‫‪23‬‬
‫‪EFFECT OF NITRIC OXIDE, VITAMIN E AND SELENIUM‬‬
‫‪ON STREPTOZOTOCIN INDUCED DIABETIC RATS‬‬
‫‪Manar M. Nader,Laila A. Eissa*, Nariman M. Gamil and El-Sayed M. Ammar‬‬
‫يتصف مرض السكري بمضاعفات خطيرة تؤثر على العديد من األعضاء‪.‬‬
‫هدف البحث‪ :‬تهدف الدراسة إلى التحقق من دور أكسيد النيتريك كوسيط فسيولوجي في الجسم باسمتادا ممادة إل‪ -‬أرجينمين‬
‫كمادة أوليمة ألكسميد النيتريمك وييتمرول إل‪ -‬أرجينمين مياايمإ إسمتر ‪ )L-NAME‬كمامبط إليميي أكسميد النيتريمك سميناا فمي‬
‫الجرذان المصابة بالسكري‪ .‬كما ت دراسة تأثير فيتامين هـ كمضاد لألكسدة ومادة سلينيو كمحاكي قوي لمادة إيسمولين فمي‬
‫الجرذان المصابة بالسكري‪.‬‬
‫الطريقة‪ :‬ت دراسة التوافق المحتمإ لمادة سيلينيو أو فيتامين هـ مع إل‪ -‬أرجنين في يفس النموذج الحيوايي لتبيان قدرة همه‬
‫العالجات على تحسين بعض التغيرات الكيميائية التي تيداد سوءا ً بظهور مرض السكري ماإ مسمتو الحمحو و وجلوكمو‬
‫البال ماو ومالويالدهيد الد و وأكسيد النيتريك في البال ماو ومستو بيتما ‪ 2‬ميكروجلوبيمولين فمي البال مما‪ .‬وقمد تم اسمتحدا‬
‫مرض السكري معمليا ً في ذكور الجرذان بواسطة الحقن الوريدي لمادة ستربتو وتوسين ‪ 50‬مغ‪/‬كغ)‪.‬‬
‫النتائج‪ :‬أظهرت الجرذان المصابة بالسكري يادة معنوية ‪ )p<0.05‬في مستو جلوكو البال ماو والجلسمريدات الاالثيمةو‬
‫والكوليستيرول الكليو والكوليستيرول منافض الكاافةو وبيتا ‪ 2‬ميكروجلوبيولينو ومالويالدهيد الد كنتيجة ال ديماد الضمغط‬
‫المؤكسد في الوقت الهي حد فيه ايافاضا ً معنويا ً في مستو الكوليستيرول عالي الكاافة في البال مما ومسمتويات النتمرات‪/‬‬
‫النيتريمت‪ .‬كمما أحمد إعطماء إل ‪ -‬آرجنمين وفيتمامين همـ وسمملينيو ايافاضما ً معنويما ً فمي مسمتو جلوكمو المد فمي الجممرذان‬
‫المصابة بالسكري ‪%13‬و ‪%29.11‬و ‪ )%61.65‬على التوالي من قيمهما المبدئيمةو كمما أظهمرت أيضما ً ايافاضما ً معنويما ً فمي‬
‫مستويات مالويالدهيد الد و والجلسريدات الاالثية في البال ماو والكوليستيرول الكلميو والكوليسمتيرول عمالي الكاافمة وبيتما ‪2‬‬
‫ميكروجلوبيولين ولكنها أظهرت يادة يوعية في مسمتويات ييترات‪/‬ييتريمت والكولسمترول عمالي الكاافمة بالمقاريمة ممع القمي‬
‫المبدئية للسكري‪ .‬ول يظهر العالج المؤلف ممن فيتمامين همـ وإل‪ -‬أرجنمين أي تغييمر معنموي فمي أي ممن المؤامرات التمي تم‬
‫قياسها ماعدا مستو النترات‪ /‬النيتريت) بالمقارية مع المجموعة المعالجة بفيتامين هـ أو تلك المعالجة بإل‪ -‬أرجنين‪.‬‬
‫االسممتنتاج‪ :‬لقممد أظهممر العممالج المؤلممف مممن مممادة سمملينيو وإل‪ -‬أرجنممين ايافاض ما ً معنوي ما ً فممي تركيممي جلوكممو البال ممما إلممى‬
‫المستو الطبيعي تقريبا ً وربما كان لهلك أهمية إكلينيكية‪.‬‬
‫‪Diabetes mellitus is characterized by a serious of complications that affect many organs.‬‬
‫‪Aim: This study aimed to investigate the role of nitric oxide (NO) as a physiological mediator in‬‬
‫‪the body via the use of L-arginine as NO precursor and NG-nitro-L-arginine methyl ester (L‬‬‫‪NAME) as Nitric oxide synthase (NOS) enzyme inhibitor in diabetic rats. The effect of vitamin E‬‬
‫‪as antioxidant and selenium as a potent insulin-mimetic agent in diabetic rats were studied.‬‬
‫‪Methods: The possible combination of selenium or vitamin E with L-arginine was studied in the‬‬
‫‪same animal model to show the ability of these treatments to ameliorate some of the biochemical‬‬
‫‪changes that are worsen with the development of diabetes such as lipid profile, plasma glucose,‬‬
‫‪blood malondialdehyde (MDA), plasma nitric oxide & plasma β2- microglobulin levels.‬‬
‫‪Experimental diabetes was induced in male rats by I.V. injection of Streptozotocin (STZ) (50‬‬
‫‪mg/kg).‬‬
‫‪Results: Diabetic rats showed a significant increase (P<0.05) in plasma level of glucose,‬‬
‫‪triglycerides, total cholesterol, LDL-cholesterol, β2 – micro globulin, blood MDA as a result of‬‬
‫‪increased oxidative stress while there was a significant decrease in plasma HDL- cholesterol, and‬‬
‫‪Department of Pharmacology & Toxicology and Department of‬‬
‫‪Biochemistry, Faculty of Pharmacy, Mansoura‬‬
‫‪University,‬‬
‫‪Mansoura 35516, Egypt.‬‬
‫*‬
‫‪To whom correspondence should be addressed.‬‬
‫‪E-mail:lailaeissa 2002@yahoo.com‬‬
‫‪Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007‬‬
24
NADER ET AL
nitrate/nitrite levels. L-arginine, vitamin E and selenium administration produced a significant
decrease in plasma glucose level of diabetic rats (13%,29.11%,61.65%) respectively from its
initial value, also they showed a significant reduction in blood MDA level, plasma triglyceride,
total cholesterol, LDL-Ch. and β2-microglobulin levels but showed a significant increase in
plasma nitrate/nitrite and HDL-Ch.. levels when compared with the initial diabetic values.
Combined therapy of vitamin E and L-arginine showed no significant change of any of the
measured parameters ( except for nitrate/nitrite level) on comparison either with vitamin E or with
L-arginine treated group.
Conclusion: The combined therapy of selenium & L-arginie showed a significance decrease
nearly to normal level in the plasma glucose concentration & may be of clinical signifiance.
Key words: Diabetes mellitus, nitric oxide, L-arginine, oxidative stress, vitamin E, selenium.
Introduction
Diabetes mellitus is a chronic metabolic disorder
characterized by a high blood glucose concentration
(fasting plasma glucose > 125 mg/dl or plasma
glucose > 180 mg/dl, 2 hours after a meal) due to
insulin deficiency and/or insulin resistance (1). The
morbidity associated with long-standing diabetes of
either types results from complications such as
microangiopathy, retinopathy, nephropathy and
neuropathy. The basis of the chronic long-term
complications is a subject of a great deal of research.
Most of the available experimental and clinical
evidence suggest that the complications of diabetes
mellitus are a consequence of the metabolic
derangements, mainly hyperglycemia (2). Hyperglycemia may lead to an increased generation of free
radicals via multiple mechanisms such as glucose
autooxidation, non-enzymatic glycation, the polyol
pathway and reduced antioxidant defense system
(3,4).
Nitric oxide (NO) is an inorganic, gaseous free
radical. It is the first gas known to act as a biological
messenger in mammals. Nitric 0xide has a dual role
as a mediator of physiological and pathophysiological processes in pancreatic islets (5). Nitric oxide
plays an important role as an effector molecule in βcell destruction in rodents. In pancreatic islets, NO
produced on exposure to cytokines mediates β-cell
injury leading to diabetes mellitus. On the other
hand, L-arginine-derived NO may participate in the
signal transduction pathway of physiological insulin
secretion. Although, NO appears to play a major role
in β- cell destruction in rodent autoimmune diabetes,
its importance as an effector molecule of β- cell
injury in human insulin- dependant diabetes mellitus
is less evident. Nitric oxide can also combine with
oxygen to produce potent cellular killer such as the
highly toxic hydroxyl radical (OH˙) and peroxynitrite (ONOO¯) which is a powerful oxidant (5). NO
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
system is upregulated in diabetes and reduced
bioavailability in diabetic patients is more likely to
be secondary to enhanced breakdown of NO .One of
the most important determinants of NO
bioavailability is the reaction of NO with reactive
oxygen species. This mechanism of inactivation of
NO may be of particular relevance for patients with
diabetes mellitus (6). The shift in redox state in
diabetes mellitus may cause increased breakdown of
NO by superoxide resulting in the formation of
peroxynitrite. Peroxynitrite is a potent volatile
oxidant that can attack many types of biological
molecules. High levels of peroxynitrite cause
initiation of lipid peroxidation, sulfhydryl oxidation,
nitration of aromatic amino acids such as tyrosine,
direct DNA damage and oxidation of antioxidants
such as ascorbate and -tocopherol. Peroxynitrite
has also been shown to react with glucose to form
NO donors. This latter mechanism probably occurs
at lower concentration of peroxynitrite as defense
mechanism against oxyradical damage (7). In
diabetes, oxidative stress has been found to be
mainly due to an increased production of free radical
and a sharp reduction of antioxidant defense. The
relationship between oxidative stress and diabetic
complications has been extensively investigated.
Briefly, oxidative stress has been suggested to be a
major factor in the genesis of both macroangiopathy
and microangiopathy (4). Nitric oxide is believed to
participate in the regulation of the oxidation
/reduction potential of various cells and may be
involved in either the protection against or the
induction of oxidative stress within various tissues
depending on its concentration. Emerging evidence
suggests that some diseases are related to either an
inadequate or excessive production of NO (8). Lipid
peroxidation (LPO) is the most studied biologically
relevant free radical chain reaction (9). The
increased plasma LPO level in diabetic individuals
was positively correlated with the duration of the
EFFECT OF NITRIC OXIDE, VITAMIN E AND SELENIUM ON DIABETES
disease and was of a higher magnitude with the
development of complications (10). Antioxidants are
our first line against free radical damage. Vitamin E,
a membrane bound lipid soluble and naturally
occurring antioxidant has been shown to protect
animal tissue against oxidative damage such as lipid
peroxidation both in vitro and in vivo.. Vitamin E
treatment also prevents the activation of protein
kinase C (PKC) and the increase in diacylglycerol
content in the glomeruli of diabetic rats (11).
Selenium(Se) is important in many biochemical and
physiological processes. Selenium is incorporated as
seleno-cysteine at the active site of a wide range of
selenoproteins. The best-known biochemical role for
Se is as a part of the active site of the enzyme
glutathione peroxidase which is known as an
endogenous antioxidant enzyme (12) and thioredoxin reductase (TR) catalyzes the NADPH- dependent
reduction of which is a major redox protein for many
enzymes/transcription factors (13). Also, selenium
possesses insulin like action (14,15).
The aim of this study is to investigate the role of
NO as a physiological mediator in the body via the
use of L-arginine as NO precursor and L-NAME as
NOS enzyme inhibitor,the effect of vitamin E as
antioxidant and selenium as a potent insulin-mimetic
agent and the combination of them in diabetic rats.
Material and Methods
Experimental diabetes was induced in male rats
by I.V. injection of streptozotocin {STZ) (50
mg/kg). Induction of diabetes was confirmed by the
presence of hyperglycemia and glucosuria within 48
hours. Two weeks after the overt of diabetes, rats
were divided into groups each of 6 animals. Rats
received the following treatment for 4 weeks except
for selenium treated group and its combination with
L-arginine treatment, lasted only for 18 days:
1- Group I received L-arginine (622 mg/kg)
dissolved in distilled water and given by stomach
tube once daily (16).
2- Group II received L-NAME (10 mg/kg)
dissolved in distilled water and given by I.P
injection every day (17).
3- Group III received vitamin E acetate (40 mg/kg)
every other day diluted with sunflower oil and
taken by I.P injection (11).
4- Group IV received sodium selenate (1.89 mg/kg)
every day dissolved in water and given by I.P
injection (18).
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
25
5- Group V received both vitamin E (40 mg/kg)
every other day by I.P injection and L-arginine
(622 mg/kg) every day by stomach tube
6- Group VI received both sodium selenate (1.89
mg/kg) every day by I.P injection and L-arginine
(622 mg/kg) every day by stomach tube for 18
days.
7- Group VII a group of 6 diabetic rats received
normal saline.
8- Group VIII of 6 non-diabetic control rats
received also normal saline.
Blood samples:
were collected from the retro-orbital plexus of
the rat by heparinized capillary tube in test tube
containing EDTA, then the packed cells were
separated for determination of lipid peroxide and
plasma was separated for determination of glucose
level immediately and other plasma samples were
stored at -20oC for determination of nitric oxide as
nitrate/nitrite, β2- microglobulin, triglycerides, total
cholesterol, high density lipoprotein (HDL)cholesterol and low density lipoprotein (LDL)cholesterol levels. Glucose was determined
according to the enzymatic method described by
Trinder (19). NO was determined according to the
method described by Tracy et al (20) as nitrate and
nitrite. Plasma total cholesterol was determined
enzymatically according to the method of Allain and
coworkers (21). Plasma high-density lipoprotein
cholesterol was determined enzymatically according
to the method of Finley et al (22). Plasma
triglycerides were estimated by the method
described by Fredrickson and his colleagues (23).
Plasma low-density lipoprotein cholesterol is
calculated according to the method described by
Friedewald and coworkers, (24). MDA (an index of
lipid peroxidation). was determined by the method
of Stocks and Donnandy (25). The β2-microglobulin
ELISA test is based on the principle of a solid phase
enzyme-linked immunosorbent assay (26).
Results
Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination on some
biochemical paramaters in diabetic rats:
a) plasma glucose level:
Streptozotocin induced diabetes in rats produced
26
b) Blood MDA level:
Diabetic rats produced a significant increase in
blood MDA level. L-arginine significantly normalized the high level of blood MDA. Also, There was
a significant difference between L-arginine and LNAME treatment on plasma glucose level. Vitamin
E alone and its combination with L-arginine
produced a significant lowering effect on blood
MDA to near the normal level. Selenium alone and
its combination with L-arginine produced a
significant decrease in blood MDA level.
c) Plasma Nitrate/Nitrile (NO(x)) level:
STZ-induced diabetes in rats produced a
significant decrease in plasma NO(x) level Larginine treatment significantly raised plasma NO(x)
level, L-NAME treatment significantly reduced
plasma NO(x) level when compared with its initial,
control non-diabetic and L- arginine treated group
values, but there was no significant difference when
compared with control diabetic value. Vitamin E and
vitamin E + L-arginine combination treatment
produced a significant increase in plasma NO(x)
when compared with their initial and the control
diabetic values. This combination is significantly
different from L-arginine treated group In selenium
treated group plasma NO(x) level was significantly
lower than normal control value.
d) Plasma triglycerides, total cholesterol and LDLcholesterol levels:
Diabetic rats produced significant increase in
plasma triglycerides, total cholesterol and LDLcholesterol levels. L-arginine treatment showed a
significant decrease in plasma triglycerides, total
cholesterol and LDL-cholesterol levels level There
was a significant difference between the effect of Larginine and L-NAME treatment on plasma
triglycerides, total cholesterol and LDL-cholesterol
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
levels. Vitamin E treatment and the combined
treatment with vitamin E and L-arginine produced a
significant reduction in plasma triglycerides, total
cholesterol and LDL-cholesterol levels. Selenium
and selenium + L-arginine combination treatedgroups showed significant decrease in plasma
triglycerides, total cholesterol and LDL-cholesterol
levels.
e) Plasma HDL-cholesterol:
Diabetic rats produced a significant decrease in
plasma HDL-cholesterol. L-arginine treatment
significantly raised plasma HDL-cholesterol level
There was a significant difference between Larginine and L-NAME treatment on plasma HDLcholesterol selenium and selenium + L-arginine
combination showed significant increase in plasma
HDL-cholesterol level.
f) Plasma β2- microglobulin level:
Diabetic rats produced significant increase in
plasma β2- microglobulin level L-arginine treatment
showed a significant decrease in plasma β2microglobulin level. There was a significant
difference between the effect of L- arginine and LNAME treatment on plasma β2- microglobulin level.
Vitamin E and vitamin E + L-arginine combinationtreated groups produced a significant reduction in
plasma β2- microglobulin level Selenium and
selenium + L-arginine combination treated-groups
showed significant decrease in plasma β2microglobulin level.
o
$p
600
Plasma glucose level (mg/dl)
a significant increase in plasma glucose level. Larginine treatment significantly decreased plasma
glucose level but it was still significantly higher than
that of the control non-diabetic value. There was a
significant difference between L-arginine and LNAME treatment on plasma glucose level.Vitamin E
alone and its combination with L-arginine showed a
significant decrease in plasma glucose level.
Selenium alone and its combination with L-arginine
produced a significant decrease in plasma glucose
level.
NADER ET AL
*p o
500
*$p
400
p*$
p*$
300
*$
200
* $o
100
0
cont
cont diab
L-arg
L-NAME
vit-E
vit-E+L-arg
Se
Se+L-arg
Figure 1. Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination treatment on plasma
glucose level in diabetic rats.
p, * significantly different from control
non diabetic and control diabetic rats respectively using
one way ANOVA followed by Tueky Kramer Multiple
comparisons test (P<0.05).
$ significantly different from initial value using paired
Student’s t- test (P<0.05). significantly different from the
corresponding mean value of L-arginine treated group
(P<0.05). o significantly different from the corresponding
mean value of L-arginine treated group (P<0.05).
12.5
After treatment
150
p$
100
*$
*$
*
$
*
*$
$
50
0
cont
10.0
7.5
27
*po
cont diab
L-arg
L-NAME
vit-E
vit-E+L-arg
Se
Se+L-arg
*$
*$
Figure 4 (A)
*p o
5.0
*$
2.5
0.0
cont
cont diab
L-arg
*$
L-NAME
vit-E
*$
vit-E+L-arg
*$
Se
*$
Se+L-arg
Figure 2. Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination treatment on blood
malondialdehyde level in diabetic rats.
Plasma triglycerides level (mg/dl)
Blood MDA level
(m mol/ml packed cell)
Before treatment;
$p
Plasma total cholesterol (mg/dl)
EFFECT OF NITRIC OXIDE, VITAMIN E AND SELENIUM ON DIABETES
500
p$
400
300
*p o
200
100
0
*$
*$
cont
cont diab
L-arg
L-NAME
vit-E
*$
vit-E+L-arg
Se
Se+L-arg
Figure 4 (B)
* $o
40
*$
*
500
$
*p $
30
$p
*$
p$o
20
10
0
cont
cont diab
L-arg
L-NAME
vit-E
vit-E+L-arg
Se
Se+L-arg
Figure 3. Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination treatment on plasma
Nitrate/ Nitrite [NO(x)] level in diabetic rats.
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
Plasma LDL-cholesterol level
(mg/dl)
Plasma total nitrate/nitrite
(m mol/l )
50
p$
400
p* o
300
200
0
*$
*$
100
cont
cont diab
L-arg
L-NAME
vit-E
Figure 4 (C)
*$
vit-E+L-arg
*$
Se
*$
Se+L-arg
28
NADER ET AL
Figure 4 (D)
Plasma  2- microglobulin m
( g/ml)
Figure 4. Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination treatment on (a)
triglycerides, (b) total cholesterol, (c) LDL-cholesterol and
(d) HDL-cholesterol levels in diabetic rats.
15.0
p
12.5
po
10.0
7.5
*$
*$
*$
*$
*$
5.0
2.5
0.0
cont
cont diab
L-arg
L-NAME
vit-E
vit-E+L-arg
Se
Se+L-arg
Figure 5. Effect of L-arginine, L-NAME, vitamin E,
vitamin E + L-arginine combination, selenium and
selenium + L-arginine combination treatment on plasma
β2- microglobulin level in diabetic rats.
Discussion
It is generally considered that hyperglycemia is
the major factor in the pathogenesis of diabetic
complications (27). The persistent hyperglycemia
and the development of diabetic complications affect
nitric oxide (NO) signaling, availability and release.
An untreated diabetic state results in exaggerated
synthesis of free radicals and defective scavenging
systems also lead to impairment of NO system.
Results of the present study showed that diabetic
rats exhibited a significant increase in plasma
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
glucose level The beneficial effect of L-arginine
administration on plasma glucose level has several
systemic consequences. Firstly, L-arginine has the
ability to form polyamines (putrescine, spermidine
and spermine) (16, 28, 29). Secondly, L-arginine
may increase insulin secretion by the pancreatic cells that were not damaged after STZ- injection
(30). Concerning the effect of vitamin E treatment in
which vitamin E has a positive effect on basal and
arginine induced insulin secretion in the pancreatic
-cells that not destroyed after STZ-injection but
vitamin E did not restore glucose-induced insulin
secretion that had been abolished by the diabetic
state (31). Treatment of diabetic rats with sodium
selenate showed significant reduction in plasma
glucose level to near the normal values. The insulin
like actions of selenium include stimulation of
glucose uptake and regulation of metabolic
processes such as glycolysis, gluconeogenesis, fatty
acid synthesis, and pentose phosphate pathway. The
mechanism by which selenium is capable of
mimicking insulin is not clear. However reports
indicate that selenium does activate a key proteins
involved in the insulin-signal cascade (32).Also
selenium cause partial restoration of mRNA levels
and activities of two key glycolytic enzymes
(glucokinase and pyruvate kinase). It also decreases
the elevated mRNA concentration and the activity of
a major gluconeogenic enzyme phosphoenol pyruvate carboxy kinase (15). Also, it has the ability to
restore the expression of both the lipogenic enzymes,
glucose-6-phosphate dehydrogenase (G6PDH) and
fatty acid synthase (FAS). Increase both G6PDH
and FAS mRNA suggesting that the regulation of
expression by the mimetics occur pretranslationally
(18). Results of our investigation indicate that
diabetic rats showed significant increase in blood
MDA level (33,34). Increased MDA level might
reflect increased reactive oxygen species (ROS)
generation (35). Treatment of diabetic rats with Larginine produced a significant reduction in blood
MDA level. This effect may be due to that Larginine can scavenge superoxide anion [O2•‾ ]
radical causing inhibition of lipid peroxidation
process (36,37). Lipid peroxidation produces a series
of reactive carbonyl mainly aldehydes as N-epsiloncarboxymethyl lysine and methyl glyoxal that were
accompanied by decreased collagen accumulation, a
phenomenon consistently linked to diabetic longterm complication. L-arginine had a beneficial effect
of reducing lipid peroxidation-derived aldehydes
EFFECT OF NITRIC OXIDE, VITAMIN E AND SELENIUM ON DIABETES
(38). Vitamin E treatment showed significant
reduction in blood MDA level. The efficacy of
vitamin E is due to the powerful antioxidant
properties, also vitamin E is known as a membrane
bound, lipid soluble, hydrogen donor so that it is
effective as a major peroxyl radical scavenger of
biomembranes and low density lipoprotein (LDL).
So it can protect polyunsaturated fatty acids through
its function as a chain breaking antioxidant, thus
inhibit the propagation of lipid peroxidation process
(35-37). Selenium treatment showed significant
reduction in blood MDA level in diabetic rats.
Selenium may function via glutathione peroxidase
(GSH-Px) as its active site contains selenium in the
form of seleno-cysteine residue that is incorporated
into the polypeptide backbone. GSH-Px has
antioxidant activity (39,40). Diabetic rats produced
a significant reduction in plasma nitrate/ nitrite level
(taken as an index for NO production) (41-43). The
decreased NO level in diabetes may be due to either
reduced nitric oxide production and/or reduced nitric
oxide bioavailability. The reduced nitric oxide
production may be due to deficiency in substrate or
cofactors or both, which are important regulators of
NOS enzyme activity (44). In the absence of
sufficient tetrahydrobiopterin, NOS changes its
functional profile: instead of oxidizing L-arginine,
the enzyme reduces molecular oxygen to superoxide
anion, even in the presence of adequate substrate
concentrations (45). In diabetes there is an
accelerated polyol pathway and this increases the
consumption of NADPH, which is required for
aldose reductase activity. This leads to NADPH
depletion that affects NOS activity (46). The reduced
NO bioavailability may be due to increased
oxidative stress observed in diabetes or increased
advanced glycation end products. Hyperglycemia
lead to over production of O2˙‾ thus NO is trapped
by superoxide anion with the generation of ONOO˙.
The over production of ONOO˙ reduces the
biological effect of NO (47,48). The effect of Larginine on plasma nitrate/nitrite can be explained by
Restoration and/or increase in NO production by
quenching the release of superoxide anion from the
endothelium, thus reducing oxidative stress (49-51)
and/or reduction of advanced glycation end product
(AGEs) formation, diminishing the cross-linked
vascular AGEs (44).
In normal physiological conditions, additional Larginine showed no effect on NO production.
However, under various pathological conditions as
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
29
diabetes mellitus, hypertension and renal failure, Larginine supplementation has been shown to increase
NO production and influence its physiological
function due to arginine paradox (52). L-arginine
supplementation may become a promising solution
to improve endothelial function in diabetics (53).
The beneficial effect of vitamin E was by improving
the activity of endothelium derived nitric oxide (35).
Vitamin E was effective in increasing NOS activity
in diabetic rats, also it increased NO efficiency by
scavenging oxygen radicals in diabetic animals. So,
vitamin E supplementation is beneficial in reducing
nitric oxide oxidation mechanisms (41) and maintains both NOS activity and availability of NO (54).
Vitamin E has the ability to preserve diacylglycerol
kinase activity and thus normalize the upregulation
in PKC which can inhibit NOS activity (51).
Selenium treatment showed a significant increase in
plasma nitrate/nitrite level. Selenium-dependant
enzymes would appear to be of particular importance
in this regard. Glutathione peroxidase is chiefly
responsible for eliminating hydrogen peroxide and
for reducing lipid peroxides and their degeneration
products (55,56). Regarding the effect of combined
treatment with selenium and L-arginine showed no
significant difference in comparison with the control
non-diabetic value. This may be attributed to the
combined beneficial effects of both selenium and Larginine on plasma nitrate/nitrite level in diabetic
rats. Diabetic rats had marked elevated levels of
plasma triglycerides, total cholesterol and LDLcholesterol but a decreased level of plasma HDLcholesterol. (33,39,56). L-arginine treatment could
be explained not only by its possible participation as
insulin secretagague but also by the antilipolytic
action of polyamines formed from L-arginine. The
efficacy of vitamin E treatment may be due to that
vitamin E inhibits lipid peroxides, increases the total
hepatic triglycerides by increasing LPL activity
possibly by protecting the membrane bound lipase
against peroxidative damage (57,58). Vitamin E, the
most abundant antioxidant in LDL, is taken up by
LDL particles and can act as a chain breaking
antioxidant by scavenging highly reactive peroxyl
and alkoxyl radicals, which otherwise would
propagate the chain reaction of lipid peroxidation.
(59,60). The efficacy of selenate on plasma
triglyceride level and plasma HDL-cholesterol level
may be attributed to its insulin mimetic action that
increases LPL activity (61). Also, selenate had the
ability to restore the expression of both lipogenic
30
enzymes G6PDH and Fibroblast associated (FAS)
(18). Selenium as an insulin mimetic element has the
ability to restore the level of HMG-CoA reductase
activity, thus selenium treatment normalizes the
alteration in cholesterol metabolism that occurs in
diabetes (62). Selenium dependent peroxidase can
detoxify a wide variety of peroxides including lipid
derived-species (LOOHs) present in (LDL ox), thus
selenium treatment can ameliorate the modification
occurring in LDL-cholesterol by oxidative injury
(63). The present study showed that diabetic rats
exhibited a significant increase in plasma 2microglobulin level. Previous studies showed a
marked increase in plasma 2-microglobulin level in
diabetic patients in comparison with non-diabetic
controls (64). Also, there was a marked increase in
urinary 2-microglobulin level in STZ-diabetic rats
(65,66). L-arginine produced a significant reduction
in plasma 2-microglobulin level The reduction in
advanced glycation end products by L-arginine
ameliorates the elevated level of plasma 2microglobulin, as there is positive correlation
between 2-microglobulin excretion and glycated
hemoglobin (67).
vitamin E treatment showed a marked reduction
in plasma 2-microglobulin level. Vitamin E
treatment could prevent early changes in diabetic
renal dysfunction by normalizing the increase in
DAG and PKC levels as vitamin E activates DAG
kinase and facilitates the conversion of DAG to
phosphatidic acid (68). The effect of vitamin E could
be partly mediated by its antioxidant action and it
has been reported that vitamin E affects plasma
membrane fluidity by its direct interaction with lipid
bilayer structure of the membrane (11). Also,
vitamin E affects glycated hemoglobin level as it
significantly lowers glycated hemoglobin through its
antioxidant function (69). About the effect of
selenium on plasma  2-microglobulin, our data
showed significant reduction in plasma 2microglobulin level. In diabetic rat, selenium
deficiency caused oxidative stress and renal injury,
the mechanism is not completely understood. Thus
selenium supplementation to diabetic rats prevents
not only oxidative stress but also renal structural
injury as well (70). Also, selenium as an insulin
mimetic element reduces plasma 2-microglobulin
level. Selenium dependant peroxidase significantly
lowers glycated hemoglobin through its antioxidant
properties and thus affecting 2-microglobulin level.
Also, the ameliorating effect of selenium on the
Saudi Pharmaceutical Journal, Vol. 15, No. 1, January 2007
NADER ET AL
kidney of diabetic rats may be due to its antioxidant
as well as its insulin mimetic action which influence
polyol pathway, thus selenium treatment may delay
or prevent the development of diabetic kidney by
affecting polyol pathway.
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