Magnesium is the most abundant intracellular divalent cation
present in living organisms. Magnesium is an essential ion involved
in glucose homeostasis at multiple levels as it plays an important
role in the activities of various enzymes involved in glucose
oxidation and may play a role in the release of insulin. Magnesium
has been reported to be mainly intracellular and its intracellular
uptake is stimulated by insulin [1]. Magnesium on the other hand
influences insulin secretion by altering the sensitivity of β cells
of islets of Langerhans to glucose [2].
In recent years there has been a growing interest in magnesium and
its correlation with development of various age related diseases
viz: hypertension, diabetes mellitus, cardiovascular diseases,
atherosclerosis, myocardial damage and cardiac arrhythmias. There
is a large volume of literature suggesting that magnesium deficit
contributes to the aging process and to the vulnerability to these
diseases [3-5]. One of the biological changes associated with aging
is an increase in free radical formation and subsequent damage to
cellular processes. It has recently been suggested that mammalian
tissues contain numerous defenses against oxidative stress some of
which may be compromised during magnesium deficiency [6].
Furthermore, magnesium itself possesses antioxidant properties,
scavenging oxygen radicals, possibly by affecting the rate of
spontaneous dismutation of the superoxide ion [7].
Oxidative stress, resulting both from over-production of reactive
oxygen radicals and decreased efficiency of antioxidant defenses is
now considered an important factor contributing to chronic diabetic
complications. The strict relation between the known pathogenic
factors involved in the development of these complications
(non-enzymatic protein glycation, activation of polyol pathway,
changes in lipid metabolism, haemostatic abnormalities) and
oxidative stress is being explored in the recent studies [8].
Keeping in view the above observations, the present study was
planned to find out the status of magnesium and oxidative stress in
experimental diabetes and to evaluate the effect of magnesium
supplementation on restoration of magnesium levels and
reversibility of oxidative stress in diabetic rats.
Alloxan, DTNB, GSH and CDNB used were procured from Sisco
Research Laboratories Pvt. Ltd. Mumbai, India. Methyl thymol blue
(MTB), Poly vinyl pyrolidine (PVP), Ethylene glycol tetra acetic
acid (EGTA), α-tocopherol and 2,4,6-tripyridyl-S-triazine (TPTZ)
were from Sigma Chemical Company, St. Louis, Mo. USA and were
kindly provided by Prof. Ronal R. MacGregor, Department of Anatomy
and Cell Biology, University of Kansas Medical Center, Kansas City,
Kansas, USA. All other chemicals used were of analytical reagent
grade.
Male Wistar rats (approx. 140 g wt.) were fasted overnight
before inducing diabetes with alloxan. Animals were anesthetized
with ether and injected intraperitoneally with freshly prepared
solution of alloxan (in normal saline) to give a final dose of
150 mg/kg body weight [9]. Control rats received an equal
amount of normal saline. Alloxan injected rats were allowed to
drink 5% glucose solution overnight to overcome drug-induced
hypoglycemia. Alloxan treated and control rats were housed
individually and allowed free access to food and water ad
libitum. During the 10-week experimental period, alloxan
treated rats were monitored by periodic testing for glucosuria with
Ames glucostix (Bayer Diagnostics India Ltd, Baroda).
After six weeks, diabetic rats were randomly divided into two
groups of six rats each. One group was supplemented with magnesium
as MgSO4 0.9 mg/ml along with drinking water for
four weeks and other was continued as such for four weeks [10].
To carry out the studies in blood, rats were fasted and blood
was drawn from the orbital sinus under light ether
anesthesia and collected into heparinized tubes, in oxalate
fluoride tube for glucose and in plain tube for magnesium and other
estimations. Heparinized blood was immediately centrifuged at
2 000 g for 15 min at 4 °C in cold centrifuge.
Plasma was separated and stored at – 18 °C until
analysis. RBCs were used for membrane preparation and magnesium
estimation. Blood glucose was estimated within half an hour. Serum
was separated from the plain sample within 1 h and was kept
for at – 18 °C until analysis.
On the penultimate day of the experiment, urine for 24-hour period
was collected in acid washed containers by placing the animals in
metabolic cages with free access to food and water.
At the end of the experiment, rats were killed by
exsanguinations from heart under diethyl ether anesthesia. Liver
was removed immediately and washed thrice with normal saline (0.89%
NaCl) to remove blood. Tissue was blotted, weighed, minced with
scissors and homogenized with ice-cold 50mM Tris-0.1 mM EDTA
buffer (10 ml/g of tissue), using a motor driven glass Teflon
homogenizer.
The homogenate was centrifuged at 1 000 g at 4 °C
for 10 min in a Remi cold centrifuge to get nuclear pellet and
supernatant. The supernatant was recentrifuged at 10,000 g for
15 min in Remi cold centrifuge. The pellet thus obtained was
suspended in Tris EDTA buffer and was used to estimate MDA, vitamin
C and non-protein thiols in mitochondrial fraction. The supernatant
was used to assay enzyme activities (SOD and GST).
Blood glucose and serum uric acid were measured by standard
reagent kits using semi auto analyzer. Magnesium was estimated
colorimetrically by dye method (methyl thymol blue) [11] Serum MDA,
vitamin E, vitamin C and total thiols were measured by the methods
of Beuge and Aust [12], Martinek [13], Roe and Kuether [14] and
Koster et al. [15] respectively. Activities of SOD and GST
were assayed by methods of Kono [16] and Habig et al. [17]
respectively. Insulin was estimated by RIA kit method procured from
BARC, Mumbai, India.
The data was statistically analyzed using Post Hoc Test (LSD)
for multiple comparisons using SPSS software to compare significant
alterations between control rats and diabetic rats and also among
the non-supplemented diabetic rats, supplemented diabetic rats and
control rats. All values are reported as mean ± SEM. A
‘p' value < 0.05 was considered as
significant.
Induction of diabetes by alloxan was confirmed by the presence
of glucosuria within 48 h as tested by glucostix (Ames India).
Within the first week of diabetes, mortality rate was ~ 30%.
Animals that passed this period survived for the remaining
10 weeks of diabetes. Marked hyperglycemia in diabetic rats
was persistent throughout the period of the experiment. Fasting
insulin levels in diabetic rats were drastically decreased
(p < 0.001) as compared with controls (Table I).
Values are expressed as; mean (SEM. Total number of rats were
six in each group. Total duration of experiment was ten weeks.
Magnesium was supplemented for four weeks.
(Ψp < 0.001 as compared to control
rats, * p < 0.001, ***
p < 0.02, **** p < 0.05,
# p non-significant, as compared to
non-supplemented diabetic rats).
|
70.2 ± 2.9Ψ
|
70.8 ± 3.9#
|
|
Total
thiols
|
352 ± 7.76
|
207 ± 4.29Ψ
|
275 ± 9.72*
|
Values are expressed as; mean (SEM. Total number of rats were
six in each group. Total duration of experiment was ten weeks.
Magnesium was supplemented for four weeks.
(Ψp < 0.001 as compared to control
rats, * p < 0.001, # p
non-significant, as compared to non-supplemented diabetic
rats).
Increased levels of MDA were also found in the liver of diabetic
rats after ten weeks (p < 0.001). There was a
significant decrease in vitamin C levels in the liver
(p < 0.001). Non-protein thiols (NPSH) were
also decreased significantly (p < 0.001) in the
liver of diabetic rats (Table III).
Table III. Effect of magnesium
supplementation on MDA, vitamin C and non-protein thiols in liver
of experimental rats.
|
Parameters
|
Control rats
|
Non-supplemented
diabetic rats
|
Supplemented diabetic
rats
|
|
MDA
(nmol/mg Pr)
|
0.91 ± 0.04
|
1.83 ± 0.09Ψ
|
1.5 ± 0.07**
|
|
Vitamin
C (mg/g Pr)
|
0.32 ± 0.01
|
0.23 ± 0.01Ψ
|
0.27 ± 0.01****
|
|
Non-Protein Thiols (mg/g Pr)
|
2.94 ± 0.05
|
2.04 ± 0.08Ψ
|
2.61 ± 0.08*
|
Values are expressed as; mean (SEM. Total number of rats were
six in each group. Total duration of experiment was ten weeks.
Magnesium was supplemented for four weeks.
(Ψp < 0.001 as compared to control
rats, * p < 0.001, **
p < 0.01,**** p < 0.05,
# p non-significant, as compared to
non-supplemented diabetic rats).
SOD activity was found to be significantly decreased in the
liver in the diabetic rats (p < 0.001) compared
to the respective control rats. The activity of GST was also
significantly decreased (p < 0.05) in the liver
of the diabetic group (Table IV).
Table IV. Effect of magnesium
supplementation on antioxidant enzymes (SOD & GST) in liver of
experimental rats.
|
Parameters
|
Control rats |
Non-supplemented diabetic
rats |
Supplemented diabetic
rats |
|
SOD
(u/mg Pr/min)
|
5.11 ± 0.11
|
3.42 ± 0.16Ψ
|
4.01 ± 0.13**
|
|
GST
(u/mg Pr/min)
|
13.47 ± 0.4
|
11.04 ± 0.39Ψ
|
12.3 ± 0.31****
|
Values are expressed as; mean (SEM. Total number of rats were
six in each group. Total duration of experiment was ten weeks.
Magnesium was supplemented for four weeks.
(Ψp < 0.001, as compared to control
rats, * p < 0.001, **
p < 0.01, **** p < 0.05,
# p non-significant, as compared to
non-supplemented diabetic rats).
Supplementation effects
Oral magnesium supplementation for four weeks caused a
significant increase in body weight in diabetic rats as compared to
unsupplemented diabetic rats. Supplementation of magnesium led to a
marginal but significant decrease in blood glucose in supplemented
diabetic rats as compared to non-supplemented rats
(p < 0.02). Marginal but significant recovery
in plasma insulin levels (p < 0.05) was also
observed as compared to non-supplemented diabetic rats (Table I).
In diabetic rats, the supplementation of magnesium restored the
serum magnesium levels to near normal levels
(p < 0.001) (Table I). RBC
magnesium was also increased significantly
(p < 0.001) after the supplementation of
magnesium in diabetic rats. However, urine magnesium excretion was
not significantly corrected as compared to unsupplemented diabetic
rats and remained higher as compared to control rats.
Plasma vitamin C (p < 0.001) and total thiols
(p < 0.001) increased significantly in the
diabetic rats with magnesium supplementation as compared to
non-supplemented diabetic rats (Table II). Magnesium supplementation however,
failed to show any significant change in vitamin E or uric acid
levels in plasma of diabetic rats. A significant decrease in plasma
MDA levels was observed after magnesium supplementation in the
diabetic rats as compared to non-supplemented diabetic rats
(p < 0.001).
In the liver of the diabetic rats, magnesium supplementation was
able to check the levels of MDA in liver
(p < 0.01), which were otherwise substantially
higher in non-supplemented diabetic rats (Table
III). Magnesium supplementation significantly increased vitamin
C levels in the liver of diabetic rats
(p < 0.05). Non-protein thiols (NPSH) levels
were also increased significantly in the liver in diabetic rats
with magnesium supplementation (p < 0.001) (Table III).
SOD activity was increased marginally but significantly in the
liver of diabetic rats (p < 0.01). Similarly,
liver responded positively to magnesium supplementation with regard
to GST activity in diabetic rats (p < 0.05) (Table IV).
Discussion
Magnesium depletion has been recognized as a common feature in
diabetes and its presence has been inversely related to glycemic
control and development of complications including cardiovascular
diseases [18, 19]. In diabetic rats, hypomagnesaemia and depleted
RBC magnesium was found in this study in confirmation with other
studies [20]. Urine excretion in the diabetic rats was increased to
approximately four times that of control rats and so was the
magnesium excretion. The renal handling of magnesium in diabetic
rats thus may be compromised and failure of the renal mechanism may
result in perpetuating hypomagnesemia and subsequently magnesium
depletion. Low levels of magnesium induce insulin resistance, which
in turn attenuates magnesium uptake by insulin-responsive tissues.
An inverse correlation between plasma magnesium and blood glucose
concentration has been demonstrated in rats with
streptozotocin-induced diabetes [20]. Thus, it can be proposed that
both extracellular and intracellular magnesium stores are being
significantly depleted in diabetes. Magnesium depletion in diabetes
represents a secondary type of magnesium deficit which requires
correction of the underlying primary cause as compared to primary
magnesium deficiency which can simply be corrected by oral
magnesium supplementation. Hypomagnesemia has been correlated with
both poor diabetic control and insulin resistance in non-diabetic
elderly patients [21]. ARIC Study [22] found a strong and inverse
independent relationship between serum magnesium levels and
subsequent development of incidents of diabetes in middle-aged
adults. It has been suggested that interpretation of serum
magnesium levels in the diabetic patients should be done with
reference to patient‘s metabolic state and ambient glucose levels.
The plasma magnesium levels are inversely related to the fasting
blood glucose and urinary magnesium excretion in the context of
hypermagnesuria. In accordance with other studies, this data
suggests that the diabetic state per se enhances urine
magnesium wasting irrespective of the degree of metabolic control.
It is believed that glycosuria that accompanies the diabetic state,
impairs renal tubular reabsorption of magnesium from the glomerular
filtrate. Glucose itself is a crucial part of cellular ion
homeostasis, increasing intracellular calcium and decreasing
intracellular magnesium [23].
Lipid peroxidation and derived oxidized products are being
intensively investigated because of their potential to cause injury
and their pathogenic role in several clinically significant
diseases [24]. The present study shows that experimental diabetes
in rats caused a marked increase in MDA levels and decrease in the
vitamin E, vitamin C, uric acid and total thiols in the blood and
antioxidant enzyme activity (SOD and GST) in liver. These results
are consistent with previous studies that oxidative stress is
increased in alloxan diabetic rats due to both increased lipid
peroxidation and decreased levels of natural antioxidants [25].
Elevated levels of MDA in the liver from diabetic animals have also
been supported by other studies [26, 27].
It has been suggested that the reduction in the antioxidant
parameters and increased free radical formation contribute to the
development of oxidative stress in diabetes [25, 28]. The sources
of oxygen-derived free radicals in diabetes are not known, but it
is possible that the sources may be from autoxidation of glucose
[29] and non-enzymatic glycation of proteins [30]. Even though it
is well known that free radicals are capable of inducing diabetic
complications, how oxidative stress in diabetes initiates
complications remain hypothetical. Elevated levels of MDA have been
identified in diabetes, and more marked in patients with poor
metabolic control. Free radical damage is increased in diabetic
patients with nephropathy and retinopathy in comparison to those
without diabetic complications [8]. Measurements of
tissue-scavenging enzymes in tissues of rats showed clearly that
liver showed a significant decrease in the activities of SOD and
GST in diabetic rats.
Large numbers of observations suggest that magnesium
supplementation may be useful in the treatment of patients with
diabetes, improving the glycemic control and preventing the
development of diabetic complications [21, 31]. Long-term magnesium
supplementation leads to improvement in both glucose-induced
insulin response and insulin action, decreasing insulin resistance
and improving glucose homeostasis in contrast to acute intravenous
supplementation which replenishes only the depleted magnesium store
in the body [21]. In diabetic rats in this study, though plasma and
RBC magnesium were replenished, the plentiful magnesium supply did
not however protect the magnesium metabolism from the adverse
effects of glycosuria characterized by intensification of the
excretion of magnesium in urine. The marginal decrease in the
glucose levels with the supplementation of magnesium confirms the
beneficial role of magnesium in diabetes. Recently it was shown
that in obese Zucker rats after 8 weeks of high dietary
magnesium intake, glycosuria and glycosylated hemoglobin, which
were present due to diabetes and not obesity, were reduced [32]. If
hyperglycemia is the main pathophysiological factor in the
development of diabetic complications, then positive impact of
magnesium supplementation may be expected. The results of the
present study indicate that supplementation of magnesium for a
period of four weeks increased the depleted vitamin C and total
thiols in the plasma and liver of diabetic rats. This may be due to
the well-known obligatory role of magnesium in the synthesis of
vitamin C and glutathione, or may be a consequence of the lowered
oxidative stress as levels of MDA were also decreased after the
supplementation of magnesium. The vitamin E and uric acid levels in
plasma showed a slight increase in magnesium supplemented compared
to untreated diabetic rats that may be due to decreased oxidative
stress in magnesium supplemented rats. Thus, supplementation of
magnesium could partly delay the oxidative stress in diabetes. In
addition to scavenging free radicals directly, magnesium
supplementation may have other benefits; magnesium can reduce the
extent of protein glycation which may also play an important role
in the development of diabetic complications, and this provides an
additional rationale for the use of magnesium in diabetes [32]. The
increased activity of antioxidant enzymes after supplementation of
magnesium supplementation indicates that magnesium may restore the
decreased overall antioxidant capacity in the diabetic animals. The
mechanism by which magnesium affects enzyme activity is not clear.
It is possible that magnesium may have been adequate to metabolize
the increased cellular peroxide to protect the enzyme activity.
Data have shown that tissue antioxidant systems are altered in
experimental diabetes and the restoration of these enzyme
activities to some extent by magnesium supplementation seems
indicative of the association of magnesium with the process of
development of oxidative stress in diabetes.
These findings raise the possibility that hypomagnesemia may
contribute in part to the oxidative stress and worsen late diabetic
complications at multiple levels. However, prospective studies are
needed to demonstrate convincingly whether supplementation with
magnesium will decrease the development of diabetes and its
complications.
Conclusion
Magnesium is a critically important nutrient and a useful
therapeutic agent. Depletion of magnesium and hypomagnesaemia are
relatively common but difficult to diagnose and have been
implicated in several disorders. The potential role of magnesium as
a therapeutic agent has not been well appreciated in the past. The
present study indicates that the mechanism responsible for the
oxidative stress in diabetes may be partly mediated through
magnesium depletion. Repletion of magnesium was associated with
restoration of antioxidant levels and decreased oxidative stress,
further supporting the viewpoint of the study. The implication of
magnesium as an important factor in glucose metabolism might at
least partially explain its postulated role in the late diabetic
stage. It seems very important to point out that magnesium
depletion and hyperglycemia aggravate each other in a true
pathogenic vicious cycle.
From this study, it may be concluded that magnesium is a
critical component of the antioxidant system and may be used as
potential therapeutic agent to reduce clinical diseases associated
with increased oxidative stress. Additional experimental and
outcome studies will continue to define the clinical scope of
therapy with magnesium and the emphasis should be to correct the
mechanism responsible for causing the deficit or depletion.
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|
Parameter (µmol/L)
|
Control rats
|
Non-supplemented
diabetic rats
|
Supplemented diabetic
rats
|
|
MDA
|
4.2 ± 0.33
|
17.9 ± 1.1Ψ
|
10.9 ± 0.8*
|
|
Vitamin
C
|
46.2 ± 1.39
|
15.3 ± 1.16Ψ
|
29.7 ± 1.49*
|
|
Vitamin
E
|
17.8 ± 0.76
|
11.7 ± 0.66Ψ
|
13.0 ± 0.53#
|
|
Uric
acid
|
102.3 ± 3.2
|
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