# The Characterization of High-Fat Diet and Multiple Low-Dose Streptozotocin Induced Type 2 Diabetes Rat Model

Authors: Ming Zhang; Xiao-Yan Lv; Jing Li; Zhi-Gang Xu; Li Chen
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Aim. Based on the previously established method, we developed a better and stable animal model of type 2 diabetes mellitus by high-fat diet combined with multiple low-dose STZ injections. Meanwhile, this new model was used to evaluate the antidiabetic effect of berberine.  Method. Wistar male rats fed with regular chow for 4 weeks received vehicle (control groups), rats fed with high-fat diet for 4 weeks received different amounts of STZ once or twice by intraperitoneal injection (diabetic model groups), and diabetic rats were treated with berberine (100 mg/kg, berberine treatment group). Intraperitoneal glucose tolerance test and insulin tolerance test were carried out. Moreover, fasting blood glucose, fasting insulin, total cholesterol, and triglyceride were measured to evaluate the dynamic blood sugar and lipid metabolism. Result. The highest successful rate (100%) was observed in rats treated with a single injection of 45 mg/kg STZ, but the plasma insulin level of this particular group was significantly decreased, and ISI has no difference compared to control group. The successful rate of 30 mg/kg STZ twice injection group was significantly high (85%) and the rats in this group presented a typical characteristic of T2DM as insulin resistance, hyperglycemia, and blood lipid disorder. All these symptoms observed in the 30 mg/kg STZ twice injection group were recovered by the treatment of berberine. Conclusion. Together, these results indicated that high-fat diet combined with multiple low doses of STZ (30 mg/kg at weekly intervals for 2 weeks) proved to be a better way for developing a stable animal model of type 2 diabetes, and this new model may be suitable for pharmaceutical screening.


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## Body

## 1. Introduction

Now there has been a tragic increase in diabetes across the world,
paralleling the overweight and obesity epidemic. There are 95 percent of those people belonging to type 2 diabetes. Therefore, it is great urgency to find better treatments and novel
prevention strategies for type 2 diabetes. To accomplish this goal, appropriate experimental models are considered as essential tools for understanding the molecular
basis, pathogenesis of the vascular and neural lesions, actions of therapeutic
agents, and genetic or
environmental influences that increase the risks of type 2 diabetes.Although there are numerous animal models (natural as well as developed) available for the study of
type 2 diabetes [1–4], the
pattern of disease establishment and progress in most of them did not appear to be similar to the clinical situation in
humans. Thus, there is a continued quest among the
investigators with respect to the
establishment of better animal model for type 2 diabetes by adjusting the existing methods, developing
new methodologies, or a combination of both.Many studies
have reported that the rats fed with
high-fat diet (HFD) develop insulin resistance but not frank hyperglycemia or diabetes [5–7]. It is
suggested that the HFD might be a better way to initiate the insulin
resistance which is one of the important features of type 2 diabetes. At the
same time, streptozotocin (STZ)
is widely used to reproducibly induce both insulin-dependent
and noninsulin-dependent diabetes mellitus presently by inducing β cell
death through alkylation of DNA [8]. Although high-dose STZ severely impairs insulin secretion mimicking
type 1 diabetes, low-dose STZ has been known to induce a mild impairment of insulin secretion which is similar to the feature of
the later stage of type 2 diabetes [1, 2]. Therefore, investigators have started to develop a rat model by feeding the animal with high-fat diet following low-dose STZ that would closely mimic the natural history of the disease events
(from insulin resistance to β cell dysfunction) as well as metabolic characteristics of human type 2
diabetes [1, 2, 4]. The successful
establishment of such a model would be cheaper, easily accessible, and practical for the investigation
as well as testing of various compounds for the treatment of type 2 diabetes. Although the appearance of the type 2 diabetes pattern was achieved
by combining the feeding of HFD and low dose of STZ treatments in nongenetic, out-bred rats, the injection dose of STZ and its methodologies
were not consistent in those studies.
Others reported that STZ may
also be given in multiple low doses. It
has been extensively used in the development of type 1 diabetes in rats and mice
to study immune response in pancreas, since the multiple low-dose
injections of STZ could induce
a gradual, autoimmune destruction of β cells instead of the rapid destruction induced by a single high-dose injection [9–14].
However, it has not been reported whether the high-fat diet has synergistic
effect on accelerating the development of type 2 diabetes with
multiple low doses of STZ.The purpose of the present study is to
develop an appropriate, stable animal
model which is analogous to the
human type 2 diabetes mellitus through
a combination of high-fat diet with multiple low-dose STZ injections. As a result, we provide
a suitable animal model to understand the
possible cellular and molecular mechanisms of type 2 diabetes. Meanwhile, the treatment has been conducted. Berberine (C20H18C1NO4) is the
major active constituent of Rhizoma
coptidis, chemically named 5,6-Dihydro-9,10-dimethoxybenzo(g)-1,3-dioxolobenzo(5,6a) quinolizine chloride. It was commonly used to treat diarrhea as an antimicrobial agent before. Recently, it has been demonstrated
that berberine is available for the treatment of diabetes
patients [12–15]. In the
present study, we will provide berberine to this model to evaluate whether it
could cure the diabetes induced by high-fat diet combined with new models of multiple low-dose
STZ injections.


## 2. Materials and Methods

### 2.1. Materials

STZ was purchased from Sigma, insulin was purchased from Eli Lilly, Changchun, China; glucose,
total cholesterol (TC), and triglyceride (TG) test kits were obtained from Beijing BHKT Clinical
Reagent Co., Ltd, Beijing, China; iodine [I125] insulin radioimmunoassay kit was
purchased from Tianjing Nine Tripods Medical & Bioengineering Co., Ltd,
Tianjing, China; Other reagents were purchased from Beijing General Chemical
Reagent Factory, Beijing, China. Berberine was a gift from Northeast drug factory.


### 2.2. Experimental Protocol

Male Wistar rats (200–250 g) were purchased
from the Experimental Animal Holding of Jilin University. The animals were
housed in standard polypropylene cages (three rats/cage) and maintained under
controlled room temperature and humidity with 12/12-hour light-dark cycle. Regular chow consisting
of 5% fat, 53% carbohydrate, 23% protein, with total calorific value 25 kJ/kg
and high-fat diet consisting of 22% fat, 48% carbohydrate, and 20% protein with
total calorific value 44.3 kJ/kg were ordered from the stoyer center of
Experimental Animal Holding. Experiments were conducted in the following three
sections.


#### 2.2.1. First Section

100 Wistar rats were randomly divided into 5 groups: control group (CON1), model group 1 (DM1),
model group 2 (DM2), model group 3 (DM3), and model group 4 (DM4); control
group was fed with regular chow, and other four groups were given high-fat diet
for 4 weeks; four model groups were injected intraperitoneally (IP) with different
doses of STZ (STZ was
injected only once, DM1:
25 mg/kg; DM2: 30 mg/kg; DM3: 35 mg/kg; DM4: 45 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg IP, respectively.
The body weight and food intake were recorded every week. After 8 weeks of STZ
injection, all the rats were fasted for 12 hours; the fasting blood glucose (FBG)
analysis was carried out. The successful rate was calculated. The fasting blood
insulin (FINS) was measured; intraperitoneal glucose tolerance test (IPGTT) and
insulin tolerance test (ITT) were carried out in control and highest successful
rate model group.


#### 2.2.2. Second Section

60 Wistar rats were randomly divided into 3
groups: control group (CON2), model
group 5 (DM5), and model group 6 (DM6); control group was fed with regular
chow, and other two groups were given high-fat diet for 4 weeks; two model
groups were
injected IP with a low dose of STZ (STZ was injected twice, DM5: 25 mg/kg; DM6: 30 mg/kg). After one
week, FBG was measured, the rats with FBG < 7.8 mmol/L were injected with STZ again (DM5: 25 mg/kg;
DM6: 30 mg/kg), while the control rats were given vehicle citrate buffer (pH
4.4) in a dose volume of 0.25 mL/kg, IP, respectively. The body weight and food
intake were recorded every week. After 8 weeks of STZ injection, all the rats
were fasted for 12 hours, FBG was carried out. The successful rate was calculated. Intraperitoneal glucose tolerance test
(IPGTT) and insulin tolerance test (ITT) were carried out in control and
highest successful rate model group.


#### 2.2.3. Third Section

100 Wistar rats were randomly divided into 5
groups: control group (CON3), control plus STZ injection group (C-STZ), high-fat
diet group (HFD), high-fat diet plus STZ injection group (HFD-STZ), Berberine treated
high-fat diet plus STZ injection group (BER); control group and control plus
STZ injection group were fed with regular chow, and other three groups were
given high-fat diet for 4 weeks; the C-STZ, HFD-STZ groups and BER group were
injected IP with a low dose of STZ (according to the second section, choosing
the dose of the group with higher
successful rate: 30 mg/kg). After one week, FBG was measured in these three
groups, the rats with FBG < 7.8 mmol/L were injected with STZ again (30 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg, IP,
respectively. The fasting blood glucose was measured every week. After 4 weeks of
STZ injection, the rats with the fasting blood glucose of ≥7.8 mmol/L twice or with nonfasting blood glucose of ≥11.1 mmol/L were considered diabetic. Berberine (100 mg/kg body weight) was
administered orally as suspension by mixing with vehicle 1% Na-CMC at a dose
volume of 0.5 mL/kg body weight of rats in treatment group for another 4 weeks.
The body weight and food intake of the animals were also measured. The rats were
allowed to continue to feed on their respective diets until the end of the
study. At the end of the study, IPGTT and ITT were conducted in the five groups;
fasting plasma was collected for further measurement of insulin, TG, TC, and
glucose. The insulin sensitivity index (ISI) was calculated according to the fasting insulin and glucose
concentration.


### 2.3. Measurement of FBG, FINS, TG, TC

Rats were fasted for 12–16 hours. Blood
was collected from tail vein; plasma was separated by centrifuge at 3500 × g for
10 minutes. Fasting blood glucose (GOD-POD, glucose oxidase-peroxidase), TC (CHOD-POD,
cholesterol oxidase peroxidase), and TG (GPO-POD, glycerol-phosphoric acid oxidase
peroxidase) were measured by using commercially available colorimetric
diagnostic kits according to the instruction. Plasma insulin was assayed by RIA
according to the instruction.


### 2.4. Diabetic Model Successful Rate and Insulin Sensitivity Index

Diabetic model successful rate referred to the percentage of diabetic rats
in the group. The glucose level and insulin level of the same rat were measured
and its insulin sensitivity index (ISI) was calculated as Ln(FBG × FINS)−1.


### 2.5. Intraperitoneal Glucose Tolerance Test

After an overnight fast (12–16 hours),
the rats were IP injected with 40% glucose (2 g/kg
body weight). Blood samples were collected from the tail at 0, 30, 60, and 120
minutes for measurement of glucose.


### 2.6. Insulin Tolerance Test

Insulin (0.75 IU/kg) was administered by
intraperitoneal injection and blood samples were collected at 0, 30, 60, and 120
minutes for the measurement of plasma
glucose. The value is presented as a percentage of
initial plasma glucose level.


### 2.7. Statistical Analysis

The data are reported as mean ± SEM (n=17–20/group).
Statistical analysis was performed by
one way ANOVA. P<.05 was considered a statistical
significance between control and experimental groups.


## 2.1. Materials

STZ was purchased from Sigma, insulin was purchased from Eli Lilly, Changchun, China; glucose,
total cholesterol (TC), and triglyceride (TG) test kits were obtained from Beijing BHKT Clinical
Reagent Co., Ltd, Beijing, China; iodine [I125] insulin radioimmunoassay kit was
purchased from Tianjing Nine Tripods Medical & Bioengineering Co., Ltd,
Tianjing, China; Other reagents were purchased from Beijing General Chemical
Reagent Factory, Beijing, China. Berberine was a gift from Northeast drug factory.


## 2.2. Experimental Protocol

Male Wistar rats (200–250 g) were purchased
from the Experimental Animal Holding of Jilin University. The animals were
housed in standard polypropylene cages (three rats/cage) and maintained under
controlled room temperature and humidity with 12/12-hour light-dark cycle. Regular chow consisting
of 5% fat, 53% carbohydrate, 23% protein, with total calorific value 25 kJ/kg
and high-fat diet consisting of 22% fat, 48% carbohydrate, and 20% protein with
total calorific value 44.3 kJ/kg were ordered from the stoyer center of
Experimental Animal Holding. Experiments were conducted in the following three
sections.


### 2.2.1. First Section

100 Wistar rats were randomly divided into 5 groups: control group (CON1), model group 1 (DM1),
model group 2 (DM2), model group 3 (DM3), and model group 4 (DM4); control
group was fed with regular chow, and other four groups were given high-fat diet
for 4 weeks; four model groups were injected intraperitoneally (IP) with different
doses of STZ (STZ was
injected only once, DM1:
25 mg/kg; DM2: 30 mg/kg; DM3: 35 mg/kg; DM4: 45 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg IP, respectively.
The body weight and food intake were recorded every week. After 8 weeks of STZ
injection, all the rats were fasted for 12 hours; the fasting blood glucose (FBG)
analysis was carried out. The successful rate was calculated. The fasting blood
insulin (FINS) was measured; intraperitoneal glucose tolerance test (IPGTT) and
insulin tolerance test (ITT) were carried out in control and highest successful
rate model group.


### 2.2.2. Second Section

60 Wistar rats were randomly divided into 3
groups: control group (CON2), model
group 5 (DM5), and model group 6 (DM6); control group was fed with regular
chow, and other two groups were given high-fat diet for 4 weeks; two model
groups were
injected IP with a low dose of STZ (STZ was injected twice, DM5: 25 mg/kg; DM6: 30 mg/kg). After one
week, FBG was measured, the rats with FBG < 7.8 mmol/L were injected with STZ again (DM5: 25 mg/kg;
DM6: 30 mg/kg), while the control rats were given vehicle citrate buffer (pH
4.4) in a dose volume of 0.25 mL/kg, IP, respectively. The body weight and food
intake were recorded every week. After 8 weeks of STZ injection, all the rats
were fasted for 12 hours, FBG was carried out. The successful rate was calculated. Intraperitoneal glucose tolerance test
(IPGTT) and insulin tolerance test (ITT) were carried out in control and
highest successful rate model group.


### 2.2.3. Third Section

100 Wistar rats were randomly divided into 5
groups: control group (CON3), control plus STZ injection group (C-STZ), high-fat
diet group (HFD), high-fat diet plus STZ injection group (HFD-STZ), Berberine treated
high-fat diet plus STZ injection group (BER); control group and control plus
STZ injection group were fed with regular chow, and other three groups were
given high-fat diet for 4 weeks; the C-STZ, HFD-STZ groups and BER group were
injected IP with a low dose of STZ (according to the second section, choosing
the dose of the group with higher
successful rate: 30 mg/kg). After one week, FBG was measured in these three
groups, the rats with FBG < 7.8 mmol/L were injected with STZ again (30 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg, IP,
respectively. The fasting blood glucose was measured every week. After 4 weeks of
STZ injection, the rats with the fasting blood glucose of ≥7.8 mmol/L twice or with nonfasting blood glucose of ≥11.1 mmol/L were considered diabetic. Berberine (100 mg/kg body weight) was
administered orally as suspension by mixing with vehicle 1% Na-CMC at a dose
volume of 0.5 mL/kg body weight of rats in treatment group for another 4 weeks.
The body weight and food intake of the animals were also measured. The rats were
allowed to continue to feed on their respective diets until the end of the
study. At the end of the study, IPGTT and ITT were conducted in the five groups;
fasting plasma was collected for further measurement of insulin, TG, TC, and
glucose. The insulin sensitivity index (ISI) was calculated according to the fasting insulin and glucose
concentration.


## 2.2.1. First Section

100 Wistar rats were randomly divided into 5 groups: control group (CON1), model group 1 (DM1),
model group 2 (DM2), model group 3 (DM3), and model group 4 (DM4); control
group was fed with regular chow, and other four groups were given high-fat diet
for 4 weeks; four model groups were injected intraperitoneally (IP) with different
doses of STZ (STZ was
injected only once, DM1:
25 mg/kg; DM2: 30 mg/kg; DM3: 35 mg/kg; DM4: 45 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg IP, respectively.
The body weight and food intake were recorded every week. After 8 weeks of STZ
injection, all the rats were fasted for 12 hours; the fasting blood glucose (FBG)
analysis was carried out. The successful rate was calculated. The fasting blood
insulin (FINS) was measured; intraperitoneal glucose tolerance test (IPGTT) and
insulin tolerance test (ITT) were carried out in control and highest successful
rate model group.


## 2.2.2. Second Section

60 Wistar rats were randomly divided into 3
groups: control group (CON2), model
group 5 (DM5), and model group 6 (DM6); control group was fed with regular
chow, and other two groups were given high-fat diet for 4 weeks; two model
groups were
injected IP with a low dose of STZ (STZ was injected twice, DM5: 25 mg/kg; DM6: 30 mg/kg). After one
week, FBG was measured, the rats with FBG < 7.8 mmol/L were injected with STZ again (DM5: 25 mg/kg;
DM6: 30 mg/kg), while the control rats were given vehicle citrate buffer (pH
4.4) in a dose volume of 0.25 mL/kg, IP, respectively. The body weight and food
intake were recorded every week. After 8 weeks of STZ injection, all the rats
were fasted for 12 hours, FBG was carried out. The successful rate was calculated. Intraperitoneal glucose tolerance test
(IPGTT) and insulin tolerance test (ITT) were carried out in control and
highest successful rate model group.


## 2.2.3. Third Section

100 Wistar rats were randomly divided into 5
groups: control group (CON3), control plus STZ injection group (C-STZ), high-fat
diet group (HFD), high-fat diet plus STZ injection group (HFD-STZ), Berberine treated
high-fat diet plus STZ injection group (BER); control group and control plus
STZ injection group were fed with regular chow, and other three groups were
given high-fat diet for 4 weeks; the C-STZ, HFD-STZ groups and BER group were
injected IP with a low dose of STZ (according to the second section, choosing
the dose of the group with higher
successful rate: 30 mg/kg). After one week, FBG was measured in these three
groups, the rats with FBG < 7.8 mmol/L were injected with STZ again (30 mg/kg), while the control rats were
given vehicle citrate buffer (pH 4.4) in a dose volume of 0.25 mL/kg, IP,
respectively. The fasting blood glucose was measured every week. After 4 weeks of
STZ injection, the rats with the fasting blood glucose of ≥7.8 mmol/L twice or with nonfasting blood glucose of ≥11.1 mmol/L were considered diabetic. Berberine (100 mg/kg body weight) was
administered orally as suspension by mixing with vehicle 1% Na-CMC at a dose
volume of 0.5 mL/kg body weight of rats in treatment group for another 4 weeks.
The body weight and food intake of the animals were also measured. The rats were
allowed to continue to feed on their respective diets until the end of the
study. At the end of the study, IPGTT and ITT were conducted in the five groups;
fasting plasma was collected for further measurement of insulin, TG, TC, and
glucose. The insulin sensitivity index (ISI) was calculated according to the fasting insulin and glucose
concentration.


## 2.3. Measurement of FBG, FINS, TG, TC

Rats were fasted for 12–16 hours. Blood
was collected from tail vein; plasma was separated by centrifuge at 3500 × g for
10 minutes. Fasting blood glucose (GOD-POD, glucose oxidase-peroxidase), TC (CHOD-POD,
cholesterol oxidase peroxidase), and TG (GPO-POD, glycerol-phosphoric acid oxidase
peroxidase) were measured by using commercially available colorimetric
diagnostic kits according to the instruction. Plasma insulin was assayed by RIA
according to the instruction.


## 2.4. Diabetic Model Successful Rate and Insulin Sensitivity Index

Diabetic model successful rate referred to the percentage of diabetic rats
in the group. The glucose level and insulin level of the same rat were measured
and its insulin sensitivity index (ISI) was calculated as Ln(FBG × FINS)−1.


## 2.5. Intraperitoneal Glucose Tolerance Test

After an overnight fast (12–16 hours),
the rats were IP injected with 40% glucose (2 g/kg
body weight). Blood samples were collected from the tail at 0, 30, 60, and 120
minutes for measurement of glucose.


## 2.6. Insulin Tolerance Test

Insulin (0.75 IU/kg) was administered by
intraperitoneal injection and blood samples were collected at 0, 30, 60, and 120
minutes for the measurement of plasma
glucose. The value is presented as a percentage of
initial plasma glucose level.


## 2.7. Statistical Analysis

The data are reported as mean ± SEM (n=17–20/group).
Statistical analysis was performed by
one way ANOVA. P<.05 was considered a statistical
significance between control and experimental groups.


## 3. Results

### 3.1. FBG and Diabetic Successful Rate in the Groups of the First Section

Table1 illustrated the
diabetic successful rate (DSR) and FBG level in rats which were
fed with high-fat diet combined with STZ 25 mg/kg, 30 mg/kg,
35 mg/kg, and 45 mg/kg. As demonstrated in Table 1,
the successful rate in 25 mg/kg, 30 mg/kg, and 35 mg/kg
STZ injection groups was low (10%, 35%, and 40%, resp.). However, 45 mg/kg STZ
injection had the highest successful rate (100%), its FBG level was also
significantly high compared to control group (P<.001).Table 1
The diabetic successful rate and FBG level in the groups of the first section.
GROUPNN1FBG (mmol/L)DSRCON120—5.18±0.55—DM12025.54±0.9910%DM22077.92±1.3735%DM32088.48±1.5340%DM4202024.5±3.75*100%N: number of rats in each group; N1: number of rats with FBG > 7.8 mmol/L which were injected with STZ after 8 weeks, CON1: control group; DM1:
STZ 25 mg/kg IP; DM2: STZ 30 mg/kg IP; DM3: STZ 35 mg/kg IP; DM4: STZ 45 mg/kg IP;
FBG: fasting blood glucose; DSR: diabetic successful rate. *P<.001
versus control group.


### 3.2. IPGTT, ITT, and ISI in the Groups of the First Section

Our data showed that 45 mg/kg STZ injection
(DM4) had the highest successful rate and higher FBG level. Therefore, IPGTT
and ITT were carried out in DM4 and
control groups to measure glucose tolerance and insulin
sensitivity. As shown in Figure1(a), DM4 showed hyperglycemia compared to
control rats during 120 minutes after glucose injection. The areas under the
glucose curves (mmol/L·min) were
significantly greater in the DM4 group compared with controls [(4000 ± 153)
mmol/L·min versus (967 ± 46)
mmol/L·min, P<.001]. To investigate differences in insulin
sensitivity, we performed an ITT at different time points (Figure 1(b)). Insulin
was given intraperitoneally and blood was collected for the measurement of
glucose. In control group, glucose concentrations declined rapidly after
insulin administration, and the decrease became significant by 30 minutes. However,
there was no significant difference between the STZ 45 mg/kg injection group
and control groups during ITT. This result demonstrated that the rats after STZ
45 mg/kg injection were sensitive to insulin; the ISI also supported this point
(Table 2). All the data indicated that high-fat diet associated with 45 mg/kg
STZ injection developed a diabetic model which was prone to type
1 diabetes mellitus.Table 2
FBG, FINS, and ISI in STZ (45 mg/kg, IP) group and control group.
GroupNFBG (mmol/L)FINS (mIU/L)ISICON1205.18±0.5511.8±2.93−3.85±0.27DM42024.5±3.75**3.97±0.86*−4.10±0.32CON1: control group; DM4: 8 weeks after STZ 45 mg/kg IP once,N: number of rats in each group; FBG: fasting blood glucose at 8
weeks after STZ injection; ISI = ln (FBG × FINS)−1; FINS: fasting
plasma insulin level; **P<.001, *P<.05, DM4 versus control
group.(a) Plasma glucose  during intraperitoneal
glucose tolerance test (IPGTT) in STZ (45 mg/kg, IP) group (DM4) and control rats after 8 weeks of injection.
(b) Percentage of initial glucose level  during insulin tolerance test (ITT) in
DM4 and control groups after 8 weeks of injection. Data shown are means ± SE
(n=20 rats/group per time point). *P<.001, DM3
versus control, by t-test.
(a)(b)


### 3.3. FBG and Diabetic Successful Rate in the Groups of the Second Section

Table3 showed the diabetic successful rate (DSR) and FBG level
of the rats which were fed with high-fat diet combined with STZ 25 mg/kg, 30 mg/kg
twice injection. It was observed that the successful rate in 25 mg/kg
STZ twice injection group was low (25%). However, the successful rate of
30 mg/kg STZ twice injection group was relatively high (85%), and the number of diabetic rats has been stable
until the end of the study. FBG level of DM6 group was also significantly increased compared to control group (P<.01).Table 3
The diabetic successful rate and FBG level in the groups of the second section.
GroupNN1N2N3N4N5FBG (mmol/L)DSRCON220—————4.27±0.41—DM520023555.93±0.9325%DM62041012171714.26±0.57*85%N: number of rats in the group; N1: number of rats with FBG > 7.8 mmol/L
at one week after once STZ injection; N2: number of rats with FBG > 7.8 mmol/L
at one week after twice STZ injection; N3: number of rats with FBG > 7.8 mmol/L at two weeks after twice STZ injection; N4: number of rats with
FBG > 7.8 mmol/L at three weeks after twice STZ injection; N5: number of rats
with FBG > 7.8 mmol/L at four weeks after twice STZ injection; FBG: fasting
blood glucose; DSR: diabetic successful rate; CON2: control group; DM5: twice
STZ 25 mg/kg IP group; DM6: twice STZ 30 mg/kg IP group; *P<.01 versus control group.


### 3.4. IPGTT and ITT in the Groups of the Second Section

Since 30 mg/kg STZ twice injection
group (DM6) had the higher successful rate and higher FBG level, IPGTT and ITT were
performed further in this group at 4 weeks (Figure2) and 8 weeks (Figure 3)
after STZ injection. As shown in Figure 2(a), 4 weeks after STZ injection, the
glucose level in DM6 and control group reached the highest level at 30 minutes
after glucose injection, and slowly decreased in the following 90 minutes. But
the glucose level in DM6 showed hyperglycemia compared to control rats during
120 minutes. The areas under the glucose curves (mmol/L·min) were
significantly greater in the STZ injection group compared with controls [(3278 ± 274)
mmol/L·min versus (1103 ± 81) mmol/L·min, P<.01]. Meanwhile at 8 weeks after STZ injection (Figure 3(a)), the areas under the glucose curves (mmol/L·min) were still significantly
greater in the STZ injection group compared with controls [(4498 ± 333) mmol/L·min
versus (913 ± 47) mmol/L·min, P<.001]. To investigate differences in
insulin sensitivity, we performed ITT after 4 weeks (Figure 2(b)) and 8 weeks (Figure
3(b)) STZ injections.
We found that, after insulin administration in control group, glucose concentrations
declined rapidly; however, the glucose concentrations declined slowly or even
not declined in DM6 group within 30 minutes. After 30 minutes, the slopes of
these two curves were similar (Figure 2(b)). However, the percentage of initial
glucose level in DM6 was shown
to be significantly higher than that of control group during 120 minutes.
These changes were still significant after 8 weeks STZ injection (Figure 3(b)).
This result demonstrated that the rats after STZ 30 mg/kg twice injection presented
insulin resistance. All these data indicated that high-fat diet associated with
30 mg/kg STZ twice injection developed a diabetic model which was an analogue to
type 2 diabetes mellitus with insulin resistance and hyperglycemia.(a) Plasma glucose  during
intraperitoneal glucose tolerance test (IPGTT) in STZ (30 mg/kg, twice, IP) group (DM6) and control rats after 4 weeks of injection.
(b) Percentage of initial glucose level  during insulin tolerance test (ITT) in
DM6 and control group after 4 weeks of injection. Data shown are means ± SE
(n=17–20 rats/group per
time point). *P<.01,
DM6 versus control, by t-test.
(a)(b)(a) Plasma glucose  during intraperitoneal
glucose tolerance test (IPGTT) in STZ (30 mg/kg, twice, IP) group (DM6) and
control rats after 8 weeks of injection. (b) Percentage of initial glucose level
during insulin tolerance test (ITT) in DM6 and control group after 8 weeks
injection. Data shown are means ± SE (n=17–20
rats/group per time point). *P<.001, DM6 versus control,
by t-test.
(a)(b)


### 3.5. Changes of Body Weight and Food Intake in  30 mg/kg STZ Twice Injection Group and  Control Groups

According to the data shown above, 30 mg/kg STZ twice injection developed
a diabetic rat model with insulin resistance and hyperglycemia. Therefore, we measured
the body weight and food intake between this group and the control group. As shown in Table4, the body weight gain
during the study was not statistically different between two groups. Caloric
intake of 30 mg/kg STZ twice injection group was significantly higher compared to control group (P<.05).Table 4
Body weight and food intake of control group and DM6.
WeekBody weight (g)Food intake (KJ/d)CON2DM6CON2DM61272.3±4.9272.5±5.954.5±1.073.6±1.1*2291.6±5.5294.3±6.948.2±1.265.4±2.2*3294.4±6.3296.9±7.447.7±0.759.3±0.7*4300.6±5.8304.4±7.748.8±1.563.1±1.3*5306.4±7.1314.9±7.153.4±1.367.5±1.2*6329.4±7.5334.2±9.854.9±1.964.1±1.7*7356.2±8.1363.1±9.551.9±1.067.0±0.6*8374 ± 9.0373.0±9.451.7±1.069.4±1.0*9384.8±8.9383.6±10.158.6±1.773.4±4.2*10385.2±8.9379.2±11.060.9±2.291.4±2.7*11389.9±10.0375.6±10.169.2±1.595.3±4.0*12415.6±11.8382.1±17.372.3±3.699.5±3.0*CON2: control group; DM6: high-fat diet with STZ 30 mg/kg twice injection group.
Values are means ± SE;N: number of rats, *P<.05
versus control group.


### 3.6. Stability of Experimental Diabetic Model

From the data shown in the second section, 30 mg/kg STZ twice injection
might develop a better type 2 diabetic rat model. Therefore, in the third section
we investigated the stability of this experimental type 2 diabetic model.As shown in Figure4, the FBG of the model group has been measured every week;
this result indicated that the hyperglycemia was stable around 14 mmol/L from 4 weeks after STZ injection to 8 weeks after
STZ injection.Figure 4
Plasma glucose curve in STZ (30 mg/kg, twice, IP) group (DM6) from 3 weeks after STZ injection to
the end of the study (n=17–20 rats).The biochemical parameter and ISI at the end of study were presented in Table5. As
can be seen, the high-fat diet group increased the body weight
significantly compared with control group; the body weight from STZ injected
chow diet rats had no significant difference compared with control group. High-fat diet group presented higher FINS, TG, and
TC levels, but there was no significant difference in blood glucose level
compared with control groups. FBG, FINS,
TG, and TC of HFD-STZ group were significantly increased compared with control
group, the ISI was much lower than the control group, which indicated that the
insulin sensitivity was remarkably decreased in HFD-STZ group compared to the
control group. Moreover, biochemical parameters of the control plus STZ
injection group were not significantly changed compared to control group.Table 5
The biochemical parameter and ISI in the groups of the third section.
GroupCON3C-STZHFDHFD-STZBERN2020201717Body weight (g)415.6±11.8397.9±14.2477.3±23.1*Δ382.1±17.3368.6±19.8FBG (mmol/L)4.49±0.415.25±0.445.56±0.5214.79±0.32**6.48±0.56ΔFINS (mU/L)11.03±1.6812.04±0.2619.64±6.83*9.17±1.3410.04±0.91ISI−3.87±0.15−3.93±0.20−4.69±0.21*−4.87±0.15*−3.68±0.09ΔTG (mmol/L)0.66±0.090.63±0.081.91±0.33**1.70±0.21**0.96±0.17*ΔTC (mmol/L)1.72±0.111.78±0.193.35±0.26**2.99±0.53**2.67±0.58*CON3: control group; C-STZ: regular chow feed group with STZ 30 mg/kg twice IP; HFD:
high-fat diet group; HFD-STZ: model group by high-fat diet with STZ 30 mg/kg
twice IP; BER: berberine treatment group; FBG: fasting blood glucose; FINS:
fasting blood insulin; ISI = ln(FBG×FINS)−1;
TG: triglyceride; TC: total cholesterol. Values are means ± SE, **P<.01, *P<.05 versus
control group, ΔP<.05
versus HFD-STZ group.


### 3.7. Beneficial Effect of Berberine on the Diabetic Rats

Furthermore, we observed whether this animal model was suitable for
pharmaceutical research. As
shown in Figure5, Berberine (100 mg/kg) orally administration improved
impaired glucose tolerance, and enhanced insulin sensitivity. After 4 weeks of treatment, the areas
under the glucose curves (mmol/L·min) were still significantly
lower in the berberine treatment group compared with diabetes model group [(2499
± 167) mmol/L·min versus (3822 ±  344)
mmol/L·min, P<.05]. In the treatment group, the glucose concentration
declined rapidly during ITT. Meanwhile, berberine administration significantly
decreased FBG, TC, and TG levels compared to diabetic model group (P<.05),
the fasting insulin was changed but not significantly, ISI was higher than the model
group, and the result indicated that berberine improved insulin sensitivity and
abnormal blood lipid. The berberine treatment
decreased the body weight slightly but with no significant difference compared with HFD-STZ group
(Table 5). This data also demonstrated that the diabetic model we developed might
be suitable for pharmaceutical research.(a) Plasma glucose  during intraperitoneal glucose tolerance test (IPGTT) in STZ
(30 mg/kg, twice, IP) group (HFD-STZ), berberine trearment group (BER)
and control rats (CON3) after 8 weeks injection. (b)
Percentage of initial glucose
level  during insulin tolerance test (ITT) in these three groups after 8 weeks
injection. Data shown are means ± SE (n=17–20 rats/group per time point). *P<.01, versus control group, #P<.05,
versus HFD-STZ group.
(a)(b)


## 3.1. FBG and Diabetic Successful Rate in the Groups of the First Section

Table1 illustrated the
diabetic successful rate (DSR) and FBG level in rats which were
fed with high-fat diet combined with STZ 25 mg/kg, 30 mg/kg,
35 mg/kg, and 45 mg/kg. As demonstrated in Table 1,
the successful rate in 25 mg/kg, 30 mg/kg, and 35 mg/kg
STZ injection groups was low (10%, 35%, and 40%, resp.). However, 45 mg/kg STZ
injection had the highest successful rate (100%), its FBG level was also
significantly high compared to control group (P<.001).Table 1
The diabetic successful rate and FBG level in the groups of the first section.
GROUPNN1FBG (mmol/L)DSRCON120—5.18±0.55—DM12025.54±0.9910%DM22077.92±1.3735%DM32088.48±1.5340%DM4202024.5±3.75*100%N: number of rats in each group; N1: number of rats with FBG > 7.8 mmol/L which were injected with STZ after 8 weeks, CON1: control group; DM1:
STZ 25 mg/kg IP; DM2: STZ 30 mg/kg IP; DM3: STZ 35 mg/kg IP; DM4: STZ 45 mg/kg IP;
FBG: fasting blood glucose; DSR: diabetic successful rate. *P<.001
versus control group.


## 3.2. IPGTT, ITT, and ISI in the Groups of the First Section

Our data showed that 45 mg/kg STZ injection
(DM4) had the highest successful rate and higher FBG level. Therefore, IPGTT
and ITT were carried out in DM4 and
control groups to measure glucose tolerance and insulin
sensitivity. As shown in Figure1(a), DM4 showed hyperglycemia compared to
control rats during 120 minutes after glucose injection. The areas under the
glucose curves (mmol/L·min) were
significantly greater in the DM4 group compared with controls [(4000 ± 153)
mmol/L·min versus (967 ± 46)
mmol/L·min, P<.001]. To investigate differences in insulin
sensitivity, we performed an ITT at different time points (Figure 1(b)). Insulin
was given intraperitoneally and blood was collected for the measurement of
glucose. In control group, glucose concentrations declined rapidly after
insulin administration, and the decrease became significant by 30 minutes. However,
there was no significant difference between the STZ 45 mg/kg injection group
and control groups during ITT. This result demonstrated that the rats after STZ
45 mg/kg injection were sensitive to insulin; the ISI also supported this point
(Table 2). All the data indicated that high-fat diet associated with 45 mg/kg
STZ injection developed a diabetic model which was prone to type
1 diabetes mellitus.Table 2
FBG, FINS, and ISI in STZ (45 mg/kg, IP) group and control group.
GroupNFBG (mmol/L)FINS (mIU/L)ISICON1205.18±0.5511.8±2.93−3.85±0.27DM42024.5±3.75**3.97±0.86*−4.10±0.32CON1: control group; DM4: 8 weeks after STZ 45 mg/kg IP once,N: number of rats in each group; FBG: fasting blood glucose at 8
weeks after STZ injection; ISI = ln (FBG × FINS)−1; FINS: fasting
plasma insulin level; **P<.001, *P<.05, DM4 versus control
group.(a) Plasma glucose  during intraperitoneal
glucose tolerance test (IPGTT) in STZ (45 mg/kg, IP) group (DM4) and control rats after 8 weeks of injection.
(b) Percentage of initial glucose level  during insulin tolerance test (ITT) in
DM4 and control groups after 8 weeks of injection. Data shown are means ± SE
(n=20 rats/group per time point). *P<.001, DM3
versus control, by t-test.
(a)(b)


## 3.3. FBG and Diabetic Successful Rate in the Groups of the Second Section

Table3 showed the diabetic successful rate (DSR) and FBG level
of the rats which were fed with high-fat diet combined with STZ 25 mg/kg, 30 mg/kg
twice injection. It was observed that the successful rate in 25 mg/kg
STZ twice injection group was low (25%). However, the successful rate of
30 mg/kg STZ twice injection group was relatively high (85%), and the number of diabetic rats has been stable
until the end of the study. FBG level of DM6 group was also significantly increased compared to control group (P<.01).Table 3
The diabetic successful rate and FBG level in the groups of the second section.
GroupNN1N2N3N4N5FBG (mmol/L)DSRCON220—————4.27±0.41—DM520023555.93±0.9325%DM62041012171714.26±0.57*85%N: number of rats in the group; N1: number of rats with FBG > 7.8 mmol/L
at one week after once STZ injection; N2: number of rats with FBG > 7.8 mmol/L
at one week after twice STZ injection; N3: number of rats with FBG > 7.8 mmol/L at two weeks after twice STZ injection; N4: number of rats with
FBG > 7.8 mmol/L at three weeks after twice STZ injection; N5: number of rats
with FBG > 7.8 mmol/L at four weeks after twice STZ injection; FBG: fasting
blood glucose; DSR: diabetic successful rate; CON2: control group; DM5: twice
STZ 25 mg/kg IP group; DM6: twice STZ 30 mg/kg IP group; *P<.01 versus control group.


## 3.4. IPGTT and ITT in the Groups of the Second Section

Since 30 mg/kg STZ twice injection
group (DM6) had the higher successful rate and higher FBG level, IPGTT and ITT were
performed further in this group at 4 weeks (Figure2) and 8 weeks (Figure 3)
after STZ injection. As shown in Figure 2(a), 4 weeks after STZ injection, the
glucose level in DM6 and control group reached the highest level at 30 minutes
after glucose injection, and slowly decreased in the following 90 minutes. But
the glucose level in DM6 showed hyperglycemia compared to control rats during
120 minutes. The areas under the glucose curves (mmol/L·min) were
significantly greater in the STZ injection group compared with controls [(3278 ± 274)
mmol/L·min versus (1103 ± 81) mmol/L·min, P<.01]. Meanwhile at 8 weeks after STZ injection (Figure 3(a)), the areas under the glucose curves (mmol/L·min) were still significantly
greater in the STZ injection group compared with controls [(4498 ± 333) mmol/L·min
versus (913 ± 47) mmol/L·min, P<.001]. To investigate differences in
insulin sensitivity, we performed ITT after 4 weeks (Figure 2(b)) and 8 weeks (Figure
3(b)) STZ injections.
We found that, after insulin administration in control group, glucose concentrations
declined rapidly; however, the glucose concentrations declined slowly or even
not declined in DM6 group within 30 minutes. After 30 minutes, the slopes of
these two curves were similar (Figure 2(b)). However, the percentage of initial
glucose level in DM6 was shown
to be significantly higher than that of control group during 120 minutes.
These changes were still significant after 8 weeks STZ injection (Figure 3(b)).
This result demonstrated that the rats after STZ 30 mg/kg twice injection presented
insulin resistance. All these data indicated that high-fat diet associated with
30 mg/kg STZ twice injection developed a diabetic model which was an analogue to
type 2 diabetes mellitus with insulin resistance and hyperglycemia.(a) Plasma glucose  during
intraperitoneal glucose tolerance test (IPGTT) in STZ (30 mg/kg, twice, IP) group (DM6) and control rats after 4 weeks of injection.
(b) Percentage of initial glucose level  during insulin tolerance test (ITT) in
DM6 and control group after 4 weeks of injection. Data shown are means ± SE
(n=17–20 rats/group per
time point). *P<.01,
DM6 versus control, by t-test.
(a)(b)(a) Plasma glucose  during intraperitoneal
glucose tolerance test (IPGTT) in STZ (30 mg/kg, twice, IP) group (DM6) and
control rats after 8 weeks of injection. (b) Percentage of initial glucose level
during insulin tolerance test (ITT) in DM6 and control group after 8 weeks
injection. Data shown are means ± SE (n=17–20
rats/group per time point). *P<.001, DM6 versus control,
by t-test.
(a)(b)


## 3.5. Changes of Body Weight and Food Intake in  30 mg/kg STZ Twice Injection Group and  Control Groups

According to the data shown above, 30 mg/kg STZ twice injection developed
a diabetic rat model with insulin resistance and hyperglycemia. Therefore, we measured
the body weight and food intake between this group and the control group. As shown in Table4, the body weight gain
during the study was not statistically different between two groups. Caloric
intake of 30 mg/kg STZ twice injection group was significantly higher compared to control group (P<.05).Table 4
Body weight and food intake of control group and DM6.
WeekBody weight (g)Food intake (KJ/d)CON2DM6CON2DM61272.3±4.9272.5±5.954.5±1.073.6±1.1*2291.6±5.5294.3±6.948.2±1.265.4±2.2*3294.4±6.3296.9±7.447.7±0.759.3±0.7*4300.6±5.8304.4±7.748.8±1.563.1±1.3*5306.4±7.1314.9±7.153.4±1.367.5±1.2*6329.4±7.5334.2±9.854.9±1.964.1±1.7*7356.2±8.1363.1±9.551.9±1.067.0±0.6*8374 ± 9.0373.0±9.451.7±1.069.4±1.0*9384.8±8.9383.6±10.158.6±1.773.4±4.2*10385.2±8.9379.2±11.060.9±2.291.4±2.7*11389.9±10.0375.6±10.169.2±1.595.3±4.0*12415.6±11.8382.1±17.372.3±3.699.5±3.0*CON2: control group; DM6: high-fat diet with STZ 30 mg/kg twice injection group.
Values are means ± SE;N: number of rats, *P<.05
versus control group.


## 3.6. Stability of Experimental Diabetic Model

From the data shown in the second section, 30 mg/kg STZ twice injection
might develop a better type 2 diabetic rat model. Therefore, in the third section
we investigated the stability of this experimental type 2 diabetic model.As shown in Figure4, the FBG of the model group has been measured every week;
this result indicated that the hyperglycemia was stable around 14 mmol/L from 4 weeks after STZ injection to 8 weeks after
STZ injection.Figure 4
Plasma glucose curve in STZ (30 mg/kg, twice, IP) group (DM6) from 3 weeks after STZ injection to
the end of the study (n=17–20 rats).The biochemical parameter and ISI at the end of study were presented in Table5. As
can be seen, the high-fat diet group increased the body weight
significantly compared with control group; the body weight from STZ injected
chow diet rats had no significant difference compared with control group. High-fat diet group presented higher FINS, TG, and
TC levels, but there was no significant difference in blood glucose level
compared with control groups. FBG, FINS,
TG, and TC of HFD-STZ group were significantly increased compared with control
group, the ISI was much lower than the control group, which indicated that the
insulin sensitivity was remarkably decreased in HFD-STZ group compared to the
control group. Moreover, biochemical parameters of the control plus STZ
injection group were not significantly changed compared to control group.Table 5
The biochemical parameter and ISI in the groups of the third section.
GroupCON3C-STZHFDHFD-STZBERN2020201717Body weight (g)415.6±11.8397.9±14.2477.3±23.1*Δ382.1±17.3368.6±19.8FBG (mmol/L)4.49±0.415.25±0.445.56±0.5214.79±0.32**6.48±0.56ΔFINS (mU/L)11.03±1.6812.04±0.2619.64±6.83*9.17±1.3410.04±0.91ISI−3.87±0.15−3.93±0.20−4.69±0.21*−4.87±0.15*−3.68±0.09ΔTG (mmol/L)0.66±0.090.63±0.081.91±0.33**1.70±0.21**0.96±0.17*ΔTC (mmol/L)1.72±0.111.78±0.193.35±0.26**2.99±0.53**2.67±0.58*CON3: control group; C-STZ: regular chow feed group with STZ 30 mg/kg twice IP; HFD:
high-fat diet group; HFD-STZ: model group by high-fat diet with STZ 30 mg/kg
twice IP; BER: berberine treatment group; FBG: fasting blood glucose; FINS:
fasting blood insulin; ISI = ln(FBG×FINS)−1;
TG: triglyceride; TC: total cholesterol. Values are means ± SE, **P<.01, *P<.05 versus
control group, ΔP<.05
versus HFD-STZ group.


## 3.7. Beneficial Effect of Berberine on the Diabetic Rats

Furthermore, we observed whether this animal model was suitable for
pharmaceutical research. As
shown in Figure5, Berberine (100 mg/kg) orally administration improved
impaired glucose tolerance, and enhanced insulin sensitivity. After 4 weeks of treatment, the areas
under the glucose curves (mmol/L·min) were still significantly
lower in the berberine treatment group compared with diabetes model group [(2499
± 167) mmol/L·min versus (3822 ±  344)
mmol/L·min, P<.05]. In the treatment group, the glucose concentration
declined rapidly during ITT. Meanwhile, berberine administration significantly
decreased FBG, TC, and TG levels compared to diabetic model group (P<.05),
the fasting insulin was changed but not significantly, ISI was higher than the model
group, and the result indicated that berberine improved insulin sensitivity and
abnormal blood lipid. The berberine treatment
decreased the body weight slightly but with no significant difference compared with HFD-STZ group
(Table 5). This data also demonstrated that the diabetic model we developed might
be suitable for pharmaceutical research.(a) Plasma glucose  during intraperitoneal glucose tolerance test (IPGTT) in STZ
(30 mg/kg, twice, IP) group (HFD-STZ), berberine trearment group (BER)
and control rats (CON3) after 8 weeks injection. (b)
Percentage of initial glucose
level  during insulin tolerance test (ITT) in these three groups after 8 weeks
injection. Data shown are means ± SE (n=17–20 rats/group per time point). *P<.01, versus control group, #P<.05,
versus HFD-STZ group.
(a)(b)


## 4. Discussion

Type 2 diabetes is a complex, heterogeneous, and polygenic disease. There are many underlying factors that
contribute to the high blood glucose levels in these type 2 diabetes patients. An important factor is the body's
resistance to insulin, essentially ignoring its insulin secretions. A second
factor is the falling production of insulin by theβ cells of
the pancreas. Therefore, an individual with type 2 diabetes may have a
combination of deficient secretion and action of insulin. Hence, an
experimental animal model which aims at mimicking the pathogenesis and clinical feature
of human type 2 diabetes mellitus should preferably have these two traits. Among
the animal models available, inherited
hyperglycaemia and/or obesity in certain strains have been wildly used in the investigations, such as ob/ob mouse,
Zucker rats, and OLETF rats. However, those inbred diabetic models are comparatively
expensive and not easy to breed. Thus, type 2 diabetic model developed in rodents has been studied for
reasons such as short generation time and economic considerations.Currently, many
studies have reported that the high-fat diet (HFD) feeding rats develop insulin
resistance [5–7]. At the same time, low-dose STZ has been known to induce a mild impairment of insulin secretion which is similar to the feature of
the later stage of type 2 diabetes [1, 2]. Therefore, investigators have started to develop a rat model by high-fat diet
following low-dose STZ that
would closely mimic the natural history of the disease [1, 2, 4]. Although the appearance of the type 2 diabetes pattern was achieved by combining the feeding of
HFD and low dose of STZ treatments
in nongenetic, out-bred rats, the
injection dose of STZ and its methodologies were not consistent in those studies.The purpose of the present study was to develop an animal model of type 2
diabetes that would imitate the natural history and the metabolic
characteristics of the human syndrome and be responsive to the pharmaceutical treatment. On the other hand, the goal of the present
study was to develop an animal model which is neither inherited nor genetically
obese, and which is easily accessible, fairly economical, and with high successful
rate. The results demonstrated that we had achieved our goals.The primary attempts of the present study were to identify the dose of
STZ that was low enough to develop type 2 diabetes models in HFD rats with
higher successful rate and without much insulin deficiency. The different doses
of STZ (25, 30, 35, 45 mg/kg, IP) were studied. Injection of STZ (45 mg/kg, IP)
after 4 weeks of high-fat diet caused frank hyperglycemia with 100% success
rate, which is consistent with literature reports [1]. Further, these rats were
insulin sensitive, presenting an insulin-deficient symptom as compared to the control
rats. Thus, these fat-fed rats with high dose of STZ (45 mg/kg) were considered
resembling type 1 diabetes. In contrast, STZ (30 mg/kg and 35 mg/kg, IP) failed
to generate a significant hyperglycemia in HFD-fed rats. Srinivasan et
al. have reported the similar results showing that STZ (25 mg/kg, 35 mg/kg, 45 mg/kg,
and 55 mg/kg, once injected) could be used to develop diabetic model. Since the
dose of STZ (25 mg/kg) did not produce significant hyperglycemia, and fat-fed/STZ
(45 and 55 mg/kg, IP) diabetic rats exhibited fairly high glucose and a drastic
reduction in the body weights, they finally chose 35 mg/kg STZ injection as the
optimum dose, but the successful rate of this method has not been reported [2].Our second study attempted to find if multiple low doses
of STZ could achieve our goal (high successful rate and performance of type 2
diabetes). Multiple low-dose STZ injection to induce diabetes and its
complication model have been reported in many studies [9–14].  Multiple
low doses of STZ in rats and mice could induce a combination of poisonous and immunological response
presenting progressively hyperglycemia. Investigator from Korea
has reported that male Sprague-Dawley rats showed rapid chemical destruction of the pancreatic β cells when they were given a single
high-dose injection of STZ (80 mg/kg, IP); interestingly, multiple
low-dose injections of STZ (20 mg/kg for 5 consecutive days, IP) could induce a gradual, autoimmune
destruction of β cells [13].  Wright
Jr. and Lacy reported that rats receiving multiple low doses of STZ (25 mg/kg IP at weekly intervals
for 3 weeks) only did not develop diabetes. Immunologic adjuvants played the synergistic
role in prompting the induction of diabetes with multiple low doses of STZ in
rats [14]. Therefore, it can be proved that multiple low doses of STZ acted to induce
a gradual destruction of β-cell. This might happen in decompensated phase of type 2 diabetes. High-fat
diet has been extensively used to develop insulin resistance [5–7]. Therefore, high-fat
diet combined with multiple low dose
of STZ might develop a suitable type 2 diabetes animal model which presents not
only insulin resistance but also insulin deficiency. The current study proved this point. Our data demonstrated that multiple
low doses of STZ (30 mg/kg IP at weekly interval for 2 weeks) produced frank
hyperglycemia in HFD-fed rats with highly successful rate, but did not produce
the same in regular chow-fed rats. The ISI and ITT all demonstrate the insulin
resistance of these diabetic rats. Hence, HFD in combination with multiple low doses
of STZ (30 mg/kg, twice injection at weekly interval) can be more considered to
characterize the pathophysiology of type 2 diabetes.Furthermore, the diabetic model we developed produces hyperglycemia around 14.5 mmol/L, which
is reasonable to be treated by therapeutic compound as practiced clinically. Therapeutically,
it is difficult to reduce elevated blood glucose except for administration of
insulin. Berberine, the major active constituent of Rhizoma coptidis, is used clinically
in the treatment of diarrhea as an antimicrobial agent. Early as 1986, investigators
from china have started to report the hypoglycemic effect of berberine. In 1999,
Yuan reported that berberine exerted beneficial effect on the treatment of
diabetes clinically; other investigators also proved its role in the treatment
of type 2 diabetes in clinic [15–18]. The mechanisms of the antidiabetic effect
of berberine involved multiple factors. Yin et al. reported that berberine
improved glucose metabolism through induction of glycolysis in many cell lines
including 3T3-L1 adipocytes, L6 myotubes, C2C12
myotubes, and H4IIE hepatocytes, which might be related to inhibition of
glucose oxidation in mitochondria [19, 20]. Others revealed that the underlying
mechanism for berberine improves
insulin resistance and lowers blood sugar possibly through activating
the AMP-activated protein kinase (AMPK) pathway [21]. It has also been demonstrated
that inhibiting phosphorylation of IKKβ might be a cofactor
of berberine in achieving its anti-inflammation and insulin-resistance-improving
effects [22]. The latest study also proved that berberine could inhibit fructose-induced
insulin resistance in rats possibly by increasing the expression HNF-4α  in
liver [23]. HNF-4α is
a positive regulator of PEPCK so an increase of HNF-4α  would result in
increased gluconeogenesis. Hence, we administrated berberine as
antidiabetic drug in the present
study, and our result also demonstrated
that berberine treatment could decrease the insulin resistance and improve
impaired glucose tolerance. Meanwhile, we also found that berberine could correct
lipid metabolism disorders, which indicated that the treatment of berberine
might play an important role in diabetic complication. Altogether, the diabetic
model we developed was suitable to investigate not only the pathogenesis but also the pharmaceutical selection.
Meanwhile, the current study considered the incidence of diabetes in the group.
This method will successfully produce type 2 diabetic rat models, as well as
provide the enough number of diabetic rats once which will make the
investigation more convincing.Conclusively, our study demonstrates
that a combination of HFD and multiple low doses of STZ injection could be
effectively used to generate a rat model that mimics the natural history and
metabolic characteristics of type 2 diabetes in humans. It was also useful in
evaluating the effect of therapeutic compounds on the treatment of type 2 diabetes.



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*Source: 704045-2009-01-04.xml*