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Protective effect of vanadyl sulfate on the pancreas of streptozotocin-induced diabetic rats
Diabetes Research and Clinical Practice 70 (2005) 103–109 www.elsevier.com/locate/diabres
Protective effect of vanadyl sulfate on the pancreas of streptozotocin-induced diabetic rats§ Sema Bolkent a,*, Sehnaz Bolkent b, Refiye Yanardag c, Sevim Tunali c a
Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, 34098-Cerrahpasa, Istanbul, Turkey b Department of Biology, Faculty of Science, Istanbul University, 34459-Vezneciler, Istanbul, Turkey c Department of Chemistry, Faculty of Engineering, Istanbul University, 34320-Avcilar, Istanbul, Turkey Accepted 9 February 2005 Available online 5 March 2005
Abstract The aim of this study is to examine from a biochemical and histological perspective, whether vanadium has a protective effect on the pancreas of diabetic rats. Male, 6–6.5 months old, Swiss albino rats were divided into four groups. Group I: control (intact) animals (n = 13). Group II: control rats given vanadyl sulfate (n = 5). Group III: streptozotocin-induced diabetic animals (n = 11). Group IV: streptozotocin-induced diabetic animals given vanadyl sulfate (n = 11). Vanadyl sulfate was given by gavage technique to rats in a dose of 100 mg/kg daily for 60 days, after experimental animals were made diabetic. On day 60, the pancreas tissue and blood samples were taken from the animals. In the streptozotocin-induced diabetic group, blood glucose levels significantly increased in contrast to the loss of body weight, but vanadyl sulfate in streptozotocin-diabetic rats reduced blood glucose levels and increased both blood glutathione levels and body weight. Tissue sections were immunostained using an insulin antibody. The control group given vanadyl sulfate was no different from the other intact control group considering the insulin immunoreactivity in B cells. In pancreatic islets of the diabetic group, a decrease in the number of immunoreactive B cells was observed in comparison to the control group. On the other hand, pancreatic islets of the diabetic group given vanadyl sulfate showed a higher number of immunoreactive B cells in comparison to the diabetic group. According to the immunohistochemical and biochemical results obtained, it was concluded that vanadyl sulfate can regenerate B cells of endocrine pancreas in experimental diabetes. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Pancreas; Streptozotocin; Diabetic rats; Vanadyl sulfate
1. Introduction §
This study has been presented in part at Joint meeting of the British Association of Clinical Anatomists and the Spanish Anatomical Society, Barcelona, Spain, July 22–23, 2002. Abstracts published European Journal of Anatomy, July 2002, vol. 6, Supplement 1, p. 40. * Corresponding author. Fax: +90 212 632 00 50. E-mail address: [email protected] (S. Bolkent).
Vanadium is an element found in low concentrations in mammals. Although vanadium has been reported to be essential for rats and chickens, the exact in vivo mechanism(s) remains elusive [1–3]. The chemistry of vanadium is complex, because the
0168-8227/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2005.02.003
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element can exist in several oxidation states. There is evidence that the element accumulates into cells in vanadyl form in contrast to that found in extracellular tissues where it is present as vanadate [2,4–6]. Vanadium (including vanadyl and vanadate), has been shown to increase glucose uptake and stimulate glycogen synthesis, also to enhance glucose transport and oxidation, insulin-receptor tyrosine kinase activity, and inhibit glyconeogenesis, glycogenolysis and lipolysis and activate lipogenesis in rats [5–11]. The exact cellular mechanism action of vanadium appears to involve a combination of several post-receptor events in the insulin-signalling cascade [1,5,11]. Many studies have demonstrated that the oral administration of vanadium compounds to diabetic rats normalizes their blood glucose levels [12–14]. Vanadium may control glucose metabolism either by an insulin-dependent or independent biochemical pathways [15,16]. Vanadium has been shown to oxidize a variety of biochemical substrates including glutathione, ascorbate, NADH, and phenolic compounds [6]. Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine. It has been found that vanadium and vanadium compounds exhibit an insulin-like activity [5,8,17,18]. Vanadium compounds can mimic the actions of insulin through alternative signalling pathways [1,6,7,19]. Therefore, vanadyl sulfate has been suggested as a therapeutic agent for the treatment of type I diabetics [12,17,20,21]. Streptozotocin (STZ) treatment destroys the insulin-producing cells of the pancreas and STZinduced diabetic rats are considered a model of type I (IDDM = insulin dependent diabetes mellitus) diabetes mellitus [22–24]. In diabetes, glucose uptake into peripheral tissues is impaired. Diabetes is often a consequence of defective insulin binding. The trace element vanadium may be important in insulin action. This study examined whether vanadium has a protective effect on the pancreas of diabetic rats.
2. Material and method 2.1. Preparation of diabetic rats Experimental animals were made diabetic by an intraperitoneal injection of streptozotocin (STZ) in a single dose of 65 mg/kg. STZ was dissolved in a freshly prepared 0.01 M citrate buffer (pH 4.5) [25].
2.2. Animals and experimental design Male, 6–6.5 months old, clinically healthy Swiss albino rats were used. The experiments were reviewed and approved by the Local Institute’s Animal Care and use Committees. They were maintained in standard environmental conditions and fed with laboratory pellet chow and given water ad libitum. The animals were randomly divided into four groups. Group I: control (intact) animals (n = 13). Group II: control animals given vanadyl sulfate (n = 5). Group III: STZ-diabetic animals (n = 11). Group IV: STZ-diabetic animals given vanadyl sulfate (n = 11). Rats with blood glucose > 200 mg/dl were considered diabetic. A 100 mg/kg daily dose of vanadyl sulfate was given by gavage technique to rats for 60 days, after the experimental animals were made diabetic. Five animals from group I and II, seven animals from group III and IV were randomly selected for immunohistochemical study. 2.3. Immunohistochemical study On day 60, pancreas tissues were taken, under ethereal anesthesia, from animals, which had been fasting overnight. The tissue samples were fixed in Bouin, dehydrated in a graded series of ethanol, and embedded in paraffin wax before sectioning. Sections were dewaxed and rehydrated. After the step of washing in phosphate-buffered saline (PBS), sections were immersed in a solution of 3% H2O2 for 10 min. The sections were then pre-incubated with nonimmune serum for 20 min. They were labelled with streptavidin biotin following incubation with primary monoclonal anti-insulin clone antibody (dilution 1/ 1000 mg/ml). The localization of the antigen was indicated by a red color obtained with 3-amino-9ethyl-carbazole (AEC) as chromogenic substrate for peroxidase activity. Primary anti-insulin antibody (Sigma I-2018) and Histostain-Plus kit (Zymed Code 85-9943) were used for immunohistochemistry. Slides were counterstained with hematoxylin. The specificity of the immunohistochemical staining was checked by omission of the primary antibody, or by using an inappropriate antibody (anti-gastrin). All these controls gave negative results. Control pancreas sections with (+) signals were used as a positive control.
S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109
2.4. Biochemical study After the STZ injections, blood samples from the rats were collected from the tail vein of both control and diabetic animals at days 0, 1, 30, and 60. Fasting blood glucose levels after 18 h fasting were measured by the o-toluidine method [26]. The body weight of all rats was measured at days 0, 1, 30, and 60. The blood glutathione levels were measured using Beutler Duron Kelly Ellman’s reagent [27]. 2.5. Statistical analysis The results were evaluated using an unpaired t-test and ANOVA variance analysis using the NCSS statistical computer package [28].
3. Results 3.1. Immunohistochemistry In the control (intact) and vanadium-treated control animals, the islets showed the same structure with a large central core formed by insulin secreting cells. The control group given vanadyl sulfate was no different from the other intact control group considering the insulin immunoreactivity in B cells (Figs. 1 and 2). In the pancreatic islets of the diabetic group, a decrease in the number of immunoreactive B cells was observed by comparison with the control group. On the other hand, in the pancreatic islets of the diabetic group given vanadyl sulfate, an increase in the number of immunoreactive B cells was observed as compared with untreated diabetic group (Figs. 3 and 4). 3.2. Biochemical results The effect of vanadyl sulfate on the blood glucose levels of diabetic and nondiabetic rats is shown in Table 1. As seen in Table 1 there was no significant change in the blood glucose levels between four groups at day 0 (PANOVA = 0.324). In the control (intact) and control animals given vanadyl sulfate, the blood glucose levels did not change remarkably at days 0, 1, 30 and 60 (Pt-test = 0.107 and 0.401). After the STZ injection, a notable increase was observed in
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the blood glucose levels of diabetic rats at days 1, 30 and 60 compared to day 0 (Pt-test = 0.0001). However, in the diabetic animals given vanadyl sulfate, the blood glucose levels reduced significantly compared with the untreated diabetic groups (Pt-test = 0.0001). The mean body weight of rats is shown in Table 2. There was no significant difference in the body weight between the four groups at day 0 (PANOVA = 0.571). In the control and control animals given vanadyl sulfate, body weight considerably changed on days 30 and 60 compared to days 0 and 1 (Pt-test = 0.016 and 0.007). Body weight, in the diabetic group showed a notable decrease at days 1, 30 and 60 compared to day 0 (Pttest = 0.002). In turn, the diabetic group given vanadyl sulfate maintained weight gain in the experiments. The blood glutathione levels of the groups are given in Table 3. There was no difference in the blood glutathione levels among the four groups at day 0 (PANOVA = 0.991). The blood glutathione levels in diabetic animals significantly reduced at days 1, 30 and 60 compared to day 0 (Pt-test = 0.001). The blood glutathione levels significantly increased in the diabetic group given vanadyl sulfate at days 30 and 60 compared to day 1 (Pt-test = 0.006).
4. Discussion The effect of vanadyl sulfate as an antidiabetic agent on the endocrine pancreas of normal and diabetic rats was studied. Earlier studies have demonstrated that STZ destroys B cells and the duration of hyperglycaemia caused permanent damage [20]. The present study showed that in the diabetic group, a decrease in the number of immunoreactive B cells was observed as distinguished from the control group. It is possible that vanadium treatment protected some pancreatic B cells from STZ-induced cytotoxicity. First, in the pancreatic islets of the diabetic group given vanadyl sulfate, an increase in the number of immunoreactive B cells was observed in comparison to the diabetic group. This result suggests that vanadyl sulfate could be a potent mitogen for B cells and that it has a direct pancreatic insulinotropic activity. According to the immunohistochemical results obtained, vanadyl sulfate can regenerate the B cells of insulin dependent diabetic rats. The protective effect of vanadyl sulfate could be
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Fig. 1. Insulin immunoreactivity (!) in intact rat pancreatic islets. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 2. Immunoreactive B cells (!) in pancreatic islets of the controls rats given vanadyl sulphate. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 3. Several insulin immunoreactive B cells (!) in pancreatic islets of the diabetic rat. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 4. Insulin immunoreactivity (!) in pancreatic islets of the diabetic rat given vanadyl sulphate. Streptavidin–biotin-peroxidase technique. Magnification: 320.
Table 1 Mean levels of blood glucose for all groups (mg/dl)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
74.52 14.69 72.92 9.40 65.52 5.76 71.88 13.78
78.19 12.74 85.05 19.30 211.75 43.12 253.93 38.62
80.92 13.31 76.06 16.19 216.71 36.61 135.76 46.16
69.05 9.15 86.40 11.03 323.39 131.29 131.68 72.30
0.107 0.401 0.0001 0.0001
PANOVA n = number of animals. a Mean S.D.
0.324
0.0001
0.0001
0.0001
S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109
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Table 2 Mean levels of weight parameters (g)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
246.64 44.43 224.17 16.13 231.67 37.71 229.65 32.14
243.35 39.50 221.03 12.58 197.02 37.47 203.93 30.57
275.51 34.40 253.14 14.06 175.74 38.48 197.46 34.40
283.48 30.35 251.91 20.41 171.70 34.67 199.16 36.20
0.016 0.007 0.002 0.101
0.571
PANOVA
0.011
0.0001
0.0001
n = number of animals. a Mean S.D.
due to its direct influence on the endocrine pancreatic function in diabetic animals. Vanadate elicits a mitogenic response in human fibroblasts and in the 3T3 and 3T6 cell lines [29]. Cadene et al [15] observed that in isolated islets vanadyl sulfate did not induce insulin release, but markedly potentiated glucosestimulated insulin secretion. These results indicate that vanadyl sulfate was effective in ameliorating the diabetic state by replacing insulin. This study agrees with previous investigations concerning the effectiveness of vanadyl treatment in diabetic rats [12,13,30]. Several investigations have reported that vanadyl sulfate in STZ-induced diabetic rats alleviates some signs of diabetes, the most marked one being a normalization of blood glucose levels [12,22,30]. In the present study, the administration of vanadyl sulfate significantly reduced the mean blood glucose levels after the 30th and 60th day of treatment in the diabetic rats. On the other hand, non-treated diabetic rats remained hyperglycaemic throughout the experiment. Vanadium reduces plasma glucose by enhancing insulin sensitivity [31,32]. Vanadium acts on the stimulation of glucose intake into cells and thus a lowering of the blood glucose level is encountered [6]. Vanadium treatment diminishes hyperglycemia.
This phenomenon was associated with an increased number of B cells in each islet. In the control and control animals given vanadyl sulfate groups, body weight was significantly increased at days 30 and 60 compared to days 0 and 1. Body weight in the diabetic group was significantly lower than in the controls and than in the diabetic group given vanadyl sulfate. Previous studies have shown that vanadium treatment causes body weight gain, which was not increased in non-diabetic and diabetic rats [30,33]. Diabetes affects a variety of metabolic processes and is characterized by alterations in the tissue in terms of antioxidant concentrations such as glutathione [34,35]. Vanadate is reduced to the vanadyl ion by endogenous glutathione [2,13,17]. Glutathione plays a central role in coordinating the body’s antioxidant defense process [35]. The blood glutathione levels have been found to decrease in diabetes [36]. On the other hand, Oster et al. [34] reported that sodium metavanadate did not cause significant alterations in the antioxidant defense system of STZ-diabetic rats. In our study, the blood glutathione levels were not different in control groups. The blood glutathione levels in diabetic animals were signifi-
Table 3 Mean levels of blood glutathione for all groups (mg/dl)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
39.06 5.13 38.17 5.05 38.68 4.8 38.56 4.59
39.36 5.03 36.26 4.02 25.67 5.83 30.40 6.20
38.53 3.97 40.63 9.24 29.73 4.98 38.01 4.54
37.36 6.73 37.43 3.11 23.16 9.29 39.03 7.31
0.982 0.611 0.001 0.006
PANOVA n = number of animals. a Mean S.D.
0.991
0.0001
0.007
0.001
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cantly reduced at days 1 and 60. The blood glutathione levels significantly increased in the diabetic group given vanadyl sulfate at days 30 and 60 when compared to day 1. This result indicates that vanadyl sulfate increased the glutathione levels in diabetic rats. We conclude that vanadyl sulfate treatment causes significant alterations in the antioxidant defense system. The present study demonstrates that vanadyl sulfate could be useful as a potential antidiabetic agent. Dietary vanadyl sulfate supplementation may be an alternative to insulin in the near future for some diabetics. In addition, we can say that vanadyl sulfate has a protective effect on the pancreas of diabetic rats.
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[13] J. Meyerovitch, Z. Farfel, J. Sack, Y. Shechter, Oral administration of vanadate normalizes blood glucose levels in streptozotocin-treated rats, J. Biol. Chem. 262 (1987) 6658–6662. [14] R.M. Brand, F.G. Hamel, Transdermally delivered peroxovanadium can lower blood glucose levels in diabetic rats, Int. J. Pharm. 183 (1999) 117–123. [15] A. Cadene, R. Gross, P. Poucheret, et al. Vanadyl sulphate differently influences insulin response to glucose in isolated pancreas of normal rats after in vivo or in vitro exposure, Eur. J. Pharm. 318 (1996) 145–151. [16] A.B. Goldfine, M.E. Patti, L. Zuberi, et al. Metabolic effects of vanadyl sulfate in humans with non-insulin-dependent diabetes mellitus: in vivo and in vitro studies, Metabolism 49 (2000) 400–410. [17] H. Sakurai, K. Tsuchiya, M. Nukatsuka, M. Sofue, J. Kawada, Insulin-like effect of vanadyl ion on streptozotocin-induced diabetic rats, J. Endocrinol. 126 (1990) 451–459. [18] Y. Shechter, I. Goldwase, M. Mironchik, M. Fridkin, D. Gehel, Historic perspective and recent developments on the insulinlike actions of vanadium; toward developing vanadium-based drugs for diabetes, Coord. Chem. Rev. 237 (2003) 3–11. [19] N. Venkatesan, A. Avidan, M.B. Davidson, Antidiabetic action of vanadyl in rats independent of in vivo insulin-receptor kinase activity, Diabetes 40 (1991) 492–498. [20] G.A. Dehghani, M. Sotoodeh, Gh.R. Omrani, Trophic effects of vanadium on beta-cells of STZ-induced insulin dependent diabetic rats and evidence for long term relief of diabetes mellitus, Indian J. Med. Res. 110 (1999) 70–75. [21] A.K. Srivastava, Anti-diabetic and toxic effects of vanadium compounds, Mol. Cell. Biochem. 206 (2000) 177–182. [22] J.J. Mongold, G.H. Cros, L. Vian, et al. Toxicological aspects of vanadyl sulphate on diabetic rats: effects on vanadium levels and pancreatic B-cell morphology, Pharm. Toxicol. 67 (1990) 192–198. [23] M.C. Cam, W.M. Li, J.H. McNeill, Partial preservation of pancreatic b-cells by vanadium: evidence for long-term amelioration of diabetes, Metabolism 46 (1997) 769–778. [24] J. Yao, M.L. Battell, J.H. McNeill, Acute and chronic response to vanadium following two methods of streptozotocin-diabetes induction, Can. J. Physiol. Pharm. 75 (1997) 83–90. [25] A. Jound, A.E. Lambert, W. Stauffacher, A.E. Renold, Diabetogenic action of streptozotocin. Relationship of dose to metabolic response, J. Clin. Invest. 48 (1969) 2129–2139. [26] A. Relander, C.E. Raiha, Differences between the enzymatic and toluidine methods of blood glucose determination, Scand. J. Clin. Lab. Invest. 15 (1963) 221–224. [27] E. Beutler, O. Duron, B.M. Kelly, Improved method for the determination of blood glutathione, J. Lab. Clin. Med. 61 (1963) 882–888. [28] J.L. Hintze, Copyright C, 865, East 400, North Kaysville, UT, 84 (1986) 037 (801), 546-0445. [29] J.B. Smith, Vanadium ions stimulate DNA synthesis in Swiss mouse 3T3 and 3T6 cells, Proc. Natl. Acad. Sci. USA 80 (1983) 6162–6166. [30] S. Dai, K.H. Thompson, J.H. McNeill, One-year treatment of streptozotocin-induced diabetic rats with vanadyl sulphate, Pharm. Toxicol. 74 (1994) 101–109.
S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109 [31] M.C. Cam, B. Rodrigues, J.H. McNeill, Distinct glucose lowering and beta cell protective effects of vanadium and food restriction in streptozotocin-diabetes, Eur. J. Endocrinol. 141 (1999) 546–554. [32] I.G. Fantus, E. Tsiani, Multifunctional actions of vanadium compounds on insulin signaling pathways: evidence for preferential enhancement of metabolic versus mitogenic effects, Mol. Cell. Biochem. 182 (1998) 109–119. [33] M.C. Cam, R.A. Pederson, R.W. Brownsey, J.H. McNeill, Long-term effectiveness of oral vanadyl sulphate in streptozotocin-diabetic rats, Diabetologia 36 (1993) 218–224.
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[34] M.H. Oster, J.M. Llobet, J.L. Domingo, J.B. German, C.L. Keen, Vanadium treatment of diabetic Sprague-Dawley rats results in tissue vanadium accumulation and pro-oxidant effects, Toxicology 83 (1993) 115–130. [35] N. Sekar, A. Kanthasamy, S. William, N. Balasubramaniyan, S. Govindasamy, Antioxidant effect of vanadate on experimental diabetic rats, Acta Diabetol. Lat. 27 (1990) 285– 293. [36] A. Yarat, T. Tunali, R. Yanardag, et al. The effect of glurenom (gliquidone) on lenses and skin in experimental diabetes, Free Radic. Biol. Med. 31 (2001) 1038–1042.
Protective effect of vanadyl sulfate on the pancreas of streptozotocin-induced diabetic rats§ Sema Bolkent a,*, Sehnaz Bolkent b, Refiye Yanardag c, Sevim Tunali c a
Department of Medical Biology, Cerrahpasa Faculty of Medicine, Istanbul University, 34098-Cerrahpasa, Istanbul, Turkey b Department of Biology, Faculty of Science, Istanbul University, 34459-Vezneciler, Istanbul, Turkey c Department of Chemistry, Faculty of Engineering, Istanbul University, 34320-Avcilar, Istanbul, Turkey Accepted 9 February 2005 Available online 5 March 2005
Abstract The aim of this study is to examine from a biochemical and histological perspective, whether vanadium has a protective effect on the pancreas of diabetic rats. Male, 6–6.5 months old, Swiss albino rats were divided into four groups. Group I: control (intact) animals (n = 13). Group II: control rats given vanadyl sulfate (n = 5). Group III: streptozotocin-induced diabetic animals (n = 11). Group IV: streptozotocin-induced diabetic animals given vanadyl sulfate (n = 11). Vanadyl sulfate was given by gavage technique to rats in a dose of 100 mg/kg daily for 60 days, after experimental animals were made diabetic. On day 60, the pancreas tissue and blood samples were taken from the animals. In the streptozotocin-induced diabetic group, blood glucose levels significantly increased in contrast to the loss of body weight, but vanadyl sulfate in streptozotocin-diabetic rats reduced blood glucose levels and increased both blood glutathione levels and body weight. Tissue sections were immunostained using an insulin antibody. The control group given vanadyl sulfate was no different from the other intact control group considering the insulin immunoreactivity in B cells. In pancreatic islets of the diabetic group, a decrease in the number of immunoreactive B cells was observed in comparison to the control group. On the other hand, pancreatic islets of the diabetic group given vanadyl sulfate showed a higher number of immunoreactive B cells in comparison to the diabetic group. According to the immunohistochemical and biochemical results obtained, it was concluded that vanadyl sulfate can regenerate B cells of endocrine pancreas in experimental diabetes. # 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Pancreas; Streptozotocin; Diabetic rats; Vanadyl sulfate
1. Introduction §
This study has been presented in part at Joint meeting of the British Association of Clinical Anatomists and the Spanish Anatomical Society, Barcelona, Spain, July 22–23, 2002. Abstracts published European Journal of Anatomy, July 2002, vol. 6, Supplement 1, p. 40. * Corresponding author. Fax: +90 212 632 00 50. E-mail address: [email protected] (S. Bolkent).
Vanadium is an element found in low concentrations in mammals. Although vanadium has been reported to be essential for rats and chickens, the exact in vivo mechanism(s) remains elusive [1–3]. The chemistry of vanadium is complex, because the
0168-8227/$ – see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.diabres.2005.02.003
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S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109
element can exist in several oxidation states. There is evidence that the element accumulates into cells in vanadyl form in contrast to that found in extracellular tissues where it is present as vanadate [2,4–6]. Vanadium (including vanadyl and vanadate), has been shown to increase glucose uptake and stimulate glycogen synthesis, also to enhance glucose transport and oxidation, insulin-receptor tyrosine kinase activity, and inhibit glyconeogenesis, glycogenolysis and lipolysis and activate lipogenesis in rats [5–11]. The exact cellular mechanism action of vanadium appears to involve a combination of several post-receptor events in the insulin-signalling cascade [1,5,11]. Many studies have demonstrated that the oral administration of vanadium compounds to diabetic rats normalizes their blood glucose levels [12–14]. Vanadium may control glucose metabolism either by an insulin-dependent or independent biochemical pathways [15,16]. Vanadium has been shown to oxidize a variety of biochemical substrates including glutathione, ascorbate, NADH, and phenolic compounds [6]. Glutathione is a tripeptide consisting of glutamic acid, cysteine, and glycine. It has been found that vanadium and vanadium compounds exhibit an insulin-like activity [5,8,17,18]. Vanadium compounds can mimic the actions of insulin through alternative signalling pathways [1,6,7,19]. Therefore, vanadyl sulfate has been suggested as a therapeutic agent for the treatment of type I diabetics [12,17,20,21]. Streptozotocin (STZ) treatment destroys the insulin-producing cells of the pancreas and STZinduced diabetic rats are considered a model of type I (IDDM = insulin dependent diabetes mellitus) diabetes mellitus [22–24]. In diabetes, glucose uptake into peripheral tissues is impaired. Diabetes is often a consequence of defective insulin binding. The trace element vanadium may be important in insulin action. This study examined whether vanadium has a protective effect on the pancreas of diabetic rats.
2. Material and method 2.1. Preparation of diabetic rats Experimental animals were made diabetic by an intraperitoneal injection of streptozotocin (STZ) in a single dose of 65 mg/kg. STZ was dissolved in a freshly prepared 0.01 M citrate buffer (pH 4.5) [25].
2.2. Animals and experimental design Male, 6–6.5 months old, clinically healthy Swiss albino rats were used. The experiments were reviewed and approved by the Local Institute’s Animal Care and use Committees. They were maintained in standard environmental conditions and fed with laboratory pellet chow and given water ad libitum. The animals were randomly divided into four groups. Group I: control (intact) animals (n = 13). Group II: control animals given vanadyl sulfate (n = 5). Group III: STZ-diabetic animals (n = 11). Group IV: STZ-diabetic animals given vanadyl sulfate (n = 11). Rats with blood glucose > 200 mg/dl were considered diabetic. A 100 mg/kg daily dose of vanadyl sulfate was given by gavage technique to rats for 60 days, after the experimental animals were made diabetic. Five animals from group I and II, seven animals from group III and IV were randomly selected for immunohistochemical study. 2.3. Immunohistochemical study On day 60, pancreas tissues were taken, under ethereal anesthesia, from animals, which had been fasting overnight. The tissue samples were fixed in Bouin, dehydrated in a graded series of ethanol, and embedded in paraffin wax before sectioning. Sections were dewaxed and rehydrated. After the step of washing in phosphate-buffered saline (PBS), sections were immersed in a solution of 3% H2O2 for 10 min. The sections were then pre-incubated with nonimmune serum for 20 min. They were labelled with streptavidin biotin following incubation with primary monoclonal anti-insulin clone antibody (dilution 1/ 1000 mg/ml). The localization of the antigen was indicated by a red color obtained with 3-amino-9ethyl-carbazole (AEC) as chromogenic substrate for peroxidase activity. Primary anti-insulin antibody (Sigma I-2018) and Histostain-Plus kit (Zymed Code 85-9943) were used for immunohistochemistry. Slides were counterstained with hematoxylin. The specificity of the immunohistochemical staining was checked by omission of the primary antibody, or by using an inappropriate antibody (anti-gastrin). All these controls gave negative results. Control pancreas sections with (+) signals were used as a positive control.
S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109
2.4. Biochemical study After the STZ injections, blood samples from the rats were collected from the tail vein of both control and diabetic animals at days 0, 1, 30, and 60. Fasting blood glucose levels after 18 h fasting were measured by the o-toluidine method [26]. The body weight of all rats was measured at days 0, 1, 30, and 60. The blood glutathione levels were measured using Beutler Duron Kelly Ellman’s reagent [27]. 2.5. Statistical analysis The results were evaluated using an unpaired t-test and ANOVA variance analysis using the NCSS statistical computer package [28].
3. Results 3.1. Immunohistochemistry In the control (intact) and vanadium-treated control animals, the islets showed the same structure with a large central core formed by insulin secreting cells. The control group given vanadyl sulfate was no different from the other intact control group considering the insulin immunoreactivity in B cells (Figs. 1 and 2). In the pancreatic islets of the diabetic group, a decrease in the number of immunoreactive B cells was observed by comparison with the control group. On the other hand, in the pancreatic islets of the diabetic group given vanadyl sulfate, an increase in the number of immunoreactive B cells was observed as compared with untreated diabetic group (Figs. 3 and 4). 3.2. Biochemical results The effect of vanadyl sulfate on the blood glucose levels of diabetic and nondiabetic rats is shown in Table 1. As seen in Table 1 there was no significant change in the blood glucose levels between four groups at day 0 (PANOVA = 0.324). In the control (intact) and control animals given vanadyl sulfate, the blood glucose levels did not change remarkably at days 0, 1, 30 and 60 (Pt-test = 0.107 and 0.401). After the STZ injection, a notable increase was observed in
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the blood glucose levels of diabetic rats at days 1, 30 and 60 compared to day 0 (Pt-test = 0.0001). However, in the diabetic animals given vanadyl sulfate, the blood glucose levels reduced significantly compared with the untreated diabetic groups (Pt-test = 0.0001). The mean body weight of rats is shown in Table 2. There was no significant difference in the body weight between the four groups at day 0 (PANOVA = 0.571). In the control and control animals given vanadyl sulfate, body weight considerably changed on days 30 and 60 compared to days 0 and 1 (Pt-test = 0.016 and 0.007). Body weight, in the diabetic group showed a notable decrease at days 1, 30 and 60 compared to day 0 (Pttest = 0.002). In turn, the diabetic group given vanadyl sulfate maintained weight gain in the experiments. The blood glutathione levels of the groups are given in Table 3. There was no difference in the blood glutathione levels among the four groups at day 0 (PANOVA = 0.991). The blood glutathione levels in diabetic animals significantly reduced at days 1, 30 and 60 compared to day 0 (Pt-test = 0.001). The blood glutathione levels significantly increased in the diabetic group given vanadyl sulfate at days 30 and 60 compared to day 1 (Pt-test = 0.006).
4. Discussion The effect of vanadyl sulfate as an antidiabetic agent on the endocrine pancreas of normal and diabetic rats was studied. Earlier studies have demonstrated that STZ destroys B cells and the duration of hyperglycaemia caused permanent damage [20]. The present study showed that in the diabetic group, a decrease in the number of immunoreactive B cells was observed as distinguished from the control group. It is possible that vanadium treatment protected some pancreatic B cells from STZ-induced cytotoxicity. First, in the pancreatic islets of the diabetic group given vanadyl sulfate, an increase in the number of immunoreactive B cells was observed in comparison to the diabetic group. This result suggests that vanadyl sulfate could be a potent mitogen for B cells and that it has a direct pancreatic insulinotropic activity. According to the immunohistochemical results obtained, vanadyl sulfate can regenerate the B cells of insulin dependent diabetic rats. The protective effect of vanadyl sulfate could be
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Fig. 1. Insulin immunoreactivity (!) in intact rat pancreatic islets. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 2. Immunoreactive B cells (!) in pancreatic islets of the controls rats given vanadyl sulphate. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 3. Several insulin immunoreactive B cells (!) in pancreatic islets of the diabetic rat. Streptavidin–biotin-peroxidase technique. Magnification: 320. Fig. 4. Insulin immunoreactivity (!) in pancreatic islets of the diabetic rat given vanadyl sulphate. Streptavidin–biotin-peroxidase technique. Magnification: 320.
Table 1 Mean levels of blood glucose for all groups (mg/dl)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
74.52 14.69 72.92 9.40 65.52 5.76 71.88 13.78
78.19 12.74 85.05 19.30 211.75 43.12 253.93 38.62
80.92 13.31 76.06 16.19 216.71 36.61 135.76 46.16
69.05 9.15 86.40 11.03 323.39 131.29 131.68 72.30
0.107 0.401 0.0001 0.0001
PANOVA n = number of animals. a Mean S.D.
0.324
0.0001
0.0001
0.0001
S. Bolkent et al. / Diabetes Research and Clinical Practice 70 (2005) 103–109
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Table 2 Mean levels of weight parameters (g)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
246.64 44.43 224.17 16.13 231.67 37.71 229.65 32.14
243.35 39.50 221.03 12.58 197.02 37.47 203.93 30.57
275.51 34.40 253.14 14.06 175.74 38.48 197.46 34.40
283.48 30.35 251.91 20.41 171.70 34.67 199.16 36.20
0.016 0.007 0.002 0.101
0.571
PANOVA
0.011
0.0001
0.0001
n = number of animals. a Mean S.D.
due to its direct influence on the endocrine pancreatic function in diabetic animals. Vanadate elicits a mitogenic response in human fibroblasts and in the 3T3 and 3T6 cell lines [29]. Cadene et al [15] observed that in isolated islets vanadyl sulfate did not induce insulin release, but markedly potentiated glucosestimulated insulin secretion. These results indicate that vanadyl sulfate was effective in ameliorating the diabetic state by replacing insulin. This study agrees with previous investigations concerning the effectiveness of vanadyl treatment in diabetic rats [12,13,30]. Several investigations have reported that vanadyl sulfate in STZ-induced diabetic rats alleviates some signs of diabetes, the most marked one being a normalization of blood glucose levels [12,22,30]. In the present study, the administration of vanadyl sulfate significantly reduced the mean blood glucose levels after the 30th and 60th day of treatment in the diabetic rats. On the other hand, non-treated diabetic rats remained hyperglycaemic throughout the experiment. Vanadium reduces plasma glucose by enhancing insulin sensitivity [31,32]. Vanadium acts on the stimulation of glucose intake into cells and thus a lowering of the blood glucose level is encountered [6]. Vanadium treatment diminishes hyperglycemia.
This phenomenon was associated with an increased number of B cells in each islet. In the control and control animals given vanadyl sulfate groups, body weight was significantly increased at days 30 and 60 compared to days 0 and 1. Body weight in the diabetic group was significantly lower than in the controls and than in the diabetic group given vanadyl sulfate. Previous studies have shown that vanadium treatment causes body weight gain, which was not increased in non-diabetic and diabetic rats [30,33]. Diabetes affects a variety of metabolic processes and is characterized by alterations in the tissue in terms of antioxidant concentrations such as glutathione [34,35]. Vanadate is reduced to the vanadyl ion by endogenous glutathione [2,13,17]. Glutathione plays a central role in coordinating the body’s antioxidant defense process [35]. The blood glutathione levels have been found to decrease in diabetes [36]. On the other hand, Oster et al. [34] reported that sodium metavanadate did not cause significant alterations in the antioxidant defense system of STZ-diabetic rats. In our study, the blood glutathione levels were not different in control groups. The blood glutathione levels in diabetic animals were signifi-
Table 3 Mean levels of blood glutathione for all groups (mg/dl)a Groups
n
0 Day
1 Day
30 Day
60 Day
Pt-test
Control Control + vanadyl sulfate Diabetic Diabetic + vanadyl sulfate
13 5 11 11
39.06 5.13 38.17 5.05 38.68 4.8 38.56 4.59
39.36 5.03 36.26 4.02 25.67 5.83 30.40 6.20
38.53 3.97 40.63 9.24 29.73 4.98 38.01 4.54
37.36 6.73 37.43 3.11 23.16 9.29 39.03 7.31
0.982 0.611 0.001 0.006
PANOVA n = number of animals. a Mean S.D.
0.991
0.0001
0.007
0.001
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cantly reduced at days 1 and 60. The blood glutathione levels significantly increased in the diabetic group given vanadyl sulfate at days 30 and 60 when compared to day 1. This result indicates that vanadyl sulfate increased the glutathione levels in diabetic rats. We conclude that vanadyl sulfate treatment causes significant alterations in the antioxidant defense system. The present study demonstrates that vanadyl sulfate could be useful as a potential antidiabetic agent. Dietary vanadyl sulfate supplementation may be an alternative to insulin in the near future for some diabetics. In addition, we can say that vanadyl sulfate has a protective effect on the pancreas of diabetic rats.
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