Effects of glutamine deprivation on protein synthesis in a model of human enterocytes in culture

To assess the effect of glutamine availability on rates of protein synthesi s in human enterocytes, Caco-2 cells were grown until differentiation and t hen submitted to glutamine deprivation produced by exposure to glutamine-fr ee medium or methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methyl sulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incub ated with H-2(3)-labeled leucine with or without glutamine, and the fractio nal synthesis rate (FSR) of total cell protein was determined from H-2(3)-l abeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 +/- 1.5%/day ) and intracellular glutamine concentration (6.1 +/- 0.6 mmol/g protein) re mained unaltered when cells were grown in glutamine-free medium. In contras t, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 +/- 0.6 vs. 20.2 +/- 0.8%/day, P < 0.01). Supplementation with 0.5-2 mM gl utamine for 4 h after MSO incubation, but not with glycine nor glutamate, r estored protein FSR to control values (24 +/- 1%/day). These results demons trate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active , since it can maintain intracellular glutamine pool during glutamine depri vation, 2) inhibition of glutamine synthesis is associated with reduced pro tein synthesis, and 3) when glutamine synthesis is depressed, exogenous glu tamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevanc e of these findings for the human intestine in vivo remains to be determine d.