The in vitro manipulation of carbohydrate metabolism: a new strategy for deciphering the cellular defence mechanisms against nitric oxide attack

This study was aimed at examining the effects of manipulating the carbohydr ate source of the culture medium on the cellular sensitivity of epithelial cells to an oxidative attack. Our rationale was that substituting galactose for glucose in culture media would remove the protection afforded by gluco se utilization in two major metabolic pathways, i.e. anaerobic glycolysis a nd/or the pentose phosphate pathway (PPP), which builds up cellular reducin g power. Indeed, we show that the polarized human colonic epithelial cell l ine HT29-C1.16E was sensitive to the deleterious effects of the NO donor PA PANONOate [3-(2-hydroxy-2-nitroso-1-propylhydrazino)-1-propanamine] only in galactose-containing medium. In such medium NO attack led to cytotoxic and apoptotic cell death, associated with formation of derivatives of NO auto- oxidation (collectively termed NOx) and peroxynitrite, leading to intracell ular GSH depletion and nitrotyrosine formation. The addition of 2-deoxygluc ose, a non-glycolytic substrate, to galactose-fed cells protected HT29-C1.1 6E cells from NO attack and maintained control GSH levels through its metab olic utilization in the PPP, as shown by (CO2)-C-14 production from 2-deoxy [1-C-14]glucose. Therefore, increasing the availability of reducing equival ents without interfering with energy metabolism is able to prevent NO-induc ed cell injury. Finally, this background provides the conceptual framework for establishing nutritional manipulation of cellular metabolic pathways th at could provide new means for (i) deciphering the mechanisms of cell injur y by reactive nitrogen species and reactive oxygen species at the whole-cel l level and (ii) establishing the hierarchy of intracellular defence mechan isms against these attacks.