Endotoxin stimulates lymphocyte glutaminase expression
P. Sarantos, K. Ockert and W. W. Souba
Department of Surgery, University of Florida College of Medicine, Gainesville.
BACKGROUND/HYPOTHESIS: Glutamine is the principal fuel used by lymphocytes.
It is hydrolyzed by the glutaminase enzyme, which regulates the rate of
intracellular glutamine metabolism. Since lymphocyte glutamine utilization
is increased during infection to support cellular proliferation, we
hypothesized that endotoxin regulates lymphocyte glutaminase expression at
the molecular level. METHODS: Adult rats received Escherichia coli
endotoxin (one dose of 7.5 mg/kg) or saline. Total RNA from lymphocytes in
the ileocolic lymph node chain was extracted for Northern hybridization and
labeled with an alpha-phosphorus 32 rat glutaminase cDNA probe. The mRNA of
the constitutively expressed gene beta-actin was the control for RNA
loading. Quantitation of glutaminase transcripts was determined by
densitometric scanning and values were normalized to actin.
Glutaminase-specific activity (nanomoles per milligram of protein per hour)
and glutaminase kinetic parameters were also determined. RESULTS: Treatment
with a single dose of endotoxin resulted in a 53% increase in glutaminase
activity at 4 hours. Kinetic analysis showed that the increase in
glutaminase activity was due to an 84% increase in Vmax (maximal enzyme
velocity) with no change in Km (enzyme affinity). Endotoxin increased
glutaminase mRNA twofold at 2 hours and more than fourfold at 4 hours. The
increase in message preceded the increase in activity consistent with gene
transcription prior to enzyme biosynthesis. CONCLUSION/CLINICAL RELEVANCE:
The increase in glutaminase activity provides lymphocytes in the mesenteric
lymph nodes with more glutamine for energy and cellular proliferation
during times of infection when the gut mucosal barrier may become
compromised.
