Gama-aminobutyric Acid Enriched Rice Bran Diet ... - ACS Publications

Supplementary Tables S1 and S2 show concentrations of GABA and phenolic .... Tabbaa, D.; Highlander, S. K.; Sodergren, E.; Methé, B.; DeSantis, T. Z...
0 downloads 0 Views 2MB Size
Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

Gama-aminobutyric Acid Enriched Rice Bran Diet Attenuates Insulin Resistance and Balances Energy Expenditure via Modification of Gut Microbiota and SCFAs Xu Si, Wenting Shang, Zhongkai Zhou, Guanghou Shui, Sin Man Lam, Christopher L. Blanchard, and Padraig Strappe J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04994 • Publication Date (Web): 12 Jan 2018 Downloaded from http://pubs.acs.org on January 12, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 38

Journal of Agricultural and Food Chemistry

Gama-aminobutyric Acid Enriched Rice Bran Diet Attenuates Insulin Resistance and Balances Energy Expenditure via Modification of Gut Microbiota and SCFAs Xu Si a, Wenting Shang a, Zhongkai Zhou a, b, *, Guanghou Shui c, Sin Man Lam c, Chris Blanchard b, Padraig Strappe d

a

Key Laboratory of Food Nutrition and Safety, Ministry of Education, Tianjin University of Science and Technology, Tianjin 300457, China

b

ARC Industrial Transformation Training Centre for Functional Grains, Charles Sturt University, Wagga Wagga, NSW 2678, Australia

c

Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China

d

School of Medical and Applied Sciences, Central Queensland University, Rockhampton, Qld 4700, Australia

*Corresponding author Prof. Zhongkai Zhou (PhD), Telephone number: +86 18812697366 E-mail: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 38

1

ABSTRACT: In this study, gama-aminobutyric acid (GABA) enriched rice bran (ERB) was

2

supplemented to obese rats to investigate the attenuation of metabolic syndromes induced

3

by high-fat diet. ERB-containing diet stimulated butyrate and propionate production by

4

promoting Anaerostipes, Anaerostipes sp. and associated synthesizing enzymes. This altered

5

SCFA distribution further enhanced circulatory levels of leptin and glucagon-like peptide-1,

6

controlling food intake by down-regulating orexigenic factors. Together with the enhanced

7

fatty acid β-oxidation highlighted by Prkaa2, Ppara, Scd1 expression via AMPK signaling

8

pathway and Non-alcoholic fatty liver disease pathway, energy expenditure was positively

9

modulated. Serum lipid compositions showed ERB supplement exhibited a more efficient

10

effect on lowering serum sphingolipids, which was closely associated with status of insulin

11

resistance. Consistently, genes of Ppp2r3b and Prkcg, involved in the function of ceramides in

12

blocking insulin action were also down-regulated following ERB intervention. Enriched GABA

13

and phenolic acids were supposed to be responsible for the health-beneficial effects.

14

KEYWORDS:

15

hyperinsulinism

gama-aminobutyrate

acid,

sphingolipids,

Anaerostipes,

butyrate,

2

ACS Paragon Plus Environment

Page 3 of 38

Journal of Agricultural and Food Chemistry

16

INTRODUCTION

17

Metabolic syndrome is considered as a multifactorial pathological state, associated with a

18

long-term imbalance of diet and physical activity, genetic predisposition, and a disordered

19

gut microbiota influencing several metabolic pathways.1 Excess energy intake and

20

accompanying obesity are the main drivers of the syndrome.2 The rapidly increasing

21

prevalence of metabolic syndrome has made it a major public health concern.3 The

22

applications of natural components with health-promoting functions in foods for therapeutic

23

use have drawn attention from both researchers and consumers.

24

Rice bran is a nutrient-dense byproduct derived from the milling process of rice. With the

25

most abundant production of rice in Asia (90% of the world total rice production), rice bran

26

is readily available at a low cost in Asian countries.4 Moreover, its high nutritional value has

27

highlighted its potential as a food supplement for improvement of body health.5

28

Gamma-aminobutyric acid (GABA) is a ubiquitous non-protein amino acid, and its utilization

29

has been related to improvements in brain function, decrease in blood pressure, and

30

regulation on pancreatic secretion.6-8 Rice bran byproduct contains glutamic acid

31

decarboxylase (GAD), with the ability to convert glutamic acid (GA) into GABA,9 thus rice

32

bran is an important potential source of GABA.10 Moreover, the enhanced recovery of other

33

bioactive compounds such as polyphenols during the GABA enrichment process will further

34

strengthen the health-promoting feature of rice bran. Previous studies have demonstrated

35

the regulatory role of oral rice bran administration in maintenance of gut health is through

36

modulation of mucosal immunity and promotion of probiotic growth, as well as colorectal

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 4 of 38

37

cancer prevention by ameliorating oxidative stress and inflammation.11-13 In this study, GABA

38

enriched rice bran (ERB) was supplemented to high fat diet (HFD) induced obese rats, to

39

investigate the resulting alterations in obesity control and further reveal the underlying

40

mechanism through alterations of host’s gut microbiota and peripheral signaling pathways.

41

MATERIALS AND METHODS

42

GABA enriched rice bran preparation. Fresh rice bran (FRB) was supplied from Sunrice

43

Co. (Leeton, Australia). The GABA enriched rice bran (ERB) was prepared as follows: the

44

moisture content of FRB was modified to 30% using a 5 mmol/L glutamic solution, followed

45

by oxygen-free incubation at 40 °C for 5 h. The FRB and ERB were subjected to vacuum

46

drying (50 °C, 12 h) and grinding treatment, then stored at -80 °C for feed preparation and

47

further analysis. The nutritive indexes are presented in Table S1 (Supporting information).

48

Physicochemical properties of rice bran. The concentration of GABA in rice bran was

49

determined followed the descriptions of Kim, Lee, Lim & Han10 with some modifications.

50

Derivatized samples were measured using a HPLC (Agilent, USA) equipped with a

51

chromatographic column of Venusil MP-C18 (4.6 × 250 mm) and an ultraviolet detector. The

52

detection conditions were as follows: column temperature of 30 °C, mobile phase A

53

containing 7 mmol/L sodium acetate trihydrate at pH 7.2, and mobile phase B containing

54

50% methanol and 50% acetonitrile, flow rate of 0.9 mL/min and measurement at a

55

wavelength of 338 nm. Phenolic acid measurement was performed according to Wanyo,

56

Meeso & Siriamornpun.14 Briefly, three grams of rice sample was subjected to a defatting

57

treatment using n-hexane (1:5, w:v) with one hour’s magnetic stirring. The residue after

4

ACS Paragon Plus Environment

Page 5 of 38

Journal of Agricultural and Food Chemistry

58

filtration was fully extracted twice using 15 mL of 80% methanol, and the filtrate was mixed

59

for further analysis. HPLC was performed with a chromatographic column of Venusil MP-C18

60

(4.6 × 250 mm) and a diode assay detector was used for concentration detection at 280 nm

61

and 320 nm. The column temperature was 40 °C, and the eluant (containing mobile phase A

62

of acetic acid and water (3:97) and phase B of acetic acid, acetonitrile and water (3:25:72))

63

was used with a gradient program: 1-40 min, 70% B, flow rate of 1 mL/min; 40-45 min,

64

70%-80% B, flow rate of 1 mL/min-1.2 mL/min; 45-55 min, 80%-85% B, flow rate of 1.2

65

mL/min.

66

Animals. Twenty four healthy male Sprague-Dawley (SD) rats of 90 ± 10 g body weight

67

were provided by National Institutes for Food and Drug Control (SCXK 2014-0013). The study

68

was approved by the Ethical Committee for the Experimental Use of Animals at the Center

69

for Drug Safety Evaluation, Tianjin University of Science & Technology (approval No:

70

13/051/MIS). The rats were housed in plastic cages (4 rats/ cage) with free access to drinking

71

water, under controlled conditions of humidity (50%-55%), light (12/ 12 h light/dark cycle)

72

and temperature (20-25 °C). Diet-induced obese rats were obtained after 7 weeks of HFD

73

feeding and equally divided into three groups randomly: MC (Model Control, a high-fat diet)

74

group, FRB group and ERB group. The compositions of high-fat diet included 63.8% AIN-93

75

diet15, 15% lard oil, 10% saccharose, 1% cholesterol, 0.2% sodium cholate and 10% egg yolk

76

powder. Then rice bran intervention was performed, with either FRB or ERB occupying 15%

77

in the high-fat diet by replacing part of basal diet. Body mass was recorded once a week and

78

fecal samples were collected at the end of the experimental course of 6 weeks. The feeding

5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 38

79

process is presented in Figure S1. At the end of the experimental period, the rats were fasted

80

overnight and sacrificed by cervical dislocation. Blood samples were taken from arteria

81

femoralis. The serum was separated by a centrifugation at 4 °C and 3 000 r/min for 10 min

82

(Sorvall ST 8R, Thermo Fisher Scientific, USA). Epididymal white adipose tissues, perirenal

83

white adipose tissues and liver tissues were weighed after removing superficial bloods with

84

physiological saline solution, and subjected to a rapid frozen with liquid nitrogen. Samples of

85

serum, liver, adipose tissue and feces were stored at -80 °C for further analysis.

86

Serum hormone levels. Serum levels of insulin, leptin, glucagon-like peptide-1 (GLP-1),

87

gastrin and ghrelin were measured using commercial kits (Biolab Science and Technology Ltd.

88

China) according to the manufacturer’s instructions, respectively. Briefly, serum sample was

89

added onto the plate well coated with specific rat antibody, followed by the addition of HRP

90

labeled corresponding antibody, obtaining antibody-antigen-enzyme-antibody complex.

91

After washing completely, 3,3',5,5'-Tetramethylbenzidine was added, followed by

92

terminated catalysis by the addition of sulphuric acid solution. The OD values were then

93

measured at a wavelength of 450 nm using a microplate reader (Thermo, USA). The

94

concentration of hormones in the serum samples was calculated according to the standard

95

curves.

96

Lipidomics analysis. Lipid extractions from serum and fecal samples were carried out

97

using a modified version of the Bligh and Dyer’s protocol as previously described for serum

98

lipid composition and fecal bile acids determination.16 Extracted organic fractions were

99

pooled and dried in a miVac (Genevac, UK). Samples were stored at -80 °C until further

6

ACS Paragon Plus Environment

Page 7 of 38

Journal of Agricultural and Food Chemistry

100

analyses. Lipids were analyzed using an Exion UPLC coupled with a triple quadrupole/ion

101

trap mass spectrometer (QTRAP 6500 Plus, Sciex). Normal-phase LC/MS was applied for

102

individual lipid classes of polar lipids. Separation of individual lipid classes of polar lipids was

103

performed using a Phenomenex Luna 3u silica column (i.d. 150x2.0 mm) with the following

104

conditions: mobile phase A (chloroform:methanol:ammonium hydroxide, 89.5:10:0.5), B

105

(chloroform:methanol:ammonium

106

GluCer-d18:1/8:0, LacCer-d18:1/8:0, Sph d17:0 and SM d18:1/12:0 obtained from Avanti Polar

107

Lipids were used for quantification, and FFA 19:0 purchased from Cayman Chemicals for free

108

fatty acid quantitation. A reverse phase LC/MS for glyerol lipids using a modified version was

109

described previously.18 Separation was carried out on a Phenomenex Kinetex 2.6 µm-C18

110

column (i.d. 4.6x100mm) using an isocratic mobile phase chloroform:methanol:0.1M

111

ammonium acetate (100:100:4) at a flow rate of 150 µL/min for 22 min. The levels of CEs

112

and TAGs were calculated as relative contents to the levels of spiked d6-CE, d29-TAG(15:0),

113

d5-TAG (16:0) internal standards (CDN isotopes), while DAG species were quantified using

114

4ME 16:0 Diether DG as an internal standard (Avanti Polar Lipids, Alabaster, AL, USA). Bile

115

acids were separated on a Phenomenex Kinetex 2.6 µm-C18 column (i.d. 2.1x100 mm) using

116

a gradient elution consisting of methanol as mobile phase A and 10 mmol/L ammonium

117

acetate buffer at pH 6.5 as mobile phase B.19 Internal standard cocktail consisted of

118

d4-glycocholic acid, d9-glycochenodeoxycholic acid, d4-glycodeoxycholic acid, d4-cholic acid,

119

d4-ursodeoxycholic acid, d4-chenodeoxycholic acid, d4-deoxycholic acid and d4-lithocholic

120

acid (Cambridge Isotopes Laboratories, Tewksbury, MA, USA).

hydroxide:water,

55:39:0.5:5.5).17

Cer-d18:1/17:0,

7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 8 of 38

121

Short-chain fatty acid (SCFA) measurement. The serum concentration of SCFAs was

122

measured following the method of Frost et al.20 with some modifications. Briefly, a 200 μL

123

aliquot of rat serum was filtered through a 30 kDa micropartition system Vivaspin RC filters

124

(Sartorius Inc., Canada) by centrifugation at 5000 g at 4 °C for 90 min. One microlitre of each

125

sample was injected into a GC2010 Plus gas chromatography (GC) system (Shimadzu, Japan)

126

fitted with a NukolTM Capilllary Column (30 m × 0.53 mm × 1.0 mm, SUPELCOTM Analytical,

127

UK) and flame ionization detector. Nitrogen, was used as carrier gas, with a flow rate of 2

128

mL/min. The head pressure was set at 11.6 kPa with split injection. Run conditions were as

129

follows: initial temperature 60 °C, 3 min; 10 °C/min to 190 °C, hold 25 min. Peaks were

130

integrated using the Shimadzu GC Postrun software (Shimadzu, Japan) and SCFA content was

131

quantified using a standard cocktail including acetate, propionate and butyrate.

132

DNA amplification sequence analysis. Total genome DNA from feces was extracted by

133

Cetyltrimethyl Ammonium Bromide (CTAB) method according to Geel et al.21 with some

134

modifications. Briefly, freshly prepared CTAB buffer was added to 0.2 g feces before

135

incubation at 65 °C for 1 h under agitation. DNA was subsequently extracted using an equal

136

volume of chlor-oform:isoamyl alcohol (24:1), precipitated with ice-cold iso-propanol. The

137

DNA precipitation was washed twice in 76% ethanol. The resulting DNA was air dried and

138

resuspended in ddH2O. 16S rRNA genes of regions in V4 were amplified using the specific

139

primers 515F-806R, with barcodes. PCR products with bright main bands between 400~450

140

bp by 2% agarose gel electrophoresis were chosen for the following library preparation and

141

sequencing. A mixture of PCR products in equidensity ratios was purified using a Qiagen Gel

8

ACS Paragon Plus Environment

Page 9 of 38

Journal of Agricultural and Food Chemistry

142

Extraction Kit (Qiagen, Germany), and the sequencing libraries were established using

143

TruSeq® DNA PCR-Free Sample Preparation Kit (Illumina, USA) according to the

144

manufacturer's instructions, and index codes were added. The library quality was evaluated

145

using a Qubit@ 2.0 Fluorometer (Thermo Scientific) and Agilent Bio-analyzer 2100 system.

146

Further, the library was sequenced using an IlluminaHiSeq2500 platform, generating 250 bp

147

paired-end reads which were then assigned to samples identified by their unique barcode

148

and truncated by cutting off the barcode and primer sequence. For sequencing assembly,

149

paired-end reads were merged using FLASH (V1.2.7), when at least some of the reads

150

overlap the read generated from the opposite end of the same DNA fragment, and the

151

splicing sequences were called raw tags. Effective Tags were finally obtained by removing

152

chimera sequences from high-quality clean tags.22,23 Sequences with ≥97% similarity

153

analyzed by Uparse software (Uparse v7.0.1001) were assigned to the same OTUs,24 and

154

species annotation was performed on the representative sequence for each OUT using the

155

GreenGene Database based on RDP 3 classifier (Version 2.2).

156

Metagenomics analysis. A total amount of 1 μg DNA (extracted using CTAB extraction

157

method as stated above) per fecal sample was used as input material for the DNA sample

158

preparation. Sequencing libraries were generated using NEBNext® Ultra™ DNA Library Prep

159

Kit for Illumina (NEB, USA) according to the manufacturer’s instructions and index codes

160

were added to attribute sequences to each sample. The clustering of the index-coded

161

samples was implemented on a cBot Cluster Generation System following the

162

manufacturer’s recommendations. After cluster generation, the library preparations were

9

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 10 of 38

163

sequenced on an Illumina HiSeq platform and 350 base-paired end reads were generated.

164

Sequences for each sample were subjected to filtration for contaminants from the rat

165

genome using SoapAligner (Version 2.21). Reads were subjected to gene prediction using

166

MetaGeneMark (Version 2.10) with default parameters25,26 and species annotation by

167

aligning with Bacteria, Fungi, Archaea and Viruses in NCBI NR database (Version: 2014-10-19)

168

using DIAMOND,27 obtaining abundance information for different levels. Metagenomic reads

169

from each sample were searched against the Kyoto Encyclopedia of Genes and Genomes

170

(KEGG) gene database (version 58) using DIAMOND.28 The search results underwent a

171

filtration (one HSP > 60 bitsa) for further obtaining enzyme and pathway abundance and

172

coverage from metagenomics communities.29

173

Quantitative PCR. Total RNA was extracted from adipose, hypothalamus and liver tissues

174

using RNAprep Pure Tissue Kit (TIANGEN, China), and transcribed to cDNA with a RevertAid

175

First Strand cDNA Synthesis Kit (Thermo Scientific, USA) according to the manufacturer’s

176

instructions. Real-time quantitative PCR reactions were performed using a SYBR® Premix Ex

177

Taq™ II (Takara, Japan) on a StepOnePlusTM Real-Time PCR System (Thermo Scientific, USA).

178

Real-time PCR was conducted with the following parameters: initial denaturation at 95 °C for

179

30 s, and then 40 cycles of 95 °C for 5 s and 60 °C for 30 s. 18S rRNA gene was used as an

180

internal control to normalize target gene expression. Three replicates of each reaction were

181

carried out, and the relative transcript quantity was calculated according to the method of

182

2-ΔΔCT.30 The primer sequences are shown in supplement document (Table S2, in Supporting

183

information).

10

ACS Paragon Plus Environment

Page 11 of 38

Journal of Agricultural and Food Chemistry

184

Transcriptome sequencing of liver tissue. A total amount of 3 μg RNA extracted from

185

each liver sample using TRIzol reagent was used as input material for the RNA sample

186

preparations. Sequencing libraries were established by NEBNext® Ultra™ RNA Library Prep

187

Kit for Illumina® (NEB, USA) based on the manufacturer’s recommendations and index codes

188

were added to assign sequences to each sample. Then PCR was performed with Phusion

189

High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. Finally, library

190

quality was assessed on the Agilent Bioanalyzer 2100 system after the purification of PCR

191

products by AMPure XP system. Index-coded samples were clustered on a cBot Cluster

192

Generation System using TruSeq PE Cluster Kit v3-cBot-HS (Illumia) according to the

193

manufacturer’s specifications. After cluster generation, the library preparations were

194

sequenced on an Illumina Hiseq platform and 150 bp paired-end reads were generated.

195

Differential expression analysis of FRB and ERB groups versus MC group was implemented

196

using the DESeq R package (1.18.0), respectively. The P values were adjusted using the

197

Benjamini & Hochberg method, setting an adjusted P-value of 0.05 for significantly

198

differential expression.31 The enrichment of differential expression genes in KEGG pathways

199

was analyzed using KOBAS software.

200

Tissue histology. The histology analysis for tissues were based on the method previously

201

reported.32 After twelve hours’ fixation with 10% neutral buffered formalin, the liver tissues

202

were dehydrated using a gradient of ethanol solutions (70%, 80%, 90%, 95% and 100%,

203

respectively) and xylene. After three cycles of a waxdip process at 55 °C for 30 min, paraffin

204

embedding was performed, followed by H and E staining. Colonic tissue histology was

11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 12 of 38

205

observed and imaged using a computer-integrated microscope and an image analysis system

206

(Leica Microsystems Imaging Solutions, UK). An ocular micrometer was used for random

207

thickness determination.

208

Statistical analysis. Quantitative data is represented as mean ± standard deviation (SD).

209

Statistical analyses were completed using the Statistical Package for Social Science (SPSS)

210

program (Inc. Chicago). Comparison between groups was carried out by Kruskal-Wallis test

211

with Bonferroni correction. Results were considered statistically significant when P < 0.05,

212

with the significance level indicated as *P < 0.05, ** P < 0.01, respectively.

213

RESULTS

214

The effect of rice bran on obesity syndromes. The rats supplemented with ERB

215

showed lower body weight gain and fat weight compared to the HFD group after six weeks

216

(Figure 1A, B). An insulin resistant status was observed in obese rats, whilst rice bran

217

treatments, in particular ERB administration, significantly attenuated insulin resistance

218

(Figure 1C). Rats supplemented with ERB also consumed the least amount of food (Figure

219

1D). Both of the rice bran administrations positively altered the composition of serum lipids

220

(Figure 1E, F), resulting in a lowered TAG and cholesterol status. Remarkably, ERB exerted a

221

more efficient effect on suppressing synthesis of sphingolipids (including sphingomyelins-SM,

222

glucosylceramides-GluCer, lactosylceramides-LacCer, monosialo-dihexosylceramides-GM3),

223

which have been demonstrated to be closely associated with insulin resistance. Furthermore,

224

ERB consumption significantly enhanced leptin (Figure 1G) and GLP-1 (Figure 1H) levels and

225

suppressed parasympathetic activity by lowering gastrin secretion (Fig. 1I). Hypothalamus

12

ACS Paragon Plus Environment

Page 13 of 38

Journal of Agricultural and Food Chemistry

226

mRNA expression analysis showed a significant increase in the expression of the long form of

227

the leptin receptor (OBRb) (Figure 2A), as well as decreases in the expressions of orexigenic

228

agouti-related peptide (AgRP), suppressor of cytokine signalling-3 (SOCS-3), adenosine

229

monophosphate-activated protein kinase alpha 2 (AMPKα2) and neuropeptide Y (NPY) in

230

interventional groups compared to MC group (Figure 2B-F). This indicated that rice bran

231

administration, especially ERB contributed to body weight control through leptin signaling

232

and appetite control.

233

Improvements in lipid and energy metabolism in both liver and adipose

234

tissues. Significantly expressed and obesity/metabolic syndrome associated genes detected

235

by RNA sequencing method in liver tissues were collected according to the Rat Genomic

236

Database, and arranged for Local Gene Network linkage pathways, presented as Figure S2A

237

for FRB versus MC and Figure S2B for ERB versus MC (in Supporting information). In the

238

network established by FRB group, genes Foxo1, Pdgfα, Pck1, Rxrα and Acox1, participating

239

in PPAR signaling pathway or Adipocytokine signaling pathway were responsible for

240

regulating lipid and glucose metabolism and acted as bridges for linking other pathways. In

241

contrast, a relatively dispersive network was exhibited in the ERB-treated group, highlighting

242

the important role of Pparα, Foxo1, Pdgfα and Il6r, which modified energy homeostasis via

243

AMPK signaling pathway and Non-alcoholic fatty liver disease (NAFLD) pathway. Fifteen

244

bridge-linking genes from gene network and enriched KEGG pathway revealed by RNA

245

sequencing were showed in Figure 3A with fold change values. Remarkably in the ERB

246

administration group, Ppp2r3b and Prkcg expressions through which ceramides block insulin

13

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 38

247

action, were only observed to be down-regulated (Figure 3A), and might be one effect of

248

ERB which may contribute to promoting insulin resistance improvement. Also GM3

249

accumulation was inhibited by the up-regulated expression of St8sia1 (Figure 3A) which

250

converts GM3 finally to GT3 ganglioside sugar. These results indicated that rice bran

251

treatments contributed to a promotion in lipid oxidation and an inhibition in insulin

252

over-secretion. Additionally, ceramide signaling was modified resulting in an improvement

253

in insulin sensitivity following ERB consumption, which was validated by the RT-PCR

254

expression analysis of the selected genes shown in Figure S3 (Supporting information).

255

Interestingly, histological analysis of liver tissue revealed a decreased degree of steatosis

256

following rice bran supplement whilst the HFD induced hepatocytes with large fat vacuoles,

257

numerous fat droplets and infiltration of inflammatory cells and these features were

258

attenuated by ERB administration (Figure 3B), suggesting a role of rice bran diet in the

259

protection of liver integrity and function.

260

The mRNA levels of some key genes involved in gluco-lipid metabolism in adipose tissue

261

showed that the expressions of the energy expenditure control and metabolic rate modifying

262

associated genes of CIDEA and COX4 were significantly down regulated in ERB-treated rats

263

compared to either MC group or FRB group (Figure 3C, D). The administration of FRB showed

264

the highest expression of PAI-1, and the value of which is approximately four times higher

265

than that of obese rats (Figure 3E). In contrast, diet supplemented with ERB declined the

266

PAI-1 gene expression six times compared to HFD, indicating its suppression in adipose tissue

267

development. Meanwhile, the significantly increased gene expressions of FATP1 and GLUT4

14

ACS Paragon Plus Environment

Page 15 of 38

Journal of Agricultural and Food Chemistry

268

in both interventional groups (Figure 3F, H) provided an explanation for the suppression of

269

insulin resistance and increased triglyceride hydrolysis at the molecular level. Furthermore,

270

lipogenesis and energy harvest was restrained by ERB supplement through the up-regulation

271

of LPL (Figure 3G) and down-regulation of SREBP and PPARγ (Figure 3I, J).

272

Alterations in intestinal environment are responsible for obesity attenuation.

273

Distribution of intestinal metabolites was altered following the rice bran administration,

274

associated with an increased secondary bile acids secretion and modified short chain fatty

275

acids profile. An elevated total bile acid secretion was induced by the ERB supplemented diet

276

compared to either HFD or FRB-containing diet, which was reflected in the increased levels

277

of fecal chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, and muricholic acid

278

following ERB intervention (Figure 4A-F). The serum concentrations of the three primary

279

short chain fatty acids showed a significant decrease in acetate, whilst an opposite tendency

280

was seen for propionate and butyrate (Figure 4G-I). We hypothesized that the alterations in

281

these metabolites resulted from the changes in the gut microbiota profile.

282

The metagenomics data showed that the highest total predicted gene number was

283

observed in the ERB group which also demonstrated the maximum specific genes,

284

approximately four-fold more than MC group and FRB group (Figure 5A), indicating a

285

remarkably increased microbial variety in rats subjected to the ERB intervention. The ratio of

286

Bacteroidetes:Firmicutes in the fecal samples was efficiently increased by ERB treatment

287

(Figure 5B). Among the top ten most abundant genus, Bifidobacterium, Lactobacillus and

288

Anaerostipes were observed to be significantly enhanced following ERB administration

15

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 16 of 38

289

(Figure 5C-E). At the level of species, the growth of Bifidobacterium animalis, Anaerostipes

290

sp. and Clostridium leptum was significantly promoted by either FRB or ERB-containing diet

291

(Figure 5F-H), and more importantly the latter two species are butyrate producing bacteria.

292

The supplementation of FRB significantly elevated the abundance of Clostridium leptum

293

compared to either HFD or ERB diet, and the concentration magnitude was four times lower

294

than the ERB diet-enhanced Anaerostipes sp., which could produce butyrate with acetate

295

serving as substrate. Furthermore, the relative abundance of enzymes participating in

296

butyrate and propionate synthesis was greatly up-regulated following ERB treatment

297

compared to either MC group or FRB group (Figure 5I), in particular the enzymes involved in

298

the final synthesis steps, such as phosphate butyryltransferase (2.3.1.9), butyrate kinase

299

(2.7.2.7), acetate CoA-transferase (2.8.3.8) and propionate CoA transferase (3.8.3.1). The

300

alterations in the SCFA distribution was associated with lower levels of acetate whilst the

301

higher butyrate and propionate levels seen in the ERB fed rats, and was a result of

302

enrichment of butyrate producing bacteria and up-regulations of enzymes in related

303

biosynthetic pathways, together with conversion between individual SCFAs (primarily from

304

acetate to butyrate).

305

DISCUSSION

306

In the current study, we employed multi-dimensional approaches to gain an insight into the

307

anti-obesity mechanisms associated with the observations in which rats fed a GABA enriched

308

rice bran diet attenuated metabolic disorders. This study revealed that rice bran treatments,

309

in particular ERB diet, significantly decreased the HFD induced body weight gain and insulin

16

ACS Paragon Plus Environment

Page 17 of 38

Journal of Agricultural and Food Chemistry

310

resistance, as well as energy metabolism abnormity. Complex cellular and biochemical

311

pathways lead to the development of metabolic syndromes such as increased weight gain,

312

but recent studies have suggested that the gut microbiota can be predominant players.33,34

313

Here, we demonstrated that the alterations in gut microbiota composition and intestinal

314

metabolites distribution contributed to the ability of a ERB diet to modulate a reversal in

315

insulin resistance and food over-intake induced by HFD in rats. Rice bran administration

316

results in increased total dietary fiber intake, which was reported to increase the production

317

of the health-promoting SCFAs.35 This is the first time to reveal that the rice bran

318

supplemented diet demonstrated a significant reduction in acetate, but increased butyrate

319

and propionate levels in the serum compared to HFD, in particular ERB diet. Consistently, the

320

accumulation of gut microbial produced acetate has also been reported to trigger insulin

321

over-secretion.36 The current metagenomics analysis provided plausible interpretations for

322

the altered SCFA distribution through the promotion in growth of butyrate-producing

323

bacteria of Anaerostipes and Anaerostipes sp. which enhanced butyrate synthesis via the

324

conversion of acetate into butyrate.37 The enhanced growth of Bifidobacterium and

325

Lactobacillus also contributed to the production of butyrate and other SCFA.38 Meanwhile,

326

the stimulation of key enzymes involved in both butyrate and propionate syntheses also

327

contributed to the modified SCFA distribution under ERB supplementation. The

328

administration of FRB led to a decreased microbial acetate production but not increased

329

butyrate production. The significantly promoted production of SCFA in particular butyrate

330

under ERB administration might be primarily attributed to the synergistic action of phenolic

17

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 18 of 38

331

acids and GABA, which have been demonstrated to facilitate butyrate synthesis in the

332

animals.39,40 Furthermore, Phenolic acids and GABA were supposed to facilitate the

333

maintenance of gastrointestinal health by the positive modulation in the enhancement of

334

intestinal antioxidant status.41 The improved gut environment boosted bile acids circulation,

335

especially highly hydrophilic secondary bile acids of ursodeoxycholic acid and muricholic acid,

336

which could increase transhepatic bile acid flux, thus reducing hypertriglyceridaemia and

337

improving cholesterol homeostasis.

338

Butyrate and propionate are known for their stimulating effect on gut hormones in

339

particular anorexigenic peptides,42 thus regulating appetite and energy expenditure. Under

340

ERB administration, the accumulation of butyrate and propionate in the gut activated G

341

protein-coupled receptors which exist in intestinal epithelial cells, further releasing

342

anorexigenic peptide GLP-1. Stimulation of receptors by butyrate and propionate also results

343

in the production of the hormone leptin in adipose tissue,43 and this was evident in the

344

increased circulating leptin concentration in ERB diet fed rats. Leptin can result in appetite

345

suppression and hypothalamic neuronal activation after crossing the blood-brain barrier and

346

binding to ObRb, in particular inhibiting the expression of orexigenic AgRP/NPY. The

347

inhibitions of hypothalamic AMPK (a monitor on cellular energy status44) and SOCS3 (a

348

negative-feedback regulator of leptin receptor signaling45) were observed under ERB

349

administration, which are necessary for leptin’s effects on energy consumption.46

350

Moreover, butyrate and propionate also exert effects elsewhere in the body due to their

351

ability to transit through enterocytes and into the circulation. Previous research has

18

ACS Paragon Plus Environment

Page 19 of 38

Journal of Agricultural and Food Chemistry

352

reported an important role for butyrate and propionate in reducing lipid accumulationand

353

modulating glucose homeostasis.47-50 This study found that the enhanced butyrate and

354

propionate circulation in ERB-containing diet greatly improved lipid metabolism and insulin

355

resistance. Lipidomic analysis in this study indicated that the improved insulin insensitivity

356

was associated with suppressed synthesis of DAG and ceramides species, which have long

357

been suspected to be key lipid intermediates linking nutrient excess to the antagonism of

358

insulin signaling.51 The high fat diet induced higher serum levels of DAG and ceramides,

359

which can inhibit insulin receptor substrates and block the activation of Akt/PKB,52,53

360

respectively. Ceramides inhibit insulin action via two primary mechanisms: direct activation

361

of protein kinase C (PKC) which phosphorylates and inhibits the translocation of Akt/PKB;

362

stimulation of protein phosphatase 2A (PP2A), and the primary phosphatase responsible for

363

dephosphorylating Akt/PKB.54 While the undesirable status above caused by the HFD was

364

alleviated by rice bran supplementation, in particular ERB treatment which performed more

365

efficiently in relation to reducing serum concentrations of GluCer, LacCer and GM3.

366

Furthermore, the gene network established by differentially expressed genes in both rice

367

bran groups indicated a bridge linking effects of Pdgfa, which is involved in the ceramide

368

signaling pathway. Furthermore, ERB significantly down-regulated the expressions of PP2A

369

(Ppp2r3b) and PCK (Prkcg), reconfirming its ability to efficiently suppress the blocking effect

370

of ceramides on insulin signaling. Meanwhile, the alleviated insulin resistance might also be

371

associated with the activation of GABA receptors by the enhanced levels of GABA in

372

circulation, further improving glucose tolerance and insulin sensitivity as reported by Tian et

19

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 20 of 38

373

al.55 Moreover, the RNA sequencing analysis in liver tissue and the mRNA relative expression

374

in adipose tissue revealed a positive energy harvest modulation associated with ERB through

375

NAFLD pathway and AMPK signaling pathway, which was reflected in the balanced

376

lipogenesis and lipolysis via regulating expressions of LPL, CIDEA, FATP1 and SREBP as well as

377

the enhanced fatty acid β-oxidation via Prkaa2, Ppara, Scd1 and Foxo1. Therefore, the ERB

378

supplement drives the shift of gut microbiota and re-distribution of gut microbiome

379

produced SCFAs to stimulate the release of gut hormones (leptin, GLP-1 and gastrin),

380

resulting in a decreased food intake via controlling hypothalamus/appetite pathways and

381

attenuated insulin resistance via suppressing ceramides synthesis.

382

In conclusion, this study shows that ERB supplement leads to a remarkable attenuation in

383

HFD induced body weight gain and insulin resistance. The administration of ERB altered

384

intestinal metabolites distribution, characterized by a decrease in acetate coupled with an

385

increase in butyrate and propionate. The modified SCFA distribution was attributed to the

386

increase in butyrate producing bacteria in particular those utilizing acetate as a substrate,

387

together with an enhancement in the levels of enzymes involved in butyrate and propionate

388

syntheses. Furthermore, the elevated microbiome produced butyrate and propionate

389

stimulated the release of gut hormones such as GLP-1 and the leptin adipocytokine, further

390

contributing to the appetite inhibition by suppressing orexigenic factors (AgPR/NPY),

391

promoting leptin receptor expression and regulating AMPKα and SOCS3. Moreover, the

392

increased blood circulatory levels of butyrate and propionate demonstrated their positive

393

effects on the regulation of ceramide biosynthesis and lipid homeostasis, which also

20

ACS Paragon Plus Environment

Page 21 of 38

Journal of Agricultural and Food Chemistry

394

contributed to a reduced insulin resistance. These findings highlight a potential therapeutic

395

approach for attenuating metabolic syndrome such as obesity.

396

ABBREVIATIONS USED

397

GABA, gama-aminobutyric acid; ERB, GABA enriched rice bran; FRB, fresh rice bran; MC,

398

model control; HFD, high-fat diet; FFA, free fatty acid; TAG, triacylglycerol; Cho, cholesterol;

399

DAG, diacylglycerols; CE, cholesteryl esters; SM, sphingomyelins; Cer, ceramides; GluCer,

400

glucosylceramides;

401

monosialo-dihexosylceramides; GLP-1, glucagon-like peptide-1; SCFA, short chain fatty acid;

402

OBRb, leptin receptor; AgRP, agouti-related peptide; POMC, proopiomelanocortin; SOCS-3,

403

suppressor of cytokine signalling-3; AMPKα2, adenosine monophosphate-activated protein

404

kinase alpha 2; NPY, neuropeptide Y.

405

ACKNOWLEDGEMENTS

406

We appreciate Sunrice Co. (Leeton, NSW, Australia) for supporting rice bran samples.

407

SUPPORTING INFORMATION DESCRIPTION

408

Supplementary Tables S1 and S2 show concentrations of GABA and phenolic acids in rice

409

bran before and after enrichment and primer sequences used for PCR analysis, respectively.

410

Supplementary Figure S1 shows feeding progress for rats. Supplementary Figure S2 shows

411

local gene networks (LGN) of obesity-associated up/down-regulated genes connected by

412

indicated pathways. Supplementary Figure S3 shows validation of RNA sequencing data by

413

RT-PCR.

LacCer,

lactosylceramides;

Sph,

sphingosines;

GM3,

21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 22 of 38

414

REFERENCES

415

1. Remely, M.; Haslberger, A. G. The microbial epigenome in metabolic syndrome. Molecul.

416

Aspects Med. 2017, 54, 71-77.

417

2. Grundy, S. M. Metabolic syndrome update. Trends Cardiovas. Med. 2016, 26, 364-373.

418

3. Amiot, M. J.; Riva, C.; Vinet, A. Effects of dietary polyphenols on metabolic syndrome

419

features in humans: a systematic review. Obes. Rev. 2016, 17, 573-586.

420

4. Muthayya, S.; Sugimoto, J. D.; Montgomery, S.; Maberly, G. F. An overview of global rice

421

production, supply, trade, and consumption. Ann. N. Y. Acad. Sci. 2014, 1324, 7-14.

422

5. So, W. K.; Law, B. M.; Law, P. T.; Chan, C. W. Current Hypothesis for the Relationship

423

between Dietary Rice Bran Intake, the Intestinal Microbiota and Colorectal Cancer

424

Prevention. Nutrients 2016, 8, 569.

425

6. Tabassum, S.; Ahmad, S.; Madiha, S.; Khaliq, S.; Shahzad, S.; Batool, Z.; Haider, S. Impact

426

of oral supplementation of glutamate and gaba on memory performance and neurochemical

427

profile in hippocampus of rats. Pak. J. Pharm. Sci. 2017, 30(3(Suppl.)), 1013.

428

7. Nishimura, M.; Yoshida, S.; Haramoto, M.; Mizuno, H.; Fukuda, T.; Kagami-Katsuyama, H.;

429

Tanaka, A.; Ohkawara, T.; Sato, Y.; Nishihira J. Effects of white rice containing enriched

430

gamma-aminobutyric acid on blood pressure. J. Tradition. Complemen. Med. 2016, 6, 66-71.

431

8. Bansal, P.; Wang, S.; Liu, S.; Xiang, Y. Y.; Lu, W. Y.; Wang, Q. GABA coordinates with insulin

432

in regulating secretory function in pancreatic INS-1 β-cells. PloS One 2011, 6, e26225.

433

9. Bouché, N.; Fromm, H. GABA in plants: Just a metabolite? Trends Plant Sci. 2004, 9,

434

110-115.

22

ACS Paragon Plus Environment

Page 23 of 38

Journal of Agricultural and Food Chemistry

435

10. Kim, H. S.; Lee, E. J.; Lim, S. T.; Han, J. A. Self-enhancement of GABA in rice bran using

436

various stress treatments. Food Chem. 2015, 172, 657-662.

437

11. Henderson, A. J.; Kumar, A.; Barnett, B.; Dow, S. W.; Ryan, E. P. Consumption of rice bran

438

increases mucosal immunoglobulin A concentrations and numbers of intestinal Lactobacillus

439

spp. J. Med. Food 2012, 15, 469-475.

440

12. Cherukuri, R. S. V.; Cheruvanky, R. METHOD FOR TREATING COLON CANCER WITH RICE

441

BRAN COMPOSITION. 2007, US20070243272.

442

13. Komiyam, Y.; Andoh, A.; Fujiwara, D.; Ohmae, H.; Araki, Y.; Fujiyama, Y.; Mitsuyama, K.;

443

Kanauchi, O. New prebiotics from rice bran ameliorate inflammation in murine colitis models

444

through the modulation of intestinal homeostasis and the mucosal immune system. Scand. J.

445

Gastroentero. 2011, 46, 40-52.

446

14. Wanyo, P.; Meeso, N; Siriamornpun, S. Effects of different treatments on the antioxidant

447

properties and phenolic compounds of rice bran and rice husk. Food Chem. 2014, 157,

448

457-463.

449

15. Bieri, J. G. Second report of the ad hoc committee on standards for nutritional studies. J.

450

Nutr. 1980, 110, 1726-1726.

451

16. Lam, S. M.; Wang, Y. T.; Duan, X. R.; Wenk, M. R.; Kalaria, R. N.; Chen, C. P.; Lai, M. K. P.;

452

Shui, G. H. The brain lipidomes of subcortical ischemic vascular dementia and mixed

453

dementia. Neurobiol. Aging. 2014, 35, 2369-2381.

454

17. Guan, X. L. Cestra, G.; Shui, G.; Kuhrs, A.; Schittenhelm, R. B.; Hafen, E.; van der Goot, F.

455

G.; Robinett, C. C.; Gatti, M.; Gonzalez-Gaitan, M.; Wenk, M. R. Biochemical membrane

23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 24 of 38

456

lipidomics during Drosophila development. Dev. Cell. 2013, 24, 98-111.

457

18. Shui, G.; Guan, X. L.; Low, C. P.; Chua, G. H.; Goh, J. S.; Yang, H.; Wenk, M. R. Toward one

458

step analysis of cellular lipidomes using liquid chromatography coupled with mass

459

spectrometry: application to Saccharomyces cerevisiae and Schizosaccharomyces pombe

460

lipidomics. Mol. BioSyst. 2010, 6, 1008-1017.

461

19. Steiner, C.; Von Eckardstein, A.; Rentsch, K. M. Quantification of the 15 major human

462

bile acids and their precursor 7α-hydroxy-4-cholesten-3-one in serum by liquid

463

chromatography–tandem mass spectrometry. J. Chromatogr. B 2010, 878, 2870-2880.

464

20. Frost G. Sleeth, M. L.; Sahuri-Arisoylu, M.; Lizarbe, B.; Cerdan, S.; Brody, L.; Anastasovska,

465

J.; Ghourab, S.; Hankir, M.; Zhang, S.; Carling, D.; Swann, J. R.; Gibson, G.; Viardot, A.;

466

Morrison, D.; Thomas, E. L.; Bell, J. D. The short-chain fatty acid acetate reduces appetite via

467

a central homeostatic mechanism. Nat. Commun. 2014, 5, 3611.

468

21. Geel, B. V.; Guthrie, R. D.; Altmann, J. G.; Broekens, P.; Bull, I. D.; Gill, F. L., Jansen, B.;

469

Nieman, A. M.; Gravendeel, B. Mycological evidence of coprophagy from the feces of an

470

alaskan late glacial mammoth. Quaternary Sci. Rev. 2011, 30, 2289-2303.

471

22. Haas, B. J.; Gevers, D.; Earl, A. M.; Feldgarden, M.; Ward, D. V.; Giannoukos, G.; Ciulla, D.;

472

Tabbaa, D.; Highlander, S. K.; Sodergren, E.; Methé, B.; DeSantis, T. Z.; Human Microbiome

473

Consortium; Petrosino, J. F.; Knight, R.; Birren, B. W. Chimeric 16S rRNA sequence formation

474

and detection in Sanger and 454-pyrosequenced PCR amplicons. Genome Res. 2011, 21,

475

494-504.

476

23. Bokulich, N. A.; Subramanian, S.; Faith, J. J.; Gevers, D.; Gordon, J. I.; Knight, R.; Mills, D.

24

ACS Paragon Plus Environment

Page 25 of 38

Journal of Agricultural and Food Chemistry

477

A., Caporaso, J. G. Quality-filtering vastly improves diversity estimates from Illumina

478

amplicon sequencing. Nat. Methods 2013, 10, 57-59.

479

24. Edgar, R. C. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat.

480

Methods 2013, 10, 996-998.

481

25. Karlsson, F. H.; Tremaroli, V.; Nookaew, I.; Bergström, G.; Behre, C. J.; Fagerberg, B.;

482

Nielsen, J.; Bäckhed. F. Gut metagenome in European women with normal, impaired and

483

diabetic glucose control. Nature 2013, 498, 99-103.

484

26. Nielsen, H. B.; Almeida, M.; Juncker, A. S; Rasmussen, S.; Li, J.; Sunagawa, S.; Plichta, D.

485

R.; Gautier, L.; Pedersen, A. G.; Le Chatelier, E.; Pelletier, E.; Bonde, I.; Nielsen, T.; Manichanh,

486

C.; Arumugam, M.; Batto, J. M.; Quintanilha Dos Santos, M. B.; Blom, N.; Borruel, N.;

487

Burgdorf, K. S.; Boumezbeur, F.; Casellas, F.; Doré, J.; Dworzynski, P.; Guarner, F.; Hansen, T.;

488

Hildebrand, F.; Kaas, R. S.; Kennedy, S.; Kristiansen, K.; Kultima, J. R.; Léonard, P.; Levenez, F.;

489

Lund, O.; Moumen, B.; Le Paslier, D.; Pons, N.; Pedersen, O.; Prifti, E.; Qin, J.; Raes, J.;

490

Sørensen, S.; Tap, J.; Tims, S.; Ussery, D. W.; Yamada, T.; MetaHIT Consortium; Renault, P.;

491

Sicheritz-Ponten, T.; Bork, P.; Wang, J.; Brunak, S.; Ehrlich, S. D. Identification and assembly of

492

genomes and genetic elements in complex metagenomic samples without using reference

493

genomes. Nat. Biotechnol. 2014, 32, 822-828.

494

27. Buchfink, B.; Xie, C; Huson, D. H. Fast and sensitive protein alignment using DIAMOND.

495

Nat. Methods 2015, 12, 59-60.

496

28. Li, J. Jia, H.; Cai, X.; Zhong, H.; Feng, Q.; Sunagawa, S.; Arumugam, M.; Kultima, J. R.;

497

Prifti, E.; Nielsen, T.; Juncker, A. S.; Manichanh, C.; Chen, B.; Zhang, W.; Levenez, F.; Wang, J.;

25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 26 of 38

498

Xu, X.; Xiao, L.; Liang, S.; Zhang, D.; Zhang, Z.; Chen, W.; Zhao, H.; Al-Aama, J. Y.; Edris, S.;

499

Yang, H.; Wang, J.; Hansen, T.; Nielsen, H. B.; Brunak, S.; Kristiansen, K.; Guarner, F.; Pedersen,

500

O.; Doré, J.; Ehrlich, S. D.; MetaHIT Consortium; Bork, P.; Wang, J. An integrated catalog of

501

reference genes in the human gut microbiome. Nat. Biotechnol. 2014, 32, 834-841.

502

29. Feng, Q.; Liang, S.; Jia, H.; Stadlmayr, A.; Tang, L.; Lan, Z.; Zhang, D.; Xia, H.; Xu, X.; Jie, Z.;

503

Su, L.; Li, X.; Li, X.; Li, J.; Xiao, L.; Huber-Schönauer, U.; Niederseer, D.; Xu, X.; Al-Aama, J. Y.;

504

Yang, H.; Wang, J.; Kristiansen, K.; Arumugam, M.; Tilg, H.; Datz, C.; Wang, J. Gut microbiome

505

development along the colorectal adenoma-carcinoma sequence. Nat. Commun. 2015, 6,

506

6528.

507

30. Lavie, C. J.; Mcauley, P. A.; Church, T. S.; Milani, R. V.; Blair, S. N. Obesity and

508

cardiovascular diseases: implications regarding fitness, fatness, and severity in the obesity

509

paradox. J. Am. Coll. Cardiol. 2014, 63, 1345-1354.

510

31. Benjamini, Y.; Hochberg, Y. Controlling the false discovery rate: a practical and powerful

511

approach to multiple testing. J. Roy. Stat. Soc. Series B (Methodological) 1995, 57, 289-300.

512

32. Si, X.; Shang, W.; Zhou, Z.; Strappe, P.; Wang, B.; Bird, A.; Blanchard, C. Gut

513

Microbiome-Induced Shift of Acetate to Butyrate Positively Manages Dysbiosis in High Fat

514

Diet. Mol. Nutr. Food Res. 2017, DOI: 10.1002/mnfr.201700670.

515

33. Clavel, T.; Desmarchelier, C.; Haller, D.; Gérard, P.; Rohn, S.; Lepage, P.; Daniel, H.

516

Intestinal microbiota in metabolic diseases: from bacterial community structure and

517

functions to species of pathophysiological relevance. Gut Microb. 2014, 5, 544-551.

518

34. Goulet, O. Potential role of the intestinal microbiota in programming health and disease.

26

ACS Paragon Plus Environment

Page 27 of 38

Journal of Agricultural and Food Chemistry

519

Nutr. Rev. 2015, 73(suppl_1), 32-40.

520

35. Sheflin, A. M.; Borresen, E. C.; Kirkwood, J. S.; Boot, C. M.; Whitney, A. K.; Lu, S., Brown;

521

R. J.; Broeckling, C. D.; Ryan, E. P.; Weir, T. L. Dietary supplementation with rice bran or navy

522

bean alters gut bacterial metabolism in colorectal cancer survivors. Mol. Nutr. Food Res.

523

2017, 61, DOI: 10.1002/mnfr.201500905.

524

36. Perry, R. J.; Peng, L.; Barry, N. A.; Cline, G. W.; Zhang, D.; Cardone, R. L.; Petersen, K. F.;

525

Kibbey, R. G.; Goodman, A. L.; Shulman, G. I. Acetate mediates a microbiome-brain-β-cell

526

axis to promote metabolic syndrome. Nature 2016, 534, 213-217.

527

37. Duncan, S. H.; Barcenilla, A.; Stewart, C. S.; Pryde, S. E.; Flint, H. J. Acetate utilization and

528

butyryl coenzyme a (coa):acetate-coa transferase in butyrate-producing bacteria from the

529

human large intestine. Appl. Environ. Microb. 2002, 68, 5186-5190.

530

38. Araki, Y.; Andoh, A.; Koyama, S.; Fujiyama, Y.; Kanauchi, O.; Bamba, T. Effects of

531

germinated barley foodstuff on microflora and short chain fatty acid production in dextran

532

sulfate sodium-induced colitis in rats. Biosc. Biotech. Bioch. 2000, 64, 1794-1800.

533

39. Xie, M.; Chen, H. H.; Nie, S. P.; Yin, J. Y.; Xie, M. Y. Gamma-aminobutyric acid increases

534

the production of short-chain fatty acids and decreases pH values in mouse colon. Molecules

535

2017, 22, 653, DOI:10.3390/molecules22040653.

536

40. Parkar, S. G.; Trower, T. M.; Stevenson, D. E. Fecal microbial metabolism of polyphenols

537

and its effects on human gut microbiota. Anaerobe 2013, 23, 12-19.

538

41. Cardona, F.; Andrés-Lacueva, C.; Tulipani, S.; Tinahones, F. J.; Queipo-Ortuño, M. I.

539

Benefits of polyphenols on gut microbiota and implications in human health. J. Nutr.

27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 28 of 38

540

Biochem. 2013, 24, 1415-1422.

541

42. Lin, H. V.; Frassetto, A.; Kowalik Jr, E. J.; Nawrocki, A. R.; Lu, M. M.; Kosinski, J. R.; Hubert,

542

J. A.; Szeto, D.; Yao, X.; Forrest, G.; Marsh, D. J. Butyrate and propionate protect against

543

diet-induced obesity and regulate gut hormones via free fatty acid receptor 3-independent

544

mechanisms. PloS One 2012, 7, e35240.

545

43. Xiong, Y.; Miyamoto, N.; Shibata, K.; Valasek, M. A.; Motoike, T.; Kedzierski, R. M.;

546

Yanagisawa, M. Short-chain fatty acids stimulate leptin production in adipocytes through the

547

G protein-coupled receptor GPR41. P. NATL. ACAD. SCI. USA 2004, 101, 1045-1050.

548

44. Hardie, D. G.; Scott, J. W.; Pan, D. A.; Hudson, E. R. Management of cellular energy by

549

the AMP-activated protein kinase system. FEBS Lett. 2003, 546, 113-120.

550

45. Gamber, K. M.; Huo, L.; Ha, S.; Hairston, J. E.; Greeley, S.; Bjørbæk, C. Over-expression of

551

leptin receptors in hypothalamic POMC neurons increases susceptibility to diet-induced

552

obesity. PloS One 2012, 7, e30485.

553

46. Minokoshi, Y.; Alquier, T.; Furukawa, N.; Young-Bum, K. AMP-kinase regulates food intake

554

by responding to hormonal and nutrient signals in the hypothalamus. Nature 2004, 428,

555

569-574.

556

47. Khan, S.; Jena, G. Sodium butyrate reduces insulin-resistance, fat accumulation and

557

dyslipidemia in type-2 diabetic rat: A comparative study with metformin. Chem-Bio. Interact.

558

2016, 254, 124-134.

559

48. Higashimura, Y.; Naito, Y.; Takagi, T.; Uchiyama, K.; Mizushima, K.; Yoshikawa, T.

560

Propionate promotes fatty acid oxidation through the up-regulation of peroxisome

28

ACS Paragon Plus Environment

Page 29 of 38

Journal of Agricultural and Food Chemistry

561

proliferator-activated receptor α in intestinal epithelial cells. J. Nutr. Sci. Vitaminol. 2016, 61,

562

511-515.

563

49. Gao Z. Yin, J.; Zhang, J.; Ward, R. E.; Martin, R. J.; Lefevre, M.; Cefalu, W. T.; Ye, J.

564

Butyrate improves insulin sensitivity and increases energy expenditure in mice. Diabetes

565

2009, 58, 1509-1517.

566

50. Zhang, Q.; Koser, S. L.; Bequette, B. J.; Donkin, S. S. Effect of propionate on mRNA

567

expression of key genes for gluconeogenesis in liver of dairy cattle. J. Dairy Sci. 2015, 98,

568

8698-8709.

569

51. Holland, W. L.; Knotts, T. A.; Chavez, J. A.; Wang, L.; Hoehn, K. L.; Summers, S. A. Lipid

570

mediators of insulin resistance. Nutr. Rev. 2007, 65, S39-S46.

571

52. Dobbins, R. L.; Szczepaniak, L. S.; Bentley, B.; Esser, V.; Myhill, J.; Mcgarry, J. D. Prolonged

572

inhibition of muscle carnitine palmitoyltransferase-1 promotes intramyocellular lipid

573

accumulation and insulin resistance in rats. Diabetes 2001, 50, 123-130.

574

53. Summers, S. A. Ceramides in insulin resistance and lipotoxicity. Prog. Lipid Res. 2006, 45,

575

42-72.

576

54. Chavez, J. A.; Summers, S. A. A ceramide-centric view of insulin resistance. Cell Metab.

577

2012, 15, 585-594.

578

55. Tian, J.; Dang, H. N.; Yong, J.; Chui, W. S.; Dizon, M. P.; Yaw, C. K.; Kaufman, D. L. Oral

579

treatment with γ-aminobutyric acid improves glucose tolerance and insulin sensitivity by

580

inhibiting inflammation in high fat diet-fed mice. PLoS One 2011, 6, e25338.

581

29

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 30 of 38

582

Funding

583

This work was supported by National Key Research and Development Program

584

(2016YFD0400104-4, 2016YFD0400401-2), the NSFC (U1501214, 31471701), National Spark

585

Program (2015GA610003), Tianjin Research Program of Application Foundation and

586

Advanced Technology (15JCZDJC34300), the China-European research collaboration program

587

(SQ2013ZOA100001), and ARC Industrial Transformation Training Centre for Functional

588

Grains, Charles Sturt University.

30

ACS Paragon Plus Environment

Page 31 of 38

Journal of Agricultural and Food Chemistry

589

FIGURE CAPTIONS

590

Figure 1. Rice bran attenuated obesity syndromes. (A) Body weight gain and (B) fat-body

591

weight ratio of obese rats fed with FRB and ERB. (C) Serum insulin secretion in the rats with

592

different treatments. (D) Average food intake was reduced following rice bran administration.

593

(E-F) Serum lipid composition was significantly altered. Different lowercase letters above

594

each column represents a significant difference (P