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Effect of sodium selenite treatment on platelet aggregation of streptozotocin-induced diabetic rats
Thrombosis Research 111 (2003) 363 – 367
Brief Communication
Effect of sodium selenite treatment on platelet aggregation of streptozotocin-induced diabetic rats Gu¨lriz Erso¨z a,*, Ali Yakaryılmaz a, Belma Turan b a b
Department of Physiology, Faculty of Medicine, Ankara University, 06100 Sihhiye, Ankara, Turkey Department of Biophysics, Faculty of Medicine, Ankara University, 06100, Sihhiye, Ankara, Turkey Received 25 March 2003; received in revised form 5 May 2003; accepted 8 May 2003
Abstract Introduction: There is a well-known association between diabetes and atherosclerosis. Platelets are involved in the development of atherosclerotic vascular diseases and play a key role in atherosclerotic complications. Diabetes mellitus is related to alteration in the homeostasis of selenium and the protective role of selenium against lipid peroxidation in diabetes is reported. In the present study, thrombin-induced platelet aggregation and thromboxane A2 (TxA2) formation in diabetes and the effect of sodium selenite were evaluated. Materials and methods: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in Wistar rats (n = 21). Thirty of them were used as control rats. A week after streptozotocin injection, 11 of the control rats and 12 of the diabetics were injected with 5 Amol/kg/day of sodium selenite for 4 weeks. Thrombin-induced aggregation of the platelets was evaluated by optical technique. Thromboxane B2 (TxB2), TxA2 metabolite, was measured by enzyme-linked immunoassay (EIA) in thrombin-induced platelets. Results and conclusion: The platelet aggregation and TxB2 level increased in diabetic rats. Sodium selenite reversed the increase in platelet aggregation and TxB2 and caused a small but significant ( p < 0.05) decrease in the glucose level. The hyperaggregability of platelets in STZ-induced diabetic rats was thought to be related to the enhanced TxA2 formation of platelets. Increase in TxA2 formation implies lipid peroxidation. Sodium selenite decreased the TxA2 formation. Besides its antioxidative effect, further studies are needed to establish the insulin-like effect of selenite because of a small decrease in blood glucose. D 2003 Elsevier Ltd. All rights reserved. Keywords: Diabetes; Platelet aggregation; Thromboxane A2; Selenium; Antioxidant
1. Introduction There is a well-known association between diabetes and atherosclerotic cardiovascular and microangiopathic diseases. Since platelets contribute to the development of these vascular events, alteration in platelet function may be a key factor in these complications in diabetes. Several platelet abnormalities have been reported in insulin- and noninsulindependent diabetes; increase in platelet adhesion, increased sensitivity to aggregating agents, changes related to membrane lipids and glycoproteins and several metabolic changes, particularly of arachidonic acid and abnormal Ca2 + homeostasis [1,2]. Hemodynamic disturbances and
Abbreviations: TxA2, thromboxane A2; TxB2, thromboxane B2; STZ, streptozotocin; PGI2, prostacyclin; PRP, platelet-rich plasma; EIA, enzymelinked immunoassay; RIA, radioimmunoassay; PPP, platelet-poor plasma. * Corresponding author. Tel.: +90-312-310-3010; fax: +90-312-3097404. E-mail address: [email protected] (G. Erso¨z). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00338-4
oxidative mechanisms may also be involved in diabetesinduced platelet abnormalities [1,3,4]. Chronic hyperglycemia causes an imbalance between radical oxygen species and scavenging system activity, and leads to oxidative stress [5,6]. It has been reported that supplementation with antioxidants prevents diabetic and hyperglycemic cardiovascular disease [4,7 –12]. Selenium is a trace element that plays an important role in many physiological mechanisms. It is an integral part of glutathione peroxidase and protects various cells against oxidative damage. It is suggested that diabetes mellitus is related to alteration in the homeostasis of selenium [13]. The protective role of selenium administration against lipid peroxidation and diabetes-induced injury in several tissues was reported [14,15]. It has also been suggested that selenium affects carbohydrate metabolism and has insulinlike actions [13,16]. The effect of selenium supplementation showed a strong dependency on the type of the selenium compound, its concentration and the way of administration. In our previous
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study, we treated STZ-induced diabetic rats with a much lower concentration of selenite (5 Amol/kg, i.p.) than that used by other groups [17,18]. We observed that the treatment protected the heart tissue against diabetes-induced injury [14]. Accordingly, we aimed to examine the thrombin-induced platelet aggregation and lipid peroxidation in diabetic rats and the role of sodium selenite administration. The thromboxane A2 (TxA2) formation was planned to measured as a marker of platelet activation and lipid peroxidation [19,20].
group, 440 000 F 37 700/mm3 of diabetic group, 450 000 F 46 600/mm3 of diabetic + Se-treated group.). 2.3. Platelet aggregation
2. Materials and methods
Platelet resuspension was incubated at 37 jC for 5 min. Thrombin (Chronolog, PA, USA) at a final concentration of 0.8 U/ml was used for induction of platelet aggregation. The aggregation was performed on a Chronolog 560 WB Aggregometer by the optical method [21]. Maximal amplitude of aggregation curve (the percentage of platelet-poor plasma (PPP) being 100%) was calculated as the indicator of platelet aggregation.
2.1. Animals and treatment
2.4. Platelet TxA2 Formation
Twelve- to fourteen-week-old Wistar rats (initial body weight of 200 – 250 g) were used in this study. They were housed three in a cage at a temperature of 22 F 1 jC with a fixed 12 h light– dark cycle. They were fed ad libitum with a standard rat diet. The procedures used in the study were approved by the Ethics Committee of Ankara University Faculty of Medicine. The rats were randomly divided into two groups. Streptozotocin (STZ: 50 mg/kg body weight; dissolved in 0.02 M sodium citrate, pH 4.5, Sigma) was intraperitoneally (i.p.) injected to the first group. The rats with glycemia > 2.5 g/l, a week after STZ injection were considered as diabetic (n = 21). Control rats (n = 30) were injected with a corresponding volume of citrate buffer (i.p.). Plasma insulin level was measured by radioimmunoassay (RIA). A week after the STZ injection, 11 of the control rats and 12 of the diabetics were injected (i.p.) with 5 Amol/kg/day of sodium selenite for 4 weeks (Se-treated groups). Other rats (19 controls and 9 diabetics) were injected with a corresponding volume of saline (untreated groups). The mean blood glucose, insulin levels and the final body weight of the rats were measured at the beginning and after the treatments.
Thromboxane B2 (TxB2), a metabolite of TxA2 was measured in the supernatant samples prepared by centrifugation of thrombin-induced platelet suspension for 3 min at 10 000 rpm. Enzyme-linked immunoassay (EIA) was used for the measurement. The TxB2 EIA kit was obtained from Cayman Chemical (MI, USA). 2.5. Statistical analyses Results are given as the mean F SE. Comparisons between control untreated and Se-treated, diabetic untreated and Se-treated rats were carried out by variance analysis followed by the Newman – Keuls test. Differences were considered significant at p < 0.05. The Pearson correlation coefficient was used to measure the relationship between platelet aggregation and platelet TxA2 formation.
3. Results The mean blood glucose, the final body weight and insulin levels of the Se-treated and untreated groups of controls and diabetics are given in Table 1. The blood
2.2. Blood samples and platelet preparation After the selenite and placebo treatments, rats were anesthetised by sodium pentobarbital (30 mg/kg) and blood samples were collected by cardiac puncture into 5 ml of siliconized tubes containing 0.5 ml of 3.8% sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation of sodium citrated blood samples at 300 g for 15 min. Platelet count was determined by the Coulter counter T890. The platelet pellet was obtained by centrifugation of PRP at 900 g for 15 min. The platelet pellet was washed and resuspended to 300 000 –500 000 platelet/mm3 with Hepesbuffered Tyrode solution containing 138 mM NaCl, 0.36 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, 20 mM Hepes (pH 7.4). There was no significant difference between platelet counts of both groups (452 000 F 28 300/ mm3 of control group, 439 000 F 37 000/mm3 of Se-treated
Table 1 Final body weights, blood glucose and insulin levels of control and diabetic rats Rats
Control group Untreated Se-treated
Body weight (g)
Blood glucose (mg/dl)
Blood insulin level (fmol/ml)
238 F 5 251 F 7
103.5 F 2.3 129.0 F 5.1*
281.6 F 57.3 109.2 F 15.2**
Diabetic group Untreated 188 F 5** Se-treated 204 F 11**
437.1 F 15.7** 390.1 F 30.8**,#
76.1 F 15.9** 58.1 F 14.9**
Untreated rats were saline-injected group and Se-treated rats were sodium selenite-injected group. Values are mean F SD. * p < 0.05 compared with untreated control group. ** p < 0.001 compared with untreated control group. # p < 0.05 compared with untreated diabetic group.
G. Erso¨z et al. / Thrombosis Research 111 (2003) 363–367 Table 2 Maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels of control and diabetic rats (mean F SD) Rats
Maximal amplitude of platelet aggregation (%)
TxB2 level (pg/ml)
Control group Untreated Se-treated
7.6 F 5.8 6.7 F 5.3
43.4 F 18.1 53.4 F 16.9
Diabetic group Untreated Se-treated
19.3 F 9.2* 10.9 F 5.5
91.2 F 22.8* 53.7 F 9.7**
Untreated rats were saline-injected group and Se-treated rats were sodium selenite-injected group. Values are mean F SD. * p < 0.05 compared with untreated and Se-treated control group. ** p < 0.05 compared with untreated diabetic group.
glucose of the diabetic group was significantly higher than that of the control group ( p < 0.001). Sodium selenite treatment caused a slight but significant ( p < 0.05) decrease in the blood glucose of the diabetic rats ( p < 0.05) but a slight increase in the non-diabetic animals ( p < 0.05). Blood insulin levels of the Se-treated controls and the diabetics were significantly lower than those of untreated controls ( p < 0.001). The final body weight of the diabetic group was significantly lower than of the control group ( p < 0.001). Sodium selenite treatment did not significantly affect the weight gain in the diabetic and control groups. Maximal amplitude of thrombin-induced aggregation of washed platelets was significantly higher in diabetic rats than that of the control group ( p < 0.05). Selenite treatment caused a significant decrease in the platelet aggregation of the diabetic group ( p < 0.05). Selenite did not affect platelet aggregation in the control group. Thrombin-induced platelet TxA2 formation was significantly higher in the diabetic group than the control group. Selenite administration caused a significant decrease in the TxB2 level of diabetic group but it had no effect on the control group. There was also a significant correlation between maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels (r = 0.43, p = 0.05). Maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels of control and diabetic groups are summarized in Table 2.
4. Discussion In the present study, we examined the effect of selenite treatment on the platelet aggregation of STZ-induced diabetic rats. The major findings of our study are: (1) maximal amplitude of platelet aggregation was higher in STZ-induced diabetic rats. The increase in platelet aggregation was accompanied by an increase in TxA2 formation. (2) Selenite treatment clearly and significantly reversed the increase in the maximum amplitude of platelet aggregation and decreased the level of TxB2. (3) Sodium selenite had a small
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but significant effect on the blood glucose levels of the diabetic rats and caused a small increase in blood glucose level of the control group. Blood insulin levels of the Setreated control group and both diabetic groups were lower than those of untreated controls. Thrombin-induced hyperaggregability in diabetic rats confirms the results of several investigations [2,20,22]. The abnormalities of diabetic platelets such as, changes in the platelet membrane lipids, reduced membrane fluidity, abnormal glycoprotein expression, increase in platelet calcium and prostacyclin (PGI2)/TxA2 imbalance may result in hyperaggregability [2,20,22,23]. We observed that the increase in platelet aggregation was accompanied with formation of TxA2. Among the mechanisms proposed as mediators of diabetic cardiovascular complications, the increase in oxygenderived free radicals is emerging as a key factor. Oxidative stress has been due to the increased production of free radicals and decrease in antioxidant activity [6]. The imbalance between generation of oxygen-derived free radicals and the activity of scavenging systems damages lipid membranes and increases arachidonic acid peroxidation. It has been suggested that increase in phospholipid deacylation by lipid peroxidation increased TxA2 formation. In platelets, cyclooxygenase/thromboxane synthase converts it to TxA2, a potent aggregant [24,25]. It has been reported that supplementation with antioxidants prevents diabetic and hyperglycemic cardiovascular disease [4,7 – 12]. Stefek et al. [6] found that stobadine attenuated angiopathic and atherogenic processes in the myocardium by normalization of a-tocopherol and coenzyme Q9 levels. Karpen et al. [19] observed that dietary vitamin E restored PGI2/TxA2 balance in the diabetic rats. Selenium, as an integral part of glutathione peroxidase, protects various cells against oxidative damage. Animal experiments have suggested that a low Se could play a role in the development of cardiovascular disease and was associated with higher platelet aggregation [19,26]. Diabetes mellitus has also been related to alteration in the homeostasis of certain elements such as selenium, and selenate administration improved glucose homeostasis [13]. In the present study, selenite administration reversed hyperaggregability in the diabetic rats, while it did not affect platelet aggregation in controls. Decrease in maximal amplitude of platelet aggregation was accompanied by a decrease in TxB2 level. Besides well-known antioxidant role of selenium, its insulin-like role has recently been suggested [16,27,28]. It is well known that insulin deficiency changes cardiac energy metabolism [29] and this is reversed by insulin, and insulin-mimetic substances [30]. Basically, two promising insulin-mimetics, vanadate and selenate, have been studied [16,17,31,32]. Their insulin mimicry has been shown in the areas of glucose uptake [28,30,33], glucose metabolism [27,28,31] and signal transduction [16,30,31]. Ghosh et al. [33] have shown that oral administration of
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sodium selenite to the STZ-induced diabetic mice restored the liver glycogen level, plasma glucose and insulin levels and glucose-6-phosphatase activity to the control levels without having any effects on the control rats. We observed that selenite administration caused a small but significant decrease in the blood glucose levels of the diabetics and a mild hyperglycemia in controls. Lopaschuk and Spafford [29] also indicated that acute sodium selenite (i.p.) administration to normal rats induced hyperglycemia, at least when high doses of sodium selenite were used. Additional studies are needed to establish the metabolic effects of selenium. In our previous study, we observed that selenite treatment normalized cardiomyopathy in STZ-induced diabetic rats and the effect did not relate to plasma insulin levels [34]. This study also showed that sodium selenate decreased the platelet aggregation and TxA2 formation of diabetic rats without an increase in insulin levels, although blood glucose levels decreased significantly. In conclusion, this study confirms the hyperaggregability of platelets in STZ-induced diabetic rats. Hyperaggregability was related to enhanced TxA2 formation. Increase in TxA2 formation implies lipid peroxidation in platelets. Sodium selenite administration decreased thrombin-induced platelet aggregation and TxA2 formation. It was thought that selenium decreased lipid peroxidation as an antioxidant. Selenium also decreased blood glucose levels without a change in the insulin level. Besides its antioxidant effect, further studies are necessary to establish the insulin-like effect of selenite and the underlying mechanisms of this beneficial effect.
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G. Erso¨z et al. / Thrombosis Research 111 (2003) 363–367 abnormal expression of liver glycolytic and gluconeogenic enzymes in diabetic rats. Diabetologia 1996;39:3 – 11. [28] Berg EA, Wu JY, Campbell L, Kagey M, Stapleton SR. Insulin-like effects of vanadate and selenate on the expression of glucose-6-phosphate dehydrogenase and fatty acid synthase in diabetic rats. Biochimie 1995;77:919 – 24. [29] Lopaschuk GD, Spafford M. Response of isolated working hearts to fatty acids and carnitine palmitoyltransferase I inhibition during reduction of coronary flow in acutely and chronically diabetic rats. Circ Res 1989;65:378 – 87. [30] Stapleton SR. Selenium: an insulin-mimetic. Cell Mol Life Sci 2000;57:1874 – 9.
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[31] Ezaki O. The insulin-like effects of selenate in rat adipocytes. J Biol Chem 1990;265:1124 – 30. [32] Orvig C, Thompson KH, Battell M, McNeill JH. Vanadium compounds as insulin-mimics. Met Ions Biol Syst 1995;31:575 – 94. [33] Ghosh R, Mukherjee B, Chatterjee M. A novel effect of selenium on streptozotocin-induced diabetic mice. Diabetes Res 1994;25: 165 – 71. [34] Turan B, Ayaz M, Ozdemir S, Ugur M. Selenium shortens the prolonged action potential in experimental diabetic rat hearts. Abstract in: ISHO Montreal. The cardiovascular system and inflammatory mediators: Interactions and therapeutics, Montreal, July 2001, 12 – 14, A0.14.
Brief Communication
Effect of sodium selenite treatment on platelet aggregation of streptozotocin-induced diabetic rats Gu¨lriz Erso¨z a,*, Ali Yakaryılmaz a, Belma Turan b a b
Department of Physiology, Faculty of Medicine, Ankara University, 06100 Sihhiye, Ankara, Turkey Department of Biophysics, Faculty of Medicine, Ankara University, 06100, Sihhiye, Ankara, Turkey Received 25 March 2003; received in revised form 5 May 2003; accepted 8 May 2003
Abstract Introduction: There is a well-known association between diabetes and atherosclerosis. Platelets are involved in the development of atherosclerotic vascular diseases and play a key role in atherosclerotic complications. Diabetes mellitus is related to alteration in the homeostasis of selenium and the protective role of selenium against lipid peroxidation in diabetes is reported. In the present study, thrombin-induced platelet aggregation and thromboxane A2 (TxA2) formation in diabetes and the effect of sodium selenite were evaluated. Materials and methods: Diabetes was induced by intraperitoneal injection of streptozotocin (STZ) in Wistar rats (n = 21). Thirty of them were used as control rats. A week after streptozotocin injection, 11 of the control rats and 12 of the diabetics were injected with 5 Amol/kg/day of sodium selenite for 4 weeks. Thrombin-induced aggregation of the platelets was evaluated by optical technique. Thromboxane B2 (TxB2), TxA2 metabolite, was measured by enzyme-linked immunoassay (EIA) in thrombin-induced platelets. Results and conclusion: The platelet aggregation and TxB2 level increased in diabetic rats. Sodium selenite reversed the increase in platelet aggregation and TxB2 and caused a small but significant ( p < 0.05) decrease in the glucose level. The hyperaggregability of platelets in STZ-induced diabetic rats was thought to be related to the enhanced TxA2 formation of platelets. Increase in TxA2 formation implies lipid peroxidation. Sodium selenite decreased the TxA2 formation. Besides its antioxidative effect, further studies are needed to establish the insulin-like effect of selenite because of a small decrease in blood glucose. D 2003 Elsevier Ltd. All rights reserved. Keywords: Diabetes; Platelet aggregation; Thromboxane A2; Selenium; Antioxidant
1. Introduction There is a well-known association between diabetes and atherosclerotic cardiovascular and microangiopathic diseases. Since platelets contribute to the development of these vascular events, alteration in platelet function may be a key factor in these complications in diabetes. Several platelet abnormalities have been reported in insulin- and noninsulindependent diabetes; increase in platelet adhesion, increased sensitivity to aggregating agents, changes related to membrane lipids and glycoproteins and several metabolic changes, particularly of arachidonic acid and abnormal Ca2 + homeostasis [1,2]. Hemodynamic disturbances and
Abbreviations: TxA2, thromboxane A2; TxB2, thromboxane B2; STZ, streptozotocin; PGI2, prostacyclin; PRP, platelet-rich plasma; EIA, enzymelinked immunoassay; RIA, radioimmunoassay; PPP, platelet-poor plasma. * Corresponding author. Tel.: +90-312-310-3010; fax: +90-312-3097404. E-mail address: [email protected] (G. Erso¨z). 0049-3848/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0049-3848(03)00338-4
oxidative mechanisms may also be involved in diabetesinduced platelet abnormalities [1,3,4]. Chronic hyperglycemia causes an imbalance between radical oxygen species and scavenging system activity, and leads to oxidative stress [5,6]. It has been reported that supplementation with antioxidants prevents diabetic and hyperglycemic cardiovascular disease [4,7 –12]. Selenium is a trace element that plays an important role in many physiological mechanisms. It is an integral part of glutathione peroxidase and protects various cells against oxidative damage. It is suggested that diabetes mellitus is related to alteration in the homeostasis of selenium [13]. The protective role of selenium administration against lipid peroxidation and diabetes-induced injury in several tissues was reported [14,15]. It has also been suggested that selenium affects carbohydrate metabolism and has insulinlike actions [13,16]. The effect of selenium supplementation showed a strong dependency on the type of the selenium compound, its concentration and the way of administration. In our previous
364
G. Erso¨z et al. / Thrombosis Research 111 (2003) 363–367
study, we treated STZ-induced diabetic rats with a much lower concentration of selenite (5 Amol/kg, i.p.) than that used by other groups [17,18]. We observed that the treatment protected the heart tissue against diabetes-induced injury [14]. Accordingly, we aimed to examine the thrombin-induced platelet aggregation and lipid peroxidation in diabetic rats and the role of sodium selenite administration. The thromboxane A2 (TxA2) formation was planned to measured as a marker of platelet activation and lipid peroxidation [19,20].
group, 440 000 F 37 700/mm3 of diabetic group, 450 000 F 46 600/mm3 of diabetic + Se-treated group.). 2.3. Platelet aggregation
2. Materials and methods
Platelet resuspension was incubated at 37 jC for 5 min. Thrombin (Chronolog, PA, USA) at a final concentration of 0.8 U/ml was used for induction of platelet aggregation. The aggregation was performed on a Chronolog 560 WB Aggregometer by the optical method [21]. Maximal amplitude of aggregation curve (the percentage of platelet-poor plasma (PPP) being 100%) was calculated as the indicator of platelet aggregation.
2.1. Animals and treatment
2.4. Platelet TxA2 Formation
Twelve- to fourteen-week-old Wistar rats (initial body weight of 200 – 250 g) were used in this study. They were housed three in a cage at a temperature of 22 F 1 jC with a fixed 12 h light– dark cycle. They were fed ad libitum with a standard rat diet. The procedures used in the study were approved by the Ethics Committee of Ankara University Faculty of Medicine. The rats were randomly divided into two groups. Streptozotocin (STZ: 50 mg/kg body weight; dissolved in 0.02 M sodium citrate, pH 4.5, Sigma) was intraperitoneally (i.p.) injected to the first group. The rats with glycemia > 2.5 g/l, a week after STZ injection were considered as diabetic (n = 21). Control rats (n = 30) were injected with a corresponding volume of citrate buffer (i.p.). Plasma insulin level was measured by radioimmunoassay (RIA). A week after the STZ injection, 11 of the control rats and 12 of the diabetics were injected (i.p.) with 5 Amol/kg/day of sodium selenite for 4 weeks (Se-treated groups). Other rats (19 controls and 9 diabetics) were injected with a corresponding volume of saline (untreated groups). The mean blood glucose, insulin levels and the final body weight of the rats were measured at the beginning and after the treatments.
Thromboxane B2 (TxB2), a metabolite of TxA2 was measured in the supernatant samples prepared by centrifugation of thrombin-induced platelet suspension for 3 min at 10 000 rpm. Enzyme-linked immunoassay (EIA) was used for the measurement. The TxB2 EIA kit was obtained from Cayman Chemical (MI, USA). 2.5. Statistical analyses Results are given as the mean F SE. Comparisons between control untreated and Se-treated, diabetic untreated and Se-treated rats were carried out by variance analysis followed by the Newman – Keuls test. Differences were considered significant at p < 0.05. The Pearson correlation coefficient was used to measure the relationship between platelet aggregation and platelet TxA2 formation.
3. Results The mean blood glucose, the final body weight and insulin levels of the Se-treated and untreated groups of controls and diabetics are given in Table 1. The blood
2.2. Blood samples and platelet preparation After the selenite and placebo treatments, rats were anesthetised by sodium pentobarbital (30 mg/kg) and blood samples were collected by cardiac puncture into 5 ml of siliconized tubes containing 0.5 ml of 3.8% sodium citrate. Platelet-rich plasma (PRP) was obtained by centrifugation of sodium citrated blood samples at 300 g for 15 min. Platelet count was determined by the Coulter counter T890. The platelet pellet was obtained by centrifugation of PRP at 900 g for 15 min. The platelet pellet was washed and resuspended to 300 000 –500 000 platelet/mm3 with Hepesbuffered Tyrode solution containing 138 mM NaCl, 0.36 mM NaH2PO4, 2.9 mM KCl, 1 mM MgCl2, 5 mM glucose, 20 mM Hepes (pH 7.4). There was no significant difference between platelet counts of both groups (452 000 F 28 300/ mm3 of control group, 439 000 F 37 000/mm3 of Se-treated
Table 1 Final body weights, blood glucose and insulin levels of control and diabetic rats Rats
Control group Untreated Se-treated
Body weight (g)
Blood glucose (mg/dl)
Blood insulin level (fmol/ml)
238 F 5 251 F 7
103.5 F 2.3 129.0 F 5.1*
281.6 F 57.3 109.2 F 15.2**
Diabetic group Untreated 188 F 5** Se-treated 204 F 11**
437.1 F 15.7** 390.1 F 30.8**,#
76.1 F 15.9** 58.1 F 14.9**
Untreated rats were saline-injected group and Se-treated rats were sodium selenite-injected group. Values are mean F SD. * p < 0.05 compared with untreated control group. ** p < 0.001 compared with untreated control group. # p < 0.05 compared with untreated diabetic group.
G. Erso¨z et al. / Thrombosis Research 111 (2003) 363–367 Table 2 Maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels of control and diabetic rats (mean F SD) Rats
Maximal amplitude of platelet aggregation (%)
TxB2 level (pg/ml)
Control group Untreated Se-treated
7.6 F 5.8 6.7 F 5.3
43.4 F 18.1 53.4 F 16.9
Diabetic group Untreated Se-treated
19.3 F 9.2* 10.9 F 5.5
91.2 F 22.8* 53.7 F 9.7**
Untreated rats were saline-injected group and Se-treated rats were sodium selenite-injected group. Values are mean F SD. * p < 0.05 compared with untreated and Se-treated control group. ** p < 0.05 compared with untreated diabetic group.
glucose of the diabetic group was significantly higher than that of the control group ( p < 0.001). Sodium selenite treatment caused a slight but significant ( p < 0.05) decrease in the blood glucose of the diabetic rats ( p < 0.05) but a slight increase in the non-diabetic animals ( p < 0.05). Blood insulin levels of the Se-treated controls and the diabetics were significantly lower than those of untreated controls ( p < 0.001). The final body weight of the diabetic group was significantly lower than of the control group ( p < 0.001). Sodium selenite treatment did not significantly affect the weight gain in the diabetic and control groups. Maximal amplitude of thrombin-induced aggregation of washed platelets was significantly higher in diabetic rats than that of the control group ( p < 0.05). Selenite treatment caused a significant decrease in the platelet aggregation of the diabetic group ( p < 0.05). Selenite did not affect platelet aggregation in the control group. Thrombin-induced platelet TxA2 formation was significantly higher in the diabetic group than the control group. Selenite administration caused a significant decrease in the TxB2 level of diabetic group but it had no effect on the control group. There was also a significant correlation between maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels (r = 0.43, p = 0.05). Maximal amplitude of thrombin-induced platelet aggregation and TxB2 levels of control and diabetic groups are summarized in Table 2.
4. Discussion In the present study, we examined the effect of selenite treatment on the platelet aggregation of STZ-induced diabetic rats. The major findings of our study are: (1) maximal amplitude of platelet aggregation was higher in STZ-induced diabetic rats. The increase in platelet aggregation was accompanied by an increase in TxA2 formation. (2) Selenite treatment clearly and significantly reversed the increase in the maximum amplitude of platelet aggregation and decreased the level of TxB2. (3) Sodium selenite had a small
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but significant effect on the blood glucose levels of the diabetic rats and caused a small increase in blood glucose level of the control group. Blood insulin levels of the Setreated control group and both diabetic groups were lower than those of untreated controls. Thrombin-induced hyperaggregability in diabetic rats confirms the results of several investigations [2,20,22]. The abnormalities of diabetic platelets such as, changes in the platelet membrane lipids, reduced membrane fluidity, abnormal glycoprotein expression, increase in platelet calcium and prostacyclin (PGI2)/TxA2 imbalance may result in hyperaggregability [2,20,22,23]. We observed that the increase in platelet aggregation was accompanied with formation of TxA2. Among the mechanisms proposed as mediators of diabetic cardiovascular complications, the increase in oxygenderived free radicals is emerging as a key factor. Oxidative stress has been due to the increased production of free radicals and decrease in antioxidant activity [6]. The imbalance between generation of oxygen-derived free radicals and the activity of scavenging systems damages lipid membranes and increases arachidonic acid peroxidation. It has been suggested that increase in phospholipid deacylation by lipid peroxidation increased TxA2 formation. In platelets, cyclooxygenase/thromboxane synthase converts it to TxA2, a potent aggregant [24,25]. It has been reported that supplementation with antioxidants prevents diabetic and hyperglycemic cardiovascular disease [4,7 – 12]. Stefek et al. [6] found that stobadine attenuated angiopathic and atherogenic processes in the myocardium by normalization of a-tocopherol and coenzyme Q9 levels. Karpen et al. [19] observed that dietary vitamin E restored PGI2/TxA2 balance in the diabetic rats. Selenium, as an integral part of glutathione peroxidase, protects various cells against oxidative damage. Animal experiments have suggested that a low Se could play a role in the development of cardiovascular disease and was associated with higher platelet aggregation [19,26]. Diabetes mellitus has also been related to alteration in the homeostasis of certain elements such as selenium, and selenate administration improved glucose homeostasis [13]. In the present study, selenite administration reversed hyperaggregability in the diabetic rats, while it did not affect platelet aggregation in controls. Decrease in maximal amplitude of platelet aggregation was accompanied by a decrease in TxB2 level. Besides well-known antioxidant role of selenium, its insulin-like role has recently been suggested [16,27,28]. It is well known that insulin deficiency changes cardiac energy metabolism [29] and this is reversed by insulin, and insulin-mimetic substances [30]. Basically, two promising insulin-mimetics, vanadate and selenate, have been studied [16,17,31,32]. Their insulin mimicry has been shown in the areas of glucose uptake [28,30,33], glucose metabolism [27,28,31] and signal transduction [16,30,31]. Ghosh et al. [33] have shown that oral administration of
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sodium selenite to the STZ-induced diabetic mice restored the liver glycogen level, plasma glucose and insulin levels and glucose-6-phosphatase activity to the control levels without having any effects on the control rats. We observed that selenite administration caused a small but significant decrease in the blood glucose levels of the diabetics and a mild hyperglycemia in controls. Lopaschuk and Spafford [29] also indicated that acute sodium selenite (i.p.) administration to normal rats induced hyperglycemia, at least when high doses of sodium selenite were used. Additional studies are needed to establish the metabolic effects of selenium. In our previous study, we observed that selenite treatment normalized cardiomyopathy in STZ-induced diabetic rats and the effect did not relate to plasma insulin levels [34]. This study also showed that sodium selenate decreased the platelet aggregation and TxA2 formation of diabetic rats without an increase in insulin levels, although blood glucose levels decreased significantly. In conclusion, this study confirms the hyperaggregability of platelets in STZ-induced diabetic rats. Hyperaggregability was related to enhanced TxA2 formation. Increase in TxA2 formation implies lipid peroxidation in platelets. Sodium selenite administration decreased thrombin-induced platelet aggregation and TxA2 formation. It was thought that selenium decreased lipid peroxidation as an antioxidant. Selenium also decreased blood glucose levels without a change in the insulin level. Besides its antioxidant effect, further studies are necessary to establish the insulin-like effect of selenite and the underlying mechanisms of this beneficial effect.
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