Alpha Linolenic Acid-enriched Diacylglycerol Consumption Enhances Dietary Fat Oxidation in Healthy Subjects: A Randomized Double-blind Controlled Trial

Abstract

Consumption of alpha linolenic acid-enriched diacylglycerol (ALA-DAG) reduces visceral fat area. In this study, we performed a randomized, placebo-controlled, double-blind, crossover intervention trial to investigate the effect of ALA-DAG on dietary fat oxidation in comparison with control triacylglycerol (TAG). Each subject (n=16) consumed either 2.5 g/d of ALA-DAG or TAG for 14-d, separated by a 21-d washout period. At the end of each consumption period, we assessed dietary fat oxidation. ALA-DAG consumption significantly enhanced dietary fat utilization as energy compared to TAG consumption.

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181
Journal of Oleo Science
Copyright ©2017 by Japan Oil Chemists’ Society
doi : 10.5650/jos.ess16183
J. Oleo Sci. 66, (2) 181-185 (2017)
Alpha Linolenic Acid-enriched Diacylglycerol
Consumption Enhances Dietary Fat Oxidation in
Healthy Subjects: A Randomized Double-blind
Controlled Trial
Yasutoshi Ando1, Shinichiro Saito1＊, Nami Yamanaka1, Chizuka Suzuki1, Takahiro Ono2,
Noriko Osaki1 and Yoshihisa Katsuragi1
1 Healthcare Food Research Laboratories, Kao Corporation 2-1-3 Bunka Sumida-ku Tokyo 131-8501, JAPAN
2 Medical Corporation Wakei-kai Medics Hongo Clinic, Bunkyo-ku, Tokyo 113-0023, JAPAN
1 INTRODUCTION
Visceral obesity is strongly associated with metabolic
risk factors, such as hyperglycemia, hypertension, and hy-
perlipidemia1−3）
. The development of obesity is related to
genetic background, physical activity, and diet composi-
tion. In particular, a chronic imbalance between fat intake
and fat expenditure is an important regulator of body fat4）
.
Moreover, dietary fat ingestion may be related to fat oxida-
tion5−7）
.
Alpha linolenic acid-enriched diacylglycerol（ALA-DAG）
is a minor natural component of many edible oils and has
long been consumed by humans. ALA-DAG mainly occurs
with the chemical structure 1,3-diacyl-sn-glycerol（1,3-
DAG）and alpha-linolenic acid as the fatty acid. Previous
human studies demonstrated that the long-term consump-
tion of ALA-DAG significantly decreases body weight and
visceral fat area compared to consuming the control triac-
ylglycerol（TAG）
8−10）
. ALA-DAG consumption enhances
both fat oxidation and energy expenditure in healthy
humans11）
. Thus, ALA-DAG consumption could be useful
for controlling body weight by maintaining or improving fat
＊Correspondence to: Shinichiro Saito, Healthcare Food Research Laboratories, Kao Corporation, 2-1-3 Bunka Sumida-ku Tokyo
131-8501, JAPAN
E-mail: saito.shinichiro@kao.co.jp
Accepted September 25, 2016 (received for review September 15, 2016)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs
and energy metabolism. This underlying mechanism is sup-
ported by reports showing enhanced expression of
β-oxidation related enzymes and genes in the small intes-
tine12）
and in the liver13）
in rodents. Based on these reports,
ALA-DAG is expected to enhance oxidation of diet-derived
fat as well as body fat. Indeed, in rodents, dietary fat oxida-
tion is enhanced after repeated ingestion of ALA-DAG
compared to TAG14）
. Its effect in humans, however, remains
unclear. Therefore, this study evaluated whether repeated
ALA-DAG consumption affects oxidation of diet-derived fat
in healthy human subjects.
2 EXPERIMENTAL
2.1 Ethics and registration
This study was performed in accordance with the princi-
ples of the Declaration of Helsinki and was approved by the
clinical ethics committee of Oriental Ueno Kenshin Center
（Tokyo, Japan）. The study protocol was registered in
advance with the University hospital Medical Information
Abstract: Consumption of alpha linolenic acid-enriched diacylglycerol (ALA-DAG) reduces visceral fat
area. In this study, we performed a randomized, placebo-controlled, double-blind, crossover intervention
trial to investigate the effect of ALA-DAG on dietary fat oxidation in comparison with control
triacylglycerol (TAG). Each subject (n=16) consumed either 2.5 g/d of ALA-DAG or TAG for 14-d,
separated by a 21-d washout period. At the end of each consumption period, we assessed dietary fat
oxidation. ALA-DAG consumption significantly enhanced dietary fat utilization as energy compared to
TAG consumption.
Key words: ALA-DAG, alpha-linolenic acid, diacylglycerol, dietary fat oxidation, human
NOTE

Y. Ando, S. Saito and N. Yamanaka et al.
J. Oleo Sci. 66, (2) 181-185 (2017)
182
Network Clinical Trials Registry（registration no.
UMIN000021181）, and the execution of this study was out-
sourced to TES Holdings Co., Ltd（Tokyo, Japan）. After re-
ceiving an explanation of the study, all subjects provided
written informed consent to participate in the study.
2.2 Subjects
Twenty subjects were enrolled in the study according to
the inclusion and exclusion criteria. Inclusion criteria were
1）age 35 to 64 years and 2）body mass index 23.0 to 29.9
kg/m2. Exclusion criteria included the presence of severe
disease, surgery within 2 months, taking medication, taking
supplements or foods with health claims, premenopausal
woman, and food allergy. The sample size was estimated
based on our previous study showing that ALA-DAG signifi-
cantly enhanced postprandial total fat oxidation in 19 sub-
jects11）
. Because the dose and the consumption duration of
ALA-DAG in this present study were the same as the previ-
ous study, we estimated that a similar sample size was ap-
propriate.
2.3 Design and protocol
The study had a randomized, double-blind, placebo-con-
trolled crossover design with two 14-d consumption
periods of ALA-DAG or control TAG, separated by a 21-d
washout period. The subjects were assigned to each order
of the consumption period by stratified block randomiza-
tion using computer-generated random numbers under
blinded condition. During each consumption period, the
subjects consumed shortbreads containing 2.5 g/d of
ALA-DAG or TAG each day. During the consumption
period, the subjects were instructed to maintain their ha-
bitual lifestyle, including their usual physical activity and
dietary intake. The subjects recorded their meals for 3-d
before the measurements, and nationally registered dieti-
tians analyzed the dietary records. Alcohol intake and
strenuous exercise were not allowed for the 3-d before
each measurement. One day before the measurements, the
subjects ingested specified meals for breakfast, lunch, and
dinner（total calories: 2173 kcal/day for men, 1818 kcal/day
for women）. At the end of the consumption period, we
measured the subjects’ body composition and dietary fat
oxidation, and collected serum samples after they had
fasted for at least 12 h.
2.4 Test diet
We used a previously reported method15）
to prepare the
ALA-DAG from flaxseed oil（Summit Oil Corporation,
Chiba, Japan）and rapeseed oil（The Nisshin OilliO Group,
Ltd, Tokyo, Japan）using equipment owned by Kao Corpo-
ration（Tokyo, Japan）. Each 2.5 g of ALA-DAG contained
0.9 g DAG-bound ALA. Rapeseed oil was used as the
source of the TAG（The Nisshin OilliO Group, Ltd, Tokyo,
Japan）. Table 1 shows the components of the TAG and
ALA-DAG. We produced a cooking oil by mixing the
ALA-DAG with rapeseed oil, anti-oxidants, and emulsifying
agents. We made a test shortbread（hard flour, soft flour,
superfine sugar, salt, egg, pullulan, water, and the prepared
cooking oil）to ensure accurate ingestion of the ALA-DAG
or TAG. Each shortbread（60 g）contained 291 kcal
（protein:fat:carbohydrate＝8:39:53）per serving（2.5 g
ALA-DAG or TAG）and were individually packaged. The
shortbreads containing TAG and ALA-DAG could not be
distinguished from each other by appearance, taste, or
odor.
2.5 Dietary fat oxidation assessment
We synthesized the 13C-labelled triolein probes from
［1-13C］oleic acid（purity＞99％, 13C＞99％; Isotec, Miamis-
burg, OH, US）and free glycerol using an enzymatic method
and purified the probes using silica gel liquid chromatogra-
phy. The assessment of dietary fat oxidation was performed
as reported previously16−18）
. Briefly, before and after inges-
tion of the test meal（555 kcal, protein: fat: carbohydrate＝
17:30:52 as energy value）containing 13C-labelled triolein
（400 mg）, we collected breath samples in aluminum bags
（GL Science Inc., Tokyo, Japan）every hour for 6 h. As the
primary outcome, oxidation of dietary fat was assessed by
measuring recovery rate of ingested 13C-labelled triolein to
13CO2 in the breath. Recovery rate of 13C was assessed by
the combined use of an indirect calorimeter（Arco Systems
Inc., Chiba, Japan）and an stable isotope ratio mass spec-
trometer （ANCA-GSL; Sercon, Crewe, UK）.
Tabl e 1
Glycerides and fatty acids composition
of TAG and ALA-DAG.
TAG ALA-DAG
Glyceride (g/100 g)
DAG 1.5 80.2
DAG-bound ALA 0.1 35.3
Monoacylglycerol 0.0 0.5
Free fatty acid 0.0 0.1
TAG and others 98.5 19.4
Fatty acid (wt, %)
C16:0 4.1 2.6
C18:0 1.9 1.5
C18:1 61.0 26.9
C18:2 20.4 16.9
C18:3 9.3 50.7
C20:0 0.6 0.1
C20:1 1.1 0.4
C22:0 0.4 0.3
Others 1.1 0.8

ALA-DAG enhances dietary fat oxidation
J. Oleo Sci. 66, (2) 181-185 (2017)
183
2.6 Statistical analysis
Data are expressed as mean±SD. Comparisons of the
difference in two periods between the ALA-DAG treatment
followed by the TAG treatment and the TAG treatment fol-
lowed by the ALA-DAG treatment were performed using a
two-sample t-test. A p-value of less than 0.05 was consid-
ered statistically significant.
3 RESULTS
3.1 Subjects and characteristics
Sixteen subjects（11 men and 5 women）completed the
measurements and were included in the analyses, and 4
subjects did not complete the measurements. The baseline
physical characteristics, including body composition,
serum triglyceride, glucose, insulin, non-esterified fatty
acid, total-, low-density-, and high-density-cholesterol did
not significantly differ between the treatment orders. Table
2 shows the physical characteristics of the subjects after
the treatments. Body fat ratio and fat mass were signifi-
cantly lower after the ALA-DAG treatment compared with
the TAG treatment. The serum parameters did not signifi-
cantly differ after the treatments between ALA-DAG and
TAG（data not shown）.
3.2 Outcomes
The 13C recovery at the 1 h time-point（Fig. 1A）and the
cumulative 13C recovery at 6 h（Fig. 1B）were significantly
higher in the ALA-DAG treatment compared to the TAG
treatment. The cumulative recovery of 13C was 14.8±4.3％
in the TAG treatment and 17.1±4.0％ in the ALA-DAG
treatment（Fig. 1B）.
4 DISCUSSION
In the present study, to gain further insight into the
mechanism underlying the anti-visceral obesity effect of
ALA-DAG, the recovery rate of 13C-labelled dietary fat to
13CO2 in the breath was assessed as an indicator of dietary
fat oxidation during either ALA-DAG or TAG treatment.
The ALA-DAG treatment enhanced dietary fat utilization
as energy compared to the TAG treatment in humans.
Taken together with our previous data showing increased
postprandial total fat oxidation and energy expenditure
after treatment with ALA-DAG11）
, these findings suggest
that ALA-DAG treatment induces up-regulation of dietary
fat metabolism and contributes to prevent visceral obesity.
Indeed, significantly decreased fat mass（Table 2）and sig-
nificantly increased dietary fat oxidation（Fig. 1）were both
observed in this present study.
Repeated consumption of ALA-DAG enhances
β-oxidation in the small intestine12）
and liver13）
in rodents.
Fig. 1
13C recovery in the breath for up to 6 h（A）, and its cumulative value（B）. Time zero corresponds to the ingestion time
of the test meal containing 13C-triolein. Data are presented as means±SD. *, p＜0.05 between TAG and ALA-DAG.
Table 2
Physical status of the subjects after the
treatment with either TAG or ALA-DAG.
Parameter TAG ALA-DAG
Sex (M/F) 11/5
Age (year) 49±9
Body weight (kg) 70.8±8.9 70.4±8.6
Body mass index (kg/m2) 26.0±2.0 25.8±2.0
Body fat ratio (%) 29.2±7.5 28.9±7.6*
Fat mass (kg) 20.4±4.4 20.0±4.3*
Fat-free mass (kg) 50.4±9.7 50.4±9.7
Waist (cm) 92.4±6.2 92.1±6.3
Mean±SD; n＝16. *, p＜0.05 between TAG and ALA-
DAG.

Y. Ando, S. Saito and N. Yamanaka et al.
J. Oleo Sci. 66, (2) 181-185 (2017)
184
Although the precise mechanism by which ALA-DAG con-
sumption stimulates gene expression is not clear, it may be
related to the metabolic pathway of ALA-DAG. In general,
the TAG structure is hydrolyzed by lipase to 2-monoacylg-
lycerol（MAG）and fatty acids and then resynthesized into
TAG in the intestinal mucosa cells. In contrast, the DAG
structure is hydrolyzed to 1-（or 3）MAG and fatty acids.
Because 1-MAG is not easily resynthesized to TAG in the
small intestine19, 20）
, the 1-MAG and free fatty acids may
remain in the intestinal mucosa cells after DAG consump-
tion21）
. Omega 3 fatty acids, such as ALA, eicosapentaenoic
acid, and docosahexaenoic acid increase the gene expres-
sions including farnesoid X receptor and peroxisome pro-
liferator-activated receptor compared to other fatty acids,
such as saturated and omega 6 fatty acids22）
, suggesting
that the free ALA derived from ALA-DAG activates fat oxi-
dation and suppress fat synthesis, particularly in the small
intestine and the liver. Thus, DAG and ALA can synergisti-
cally affect fat oxidation. Indeed, Murase et al. suggested
that β-oxidation is higher in rodents fed ALA-DAG than in
those fed ALA-TAG12）
. Additionally, ALA-DAG can enhance
fat oxidation at 25％ of the dose compared to oleic and lin-
olenic acid-riched DAG16）
.
A limitation of the present study is that the ALA-DAG
shortbread contained more ALA than the control TAG
shortbread because we did not adjust the fatty acids com-
ponent. Therefore, we cannot determine whether the DAG
structure or the fatty acid composition was the major
factor inducing the ALA-DAG effects. Additional studies
with a similar fatty acid composition are required.
5 CONCLUSION
Treatment with ALA-DAG for 14-d enhanced the utiliza-
tion of dietary fat as energy in healthy humans. This result
indicates that ALA-DAG consumption has beneficial effects
for preventing visceral obesity by upregulating dietary fat
metabolism.
ACKNOWLEDGMENTS
We thank Dr. Masanobu Hibi, Mr. Takuya Wakisaka and
Dr. Tohru Yamaguchi for technical and scientific advice re-
garding this study. We thank Dr. Haruo Nakamura（Mitsu-
koshi Health and Welfare Foundation）for instruction and
advice regarding the study design, protocol, and discus-
sion.
CONFLICT OF INTEREST
This study was financially supported by Kao Corpora-
tion. ALA-DAG was prepared using equipment owned by
Kao Corporation. Study management, sample collection,
and data analysis were performed independently, and thus
there are no conflicts of interest that would affect the
study results.
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