S Abcouwer | Search PubMed

PubMed1995

Attenuation of the endotoxin-stimulated increase in hepatic amino acid transport with a glucocorticoid receptor antagonist.

The role of the glucocorticoid hormones in mediating the accelerated hepatic amino acid transport that is characteristic of endotoxemia was investigated. To determine the role of these steroid hormones, rats that received endotoxin (LPS) were pretreated with the glucocorticoid receptor antagonist RU38486. The activities of the Na(+)-dependent amino acid transport systems A, ASC, and N and the Na(+)-independent systems L, y+, n, b0,+, and asc in hepatic plasma membrane vesicles were measured 4 hr after exposure to LPS. Endotoxin treatment resulted in time- and dose-dependent 5-fold (System A), 2.5-fold (System N), 2.6-fold (System ASC), and 2-fold (System y+ and b0,+) increases in transport activity attributable to an increase in carrier Vmax. The activities of L, asc, and n were unchanged by LPS administration. Pretreatment of endotoxemic animals with RU38486 attenuated the LPS-induced enhancement in transport activity by 20-60% by diminishing carrier Vmax, with no effect on transport Km. We conclude that the marked increase in hepatic amino acid transport activity that occurs during endotoxemia requires participation of the glucocorticoid hormones.

Amino Acids↗

PubMed1994

Cytokines decrease glutaminase expression in human fibroblasts.

BACKGROUND: Glutamine metabolism in fibroblasts is essential for energy production, nucleotide biosynthesis, and growth during wound healing. Because cytokines can impair fibroblast proliferation, we tested the hypothesis that cytokines impair glutamine metabolism. We studied the influence of several cytokines on the expression of glutaminase, the major enzyme of intracellular glutamine metabolism in fibroblasts. METHODS: Human foreskin fibroblasts were incubated for 6 and 12 hours with varying doses (10, 100, or 1000 units/ml) of interleukin (IL)-1, IL-6, tumor necrosis factor-alpha, or gamma-interferon. Cell lysates were assayed for glutaminase-specific activity, and glutaminase protein content was measured by Western blotting with a polyclonal antibody. Total cellular RNA was extracted, and relative glutaminase messenger RNA levels were determined by Northern blotting with a 32P-labeled glutaminase complement DNA-derived probe. These mRNA levels were normalized by blotting with a beta-actin cDNA-derived probe as control. Cell nuclei were isolated, and nuclear run-ons were used to determine relative glutaminase mRNA transcription rates. RESULTS: IL-1, IL-6, tumor necrosis factor-alpha, and gamma-interferon decreased glutaminase activity and protein concentration after a 12-hour incubation in a dose-independent fashion. No difference was noted at 6 hours. Western blot analysis showed a 30% to 60% reduction in glutaminase protein in treated cells. These cytokines also decreased glutaminase mRNA levels, consistent with transcriptional regulation. This was confirmed by nuclear run-on assays that showed a decrease in the number of glutaminase transcripts. CONCLUSIONS: A variety of different pro-inflammatory cytokines decrease glutaminase expression in cultured human fibroblasts. This cytokine-mediated inhibition of glutamine metabolism may limit the availability of key glutamine-derived intermediates and impair fibroblast proliferation in certain patients.

Cells, Cultured↗