Wang J Y, Viar M J, Blanner P M, Johnson L R
Department of Surgery, University of Maryland Medical School, Baltimore, USA.
Am J Physiol. 1996 Jul;271(1 Pt 1):G164-71. doi: 10.1152/ajpgi.1996.271.1.G164.
Refeeding fasted rats significantly stimulates mucosal growth and ornithine decarboxylase (ODC), the rate-limiting enzyme in the biosynthesis of polyamines, but the exact mechanism responsible for induction of ODC at the molecular level is unknown. Of normal dietary constituents, the amino acid asparagine markedly increases ODC activity and mucosal growth when administered intragastrically. The current study examined the expression of the ODC gene in IEC-6 cells (a line of normal rat small intestinal crypt cells) after exposure to asparagine. Cells were grown in Dulbecco's minimal essential medium containing 5% dialyzed fetal bovine serum. They were deprived of serum for 24 h before experiments. Exposure to asparagine at the dose of 10 mM resulted in the rapid increase in ODC mRNA levels. The increased expression of the ODC gene began 1 h after and peaked between 3 and 5 h after treatment with asparagine. Maximum increases in ODC mRNA levels were approximately fivefold the normal value. Increased levels of ODC mRNA in cells exposed to asparagine were paralleled by increases in ODC protein and enzyme activity and cellular polyamine levels. The half-life of mRNA for ODC in unstimulated IEC-6 cells was approximately 30 min and increased to > 2 h in cells exposed to 10 mM asparagine. The half-life of ODC activity also was increased in asparagine-treated cells. When cellular protein synthesis was inhibited by cycloheximide, asparagine superinduced ODC mRNA levels. Furthermore, asparagine also significantly stimulated DNA synthesis in IEC-6 cells. These results indicate that 1) asparagine stimulates ODC in IEC-6 cells through multiple pathways and 2) increased ODC mRNA levels result partly from a delay in the rate of degradation. These findings suggest that luminal amino acids stimulate gut mucosal growth in association with their ability to regulate ODC gene expression.
对禁食大鼠重新喂食可显著刺激黏膜生长以及鸟氨酸脱羧酶（ODC，多胺生物合成中的限速酶），但在分子水平上负责诱导ODC的具体机制尚不清楚。在正常饮食成分中，氨基酸天冬酰胺经胃内给药时可显著增加ODC活性和黏膜生长。本研究检测了天冬酰胺作用于IEC-6细胞（一种正常大鼠小肠隐窝细胞系）后ODC基因的表达。细胞在含有5%透析胎牛血清的杜尔贝科改良 Eagle 培养基中培养。实验前将它们血清饥饿24小时。以10 mM的剂量暴露于天冬酰胺会导致ODC mRNA水平迅速升高。ODC基因表达的增加在天冬酰胺处理后1小时开始，并在处理后3至5小时达到峰值。ODC mRNA水平的最大增加约为正常值的五倍。暴露于天冬酰胺的细胞中ODC mRNA水平的增加与ODC蛋白、酶活性和细胞多胺水平的增加平行。未刺激的IEC-6细胞中ODC mRNA的半衰期约为30分钟，而在暴露于10 mM天冬酰胺的细胞中增加到>2小时。天冬酰胺处理的细胞中ODC活性的半衰期也增加。当用环己酰亚胺抑制细胞蛋白质合成时，天冬酰胺超诱导ODC mRNA水平。此外，天冬酰胺还显著刺激IEC-6细胞中的DNA合成。这些结果表明：1）天冬酰胺通过多种途径刺激IEC-6细胞中的ODC；2）ODC mRNA水平的增加部分是由于降解速率延迟所致。这些发现表明，肠腔氨基酸与其调节ODC基因表达的能力相关联，从而刺激肠道黏膜生长。
