EFFECT OF OLIGODEOXYNUCLEOTIDE IMT504 ON MURINE β-CELLS

AYELÉN CONVERTI; MARÍA SILVIA BIANCHI ; ANDREA MONTANER; VICTORIA LUX-LANTOS; MARÍA MARTA BONAVENTURA; DELGUI, LAURA RUTH

Mendoza

Congreso; LVIII Annual Meeting of the Argentine Society for Biochemistry and Molecular Biology Research; 2022

SAIB

IMT504 is an oligodeoxynucleotide (ODN) that exerts regenerative properties. We demonstrated that the presence of IMT504 significantly promotes metabolic improvement in the diabetic condition in diverse animal models. Using diabetic IMT504-treated animals we showed a different gene expression profile and protective effects against apoptosis in pancreatic cells. Then, we investigated if these effects were due to its direct effects on those cells. In the same way, IMT504-treated in vitro β-cells showed a different gene expression profile of genes related to their functionality and a modification on glycogen synthase kinase-3 (GSK3) phosphorylation, a protein that participates in the regulation on β-cell function. Based on these results, here we evaluated the ODN internalization and protective effects against apoptosis in a pancreatic β-cell line.For internalization of the ODN, we analyzed the IMT504-Texas Red incorporation into Beta-TC-6 cells by immunofluorescence at different time points. At 5 and 15 min post-internalization (p.i.) the IM504 was found both, associated to the cytoplasmic membrane and in the cytoplasm. At 30 and 60 min p.i., when the percentage of cells with IMT504 reached the maximum, IMT504 was almost exclusively in the cytoplasm of the cells. Complementary, we used flow cytometry to monitor IMT504 internalization. We showed that 69% of the cells treated with IMT504-Texas Red for 60 min incorporated the ODN. The cytoplasmic destiny of the ODN was confirmed by the treatment of the cells with methanol, which promotes the removal of soluble proteins found in the cytoplasm enabling us to differentiate cytoplasmic proteins from those anchored to membranes. We demonstrated that this ODN remains as a soluble molecule in the cytoplasm.On the other hand, the protective effects of IMT504 against H2O2-induced cell apoptosis was analyzed in MIN6B1 cells. We observed that the IMT504 reversed the H2O2-induced apoptosis in a concentration dependent manner. Apoptosis is regulated by Bcl-2 proteins, comprising pro-apoptotic and anti-apoptotic members. In an effort to better understand the effect of IMT504 in H2O2-induced apoptosis, we evaluated the expression of apoptosis regulator genes Bcl2 and Bax. We observed that Bcl2, an anti-apoptotic factor, was unaltered. In contrast, the pre-treatment of the cells with IMT504 attenuated the increase of Bax induced by the exposition of the cells to the apoptotic stimulus. Likewise, the Bax/Bcl2 ratio was significantly decreased after IMT504 treatment.Our results indicate that IMT504 internalizes shortly after contacting the β-cells, reaching the cytosol of the cells. Furthermore, IMT504 lead to the inhibition of H2O2-induced β-cell apoptosis suggesting the IMT504 treatment as a promising strategy in the prevention/amelioration of diabetes.