Maintenance of cellular NADPH is critical to intestinal cell survival during oxidative challenge: Role of glucose and pentose phosphate shunt activity *

Cellular redox balance is maintained by the status of reduced glutathiona (GSH) and glutathione disulfide (GSSG) through the function of GSH pemxidase and GSSG reductase coupled to the NADP+/NADPH redox pair. DUring oxidative challenge enhanced glucose supply and pentose phosphate shunt activity to support NADPH production for GSSG reduction is critical to restonng redox homeostasis and maintaining cell survival. The current objective was to test the hypothesis using two intestinal cell lines with distinct cellular redox status, viz., the normal human colon epithelial NCM 460 and the human coIon adenocarcinoma G'~Co2 cell lines Cells were treated with the thiol oxidam, diamide, to induce cellular GSH/ GSSG imbalance without oxyradical generation. Cellular redox, NADPH, redox cycle enzymes and cell apopmsis were determined. Cells were cultured in normal (25 raM) or reduced (5 raM) glucose DMEM to evaluate the role of glucose in diamideqnduced apoptosis. NCM 460 cells exhibited 50% more cellular GSH and a 10-fold higher glucose 6-phosphate dehydrogenase (~PD) activity. Diamide at 200, 500 or 750 ~M significantly increase cell apoptosis at 24h in CaCo-2 (15%. 25% & 55%, versus 5%, 10% & 20% in NCM460, respectively). Diamide initiated an early ( i 5 rain) GSH loss with GSSG increase in both cell lines, but NCM460 ceils recovered more rapidly fi'om this imbalance. Basehne NADPH levels were similar in tbe two cell lines, but fell markedly after diamide treatment in CaCo-2 cells, indicating a decreased capacity for NADPH production, consistent with higher GSSG and reduced G6PD activity in these cells. CaCo-2 cells grown in reduced glucose DMEM (5 raM), were more vulnerable to diamide challenge (45% at t.500 M diamide), confirnung the importance ot glucose supply in NADPH generation to sustain GSSG reduction and cell survival These results show that intestinal cells like NCM460, which possess higher redox and antioxidant potentials, are more resistant to diamide-induced apoptosis. This resistance is related to the cell's ability to maintain intracdlular GSH redox balance during diantide stress through enhanced NADPH produetinn by the pentose phosphate pathway-. The results underscore tim rate limiting role of G6PD and glucose availability in the function of the shunt Supported hy NIH grant DK 44510,