Glutamine protects intestinal epithelial cells: role of inducible HSP70.

Abstract

In this in vitro study, the authors test the premise that glutamine (Gln) protects intestinal epithelial cells under conditions of stress via induction of heat shock proteins. Gln (0–20 mM) or its non‐metabolizable analogue, 6‐diazo‐5‐oxo‐L‐norleucine (DON, 0400 μM), was added for 2 hours to IEC‐18 cells that had previously been grown for two days to near confluence in Gln‐free and serum‐free culture media (Dulbecco's modified Eagle's medium). Induction of heat shock protein 70 (HSP70) under these conditions was assessed by Western blots using the monoclonal antibody C92, which recognizes only the inducible form of HSP70. HSP70 mRNA abundance was assessed with Northern blot analysis using a cDNA probe specific for inducible HSP70. Radiolabeled cells (51Cr) treated with Gln or DON were subsequently exposed to heat (49°C for 30–240 minutes) or an oxidant, monochloramine (NH2Cl, 2 μM), and cell injury was quantitated by calculating the percent of 51Cr release. Finally, Gln or DON was added to cells cultured in quercetin, a known inhibitor of HSP70 production. The cells were then analyzed for HSP70 induction, HSP70 mRNA abundance, and cellular protection from heat and oxidation.Gln added to IEC‐18 cells induced an increase in HSP70 and HSP70 mRNA in a concentration‐dependent manner. Gln administration caused a statistically significant 50% and 65% reduction in cytolysis of cells exposed to lethal heat and oxidation, respectively. The non‐metabolizable analogue of Gln, DON, also induced an increase in HSP70 and HSP mRNA but less than that induced by Gln. DON caused a statistically significant 35% and 25% reduction in cytolysis of cells exposed to lethal heat and oxidation. This suggests that the observed effects of Gln are mediated by both metabolic and non‐metabolic mechanisms. Incubation of cells with quercetin prior to Gln and DON administration not only blocked HSP70 protein and mRNA induction but abrogated the protective effects of Gln and DON on cells exposed to lethal heat and oxidation. The authors conclude that Gln protects intestinal epithelial cells from heat and oxidation via mechanisms which are in part mediated by HSP70 induction.