Daniel Strauss

Glutamine Synthetase: A Key Enzyme for Intestinal Epithelial Differentiation?

BACKGROUND We have previously shown that glutamine synthetase protein and mRNA are concentrated in the crypt region of the rat small intestine and that the activity of this enzyme is highest around the time of weaning. This anatomical location and time of peak activity are sites and periods of active enterocyte differentiation. This led to our current hypothesis that glutamine synthetase is important in the differentiation of enterocytes. METHODS To test our hypothesis, we treated Caco-2 cells with physiologic (0.6 mM) glutamine concentrations in cell culture medium. The experimental group was treated with methionine sulfoximine, an irreversible glutamine synthetase inhibitor, and the control group with phosphate buffered saline. Three standard and well-defined markers of intestinal differentiation-sucrase-isomaltase activity, microvillus formation, and electrical impedance in transwell plates-were compared between the two groups. RESULTS The methionine-sulfoximine-inhibited group was found to have lower sucrase-isomaltase activity, a lower density of microvilli, and lower electrical impedance values over time compared with the control group. CONCLUSION The experimental group was found to be less differentiated by all three markers of differentiation. Therefore, glutamine synthetase is important for Caco-2 cell differentiation.

Characterization of glutamine synthetase transcript, protein, and enzyme activity in the human placenta

This study characterizes the molecular mechanisms necessary for glutamine synthesis in the human placenta. RNA hybridization and protein immunoblotting were used to verify the presence of glutamine synthetase (GS) transcripts and protein, respectively. Additionally, the presence of GS was determined by immunohistochemistry. RNA hybridization demonstrated the presence of 1.8- and 2.8-kB transcripts and protein immunoblotting yielded a single 49-kDa band, characteristics of GS transcripts and protein, respectively. The mean (+/- s.d.) specific activity of placental GS, expressed as mumol gamma-glutamyl hydroxamic acid/mg protein/h was 1.80 +/- 0.59, which is comparable to other organs which are net glutamine producers. Immunohistochemical analysis indicated the presence of GS within the cytotrophoblast and mesenchyme layers of placental villi, but not in the syncytiotrophoblast. Although these results suggest that the human placenta is capable of synthesizing glutamine, the fate of glutamine produced by this organ remains speculative.

GLUTAMINE SYNTHETASE AND GLUTAMINASE IN THE HUMAN PLACENTA: CLUES TO A GLUTAMINE-GLUTAMATE SHUTTLE? • 1830

Based on studies of glutamine flux in the ovine placenta, we hypothesize that the human placenta is capable of substantial glutamine synthesis via glutamine synthetase (GS) and has little capacity to deaminate glutamine via glutaminase (GA). GS and GA activities and GS transcript were examined on freshly obtained homogenates from term human placenta and compared to values obtained from other organs known to be primarily producers or consumers of glutamine. The activity of GS and transcript size (1.8 and 2.8 kB species) in human placenta is comparable to that observed in adult rat lung, an organ known to be an important glutamine producer involved in glutamine homeostasis. GS activity in the placenta is higher than that in adult rat small intestine which is a major glutamine consumer rather than a glutamine producer. Placental GA shows low activity (0.21 ± 0.29 μmoles • mg protein-1 indicating that the human placenta converts very little glutamine to glutamate. Low activity of GA suggests that the placenta has little need to convert glutamine to glutamate. The human placenta may, like the ovine placenta, derive sufficient glutamate from the fetus for incorporation into the citric acid cycle. These results: 1) suggest that the human placenta, like the rat lung, is a glutamine producing organ; and 2) are consistent with the presence of an ovine-like glutamine-glutamate shuttle between the human placenta and fetal liver. Supported by NIH Grant#RO1-HD29279. Table

Intestinal Glutamine Synthetase (Gs) and Dna Synthesis are Concurrently Upregulated By Dexamethasone (Dex) in Vitro † 571

Intestinal Glutamine Synthetase (Gs) and Dna Synthesis are Concurrently Upregulated By Dexamethasone (Dex) in Vitro † 571

GLUTAMINE DEPRIVATION IN RAT INTESTINAL CELLS. 476

Our objective is to establish the importance of both endogenous and exogenous sources of glutamine (GLN) for proliferation and differentiation of small intestinal epithelium. We hypothesize that exposure to sub-physiologic concentrations of GLN (<0.4 mM) in cell culture media (exogenous GLN source) results in upregulation of glutamine synthetase (GS) activity(endogenous GLN source). Increased GS activity should compensate for the lack of exogenous GLN and result in no differences in variables indicative of cell growth. IEC-6 cells were grown in GLN-free DMEM containing 10% FBS. Confluent stock cultures were trypsinized and seeded (1 × 105 cells/well) onto plates grown in media containing 1 of 6 different concentrations of supplemental GLN (4, 2, 0.4, 0.2, 0.02, and 0 mM). The approximate percentage of area covered by cells, an indicator of cell growth, was monitored each day and time to confluency was noted. Cells were trypsinized and processed for determination of protein concentration, GS activity (μ M•mg-1protein•h-1), and 3H-thymidine incorporation (dp m•mg protein-1). Results (means± SD, *p<0.05): All variables decreased in response to decreased [GLN] in a dose-dependent manner. Table

GLUTAMINE DEPRIVATION DURING PREGNANCY: COMPENSATORY MECHANISMS AND ADVERSE EFFECTS. † 1368

GLUTAMINE DEPRIVATION DURING PREGNANCY: COMPENSATORY MECHANISMS AND ADVERSE EFFECTS. † 1368

DEPRIVATION OF EXOGENOUS GLUTAMINE AND GLUTAMINE SYNTHETASE INHIBITION ALTER GROWTH AND MORPHOLOGY OF HT-29 CELLS. † 477

DEPRIVATION OF EXOGENOUS GLUTAMINE AND GLUTAMINE SYNTHETASE INHIBITION ALTER GROWTH AND MORPHOLOGY OF HT-29 CELLS. † 477

GLUTAMINE SYNTHETASE EXPRESSION AND IMMUNOLOCALIZATION IN THE DEVELOPING RAT LUNG. |[dagger]| 1831

Although the lung is known to be a major producer of glutamine and contributes to glutamine homeostasis, little is known about glutamine during development. We hypothesize that glutamine synthetase (GS) undergoes changes in activity and steady state mRNA from infancy to adulthood. GS activity and RNA were measured in the lungs of rat term fetuses, 5, 10, and 19-day-old infants, and adults following treatment with saline (SAL) or 0.2 mg/kg dexamethasone (DEX). Significant interactions between treatment and age effects were noted for both mRNA and specific activity indicating that the degree of difference between SAL and DEX treatment groups depended on age (2 way ANOVA). Pairwise comparisons indicate that mRNA quantity and specific activity were greater in the 5-day DEX group. Within the SAL treatment set, mRNA levels did not differ among age groups; however, specific activity of the 19-day group was greater than fetal, 5-day and 10-day specific activities. Within the DEX treatment set, the mean mRNA level of the 5-day group was greater than in the other age groups; however, DEX treatment did not alter the specific activities among age groups. Immunohistochemical analysis demonstrates that GS enzyme is located primarily in the smooth muscle layer of the pulmonary vasculature. These data demonstrate that: 1) GS responds to exogenous glucocorticoids prior to the endogenous glucocorticoid surge; and 2) the effects of glucocorticoid treatment are blunted when endogenous glucocorticoids are elevated. Our data suggest that: 1) glucocorticoid receptors are unavailable to exogenous glucocorticoids when endogenous glucocorticoids are elevated; and 2) localization of GS in the pulmonary smooth muscle layer permits rapid release of glutamine into the circulation during periods of high glutamine demand. Supported by NIH Grant#RO1-HD29279.