nutrients
ArticleEffects of Paramylon Extracted from Euglena gracilis
EOD-1 on Parameters Related to Metabolic Syndrome
in Diet-Induced Obese Mice
Seiichiro Aoe 1,2, *, Chiemi Yamanaka 2 , Kotone Koketsu 1 , Machiko Nishioka 3 ,Nobuteru Onaka 3 , Norihisa Nishida 3 and Madoka Takahashi 3
1
2
3

*

Studies in Human Life Sciences, Graduate School of Studies in Human Culture,
Otsuma Women’s University, Chiyoda-ku, Tokyo 102-8357, JapanThe Institute of Human Culture Studies, Otsuma Women’s University Chiyoda-ku, Tokyo 102-8357, Japan
Kobelco Eco-Solutions Co., Ltd., Kobe, Hyogo 651-2241, Japan
Correspondence: s-aoe@otsuma.ac.jp; Tel.: +81-3-5275-6048

Received: 23 June 2019; Accepted: 18 July 2019; Published: 21 July 2019


Abstract: Paramylon (PM), a type of β-glucan, functions like dietary fiber, which has been suggested
to exert a protective effect against obesity. We evaluated the potential beneficial effects of PM powder
on obesity in mice. Male C57BL/6J mice were fed a high-fat diet supplemented with either 2.5 or
5% PM powder, extracted from Euglena gracilis, for 74 days. Growth parameters, abdominal fat
content, serum biochemical markers, hepatic lipid accumulation and hepatic mRNA expression
were measured. Dietary supplementation with PM resulted in decreased food efficiency ratios andabdominal fat accumulation. Dose-dependent decreases were observed in postprandial glucose
levels, serum low-density lipoprotein (LDL)-cholesterol, and serum secretary immunoglobulin A(sIgA) concentrations. PM supplementation increased peroxisome proliferator-activated receptor α(PPARα) mRNA expression in the liver which is suggested to induce β-oxidation through activationof acyl-coenzyme A oxidase (ACOX), carnitine palmitoyltransferase (CPT) and fatty acid transport
protein 2 (FATP2) mRNA expression. Changes in fatty acid metabolism may improve lipid andglucose metabolism. In conclusion, a preventive effect against obesity was observed in mice given a
PM-enriched diet. The mechanism is suggested to involve a reduction in both serum LDL-cholesterollevels and the accumulation of abdominal fat, in addition to an improvement in postprandial
glucose concentration.
Keywords: Euglena gracilis; paramylon; obesity; abdominal fat; PPARα

1. Introduction
Metabolic syndrome is a world wide problem; it is a medical term referring to lifestyle related risk
factors for arteriosclerosis-related diseases [1]. Obesity and insulin resistance are at the core of mostcases of metabolic syndrome; therefore, lifestyle changes, including diet, are strongly recommended.
Dietary fiber is one of the best candidates for improving the components of metabolic syndrome [2].
Some dietary fibers reduce the postprandial rise in plasma glucose [3], reduce body weight [4], andimprove lipid metabolism [5,6].
Dietary fiber is characterized by water solubility and has different functions. Insoluble dietaryfiber has shown favorable results in the treatment of irritable bowel disease [7] and induced a significant
increase in fecal bulk, a reduction in intestinal transit time, and a significant increase in the frequency ofbowel movements in healthy people [8]. In another study, soluble dietary fiber inhibited carbohydrate
and lipid digestion, lipid absorption, reduced the postprandial glucose response, and improved serum
lipid levels [9].
Nutrients 2019, 11, 1674; doi:10.3390/nu11071674

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Paramylon (PM) is a new source of dietary fiber generated as a characteristic cellular reserve
material in Euglena gracilis; it is a β-1, 3-glucan arranged in a triple helix and is considered to be an
insoluble dietary fiber [10]. Beta-glucan is commonly found in nature, in cereals, fungi and algae, but
the structural and physical properties of β-glucans are quite different among the sources. Evidence ofany beneficial effects of PM supplementation has not yet been reported. Anti-diabetic effects were notobserved in genetically diabetic rats fed a 2% PM diet [11] and recent experiments have shown that
PM did not improve obesity or pro-inflammatory status in mice fed a high fat diet [12,13]. The lack ofevidence for the beneficial effects of PM is suggested to be caused by insufficient dosage; a recent reportsuggested that the amount of PM used in the diets of previous studies were insufficient to detect any
effectiveness [14]. The dosage of dietary fiber administered via experimental diets is usually 5%, andsignificant effects have been observed on glucose and lipid metabolism in animal experiments at thisdosage [15–17]. The cellulose content in the AIN-93 diet is also 5% [18]. Therefore, experiments using
adequate amounts of PM in the diet are needed to elucidate the functions of PM as a new dietary fiber.
The purpose of this study was to investigate the effects of 2.5% and 5% PM, extracted from Euglena
gracilis, in diet-induced obese mice fed a high-fat diet; we measured visceral fat accumulation, cholesteroland glucose metabolism and serum serum secretary immunoglobulin A (sIgA) concentrations. This is
the first study to report on the preventive effects of dietary PM against obesity.
2. Materials and Methods
2.1. Sample Preparation and Chemical Analysis
PM extracted from Euglena gracilis EOD-1 was obtained from Kobelco Eco-Solutions Co., Ltd.
(Kobe, Japan). The Euglena gracilis EOD-1 strain produces high PM yields (70–80%) depending on
culture conditions [19]. PM was obtained from cultured Euglena gracilis EOD-1 with glucose as the
carbon source, under heterotrophic conditions in the dark. Euglena gracilis EOD-1 was collected bycentrifugation (500× g, 4 min) at room temperature and treated with 5 g/L protease (YP-SS; Yakult
Pharmaceutical Industry Co., Ltd, Tokyo, Japan) at 50 ◦ C for 2 h before being washed with 1% sodium
dodecyl sulfate and distilled water, and then dried. The total dietary fiber (TDF) content of PM wasdetermined to be 97.8% using the method of Prosky et al. [20].
2.2. Animals and Study Design
Thirty 4-week-old male C57BL/6J mice were purchased from Charles River Laboratories Japan(Yokohama, Japan) and were maintained on a 12-h light/dark cycle (lights on at 08:00 h). Mice werehoused individually in plastic carbonate cages and given free access to water. The animal protocols
used were reviewed and approved by the Animal Research Committee of Otsuma Women’s University(Tokyo, Japan) and are in accordance with their animal experimentation regulations. Mice wereacclimatized for 7 days before allocation into three experimental dietary groups (n = 10): mice were
fed either a high-fat diet supplemented with 0% (control), 2.5%, or 5% PM. The control and 2.5%
PM diets were supplemented with cellulose to give a total dietary fibre content of 5%. Test diets
were prepared according to Table 1. Mice received the experimental diets ad libitum for 74 days.
Food intake and body weights were monitored three times a week during the study. Feces werecollected over the final 3 days of the study period. The feces were freeze dried after washing thesurface with distilled water, milled, and kept at −20 ◦ C until measurement. After fasting for 8 h, mice
were sacrificed by isoflurane/CO2 anesthesia, then the cecum with digesta, adipose tissue depots:
retroperitoneal (including prerenal), mesenteric, epididymal, and liver were dissected and weighed.
Small samples of liver tissue (150 mg) were suspended in RNA Stabilization Reagent (RNAlater, Qiagen,Hilden, Germany), and the remainder was freeze-dried, milled, and stored at −20◦ C until requiredfor cholesterol and triglyceride analysis. Cecum with digesta were stored at −20◦ C until required forshort chain fatty acid analysis. Blood samples, taken from the postcaval vein under isoflurane/CO2
anesthesia at sacrifice, were centrifuged, and serum was collected for biochemical analysis.

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Table 1. Composition of the experimental diets (g/kg diet).

Caseinl-cystine
Corn starch
Dextrinized corn starch
SucroseSoybean oil
Lard
CellulosePM
AIN-93G mineral
mixtureAIN-93 vitamin mixture
Choline bitartrate
t-Butylhydroquinone

Control

2.5% PM

5% PM

200
3
197.486
132
100
70
200
50
-

200
3
196.886
132
100
70
200
25
25.6

200
3
196.286
132
100
70
200
51.2

35

35

35

10
2.5
0.014

10
2.5
0.014

10
2.5
0.014

PM: Paramylon, TDF 97.7%.

2.3. Biochemical Analysis of the Serum and Concentration of Liver Lipids
Total, low-density lipoprotein (LDL), and high-density lipoprotein (HDL)-cholesterol, triglycerides
and free fatty acids were measured in mouse serum using Hitachi 7180 auto-analyzers at the Nagahama
Research Institute (Oriental Yeast Co., Ltd., Shiga, Japan). Enzyme-linked immunosorbent assay(ELISA) was used to measure serum insulin (mouse insulin ELISA kit, Shibayagi Co., Ltd., Gunma,
Japan) and sIgA concentrations (ELISA Kit for Secretory Immunoglobulin A, Cloud-Clone Corp., Texas,
USA). A 2:1 (v/v) chloroform-methanol solution was used to extract lipids from the liver [21], whichwere then dissolved in isopropanol containing 10% polyoxyethylene octylphenyl ether (Triton X-100,
FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan). Cholesterol and triglyceride concentrationin the lipid extracts were measured enzymatically using the Cholesterol E-test and Triglyceride E-test,
respectively (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan).
2.4. Total Fecal Lipid Analysis
Fecal lipids were extracted using a 2:1 (v/v) chloroform: methanol mixture under acidic conditions(acetic acid was added to a final concentration of 4%) [22] and determined gravimetrically.
2.5. Detection of PM Ultrastructure Recovered from Feces by Scanning Electron Microscopy (SEM)
Intact PM and PM recovered from feces were examined using SEM. Feces were collected and pooled
from ten C57BL/6J mice fed a 5% PM diet for 3 days under the same experimental conditions before
sacrifice. Lyophilized and powdered feces were treated according to the method of Prosky et al. [20] tocollect the dietary fiber. Extracts were washed with distilled water and ethanol, before being dried andcoated with osmium. Intact PM was also observed by SEM. Microscopy was performed using a fieldemission scanning electron microscope (FE-SEM) SU−70–scanning electron microscope (Hitachi HighTechnologie, Tokyo, Japan) at −1–2 kV accelerating voltage.
2.6. Oral Glucose Tolerance Test
Mice were fed the experimental diets for 71 days. After which, they were fasted for 8 h before anoral glucose tolerance test (OGTT) was performed. Blood was collected from the tail tip at 0, 15, 30, 60
and 120 min after oral glucose gavage (1.5 g/kg) and analyzed using a glucose meter (Glutest Ace R,
Sanwa Kagaku Kenkyusho Co., Ltd., Aichi, Japan).

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The concentration of cecal short chain fatty acids (SCFAs) was determined using a gas
chromatography-mass spectrometer system [23]. Ten milligrams of the cecal digesta were homogenizedin extraction solution (100 µL of internal standard (100 µM crotonic acid), 50 µL of HCl, and 20 µL
of ether) using 5 mm stainless beads (AS ONE Corp., Osaka, Japan). SCFAs in the cecal samples
were extracted using a TissueLyser II (Qiagen, Hilden, Germany) at 2000 rpm for 15 min and thencentrifuged at 1000× g at 25 ◦ C for 10 min. Aliquots (80 µL) of the upper phase (ether layer) weremixed with 16 µL of N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide and heated at 80 ◦ C for
20 min, and then derivatized for 48 h. An aliquot of sample was automatically injected into the inletof a 7890B GC system (Agilent, Tokyo, Japan) equipped with a 5977A mass selective detector (MSD;
Agilent). A DB-5MS capillary column (30 m × 0.53 mm) (Agilent) was used to separate the SCFAs. Theoven was programmed to 60 ◦ C for 3 min and increased to 120 ◦ C at the rate of 5 ◦ C/min and then to300 ◦ C at a rate of 20 ◦ C/min, and finally held at 300 ◦ C for 2 min. Helium was used as a carrier gas at
1.2 mL/min. The temperatures of the front inlet, transfer line, and electron impact ion source wereset at 250, 260, and 230 ◦ C, respectively. The ion mass of each SCFA was determined in the selected
ion mode. SCFA concentrations were determined by comparing their peak areas with the internal
standards, and were expressed as µmol/g of the cecal digesta.
2.8. Expression Analysis of mRNAs Related to Lipid and Glucose Metabolism in Liver
Primer sequences are presented in Table 2. Total RNA in the liver was prepared using RNeasy minikits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. mRNA expression wasmeasured with an Applied Biosystems Quant3 Real-Time polymerase chain reaction (PCR) System andSYBR® Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) using cDNA preparedby real-time (RT)-PCR. The 2−∆∆CT method was utilized for data analysis where the threshold cycle(CT) indicates the fractional cycle number at which the amount of amplified target reaches a fixed
threshold. The ∆CT is the difference in threshold cycles for target genes, using 36B4 as the reference
gene. The ∆∆CT is the difference between the ∆CT for treatment diets and the ∆CT for control diet.
Relative expression levels are presented as fold changes to the control group (arbitrary unit).
2.9. Statistical Analysis
Sample sizes were calculated from our background mice serum data which was based on
differences in serum LDL-cholesterol concentrations of 0.06 mmol/L with a standard deviation (SD) of
0.04 mmol/L. The sample size was then calculated with the result that 29 mice were required in total(type I error (α) = 0.05, 1 − β = 0.80): thirty mice (10 mice per group) were used. Data are presented asmean ± standard error of the mean. Dose-dependent effects of paramylon were investigated using alinear regression model. Significant difference (p < 0.05) between group means was determined byWilliams’s test. The relationships between several parameters related to obesity were evaluated using
Spearman’s rank correlation coefficient. JMP (Version 14.0, SAS Institute Inc., Cary, NC, USA) and R
software were used to perform the statistical analyses.

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Table 2. Primers used in the real-time reverse transcription polymerase chain reaction.
ForwardPPARαLXR
SREBP1c
ACOX
CPT1
CPT2
CD36 (FAT)
FATP2
LPL
FGF21

Reverse50 -AGGAAGCCGTTCTGTGACAT-30
50 -AATCCCCTCCTGCAACTTCT-30
50 -CCTTCCTCAAGGACTTCAGTTACAA
50 -CATGGCTCTGGAGAACTCAAAGAT-30
50 -GGCACTAAGTGCCCTCAACCT-30
50 -GCCACATAGATCTCTGCCAGTGT-30
50 -CAGCGTTACGAGGTGGCTGTTA-30
50 -TGCCCAAGTGAAGGTCCAAAG-30
50 -GATGGAGAGGATGTTCAACACTACAC-30 50 -AGCCCTCATAGAGCCAGACCTT-30
50 -ATCGTACCCACCATGCACTA-30
50 -TGGCTGTCATTCAAGAGAGG-30
50 -TCCCTCTCTGGAGTTCTTGG-30
50 -TTGCAGCTGAGCAGAAAGAG-30
50 -TTTCCGGTGGAAAGGAGA-30
50 -AGGTACTCCGCGATGTGTTG-30
50 -AGGGCTCTGCCTGAGTTGTA-30
50 -AGAAATCTCGAAGGCCTGGT-30
50 -CAAGACACTGAAGCCCACCT-30
50 -TGCCAGGACGCGCTTGT-30

Reference
36B4

50 -GGCCCTGCACTCTCGCTTTC-30

50 -TGCCAGGACGCGCTTGT-30

PPARα, peroxisome proliferator-activated receptor α; LXR, liver X receptor; SREBP1c, sterol regulatory
element-binding transcription factor 1c; ACOX, acyl-coenzyme A oxidase; CPT1, CPT2, carnitine palmitoyltransferase
1 and 2; CD36 (FAT), fatty acid translocase; FATP2, fatty acid transport protein 2; LPL, lipoprotein lipase; FGF21,
fibroblast growth factor 21; 36B4, acidic ribosomal protein.

3. Results
3.1. Food Intake, Body Weight and Organ Weight
Food intake, body weight, and food efficiency ratio in mice fed different concentrations of PM are
shown in Table 3. There were no significant differences in final weight and body weight gain in the mice
fed differing concentrations of PM; however, food intake in the 5% PM group was significantly different
when compared with the control group (p < 0.05). The food efficiency ratio was dose dependently
reduced (p < 0.05) in the paramylon groups and a significant difference between the control and the 5%
PM group was observed (p < 0.05). The organ weights in mice fed paramylon are shown in Table 4.
The weights of the cecum with digesta were dose-dependently increased in the PM groups (p < 0.01)
and the weight was significantly higher in the 5% PM group compared to the control group (p < 0.05).
Total abdominal, retroperitoneal and mesenteric fat weights were dose dependently reduced in the PMgroups (p < 0.05) and they were significantly lower in the 5% PM group compared to the control group(p < 0.05).
3.2. Fecal Total Fat Excretion and Apparent Absorption of Fat
Total fat excretion and apparent absorption of fat into the feces are shown in Table 5. No significant
differences were observed in total fat excretion among the groups. The apparent absorption of fat in
the 5% PM group was significantly different to the control group, and dose dependency was observed;
however, the difference in apparent absorption of fat between the control and the 5% PM group wasless than 1%.
3.3. Ultrastructure of PM Recovered from Feces by SEM
The ultrastructure of intact PM and PM recovered from feces by SEM are shown in supplementary
Figure S1. The recovery of intact PM particles from feces in mice fed the 5% PM diet suggests that PM
is resistant to the digestive process and bacterial degradation.
3.4. Oral Glucose Tolerance Test (OGTT)The change in blood glucose levels over time was measured with an OGTT (Figure 1). The increasein blood glucose levels after glucose administration was dose dependently reduced at 60 min in the
PM groups (p < 0.05) and it was significantly reduced in the 5% PM group compared with the control(p < 0.05). No significant differences were observed at the other time points or in the incremental area
under the curve (IAUC; data not shown) among the groups.

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6 of 14the control (p < 0.05). No significant differences were observed at the other time points or in the
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incremental area under the curve (IAUC; data not shown) among the groups.

Δglucose (mmol/l)

10
8
6
4
2
*

0
0

15

30

45

Control

60

75

90

2.5%PM

105 120

5%PM

Time

Figure 1. Increase in blood glucose levels in the oral glucose tolerance test (OGTT). Error bars represent
Figure 1. Increase in blood glucose levels in the oral glucose tolerance test (OGTT). Error bars
standard error (n = 10). Paramylon (PM) displayed significant dose-dependent effects in reducing
represent standard error (n = 10). Paramylon (PM) displayed significant dose-dependent effects inthe blood glucose increase level at 60 min (p < 0.05). * The increase in blood glucose at 60 min was
reducing the blood glucose increase level at 60 min (p < 0.05). * The increase in blood glucose at 60
significantly reduced in the 5% PM group compared with the control group (p < 0.05).
min was significantly reduced in the 5% PM group compared with the control group (p < 0.05).
Table 3. Body weight gain, food intake, and food efficiency ratio.

Table 3. Body weight gain, food intake, and food efficiency ratio.
Control

2.5% PM

5% PM

19.5 Control± 0.2
38.319.5± 1.1± 0.2
0.25 38.3± 0.01± 1.1
2.80.25± 0.1± 0.01
9.03 ± 0.38

2.5%
PM
19.5
± 0.2
19.5±±1.0
0.2
39.4
0.27
± 0.01
39.4
± 1.0
3.1
0.27±±0.2
0.018.83 ± 0.56

5%±PM
19.5
0.2
19.5±±0.9
0.2
38.2
0.25
38.2±±0.01
0.9
3.1
*
0.25± ±0.1
0.01
8.14 ± 0.24 *p for Trend

p forn.s.Trend
n.s.
n.s.
n.s.
n.s.
n.s.
n.s.
p < 0.05
Food intake (g/day)
2.8 ± 0.1
3.1 ± 0.2
3.1 ± 0.1 *n.s.
Valuesareefficiency
means ± standard
mean(SE), n =8.83
10. ±* 0.56
Significantly
different
the control
group
Food
ratio (%)error of the
9.03± 0.388.14± 0.24 from*p < 0.05
Initial weight (g)InitialweightFinalweight(g)(g)
Body weightgain (g/day)Final weight(g)
Foodintakegain(g/day)Body
weight(g/day)Food efficiency ratio (%)(Williams’s method; p < 0.05), n.s.: not significant.

Values are means ±standard error of the mean (SE), n = 10. * Significantly different from the control
group (Williams’s method; p < 0.05), n.s.:
not4.significant.
Table
Weight of organs.
Control
2.5%
Table 4. Weightof PM
organs.
Liver (g)Cecum with digesta (g)Liver (g)Total abdominal fat
Cecum
with digesta
Retroperitonealfat(g) (g)Total abdominal
Epididymal
fat(g) fat
Mesentericfat(g) fat(g)Retroperitoneal

1.21 ± 0.04
Control
0.24 ± 0.02
1.21
± 0.044.13 ± 0.08
0.24
0.87 ± 0.02± 0.02
2.38 ±4.13
0.05± 0.08
0.88 ±0.87
0.05± 0.02

1.232.5%± 0.05PM
0.28 ± 0.01
1.23
±
0.05
3.92 ± 0.15
0.28
±
0.01
0.81 ± 0.05
3.92
± 0.152.29± 0.080.83± 0.070.81± 0.055% PM

p for Trend

1.19
± PM
0.04
5%
0.33 ± 0.02 *1.19 ± 0.04
3.57 ± 0.21 *0.33
0.02* *
0.73 ±±0.04
3.57±± 0.12
0.21 *
2.21
0.63
0.73±±0.07
0.04* *

n.s.
p for
Trend
p < 0.01
n.s.
p < 0.05
0.01
pp<<0.05
p n.s.
< 0.05pp<<0.05
0.05
Epididymal
fat(g)
2.38
±
0.05
2.29
±
0.08
2.21
±
0.12
n.s.
Values are means ± SE, n = 10. * Significantly different from the control group (Williams’s method; p < 0.05),
n.s.: not significant.
Mesenteric fat(g)0.88 ± 0.05
0.83 ± 0.07
0.63 ± 0.07 *p < 0.05

Values are means ± SE, n = 10. * Significantly different from the control group (Williams's method; p
Table 5. Fecal fat excretion and apparent digestibility of fat.
< 0.05), n.s.: not significant.
Control

2.5% PM

5% PM

Table 5. Fecal fat excretion and apparent digestibility of fat.

Fat intake (mg/day)Fecal fat excretion (mg/day)Apparent digestibility of fat (%)

711.0 ± 32.4
18.6 ±
2.4
Control
97.4 ± 0.3

775.8 ± 19.9
18.92.5%± 2.1PM
97.6 ± 0.3

747.9 ± 32.1
25.35%± 2.1
PM
96.6 ± 0.2 *p for Trend
n.s.

n.s.Trend
p for
p < 0.05
Fat intake（mg/day）
711.0 ± 32.4
775.8 ± 19.9
747.9 ± 32.1
n.s.
Valuesarefatmeans± SE, n = 10. * Significantly
different< 0.05),Fecal
excretion（mg/day）
18.6± 2.4 from the18.9control± 2.1 group (Williams’s
25.3 ± 2.1 method; pn.s.
n.s.: not significant.
Apparent digestibility of fat（%）
97.4 ± 0.3
97.6 ± 0.3
96.6 ± 0.2 *p < 0.05

Values are means ± SE, n = 10. * Significantly different from the control group (Williams's method; p< 0.05), n.s.: not significant.

3.5. Short-Chain Fatty Acid (SCFA) Concentrations in Cecal Digesta

The concentrations of short-chain fatty acid in cecal digesta are shown in Table 6. Total SCFA
concentrations, as well as acetate and n-butyrate concentrations, did not differ among the groups.
Significant dose-dependent effects of paramylon were observed in increasing propionate concentrations(p < 0.05). Propionate concentrations were significantly different in the 2.5% and 5% PM groups
compared to the control group.

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Table 6. Concentration of short-chain fatty acids (SCFA) in the cecal digesta.
SCFA (µmol/g cecum)Control

2.5% PM

5% PM

p for Trend

AcetatePropionate
n-Butyrate
Other SCFAs *Total SCFAs

11.9 ± 0.5
4.0 ± 0.2
3.8 ± 0.4
1.9 ± 0.1
22.3 ± 1.0

12.2 ± 0.5
5.0 ± 0.2 *4.4 ± 0.4
2.3 ± 0.1
24.2 ± 0.9

11.9 ± 0.4
5.0 ± 0.2 *4.0 ± 0.3
2.5 ± 0.1
24.8 ± 0.9n.s.
p < 0.05
n.s.
n.s.
n.s.

Values are means ± SE, n = 10. * Other SCFAs, the sum of the concentrations of formate, iso-butyrate, iso-varerate, andvarerate is shown. * Significantly different from the control group (Williams’s method; p < 0.05), n.s.: not significant.

3.6. Biochemical Analysis of the Serum and Liver Lipids
Serum biochemical concentrations are shown in Table 7. PM dose dependently reduced serum
LDL-cholesterol levels (p < 0.05). In addition, levels were significantly reduced in the 5% PM groupcompared with the control (p < 0.05). There were no significant differences in serum total and
HDL-cholesterol, triglyceride, free fatty acid, and insulin concentrations among the experimentalgroups. Liver lipid levels are shown in Table 8; cholesterol and triglyceride accumulation (mg/liver)and triglyceride concentration (mg/g liver) were not statistically different among the groups.
Table 7. Biochemical analysis of the serum.

Total cholesterol (mmol/L)
LDL-cholesterol (mmol/L)
HDL-cholesterol (mmol/L)
Triglyceride (mmol/L)
NEFA (µmol/L)
Insulin (ng/mL)

Control

2.5% PM

5% PM

p for Trend

4.36 ± 0.12
0.21 ± 0.01
2.21 ± 0.04
0.56 ± 0.02
521.6 ± 28.2
1.96 ± 0.32

4.56 ± 0.12
0.19 ± 0.01
2.30 ± 0.03
0.52 ± 0.04
477.0 ± 15.8
1.80 ± 0.34

4.24 ± 0.13
0.17 ± 0.01 *2.19 ± 0.04
0.58 ± 0.07
500.1 ± 11.7
1.34 ± 0.18

n.s.
p < 0.05
n.s.
n.s.
n.s.
n.s.

Values are means ± SE, n = 10. * Significantly different from the control group (Williams’s method; p < 0.05),
n.s.: not significant.

Table 8. Liver lipid levels.

Cholesterol (µmol/liver)(µmol/g liver)
Triglyceride (µmol/liver)(µmol/g liver)Control

2.5% PM

5% PM

p for Trend

2.12 ± 0.10
1.76 ± 0.07
8.56 ± 1.15
6.90 ± 0.78

2.07 ± 0.11
1.68 ± 0.03
8.72 ± 1.41
6.82 ± 0.81

2.03 ± 0.08
1.72 ± 0.03
7.29 ± 0.96
6.00 ± 0.59

n.s.
n.s.
n.s.
n.s.

Values are means ± SE, n = 10.

3.7. Expression of mRNAs Related to Liver Lipid Metabolism
mRNA expression levels are shown in Figure 2 and Table 9. Significant dose-dependent effectsof PM were observed in up-regulating the mRNA expression of peroxisome proliferator-activatedreceptor α (PPARα; p < 0.05): the mRNA expression levels of PPARα were significantly higher in the
2.5% and 5% PM groups compared to the control group (p < 0.05; Figure 2). No significant differences in
the mRNA expression levels of sterol regulatory element-binding transcription factor 1c (SREBP1c) andliver X receptor (LXR) were observed. PM dose dependently up-regulated the mRNA expression levelsof acyl-coenzyme A oxidase (ACOX), carnitine palmitoyltransferase 2 (CPT2) and fatty acid transport
protein 2 (FATP2) (p < 0.05; Table 9): the mRNA expression levels of ACOX, CPT2, and FATP2 weresignificantly higher in the 2.5% and 5% PM groups when compared with the control group (p < 0.05;
Table 9). No significant differences in the mRNA expression levels of carnitine palmitoyltransferase
1 (CPT1), fatty acid translocase (cluster of differentiation36; CD36), lipoprotein lipase (LPL), andfibroblast growth factor 21 (FGF21) were observed. Spearman’s rank correlation coefficients for theNutrients 2019, 11, 1674

8 of 13relationships between PPARα or serum LDL-cholesterol levels and parameters related to obesity
are shown in Table 10. Significant correlations (Spearman’s rank correlation coefficient) between
PPARα and each parameter regulated by PPARα, except FGF21, were observed. Significant negative
correlations were observed for PPARα vs. LDL-cholesterol levels. Significant positive correlations
2019,for11, xabdominal
FOR PEER REVIEW
9 of 14
wereNutrientsobserved
fat weights and liver lipid levels vs. serum LDL-cholesterol levels.

Figure2. Box-whisker
plot
levelsofofmRNAmRNA
liver.
* Significant
dependent
Figure2. Box-whisker
plotofofthe
theexpression
expression levels
ininliver.
* Significant
dosedosedependent
up-regulationof PPARαmRNA
wasobserved
observedininthegroups< 0.05).
up-regulationof PPARαmRNAexpression
expression levels
levels wasthe
PMPM
groups(p <(p0.05).

Nutrients 2019, 11, 1674

9 of 13

Table 9. Expression of mRNAs under the control of PPARα in liver.

ACOX
CPT1
CPT2
CD36
FATP2
LPL
FGF21

Control

2.5% PM5% PM

1.0 ± 0.2
1.0 ± 0.2
1.0 ± 0.1
1.0 ± 0.2
1.0 ± 0.1
1.0 ± 0.2
1.0 ± 0.2

2.1 ± 0.1 *0.6 ± 0.1
1.3 ± 0.1 *1.3 ± 0.2
2.3 ± 0.2 *1.4 ± 0.2
1.5 ± 0.3

2.0 ± 0.1 *0.5 ± 0.1
1.3 ± 0.1 *1.4 ± 0.2
2.0 ± 0.4 *1.3 ± 0.1
1.5 ± 0.3

Arbitrary Unit (/36B4)
p for Trendp < 0.05
n.s.
p < 0.05
n.s.
p < 0.05
n.s.
n.s.

Values are means ± SE, n = 10. * Significantly different from the control group (Williams’s method; p < 0.05).
n.s.: not significant.
Nutrients 2019, 11, x FOR PEER REVIEW

10 of 14

Table 10. Spearman’s rank correlation coefficients for the relationship between PPARα or
LDL-cholesterollevels andrankparameters
relatedto obesity.
Table 10. Spearman’s
correlation
coefficientsfor the relationship between PPARα or LDLcholesterol levels and parameters related to obesity.
ACOX mRNA
ACOX
mRNA
CPT1
mRNA
mRNA
CPT2CPT1
mRNA
CPT2
mRNA
FATP2
mRNA
FATP2
mRNA
CD36
mRNA
mRNA
LPLCD36
mRNA
FGF21LPL
mRNA
mRNA
Serum LDL-cholesterollevels
FGF21 mRNA
Abdominal
fat weight levels
Serum LDL-cholesterol
BloodAbdominal
glucose at 60
fatminweightvs. PPARα mRNAvs. PPARα mRNA
0.68 *
0.68
0.55
* *
0.55
0.38
* *
0.38
0.64 * *
0.64
0.38
* *
0.38
0.39
* *
0.18
0.39 *
−0.43
*
0.18
−0.16
−0.43 *
−0.12
−0.16

Blood glucose at 60 min−0.12
vs. LDL-cholesterolvs.
LDL-cholesterol
Retroperitoneal fat weight
0.58 *
Retroperitoneal
fat weight
0.58
Mesentericfat weight
0.53* *weight0.53
LiverMesentericcholesterolfat
levels0.59* *cholesterollevels0.59
LiverLiver
triglyceride
levels
0.46
* *
Liver
triglyceride
levels0.46** p < 0.05 for Spearman’s rank correlation coefficient.
* p < 0.05 for Spearman’s rank correlation coefficient.

3.8. Serum sIgA Concentrations

3.7. Serum sIgA Concentrations

Serum
sIgAsIgA
concentrations
areareshown
inin
FigureincreasedsIgA
sIgA
levels (pSerumconcentrations
shown
Figure3.3.PM
PMdosedose dependently
dependently increased
levels< 0.05).
sIgA sIgA
concentrations
weresignificantly
inthe
the2.5%2.5%
andgroups(p <Serum0.05). Serum
concentrations
weresignificantlyhigher
higher in
and
5%5%
PMPM
groupswhenwhencompared
withwiththe control
group
(p (p
< <0.05).
comparedthe control
group0.05).

Figure3. Box-whisker
plot
ofofserumPM
increasedsIgA
levelsa doseFigure3. Box-whisker
plot
serumsIgA
sIgA concentrations.
concentrations. * *PM
increasedsIgA
levelsin a indose
dependent
manner
< 0.05).
dependent
manner(p <(p0.05).

4. Discussion
We investigated the effects of PM extracted from Euglena gracilis EOD-1 on both lipid andglucose metabolism and abdominal fat accumulation in diet-induced obese mice. This is the firststudy to report on the preventive effects of dietary PM against obesity. The mechanism involves aNutrients 2019, 11, 1674

10 of 13

4. Discussion
We investigated the effects of PM extracted from Euglena gracilis EOD-1 on both lipid and glucose
metabolism and abdominal fat accumulation in diet-induced obese mice. This is the first study toreport on the preventive effects of dietary PM against obesity. The mechanism involves a reduction inboth abdominal fat accumulation and serum LDL-cholesterol concentrations and an improvement in
postprandial glucose concentrations. We also determined the characteristics of PM as a dietary fiber
source; lipid absorption was hardly influenced by PM intake, and the intestinal fermentability of PM
was low. Examination using electron microscopy revealed that PM recovered from feces retained its
shape and was resistant to bacterial degradation.
Growth data showed that the reduction in the food efficiency ratio in mice fed PM wasdose-dependent; a significant difference was also observed between the control and 5% PM group. It
is suggested that reduced lipid accumulation in the abdominal fat organs leads to a reduction in thefood efficiency ratio. Significant dose dependent reductions in total, retroperitoneal and mesentericfat weight were observed in mice fed 5% PM. Dietary fiber has been reported to reduce abdominal
fat accumulation through the inhibition of dietary fat absorption [24]. A significant decrease inapparent digestibility of fat was observed with 5% PM supplementation; however, the difference infat digestibility between the control and 5% PM group was less than 1%. The contribution of PM
supplementation to the inhibition of fat absorption may be weak. The lack of reduction in hepatic lipid
accumulation is suggested to be partially due to a poor reduction in apparent lipid digestibility.
Sixty minutes after oral glucose administration in mice, the blood glucose level was significantly
lower in the 5% PM group compared with the control group. It is suggested that insulin resistanceoccurs in mice fed PM due to the retardation of gastric emptying [16]. It is also suggested that anincrease in colonic fermentation in PM-fed mice may lower the increase in blood glucose due tothe increase in SCFAs [12,13]. The production of incretins, such as glucagon-like peptide-1 (GLP-1),
promotes insulin secretion and may contribute to improved insulin sensitivity [25]. Significant increases
in the concentration of cecal propionate were observed in the 2.5% and 5% PM supplementationgroups; however, the contribution of propionate production to the total SCFA concentrations was
relatively small. Further studies are needed to clarify the mechanism of improved glucose tolerance by
PM intake.
It has been reported that curdlan, produced by Alcaligenes faecalis var. myxogenes, is also a β-1,3glucan, but it is composed of linear single chains [26]. Dietary curdlan is reported to be degraded andfermented by intestinal bacteria in the cecum, and may act to improve the intestinal environment via
modification in the lower intestine [27]. Therefore, the physiological characteristics of PM and curdlan
differ, even though they are both β-1,3 glucans.
Serum sIgA concentrations were dose dependently increased in the PM-supplemented mice,in addition, significant differences were observed between the control group and the 2.5% and 5%
PM supplementation groups. The insoluble PM particle itself may exert a direct stimulatory effecton intestinal epithelial cells, which may promote slgA content [28]. PM might promote intestinal
immunological activity: Euglena gracilis EOD-1 biomass ingestion led to the production of a PM-specific
IgA antibody and increased salivary IgA antibody titers in humans [29]. Our data supports theseprevious reports.
PPARα is involved in many aspects of lipid metabolism [30,31], including fatty acid β-oxidation,
synthesis, transport, storage, and lipoprotein metabolism during fasting [32,33]. Activation of PPARα by
endogenous ligands or synthetic agonists moderately lowers LDL levels [34]. Serum LDL-cholesterol
concentrations were dose dependently reduced in mice supplemented with PM, and significant
differences were observed between the 0% group and 5% PM supplementation group. The reduction inserum LDL-cholesterol might be caused by increasing hepatic PPARα expression; this is supported by
the significant negative correlation of PPARα mRNA expression vs. LDL-cholesterol levels. Significant
positive correlations between retroperitoneal and mesenteric fat weights vs. LDL-cholesterol levels
were observed, suggesting that dose dependent reductions in serum LDL-cholesterol levels might

Nutrients 2019, 11, 1674

11 of 13cause a reduction in abdominal fat weights. However, we measured only PPARα mRNA expression;qualitative analysis of protein levels will be needed to help elucidate the mechanism of PM’s activationof PPARα mRNA expression.
Liver triacylglycerol production and secretion into VLDL are mainly determined by the fatty acidsynthesis rate, which is controlled to a large extent at the level of transcription by both PPARα [35],which stimulates fatty acid β-oxidation, and SREBP-1c [36], which controls fatty acid synthesis. Our
results indicated that activation of PPARα might decrease triglyceride accumulation in the abdominal
fat tissues by reducing secretion into VLDL, whereas SREBP-1c may not be involved in fatty acidsynthesis activation after PM supplementation. Previous reports showed that PPARα regulates theexpression of fibroblast growth factor 21 (FGF21) during starvation [37,38]. FGF21 acts as an endocrine
hormone targeting various functions, including glucose and lipid metabolism [39]. Our results
indicated that mRNA expression of hepatic FGF21 in the 2.5 and 5% PM groups increased by 1.5 times
compared to the control group; however, the differences were not statistically significant. Further
analyses of serum FGF21 concentrations are needed to confirm the relationship between PPARα mRNA
expression and serum FGF21 secretion. It has been reported that PPAR activators inhibit the activationof inflammatory response genes by interfering with nuclear factor-κB (NF-κB) and transcription factor
activator protein 1 (AP1) signaling pathways, thereby promoting insulin sensitivity [40]. Improvements
in insulin sensitivity caused by PM supplementation might activate PPARα by the same mechanism.
Although PPARα is suggested to be activated by high levels of fatty acids present in the fasted liver [41],the exact nature of the endogenous activation signal remains unknown [42]. Further research is needed
to elucidate the endogenous ligands promoted by direct PM stimulation of the epithelial cell.
5. Conclusions
Dietary fiber has been suggested to exert a protective effect against obesity. PM, from Euglena
gracilis, is a dietary fibre; however, there has been very little research on its effects. We investigatedthe effects of 2.5 or 5% PM in diet-induced obese mice fed a high-fat diet; we observed a preventive
effect against obesity, which may involve a reduction in both abdominal fat accumulation and serum
LDL-cholesterol concentrations and an improvement in postprandial glucose concentrations. Hepaticinduction of PPARα mRNA expression is suggested to increase mRNA expression of ACOX, CPT2,
FATP2 and induce β-oxidation. Subsequent changes to fatty acid metabolism might cause a series ofimprovements in lipid and glucose metabolism.
Supplementary Materials: The following are available online at http://www.mdpi.com/2072-6643/11/7/1674/s1.
Figure S1: Intact PM and PM recovered from feces by scanning electron microscopy.
Author Contributions: Conceptualization, S.A., M.N., N.O., N.N. and M.T.; methodology, S.A.; formal analysis,
C.Y. and K.K.; resources, M.N., N.O., N.N. and M.T.; original draft preparation and writing, S.A.; review andediting, S.A., M.N., N.O., N.N. and M.T.; supervision, S.A. and M.T.
Funding: This study was financially supported by Kobelco Eco-Solutions Co.,Ltd. (Kobe, Japan).
Conflicts of Interest: Four of the authors (M.N., N.O., N.N. and M.T.) are salaried employees of the he Kobelco
Eco-Solutions Co., Ltd. The remaining authors (S.A., C.Y., and K.K.) have no conflict of interest to disclose. The
PM used in this study was provided by Kobelco Eco-Solutions Co., Ltd.

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