Biomedical subjects

P Sarantos

Publications and source records attributed to P Sarantos.

7 recordsLinked to original sources

PubMed1994

Glucocorticoids regulate intestinal glutamine synthetase gene expression in endotoxemia.

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.

Animals↗

PubMed1994

Glucocorticoids regulate glutaminase gene expression in human intestinal epithelial cells.

Glutamine is essential for intestinal metabolism and function, but its circulating and luminal availability to the mucosa may be diminished during critical illness. We hypothesized that glucocorticoids, which are produced in increased amounts during critical illness, accelerate mucosal glutamine metabolism. We studied intestinal glutamine utilization by examining the regulation of glutaminase in vitro, the enterocyte's principal enzyme of glutamine metabolism. Differentiated confluent human enterocytic cells (Caco-2 cells) were incubated with dexamethasone. Glutaminase activity was assayed and mRNA was extracted. Glutaminase transcripts were labeled with a 32P-labeled glutaminase cDNA probe, quantitated by phosphoimaging, and normalized to beta-actin. Dose- and time-response studies were performed. Dexamethasone-treated cells were also incubated with actinomycin D and cycloheximide. Dexamethasone (DEX) increased mucosal glutaminase activity by 45%, with maximal response at 12 hr. This increase was dose-dependent and was significant at doses of 1 and 10 microM. The dexamethasone-mediated increase in glutaminase activity was associated with a 40% increase in glutaminase mRNA. The DEX-induced increase in glutaminase activity was inhibited by actinomycin D and cycloheximide, indicating the requirement for de novo RNA and protein synthesis. Glucocorticoids stimulate glutamine metabolism in these human enterocytic cells by increasing the activity of glutaminase, a response that is preceded by an increase in gene transcription. This glucocorticoid-mediated increase in glutaminase activity may be a mechanism by which gut glutamine metabolism is maintained during critical illness when blood glutamine levels are diminished and food intake is often interrupted.

Autoradiography↗

PubMed1994

Decrease of glutaminase expression by interferon-gamma in human intestinal epithelial cells.

BACKGROUND: Glutaminase, the principal enzyme of glutamine hydrolysis, breaks down glutamine to supply energy and intermediates for cell growth and is present in high concentrations in replicating tissues such as intestinal epithelium and malignant tumors. In the host with cancer, glutaminase activity in the gut mucosa diminishes as the tumor grows, but the regulation of this response is unknown. Because cytokines may regulate the altered glutamine metabolism that is characteristic of the host with cancer, we studied the effects of cytokines on gut mucosal glutaminase expression in vitro using the human enterocytic Caco-2 cell line. METHODS: Differentiated confluent cells were incubated with interleukin (IL)-1, IL-6, tumor necrosis factor, or interferon-gamma (IFN-gamma). After a 12-h incubation, glutaminase-specific activity and kinetic parameters (maximal enzyme activity [Vmax] and enzyme affinity [Km]) were determined. Glutaminase protein concentration was determined by Western blot analysis using a rabbit antirat polyclonal antibody. Total cellular RNA was extracted for Northern hybridization and radiolabeled with a glutaminase cDNA probe. RESULTS: Of the cytokines studied, only IFN-gamma altered glutaminase activity. Kinetic studies indicated a decrease in activity secondary to a 25% decrease in Vmax with no change in Km, consistent with a reduction in the number of glutaminase molecules rather than a change in enzyme affinity. Glutaminase protein was decreased 50% in IFN-gamma-treated cells when compared with controls. This decrease was dose-independent and was associated with a concomitant 75% decrease in glutaminase messenger RNA levels. These reductions in message and protein translated into a 60-80% decrease in functional glutaminase-specific activity. CONCLUSIONS: This IFN-gamma-mediated decrease in glutaminase activity may be one mechanism by which gut glutamine metabolism is diminished as the tumor grows and becomes the principal organ of glutamine use.

Adenocarcinoma↗

PubMed1994

Dexamethasone increases jejunal glutamine synthetase expression via translational regulation.

Glutamine provides energy and precursors for nucleotide biosynthesis for the gut mucosa, and it is essential for intestinal metabolism and function. During stress states, glutamine uptake of circulating and luminal glutamine may be diminished, but the ability of the gut mucosa to synthesize glutamine de novo in response to this decreased delivery remains undefined. Since the glucocorticoids play an important role in regulating interorgan glutamine metabolism during catabolic states, we hypothesized that these hormones induce the expression of gut mucosal glutamine synthetase (GS), the enzyme that catalyzes the intracellular biosynthesis of glutamine. Adult rats were treated with dexamethasone (DEX, 0.5 mg/kg intraperitoneally) or saline (controls). At various times after treatment (4, 12, 24, 48, and 72 hours), jejunal mucosal GS-specific activity was assayed, and total RNA was extracted. GS transcripts were detected by Northern blot analysis, using a radiolabeled rat GS cDNA probe. Transcripts were quantitated by phospho-imaging and normalized to beta-actin. An anti-GS polyclonal antibody was used to quantitate GS protein concentrations by Western blot analysis. The relative quantities of GS translated were measured using a cell-free protein-synthesizing system (reticulocyte lysate assay). Data were analyzed using analysis of variance and were considered statistically significant for p < 0.05. DEX increased GS activity by 45% 12 hours after administration. Western blot analysis revealed an increase in the concentration of the GS protein in the jejunum of DEX-treated animals. Northern blot analysis demonstrated no significant change in GS mRNA levels after DEX treatment, indicating the possibility of post-transcriptional regulation. In vitro translational experiments demonstrated that the quantity of GS translated was increased by 25% after the administration of DEX. These data suggest that glucocorticoids may increase jejunal mucosal GS levels by accelerating protein translation. This adaptive response could provide glutamine for the gut mucosa during stress, when exogenous glutamine supplies may be rate limiting.

Animals↗

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↗

PubMed1993

Dexamethasone regulates glutamine synthetase expression in rat lung.

Since the lungs play a central role in maintaining glutamine homeostasis in normal and catabolic disease states, we studied the regulation of glutamine synthetase (GS) expression by dexamethasone in rat lung. Adult rats received saline (controls) or dexamethasone (0.5 mg/kg). Lung total RNA was extracted for Northern hybridization and labeled with an alpha-32P rat GS cDNA probe. The mRNA of the constitutively expressed gene beta-actin was the control for RNA loading. GS transcripts were measured by laser densitometry and normalized to actin, and GS specific activity was also determined. Following a single injection of dexamethasone (0.5 mg/kg), lung GS activity increased by 40% at 4 hours and by 75% at 8 hours. The dexamethasone-mediated increase in GS activity was associated with a marked increase in GS mRNA levels, which preceded the increase in enzyme activity by approximately 2 hours. Serial daily dexamethasone administration for 3 and 6 days caused an even greater increase in GS mRNA levels and specific activity. No effect was seen on beta-actin levels, demonstrating that the expression of GS was not part of a global response to steroids. Therefore, glucocorticoids stimulate GS expression in rat lung. This regulation appears to be one mechanism by which lung glutamine release is augmented during critical illness.

Analysis of Variance↗

PubMed1992

Glucocorticoids regulate intestinal glutaminase expression.

BACKGROUND: The metabolism of glutamine by the small intestinal mucosal cells is highly dependent on the glutaminase enzyme. Because mucosal glutamine utilization is increased after operation, we hypothesized that the elevated glucocorticoid hormones that occur after surgical stress regulate expression of mucosal glutaminase at the molecular level. METHODS: Adult rats received saline solution or dexamethasone (0.5 mg/kg, one dose) and were sacrificed at various times after treatment. Jejunal mucosal total RNA was extracted for Northern hybridization with an alpha-32P-labeled rat glutaminase cDNA. 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 normalized to actin. Glutaminase activity (micromoles per milligram of protein per hour) and its kinetic parameters, maximal transport velocity (micromoles per milligram of protein per hour) and Michaelis-Menten constant (micromoles per liter), were also determined. RESULTS: Dexamethasone increased glutaminase mRNA (twofold at 4 hours, sixfold at 12 hours; p less than 0.01) and glutaminase-specific activity. The increase in message preceded the increase in activity by 4 hours, consistent with de novo RNA synthesis followed by protein synthesis. The increase in glutaminase activity was the result of a 21% increase in the maximal enzyme capacity (maximal transport velocity = 8.6 +/- 0.5 mumol/mg protein/hr in control rats vs 10.4 +/- 0.3 mumol/mg protein/hr in rats treated with dexamethasone; p less than 0.01) rather than a change in enzyme affinity (Michaelis-Menten constant). CONCLUSION: Glucocorticoids may accelerate intestinal glutamine utilization by increasing glutaminase expression, an adaptive response that could provide more energy for mucosal cells in stress states.

Animals↗