Anti-diabetic effect of organo-chalcogen (sulfur and selenium) zinc complexes with hydroxy-pyrone derivatives on leptin-deficient type 2 diabetes model ob/ob mice

Article abstract of DOI:10.3390/ijms18122647

Since the discovery of the anti-diabetic effects of zinc (Zn) complex, we synthesized several Zn complexes and evaluated their effects using the KKA^y type 2 diabetes mouse model. Recently, we demonstrated that organo-chalcogen (sulfur and selenium) Zn complexes elicit strong anti-diabetic effects. In this study, we treated leptin-deficient ob/ob mice with organo-chalcogen Zn complexes, and evaluated the resulting anti-diabetic effects in a mouse model of diabetes arising from pathogenic mechanisms different from those in KKA^y mice. C57BL/6J ob/ob mice orally received either bis(3-hydroxy-2-methyl-4(H)-pyran-4-thiono)Zn, [Zn(hmpt)2] or bis(3-hydroxy-2-methyl-4(H)-pyran-4-seleno)Zn, [Zn(hmps)₂], daily for 28 days. Both Zn complexes elicited potent blood glucose-lowering effects and improved HbA1c values. Moreover, glucose intolerance improved as evidenced by the oral glucose tolerance test, and fasting plasma insulin levels decreased in both types of Zn complex-treated mice. Zn concentrations in the liver and pancreas of [Zn(hmpt)₂]-treated mice and in the pancreas of [Zn(hmps)₂]-treated mice were increased, respectively. The results suggest that the present Zn complexes mainly exerted an anti-diabetic effect in the liver or pancreas. This study is the first to demonstrate that potent Zn complexes elicit anti-diabetic effects in not only KKAy but also ob/ob mice via a normalizing effect on insulin secretion and fasting blood glucose levels.

Full text of DOI:10.3390/ijms18122647

International Journal of
Molecular Sciences
Article
Anti-Diabetic Effect of Organo-Chalcogen
(Sulfur and Selenium) Zinc Complexes with
Hydroxy-Pyrone Derivatives on Leptin-Deficient
Type 2 Diabetes Model ob/ob Mice
Takayuki Nishiguchi 1, Yutaka Yoshikawa 2,* and Hiroyuki Yasui 1,
*
ID
1
Department of Analytical and Bioinorganic Chemistry, Division of Analytical and Physical Sciences,
Kyoto Pharmaceutical University, 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan;
kd14006@poppy.kyoto-phu.ac.jp
Department of Health, Sports, and Nutrition, Faculty of Health and Welfare, Kobe Women’s University,
4-7-2 Minatojima-nakamachi, Chuo-ku, Kobe 650-0046, Japan
2
*
Correspondence: y-yoshikawa@yg.kobe-wu.ac.jp (Y.Y.); yasui@mb.kyoto-phu.ac.jp (H.Y.);
Tel.: +81-78-303-4826 (Y.Y.); +81-75-595-4629 (H.Y.)
Received: 13 November 2017; Accepted: 2 December 2017; Published: 7 December 2017
Abstract: Since the discovery of the anti-diabetic effects of zinc (Zn) complex, we synthesized
several Zn complexes and evaluated their effects using the KKAy type 2 diabetes mouse
model. Recently, we demonstrated that organo-chalcogen (sulfur and selenium) Zn complexes
elicit strong anti-diabetic effects. In this study, we treated leptin-deficient ob/ob mice with
organo-chalcogen Zn complexes, and evaluated the resulting anti-diabetic effects in a mouse model
of diabetes arising from pathogenic mechanisms different from those in KKAy mice. C57BL/6J
ob/ob mice orally received either bis(3-hydroxy-2-methyl-4(H)-pyran-4-thiono)Zn, [Zn(hmpt)2]
or bis(3-hydroxy-2-methyl-4(H)-pyran-4-seleno)Zn, [Zn(hmps)2], daily for 28 days. Both Zn
complexes elicited potent blood glucose-lowering effects and improved HbA1c values. Moreover,
glucose intolerance improved as evidenced by the oral glucose tolerance test, and fasting plasma
insulin levels decreased in both types of Zn complex-treated mice. Zn concentrations in the liver and
pancreas of [Zn(hmpt)2]-treated mice and in the pancreas of [Zn(hmps)2]-treated mice were increased,
respectively. The results suggest that the present Zn complexes mainly exerted an anti-diabetic
effect in the liver or pancreas. This study is the first to demonstrate that potent Zn complexes elicit
anti-diabetic effects in not only KKAy but also ob/ob mice via a normalizing effect on insulin secretion
and fasting blood glucose levels.
Keywords: diabetes mellitus; organo-chalcogen zinc complexes; ob/ob mice; anti-diabetic effect;
inductively coupled plasma mass spectrometry; morphological analysis
1. Introduction
The number of adults with diabetes mellitus (DM) has been increasing annually and is expected
to reach 642 million worldwide by 2040 [1]. DM is generally classified into two main types (1 and 2),
with type 2 DM accounting for more than 90–95% of the cases [
2]. To treat DM, diet modification,
exercise therapy, and administration of various anti-diabetic agents are utilized. However, the agents
available for clinical use with DM are associated with problems such as physical and mental discomfort
owing to daily insulin injections; furthermore, some of these agents have side effects [3]. Therefore,
the development of new types of anti-diabetic agents is essential not only to treat DM but also to
improve the quality of life (QOL) in patients with this disorder.
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www.mdpi.com/journal/ijms

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Zinc (Zn) constitutes an essential trace element in humans and plays a pivotal role in health
maintenance as a cofactor of several proteins and enzymes [ ]. In 1980, Coulston and Dandona reported
the first insulin-mimetic activity of Zn ions on rat adipocytes [ ]. Subsequently, several research groups
4
5
have attempted to confirm the insulin-mimetic activity of Zn ion via analysis of the anti-diabetic effects
of Zn ions in mouse models of type 1 and type 2 DM [6–8].
In our laboratory, we synthesized Zn complexes ([Zn(mal)2]) with maltol, which is used as a
food additive, and found that the insulin-mimetic activity was higher than that of Zn ion alone [
Subsequent to this study, we synthesized and evaluated the anti-diabetic effect of several additional
Zn complexes with various ligands, such as picolinic acid, amino acid, and vitamin derivatives [10 12].
9].
–
Overall, we found that Zn complexes with S2O2 and Se2O2 coordination modes exhibited the most
potent insulin-mimetic activities and anti-diabetic effects [13,14].
In our previous studies, we evaluated the anti-diabetic effect of Zn complexes in vivo using KKAy
mice as a model of type 2 DM. KKAy mice are polygenic mice generated by transferring the yellow
obese gene (Ay) into KK mice [15]. KKAy mice spontaneously develop type 2 DM that recapitulates the
characteristics of human type 2 DM, such as hyperglycemia, obesity, and insulin resistance. However,
a major disadvantage of this model is the lack of an appropriate non-diabetic control. Furthermore,
although we have evaluated the anti-diabetic effects of Zn complexes using KKAy mice, few Zn
complexes have been evaluated for their anti-diabetic effects using mouse models of type 2 DM arising
from different pathogenic mechanisms.
Thus, we consider that it is important to confirm the generality of the anti-diabetic effects of Zn
complexes through evaluation using other diabetes mouse models. The ob/ob mouse strain constitutes
a genetic leptin-deficient monogenic model of type 2 DM that lacks functional leptin, leading to
hyperphagia, obesity, hyperglycemia, hyperinsulinemia, and insulin resistance, thus demonstrating
features of human type 2 DM [16]. Therefore, in the present study we evaluated the anti-diabetic effects
of Zn complexes, previously confirmed as potent anti-diabetic agents in KKAy mice [17], using the
ob/ob type 2 DM mouse model. Our findings indicated that Zn complexes exert potent anti-diabetic
effects in ob/ob mice as well as in KKAy mice, and that these likely occur via a normalizing effect on
insulin secretion and fasting blood glucose level.
2. Results
2.1. Syntheses of [Zn(hmpt)2] and [Zn(hmps)2]
Based on results from elemental analyses and mass spectra of [Zn(hmpt)2] and [Zn(hmps)2],
the Zn complex structure was identified as Zn:ligand at a 1:2 ratio; 1H-nucleic magnetic resonance
(NMR) spectroscopy yielded hydroxy group (OH) peaks of hmpt and hmps at 7.78 and 7.83 ppm,
respectively. However, these peaks were not present in the corresponding Zn complexes, indicating
that the hydroxy group was coordinated to the Zn. The 13C-NMR data of hmpt and hmps and their
corresponding Zn complexes are indicated in Table S1. The peaks for C1, C2 (hydroxy group-linked
carbon), and C3 (carbonyl group) were significantly shifted in the complexes compared with those
in the corresponding ligands. The downfield shifts in C1 and C2 positions of the Zn complexes were
thought to reflect a decrease in electron density in these positions because of a decrease in resonance
stabilization by the phenolic hydroxyl group (OH) owing to hydrate group (O−) coordination to Zn.
In contrast, we considered that the upfield shift of the C3 position was due to an increase in electron
density at S and Se atoms by donor-acceptor interaction. In other words, at the polarized bonds
generated between Zn of the acceptor atoms and S and Se of the donor atoms, the electron density
of the S and Se atoms is increased by a pile-up of electron density at S and Se atoms and spillover
of electron density at the Zn atom [18]. These data support a structure wherein these ligands are
coordinated to Zn with a hydrate group (C2) and sulfur- and seleno-carbonyl group (C3). From the
above results, we confirmed the structures of both [Zn(hmpt)2] and [Zn(hmps)2] (Figure 1).

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Figure 1. Chemical structures of organo-sulfur (S) and -selenium (Se) zinc (Zn) complexes.
2.2. In Vitro Insulin-Mimetic Activity Assay
The in vitro insulin-mimetic activity of [Zn(hmpt)2] was determined by assessing both free fatty
acid (FFA) release-inhibitory and glucose uptake-enhancing effects (Table 1). The activities of both
[Zn(hmpt)2] and [Zn(hmpt)2] were more effective than those of ZnSO4 (Zn ion). In addition, the IC50
of [Zn(hmpt)2] was lower than that of [Zn(hmps)2], whereas glucose uptake at 20 µM [Zn(hmps)2]
was greater than that with [Zn(hmpt)2]. Based on the results of in vitro insulin mimetic activities,
these complexes were expected to exhibit an anti-diabetic effect in type 2 DM model mice, including in
ob/ob mice.
Table 1. IC50 values of free fatty acid release and glucose uptake using 20 µM Zn complexes.
Zn Complex
IC50 (µM)
Glucose Uptake (µg/106 Cells)
[Zn(hmpt)2]
[Zn(hmps)2] [17]
ZnSO4 [17]
2 ± 1
8 ± 1
79 ± 26
138 ± 6
none
585 ± 305
Data are expressed as the means ± SDs for three runs.
2.3. In Vivo Oral Administration of [Zn(hmpt)2] and [Zn(hmps)2] to ob/ob Mice
We evaluated the anti-diabetic effect of both [Zn(hmpt)2] and [Zn(hmps)2] in vivo. Both complexes
exhibited potent daily blood glucose-lowering effect during oral administration (Figure 2). The blood
glucose levels in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice were lowered to approximately
200 mg/dL by the end of treatment (Figure 2). Moreover, [Zn(hmps)2] exhibited a daily blood
glucose-lowering effect at doses lower than those of [Zn(hmpt)2].
Figure 2. Changes in the daily blood glucose levels over 28 days in normal, control (PEG-400
administered), [Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice; doses of both [Zn(hmpt)2]
and [Zn(hmps)2] are shown in the top part of the figure. Data are expressed as the means
6–8 mice.
±
SDs for

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Body weight gains in control and [Zn(hmps)2]-treated mice were gradually observed. Although
body weight loss in [Zn(hmpt)2]-treated mice was observed during day 22 to 25 (Figure 3A), the body
weight subsequently increased after reduction of the [Zn(hmpt)2] dose. It is considered that the
body weight loss in [Zn(hmpt)2]-treated mice was likely due to decreased food intake caused
by gastrointestinal disorder (Figure 3B). In addition, water intake in control mice significantly
increased compared with the intake in normal mice, whereas the intake in both [Zn(hmpt)2]- and
[Zn(hmps)2]-treated mice significantly decreased compared with the intake in control mice on the
final day (control: 12.6
±
1.6 g, [Zn(hmpt)2]-treated mice: 7.5
±
2.5 g, and [Zn(hmps)2]-treated
mice: 6.9 0.5 g. p < 0.01 vs. control for each) (Figure 3C). These results indicated that excessive
±
drinking, which is one of the symptoms of DM, improved in the mice treated with either [Zn(hmpt)2]
or [Zn(hmps)2].
Figure 3. Changes in body weight (A); food intake (B); and water intake (C) over 28 days in normal,
control (PEG-400 administered), [Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice; data are expressed
as the means ± SDs for 6–8 mice.
We additionally performed oral glucose tolerance tests (OGTT) after oral treatment with Zn complexes
for 28 days. Fasting blood glucose levels (the levels in 0 min) in mice treated with either Zn complex
decreased, compared with the level in control mice (Control mice: 237
±
52 mg/dL, [Zn(hmpt)2]-treated
38 mg/dL, respectively
mice: 143 75 mg/dL (p < 0.05 vs. control), and [Zn(hmps)2]-treated mice: 172
±
±
(Figure 4A). We next calculated the area under the blood glucose concentration-time curve (AUC) from the
results of changing blood glucose levels after oral administration of 1 g glucose/kg body weight. AUC in
both [Zn(hmpt)2]-treated and [Zn(hmps)2]-treated mice significantly decreased compared with that in
control mice (Figure 4B). These results suggested that glucose intolerance improved in the mice treated
with either [Zn(hmpt)2] or [Zn(hmps)2].

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Figure 4. Oral glucose tolerance test (OGTT) after 28 days in normal, control (PEG-400 administered),
[Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice. ( ) Changes in blood glucose levels after gastric
gavage of 1 g glucose/kg body weight and ( ) Area under the blood glucose concentration-time
curve (AUC) in OGTT; data are expressed as the means SDs for 6–8 mice. Statistical analysis was
A
B
±
performed using the Tukey–Kramer test; significance: * p < 0.05 and ** p < 0.01 vs. normal mice,
#
p < 0.05 and ## p < 0.01 vs. control mice.
Table 2 shows the HbA1c level, which reflects the average blood glucose levels over a long period
of time, and plasma parameters after oral treatment with Zn complexes for 28 days. The HbA1c level
in control mice significantly increased compared with the level in normal mice, whereas the HbA1c
levels in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice significantly decreased compared with those
in control mice. Plasma insulin levels in control mice were also significantly elevated compared with
those in normal mice, whereas both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice showed reduced levels
compared with controls. The results of OGTT, HbA1c, and plasma insulin level assessments suggested
that the anti-diabetic effects of the Zn complexes in ob/ob mice were realized owing to improvements
in the chronic high glucose state by normalizing insulin secretion. In comparison, plasma adiponectin
levels in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice did not significantly change compared with
the level in control mice.
Both aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels, which comprise
plasma parameters related to hepatic disturbance, were significantly elevated in control compared
with normal mice because of fatty liver [19] (Table 2). Both AST and ALT levels in [Zn(hmps)2]-treated
mice were significantly elevated compared with the levels in control mice, suggesting that hepatic
disturbance in [Zn(hmps)2]-treated mice may have occurred. Moreover, blood urea nitrogen (BUN)
levels in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice were significantly elevated compared with
control mice, thus suggesting that renal damage in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice
may have occurred. Triglyceride (TG) and total cholesterol (T-CHO) levels in both [Zn(hmpt)2]
and [Zn(hmps)2]-treated mice were not significantly changed. Alkaline phosphatase (ALP) level in
control mice was significantly elevated compared with that in normal mice; however, ALP levels in
both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice significantly decreased compared with the level in
control mice.

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Table 2. HbA1c levels and plasma parameters in normal, control (PEG-400 administered),
[Zn(hmpt)2]-treated and [Zn(hmps)2]-treated mice.
Normal
Control
[Zn(hmpt)2]
[Zn(hmps)2]
6.5 ± 0.7 **,##
0.9 ± 0.3 ##
9.4 ± 0.8 **
103 ± 25 **
118 ± 68
6.7 ± 0.4 **,##
HbA1c (%)
Insulin (ng/mL)
Adiponectin (µg/mL)
AST (U/L)
4.1 ± 0.3
0.3 ± 0.0
12.8 ± 2.7
34 ± 6
7.9 ± 0.8 **
2.2 ± 1.0 **
8.2 ± 1.1 **
93 ± 19 *
1.4 ± 0.6 **
9.0 ± 0.8 **
200 ± 55 **,##,††
355 ± 138 **,##,††
24 ± 3 **,##
ALT (U/L)
BUN (mg/dL)
TG (mg/dL)
13 ± 2
164 ± 64 *
17 ± 4
23 ± 2 **,##
76 ± 25 **
189 ± 42 **
360 ± 88 ##
16 ± 1
30 ± 3
83 ± 16 **
195 ± 16 **
723 ± 126 **
87 ± 11 **
T-CHO (mg/dL)
76 ± 12
257 ± 28
186 ± 16 **
550 ± 89 **,##,††
ALP (U/L)
Data are expressed as the means
±
SDs for 6–8 mice; statistical analysis was performed using a Tukey-Kramer test;
significance: * p < 0.05 and ** p < 0.01 vs. normal mice, ## p < 0.01 vs. control mice, †† p < 0.01 vs. [Zn(hmpt)2]-treated
mice. AST, aspartate aminotransferase; ALT, alanine aminotransferase; BUN, blood urea nitrogen; TG, triglyceride;
T-CHO, total cholesterol; ALP, alkaline phosphatase.
2.4. Determination of Zn and Se Concentrations in the Organs of ob/ob Mice
We investigated the organ distributions of Zn complexes to determine the concentrations of both
Zn and Se simultaneously in the organs (plasma, liver, kidney, muscle, pancreas, spleen, and bone)
of treated mice using inductively coupled plasma mass spectrometry (ICP-MS). Zn concentrations
in the liver significantly increased after treatment with [Zn(hmpt)2] (Table 3). In contrast, only Zn
concentrations in the plasma significantly increased after oral treatment with [Zn(hmps)2].
Table 3. Zn and Se concentrations in dry tissues (µg/g) and plasma (µg/mL) in normal, control
(PEG-400 administered), [Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice.
Zn
Se
Organ
Normal
Control
[Zn(hmpt)2]
[Zn(hmps)2]
Normal
Control
[Zn(hmpt)2]
[Zn(hmps)2]
2.5 ± 0.2 **,##
46 ± 6 ##,†
68 ± 4 **
1.1 ± 0.2 **,##,††
5.1 ± 0.6 ##,††
12.7 ± 2.0 **,##,††
2.5 ± 0.4 **,#,††
3.4 ± 0.8 **,##,††
9.1 ± 2.4 **,##,††
1.9 ± 0.6
Plasma
Liver
1.8 ± 0.2
99 ± 10
77 ± 6
2.1 ± 0.1
44 ± 5 **
67 ± 3 **
47 ±10
2.3 ± 0.2 **
74 ± 30 #
73 ± 4
0.4 ± 0.1
5.4 ± 0.6
6.6 ± 0.2
1.2 ± 0.4
1.7 ± 0.5
2.4 ± 0.9
0.6 ± 0.3
0.6 ± 0.1 *
2.1 ± 0.3 **
6.4 ± 0.2
1.3 ± 0.4
2.3 ± 0.6
2.7 ± 0.8
0.5 ± 0.2
0.5 ± 0.1
2.4 ± 0.7 **
5.8 ± 0.5
1.2 ± 0.2
1.6 ± 0.9
2.2 ± 0.3
0.5 ± 0.2
Kidney
Muscle
Pancreas
Spleen
Bone
44 ± 6
45 ± 8
41 ± 2
121 ± 14
87 ± 9
133 ± 19
100 ± 17
154 ± 7 **
153 ± 18
95 ± 11
158 ± 28 *
95 ± 12
188 ± 8 ##
162 ± 10 *,††
177 ± 8
Data are expressed as the means
±
SDs for 4–8 mice; statistical analysis was performed using the Tukey-Kramer
test; significance: * p < 0.05 and ** p < 0.01 vs. normal mice, # p < 0.05 and ## p < 0.01 vs. control mice, † p < 0.05 and
††
p < 0.01 vs. [Zn(hmpt)2]-treated mice.
Table 4 shows the increased concentrations of
mice, as compared with control mice, as well as the increased concentrations of
and [Zn]/ [Se] molar ratios in the organs of [Zn(hmps)2]-treated mice. The large increase in
in the liver and pancreas of [Zn(hmpt)2]-treated mice suggested that [Zn(hmpt)2] elicits an anti-diabetic
effect in these organs (Table 4A). In comparison, because [Zn] only exhibited a large increase in
∆
[Zn] in the organs of [Zn(hmpt)2]-treated
[Zn], [Se],
[Zn]
∆
∆
∆
∆
∆
∆
the pancreas of [Zn(hmps)2]-treated mice, it was thought that [Zn(hmps)2] preferentially induces an
anti-diabetic effect in the pancreas (Table 4B).

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Table 4. Increased concentrations of Zn,
∆
[Zn] (nmol/g), in organs of [Zn(hmpt)2]-treated mice
[Zn] (nmol/g) and [Se]
compared with control mice (
A), and increased concentrations of Zn and Se,
∆
∆
(nmol/g), and
∆
[Zn]/
∆
[Se] molar ratios, in organs of [Zn(hmps)2]-treated mice compared with control
mice (B).
(A)
Plasma Liver
453
(B)
Plasma Liver
Kidney Bone
86 517
Pancreas
∆
∆
[Zn] (nmol/g)
[Zn] (nmol/g)
3
302
Kidney Bone
Pancreas
6
6
23
38
20
81
124
18
375
14
27
∆[Se] (nmol/g)
∆[Zn]/∆[Se]
1.0
0.61
0.25
6.9
[Zn(L)]: ∆[Zn]/∆[Se] = 1.0, [Zn(L)2]: ∆[Zn]/∆[Se] = 0.5.
Prior to oral treatment, the [Zn(hmps)2] consisted of Zn and hmps at a molar ratio of 1:2, with a
[Zn]/ [Se] molar ratio = 0.5. If Zn and the ligand formed [Zn-hmps] in an organ, the complex of
Zn and hmps would have a molar ratio of 1:1, with a ∆[Zn]/∆[Se] molar ratio = 1. We found that the
[Zn]/ [Se] molar ratios in the plasma and liver were approximately 1.0 and 0.6, respectively. Thus,
∆
∆
∆
∆
[Zn(hmps)2] in the plasma might exist in [Zn(L)] (L = ligand; hmps) chemical form, whereas in the
liver it may exist as a mixture of both [Zn(L)2] and [Zn(L)] chemical forms. The ratios in the kidney,
bone, and pancreas were 0.25, 6.9, and 27, respectively. These results indicate that dissociated hmps is
mainly distributed to the kidney, and that zinc ion (Zn2+) dissociated from [Zn(hmps)2] is primarily
distributed to both the bone and pancreas.
2.5. Histopathological Changes in the Pancreas and Liver Following Treatment with Zn Complexes
To evaluate morphological changes in the pancreas and liver, we investigated the effect of
both [Zn(hmpt)2] and [Zn(hmps)2] on pancreatic islet hypertrophy and hepatic glycogen and fat
deposition by hematoxylin and eosin (HE) staining, and compared these with the levels in control
mice. Figure 5 shows the results of evaluation of pancreatic morphology. Although the pancreatic
islet numbers per section among normal, control, and [Zn(hmpt)2] and [Zn(hmps)2]-treated mice
were not significantly changed (Figure 5B), the pancreatic islet area ratio and size in control and
[Zn(hmpt)2] and [Zn(hmps)2]-treated mice significantly increased compared with those of normal
mice (Figure 5C,D). These results demonstrate that pancreatic hypertrophy had occurred in control
and [Zn(hmpt)2] and [Zn(hmps)2]-treated mice, indicating that Zn complex treatment did not exert a
protective effect on pancreatic islets.
Glycogen and fat deposition area ratios of hepatic sectioned tissue are shown in Figure 6A,B.
Excess glycogen and fat deposition in control mice and [Zn(hmpt)2] and [Zn(hmps)2]-treated mice
were observed relative to normal mice. The ratios in control mice, [Zn(hmpt)2] and [Zn(hmps)2]-treated
mice significantly increased compared with those in normal mice (Figure 6B). However, no significant
differences in the glycogen and fat deposition area ratio in the liver were observed between control
and [Zn(hmpt)2]-treated and [Zn(hmps)2]-treated mice (Figure 6B).

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Figure 5.
) [Zn(hmpt)2]-treated; and (
(scale bar = 200 m); (
) Pancreatic islet per section (number/mm2) (normal mice: n = 57, control
mice: n = 72, [Zn(hmpt)2]-treated mice: n = 92, [Zn(hmps)2]-treated mice: n = 183 pancreatic islets);
) Pancreatic islet area ratio (%); ( ) Pancreatic islet size ( SDs
m2). Data are expressed as the means
(
A
) Pancreatic morphology in 14-week-old (
a
) normal; (
b
) control (PEG-400 administered);
(
c
d) [Zn(hmps)2]-treated mice. Hematoxylin and eosin staining,
×
100
µ
B
(
C
D
µ
±
for 6–8 mice. Statistical analysis was performed using the Steel-Dwass test; significance: * p < 0.05 and
** p < 0.01 vs. normal mice.
Figure 6.
[Zn(hmpt)2]-treated (
(scale bar = 200 m); (
(
A
) Hepatic morphology in 14-week-old normal (
); and [Zn(hmps)2]-treated mice ( ). Hematoxylin and eosin staining,
) Glycogen and fat deposition area ratios in the hepatic sectioned tissue of
a
); control (PEG-400 administered) (
b);
c
d
×
100
µ
B
normal, control (PEG-400 administered), [Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice. Data are
expressed as the means ± SDs for 6–8 mice. Statistical analysis was performed using the Steel-Dwass
test; significance: ** p < 0.01 vs. normal mice.

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3. Discussion
Several reports investigating the anti-diabetic effect of Zn supplementation in ob/ob mice
have been published previously [ 20]. However, to the best of our knowledge, the present study
8,
is the first to report the anti-diabetic effect of oral treatment with Zn complexes in ob/ob mice.
Our prior studies demonstrated that Zn complexes with hydroxy-pyrone derivatives, including
[Zn(hmps)2], elicit an anti-diabetic effect in KKAy mice, which constitute a different type of 2 DM
model mouse that is accompanied by obesity [17,21]. These anti-diabetic effects of Zn complexes with
hydroxy-pyrone derivatives suggest that Zn complexes constitute promising seed compounds for
anti-diabetic therapeutics.
Based on the results from in vitro experiments in the present study, marked anti-diabetic effects
of both [Zn(hmpt)2] and [Zn(hmps)2] on ob/ob mice were expected. We tested this hypothesis using
the dosages ([Zn(hmpt)2]: 2.5–10 mg Zn/kg body weight and [Zn(hmps)2]: 1.0–5.0 mg Zn/kg body
weight), based on the results of preliminary experiments. [Zn(hmpt)2] and [Zn(hmps)2] exerted potent
daily blood glucose lowering effect during oral treatment (Figure 2). Moreover, [Zn(hmps)2] was used
at a lower oral dose than the dose (2.0–10 mg Zn/kg body weight) utilized in our previous study
with KKAy mice [17]. Our results suggest that Zn complexes with hydroxy-pyrone derivatives may
be effective for type 2 DM caused by insulin resistance based on obesity. In contrast, two adverse
effects of treatment with Zn complexes occurred in the present study. The first was body weight loss
in [Zn(hmpt)2]-treated mice. Zn is a relative safe trace element; however, the tendency of excess Zn
intake to induce gastrointestinal disorder has been well documented [22]. It was therefore considered
that the body weight loss in [Zn(hmpt)2]-treated mice might be caused by gastrointestinal disorder
based on excess Zn intake.
The second adverse effect comprised liver disturbance in [Zn(hmps)2]-treated mice, as suggested
by the observed increase of AST and ALT levels in these animals (Table 2). Conversely, glycogen and
lipid deposition area ratios in the liver following [Zn(hmps)2] treatment were not significantly altered
compared with those of control mice (Figure 6A,B). Further, as shown in Table S2, the deposition
area may consist of TG. These results suggested that the liver disturbance was not an indirect result
of the promotion of glycogen and fat accumulation but rather a direct disturbance by [Zn(hmps)2]
or hmps. Moreover, it has been reported that necrotic areas in hepatic sections were observed in male
Wistar rats fed food containing sodium selenite, which is an inorganic selenium compound (10
µg
sodium selenite/kg/day, 4.6 g Se/kg/day), for 3 months [23]. Furthermore, it was also reported that
µ
100% lethality and vacuolation of centrilobular and peripheral hepatocytes were observed in male
ICR mice orally administered D,L-selenocysteine, an organic selenium compound, once daily (30 mg
D,L-selenocysteine/kg/day, 13.9 mg Se/kg/day), six days per week, for 30 days [24]. Taking these
results into consideration, we speculate that the liver disturbance was caused by hmps.
From the results of ∆[Zn] in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice, it was thought that
[Zn(hmpt)2] or [Zn(hmps)2] might exhibit an anti-diabetic effect in both the liver and the pancreas or the
pancreas, respectively. In the liver of ob/ob mice, it has been reported that both a decrease in glycogen
synthesis and an increase in gluconeogenesis caused by insulin-resistance occurred [25,26]. Moreover,
Zn ion has been shown to exhibit an anti-diabetic effect by inhibiting phosphatases, which negatively
regulate insulin signaling cascades, such as protein tyrosine phosphatase 1B (PTP-1B) and phosphatase
and tensin homolog deleted from chromosome 10 (PTEN) [27]. In addition, we reported that [Zn(hkt)2]
(hkt; hinokitiol) might exert an anti-diabetic effect in KKAy mice to improve insulin sensitivity by
inhibiting both PTP-1B and PTEN [28,29]. In comparison, another study found that in HepG2 cells,
Zn ion enhanced the phosphorylation of FoxO1a, which is related to gluconeogenesis, by activation of
the PI3-Akt pathway in the insulin signaling cascade, during which FoxO1a is transferred from the
nucleus to the cytoplasm [30]. Thus, [Zn(hmpt)2] might exhibit an anti-diabetic effect by the same
mechanism in the liver of ob/ob mice. Therefore, we considered that investigating the influence of
Zn complexes on gluconeogenesis-related-genes and the corresponding enzyme activities, such as

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phosphoenolpyruvate carboxykinase (PEPCK) or glucose-6-phosphatase (G6Pase), is required in
future studies.
In addition, it has recently been reported that Zn, which is expelled together with insulin from
the pancreas, protects insulin from degradation owing to its uptake into hepatic cells [31]. Thus,
it is thought that as both Zn complexes showed an anti-diabetic effect in the pancreas, [Zn(hmpt)2]
and [Zn(hmps)2] may each have supplied Zn ions to the pancreas and might exhibit an anti-diabetic
effect owing to enhancement of peripheral insulin utilization through inhibition of the insulin
degradation in the liver. This supports the potential utility of these compounds as the basis for
developing effective therapeutics for type 2 DM targeting the pancreas.
In our previous study, preservation effects on the pancreas using Zn complexes, including
[Zn(hmps)2], were observed in KKAy mice [17]. In ob/ob mice, because insulin section is increased to
compensate for the peripheral insulin insufficiency caused by insulin resistance, these animals develop
hyperinsulinemia compared with normal +/+ mice. The mechanism of increased insulin secretion
has been shown to be caused by increases of
β cells resulting from hyperplasia of pancreatic islet [32].
Thus, in the present study, we expected that the islet area in both [Zn(hmpt)2] and [Zn(hmps)2]-treated
mice would be smaller than that in control mice. However, although significant differences of the
islet area among control, [Zn(hmpt)2], and [Zn(hmps)2]-treated mice were not observed, the areas
in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice tended to be larger than that in control mice
(Figure 5). Insulin signaling in pancreatic
enlargement of islets [33 34]. Alternatively, Zn ion and Zn complexes improve DM to enhance insulin
signaling [28 29]. Moreover, the results of ICP-MS indicated that although significant differences were
β cells is known to play an important role in the
,
,
not observed, pancreatic Zn concentrations in [Zn(hmpt)2] and [Zn(hmps)2]-treated mice tended to
increase compared to those in control mice (Table 3). Thus, an enlargement of the islet area may have
occurred owing to an increase in
β cells mediated by enhancement of insulin signaling by Zn ions in
the pancreas. Furthermore, we reported that [Zn(hkt)2] increased pancreatic duodenal homeobox-1
(PDX-1), which is a transcription factor that translocates into the nucleus and induces the transcription
of various gene including insulin, glucokinase (GK) and glucose transporter type 2 (GLUT 2) [35],
as well as mRNA expression in RIN-5F cells, which are insulinoma
β cells derived from rat [29]. It has
also been reported that insulin productive cells (i.e., cells) are newly produced by overexpression
β
of PDX-1 in the pancreas via adenovirus vector [36]. Together, these observations suggest that the
islet hypertrophy in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice may also have occurred through
enhancing the increase in β cells owing to increased PDX-1 expression in the pancreas.
In contrast, the fasting plasma insulin levels in both [Zn(hmpt)2] and [Zn(hmps)2]-treated mice
decreased compared to the levels in control mice (Table 2). Considering the results regarding the
characteristics of the anti-diabetic effect including lowered daily blood glucose and improvements of
HbA1c and glucose intolerance in OGTT, although islet hypertrophy may have occurred by treatment
with both [Zn(hmpt)2] and [Zn(hmps)2], it is likely that hyperinsulinemia did not occur in neither
[Zn(hmpt)2] nor [Zn(hmps)2]-treated mice owing to the normalization of insulin secretion.
4. Materials and Methods
4.1. Chemicals
Zinc sulfate heptahydrate (ZnSO4
and polyethylene glycol 400 (PEG-400) were obtained from Wako Pure Chemicals Co. (Osaka, Japan);
3-Hydroxy-2-methyl-4(H)-pyran-4-one (maltol) was from Tokyo Chemical Industry Co. (Tokyo, Japan).
·
7H2O), lithium hydroxide monohydride (LiOH H2O),
·
Lawesson’s reagent, ( )-adrenaline hydrochloride, and bovine serum albumin (BSA: fraction V)
±
were obtained from Sigma Aldrich Inc. (St. Louis, MO, USA). Dry toluene and dichloromethane were
obtained from Nacalai Tesque Inc. (Kyoto, Japan). Acetone was obtained from JUNSEI Chemical Co.,
Ltd. (Kyoto, Japan). Dimethyl sulfoxide was obtained from Kishida Chemical Co. (Osaka, Japan).

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4.2. Analytical Instrumentation
Elemental analyses were performed using a Perkin-Elmer 240CHN elemental analyzer
(PerkinElmer Japan Co., Ltd., Tokyo, Japan). Mass spectra were recorded by a JEOL JMS-SX 102
AQQ mass spectrometer (JEOL Ltd., Tokyo, Japan). 1H- and 13C-NMR were measured using a
Varian Unity Inova spectrometer (Varian, Inc., Palo Alto, CA, USA), with tetramethylsilane as an
internal standard.
4.3. Experimental Animals
Male Wistar rats (8 weeks old), type 2 diabetic ob/ob mice (8 weeks old, n = 30), and non-diabetic
+/+ mice (8 weeks old, n = 6) were purchased from Shimizu Experimental Laboratory Inc. (Kyoto,
Japan). The ob/ob and +/+ mice were used for in vivo studies when they were 10 weeks old. They were
◦
housed 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. The animal studies were approved by the Bioscience Research Center
at the Kyoto Pharmaceutical University (KPU) and performed according to the Guidelines for Animal
Experimentation at the KPU. The number and detailed date of this approval for animal studies were
15-13-013 and 31 March 2015, respectively.
4.4. Syntheses of [Zn(hmpt)2] and [Zn(hmps)2]
4.4.1. HMPT: 3-Hydroxy-2-Methyl-4(H)-Pyran-4-Thione
Lawesson’s reagent was added to a hot solution of maltol in dry toluene at 80 ◦C under nitrogen
gas. The resulting solution was heated with stirring for 1.5 h, cooled to room temperature (22–25 ◦C),
and clarified by filtration. The filtrate was evaporated to yield a dark orange oil that was purified
using a silica gel column (4.0–6.3 µm mesh, dichloromethane solvent). After evaporation of the solvent,
the product was obtained as a yellow solid substance (yield: 53%). Anal. found (Calcd.) for C6H6O2S:
1
C, 50.70 (50.68); H, 4.02 (4.25). EI(+)-MS: m/z 142 [M+]. H-NMR (400 MHz, CDCl3): δH 7.78 (s, 1H),
7.58 (d, J = 5.04 Hz, 1H), 7.32 (d, J = 4.99 Hz, 1H), 2.45 (s, 3H). 13C-NMR (100 MHz, CDCl3): δC 185.88,
150.61, 146.97, 145.27, 124.20, 15.07.
4.4.2. HMPS: 3-Hydroxy-2-Methyl-4(H)-Pyran-4-Selenone [17]
Woollins’ reagent was added to a hot solution of maltol in dry toluene at 80 ◦C under nitrogen
gas. The resulting solution was heated with stirring for 12 h, cooled to room temperature, and clarified
by filtration. The filtrate was evaporated to yield a dark red oil that was purified by silica gel column
(4.0–6.3 µm mesh, dichloromethane solvent). The oil cooled in freezer was converted into a dark
red solid substance (yield: 57%). Anal. found (Calcd.) for C6H6O2Se: C, 38.11 (38.11); H, 2.98 (3.20).
EI(+)-MS: m/z 190 [M+]. 1H-NMR (400 MHz, CDCl3): δH 7.83 (s, 1H), 7.68 (d, J = 4.90 Hz, 1H),
7.56 (d, J = 4.90 Hz, 1H), 2.32 (s, 3H). 13C-NMR (100 MHz, CDCl3): δC 185.83, 154.21, 145.49, 145.03,
129.55, 15.63.
4.4.3. [Zn(hmpt)2]: bis(3-Hydroxy-2-Methyl-4(H)-Pyran-4-Thiono)Zn
A methanol solution of LiOH
30 min at room temperature under nitrogen gas. After 30 min, a methanol solution of ZnSO4
·
H2O was added to a solution of hmpt, followed by stirring for
7H2O
·
was added to the mixture, followed by stirring for 2.5 h under nitrogen gas. After removing the solvent,
the obtained yellow precipitate was recrystallized with acetone (yield: 32%). Anal. found (Calcd.)
for C12H10O4S2Zn: C, 41.41 (41.44); H, 2.62 (2.90). EI(+)-MS: m/z 346 [M+]. 1H-NMR (400 MHz,
CDCl3): δH 7.72 (d, J = 4.58 Hz, 1H), 7.59 (d, J = 4.58 Hz, 1H), 2.62 (s, 3H). 13C-NMR (100 MHz, CDCl3):
δC 176.83, 159.99, 154.23, 144.60, 122.54, 16.64.

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4.4.4. [Zn(hmps)2]: bis(3-Hydroxy-2-Methyl-4(H)-Pyran-4-Seleno)Zn [17]
A methanol solution of ZnSO4
5 min at room temperature under nitrogen gas. After 5 min, a methanol solution of LiOH
·
7H2O was added to a solution of hmps, followed by stirring for
H2O was
·
added to the mixture, followed by stirring for 1 h under nitrogen gas. After removing the solvent,
the obtained orange precipitate was washed with cold methanol (yield: 89%). Anal. found (Calcd.)
for C12H10O4Se2Zn: C, 32.72 (32.64); H, 2.15 (2.28). EI(+)-MS: m/z 442 [M+]. 1H-NMR (400 MHz,
CDCl3): 7.88 (d, J = 4.53 Hz, 1H), 7.63 (d, J = 4.49 Hz, 1H), 2.57 (s, 3H). 13C-NMR (100 MHz, CDCl3):
δC 175.17, 162.38, 154.42, 143.27, 126.19, 17.18.
4.5. In Vitro Insulin Mimetic Activity Assay
The in vitro insulin-mimetic activities of Zn complexes were determined by the inhibition of FFA
release from adipocytes and increase of glucose uptake into adipocytes [37]. Isolated male Wistar
rat epididymal adipocytes (1.0
×
106 cells/mL) were prepared and pre-incubated separately with
various concentrations of the different Zn complexes in 5 mM glucose in Krebs-Ringer bicarbonate
buffer (120 mM NaCl, 1.3 mM CaCl2, 1.2 mM MgSO4, 4.8 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3,
pH 7.4) containing 2% BSA, with shaking at 100 cycles/min for 30 min at 37 ◦C. Adrenaline (15
µ
L,
0.2 mM) was then added to the mixtures, and the resultant solutions were then incubated for 3 h at
37 ◦C. The reaction mixture was subsequently centrifuged at 650 g for 10 min at 4 ◦C. FFA levels in
×
the extracellular solutions were determined using an FFA measuring kit (NEFA C-test Wako) and the
IC50 value, which is the 50% inhibitory concentration of FFA-release in each complex, was calculated.
Glucose concentrations in the solutions with 20 µM Zn complexes were determined using a Fuji Dry
Chem system (Fuji Medical Co., Tokyo, Japan) and then the glucose uptake quantities/(1.0
in the 20 µM Zn complexes were calculated.
×
106 cells)
4.6. In Vivo Oral Treatment with both [Zn(hmpt)2] and [Zn(hmps)2] of ob/ob Mice
The anti-diabetic effects of both [Zn(hmpt)2] and [Zn(hmps)2] were examined in ob/ob mice.
The ob/ob and +/+ mice were used as type 2 diabetic and normal (non-diabetic) mice, respectively.
The ob/ob (10-week-old) and +/+ (10-week-old) mice received daily oral treatment of either Zn
complexes dissolved in PEG-400 or PEG-400 only, respectively. The mice received oral treatments of
[Zn(hmpt)2] or [Zn(hmps)2] at doses of 2.5–10 mg Zn/kg or 1.0–5.0 mg Zn/kg for 28 days, respectively.
Blood glucose levels (BGL) were measured using a Glucocard (Arkray, Kyoto, Japan) just before
treatment with the Zn complexes, and the doses were regulated based on the BGL. Over 28 days, BGL,
body weight, food intake, and water intake were measured every day. The number of normal mice
(+/+ mice), control mice (ob/ob mice), [Zn(hmpt)2]-treated, and [Zn(hmps)2]-treated mice was 6, 8, 8,
and 8 in the initial animal experiments, respectively. This was modified to 6, 6, 7, and 8 after the end of
experiments as two control mice died naturally by the end of experiments, and one [Zn(hmpt)2]-treated
mouse died from an accident during the oral glucose tolerance test.
4.7. OGTT to ob/ob Mice after Treatment with Zn Complexes
After treatment with [Zn(hmpt)2] or [Zn(hmps)2] for 28 days, OGTTs were performed. The ob/ob mice
and +/+ mice were fasted for 12-h and glucose (1 g/kg body weight) was given orally. Blood samples
were obtained from the tail vein at 0, 15, 30, 45, 60, 90, and 120 min after glucose administration.
Blood glucose concentrations were measured using a Glucocard.
4.8. Determination of Blood Parameters and Organ Distributions of Zn and Se in ob/ob Mice after Treatment
with Zn Complexes
After OGTT, HbA1c levels were measured in blood obtained from the mouse tail vein using
an immunoassay with a DCA 2000 analyzer (Bayer-Sankyo Co., Ltd., Tokyo, Japan). The mice were
subjected to a 12-h fast and blood samples were collected from the orbital vein or postcaval vein

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under isoflurane anes◦thesia using heparinized tools. Collected samples were then centrifuged at
g for 10 min at 4 C, and the resultant plasma samples were used to analyze various biochemical
650
×
parameters. The levels of AST, ALT, BUN, TG, T-CHO, and ALP in plasma samples were determined
using a Fuji Dry Chem system. The plasma levels of insulin and adiponectin were determined using
an ultra-sensitive Mouse Insulin ELISA Kit (Morinaga Institute of Biological Science, Inc., Kanagawa,
Japan) and an adiponectin immunoassay kit (R&D Systems Inc., Minneapolis, MN, USA), respectively.
After collecting the blood samples, mice were subjected to dissection under isoflurane anesthesia,
and the resultant tissue samples were washed in saline. Each of the samples was d◦ehydrated for
a week in desiccators, weighed, and then heated in 5% HNO3 (5 mL) for 5 h at 110 C in a closed
system. The resultant solution was centrifuged at 1250
×
g for 15 min, and the supernatant was diluted
using 5% HNO3. Both Zn and Se concentrations in organs were determined with an ICP-MS Agilent
7700x/Mass Hunter instrument (Agilent Technologies Inc., Santa Clara, CA, USA). The Zn and Se
concentrations in each sample were calculated using linear regression of the standard curves prepared
from standard solutions of the respective trace elements.
4.9. Histopathology and Morphometric Analysis
Excised pancreas and liver from 14-week-old mice were immediately fixed in 10% buffered
formalin for a week, sectioned at a thickness of 3 µm, and stained with HE for histopathological
examination. HE-stained tissue sections were scanned using a Moticam T2 (Shimadzu Co., Kyoto,
Japan) to prepare digital images. Both pancreatic islet area and glycogen and fat deposition areas were
calculated with the dedicated image processing software WinROOF (Mitani Corp., Tokyo, Japan).
4.10. Detection of Glycogen and TG in the Liver
Lipids in the liver were extracted according to modified Folch method [38]. TG in the extracted
lipids was detected using the triglyceride E test wako (Wako Pure Chemicals Co., Osaka, Japan).
Glycogen in the liver was extracted with a strong base (30% KOH aq). Then, ethanol precipitation
methods (using 95% ethanol and saturated Na2SO4), hydrolysis, and neutralization (with 2.4 M HCl
aq and 0.8 M NaOH aq) of the glycogen extracts were performed, resulting in degradation to glucose
solution. To detect the glucose levels in the solution using the glucose CII test wako (Wako Pure
Chemicals Co., Osaka, Japan), glycogen levels in the liver were estimated. Because the liver sample
of normal mice was used up during TG detection and ICP-MS analysis, glycogen content in the liver
could not be detected.
4.11. Statistical Analysis
Data are expressed as the means
one-way analysis of variance and Tukey–Kramer, or Steel-Dwass multiple comparison tests. Differences
±
SDs. Significant differences among groups were analyzed by
were considered significant at p < 0.05 or p < 0.01.
5. Conclusions
In this study, it was found that Zn complexes with hydroxy-pyrone derivatives elicited an
anti-diabetic effect in leptin-deficient ob/ob mice, as previously shown for KKAy mice, via a
normalizing effect on insulin secretion and fasting blood glucose level. Moreover, the Zn and
Se concentrations in each organ, as determined by ICP-MS, indicated that [Zn(hmpt)2] induces
anti-diabetic effects in the liver and pancreas, whereas [Zn(hmps)2] may exert its effects in the pancreas.
Although further research is needed, the findings regarding the Zn complexes used in this study
support these as suitable seed compounds for anti-diabetic therapeutics.

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Supplementary Materials: Supplementary materials can be found at www.mdpi.com/1422-0067/18/12/2647/s1.
Acknowledgments: The authors are grateful to the members of the Analytical Center of Kyoto Pharmaceutical
University (KPU) for the elemental analysis and mass spectra measurements. This study was financially supported
in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (MEXT)
Supported Program for the Strategic Research Foundation at Private Universities, 2012–2016 (S1201008). This work
was also supported by JSPS KAKENHI, Grant Numbers 25460048 (Yutaka Yoshikawa) and 26460636 (Hiroyuki
Yasui). We would like to thank Editage (available online: www.editage.jp) for English language editing.
Author Contributions: Takayuki Nishiguchi, Yutaka Yoshikawa, and Hiroyuki Yasui conceived and designed
the experiments; Takayuki Nishiguchi and Yutaka Yoshikawa performed the experiments; Takayuki Nishiguchi
analyzed the data; Takayuki Nishiguchi, Yutaka Yoshikawa, and Hiroyuki Yasui wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ALP
alkaline phosphatase
AST
aspartate aminotransferase
ALT
alanine aminotransferase
BUN
blood urea nitrogen
G6Pase
GK
glucose-6-phosphatase
glucokinase
GLUT2
HE
glucose transporter type 2
hematoxylin and eosin
hmps
hmpt
3-hydroxy-2-methyl-4(H)-pyran-4-selenone
3-hydroxy-2-methyl-4(H)-pyran-4-thione
inductively coupled plasma mass spectrometry
oral glucose tolerance test
pancreatic duodenal homeobox-1
phosphoenolpyruvate carboxykinase
phosphatase and tensin homolog deleted from chromosome 10
protein tyrosine phosphatase 1b
total cholesterol
ICP-MS
OGTT
PDX-1
PEPCK
PTEN
PTP-1B
T-CHO
TG
triglyceride
[Zn(hmps)2]
[Zn(hmpt)2]
bis(3-hydroxy-2-methyl-4(H)-pyran-4-seleno)Zn
bis(3-hydroxy-2-methyl-4(H)-pyran-4-thiono)Zn
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