Differential action of methylselenocysteine in control and alloxan-diabetic rabbits

Chemico-Biological Interactions 177 (2009) 161–171

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Differential action of methylselenocysteine in control and alloxan-diabetic rabbits Anna Kiersztan a , Anna Baranska a , Michal Hapka a , Magdalena Lebiedzinska a , Katarzyna Winiarska a , Marta Dudziak b , Jadwiga Bryla a,∗ a b

Department of Metabolic Regulation, Institute of Biochemistry, Faculty of Biology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warsaw, Poland Department of Animal Physiology, Institute of Zoology, Faculty of Biology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warsaw, Poland

a r t i c l e

i n f o

Article history: Received 29 July 2008 Received in revised form 2 October 2008 Accepted 6 October 2008 Available online 1 November 2008 Keywords: Methylselenocysteine Diabetes Glutathione redox state Hypoglycemic action Blood glucose Kidney injury

a b s t r a c t Antidiabetic action of inorganic selenium compounds is commonly accepted. Since in diet selenium mainly exists as selenoamino acids, potential hypoglycemic properties of methylselenocysteine (MSC) were investigated in four groups of rabbits: untreated and MSC-treated control animals as well as alloxan-diabetic and MSC-treated diabetic rabbits. MSC (at a dose of 1 mg/kg body weight) was administered daily for 3 weeks via intraperitoneal injection. The data show, that in MSC-treated control animals plasma glucose concentration was diminished, while plasma urea and creatinine levels as well as urine albumin content were elevated and necrotic changes occurred in kidney-cortex. Decreased GSH/GSSG ratios in blood, liver and kidney-cortex were accompanied by increased glutathione peroxidase and glutathione reductase activities and a diminished renal ␥-glutamylcysteine synthetase activity. Death of 50% of control animals was preceded by a dramatic decline in blood glucose concentration. Surprisingly, in MSC-treated diabetic rabbits, plasma glucose levels were either normalized or significantly decreased. Blood and liver GSH/GSSG ratios were increased and renal functions were markedly improved, as indicated by a diminished albuminuria and attenuated histological changes characteristic of diabetes. However, after administration of MSC to diabetic rabbits plasma urea and creatinine levels as well as renal GSH/GSSG ratios were not altered. In view of MSC-induced marked accumulation of selenium in kidneys and liver of control rabbits, accompanied by a decline in blood glucose level, disturbance of glutathione homeostasis and kidney-injury, application of MSC in chemotherapy needs a careful evaluation. On the contrary, MSC supplementation might be beneficial for diabetes therapy due to an improvement of both glycemia and renal function. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction As selenium [Se] is a dietary essential trace element required for many cellular processes, its importance for human health has been the subject of numerous studies. It is commonly accepted that physiological effects of selenium occur mainly through over 30 selenoproteins detected in mammalian systems [1]. As a constituent of selenoproteins, selenium exhibits structural and enzymatic roles being the best-known antioxidant and catalyst for the production of active thyroid hormones. It is also required for the proper functioning of the immune system and appears to be a key nutrient in counteracting the development of virulence and inhibiting HIV progression to AIDS. It is also required for sperm motility and may reduce the risk of miscarriage. Selenium deficiency has been linked to adverse mood states [2].

∗ Corresponding author. Tel.: +48 22 5543213; fax: +48 22 5543221. E-mail address: [email protected] (J. Bryla). 0009-2797/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.cbi.2008.10.022

In diet, Se exists mostly in organic forms, whereas inorganic Se compounds such as selenite or selenate occur much less frequently and at very low amounts. Organic Se is present mainly in the form of selenoamino acids (l-selenomethionine, selenocysteine and methylselenocysteine) in cereal grains, plants and animal proteins, where the Se content depends upon the area of growth. Both organic and inorganic forms of Se appear to be utilized with similar efficacy in the body to produce selenoproteins [3] but they enter metabolic pathway at different points, depending on their chemical forms. A scheme showing Se metabolism is presented in Fig. 1. Inorganic forms of selenium are reduced by glutathione and number of intermediate metabolic steps lead to the generation of hydrogen selenide (H2 Se). Most of organic selenium compounds are metabolized producing the same key intermediate—H2 Se, which either serves as a precursor for the synthesis of selenoproteins or undergoes stepwise methylation to generate the mono-, di- and trimethylated forms of selenium [4]. In contrast to selenomethionine, methylselenocysteine (MSC) is not incorporated into proteins but it may be converted by ␤-lyase to

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2. Materials and methods 2.1. Animals and experimental design

Fig. 1. Selenium metabolic pathway in view of reports by Abdulah et al. [28], Tapiero et al. [29], Suzuki and Ogra [45] and Finley [68].

methylselenol, which is considered to be the main intermediate metabolite responsible for the anticarcinogenic action of selenium [5,6]. Many epidemiological studies have demonstrated the fundamental importance of relatively high selenium levels for cancer prevention. Several naturally occurring organoselenium compounds including methylselenocysteine exhibit a potent anticarcinogenic activity against prostate, lung, and colon cancers [7,8]. Moreover, the antiangiogenic effects of MSC have been reported to result in tumor growth inhibition, vascular maturation in vivo, and enhanced anticancer drug delivery that are associated with the observed therapeutic synergy in vivo [9]. Administration of selenate in supranutritive doses to rats with streptozotocin-induced type 1 diabetes was shown to decrease the elevated blood glucose level and considerably changed the expression of abnormally expressed glycolytic and gluconeogenic enzymes (reviewed by Ref. [10]). Moreover, antidiabetic effect of selenate reported for type 2 diabetic db/db mice, could result from reduction of insulin resistance [11,12]. On the contrary, oral administration of selenite to mice exhibiting alloxan-induced type 1 diabetes failed to reduce hyperglycemia in theses animals [13]. Furthermore, in contrast to selenate, treatment of type 2 diabetic db/db mice with selenite led to an aggravation of glucose tolerance and glucose metabolism [14]. The mechanism by which selenium is capable of mimicking of insulin action is still elusive. However, several reports indicate that selenium might increase phosphorylation of the ␤-subunit of insulin receptor and other downstream proteins involved in the insulin-signaling pathway like IRS-1, IRS-2, MAPK and PI 3 kinase [10]. Moreover, inhibition of protein tyrosine phosphatases by selenium compounds might be also responsible for maintaining the transmission of insulin signal [14]. In view of these observations it seems likely that: (i) several selenium compounds at high concentrations might be beneficial for cancer therapy [8] and (ii) methylselenocysteine might be one of the most effective chemopreventive chemicals [7]. As the latter compound inhibits glucose synthesis as significantly as selenite in isolated renal tubules, which contribute effectively to whole body glucose homeostasis [15] the aim of the present study was to investigate MSC action in both control and alloxan-diabetic rabbits.

The experiments were performed with 32 male white Termond rabbits weighing approximately 1.8–2.2 kg. The animals were housed in individual cages in an environmentally controlled room (temperature 22 ◦ C and 12-h light/dark cycle). All animals were maintained on the standard rabbit chow with free access to water and food. Rabbits were divided into four experimental groups each of eight animals: the control group, the MSC-treated control group, the diabetic group and the MSC-treated diabetic group. Diabetes was induced by the single injection of alloxan (150 mg/kg body wt) dissolved in 1 ml of 10 mM citrate buffer (pH 4.5) into the marginal vein of the ear of rabbit starved for 48 h [16]. Before the injection to animals, the alloxan solution was filtered with the use of filter with the pore size of 0.2 ␮m. Rabbits were allowed a standard diet and water ad libitum. However, to avoid hypoglycemic shock, animals were given 1% glucose solution to drink during 24 h following the alloxan administration. Only those alloxan-treated animals which exhibited decreased or stabilized body weight and blood glucose concentration in excess of 300 mg/100 ml (up to 600 mg/100 ml) 3 days after treatment were considered diabetic and used for experiments. MSC was dissolved in physiological saline. Solutions were prepared fresh daily and administered for 21 days via intraperitoneal injection at a dose of 1 mg (0.361 mg of selenium)/kg body weight. The untreated control and diabetic rabbits had injections of saline or saline plus sterile citrate buffer (pH 4.5), respectively. At the end of experiment the rabbits were killed by Morbital overdose. The liver and kidneys were immediately removed and used either for glutathione measurement or frozen at −70 ◦ C for subsequent determination of selenium level. All animal use procedures were approved by the First Warsaw Local Commission for the Ethics of Experimentation on Animals. 2.2. Sample preparation Blood (0.7 ml) of fed rabbits was withdrawn from marginal vein of the ear, collected into heparinized tubules placed on ice. Quantities of 200 ␮l of whole blood were left for both reduced (GSH) and oxidized glutathione (GSSG) determinations, while the residue was centrifuged at 2500 × g for 15 min to separate blood cells. The supernatants for selenium determination were mixed with the solution containing 0.1% nitric acid and 0.5% Triton X-100 (1:9), while glucose, urea and creatinine were determined in samples deproteinized with 1% Na2 WO4 in 30 mM H2 SO4 (1:6). Blood samples for GSH and GSSG assays were processed according to Dincer et al. [17]. In case of GSH determination, quantities of 100 ␮l of blood were added to 2.4 ml of the precipitating solution containing 0.13 M metaphosphoric acid, 4 mM EDTA and 3.2 M NaCl and thoroughly mixed. The samples were left for 5 minutes at room temperature and then centrifuged at 10,000 × g for 10 min. The supernatants obtained in this step were further used for reduced glutathione assay. For GSSG measurements, quantities of 100 ␮l of blood were added to 2.4 ml of the precipitating solution containing 0.13 M metaphosphoric acid, 4 mM EDTA, 3.2 M NaCl and 50 mM N-ethylmaleimide (to avoid nonenzymatic GSH oxidation [18]). The samples were left for 5 minutes at room temperature and then centrifuged at 10,000 × g for 10 min. Following the removal of the excess of N-ethylmaleimide by hexane extraction the samples were used for GSSG determination. Urine for albumine determination was withdrawn from bladder immediately after animal killing.

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Kidneys and livers for measurements of glutathione redox state were homogenized in ice-cold NaCl (0.5 g per 10 ml and 0.5 g per 2 ml for quantification of GSH and GSSG, respectively). The homogenates intended for GSH measurements were immediately mixed with the equal volume of 24% perchloric acid. Supernatants collected following centrifugation at 10,000 × g for 10 min were used for GSH determination. Samples intended for GSSG determination were treated with perchloric acid enriched with 100 mM N-ethylmaleimide. The supernatants obtained after centrifugation of samples at 10,000 × g for 10 min were treated with hexane to remove the excess of N-ethylmaleimide and subsequently used for GSSG measurement. Cytosol fractions for the measurements of activities of glutathione reductase, glutathione peroxidase and ␥-glutamylcysteine synthetase were prepared as described by McLellan et al. [19]. 2.3. Metabolite assays GSSG was measured fluorimetrically using glutathione reductase (1.2 U) according to Bergmeyer [20]. GSH levels were quantified by HPLC (Beckman Instruments) after derivatization with N-(1pyrenyl)maleimide (NPM) [18]. Blood glucose was determined spectrophotometrically using hexokinase (10 U) and glucose-6-phosphate dehydrogenase (1.5 U) according to Bergmeyer [20], while urea was measured in deproteinized blood serum as an ammonium following sample treatment with urease [21]. An increase in ammonium concentration was measured spectrophotometrically ( = 340 nm) using glutamate dehydrogenase. Briefly, to the cuvette (1 ml) containing 0.15 M TRA buffer, pH 8.6, 0.2 mM NADPH, 2 mM 2-oxoglutarate, 1.5 mM ADP, glutamate dehydrogenase (12 U) and 20 ␮l of deproteinized serum urease solution (4 U dissolved in 0.2 mM phosphate buffer, pH 7.4) was added to start the reaction. Creatinine was determined by the Jaffe’s reaction as described by Michalik et al. [22]. Urinary albumin level was determined with an assay kit (Sigma Chemicals, St. Louis, MO, USA) according to manufacturer’s instruction. Protein content was quantified with a spectrophotometric method of Layne [23]. 2.4. Measurements of enzyme activities Glutathione reductase activity was determined by spectrophotometric measurements of the decline in NADPH concentration at 340 nm [20]. The cuvette (2 ml) was containing 50 mM phosphate buffer, pH 6.6, 0.25 mM NADPH and the cytosol fraction (about 200 ␮g of protein). The reaction was started by the addition of GSSG solution to 1 mM final concentration. Glutathione peroxidase activity was measured spectrophotometrically ( = 340 nm) with a method of Paglia and Valentine [24]. Glutathione reductase was applied for monitoring GSSG generation accompanying glutathione peroxidase-catalysed H2 O2 decomposition. The cuvette (2 ml) contained 40 mM phosphate buffer, pH 7.0, 4 mM EDTA, 4 mM NaN3 , 0.25 mM NADPH, glutathione reductase (1.4 U) and the cytosol fraction (about 10 ␮g of protein). The reaction was started by the addition of H2 O2 solution to 75 ␮M final concentration. ␥-Glutamylcysteine synthetase activity was assayed by monitoring the rate of ATP consumption, as described by Hamilton et al. [25] and Kim et al. [26]. An increase in ADP concentration was measured spectrophotometrically ( = 340 nm) using reactions catalysed by both pyruvate kinase and lactate dehydrogenase. The cuvette (2 ml) contained 0.1 M Tris–HCl buffer, pH 8.0, 150 mM KCl, 20 mM MgCl2 , 0.25 mM NADH, 5 mM ATP, 10 mM glutamate, 10 mM 2-aminobutyrate, 2 mM phosphoenolpyruvate, pyruvate kinase (10 U) and lactate dehydrogenase (20 U). The reac-

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tion was started by addition of the cytosol fraction (about 20 ␮g of protein). All spectrophotometric measurements were performed using spectrophotometer Cary 50 Bio (Varian Ltd., Melbourne, Australia). 2.5. Determination of selenium Selenium level was determined using graphite-furnace atomic absorption spectrometry (Solaar M6, Waters) fitted with a 6F 95 graphite-furnace atomiser and a programmable sample dispenser. For determination of selenium concentration 0.5 g of tissue desiccated for 24 h at 37◦ C was dissolved in 9 ml of 65% nitric acid (Suprapur, Merck) and 0.8 ml hydrogen peroxide (Merck). Kidney and liver mineralizations were made for 15 min at 180o C in microwave labstation (Ethos D, Milestone). After mineralization 0.2 ml of hydrogen peroxide was added to the final volume of 10 ml. For determination of selenium concentration in serum, 100 ␮l samples were mixed with the solution containing 0.1% nitric acid and 0.5% Triton X-100 (1:9). The selenium concentration was estimated via comparison with the calibration curve obtained with the use of selenium standard solutions. Detection limit was 6 ␮g/l. 2.6. Histological studies Left kidney was perfused with saline and subsequently with 10% formalin in 0.1 M phosphate buffer, pH 7.4. The kidney was fixed overnight in buffered 10% formalin, washed, embedded in paraplast and sectioned at 8 ␮m. Serial sections were mounted on chrome alum–gelatine coated glass slides, deparaffinized with xylene, rehydrated and stained with either hematoxylin–eosin (H&E) to examine microanatomical details or periodic acid-Schiff (PAS) to estimate detailed glomerulal changes. Both examination and documentation of stained sections were performed using a light microscope (Nikon Eclipse 600) linked to a digital camera (Panasonic GP-KR222E with computer image analysis system Lucia GTM ). 2.7. Chemicals Enzymes, coenzymes and nucleotides for metabolite determinations were obtained from Roche (Mannheim, Germany). Methylselenocysteine and all others chemicals were purchased from Sigma–Aldrich–Fluka (USA). HPLC solvents came from either Merck (Germany) or POCH (Poland) and were of HPLC grade. Deionized water was obtained using the Elix 5 UV and Milli-Q filter systems (Millipore, USA). 2.8. Expression of results Data shown are means ± S.D. for at least four separate experiments performed with different animals. The results were statistically evaluated using ANOVA two-factors test. Values of P less than 0.05 were considered as significant. 3. Results 3.1. Body weight and plasma glucose, creatinine and urea Since a broad range of selenium doses (from 0.079 up to 4 mg/kg body weight) was used in studies regarding hypoglycemic action of various selenium compounds [27,28], in the current investigation the effect of MSC, an organic selenium compound, has been evaluated at a dose of 1 mg/kg body weight in control and diabetic rabbits. This dose was chosen due to following reasons: (i) in our recent studies the use of 0.182 mg of MSC/kg of rabbit body

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Fig. 2. The effect of MSC administration to control and diabetic rabbits on plasma glucose level (A and B) and body weight of animals (C and D). The treatment of diabetic rabbits was started 3 days after alloxan injection. MSC was administrated intraperitoneally once a day for 21 days at the dose of 1 mg (0.361 mg of selenium)/kg body weight. MSC-1—the group of rabbits exhibiting significant decline of plasma glucose level following MSC treatment (n = 4 and n = 5 for control and diabetic animals, respectively); MSC-2—the group of rabbits exhibiting only a slight decline in plasma glucose level following MSC treatment (n = 4 and n = 3 for control and diabetic animals, respectively). Values are means ± S.D. for the number of rabbits shown for each group. Statistical significance, a P < 0.05; b P < 0.01; c P < 0.005 versus values for control rabbits. A P < 0.05; B P < 0.01; C P < 0.005 versus values for untreated diabetic rabbits.

Table 1 Selenium levels in liver, kidney-cortex and plasma of control and diabetic rabbits untreated and treated with MSC. Selenium level Liver (␮g Se/g dry weight of tissue) Control Diabetes Control + MSC Diabetes + MSC

3.29 4.74 18.75 26.13

± ± ± ±

0.35 0.78 1.98a 0.10A

Kidney 4.73 4.75 48.10 56.50

± ± ± ±

Plasma (␮g Se/ml) 0.21 0.39 7.21a 8.47A

0.168 0.107 1.200 0.680

± ± ± ±

0.003 0.008a 0.230a 0.069A

MSC was administrated daily as intraperitoneal injection at a dose of 1 mg/kg body weight to both control and diabetic rabbits, where indicated. Selenium levels were determined following 21 days of experiment. Values are means ± S.D. for 8 rabbits in each group. Statistical significance versus values for control and diabetic animals, respectively: a P < 0.01 versus values for control rabbits; A P < 0.01 versus values for untreated diabetic rabbits.

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Fig. 3. The effect of MSC administration to control and diabetic rabbits on their plasma urea (A and B) and creatinine (C and D) levels. The treatment of diabetic rabbits was started 3 days after alloxan injection. MSC was administrated intraperitoneally once a day for 21 days at the dose of 1 mg (0.361 mg of selenium)/kg body weight. MSC-1—the group of rabbits exhibiting significant decline of plasma glucose level following MSC treatment (n = 4 and n = 5 for control and diabetic animals, respectively); MSC-2—the group of rabbits exhibiting only a slight decline of plasma glucose level following MSC treatment (n = 4 and n = 3 for control and diabetic animals, respectively). Values are means ± S.D. for the number of rabbits shown for each group. Statistical significance, a P < 0.05; b P < 0.01; c P < 0.005 versus values for control rabbits.

weight resulted in a decrease in blood glucose concentration in 30% of diabetic animals [15] and (ii) at high supranutritive doses MSC exhibited a potent anticarcinogenic activity [7,8]. As shown in Fig. 2, after administration of MSC to control and diabetic rabbits the plasma glucose level changed either significantly (marked as the MSC-1 group) or slightly (marked as the MSC-2 group). Surprisingly, in control rabbits MSC resulted in a dramatic decline in blood glucose level in 50% of animals (MSC-1 group), leading to their death while in the rest animals (the MSC-2 group) plasma glucose level was decreased by about 20%. After 3 weeks of MSC treatment of diabetic rabbits normalization of plasma glucose level was observed only in 60% of diabetic animals (the MSC-1 group). The rest diabetic animals (the MSC-2 group) exhibited a diminished plasma glucose level (by about 25%) already after 2 weeks of treatment and this glucose concentration was maintained during the third week of experiment. In contrast to untreated control rabbits, body mass of control animals treated with the selenium compound continually decreased in the MSC-1 group and only slightly

diminished in the MSC-2 group. Moreover, to check the kidney functions during MSC-administration the level of blood creatinine and urea were determined. In control animals of the MSC-1 group almost 2-fold elevation of urea and creatinine levels was noticed (Fig. 3), whereas in all diabetic rabbits (both treated and not treated with MSC) the body weight as well as high plasma concentrations of both urea and creatinine were not altered during 3 weeks of experiment. 3.2. Liver and kidney selenium Selenium levels in kidneys, liver and plasma did not differ significantly between MSC-1 and MSC-2 groups. Therefore, the data shown in Table 1 present mean values determined for both MSC1 and MSC-2 groups. The selenium content in plasma was lower by about 35% in diabetic animals than in control ones. Administration of MSC resulted in elevation of the plasma selenium level in both diabetic and control rabbits by about 6- and 7-fold,

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Fig. 4. The effect of MSC administration on glutathione redox state in kidney-cortex and liver of control and diabetic rabbits. MSC was injected for 21 days at the dose of 1 mg (0.361 mg of selenium)/kg body weight. Values are means ± S.D. for 8 rabbits in each group. Statistical significance, a P < 0.05; b P < 0.01; c P < 0.005 versus values for control rabbits. A P < 0.05; B P < 0.01 versus values for untreated diabetic rabbits.

respectively. However, the selenium concentration in plasma of MSC-treated control animals was almost 2-fold higher than in MSC-treated diabetic animals. Similarly, after 3 weeks of MSC administration to control and diabetic rabbits, approximately 5and 10-fold accumulation of selenium was observed in liver and kidney, respectively. 3.3. Glutathione redox state and activities of glutathione metabolic enzymes As selenium compounds are considered to exhibit antioxidative properties [29], their action on glutathione redox state was

also investigated. As in the case of selenium levels, the data for changes in glutathione concentrations are means for both MSC-1 and MSC-2 groups, since none significant differences were found in these two groups. Thus, it seems likely that selenium-induced changes in glutathione redox state might precede those in glucose metabolism and renal function. MSC treatment of control animals diminished blood GSH/GSSG ratio by about 25% due to a decrease in GSH level accompanied by an increase in GSSG content. Diabetes induced a decrease in GSH level accompanied by a rise in GSSG content, hence GSH/GSSG ratio was for 50% lower than the value achieved in control animals. However, blood GSH level in diabetic animals increased by about 40%, following MSC

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Fig. 5. Light microscopic photographs of kidney-cortex sections of control rabbits (A–C), control rabbits treated with MSC (MSC-1 group) (D– F), untreated diabetic rabbits (G–I) and diabetic rabbits treated with MSC (J–L). MSC was administrated intraperitoneally once a day for 21 days at the dose of 1 mg (0.361 mg of selenium)/kg body weight. Only in proximal tubules of control animals receiving MSC (the MSC-1 group) necrotic areas were observed (part D). In glomeruli of diabetic animals mesangial matrix expansion and compressed capillaries were observed (part I). These changes were partially attenuated following MSC treatment of diabetic rabbits (part L). Moreover, no MSC-induced tubular necrosis was noticed in kidneys of diabetic animals (part J). A, D, G, J—H&E staining, original magnification 100×; B, E, H, K—H&E staining, original magnification 400×; C, F, I, L—PAS staining, original magnification 400×.

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Table 2 Reduced glutathione (GSH) and oxidized glutathione (GSSG) levels as well as GSH/GSSG ratios in blood of control and diabetic rabbits untreated and treated with MSC. Animals

MSC

GSH (nmol/ml)

Control

– + – +

663.1 534.6 391.0 558.4

Diabetes

± ± ± ±

GSSG (nmol/ml)

40.4 17.6a 36.9c 27.9C

28.1 32.2 36.8 37.6

± ± ± ±

GSH/GSSG ratio

2.0 2.0a 4.9a 1.2

23.6 17.4 10.6 14.8

± ± ± ±

2.6 0.9b 0.8a 1.2B

MSC was administered daily to both control and diabetic animals for 21 days at the dose of 1 mg/kg body weight. Values are means ± S.D. for 8 rabbits in each group. Statistical significance: a P < 0.05; b P < 0.01; c P < 0.005 versus values for control rabbits. B P < 0.01; C P < 0.005 versus values for untreated diabetic rabbits.

administration, resulting in consequence an elevation of GSH/GSSG ratio. The data of Fig. 4 show that in control animals treated with MSC kidney GSH levels were diminished by about 45%, while GSSG contents were increased in both liver and kidney (by about 30 and 100%, respectively), resulting in a decrease in GSH/GSSG ratios in liver and kidney by about 30 and 65%, respectively. Diabetes did not induce changes in GSH levels in the two organs, while it evoked an increase in GSSG contents, causing a decrease in GSH/GSSG ratios by about 25–30%. Surprisingly, MSC administration to diabetic animals did not affect glutathione redox state in kidneys, while it elevated GSH level in liver leading to the significant increase in GSH/GSSG ratio. In order to elucidate reasons of MSC-induced changes in glutathione redox state, activities of glutathione reductase and glutathione peroxidase (enzymes of glutathione redox state) as well as ␥-glutamylcysteine synthetase (the first enzyme in the glutathione biosynthesis pathway) in both kidney-cortex and liver were measured. Data of the Table 3 show, that activities of these enzymes were similar under both control and diabetic conditions. However, treatment of control rabbits with MSC increased activities of both glutathione reductase and glutathione peroxidase in kidneys and reduced the renal ␥-glutamylcysteine synthetase activity by about 50%. In consequence, the renal GSH/GSSG ratio in MSCtreated control animals could decrease (cf. Fig. 4). By contrast, the administration of MSC to diabetic rabbits increased activity of the liver glutathione reductase by about 30% and decreased activity of the liver glutathione peroxidase by about 45%. Hence, MSCinduced increase in liver GSH/GSSG ratio in diabetic animals might be caused by changes in activities of glutathione metabolic enzymes in this tissue. 3.4. Urinary albumin levels and renal histology As appearance of albumin in the urine is a marker of early renal injury the amount of this protein was evaluated at the end of experiment. Urinary albumin content was 3-fold higher in diabetic

than in control rabbits (32.6 ± 4.2 and 10.2 ± 1.9 mg/l, respectively, P < 0.05 versus the value determined for control rabbits). However, MSC administration to control rabbits for 3 weeks resulted in the 7-fold elevation of urinary albumin level in MSC-1 group (up to 74.9 ± 8.6 mg/l, P < 0.05 versus the value determined for untreated control rabbits), while it did not change the level of this compound in MSC-2 group (12.2 ± 1.1 mg/l). Surprisingly, treatment with MSC of diabetic animals significantly diminished albuminuria by about 50% (down to 17.3 ± 2.1 mg/l, P < 0.05 versus the value determined for untreated diabetic rabbits), suggesting an amelioration of diabetes-evoked kidney-injury. These data are in agreement with those obtained during histological studies (Fig. 5) indicating that after MSC administration extent of renal injury was more marked in kidneys of control rabbits of the MSC-1 group. The tubules of these animals showed necrosis and subsequent sloughing of damaged tubular epithelial cells exhibiting an increased tubular luminal size. However, renal tubules in the MSC-2 group of animals seemed to be not changed. Moreover, kidneys withdrawn from diabetic rabbits treated with MSC showed a partial attenuation of abnormalities such as mesangial matrix expansion and compression of capillaries in glomeruli characteristic of the disease. 4. Discussion 4.1. The action of selenium on blood glucose levels Since oxidative stress is considered to be the main reason of diabetes mellitus complications [30,31], supplementation with antioxidants might be beneficial for diabetes therapy. Recently, numerous studies indicate both hypoglycemic and antioxidant properties of selenium. However, the current knowledge concerning the hypoglycemic action of selenium compounds is limited to inorganic derivatives despite their different bioavailability, anticancer action and toxic effects in comparison with those evoked by the organic selenium derivatives [32]. In view of observations that: (i) methylselenocyteine is naturally occurring in garlic, onion [33,34] and broccoli [35], (ii) it exhibits potent anticarcinogenic properties [7,8] and (iii) monomethylated forms of Se do not show toxic effects generated by other derivatives, e.g. inorganic selenite [28], in the current investigation the action of MSC administered to control and diabetic rabbits at a dose of 1 mg MSC/kg body weight (0.361 mg of selenium/kg body weight) was evaluated. MSC-induced normalization of glycemia in 60% of diabetic rabbits while others showed a slight decrease in the plasma glucose content (cf. Fig. 2). The latter data are in agreement with those reported by Battell et al. [36] who observed no improvement of fed blood glucose level despite ameliorated glucose tolerance in

Table 3 Glutathione reductase, glutathione peroxidase and ␥-glutamylcysteine synthetase activities in kidney-cortex and liver of control and diabetic animals either untreated or treated with MSC. Enzyme activity (nmol min−1 mg−1 protein)

Enzyme

Organ

Glutathione reductase

Liver Kidney

52 ± 12 63 ± 7

48 ± 8 94 ± 6b

67 ± 9 73 ± 10

86 ± 4B 70 ± 6

Glutathione peroxidase

Liver Kidney

2461 ± 193 967 ± 52

2499 ± 125 1139 ± 49b

2131 ± 260 947 ± 103

1149 ± 138A 1149 ± 57

␥-Glutamylcysteine synthetase

Liver Kidney

302 ± 33 1369 ± 113

291 ± 38 655 ± 40c

293 ± 26 1017 ± 145

263 ± 15 961 ± 117

Control rabbits

Control rabbits treated with MSC

Diabetic rabbits

Diabetic rabbits treated with MSC

The measurement were made following 3 weeks of diabetes and/or MSC treatment. MSC was administered to both control and diabetic rabbits as described in Methods. Values are means ± SD for 5–9 animals. a P < 0.05; b P < 0.025; c P < 0.005 versus values for control rabbits. A P < 0.05; B P < 0.025 versus values for untreated diabetic rabbits.

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63% of diabetic rats treated with selenite. In rats selenium doses close to the LD50 value, i.e. 3.5 [37] and 4.5 mg/kg body weight [36] were applied in the form of sodium selenate to attenuate the diabetic status. Moreover, selenite at a dose over 2 mg/kg body weight per day diminished (but it did not normalize) blood glucose level in alloxan-diabetic mice [38]. In contrast to reports concerning type 1 diabetic animals [36,39,40], Mueller et al. [14] demonstrated no action of selenate (0.52 mg/kg body weight) on blood glucose concentration in db/db mice exhibiting type 2 diabetes, whereas this treatment attenuated insulin resistance. Moreover, selenite at the same dose showed insulinomimetic properties with respect to the type 2 diabetic db/db mice [14]. Clinical trials using selenium doses of 200–800 ␮g daily showed no obvious toxic effects [41]. Investigation of chemoprevention of prostate cancer progression was discontinued due to a lack of information concerning safety of doses applied [42]. However, the administration to dogs and rats of MSC at a dose of 0.6 and 2 mg/kg, respectively, induced several side effects (hepatotoxicity, arrested spermatogenesis and anemia) [43]. Surprisingly, toxic action of MSC at a dose of 8 mg/kg was not observed in mice [44]. A dose of 2 mg/kg of selenite, containing 913 ␮g of selenium, was reported to induce no changes in the blood glucose level in control mice [38]. In our hands, however, treatment of control rabbits with MSC at a dose of 1 mg/kg (361 ␮g of selenium), caused a significant decline in blood glucose level and disturbance of kidney function and structure, leading to the death of 50% of control rabbits (cf. Figs. 2, 3 and 5). Hence, in contrast to selenite, organic selenium compounds such as MSC, which could be very effectively converted to their active metabolites [45,46] might cause a potent action. According to Becker et al. [39] and Sheng et al. [38], hypoglycemic action of selenium does not seem to be dependent on the increased serum insulin level in diabetic animals. This is supported by diminished insulin levels in control animals treated with either selenite [38] or selenate [37]. Therefore, it seems likely that the selenium effects could result from insulin action rather than insulin release from the pancreatic islets. Selenate was shown to increase in phosphorylation of the ␤-subunit of insulin receptor and other downstream components of the insulin-signaling pathway (IRS-1, IRS-2, MAPK, PI 3 kinase and translocation of the glucose transporters GLUT 1 and 2 to the membrane surface), resulting in elevation of the rate of glucose transport into adipocytes and muscles [10]. Furthermore, selenium compounds are strong inhibitors of protein tyrosine phosphatases [14,47], e.g. PTP 1B, which is involved in the negative regulation of insulin signaling. A significant decrease in liver cytosolic PTPs activities was evoked by oral selenate administration to db/db mice [11]. A similar mode of action has been demonstrated previously for vanadate, similar to selenium in terms of chemical structure and a mechanism of action within a cell [10]. In agreement with our results, diminished blood selenium levels were also observed in diabetic animals after 3 and 5 weeks of diabetes [48] and in diabetic humans [49]. However, they were elevated after selenium treatment of control and diabetic rabbits (cf. Table 1) and mice [48]. Hence, application of selenium in the therapy needs a careful evaluation of its dose to limit its accumulation in kidneys and liver as much as possible (cf. Table 1). 4.2. Selenium-induced renal injury Protection against nephrotoxicity of various toxicants in diabetic animals might result from diabetes-evoked changes in bioactivation and metabolism of the toxic compound and/or stimulation of compensatory repair mechanism in the renal tissue [50–52]. Protection against drug- and toxicant-induced nephrotoxicity has been

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investigated in diabetic rats and rabbits treated with gentamycin [53,54], cisplatin [55,56] and mercuric chloride [57]. Similarly, in contrast to only 10% survivals among control mice receiving 2dichlorovinyl-l-cysteine (DCVC, a compound generating a model of acute renal failure), 90% of survivals occurred among DCVC-treated diabetic mice [52]. Oxidative stress is considered as the main reason of diabetic nephropathy [58]. Hence, the administration of antioxidants appears to be the most reasonable therapeutic approach. Although the MSC treatment did not affect elevated plasma creatinine and urea concentrations in diabetic rabbits (cf. Fig. 3), however, it improved renal function as indicated by a diminished albuminuria and attenuated histological changes characteristic of diabetes (cf. Fig. 5). On the contrary, control animals treated with MSC showed increased plasma creatinine and urea levels (cf. Fig. 3), an elevated urinary albumin concentration as well as necrotic changes in tubules (cf. Fig. 5). Although a low renal accumulation of the toxicants in the diabetic kidney was considered to be of importance, however, in our hands selenium content was of the same order of magnitude in kidneys of both control and diabetic rabbits treated with MSC (cf. Table 1). Hence, the discrepancy in responses from control and diabetic animals might reflect different sensitivities against toxicity of a supranutritional MSC doses and/or different mechanisms of nephrogenic tissue repair. 4.3. The action of selenium on glutathione redox state Our present knowledge concerning selenium action on the cellular redox state is rather controversial and it seems to depend on a dose of selenium compound that can evoke either pro-oxidative or antioxidative effects [29,48,59,60–62]. As oxidative stress plays a crucial role in the development of diabetes-related complications, drugs applied in the therapy are expected to normalize blood glucose concentration as well as to show antioxidative properties, as attributed to metformin [63]. Diabetes-induced oxidative stress, is manifested by a disturbed glutathione homeostasis (cf. Table 2 and Fig. 4) and in consequence an impairment of antioxidative defence. Diminished GSH levels have been reported in blood of alloxan-diabetic rabbits [64] and mice [38]. Moreover, a decreased GSH concentration has been found in erythrocytes and plasma of diabetic humans [65,66]. In addition, diminished blood GSH/GSSG ratios in diabetic rabbits are accompanied by reduced GSH/GSSG ratios in both liver and kidney. However, in contrast to plasma, a decrease in GSH/GSSG ratio in these two organs seems to be due to elevation of GSSG content (cf. Table 2 and Fig. 4). MSC slightly improved glutathione homeostasis in both the blood (cf. Table 2) and liver (cf. Fig. 4) of diabetic animals due to elevation of GSH level, while it did not influence kidney GSH/GSSG ratio (cf. Fig. 4). On the other hand, MSC administration to control rabbits diminished GSH/GSSG ratios in blood, liver and kidneys, mainly due to increase in GSSG levels (cf. Table 2 and Fig. 4). Both elevated GSSG and not altered GSH contents in liver were also observed in type 2 diabetic mice treated with either selenite or selenate [11]. Selenite-induced changes in glutathione redox status are not surprising as selenite undergoes a thiol-dependent reduction to hydrogen selenide, consuming 4–6 equivalents of GSH before being incorporated into selenocysteine during synthesis of specific Se-proteins [45,67]. As in the case of other cysteine precursors: NAC and OTZ, which elevate GSH and GSSG levels in kidney-cortex tubules [18], MSC can also be a source of cysteine for GSH synthesis. Since administration of selenium at high concentrations may result in ROS generation via reaction of methylselenol with O2 [62], a decline in glutathione redox state due to pro-oxidative action of MSC is not excluded. MSC-evoked dramatic changes in both the renal glutathione content and the glutathione redox state

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in control animals result probably from a significant decline in renal ␥-glutamylcysteine synthetase (cf. Fig. 4 and Table 3). On the other hand, MSC-induced improvement of GSH/GSSG ratio in liver of diabetic rabbits is achieved probably due to normalization of GSH content, as concluded from reciprocal changes of glutathione reductase and glutathione peroxidase activities (cf. Fig. 4 and Table 3). In summary, since administration of supranutritional dose of methylselenocysteine to control animals might result in an accumulation of selenium in kidneys and liver and disturbance of glutathione homeostasis and renal function, further studies are required before its application in chemotherapy. To date, the use of selenium in human trials is limited. In view of recent observations Se is among of the most promising agents. However, the optimum doses for both dietary and pharmacological applications as well as chemical formulation of selenium need to be evaluated for the safe usage of this element in the therapy. On the contrary, MSC supplementation might be beneficial for the treatment of diabetic patients as it might improve both glycemia and renal function similarly to those observed in diabetic animals. However, reasons of the discrepancy in responses from control and diabetic rabbits to MSC treatment have yet to be elucidated. Conflicts of interest None declare. Acknowledgments The authors would like to thank Professor A. Sklodowska and Mr. B. Rewerski for selenium determinations as well as to Mr. K. Szymanski for arrangement of Fig. 5. The technical assistance of Miss B. Dabrowska is also acknowledged. This investigation was supported by the grants of the Ministry of Science and Higher Education -BW 1720/29, BW 1755/25 and 115/01/E-343/S/2007. References [1] C.D. Thomson, Assessment of requirements for selenium and adequacy of selenium status, a review, Eur. J. Clin. Nutr. 58 (2004) 391–402. [2] M.P. Rayman, The importance of selenium to human health, Lancet 356 (2000) 233–241. [3] Y. Shiobara, T. Yoshida, K.T. Suzuki, Effects of dietary selenium species on Se concentrations in hair, blood, and urine, Toxicol. Appl. Pharmacol. 152 (1998) 309–314. [4] L. Letavayova, V. Vlckova, J. Brozmanova, Selenium, from cancer prevention to DNA damage, Toxicology 227 (2006) 1–14. [5] K. El-Bayoumy, R. Sinha, Molecular chemoprevention by selenium, a genomic approach, Mutat. Res. 591 (2005) 224–236. [6] K.T. Suzuki, Y. Tsuji, Y. Ohta, N. Suzuki, Preferential organ distribution of methylselenol source Se-methylselenocysteine relative to methylseleninic acid, Toxicol. Appl. Pharmacol. 227 (2008) 76–83. [7] S.M. Lyi, L.I. Heller, M. Rutzke, R.M. Welch, L.V. Kochian, L. Li, Molecular and biochemical characterization of the selenocysteine Se-methyltransferase gene and Se-methylselenocysteine synthesis in broccoli, Plant Physiol. 138 (2005) 409–420. [8] P.D. Whanger, Selenium and its relationship to cancer, an update dagger, Br. J. Nutr. 91 (2004) 11–28. [9] A. Bhattacharya, M. Seshadri, S.D. Oven, K. Tałth, M.M. Vaughan, Y.M. Rustum, Tumor vascular maturation and improved drug delivery induced by methylselenocysteine leads to therapeutic synergy with anticancer drugs, Clin Cancer Res. 14 (2008) 3926–3932. [10] S.R. Stapleton, Selenium, an insulin-mimetic, Cell. Mol. Life Sci. 57 (2000) 1874–1879. [11] A.S. Mueller, J. Pallauf, Compendium of the antidiabetic effects of supranutritional selenate doses. In vivo and in vitro investigations with type II diabetic db/db mice, J. Nutr. Biochem. 17 (2006) 548–560. [12] A.S. Muller, E. Most, J. Pallauf, Effects of a supranutritional dose of selenate compared with selenite on insulin sensitivity in type II diabetic dbdb mice, J. Anim. Physiol. Anim. Nutr. (Berl.) 89 (2005) 94–104. [13] X.Q. Sheng, K.X. Huang, H.B. Xu, New experimental observation on the relationship of selenium and diabetes mellitus, Biol. Trace Elem. Res. 99 (2004) 241–253.

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