUs for ob/ob control mice. And a total of 30 OTUs were identified to be

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different in abundance between the mice in FA 0 and FA 0.5 group (Figure 6B). The

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largest effect size was computed for the family Muribaculaceae, which was more

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abundant in the ob/ob control mice; and for the species Parabacteroides_merdae

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(phylum Bacteroidetes), which was more abundant in the ob/ob mice fed 0.5% FA.

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To identify the genera potentially have the anti-obesity and anti-hyperglycemia

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effects, we assessed the correlation between the dominant gut microbial genera and

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metabolic parameters using Spearman’s correlation analysis (Supplemental Figure

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S1). The results indicated that the genera, such as Helicobacter, Faecalibaculum

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(phylum Firmicutes), Alloprevotella (phylum Bacteroidetes), etc. were positively

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correlated with the obesity-associated parameters, including body weight, lipogenesis



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in liver and lipid profiles (Figure 7A). In contrast, the microbiota, such as Alistipes

214

(phylum Bacteroidetes), unidentified_Ruminococcaceae (phylum Firmicutes), and

215

Ruminiclostridium (phylum Firmicutes), were negatively correlated with the

216

obesity-associated parameters (Figure 7B, all P < 0.05). However, the abundances of

217

these functional groups had no significant differences between the ob/ob mice fed FA

218

or not.

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Discussion

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C57BL-6J-ob/ob mice having mutations in leptin gene become profoundly obese

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at a young age. These leptin-deficient mice have been widely used as a model to study

222

obesity and obesity-related syndromes, such as hyperglycemia, hyperlipidemia, fatty

223

liver and so on27, 28. In the present study, body weight gain and feed efficiency were

224

all 1.5 folds greater in ob/ob control mice compared with those in WT mice. However,

225

the 0.5% FA-supplemented diet effectively suppressed the weight gain and WAT

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accumulation (Table 1; -13.4% and -13.7%, respectively) in ob/ob mice. Consistently,

227

we also found that dietary supplementation with 0.5% FA restrained the weight gain

228

and body fat accumulation in high-fat diet-induced obese mice. Meanwhile, dietary

229

supplementation with 0.5% FA had no influences on the weight gain, WAT, hepatic

230

lipid accumulation, blood glucose and inflammatory cytokines release in WT

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C57BL/J mice29. Polyphenols extracted from grape seed could inhibit WAT

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deposition and control dyslipidemia in obesity30. Similarly, we found that FA

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decreased WAT index in ob/ob mice by about 11.3% compared with ob/ob control

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mice. Besides, we demonstrated that ob/ob mice had higher content of palmitic acid


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(C16:0), palmitoleic acid (C16:1), stearic acid (C18:0), and oleic acid (C18:1) content

236

in WAT than WT mice (Table 3). The fatty acid desaturation index (C16:1/C16:0 and

237

C18:1/C18:0), which is a sensitive biochemical indicator correlating with body mass

238

index (BMI) and WAT index, would keep high in adipose tissue in obese rat models31,

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32.

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of C16:1 and C18:1, further reflecting a lower desaturation index, compared with the

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ob/ob control mice. Above data indicated that 0.5% dietary FA was the recommended

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level for weight control in leptin-deficient obese mice when growth tendency was

243

considered as the sole criterion.

However, ob/ob mice fed the 0.25% or 0.5% FA diet had significantly lower levels

244

Previous studies have indicated that liver steatosis is generally occurred in

245

obese33. In the present study, ob/ob control mice had both higher liver weight

246

(+152%) and higher liver index (liver weight/body weight, +47%) compared with

247

those in WT mice, which was consistent with other reports23. The FA supplementation

248

has not affected the liver weight and liver index in ob/ob mice. However, it decreased

249

the hepatic TC and TG contents (Figure 2) in ob/ob mice, which was confirmed by the

250

histopathological results (Figure 3), suggesting a reduced fatty liver probability.

251

Particularly, when the ob/ob mice fed the diet supplemented with 0.25% or 0.5% FA,

252

the lipid accumulation in liver were restored to similar level as WT mice (Figure 2).

253

Similarly, the wheat bran with bound or unbound FA was reported to reduce hepatic

254

lipid levels34. Correspondingly, lesser and smaller fat vacuoles were observed in ob/ob

255

mice fed with 0.25% and 0.5% FA compared to ob/ob control mice (Figure 3).



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HDL has the role in esterifying and transporting cholesterol to the liver and then

257

secreted into the bile. Therefore, it was documented to be beneficial for protecting

258

against arterial disease and atherosclerosis35. In the present study, 0.25% FA

259

up-regulated the serum content of HDL, which was accordant with the

260

down-regulation of total cholesterol in blood in ob/ob mice (Table 2). Besides, a low

261

HDL/LDL ratio is a forceful predictor of cardiovascular events, such as coronary

262

atherosclerosisand carotid intima-media thickness progression36, 37. A depressed level

263

of HDL/LDL ratio was shown in ob/ob mice compared with that in WT mice.

264

However, the ratio was elevated when the ob/ob mice was fed 0.25% FA in the

265

present study. Previous reports have demonstrated that FA exerts hypolipidemic

266

ability by suppressing cholesterol synthesis and cholesterol esterification through

267

reducing HNG-CoA reductase and acyl-CoA: cholesterol transferase in tissues, and by

268

elevating the acidic sterol excretion38. Consistently, FA and its steryl esters,

269

γ-oryzanol have been reported to exhibit strong anti-atherogenic properties39. These

270

data suggested that FA could be a potential therapy for treating or preventing the

271

obesity-related diseases. Accordingly, in our present study, the 0.25% dietary FA

272

level ameliorated hyperlipidemiain the leptin-deficient obese C57BL/6J-ob/ob mice

273

and had the potential to prevent the atherosclerosis.

274

An increasing concerning is involved in the influence of dietary polyphenols on

275

the gut microbiota and the probable correlation between this influence and the

276

progression of obesity. Nevertheless, seldom information is available about how the

277

polyphenols influence gut microbiota in obese human or animals. To identify whether


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the FA-mediated decrease in body weight and fat deposition in liver and WAT had

279

correlation with the change in gut microbiota, the gut microbial structure and

280

composition in WT mice, ob/ob control mice and 0.5% FA-fed ob/ob mice were

281

profiled. Rearrangements of both the structure and composition of gut microbiota

282

were observed in the ob/ob control mice with comparison with those of WT mice

283

(Figure 4-5). Previous research had demonstrated that green tea polyphenols

284

administration resulted in the changes in diversity of colonic microbiota, resulting in

285

redunced body weight and hyperlipemia in high fat-induced obese mice18. Blueberry,

286

which is high in anthocyanins and other polyphenolic compounds, led to change of

287

composition in the gut microbiota associated with elevated systemic inflammation and

288

insulin signaling in high fat-induced obese rats40. However, the diversity of the gut

289

microbiota was not significantly influenced under intervention with FA in ob/ob obese

290

mice, and the structure of gut microbiota was only slightly different between ob/ob

291

mice in FA 0 group and FA 0.5 group (Figure 4D). In Spearman’s correlation analysis

292

(Supplementary Figure S1), the association of the gut microbiota with metabolic

293

parameters was displayed. We found that the abundances of genera Helicobacter,

294

Dubosiella,

295

Parabacteroides, Alloprevotella, and Faecalibaculum, have a positive correlation

296

with body weight gain and other obese-related parameters. In these genera, Dubosiella

297

and Negativibacillus abundances were decreased in ob/ob mice fed with 0.5% FA

298

after FA intervention compared to those in ob/ob control mice (P < 0.05). However,

Desulfovibrio,

Bifidobacterium,

Negativibacillus,


Angelakisella,


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299

the total abundances of this functional genera group had no significant differences

300

between the ob/ob control mice and ob/ob mice fed 0.5% FA.

301

Besides,

it

is

controversial

about

the

relationship

between

the

302

Firmicutes/Bacteroidetes ratio and obesity. Some reports declared an increased ratio

303

was associated with obesity14, while others indicated the inverse results41. Meanwhile,

304

there also have studies revealed no correlation between this ratio and obesity42, 43. Our

305

study found the Firmicutes/Bacteroidetes ratio had no difference among WT mice,

306

ob/ob obese mice and ob/ob mice fed 0.5% FA. This discrepancy may be owing to

307

different obese models used (high fat diet-induced obese mice vs leptin-deficient

308

ob/ob obese mice, mice vs rat, etc.), different lifestyles and different methodology in

309

DNA extraction protocols as well as primer design41. The 16S rRNA data indicated

310

that 0.5% dietary FA could not change the structure of the gut microbiota and the total

311

abundance of genera associated with obesity and anti-obesity in leptin-deficient ob/ob

312

obese mice. Consequently, our study showed that there was no association between

313

the gut microbes and the anti-obesity effects of FA.

314

In conclusion, 0.25% or 0.5% dietary FA ameliorated lipogenesis and fat

315

deposition in WAT and lipid accumulation in liver in obese C57BL/6J-ob/ob mice.

316

Moreover, 0.5% FA-supplemented diet suppressed weight gain in obese ob/ob mice.

317

Overall, our results indicates that sufficient intake of FA could be useful for treating

318

obesity or obesity-correlated diseases, and this weight-control effect possibly not

319

correlated with the gut-brain axis. However, the exact mechanisms still require more

320

investigations.


Page 17 of 39


321

Abbreviations

322

C16:0, palmitic acid; C16:1, palmitoleic acid; C18:0, stearic acid; C18:1, oleic

323

acid; CTAB, cetyltrimethylammonium bromide; FA, ferulic acid; HDL, high-density

324

lipoprotein cholesterol; LDA, linear discriminant analysis; LDL, low-density

325

lipoprotein cholesterol; LEfSe, LDA effect size pipeline; OTU, Operational

326

Taxonomic Unit; PCoA, principal coordinate analysis; ROS, reactive oxygen species;

327

TC, total cholesterol; TG, total triglyceride; WAT, white adipose tissue; WT, wild

328

type.

329

Acknowledgments

330

W. W., J. L., and A. L. designed research; W. W., Y. P., L. W., H. Z., G. S., Y.

331

W. and A. L. conducted research; W. W., Y. P., and A. L. analyzed data; W. W., and

332

Y. P. wrote the paper. A. L. had primary responsibility for final content. All authors

333

have read and approved the final manuscript.



Page 18 of 39 18

320

Supporting Information

321

The manuscript includes one supplementary table and one supplementary figure.

322

Table S1. Composition of the experimental basal diet.

323

Figure S1. Spearman correlation heatmap. The relationship between gut microbiota

324

(genus level) and metabolic parameters.*P< 0.05 and **P< 0.01.


Page 19 of 39


320 321 322

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Funding This work was supported by the National key R&D Program of China (2017YFD0500505), the National Finance Project of China (164007000000150001), the National Natural Science Foundation of China (No. 31471591), and the Non-profit Industry (grain) Scientific Research Special Fund Agreement (No. 201513003-8, 201313011-6).


Page 27 of 39


Figure captions Figure 1. Body weight in C57BL/6J wild type (WT control) and C57BL/6J-ob/ob mice fed the 0 (ob/ob control), 0.05%, 0.125%, 0.25% or 0.5% ferulic acid (FA) diet from wk 3 to wk 9. Mean body weight of 8 replicates at each time point. Values are means ± SEM, n = 8. *P < 0.05 vs. ob/ob control. Figure 2. Liver triglyceride (A) and total cholesterol (B) in C57BL/6J wild type (WT control) and C57BL/6J-ob/ob mice fed the 0 (ob/ob control), 0.05%, 0.125%, 0.25% or 0.5% ferulic acid (FA) diet for 9 weeks. Values are means ± SEM, n = 8. Means in a list without a common letter differ, P < 0.05. Figure 3. Light photomicrographs of the liver sections in C57BL/6J wild type (WT control, A) and C57BL/6J-ob/ob mice fed the 0 (ob/ob control, B), 0.05% (C), 0.125% (D), 0.25% (E) or 0.5% (F) ferulic acid (FA) diet for 9 weeks. (hematoxylin-eosin staining, original magnification × 250). Figure 4. The structure of gut microbiota in C57BL/6J wild type (WT), C57BL/6J-ob/ob mice fed the 0 (ob/ob control, OB) and 0.5% ferulic acid (FA) diet for 9 weeks (n = 6). (A) The chao 1 curves. (B) The chao1 index levels. (C) The PD_whole_tree index levels. (D) Principal coordinate analysis (PCoA) generated using a weighted UniFrac distance-metrics. Figure 5. The composition of gut microbiota in C57BL/6J wild type (WT), C57BL/6J-ob/ob mice fed the 0 (ob/ob control, OB) and 0.5% ferulic acid (FA) diet for 9 weeks (n = 6). (A) Composition of gut microbiota at the phylum level. (B) Composition of gut microbiota at the family level.



Page 28 of 39 28

Figure 6. LEfSe analysis, indicating significantly differential distribution of taxa between C57BL/6J wild type (WT) and C57BL/6J-ob/ob control (OB) mice (A); and between C57BL/6J-ob/ob mice fed the 0.5% ferulic acid (FA) diet and OB group (B). (A) OTUs are presented in green when the taxon is significantly more abundant in the WT group and in red when it is significantly more abundant in the OB group. (B) OTUs are presented in green when the taxon is significantly more abundant in the OB group and in red when it is significantly more abundant in the FA group (n = 6). Figure 7. The relative abundance of the functional bacterial groups in the gut microbiota of mice. (A) Obesity-associated genera. (B) Anti-obesity-associated genera. WT, C57BL/6J wild type mice; OB, C57BL/6J-ob/ob control mice; FA, C57BL/6J-ob/ob mice fed the 0.5% ferulic acid diet. Values are means ± SEM, n = 6. Means without a common letter differ, P < 0.05.


Page 29 of 39


Table 1 Growth performance and tissue weights in WT and ob/ob mice fed the 0, 0.05%, 0.125%, 0.25% or 0.5% ferulic acid diet for 9 weeksa ob/ob Items

WT FA 0

Weight gain (g)

FA 0.05

FA 0.125

FA 0.25

FA 0.5

6.39 ± 0.33c

12.43 ± 0.40a

11.40 ± 0.37ab 12.17 ± 0.35ab 11.14 ± 0.42ab

10.77 ± 0.46b

43.42 ± 0.58b

54.22 ± 1.69a

53.61 ± 2.08a

54.55 ± 1.46a

52.43 ± 0.98a

52.48 ± 0.97a

(g/100 g)

3.72 ± 0.19c

5.79 ± 0.11a

5.40 ± 0.19ab

5.65 ± 0.17ab

5.35 ± 0.13ab

5.19 ± 0.22b

Liver weight (g)

0.89 ± 0.11c

2.24 ± 0.10ab

2.07 ± 0.11b

2.42 ± 0.10a

2.14 ± 0.10b

2.14 ± 0.04b

WAT weight (g)

0.87 ± 0.20c

4.08 ± 0.12a

3.50 ± 0.13b

3.59 ± 0.12b

3.57 ± 0.08b

3.52 ± 0.09b

Food intake (kJ/d) Feed efficiency

Relative weight of tissueb Liver

3.23 ± 0.18c

4.76 ± 0.22ab

4.51 ± 0.24b

5.14 ± 0.19a

4.65 ± 0.21ab

4.71 ± 0.11ab

WATc

2.96 ± 0.33c

8.65 ± 0.24a

7.59 ± 0.24b

7.61 ± 0.19b

7.74 ± 0.16b

7.75 ± 0.35b

aValues

are means ± SEM, n = 8. Means in a row without a common letter differ, P

Optimal Dietary Ferulic Acid for Suppressing the Obesity-Related

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