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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

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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


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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


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INTRODUCTION

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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

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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

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

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

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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

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

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

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

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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.

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MATERIALS AND METHODS

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

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

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

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

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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).

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

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

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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

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

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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,

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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

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

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

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

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

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

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

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

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mL/min.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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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

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

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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.

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

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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

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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.

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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

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

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curves.

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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

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

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

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

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

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

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

105

(chloroform:methanol:ammonium

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

hydroxide:water,

55:39:0.5:5.5).17

Cer-d18:1/17:0,

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

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

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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

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

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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

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

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

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

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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

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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

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

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

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

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

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

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

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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.

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

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

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

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

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

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

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

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

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

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

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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

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

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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

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samples was implemented on a cBot Cluster Generation System following the

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

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

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

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

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

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

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

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

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

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

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coverage from metagenomics communities.29

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

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

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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

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

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

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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

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

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

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

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information).

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

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

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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

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

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

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

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

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

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was analyzed using KOBAS software.

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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

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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

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

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

207

thickness determination.

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

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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

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

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

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RESULTS

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

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

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

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treatments, in particular ERB administration, significantly attenuated insulin resistance

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

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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,

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

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

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

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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

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

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

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and appetite control.

233

Improvements in lipid and energy metabolism in both liver and adipose

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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

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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

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

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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.

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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

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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

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(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


Page 17 of 38


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



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


Page 19 of 38


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



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


Page 21 of 38


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



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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.

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FIGURE CAPTIONS

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

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

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

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

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each column represents a significant difference (P

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

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