Article

Rice Bran Reduces Weight Gain and Modulates

Lipid Metabolism in Rats with High-Energy-Diet-
Induced Obesity
Suh-Ching Yang 1,†, Wen-Ching Huang 2,†, Xin Er Ng 3,†, Mon-Chien Lee 4, Yi-Ju Hsu 4,
Chi-Chang Huang 4, Hai-Hsin Wu 2, Chiu-Li Yeh 1, Hitoshi Shirakawa 5, Slamet Budijanto 6,
Te-Hsuan Tung 1 and Yu-Tang Tung 3,7,8,*
1 School of Nutrition and Health Sciences, Taipei Medical University, Taipei 110, Taiwan
2 Department of Exercise and Health Science, National Taipei University of Nursing and Health Sciences,
Taipei City 112, Taiwan
3 Graduate Institute of Metabolism and Obesity Sciences, Taipei Medical University, Taipei 110, Taiwan

4 Graduate Institute of Sports Science, National Taiwan Sport University, Taoyuan 333, Taiwan

5 Graduate School of Agricultural Science, Tohoku University, City 981-8555, Japan

6 Faculty of Agricultural Engineering and Technology, Bogor Agricultural University, City 16680, Indonesia

7 Nutrition Research Center, Taipei Medical University Hospital, Taipei 110, Taiwan

8 Cell Physiology and Molecular Image Research Center, Wan Fang Hospital, Taipei Medical University,

Taipei City 116, Taiwan

* Correspondence: f91625059@tmu.edu.com; Tel.: +886-227361661
† These authors contributed equally to this work.

Received: 9 August 2019; Accepted: 28 August 2019; Published: 30 August 2019

Abstract: Obesity has become an epidemic worldwide. It is a complex metabolic disorder associated
with many serious complications and high morbidity. Rice bran is a nutrient-dense by product of
the rice milling process. Asia has the world’s highest rice production (90% of the world’s rice
production); therefore, rice bran is inexpensive in Asian countries. Moreover, the high nutritional
value of the rice bran suggests its potential as a food supplement promoting health improvements,
such as enhancing brain function, lowering blood pressure, and regulating pancreatic secretion. The
present study evaluated the anti-obesity effect of rice bran in rats with high-energy diet (HED)-
induced obesity. Male Sprague–Dawley rats were randomly divided into one of five diet groups (n
= 10 per group) and fed the following for eight weeks: Normal diet with vehicle treatment, HED
with vehicle, rice bran-0.5X (RB-0.5X) (2% wt/wt rice bran), RB-1.0X (4% wt/wt rice bran), and RB-
2.0X (8% wt/wt rice bran). Rice bran (RB-1.0X and RB-2.0X groups) markedly reduced obesity,
including body weight and adipocyte size. In addition, treating rats with HED-induced obesity
using rice bran significantly reduced the serum uric acid and glucose as well as the liver triglyceride
(TG) and total cholesterol (TC). Furthermore, administration of an HED to obese rats significantly
affected hepatic lipid homeostasis by increasing phosphotidylcholine (PC; 18:2/22:6), diacylglycerol
(DG; 18:2/16:0), DG (18:2/18:1), DG (18:1/16:0), cholesteryl ester (CE; 20:5), CE (28:2), TG
(18:0/16:0/18:3), and glycerol-1-2-hexadecanoate 3-octadecanoate. However, the rice bran treatment
demonstrated an anti-adiposity effect by partially reducing the HED-induced DG (18:2/18:1) and
TG (18:0/16:0/18:3) increases in obese rats. In conclusion, rice bran could act as an anti-obesity
supplement in rats, as demonstrated by partially reducing the HED-induced DG and TG increases
in obese rats, and thus limit the metabolic diseases associated with obesity and the accumulation of
body fat and hepatic lipids in rats.

Keywords: high-energy diet (HED); lipid metabolism; lipidomics; obesity; rice bran

Nutrients 2019, 11, 2033; doi:10.3390/nu11092033 www.mdpi.com/journal/nutrients

Nutrients 2019, 11, 2033 2 of 10

1. Introduction
The prevalence of obesity is rising worldwide. If this trend continues, 57.8% of the adult
population worldwide (3.3 billion people) may be overweight or obese by 2030 [1]. Obesity is a
serious global health problem leading to many health complications, such as type 2 diabetes, fatty
liver disease, dyslipidemia, hypertension, coronary artery disease, stroke, heart failure, reproductive
and gastrointestinal cancers, osteoarthritis, obstructive sleep apnea, and gallstones [2,3]. However,
most of the approved and marketed anti-obesity drugs have been withdrawn due to serious side
effects [4–6]. Therefore, natural products such as anti-obesity agents are indispensable for combating
obesity.
Rice is the second leading cereal crop, and the highest rice production is found in Asia (90% of
the world’s total rice production) [7]. Therefore, in Asian countries, rice bran (RB) is inexpensive. RB,
one of the most abundant byproducts produced in the rice milling industry, contains appreciable
quantities of nutrients (protein, fat, unsaturated fatty acids, dietary fiber, K, Ca, Mg, and Fe) and
antioxidants (γ-oryzanol, tocopherols, tocotrienols, and ferulic acid) [7–11]. Due to its nutritional
value, it can also be used as a health food supplement that improves brain function, lowers blood
pressure and cholesterol concentration, and regulates pancreatic secretion [12,13].
The lipidomic analysis provides a powerful approach for assessing a wide range of lipid species
in biological systems. The liquid chromatography-mass spectrometry (LC-MS) was successfully
applied to determine the metabolomic alternations in the plasma or serum of rodents fed with a high-
fat and high-carbohydrate (fructose and sucrose) diet [14]. We investigated the anti-obesity effect of
RB through an ultraperformance liquid chromatography coupled with mass spectrometry (UPLC-
QTOF/MS) in diet-induced-obesity rat models by determining the serum lipid profiles (i.e.,
lipidomics).

2. Materials and Methods

2.1. Animals and Study Design

Fifty male Sprague–Dawley (SD) rats (six weeks old; 200–250 g) obtained from BioLASCO (A
Charles River Licensee Corp., Yi-Lan, Taiwan) were used in this study. The rats were maintained in
a laboratory under controlled conditions of a 12-h light/dark cycle, 65% ± 5% relative humidity, and
24 °C ± 2 °C. The care and use of animals followed the guidelines of the Institutional Animal Care
and Utilization Committee of the National Taiwan Sport University (IACUC-10707). The animals
were acclimatized to the laboratory conditions for two weeks.
After two weeks of acclimatization, all 50 rats were randomly divided into one of five treatment
groups based on diet: (1) Chow diet (control; n = 10), (2) High-energy diet (HED/control; n = 10), (3)
HED and RB-0.5X (HED/0.5X; n = 10), (4) HED and RB-1.0X (HED/1.0X; n = 10), or (5) HED and RB-
2.0X (HED/2.0X; n = 10). The body weight change and diet consumption were measured every week.
The rats were sacrificed after eight weeks of intervention. The compositions of each diet were as
follows: The chow diet (LabDiet Rodent 5001) contained 3.35 kcal/g with 28.5% protein, 13.4% fat,
and 58.1% carbohydrates. The HED contained 4.22 kcal/g with 8% (wt/wt) lard oil, 44% (wt/wt) high-
fructose syrup, and 48% (wt/wt) standard chow. The RB-0.5X diet contained 8% (wt/wt) lard oil, 44%
(wt/wt) high-fructose syrup, 46% (wt/wt) standard chow, and 2% (wt/wt) RB for 4.24 kcal/g. The RB-
1.0X diet contained 8% (wt/wt) lard oil, 44% (wt/wt) high-fructose syrup, 44% (wt/wt) standard chow,
and 4% (wt/wt) RB for 4.26 kcal/g. The RB-2.0X diet contained 8% (wt/wt) lard oil, 44% (wt/wt) high-
fructose syrup, 40% (wt/wt) standard chow, and 8% (wt/wt) RB for 4.29 kcal/g. At the end of the
experiment, all rats were fasted for 12 h and anesthetized, and blood samples were collected through
a cardiac puncture. The serum was obtained by centrifugation at 1500× g 4 °C for 10 min. The liver,
omental fat, and epididymal fat were resected and immediately weighed. Additionally, the
pathological histology of epididymal fat and liver tissues was performed. All of the samples were
snap-frozen and stored at −80 °C until further analysis.

2.2. Determination of Biochemical Markers

Nutrients 2019, 11, 2033 3 of 10

The collected blood samples were used to detect the serum levels of the uric acid, glutamate
oxaloacetate transaminase (GOT), free fatty acid (FFA), total cholesterol (TC), triglyceride (TG), high-
density lipoprotein (HDL), low-density lipoprotein (LDL), and glucose content using an autoanalyzer
(Hitachi 7060, Company, City, Country). Total lipids were extracted from the liver through the Folch
method [15]. For the liver TC and TG determination, 20 mg of liver tissue was homogenized in a 200
μL solvent (chloroform : isopropanol: nonyl phenoxypolyethoxylethanol, NP40  = 7:11:0.1).
Centrifuged at 12,000× g for 10 min, an aliquot of 100 μL was extracted and dried. The pellet was
reconstituted with a buffer (1 M of potassium phosphate, pH  =  7.4, 500 mM of sodium chloride, and
50 mM of cholic acid), and a water bath sonication was employed to dissolve the precipitate [16]. The
cholesterol fluorometric assay kit (Cayman, Ann Arbor, MI, USA) and colorimetric triglyceride assay
kit (Cayman, Ann Arbor, MI, USA) were further applied to the liver TC and TG contents analysis.

2.3. Hematoxylin and Eosin Staining

The liver tissue and epididymal fat were fixed in a 10% buffered formaldehyde. The tissues were
then soaked in absolute ethanol overnight, embedded in paraffin, and cut into 4-μm-thick slices.
Sections were then stained with hematoxylin and eosin and examined by a clinical pathologist under
a light microscope equipped with a CCD camera (BX-51, Olympus, Tokyo).

2.4. Serum Lipidomics Analyses

2.4.1. Serum Lipid Extraction

The Folch method [13] was used with a slight modification for extraction of the serum lipids
from six samples of the vehicle, HED, and 2.0X groups. Briefly, 1.5 mL of methanol was added into
40 μL of the plasma sample, followed by the addition of 3 mL of CHCl3 and incubation for 1 h at
room temperature with occasional vortex mixing. Then, 1.25 mL of purified water was added and
allowed to stand for 10 min to facilitate the phase separation. The sample was centrifuged at 1000× g
for 10 min at 4 °C, and aliquots of 2000 μL of the organic phase were collected. Finally, the aliquots
were vacuum-dried and stored at −80 °C until further analysis.

2.4.2. UPLC-QTOF/MS
The lipid extracts were reconstituted with 250 μL of an isopropanol-acetonitrile-water (2:1:1),
and SYNAPT G2 QTof (Waters MS Technologies, Manchester, UK) was used for the UPLC-QTOF/MS
analysis. The parameters of the mass spectrometer for positive ionization mode detection were as
follows: Desolvation gas flow of 900 L/h, desolvation temperature of 550 °C, cone gas flow of 15 L/h,
source temperature of 120 °C, capillary voltage of 2.8 kV, cone voltage of 40 V, and TOF-MS scan
range of 100–2000 m/z. The data acquisition rate was set to 1.2 s with a 0.02-s interscan delay using
the Waters MSE acquisition mode, and the full exact masses were collected simultaneously by rapidly
alternating between two functions. Function 1 acquired data with a low collision energy of 4 and 2
eV for trap and transfer collision cells, whereas function 2 acquired data using a transfer collision
energy ramp from 15 to 35 eV. All analyses were performed using a LockSpray to ensure the accuracy
and reproducibility. Leucine–enkephalin was used as the lock mass at a concentration of 1 ng/μL and
a flow rate of 5 μL/min. Data were collected in a continuum mode, and the LockSpray frequency was
set at 20 s. All the data acquisition was controlled by the Waters MassLynx v4.1 software. The data
was obtained from six individual samples, and each data is triplicated.

2.4.3. Lipid Identification

The raw data were imported into the Progenesis QI software (Waters Corporation , MA, USA)
for alignment. Further, the peak picking and identification of polar lipids were carried out using a
high-resolution positive-ion MS, and the absolute intensities of all identified compounds were
recalculated to determine the relative abundances and to normalize the values of the lipid molecules.
The data were then exported into the EZinfo 2.0 software (Sartorius Stedim Biotech , Umeå, Sweden)
Nutrients 2019, 11, 2033 4 of 10

for the multivariate statistical analysis, and the principal component analysis (PCA) and orthogonal
projections to latent structures discriminant analysis (OPLS-DA) were used to create the final
statistical models to obtain the group clusters. Lipid molecules with the strongest effect on the group
clustering were identified as those with a VIP greater than one. In addition, potential ions with p <
0.05 and FC > 2 were selected for further metabolite relationship pathway characterization using the
MetaboAnalyst web server. Human Metabolome Database (HMDB) IDs were matched with the
Kyoto Encyclopedia of Genes and Genomes (KEGG) IDs for the KEGG mapping. IDs without a match
were excluded from the analysis, and the Rattus norvegicus (rat) pathway library in the KEGG was
selected for analysis.

2.5. Statistical Analysis

Data are expressed as the mean ± standard error (SE). Statistical differences were analyzed
through the one-way analysis of variance (ANOVA) with Duncan’s test using the SPSS version 20.0
(SPSS Inc., Chicago, IL, USA). Differences were considered statistically significant where p < 0.05.

3. Results

3.1. Effect of RB on Body Weight and Masses of Liver, Omental Fat, and Epididymal Fat in Rats with HED-
induced Obesity
The effects of RB on the body weight and the masses of the liver tissue, omental fat, and
epididymal fat are presented in Table 1. After eight weeks, the body weight and the masses of liver,
omental, epididymal, and total fat of the HED groups significantly increased compared with the
vehicle group until end of the study (p < 0.05). Although the initial body weight did not significantly
differ among the groups, the final body weight was lower in the 1.0X (466 ± 8 g) and 2.0X (463 ± 5 g)
groups by 4% and 5%, respectively, compared with the HED group (487 ± 5 g) (p < 0.05). However,
the daily energy intake did not significantly differ between the HED and RB-0.5X, RB-1.0X, or RB-
2.0X groups. The fat around the omental and epididymis of the vehicle, HED, RB-0.5X, RB-1.0X, and
RB-2.0X groups weighed 3.74 ± 0.33, 7.03 ± 0.57, 6.40 ± 0.40, 7.10 ± 1.14, and 6.48 ± 0.64 g, respectively.
The fat around the epididymis of the vehicle, HED, RB-0.5X, RB-1.0X, and RB-2.0X groups weighed
3.33 ± 0.36, 7.56 ± 0.53, 6.62 ± 0.26, 6.75 ± 0.75, and 6.96 ± 0.65 g, respectively. The omental and
epididymal fat masses were greater in the HED group than the vehicle group by 88% (p < 0.05) and
127% (p < 0.05), respectively. However, the RB groups exhibited slightly lower epididymal fat
compared with the HED group.

Table 1. Comparison of the physical and metabolic effects of rice bran (RB) in rat groups.

Vehicle HED RB-0.5X RB-1.0X RB-2.0X

Water (mL) 45.3 ± 0.8 b 26.3 ± 0.4 a 25.8 ± 0.2 a 26.7± 0.3 a 25.0 ± 0.3 a
Food intake (g/day) 23.3 ± 0.4 a 23.5 ± 0.4 a 23.6 ± 0.4 a 23.0 ± 0.3 a 23.2 ± 0.4 a
Energy intake (kcal/day) 77.9 ± 1.3 a 99.2 ± 1.6 b 99.9 ± 1.9 b 98.2 ± 1.5 b 99.4 ± 1.7 b
Feed efficiency 1 0.5 ± 0.2 a 0.9 ± 0.2 a 0.8 ± 0.2 a 0.8 ± 0.2 a 0.8 ± 0.1 a
Final body weight (g) 416 ± 8 a 487 ± 5 c 472 ± 4b c 466 ± 8 b 463 ± 5 b
Body weight gain (g) 97.1 ± 4.4a 168.1 ± 5.4 b 149.1 ± 2.5 b 148.4 ± 6.1 b 146.8 ± 14.3 b
Liver 11.55 ± 0.41 a 15.34 ± 0.28 b 14.09 ± 0.23 b 14.09 ± 0.23 b 14.24 ± 0.42 b
Omental fat 3.74 ± 0.33 a 7.03 ± 0.57 b 6.40 ± 0.40 b 7.10 ± 1.14 b 6.48 ± 0.64 b
Epididymal fat 3.33 ± 0.36 a 7.56 ± 0.53 b 6.62 ± 0.26 b 6.75 ± 0.75 b 6.96 ± 0.65 b
Total fat 9.51 ± 0.88 a 24.40 ± 1.50 b 22.65 ± 0.76 b 23.76 ± 1.81 b 23.36 ± 1.80 b
Data are expressed as the mean ± standard error (SE) (n = 10). Different letters (a, b, and c) indicate
significant differences at p < 0.05 in the one-way ANOVA. Column titles indicate the following:
Vehicle, Sprague–Dawley (SD) rat fed chow diet; high-energy diet (HED), SD rat fed HED; RB-0.5X,
SD rat fed HED with RB-0.5X; RB-1.0X, SD rat fed HED with RB-1.0X; RB-2.0X, SD rat fed HED with
RB-2.0X. 1 Feed efficiency = weight gain/food intake.
Nutrients 2019, 11, 2033 5 of 10

3.2. Effect of RB on Serum Biochemical Parameters of Rats with HED-induced Obesity

The effects of RB on the uric acid, GOT, FFA, TC, TG, HDL, LDL, and glucose are presented in
Table 2. The HED-fed SD rats had significantly higher serum levels of the uric acid, FFA, TC, TG, and
glucose by 113% (p < 0.05), 68% (p < 0.05), 27% (p < 0.05), 288% (p < 0.05), and 123% (p < 0.05),
respectively, compared with the SD rats fed with a normal diet. Significantly lower serum levels of
the uric acid (30%, 41%, and 59% lower in the RB-0.5X, RB-1.0X, and RB-2.0X groups, respectively)
and glucose (27%, 17%, and 17% lower in the RB-0.5X, RB-1.0X, and RB-2.0X groups, respectively)
were exhibited by the RB-treated groups compared with the HED group.

Table 2. Effect of RB on the serum biochemical parameters in rats with high-energy diet (HED)-
induced obesity.

Vehicle HED RB-0.5X RB-1.0X RB-2.0X

Uric acid (mg/dL) 3.0 ± 0.4 a 6.4 ± 0.6 c 4.5 ± 0.2 b 3.8 ± 0.6 ab 2.6 ± 0.3 a
GOT (U/L) 64 ± 2 a 66 ± 2 a 63 ± 2 a 62 ± 1 a 64 ± 1 a
FFA (mmol/L) 0.62 ± 0.03 a 1.04 ± 0.06 b 0.95 ± 0.07 b 0.88 ± 0.09 b 1.03 ± 0.07 b
TC (mg/dL) 59 ± 4 a 75 ± 4 b 72 ± 5 b 68 ± 4 ab 69 ± 6 ab
TG (mg/dL) 40 ± 3 a 155 ± 13 b 135 ± 13 b 137 ± 16 b 133 ± 10 b
HDL (mg/dL) 35.3 ± 2.3 a 35.4 ± 0.8 a 37.8 ± 2.5 a 34.8 ± 1.8 a 36.1 ± 2.6 a
LDL (mg/dL) 14.0 ± 1.1 a 11.5 ± 0.8 a 11.8 ± 1.1 a 11.4 ± 1.1 a 11.4 ± 1.1 a
Glucose (mg/dL) 153 ± 8 a 341 ± 25 c 250 ± 14 b 283 ± 24 b 283 ± 8 b
Data are expressed as the mean ± SE (n = 10). Different letters (a, b, and c) indicate significant
differences at p < 0.05 in one-way ANOVA. Vehicle, SD rat fed chow diet; HED, SD rat fed HED; RB-
0.5X, SD rat fed HED with RB-0.5X; RB-1.0X, SD rat fed HED with RB-1.0X; RB-2.0X, SD rat fed HED
with RB-2.0X.

3.3. Effect of RB on Hepatic Lipid Accumulation in Rats with HED-induced Obesity

Liver tissue biopsies performed through the hematoxylin and eosin staining revealed that rats
fed with an HED for eight weeks developed a higher degree of steatosis and microvesicular fatty
changes. The extent of steatosis was diminished by the RB intervention (Figure 1A). In addition, the
HED-fed to rats accumulated more hepatic TG (49% higher compared with the vehicle group, p <
0.05) and hepatic TC (102% higher compared with the vehicle group, p < 0.05). In the liver tissues of
the RB-0.5X, RB-1.0X, and RB-2.0X groups, the TG (10.0 ± 1.4, 7.5 ± 1.1, and 7.2 ± 1.0 mg/g, respectively)
and TC (1.0 ± 0.1, 1.0 ± 0.1, and 0.8 ± 0.0 mg/g) levels were significantly lower than those (12.7 ± 2.4
and 1.7 ± 0.1 mg/g, respectively) of the HED only group (p < 0.05) (Figure 1B and 1C). Thus, rats with
HED-induced obesity fed RB-0.5X, RB-1.0X, and RB-2.0X diets accumulated significantly less hepatic
TG and TC (21%, 41%, 43%, and 41%, 41%, 53%, respectively) compared with those fed only HED.
Thus, strong dose-dependent effects were observed of the hepatic TG and TC levels. These results
suggest that RB may have an inhibitory effect on lipid accumulation in the liver.
Nutrients 2019, 11, 2033 6 of 10

Figure 1. Effect of rice bran (RB) on the hepatic lipid accumulation in rats with high-energy diet
(HED)-induced obesity. (A) Hematoxylin and eosin staining of liver sections (100× magnification).
Scale bar: 40 μm. (B) Liver triglyceride (TG) concentration and (C) liver total cholesterol (TC)
concentration. The x-axis parameters are as follows: Vehicle, SD rat fed with chow diet; HED, SD rat
fed with HED; RB-0.5X, SD rat fed with HED and RB-0.5X; RB-1.0X, SD rat fed with HED and RB-
1.0X; and RB-2.0X, SD rat fed with HED and RB-2.0X. The data are expressed as the mean ± SE (n =
10). Different letters (a, b, and c) indicated significant differences at p < 0.05 by one-way ANOVA.

3.4. Effect of RB on Adipocyte Size Distribution in Rats with HED-induced Obesity

Rats in the intervention groups were significantly heavier than those in the vehicle group.
However, the body weights of the rats in the RB treatment groups increased more slowly than those
of rats in the HED group (Figure 2A). Histological analyses revealed larger white adipocytes around
the epididymis of the HED group compared with the vehicle group (Figure 2B). Nevertheless, the
epididymal white adipocytes of the rats in the RB treatment groups were smaller than those in the
HED group (Figure 2B). The results indicate that the RB-0.5X, RB-1.0X, and RB-2.0X intervention
reduced the HED-induced adiposity.

Figure 2. Effect of RB on body weight and epididymal white adipose tissue (eWAT) change in rats
with HED-induced obesity. (A) Body weight change over time; (B) hematoxylin and eosin staining of
eWAT sections (×100 magnification). Scale bar: 40 μm. Vehicle, SD rat fed chow diet; HED, SD rat fed
HED; RB-0.5X, SD rat fed HED with RB-0.5X; RB-1.0X, SD rat fed HED with RB-1.0X; RB-2.0X, SD rat
fed HED with RB-2.0X. The data are expressed as the mean ± SE (n = 10).
Nutrients 2019, 11, 2033 7 of 10

3.5. Effect of RB on Lipidomics in Rats with HED-induced Obesity Analyses of Lipidomic Profiles
PCA and supervised OPLS-DA score plots were used to determine which dominant lipid species
were reduced by the RB amelioration of HED-induced obesity in rats. The vehicle and HED groups
were compared to determine the obesity-related changes in serum lipids. A distinct clustering of the
vehicle and HED groups was observed in the plots (Figure 3), suggesting that an HED induced severe
lipid biosynthesis in SD rats. Therefore, we selected 29 variables determined based on the three
thresholds (p < 0.05, VIP > 1, and FC > 2), among which eight lipid species were significantly increased
by an HED: phosphotidylcholine (PC; 18:2/22:6), diacylglycerol (DG; 18:2/16:0), DG (18:2/18:1), DG
(18:1/16:0), cholesteryl ester (CE; 20:5), CE (28:2), TG (18:0/16:0/18:3), and glycerol-1-2-hexadecanoate
3-octadecanoate. The PCA and OPLS-DA score plots were used to determine changes in the serum
lipids due to the RB effect, revealing that the HED and RB-2.0X groups (Figure 4) were well separated.
The results indicate that the RB treatment led to a limited reduction of the increment in two lipid
species induced by the HED, namely DG (18:2/18:1/0:0) and TG (18:0/16:0/18:3).

Figure 3. Score plots of the serum samples from the vehicle and HED groups. (A) principal component
analysis (PCA) score plot and (B) orthogonal projections to latent structures discriminant analysis
(OPLS-DA) score plot. Vehicle, SD rat fed chow diet; HED, SD rat fed HED.

3.6. Metabolic Pathway Analysis

The identified metabolites were exported to the MetaboAnalyst web server
(https://www.metaboanalyst.ca/) for the metabolic pathway analysis to determine the obesity-related
metabolic pathways involved. The pathway analysis results indicated that these metabolites were
involved in the linoleic acid metabolism, alpha-linolenic acid metabolism, glycerophospholipid
metabolism, steroid biosynthesis, and arachidonic acid metabolism.

Figure 4. Score plots of the serum samples from the HED and RB-2.0X groups. (A) PCA score plot and
(B) OPLS-DA score plot. HED, SD rat fed HED; RB-2.0X, SD rat fed HED with RB-2.0X.
Nutrients 2019, 11, 2033 8 of 10

4. Discussion
The abundance of phytonutrients in the RB has made RB a potential functional food for disease
prevention [9]. Our results revealed that dietary RB supplementation provided a beneficial effect of
the ameliorating body weight, morphometry, and biochemical alternations related to diet-induced
obesity. The serum lipidomic changes revealed potential biomarkers, namely PC (18:2/22:6), DG
(18:2/16:0), DG (18:2/18:1), DG (18:1/16:0), CE (20:5), CE (28:2), TG (18:0/16:0/18:3), and glycerol-1-2-
hexadecanoate 3-octadecanoate for understanding HED-induced obesity. The RB treatment
presented an anti-obesity effect by partially reducing the HED-induced DG (18:2/18:1) and TG
(18:0/16:0/18:3) increases in obese rats.
Obesity is a major risk factor for the development of chronic diseases, such as type 2 diabetes
and cardiovascular diseases. Excess consumption of high-energy-density food, such as eating a high-
fat or high-sugar diet, leads to an increase in the white adipose tissue, which may cause metabolic,
hormonal, and inflammatory changes resulting in organ damage [17]. Our study revealed that the
HED intervention group had higher body weight as well as serum concentrations of uric acid, FFA,
TC, TG, and glucose. Moreover, the morphometric analysis revealed liver steatosis and enlargement
of adipocytes due to the HED.
RB is a rich source for dietary fiber, oligosaccharides, hemicelluloses, and nonstarchy
polysaccharides as well as some water-soluble phytochemicals that may be beneficial to health
[18,19]. A previous clinical study revealed that the RB supplementation could reduce blood glucose
and lipid concentrations [20]. We found that the RB-1.0X and RB-2.0X treatment groups had lower
HED-induced elevation of body weight and blood glucose and less hepatic accumulation of TC and
TG, indicating that the RB supplementation had a protective effect against the HED-induced obesity.
However, the increase in the rice bran dose (from RB-1.0X to RB-2.0X) has little effect and that the
efficacy of rice bran is therefore not dose dependent. The main bioactive compound in RB is γ-
oryzanol, which has demonstrated antioxidative, anti-inflammatory, antidiabetic, and anticancer
effects [21,22]. Moreover, γ-oryzanol tends to reduce high-fat-diet-induced cholesterol accumulation
in the liver, and our findings supported this [21]. Thus, we propose that RB tends to reduce the
accumulation of lipids in the liver because of its major bioactive compound, γ-oryzanol.
We investigated the serum lipidomic profiles of SD rats with the HED-induced obesity using
HPLC-QTOF-MS. To avoid bias, an untargeted analysis was carried out to obtain novel biological
insight regarding the lipid species affected by HEDs [23]. High blood lipids were found to be
associated with obesity [24]. We found that concentrations of CE, PC, DG, and TG were increased in
the HED group compared with the vehicle group. The PC acyl chain length was found to be
associated with the TC and hepatic cholesterol concentrations [23], whereas the long chain of the PC
was found to be positively associated with the glucose, insulin, and leptin concentrations [25]. CE
was associated with liver cholesterol homeostasis [26]. Our data revealed that SD rats with HED-
induced obesity exhibited significant increases in the lipid accumulation and glucose levels in the
liver. Thus, we propose that PC and CE are both involved in the glucose and cholesterol regulation.
Obesity has been associated with glucose intolerance and insulin resistance in animal [27,28] and
human studies [29–32], suggesting that lipids play an important role in modulating energy
metabolism in the body. An increase in the concentration of DG may decrease liver insulin sensitivity
and therefore lead to fat accumulation in hepatocytes [33]. Similarly, in our study, the obese rats of
the HED group had a higher concentration of DG than their lean littermates. Lipid droplet infiltration
in hepatocytes detected through the liver resection and the elevation in hepatic TG and TG revealed
consistent results. However, the RB intervention groups exhibited lower concentrations of blood
glucose and accumulation of hepatic lipids than the HED group, and a significantly lower serum DG
in the RB-2.0X group was observed. Thus, these results reveal that the RB supplementation can
ameliorate adiposity by partially reducing HED-induced DG and TG increases in obese rats.

5. Conclusions
Nutrients 2019, 11, 2033 9 of 10

In conclusion, rice bran could act as an anti-obesity supplement in rats, as demonstrated by

partially reducing the HED-induced DG and TG increases in obese rats, and thus limit the metabolic
diseases associated with obesity and accumulation of body fat and hepatic lipids in rats.
Author Contributions: S.C.Y., W.C.H., H.S., S.B., and C.C.H. designed the experiments. X.E.N., W.C. H., M.C.L.,
Y.J.H., H.H.W., and C.C.H. performed the laboratory experiments. Y.T.T., X.E.N., W.C.H., and T.H.T. analyzed
the data, and interpreted the results. Y.T.T., X.E.N., and W.C.H. prepared the figures and wrote the manuscript.
S.C.Y., C.L.Y., and W.C. H. contributed reagents, materials, and analysis platforms.

Funding: Grants MOST-106-2313-B-038-003-MY2, and MOST-107-2628-H-038-001-MY3 from the Ministry of

Science, and Technology, and TMU-106-AE1-B15, TMU-106-3805-012-110 (Office of Global Engagement), and
TMU-106-6820-001-112 from Taipei Medical University as well as The New Southbound Talent Development
Program of the Ministry of Education.

Acknowledgments: The authors are grateful to the graduate students at the Sport Nutrition Laboratory of the
National Taiwan Sport University for their technical assistance in conducting the animal experiments. We thank
the technical support provided by the TMU Core Facility, and Ms. Y.C.H. for her excellent technical support at
the TMU Core Facility.

Conflicts of Interest: The authors declare no conflict of interest.

References
1. Kelly, T.; Yang, W.; Chen, C.S.; Reynolds, K.; He, J. Global burden of obesity in 2005 and projections to
2030. Int. J. Obes. (Lond.) 2008, 32, 1431–1437.
2. National Task Force on the Prevention and Treatment of Obesity. Overweight, Obesity, And Health Risk.
Arch. Intern. Med. 2000, 160, 898–904.
3. Park, H.S.; Park, C.Y.; Oh, S.W.; Yoo, H.J. Prevalence of obesity and metabolic syndrome in Korean adults.
Obes. Rev. 2008, 9, 104–107.
4. Kang, J.G.; Park, C.Y. Anti-Obesity Drugs: A Review about Their Effects and Safety. Diabetes Metab. J. 2012,
36, 13–25.
5. Cheung, B.C.; Cheung, T.T.; Samaranayake, N.R. Safety of antiobesity drugs. Eur. PMC. 2013, 4, 171–181.
6. Onakpoya, I.J.; Heneghan, C.J.; Aronson, J.K. Post-marketing withdrawal of anti-obesity medicinal
products because of adverse drug reactions: A systematic review. BMC Med. 2016, 14, 191.
7. Sharif, M.K.; Butt, M.S.; Anjum, F.M.; Khan, S.H. Rice bran: A novel functional ingredient. Crit. Rev. Food.
Sci. Nutr. 2014, 54, 807–816.
8. Fabian, C.; Ju, Y.H. A review on rice bran protein: Its properties and extraction methods. Crit. Rev. Food.
Sci. Nutr. 2011, 51, 816–827.
9. Ha, T.Y.; Han, S.Y.; Kim, S.R.; Kim, I.H.; Lee, H.Y.; Kim, H.K. Bioactive components in rice bran oil improve
lipid profiles in rats fed a high-cholesterol diet. Nutr. Res. 2005, 25, 597–606.
10. Islam, M.S.; Nagasaka, R.; Ohara, K.; Hosoya, T.; Ozaki, H.; Ushio, H.; Hori, M. Biological abilities of rice
bran-derived antioxidant phytochemicals for medical therapy. Curr. Top. Med. Chem. 2011, 11, 1847–1853.
11. Jariwalla, R.J. Rice-bran products: Phytonutrients with potential applications in preventive and clinical
medicine. Drugs Exp. Clin. Res. 2001, 27, 17–26.
12. Hagl, S.; Berressem, D.; Grewal, R.; Sus, N.; Frank, J.; Eckert, G.P. Rice bran extract improves mitochondrial
dysfunction in brains of aged NMRI mice. Nutr. Neurosci. 2016, 19, 1–10.
13. McPeak, P.; Cheruvanky, R.; Cherukuri, R.S.V. Supportive therapy for diabetes, hyperglycemia and
hypoglycemia. 2001, Google Patents.
14. Lin, S.; Yang, Z.; Liu, H.; Tang, L.; Cai, Z. Beyond glucose: Metabolic shifts in responses to the effects of the
oral glucose tolerance test and the high-fructose diet in rats. Mol. Biosyst. 2011, 7, 1537–1548.
15. Folch, J.; Lees, M.; Sloane Stanley, G.H. A simple method for the isolation and purification of total lipides
from animal tissues. J. Biol. Chem. 1957, 226, 497–509.
16. Huang, C.C.; Tung, Y.T.; Huang, W.C.; Chen, Y.M.; Hsu, Y.J.; Hsu, M.C. Beneficial effects of cocoa, coffee,
green tea, and garcinia complex supplement on diet induced obesity in rats. BMC Complement. Altern. Med.
2016, 16, 100.
17. Bray, G.A.; Kim, K.K.; Wilding, J.P.H. Obesity: A chronic relapsing progressive disease process. A position
statement of the World Obesity Federation. Obes. Rev. 2017, 18, 715–723.
Nutrients 2019, 11, 2033 10 of 10

18. Faria, S.A.D.S.C.; Bassinello, P.Z.; Penteado, M.d.V.C.J. Nutritional composition of rice bran submitted to
different stabilization procedures. Braz. J. Pharm. Sci. 2012, 48, 651–657.
19. Yamagishi, T.; Tsuboi, T.; Kikuchi, K. Potent natural immunomodulator, rice water-soluble polysaccharide
fractions with anticomplementary activity. Cereal Chem. 2003, 80, 5–8.
20. Qureshi, A.A.; Sami, S.A.; Khan, F.A. Effects of stabilized rice bran, its soluble and fiber fractions on blood
glucose levels and serum lipid parameters in humans with diabetes mellitus Types I and II. J. Nutr. Biochem.
2002, 13, 175–187.
21. Sugano, M.; Tsuji, E. Rice bran oil and cholesterol metabolism. J. Nutr. 1997, 127, 521s–524s.
22. Xu, Z.; Hua, N.; Godber, J.S. Antioxidant activity of tocopherols, tocotrienols, and gamma-oryzanol
components from rice bran against cholesterol oxidation accelerated by 2, 2’-azobis(2-
methylpropionamidine) dihydrochloride. J. Agric. Food Chem. 2001, 49, 2077–2081.
23. Alan Saghatelian; Sunia, A.; Trauger Elizabeth; J. Want; Edward, G.; Hawkins Gary; Siuzdak Benjamin; F.
Cravatt. Assignment of endogenous substrates to enzymes by global metabolite profiling. Biochemistry
2004, 43, 14332–14339.
24. Szczygielska, A.; Widomska, S.; Jaraszkiewicz, M.; Knera, P.; Muc, K. Blood lipids profile in obese or
overweight patients. Ann. Univ. Mariae. Curie. Sklodowska. Med. 2003, 58, 343–349.
25. Nam, M.; Choi, M.S.; Jung, S.H.; Jung, Y.G.; Choi, J.Y.; Ryu, D.H.; Hwang, G.S. Lipidomic profiling of liver
tissue from obesity-prone and obesity-resistant mice fed a high fat diet. Sci. Rep. 2015, 5, 16984.
26. Eisinger, K.; Krautbauer, S.; Hebel, T.; Schmitz, G.; Aslanidis, C.; Liebisch, G.; Buechler, C. Lipidomic
analysis of the liver from high-fat diet induced obese mice identifies changes in multiple lipid classes. Exp.
Mol. Pathol. 2014, 97, 37–43.
27. Holland, W.L.; Brozinick, J.T.; Wang, L.P.; Hawkins, E.D.; Sargent, K.M.; Liu, Y.; Narra, K.; Hoehn, K.L.;
Knotts, T.A.; Siesky, A.; et al. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-,
and obesity-induced insulin resistance. Cell Metab. 2007, 5, 167–179.
28. Khamzina, L.; Veilleux, A.; Bergeron, S.; Marette, A. Increased activation of the mammalian target of
rapamycin pathway in liver and skeletal muscle of obese rats: Possible involvement in obesity-linked
insulin resistance. J. Endocrinol. 2005, 146, 1473–1481.
29. Murphy, N.J.; Schraer, C.D.; Thiele, M.C.; Boyko, E.J.; Bulkow, L.R.; Doty, B.J.; Lanier, A.P. Dietary change
and obesity associated with glucose intolerance in Alaska Natives. J. Am. Diet. Assoc. 1995, 95, 676–682.
30. Qatanani, M.; Lazar, M.A. Mechanisms of obesity-associated insulin resistance: Many choices on the menu.
Genes. Dev. 2007, 21, 1443–1455.
31. Steppan, C.M.; Lazar, M.A. Resistin and obesity-associated insulin resistance. Trends. Endocrinol. Metab.
2002, 13, 18–23.
32. Winer, S.; Chan, Y.; Paltser, G.; Truong, D.; Tsui, H.; Bahrami, J.; Dorfman, R.; Wang, Y.; Zielenski, J.;
Mastronardi, F.; et al. Normalization of obesity-associated insulin resistance through immunotherapy. Nat.
Med. 2009, 15, 921–929.
33. Magkos, F.; Su, X.; Bradley, D.; Fabbrini, E.; Conte, C.; Eagon, J.C.; Varela, J.E.; Brunt, E.M.; Patterson, B.W.;
Klein, S. Intrahepatic diacylglycerol content is associated with hepatic insulin resistance in obese subjects.
Gastroenterology 2012, 142, 1444–1446.e2.

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