In search of the mechanism of action of IMT504 in beta cells (MIN6B1)

CONVERTI AYELÉN; BIANCHI, STEFANIA; MONTANER, ALEJANDRO; LIBERTUN, CARLOS; LUX-LANTOS, VICTORIA A.; BIANCHI, MARÍA S.

Buenos Aires

Jornada; XX Jornadas anuales de la Sociedad Argentina de Biología y XVII Jornada de la Sociedad Uruguaya de Biociencias; 2018

Sociedad Argentina de Biología

We have previously demonstrated that treatment with IMT504 promotes significant improvement in the diabetic condition in diverse animal models. We have also shown effects on gene expression in freshly isolated islets from diabetic, in vivo IMT504-treated animals and in in vitro IMT504-treated beta cells (MIN6B1). Based on these results, here we started to investigate the possible mechanism ofaction of IMT504 on beta cells. Other authors have previously demonstrated that IMT504 modifies GSK3β and AKT phosphorylation in other cell types. Since GSK3β and AKT participate in the regulation of beta cell function, we started to evaluate this signaling pathway in MIN6B1 cells.MIN6B1 cells were cultured in DMEM with 20 mM glucose, 15% SFB, 71 µM β-mercaptoethanol. Phosphorylation of proteins of interest was analyzed by Western Blot. To study enzyme activation, cells were stimulated for 0, 5, 15, 30 and 60 min with 4 µg/µl of IMT504 (IMT4) in DMEM, 20 mM glucose, 0,5% BSA, 71 µM β-mercaptoethanol (n=6). For more prolonged stimulation, cells were incubated for 24 or 48 hs with 0 (C), 2 (IMT2), 4 (IMT4) and 8 µg/µl (IMT8) of IMT504 in the same medium (n=3-4). At each time point cells were lysed in sample buffer for Western Blot analysis. pGSK3β/GSK3β ratio (A.U.) and pAkt/Akt ratio (A.U.) were calculated and informed as fold increase with regard to 0. At 60 min, pGSK3β /GSK3β ratio was increased compared to 0 min (ANOVA with repeated measures: pGSK3β/GSK3β ratio (A.U.) 0 min = 1, 5 min = 1.15 ± 0.06, 15 min = 1.20 ± 0.05; 30 min = 1.54 ±0.14; 60 min = 2.04 ± 0.17, 60 min different from 0 min, p