INTRODUCTION:
Diabetes Mellitus is a clinical
syndrome, characterized by hyperglycemia caused by a relative or absolute
deficiency of insulin at the cellular level. It is the most common endocrine
disorder, affecting mankind all over the world, prevalence of which is
increasing, daily (Tong and Cockrum, 2003). Experimental diabetes in animals
has provided considerable insight into the physiologic and biochemical
derangement of the diabetic state. Many of the derangement have been
characterized in hyperglycemic animals. Significant changes in lipid metabolism
and structure also occur in diabetes (Socharet al., 1985).
In these cases the structural
changes are clearly oxidative in nature and are associated with development of
vascular disease in diabetes (Baynes and Thropes, 1999). In diabetic rats,
increased lipid peroxidation was also associated with hyperlipidemia (Morel and
Chisolm, 1989).
Diabetes mellitus (DM) is associated with
an increased risk of thrombotic, atherosclerotic and cardiovascular disease.
Hyperlipidemia is metabolic complication of both clinical and experimental
diabetes (Gandh, 2001). Low-density lipoprotein in diabetic patients leads to
abnormal metabolism and is associated with increase in very low-density
lipoprotein (VLDL) secretion and impaired VLDL catabolism. Ultimately this
leads to atherosclerotic plaque (Howard, 1987). A number of known factors for
coronary artery disease such as hypertension, obesity and dyslipidemia are more
common in diabetics than in the general population. The World Health
Organization (WHO) predicts that the number of cases worldwide for diabetes,
now as of 171 million, will touch 366 million or more by the year 2030 (Wild et
al., 2004). Patients with DM are more likely to develop microvascular and
macrovascular complications than the non diabetic population (Baynes, 1991).
Dyslipidemia is a frequent complication of DM and is characterized by low
levels of high density lipoprotein-cholesterol (HDL-C) and high levels of low
density lipoprotein-cholesterol (LDLC) and triglyceride (TG).
Hyperlipidemia is the presence of high
levels of cholesterol in the blood. It is not a disease but a derangement that
can be secondary to many diseases and can contribute to many forms of diseases,
most notably cardiovascular disease. The treatment of hyperlipidemia depends on
the patients’ lipid profile. Many antihyperlipidemic agents like statin,
fibrates, niacins, bile acids, ezitimibe etc., reduce cholesterol level with
different conditions. Currently, available drugs have been associated with
number of side effects (Brown, 1996).
Recent studies have shown that lipid
associated disorders are not only attributed to the total serum cholesterol,
but also to its distribution among different lipoproteins. The low density
lipoprotein (LDL) are the major carriers of cholesterol towards tissues having
atherogenic potential while the high density lipoprotein (HDL) carry
cholesterol from peripheral tissues to the liver. HDL, thus gives protection
against many cardiac problems and obesity.
The injection of streptozotocin and alloxan
leads to development of diabetes and also development of hyperlipidemia
(O’mearaet al., 1991 and Agardhet al., 1999). Treatment of
hyperlipidemia in diabetes involves improving glycemic control, exercise and
the use of lipid lowering diets, drugs and hypoglycemic agents (Betterridge,
1997; Miller et al., 2001).
Atherosclerosis is the most prevalent
long-term diabetic complication. The increased mortality and morbidity in
diabetics caused by atherosclerosis has been related to the frequent occurrence
of hyperlipidemia in diabetes.
This study is designed to evaluate the
effect combined therapy of metformin and reducdyn in streptozotocin induced
diabetic rats.
MATERIALS AND METHOD:
Drugs and Equipment.
Glucophage (metformin) and reducdyn were
obtained from DookaPharmarcy Ltd, opposite the main gate of University of Port
Harcourt teaching hospital, Alakahia, Port Harcourt while Streptozotocin was
obtained from glaxo smith line Ltd, Ibadan. All other reagents were of
analytical grade. Optical densities were measured using digital
spectrophotometer (model 752S).
Treatment of Animals
Adult male wistar albino rats weighing
170-200g were used in this study. They were obtained from the animal House of
the Department of Biochemistry, University of Port Harcourt, Port Harcourt,
Nigeria. The guidelines of the Ethical committee for the use of experimental
animals of the University of Port Harcourt were followed. The animals were
housed in polypropylene cages in the animal house of the department, under room
temperature and pressure and were given normal feedi.eguinea growers mash (Port
Harcourt Flour Mills, Port Harcourt, Nigeria) and water ad libitum.
Anti-Diabetic
Study
After
one week of acclimatization of the animals. They were fasted overnight and
diabetes was induced by intraperitoneal injection of freshly prepared solution
of streptozotocin (160mg/kg body weight) in distilled water, while the normal
control rats (NCR) were injected with distilled water alone. Seven days after
administration of streptozotocin, the animals were again fasted and blood
collected via tail cutting (Burcelinet al., 1995), for the determination
of their fasting glucose levels. Then the rats were kept for 3 days to
stabilize the diabetic condition (Jyotyet al., 2002) before commencing
treatment, which lasted for 9 weeks.
Drug Administration
The drugs were administered as follows
Group-I--Normal control rats-received water
throughout the study.
Group-II --Diabetic control rats- treated with
water throughout the study.
Group-III-- Diabetic rats treated with a standard
drug - Glucophage(Metformin) (1.4mg/kg)
Group-IV-- Diabetic rats treatedwith 1.4mg/kg of
Glucophage and 0.25mg/kg of Reducdyn combined.
Group-V--Diabetic rats treated with 1.4mg/kg of
Glucophage and 0.5mg/kg of Reducdyn combined.
Lipid Profile Analysis
Serum total cholesterol (TC), high-density
lipoprotein cholesterol (HDL-C), and triglyceride (TG) were determined using
enzymatic methods with commercial test kits (Randox Laboratories, Crumlin,
England). The low-density lipoprotein cholesterol (LDL-C) was calculated using
the Friedewald et al (1972) formula.
Statistical Analysis of Data
The Data were analyzed by means
of one-way ANOVA and Post hoc LSD, on SPSS 19. In all, p<0.05 was considered
significant. Data were presented as Mean
S.D (Standard deviation).
RESULTS:
Table 1 below shows the results
of the effect of co-administration of metformin and reducdyn onplasma glucose
level of normal and streptozotocin induced diabetic male rats. Rats in group 1
maintained a fairly stable level of glucose throughout the study period with
the values 4.2±0.26, 3.9±0.85 and 4.8±0.91mmol/l after 3, 6 and 9 weeks of
treatment respectively. There was however significant (P˂0.05)
increase in the level of glucose concentration for the diabetic control rats
reaching a hyperglycemic level of 8.1±0.32, 7.0±0.40 and 6.3±0.43mmol/l for 3,
6 and 9 weeks of treatment respectively.
Group III rats showed
significant (P˂0.05) decrease in serum glucose levels on the 3rd,
6th and 9th week of treatment when compared with Group II
as shown in table 1.When metformin (1.4mg/kg body weight) was co-administered
with different doses of reducdyn, there was a significant (P˂0.05)
decrease in the level of glucose at the 6th and 9th week
of treatment than the first 3 weeks of treatment.
Table 1: Effect of
Co-administration of Metformin and Reducdyn on Plasma glucose level of Normal and Streptozotocin - induced diabetic male
rats.
|
Group
|
Treatment
|
Serum glucose level (mmol/l)
|
|
|
|
3 weeks
|
6 weeks
|
9 weeks
|
|
I
|
Normal Control Rats (NCR)
|
4.2±0.26a,b
|
3.9±0.85a,b
|
4.8±0.91a,b
|
|
II
|
Diabetic Control Rats (DCR)
|
8.1±0.32a,b
|
7.0±0.40a,b
|
6.3±0.43a,b
|
|
III
|
DCR on Metformin (1.4mg/kg)
|
4.6±0.97b
|
4.2±0.80b
|
4.5±0.20b
|
|
IV
|
DCR on Metformin (1.4mg/kg) + Reducdyn
(0.25mg/kg)
|
5.6±0.41a,b
|
3.8±0.20b
|
4.2±0.05b
|
|
V
|
DCR on Metformin (1.4mg) + Reducdyn
(0.5mg/kg)
|
6.3±0.77a,b
|
4.9±0.98b
|
4.4±0.98b
|
Values are expressed as Mean±SD;
n=3, per group/week. Values in the same column with common superscript letters
(a,b,) are significant at P< 0.05.
Superscript A(a) represents
significant difference when group I (normal control rats) are compared with
other groups at P<0.05.
Superscript B(b) represents
significant difference when group II (diabetic control rats) are compared with
other groups at P<0.05.
Values without superscripts
indicates no significant difference when compared with the normal and diabetic
control groups (groups I and II)
Table 2: Effect of
Co-administration of Metformin and Reducdyn on Plasma Triglyceride level of
Normal and Streptozotocin- induced Diabetic Male rats.
|
Group
|
Treatment
|
Plasma Concentration (mmol/L)(mean±S.D)
|
|
|
|
3 weeks
|
6 weeks
|
9 weeks
|
|
I
|
Normal Control Rats (NCR)
|
0.73±0.05a
|
0.80±0.20a
|
0.83±0.05a
|
|
II
|
Diabetic Control Rats (DCR)
|
0.80±0.00b
|
0.86±0.05b
|
0.90±0.10b
|
|
III
|
DCR on Metformin (1.4mg/kg)
|
0.70±0.10
|
0.80±0.10
|
0.90±0.43
|
|
IV
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.25mg/kg)
|
0.60±0.10b
|
0.73±0.05
|
0.76±0.37
|
|
V
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.5mg/kg)
|
0.60±0.00b
|
0.66±0.05b
|
0.70±0.26
|
Values are expressed as Mean
±SD; n=3, per group/week. Values in the same column with common superscript letters
(a,b,…) are significantly different at P<0.05.
Superscript A(a) represents
significant difference when group I (normal control rats) are compared with
other groups at P<0.05.
Superscript B(b) represents
significant difference when group II (diabetic control rats) are compared with
other groups at P<0.05.
Values without superscripts
indicates no significant difference when compared with the normal and diabetic
control groups (groups I and II)
Table 3: Effect of
Co-administration of Metformin and Reducdyn on Plasma Total Cholesterol level
of normal and Streptozotocin- induced Diabetic male rats.
|
Group
|
Treatment
|
Plasma Concentration
(mmol/L)
|
|
|
|
3 weeks
|
6 weeks
|
9 weeks
|
|
I
|
Normal Control Rats (NCR)
|
1.60±0.00a,b
|
1.73±0.11a,b
|
1.83±0.28a,b
|
|
II
|
Diabetic Control Rats (DCR)
|
2.13±0.11a,b
|
2.30±0.30a,b
|
2.43±0.11a,b
|
|
III
|
DCR on Metformin (1.4mg/kg)
|
2.13±0.30a
|
1.80±0.20b
|
1.56±0.05b
|
|
IV
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.25mg/kg)
|
1.80±0.00b
|
1.63±0.15b
|
1.60±0.00b
|
|
V
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.5mg/kg)
|
2.00±0.30a
|
2.00±0.20b
|
2.03±0.25b
|
Values are expressed as Mean
±SD; n=3, per group/week. Values in the same column with common superscript
letters (a,b,…) are significantly different at P<0.05.
Superscript A(a) represents
significant difference when group I (normal control rats) are compared with
other groups at P<0.05.
Superscript B(b) represents
significant difference when group II (diabetic control rats) are compared with
other groups at P,0.05.
Values without superscripts
indicates no significant difference when compared with the normal and diabetic
control groups (groups I and II)
Table 4: Effect of
co-administration of metformin and reducdyn on plasma HDL level of normal and
streptozotocin induced diabetic male rats.
|
Group
|
Treatment
|
Plasma Concentration
(mmol/L)
|
|
|
|
3 weeks
|
6 weeks
|
9 weeks
|
|
I
|
Normal Control Rats (NCR)
|
0.80±0.00
|
0.76±0.05
|
0.73±0.05
|
|
II
|
Diabetic Control Rats (DCR)
|
0.80±0.10
|
0.80±0.20
|
0.83±0.05
|
|
III
|
DCR on Metformin (1.4mg/kg)
|
0.86±0.05
|
0.83±0.05
|
0.80±0.10
|
|
IV
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.25mg/kg)
|
0.76±0.05
|
0.80±0.00
|
0.80±0.10
|
|
V
|
DCR on Metformin (1.4mg) +
Reducdyn (0.5mg/kg)
|
0.80±0.10
|
0.86±0.05
|
0.86±0.11
|
Values are expressed as Mean
±SD; n=3, per group/week. Values in the same column with common superscript letters
(a,b,…) are significantly different at P<0.05.
Superscript A(a) represents
significant difference when group I (normal control rats) are compared with
other groups at P<0.05.
Superscript B(b) represents
significant difference when group II (diabetic control rats) are compared with
other groups at P<0.05.
Values without superscripts
indicates no significant difference when compared with the normal and diabetic
control groups (groups I and II)
Table 5: Effect of
co-administration of metformin and reducdyn on plasma LDL level of normal and
streptozotocin induced diabetic male rats.
|
Group
|
Treatment
|
Plasma Concentration (mmol/L)
|
|
|
|
3 weeks
|
6 weeks
|
9 weeks
|
|
I
|
Normal Control Rats (NCR)
|
0.76±0.05a,b
|
0.80±0.20a,b
|
0.80±0.30a,b
|
|
II
|
Diabetic Control Rats (DCR)
|
1.16±0.15a,b
|
1.30±0.10a,b
|
1.40±0.10a,b
|
|
III
|
DCR on Metformin (1.4mg/kg)
|
1.13±0.20a
|
0.76±0.15b
|
0.60±0.10b
|
|
IV
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.25mg/kg)
|
0.93±0.05a
|
0.86±0.30b
|
0.66±0.05b
|
|
V
|
DCR on Metformin (1.4mg/kg) +
Reducdyn (0.5mg/kg)
|
1.16±0.20a
|
1.20±0.20a
|
1.30±0.26a
|
Values are expressed as Mean
±SD; n=3, per group/week. Values in the same column with common superscript
letters (a,b,…) are significantly different at P<0.05.
Superscript A(a) represents
significant difference when group I (normal control rats) are compared with
other groups at P<0.05.
Superscript B(b) represents
significant difference when group II (diabetic control rats) are compared with
other groups at P<0.05.
Values without superscripts
indicates no significant difference when compared with the normal and diabetic
control groups (groups I and II)
There was no significant (P<0.05)
difference in the triglyceride levels of the normal and diabetic control groups
treated for 3, 6 and 9 weeks but the level of triglyceride in the diabetic
control group increased more than the normal control group with the values
0.80±0.00 and 0.73±0.05, 0.80±0.20 and 0.86±0.05, 0.83±0.05 and 0.90±0.10
respectively
DISCUSSION:
The success recorded in the use
of streptozotocin (STZ) for the induction of diabetes mellitus through the
administration of 160mg/kg body weight can be attributed to the work of
Ferreira et al., 2002. This achievement was confirmed by evaluation of
fasting blood glucose concentration in the present study. There was significant
(P<0.05) increase in the level of glucose concentration for the
diabetic control rats reaching a hyperglycemic level of 8.1±0.32, 7.0±0.40 and
6.3±0.43 for 3, 6 and 9 weeks of treatment respectively.
Group III treated with standard
drug (metformin 1.4mg/kg) showed a significant (P<0.05) decrease in
serum glucose levels on the 3rd, 6th and 9th
week of treatment when compared with the diabetic control group (group II) as
shown in table 1.When metformin 1.4mg/kg was co-administered with different
doses of reducdyn, there was a significant (P<0.05) decrease in the
level of glucose at the 6th and 9th week of treatment
than the first 3 weeks of treatment.
There was no significant (P<0.05)
difference in the triglyceride levels of the normal and diabetic control groups
treated for 3, 6 and 9 weeks but the level of triglyceride in the diabetic
control group increased more than the normal control group with the values
0.80±0.00 and 0.73±0.05, 0.80±0.20 and 0.86±0.05, 0.83±0.05 and 0.90±0.10
respectively. This was however consistent with other workers that alloxan and
streptozotocin injection led to the development of hyperlipidemia (O’mearaet
al., 1991 and Agardhet al., 1991). Groups IV and V had a significant
(P<0.05) decrease in the triglyceride level when compared with the
diabetic control group after 3 weeks of treatment while group III had no
significant (P<0.05) difference in the triglyceride level when
compared with the normal and diabetic control groups after 3 weeks of
treatment. After 6 weeks of treatment, only groups IV had a significant (P<0.05)
decrease in the triglyceride level when compared with the diabetic control
group.
It was observed that there was a
significant (P<0.05) difference in the level of total cholesterol
between the normal and diabetic control groups after 3, 6 and 9 weeks of
treatment. Apart from group IV that has a significant (P<0.05)
difference in the level of triglyceride when compared with the diabetic control
group, every other group after 3 weeks of treatment has a significant (P<0.05)
difference in the level of total cholesterol when compared with the normal
control group. After 6 and 9 weeks of treatment, all the groups had a
significant (P<0.05) decrease in the total cholesterol level when
compared with the diabetic control group. There was no significant (P<0.05)
difference in the level of HDL cholesterol levels between the groups treated
for 3, 6 and 9 weeks when compared with the normal and diabetic control groups.
It is also pertinent to observe that the administration of the drugs at
different dosages along the period of treatment increased the plasma HDL
cholesterol.
The plasma low density
lipoprotein cholesterol levels of the normal and diabetic control groups were
significantly (P˂0.05) raised from 0.76±0.05 to 1.16±0.15,
0.80±0.20 to 1.30±0.10 and 0.80±0.30 to 1.40±0.10 for 3,6 and 9 weeks of
treatment respectively. This result practically showed that the induction of
diabetes mellitus automatically elevates hyperlipidemia which was shown here by
an increase in the LDL cholesterol (Nikkila and Hormila, 1978 and Bopannaet
al., 1997). After 3 weeks of treatment, there was a significant (P<0.05)
difference in the level of plasma LDL cholesterol all the groups when compared
with the normal control group. After 6 and 9 weeks of treatment, groups III and
IV had a significant (P<0.05) decrease in the level of LDL
cholesterol when compared with the diabetic control group. Only group V had a
significant (P˂0.05) difference in the level of LDL cholesterol
when compared with the normal control groups after 6 and 9 weeks of treatment
(table 5). Decreases in the plasma LDL cholesterol have been considered to
reduce risk of coronary heart disease (Rang et al., 2005; Shen, 2007);
while high plasma levels of LDL cholesterol is a risk factor for cardiovascular
diseases (Ademuyiwaet al., 2005; Lichtennstienet al., 2006) and
often accompanies diabetes mellitus (Rang et al., 2005; Brunzellet
al., 2008; Shepherd, 1998; Shen, 2007).
This present investigation shows
that all the streptozotocin induced rats displayed hyperglycemia and
hyperlipidemiaas shown by their elevated fasting glucose level, total
cholesterol, triglyceride, LDL and reduction in the HDL level. It can be
concluded that the co-administration of metformin and reducdyn was effective in
the reduction of glucose level, cholesterol, TG, LDL and increase HDL in a dose
dependent manner.
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