Reduced glucose transport sensitizes Iec-6 cells to peroxynitrite-induced apoptosis

Abstract

Introduction: Formula-fed infants develop necrotizing enterocolitis at higher rates than breast-fed infants. We hypothesize that diminished enterocyte glucose transport sensitizes enterocytes to nitrosative stress and this results in mitochondrial-dependent apoptosis. Methods: IEC6 cells were treated with 3-morpholinosydnonimine (SIN-1) (500 uM-2 mM), a peroxynitrite generator, in either normal (NM) (4.5 g/ml) and low (LM) (1 g/ml) glucose media. IEC6 cell apoptosis was measured by 7-aminoactinomycin-D FACS analysis and histology. In order to test the role of glucose transport, we pretreated the cells with phloretin (100 uM), a specific GLUT-2 transport inhibitor. Cells were treated with SIN-1 in combination with phloretin in NM for 4 hours. Mitochondrial respiration was tested with tetrazolium salts (MTT) and transmembrane potential with 5,5′, 6,6′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1), ratiometric dye. ANOVA or Student t-test was used where appropriate; p < 0.05 is considered significant. Results: IEC6 cells exposed to the combination of SIN-1 and LM experienced a 2-fold increase in apoptosis at 24 hours compared to NM and SIN-1 (22.2 ± 3.2, 10.7 ± 2.4, P < 0.05). Impaired GLUT-2 transport plus SIN-1 increased apoptosis compared to SIN-1 alone in NM. (Table)TABLE—ABSTRACT 74GroupsApoptosis (%)MTTNM5.4 ± 0.6100.5 ± 0.4NM + SIN-14.3 ± 2.678.0 ± 5.9NM + SIN-1 and Phloretin33.7 ± 9.6∗43.6 ± 4.4∧NM and phloretin3.2 ± 0.487.4 ± 10.8LM4.4 ± 1.594.9 ± 6.0LM and SIN-133.2 ± 13.7∗78.4 ± 7.2Apop. and MTT (∗p < 0.05 v NM + SIN-1;∧p < 0.01 v all). Mitochondrial respiration was impaired in NM plus SIN-1 and phloretin but not in NM and SIN-1 alone; transmembrane potential findings were consistent with these results (dns). Conclusions: These findings support the hypothesis that decreased glucose in the local enterocyte environment may render enterocytes susceptible to reactive nitrogen species.