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.