Download The effect of acetylsalicylic acid on vasopressin, serum insulin levels

Turkish Journal of Medical Sciences
http://journals.tubitak.gov.tr/medical/
Research Article
Turk J Med Sci
(2017) 47: 996-1001
© TÜBİTAK
doi:10.3906/sag-1503-90
The effect of acetylsalicylic acid on vasopressin, serum insulin levels, insulin resistance,
and biochemical parameters in rats induced with experimental diabetes type 2
1,
1
2
Serkan ŞEN *, Sefa ÇELİK
Department of Medical Laboratory Techniques, Atatürk Vocational School of Health Services, Afyon Kocatepe University,
Afyonkarahisar, Turkey
2
Department of Medical Biochemistry, Faculty of Medicine, Afyon Kocatepe University, Afyonkarahisar, Turkey
Received: 16.03.2015
Accepted/Published Online: 19.11.2015
Final Version: 12.06.2017
Background/aim: Acetylsalicylic acid (ASA) treatment in diabetic patients is very important owing to the increasing hyperactivity
of thrombocytes and atherosclerosis. In several investigations, it was reported that diabetes caused increased coronary artery disease,
cerebrovascular disease, and death. In this study, we aimed to investigate the effect of ASA on osmoregulation, glycemic control, and
some biochemical parameters in rats induced with experimental diabetes type 2.
Materials and methods: Twenty-four rats were randomly divided in four groups: control (I), ASA control (II), diabetic (III), and ASA
diabetic (IV). Diabetes was induced by streptozotocin treatment (30 mg/kg, twice, intraperitoneal injection) in obese rats. ASA (150
mg/kg body weight, orally) was administered for 5 weeks in the ASA control and ASA diabetic groups. Serum electrolytes, creatinine,
albumin, and total protein levels were analyzed with an autoanalyzer. Arginine vasopressin (AVP) and insulin were analyzed by ELISA
techniques.
Results: At the end of the study ASA treatments had decreased the fasting blood glucose levels but had interestingly increased the serum
AVP levels in diabetics rats.
Conclusion: AVP levels were increased 2-fold by ASA treatment in diabetic rats. For the first time in this study, the hypoglycemic effect
of ASA was attributed to an increase in blood volume by AVP levels. This explanation may be a new approach to the literature on this
topic.
Key words: Type 2 diabetes, arginine vasopressin, insulin resistance, osmoregulation
1. Introduction
Diabetes mellitus is characterized by chronic hyperglycemia
developed as a result of disorders in carbohydrate, fat,
and protein metabolisms related to disorders in insulin
secretion, insulin action, or both (1) .
Type 2 diabetes is the most common form of diabetes.
Its main disorders are characterized by insulin secretion
and activity. Type 2 diabetic patients usually have an
excess of insulin and insulin resistance rather than insulin
deficiency (2).
Morbidity and mortality rates of cardiovascular
disease in diabetic patients are high, and cardiovascular
complications form the most extensive area in the cost
tables caused by diabetes complications (3). The risk of
cardiovascular diseases for type 2 diabetics is 2.32 times
higher than that of nondiabetics (4). Additionally, there is
a link between cardiovascular diseases and glucose levels
*Correspondence: [email protected]
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in diabetics. A 1% increase in HbA1c levels doubles the
risk of cardiovascular diseases (5).
Inflammation plays a key role in the development
of atherosclerosis (6). It was also suggested that type 2
diabetes has an effective role in atherosclerosis with its
NOS, NO, redox stress, and inflammatory components (7).
Today it is clearly known that there are close relations
between diabetes, inflammation, atherosclerosis, and
thrombosis (8). In the global treatment strategies for type 2
diabetes, acetylsalicylic acid (ASA) is used as a therapeutic
agent and its use is recommended (9). Therefore, ASA
treatment for type 2 diabetics in order to minimize the risk
of cardiovascular diseases is essential.
Arginine vasopressin (AVP) is produced in the
supraoptic and paraventricular cores found in the
hypothalamus and is transferred to the posterior pituitary
gland via the supraoptic hypophyseal channel and stored
ŞEN and ÇELİK / Turk J Med Sci
there. It is released by exocytosis in response to osmotic
and nonosmotic stimuli (10).
In this study, it was aimed to investigate the effect of
ASA on osmoregulation, glycemic control, and some
biochemical parameters in rats induced with experimental
diabetes type 2.
2. Materials and methods
2.1. Animals
The 24 male Wistar rats of 6–8 weeks old (140–200 g) used
in this study were obtained from the Süleyman Demirel
University Experimental Animal Research Center (Isparta,
Turkey). Care and feeding of rats was carried out at 21 ± 2
°C ambient temperature and 55%–60% relative humidity,
with a 12/12-h light/dark cycle. High-energy normal feed
and daily fresh water supply was provided continuously
according to the groups. Rats were randomly divided in
four groups: control (I), ASA control (II), diabetic (III),
and ASA diabetic (IV).
2.2. Preparation of a high-fat diet
In order to cause experimental type 2 diabetes, insulin resistance should be developed in diabetic rats. High-energy diets are used for this purpose. However, the high-energy feed
used for this purpose has not been found from the manufacturers in Turkey. Therefore, studies on type 2 diabetes
formation with streptozotocin (STZ) injection following
a high-energy diet were reviewed and the feeds used, energy contents, and foods that increase the energy contents
were determined. The high-fat diet (HFD) administered to
test animals in order to create obesity or insulin resistance
varies depending on the study and the fat content changed
between 22% and 60% (11–13). HFD content was prepared
according to the average feed contents in studies using a
standard diet and is given in Table 1.
2.3. Formation of insulin resistance in rats
Because the fastest weight gain in HFD-applied groups
started at the 5th week and continued until the rats were 20
weeks old, the study was initiated with rats of 6–8 weeks old
(14). Rats were brought to the Afyon Kocatepe University
Experimental Animal Research and Application Center
and were adapted to the environment by feeding with
standard pellet feed for 1 week. Rats, except the control
and ASA control groups, were administered the HFD for 6
weeks and the changes in weights of the rats in the control
and HFD-applied groups were monitored weekly.
At the end of the HFD administration period (6 weeks),
an insulin tolerance test (ITT) was performed on all rats
and the rats with insulin resistance were determined.
As explained in the literature, HFD-administered rats
that had a weight gain of 5% compared to control group
rats and additionally developed insulin resistance were
regarded as obese (15), and type 2 diabetes formation was
carried out with STZ injection.
2.4. Formation of type 2 diabetes model
In the 8th week of the study, obese rats were starved for
12 h and fasting blood sugar (FBS) levels were measured
(Accu-Check Go, Bayer, Germany) in order to compare
the blood glucose levels after STZ injection. Following
ketamine (65 mg/kg) and xylazine (7 mg/kg) anesthesia,
rats were injected with STZ dissolved in citrate buffer
(pH 4.5) with a 30 mg/kg dosage twice in weekly intervals
with i.p. injection. FBS levels of the rats were measured
one week after the last injection and the rats with levels
higher than 300 mg/dL FBS were regarded as having type
2 diabetes (16). Following the development of obesity, the
rats in the type 2 diabetes model injected with a low dose
(30 mg/kg) of double STZ injections (i.p.) were divided
into groups for the following phases of the study.
2.5. Insulin tolerance test
Insulin solutions prepared for the rats were administered
at a dose of 0.50 IU/kg by i.p. injection, and the plasma
glucose levels before the injection (minute 0) and at
minutes 15, 30, and 60 following the injection were
measured and insulin resistance was determined.
Very low or no decreases in FBS levels show that
insulin resistance is established and the diabetes model is
type 2 (16,17). In many studies that applied the ITT, it was
reported that the glucose-lowering effect of insulin lasted
until the 30th minute; however, plasma glucoses started
to increase after the 30th minute due to gluconeogenesis
Table 1. The content of the high-fat diet and standard diet.
Standard pellet feed ingredients
High-energy (fatty) feed ingredients
Fat ratio (%)
4.1
57.3
Protein ratio (%)
17
13.6
Carbohydrate ratio (%)
76.4
30.1
Other substances (%)
2.5
1.5
Energy level (cal/kg)
2600
4930
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ŞEN and ÇELİK / Turk J Med Sci
3. Results
3.1. Effects of ASA treatment on blood biochemical parameters in type 2 diabetic rats
Serum sodium, chloride, potassium, creatinine, albumin,
and total protein levels are shown in Table 2. According
to these data, a significant difference was found in serum
sodium levels between the ASA-applied diabetics group
and non-ASA-applied diabetics group (Figures 1A–1F;
Table 2).
It was determined that serum sodium levels decreased
following the ASA administration. Serum potassium levels
between the ASA-applied diabetics group and other groups
were statistically significant; in the ASA-applied diabetics
group serum potassium levels increased (Figure 1).
Regarding the serum chloride levels, no significant
difference was found between the diabetic group and ASA
diabetic group, or between the control and ASA control
group. However, the difference in serum chloride levels
between the nondiabetic group (control and ASA control)
and diabetic group (diabetic and ASA diabetic) was
statistically significant and serum chloride levels in the
diabetic group were lower (Figure 1; Table 2).
Serum creatinine levels in ASA-applied diabetic
groups were lower than those of non-ASA-applied
diabetic groups (Table 2). Serum albumin levels were also
significantly lower in the ASA diabetic group than in other
groups, as was the case for all biochemical parameters
except serum potassium (Table 2). As a result of serum
total protein analyses, it was found that the control group
had significantly higher total protein values than the other
groups.
3.2. Serum arginine vasopressin (antidiuretic hormone)
levels
Vasopressin levels in the control group, ASA control
group, diabetic group, and ASA diabetic group were
2.33 ng/mL, 14.38 ng/mL, 37.03 ng/mL, and 70.4 ng/
mL, respectively. Statistically significant differences were
and hepatic glycogenolysis. In this manner, the peak in
the antihyperglycemic activity of insulin is considered to
occur at the 30th minute and 30-min data were used for
the ITT (18), and data obtained at the end of this time were
used for the statistical analyses.
2.6. Analysis of the biochemical parameters
The rats were sacrificed under ketamine and xylazine
anesthesia and intracardiac blood was taken with 10 mL
syringes. The blood samples were centrifuged for 5 min
at 4000 rpm and serum samples were obtained. Sodium,
potassium, chloride, creatinine, total protein, and albumin
values of serum samples as biochemical parameters were
analyzed at the Afyon Kocatepe University Ahmet Necdet
Sezer Research and Training Hospital in the biochemistry
laboratories using a Roche Cobas C501 autoanalyzer.
2.7. Determination of serum insulin and vasopressin levels by ELISA technique
Both insulin and vasopressin were analyzed from blood
samples. Analyses were carried out by ELISA techniques
using commercial kits for insulin (EMD Millipore
Corporation, USA) and vasopressin (Bachem, Switzerland)
determinations by ELISA reader (Trinity Biotech, Ireland).
2.8. Assessment of beta cell function (HOMA-β: Homeostasis Model Assessment, Beta Cell Function)
With the blood glucose and insulin levels obtained at the
end of the study, HOMA-β values were calculated with the
following formula:
HOMA-β = 20 × fasting insulin level (mU/L) / fasting
glucose level (mmol/L) – 3.5 (19).
2.9. Assessment of insulin resistance (HOMA-IR: Homeostasis Model Assessment, Insulin Resistance)
With the plasma glucose and insulin levels obtained at the
end of the study, HOMA-IR values were calculated with
the following formula:
HOMA-IR = 20 × fasting insulin level (mU/L) / fasting
glucose level (mmol/L) / 22.5 (19).
Table 2. Changes in biochemical parameters.
Control
ASA control
Diabetic
ASA diabetic
P
Sodium (mEq/L)
141.17 ± 0.65b
142.60 ± 0.68b
140.50 ± 0.85b
133.5 ± 0.50a
0.001
Postassium (mEq/L)
5.18 ± 0.24a
5.46 ± 0.29a
5.28 ± 0.15a
6.28 ± 0.03b
0.003
Chlorine (mEq/L)
100.27 ± 0.92
100.64 ± 0.80
96.95 ± 1.27
95.82 ± 1.18
0.011
Creatinine (mg/dL)
0.39 ± 0.03
0.35 ± 0.02
0,45 ± 0.01
a
0.33 ± 0.01
0.001
Albumin (g/dL)
3.45 ± 0.01b
3.47 ± 0.06b
3.77 ± 0.06c
3.18 ± 0.09a
0.001
T. protein (g/dL)
6.22 ± 0.043b
5.47 ± 0.15a
5.63 ± 0.06a
5.46 ± 0.07a
0.001
b
b
b
a,b
a
c
a
: The differences between values with different superscripted letters in the same line are statistically significant (P < 0.005).
a, b, c
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ŞEN and ÇELİK / Turk J Med Sci
145
b
A. Sodium (mEq/L)
b
b
B. Potassium (mEq/L)
8
6
140
a
135
a
a
4
0
125
Control
ASA
Control
Diabetic
Control
ASA
Diabetic
b
0.5
b
0.4
100
a
98
96
ASA Control
Diabetic
ASA
Diabetic
D. Creatinine (mg/dL)
C. Chlorine (mEq/L)
a
b
c
a,b
a
0.3
0.2
0.1
94
0
92
Control
ASA
Control
Diabetic
c
3.8
b
b
3.4
a
3.2
3
2.8
Control
ASA
Control
Diabetic
Control
ASA
Diabetic
E. Albumin (g/dL)
4
3.6
b
2
130
102
a
ASA
Diabetic
6.4
6.2
6
5.8
5.6
5.4
5.2
5
b
ASA
Control
ASA
Diabetic
F. Total Protein (g/dL)
a
Control
Diabetic
ASA
Control
a
Diabetic
a
ASA
Diabetic
Figure 1. Changes in serum biochemical parameters. A) Changes in serum sodium values between the groups. B) Changes in serum
potassium values between the groups. C) Changes in serum chlorine values between the groups. D) Changes in serum creatinine values
between the groups. E) Changes in serum albumin values between the groups. F) Changes in serum total protein values between the
groups.
determined between all groups except the control group
and ASA control group (Table 3).
3.3. Serum insulin levels
There was no significant difference in insulin levels
between any groups.
3.4. HOMA-β and HOMA-IR results
According to HOMA-β data that show the beta cell
functionality, the differences between control groups
and diabetic groups were statistically significant. In this
manner, beta cell functionality of the diabetic groups was
lower than that of control groups. Additionally, statistically
nonsignificant decreases were observed in beta cell
functionality in both control groups and diabetic groups
with ASA administration (Table 3).
Regarding the HOMA-IR results that show the insulin
resistance coefficients, similar to the HOMA-β results,
statistically significant differences were determined
between control groups and diabetic groups. In other
words, the insulin resistance coefficient was found
significantly higher in diabetic groups than in control
groups. Additionally, statistically insignificant decreases
were observed in insulin resistance coefficients in
both control groups and diabetic groups with ASA
administration (Table 3).
FBS findings show that the differences between diabetic
groups and nondiabetic groups were not significant (Table
3). As a result of ASA administration, a significant decrease
was found in the FBS levels in the ASA diabetic group
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ŞEN and ÇELİK / Turk J Med Sci
Table 3. Serum arginine vasopressin, insulin, and FBS levels, and results of insulin resistance and beta cell function.
Control
Vasopressin (ng/mL)
2.33 ± 0.44
Insulin (ng/mL)
0.328 ± 0.059
HOMA-β
a
ASA Control
Diabetic
14.38 ± 2.95
37.03 ± 7.11
a
b
ASA Diabetic
P
70.40 ± 3.86
0.001
c
0.242 ± 0.022
0.369 ± 0.048
0.253 ± 0.024
0.157
0.075 ± 0.014a
0.060 ± 0.004a
0.023 ± 0.002b
0.017 ± 0.002b
0.001
HOMA-IR
1.333 ± 0.245a
0.929 ± 0.144a
5.323 ± 0.778b
4.113 ± 0.437b
FBS (mg/dL)
90.83 ± 2.86
85.00 ± 6.70
405.33 ± 17.25
a
a
a
a
a
b
a
0.001
341.00 ± 27.22
c
0.001
: The differences between values with different superscripted letters in the same line are statistically significant (P < 0.005). FBS:
Fasting blood sugar. HOMA-β: Homeostasis Model Assessment, Beta Cell Function. HOMA-IR: Homeostasis Model Assessment,
Insulin Resistance.
a, b, c
compared to the diabetic group. The FBS levels decreased
directly proportional to the insulin resistance coefficients
(Table 3).
3.5. Insulin tolerance test results
Very low or no decreases in FBS levels show that insulin
resistance was established and that the diabetes model
was type II (16,17). In many studies that applied the ITT,
it was reported that the glucose-lowering effect of insulin
lasted until the 30th minute; however, the plasma glucose
level started to increase after the 30th minute due to
gluconeogenesis and hepatic glycogenolysis.
In this manner, the peak in the antihyperglycemic
activity of insulin is considered to occur at the 30th minute
and 30-min data were used for ITT (18). In this manner,
30-min data obtained during ITT studies were used for the
statistical analyses.
According to the 30-min data, insulin sensitivity in the
HFD group (diabetic group) was found to be higher than
that in the control group, meaning that insulin resistance
had developed.
4. Discussion
The main objective of diabetes treatment is to reach
normal blood glucose levels, eliminate the symptoms, and
prevent or delay the complications. In this study, the effect
of ASA on osmoregulation and glycemic control in terms
of diabetes has been discussed, considering the relations
between ASA, obesity, insulin resistance, diabetes,
cardiovascular disease, and AVP secretion.
In the literature, it was reported that salicylates
decreased the FBS by 13% (P < 0.002) and HbA1c by 17%
(20). In another study, it was reported that salicylates
decreased the HbA1c levels and therefore might be a
new way of treatment for glycemic control in type II
diabetics; however, it was emphasized that further studies
are required for renal and cardiac safety (21). Parallel to
the literature results mentioned above, in this study, it
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was found that glycemic states were suppressed in ASA
diabetics by making significant differences in FBS with
ASA administration between the diabetic group and ASA
diabetic group.
It was shown that AVP was synthesized 70% more in
diabetic rats compared to nondiabetic rats (22). Similarly,
it was reported that AVP levels were higher in diabetics
than in nondiabetics (23). In this manner, AVP results
obtained in this study were found to be parallel to the data
reported in the literature.
The reason for higher AVP synthesis in diabetics
compared to nondiabetics is still unknown. However,
it was suggested that this increase is associated with the
regulation of the osmotic balance (24). Examining the
control and ASA control groups and the diabetic and
ASA diabetic groups in this study, it was found that ASA
application contributed to the increase of AVP levels and
this increase was statistically significant between ASA
diabetic and diabetic groups.
It was shown that potent inhibition of AVP activity
occurred due to the increase in PGE2 production, and this
also caused nephrogenic diabetes insipidus (NDI) (25).
It was also found that as a result of the PGE2 synthesis
inhibition caused by using NSAIDs such as indomethacin,
polyuria caused by NDI was decreased (26). In light of
these two mutually supportive studies, it is understood
that PGE2 is an inhibitor of AVP. In the literature, it was
also clearly reported that PG synthesis was inhibited by
ASA by inhibiting the COX pathway (27). In light of this
information, it can be concluded that ASA has a positive
effect on AVP secretion by inhibiting the AVP inhibitor
PGE2.
With this study, a new approach is presented for
the subject. In diabetic groups, it was found that AVP
decreased significantly with ASA administration and this
indicates that blood glucose levels were relatively increased
ŞEN and ÇELİK / Turk J Med Sci
as a result of high AVP concentrations of hypoglycemic
activity caused by ASA. Additionally, the low results
of other biochemical parameters including chloride,
albumin, total protein, and creatine, similar to the blood
glucose results found in the ASA-administered diabetic
group, indicate that high AVP levels were dilutional.
Acknowledgement
The authors acknowledge with gratitude the cooperation
of the people who collected and managed the database of
their institution. This study was supported by the Afyon
Kocatepe University Scientific Research Projects Unit
(Project No: 10.Tip.18).
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