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Global Journal of Pharmacology 8 (4): 525-531, 2014
ISSN 1992-0075
© IDOSI Publications, 2014
DOI: 10.5829/idosi.gjp.2014.8.4.84174
Regulation of Hyperglycemia and Dyslipidemia by Exogenous
L-Arginine in Streptozotocin-induced Diabetic Rats
1
Omnia Aly, 2Dina Abo El-Matty, 1Ehsan Aly Badawy,
1
Hanna Wafaay El Sherif and 1Hoda Aly Megahed
Department of Medical Biochemistry, National Research Center, Egypt
Department of Biochemistry, Faculty of Pharmacy Suez Canal University, Egypt
1
2
Abstract: L-arginine is a conditionally essential amino acid in human diet that serves as the substrate for nitric
oxide synthases (NOS) enzymes that generate NO, a key chemical involved in normal endothelial function. The
study evaluate role of L-arginine in improvement of insulin resistance and lipid profile in diabetic rats. Seventy
five male albino rats weighting 180-200 g were divided into five groups including; Control group, L-arginine
group, Diabetic group, Treated group and Prophylactic group. Fasting blood samples were collected from all
groups for determination of fasting blood glucose, insulin, insulin resistance, HDL, LDL, cholesterol and
triglyceride. The results showed that groups received L-arginine have a significant increase in insulin and HDL.
While they showed significant decrease in glucose, insulin resistance LDL, cholesterol and triglyceride. In
conclusion L-arginine may be a novel nutrient, which has important implications for the prevention and
treatment of diabetic patients.
Key words: Diabetes Mellitus
L-Arginine
Endothelial Function And Nitric Oxide
INTRODUCTION
improved vascular reactivity, reduced blood pressure and
normalized lipid peroxidation. Further, concentrations of
malondialdehyde, a product of lipid peroxidation, may be
reduced by arginine in diabetic patients and diabetic rats
[3].
L-arginine is engaged in several metabolic pathways
within the human body. It serves as a precursor for the
synthesis not only of proteins but also of urea,
polyamines, proline, glutamate, creatine and agmatine.
L-arginine is an essential component of the urea cycle, the
only pathway in mammals that allows the elimination of
toxic ammonia from the body. Ornithine, the by-product of
this reaction, is a precursor for the synthesis of
polyamines, molecules essential for cell proliferation and
differentiation [4]. L-arginine is also required for the
synthesis of creatine, an essential energy source for
muscle contraction. Agmatine, which has a clonidine-like
action on blood pressure, is also formed from L-arginine,
though its physiological function is not yet fully
understood. However, current interest in L-arginine is
focused mainly on its close relationship with the
Diabetes mellitus is a complex of metabolic disease
characterized by hyperglycemia, diminished insulin
production, impaired insulin action, or a combination of
both resulting in the inability of glucose to be transported
from the blood stream into the tissues, which in turn
results in high blood glucose levels and excretion of
glucose in the urine [1].
Diabetes is associated with reduced plasma
concentrations of arginine. Thus, dietary supplementation
of L-arginine could be beneficial for the treatment of
endothelial dysfunction in diabetic patients. L-arginine
supplementation normalized the endothelium-dependent
relaxation in diabetic aorta by enhancing NO availability
and restoring the acetylcholine-stimulated cGMP
generation [2].
In the diabetic state, it is well known that oxidative
stress is increased due to excessive production of oxygen
free radicals and impaired antioxidant defense
mechanisms. Increasing arginine supply to diabetic rats
Corresponding Author: Omnia Aly Abd El-Fattah, Medical Biochemistry Department, National Research Center, Giza, Egypt.
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Global J. Pharmacol., 8 (4): 525-531, 2014
Group V: (Prophylactic group): healthy rats received Larginine before and after induction of diabetes (10 mM Larginine /Kg b.w./day orally) according to Méndez and
Balderas [7].
important signal molecule nitric oxide. L-Arginine is the
only substrate in the biosynthesis of NO, which plays
critical roles in diverse physiological processes in the
human body including neurotransmission, vasorelaxation,
cytotoxicity and immunity [4].
L-arginine is reported to have beneficial effects on
several complications including pulmonary hypertension,
type-1 diabetes, cell neogenesis, insulin sensitivity and
improvement of endothelial function and reduction of fat
mass in diabetic rats [5]. This work aims to evaluate
L-arginine supplementations will improves insulin
resistance and serum lipid levels in streptozotocin
induced diabetic rats.
After 8 weeks, animals were kept fasting for 12 hours
before blood sampling, blood was withdrawn from the
retro-orbital venous plexus of the eye using a capillary
tube, blood collected in tubes contain sodium florid for
blood glucose and insulin estimation. The remaining part
of blood then left to clot and centrifuge at 3000 r.p.m.
using cooling centrifuge for 15 minutes, serum was
separated and divided into aliquots and stored at -20°C
until assayed.
MATERIALS AND METHODS
Biochemical Assay: Fasting glucose in serum was
performed according to the method of Passing and Bablok
[8] the kit was supplied by Biocon Diagnostic, Germany.
Serum insulin was performed according to Judzewitsch
et al. [9] the kit was provided from DRG, USA. Insulin
resistance was calculated from the equation according to
Mathews et al. [10].
Insulin resistance = fasting glucose (mg dl-1) x fasting
insulin (µIU ml 1)/405.
Serum HDL-cholesterol was performed according to
the method of Lopez-Virella et al. [11], the kit was
supplied from Biocon Diagnostic, Germany. Serum
LDL-cholesterol was calculated from equation developed
by Friedewald et al. [12]. Serum triglycerides were
determined using Kit from Centronic, Germany, according
to Fossati [13]. Cholesterol in serum was performed
according to the method of Allain et al. [14].The kit was
supplied from Biocon Diagnostic, Germany.
Material: L-arginine and streptozotocin
were
purchased from Sigma Aldrich Medical Company St.Louis
USA.
Experimental Animals: Male albino rats (Sprague Dawely
strain) weighting 180-200 g were obtained from the animal
house of National Research Center, Giza, Egypt. The
animals were housed in individual suspended stainless
steel cages in a controlled environment (22-25°C) and
12 hour light, 12 hour dark with food and water freely
available.
Methods
Induction of Diabetes: Streptozotocin (STZ) was
dissolved in 50 mM sodium citrate (pH 4.5) solution
containing 150 mM NaCl. The final concentration of the
injectable solution was containing (6.0 mg/100g body
weight) was subcutaneously administrated in rats; fasting
blood sugar was estimated after 3 days to confirm the
induction of diabetes mellitus according to Uchiyama and
Yamaguchi [6].
Statistical Analysis: Statistical analysis using SPSS
(Statistical package for social science) version 12,
software package for data analysis was done [15].
The quantitative data were presented in the form of
mean and standard error (SE) and the following tests were
used:-
Experimental Design: Seventy five male albino rats were
divided into five groups (15 rats each).
Group I: (Control group): healthy rats.
Group II: (L-arginine group): healthy rats
(10 mM L- arginine/Kg b.w./day orally).
Test of Significance: One way ANOVA was used to
compare between the means. P value < 0.05 is considered
to be significant.
received
Group III: (Diabetic group): diabetic rats.
Correlation Coefficient: Pearson's correlation coefficient
was done between each two variables to study the
relation between them.
Group IV: (Treated group): diabetic rats received
(10 mM L-arginine /Kg b.w./day orally)
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Global J. Pharmacol., 8 (4): 525-531, 2014
RESULTS
level was significantly decreased in diabetic group
compared to control group while this value increased
by L-arginine administration in treated groups compared
to diabetic group as shown (Table 2).
In the present study, Insulin resistance is directly
proportional with glucose, LDL, cholesterol and
triglyceride but it inversely proportional with insulin and
HDL.
Pearson's correlation was calculated
with
insulin resistance and
the
other
evaluated
parameters. It reveals a negative correlation with insulin
and HDL as shown in Figures 2, 3, however a positive
correlation was demonstrated with glucose, LDL,
cholesterol and triglyceride as presented I n
Figures 1, 4,5,6.
In the present study, fasting blood glucose and
insulin resistance levels were significantly increased in
diabetic group compared to control group, while this
value was improved by L-arginine administration in
treated groups compared to diabetic group. The insulin
level was significantly decreased in diabetic group
compared to control group while this value increased by
L-arginine administration in treated groups compared to
diabetic group (Table 1).
The LDL, cholesterol and triglycerides levels were
elevated in diabetic group compared to control group,
while these values decreased by L-arginine administration
in treated groups compared to diabetic group. The HDL
Fig. 1: Correlation between Insulin resistance and Glucose
Fig. 2: Correlation between Insulin resistance and Insulin
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Global J. Pharmacol., 8 (4): 525-531, 2014
Table 1: Blood glucose, insulin and insulin resistance levels in different groups.
Parameters
--------------------------------------------------------------------------------------------------------------------Glucose
Insulin
Insulin resistance
(mg/dl)
(µIU/ml)
(mgdl 1 µIU ml 1 )
Groups
Control
L-arginine
Diabetic
Prophylactic
Treated
Mean ± S.E
Mean ± S.E
Mean ± S.E
Mean ± S.E
Mean ± S.E
79.7±1.1
79.2±1.4b
243.2±2.7a
180.3±1.6a,b
203.5±1.7a,b
11.7±0.4
11.4±0.4b
8.5±0.2a
9.5±0.3a,b
9.0±0.3a
2.3±0.1
2.2±0.1b
5.1±0.9a
4.2±0.1a, b
4.5±0.1a,b
Significant p value 0.05
a = significant difference compared to control group
b = significant difference compared to diabetic group
Table 2: Serum Lipid profile in different studied groups
Parameters
---------------------------------------------------------------------------------------------------------------------------------HDL- cholesterol
LDL- cholesterol
Cholesterol
Triglyceride
(mg/dl)
(mg/dl)
(mg/dl)
(mg/dl)
Groups
Control
L-arginine
Diabetic
Prophylactic
Treated
Mean ± S.E
Mean ± S.E
Mean ± S.E
Mean ± S.E
Mean ± S.E
55.7±1.3
59.8±1.5a,b
39.0±1.4a
49.4±1.0a,b
45.5±1.0a,b
55.3±0.9
52.8±1.0b
119.9±1.0a
70.2±0.9 a,b
80.9±1.1 a,b
73.5±2.9
70.2±2.3b
150.7±3.2a
107.3±3.9a, b
120.8±2.8a, b
89.9±1.9
84.2±1.5a,b
197.5±1.8a
166.8±1.4a,b
175.7±1.8a, b
Significant p value 0.05
a = significant difference compared to control group
b = significant difference compared to diabetic group
Fig. 3: Correlation between Insulin resistance and HDL
Fig. 4: Correlation between Insulin
Cholesterol
DISCUSSION
resistance and
In the present study, the elevation in
the
serum glucose level and decline in serum insulin level
of diabetic group may be attributed to the specific
destruction of ß-cells by STZ which produces the
hormone insulin for normal glucose homeostasis
[16,17].
L-arginine is a basic natural amino acid. It is engaged
in several metabolic pathways within the human body. It
serves as a precursor for the synthesis not only of
proteins but also of urea, polyamines, proline, glutamate,
creatine and agmatine [4].
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Global J. Pharmacol., 8 (4): 525-531, 2014
Fig. 5: Correlation between
Triglyceride
Insulin
resistance
and
Fig. 6: Correlation between
LDL
Insulin enables the cells to absorb glucose from the
blood and also helps in the utilization of the glucose in
the cells by glycolysis, tricarboxylic acid cycle, hexose
monophosphate shunt and glycogenesis. In STZ induced
diabetes, cells fail to produce insulin which causes excess
glucose accumulation in the blood instead of being
utilized or stored. The decline in the mean insulin values
observed in the present study has also been reported by
many earlier workers [18,19].
In the present study, it was found that the treated
groups received L-arginine (Prophylactic and treated
groups) showed significant decrease in glucose and
insulin resistance levels as compared to diabetic group
and increased significantly in insulin compared to diabetic
group these results were in agreement with Young et al.
[20], Flynn et al. [21] and Salt et al.[22]. Also this group
showed significantly increase in glucose and insulin
resistance compared to control group.
L-Arginine is known to stimulate the secretion of
insulin from beta cells of the pancreas [21]. Dietary
supplementation of L-arginine resulted in an increase in
the plasma insulin levels in both STZ-induced diabetic
and non-diabetic rats. Note that in the STZ-diabetic rat
model, not all the ß-cells are destroyed and the remaining
cells can secret a physiologically significant quantity of
sufficient insulin to keep the animals alive for up to 2
months and also would promote net protein synthesis and
glucose utilization in skeletal muscle. The available
evidence suggests that the action of insulin and arginine
involves the following mechanisms. First, both insulin and
arginine stimulate NO production by endothelial cells,
Insulin resistance
and
which would contribute to increase in blood flow and,
therefore, glucose and amino acid uptake by skeletal
muscle in vivo [22]. Second, NO itself stimulates glucose
transport and oxidation by skeletal muscle [20]. Third,
physiological concentrations of NO may inhibit muscle
proteolysis [21].
In the present study, it was found that the group
received L-arginine showed significant increased in HDL
and also showed significant decrease in LDL, cholesterol
and triglyceride compared to control group. These results
were in agreement with Gad [4], Méndez and Balderas [7]
and Méndez and Zarzoza[23] who stated that L-arginine
could be explained not only by its possible participation
as insulin secretagogue, but also, by antilipolytic action
of polyamines formed from L-arginine. The effects of
polyamines have been observed for spermidine and
spermine, which enhance glucose oxidation and inhibit
lipolysis by suppressing endogenous cyclic AMP levels
in a manner similar to insulin in isolated rat fat cells.
In the present study, it was found that the diabetic
group showed significant decreased in HDL and also
showed significant increased in LDL, cholesterol and
triglyceride compared to control group. These results
were in agreement with the previous studies which
suggest that lipoprotein abnormalities are higher in
diabetics than in non-diabetics [24-26].
Veiraiah [27] suggested that hyperglycemia leads to
an increase in LDL cholesterol by reducing the ability of
the body to remove cholesterol. When blood sugars are
too high, LDL cholesterol and the receptors for LDL in the
liver become coated with sugar (Glycosylated), impairing
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Global J. Pharmacol., 8 (4): 525-531, 2014
ACKNOWLEDGEMENT
the liver's ability to remove cholesterol from the
bloodstream. In addition, hyperglycemia also leads to
inhibition of lipoprotein lipase and further aggravating
hyperlipidemia.
When diabetes is not under good control, there are
high levels of glucose in the body, so, the conversion of
glucose into glycogen increased and stored in the liver,
when the liver becomes too saturated with glycogen,
though, glucose is instead used to synthesized fatty acids
that are released into the bloodstream; these fatty acids
are used to produce triglycerides, which build up in fat
cells and contribute to body fat [28].
Hyperlipidemia is a recognized complication of DM
characterized by elevated levels of cholesterol,
triglycerides and LDL. One of the major pathogenesis of
lipid metabolism disturbances in diabetes is the increased
mobilization of free fatty acids from adipose tissue and
secondary elevation of free fatty acid level in the
blood due to insulin deficiency or insulin resistance.
The excessive lipolysis in diabetic adipose tissue may
lead to increased free fatty acids in circulation which enter
the liver and are esterified to form triglycerides. The fatty
acid compositions of various tissues are altered in both
experimental and human diabetes. The finding in the
present study is in correlation with the findings of
Krishna et al. [17] and Sharma et al. [29].
In the present study, it was found that the treated
groups received L-arginine (Prophylactic and treated
groups) decreased significantly in HDL and also showed
significant increase in LDL, cholesterol and triglyceride
compared to control group these results were in
agreement with Méndez and Balderas [7]. Also, this group
showed significant increase in HDL and significant
decreased in LDL, cholesterol and triglyceride compared
to diabetic group.
In this study, the tendency to normalization of lipid
and lipoprotein, levels in diabetic rats treated with
L-arginine could be explained according to Kawano et al.
[30] who stated that its accelerated formation from
L-arginine in the pancreas of diabetic rats could, together
with spermidine and spermine, not only stimulate glucose
uptake but also inhibit lipolysis in diabetic rats these
results were in agreement with.
Authors are grateful to the National Research Center,
Giza, Egypt for unlimited help and support to carry out
this work.
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L-Arginine may be a novel nutrient, which improves
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important implication for the prevention and treatment of
diabetic patients.
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