Sie sind auf Seite 1von 9

2008 The Authors

Doi: 10.1111/j.1742-7843.2008.00321.x
Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

Blackwell Publishing Ltd

Hypoglycaemic Effects of Antidiabetic Drugs in


Streptozotocin-Nicotinamide-Induced Mildly Diabetic and
Streptozotocin-Induced Severely Diabetic Rats
Atsuo Tahara, Akiko Matsuyama-Yokono, Ryosuke Nakano, Yuka Someya and Masayuki Shibasaki
Drug Discovery Research, Astellas Pharma Inc., Ibaraki, Japan
(Received December 25, 2007; Accepted May 20, 2008)
Abstract: In this study, streptozotocin-induced severely diabetic rats and streptozotocin-nicotinamide-induced mildly
diabetic rats were established to compare their characteristics and to investigate the hypoglycaemic effects of antidiabetic
drugs. Results show that in streptozotocin-induced severely diabetic rats, the pancreatic insulin content decreased to
approximately 10% of that in normal rats. These severely diabetic rats also exhibited marked hyperglycaemia and impaired
glucose tolerance due to insulin secretory deficiency. In contrast, the pancreatic insulin content was approximately 50%
of normal levels in streptozotocin-nicotinamide-induced mildly diabetic rats. These mildly diabetic rats exhibited
moderate hyperglycaemia and impaired glucose tolerance due to loss of early-phase insulin secretion. Voglibose (-glucosidase
inhibitor), metformin (biguanide), glibenclamide (sulfonylurea), sitagliptin (dipeptidyl peptidase-4 inhibitor) and insulin
significantly improved glucose tolerance in streptozotocin-nicotinamide-induced mildly diabetic rats. In contrast, in streptozotocininduced severely diabetic rats, voglibose, metformin and insulin significantly improved glucose tolerance, but no significant
effect was observed for glibenclamide and sitagliptin due to a decreased insulinotropic effect. These results suggest that
streptozotocin-nicotinamide-induced mildly diabetic rats, which exhibit a mild decline in glucose tolerance due to loss of
early-phase insulin secretion, are sensitive to the hypoglycaemic effects of insulinotropic agents and have many pathological
features resembling type 2 diabetes, which may be useful in the pharmacological investigation of numerous antidiabetic
drugs.

Type 2 diabetes is characterized by abnormal insulin


secretion and the inability of peripheral tissue to respond to
insulin. To better understand the pathogenesis, genetic
environmental factors and biological complications involved
in type 2 diabetes, and to better test the various therapeutic
agents, appropriate experimental models are necessary. To date,
many animal models can be obtained either spontaneously
or through diet, surgical manipulation, use of inducing
chemicals or a combination of these techniques. Some are
also known to posses pathological syndromes resembling
those of type 2 diabetes [1]. Although these syndromes are
unable to reproduce the complexity of human diabetes,
they can provide insight into some aspects of the diabetes
pathogenesis and progression, as well as facilitate the testing
of antidiabetic drugs. The diabetic models used to evaluate
insulin secretagogues, such as sulfonylureas and dipeptidyl
peptidase-IV inhibitors, include Goto-Kakizaki rats and
neonatal streptozotocin-induced diabetic rats, which exhibit
impaired insulin secretion [2 4], as well as Zucker fatty rats,
which exhibit abnormal glucose tolerance [5 7]. These models
maintain pancreatic insulin contents without developing a
marked insulin resistance, but exhibit abnormal glucose
tolerance associated with reduced postprandial early-phase
insulin secretion or obesity. In contrast, streptozotocin- and
Author for correspondence: Atsuo Tahara, Drug Discovery Research,
Astellas Pharma Inc., 5-2-3, Toukoudai, Tsukuba, Ibaraki 300-2698,
Japan (fax +81-29-847-1536, e-mail atsuo.tahara@jp.astellas.com).

alloxan-induced diabetic rats that exhibit a marked decrease


in pancreatic insulin content, or KK/Ay, db/db and ob/ob
mice, which show a marked insulin resistance, are unsuitable
for the evaluation of insulin secretagogues based on their
insulin deficiency or insulin resistance.
Recently, streptozotocin-induced diabetic rats and mice
pre-treated with nicotinamide have been reported as
models that allow for the evaluation of insulin secretagogues.
Streptozotocin, an antibiotic derived from Streptomyces
achromogenes and a structural glucosamine derivative of
nitrosourea, has a selective toxic effect on pancreatic -cells
and induces diabetes. Although the exact mechanism of its
toxicity is still unclear, a possible site of action is nuclear
DNA. Streptozotocin is taken up by -cells through glucose
transporter GLUT-2 and increases free radical production
causing DNA damage, followed by activation of nuclear
enzyme poly(ADP-ribose) synthetase, which is involved
in DNA repair. This leads to the depletion of the substrate
of nuclear enzyme poly(ADP-ribose) synthetase, nicotinamide
adenine dinucleotide (NAD), which results in reduced insulin
synthesis in -cells and cell death [810]. This streptozotocininduced cytotoxicity is alleviated by nicotinamide, a component of NAD, which inhibits poly(ADP-ribose) synthetase
activity and prevents NAD depletion in pancreatic -cells
[11,12]. Streptozotocin-induced diabetic rats pre-treated with
nicotinamide exhibited moderate hyperglycaemia associated
with the loss of postprandial early-phase insulin secretion,
as well as an approximate 50% decrease in pancreatic insulin

561

EFFECTS OF ANTIDIABETIC DRUGS IN RAT MODELS

content. In addition, some insulin secretagogues allowed


these rats to experience improved glucose tolerance without
developing a marked insulin resistance [13,14]. This model
is an advantageous tool for investigation of insulinotropic
agents in the treatment of diabetes.
Recently, many studies on the development of pancreatic
lesions associated with diabetes mellitus, as well as basic studies
related to the research and development of compounds which
possess antidiabetic effect have used these streptozotocinnicotinamide-induced diabetic rat and mouse models. However, few studies have evaluated in detail the characteristics
of glucose metabolism or the effects of antidiabetic drugs
which are already used in clinical setting on glucose tolerance.
Here, we established streptozotocin-induced severely diabetic
rats and streptozotocin-nicotinamide-induced mildly diabetic
rats, and compared their characteristics. The effects of marketed
antidiabetic drugs on glucose tolerance were also investigated.
Materials and Methods
Materials. Streptozotocin, metformin and glibenclamide were
purchased from Sigma-Aldrich Co. (St. Louis, MO, USA), insulin
(NovolinR 100) was purchased from Novo Nordisk Pharma Ltd.
(Tokyo, Japan), voglibose (BASEN) and sitagliptin (JANUVIA)
were purchased from Takeda Pharmaceutical Company Ltd. (Osaka,
Japan) and Merck & Co. Inc. (Whitehouse Station, NJ, USA), respectively, and purified at Astellas Pharma Inc. (Ibaraki, Japan). Insulin was
diluted with physiological saline, and administered intraperitoneally. The
other compounds were dissolved or suspended in 0.5% methylcellulose
solution, and administered orally. The vehicle treatment group received
an oral 0.5% methylcellulose solution.
Animal models. Male Sprague-Dawley rats were purchased from
Japan SLC Inc. (Shizuoka, Japan) at age 7 weeks and used in the
study at 8 weeks. Streptozotocin-induced and streptozotocinnicotinamide-induced diabetic rats were established by overnight
fasting before receiving chemicals. Streptozotocin was dissolved in
50 mM citric acid buffer before intravenous administration at 50 mg/kg.
Nicotinamide was dissolved in physiological saline and intraperitoneally administered to at 50, 100, 150 and 200 mg/kg, 20 min. before
streptozotocin administration. Normal control rats were administered
physiological saline. One week later, the characteristics of diabetic
rats were investigated. In addition, to investigate the effects of
antidiabetic drugs, the diabetic rats were uniformly grouped for
plasma glucose levels among groups and for each model.
Evaluation of the characteristics of mildly and severely diabetic rats.
The body weight was measured in normal and diabetic rats under
fasting and non-fasting conditions. To measure plasma glucose,
insulin and glucagon-like peptide-1 (GLP-1) levels, blood samples
from the tail veins of rats were collected. To measure insulin content,
a portion of the pancreas was removed. After being weighed, it was
transferred to a tube and stored on ice. For glucose tolerance testing,
a glucose solution (2 g/kg) was orally administered to normal and
diabetic rats that had been fasted overnight, and blood samples
were collected over time to assess plasma glucose, insulin and GLP-1
levels.
Evaluation of the effects of antidiabetic drugs on glucose tolerance.
Group compositions for both mildly and severely diabetic rats were
as follows: normal rats: (1) normal (vehicle treatment); diabetic
rats: (2) vehicle, (3) voglibose (0.1 mg/kg), (4) voglibose (0.3 mg/kg),
(5) voglibose (1 mg/kg), (6) metformin (100 mg/kg), (7) metformin
(300 mg/kg), (8) metformin (1000 mg/kg), (9) glibenclamide (1 mg/kg),

(10) glibenclamide (3 mg/kg), (11) glibenclamide (10 mg/kg), (12)


sitagliptin (0.3 mg/kg), (13) sitagliptin (1 mg/kg), (14) sitagliptin
(3 mg/kg), (15) insulin (0.1 IU/kg), (16) insulin (0.2 IU/kg) and (17)
insulin (0.5 IU/kg). After overnight fasting, plasma glucose levels
(0.5 hr value) of normal and diabetic rats were measured. Diabetic
rats received either vehicle or test compound and normal rats
received the vehicle, orally or intraperitoneally. After 30 min.,
plasma glucose levels (0 hr value) were measured again, followed by
oral administration of a liquid meal (ENSUREH; Meiji Dairies
Corporation, Tokyo, Japan; 20 ml/kg). At 0.5, 1 and 2 hr after liquid
meal administration, plasma glucose levels (0.5, 1 and 2 hr values)
were measured.
Evaluation of the effects of antidiabetic drugs on insulin and GLP-1
secretion. Group compositions of both mildly and severely diabetic
rats were as follows: normal rats: (1) normal (vehicle treatment);
diabetic rats: (2) vehicle, (3) voglibose (1 mg/kg), (4) metformin
(1000 mg/kg), (5) glibenclamide (10 mg/kg), (6) sitagliptin (3 mg/kg)
and (7) insulin (0.5 IU/kg). For the measurement of basal plasma
insulin and GLP-1 levels, blood samples were collected from the tail
vein of normal and diabetic rats that had been fasted overnight.
Diabetic rats received either vehicle or test compound and normal
rats received the vehicle, orally or intraperitoneally. After 30 min.,
a liquid meal was orally administered and blood samples were
collected 15 min. later.
Measurement of plasma glucose, insulin and GLP-1 levels and pancreatic
insulin content. Blood samples collected from the tail veins were
centrifuged at 2,000 g. for 10 min., and the supernatant plasma
was used for the measurement of glucose, insulin and GLP-1 levels.
A 2-ml aliquot of acid-ethanol solution (75% ethanol, 23.5% purified
water and 1.5% concentrated hydrochloric acid) was added to the
pancreas samples stored on ice. To extract the insulin, the mixture
was homogenized and incubated at 4 for 1 hr. The supernatant
samples obtained by centrifugation at 15,000 g. for 10 min. were
used for measurement of pancreatic insulin content. Plasma
glucose, insulin and GLP-1 levels were determined using a Glucose
CII-Test reagent (Wako Pure Chemical Industries, Osaka, Japan),
an insulin EIA test kit (Morinaga Institute of Biological Science
Inc., Kanagawa, Japan) and a GLP-1 active ELISA kit (Linco
Research Inc., St. Charles, MO, USA), respectively.
Statistical analysis. Results are expressed as the mean S.E.M. The
areas under the plasma glucose and insulin concentration-time
curves were calculated from plasma glucose and insulin concentrations
measured over time. To evaluate the characteristics of diabetic rats,
the significance of differences among the normal and diabetic groups
was assessed using Dunnetts multiple range test. To evaluate the
effects of antidiabetic drugs on glucose tolerance, as well as insulin
and GLP-1 secretion, the significance of differences between the
normal and diabetic groups was assessed using Students t-test. The
significance of differences between the vehicle and the three dose
groups for each antidiabetic drug, and between the vehicle and the
five antidiabetic drug-treated groups was assessed using Dunnetts
multiple range test. P-values of <5% were considered statistically
significant.

Results
Evaluation of the characteristics of streptozotocin-induced
and streptozotocin-nicotinamide-induced diabetic rats.
Table 1 shows the main metabolic parameters of rats
established 1 week after the administration of streptozotocin
alone or streptozotocin and several doses of nicotinamide.
Compared to the normal rats, streptozotocin-induced diabetic
rats showed a marked increase in plasma glucose levels and

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

562

ATSUO TAHARA ET AL.


Table 1.

Body weight, plasma glucose and insulin levels, and pancreatic insulin content in the normal, streptozotocin (STZ)-induced and streptozotocinnicotinamide (STZ-NA)-induced diabetic rats.
STZ + NA
Normal
Body weight (g)
Fasting plasma glucose (mg/dl)
Non-fasting plasma glucose (mg/dl)
Fasting plasma insulin (ng/ml)
Non-fasting plasma insulin (ng/ml)
Pancreatic insulin content
(ng/mg pancreas)

STZ

50 mg/kg
1

100 mg/kg

150 mg/kg

200 mg/kg

308 8
94.6 5.3
124 2
0.55 0.04
1.85 0.08

243 4
249 171
643 121
0.27 0.031
0.54 0.071

264 13
179 151
386 181
0.50 0.06
0.91 0.091

295 6
125 8
206 31
0.52 0.05
1.42 0.081

302 10
117 5
166 31
0.61 0.05
1.66 0.11

313 4
102 8
137 10
0.70 0.09
1.85 0.15

99.5 7.8

9.7 0.91

30.9 7.01

53.3 2.01

76.7 8.7

91.3 8.3

Data are expressed as the mean S.E.M. from five animals in each group. The significance of differences between the normal and diabetic
groups was assessed using Dunnetts multiple range test (1P < 0.05 versus the normal group).

a significant decrease in plasma insulin levels under


fasting and non-fasting conditions. In addition, body weight
significantly decreased and the amount of pancreatic insulin
markedly decreased to approximately 10% of that found in
normal rats. However, the streptozotocin-induced hyperglycaemia, body weight loss and decrease in pancreatic insulin
content were depressed by nicotinamide in a dose-dependent
manner. At the low dosage of 50 mg/kg, nicotinamide exerted
only minor protection against the effect of streptozotocin.
At 100 mg/kg, nicotinamide substantially limited the severe
decrease in pancreatic insulin content, and consequently
largely prevented streptozotocin-induced hyperglycaemia
and body weight loss. At 150 mg/kg, only plasma glucose
levels under non-fasted conditions were significantly increased.
Finally, at the highest nicotinamide dosage of 200 mg/kg,
streptozotocin-induced alterations were completely prevented.
In the oral glucose tolerance test in streptozotocin-induced
diabetic rats, the results showed that glucose tolerance
was markedly impaired due to severely impaired insulin
secretion (fig. 1). However, these streptozotocin-induced
impairments in glucose tolerance and insulin secretion were
countered by nicotinamide treatment in a dose-dependent
manner. In streptozotocin and nicotinamide (100 mg/kg)induced diabetic rats, glucose tolerance was mildly impaired
due to the loss of early-phase insulin secretion. In contrast,
no significant change in plasma GLP-1 levels was observed
between normal, streptozotocin-induced and streptozotocinnicotinamide-induced diabetic rats (data not shown). Based
on these observations, streptozotocin-induced diabetic rats
were used as a severely diabetic model, and streptozotocinand nicotinamide (100 mg/kg)-induced diabetic rats were used
as a mildly diabetic model in the following experiments.
Effect of antidiabetic drugs on glucose tolerance in mildly
diabetic rats.
In the liquid meal tolerance test for mildly diabetic rats,
voglibose (0.11 mg/kg), metformin (1001000 mg/kg),
glibenclamide (110 mg/kg), sitagliptin (0.33 mg/kg) and
insulin (0.10.5 IU/kg) dose-dependently and significantly
improved glucose tolerance (fig. 2). In addition, plasma

insulin levels were significantly elevated in groups treated


with glibenclamide or sitagliptin, which exhibit insulinreleasing activity, or with insulin. In contrast, plasma insulin
levels were low with statistical significance in groups treated
with voglibose or metformin (fig. 3). Plasma GLP-1 levels did
not significantly differ between normal and mildly diabetic
rats, but significantly increased in the sitagliptin-treated group,
while the other drug-treated groups showed no significant
changes.
Effect of antidiabetic drugs on glucose tolerance in severely
diabetic rats.
In the liquid meal tolerance test in severely diabetic rats,
voglibose (0.11 mg/kg), metformin (1001000 mg/kg) and
insulin (0.10.5 IU/kg) dose-dependently and significantly
improved glucose tolerance, whereas glibenclamide (1
10 mg/kg) and sitagliptin (0.33 mg/kg) did not (fig. 4). In
addition, the plasma insulin levels were significantly increased
in the insulin-treated group, while the other groups showed
no significant changes (fig. 5). Plasma GLP-1 levels did not
significantly differ between normal and severely diabetic rats,
but significantly increased in the sitagliptin-treated group,
while the other drug-treated groups showed no significant
changes.
Discussion
In this study, streptozotocin-induced severely diabetic and
streptozotocin-nicotinamide-induced mildly diabetic rats were
established to compare their characteristics and to investigate the effects of antidiabetic drugs on glucose tolerance.
Streptozotocin-induced diabetic rats exhibited a marked
decrease in pancreatic insulin contents to approximately
10% of those in normal rats, a severe decline in glucose
tolerance associated with insulin secretion impairment,
frank hyperglycaemia and body weight loss. These results
suggest that these severely diabetic rats represent a model of
insulin-dependent diabetes due to their marked decrease in
pancreatic insulin content. In contrast, the streptozotocininduced hyperglycaemia, body weight loss and a decrease in

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

EFFECTS OF ANTIDIABETIC DRUGS IN RAT MODELS

563

Fig. 1. (A) Plasma glucose and (B) insulin levels during an oral glucose tolerance test in the normal, streptozotocin (STZ)- and
streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats. Values are expressed as the mean S.E.M. from five animals in each group.
The significance of differences between the normal and diabetic groups was assessed using Dunnetts multiple range test (*P < 0.05 versus the
normal group).

pancreatic insulin content were depressed by nicotinamide


in a dose-dependent manner. In particular, diabetic rats exposed
to streptozotocin and nicotinamide (100 mg/kg) exhibited only
a mild decline in glucose tolerance associated with reduced
early-phase insulin secretion after glucose loading. Compared
to normal rats, these rats showed an approximately 50%
decrease in pancreatic insulin content. In addition, because
insulin resistance index values (HOMA-R), calculated from
plasma glucose and insulin levels under fasting conditions,
were similar to those of normal rats (HOMA-R: normal,
3.37 0.32; mildly diabetic rats, 4.20 0.49), insulin
resistance was barely detected. These results suggest that
these mildly diabetic rats represent a model that exhibits a
moderate decline in glucose tolerance associated with the
loss of early-phase insulin secretion.
Current therapeutic strategies for type 2 diabetes are
limited and involve insulin and four main classes of oral

antidiabetic agents that stimulate pancreatic insulin secretion


[sulfonylureas, e.g. glibenclamide; rapid-acting insulinotropics, e.g. nateglinide; and dipeptidyl peptidase-4 (DPP-IV)
inhibitors, e.g. sitagliptin], reduce hepatic glucose production
(biguanides, e.g. metformin), delay digestion and absorption
of intestinal carbohydrate (-glucosidase inhibitors, e.g.
voglibose) or improve insulin action (thiazolidinediones,
e.g. rosiglitazone). In this study, the effects of voglibose,
metformin, glibenclamide, sitagliptin and insulin on glucose
tolerance were examined in both diabetic rat models. Results
showed that all drugs significantly improved glucose tolerance
in streptozotocin-nicotinamide-induced mildly diabetic rats.
In particular, the insulin secretagogues, glibenclamide and
sitagliptin, caused a significant increase in plasma insulin
levels. This is in agreement with Masiello et al., who reported
that the insulin-releasing activity of pancreas isolated from
streptozotocin-nicotinamide-induced mildly diabetic rats is

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

564

ATSUO TAHARA ET AL.

Fig. 2. Hypoglycaemic effect of antidiabetic drugs on glucosetolerance in mildly diabetic rats during the liquid meal tolerance test (LMTT).
Effect of (A) voglibose, (B) metformin, (C) glibendamide, (D) sitagliptin and (E) insulin on plasma glucose levels during the LMTT. (F)
Plasma glucose AUC during the LMTT. Values are expressed as the mean S.E.M. from four animals in each group. The significance of
differences between the normal and diabetic vehicle group was assessed using Students t-test (*P < 0.05 versus the normal group). The
significance of differences between the vehicle and each antidiabetic drug-treated groups was assessed using Dunnetts multiple range test
(P < 0.05 versus the vehicle group).

glucose-dependent and that sulfonylurea tolbutamide causes


an insulin-releasing effect similar to that of normal rats [13].
In addition, the present study confirmed that in mildly
diabetic rats, plasma GLP-1 levels after liquid meal loading
are similar to those in normal rats. GLP-1, the most potent
endogenous insulinotropic peptide, is secreted from L cells
in the intestine in response to food ingestion, and is rapidly
degraded by DPP-IV. Therefore, DPP-IV inhibitors, including
sitagliptin, delay proteolytic degradation of GLP-1 and
enhance glucose-dependent insulin secretion from pancreatic
-cells, leading to a reduction in glucose excursion [1517].
Our results show that sitagliptin significantly increases
plasma GLP-1 and insulin levels in mildly diabetic rats.

Although mildly diabetic rats exhibited a decrease in


pancreatic insulin content and a loss of early-phase insulin
secretion, the insulin-releasing activity of glibenclamide through
sulfonylurea receptors and that of sitagliptin through the
incretin effect was maintained without severe insulin resistance
development. These findings indicate that streptozotocinnicotinamide-induced mildly diabetic rats represent a
model in which glucose tolerance can be improved by glucosidase inhibitors, biguanides, sulfonylureas, DPP-IV
inhibitors and insulin.
Similarly to the case in mildly diabetic rats, voglibose,
metformin and insulin significantly improved glucose
tolerance in streptozotocin-induced severely diabetic rats,

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

EFFECTS OF ANTIDIABETIC DRUGS IN RAT MODELS

565

Fig. 3. Effect of antidiabetic drugs on plasma insulin and GLP-1 levels in mildly diabetic rats during the liquid meal tolerance test (LMTT).
(A) Plasma insulin and (B) GLP-1 levels under basal (fasted) conditions and 15 min. after liquid meal loading. Values are expressed as the mean
S.E.M. from four animals in each group. The significance of differences between the normal and diabetic vehicle groups was assessed using
Students t-test (*P < 0.05 versus the normal group). The significance of differences between the vehicle and antidiabetic drug-treated groups was
assessed using Dunnetts multiple range test (P < 0.05 versus the vehicle group).

while glibenclamide and sitagliptin had no significant effect.


In addition, the plasma GLP-1 levels after liquid meal loading
were similar to those of normal rats and significantly increased
after sitagliptin administration. Furthermore, similarly to
mildly diabetic rats, the HOMA-R of severely diabetic rats
was closely comparable to that of normal rats (HOMA-R:
normal, 3.37 0.32; severely diabetic rats, 4.37 0.62),
suggesting that insulin resistance does not occur in severely
diabetic rats. However, sitagliptin and glibenclamide did not
significantly increase insulin secretion. It has been reported that
glucose-dependent and tolbutamide-induced insulin-releasing
activity of pancreas isolated from streptozotocin-induced
severely diabetic rats had almost disappeared [13]. These
results indicate that insulin secretagogues have no effect
in severely diabetic rats due to the marked decrease or
depletion in the amount of pancreatic insulin content
and severe pancreatic -cell dysfunction. -Glucosidase
inhibitors, including voglibose, act on the small intestinal
disaccharidases sucrase and maltase, and reduce postprandial hyperglycaemia not only in streptozotocin-induced
severely diabetic mice, but also in normal and various
diabetic animal models, including Wistar fatty rats, Zucker
fatty rats, Goto-Kakizaki rats and KK/Ay mice [1821].
This suggests that irrespective of the type of pathological
syndrome, the use of -glucosidase inhibitors is effective
in many diabetic models. Previous reports show that metformin produces a hypoglycaemic effect not only in db/db
mice, a type 2 diabetic model exhibiting severe obesity
and insulin resistance, but also in streptozotocin-induced
insulin-dependent diabetic models [22,23]. The mechanism of
action is assumed to involve the insulin-independent inhi-

bition of hepatic gluconeogenesis, an increase in peripheral


glucose uptake, and the inhibition of small intestinal glucose
absorption [24,25]. These findings support the hypoglycaemic
effects of voglibose and metformin in streptozotocin-induced
severely diabetic rats. These results indicate that streptozotocininduced severely diabetic rats represent a model in which glucose
tolerance can be improved by -glucosidase inhibitors,
biguanides and insulin, but not by insulin secretagogues.
It has been reported that administration of low dose
(35 mg/kg) of streptozotocin also induced mildly diabetic
syndrome [26,27]. Our preliminary examinations show
that administration of low doses of streptozotocin causes
moderate pancreatic injury, but the incidence of injury also
decreased. When 30 mg/kg streptozotocin was administered,
about 40% of rats exhibited no significant decrease in the
pancreatic insulin content or accompanying abnormal
glucose tolerance. In addition, about half of the remaining
rats (30% of the total) exhibited a moderate decrease in
pancreatic insulin content (3070% of normal) and abnormal
glucose tolerance. However, the ability to secrete insulin
varied greatly among individual rats. Those animals affected
included not only the rats exhibiting impaired first-phase
insulin secretion, but also those which experienced an overall
decrease in insulin secretion. In addition, the remaining
half (30% of the total) exhibited severely abnormal glucose
tolerance and body weight loss accompanied by the depletion
of pancreatic insulin content (<20% of normal), which was
also the case when 50 mg/kg streptozotocin alone was
administered. In these rats, sulfonylureas and DPP-IV
inhibitors did not induce any increase in insulin secretion or
improvement of glucose tolerance. On the other hand, in

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

566

ATSUO TAHARA ET AL.

Fig. 4. Hypoglycemic effect of antidiabetic drugs on glucose tolerance in severely diabetic rats during the liquid meal tolerance test (LMTT).
Effect of (A) voglibose, (B) metformin, (C) glibenclamide, (D) sitagliptin and (E) insulin on plasma glucose levels during the LMTT. (F)
Plasma glucose AUC during the LMTT. Values are expressed as the mean S.E.M. from four animals in each group. The significance of
differences between the normal and diabetic vehicle groups was assessed using Students t-test (*P < 0.05 versus the normal group). The
significance of differences between the vehicle and each antidiabetic drug-treated groups was assessed using Dunnetts multiple range test
(P < 0.05 versus the vehicle group).

this study, administration of 50 mg/kg streptozotocin caused


serious pancreatic injury in approximately 100% of the rats.
In addition, co-administration of 50 mg/kg streptozotocin and
100 mg/kg nicotinamide uniformly caused glucose tolerance
abnormality and an approximately 4060% decrease in the
pancreatic insulin content was obtained in more than 90%
of administered rats. Furthermore, almost all of these rats
exhibited impaired first-phase insulin secretion. Therefore,
uniformly diabetic rats that maintain a pancreatic insulin
content, to some extent, exhibit impairment of first-phase
insulin secretion and have a moderate glucose tolerance
abnormality, can be easily obtained in this way.
Our results show that streptozotocin-nicotinamideinduced mildly diabetic rats develop a diabetic syndrome

characterized by a mild decline in glucose tolerance


associated with the loss of early-phase insulin secretion and
reduced pancreatic insulin stores. On the basis of insulin
responsiveness to glucose and insulin secretagogues, we
demonstrated that this experimental rat syndrome
resembles type 2 diabetes; it may prove useful in the evaluation of numerous antidiabetic agents that possess potential
insulinotropic action.
Acknowledgements
The authors would like to thank Drs Toichi Takenaka,
Isao Yanagisawa, Yasuaki Shimizu and Yutaka Yanagita
(Astellas Pharma Inc.) for their valuable comments and
continuing encouragement.

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

EFFECTS OF ANTIDIABETIC DRUGS IN RAT MODELS

567

Fig. 5. Effect of antidiabetic drugs on plasma insulin and GLP-1 levels in severely diabetic rats during the liquid meal tolerance test (LMTT).
(A) Plasma insulin and (B) GLP-1 levels under basal (fasted) conditions and 15 min. after liquid meal loading. Values are expressed as the
mean S.E.M. from four animals in each group. The significance of differences between the normal and diabetic vehicle groups was assessed
using Students t-test (*P < 0.05 versus the normal group). The significance of differences between the vehicle and antidiabetic drug-treated groups
was assessed using Dunnetts multiple range test (P < 0.05 versus the vehicle group).

References
1 Srinivasan K, Ramarao P. Animal models in type 2 diabetes
research: an overview. Indian J Med Res 2007;125:45172.
2 Ladriere L, Malaisse-Lagae F, Fuhlendorff J, Malaisse WJ.
Repaglinide, glibenclamide and glimepiride administration to
normal and hereditarily diabetic rats. Eur J Pharmacol
1997;335:22734.
3 Efanov AM, Appelskog IB, Abdel-Halim SM, Khan A, Branstrom
R, Larsson O et al. Insulinotropic activity of the imidazoline
derivative RX871024 in the diabetic GK rat. Am J Physiol
Endocrinol Metab 2002;282:E117 24.
4 Portha B, Serradas P. Improvement in glucose-induced insulin
secretion in diabetic rats after long-term gliclazide treatment:
a comparative study using different models of non-insulindependent diabetes mellitus induced by neonatal streptozotocin.
Am J Med 1991;90:S15 S21.
5 Balkan B, Kwasnik L, Miserendino R, Holst JJ, Li X. Inhibition
of dipeptidyl peptidase IV with NVP-DPP728 increases plasma
GLP-1 (7-36 amide) concentrations and improves oral glucose
tolerance in obese Zucker rats. Diabetologia 1999;42:1324 31.
6 Mine T, Miura K, Kitahara Y, Okano A, Kawamori R.
Nateglinide suppresses postprandial hypertriglyceridemia in
Zucker fatty rats and Goto-Kakizaki rats: comparison with
voglibose and glibenclamide. Biol Pharma Bull 2002;25:14126.
7 Farrar NS, Chambers NJ, Carlsson AR, Denyer G, Johnston
GA. Effect of a series of novel sulphonylthioureas on glucose
tolerance in the obese fa/fa Zucker rat. Clin Exp Pharmacol
Physiol 2001;28:38691.
8 Schnedl WJ, Ferber S, Johnson JH, Newgard CB. STZ transport
and cytotoxicity. Specific enhancement in GLUT2-expressing cells.
Diabetes 1994;43:1326 33.
9 Turk J, Corbett JA, Ramanadham S, Bohrer A, McDaniel
ML. Biochemical evidence for nitric oxide formation from
streptozotocin in isolated pancreatic islets. Biochem Biophys
Res Commun 1993;197:145864.

10 Burkart V, Wang ZQ, Radons J, Heller B, Herceg Z, Stingl L


et al. Mice lacking the poly(ADP-ribose) polymerase gene
are resistant to pancreatic beta-cell destruction and diabetes
development induced by streptozotocin. Nat Med 1999;5:3149.
11 Oguri S, Motegi K, Endo Y. Augmented lipopolysaccharideinduction of the histamine-forming enzyme in streptozotocininduced diabetic mice. Biochim Biophys Acta 2003;1637:83
90.
12 LeDoux SP, Hall CR, Forbes PM, Patton NJ, Wilson GL.
Mechanisms of nicotinamide and thymidine protection from
alloxan and streptozocin toxicity. Diabetes 1988;37:1015 9.
13 Masiello P, Broca C, Gross R, Roye M, Manteghetti M, HillaireBuys D et al. Experimental NIDDM: development of a new
model in adult rats administered streptozotocin and nicotinamide.
Diabetes 1998;47:224 9.
14 Ichikawa K, Yamato T, Ojima K, Tsuji A, Ishikawa K, Kusama
H et al. Effect of KAD-1229, a novel hypoglycaemic agent, on
plasma glucose levels after meal load in type 2 diabetic rats.
Clin Exp Pharmacol Physiol 2002;29:423 7.
15 Holst JJ, Deacon CF. Glucagon-like peptide 1 and inhibitors of
dipeptidyl peptidase IV in the treatment of type 2 diabetes mellitus. Curr Opin Pharmacol 2004;4:589 96.
16 Mentlein R, Gallwitz B, Schimdt WE. Dipeptidyl-peptidase
IV hydrolyses gastric inhibitory polypeptide, glucagon-like
peptide-1(7-36)amide, peptide histidine methionine and is
responsible for their degradation in human serum. Eur J Biochem
1993;214:829 35.
17 Ahren B, Holst JJ, Martensson H, Balkan B. Improved glucose
tolerance and insulin secretion by inhibition of dipeptidyl
peptidase IV in mice. Eur J Pharmacol 2000;404:239 45.
18 Casirola DM, Ferraris RP. Alpha-glucosidase inhibitors prevent
diet-induced increases in intestinal sugar transport in diabetic
mice. Metabolism 2006;55:832 41.
19 Matsuo T, Odaka H, Ikeda H. Effect of an intestinal disaccharidase
inhibitor (AO-128) on obesity and diabetes. Am J Clin Nutr
1992;55:314S 7.

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

568

ATSUO TAHARA ET AL.

20 Odaka H, Shino A, Ikeda H, Matsuo T. Antiobesity and


antidiabetic actions of a new potent disaccharidase inhibitor
in genetically obese-diabetic mice, KKA(y). J Nutr Sci Vitaminol
1992;38:2737.
21 Azuma K, Toyofuku Y, Iesaki T, Otsuka A, Tanaka A, Mita T
et al. Acarbose, an alpha-glucosidase inhibitor, improves
endothelial dysfunction in Goto-Kakizaki rats exhibiting repetitive blood glucose fluctuation. Biochem Biophys Res Commun
2006;345:68893.
22 Fujita H, Fujishima H, Koshimura J, Hosoba M, Yoshioka N,
Shimotomai T et al. Effects of antidiabetic treatment with
metformin and insulin on serum and adipose tissue adiponectin
levels in db/db mice. Endocr J 2005;52:427 33.
23 Cheng JT, Huang CC, Liu IM, Tzeng TF, Chang CJ. Novel
mechanism for plasma glucose-lowering action of metformin in
streptozotocin-induced diabetic rats. Diabetes 2006;55:81925.

24 El-Atat F, McFarlane SI, Sowers JR. Diabetes, hypertension,


and cardiovascular derangements: pathophysiology and management. Curr Hypertens Rep 2004;6:215 23.
25 Santeusanio F, Di Loreto C, Lucidi P, Murdolo G, De Cicco A,
Parlanti N et al. Diabetes and exercise. J Endocrinol Invest
2003;26:937 40.
26 Srinivasan K, Viswanad B, Asrat L, Kaul CL, Ramarao P.
Combination of high-fat diet-fed and low-dose streptozotocin
rat: a model for type 2 diabetes and pharmacological screening.
Pharmacol Res 2005;52:313 20.
27 Suryanarayana P, Saraswat M, Mrudula T, Krishna TP, Krishnaswamy K, Reddy GB. Curcumin and turmeric delay streptozotocininduced diabetic cataract in rats. Invest Ophthalmol Vis Sci
2005;46:20929.

2008 The Authors


Journal compilation 2008 Nordic Pharmacological Society. Basic & Clinical Pharmacology & Toxicology, 103, 560568

Das könnte Ihnen auch gefallen