Effects of diabetes and insulin resistance in pregnant rats on ex vivo vascular reaction to magnesium

Magnesium Research. Volume 17, Numéro 4, 270-5, December 2004, Original article

Auteur(s) : Remare R Ettarh, Adesina P Arikawe , Department of Physiology, College of Medicine, University of Lagos, PMB 12003, Idi-Araba, Lagos, Nigeria.

Illustrations

ARTICLE

Auteur(s) :, Remare R Ettarh^*, Adesina P Arikawe

Department of Physiology, College of Medicine, University of Lagos, PMB 12003, Idi-Araba, Lagos, Nigeria

Introduction

Diabetes mellitus is characterised by vascular and endothelial dysfunction and these changes may underlie some of the complications associated with the disorder [1]. The development of endothelial dysfunction has been attributed to hyperglycaemia and accelerated formation of advanced glycosylation end-products. Sustained hyperglycaemia may lead to an increased generation of oxygen-derived free radicals that reduce nitric oxide availability [1]. Advanced glycosylation end-products also directly lower nitric oxide availability and increase the formation of oxidised LDL [2]. Studies have shown impairment of endothelium-dependent vasodilation in insulin-resistant animals [3]. Diabetes and insulin-resistant states are characterised by magnesium depletion [4], and the extracellular magnesium concentration is a determinant of vascular reactivity [5].

The relaxant effect of magnesium on vascular smooth muscle is well recognised. Magnesium modulates vascular tone via mechanisms in the smooth muscle cell or in the endothelial cell. In smooth muscle, magnesium blocks calcium entry through voltage-gated calcium channels [6], and inhibits calcium release from intracellular stores [7]. These effects result in a reduction in intracellular calcium, inhibition of intracellular signal transduction pathways, and ultimately vascular relaxation. In endothelial cells, magnesium activates Ca²⁺-activated K⁺ channels and elevates intracellular calcium, thereby inducing nitric oxide release [8]. The relaxant effect of magnesium is reduced in the absence of the endothelium suggesting a role for endothelium-dependent mechanisms [9].

The physiological adjustments that occur during pregnancy include enhanced endothelial function, probably due to increased synthesis and release of endothelium-derived vasodilators [10, 11]. Vascular contraction to phenylephrine is reduced in pregnant rats compared with non-pregnant rats, and this difference is absent in endothelium-denuded vessels [12]. Studies on vascular reactivity in pregnant women with diabetes have shown impaired flow-mediated vasodilatory responses [13]. This suggests that the enhancement of endothelial function in pregnancy may not prevent the impairment associated with diabetes and hyperglycaemia.

This study aimed to investigate the effects of alloxan-induced diabetes and fructose-induced insulin resistance on the vascular relaxation response to magnesium in pregnant rats. We hypothesized that isolated aortae from pregnant rats with diabetes or insulin resistance would demonstrate differences in responsiveness to magnesium when compared with normal pregnant rats.

Materials and methods

Animals

Virgin female Sprague-Dawley rats aged 6 weeks (120-150 g) were obtained from the Laboratory Animal Department and randomly divided into four groups. Group 1 served as control and was not exposed to male rats throughout the duration of the experiment. Group 2 was exposed to male rats and the day that a positive sperm smear was observed was regarded as Day 1 of pregnancy. Group 3 received a single intravenous injection of alloxan monohydrate into the lateral tail vein (40 mg/kg). The presence of hyperglycaemia was confirmed 48 hours later using Dextrostix Test strips (Bayer Corporation, U.K.). One week after induction of diabetes the rats were allowed to mate. Group 4 was fed ad libitum on a diet containing 25 % fructose for 12 weeks. Hyperglycaemia was confirmed using Dextrostix Test strips before the rats were allowed to mate. All the animals had free access to food and drinking water throughout the duration of the study.

Isolated tissue experiments

On Day 19 of pregnancy, the rats were anaesthetized and blood was obtained by cardiac puncture. Blood glucose was immediately determined by the glucose oxidase method [14]. The thoracic aorta was isolated and placed in cold Krebs bicarbonate solution containing (mM) NaCl 119, KCl 4.7, KH₂PO₄ 1.2, MgSO₄ 1.2, NaHCO₃ 14.9, CaCl₂ 1.6, glucose 11.5. The aorta was freed of adhering connective tissue and cut into 3 mm ring segments. The rings were suspended between a stainless steel rod and a hook attached to the base of the 20 mL organ bath. The stainless steel rod was attached to an isometric force transducer (Grass model FT 03) and connected to a Grass model 7D polygraph. The bath contained Krebs solution maintained at 37 °C, pH 7.4 and gassed continuously with 95 % O₂-5 % CO₂. The rings were equilibrated under 2 g tension. Stimulation of the rings with 10^-7 M phenylephrine was repeated every 30 min thrice to ensure contractile responses were stable. Incubation media were changed at 15 min intervals to avoid the influence of metabolites. After equilibration, the rings were contracted with 10^-7 M phenylephrine. After contraction had reached a plateau, the relaxation responses to cumulative concentrations of magnesium (1–12 mM) were determined. Successive concentrations were applied to the bath only when the effect of the previous concentration had stabilized. Relaxation responses to magnesium were also studied in aortic rings pre-incubated for 30 min with either the nitric oxide synthase inhibitor L-NAME (10^-4 M) or the cyclooxygenase inhibitor indomethacin (10^-5 M).

Drugs

The drugs used in the experiments were alloxan monohydrate, phenylephrine hydrochloride, N^ω-nitro-L-arginine methyl ester (L-NAME) and indomethacin, all purchased from Sigma-Aldrich. All solutions were freshly prepared daily. A stock solution of indomethacin was prepared in a 150 mM solution of Na₂CO₃.

Statistics

The data are expressed as mean ± SEM. EC₅₀ values (concentrations of MgSO₄ inducing 50 % relaxation of phenylephrine-induced contraction) for the concentration-response curves were calculated by regression. One-way Anova with post-hoc multiple comparisons using the Newman-Keuls test was used to compare mean values of the different groups. The results were considered significant at p < 0.05.

Results

Blood glucose level in pregnant rats (149 ± 12.3 mg/dL) was significantly higher than that in non-pregnant rats (86.0 ± 9.8 mg/dL, p < 0.05). The alloxan-treated pregnant rats (290.6 ± 10.2 mg/dL) and fructose-fed pregnant rats (270.0 ± 12.2 mg/dL) had significantly higher blood glucose levels compared with pregnant rats (p < 0.05).

Addition of cumulative concentrations of magnesium caused concentration-dependent relaxation in pre-contracted aortic rings from rats in all the groups. The relaxation-response to magnesium in rings from pregnant rats was significantly lower than that in rings from non-pregnant rats (( figure 1 )). The EC₅₀ was significantly higher in pregnant rats than in non-pregnant rats (table 1( Table 1 )). The maximum relaxation in rings from the pregnant rats (61.2 ± 1.9 %) was lower (p < 0.05) than that in rings from non-pregnant rats (86.8 ± 1.4 %). The relaxation responses to magnesium in rings from alloxan-treated pregnant rats and fructose-fed pregnant rats were significantly lower than that in rings from normal pregnant rats (( figure 1 )). EC₅₀ values in pregnant rats were significantly increased by alloxan treatment and fructose feeding (table 1). The maximum relaxation responses were significantly lower in the alloxan-treated pregnant rats (49.0 ± 3.6 %) and fructose-fed pregnant rats (44.6 ± 2.8 %) than in the normal pregnant rats (p < 0.05).

Relaxation responses to magnesium were significantly decreased in all the groups after incubation with indomethacin or L-NAME (table 1). The differences between the mean EC₅₀ values and maximum relaxation responses in pregnant and non-pregnant rats were not significantly altered after pre-treatment with indomethacin or L-NAME (table 1). Similarly, incubation with indomethacin or L-NAME did not significantly alter the differences in relaxation responses between rings from alloxan-treated pregnant rats or fructose-fed pregnant rats and rings from normal pregnant rats (table 1; figures 2, 3).
Table 1 EC₅₀ values (mM) for the relaxation response to magnesium in aortic rings of the different groups in the absence and presence of 10^-5 M indomethacin or 10^-4 M L-NAME

	Normal	Indomethacin	L-NAME
Non-pregnant	4.77 ± 0.14	6.52 ± 0.19¹	7.00 ± 0.59¹
Pregnant	8.92 ± 0.25²	9.59 ± 0.84^1,2	9.94 ± 0.44^1,2
Alloxan pregnant	11.77 ± 0.94³	12.78 ± 1.23³	14.87 ± 1.07³
Fructose pregnant	13.33 ± 1.23³	13.59 ± 1.18³	13.93 ± 1.29³

Discussion

Our main findings in this study were significant impairment in the vasodilatory response to magnesium in pregnant compared with non-pregnant rats, and greater impairment in the response to magnesium in pregnant rats with diabetes or insulin resistance compared with that in normal pregnant rats. These differences were not altered in the absence of endothelium-derived nitric oxide and prostaglandins.

The vascular smooth muscle relaxing effect of magnesium is well established [9, 15]. Increases in extracellular magnesium cause vascular smooth muscle relaxation, whereas lowering extracellular magnesium results in vasoconstriction [16]. Recent studies suggest that the effects of magnesium involve endothelium-dependent and endothelium-independent mechanisms [9]. Nitric oxide synthase inhibitors attenuate magnesium-induced endothelium-dependent relaxation [9, 17]. Nitric oxide causes relaxation of vascular smooth muscle mainly by activation of soluble guanylate cyclase thereby increasing cyclic guanosine monophosphate (cGMP) formation [18]. Increased cGMP formation causes inhibition of calcium influx and ultimately relaxation of vascular smooth muscle [18]. One study has suggested a synergistic interaction between magnesium and the nitric oxide-cGMP pathway [17]. Others propose that magnesium directly activates Ca²⁺-activated K⁺ channels in endothelial cells, inducing Ca²⁺ influx and consequently nitric oxide production and release [9]. In addition to nitric oxide, prostacyclin has been suggested to mediate the vasodilatory effect of magnesium [17]. Prostacyclin causes vascular smooth muscle relaxation via cAMP-dependent mechanisms [19]. In this study, the relaxation response to magnesium in non-pregnant rats was reduced with inhibition of the nitric oxide-cGMP and cyclooxygenase pathways.

Changes in vascular reactivity in pregnancy have been attributed to alterations in endothelium-dependent mechanisms and not smooth muscle function [20]. Increased synthesis and release of endothelium-derived relaxing factors, nitric oxide and prostacyclin, may underlie the enhanced vasodilatory responses observed in pregnancy [10, 11]. Magnesium-induced relaxation is however impaired in phenylephrine-contracted vessels in pregnant rats [15]. This effect has been attributed to altered regulation of receptor-operated calcium channels [15]. Our results also show significant impairment in the relaxation response to magnesium in pregnant rats compared with that in non-pregnant rats. Although, the relaxation response to magnesium in pregnant rats was also reduced with inhibition of the nitric oxide-cGMP and cyclooxygenase pathways, the diminution in the response to magnesium in pregnant rats compared with non-pregnant rats was unchanged in the absence of nitric oxide and prostaglandins. This suggests that the basis of the impairment in magnesium-induced relaxation in pregnant rats may be unrelated to defective action of endothelium-derived factors. Reports indicate that pregnancy is associated with magnesium depletion [21]. However, it is not clear whether magnesium depletion is responsible for the impaired vascular response to magnesium observed in pregnant rats.

Studies have shown that vascular relaxation is adversely altered in diabetes and insulin resistance [22, 23]. Endothelium-dependent vasodilation induced by acetylcholine, bradykinin and blood flow is impaired in resistance arteries of diabetic rats [22]. Insulin resistance induced by long-term fructose feeding also impairs endothelium-dependent vasodilation in rat mesenteric arteries [23]. These changes have been attributed partly to decreased production of endothelium-derived relaxing factors, and enhanced synthesis of endothelium-derived contracting factors [20]. The hyperglycaemia in diabetes and insulin resistant states is likely to be involved in the development of endothelial dysfunction. Hyperglycaemia may induce metabolic changes leading to increased generation of oxygen-derived free radicals and consequently increased inactivation of nitric oxide [1]. Formation of advanced glycosylation end-products may also impair endothelium-dependent vasodilation by directly quenching nitric oxide, or indirectly by increasing the formation of oxidised LDL which reduces nitric oxide bioavailability [2].

Pregnant women with insulin-dependent diabetes or gestational diabetes have impaired endothelium-dependent vasodilatory responses [13, 24]. It has been suggested that the severity of diabetic complications is greater in pregnancy, and this may partly be due to abnormal vascular function [20]. We observed that the impairment in the relaxation response to magnesium in pregnant rats with diabetes or insulin resistance was significantly greater than that in normal pregnant rats. This suggests that the effects of diabetes or insulin resistance on the vasculature may prevent the positive effects of pregnancy on production of endothelium-derived relaxing factors. Magnesium depletion has been reported in diabetes and pregnancy [4]. However, no studies have examined the relationship between the vascular dysfunction in pregnancy on diabetes and the magnesium depletion observed in these conditions. Incubation of magnesium-depleted tissues in a medium of elevated magnesium concentration should restore the intracellular magnesium concentration, unless there is impaired magnesium uptake by the cells. Studies using liver cells from diabetic rats have shown impaired magnesium uptake [25]. It is therefore plausible that the reduced relaxation to magnesium in diabetic and insulin resistant pregnant rats may be related to impaired magnesium uptake into the vascular smooth muscle cell. This is supported by the observation that the impaired relaxation caused by alloxan treatment and fructose feeding in pregnant rats was still evident in the absence of nitric oxide and prostaglandins.

Conclusion

In summary, the findings of this study indicate that the relaxation response to magnesium in ex vivo phenylephrine-contracted aortic rings is impaired in pregnant rats, and the degree of impairment is significantly greater in pregnant rats with diabetes or insulin resistance. Our observations suggest that these effects of diabetes and insulin resistance in pregnancy may be due in part to impairment of mechanisms other than the nitric oxide-cGMP and cyclooxygenase pathways.

References

1 Poston L. Endothelial control of vascular tone in diabetes mellitus. Diabetologia 1997; 40: S113-S114.

2 Bucala R, Tracey KJ, Cerami A. Advanced glycosylation products quench nitric oxide and mediate defective endothelium-dependent vasodilation in experimental diabetes. J Clin Invest 1991; 87: 432-8.

3 Verma S, Skarsgard P, Bhanot S, Yao L, Laher I, McNeill JH. Reactivity of mesenteric arteries from fructose hypertensive rats to endothelin-1. Am J Hypertens 1997; 10: 1010-9.

4 Durlach J. Magnesium deficit in diabetes mellitus. In: Durlach J, ed. Magnesium in Clinical Practice. London, Paris: John Libbey, 1988: 156-70.

5 Altura BM, Zhang A, Altura BT. Magnesium, hypertensive vascular diseases, atherogenesis, subcellular compartmentation of Ca²⁺ and Mg²⁺ and vascular contractility. Miner Electrolyte Metab 1993; 19: 323-36.

6 Fleckenstein-Grun GS, Matyas S, Dumont L. Voltage dependence of the pharmacological Mg²⁺ block of the Ca²⁺ entry into vascular smooth muscle cells. Magnes Res 1997; 10: 101-6.

7 Sjogren A, Edvinsson L. The influence of magnesium on the release of calcium from intracellular depots in vascular smooth muscle cells. Pharmacol Toxicol 1988; 62: 17-21.

8 Vaca L, Schilling WP, Kunze DL. G-protein-mediated regulation of a Ca²⁺-dependent K⁺ channel in cultured vascular endothelial cells. Pflügers Arch Eur J Physiol 1992; 422: 66-74.

9 Yang ZW, Gebrewold A, Nowakowski M, Altura BT, Altura BM. Mg²⁺-induced endothelium-dependent relaxation of blood vessels and blood pressure lowering: role of NO. Am J Physiol 2000; 278: R628-R639.

10 Williams DJ, Vallance PJT, Neild GH, Spencer JAD, Imms FJ. Nitric oxide-mediated vasodilation in pregnancy. Am J Physiol 1997; 272: H748-H752.

11 Greer IA, Walker JJ, McLaren M, Bonduelle M, Cameron AD, Calder AA, Forbes CD. Immunoreactive prostacyclin and thromboxane metabolites in normal pregnancy. Br J Obstet Gynaecol 1985; 92: 581-5.

12 Aloamaka CP, Ezimokhai M, Cherian T, Morrison J. Mechanism of pregnancy-induced attenuation of contraction to phenylephrine in rat aorta. Exp Physiol 1993; 78: 403-10.

13 Savvidou MD, Geerts L, Nicolaides KH. Impaired vascular reactivity in pregnant women with insulin-dependent diabetes mellitus. Am J Obstet Gynecol 2002; 186: 84-8.

14 Huggett AS, Nixon DA. Use of glucose oxidase, peroxidase, and O-dianisidine in determination of blood and urinary glucose. Lancet 1957; 273: 368-70.

15 Aloamaka CP, Ezimokhai M, Morrison J, Cherian T. Effect of pregnancy on relaxation of rat aorta to magnesium. Cardiovasc Res 1993; 27: 1629-33.

16 Altura BM. Magnesium and regulation of contractility of vascular smooth muscle. Adv Microcirc 1982; 11: 77-113.

17 Longo M, Jain V, Vedernikov YP, Facchinetti F, Saade GR, Garfield RE. Endothelium dependence and gestational regulation of inhibition of vascular tone by magnesium sulfate in rat aorta. Am J Obstet Gynecol 2001; 184: 971-8.

18 Ignarro L, Kadowitz PJ. The pharmacological and physiological role of cyclic GMP in vascular smooth muscle relaxation. Annu Rev Pharmacol Toxicol 1985; 25: 171-91.

19 Busse R, Hecker M, Fleming I. Control of nitric oxide and prostacyclin synthesis in endothelial cells. Arneim-Forsch Drug Res 1994; 44: 392-6.

20 Ang C, Lumsden MA. Diabetes and the maternal resistance vasculature. Clin Sci 2001; 101: 719-29.

21 Kurzel RB. Serum magnesium levels in pregnancy and preterm labor. Am J Perinatol 1991; 8: 119-27.

22 Tribe RM, Thomas CR, Poston L. Flow-induced dilatation in isolated resistance arteries from control and streptozotocin-diabetic rats. Diabetologia 1998; 41: 34-9.

23 Takagawa Y, Berger ME, Hori MT, Tuck ML, Golub MS. Long-term fructose feeding impairs vascular relaxation in rat mesenteric arteries. Am J Hypertens 2001; 14: 811-7.

24 Knock GA, McCarthy AL, Lowy C, Poston L. Association of gestational diabetes with abnormal vascular endothelial function. Br J Obstet Gynaecol 1997; 104: 229-34.

25 Fagan TE, Cefaratti C, Romani A. Streptozotocin-induced diabetes impairs Mg2+ homeostasis and uptake in rat liver cells. Am J Physiol Endocrinol Metab 2004; 286: E184-E193.