Effect of magnesium supplementation on oxidative stress in alloxanic diabetic rats

Magnesium Research. Volume 16, Number 1, 13-9, March 2003, ORIGINAL ARTICLE

Author(s) : Chetan P. Hans, Dharam P. Chaudhary, Devi D. Bansal , Department of Biochemistry, Panjab University, Chandigarh, India 160014 .

Summary : Magnesium deficit and oxidative stress are common features of the diabetic state. This concept supported by another observation that magnesium deficiency is also a state of increased oxidative stress prompted us to study the effect of magnesium supplementation on magnesium status and oxidative stress in diabetic rats. For this purpose, male Wistar rats were made diabetic with a single intraperitoneal injection of Alloxan. Experimental diabetes caused a significant decrease in serum and red blood cell magnesium levels and increased urinary excretion of magnesium. Marked increase in plasma malondialdehyde and corresponding decrease in vitamins C &\; E, uric acid and total thiols was observed in the diabetic rats as compared to control group. In liver, MDA levels were increased significantly with concomitant decrease in vitamin C, non‐protein thiols and antioxidant enzymes (SOD &\; GST). Magnesium supplementation for four weeks restored serum and RBC magnesium levels to near normal levels with marginal but significant decrease in blood glucose levels. Plasma and liver MDA levels were reduced significantly and vitamin C and total thiols were increased significantly with magnesium supplementation. Antioxidant enzyme activity was also increased significantly with magnesium supplementation in diabetic rats. Our data clearly demonstrates that alloxanic diabetes is associated with decreased magnesium status and increased oxidative stress and that magnesium supplementation can in part restore the antioxidant parameters and decrease the oxidative stress in experimental diabetic rats.

Keywords : magnesium, diabetes mellitus, antioxidants, oxidative stress

ARTICLE

Auteur(s) : Chetan P. Hans, Dharam P. Chaudhary, Devi D. Bansal

Department of Biochemistry, Panjab University, Chandigarh, India 160014

Address for correspondence: Bansal DD, Professor, Department of Biochemistry, Panjab University, Chandigarh, India 160014. Tel.: 0172-534133; E-mail: bansal_devi@indiatimes.com

Introduction

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.

Materials and Methods

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.

Induction of Diabetes

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].

Blood and urine collection

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.

Tissue preparation

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).

Biochemical analysis

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.

Statistical analysis

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.

Results

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).

Table I. Effect of magnesium supplementation on blood glucose, plasma insulin and serum, RBC & urine magnesium in experimental rats.

Parameter	Control rats	Non-supplemented diabetic rats	Supplemented diabetic rats
Glucose (mg/dl)	99.2 ± 2.4	252 ± 10.8^Ψ	217 ± 7.5***
Plasma Insulin (µu/ml)	12.8 ± 0.65	7.3 ± 0.53^Ψ	9.2 ± 0.37****
Serum Mg (mg/dl)	2.21 ± 0.04	1.54 ± 0.03^Ψ	1.97 ± 0.06*
RBC Mg (mg/dl)	4.20 ± 0.06	3.48 ± 0.06^Ψ	3.92 ± 0.07*
Urine Mg (mg/24 h)	3.92 ± 0.08	12.5 ± 1.10^Ψ	10.81 ± 0.42^#

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).

Serum magnesium levels decreased significantly in diabetic rats (p < 0.001) after ten weeks as compared to their respective controls (Table I). RBC magnesium was also significantly reduced in diabetic rats (p < 0.001) after ten weeks of experiment. Urinary excretion of magnesium in the diabetic rats was increased substantially as compared to normal controls (p < 0.001).
The plasma MDA levels, an index of lipid peroxidation, increased significantly from 5.46 ± 0.35 µmol/L in the control rats to 17.94 ± 1.05 µmol/ in diabetic rats (p < 0.001) after ten weeks of experiment (Table II). The levels of plasma vitamin C (p < 0.001), vitamin E (p < 0.001), total thiol (p < 0.001) and uric acid (p < 0.001) deceased significantly in the diabetic rats as compared to control rats.

Table II. Effect of magnesium supplementation on plasma MDA and antioxidants parameters in experimental rats.

70.2 ± 2.9^Ψ	70.8 ± 3.9^#
Total thiols	352 ± 7.76	207 ± 4.29^Ψ	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 ± 0.09^Ψ	1.5 ± 0.07**
Vitamin C (mg/g Pr)	0.32 ± 0.01	0.23 ± 0.01^Ψ	0.27 ± 0.01****
Non-Protein Thiols (mg/g Pr)	2.94 ± 0.05	2.04 ± 0.08^Ψ	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 ± 0.16^Ψ	4.01 ± 0.13**
GST (u/mg Pr/min)	13.47 ± 0.4	11.04 ± 0.39^Ψ	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 ± 1.1^Ψ	10.9 ± 0.8*
Vitamin C	46.2 ± 1.39	15.3 ± 1.16^Ψ	29.7 ± 1.49*
Vitamin E	17.8 ± 0.76	11.7 ± 0.66^Ψ	13.0 ± 0.53^#
Uric acid	102.3 ± 3.2