L-Arginine stimulates proliferation and prevents endotoxin-induced death of intestinal cells

Abstract

This study tested the hypothesis that L-arginine (Arg) may stimulate cell proliferation and prevent lipopolysaccharide (LPS)-induced death of intestinal cells. Intestinal porcine epithelial cells (IPEC-1) were cultured for 4 days in Arg-free Dulbecco's modified Eagle's-F12 Ham medium (DMEM-F12) containing 10, 100 or 350 microM Arg and 0 or 20 ng/ml LPS. Cell numbers, protein concentrations, protein synthesis and degradation, as well as mammalian target of rapamycin (mTOR) and Toll-like receptor 4 (TLR4) signaling pathways were determined. Without LPS, IPEC-1 cells exhibited time- and Arg-dependent growth curves. LPS treatment increased cell death and reduced protein concentrations in IPEC-1 cells. Addition of 100 and 350 microM Arg to culture medium dose-dependently attenuated LPS-induced cell death and reduction of protein concentrations, in comparison with the basal medium containing 10 microM Arg. Furthermore, supplementation of 100 and 350 microM Arg increased protein synthesis and reduced protein degradation in both control and LPS-treated IPEC-1 cells. Consistent with the data on cell growth and protein turnover, addition of 100 or 350 microM Arg to culture medium increased relative protein levels for phosphorylated mTOR and phosphorylated ribosomal protein S6 kinase-1, while reducing the relative levels of TLR4 and phosphorylated levels of nuclear factor-kappaB in LPS-treated IPEC-1 cells. These results demonstrate a protective effect of Arg against LPS-induced enterocyte damage through mechanisms involving mTOR and TLR4 signaling pathways, as well as intracellular protein turnover.

Fig. 1

The number of IPEC-1 cells cultured in Arg-free DMEM containing 10, 100, 350 or 500 μM arginine on day 0, 2, 4 and 6. Data are expressed as means ± SEM, n = 8

Fig. 2

The cell numbers (A) and protein concentrations (B) of normal and LPS (20 ng/ml)-treated IPEC-1 cells. Cells were cultured for 96 h in Arg-free DMEM containing 10, 100 or 350 μM arginine and 0 or 20 ng/ml LPS. Data are expressed as means ± SEM, n = 8. a–e Means sharing different letters differ (P < 0.05)

Fig. 3

Protein synthesis (A nmol Phe/mg protein/3 h) and protein degradation (B; %) in IPEC-1 cells treated with or without 20 ng/ml LPS. Cells were cultured for 96 h in Arg-free DMEM containing 10, 100, or 350 μM arginine and 0 or 20 ng/ml LPS. Data are expressed as means ± SEM, n = 8. a–d Means sharing different letters differ (P < 0.001)

Fig. 4

Relative protein levels for total mTOR (A), phosphorylated mTOR (B), 4EBP1 (C), phosphorylated 4EBP1 (D), S6K1 (E) and phosphorylated S6K1 (F) in IPEC-1 cells. Cells were cultured for 96 h in Arg-free DMEM containing 10, 100 or 350 μM arginine and 20 ng/ml LPS. Data are expressed as means ± SEM, n = 4. a, b Means sharing different letters differ (P < 0.01). Levels of phosphorylated 4EBP1 were higher (P < 0.05) in cells treated with 100 and 350 μM arginine, compared with the control (10 μM arginine)

Fig. 5

Relative protein levels for TLR4 (A), NFκB (B) and phosphorylated NFκB (C) in IPEC-1 cells. Cells were cultured for 96 h in Arg-free DMEM containing 10, 100 or 350 μM arginine and 20 ng/ml LPS. Data are expressed as means ± SEM, n = 4. a, b Means sharing different letters differ (P < 0.01)

Fig. 6

Representative western blots of mTOR (A), phosphorylated mTOR (Ser2448) (B), 4EBP1 (C), phosphorylated 4EBP1 (Ser65) (D), S6K1 (E), phosphorylated S6K1 (Thr389) (F), TLR4 (G), NFκB (H), phosphorylated NFκB (Ser536) (I), and β-actin (J) in IPEC-1 cells. Cells were cultured for 96 h in Arg-free DMEM containing 20 ng/ml LPS and 10 (column I), 100 (column II) or 350 (column III) μM arginine