Glutamine prevents cytokine-induced apoptosis and oxidation of glutathione redox in HT-29 cells

Abstract

Dietary glutamine (GLN) stimulates proliferation of intestinal cells in animal models of catabolic stress. In cultured intestinal cells, GLN deprivation induces apoptosis while GLN supplementation prevents heat-shock and oxidant-induced cell death. GLN also functions as a precursor to ghitathione (GSH) which detoxifies reactive oxygen species (ROS). We examined the role of GLN in prevention of apoptnsis induced by tumor necrosis factoralpha related apoptnsis-inducing hgand (TRAIL) in human colonic cells. Proliferating HT29 cells were serum-starved, with or without GLN (0, 5, 50 and 500 uM), for 24 h. Cells were then treated with TRAIL (100 ng/ml) for 8 h, with or wlthont GLN. Indices of apoptosis (caspase-3 activity, mitochondrial cytochrome c release, and caspase-8 cleavage), intracelhilar ROS production (dichlorofiuoroscein oxidation) and intracelhilar GSH redox status (GSH, ghitathione disulfide, GSSG, and the GSH/GSSG pool redox potential, Eh) were determined. TRAIL increased the percentage of HT-29 cells undergoing apoptosis 16-fold (from 0.9 to 15.6%), as assessed by % of cells in the sub-G1 peak. However, concurrent treatment with GkN decreased the percentage of apoptotic cells in a dose-dependent manner, such that TRAIL-induced apoptosis was completely prevented with 500 uM GLN. Equimolar doses of alanine (AI..A) had no effect on apoptosis. GkN metabolism was required for the mammal anti-apoptotic response, as 6-diazo-5-norleucine (DON; an inhibitor of glutaminase, the rate-limiting enzyme for GLN metabolism) only slightly decreased apoptosis. GLN, but not ALA or DON, prevented the TRAIL-induced increase in caspase-3 activity, cytochrome c release and caspase-8 cleavage. TRAIL decreased the GSH/GSSG ratio (by 38%) and induced a 9 mV oxidation of GSH/GSSG Eh. In contrast, GLN supplementation at 500 uM completely prevented the oxidative effect, as the GSH/GSSG ratio increased 4-fold with a consequent 40 mV reduction in GSH/GSSG Eh. ALA or DON did not alter the TRAIL-induced oxidation of the GSH/GSSG ratio and Eh. Treatment with TRAIL + GLN + DON prevented the antiapoptotic effects of GLN. In conclusion, GLN supplementation of TRAIL-treated human colonic HT-29 cells prevents both mitochondrial-dependem and receptor-mediated pathways of apoptosis. The anti-apoptotic effects of GLN appear to require metabolism of the amino acid and may be mediated, in part, by reduction of the intracellular GSH redox pool. Supported by NIH DK07298, DK55850, and RR00039.