JNSJOURNAL OF NUTRITIONAL SCIENCEMETABOLISM AND METABOLIC STUDIES
Enzamin ameliorates adipose tissue inflammation with impaired
adipocytokine expression and insulin resistance in db/db mice
Yukinori Tamura1, Masato Yano1, Naoyuki Kawao1, Katsumi Okumoto2, Shigeru Ueshima3, Hiroshi Kaji1and Osamu Matsuo4*
1

Department of Physiology and Regenerative Medicine, Kinki University Faculty of Medicine, Osakasayama, Japan
Life Science Research Institute, Kinki University, Osakasayama, Japan3
Department of Food Science and Nutrition, Kinki University Faculty of Agriculture, Nara, Japan
4
Kinki University Faculty of Medicine, Osakasayama, Japan
2(Received 2 May 2013 – Final revision received 29 September 2013 – Accepted 7 October 2013)

Journal of Nutritional Science (2013), vol. 2, e37, page 1 of 10

doi:10.1017/jns.2013.34

Abstract
The effects of Enzamin on obesity-related metabolic disorders in obese db/db mice were examined to explore a novel agent for the prevention of insulin
resistance. Db/db mice were treated with water containing Enzamin (0·1 and 1·0 %) for 8 weeks from 6 weeks of age. Enzamin treatment at 1·0 %, but not
at 0·1 %, signiﬁcantly decreased the fasting plasma glucose, serum total cholesterol and TAG levels in db/db mice, without affecting body weight gain andbody fat composition. Furthermore, insulin sensitivity and glucose tolerance were improved by the treatment of db/db mice with 1·0 % Enzamin.
Immunohistochemical studies and gene expression analysis showed that 1·0 % Enzamin treatment suppressed macrophage accumulation and inﬂammationin the adipose tissue. In addition, 1·0 % Enzamin treatment increased serum adiponectin in db/db mice. Treatment with 1·0 % Enzamin also signiﬁcantly
suppressed the expression of NADPH oxidase subunits, suggesting an antioxidative effect for Enzamin in the adipose tissue. Furthermore, in vitro experiments demonstrated that the lipopolysaccharide-induced inﬂammatory reaction was signiﬁcantly suppressed by Enzamin treatment in macrophages.
Enzamin treatment increased the expression of GLUT4 mRNA in muscle, but not GLUT2 mRNA in the liver of db/db mice. Enzamin also increased
the mRNA expression of carnitine palmitoyltransferase 1a (CPT1a, muscle isoform) in db/db mice, whereas Enzamin treatment did not affect the mRNA
expression of CPT1b (liver isoform) in db/db mice. In conclusion, our data indicate that Enzamin can improve insulin resistance by ameliorating impaired
adipocytokine expression, presumably through its anti-inﬂammatory action, and that Enzamin possesses a potential for preventing the metabolic syndrome.
Key words: Insulin resistance: Adipocytokines: Macrophages: Enzamin

The metabolic syndrome, which is characterised by a clusteringof visceral obesity, impaired glucose tolerance, hypertension
and dyslipidaemia, is a major cause of type 2 diabetes andatherothrombosis(1). Visceral obesity and insulin resistanceare thought to represent common underlying factors of themetabolic syndrome(2). It is critical therefore to develop
novel agents for the prevention or treatment of obesity andinsulin resistance.

Many reports have indicated that obesity is associatedwith a state of chronic, low-grade inﬂammation, suggestingthat inﬂammation may be a potential mechanism whereby
obesity can lead to insulin resistance(3). Indeed, obesity
and insulin resistance are strongly associated with systemic
markers of inﬂammation, and inﬂammation has beenrecognised clinically as a major predictor of atherosclerotic
diseases(4).

Abbreviations: ATCC, American Type Culture Collection; CPT, carnitine palmitoyltransferase; CPT1a, carnitine palmitoyltransferase 1 (liver); CPT1b, carnitine palmitoyltransferase 1 (muscle); CT, computed tomography; LPS, lipopolysaccharide; MCP-1, monocyte chemoattractant protein 1; Nox2, NADPH oxidase 2; PAI-1, plasminogenactivator inhibitor 1; t-PA, tissue-type plasminogen activator.
* Corresponding author: Dr Osamu Matsuo, fax +81 6 7635 9366, email matsuo-o@med.kindai.ac.jp© The Author(s) 2013. The online version of this article is published within an Open Access environment subject to the conditions of the Creative
Commons Attribution license <http://creativecommons.org/licenses/by/3.0/>.

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The adipose tissue is an important endocrine organ that
secretes numerous biologically active molecules, such as leptin,
adiponectin, TNF-α, monocyte chemoattractant protein 1(MCP-1) and plasminogen activator inhibitor 1 (PAI-1),
which are collectively termed adipocytokines(5,6). The impaired
production of pro-inﬂammatory and anti-inﬂammatory adipocytokines seen in visceral fat obesity is associated with themetabolic syndrome(7), indicating that inﬂammatory changes
in the adipose tissue may contribute to the development ofseveral aspects of the metabolic syndrome and result in type2 diabetes and thrombotic diseases(8).
Enzamin is a product from Bacillus subtilis AK and
Lactobacillus, treated under physically adverse conditions suchas heat treatment, and acid treatment(9). We have shown previously that Enzamin displays proﬁbrinolytic anti-thrombotic
properties both in vitro and in vivo(9). However, the effects ofEnzamin on the metabolic syndrome have not yet been elucidated. In the present study, we therefore examined the inﬂuence of Enzamin on insulin resistance and adipocytokine
expression in obese diabetic db/db mice.

Experimental methods
Materials

The following materials were obtained from the commercial
sources indicated: human regular insulin (Eli Lilly), anti-F4/
80 antibody (AbD Serotec), Glutest Ace (Sanwa Kagaku
Kenkyusho), RNeasy mini kits (Qiagen), rat monoclonalanti-F4/80 antibody (AbD Serotec), anti-rat secondary antibodies conjugated with horseradish peroxidase (Cell
Signaling Technology Japan), fetal bovine serum (American
Type Culture Collection (ATCC)) and lipopolysaccharides(LPS) from Escherichia coli 0111:B4 (Sigma Aldrich Japan).
All other reagents and chemicals were of the highest grade
available. Enzamin, which was produced by the long-term
fermentation and self-digestion of Bacillus subtilis AK and
Lactobacillus(10), was provided by the Enzamin ResearchInstitute, Osaka, Japan. Japan Food Research Laboratories,
which issue the ofﬁcial documents for the food export, analysed the molecular distribution of Enzamin by HPLC witha TSKgel G2500PWXL column (TOSOH Corporation). The
standard molecular markers were cytochrome c (12500 Da),aprotinin (6152 Da), bacitracin (1450 Da), angiotensin II(1046 Da), Gly-Gly-Tyr-Arg (451 Da) and Gly-Gly-Gly(189 Da). There was an abundance of peaks and the majority
were below the molecular weight of 500 Da (75 %). There
were peaks with the molecular weight of 500–1000 Da(10 %), 1000–3000 Da (11 %), 3000–6000 Da (2 %) andover 6000 Da (2 %), respectively.

The db/ + m mice were fed on normal chow and water ad libitum as lean mice. All animals were maintained on a 12 h light–
dark cycle. The animal experiments were conducted according
to the Guidelines for Animal Experiments at Kinki University
Faculty of Medicine.
Body fat composition analysis

Body fat composition was estimated by computed tomography(CT) analysis in mice that were anaesthetised with forane(Isoﬂurane; Abbott Japan) and then scanned using a
LaTheta (LCT-200) experimental animal CT system(Hitachi-Aloka Medical)(11). Contiguous 1-mm slice images
between L1 and L5 were used for quantitative assessment
employing LaTheta software (version 3·40). Visceral fat andsubcutaneous fat were distinguished, and the total fat content,
visceral fat weight and subcutaneous fat weight were calculated
from all slice images and evaluated quantitatively.
Analysis of metabolic parameters

The plasma insulin, serum total cholesterol, TAG, adiponectin
and TNF-α levels were measured using an insulin assay kit(Morinaga Institute of Biological Science), Cholesterol E test,
Triglyceride E test (Wako Pure Chemical Industries), adiponectin ELISA kit (Otsuka Pharmaceutical) and a Quantikine
TNF-α ELISA kit (R&D Systems), respectively. For glucose
tolerance tests, mice were deprived of food for 16 h and glucose (1·5 g/kg body mass) was then injected intraperitoneally.
For insulin tolerance tests, mice were injected intraperitoneallywith human regular insulin (10·5 μg/kg body mass for db/+
m mice and 35·0 μg/kg body mass for db/db mice). Blood
samples were collected before and after each injection, andthe plasma glucose concentration was measured with aGlutest Ace.
Cell culture

RAW 264.7 (ATCC number: TIB-71), a murine macrophage cellline, was purchased from ATCC and maintained in Dulbecco’s
modiﬁed essential medium supplemented with 10 % fetal
bovine serum and 100 mg/ml penicillin–streptomycin at 37°C
with 5 % CO2. RAW 264.7 cells (1 × 106 cells/well) were plated
on six-well cell culture plates and incubated for 24 h. Cells weretreated with 0·01 or 0·1 % concentrations of Enzamin in thepresence or absence of 1 µg/ml LPS for 12 h. After washing
twice with PBS, the total cellular RNA was extracted for gene
expression analysis employing real-time PCR.
Quantitative real-time PCR

Animal preparation and experimental design

Male db/db mice and db/ + m mice were obtained fromCharles River at 5 weeks of age. The db/db mice were fed
on normal chow and water ad libitum supplemented with 0,0·1 or 1·0 % Enzamin for 8 weeks from 6 weeks of age.
Each 0 % Enzamin group is referred to below as the control.

Total RNA was extracted from the frozen adipose tissue (100mg), liver tissue (30 mg) and muscle tissue (30 mg) of miceand from RAW 264.7 cells using an RNeasy mini kit(Qiagen). The cDNA was synthesised from the total RNA
using Super Script III (Life Technologies Japan). The real-time
PCR was performed on a StepOne Plus using the SYBR®
Green PCR Master Mix (Life Technologies Japan). The primer
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Table 1. Primers used for real-time PCR
Primer
GLUT2
GLUT4
G6Pase
CPT1a
CPT1b
TNF-α
MCP-1
IL-6
PAI-1
Emr-1
CD68
TLR-4
Nox2
P22phox
P47phox
18S rRNA

Sequence
Forward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
ReverseForward
Reverse5′-GGCTAATTTCAGGACTGGTT-3′5′-TTTCTTTGCCCTGACTTCCT-3′5′-CATGGCTGTCGCTGGTTTC-3′
5′-AAACCCATGCCGACAATGA-3′5′-CTGTGAGACCGGACCAGGA-3′5′-GACCATAACATATACACCTGCTGC-3′
5′-GTCCCAGCTGTCAAAGATAC-3′5′-GGAAGTATTGAAGAGTCGC-3′
5′-CAAGTTCAGAGACGAACGCC-3′
5′-TCAAGAGCTGTTCTCCGAACTG-3′5′-CCCAGACCCTCACACTCAGATC-3′
5′-GCCACTCCAGCTGCTCCTC-3′
5′-CCACTCACCTGCTGCTACTCA-3′
5′-TGGTGATCCTCTTGTAGCTCTCC-3′5′-GTTCTCTGGGAAATCGTGGA-3′5′-GGAAATTCGGGGTAGGAAGGA-3′5′-TTCAGCCCTTGCTTGCCTC-3′5′-ACACTTTTACTCCGAAGTCGGT-3′5′-CTTTGGCTATGGGCTTCCAGTC-3′
5′-GCAAGGAGGACAGAGTTTATCGTG-3′5′-CTTCCCACAGGCAGCACAG-3′5′-AATGATGAGAGGCAGCAAGAGG-3′5′-TATCCAGGTGTGAAATTGAAACAATT-3′5′-GGGTTTCCTGTCAGTATCAAGTTTG-3′5′-TTGGGTCAGCACTGGCTCTG-3′5′-TGGCGGTGTGCAGTGCTATC-3′
5′-GTCCACCATGGAGCGATGTG-3′
5′-CAATGGCCAAGCAGACGGTC-3′
5′-GATGTTCCCCATTGAGGCCG-3′5′-GTTTCAGGTCATCAGGCCGC-3′
5′-CGGCTACCACATCCAAGGAA-3′
5′-GCTGGAATTACCGCGGCT-3′

G6Pase, glucose-6-phosphatase; CPT1a, carnitine palmitoyltransferase 1 (liver); CPT1b, carnitine palmitoyltransferase 1 (muscle); MCP-1, monocyte chemoattractant protein 1;
PAI-1, plasminogen activator inhibitor 1; Emr-1, EGF-like module-containing mucin-like hormone receptor-like 1; TLR-4, Toll-like receptor 4; Nox2, NADPH oxidase 2; rRNA,
ribosomal RNA.

sets are listed in Table 1. The mRNA levels were normalised
relative to the amount of 18s ribosomal RNA, and expressedin arbitrary units.

Histological analysis

Adipose tissue was ﬁxed for 12–16 h at 4°C with 4 % paraformaldehyde, and embedded in parafﬁn. Sections of 4 µm
thickness were incubated with rat monoclonal anti-F4/80 antibody. The sections were then incubated with the appropriate
anti-rat secondary antibodies conjugated with horseradish peroxidase. Positive signals were visualised employing a tyramide
signal ampliﬁcation system (PerkinElmer). The sections werecounterstained with 4′,6-diamidino-2-phenylindole (DAPI)
and photographed using a ﬂuorescence microscope (E800;
Canon) with a CCD camera (Keyence Japan). For each individual mouse tissue block, ten ﬁelds from each section wereanalysed. The total number of nuclei and the number of nuclei
of F4/80-expressing cells were counted for each ﬁeld byemploying an image processing program (NIH image;National Institutes of Health) in a blinded evaluation. The
fraction of F4/80-expressing cells for each section was calculated as the sum of the number of nuclei of F4/80-expressing
cells divided by the total number of nuclei in the sections.

Statistical analysis

All data were expressed as mean values with their standard
errors. The statistical signiﬁcance of differences was assessedby the unpaired t test and one-way ANOVA. Differenceswith P < 0·05 were regarded as signiﬁcant. All statistical analyses were performed using StatView version 5.0 software(SAS Institute Inc.).
Results
Effect of Enzamin on body fat composition in db/db mice

Treatment with 1 % Enzamin exerted no effect on water
intake, whereas treatment with 0·1 % Enzamin increased
water intake slightly. Enzamin had no effect on food intake(data not shown) or body weight gain in db/db mice (Fig. 1(a)). To examine the effect of Enzamin treatment on body
fat composition, we next performed CT scan analysis.
Abdominal CT images at L2 demonstrated that both the visceral and subcutaneous fat areas were markedly increased in
db/db mice as compared with db/+ m mice, but that
Enzamin treatment exerted no effect on the visceral and subcutaneous fat areas in db/db mice (Fig. 1(b)). The total fat
content, visceral fat weight and subcutaneous fat weight calculated from the CT scan analysis were not affected by Enzamin3

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Fig. 1. Effect of Enzamin (Enm) treatment for 8 weeks on body weight and fat composition. (a) Growth curves during the experiment (left) and body weight at the endof the experiment (right) of non-treated db/ + m (lean control) and db/db (obese control) mice, and 0·1 and 1·0 % Enzamin-treated db/db mice. ○, db/ + m mice (n 6);, non-treated db/db mice (n 10); Δ, 0·1 % Enzamin-treated db/db mice (n 9); □, 1·0 % Enzamin-treated db/db mice (n 8). (b) Representative computed tomography(CT) sections of abdominal regions. con, Control. Pink areas show visceral fat; yellow areas show subcutaneous fat. (c) Total fat content, and weights of visceral (d)and subcutaneous (e) fat in db/ + m and db/db mice following each treatment, calculated from CT scan data. m, db/ + m; db, db/db. Data are means (n 6–10 pergroup), with standard errors represented by vertical bars.

†

treatment in db/db mice (Fig. 1(c)–(e)). Enzamin treatment
also had no effect on the liver fat content estimated by CT
scan analysis in db/db mice (data not shown).
Effect of Enzamin on lipid and glucose metabolism in db/db
mice

To evaluate the effect of Enzamin treatment on lipid metabolism, we measured serum TAG and total cholesterol levels
in db/db mice. The fasting serum TAG levels in db/db
mice were higher than those in db/+ m mice (Fig. 2(a)).
Treatment with Enzamin at 0·1 % slightly reduced theserum TAG level, and 1·0 % Enzamin treatment signiﬁcantly
reduced the fasting serum TAG level in db/db mice (Fig. 2(a)).
Similarly, the fasting serum total cholesterol level was slightly
but not signiﬁcantly decreased by 0·1 % Enzamin, and wassigniﬁcantly decreased by 1·0 % Enzamin in db/db mice
(Fig. 2(b)). Thus, Enzamin treatment reduced fasting serum
TAG and cholesterol levels dose-dependently in db/db

mice. Taken together, these ﬁndings suggest that Enzamin
treatment improves lipid metabolism in db/db mice.
We next evaluated the effect of Enzamin treatment on glucose metabolism in db/db mice. The fasting plasma glucose
was markedly increased in db/db mice as compared withdb/+ m mice (Fig. 2(c)). Enzamin treatment at 1·0 %, but
not at 0·1 %, signiﬁcantly reduced the fasting plasma glucose
level in db/db mice (Fig. 2(c)). Although the fasting plasma
insulin was also markedly elevated in db/db mice as comparedwith db/+ m mice (Fig. 2(d)), there were no differences infasting plasma insulin levels between non-treated db/db
mice and Enzamin-treated db/db mice (Fig. 2(d)).
To evaluate further the effects of Enzamin on glucose
metabolism, we next performed an intraperitoneal glucose tolerance test and intraperitoneal insulin tolerance test. The
plasma glucose level after intraperitoneal glucose injection
was dramatically elevated in db/db mice as compared withdb/+ m mice (Fig. 3(a)), indicating severe glucose intolerance.
Enzamin treatment at 1·0 %, but not at 0·1 %, signiﬁcantly
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Fig. 2. Effect of Enzamin treatment for 8 weeks on lipid and glucose metabolism. Fasting serum TAG (a), total cholesterol (T-Cho) (b), plasma glucose(c) and insulin (d) concentrations in non-treated db/+ m (lean control, n 6)
and db/db (obese control, n 10) mice, and 0·1 % (n 9) and 1·0 % (n 8)
Enzamin-treated db/db mice. m, db/ + m; db, db/db. Data are means (n 6–10
per group), with standard errors represented by vertical bars. *Mean value
was significantly different from that of the non-treated db/db mice (P < 0·05).

suppressed the elevation of the plasma glucose level in db/db
mice at 30 min after glucose injection. Furthermore, a similar
suppressive effect was also noted at 60 and 90 min after intraperitoneal glucose injection (Fig. 3(a)). These data suggest that
Enzamin can improve glucose intolerance in db/db mice.
Severe impairment of insulin sensitivity was also observed in
db/db mice as compared with db/+ m mice (Fig. 3(b)).
Although 0·1 % Enzamin treatment exerted no effect on insulin sensitivity in db/db mice, 1·0 % Enzamin treatment signiﬁcantly decreased the plasma glucose level in response to insulin
at 30, 60, 90 and 120 min after intraperitoneal insulin injection
in db/db mice (Fig. 3(b)). Taken together, these data suggestthat Enzamin treatment can ameliorate insulin resistance in
db/db mice.
We next evaluated the gene expression of GLUT2 (themain GLUT in liver) and glucose-6-phosphatase (G6Pase,an enzyme related to gluconeogenesis) in the liver, and thegene expression of GLUT4 (a muscular GLUT) in the muscle
of db/db mice. Enzamin treatment did not affect the mRNA
expression of GLUT2 and G6Pase in the liver of db/db mice
(Fig. 4(a) and (b)), suggesting that Enzamin does not affectglucose metabolism in the liver of db/db mice. The mRNA
expression of GLUT4 in muscle was markedly decreased in
db/db mice as compared with db/+ m mice (Fig. 4(d)).
Enzamin treatment at 1·0 % signiﬁcantly increased themRNA expression of GLUT4 in the muscle of db/db miceas compared with non-treated db/db mice (Fig. 4(d)). We
evaluated the gene expression of carnitine palmitoyltransferase
1 (CPT1, a mitochondrial enzyme related with lipid oxidation)in the liver and muscle of db/db mice. The mRNA expressionof CPT1a (liver isoform) and CPT1b (muscle isoform) was signiﬁcantly decreased in db/db mice as compared with db/+ mmice (Fig. 4(c) and (e)). Enzamin treatment at 1·0 % increased
the mRNA expression of CPT1b in the muscle of db/db miceas compared with non-treated db/db mice (Fig. 4(e)), whereas
Enzamin treatment did not affect the mRNA expression of
CPT1a in the liver of db/db mice (Fig. 4(c)). These datasuggest that Enzamin treatment improves glucose uptake
and lipid oxidation in the muscle of db/db mice.
Effect of Enzamin on adipocytokine expression in db/db mice

To clarify the mechanism whereby Enzamin improves insulin
resistance, we examined the effect of Enzamin treatment on
pro-inﬂammatory adipocytokine expression in the adipose tissue of db/db mice. The mRNA expression of TNF-α in the
adipose tissue of db/db mice was 5-fold higher than that in
db/+ m mice, indicating adipose tissue inﬂammation in
db/db mice (Fig. 5(a)). Enzamin treatment suppressed the
TNF-α mRNA expression dose-dependently in the adipose
tissue of db/db mice. In particular, 1·0 % Enzamin treatment
signiﬁcantly suppressed TNF-α mRNA expression by 40 % in
the adipose tissue of db/db mice (Fig. 5(a)). Consistent with

Fig. 3. Effects of Enzamin treatment for 8 weeks on glucose tolerance and insulin sensitivity. Responses of plasma glucose to a single intraperitoneal injection of
glucose (a) or insulin (b) in non-treated db/ + m (○, n 6) and db/db ( , n 10) mice, and 0·1 % (Δ, n 9) and 1·0 % (□, n 8) Enzamin-treated db/db mice. m, db/ + m; db,
db/db. Data are means (n 6–10 per group), with standard errors represented by vertical bars. *Mean value was significantly different from that of the non-treated db/db
mice (P < 0·05).

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Fig. 4. Effect of Enzamin treatment for 8 weeks on glucose and lipid metabolism in liver and muscle. Messenger RNA (mRNA) expression of GLUT2 (a), glucose-6phosphatase (G6Pase) (b) and carnitine palmitoyltransferase (CPT) 1a (c) in the liver, and GLUT4 (d) and CPT1b (e) in the muscle of non-treated db/ + m (leancontrol, n 6) and db/db (obese control, n 10) mice, and 0·1 % (n 9) and 1·0 % (n 8) Enzamin-treated db/db mice. AU, arbitrary units; m, db/ + m; db, db/db. Data
are means (n 6–10 per group), with standard errors represented by vertical bars. **Mean value was significantly different from that of the non-treated db/db mice
(P < 0·01).

Fig. 5. Effect of Enzamin treatment for 8 weeks on adipocytokine expression. Messenger RNA (mRNA) expressions of TNF-α (a), monocyte chemoattractant protein
1 (MCP-1) (b), IL-6 (c) and plasminogen activator inhibitor 1 (PAI-1) (d) in epididymal white adipose tissue, and serum concentrations of TNF-α (e) and adiponectin (f)in non-treated db/ + m (lean control, n 6) and db/db (obese control, n 10) mice, and 0·1 % (n 9) and 1·0 % (n 8) Enzamin-treated db/db mice. AU, arbitrary units; m, db/+ m; db, db/db. Data are means (n 6–10 per group), with standard errors represented by vertical bars. Mean value was significantly different from that of the nontreated db/db mice: *P < 0·05, **P < 0·01.

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Fig. 6. Effect of Enzamin (Enm) treatment for 8 weeks on macrophage accumulation in adipose tissue. (a) Macrophage content of epididymal white adipose tissue(WAT) as assessed by F4/80 staining (bar, 100 µm) and (b) fraction of adipose tissue macrophages (F4/80-stained cells/total cells) in epididymal WAT of non-treateddb/ + m (lean control) and db/db (obese control) mice, and 0·1 % and 1·0 % Enzamin-treated db/db mice (n 5 for each group). con, Control. Messenger RNA (mRNA)
expressions of EGF-like module-containing mucin-like hormone receptor-like 1 (Emr-1) (c), CD68 (d) and Toll-like receptor 4 (TLR-4) (e) in adipose tissue of nontreated db/ + m (lean control, n 6) and db/db (obese control, n 10) mice, and 0·1 % (n 9) and 1·0 % (n 8) Enzamin-treated db/db mice. AU, arbitrary units; m, db/ + m;
db, db/db. Data are means, with standard errors represented by vertical bars. Mean value was significantly different from that of the non-treated db/db mice: *P < 0·05,**P < 0·01.

this, 1·0 % Enzamin also suppressed the mRNA expression of
MCP-1 and IL-6 by 50 and 60 %, respectively, in the adipose
tissue of db/db mice (Fig. 5(b) and (c)). Furthermore, thePAI-1 mRNA level was signiﬁcantly reduced by both 0·1
and 1·0 % Enzamin in the adipose tissue of db/db mice
(Fig. 5(d)). These data suggest that Enzamin treatment can
suppress adipose tissue inﬂammation in db/db mice.
To evaluate the effect of Enzamin on systemic inﬂammation,
we measured the serum TNF-α level in db/db mice. Enzamin
treatment at 1·0 %, but not at 0·1 %, signiﬁcantly suppressed
the circulating TNF-α level in db/db mice (Fig. 5(e)), suggestingthat Enzamin could suppress systemic inﬂammation as well as
adipose tissue inﬂammation in db/db mice. In contrast to
pro-inﬂammatory adipocytokines, the level of serum adiponectin was signiﬁcantly increased by treatment with Enzamin in adose-dependent manner. In particular, 1·0 % Enzamin treatment increased serum adiponectin by 1·4-fold in db/db mice
(Fig. 5(f)). These data suggest that Enzamin can improve theimpaired adipocytokine expression in obese mice.
Effect of Enzamin on macrophage accumulation andactivation in db/db mice

To evaluate the effect of Enzamin on macrophage accumulation in adipose tissue, we performed immunohistochemical
staining for F4/80, a mature macrophage marker, in adipose

tissue sections from obese mice. The histological photographs
indicated that macrophage accumulation was dramaticallyincreased in db/db mice as compared with db/+ m mice,and Enzamin treatment appeared to reduce dose-dependently
the F4/80-positive area in the adipose tissue of db/db mice
(Fig. 6(a)). Consistent with the histological photographs, thefraction of F4/80-positive cells in the adipose tissue wasslightly, but not signiﬁcantly, suppressed by 0·1 % Enzamin,
and signiﬁcantly suppressed by 1·0 % Enzamin treatment in
db/db mice (Fig. 6(b)). Furthermore, 1·0 % Enzamin treatment signiﬁcantly decreased the mRNA expression of
EGF-like module-containing mucin-like hormone receptor-like
1 (Emr-1; an F4/80 antigen) in the adipose tissue of db/db
mice by 40 % (Fig. 6(c)). In addition, the mRNA expressionsof CD68 and Toll-like receptor 4 (TLR-4), a marker of activated and pro-inﬂammatory macrophages, were decreased byEnzamin treatment in the adipose tissue of db/db mice(Figs. 6(d) and (e)). These data indicate that Enzamin may suppress pro-inﬂammatory macrophage accumulation and activation in the adipose tissue of db/db mice.
Effect of Enzamin on oxidative stress in adipose tissue of db/
db miceTo evaluate the effect of Enzamin on oxidative stress in adipose tissue, we measured the expression of NADPH oxidase7

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Fig. 7. Effect of Enzamin treatment for 8 weeks on oxidative stress in adipose tissue. Messenger RNA (mRNA) expressions of NADPH oxidase (Nox) subunits, Nox2(a), p22phox (b) and p47phox (c) in adipose tissue of non-treated db/ + m (lean control, n 6) and db/db (obese control, n 10) mice, and 0·1 % (n 9) and 1·0 % (n 8)
Enzamin-treated db/db mice. AU, arbitrary units; m, db/ + m; db, db/db. Data are means (n 6–10 per group), with standard errors represented by vertical bars.
*Mean value was significantly different from that of the non-treated db/db mice (P < 0·05).

subunits in adipose tissue from db/db mice. The expressionsof NADPH oxidase subunits, such as NADPH oxidase 2(Nox2), p22phox and p47phox, in non-treated db/db mice
were markedly higher than those in db/+ m mice (Fig. 7(a)-(c)). Although there was no signiﬁcant effect, 0·1 %Enzamin treatment tended to suppress the expressions of
NADPH oxidase subunits. Furthermore, treatment with
1·0 % Enzamin signiﬁcantly suppressed the mRNA expressions of Nox2, p22phox and p47phox by 40, 30 and 40 %, respectively, in the adipose tissue of db/db mice (Fig. 7(a)–(c)). These
data suggest that Enzamin treatment can suppress oxidative
stress in the adipose tissue of obese mice.
Effect of Enzamin on lipopolysaccharide-induced
inflammatory response in macrophages in vitro

To evaluate the inﬂuence of Enzamin on the inﬂammatory
response in macrophages, we investigated the effect ofEnzamin on LPS-induced TNF-α expression in RAW 264.7
cells, a murine macrophage cell line. Although Enzamin treatment slightly increased the mRNA expression of TNF-α inmacrophages without LPS stimulation, the LPS-induced
elevation of TNF-α expression was signiﬁcantly suppressed

Fig. 8. Effect of Enzamin (Enm) treatment on lipopolysaccharide(LPS)-induced inflammatory response in macrophage in vitro. Messenger
RNA (mRNA) expressions of TNF-α in RAW 264.7 cells treated without orwith Enzamin (0·01 and 0·1 %) for 12 h, in the absence or presence of LPS(1 µg/ml). AU, arbitrary units. Data are means (n 3 per group), with standard
errors represented by vertical bars. **P < 0·01.

by both the 0·01 and 0·1 % Enzamin treatments (Fig. 8),
suggesting that Enzamin tends to suppress the
pro-inﬂammatory response in macrophages.

Discussion

In the present study, we demonstrated that Enzamin can
improve the insulin resistance and impaired adipocytokine
expression in db/db mice. We found that Enzamin treatment
suppressed macrophage accumulation in the adipose tissue aswell as adipose tissue inﬂammation in db/db mice. Several
studies have shown that the expression of pro-inﬂammatory
cytokines by adipose tissue is in part attributable to theexpression of these cytokines by non-adipocytes, including
macrophages(12,13). Furthermore, Suganami et al.(14,15) developed an in vitro co-culture system composed of adipocytes
and macrophages, and demonstrated that a paracrine loop
involving NEFA and TNF-α derived from the adipocytes
and macrophages, respectively, leads to a vicious cycle thataugments the inﬂammatory changes in both adipocytes andmacrophages, i.e. marked up-regulation of pro-inﬂammatory
cytokines such as MCP-1 and TNF-α and down-regulationof the anti-inﬂammatory cytokine adiponectin. Moreover,in a previous study, we found that pharmacological inhibitionof macrophage inﬁltration suppressed adipose tissue inﬂammation and improved the impairment of adipocytokine production(16). In the present experiments, therefore, Enzaminmight have suppressed adipose tissue inﬂammation by inhibiting
macrophage inﬁltration into the adipose tissue of obese mice.
Many reports have suggested that adipose tissue inﬂammatory changes contribute to the development of insulin resistance. It has been demonstrated that adipose tissue-derived
pro-inﬂammatory cytokines such as TNF-α(17,18), andIL-6(19,20) can actually cause insulin resistance in experimental
models. In the present study, Enzamin treatment at 1·0 %reduced the serum TNF-α level in db/db mice, suggestingthat Enzamin can suppress systemic inﬂammation in db/db
mice. Furthermore, TNF-α dose-dependently reduces theexpression of adiponectin in adipocytes by suppressing its promoter activity(21,22). Adiponectin serves as an insulinsensitising agent(6,23), so that a decrease in plasma adiponectin
is related to insulin resistance in obesity. In the present study,
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we observed that the serum adiponectin level was decreased in
db/db mice as compared with db/ + m mice, in addition to an
impaired insulin sensitivity and glucose tolerance. However,Enzamin treatment improved hypoadiponectinaemia in db/
db mice. The increase in adiponectin by Enzamin treatment
therefore may be responsible for the improvement of insulin
resistance in db/db mice. Furthermore, we also found that
Enzamin treatment increased the expression of GLUT4mRNA, which is associated with glucose uptake in muscle
of db/db mice. Previous reports suggest that the impairmentof insulin signalling through the reduction of GLUT4
expression and translocation in muscle is responsible for the
insulin resistance in db/db mice(24,25). It has been shownthat TNF-α impairs insulin receptor signalling through the
impairment of GLUT4 expression and translocation to theplasma membrane(26). Therefore, Enzamin might improve glucose uptake in the muscle of db/db mice, presumably throughthe reduction of circulating TNF-α.
Enzamin treatment could also suppress the gene expressionof the NADPH oxidase subunit, which is associated withoxidative stress, in the adipose tissue of db/db mice. In
addition to inﬂammation, oxidative stress plays a critical rolein insulin resistance(27). In fact, antioxidants such as vitamins
C and E, and α-lipoic acid ameliorate insulin resistance(28,29).
Oxidative stress is known to be increased in obesity via
NADPH oxidase activation(30,31). NADPH oxidase is amajor source of reactive oxygen species in various organs,especially in white adipose tissue(30,31). NADPH oxidase consists of the membrane-associated ﬂavocytochrome b558 familyof proteins, which include Nox2 (gp91phox) and p22phox, as
well as the cytosolic components p47phox, p67phox andp40phox(32). In the present study, we observed that Enzamin
treatment suppressed the gene expressions of these subunits.
Our ﬁndings suggest that an antioxidative effect of Enzaminmay also be involved in the improvement of insulin resistance.
We found that Enzamin treatment suppressed LPS-induced
TNF-α expression in macrophages in vitro, indicating that
Enzamin can directly suppress the pro-inﬂammatory response
in macrophages. Furthermore, we observed that Enzamin
treatment suppressed activated macrophage markers, such as
CD68 and Toll-like receptor 4 (TLR-4), in the adipose tissueof db/db mice. These ﬁndings suggest that Enzamin may suppress the inﬂammatory response in macrophages.
Morange et al.(33) reported that PAI-1 deﬁciency improved
insulin resistance and markedly increased tissue-type plasminogen activator (t-PA) activity, but not urokinase-type plasminogen activator (u-PA) activity in the adipose tissue of obese
mice. Furthermore, the levels of plasma insulin and blood glucose in obese t-PA-deﬁcient mice were higher than those inobese wild-type (WT) mice, suggesting that t-PA is associatedwith insulin sensitivity(34). We observed that Enzamin treatment markedly decreased the levels of PAI-1 mRNA in the
adipose tissue of db/db mice. Furthermore, we previously
demonstrated that Enzamin treatment increased t-PA activity,
but not u-PA activity, in the blood of mice. We also reported
that Enzamin contains the substances that strongly bind t-PA
assessed by the IAsys resonance assay(9). Therefore, it isassumed that the pro-ﬁbrinolytic state induced by Enzaminmight be associated with the improvement of insulin resistancein obese mice. However, further study will be required to clarify the mechanisms by which Enzamin improves insulin resistance in db/db mice.
In conclusion, the present data suggest that Enzamin canameliorate insulin resistance presumably through suppressionof the inﬂammatory response and oxidative stress in adipose
tissue. Since Enzamin has been utilised in health care andno side effects have been reported, it may represent a beneﬁcial supplement for the prevention of the metabolic
syndrome.
Acknowledgements

We appreciate the animal care facility at Kinki University.
There is no funding available to the present research. All
issues in the present study have been planned, performed
and evaluated by the authors themselves.
O. M. and Y. T. designed the research; Y. T. and M. Y. conducted the research; O. M., K. O., S. U., H. K. and
Y. T. analysed the data; O. M. and Y. T. wrote the manuscript.
All of the authors read and approved the ﬁnal manuscript.
There are no conﬂicts of interest to declare.
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