- Email: [email protected]
Antidiabetic efficacy of bradykinin antagonist R-954 on glucose tolerance test in diabetic type 1 mice
Neuropeptides 44 (2010) 187–189
Contents lists available at ScienceDirect
Neuropeptides journal homepage: www.elsevier.com/locate/npep
Special Issue on Peptide Receptors: Focus on Neuropeptides and Kinins
Antidiabetic efficacy of bradykinin antagonist R-954 on glucose tolerance test in diabetic type 1 mice Orlando L. Catanzaro a,b,*, Damian Dziubecki a, Pablo Obregon a, Ricardo R. Rodriguez b, Pierre Sirois c a
Direccion de Investigaciones Cientificas Bioquimico-Odontologicas, Universidad Argentina John F Kennedy, Argentina Escuela de Medicina y Odontología, Universidad Del Salvador, Argentina c Sherbrooke University Medical School, Sherbrooke, Canada b
a r t i c l e
i n f o
Article history: Available online 21 January 2010 Keywords: Type 1 diabetes DBK BKB1-R antagonist R-954
a b s t r a c t Insulin-dependent diabetes mellitus (type 1 diabetes) is an inflammatory autoimmune disease associated with many complications including nephropathy, retinopathy, neuropathy and hyperalgesia. Experimental evidence has shown that the bradykinin B1 receptor (BKB1-R) is involved in the development of type 1 diabetes and found to be upregulated alongside the disease. In the present study the effects of the selective BKB1-R antagonist the R-954 (Ac-Orn-[Oic2, a-MePhe5, D-b Nal7, Ile8 ]des-Arg9-BK and the BKB1-R agonist des Arg9-BK (DBK) were studied on diabetic hyperglycemia. Diabetic type 1 was induced in C57 BL/KsJ mdb male mice by five consecutives doses of STZ (45 mg/kg i.p.). A glucose tolerance test (GTT) was performed by an intraperitoneal administration of glucose, 8, 12 and 18 days after the diabetes induction. The induction of type 1 diabetes provoked a significant hyperglycemia levels in diabetic mice at 12 and 18 days after STZ. The administration of R-954 (400 lg/kg i.p.) at 12 and 18 days after STZ returned the glycemia levels of this animals to normal values. In addition the administration of DKB (300 lg/kg i.p.) significantly potentiated the diabetes-induced hyperglycemia; this effect that was totally reversed by R-954. These results provide further evidence for the implication of BKB1-R in the type 1 diabetes mellitus (insulitis). Ó 2009 Published by Elsevier Ltd.
1. Introduction Type 1 diabetes is caused by the progressive autoimmune destruction of pancreatic b-cells and the autoimmune process begins years before the b-cells destruction became complete, thereby providing a window of opportunity for intervention (Mauricio and Mandrup-Poulsen, 1998; Yoon et al., 1998). The loss of b-cells is associated with local and circulating signs of an autoimmune reactivity against the b-cell antigens (Gepts, 1965; Bottazo et al., 1978). Islet inflammation or insulitis is followed by the selective destruction of insulin secreting b-cells. Experimental evidence which suggests that kinins are involved in the development of an insulin-dependent form of diabetes mellitus in mice was reported by Zuccollo et al. (1999). The kallikrein–kinin system is made of a group of peptides and proteins involved in the control of blood pressure, gastrointestinal tract motility, inflammation and pain induction (Calixto et al., 2004; Leeb-Lundberg et al., 2005). The objective of the present study was to investigate the role of the bradykinin B1 (BKB1) receptor in the insulitis associated with * Corresponding author. Address: Universidad Argentina, JFK, School of Chemistry, Carlos Calvo 3660, Buenos Aires, Argentina. Tel.: +54 011 4901 3752; fax: +54 011 54 4901/1935. E-mail address: [email protected] (O.L. Catanzaro). 0143-4179/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.npep.2009.12.010
the development of diabetes-induced by streptozotocin (STZ) treatment in mice. The effect of the pharmacological BKB1 receptor, agonist des-Arg9-bradykinin (DBK), on the early development of diabetic hyperglycemic mice were evaluated. 2. Material and methods Male C57 BL/KsJ mdb mice of 6 weeks of age weighing 25–30 g were used (Institute Malbran Laboratory, Argentina). The mice were housed, five per cage and maintained under conditions of standard lighting (12 h light/dark), temperature (22 ± 0.5 °C) and humidity (60 ± 10%) with food and water ad libitum. All protocol and procedures were approved by the committee for laboratory animals care (USAL). 2.1. Experimental protocol Insulin-dependent diabetes mellitus (IDDM) was induced in mice, by the injection of five consecutives doses of STZ (45 mg/ kg). The mice were divided in the following groups:(i) control, (saline); (ii) treated with STZ; (iii) treated with STZ (SIGMA, Chem Co., St Louis, MO, USA) and compound R-954 (Ac-Orn-[Oic2, a-MePhe5, D-b Nal7, Ile8]des-Arg9-BK) (IPS Pharma Inc., Sherbrooke, Canada)
O.L. Catanzaro et al. / Neuropeptides 44 (2010) 187–189
(Neugebauer et al., 2002) a BKB1 antagonist(400 lg/kg i.p.) from the 3rd day, daily, during 16 days; (iv) treated with STZ and DBK (des-Arg9-BK) (300 lg/kg, i.p.) a BKB1 agonist (SIGMA Chem. Co., St Louis, MO, USA), from the 1st day during, 16 days; (v) treated with STZ, DBK agonist and R-954 from the 1st day, and during 15 days.
A Glucose mM
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2.2. Glucose tolerance test
Glucose Tolerance Test-8Days Control
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Glucose Tolerance Test-12 Days
30
Glucose mM
A glucose tolerance test was performed following an intraperitoneal administration of glucose, 8, 12 and 18 days after the induction of diabetes with STZ. Mice from groups (i), (ii) and (iii) were fasted for 6 h. On the day of the experiments, a solution of glucose (1.5 mg/g of body weight) was administered via the intraperitoneal route, for the intraperitoneal glucose tolerance test (IPGTT). Blood samples were drawn from the tail vein at 0, 10, 20, 30, 60, 90, and 120 min after the glucose administration. Blood glucose levels were measured using a glucometer (Accu-chek-Performa, Roche, Buenos Aires, Argentina).
20 18 16 14 12 10 8 6 4 2 0
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*
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2.3. Effects of a selective agonist and a selective antagonist of BKB1-R
C Glucose mM
Groups (iv) and (v) were injected with the selective BKB1-R agonist, DBK, and the selective antagonist R-954 on the 1st day of STZ treatment and, during 16 days. After 8, 12, and 18 days of, STZ, treatment, the blood glucose level was determined using a glucometer (Roche, Buenos Aires, Argentina).
5 0
2.4. Statistical analysis
Glucose Tolerance Test-18 Days
40 35 30 25 20 15 10
Control
Dbt 18 days
Dbt 18 days+R-954
* *
*
*
*
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60
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120
140
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The results presented as means ± S.E.M. were submitted to analyses of variance (ANOVA) followed by the Student t-test. P < 0.05 was considered significant. 3. Results
Fig. 1. Effects of the R-954 antagonist on STZ-treated mice. (A) 8 days; (B) 12 days; (C) 18 days after STZ treatment. Intraperitoneal glucose tolerance test were performed at 0–120 min after glucose challenge. All data represent the means ± SE and significance was tested using ANOVA test, n = 6–8 mice for group. *P < 0.05 vs STZ.
Plasma glucose levels in STZ mice increased significantly at all time points, 12 and 18 days after the STZ treatment (P < 0.05) but did not increase 8 days after the treatment. The administration of the BKB1-R antagonist significantly reduced the IPGTT (P < 0.05) of diabetic mice at the treatment periods of 12 and 18 days (Fig. 1A–C) The reduction of blood glucose by compound R-954 was statistically significant (AUC glucose) (*P < 0.05) (Fig. 2).
AUC(Glucose)
3.1. Effect of the BKB1-R antagonist on STZ-treated mice AUC
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
Control
Diab+R-954
Diabetics
*
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Days
*
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18
3.2. Effect of the selective agonist DBK and compound R-954
4. Discussion Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by a local inflammatory reactions which occur in and around the pancreas islets. Islet inflammation or insulitis is followed by selective destruction of insulin secreting b-cells. These initial events are associated with the release of inflammatory mediators, like interleukin 1b (IL-1b), TNF-a and kinins (Hussain
Fig. 2. The area under the glucose curve (AUC) was calculated from 0 to 120 min. All data represent the means ± SE and significance was tested using ANOVA test, n = 6–8 mice per group *P < 0.05 vs STZ.
Glycemias (mM)
30
Glucose mM
To further confirm the role of BKB1 receptor in this phenomenon, DBK was administered to the animals from the first to the eighteenth days and the blood glucose levels were measured every day, after the initial STZ treatment. The results showed that the blood glucose levels of diabetic mice increased significantly from the 3rd to 18 days (P < 0.05) The potent BKB1-R antagonist, R954 administered to the animals treated with DKB inhibited significantly the blood glucose levels in these animals (Fig. 3; P < 0.05).
Control STZ+DBK1
25
Diabetics STZ+ DBK1+BKB1-R
20
* *
15
* *
10
*
*
*
*
5 0
1
3
8
12
18 Days
Fig. 3. Effects of selective agonist DBK and compound R-954 on glucose levels in STZ-treated mice 8, 12 18 days after the induction of diabetes. The agonist DBK was administered from the 1st to 18 days. All data represent the means ± SE and significance was tested using ANOVA test, n = 8–10 mice per group. *P < 0.05 vs control or STZ.
O.L. Catanzaro et al. / Neuropeptides 44 (2010) 187–189
et al., 1996; Rabinovitch, 1998; Rabinovitch and Suarez-Pinzon, 1998; Couture et al., 2001). Experimental evidences suggest that the bradykinin B1 receptors play a significant role in the pathogenesis of experimental diabetes and the development of complications (Zuccollo et al., 1996, 1999; Simar et al., 2002). In the present study, the development of the time – dependent increase of the blood glucose tolerance test in a model of STZ-induced diabetes in mice and the active participation of BKB-1 receptor in the early development of type 1 diabetes was confirmed. Our results showed that levels of the blood glucose tolerance, after treatment of STZ-treated mice with the BKB1 receptor antagonist were significantly reduced after 12 and 18 days of treatment. These observations are in agreement with our previous data (Zuccollo et al., 1996, 1999). Moreover, after 8 days of the STZ treatment we did not observe modifications in the IPGTT, although our previous studies (Catanzaro et al., 2004) suggested that the expression profile of bradykinin B1 receptor in IDDM was upregulated early in the development of the disease state and preceded the occurrence of hyperglycemia (Ongali et al., 2004; Gabra and Sirois, 2005). In addition the administration of DBK to diabetic mice significantly potentiated the diabetes-induced hyperglycemia. Interestingly, the selective activated BKB1 receptor subtype by B1 receptor agonist is clearly absent or of little impact under normal conditions (Couture et al., 2001), but is over-expressed in diabetic state (Catanzaro et al., 2004). Hyperglycemia and the resulting oxidative stress observed alongside diabetes could activate NF-jB (Yemeni et al., 1999), a factor which is known to induce the expression of the BKB1 receptors (Marceau et al., 1998). According to Tiffany and Burch (1989), bradykinin stimulated the release of inflammatory cytokines (TNF-a and IL-1b) from macrophages. On the other hand macrophages play a pivotal role in the immune attack against the pancreatic islets in several animal models of type 1 diabetes (Kolb-Bachofen et al., 1988; Zuccollo et al., 1997). It is suggested that the overproduction of IL-1b and TNF-a, iNOS and the diabetic hyperglycemia could trigger the expression of BKB1-R through NF-jB in diabetes. The accumulation of des Arg9-BK and other metabolites resulting from the degradation of kinins at the site of inflammation can amplified the expression of the inductible B1 receptors in diabetes (Schanstra et al., 1998). In conclusion, the present study confirms that the BKB1 receptor subtype could play a major role in the diabetic hyperglycemia associated with type 1 diabetes in mice. Since the BKB1 receptor antagonist reduced the blood glucose and improved the glucose tolerance test, and since the selective agonist DBK amplified the diabetic hyperglycemia, our observations strongly suggest that the bradykinin B1 receptor antagonist could became novel pharmacological tools for the treatment of diabetes complications. Acknowledgments This work was supported by Grants from the UAJFK and the Univ. Del Salvador-Argentina. We thank IPS-Canada for critical reading of the manuscript and for the generous gift of the R-954. We also thank Lic Irene Di Martino for technical assistance.
189
References Bottazo, G.F., Mann, J.L., Thorogood, M., Baum, J.D., Doniach, D., 1978. Autoimmunity in juvenile diabetes and their families. Br. Med. J. 6131, 165– 168. Calixto, J.B., Fernandez, E.S., Medeiros, R., Ferreira, J., Cabrini, D.A., Campos, N.M., 2004. Kinin B1 receptors key G-protein coupled receptors and their role in inflammatory and painful process. Br. J. Pharmacol. 7, 803–818. Catanzaro, O., Dziubecki, D., Frontera, M., Rodríguez, R.R., Pavetto, C., Zuccollo, A., 2004. Evidence for the involvement of kinin B1 receptors in the early status of insulitis. In: Proceedings of the International Symposium on Peptide receptors, Montreal, Canada, p.69. Couture, R., Harrison, M., Vianna, R.M., Cloutier, F., 2001. Kinin receptors in pain and inflammation. Eur. J. Pharmacol. 429, 161–176. Gabra, B.H., Sirois, P., 2005. Hyperalgesia in non diabetic (NOD) mice. A role for the inductible bradykinin B1 receptor. Eur. J. Pharmacol. 514, 61–67. Gepts, W., 1965. Pathological anatomy of pancreas in juvenile diabetes mellitus. Diabetes 14, 619–633. Hussain, M.J., Peakman, M., Gallati, H., Lo, S.S., Hawa, M., Viberti, G.C., Watkins, P.J., Leslie, R.D., Vergani, D., 1996. Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM. Diabetology 39, 60–69. Kolb-Bachofen, V., Epstein, S., Kiesel, U., Kolb, H., 1988. Low-dose streptozotocininduced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset. Diabetes 37, 249–252. Leeb-Lundberg, L.M., Marceau, F., Muller-Esterl, W., Petibone, D.J., Zuraw, R.L., 2005. International Union of Pharmacology XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol. Rev. 57, 27–37. Marceau, F., Hess, J.F., Bacaharov, D.R., 1998. The B1 receptors for kinins. Pharm. Rev. 50, 157–186. Mauricio, F., Mandrup-Poulsen, T., 1998. Apoptosis and the pathogénesis of IDDM: a question of life and death. Diabetes 47, 1537–1543. Neugebauer, W., Blais, P.H., Halle, S., Filteau, C., Regoli, D., Gobeil, F., 2002. Kinin receptor antagonist with multi-enzymatic resistance properties. Can. J. Physiol. Pharmacol. 80, 287–292. Ongali, B., Campos, M.M., Petcu, M., Rodi, M., Cloutier, F., Chabot, J.G., Thibault, G., Couture, R., 2004. Expression of kinin B1 receptors in the spinal cord of streptozotocin diabetic rat. Neuro Report 15, 2463–2466. Rabinovitch, A., 1998. An update on cytokines in the pathogénesis of insulindependent diabetes mellitus. Diabetes Metab. Rev. 14, 129–151. Rabinovitch, A., Suarez-Pinzon, W.L., 1998. Cytokines and their roles in pancreatic islet beta-cells destruction and insulin-dependent diabetes mellitus. Biochem. Pharmacol. 55, 1139–1149. Schanstra, J.P., Bataille, E., Marin Castano, M.E., Barascud, Y., Hertz, C., Pesquero, J.B., Pecher, C., Gauthier, F., Girolami, J.P., Bascands, J.L., 1998. The B1-agonist[des– Arg 10] kallidin activates transcription factor NF-kappa B and induces homologous upregulation of the bradykinin B1-receptor in culture human lung fibroblasts. J. Clin. Invest. 101, 2080–2091. Simar, B., Gabra, B.H., Sirois, P., 2002. Inhibitory effect of a novel bradykinin receptor B1 receptor antagonist R-954 on enhanced vascular permeability in type 1 diabetic mice. Can. J. Physiol. Pharmacol. 80, 1203–1207. Tiffany, C., Burch, R.M., 1989. Bradykinin stimulates tumor necrosis factor and interleukin-1 release from macrophages. FEBS Lett. 247, 189–192. Yemeni, K.K., Bai, W., Khan, B.V., Medford, R.M., Natarajan, R., 1999. Hyperglycemiainduced activation nuclear transcription factor kappa B in vascular smooth muscle cells. Diabetes 48, 855–864. Yoon, J.W., Jun, H.S., Santamaría, P., 1998. Cellular and molecular mechanisms resulting from the collaboration between macrophages and T cells. Autoimmunity 27, 109–122. Zuccollo, A., Navarro, M., Catanzaro, O., 1996. Effects of B1 and B2 kinin receptor antagonist in diabetic mice. Can. J. Physiol. Pharmacol. 74, 586–589. Zuccollo, A., Frontera, M., Cueva, F., Navarro, M., Catanzaro, O.L., 1997. Effects of aprotinin on the kallikrein–kinin system in type 1 diabetes (insulitis). Immunopharmacology 37, 251–256. Zuccollo, A., Navarro, M., Frontera, M., Cueva, F., Carattino, M., Catanzaro, O., 1999. The involvement of kallikrein–kinin system in diabetes type 1 (insulitis). Immunopharmacology 45, 69–74.
Contents lists available at ScienceDirect
Neuropeptides journal homepage: www.elsevier.com/locate/npep
Special Issue on Peptide Receptors: Focus on Neuropeptides and Kinins
Antidiabetic efficacy of bradykinin antagonist R-954 on glucose tolerance test in diabetic type 1 mice Orlando L. Catanzaro a,b,*, Damian Dziubecki a, Pablo Obregon a, Ricardo R. Rodriguez b, Pierre Sirois c a
Direccion de Investigaciones Cientificas Bioquimico-Odontologicas, Universidad Argentina John F Kennedy, Argentina Escuela de Medicina y Odontología, Universidad Del Salvador, Argentina c Sherbrooke University Medical School, Sherbrooke, Canada b
a r t i c l e
i n f o
Article history: Available online 21 January 2010 Keywords: Type 1 diabetes DBK BKB1-R antagonist R-954
a b s t r a c t Insulin-dependent diabetes mellitus (type 1 diabetes) is an inflammatory autoimmune disease associated with many complications including nephropathy, retinopathy, neuropathy and hyperalgesia. Experimental evidence has shown that the bradykinin B1 receptor (BKB1-R) is involved in the development of type 1 diabetes and found to be upregulated alongside the disease. In the present study the effects of the selective BKB1-R antagonist the R-954 (Ac-Orn-[Oic2, a-MePhe5, D-b Nal7, Ile8 ]des-Arg9-BK and the BKB1-R agonist des Arg9-BK (DBK) were studied on diabetic hyperglycemia. Diabetic type 1 was induced in C57 BL/KsJ mdb male mice by five consecutives doses of STZ (45 mg/kg i.p.). A glucose tolerance test (GTT) was performed by an intraperitoneal administration of glucose, 8, 12 and 18 days after the diabetes induction. The induction of type 1 diabetes provoked a significant hyperglycemia levels in diabetic mice at 12 and 18 days after STZ. The administration of R-954 (400 lg/kg i.p.) at 12 and 18 days after STZ returned the glycemia levels of this animals to normal values. In addition the administration of DKB (300 lg/kg i.p.) significantly potentiated the diabetes-induced hyperglycemia; this effect that was totally reversed by R-954. These results provide further evidence for the implication of BKB1-R in the type 1 diabetes mellitus (insulitis). Ó 2009 Published by Elsevier Ltd.
1. Introduction Type 1 diabetes is caused by the progressive autoimmune destruction of pancreatic b-cells and the autoimmune process begins years before the b-cells destruction became complete, thereby providing a window of opportunity for intervention (Mauricio and Mandrup-Poulsen, 1998; Yoon et al., 1998). The loss of b-cells is associated with local and circulating signs of an autoimmune reactivity against the b-cell antigens (Gepts, 1965; Bottazo et al., 1978). Islet inflammation or insulitis is followed by the selective destruction of insulin secreting b-cells. Experimental evidence which suggests that kinins are involved in the development of an insulin-dependent form of diabetes mellitus in mice was reported by Zuccollo et al. (1999). The kallikrein–kinin system is made of a group of peptides and proteins involved in the control of blood pressure, gastrointestinal tract motility, inflammation and pain induction (Calixto et al., 2004; Leeb-Lundberg et al., 2005). The objective of the present study was to investigate the role of the bradykinin B1 (BKB1) receptor in the insulitis associated with * Corresponding author. Address: Universidad Argentina, JFK, School of Chemistry, Carlos Calvo 3660, Buenos Aires, Argentina. Tel.: +54 011 4901 3752; fax: +54 011 54 4901/1935. E-mail address: [email protected] (O.L. Catanzaro). 0143-4179/$ - see front matter Ó 2009 Published by Elsevier Ltd. doi:10.1016/j.npep.2009.12.010
the development of diabetes-induced by streptozotocin (STZ) treatment in mice. The effect of the pharmacological BKB1 receptor, agonist des-Arg9-bradykinin (DBK), on the early development of diabetic hyperglycemic mice were evaluated. 2. Material and methods Male C57 BL/KsJ mdb mice of 6 weeks of age weighing 25–30 g were used (Institute Malbran Laboratory, Argentina). The mice were housed, five per cage and maintained under conditions of standard lighting (12 h light/dark), temperature (22 ± 0.5 °C) and humidity (60 ± 10%) with food and water ad libitum. All protocol and procedures were approved by the committee for laboratory animals care (USAL). 2.1. Experimental protocol Insulin-dependent diabetes mellitus (IDDM) was induced in mice, by the injection of five consecutives doses of STZ (45 mg/ kg). The mice were divided in the following groups:(i) control, (saline); (ii) treated with STZ; (iii) treated with STZ (SIGMA, Chem Co., St Louis, MO, USA) and compound R-954 (Ac-Orn-[Oic2, a-MePhe5, D-b Nal7, Ile8]des-Arg9-BK) (IPS Pharma Inc., Sherbrooke, Canada)
O.L. Catanzaro et al. / Neuropeptides 44 (2010) 187–189
(Neugebauer et al., 2002) a BKB1 antagonist(400 lg/kg i.p.) from the 3rd day, daily, during 16 days; (iv) treated with STZ and DBK (des-Arg9-BK) (300 lg/kg, i.p.) a BKB1 agonist (SIGMA Chem. Co., St Louis, MO, USA), from the 1st day during, 16 days; (v) treated with STZ, DBK agonist and R-954 from the 1st day, and during 15 days.
A Glucose mM
188
2.2. Glucose tolerance test
Glucose Tolerance Test-8Days Control
0
20
Dbt 8 days
40
Dbt 8 days+R-954
60
80
100
120
140
Minutes
B
Glucose Tolerance Test-12 Days
30
Glucose mM
A glucose tolerance test was performed following an intraperitoneal administration of glucose, 8, 12 and 18 days after the induction of diabetes with STZ. Mice from groups (i), (ii) and (iii) were fasted for 6 h. On the day of the experiments, a solution of glucose (1.5 mg/g of body weight) was administered via the intraperitoneal route, for the intraperitoneal glucose tolerance test (IPGTT). Blood samples were drawn from the tail vein at 0, 10, 20, 30, 60, 90, and 120 min after the glucose administration. Blood glucose levels were measured using a glucometer (Accu-chek-Performa, Roche, Buenos Aires, Argentina).
20 18 16 14 12 10 8 6 4 2 0
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*
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*
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*
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2.3. Effects of a selective agonist and a selective antagonist of BKB1-R
C Glucose mM
Groups (iv) and (v) were injected with the selective BKB1-R agonist, DBK, and the selective antagonist R-954 on the 1st day of STZ treatment and, during 16 days. After 8, 12, and 18 days of, STZ, treatment, the blood glucose level was determined using a glucometer (Roche, Buenos Aires, Argentina).
5 0
2.4. Statistical analysis
Glucose Tolerance Test-18 Days
40 35 30 25 20 15 10
Control
Dbt 18 days
Dbt 18 days+R-954
* *
*
*
*
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40
60
80
*
100
120
140
Minutes
The results presented as means ± S.E.M. were submitted to analyses of variance (ANOVA) followed by the Student t-test. P < 0.05 was considered significant. 3. Results
Fig. 1. Effects of the R-954 antagonist on STZ-treated mice. (A) 8 days; (B) 12 days; (C) 18 days after STZ treatment. Intraperitoneal glucose tolerance test were performed at 0–120 min after glucose challenge. All data represent the means ± SE and significance was tested using ANOVA test, n = 6–8 mice for group. *P < 0.05 vs STZ.
Plasma glucose levels in STZ mice increased significantly at all time points, 12 and 18 days after the STZ treatment (P < 0.05) but did not increase 8 days after the treatment. The administration of the BKB1-R antagonist significantly reduced the IPGTT (P < 0.05) of diabetic mice at the treatment periods of 12 and 18 days (Fig. 1A–C) The reduction of blood glucose by compound R-954 was statistically significant (AUC glucose) (*P < 0.05) (Fig. 2).
AUC(Glucose)
3.1. Effect of the BKB1-R antagonist on STZ-treated mice AUC
5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0
Control
Diab+R-954
Diabetics
*
8
Days
*
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18
3.2. Effect of the selective agonist DBK and compound R-954
4. Discussion Insulin-dependent diabetes mellitus (IDDM) is an autoimmune disease characterized by a local inflammatory reactions which occur in and around the pancreas islets. Islet inflammation or insulitis is followed by selective destruction of insulin secreting b-cells. These initial events are associated with the release of inflammatory mediators, like interleukin 1b (IL-1b), TNF-a and kinins (Hussain
Fig. 2. The area under the glucose curve (AUC) was calculated from 0 to 120 min. All data represent the means ± SE and significance was tested using ANOVA test, n = 6–8 mice per group *P < 0.05 vs STZ.
Glycemias (mM)
30
Glucose mM
To further confirm the role of BKB1 receptor in this phenomenon, DBK was administered to the animals from the first to the eighteenth days and the blood glucose levels were measured every day, after the initial STZ treatment. The results showed that the blood glucose levels of diabetic mice increased significantly from the 3rd to 18 days (P < 0.05) The potent BKB1-R antagonist, R954 administered to the animals treated with DKB inhibited significantly the blood glucose levels in these animals (Fig. 3; P < 0.05).
Control STZ+DBK1
25
Diabetics STZ+ DBK1+BKB1-R
20
* *
15
* *
10
*
*
*
*
5 0
1
3
8
12
18 Days
Fig. 3. Effects of selective agonist DBK and compound R-954 on glucose levels in STZ-treated mice 8, 12 18 days after the induction of diabetes. The agonist DBK was administered from the 1st to 18 days. All data represent the means ± SE and significance was tested using ANOVA test, n = 8–10 mice per group. *P < 0.05 vs control or STZ.
O.L. Catanzaro et al. / Neuropeptides 44 (2010) 187–189
et al., 1996; Rabinovitch, 1998; Rabinovitch and Suarez-Pinzon, 1998; Couture et al., 2001). Experimental evidences suggest that the bradykinin B1 receptors play a significant role in the pathogenesis of experimental diabetes and the development of complications (Zuccollo et al., 1996, 1999; Simar et al., 2002). In the present study, the development of the time – dependent increase of the blood glucose tolerance test in a model of STZ-induced diabetes in mice and the active participation of BKB-1 receptor in the early development of type 1 diabetes was confirmed. Our results showed that levels of the blood glucose tolerance, after treatment of STZ-treated mice with the BKB1 receptor antagonist were significantly reduced after 12 and 18 days of treatment. These observations are in agreement with our previous data (Zuccollo et al., 1996, 1999). Moreover, after 8 days of the STZ treatment we did not observe modifications in the IPGTT, although our previous studies (Catanzaro et al., 2004) suggested that the expression profile of bradykinin B1 receptor in IDDM was upregulated early in the development of the disease state and preceded the occurrence of hyperglycemia (Ongali et al., 2004; Gabra and Sirois, 2005). In addition the administration of DBK to diabetic mice significantly potentiated the diabetes-induced hyperglycemia. Interestingly, the selective activated BKB1 receptor subtype by B1 receptor agonist is clearly absent or of little impact under normal conditions (Couture et al., 2001), but is over-expressed in diabetic state (Catanzaro et al., 2004). Hyperglycemia and the resulting oxidative stress observed alongside diabetes could activate NF-jB (Yemeni et al., 1999), a factor which is known to induce the expression of the BKB1 receptors (Marceau et al., 1998). According to Tiffany and Burch (1989), bradykinin stimulated the release of inflammatory cytokines (TNF-a and IL-1b) from macrophages. On the other hand macrophages play a pivotal role in the immune attack against the pancreatic islets in several animal models of type 1 diabetes (Kolb-Bachofen et al., 1988; Zuccollo et al., 1997). It is suggested that the overproduction of IL-1b and TNF-a, iNOS and the diabetic hyperglycemia could trigger the expression of BKB1-R through NF-jB in diabetes. The accumulation of des Arg9-BK and other metabolites resulting from the degradation of kinins at the site of inflammation can amplified the expression of the inductible B1 receptors in diabetes (Schanstra et al., 1998). In conclusion, the present study confirms that the BKB1 receptor subtype could play a major role in the diabetic hyperglycemia associated with type 1 diabetes in mice. Since the BKB1 receptor antagonist reduced the blood glucose and improved the glucose tolerance test, and since the selective agonist DBK amplified the diabetic hyperglycemia, our observations strongly suggest that the bradykinin B1 receptor antagonist could became novel pharmacological tools for the treatment of diabetes complications. Acknowledgments This work was supported by Grants from the UAJFK and the Univ. Del Salvador-Argentina. We thank IPS-Canada for critical reading of the manuscript and for the generous gift of the R-954. We also thank Lic Irene Di Martino for technical assistance.
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References Bottazo, G.F., Mann, J.L., Thorogood, M., Baum, J.D., Doniach, D., 1978. Autoimmunity in juvenile diabetes and their families. Br. Med. J. 6131, 165– 168. Calixto, J.B., Fernandez, E.S., Medeiros, R., Ferreira, J., Cabrini, D.A., Campos, N.M., 2004. Kinin B1 receptors key G-protein coupled receptors and their role in inflammatory and painful process. Br. J. Pharmacol. 7, 803–818. Catanzaro, O., Dziubecki, D., Frontera, M., Rodríguez, R.R., Pavetto, C., Zuccollo, A., 2004. Evidence for the involvement of kinin B1 receptors in the early status of insulitis. In: Proceedings of the International Symposium on Peptide receptors, Montreal, Canada, p.69. Couture, R., Harrison, M., Vianna, R.M., Cloutier, F., 2001. Kinin receptors in pain and inflammation. Eur. J. Pharmacol. 429, 161–176. Gabra, B.H., Sirois, P., 2005. Hyperalgesia in non diabetic (NOD) mice. A role for the inductible bradykinin B1 receptor. Eur. J. Pharmacol. 514, 61–67. Gepts, W., 1965. Pathological anatomy of pancreas in juvenile diabetes mellitus. Diabetes 14, 619–633. Hussain, M.J., Peakman, M., Gallati, H., Lo, S.S., Hawa, M., Viberti, G.C., Watkins, P.J., Leslie, R.D., Vergani, D., 1996. Elevated serum levels of macrophage-derived cytokines precede and accompany the onset of IDDM. Diabetology 39, 60–69. Kolb-Bachofen, V., Epstein, S., Kiesel, U., Kolb, H., 1988. Low-dose streptozotocininduced diabetes in mice. Electron microscopy reveals single-cell insulitis before diabetes onset. Diabetes 37, 249–252. Leeb-Lundberg, L.M., Marceau, F., Muller-Esterl, W., Petibone, D.J., Zuraw, R.L., 2005. International Union of Pharmacology XLV. Classification of the kinin receptor family: from molecular mechanisms to pathophysiological consequences. Pharmacol. Rev. 57, 27–37. Marceau, F., Hess, J.F., Bacaharov, D.R., 1998. The B1 receptors for kinins. Pharm. Rev. 50, 157–186. Mauricio, F., Mandrup-Poulsen, T., 1998. Apoptosis and the pathogénesis of IDDM: a question of life and death. Diabetes 47, 1537–1543. Neugebauer, W., Blais, P.H., Halle, S., Filteau, C., Regoli, D., Gobeil, F., 2002. Kinin receptor antagonist with multi-enzymatic resistance properties. Can. J. Physiol. Pharmacol. 80, 287–292. Ongali, B., Campos, M.M., Petcu, M., Rodi, M., Cloutier, F., Chabot, J.G., Thibault, G., Couture, R., 2004. Expression of kinin B1 receptors in the spinal cord of streptozotocin diabetic rat. Neuro Report 15, 2463–2466. Rabinovitch, A., 1998. An update on cytokines in the pathogénesis of insulindependent diabetes mellitus. Diabetes Metab. Rev. 14, 129–151. Rabinovitch, A., Suarez-Pinzon, W.L., 1998. Cytokines and their roles in pancreatic islet beta-cells destruction and insulin-dependent diabetes mellitus. Biochem. Pharmacol. 55, 1139–1149. Schanstra, J.P., Bataille, E., Marin Castano, M.E., Barascud, Y., Hertz, C., Pesquero, J.B., Pecher, C., Gauthier, F., Girolami, J.P., Bascands, J.L., 1998. The B1-agonist[des– Arg 10] kallidin activates transcription factor NF-kappa B and induces homologous upregulation of the bradykinin B1-receptor in culture human lung fibroblasts. J. Clin. Invest. 101, 2080–2091. Simar, B., Gabra, B.H., Sirois, P., 2002. Inhibitory effect of a novel bradykinin receptor B1 receptor antagonist R-954 on enhanced vascular permeability in type 1 diabetic mice. Can. J. Physiol. Pharmacol. 80, 1203–1207. Tiffany, C., Burch, R.M., 1989. Bradykinin stimulates tumor necrosis factor and interleukin-1 release from macrophages. FEBS Lett. 247, 189–192. Yemeni, K.K., Bai, W., Khan, B.V., Medford, R.M., Natarajan, R., 1999. Hyperglycemiainduced activation nuclear transcription factor kappa B in vascular smooth muscle cells. Diabetes 48, 855–864. Yoon, J.W., Jun, H.S., Santamaría, P., 1998. Cellular and molecular mechanisms resulting from the collaboration between macrophages and T cells. Autoimmunity 27, 109–122. Zuccollo, A., Navarro, M., Catanzaro, O., 1996. Effects of B1 and B2 kinin receptor antagonist in diabetic mice. Can. J. Physiol. Pharmacol. 74, 586–589. Zuccollo, A., Frontera, M., Cueva, F., Navarro, M., Catanzaro, O.L., 1997. Effects of aprotinin on the kallikrein–kinin system in type 1 diabetes (insulitis). Immunopharmacology 37, 251–256. Zuccollo, A., Navarro, M., Frontera, M., Cueva, F., Carattino, M., Catanzaro, O., 1999. The involvement of kallikrein–kinin system in diabetes type 1 (insulitis). Immunopharmacology 45, 69–74.