Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia
P. Sarantos, A. Abouhamze, R. Chakrabarti and W. W. Souba
Department of Surgery, University of Florida College of Medicine, Gainesville.
PURPOSE: Although glutamine is required to maintain gut mucosal metabolism
and function, intestinal glutamine uptake from the gut lumen and from the
bloodstream is decreased during sepsis. We hypothesized that endogenous
mucosal glutamine biosynthesis is increased during endotoxemia, and we
attempted to define the "stress" mediators that regulate the activity of
small intestinal glutamine synthetase (GS), the principal enzyme of de novo
glutamine biosynthesis in the gut. METHODS: Adult rats received Escherichia
coli lipopolysaccharide (LPS) (7.5 mg/kg intraperitoneally), RU 38486 (a
glucocorticoid antagonist; 10 mg/kg by gavage) 2 hours prior to LPS
administration, antibody to tumor necrosis factor (TNF) (4 mg/kg
intraperitoneally) prior to LPS administration, or ketorolac tromethamine
(a prostaglandin synthesis inhibitor; 1 mg/kg intraperitoneally) followed
by LPS administration. Mucosal GS activity was assayed 12 hours after LPS
administration. In a separate set of studies, cultured intestinal mucosal
cells (Caco-2) were exposed to LPS, interleukin 1 (IL-1), IL-6, TNF-alpha,
interferon-gamma, prostaglandin E2, or dexamethasone. Twelve hours later,
GS activity was assayed and messenger RNA was extracted. The GS transcripts
were labeled with a GS complementary DNA probe radiolabeled with phosphorus
32, were quantitated by phosphoimaging, and were normalized to beta-actin.
RESULTS: In vivo LPS treatment increased mucosal GS activity by 250%.
Pretreatment with antibody to TNF or ketorolac did not inhibit the
LPS-induced increase in mucosal GS, whereas pretreatment with RU 38486
attenuated the increase in gut GS activity by 60%. Lipopolysaccharide,
IL-1, IL-6, TNF-alpha, gamma-interferon, and prostaglandin E2 did not
increase GS activity in Caco-2 cells, whereas dexamethasone increased GS
activity and messenger RNA 2.5-fold and threefold, respectively. These data
indicate that cytokines and prostaglandins (prostaglandin E2) do not
regulate mucosal GS expression during endotoxemia. Glucocorticoids,
however, stimulate GS gene expression directly. CONCLUSIONS: This
hormonally mediated response may support de novo mucosal GS during septic
states when uptake of glutamine from the lumen and blood is decreased.