Glutamine is essential for epidermal growth factor-stimulated intestinal cell proliferation.

Abstract

In this study, Ko et al determined whether L‐glutamine (GLN) is essential for epidermal growth factor (EGF)‐stimulated enterocyte proliferation in an in vitro cell culture system by using the nontransformed rat small intestinal crypt cell line IEC‐6. In addition, specific mitogenic actions of EGF that require GLN were investigated.IEC‐6 cells were maintained in monolayer culture by incubation in standard culture medium (Dulbecco's minimum essential medium) containing 5% bovine serum, grown to quiescence, and then incubated in serum‐free Dulbecco's minimum essential medium containing 0.1 to 0.2 mmol/L GLN for 36 hours (as in almost all other cell culture systems, GLN is required to prevent IEC‐6 cell death in serum‐free medium). Cells were then stimulated with a previously determined maximal trophic dose of EGF (20 ng/mL) plus varying concentrations of L‐GLN (0 to 10 mmol/L) added to the culture medium. Under these conditions, DNA, RNA, and protein synthesis were quantitated over time (0 to 30 hours) by using tritiated thymidine, tritiated uridine, and 14C‐leucine, respectively. Cell number was determined after 72 hours of treatment with varying amounts of added GLN with or without EGF. Total RNA was isolated from the EGF/GLN‐treated cells in other experiments for determination of messenger RNA (mRNA) levels of the “early response” proto‐oncogene transcription factors zif268, jun‐B, and c‐myc by Northern blotting.GLN was required for EGF‐stimulated DNA synthesis in IEC‐6 cells. Thus, EGF (20 ng/mL) added to quiescent cells without additional GLN (ie, not more than the 0.1 mmol/L required to prevent cell death) did not stimulate DNA synthesis at any time from 0 to 30 hours of incubation. However, addition of a larger amount of GLN (1.0 mmol/L) to EGF significantly stimulated DNA synthesis beginning 9 hours after incubation, and this effect on DNA synthesis steadily increased over time. The increased DNA synthesis in cells treated with EGF was related to L‐GLN dose; stimulation occurred only after 0.3 mmol/L GLN. In contrast, DNA synthesis was not stimulated significantly by addition of increasing amounts of GLN alone to the medium and was maximal at 1 to 3 mmol/L GLN. The requirement for GLN under these conditions was amino acid specific and stereospecific, inasmuch as D‐GLN, L‐arginine, and L‐alanine were not effective in enhancing EGF‐stimulated DNA synthesis.The addition of 1 mmol/L GLN to EGF also significantly increased RNA and protein synthesis beginning 3 hours after incubation. Without the addition of L‐GLN to standard media, EGF‐induced protein synthesis was reduced 80% and RNA synthesis 70%. Cell growth studies revealed that GLN added at a dose of either 1.0 or 10 mmol/L significantly increased (~fourfold) cell number compared with control cells (without EGF in standard medium containing 0.1 mmol/L GLN) and cells treated with EGF alone in standard medium.Northern analysis for mRNA levels of the proto‐oncogene transcription factors zif268, jun‐B, and c‐myc showed significantly increased mRNA expression of all three genes after 30 minutes of exposure to EGF; however, these responses were similar with or without supplemental L‐GLN. Interestingly, mRNA levels for lf268, jun‐B, and c‐myc remained persistently elevated at 2 hours after treatment with EGF alone, but levels decreased to a variable degree with EGF plus GLN at this time point.