Oral administration of Bis(aspirinato)zinc(II) complex ameliorates hyperglycemia and metabolic syndrome-like disorders in spontaneously diabetic KK-A(y) mice: structure-activity relationship on zinc-salicylate complexes.

Abstract

In recent years, the number of patients suffering from diseases, such as cancer, apoplexy, osteoporosis, hypertension, and type 2 diabetes mellitus is increasing worldwide. Type 2 diabetes, a lifestyle-related disease, is recognized as a serious disease. Various types of pharmaceutics for diabetes have been used. Since the relationship between diabetes and biometals such as vanadium, copper, and zinc ions has been recognized for many years, we have been developing the anti-diabetic metal complexes as new candidates. We found that several zinc(II) (Zn) complexes exhibit glucose-lowering activity for treating type 2 diabetes. High doses of salicylates have been known to reverse hyperglycemia and hyperinsulinemia in type 2 diabetic patients. These findings strongly suggest that the combined use of Zn and salicylates achieves the synergism in treating type 2 diabetes. Because aspirin, acetyl salicylic acid, has a chelating ability, we used it as a ligand to Zn. Several Zn-salicylate complexes were prepared and their biological activities were examined in this study. The complexes with an electron-withdrawing group in the ligand exhibited higher in vitro insulinomimetic activity than those of Zn complexes with an electron-donating group in the ligand. When bis(aspirinato)Zn (Zn(asp)₂) complex was orally administered on KK-A(y) mice with hereditary type 2 diabetes, the diabetic state was improved. In addition, this complex exhibited normalizing effects on serum adiponectin level and high blood pressure in metabolic syndrome. In conclusion, Zn(asp)₂ complex is newly proposed as a potent anti-diabetic and anti-metabolic syndrome agent.

Full text

Page 1

The number of patients suffering from diabetes mellitus in
the 21st century is increasing worldwide.1)Several types of
injectable insulin preparations are available for patients with
type 1 diabetes, characterized by progressive b-cell destruc-
tion and deficiency of intrinsic insulin secretion. On the other
hand, various types of synthetic pharmaceutics are clinically
used for the treatment of type 2 diabetes, characterized by in-
sulin resistance. In recent years, new types of anti-diabetic
pharmaceutics such as glucagon-like peptide-1 (GLP-1) re-
ceptor agonist and dipeptidyl peptidase-4 (DPP-4) inhibitors
are developed and clinically available for treatment.2)
For many years, the relationship between diabetes and bio-
metals such as vanadium, copper, and zinc ions has been rec-
ognized and discussed.3)We have developed anti-diabetic
metal complexes as new candidates for hypoglycemic agents.
Since 2000, we have proposed several types of zinc(II) (Zn)
complexes with various coordination modes,4—8)in which Zn
complexes with Zn(O4) and Zn(N2O2) coordination environ-
ments showed anti-diabetic effect on oral administration.9)
Coincidently, a high dose of salicylates was found to reverse
hyperglycemia and hyperinsulinemia in type 2 diabetic pa-
tients.10)These findings strongly suggest that the combined
use of Zn and salicylates achieves the synergism in treating
diabetes. We then tried to use aspirin (acetylsalicylic acid)
and its related compounds with two oxygen atoms as ligand
to Zn.
Salicylic acid, a colorless crystalline organic acid, func-
tions as a plant hormone.11)It is probably the best known
compound that is chemically similar but not identical to the
active component of aspirin. The medicinal property of sali-
cylic acid has been known mainly for pain and fever relieves
since ancient times. A side effect of salicylic acid such as
stomachache, however, was replaced by aspirin with low side
effect. Aspirin, which is a medicine in the family of salicyl-
ates, has been used as an analgesic, anti-pyretic, and anti-
inflammatory.12,13)It has also an anti-platelet effect with
low doses to prevent both heart attacks and thrombus forma-
tion in hyper-coagulate states.14,15)On the other hand, over 100
years ago, a high-dose salicylic acid treatment was reported
to reduce the glycosuria in type 2 diabetic patients.16)Later in
1957, treatment of diabetes with aspirin for 10—14d was
found to improve the impaired oral glucose tolerance test in
the diabetic patients.17)The mechanism how salicylic acid
may affect the whole-body glucose homeostasis remains
unknown until a recent finding that salicylic acid inhibits
the activity of inhibitory kB kinase-b (IKKb), a known ser-
ine kinase.10,18,19)
In light of these findings, we hypothesized that Zn–salicy-
late complexes might be effective for not only treating type 2
diabetes and its complications but also improving metabolic
syndrome. To verify this hypothesis, we have examined
whether Zn–salicylate complexes containing Zn–aspirin
complex show the anti-diabetic activity in vitro and in vivo.
This paper reports here that daily oral administrations of the
Zn–aspirin complex improve diabetes and other metabolic
abnormalities in obesity-linked type 2 diabetic KK-Aymice.
972 Vol. 59, No. 8Regular Article
Oral Administration of Bis(aspirinato)zinc(II) Complex Ameliorates
Hyperglycemia and Metabolic Syndrome-Like Disorders in
Spontaneously Diabetic KK-AyMice: Structure–Activity
Relationship on Zinc–Salicylate Complexes
Yutaka YOSHIKAWA,*,aYusuke ADACHI,aHiroyuki YASUI,aMasakazu HATTORI,band Hiromu SAKURAI*,c
aDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University; 5 Nakauchi-cho, Misasagi,
Yamashina-ku, Kyoto 607–8414, Japan: bDivision of Diabetes, Clinical Research Institute for Endocrine and Metabolic
Diseases, National Hospital Organization, Kyoto Medical Center; 1–1 Mukaihata-cho, Fushimi-ku, Kyoto 612–8555,
Japan: and cDepartment of Pharmaco-analytical and Biocoordination Chemistry, Faculty of Pharmaceutical Sciences,
Suzuka University of Medical Science; 3500–3 Minami-Tamagaki-cho, Suzuka, Mie 513–8670, Japan.
Received February 24, 2011; accepted May 17, 2011; published online May 19, 2011
In recent years, the number of patients suffering from diseases, such as cancer, apoplexy, osteoporosis, hy-
pertension, and type 2 diabetes mellitus is increasing worldwide. Type 2 diabetes, a lifestyle-related disease, is
recognized as a serious disease. Various types of pharmaceutics for diabetes have been used. Since the relation-
ship between diabetes and biometals such as vanadium, copper, and zinc ions has been recognized for many
years, we have been developing the anti-diabetic metal complexes as new candidates. We found that several
zinc(II) (Zn) complexes exhibit glucose-lowering activity for treating type 2 diabetes. High doses of salicylates
have been known to reverse hyperglycemia and hyperinsulinemia in type 2 diabetic patients. These findings
strongly suggest that the combined use of Zn and salicylates achieves the synergism in treating type 2 diabetes.
Because aspirin, acetyl salicylic acid, has a chelating ability, we used it as a ligand to Zn. Several Zn–salicylate
complexes were prepared and their biological activities were examined in this study. The complexes with an elec-
tron-withdrawing group in the ligand exhibited higher in vitro insulinomimetic activity than those of Zn com-
plexes with an electron-donating group in the ligand. When bis(aspirinato)Zn (Zn(asp)2) complex was orally
administered on KK-Aymice with hereditary type 2 diabetes, the diabetic state was improved. In addition,
this complex exhibited normalizing effects on serum adiponectin level and high blood pressure in metabolic
syndrome. In conclusion, Zn(asp)2complex is newly proposed as a potent anti-diabetic and anti-metabolic syn-
drome agent.
Key words
inorganic medicine; Zn–aspirin complex; anti-diabetic agent; anti-metabolic syndrome agent
Chem. Pharm. Bull. 59(8) 972—977 (2011)
© 2011 Pharmaceutical Society of Japan
∗ To whom correspondence should be addressed.e-mail: yutaka@mb.kyoto-phu.ac.jp; sakuraih@suzuka-u.ac.jp

Page 2

Experimental
Materials
pirin), 5-aminosalicylic acid, 5-bromosalicylic acid, 5-chlorosalicylic acid, 5-
fluorosalicylic acid, 5-iodosalicylic acid, FFA-kit WAKO, and polyethylene
glycol 400 (PEG) were purchased from Wako Pure Chemicals Co., Osaka,
Japan. 5-Nitrosalicylic acid was purchased from Tokyo Kasei Inc., Tokyo,
Japan. 5-Methylsalicylic acid was obtained from Kanto Chemical Co., Inc.,
Tokyo, Japan. (?)Epinephrine hydrochloride, collagenase (from Clostridium
histolyticum, Type II), and bovine serum albumin (BSA; fraction V) were
obtained from Sigma Chemical Co., St. Louis, MO, U.S.A.
Animals
Male KK-Aymice (4 weeks old and weighing 22—25g) were
obtained from CLEA Japan, Inc. (Osaka, Japan) and were used for in vivo
study when they were 12 weeks old. KK-Aymice, obtained by crossing be-
tween glucose-intolerant black KK female mice and yellow male obese Ay
mice, are characterized by hyperphagia due to leptin resistance, obesity, and
developments of hyperleptinaemia, hyperinsulinaemia, hypoadiponectine-
mia, diabetes mellitus (DM), and hypertension after approximately 8 weeks
of age.20—23)Therefore, KK-Aymice are known to serve as an excellent
model that closely resembles the obesity-linked type 2 DM in human. All
mice were allowed free access to solid food (CLEA Rodent Diet CE-2,
CLEA Japan Inc., Tokyo, the main components included are as follows;
crude protein 25.1%, crude fat 4.8%, crude fiber 4.2%, crude ash 6.7%,
nitrogen-free extract 50.0%, energy 343.1kcal, Fe 32.22mg/100g, Zn
5.68mg/100g, Cu 0.75mg/100g and other inorganic compounds and
vitamins) and tap water. They were reared in an air-conditioned room at a
temperature of 23 ?1°C and a humidity of 60?10%, with lights on from
8:00 to 20:00. All animal experiments were approved by the Experimental
Animal Research Committee of Kyoto Pharmaceutical University (KPU)
and performed according to the guidelines for animal experimentation
developed by KPU.
Preparation and Characterization of Zinc Complexes
Zn–salicylate complexes were prepared according to the methods described
previously.4—6,24)These complexes were characterized for physicochemical
properties determined by elemental analysis, IR spectrometry, and mass
spectrometry. Elemental analyses for C, H, and N were performed by a
Perkin-Elmer 240C elemental analyzer (Perkin-Elmer, Tokyo, Japan). IR
spectra were measured using a Shimadzu IR-408 spectrometer with a KBr
disk. Mass spectra were measured using a JEOL JMS-SX 102 AQQ mass
spectrometer (JEOL, Tokyo, Japan).
In Vitro Insulinomimetic Activity of Zinc Complexes
rat adipocytes (1.0?106cells/ml) were prepared as described previously
elsewhere25)and preincubated at 37°C for 30min at various concentrations
(0.1—1.0mM) of Zn complexes in Krebs–Ringer Bicarbonate (KRB) buffer
(120mM NaCl, 1.27mM CaCl2, 1.2mM MgSO4, 4.75mM KCl, 1.2mM
KH2PO4, 24mM NaHCO3, and 5mM glucose: pH 7.4) containing 2% BSA at
a total volume of 200ml. Epinephrine was then added to the reaction mix-
tures at the concentration of 0.1mM, and the resulting solutions were incu-
bated at 37°C for 3h. The reactions were stopped by soaking in ice water,
and the mixtures were centrifuged at 3000rpm for 10min. After centrifuga-
tion, the cell free supernatant was subjected to measure free fatty acids
(FFA) levels with an FFA kit WAKO. In vitro insulinomimetic activity of the
complexes was evaluated by IC50value, which defines 50% inhibition con-
centration of the complex on FFA release from the isolated rat adipocytes
treated with epinephrine.
Administration of Zinc Complexes in KK-AyMice
received daily intraperitoneal (i.p.) injections or oral administrations of Zn
complexes at various doses at 10:00 a.m. without fasting after measuring
blood glucose levels. The KK-Aymice were given Zn complexes suspended
in 5% acasia by daily i.p. injections at the doses of 1.5—3mg Zn/kg body
weight (BW) for 14d. Other mice received daily oral administrations of the
same Zn complexes dissolved in polyethylene glycol (PEG) at a dose of
15mg Zn/kg BW for 24d. Acasia and PEG were used to increase the solu-
bility of the complexes. The blood samples were obtained from the tail vein
of each mouse and subjected to measuring blood glucose levels with a Glu-
cocard (Arkray, Kyoto, Japan). The body weight of the KK-Aymice was
measured daily during the administration of Zn complexes. The intake of
solid food and drinking water in each mouse was monitored daily through-
out the course of the experiments. After the administrations of the com-
plexes for 24d, blood samples were withdrawn from the retro-orbital cav-
ernous sinus of the animals for the analyses of serum urea nitrogen (UN),
glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase
(GPT), total cholesterol (TCHO), and triglyceride (TG) under ether anesthe-
sia using a capillary at 24h after the final Zn complex administrations. Con-
centrations of UN, GOT, GPT, TCHO, and TG in the serum were determined
Zinc sulfate (ZnSO4), salicylic acid, acetyl salicylic acid (as-
Nine kinds of
Isolated male
The KK-Aymice
with the Fuji Dry Chem system (Fuji Medical Co., Tokyo, Japan). The level
of hemoglobin A1C(HbA1C) was measured 24h after the final administration
of the complex by an immunoassay method using the DCA 2000, Bayer-
Sankyo Co., Ltd., Tokyo, Japan. Serum levels of insulin, leptin, and
adiponectin were determined by a Glazyme insulin-EIA test (Wako Pure
Chemicals Co., Osaka, Japan), the leptin immunoassay kit, and the
adiponectin immunoassay kit (R&D Systems Inc., Minneapolis, MN,
U.S.A.), respectively.
Oral Glucose Tolerance Test (OGTT) on Zinc Complexes
administration of Zn complexes for 24d, OGTT was performed. The KK-Ay
mice were subjected to 12-h-fasting followed by OGTT at a dose of 1g glu-
cose/kg body weight. Blood samples were obtained without anesthesia from
the tail vein at 0, 0.25, 0.5, 0.75, 1, 1.5, 2, and 3h after the glucose adminis-
tration.
Measurement of Systolic Blood Pressure
measured in animals under conscious conditions on the final day of the com-
plex administration by the indirect tail-cuff method using the MK2000 BP
monitor (Muromachi-Kikai, Tokyo, Japan) according to the manufacture’s
instructions. The measurements were carried out at room temperature be-
cause the instrument does not require pre-warming of the animals.
Determination of Total Zinc Concentrations in KK-AyMice Treated
with the Zinc Complexes
KK-Aymice were given a suspension of Zn
complexes in PEG by oral administration at a dose of 15mg Zn/kg body
weight for 24d. After administration for 24d, the mice were sacrificed under
anesthesia with ether. Following collection of plasma samples, organs (liver,
muscle, adipose, pancreas, femur, and kidney) were dissected, weighed and
heated repeatedly at approximately 200°C with 60% HNO3, 60% HClO4,
and 30% H2O2in 50ml tall beakers. When the residues became white pow-
der, the samples were dissolved in 1% HNO3. The Zn concentrations were
determined by a graphite furnace atomic absorption spectrometer (AAS;
AA-630 and GFA-EX7i, Shimadzu Co., Kyoto, Japan).
Statistical Analysis
All results are expressed as the mean values?stan-
dard deviations (S.D.). Differences were analyzed by either the paired Stu-
dent’s t test or a one-way analysis of variance followed by Dunnett’s multi-
ple-comparison test and considered to be statistically significant when p val-
ues were ?0.01 or 0.05.
After the
Systolic blood pressure was
Results and Discussion
Physicochemical Properties of Zinc–Salicylate Com-
plexes
All prepared Zn complexes were identified by ele-
mental analyses, mass spectra, and IR spectra. The analytical
and physicochemical properties of the complexes are sum-
marized in Table 1. For the elemental analysis, both the theo-
retical and measured values of the concentrations of C, H,
and N were identical within the estimated range of experi-
mental errors. On the other hand, IR spectra of the C?O
stretching with the Zn–salicylate complexes shifted to lower
wave number than that of the ligand. From both elemental
analysis and mass spectra for the Zn–salicylate complexes,
the complex structure consisting of ligand:Zn?2:1 was
suggested. By using these complexes, we examined the in
vitro insulinomimetic activity in the isolated rat adipocytes,
which followed by the study on structure–activity relation-
ship.
In Vitro Insulinomimetic Activity of Zinc Complexes
To evaluate the in vitro insulinomimetic activity of Zn com-
plexes, we examined the effects of the complexes on FFA re-
lease from the isolated rat adipocytes, which has been pro-
posed to be a simple and convenient method free from any
radioactive materials.25,26)The activity of complexes was esti-
mated in terms of IC50value (Table 2).
Among these complexes, Zn(5NO2sal)2with a strong elec-
tron-withdrawing nitro group in the ligand showed the high-
est insulinomimetic activity, followed by Zn(5Isal)2and
Zn(asp)2. It is noteworthy that because both ZnSO4and asp
each exhibited essentially no insulinomimetic effects, the
synergic effect of Zn(asp)2was apparently observed.
August 2011973

Page 3

When 1/IC50values were plotted against the Hammett’s
substituent constants (s) of functional group in the Zn–sali-
cylate systems (Fig. 1), the insulinomimetic activity in terms
of 1/IC50value of the complexes was found to increase with
an increase of the electron-withdrawing property of the
group. In general, the complexes with electron-withdrawing
have low stability compared with those with electron-donat-
ing. Thus, the complex with an electron-withdrawing group
974 Vol. 59, No. 8
Table 1. Analytical and Physico-chemical Features of Zn Complexes with Salicylic-Acid Derivatives
Elemental analysis (%) (Calcd (Found))
IR FAB (?) MS m/z
[M?H]?
CHN
n (C?O)
Zn(sal)2·2H2O
sal
Zn(5msal)2·2H2O
5msal
Zn(5Fsal)2·1.8H2O
5Fsal
Zn(5Clsal)2·2H2O
5Clsal
Zn(5Brsal)2·2H2O
5Brsal
Zn(5Isal)2·3.2H2O
5Isal
Zn(5NO2sal)2·5.8H2O
5NO2sal
Zn(5NH2sal)2·2.1H2O
5NH2sal
Zn(asp)2·2H2O
asp
44.76 (44.82) 3.76 (3.72)1623
1657
1647
1661
1635
1655
1625
1673
1648
1659
1622
1665
1658
1671
1664
1695
1735
1754
337
47.60 (47.72) 4.49 (4.59)365
41.21 (41.35) 2.87 (3.14)373
37.83 (37.74)2.72 (2.80)405
31.52 (31.53)2.30 (2.27) 493
25.91 (25.83)1.98 (2.24) 589
31.27 (31.09)3.75 (3.74)5.21 (5.21)427
41.27 (41.33)4.01 (3.98)6.87 (6.72)369 [M?H]?
47.03 (46.96)3.95 (4.04)443 [M?Na]?
Table 2.
from Isolated Rat Adipocytes
Insulinomimetic Activity of Zn Complexes in the FFA Release
No.Compound IC50(mM)
1
2
3
4
5
6
7
8
Zn(5NH2sal)2
Zn(sal)2
Zn(5Fsal)2
Zn(5msal)2
Zn(5Brsal)2
Zn(5Clsal)2
Zn(5Isal)2
Zn(5NO2sal)2
Zn(asp)2
ZnSO4
1.12?0.09
1.00?0.04
0.99?0.09
0.99?0.06
0.91?0.06
0.88?0.05*
0.84?0.05*
0.75?0.05*
0.86?0.02*
1.22?0.05
Asp, 5NO2salNone
Data are presented as the means?S.D. for 3 experiments. Significance: ∗p?0.05 vs.
ZnSO4or Zn(sal)2.
Fig. 1.
Data for IC50were based on the results of Table 2, and 1/IC50values are presented as
the means?S.D. for 3 experiments.
Relationship between 1/IC50Values and Hammett’s Values
Fig. 2.
and Zn(asp)2(?) at the Doses of 1.5—3.0mg/kg Body Weight for 14d by Daily i.p. Injections
Data are presented as the means?S.D. for 5 mice. Significance: ∗∗p?0.01 vs. control, ∗p?0.05 vs. control.
Changes of Blood Glucose Levels (a) and Body Weight (b) in Control Diabetic KK-AyMice (?) and KK-AyMice Treated with Zn(5NO2sal)2(?)

Page 4

is softly-coupled between Zn and ligands. Moreover, it was
reported that Zn(asp)2is four-coordinated complex,24)indi-
cating that the complex has low stability.
The present result is the first example showing the correla-
tion between the in vitro insulinomimetic activity of Zn com-
plexes and the Hammett’s substituent constants. In connec-
tion to the result, the similar correlation has been reported in
the vanadyl (VO(II)) complexes with hydroxythiazolethione
ligands.27)These correlations suggest that (1) both Zn and
vanadyl complexes have common action mechanism in the
isolated rat adipocytes and (2) high stability of the complex
does not require to exhibit its high insulinomimetic activity,
but moderate stability might be necessary.
In Vivo Anti-diabetic Effect of Zinc Complexes
on the results from in vitro experiment, we examined the ef-
fects of Zn(asp)2and Zn(5NO2sal)2on the changes of blood
glucose levels in KK-Aymice by daily i.p. injections and oral
administrations. When KK-Aymice received daily i.p. injec-
tions of the Zn complexes for the 14d, the blood glucose
level at 10 a.m. lowered to the normal range, as shown in Fig.
2. The efficacy of glucose-lowering
Zn(asp)2?Zn(5NO2sal)2in order (Fig. 2a). The body weight
of the animals hardly changed even under the complex ad-
ministrations (Fig. 2b).
Based
activity was
We then examined whether the orally administered Zn
complexes show hypoglycemic effect in KK-Aymice. Ad-
ministration of Zn(asp)2at a dose of 15mg Zn/kg body
weight for 24d lowered the blood glucose levels, being sig-
nificantly greater than the other complexes (Fig. 3). The Zn
complexes have little impact on the body weight change
(data not shown). In this study, both Zn(sal)2and aspirin
were used as the control (Fig. 3). The diabetic state of the
mice was clearly improved by administration of the Zn(asp)2
for 24d, as evaluated by the OGTT (Fig. 4). The change in
HbA1clevels, which shows the number of glucose molecules
attached to hemoglobin (glycated hemoglobin), was also ex-
amined in KK-Aymice treated with PEG alone (control),
Zn(sal)2, Zn(5NO2sal)2, sal, or Zn(asp)2for 24d. The change
in HbA1clevel is used as an average of blood glucose control
over 2—3 months in diabetic patients. The HbA1clevel in the
KK-Aymice treated with Zn(asp)2decreased significantly
compared with that in the PEG-treated KK-Aymice (con-
trol). In contrast, Zn(5NO2sal)2, Zn(sal)2, and sal did not im-
prove the HbA1clevel in KK-Aymice (Fig. 5). These results
indicate that Zn(asp)2treatment achieves good glycemic con-
trol in KK-Aymice compared with other complexes.
The levels of GOT, GPT, UN, and TCHO in the plasma of
the KK-Aymice are summarized in Table 3. No changes in
the serum parameters related to renal disturbance (UN) and
TCHO levels were observed between the treated and un-
treated KK-Aymice. In the GOT and GPT levels that indicate
liver damage, there was no difference between the treated
and untreated KK-Aymice. In present experiment, Zn(asp)2
showed the hypoglycemic effect. Previously, we proposed
that Zn complexes exhibited both insulinomimetic and
antidiabetic effects by activating insulin signaling cascade
through Akt/PKB phosphorylation.28,29)The Zn(asp)2is also
speculated to exhibit the hypoglycemic effect with the similar
mechanism.
Improvement of Insulin Resistance, Leptin Resistance
and Hypertension, Increase of Depressed Plasma
Adiponectin Levels in KK-AyMice by Oral Administra-
tion of Zinc Complexes
There is increasing evidence that
the adipose tissue is an important organ that secretes biologi-
cally active substances called adipocytokines, such as tumor
necrosis factor-a, resistin, leptin, adiponectin, and plasmino-
gen activator inhibitior-1 etc.30—32)In addition, several
August 2011975
Fig. 3.
ments in the Control KK-AyMice and KK-AyMice Treated with Zn(sal)2,
Zn(5NO2sal)2, Zn(asp)2, and aspa)at the Dose of 15mg Zn/kg Body Weight
for 24d by Daily Oral Administrations
a) Dose of asp: 82.8mg/kg body weight. Data are presented as the means?S.D. for 5
mice. Significance: ∗p?0.05 vs. before treatment.
Changes of Blood Glucose Levels before and after Several Treat-
Fig. 4.
Zn(asp)2(?), and asp (?)a)at the Dose of 15mg Zn/kg Body Weight for 24d by Daily Oral Administrations
a) Dose of asp: 82.8mg/kg body weight. Data are presented as the means?S.D. for 5 mice. Significance: ∗p?0.05 vs. control.
Oral Glucose Tolerance Tests (OGTT) in Control Diabetic KK-AyMice (?) and KK-AyMice Treated with Zn(sal)2(?), Zn(5NO2sal)2(?),

Page 5

adipocytokines have been shown to influence insulin sensi-
tivity.33,34)These reports suggest that correcting for the ab-
normality of adipocytokines is useful to treat diabetes. When
we measured the parameters indicating insulin resistance,
such as plasma insulin, leptin, adiponectin, and TG levels
after administrations of the Zn complexes for 24d (Fig. 5),
the plasma insulin (17?9mU/ml), leptin (33?14ng/ml), TG
(129?16mg/dl), and adiponectine (9175?1126mg/ml) lev-
els in the Zn(asp)2-treated KK-Aymice were improved sig-
nificantly to those of the PEG-treated control mice. It is well
known that KK-Aymice show hyperinsulinemia, hyper-
leptinemia, hypertriglycemia, and hypoadiponectinemia and
thus develop diabetes.20—23,35)Plasma insulin, leptin, TG, and
adiponectin levels were also estimated to be 51?26mU/ml,
53?13ng/ml, 192?57mg/dl, and 6175?1466mg/ml in the
PEG-treated KK-Aymice, respectively. On the other hand,
the changes of these parameters treated with other Zn com-
plexes and ligand such as Zn(5NO2sal)2and Zn(sal)2, and sal
were observed, however, their effects were weak compared
with those of Zn(asp)2-treated KK-Aymice. These findings
demonstrate that Zn(asp)2has antidiabetic potency through
not only their glucose-lowering effect but also as their ability
to attenuate the insulin resistance in type 2 diabetes. In addi-
tion, the systolic blood pressure of the KK-Aymice which re-
ceived Zn(asp)2and asp decreased compared with that of the
control KK-Aymice (Fig. 5). Hyperinsulinemia and hyper-
leptinemia may play key roles in hypertension or hypoadipo-
nectinemia that contributes partially to the development of
hypertension such as the case in KK-Aymice.36)On the basis
of these observations, Zn(asp)2may normalize the hyperten-
sion by improving hyperinsulinemia, hyperleptinemia, and
hypoadiponectinemia.
Organ Distribution of Zinc in KK-AyMice Given Zinc
Complexes by Oral Administration
administration of Zn(sal)2, Zn(5NO2sal)2, asp, and Zn(asp)2,
Zn concentrations in the plasma, adipose, kidney, spleen,
muscle, liver, pancreas, and femur were determined by
atomic absorption spectrometric method (Fig. 6). When
Zn(asp)2was given to KK-Aymice, a significant increase in
Zn concentrations was observed in the plasma, adipose, liver,
pancreas, and femur compared with those of the control KK-
Aymice. Concentrations of Zn in the plasma and muscle
have been known to be lowered in diabetic mice than those of
healthy mice.27,37,38)Thus, Zn deficiency may induce glucose
intolerance and insulin resistance.39,40)Our present results
suggest that improvement of Zn deficiency is one of the im-
portant factors for treating diabetes. Since it was reported
that both acinar cell necrosis and edema in the pancreas were
observed histopathologically after a high dose (300mg Zn/kg
BW) of Zn treatment.41)It will be needed to examine the
pancreas toxicity after the long term and high dose adminis-
After the 24d of oral
976Vol. 59, No. 8
Table 3.
lesterol) Levels in Control Diabetic KK-AyMice and KK-AyMice Treated
with Zn(sal)2, Zn(5NO2sal)2, Zn(asp)2, and aspa)at the Dose of 15mg Zn/kg
Body Weight for 24d by Oral Administrations
Serum GOT, GPT, UN (Urea Nitrogen), and TCHO (Total Cho-
GOT GPTUNTCHO
U/lmg/dl
Control
Zn(sal)2treated
Zn(5NO2sal)2treated
asp treated
Zn(asp)2treated
53?8
51?3
50?6
63?28
49?6
28?5
23?3
22?3*
27?4
24?5
27.5?4.7
22.5?2.1
25.2?1.2
24.8?12.9
28.6?4.9
139?22
135?19
136?13
145?23
148?16
a) Dose of asp: 82.8mg/kg body weight. Data are presented as the means?S.D. for 5
mice. Significance: ∗p?0.05 vs. control.
Fig. 5.
Treated with Zn(sal)2, Zn(5NO2sal)2, Zn(asp)2, and aspa)at the Dose of 15mg Zn/kg Body Weight for 24d by Daily Oral Administrations
a) Dose of asp: 82.8mg/kg body weight. Data are presented as the means?S.D. for 5 mice. Significance: ∗∗p?0.01 vs. control, ∗p?0.05 vs. control.
Hemoglobin A1cand Serum Insulin, Leptin, Adiponectin, and TG Levels and Blood Pressure of Control Diabetic KK-AyMice and KK-AyMice

Page 6

trations.
Conclusion
Synergistic effect of Zn and aspirin in the form of Zn(asp)2
was observed in vitro and in vivo studies on glucose metabo-
lism and insulin resistance. We propose here a Zn(asp)2com-
plex, which is found to exhibit more potent insulinomimetic
activity than those of Zn ion, ligand, and other Zn complexes
on the basis of in vitro activity. Oral administration of
Zn(asp)2improved not only hyperglycemia, insulin resist-
ance, leptin resistance, and hypoadiponectinemia but also hy-
pertension in type 2 diabetic KK-Aymice without lowering
their body weights. Our present study suggests that Zn(asp)2
is a new candidate for an oral therapeutic to treat type 2 dia-
betes and metabolic syndrome.
Acknowledgements
istry of Education, Culture, Sports, Science and Technology (MEXT) of
Japan (Grants-in-Aid for Scientific Research (B), Scientific Research on Pri-
ority Area, and Specially Promoted Research) for H.S.
This study was supported by Grants from the Min-
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Fig. 6.
AyMice and KK-AyMice Treated with Zn(sal)2, Zn(5NO2sal)2, Zn(asp)2,
and aspa)at the Dose of 15mg Zn/kg Body Weight for 24d by Daily Oral
Administrations
a) Dose of asp: 82.8mg/kg body weight. Data are presented as the means?S.D. for 5
mice. Significance: ∗∗p?0.01 vs. control KK-Ay, ∗p?0.05 vs. control KK-Ay.
Organ Distribution of Zn Concentration in Control Diabetic KK-