Insulinotropic and anti-inflammatory effects of rosiglitazone in experimental autoimmune diabetes

Abstract

Cytokines and nitric oxide (NO) are involved in the pathogenesis of autoimmune diabetes mellitus (DM). Rosiglitazone is an insulin-sensitizing drug that is a ligand for the nuclear receptor peroxisome proliferator-activated receptor-gamma (PPAR-gamma). The anti-inflammatory and immunomodulating properties of PPAR-gamma have been documented. The aim of this study is to investigate the effectiveness of rosiglitazone in autoimmune DM and to clarify the possible mechanism(s) involved. Autoimmune DM was induced in adult male Balb/c mice by co-administration of cyclosporin A and multiple low doses of streptozotocin. Diabetic mice were treated daily with rosiglitazone (7 mg/kg, p.o.) for 21 days. Blood glucose level (BGL), serum insulin level and pancreatic levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma) and NO were measured. Histopathological examination and immunohistochemical determination of CD4 and CD8 T lymphocytes in the pancreatic islets were performed. In addition, analysis of pancreatic protein expression was carried out. The results showed that rosiglitazone treatment resulted in a significant decrease in the BGL and the pancreatic levels of TNF-alpha, IFN-gamma and NO compared to diabetic mice. The serum insulin level was significantly increased after rosiglitazone treatment compared to diabetic mice. The destroyed pancreatic islets were regenerated and became free from both CD4 and CD8 T cells after treatment. Furthermore, many changes in pancreatic protein expression were observed. These results suggest that rosiglitazone has a beneficial effect in the treatment of autoimmune diabetes, an effect that seemed to be a secondary consequence of its anti-inflammatory and immunomodulating properties and might be reflected at the level of protein expression.

Figure 1. Time-course effect of rosiglitazone on BGL

Mice were treated with rosiglitazone (7 mg/kg/day) for 21 days. BGL was determined every other day. Data are presented as mean ± SEM, n = 13 for diabetic groups and n = 10 for control groups. Day 0: immediately before the start of treatment. Day 1: the first day of rosiglitazone treatment. Asterisk (*) indicates significant difference from the same group before treatment (day 0). p < 0.05; paired Student's t-test.

Figure 2. Effect of rosiglitazone on the serum insulin level in diabetic mice

Mice were treated with rosiglitazone (7 mg/kg/day) for 21 days. Serum insulin levels were determined at the end of the treatment period. Data are presented as mean ± SEM, n = 13 for diabetic groups and n = 10 for control groups. (*) indicates significant difference from control saline group. (**) indicates significant difference from diabetic saline group. p < 0.05; ANOVA, Tukey-Kramer test.

Figure 3. Effect of rosiglitazone on the TNF-α content in pan-creatic tissue

Mice were treated with rosiglitazone (7 mg/kg/day) for 21 days. Pancreatic TNF-α content was determined at the end of the treatment period. Data are presented as mean ± SEM, n = 13 for diabetic groups and n = 10 for control groups. (*) indicates significant difference from control saline group. (**) indicates significant difference from dia-betic saline group. p < 0.05; ANOVA, Tukey-Kramer test.

Figure 4. Effect of rosiglitazone on the IFN-γ content in pancreatic tissue

Mice were treated with rosiglitazone (7 mg/kg/day) for 21 days. Pancreatic IFN-γ content was determined at the end of the treatment period. Data are presented as mean ± SEM, n = 13 for diabetic groups and n = 10 for control groups. (*) indicates significant difference from control saline group. (**) indicates significant difference from diabetic saline group. p < 0.05; ANOVA, Tukey-Kramer test.

Figure 5. Effect of rosiglitazone on the NO content in pancreatic tissue

Mice were treated with rosiglitazone (7 mg/kg/day) for 21 days. Pancreatic NO content (measured as nitrite/nitrate) was determined at the end of the treatment period. Data are presented as mean ± SEM, n = 13 for diabetic groups and n = 10 for control groups. (*) indicates significant difference from control saline group. (**) indicates significant difference from diabetic saline group. p < 0.05; ANOVA, Tukey-Kramer test.

Figure 6. SDS-PAGE analysis of the effect of rosiglitazone on protein expression in pancreatic tissue homogenate from diabetic mice

Mice were treated with rosiglitazone (7 mg/kg) for 21 days. Protein separation of pancreatic tissue homogenate was carried out at the end of the treatment period. A: SDS-PAGE image of pancreatic tissue homogenate of control saline group. B: SDS-PAGE image of pancreatic tissue homogenate of diabetic saline group. C: SDS-PAGE image of pancreatic tissue homogenate of diabetic rosiglitazone group.

Figure 7

Photomicrograph of H&E-stained pancreatic section from diabetic mice given saline (A, B) for 21 days, showing severe islet hyalinization with fibrosis and near-complete islet destruction with severe (+++) mononuclear cell infiltration, respectively. Mice treated with rosiglitazone (7 mg/kg) for 21 days showed regenerative islets of variable sizes with residual mild (+) mononuclear cell infiltration (C). Pancreatic section of normal mice that received vehicles for 21 days, showing normal islet architecture (D). Magnification: x 250.

Figure 8

Photomicrograph of CD4 and CD8 immunostaining of pancreatic section from normal mice (A and B), diabetic mice given saline (C and D) or 7 mg/kg rosiglitazone (E and F) for 21 days. A and B show normal pancreatic islets that are negative for CD4- and CD8-stained cells. C shows positive CD4 cells infiltrating islet. D shows fibrotic islet with negative stain for CD8 cells. E and F show regenerated pancreatic islets that are free from both CD4- and CD8-stained cells, respectively. PAP, magnification: x 400.