BMC Pharmacology

BioMed Central

Research article

Open Access

Effect of green tea on blood glucose levels and serum proteomic
patterns in diabetic (db/db) mice and on glucose metabolism inhealthy humans
Hiroshi Tsuneki*1, Mitsuyo Ishizuka1,2, Miki Terasawa1, Jin-Bin Wu3,
Toshiyasu Sasaoka1 and Ikuko Kimura1
Address: 1Department of Clinical Pharmacology, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan, 2Toyama College,
Toyama 930-0193, Japan and 3China Medical College, Taichung, Taiwan Republic of China
Email: Hiroshi Tsuneki* - htsuneki@ms.toyama-mpu.ac.jp; Mitsuyo Ishizuka - ishizuka@toyama-c.ac.jp;
Miki Terasawa - pm049009@st.toyama-mpu.ac.jp; Jin-Bin Wu - jbwu@mail.cmu.edu.tw; Toshiyasu Sasaoka - tsasaoka-tym@umin.ac.jp;
Ikuko Kimura - ikukokim@ms.toyama-mpu.ac.jp* Corresponding author

Published: 26 August 2004
BMC Pharmacology 2004, 4:18

doi:10.1186/1471-2210-4-18

Received: 04 June 2004
Accepted: 26 August 2004

This article is available from: http://www.biomedcentral.com/1471-2210/4/18© 2004 Tsuneki et al; licensee BioMed Central Ltd.
This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract
Background: Green tea is widely consumed in Asian countries and is becoming increasingly
popular in Western countries. Epidemiologically, it has been suggested that green tea consumption
prevents type 2 diabetes. The present study was aimed at providing evidence of improvement inglucose metabolism in diabetic mice and healthy humans upon green tea consumption.
Results: Green tea promoted glucose metabolism in healthy human volunteers at 1.5 g/body inoral glucose tolerance tests. Green tea also lowered blood glucose levels in diabetic db+/db+ miceand streptozotocin-diabetic mice 2–6 h after administration at 300 mg/kg without affecting serum
insulin level, whereas no effect was observed in control mice (+m/+m and normal ddY mice). The
serum protein profiles of db+/db+ and +m/+m mice were analyzed for the first time by SELDI(surface-enhanced laser desorption/ionization)-TOF (time-of-flight)-MS (mass spectrometry), andthen compared to investigate any effects of oral green tea administration on serum proteins. The
protein profiles in db+/db+ mice showed that the spectral peak intensities at the mass/charge ratios(m/z) of 4119, 4203, 4206, 4211, 4579, 9311 and 18691 were >3 times lower, and those of 13075,
17406, 17407, 17418, 17622, 18431 and 26100 were >3 times higher than respective peakintensities in +m/+m mice. When green tea was administered to db+/db+ mice, the peak intensities
were markedly decreased at m/z 11651 and 11863, and slightly decreased at m/z 4212. The peakintensities at 7495, 7595, 7808, 14983, 15614, 31204 were markedly increased after theadministration.
Conclusion: The present study provides evidence that green tea has an antidiabetic effect.
Although we could not find simple reversed effect of green tea on the diabetes-induced
modifications of the levels of several serum proteins, we found that the 4211 (4212) Da protein
level that was decreased in the diabetic state was further decreased after green tea administration.
This is the first report demonstrating that a certain serum protein may be involved in theantihyperglycemic effect of green tea. The contribution of this protein should be further studied.

Page 1 of 10(page number not for citation purposes)

Background
Green tea (leaves of Camellia sinensis, Theaceae) is a popular beverage in East Asia, and also used as a herbal remedy in Europe and North America. Green tea is considered
to be antiinflammatory, antioxidative, antimutagenic,
and anticarcinogenic [1,2], and can prevent cardiac disorders. Epidemiologically, it has been suggested that green
tea consumption prevents type 2 diabetes. The amelioration of insulin resistance by green tea is associated with
the increased expression level of glucose transporter IV ina fructose-fed rat [3]. Green tea extract contains polyphenols (e.g., catechin, epicatechin, epigallocatechin, andtheir gallates), teanin and caffeine. The extract also
includes pyrroloquinoline quinone, a newly discovered
vitamin [4]. Some constituent components have beenshown to enhance the basal and insulin-stimulated glucose uptake of rat adipocytes [5], to inhibit intestinal glucose uptake by inhibiting the sodium-dependent glucose
transporter of rabbit intestinal epithelial cells [6], and to
reduce serum glucose level in alloxan-diabetic rats [7].
Controversially, caffeine acutely lowers insulin sensitivity
in humans [8].
The present study was aimed at providing evidence of the
improvement in glucose metabolism in humans and diabetic mice upon green tea consumption. Furthermore, toinvestigate whether some serum proteins mediate theeffects of green tea on hyperglycemia in the diabetic state,
proteomic analyses were performed using SELDI (surface
enhanced laser desorption/ionization)-TOF (time-offlight)-MS (mass spectrometry), because the SELDI-TOFMS enables the measurement of the relative abundance ofproteins of various sizes in a blood sample. Here, we compared the proteomic patterns of sera from diabetic db+/
db+ and wild-type mice, and further investigated theinfluence of green tea administration on the proteomic
patterns of serum in diabetic mice. If the diabetic modifications of serum protein levels are reversed by green tea
administration, these affected proteins can be used astherapeutic targets in diabetes.

Results
Oral glucose tolerance test (OGTT)
We investigated whether oral glucose tolerance isimproved in healthy volunteers by drinking a suspensionof green tea powder. Blood glucose levels were measured
before and 30, 60, and 120 min after drinking 1.5 g ofgreen tea. The effects of hot water without tea powder onblood glucose level in the same participants were also
investigated. Fig. 1 shows that glucose tolerance was substantially improved with tea administration comparedwith hot water administration. In detail, glucose metabolism increased in 14 participants, remained unchanged in
3 participants, and worsened in 5 participants. Basal

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Blood glucose level (mg/dl)

BMC Pharmacology 2004, 4:18

tea

170

water

OGTT
150

Basal

130
110
90
70
0

30

60

90

120

Time (min)FigureIncrease
tered
with
1in green
glucoseteametabolism in healthy humans adminisIncrease in glucose metabolism in healthy humans
administered with green tea. Oral glucose tolerance
curves (OGTT, n = 22) and basal blood glucose levels (Basal,n = 25) before and 30, 60 and 120 min after administration
with either a suspension of green tea powder (●) or hot
water (❍). All participants were fasted since the last supper
before starting the experiment at 9:30 a.m. In the glucose tolerance test, 225 ml of Trelan-G75 containing 75 g glucose
was perorally administered 10 min after drinking a suspension of green tea powder (1.5 g/150 ml hot water) or hot
water (150 ml). Effect of green tea or hot water on basal
blood glucose levels was examined without administrating
Trelan-G75. Blood glucose levels (mg/dl) measured wereaveraged in each treatment group, and compared by crossover test. Values represent the means ± S.E.M. *P < 0.05, **P< 0.01; significantly different from the vehicle control at eachtime-point, determined by one-way ANOVA, followed by
Scheffé's test.

blood glucose levels (BGLs) did not significantly change
in all participants (Fig. 1).
Antihyperglycemic effects of green tea in diabetic mice
The crude extract of fresh tea leaves picked from either
No.12 or No. 13 cultivar at 300 mg/kg significantly lowered BGL 2–6 h after peroral administration in streptozotocin (STZ)-diabetic ddY mice in the fasting state (Fig. 2).
In the saline-treated control group (n = 5), BGL slightly
changed from 273 ± 28 mg/dl to 241 ± 28 mg/dl after 6 h,
whereas in the green-tea (No. 12)-treated group (n = 3),
BGL markedly changed from 235 ± 15 mg/dl to 116 ± 12
mg/dl. Similarly, BGL markedly changed from 201 ± 10mg/dl to106 ± 8 mg/dl after 6 h in the green-tea (No. 13)treated group (n = 3).

The antihyperglycemic effects of green tea powder suspension at 30, 150, and 300 mg/kg were examined 2 h afteradministration to the STZ-diabetic mice in the fastingstate. As shown in Fig. 3, green tea tended to lower BGL atPage 2 of 10(page number not for citation purposes)

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Time (h)
2

4

6

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90
80

Saline (n=5)
TTES No.12 (Chinhsuan) (n=3)
TTES No.13 (Tzuiyu) (n=3)

70
60
50
40

㧖

P<0.05120

Blood glucose level(%)

Blood glucose level (%)

0

100
80

**

60
40
20
0

FigureSTZ-diabetic
Antihyperglycemic
fasting2effects
ddY miceof green tea leaf extracts in the
Antihyperglycemic effects of green tea leaf extracts
in the fasting STZ-diabetic ddY mice. The dried
extracts of fresh tea leaves prepared at the Taiwan Tea
Experiment Station [TTES, Cultivar No. 12 (Chinhsuan) andNo.13 (Tzuiyu)] were dissolved in saline and perorally administered at 300 mg/kg into STZ-diabetic ddY mice in the fasting state. Blood glucose levels 2 h, 4 h, and 6 h afteradministration of green tea leaf extracts are expressed aspercentages of the glucose levels at 0 h (before the administration) in each mouse, and then averaged. Values represent
the means ± S.E.M. (n = 3–5). *P < 0.05; significantly different
from the blood glucose levels of STZ-diabetic mice administered with saline (control) at each time-point, determined by
one-way ANOVA, followed by Scheffé's test.

150 mg/kg, and significantly lowered BGL at 300 mg/kg.
Furthermore, the effect of green tea was compared among
different groups of mice, i.e., STZ-diabetic ddY mice, normal ddY mice, diabetic db+/db+ mice and +m/+m mice
(Fig. 4). BGLs were significantly lowered in both STZ-diabetic mice and db+/db+ mice 2 h after administration ofgreen tea powder suspension (300 mg/kg), whereas no
significant changes were observed in normal ddY miceand +m/+m mice.
No elevation of blood insulin levels was observed during
the decrease in BGL either by tea leaf extract or by green
tea suspension in STZ-diabetic mice (data not shown).
These results suggest that both fresh green tea leaf extract
and green tea suspension lower BGL without changing
serum insulin concentration.
Serum protein profiles altered in diabetic db+/db+ mice
To explore the mechanisms underlying the antihyperglycemic effect of green tea, proteomic patterns of sera fromdiabetic (db+/db+) and wild-type (+m/+m) mice werefirst investigated and compared to identify peaks specificto the diabetic state using SELDI-TOF-MS. Samples of sera

0

30

150

300

Dose (mg/kg)
FigureAntihyperglycemicthefasting
3 STZ-diabetic
effectsddYof green
mice tea powder suspension in
Antihyperglycemic effects of green tea powder suspension in the fasting STZ-diabetic ddY mice. STZdiabetic ddY mice were perorally administered with a suspension of green tea powder at 30, 150, and 300 mg/kg in thefasting state. Blood glucose levels 2 h after the administrationof green tea are expressed as percentages of the glucose
level 0 h before the administration in each mouse, and thenaveraged. Values represent the means ± S.E.M. (n = 6 – 20).
**P < 0.01; significantly different from the blood glucose levels of STZ-diabetic mice administered with saline (control, 0
mg/kg of green tea), by one-way ANOVA, followed by
Scheffé's test.

from fasting +m/+m mice were loaded onto CM10 (a cationic exchanger, pH4) ProteinChip arrays (Fig. 5). The
other types of ProteinChip Array, i.e., CM10 (a cationicexchanger, pH7), Q10 (an anionic exchanger, pH8) andIMAC30-Cu2+ (immobilized metal affinity chromatography, pH7), were also used to fractionate proteins in theserum. Then, these arrays were analyzed using a ProteinChip system, and the amount of individual serum proteins was estimated from the peak intensity of the mass
spectral signal and the mass/charge ratio (m/z) equivalent
to the molecular weight of each protein. All the spectral
peaks of serum proteins from diabetic mice, which weresignificantly different (P < 0.05, by unpaired t-test) inintensities from those of serum proteins from wild-type
mice, are shown in Fig. 6A: the peak intensities of proteinsat m/z 4119, 4203, 4206, 4211, 4579, 9311 and 18691
were >3 times lower, and those at m/z 13075, 17406,
17407, 17418, 17622, 18431, 26100 were >3 times
higher than respective peak intensities of proteins of serafrom +m/+m mice. Typical spectra are shown in Figs. 6B
and 6C (see additional file 1 concerning the other peaks).

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db+/db+

ddY-STZ

Blood glucose level (mg/dl)

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MW3000-10000

0h

500

2h
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300
ddY-normal
200
100
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Tea

SalineTea

Saline

4000

+m/+m

Tea

SalineTea Saline

FigureBlood-glucose-loweringbut
not in
4 normal mice effects of green tea in diabetic mice
Blood-glucose-lowering effects of green tea in diabetic mice but not in normal mice. STZ-diabetic ddY,
normal ddY, diabetic db+/db+, and wild-type (+m/+m) mice
were perorally administered with either a suspension ofgreen tea powder at 300 mg/kg or saline in the fasting state.
Blood glucose levels (mg/dl) were measured before and 2 hafter the administration, and then averaged. Values represent
the means ± S.E.M. (n = 5 – 21). *P < 0.05, **P < 0.01; significantly different from values at 0 h (before the administration), by one-way ANOVA, followed by Scheffé's test.

Modification of serum protein profiles of diabetic db+/db+
mice by green tea
Serum protein profiles of db+/db+ mice were investigated
2 h after green tea administration, and compared withthose administered with saline (control). Again, we confirmed a significant decrease in BGL by green tea in the
blood samples used in these proteomic analyses (data notshown). All the protein signals that were significantly
changed (P < 0.05, by unpaired t-test) in terms of peakintensity by the green tea administration, but not by thesaline administration, are described in Fig. 7A. The serum
proteomic patterns of the green-tea-treated group demonstrate that the peak intensities of proteins at m/z 11651
and 11863 decreased to less than one third after 2 h of tea
administration, whereas no significant changes wereobserved in the control group. Typical spectra are shownin Fig. 7B (see additional file 2 for the change at m/z
11651). When the results in Fig. 7A are compared withthose in Fig. 6A to identify protein peaks specific for diabetes and which are sensitive to green tea administration,a peak at m/z 4211 (4212) was found (Fig. 8). The
intensity of this peak was significantly lowered in the diabetic state (Fig. 8A), and was significantly decreased 2 hafter green tea administration (Fig. 8B).

The relative peak intensities of hemoglobin-related multisignals ranging from m/z 14974 to 15638 [9] wereincreased by green tea administration (Fig. 7A). Parallel

25
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100000MW20000-100000

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40000

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TOF-MS5of proteomic profiles in mouse serum using SELDIAnalysis
FigureAnalysis of proteomic profiles in mouse serum using
SELDI-TOF-MS. Serum samples from fasting wild-type(+m/+m) mice were loaded onto the spots of CM10 (a cationic exchanger, pH4) ProteinChip arrays. The spots wereanalyzed using the SELDI ProteinChip system on three different ranges: m/z 3000 – 10000, m/z 10000 – 20000 and m/z
20000 – 100000. The y-axis of the spectra indicates the
mass-to-charge ratio (m/z) of protonated proteins, and the xaxis indicates the relative intensities of mass spectral signals.
Note: The intensity of each peak is directly proportional tothe amount of protein, but the peak intensities could not becomparable among the different proteins because of the difference in ionization.

changes were observed at half and double these m/z values (Fig. 7A, 9) (see additional file 2 concerning thesechanges). Since multicharged protein ions are apparently
observed as proteins of different sizes in the mass spectrometry, these changes seem to reflect the modificationof one group of proteins (hemoglobin-related proteins)by green tea intake.

Discussion
Green tea is widely consumed in Asian countries, while
black tea is most popular in Western countries. The manufacturing process of green tea differs from that of blacktea because freshly picked young leaves of the tea are
immediately steamed. This process destroys the enzymes
responsible for breaking down the color pigments in theleaves and allows the tea to maintain its green color during the subsequent rolling and drying processes. The
amounts of constituent compounds are slightly different
from those of black tea. Pharmacological studies using

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A

A
Decrease

Decrease

Increasem/z

m/z 4206
30
20
10
0

4200

4300

C

4200 4300

0.1
m/z 26100

32
1
026000 27000

30

**

2.0

20
10

**

0

26000 27000

1.0

1.0
1.0
Fold‫ޓ‬change

B

Saline0+m/+m

db+/db++m/+m

db+/db+Figure ofProteomic
profiles6 seraanalyses
fromdemonstrating
diabetic and wild-typethe differences
mice in protein
Proteomic analyses demonstrating the differences inprotein profiles of sera from diabetic and wild-type
mice. Serum samples from fasting diabetic (db+/db+) miceand fasting wild-type (+m/+m) mice were loaded onto ProteinChip arrays. (A) List of peaks of proteins and/or peptides
with indicated m/z values, the peak intensities of which weresignificantly changed in the diabetic state. Relative peak intensities were averaged (n = 8). The fold changes are presented
as ratios of the peak intensities at indicated m/z values in
db+/db+ to those in the +m/+m mice. Peaks at m/z 4203,
4576, 8515, 9291, 17406 and 18678 were obtained using
CM10 ProteinChip (pH4). Peaks at 8733 and 9311 m/z wereobtained using CM10 ProteinChip (pH7). Peaks at m/z 3933,
4119, 4206, 4369, 4566, 4579, 4637, 8523, 8827, 8915, 9283,
13075, 17407, 17418, 17622, 18431, 18691, 22334 and26100 were obtained using Q10 ProteinChip. Peak at m/z
4211 was obtained using IMAC30. The chips were analyzed
by SELDI-TOF-MS. (B) and (C) Typical data of relative peakintensities in +m/+m and db+/db+ mouse sera (upper, representative of 4–8 independent observations) and the peakintensity averages at m/z 4206 and 26100 (lower, n = 4 for+m/+m, n = 8 for db+/db+). The analyzed peak is indicatedby arrows in the data of mass spectral signals. **P < 0.01; significantly different from the peak in wild-type mice, by
unpaired t-test.

Peak intensity‫ ޓ‬average

B

Peak Intensity
Average

10

1.0
1.0
Fold change

after before

0.1

Increasem/z
65906 31204
15638
15614
15612
15180
15004
14983
13831 14974
13711
11863
11846
11664
11651
11637 7926
7920
7823
7808
7611
7595
7503
7499
7495
4226
4212

26100
18691 22334
18678 18431
17622
17418
17407
17406
9311 13075
9291
8915
9283
8827
8733
8523
8515
4637
4579
4576
4566
4369
4211
4206
4203
4119
3933

15
10
5
0
15
10
5
0

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4
3
2
1
0

10

Tea

11000 12000 13000
14000
15
10
5
0

11000 12000 13000

15
10
5
0

m/z 11863**

SalineTea

Figure 7of
Changes
istration
in serumgreen tea
protein profiles in db+/db+ mice by adminChanges in serum protein profiles in db+/db+ mice byadministration of green tea. Serum samples from fasting
diabetic (db+/db+) mice 2 h after administration of green tea
suspension were loaded onto ProteinChip Arrays. The chips
were analyzed using SELDI-TOF-MS. (A) List of peaks ofproteins and/or peptides with indicated m/z values, the peakintensities of which were significantly changed by the green
tea administration. Relative peak intensities were averaged (n= 4). The fold changes are presented as ratios of the peakintensities at indicated m/z values 2 h after to before green
tea administration. Peaks at m/z 7495, 7595, 7808, 7920,
14983, 15612 and 15614 were obtained using CM10 ProteinChip (pH4), whereas those at m/z 7503, 7611, 7823, 7926,
11651, 11664, 11863, 15004 and 15638 were obtained using
CM10 ProteinChip (pH7). Peaks at m/z 4212, 4226, 7499,
11637, 11846, 13711, 13831, 14974, 15180, 31204 and65906 were obtained using IMAC30 ProteinChip. (B) Typical
data of relative peak intensities (upper, representative of 4
independent observations) and the peak intensity average at
m/z 11863 (lower, n = 4) after green tea administration andsaline control. The analyzed peak is indicated by arrows in
the data of mass spectral signals. **P < 0.01; significantly different from the peak obtained before the administration, by
unpaired t-test.

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A

Peak intensity
average

1510504100 4200

4400 4100
6
4
2
0

4200

m/z 4211
12

40

10000

12500

m/z
15000
40

30
20
10

17500

15000

20000

30000

30

14980.1+H
14980.1+2H

15614.6+H
20

15616.1+H
14821.4+H

15616.1+2H

14821.4+H
10

29946.8+H

0

40000

14978.4+H

31235.5+H

0

15000
15000

8
**

4
0

after before

B

Peak intensity
averagedb+/db++m/+mRelative peak intensity

m/z
7500

3.0

6
4
2
0
6
4
2
0

+m/+m

db+/db+SalineTea4100 4200

4100 4200

4100 4200

6
4
2
0
6
4
2
0

440041004200

beforeafter

m/z 4212

2.0
**

1.0
0

SalineTea

FigureChanges
aftergreen
8in peaktea administration
intensity at m/z 4211 in diabetic state andChanges in peak intensity at m/z 4211 in diabetic
state and after green tea administration. (A) Decreasein the peak intensity at m/z 4211 in serum samples from fasting diabetic (db+/db+) mice, compared with that of the fasting wild-type (+m/+m) mice. Typical data of relative peakintensities in +m/+m and db+/db+ mouse sera (upper, representative of 4–8 independent observations) and the averaged
intensities of the peak at m/z 4211 (lower, n = 4 for +m/+m,
n = 8 for db+/db+) indicated above by arrows. **P < 0.01;
significantly different from the peak in wild-type mice, by
unpaired t-test. (B) Decrease in the peak intensity at m/z
4212 in serum samples from fasting db+/db+ mice 2 h after
green tea administration. Typical data of relative peak intensity (upper, representative of 4 independent observations)and the averaged intensities of the peak at m/z 4212 (lower,
n = 4) after administration with either green tea or saline.
**P < 0.01; significantly different from the peak obtained
before the administration, by unpaired t-test.

FigureHemoglobin-related
db+
mice9
multiple SELDI-TOF-MS signals in db+/
Hemoglobin-related multiple SELDI-TOF-MS signals
in db+/db+ mice. Samples of sera from nontreated db+/
db+ mice in the fasting state were loaded onto ProteinChip
arrays. Left panel: hemoglobin-related multi-MS signals. From
the average of 8 data, the signals were detected from m/z
14974 to 15638 (+H) using CM10 ProteinChip (pH7). The
double-charged m/z values appear to be observed from m/z
7495 to 7823 (+2H). These values may correspond to hemoglobin α- and β-chains, and other hemoglobin-related proteins. Right panel: multi-MS signals observed at approximately
double the single-charged m/z values for the hemoglobinrelated signals, using IMAC ProteinChip. These signals may
correspond to the dimers of hemoglobin α- and β-chains.

constituent compounds in green tea have been recently
reviewed by Kaszkin et al. [10]. Green tea extracts are morestable than pure epigallocatechin gallate, the major
constituents of green tea, because of the presence of otherantioxidant constituents in the extract [10]. In general,
herbal medicines are complex mixtures of different compounds that often act in a synergistic fashion and exert
their full beneficial effect as total extracts [11].
In the present study, we demonstrated that green tea produces an antihyperglycemic effect without affecting insulin secretion in STZ-diabetic mice. We therefore explore
the mechanism underlying the green tea effect byinvestigating the serum protein profiles of db+/db+ mice,a genetic model of type 2 diabetes, using SELDI-TOF-MS.
First, we performed a preliminary analysis to determine
the peaks (molecular weights) of biomarker proteins thatwere abnormally contained in the serum of diabetic mice,
without identifying individual serum proteins. As a result,
we found that the levels of several serum proteins weresignificantly altered in the diabetic state. Secondly, weinvestigated which marker proteins are affected by green
tea administration. Despite the changes in the levels of
several serum proteins after green tea administration,none of the protein peaks specific for diabetes were sensitive to the administration, except for a peak at m/z
4211(4212). The level of this 4211(4212) Da protein was
reduced both in the diabetic state and by green tea

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administration. Thus, no simple reversed effect of green
tea on the diabetes-induced modifications of serum protein levels was observed.
The 4211(4212) Da protein has not yet been identified,but only two candidate groups of vertebrate proteins arefound by scanning Swiss-Prot database (molecularweight, 4211; molecular weight ranges, 0.1% according to
the standard errors of the current MS analysis; pI, 6; pI
ranges, 10): pancreatic polypeptide (PP) (primary accession numbers P13083, P37999, P41519, P38000,
P11967) and antibacterial peptides [β-defensin C7
precursor (018815), 4 kDa defensin (P56686), cryptidine-5 precursor (P28312), antibacterial peptide BMAP34 precursor (P56425)]. PP is primarily expressed in theendocrine cells of the pancreas, and the plasma PP concentrations are elevated by food intake [12]. Peripheral PP
administration results in a reduction in food intake andan increase in energy expenditure [12]. Interestingly, aprevious study revealed that PP-containing cell
populations in the islets of Langerhans are reduced in
db+/db+ mice with mild hyperinsulinemic diabetes [13],which is consistent with the observed reduction in theintensity of the peak at m/z 4211(4212) in the diabetic
state. We speculate that the reduction of BGL by green tea
causes a decrease in serum PP concentrations as a meansof maintaining energy homeostasis, although more precise studies are required.
The changes in serum protein profiles by green tea also
demonstrate the increase in the peak intensities of hemoglobin-related multi-MS signals, suggesting the adverse
side effects of green tea, although blood samples fromdb+/db+ mice tended to exhibit a hemolytic feature compared with those from wild-type mice (data not shown).
Interestingly, the hemoglobin-related multi-MS signals
shown in Fig. 9 may include hemoglobin α-chains and βchains [9], their dimers, and possibly variously glycatedhemoglobins. Hemoglobin A1c is used as a marker of diabetes in clinical diagnostic tests. Until now, however, theextent of glycation per hemoglobin that practicallyoccurred is not clear. More detailed analyses of serum
protein profiles using SELDI-TOF-MS will provide a more
useful clinical index of the diabetic state.
We observed that green tea improved oral glucose tolerance in humans. It is therefore likely that green tea is prophylactic against diabetes and ameliorates diabetic
hyperglycemia. Green tea consumption at moderate doses
may be associated with a reduced risk of type 2 diabetes inapparently healthy individuals by controlling postprandial hyperglycemia.

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Conclusions
The control of postprandial hyperglycemia by green tea
can help reduce the risk of type 2 diabetes. In the present
study, we provide evidence showing that green tea promotes glucose metabolism in healthy humans, andproduces an antihyperglycemic effect in diabetic mice. In
addition, we analyzed the serum protein profiles of db+/
db+ and +m/+m mice for the first time using SELDI-TOFMS, and further investigated its association with any
effects of oral green tea administration on serum proteins.
Among the several proteins that were significantly lowered in the serum of diabetic mice, the 4211(4212) Da
protein was significantly decreased after green tea administration. This is the first report demonstrating that a certain serum protein is involved in the antihyperglycemic
effect of green tea. The contribution of this protein, therefore, should be further investigated in a future study.
Moreover, we speculate that the observed effects of green
tea on BGL are primarily due to the promotion of insulin
action in peripheral tissues, such as skeletal muscles andadipocytes. Indeed, a recent paper showed that green tea
supplementation for 12 weeks ameliorates insulin resistance and increases glucose transporter IV content in a fructose-fed rat model resembling the human type 2 diabetes
mellitus [3]. Since the administration of green tea produced an acute antihyperglycemic effect on BGL in diabetic mice in the present study, additional mechanisms,
such as changes in amelioration or enhancement of insulin action, should be clarified in a future study. To elucidate whether new protein synthesis is required for green
tea action, it would be useful to examine the influence ofprotein synthesis inhibitors on the acute antihyperglycemic effect of green tea.

Methods
Housing and care of animals
C57BLKS/J db+/db+ mice (male, 9 – 11 weeks old, 36.2 –46.3 g, BGL: 243–411 mg/dl) and its age-matched control
C57BLKS/J +m/+m mice (male, 20.6–23.9 g, BGL: 110–185 mg/dl) were purchased from SLC (Shizuoka, Japan)
and used as the type 2 diabetic mouse model. Male ddY
mice (4 weeks old, purchased from SLC, Shizuoka, Japan)
were singly injected with STZ (150 mg/kg, i.v.), and thenused 4–6 weeks after the injection (28.8–38.5 g, BGL:
247–600 mg/dl). The age-matched normal ddY mice
(34.7–38.6 g, BGL: 129–197 mg/dl) were also used. The
animals were housed (3–5 per cage) under a daily cycle of
12 h light and 12 h darkness, with free access to food andwater. Animals were treated as approved by the Toyama
Medical and Pharmaceutical University Animal ResearchCommittee, and according to the guidelines for animal
experiments established by the Japanese PharmacologicalSociety.

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Sampling of blood
Mice were deprived of food for 11–14 h. Blood samples(20 µl) were collected from mouse tail veins under ether
anesthesia. In experiments for proteomic analyses andserum insulin measurements, blood samples (100 µl)
were collected from the orbital venous plexus of mice
under ether anesthesia. The food deprivation was continued throughout the measurement of blood glucose levels(until 6 h after the administration with green tea). In theoral glucose tolerance test as described below, blood samples (0.3 µl) were obtained from human skin microvessels
using FreeStyle (Nipro, Osaka, Japan).
Oral glucose tolerance test (OGTT)
Healthy human volunteers (18–24 years old) were fasted
12 h before the starting point of experiments. The participants were perorally administered with either a suspension of green tea powder or hot water at 9:20 a.m. Ten min
after the administration (at 9:30 a.m.), the participants
were perorally administered with 225 ml of Trelan-G75(Shimizu Pharmaceuticals Co., Shimizu, Shizuoka) containing 75 g glucose. BGLs were measured before and 30,
60, and 120 min after the administration with Trelan-G75(glucose). All the subjects enrolled in this study were ethnic Japanese. Before participation, the purpose and risksof the study were carefully explained, and written
informed consent was obtained from all the participants.
The protocol was approved by the Toyama Medical andPharmaceutical University Ethics Committee regulating
human research.
Measurement of blood glucose levels and serum insulinlevels
BGLs were measured using ANTSENSE II (Horiba, Kyoto,
Japan) in the mouse study and using FreeStyle (Nipro,
Osaka, Japan) in the human study. Serum insulin levels
were measured using an insulin-ELISA kit (Morinaga Seikagaku, Tokyo, Japan). To prepare the serum samples,blood samples from mice (100 µl) were kept on ice for 2h, centrifuged at 16,000 × g for 1 min at 4°C, and its
supernatant was immediately separated from the pellet.

SELDI ProteinChip Analysis
Quantitative serum proteomic profiles were measured
with SELDI-TOF-MS (Ciphergen Biosystems, Yokohama,Japan). The mouse serum was prepared as describedabove. Several types of ProteinChip Array, i.e., Q10 (ananionic exchanger, pH8), CM10 (a cationic exchanger,
pH4 and pH7) and IMAC30-Cu2+ (immobilized metal
affinity chromatography, pH7) (Ciphergen Biosystems),
were used to fractionate proteins in serum. For the proteomic analyses with Q10 ProteinChip array, samples
were 10-times diluted with denaturation buffer (7 M urea/
2 M Thiourea/ 4% CHAPS/ 1% dithiothreitol/ 2%
ampholine) and incubated on ice for 10 min, and then
10-times more diluted with buffer of 50 mM Tris-HCl(pH8). When CM10 ProteinChip array was used, samples
were 10-times diluted with the denaturation buffer andincubated on ice for 10 min, and then 10-times more
diluted with buffer of 100 mM NaOAc (pH4) or 50 mM
HEPES (pH7). When IMAC30 ProteinChip array wasused, samples were 100-times diluted with phophatebuffered saline. Spots on the different arrays were equilibrated with the buffer used for sample dilution, e.g., TrisHCl buffer for spots on Q10 array, and each sample solution (70 µl) was loaded onto two separate spots on thearrays. After incubation for 20 min with rotation spots
were rinsed with water and air-dried completely. The
spots were analyzed using the SELDI ProteinChip system(PBS-IIc, Ciphergen Biosystems). In each sample, datafrom the two spots were averaged (duplicate assay). If asignal/noise ratio was larger than 2, the peak was considered to reflect the amount of a protein. Quantitative
nature of the instrument was confirmed as previously
described [14]. The m/z value of each protein peak (themolecular weight of each detected protein) was corrected,
based on the m/z values of the external standards of peptides or proteins, the molecular weights of which are
known. Standard errors in the estimated molecularweights were less than 0.1%. The axis of abscissa in spectra(Figs 6B, 7B, 8 and 9) indicates the mass-to-charge ratio(m/z) of protonated proteins.

Table 1: Contents of catechins and caffeine in green tea samples (grams per 100 g)

catechins
C
EC
ECg
EGC
EGCg
total
caffeine

Green tea powder

No. 12aNo. 13b

0.1
0.6
0.6
0.3
5.6
7.2
4.3

1.3
1.9
1.6
0.9
10.6
16.3
4.5

0
1.0
0.2
5.8
3.5
10.5
1.7

C: (+)-catechin, EC: (-)-epicatechin, ECg: (-)-epicatechin gallate, EGC: (-)-epigallocatechin, EGCg: (-)-epigallocatechin gallate, total: total catechins. a,b
The dried extract of fresh raw leaves of tea (Camellia sinensis, Theaceae) prepared at Cultivar No.12 (a, Chinhsuan) and No. 13 (b, Tzuiyu).

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Reagents
Fresh raw leaves of tea (Camellia sinensis, Theaceae) (Cultivar No.12, Chinhsuan and No.13, Tzuiyu) were prepared at the Taiwan Tea Experiment Station, picked in
May at Nan-Tou County, and immediately used without
drying. The leaves were steeped in hot water (95°C) for 30
min and the filtrate was condensed under reduced pressure. The dry weight of the extracts was determined, andthe extracts were dissolved in saline for administration
into mice. On the other hand, green tea powder was
donated by Fukuju-en (Kyoto, Japan). The particle size
(median diameter) of the green tea powder was 2.9 µm,
according to the measurement using a Centrifugal Particle
Size Analyzer (Shimadzu, Kyoto, Japan). In the humanstudy, the tea powder (1.5 g) was added to 150 ml of hot
water (80°C) and then whipped with a bamboo whisk. In
the mouse study, the tea powder was suspended in saline
at room temperature using a sonicator. The contents ofcaffeine and catechins in these tea samples were determined by HPLC: the samples were injected into an HPLC
column (TSK-GEL, ODS-80TM, Tosoh, Tokyo, Japan); theeluent was 10 mM phosphate buffer (pH2.6)/MeCN(gradient: 5 to 15%) at a flow rate of 1.3 ml/min. The data
are shown in Table 1.
Statistical analysis
The significance of differences between two groups wasassessed by Student's t-test, and the differences between
multiple groups were assessed by one-way analysis ofvariance (ANOVA) followed by the Scheffé's multiple
range test. Values of P less than 0.05 were considered to besignificant. Especially, to determine the significance of thetime-dependent effects of green tea (Fig. 1 and 2) on the
blood glucose levels, repeated measures ANOVA was performed, and then, if the statistical significance was
detected by this analysis, further statistical comparison ateach measurement time between the groups was conducted by one-way ANOVA, followed by the Scheffé test.

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Additional material
Additional File 1
Differences in serum protein profiles between diabetic and wild-typemice Typical data of relative peak intensities in +m/+m and db+/db+
mouse sera (left 2 panels, representative of 4–8 independent observations)and the peak intensity averages at m/z indicated (right panels; +m/+m:
open column, n = 4; db+/db+: closed column, n = 8). The analyzed peakis indicated by arrows in the data of mass spectral signals. **P < 0.01;
significantly different from the peak in wild-type mice, by unpaired t-test.
Types of ProteinChip used were described in the Fig. 6 legend.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712210-4-18-S1.pdf]

Additional File 2
Changes in serum protein profiles of db+/db+ mice after green tea
administration MS spectra shows typical changes in the serum protein
profiles of db+/db+ mice administered with saline (left) or green tea(right). Graph shows the peak intensity averages at m/z indicated, before(open column) and after (closed column) administration with saline (n= 4) or green tea (n = 4). The analyzed peak is indicated by arrows in the
MS spectra. **P < 0.01; significantly different from the peak obtained
before the administration, by unpaired t-test. Types of ProteinChip usedwere described in the Fig. 7 legend.
Click here for file
[http://www.biomedcentral.com/content/supplementary/14712210-4-18-S2.pdf]

Acknowledgements
The present work was supported in part by Fugaku's Grant by Charitable
Trust (Japan). Powdered green tea was kindly donated by Fukujuen (Sourakugun, Kyoto, Japan). The donator had no role in study design, data analysis, data interpretation or writing of the manuscript. We thank Dr. Y
Sugihara, Y Fukuda, and S Hara for technical assistance; Dr. R Taniguchi(CHA Research Center, Fukujuen) for measurement of particle size ofgreen tea powder and the amount of caffeine and catechins in the tea samples; and Dr. H Tanaka and R Wakatabe (Ciphergen Biosystems KK, Yokohama Laboratory, Japan) for measurement and analysis of SELDI-TOF-MS
data.

Authors' Contributions

References

IK had the original idea for the study, designed and coordinated the experiments, and wrote the manuscript. JW
prepared fresh young leaves of green tea and the hot water
extract. MT assisted with measurement of blood glucose
and insulin level. MI, HT and TS were involved in study
design, data analysis, data interpretation and writing of
the manuscript. All authors read and approved the final
manuscript.

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