Sarantos P, Chakrabarti R, Copeland E M, Souba W W
Department of Surgery, University of Florida College of Medicine, Gainesville.
Am J Surg. 1994 Jan;167(1):8-13. doi: 10.1016/0002-9610(94)90047-7.
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.
谷氨酰胺为肠道黏膜的核苷酸生物合成提供能量和前体物质，对肠道代谢和功能至关重要。在应激状态下，循环和肠腔谷氨酰胺的摄取可能会减少，但肠道黏膜对谷氨酰胺从头合成以应对供应减少的能力仍不明确。由于糖皮质激素在分解代谢状态下调节器官间谷氨酰胺代谢中起重要作用，我们推测这些激素会诱导肠道黏膜谷氨酰胺合成酶（GS）的表达，该酶催化谷氨酰胺的细胞内生物合成。成年大鼠接受地塞米松（DEX，0.5mg/kg腹腔注射）或生理盐水（对照组）处理。在处理后的不同时间点（4、12、24、48和72小时），测定空肠黏膜GS的特异性活性，并提取总RNA。使用放射性标记的大鼠GS cDNA探针通过Northern印迹分析检测GS转录本。通过磷成像对转录本进行定量，并以β-肌动蛋白进行标准化。使用抗GS多克隆抗体通过Western印迹分析定量GS蛋白浓度。使用无细胞蛋白质合成系统（网织红细胞裂解物测定）测量GS翻译的相对量。数据采用方差分析进行分析，p<0.05被认为具有统计学意义。给药12小时后，DEX使GS活性增加了45%。Western印迹分析显示，DEX处理动物的空肠中GS蛋白浓度增加。Northern印迹分析表明，DEX处理后GS mRNA水平无显著变化，表明存在转录后调控的可能性。体外翻译实验表明，给药DEX后，GS的翻译量增加了25%。这些数据表明，糖皮质激素可能通过加速蛋白质翻译来增加空肠黏膜GS水平。这种适应性反应可以在应激期间为肠道黏膜提供谷氨酰胺，此时外源性谷氨酰胺供应可能是限速的。
