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Journal of the American College of Nutrition

ISSN: 0731-5724 (Print) 1541-1087 (Online) Journal homepage: https://www.tandfonline.com/loi/uacn20

Antiobesity and Uric Acid-Lowering Effect of


Lactobacillus plantarum GKM3 in High-Fat-Diet-
Induced Obese Rats

Chin-Lin Hsu, Yu-Hsin Hou, Ci-Sian Wang, Shih-Wei Lin, Bo-Yi Jhou, Chin-Chu
Chen & Yen-Lien Chen

To cite this article: Chin-Lin Hsu, Yu-Hsin Hou, Ci-Sian Wang, Shih-Wei Lin, Bo-Yi Jhou, Chin-
Chu Chen & Yen-Lien Chen (2019): Antiobesity and Uric Acid-Lowering Effect of Lactobacillus
plantarum GKM3 in High-Fat-Diet-Induced Obese Rats, Journal of the American College of
Nutrition, DOI: 10.1080/07315724.2019.1571454

To link to this article: https://doi.org/10.1080/07315724.2019.1571454

Published online: 22 Feb 2019.

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JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
https://doi.org/10.1080/07315724.2019.1571454

Antiobesity and Uric Acid-Lowering Effect of Lactobacillus plantarum GKM3 in


High-Fat-Diet-Induced Obese Rats
Chin-Lin Hsua,b, Yu-Hsin Houc, Ci-Sian Wangc, Shih-Wei Linc, Bo-Yi Jhouc, Chin-Chu Chenc,d,e,f, and
Yen-Lien Chenc
a
School of Nutrition, Chung Shan Medical University, Taichung City, Taiwan; bDepartment of Nutrition, Chung Shan Medical University
Hospital, Taichung City, Taiwan; cGrape King Bio Ltd, Taoyuan City, Taiwan; dDepartment of Food Science, Nutrition, and Nutraceutical
Biotechnology, Shih Chien University, Taipei City, Taiwan; eInstitute of Food Science and Technology, National Taiwan University, Taipei
City, Taiwan; fDepartment of Bioscience Technology, Chung Yuan Christian University, Taoyuan City, Taiwan

ABSTRACT ARTICLE HISTORY


Objective: Obesity has become one of the world’s biggest issues. This condition has a great Received 16 October 2018
impact on several metabolic and chronic diseases. For example, obesity is often accompanied by Accepted 15 January 2019
hyperuricemia or gout. However, few drugs are available for the treatment of obesity. The present
KEYWORDS
study is to evaluate the antiobesity effect of Lactobacillus plantarum GKM3 in high-fat-diet-induced
Lactobacillus plantarum;
obese rats and whether taking L plantarum GKM3 can effectively reduce uric acid accumulation obesity; high-fat diet; uric
caused by obesity and ameliorate other harmful factors. acid; lactic acid bacteria
Method: Sixty male Wistar rats were divided into five groups as follows: (1) ND group, fed normal
diet; (2) HFC group, fed AIN93G-based high-fat diet containing 65% solids, 7% soybean oil, and
25% lard; (3) HFL group, fed AIN93G-based high-fat diet supplemented with 102.7 mg/kg/d L plan-
tarum GKM3; (4) HFM group, fed AIN93G-based high-fat diet supplemented with 205.4 mg/kg/d L
plantarum GKM3; and (5) HFH group, fed AIN93G-based high-fat diet supplemented with
513.5 mg/kg/d L plantarum GKM3. After 6 weeks, the body, organ, and fat weights; food intake;
blood serum levels; and adipocyte size were measured.
Results: Results showed that rats fed on the high-fat diet showed more body weight, increased
feed efficiency, higher fat deposition, higher total liver weight, elevated serum lipid levels, and
increased adipocyte size compared with those on the normal diet. All these effects were reversed
by supplementation of L plantarum GKM3.
Conclusions: In conclusion, we suggest that the L plantarum GKM3 supplement may have benefi-
cial antiobesity and uric acid–lowering effects.

Abbreviations: ALT: alanine aminotransferase; AST: aspartate aminotransferase; BCRC: Bioresource


Collection and Research Center; CGMCC: China General Microbiological Culture Collection Center;
Cl-: chlorine; HDL-C: high-density lipoprotein cholesterol; HFC: high-fat-diet group; HFD: high-fat
diet; HFH: high-fat-diet group supplemented with Lactobacillus plantarum GKM3-high dose; HFL:
high-fat-diet group supplemented with L plantarum GKM3-low dose; HFM: high-fat-diet group sup-
plemented with L plantarum GKM3-medium dose; Kþ: potassium; LDL-C: low-density lipoprotein
cholesterol; ND: normal diet; Naþ: sodium

Introduction their effectiveness is limited to production and maintenance


of weight loss (6). Therefore, further research is needed to
Obesity is one of the biggest problems in modern society.
discover new therapies to reduce the prevalence of obesity.
The rapid increase in world obesity prevalence points to life-
Hyperuricemia occurs when there is too much uric acid in
style changes as the main cause, especially in eating habits.
the blood. The main causes for higher plasma uric acid are
The World Health Organization recognizes obesity as the
either lower excretion, higher synthesis, or both. Some studies
greatest public health challenges of the 21st century. Obesity
showed that hyperuricemia is closely related to body fat accu-
is the result of sustained disequilibrium between energy mulation (7, 8). Obesity is associated with higher insulin
intake and energy expenditure (1), and this condition has a resistance and leptin production, and both reduce uric acid
great impact on several metabolic and chronic diseases excretion (9). Previous studies have also shown that hyperuri-
including type 2 diabetes associated with insulin resistance, cemia increases the risk for gout, cardiovascular diseases,
atherosclerosis, cardiovascular disease, nonalcoholic fatty liver hypertension, hepatic disease, diabetes, obesity, renal failure,
disease, hypertension, and hyperlipidemia (2–4). In addition, and stroke (10–15). As a result, it is necessary to prevent
obesity is associated with hyperuricemia and gout (5). To hyperuricemia-related diseases by reducing uric acid levels.
date, few drugs are available for the treatment of obesity, and

CONTACT Yen-Lien Chen, PhD lan.chen@grapeking.com.tw No.60, Sec. 3, Longgang Road, Zhongli District, Taoyuan City 320, Taiwan.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/icnv.
ß 2019 American College of Nutrition
2 C.-L. HSU ET AL.

Probiotics are live microbial dietary supplements that controlled temperature (22 ± 2  C), relative humidity
exert beneficial effects on host health (16). Probiotics attract (60%–80%), and light cycle (12 hours light/12 hours dark).
public attention because of their potential effectiveness for
both prevention and treatment of immune diseases (17).
Study design
Recent studies have also demonstrated the preventive effects
of some bacterial strains on obesity (4, 18). Lactobacillus are The protocol was approved by Chung Shan Medical
the most common probiotics in the stomach, duodenum, University Animal Care Committee (IACUC AHPP-Groval
and jejunum of humans and are a type of lactic acid bacteria NO: 1199) before the beginning of the study. The rats were
(19). Lactobacillus plantarum exhibits a variety of potential randomly divided into five groups, each comprising 12 rats.
beneficial effects on human health, such as improvement of The experiment was conducted for 6 weeks. One group (the
microbiota, immunomodulation, suppression of fat accumula- control group) continuously received the normal diet (ND),
tion, and reduction of serum triglyceride and serum choles- and four groups received an HFD supplemented with different
terol (20–25). In one study, L plantarum administration doses of L plantarum GKM3—group 1 ND: rats were fed a
significantly reduced the mean adipocyte size in female normal diet; group 2 HFC: rats were fed AIN93G-based HFD
C57BL/6 mice fed a high-fat diet (HFD) (22). Another study containing 65% solids, 7% soybean oil, and 25% lard; group 3
HFL: rats were fed AIN93G-based HFD supplemented with
found that a strain of L plantarum exhibited a significant
gavaged 102.7 mg/kg/d L plantarum GKM3; group 4 HFM:
effect in lowering serum cholesterol and triglycerides in
rats were fed AIN93G-based HFD supplemented with gavaged
hypercholesterolemic mice (24). These data suggest that some
205.4 mg/kg/d L plantarum GKM3; and group 5 HFH: rats
specific strains of L plantarum related to lipid metabolism
were fed AIN93G-based HFD supplemented with gavaged
may be a potential new candidate in the treatment of obesity. 513.5 mg/kg/d L plantarum GKM3. The body weights of rats
The aim of this study was to evaluate the antiobesity were measured prior to the study and every two days during
effect of L plantarum GKM3 in HFD-induced obese rats the study. Measurement of feed and water intake for all rats
and whether taking L plantarum GKM3 can effectively was conducted daily during the study.
reduce uric acid accumulation caused by obesity and ameli-
orate other harmful factors.
Serum biochemistry

Materials and methods After 12 hours of overnight fasting, all rats were euthanized with
CO2 asphyxiation and blood samples were collected. The follow-
Preparation of L plantarum GKM3 ing serum biochemistry parameters were analyzed by using com-
L plantarum GKM3 was isolated by Grape King Bio Ltd, mercial kits (Diasys Co Ltd.): triglycerides, high-density
Taoyuan, Taiwan. L plantarum GKM3 is preserved at the lipoprotein cholesterol (HDL-C), low-density lipoprotein choles-
Bioresource Collection and Research Center (BCRC) of the terol (LDL-C), aspartate aminotransferase (AST), alanine amino-
transferase (ALT), cholesterol, creatinine, glucose, uric acid,
Food Industry Research and Development Institute
sodium (Naþ), potassium (Kþ), and chlorine (Cl-). We used
(Hsinchu, Taiwan), with the preservation numbers of BCRC
commercial kits (Denka Seiken Co Ltd. to analyze ketone bodies.
910787, and preserved at the China General Microbiological
Culture Collection Center (CGMCC, Beijing, China) with
the preservation numbers of CGMCC 14565. Total body fat and body fat percentage
For functional assessment, L plantarum GKM3 grown in MRS
Visceral adipose tissue (epididymal, perirenal, and mesenteric
broth (DIFCO) under anaerobic conditions was transferred to a
fats) and subcutaneous tissue (retroperitoneal and inguinal
2.0-L flask containing 1.2 L of MRS medium. The whole medium
fats) were collected, washed, and weighed. Total body fat (mg/
was cultivated at 37  C for 1 day on a rotary shaker (10–20 rpm)
g rat) = [visceral adipose tissue (mg) þ subcutaneous adipose
for seed culturing prior to its scale-up production step. The fer-
tissue (mg)]  final body weight (g). Body fat percentage (%)
mented broth (1.2 L) was inoculated into a 5-ton fermentor with = Total body fat (g)  final body weight (g)  100%. Visceral
80% working volume (MRS medium; pH 6.0), and agitated at 10 adipose tissue (mg/g rat) = [perirenal adipose tissue (mg) þ
to 20 rpm at 37  C for 1 day. The obtained L plantarum GKM3 epididymal adipose tissue (mg) þ mesenteric adipose tissue
powder was ground after a freeze-drying process, with counts of (mg)]  final body weight (g). Subcutaneous adipose tissue
greater than or equal to 2  1010 CFU/g. (mg/g rat) = [retroperitoneal adipose tissue (mg) þ inguinal
adipose tissue (mg)]  final body weight (g).
Animals
Sixty 6-week-old male Wistar rats, weighing 201 to 225 g, were Determination of liver and fecal lipids
obtained from BioLASCO Taiwan Co., Ltd. After quarantine The total lipids from liver homogenates and fecal matters
and accommodation for one week, the rats were randomized were extracted according to Tzang et al. method (26). Each
into experimental and control groups. The rats had free access 0.3 g sample of liver tissue and oven-dried fecal matters was
to commercial rodent diet and sterile reverse osmosis water. homogenized with chloroform/methanol (2/1) to a final vol-
They were housed in stainless steel cages and maintained at ume 23 times the volume of the tissue sample (0.3 g in 6.9 mL
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 3

of solvent mixture). The homogenate was evaporated to con- by Duncan’s multiple range test for comparisons of group
stant weight by a nitrogen-blowing concentrator. The total means. A p value < 0.05 is considered statistically significant.
lipid from liver or fecal matters was calculated according to
the formula: The total lipid from liver or fecal matters (mg/g) Results
= constant dry weight of homogenate  0.3 g sample.
The dried extract was then dissolved in isopropanol for Effect of L plantarum GKM3 on body weights, weight
following determination. We used commercial kits (TG change, food intake, feed efficiency, energy intake, and
assay kit, Teco Diagnosis) to detect hepatic triglycerides and water intake
fecal triglycerides. Liver total cholesterol and fecal total chol- The body weight of all groups was increased every week,
esterol were analyzed by commercial kits (Cholesterol assay especially that of the HFD-fed groups compared to that of
kit, Teco Diagnostics, Rendox Laboratories Co Ltd.).
the ND group (Table 1). The HFD promoted a significant
increase in body weight over the 6-week period. At the end
Pathology of the experiment, the gain in body weight was higher in
the HFC group than in the HFD supplemented with L plan-
A histopathological test was performed to examine liver and tarum GKM3 groups. The mean body weight increased by
perirenal adipose tissues. The collected organ and tissues were 266.62 ± 8.31 g in the ND group, 318.54 ± 11.49 g in the HFC
dehydrated, clarified, infiltrated with paraffin, and embedded group, 266.89 ± 10.13 g in the HFL group, 276.41 ± 8.95 g in
after trimming, forming paraffin tissue blocks, and cut into tis- the HFM group, and 261.20 ± 12.48 g in the HFH group.
sue slices. Serial sections were stained with hematoxylin and The daily food intake of groups fed an HFD was lower
eosin. The histopathological changes were evaluated using
(p < 0.05) than that of the control group but did not differ
optical microscope and recorded by Charge Coupled Device
among the HFD-fed groups (Table 2). Feed efficiency was
and Carl Zeiss Axio Vision microscopy software 4.9.
significantly higher in all HFD groups than in the ND group,
but feed efficiency in HFD supplemented with L plantarum
In vitro experiment method on the uric acid–reducing GKM3 groups was significantly lower than in the HFC group.
effect of lactic acid bacteria There was no significant difference in energy intake and
water intake among the obese and the non-obese groups.
The following method was used to examine the purine-
decomposing ability in each type of lactic acid bacteria (27).
Using a BD Difco Lactobacilli MRS Broth medium, L planta- Effect of L plantarum GKM3 on the weights of organs
rum GKM3 was anaerobically cultured overnight at 37  C. Except for the liver of the HFC group, no significant differ-
Bacterial cells were harvested by centrifugation at 3000 rpm ences in organ weights was observed of all rats among
for 10 minutes (4  C) followed by resuspension in phosphate- HFD-fed groups and the ND group (Table 3).
buffered saline containing 1.25 mM inosine and 1.25 mM
guanosine. These 1  109 CFU/mL bacterial cell suspension
Effect of L plantarum GKM3 on the weights of total
solutions were shake-cultured for 2 hours at 120 rpm, 37  C.
body fat, body fat percentage, visceral adipose tissue,
The consumption level of nucleosides were measured by high
performance liquid chromatography (HPLC). and subcutaneous adipose tissue
The specific operational conditions of the HPLC are as fol- Throughout the 6-week experimental period, total body fat,
lows. Guanosine and inosine were determined by HPLC body fat percentage, and visceral adipose tissue were signifi-
equipped with a reverse-phase column (CAPCELL PAK C18 cantly lower (p < 0.05) in the L plantarum GKM3 low-,
SG120, 2504.6 mm) and Waters 996 photodiode array medium-, and high-dose groups compared with those in the
detector. The mobile phase used included 25 mM KH2PO4 HFC group (Table 4). There were no significant differences
(0.1% methanol) (A) and 25 mM KH2PO4 (0.1% methanol)/ in analysis of subcutaneous adipose tissue for all rats
methanol (75:25) (B) as the gradient. The gradient began at between the obese and the non-obese groups. Visceral
100% A. Composition was remained constant for 20 minutes,
then reversed to 20% A over 5 minutes, and finally 100% A Table 1. Effect of Lactobacillus plantarum GKM3 on body weights and weight
over 15 minutes. The flow rate was 1.0 mL/min, and the col- change in high-fat-diet-induced obese rats.
umn was kept at room temperature. Absorption spectra of Groups Initial body weight (g) Final body weight (g) Weight change (g)
eluted compounds were recorded at 254 nm. The degradation ND 231.55 ± 3.01a 498.17 ± 7.96b 266.62 ± 8.31b
HFC 234.24 ± 3.47a 552.78 ± 10.21a 318.54 ± 11.49a
rates were calculated according to the following equation: HFL 230.98 ± 3.52a 497.88 ± 10.68b 266.89 ± 10.13b
Degradation rate = 100 – (amount of inosine or guanosine/ HFM 231.80 ± 3.36a 508.21 ± 10.71b 276.41 ± 8.95b
amount of inosine or guanosine in the blank)  100. HFH 231.38 ± 3.43a 492.58 ± 13.98b 261.20 ± 12.48b
The reported values are the mean ± standard error of the mean (n ¼ 12).
Mean values with different letters were significantly different (p < 0.05).
Statistical analysis ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented
with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supple-
mented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supple-
Statistical analysis was performed with SPSS statistic software mented with L plantarum GKM3-high dose. Weight change (g) ¼ final body
package. Values are expressed as mean ± standard error of the weight (g)  initial body weight (g). Results of ND, HFL, HFM, and HFH
mean and analyzed using PROC analysis of variance followed were compared with HFC.
4 C.-L. HSU ET AL.

Table 2. Effect of Lactobacillus plantarum GKM3 on food intake, energy intake, water intake, and feed efficiency in high-fat-diet-induced obese rats.
Groups Food intake (g/rat/d) Feed efficiency (%) Energy intake (kcal/rat/d) Water intake (mL/rat/d)
ND 25.75 ± 0.26a 19.89 ± 0.51c 101.95 ± 1.04a 41.78 ± 1.47a
HFC 20.51 ± 0.28b 38.76 ± 1.10a 106.88 ± 1.44a 46.12 ± 1.87a
HFL 19.45 ± 0.36b 34.29 ± 1.14b 101.35 ± 1.89a 42.35 ± 1.23a
HFM 19.76 ± 0.28b 34.94 ± 0.93b 102.95 ± 1.47a 43.08 ± 2.20a
HFH 19.54 ± 0.49b 33.22 ± 0.91b 101.80 ± 2.56a 43.15 ± 1.44a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 3. Effect of Lactobacillus plantarum GKM3 on the weights of organs in high-fat-diet-induced obese rats.
Groups Heart (g/rat) Liver (g/rat) Spleen (g/rat) Lung (g/rat) Kidney (g/rat)
ND 1.49 ± 0.03a 15.92 ± 0.31b 0.92 ± 0.04a 2.33 ± 0.10a 3.51 ± 0.05a
HFC 1.51 ± 0.03a 17.65 ± 0.47a 0.91 ± 0.03a 2.31 ± 0.12a 3.47 ± 0.08a
HFL 1.49 ± 0.03a 15.13 ± 0.44b 0.87 ± 0.03a 3.57 ± 1.32a 3.30 ± 0.06a
HFM 1.48 ± 0.03a 15.98 ± 0.50b 0.86 ± 0.03a 2.07 ± 0.09a 3.33 ± 0.07a
HFH 1.47 ± 0.03a 15.05 ± 0.44b 0.87 ± 0.04a 2.15 ± 0.07a 3.33 ± 0.09a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 4. Effect of Lactobacillus plantarum GKM3 on the weights of total body fat, body fat percentage, visceral adipose tissue, and subcutaneous adipose tissue
in high-fat-diet-induced obese rats.
Groups Total body fat (mg/g rat) Body fat percentage (%) Visceral adipose tissue (mg/g rat) Subcutaneous adipose tissue (mg/g rat)
ND 107.94 ± 8.45b 10.79 ± 0.84b 73.49 ± 5.20b 34.45 ± 3.43a
HFC 136.66 ± 3.83a 13.67 ± 0.38a 94.30 ± 2.28a 42.35 ± 2.21a
HFL 117.69 ± 5.88b 11.77 ± 0.59b 80.41 ± 3.77b 37.28 ± 2.70a
HFM 113.72 ± 7.12b 11.37 ± 0.71b 79.73 ± 4.14b 33.98 ± 3.30a
HFH 113.64 ± 6.96b 11.36 ± 0.70b 80.20 ± 4.50b 33.44 ± 2.92a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Total body fat (mg/g rat) ¼ [visceral adipose tissue (mg) þ sub-
cutaneous adipose tissue (mg)]  final body weight (g). Body fat percentage (%) ¼ Total body fat (g)  final body weight (g)  100%. Visceral adipose tissue
(mg/g rat) ¼ [perirenal adipose tissue (mg) þ epididymal adipose tissue (mg) þ mesenteric adipose tissue (mg)]  final body weight (g). Subcutaneous adi-
pose tissue (mg/g rat) ¼ [retroperitoneal adipose tissue (mg) þ inguinal adipose tissue (mg)]  final body weight (g). Results of ND, HFL, HFM, and HFH were
compared with HFC.

fat mass was assessed by weighing the total perirenal, epi- HFC group. Mean uric acid levels were 5.73 ± 0.05 mg/dL
didymal, and mesenteric adipose tissues after dissection in the ND group, 7.53 ± 0.10 mg/dL in the HFC group,
(Table 5). Supplementation of L plantarum GKM3 to the 4.53 ± 0.03 mg/dL in the HFL group, 6.48 ± 0.05 mg/dL in
HFD led to a significant reduction (p < 0.05) in the perirenal the HFM group, and 5.58 ± 0.09 mg/dL in the HFH group.
and epididymal adipose tissue weights. The low-, medium-, and high-dose L plantarum GKM3
groups significantly and dose-dependently reversed the
Effect of L plantarum GKM3 on serum HFD-induced elevation of ketone body level. No significant
biochemical parameters differences in the serum Kþ levels among the ND, HFL,
HFM, and HFH groups were observed in this study.
It was observed that rats supplemented with L plantarum
GKM3 (the HFL, HFM, and HFH groups) for 6 consecutive Effect of L plantarum GKM3 on the hepatic total lipid,
weeks showed a significant decrease in the levels of serum tri- triglyceride, and cholesterol levels
glycerides and LDL-C/HDL-C and a significant increase in
the levels of serum HDL-C compared to the HFC group Hepatic lipid contents of the animals as shown in Table 8
(p < 0.05; Table 6). Triglyceride, total cholesterol, and LDL-C indicate that consumption of the HFD resulted in a significant
levels and LDL-C/HDL-C in these L plantarum GKM3 increase in hepatic total lipids, triglycerides, and cholesterol.
groups were even significantly lower than those of the ND Consumption of L plantarum GKM3 significantly and dose-
group (p < 0.05). No significant differences in the glucose lev- dependently reversed all these lipid perturbations.
els among the five groups were observed in this study.
Effect of L plantarum GKM3 on the fecal total lipid,
The AST, ALT, uric acid, creatinine, ketone body, Naþ,
þ triglyceride, and cholesterol
K , and Cl- levels in serum in each group are summarized
in Table 7. Compared with the ND group, the HFC group The effect of L plantarum GKM3 on the fecal total lipids,
had significantly increased uric acid and ketone body levels triglycerides, and total cholesterol excretions are shown in
and decreased Kþ levels. AST, ALT, creatinine, Naþ, and Table 9. Fecal total lipids, triglycerides, and cholesterol were
Cl- levels were not different. HFL, HFM, and HFH groups significantly higher in the HFD-fed groups compared with
had significantly reduced uric acid levels compared to the the ND group, and the HFD-fed groups supplemented with
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 5

Table 5. Effect of Lactobacillus plantarum GKM3 on the weights of adipose tissue in high-fat-diet-induced obese rats.
Retroperitoneal adipose
Perirenal adipose Epididymal adipose Mesenteric adipose tissue (mg/g Inguinal adipose
Groups tissue (mg/g rat) tissue (mg/g rat) tissue (mg/g rat) rat) tissue (mg/g rat)
ND 30.95 ± 2.56b 22.59 ± 1.41b 19.94 ± 1.51a 20.92 ± 2.02a 13.53 ± 1.72a
HFC 39.82 ± 1.31a 30.99 ± 0.67a 23.49 ± 1.06a 26.53 ± 1.76a 15.82 ± 1.41a
HFL 32.72 ± 1.80b 25.96 ± 1.46b 21.73 ± 1.16a 22.88 ± 1.54a 14.41 ± 1.56a
HFM 32.54 ± 2.23b 26.42 ± 1.28b 20.78 ± 1.12a 21.23 ± 1.83a 12.75 ± 2.02a
HFH 33.32 ± 1.71b 26.24 ± 2.13b 20.64 ± 1.48a 21.32 ± 2.32a 12.12 ± 1.74a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 6. Effect of Lactobacillus plantarum GKM3 on serum biochemical parameters (serum lipid profile and glucose) in high-fat-diet-induced obese rats.
Groups Triglyceride (mg/dL) Total cholesterol (mg/dL) HDL-C (mg/dL) LDL-C (mg/dL) LDL-C/HDL-C (ratio) Glucose (mg/dL)
ND 118.58 ± 12.44b 107.86 ± 4.69a 69.00 ± 2.00a 36.67 ± 1.81a 0.53 ± 0.02a 249.20 ± 4.95a
HFC 236.92 ± 7.89a 104.55 ± 2.26a,b 57.92 ± 3.65b 28.33 ± 1.63a 0.50 ± 0.03a 251.15 ± 5.49a
HFL 85.17 ± 8.43c 98.19 ± 2.55b 72.58 ± 2.71a 27.17 ± 1.78b 0.37 ± 0.02b 235.15 ± 9.47a
HFM 88.83 ± 4.61c 97.73 ± 1.98b 66.33 ± 3.14a 25.25 ± 0.92b 0.39 ± 0.02b 242.96 ± 4.24a
HFH 82.83 ± 8.72c 97.87 ± 2.47b 67.42 ± 2.73a 26.42 ± 0.78b 0.40 ± 0.01b 239.98 ± 11.41a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). HDL-C ¼ high-
density lipoprotein cholesterol; LDL-C ¼ low-density lipoprotein cholesterol; ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with
Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supplemented with L
plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 7. Effect of Lactobacillus plantarum GKM3 on serum biochemical parameters (safety) in high-fat-diet-induced obese rats.
Groups AST (U/L) ALT (U/L) Uric acid (mg/dL) Creatinine (mg/dL) Ketone body (mmol/L) Naþ (mmol/L) Kþ (mmol/L) Cl- (mmol/L)
a a c a b a a
ND 77.75 ± 6.32 32.42 ± 2.65 5.73 ± 0.05 0.58 ± 0.01 6.01 ± 0.16 144.75 ± 0.51 4.57 ± 0.04 105.67 ± 0.19a
HFC 72.75 ± 8.02a 38.00 ± 2.66a 7.53 ± 0.10a 0.57 ± 0.02a 7.32 ± 0.27a 145.25 ± 0.35a 4.43 ± 0.04b 105.50 ± 0.23a
HFL 76.25 ± 6.98a 40.58 ± 3.69a 4.53 ± 0.03d 0.58 ± 0.01a 6.13 ± 0.21b 145.25 ± 0.25a 4.53 ± 0.03a 105.25 ± 0.28a
HFM 73.50 ± 7.87a 35.75 ± 4.45a 6.48 ± 0.05b 0.54 ± 0.01a 5.81 ± 0.14b 144.25 ± 0.45a 4.50 ± 0.03a,b 105.25 ± 0.25a
HFH 70.58 ± 4.53a 33.92 ± 2.06a 5.58 ± 0.09c 0.56 ± 0.01a 5.07 ± 0.09c 145.08 ± 0.26a 4.52 ± 0.03a,b 105.25 ± 0.28a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). AST ¼ aspartate
aminotransferase; ALT ¼ alanine aminotransferase; ND ¼ normal diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-
low dose; HFM ¼ high-fat diet supplemented with L plantarum GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose.
Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 8. Effect of Lactobacillus plantarum GKM3 on the hepatic total lipid, triglyceride, and cholesterol in high-fat-diet-induced obese rats.
Groups Hepatic total lipid (mg/g tissue) Hepatic triglyceride (mg/g tissue) Hepatic cholesterol (mg/g tissue)
ND 66.13 ± 2.91c 22.79 ± 0.61c 10.56 ± 0.35b
HFC 91.19 ± 5.70a 30.04 ± 1.21a 12.36 ± 0.33a
HFL 82.05 ± 2.13a,b 25.89 ± 1.22b 10.41 ± 0.35b
HFM 80.76 ± 2.99b 21.29 ± 1.36c 9.86 ± 0.18b
HFH 77.32 ± 2.11b 19.73 ± 0.68c 9.64 ± 0.25b
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

Table 9. Effect of Lactobacillus plantarum GKM3 on the fecal total lipid, triglyceride, and cholesterol in high-fat-diet-induced obese rats.
Groups Fecal total lipid (mg/g dried feces) Fecal triglyceride (mg/g dried feces) Fecal cholesterol (mg/g dried feces)
ND 22.67 ± 1.88c 3.96 ± 0.04c 5.25 ± 0.24c
HFC 35.08 ± 1.53b 6.51 ± 0.33b 7.57 ± 0.12b
HFL 46.96 ± 4.19a 7.07 ± 0.11a 8.21 ± 0.23a
HFM 49.19 ± 5.91a 7.13 ± 0.12a 8.24 ± 0.04a
HFH 52.18 ± 2.40a 7.23 ± 0.14a 8.43 ± 0.13a
The reported values are the mean ± standard error of the mean (n ¼ 12). Mean values with different letters were significantly different (p < 0.05). ND ¼ normal
diet; HFC ¼ high-fat diet; HFL ¼ high-fat diet supplemented with Lactobacillus plantarum GKM3-low dose; HFM ¼ high-fat diet supplemented with L plantarum
GKM3-medium dose; HFH ¼ high-fat diet supplemented with L plantarum GKM3-high dose. Results of ND, HFL, HFM, and HFH were compared with HFC.

L plantarum GKM3 were significantly and dose-dependently cytoplasm, and ballooning injury was observed around the
higher than the HFC group. central vein (Figure 1). In contrast, the hepatocytes of L
plantarum GKM3-fed rats showed only mild accumulation
Effect of L plantarum GKM3 on hepatosteatosis and the of lipid droplets and ballooning injury.
size of perirenal adipocyte Histologic examination of perirenal adipose tissue
demonstrates that HFD progressively increased the size of
The hepatocytes of the rats in the HFC group showed severe
adipocytes (Figures 2 and 3). On the other hand,
macrovascular accumulation of lipid droplets within the
6 C.-L. HSU ET AL.

hematoxylin and eosin staining of perirenal adipose tissue L plantarum GKM3 has a stronger uric acid–lowering
revealed that supplementation with L plantarum GKM3 was effect than other lactic acid bacterias
associated with a significant reduction in average adipocyte
Figures 4 and 5 shows the purine-decomposing ability of each
size in perirenal adipose tissue, as compared with the
of the lactic acid bacteria when they were cultured in the
HFC group.

Figure 1. Effect of Lactobacillus plantarum GKM3 on hepatosteatosis in high-fat-diet-induced obese rats. Liver was stained with hematoxylin and eosin. Original
magnification: 200. The oil drop marked by an arrow.
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 7

presence of inosine or guanosine. Among the lactic acid bac- acid bacteria possess this activity via inhibiting the activity
teria, L plantarum GKM3 had the highest purine-degrad- of intestinal pathogens (28), suppressing the absorption of
ation rate. excess lipids (29), decreasing the level of LDL-C (30), and
inhibiting the differentiation of 3T3-L1 adipocytes (29, 30).
Discussion After 6 weeks of feeding, supplementation with L planta-
Various materials have been purported to have antiobesity rum GKM3 reversed HFD-induced body weight gain and
activity, and there is sufficient scientific evidence that lactic the increase in total body fat and body fat percentage.

Figure 2. Effect of Lactobacillus plantarum GKM3 on the size of perirenal adipocyte in high-fat-diet-induced obese rats. Perirenal adipose tissues were stained with
hematoxylin and eosin. Original magnification: 200.
8 C.-L. HSU ET AL.

However, there was no significant differences in these concentration of L plantarum GKM3 was sufficient for col-
parameters in rats among the HFL, HFM, and HFH groups. onization in the intestines. The food intake of the HFD
This can be explained on the basis that the low group was lower than that of the control group. The explan-
ation for this may be the higher calorie intake of the HFD
group, which in turn reduced food intake. In addition, the
feeding efficiency of the HFD group was significantly
improved compared with that of the ND group, suggesting
that the intake of an HFD resulted in greater body weight
gain. Treatment with L plantarum GKM3 significantly
decreased the feeding efficiency compared with the HFC
group, suggesting that L plantarum GKM3 may affect hosts’
ability to harvest energy from the diet and produce a differ-
ence in energy utilization and storage.
An HFD increased liver weight and serum lipid levels.
The reduction of triglycerides, total cholesterol, or LDL-C in
serum is reported to lower the risk of coronary heart disease
(31, 32). Recent studies showed that probiotics, including
Bifidobacterium longum (33) and Lactobacillus acidophilus
(34), possess significant hypolipidemic activity in both rats
Figure 3. Effect of Lactobacillus plantarum GKM3 on the size of perirenal adipo- and humans. Notably, it was observed that probiotic supple-
cyte in high-fat-diet-induced obese rats. The reported values are the mean-
± standard error of the mean (n ¼ 3). Mean values with different letters were mentation may promote perirenal and epididymal fat loss
significantly different (p < 0.05). and decrease serum lipid levels in rats with HFD-induced

Figure 4. Rate of guanosine decomposition (%).

Figure 5. Rate of inosine decomposition (%).


JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION 9

obesity. This result demonstrates that L plantarum GKM3 may therefore protect obese individuals against elevated
supplementation has a protective effect against obesity serum uric acid levels and resulting hyperuricemia and gout.
induced by an HFD in rats. Further clinical trials should therefore be conducted to con-
Interactions between the intestinal microbiota and miner- firm these effects.
als and the effect of this interaction on the health of the
host were shown (35). Elevation of blood ketone bodies by
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