IMT504 protects beta cells against apoptosis and maintains beta cell identity, without modifying proliferation

Abstract

We have demonstrated that oligodeoxynucleotide IMT504 promotes significant improvement in the diabetic condition in diverse animal models. Based on these results, here we evaluated whether these effects observed in vivo could be due to direct effects on β-cells. We demonstrate by immunofluorescence that IMT504 enters the cell and locates in cytoplasm where it induces GSK-3β phosphorylation that inactivates this kinase. As GSK-3β tags Pdx1 for proteasomal degradation, by inactivating GSK-3β, IMT504 induces an increase in Pdx1 protein levels, demonstrated by Western blotting. Concomitantly, an increase in Ins2 and Pdx1 gene transcription was observed, with no significant increase in insulin content or secretion. Enhanced Pdx1 is promising since it is a key transcription factor for insulin synthesis and is also described as an essential factor for the maintenance β-cell phenotype and function. Dose-dependent inhibition of H₂ O₂ -induced apoptosis determined by ELISA as well as decreased expression of Bax was also observed. These results were confirmed in another β-cell line, beta-TC-6 cells, in which a cytokine mix induced apoptosis that was reversed by IMT504. In addition, an inhibitor of IMT504 entrance into cells abrogated the effect IMT504. Based on these results we conclude that the β-cell recovery observed in vivo may include direct effects of IMT504 on β-cells, by maintaining their identity/phenotype and protecting them from oxidative stress and cytokine-induced apoptosis. Thus, this work positions IMT504 as a promising option in the framework of the search of new therapies for type I diabetes treatment.

Keywords: apoptosis; oligodeoxynucleotides; proliferation; transcription factors; β-Cells.

FIGURE 1

Confocal microscopic analysis of two examples of IMT504‐Texas Red incorporation into MIN6B1 cells after 4 h incubation; nuclei stained with DAPI (blue), 60X, bar = 20 μM.

FIGURE 2

(a). qPCR Gene expression evaluated in MIN6B1 cells treated with different concentrations of IMT504 for 48 h. Pdx1/Ppib: ANOVA with repeated measures, p < 0.02, #: IMT4 different form C, p < 0.05, n = 6. (b) Western blot analysis of Pdx1 protein expression in MIN6B1 with IMT2 and IMT4 for 48 h. B1: Pdx1/Actin ratio was significantly increased with IMT4. ANOVA with repeated measures, p < 0.03, #: IMT4 different from C and IMT2, p < 0.05, n = 4; B2: Representative blot of Pdx1 and Actin expression. (c,d). qPCR Gene expression evaluated in MIN6B1 cells with different concentrations of IMT504 for 48 h. (c) Ins2/Ppib increased in cells incubated with IMT4; ANOVA with repeated measures, p < 0.05, #: IMT4 different from C and IMT2 p < 0.05 n = 6. (d) Ins1/Ppib did not vary among the experimental groups; ANOVA with repeated measures, NS, n = 6.

FIGURE 3

Western Blot analysis demonstrating the effects of IMT504 on GSK3β and Akt phosphorylation in MIN6B1 cells. Cells were incubated with IMT4 for a short term (5–60 min). (a) At 60 min, the IMT504‐induced pGSK3β/GSK3β ratio was significantly increased compared to the basal pGSK3β/GSK3β ratio (0 min = 1). ANOVA with repeated measures, p < 0.03. #: 60 min different from 0 min, p < 0.03, n = 6–7. (b) No significant differences in the pAkt/Akt ratio were observed. ANOVA with repeated measures, NS, n = 4–6.

FIGURE 4

Effect of IMT504 on MIN6B1 cell proliferation (a). MTS. Incubation with IMT2, IMT4, and IMT8 for 24 h had no significant effect on MIN6B1 cell proliferation compared to C cells. ANOVA with repeated measures, NS, n = 5. B1‐2. WB analysis demonstrated no effect of IMT504 (IMT2, IMT4, and IMT8 for 24 h) on PCNA/Actin expression ratio. ANOVA with repeated measures, NS, n = 4.

FIGURE 5

Effects of IMT504 on β‐cell Apoptosis. (a). Cell Death Detection ELISA: MIN6B1 cells were treated with 0.75 μM H₂O₂ for 24 h with or without IMT504. ANOVA with repeated measures, p < 0.01, n = 7, @: H₂O₂‐IMT0 different from C, p < 0.05; #: H₂O₂‐IMT2, H₂O₂‐IMT4, H₂O₂‐IMT8 different from H₂O₂‐IMT0 p < 0.05. (b) Representative images of TUNEL assay 40X, C with H₂O₂ (upper panel), IMT4 with H₂O₂ (lower panel). Regular nuclei (blue) and apoptotic nuclei (turquoise), bar = 20 μM. (c–e) qPCR Gene expression evaluated in MIN6B1 cells treated with different concentrations of IMT504 and a low concentration of H₂O₂ (0.75 μM) for 24 h. (c). Bcl2/Ppib did not vary among the experimental groups; ANOVA with repeated measures, NS, n = 4. (d) Bax/Ppib increased in cells incubated with H₂O₂ and decreased to C levels with H₂O₂‐IMT4; ANOVA with repeated measures, p < 0.05. @: H₂O₂‐IMT0 different from C, p < 0.04; *: H₂O₂‐IMT4 different from H₂O₂‐IMT0 group, p < 0.04, n = 4. (e): Bax/Bcl2 ratio increased in cells incubated with H₂O₂ and decreased to C levels with H₂O₂‐IMT4 or H₂O₂‐IMT8; ANOVA with repeated measures, p < 0.05. @: H₂O₂‐IMT0 different from C, p < 0.05; *H₂O₂‐IMT4 and H₂O₂‐IMT8 different from H₂O₂‐IMT0_, p < 0.04 and 0.01, respectively, n = 4. (f) Beta‐TC‐6 cells treated with the cytokine mix for 24 h with or without 4 μg/mL IMT504, in the presence or absence of 25 μM IPS. Apoptosis was determined by nuclei staining with Annexin V with the Incucyte® Live Cell Analysis System. ANOVA with repeated measures, p < 0.02. *: Cytokine Mix (CYT) significantly different from control and CYT‐IMT054. (g): Beta‐TC‐6 cells were treated with the cytokine mix for 24 h with or without 8 μg/mL IMT504, in the presence or absence of 25 μM IPS. ANOVA with repeated measures, NS.