Antidiabetic activity of Zn(II) complexes with a derivative of L-glutamine

Article abstract of DOI:10.1246/bcsj.78.1077

The insulinomimetic activity of zinc(II) complexes Zn(gln-m)₂ and Zn(gln-e)₂, respectively, with L-γ-glutamyl-methylamide (gln-m) and -ethylamide (theanine: gln-e), components of green tea, were compared to that of Zn(gln)₂ with L-glutamine (gin) in an in vitro study using isolated rodent adipocytes treated with epinephrine in the presence of glucose, expressed as the IC₅₀ value (50% inhibition concentration) of free fatty acids (FFA) released from fat cells. The former were found to have higher inslinomimetic activity than that of the latter. The IC₅₀ values of the former were 0.61 and 0.56, respectively. Then, the anti-diabetic activity of a zinc(II) complex, Zn(gln-e)₂, was examined in an in vivo experiment on KK-A^γ mice, model animals of type-2 DM, by the intraperitoneal (ip) injection of 3-4 mg Zn/kg b.w./day for 13 days. The blood-glucose level of a group treated with a Zn complex, Zn(gln-e)₂, decreased significantly after 13 days compared to those of non-treated and gln-e treated groups. The serum concentrations of triglyceride (TG) and HbA_1C decreased significantly in Zn(gln-e) ₂ treated KK-A^γ mice compared to those of the gln-e treated group, respectively. Furthermore, the improvement in glucose tolerance was confirmed by an oral glucose tolerance test.

Full text of DOI:10.1246/bcsj.78.1077

Ó 2005 The Chemical Society of Japan
Bull. Chem. Soc. Jpn., 78, 1077–1081 (2005) 1077
Antidiabetic Activity of Zn(II) Complexes with a Derivative
of L-Glutamine
Kinuyo Matsumoto, Shuhei Yamamoto,1 Yutaka Yoshikawa,2 Matsumi Doe,1
ꢀ;1
Yoshitane Kojima, Hiromu Sakurai,2 Hiroko Hashimoto, and Naemi M. Kajiwara
Laboratory of Nutrition Physiology, Graduate School of Life Science, Kobe Women’s University,
2-1 Aoyama, Suma, Kobe 654-8585
1Department of Chemistry, Graduate School of Science, Osaka City University,
3-3-138 Sugimoto, Sumiyoshi-ku, Osaka 558-8585
2Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University,
5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414
Received February 1, 2005; E-mail: eualq002@ican.zaq.ne.jp
The insulinomimetic activity of zinc(II) complexes Zn(gln-m)2 and Zn(gln-e)2, respectively, with L-ꢀ-glutamyl-
methylamide (gln-m) and -ethylamide (theanine: gln-e), components of green tea, were compared to that of Zn(gln)2
with L-glutamine (gln) in an in vitro study using isolated rodent adipocytes treated with epinephrine in the presence
of glucose, expressed as the IC50 value (50% inhibition concentration) of free fatty acids (FFA) released from fat cells.
The former were found to have higher inslinomimetic activity than that of the latter. The IC50 values of the former were
0.61 and 0.56, respectively. Then, the anti-diabetic activity of a zinc(II) complex, Zn(gln-e)2, was examined in an in vivo
experiment on KK-Ay mice, model animals of type-2 DM, by the intraperitoneal (ip) injection of 3–4 mg Zn/kg
b.w./day for 13 days. The blood-glucose level of a group treated with a Zn complex, Zn(gln-e)2, decreased significantly
after 13 days compared to those of non-treated and gln-e treated groups. The serum concentrations of triglyceride (TG)
and HbA1c decreased significantly in Zn(gln-e)2 treated KK-Ay mice compared to those of the gln-e treated group,
respectively. Furthermore, the improvement in glucose tolerance was confirmed by an oral glucose tolerance test.
An increasing number of people suffering from diabetes
mellitus (DM: type 1 and type 2, but mainly type 2) is a world-
wide problem. The latest (2000) WHO estimate for the number
of people with diabetes worldwide is 177 million, and is pre-
dicted to increase to at least 300 million by 2025.1
adipocytes of rats, and that the oral administration of a high
dose of ZnCl2 (210 mg/kg body weight) to insulin-dependent
type 1 model diabetic rats treated by streptozotocin reduced
blood-glucose levels by as much as 50%.7 On the other hand,
Chen et al. found the hypoglycemic effect of ZnCl2 (20 mM)
in type 2 diabetic ob/ob mice.8 However, Zn(II) complexes
have never been examined.
In Japan,2 the number of people with a ‘‘high possibility of
developing diabetes’’ (HbA1c higher than 6.1%) has increased
from 6,900,000 to 7,400,000 during these five years, the prev-
alence being about 9.0% according to a 2002 report of the
Ministry of Health, Labor, and Welfare, Japan. Those with a
‘‘possible or pre-diabetic’’ tendency (HbA1c 5.6%–6.1%) has
also increased to about 30% compared to the figures in 1997.
Thus, several types of medicines for treating non-insulin-de-
pendent type 2 DM have been developed worldwide. However,
such therapeutic medicines have been reported to have some
problems, including side effects. As a consequence, it is de-
sired to develop clinically useful drugs with low toxicity.
The Zn2þ ion is known to be one of the most important es-
sential trace elements found in living organisms as well as in
many metalloproteins and metalloenzymes.3 Among the many
pharmacological and nutritional roles of the Zn2þ ion,4 this
metal ion was found in 1980 to stimulate lipogenesis in the
adipocytes of rats, which was considered to be similar to the
action of insulin.5 Following this finding, several researchers
attempted to confirm the insulinomimetic activity of the
Zn2þ ion.6–8 For example, Shisheva et al. reported that the
Zn2þ ion stimulated glucose uptake and lipid synthesis in the
It has been reported that the bis(maltolato)Zn(II) complex,
Zn(mal)2, increases zinc absorption into erythrocytes more
than a dose of free Zn2þ ion.9 Recently, we found that
Zn(mal)2 with a Zn(O4) coordination mode has a higher insu-
linomimetic activity than free Zn2þ ions, as estimated in in
vitro experiments.10 In addition, we have reported that Zn(II)
complexes with Zn(O4), Zn(N2O2), Zn(N2S2), Zn(O2S2), and
Zn(S4) coordination modes have high insulinomimetic activi-
ties with regard to in vitro experiments and blood glucose low-
ering effects.11–19 L-Glutamine (gln), an amino acid, is a com-
ponent of glutathione, the body’s primary antioxidant, which is
present in virtually every cell. If you are deficient in gln, you
are likely to be deficient in glutathione. What is fascinating
about gln is that a handful of substances can naturally boost
the level of human growth hormone secretion, essential for
normal growth and development. Recently, growth hormones
have been used experimentally for treating muscle loss, which
often occurs among invalids and elderly prone to the wasting
syndrome.20
Theanine (L-ꢀ-glutamylethylamide = gln-e) and L-ꢀ-glu-
Published on the web June 6, 2005; DOI 10.1246/bcsj.78.1077

1078 Bull. Chem. Soc. Jpn., 78, No. 6 (2005)
Antidiabetic Activity of Zn(II) Complexes
C, 36.25; H, 5.57; N, 13.76%. ½ꢁꢁD: ꢃ5:4ꢂ (H2O). Zn(gln-e)2:
Yield: 93%. mp: 281–289 ꢂC (dec.). IR (KBr): 1637 and 1606
cmꢃ1 for ꢂC=O (amide and carboxylate, respectively). 1H NMR
(D2O, pD ¼ 8:5): ꢃ 1.11 (t, 3H, J ¼ 7:3 Hz), 1.97 (m, 1H), 2.15
(m, 1H), 2.39 (t, 2H, J ¼ 7:8 Hz), 3.20 (q, 2H, J ¼ 7:3 Hz),
and 3.50 (dd, 1H, J ¼ 7:3, 5.1 Hz). Anal. Calcd for
Zn(C7H13N2O3)2: C, 40.84; H, 6.36; N, 13.61%. Found: C,
40.69; H, 6.33; N, 13.37%. ½ꢁꢁD: ꢃ11:3ꢂ (H2O).
tamylmethylamide (gln-m) are alkylated derivatives of an
amide proton of gln. The gln-e and gln-m are known to be
one of the main components of Japanese green tea, and to ex-
hibit a hypotensive effect in spontaneously hypertensive rats.21
Also, gln-e is reported to depress a rat’s liver disorder.22
In this study, we synthesized Zn(II) complexes, Zn(gln-e)2
and Zn(gln-m)2 with gln-e and gln-m, respectively, character-
ized by NMR spectral, physical, and analytical data, and esti-
mated the insulinomimetic activities in vitro in relation to the
inhibition of FFA release from isolated rat adipocytes treated
with epinephrine by comparisons with Zn(II) complexes,
Zn(asn)2 and Zn(gln)2, with L-asparagine (asn) and gln,
respectively. A moderate enhancement of the lipophilicity
of ligands can be expected to enhance insulinomimetic
activity.13,23,24
Measurement of Inhibitory Activity of Zn(II) Complexes in
vitro on the Release of Free Fatty Acid (FFA) Release from
Isolated Rodent Adipocytes by Epinephrine. Isolated adipo-
cytes of male Wistar rat (1:0 ꢅ 106 cells/mL), prepared as de-
ꢂ
scribed,26 were preincubated at 37 C for 30 min at various con-
centrations (10ꢃ4–10ꢃ3 M) of each Zn(II) complex in KRB buffer
(120 mM NaCl, 1.27 mM CaCl2, 1.2 mM MgSO4, 4.75 mM KCl,
1.2 mM KH2PO4, 24 mM NaHCO3, and 5 mM glucose: pH 7.4)
containing 2% BSA. A 10ꢃ4 M epinephrine was then added to the
reaction mixtures, and the resulting solutions were incubated at 37
ꢂC for 180 min. The reactions were stopped by soaking in ice wa-
ter, and the mixtures were centrifuged at 3000 rpm for 10 min.
The FFA levels for the outer solution of the cells were determined
with an FFA kit (Wako Pure Chemical Industries). The IC50
values were obtained based on the concentration of the Zn(II)
complex to inhibit 50% of the FFA released from the adipocytes.
Experimental Animals for in vivo Experiment. Male KK-
Ay mice aged 4 weeks (body weight 16–19 g) with type 2 diabetes
were obtained from CLEA Japan Inc. These mice were housed in-
dividually in stainless steel cages in an air conditioned room
We also observed antidiabetic activity by in vivo experi-
ments, such as by ip injection of Zn(gln-e)2 on the animal
model of type-2 DM, KK-Ay mice, and compared it to free
gln-e treated and non-treated control mice.
Experimental
Materials. All purchased reagents were of analytical or re-
agent grade, and used without purification. Elemental analyses
of the complexes were carried out on a Perkin-Elmer 240C ele-
mental analyzer (Tokyo, Japan). FT-IR spectra were recorded
on a JASCO FT/IR-420 spectrophotometer (Tokyo, Japan). 1H
and 13C NMR spectra were recorded on JEOL GX-400 FT-NMR
spectrometers (Tokyo, Japan) and DSS was used as an internal
standard in a D2O solution. A small amount of NaOD or DCl in
the D2O solution was used for pH adjustment. The melting points
were determined on a Yanaco MP-J3 apparatus (Kyoto, Japan).
Specific rotations ½ꢁꢁD were measured on a Jasco DIP 300 polar-
imeter (Tokyo, Japan).
Preparation of Ligand. A ligand, gln-m, was prepared by a
similar method to a reference.25 A Cu(II) complex with pyroglu-
tamate was prepared by pyroglutamic acid and basic cupper car-
bonate. The Cu(II) complex was treated with a 40% methyl-
amine/methanol solution at 70 ꢂC for 7 d in an autoclave. The sol-
vent was removed under reduced pressure, after which the residue
was dissolved in water, then treated with H2S gas; the deposited
copper sulfide was then filtrated out. The filtrate was evaporated
under reduced pressure and washed with methanol.
ꢂ
(23 ꢆ 2 C) with a 12-h light and dark cycle (lighting from 7:00
to 19:00) and given free access to solid food (CE-2: CLEA Japan
Inc.) and tap water. All in vivo experiments were approved by the
Experimental Animal Research Committee at Kobe Women’s
University.
These mice were divided into three groups of 5 mice each after
the onset of type 2 diabetes (9 weeks of age): an untreated group, a
gln-e treated group and a Zn(gln-e)2 treated group. They were giv-
en daily ip injections of each material at about 10:00 a.m. after
measuring their blood glucose levels, for a period of about 2
weeks from 9 to 11 weeks of age. KK-Ay mice were given daily
ip injections of Zn(gln-e)2 at a dose of 3 mg Zn/kg of body
weight27 for the first 7 days. The gln-e group was dosed with
the same amount as that of the gln-e as the Zn(gln-e)2 treated
group, and the control group was given the same amount of water
as the Zn(gln-e)2 solution.
The dose of Zn(gln-e)2 was changed, depending on the daily
changes in the blood glucose levels of the mice from the 8th
day. For example, when the blood-glucose level was <350 or
>350, the dose was changed to 3 and 4 mg Zn/kg of the body
weight, respectively. The blood sample for the analysis of the
glucose levels was obtained from the tail vein of each mouse,
and measured using a portable blood-glucose meter (Glucocard:
Arkray, Kyoto, Japan). The body weights of the KK-Ay mice
and the intakes of solid food and drinking water for each mouse
were measured daily throughout the experiments.
The compound was obtained as a white powder. Yield: 26%.
mp: 183–186 ꢂC. IR (KBr): 1649 and 1581 cmꢃ1 for ꢂC=O (amide
1
and carboxylate, respectively). H NMR (D2O, pD ¼ 8:3): ꢃ 2.11
(dd, 2H, J ¼ 14:9, 7.4 Hz), 2.34–2.46 (m, 2H), 2.73 (s, 3H), and
3.75 (t, 1H, J ¼ 6:2 Hz). Anal. Calcd for C6H12N2O3 0.2 H2O: C,
ꢄ
44.00; H, 7.63; N, 17.10%. Found: C, 44.12; H, 7.45; N, 16.83%.
½ꢁꢁD: þ7:1ꢂ (H2O). FAB MS: m=z 161 ½M þ Hꢁþ.
Preparation of Complexes. The Zn(II) complexes used in
this study were readily prepared by adding ZnCl2 to a methanol
solution of a lithium salt of an appropriate ligand (generated in
situ from ligands and lithium hydroxide) at room temperature.
All complexes were purified, washed with methanol, identified
by elemental analyses and IR and 1H NMR spectra, and found
to be molecular complexes without counter ions. Zn(gln-m)2:
Yield: 63%. mp: 255–284 ꢂC (dec.). IR (KBr): 1641 and 1604
cmꢃ1 for ꢂC=O (amide and carboxylate, respectively). 1H NMR
(D2O, pD ¼ 8:3): ꢃ 1.98 (m, 1H), 2.16 (m, 1H), 2.40 (t, 2H,
J ¼ 7:7 Hz), 2.74 (s, 3H), and 3.51 (s, 1H). Anal. Calcd for
Hematological Analysis. Blood samples for analyses of the
levels of triglyceride (TG), blood urea nitrogen (BUN), alanine
aminotranferease (ALT), aspartate aminotransferase (AST), and
total cholesterol (TCHO) were obtained from orbital exsanguina-
tion under ether anesthesia.
The serum concentrations of TG, BUN, ALT, AST, and TCHO
were determined from measurements courted by Fuji Dry Chem
(Fuji Medical Co., Tokyo, Japan). The Hemoglobin A1c level
Zn(C6H11N2O3)2 0.8H2O: C, 36.20; H, 5.97; N, 14.07%. Found:
ꢄ

K. Matsumoto et al.
Bull. Chem. Soc. Jpn., 78, No. 6 (2005) 1079
Table 1. 13C NMR Data of Each Ligand and Complexes, Zn(gln)2, Zn(gln-m)2, Zn(gln-e)2, and Zn(asn)2
ꢂ
in D2O at 27 C
Chemical shifts (ppm), ꢃðꢀꢃaÞÞ
Compound
pD
ꢁ
ꢄ
ꢀ
Ccarbonyl
C
C
C
Camide
Calkyl
Gln
Zn(gln)2
8.7
8.7
177.6
182.1
(4.5)
57.1
56.5
(ꢃ0:6)
29.5
31.4
(1.9)
33.8
34.5
(0.7)
180.6
181.2
(0.6)
Gln-m
Zn(gln-m)2
8.3
8.3
177.4
182.0
(4.6)
57.1
56.5
(ꢃ0:6)
29.6
31.6
(2.0)
34.4
35.1
(0.7)
178.1
178.6
(0.5)
28.8
28.8
(0.0)
Gln-e
Zn(gln-e)2
8.5
8.5
177.5
182.3
(4.8)
57.2
56.4
(ꢃ0:8)
29.8
31.7
(1.9)
34.6
35.3
(0.7)
177.3
177.8
(0.5)
37.4
37.4
(0.0)
16.2
16.3
(0.1)
asn
Zn(asn)2
8.2
8.2
177.0
181.7
(4.7)
54.3
53.8
(ꢃ0:5)
38.0
39.9
(1.9)
—
—
177.5
178.6
(1.1)
a) ꢀꢃ = ꢃ [Zn(II) complex] ꢃ (free ligand).
was measured by an immunoassay method (DCA 2000 System,
Bayer Co., Ltd., Tokyo, Japan) before and after administration
of Zn(glu-e)2.
Oral Glucose Tolerance Test (OGTT). After administration
of Zn(gln-e)2 for 13 days, the mice were fasted for 16 h and then
glucose at a dose of 1 g/kg body weight was given orally. Blood
samples for analysis of glucose levels were obtained from the cau-
dal vein at 0, 30, 60, 90, 120, and 180 min after glucose adminis-
tration, and blood glucose concentrations were measured with the
Glucocard.
Table 2. Estimated IC50 (mM) Value for the Free Fatty
Acids (FFA) Release from Isolated Rat Adipocytes of
Zn(II) Complexes in the Presence of Glucose
IC50/mM
(ꢆ S.D.)aÞ
IC50/mM
(ꢆ S.D.)aÞ
Complex
Complex
Zn(asn)2
Zn(gln-m)2
0.80 (ꢆ0:04)bÞ
Zn(gln)2
Zn(gln-e)2
1.04 (ꢆ0:09)bÞ
0.56 (ꢆ0:10)cÞ
0.61 (ꢆ0:07)cÞ
ZnSO4
1.00 (ꢆ0:12)
Statistical Analysis. Data are expressed as the means ꢆ stan-
dard deviations (S.D.). Statistical analysis was performed using
the Student’s t-test at 5% or 1% significant level of the difference.
a) Each value is expressed as the mean ꢆ SD for 3 experi-
ments. b) See Ref. 10. c) Significance at p < 0:05 vs ZnSO4
Results and Discussion
10ꢃ3 mol dmꢃ3 of the Zn(II) complexes. The apparent IC50
values, and the 50% inhibitory concentration of the Zn(II)
complexes upon FFA release, are given in Table 2. These
Zn(II) complexes, with the exception of Zn(gln)2, showed
higher insulinomimetic activity than that of the standard
ZnSO4. Consequently, we concluded that the Zn(II) complexes
with gln derivatives show higher insulinomimetic activities
compared with those of the Zn2þ ion, such as ZnSO4 and
the complex, Zn(gln)2. In previous research, we reported that
Zn(II) complexes with amino acid derivatives, picolinic acid
derivatives, and nicotine amide derivatives have high insulino-
mimetic activities.13,18,24
Such Zn(II) complexes have been revealed to promote the
glucose uptake into adipocytes, which in turn normalize the
blood-glucose levels in experimental diabetic animals.32 In this
paper, following up on these compounds, we have now also
confirmed insulinomimetic Zn(II) complexes with N-alkylated
amides of gln, and, moreover, the Zn(II) complexes with deriv-
atives of gln, which are the composition of green tea, could be
possible candidates for anti-diabetic materials found in natural
products.
The Structures of Zn(II) Complexes.
The molecular
structure of the [Zn(asn)2]n complex, was reported to by
Stephens et al. have a distorted octahedral environment.28
The carboxylic O atom and the ꢁ-amino N atom from each li-
gand coordinate to the Zn atom in a trans square–planar coor-
dination and octahedral environment is completed by carbonyl
O atoms from neighbouring molecules, creating infinite chains.
The Zn2þ ion induced the 13C NMR chemical shift changes of
ligands (asn, gln, gln-m, and gln-e), as shown in Table 1.29
Zn(II) complexes with gln and its derivatives indicate a
downfield shift (ꢀꢃ ¼ 4:5{4:8) for carbonyl similar to that
(ꢀꢃ ¼ 4:7) of the Zn(asn)2 complex. The other carbons of
Zn(II) complexes with gln and its derivatives also indicate
similar shifts compared with those of Zn(asn)2. These results
reveal that the Zn2þ ion links exclusively to the carboxylic
O atom and the ꢁ-amino N atom from each ligand, but the
amide O atom does not behave so.
The Insulinomimetic Activity of Zn(II) Complexes. The
in vitro insulinomimetic activity of Zn(II) complexes with gln
derivatives was examined in relation to inhibiting the release
of free fatty acid (FFA) from isolated rat adipocytes treated
with epinephrine. For evaluating the insulinomimetic activity
of compounds, we used the inhibition of FFA release from
rat adipocytes.26,30,31 The complexes were confirmed to act
dose-dependently in concentrations of 10ꢃ4, 5 ꢅ 10ꢃ4, and
Anti-diabetic Activity of Zn(II) Complex with Theanine
(gln-e) on KK-Ay Mice. Based on these finding, we exam-
ined the in vivo anti-diabetic activity of a Zn(II) complex,
Zn(gln-e)2, on KK-Ay mice, and compared it with its ligand
(gln-e).

1080 Bull. Chem. Soc. Jpn., 78, No. 6 (2005)
Antidiabetic Activity of Zn(II) Complexes
600
500
400
10
8
a
ab
ab
ab
6
a
a
a
300
200
100
0
a
a
ab
ab
4
2
0
−1
0
1
2
3
4
5
6
7
8
9
10 11 12
before after
control
before after
gln-e
before after
Zn(gln-e)2
day
Fig. 1. Effect of Zn(gln-e)2 on the glucose level of KK-Ay
mice. Values are the mean ꢆ SDs for the five mice in each
group, control ( ), gln-e treated ( ), and Zn(gln-e)2 treat-
ed ( ). (a) significance at p < 0:05 vs control KK-Ay
mice. (b) significance at p < 0:05 vs KK-Ay mice treated
with theanine.
Fig. 3. Change in HbA1c levels in KK-Ay mice before and
after treatment with Zn(gln-e)2 and gln-e. Values are the
mean ꢆ SDs for the five mice in each group. (a) signifi-
cance at p < 0:05 vs pre in each group.
450
400
350
300
200
250
b
150
b
200
150
100
50
b
b
a.b
100
50
0
0
0
30
60
min
90
120
Fig. 4. Effects of Zn(gln-e)2 on the oral glucose tolerance
of KK-Ay mice. Oral glucose tolerance tests were per-
formed on mice fasted for 16 h, then given the glucose so-
lution orally at a dose of 1 g/kg body weight. Values are
the mean ꢆ SDs for the five mice in each group, control
control
gln-e
Zn(gln-e)2
Fig. 2. Serum TG in control KK-Ay mice and KK-Ay mice
receiving daily ip injection of gln-e and Zn(gln-e)2 for 13
days. Values are the mean ꢆ SDs for the five mice in each
group. (a) significance at p < 0:05 vs control KK-Ay mice.
(b) significance at p < 0:05 vs KK-Ay mice treated with
gln-e.
(
), gln-e treated ( ), and Zn(gln-e)2 treated ( ). (a) sig-
nificance at p < 0:05 vs control KK-Ay mice. (b) signifi-
cance at p < 0:05 vs KK-Ay mice treated with gln-e.
administration showed a tendency to increase from 7:24 ꢆ
1:0 to 8:13 ꢆ 1:1 (%) and 7:24 ꢆ 0:6 to 7:28 ꢆ 0:9 (%), re-
spectively. In contrast, the HbA1c level of the KK-Ay mice
treated with Zn(gln-e)2 decreased siginificantly before and af-
ter administration, 7:21 ꢆ 1:0 to 6:1 ꢆ 1:1 (%). These results
suggest that Zn(gln-e)2 sustained the longitudinal blood glu-
cose controlling effect.
During the treatment of Zn(gln-e)2 or gln-e for about
2 weeks, the body weight of KK-Ay mice showed no differ-
ences between the three groups, including the control (data
not shown). The blood-glucose level of KK-Ay mice (Fig. 1)
was maintained under 300 mg/dL for 2 weeks in a Zn(gln-
e)2 treated group, which showed mostly a significant decrease
compared to both the gln-e and control groups. On the other
hand, it did not show any difference between the gln-e and con-
trol groups.
There were no differences in the serum BUN, ALT, AST,
and TCHO levels between the control, treated with gln-e,
and Zn(gln-e)2 treated groups (data not shown).
However, the serum TG level, 127:7 ꢆ 17:8 (mg/dL), in
KK-Ay mice treated with Zn(gln-e)2 decreased significantly
compared to that, 168:0 ꢆ 20:5, of the gln-e treated mice
(Fig. 2).
After 13 days of the administration of materials, the animals
were given an oral glucose tolerance test (OGTT).
As shown in Fig. 4, the blood-glucose levels of the control
group and the group treated with gln-e KK-Ay rose to the max-
imum concentration range of the Glucose tester, which was
300 mg/dL at 30 min after glucose administration, and then
slowly decreased; there was no difference between both
groups. In contrast, the blood glucose level of KK-Ay mice
treated with Zn(gln-e)2 also rose at first, to 197:2 ꢆ 32:5
mg/dL, which was significantly lower compared with that of
the control KK-Ay mice at each point in time of 0, 30, 60,
and 120 min after administration.
There was no difference between the control and theanine
group. The levels of HbA1c are shown in Fig. 3.
HbA1c analysis was used as an index to assess the glycemic
control levels in DM. In the control KK-Ay mice and KK-Ay
mice treated with gln-e, the HbA1c levels before and after its
These results indicate the possibility that the treatment of
Zn(gln-e)2 has a normalizing effect for blood glucose disorders
on DM by improving glucose tolerance. Also, Zn(gln-e)2 has

K. Matsumoto et al.
Bull. Chem. Soc. Jpn., 78, No. 6 (2005) 1081
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14 Y. Kojima and H. Sakurai, PCT Patent, WO 01/39769 A1
(2001).
15 E. Ueda, Y. Yoshikawa, Y. Ishino, H. Sakurai, and Y.
Kojima, Chem. Pharm. Bull., 50, 337 (2002).
16 A. Katoh, T. Tsukahara, R. Saito, K. K. Ghosh, Y.
Yoshikawa, Y. Kojima, A. Tamura, and H. Sakurai, Chem. Lett.,
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17 J. Fugono, K. Fujimoto, H. Yasui, K. Kawabe, Y.
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high insulinomimetic activity, as evaluated in vitro, and
decreased the serum TG levels and HbA1c concentrations in
KK-Ay mice in vivo. Thus, Zn(gln-e)2 could be a useful anti-
diabetic material. Recently, theanine (gln-e) has been suggest-
ed to reduce fat tissue in rodents,33 and it is known that insulin
secretion has a close relation to obesity.34 Therefore, gln-e
may strengthen the antidiabetic action of Zn(II) complex.
In conclusion, we propose new candidates of insulino-
mimetic Zn(II) complexes with N-alkylated amides of gln. In
addition, the result of Zn(II) complexes with the compositions
of Japanese green tea shows not only a high insulinomimetic
activity in in-vitro experiments by the multiplication of zinc
and the ligand, but also the possibility to develop novel, clin-
ically useful and low toxic Zn(II) complexes from natural
products. In particular, we can expect the theanine/Zn com-
plex, Zn(gln-e)2, to be a highly antidiabetic material.
18 Y. Yoshikawa, K. Kawabe, M. Tadokoro, Y. Suzuki,
N. Yanagihara, A. Nakayama, H. Sakurai, and Y. Kojima, Bull.
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19 Y. Yoshikawa, E. Ueda, H. Sakurai, and Y. Kojima, Chem.
Pharm. Bull., 51, 652 (2003).
20 E. Mindell, ‘‘Supplement Bible,’’ Simon & Schuster Inc.,
New York (1998), pp. 60–61.
The authors are grateful to the members of the analytical
center of Osaka City University for elemental analyses and
FAB MS.
21 H. Yokogoshi and M. Kobayashi, Life Sci., 62, 1065
(1998).
22 K. Sugiyama, S. Wada, and S. Saeki, Hissu Aminosan
Kenkyu, 155, 67 (1999).
23 E. Ueda, Y. Yoshikawa, N. Kishimoto, M. Tadokoro,
H. Sakurai, N. Kajiwara, and Y. Kojima, Bull. Chem. Soc. Jpn.,
in press.
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