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Am J Physiol Endocrinol Metab 299: E899–E909, 2010.
First published September 14, 2010; doi:10.1152/ajpendo.00068.2010.
Arginine-induced stimulation of protein synthesis and survival in IPEC-J2
cells is mediated by mTOR but not nitric oxide
Caroline Bauchart-Thevret,1 Liwei Cui,1 Guoyao Wu,2 and Douglas G. Burrin1
1
US Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department
of Pediatrics, Baylor College of Medicine, Houston; and 2Department of Animal Science, Texas A & M University,
College Station, Texas
Submitted 28 January 2010; accepted in final form 13 September 2010
cell growth; p70 ribosomal protein S6 kinase; eukaryotic initiation
factor 4E-binding protein 1; rapamycin; S-nitroso-N-acetylpenicillamine
amino acid in neonatal mammals
and is required for somatic growth (39). Arginine is also a
physiological precursor for nitric oxide (NO), a molecule that
has several critical functions in the body, including vasodilatation, immune responses, neurotransmission, and cell survival
(43). Thus, beyond its role in protein deposition, it is likely that
arginine is nutritionally essential for other important functions
in infants, particularly in the intestine. Although arginine
synthesis in adult mammals occurs via the intestinal-renal axis
(43), the neonatal intestine contains all the enzymes necessary
ARGININE IS AN INDISPENSABLE
Address for reprint requests and other correspondence: D. G. Burrin,
Children’s Nutrition Research Center, 1100 Bates St., Houston, TX 77030
(e-mail: [email protected]).
http://www.ajpendo.org
to synthesize arginine (20, 37, 41), and the small intestine is a
key site of net arginine synthesis in neonates (39, 42, 43). Thus
the gut appears to play a critical role in maintaining arginine
homeostasis in neonates (13, 40).
Evidence from in vitro metabolic studies with primary cells
and transformed cell lines indicates that intestinal epithelial
cells (IEC) are capable of arginine transport, catabolism, and
synthesis from precursor amino acids, such as citrulline (3, 29,
38, 43). However, studies also showed that increasing extracellular arginine in vitro and in vivo stimulates anabolic cell
pathways, such as protein synthesis, proliferation, and migration (6, 25, 26, 32). Arginine supplementation stimulated
protein synthesis and reduced protein degradation in LPStreated IPEC-1 cells (32). Studies in preconfluent Caco-2 cells
suggest that arginine deprivation decreases cell proliferation
and heat shock protein expression and enhances the susceptibility to apoptosis, and most changes induced by arginine
deficiency were restored by subsequent supplementation with
arginine or citrulline (22). The essentiality of arginine for cell
growth is mainly due to its role as a constituent amino acid for
protein synthesis in cells where arginine synthesis is negligible.
However, recent studies also suggest that arginine may directly
activate anabolic cellular signaling pathways (6, 32, 44).
One of the major amino acid-responsive signaling pathways
involved in cell growth is mammalian target of rapamycin
(mTOR) (11). mTOR is a highly conserved serine/threonine
protein kinase that controls many aspects of cellular physiology, including protein synthesis (7, 15). mTOR is a common
signaling complex that mediates the cellular anabolic responses
to insulin and amino acids; yet, among the amino acids, leucine
and arginine are the most potent activators (16). Few studies
have examined the amino acid activation of mTOR signaling in
IEC. However, recent studies in rat IEC showed that arginine
and leucine stimulate downstream targets of mTOR, namely,
p70 ribosomal protein S6 (p70 S6) kinase and eukaryotic
initiation factor 4E-binding protein 1 (4E-BP1), and this response was inhibited by glutamine (2, 24). Consistent with the
cell culture finding, a study in rotavirus-infected piglets
showed that dietary arginine treatment increased intestinal
protein synthesis in association with activation of mTOR and
p70 S6 kinase signaling (6). Thus the emerging evidence
suggests that arginine increases IEC growth and protein synthesis via activation of mTOR pathways.
A major unresolved issue regarding mTOR function is the
nature of the cell-sensing mechanism and upstream signals that
are activated by amino acids, specifically arginine. Nitric oxide
(NO) is the major cell signaling molecule and product of
arginine metabolism via NO synthases (NOS). The three forms
of NOS, namely, endothelial NOS (eNOS), neuronal NOS, and
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Bauchart-Thevret C, Cui L, Wu G, Burrin DG. Arginineinduced stimulation of protein synthesis and survival in IPEC-J2 cells
is mediated by mTOR but not nitric oxide. Am J Physiol Endocrinol
Metab 299: E899 –E909, 2010. First published September 14, 2010;
doi:10.1152/ajpendo.00068.2010.—Arginine is an indispensable amino
acid in neonates and is required for growth. Neonatal intestinal epithelial cells (IEC) are capable of arginine transport, catabolism, and
synthesis and express nitric oxide (NO) synthase to produce NO from
arginine. Our aim was to determine whether arginine directly stimulates IEC growth and protein synthesis and whether this effect is
mediated via mammalian target of rapamycin (mTOR) and is NOdependent. We studied neonatal porcine IEC (IPEC-J2) cultured in
serum- and arginine-free medium with increasing arginine concentrations for 4 or 48 h. Our results show that arginine enhances IPEC-J2
cell survival and protein synthesis, with a maximal response at a
physiological concentration (0.1– 0.5 mM). Addition of arginine increased the activation of mTOR, p70 ribosomal protein S6 (p70 S6)
kinase, and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)
in a time- and dose-dependent manner. The arginine-induced protein
synthesis response was not inhibited by the NO inhibitors nitro-Larginine methyl ester (L-NAME) and aminoguanidine, despite inducible NO synthase expression in IPEC-J2 cells. Moreover, protein
synthesis was not increased or decreased in some cases by addition of
an NO donor (S-nitroso-N-acetylpenicillamine), arginine precursors
(proline and citrulline) in the absence of arginine, or insulin; Snitroso-N-acetylpenicillamine suppressed phosphorylation of mTOR,
p70 S6 kinase, and 4E-BP1. We found a markedly higher arginase
activity in IPEC-J2 cells than in primary pig IEC. Furthermore,
mTOR inhibition by rapamycin partially (42%) reduced the arginineinduced protein synthesis response and phosphorylation of mTOR and
4E-BP1. We conclude that arginine-dependent cell survival and protein synthesis signaling in IPEC-J2 cells are mediated by mTOR, but
not by NO.
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ARGININE AND IEC PROTEIN SYNTHESIS
inducible NOS (iNOS), are differentially expressed in the cells
within the intestine, including epithelial cells (21). A recent
study in cdx2-transformed rat crypt IEC-6 cells showed that
arginine stimulated cell migration via a mechanism requiring
NO and phosphorylation of p70 S6 kinase (26). It is unknown
whether the arginine-mediated activation of protein synthesis
and mTOR is NO-dependent. In the present study, we hypothesize that arginine directly stimulates IEC growth and protein
synthesis via NO-dependent activation of the mTOR signaling
pathway. We tested this hypothesis using an established IEC
line derived from neonatal pigs by culturing cells with increasing arginine concentrations in the presence of inhibitors of
NOS and mTOR.
Cell culture and treatment. The IPEC-J2 cell line, a porcine IEC
line originally derived from the jejunal crypts of a neonatal piglet, was
kindly supplied by Bruce Shultz (Kansas State University). The cells
were seeded in six-well plates and grown in DMEM (catalog no.
11965, GIBCO, Carlsbad, CA) added with 10% (vol/vol) FBS (Hyclone, Logan, UT) and maintained at 37°C in a 5% CO2 atmosphere.
All the experiments were performed in duplicate or triplicate on cell
passages 39 –56.
For the cell growth response, the cells were grown to 50% confluence in regular growth DMEM containing 10% FBS and then changed
to a serum- and arginine-free custom-made DMEM (modified
DMEM 21013, GIBCO) that contained (mM) 5.0 D-glucose, 0.4
cysteine·HCl·H2O, 0.5 glutamine, 0.4 glycine, 0.2 histidine·HCl·H2O,
0.8 isoleucine, 0.8 leucine, 0.8 lysine·HCl, 0.4 phenylalanine, 0.4
serine, 0.8 threonine, 0.1 tryptophan, 0.4 tyrosine·2Na·2H2O, and 0.8
valine. Cells were treated for 2 days with 1) arginine (0 –1 mM) added
with 0.2 mM methionine; 2) methionine (0 or 0.5 mM) added with 0.5
mM arginine; or 3) arginine (0 or 0.5 mM) and rapamycin, an mTOR
inhibitor (0 or 10 nM) added with 0.2 mM methionine. Cell growth
was determined by counting the number of cells in each well of the
plates using a particle counter (model Z1, Beckman Coulter). Cellular
protein and DNA content was determined using bicinchoninic acid
(BCA) protein assay reagent (Pierce, Rockford, IL) and DNA assay
Hoechst reagent 33258 (Sigma, St. Louis, MO), respectively.
For cell protein synthesis response experiments and mTOR, p70 S6
kinase, and 4E-BP1 activation by immunoblot analysis, cells were
grown to 80% confluence in regular growth DMEM (catalog no.
11965, GIBCO) added with 10% FBS and then changed to a serumand arginine-free custom-made DMEM (modified DMEM 21013,
GIBCO) overnight prior to treatment for 1– 4 h with 1) arginine (0 –1
mM) in the presence or absence of 5 ␮g/ml insulin or 5 ␮g/ml
insulin-transferrin-sodium selenite (ITS); 2) in the presence or absence of 0.5 mM arginine and 0.2 mM methionine; 3) NO inhibitors
NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (0 –
2,000 ␮M) in the presence of 0.5 mM arginine; 4) NO donor
S-nitroso-N-acetylpenicillamine (SNAP, 0 –1,000 ␮M) in the absence
of arginine; 5) proline or citrulline (0, 1, or 5 mM) in the absence of
arginine; or 6) rapamycin (0 –1,000 nM) in the presence or absence of
0.5 mM arginine.
Protein synthesis. Protein synthesis was measured by incorporation
of [3H]phenylalanine into cell proteins. Preliminary experiments verified that incorporation of [3H]phenylalanine into cell protein was
linear up to 4 h of incubation (not shown). After incubation with 1 ␮Ci
of L-[4-3H]phenylalanine (Amersham Biosciences, Piscataway, NJ),
cells were washed twice with PBS, scraped, and resuspended in 1 ml
of PBS. Proteins were precipitated by addition of 250 ␮l of ice-cold
2 M perchloric acid (PCA; Fisher Scientific, Pittsburgh, PA). After 25
min of incubation in ice and centrifugation, the PCA-precipitated
pellet was washed twice with 0.2 M PCA and solubilized in 0.3 N
NaOH at room temperature. An aliquot of the solubilized pellet was
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MATERIALS AND METHODS
assayed for protein content (BCA assay), and the incorporated radioactivity was determined by scintillation counting. Protein synthesis
was calculated as disintegrations per minute per micrograms of
protein and expressed as a percentage of the control group.
Western blot analysis. For iNOS and eNOS analysis, IPEC-J2 cells
were cultivated in regular growth DMEM (catalog no. 11965,
GIBCO) added with 10% FBS. IPEC-J2 cells were homogenized in 50
mM HEPES (pH 7.4), 1 mM EDTA, 1 mM dithiothreitol, 5 mg/l
phenylmethylsulfonyl fluoride, 5 mg/l aprotinin, 5 mg/l chymostatin,
5 mg/l pepstatin, and 2 mM sodium orthovanadate. The homogenate
was sonicated and centrifuged at 12,000 g for 15 min at 4°C. Cell
protein extracts (60 ␮g protein; assayed using the BCA method;
Pierce) were separated on a 10% denatured SDS-polyacrylamide gel
and transferred to a nitrocellulose membrane. The membrane was
blocked with 5% nonfat milk in 1⫻ Tris-buffered saline (TBS) and
0.1% Tween 20 for 1 h at room temperature and then incubated with
iNOS or eNOS primary antibody (rabbit polyclonal, 1:500 dilution;
Santa Cruz Biotechnology, Santa Cruz, CA) diluted in 5% nonfat milk
and 1⫻ TBS-0.1% Tween 20 overnight at 4°C. After it was washed
with 1⫻ TBS-0.1% Tween 20, the membrane was incubated with a
secondary antibody (goat anti-rabbit IgG-horseradish peroxidase,
1:5,000 dilution; Santa Cruz Biotechnology) for 1 h at room temperature. The iNOS (130 kDa) and eNOS (140 kDa) bands were detected
with an enhanced chemiluminescence detection kit (ECL Plus, Amersham Biosciences) and analyzed with ImageQuant 5.2 software
(Molecular Dynamics). Pig endothelial cells were used as a control.
For mTOR, p70 S6 kinase, and 4E-BP1 activation analysis, the
cells were cultivated as described above (see Cell culture and treatment). Cells were scraped from culture plates and homogenized as
described above. Cell extracts (50 –100 ␮g of protein) were separated
on a 7% denatured SDS-polyacrylamide gel for mTOR and p70 S6
kinase and a 15% denatured SDS-polyacrylamide gel for 4E-BP1, as
described above for iNOS and eNOS. The membranes were first
incubated with phosphospecific antibodies of phosphorylated (Ser2448)
mTOR (human polyclonal, 1:1,000 dilution; Cell Signaling Technology, Beverly, MA), phosphorylated (Thr421/Ser424) p70 S6 kinase
(human polyclonal, 1:1,000 dilution; Cell Signaling Technology), and
phosphorylated (Thr70) 4E-BP1 (rat polyclonal, 1:1,000 dilution; Cell
Signaling Technology), stripped in stripping buffer (Pierce) at 37°C
for 15 min, washed with 1⫻ TBS-0.1% Tween 20 three times, and
reprobed with the corresponding non-phospho-specific antibodies,
i.e., mTOR (human polyclonal, 1:1,000 dilution; Cell Signaling Technology), p70 S6 kinase (human polyclonal, 1:1,000 dilution; Cell
Signaling Technology), and 4E-BP1 (human polyclonal, 1:1,000 dilution; Bethyl Laboratories, Montgomery, TX). The activation of
mTOR, p70 S6 kinase, and 4E-BP1 was determined by the band
intensity of the phosphorylated form relative to the band intensity of
the total protein. Western blots (Figs. 2, 3C, 5, and 7C) are the results
of pooled samples from two to three experiments. All membranes
were stripped and reprobed with ␣-tubulin antibody (mouse monoclonal, 1:2,000 dilution; Sigma-Aldrich) for loading control.
Arginine and total nitrite concentration. Arginine free amino acid
concentration was determined in cell medium by reverse-phase HPLC
of their phenylisothiocyanate derivatives, as described previously
(31). Total nitrite concentration, as an index of NO production, was
determined in cell medium using a total NO/nitrate/nitrite parameter
kit (R & D Systems).
Enzyme activity assay. Activities of four key arginine-metabolizing
enzymes, pyrroline-5-carboxylate synthase (P5CS), argininosuccinate
synthase (ASS), argininosuccinate lyase (ASL), and arginase, were
determined as described previously (41) on IPEC-J2 cells and neonatal pig isolated IEC. IEC were isolated from the jejunum of four
1-wk-old neonatal pigs that had been fed liquid milk-replacer formula
(Advance Liqui-Wean, Milk Specialties, Dundee, IL) using the distended intestinal sac method, as described previously (9).
The study protocol was approved by the Animal Care and Use
Committee of Baylor College of Medicine and conducted in accor-
ARGININE AND IEC PROTEIN SYNTHESIS
RESULTS
Cell growth and protein synthesis responses to arginine. In
an arginine-free DMEM, IPEC-J2 cell number decreased significantly by 29% and 52% after 1 and 2 days of incubation,
respectively, but returned to their original number in the
presence of arginine (Fig. 1A). On day 2, we found a significant
increase in IPEC-J2 cell number (⫹129%), cell protein
(⫹75%), and DNA content (⫹50%) with all arginine doses and
a maximal response at a physiological dose of arginine (0.1–
0.5 mM; Fig. 1, A and B). After 4 h of incubation, arginine
significantly increased (⫹76%) protein synthesis in IPEC-J2
cells, with a maximal response at 0.1– 0.5 mM (Fig. 1C). The
presence of 5 ␮g/ml insulin or 5 ␮g/ml ITS in the culture
medium did not affect the cell protein synthesis response to
arginine (Fig. 1C).
mTOR, p70 S6 kinase, and 4E-BP1 activation by arginine.
mTOR, p70 S6 kinase, and 4E-BP1 activation was determined
by Western blot analysis of phosphorylated (Ser2448) mTOR,
phosphorylated (Thr421/Ser424) p70 S6 kinase, and phosphorylated (Thr70) 4E-BP1 expression relative to their respective
total protein expression (Fig. 2). Overall, a significant effect of
time was found for mTOR, p70 S6 kinase, and 4E-BP1
activation when the IPEC-J2 cells were incubated with 0.5 mM
arginine, with a significant increase (⫹79%) in mTOR phosphorylated-to-total protein ratio after 4 h of incubation and a
marked increase (⫹329%) in 4E-BP1 phosphorylated-to-total
protein ratio within 1 h of incubation (Fig. 2A). In the 1-h
incubation period, there was a significant effect of arginine
dose for mTOR, p70 S6 kinase, and 4E-BP1 activation (Fig.
Fig. 1. A and B: cell growth response, i.e., cell count and protein and DNA content, to arginine in IPEC-J2 cells incubated for 2 days in arginine-free DMEM.
C: protein synthesis response to arginine in IPEC-J2 cells incubated for 4 h in arginine-free DMEM in the presence or absence of 5 ␮g/ml insulin or 5 ␮g/ml
insulin-transferrin-sodium selenite (ITS). Protein synthesis was determined by [3H]phenylalanine incorporation into protein [disintegrations per minute (dpm)/␮g
protein]. Values (means ⫾ SE) are expressed as percentage of control (0 mM arginine) in B and C. *P ⬍ 0.05, ***P ⬍ 0.001 vs. time 0 (T0) in A. *P ⬍ 0.05
vs. control (0 mM arginine) in B. NS, not significant.
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dance with the National Institutes of Health (NIH) Guide for the Care
and Use of Laboratory Animals (US Department of Health and
Human Services Publication No. NIH 85-23, revised 1985, Office of
Science and Health Reports, NIH, Bethesda, MD).
Statistical analysis. Statistical analysis was performed using
GraphPad Prism version 5.02 and MINITAB Release version 13.1
software. Values are means ⫾ SE. Data were analyzed using a
Student’s t-test for comparison of paired samples or a one-way
ANOVA for multiple comparisons. Differences among individual
means were assessed by Tukey’s comparison test. A two-way
ANOVA with Bonferroni’s post test was also performed to compare
different treatments. For Western blot data, all statistical analysis was
performed on absolute values for phosphorylated-to-total protein
ratios, and the results are expressed as percentage of control. The
proportional SE associated with the control group is shown for
comparison. Statistical significance was assigned at P ⬍ 0.05.
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ARGININE AND IEC PROTEIN SYNTHESIS
2B). Despite this main effect of arginine dose, the phosphorylated-to-total protein ratios for mTOR, p70 S6 kinase, and
4E-BP1 were not significantly increased at any arginine dose
based on Tukey’s means comparison. However, the absolute
abundances of phosphorylated and total proteins for mTOR,
p70 S6 kinase, and 4E-BP1 were markedly increased at 0.5
mM arginine compared with 0, 0.1, and 1 mM arginine (Fig.
2B). To examine the role of insulin, we incubated the cells with
0 or 0.5 mM arginine for 1 h in the presence or absence of
insulin (5 ␮g/ml). Insulin had no effect on mTOR, p70 S6
kinase, and 4E-BP1 activation in the presence or absence of
arginine (data not shown).
Effect of methionine. To determine whether the effects
observed with arginine are specific, we also examined the
effect of another indispensable amino acid, methionine, on cell
growth, protein synthesis, and mTOR, p70 S6 kinase, and
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4E-BP1 activation in IPEC-J2 cells. A significant decrease in
IPEC-J2 cell number was observed after 1 day (⫺40%) and 2
days (⫺22%) of incubation in methionine-free DMEM (Fig. 3A).
It is important to note that only methionine was removed, and
0.5 mM arginine was included. In the presence of 0.5 mM
methionine, the number of IPEC-J2 cells returned to their
original level after 1 day (⫹80%) and 2 days (⫹26%) of
incubation (Fig. 3A). Although arginine deficiency significantly decreased (⫺43%) protein synthesis, methionine-free
DMEM had no effect on protein synthesis after 4 h of incubation in IPEC-J2 cells (Fig. 3B). However, phosphorylated-tototal protein ratios for mTOR (⫺30 –37%) and p70 S6 kinase
(⫺26%) were significantly decreased by arginine and methionine deficiency (Fig. 3C).
Effect of NO. On the basis of immunoblotting, we found that
IPEC-J2 cells mainly express iNOS, since eNOS was unde-
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Fig. 2. A: time-course activation of mammalian target of rapamycin (mTOR), p70 ribosomal protein S6 (p70 S6) kinase, and eukaryotic initiation factor
4E-binding protein 1 (4E-BP1) in IPEC-J2 cells incubated with 0.5 mM arginine, as determined by Western blot analysis of phosphorylated (Ser2448) mTOR
(p-mTOR), phosphorylated (Thr421/Ser424) p70 S6 kinase (p-p70S6 kinase), and phosphorylated (Thr70) 4E-BP1 (p-4E-BP1) expression relative to their
respective total protein expression. B: arginine dose activation of mTOR, p70 S6 kinase, and 4E-BP1 in IPEC-J2 cells incubated for 1 h, as determined by Western
blot analysis of phosphorylated (Ser2448) mTOR, phosphorylated (Thr421/Ser424) p70 S6 kinase, and phosphorylated (Thr70) 4E-BP1 expression relative to their
respective total protein expression. Values (means ⫾ SE) are expressed as percentage of control (0 h or 0 mM arginine). *P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001
vs. control. Main effect of time for mTOR (P ⬍ 0.01), p70 S6 kinase (P ⬍ 0.05), and 4E-BP1 (P ⬍ 0.001) in A. Main effect of arginine dose for mTOR
(P ⬍ 0.05), p70 S6 kinase (P ⬍ 0.01), and 4E-BP1 (P ⬍ 0.01) in B.
ARGININE AND IEC PROTEIN SYNTHESIS
E903
tectable (Fig. 4A); yet protein synthesis was not affected by the
NOS inhibitors L-NAME and aminoguanidine in the presence
of 0.5 mM arginine (Fig. 4B). Moreover, addition of the NO
donor SNAP in the culture medium did not significantly affect
protein synthesis in the absence of arginine but tended to
reduce protein synthesis, and this was significant at the highest
dose (Fig. 4C). Although the NO donor SNAP significantly
increased (⫹164%) total nitrite concentration in the medium,
no difference in total nitrite concentration was observed in the
presence or absence of 0.5 mM arginine in IPEC-J2 cells (Fig.
4D). In addition, the NO donor SNAP significantly decreased
p70 S6 kinase (⫺38%) and 4E-BP1 (⫺26%) phosphorylatedto-total protein ratios in IPEC-J2 cells incubated in argininefree DMEM (Fig. 5).
Arginine metabolism in IPEC-J2 cells. Proline and citrulline,
both precursors for arginine synthesis in neonatal pigs (34, 36),
did not stimulate protein synthesis in IPEC-J2 cells in the
absence of arginine (Fig. 6A). However, we found only barely
detectable or no arginine in the medium when IPEC-J2 cells
were incubated with added proline and citrulline in argininefree medium (Fig. 6B). The enzyme activity of P5CS, ASS, and
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ASL was significantly lower in IPEC-J2 cells than in pig
primary IEC, whereas arginase enzyme activity was markedly
(1,270-fold) higher in IPEC-J2 cells than in pig primary IEC
(Fig. 6C).
Effect of rapamycin. In the absence of arginine, rapamycin
(10 nM), an mTOR inhibitor, did not affect the protein synthesis response in IPEC-J2 cells (Fig. 7A). However, rapamycin, even at a very low dose (1–10 nM), significantly decreased
(⫺42%) the protein synthesis response induced by arginine
(Fig. 7A). After 2 days of incubation, rapamycin significantly
decreased (⫺30%) IPEC-J2 cell number in the presence of
arginine (Fig. 7B). In addition, a significant main effect of
rapamycin was found for mTOR and 4E-BP1 activation, with
a significant decrease (⫺42%) in mTOR phosphorylated-tototal protein ratio in IPEC-J2 cells incubated in arginine-free
DMEM in the presence of rapamycin (Fig. 7C).
DISCUSSION
The objective of this study was to determine whether arginine directly stimulates IEC growth and protein synthesis and
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Fig. 3. A: cell growth response to methionine in IPEC-J2 cells incubated for 2 days in methionine-free DMEM. B: protein synthesis response to arginine or
methionine deficiency in IPEC-J2 cells incubated for 4 h in DMEM in the presence or absence of 0.5 mM arginine or 0.2 mM methionine. C: activation response
of mTOR, p70 S6 kinase, and 4E-BP1 to arginine or methionine deficiency in IPEC-J2 cells incubated for 1 h in DMEM in the presence or absence of 0.5 mM
arginine or 0.2 mM methionine, as determined by Western blot analysis of phosphorylated (Ser2448) mTOR, phosphorylated (Thr421/Ser424) p70 S6 kinase, and
phosphorylated (Thr70) 4E-BP1 expression relative to their respective total protein expression. Values (means ⫾ SE) are expressed as percentage of control
(complete DMEM) in B and C. *P ⬍ 0.05, ***P ⬍ 0.001 vs. T0 in A. **P ⬍ 0.01 vs. control in B and C.
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ARGININE AND IEC PROTEIN SYNTHESIS
whether this effect is NO-dependent and mediated via the
mTOR signaling pathway. The rationale for this study was
based on recent in vitro and in vivo studies showing that
arginine stimulates protein synthesis and p70 S6 kinase in
intestinal epithelium (6, 26). We used an established porcine
IEC line (IPEC-J2) originally derived from the jejunal crypts of
a neonatal piglet. Our results show that arginine enhances
IPEC-J2 cell survival and protein synthesis and that the maximal response occurs at physiological concentrations (i.e.,
0.1– 0.5 mM arginine). We also show that arginine increases
the phosphorylation of mTOR and downstream targets, i.e.,
p70 S6 kinase and 4E-BP1 and that the arginine-mediated
protein synthesis response is partially blocked by rapamycin,
an mTOR inhibitor. However, the arginine-induced protein
synthesis response was not affected by NOS inhibitors, the
presence of insulin, or the addition of an NO donor, suggesting
that production of NO and insulin is not involved in the
arginine signaling mechanism.
The trophic effects of arginine on cell growth and protein
synthesis shown here in IPEC-J2 cells are comparable to those
recently reported in other IEC lines (IPEC-1 and Caco-2 cells)
(22, 32). Both are consistent with in vivo studies showing that
an increase in arginine availability enhances body growth and
intestinal tissue protein synthesis in neonatal pigs (6, 14, 19,
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46). An especially relevant aspect of the studies with IPEC
cells in the present study and a previous study (32) is that the
effects of arginine are maximal at physiological concentrations
(0.1– 0.5 mM) normally found in the circulating plasma of
young pigs (19, 46). Other recent IEC culture studies used
higher arginine concentrations (1– 4 mM) to stimulate cell
migration and activate downstream mTOR signals (26). In the
present study, the increase in cell number and protein and DNA
content at day 2 was mainly an effect on cell survival, rather
than cell proliferation, since the number of IPEC-J2 cells
returned (increased) to their original level but was not greater
than the number in the presence of arginine. Arginine is a
precursor for protein synthesis, and in cells that are not able to
synthesize it endogenously, arginine becomes the limiting
amino acid in the absence of extracellular arginine. Thus the
decrease in cell number after 1 and 2 days of incubation in
arginine-free medium indicates that arginine is an essential
amino acid in IPEC-J2 cells. Previous reports showed that
arginine is necessary for tumor cell growth (4, 35, 45); yet the
cellular mechanisms that explain the requirement have not
been established.
Our results show that the arginine-induced protein synthesis
response is mediated by mTOR and associated with activation
of p70 S6 kinase and 4E-BP1 in a time- and dose-dependent
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Fig. 4. A: expression of inducible and endothelial nitric oxide (NO) synthase (iNOS and eNOS) in pig endothelial cells and IPEC-J2 cells by Western blot analysis.
B and C: protein synthesis response to NO inhibitors NG-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine in IPEC-J2 cells incubated for 4 h in
DMEM in the presence of 0.5 mM arginine and to the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in IPEC-J2 cells incubated for 4 h in arginine-free
DMEM. Protein synthesis was determined by [3H]phenylalanine incorporation into protein (dpm/␮g protein). D: total nitrite concentration in IPEC-J2 cell
medium after 1 h of incubation in arginine-free DMEM in the presence or absence of 0.5 mM arginine and 30 ␮M SNAP. Values (means ⫾ SE) are expressed
as percentage of control (0 ␮M L-NAME, aminoguanidine, or SNAP) in B and C. ***P ⬍ 0.001 vs. control in C. *P ⬍ 0.001 vs. no arginine-no SNAP, †P ⬍
0.001 vs. 0.5 mM arginine-no SNAP in D.
ARGININE AND IEC PROTEIN SYNTHESIS
E905
manner. Specifically, 0.5 mM arginine markedly increased
mTOR, p70 S6 kinase, and 4E-BP1 phosphorylated and total
protein abundance compared with 0.1 or 1 mM arginine. This
arginine activation of downstream mTOR targets (p70 S6
kinase and 4E-BP1) involved in protein synthesis is consistent
with several previous studies using IEC, including porcine
IPEC-1 and cdx2-tranformed IEC-6 cells (26, 27, 32). In
addition, we also tested whether the effect of arginine on IEC
protein synthesis was affected by insulin, since this is another
major hormone known to activate mTOR (1, 12). We found
that insulin in the presence or absence of arginine did not affect
the rate of protein synthesis or phosphorylation of downstream
mTOR targets, i.e., p70 S6 kinase and 4E-BP1. Taken together,
the results indicate that the arginine-induced stimulation of
protein synthesis and mTOR signaling is insulin-independent
in IPEC-J2 cells.
To determine whether the arginine effects on IPEC-J2 cell
growth and protein synthesis are specific, we also examined the
effect of another indispensable amino acid, methionine, in
IPEC-J2 cells. We tested methionine, since it is often considered the first-limiting amino acid for growth in animals. Interestingly, although the cell growth response to methionine was
similar to arginine, the protein synthesis response was not
affected by methionine deficiency. However, the activation of
mTOR, p70 S6 kinase, and 4E-BP1 was decreased similarly by
arginine and methionine deficiency. These findings indicate
that arginine-mediated cell growth or survival and activation of
mTOR signaling are not specific and reflect the essentiality of
arginine and methionine for protein synthesis. However, the
dependence of the cell protein synthesis response on arginine,
and not on methionine, suggests that arginine may be firstlimiting for this cell function. These results are consistent with
the finding that mTOR is a vital effector protein in the cell
stress response produced by removal of essential amino acids,
especially arginine (16). Deprivation of methionine and other
essential amino acids activates cell stress signals, in addition to
mTOR, that activate further-downstream signals and gene
AJP-Endocrinol Metab • VOL
transcription (17, 30). Our results suggest that suppression of
mTOR by essential amino acid deprivation is important, not
only for protein synthesis, but also for cell survival; yet the
downstream signals involved in this response are unknown.
Besides its role in protein synthesis, arginine is also a
precursor for two key molecules involved in cell function, NO
and ornithine, synthesized via NOS and arginase enzymes,
respectively. NO plays a key role in several cell functions and
activates guanylyl cyclase and production of cGMP, which
then acts as a signaling molecule to stimulate various cell
kinases and transcription factors (23). Ornithine is a precursor
for polyamines, which are essential for cell proliferation and
growth (43). Our results suggest that NO does not play a
significant role in the arginine-induced stimulation of protein
synthesis in IPEC-J2 cells. Neither NOS inhibitor (L-NAME
nor aminoguanidine) affected the arginine-induced stimulation
of protein synthesis across a wide range of concentrations,
despite the presence of iNOS in IPEC-J2 cells. However,
IPEC-J2 cells have a limited capacity for iNOS-mediated NO
production, since we found no increase in media total nitrite
concentration in the presence of 0.5 mM arginine. Thus we also
treated cells with the NO donor SNAP and verified NO
production in the cell medium. Although SNAP treatment
increased NO, the increased NO availability failed to activate
protein synthesis in the absence of arginine. Instead, it tended
to suppress protein synthesis, especially at the highest dose. In
addition, the presence of SNAP significantly decreased p70 S6
kinase and 4E-BP1 activation in the absence of arginine.
Collectively, our results suggest that NO impairs IPEC-J2 cell
protein synthesis, which implies that the arginine-induced
stimulation of protein synthesis is NO-independent in IPEC-J2
cells. These findings are in contrast to a recent study in
cdx2-transformed rat crypt IEC-6 cells in which arginine and
an NO donor stimulated IEC migration and p70 S6 kinase
phosphorylation (26). This suggests that IEC migration and
protein synthesis response to arginine are activated by different
signaling pathways. Whether NO is involved in the arginine-
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Fig. 5. Activation response of mTOR, p70 S6 kinase, and 4E-BP1 to arginine or SNAP in IPEC-J2 cells incubated for 1 h in arginine-free DMEM in the presence
or absence of 0.5 mM arginine or 30 ␮M SNAP, as determined by Western blot analysis of phosphorylated (Ser2448) mTOR, phosphorylated (Thr421/Ser424) p70
S6 kinase, and phosphorylated (Thr70) 4E-BP1 expression relative to their respective total protein expression. Values (means ⫾ SE) are expressed as percentage
of control (0.5 mM arginine-no SNAP). *P ⬍ 0.05, **P ⬍ 0.01 vs. control.
E906
ARGININE AND IEC PROTEIN SYNTHESIS
induced protein synthesis in other IEC with more robust NOS
activity remains to be tested.
To further examine the essentiality of arginine, we incubated
arginine-deprived IPEC-J2 cells with citrulline and proline,
which are arginine precursors in neonatal piglets (34, 36).
Surprisingly, neither affected protein synthesis, nor did we find
any significant arginine produced from proline or citrulline in
IPEC-J2 cells. The lack of arginine synthesis in IPEC-J2 cells
was explained by the markedly lower activity of the enzymes,
specifically ASS, ASL, and P5CS, involved in arginine synthesis from these precursors. Also, the finding that arginase
activity was an order of magnitude higher in IPEC-J2 cells than
in pig primary IEC would prevent the production of arginine,
since this enzyme catalyzes the conversion of arginine to
ornithine. These findings clearly indicate that IPEC-J2 cells,
originally derived from newborn piglets, do not have the
capacity to synthesize arginine. This is in contrast to previous
studies showing that primary IEC isolated from newborn pigs
are capable of arginine synthesis from proline and citrulline
(37, 41).
Finally, we showed that the arginine-induced increase in
protein synthesis was maximally inhibited by ⬃42% over a
wide range of rapamycin concentrations (1–1,000 nM). Moreover, the arginine-induced increase in IPEC-J2 cell survival
was partially inhibited (⫺30%) by rapamycin after 2 days of
incubation. These results suggest that part of the arginine effect
AJP-Endocrinol Metab • VOL
is rapamycin insensitivity. mTOR exists in two protein complexes, namely, mTOR complex 1 (mTORC1) and mTOR
complex 2 (mTORC2) (7). mTORC1 is considered the master
regulator of protein synthesis; it is rapamycin-sensitive and
consists of mTOR, raptor, and G protein ␤-subunit-like protein
(G␤L). This complex is known to be activated by amino acids,
hormones/growth factors, and energy signals (18). In contrast,
mTORC2 is rapamycin-insensitive and consists of mTOR,
rictor, and G␤L (7) and activates protein kinase B, a key
regulator of cell survival upstream to mTORC1 (28). Previous
studies with IEC and cultured porcine intestinal tissue explants
showed that rapamycin treatment blocks the arginine-induced
activation of p70 S6 kinase and 4E-BP1 (6, 27). However,
although our treatment with rapamycin significantly reduced
mTOR and 4E-BP1 activation, we were surprised to find no
effect of rapamycin on p70 S6 kinase activation, which may be
explained by the high variation between the samples. Rapamycin forms a complex with its intracellular receptor FKBP12
(12-kDa FK 506-binding protein), which then binds to the FRB
(FKBP12-rapamycin binding) domain of mTOR and inhibits
mTORC1 signaling through a poorly understood mechanism
(5). However, recent reports suggest a relative inefficacy of
rapamycin and rapalogs (rapamycin analogs) in cancer treatment attributed to hyperactivation of Akt (upstream of mTOR)
through loss of the mTORC1-p70 S6 kinase-insulin receptor
substrate-1-phosphoinositide 3-kinase negative-feedback loop
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Fig. 6. A: protein synthesis response to proline and citrulline in IPEC-J2 cells incubated for 4 h in arginine-free DMEM. Protein synthesis was determined by
[3H]phenylalanine incorporation into protein (dpm/␮g protein). B: arginine concentration in IPEC-J2 cell medium after 2 days of incubation in arginine-free
DMEM when cells are supplemented with arginine and 4 h of incubation in arginine-free DMEM when cells are supplemented with proline or citrulline.
C: pyrroline-5-carboxylate synthase (P5CS), argininosuccinate synthase (ASS), argininosuccinate lyase (ASL), and arginase enzyme activity in pig primary IEC
and IPEC-J2 cells. Values (means ⫾ SE) are expressed as percentage of control (0 mM proline or citrulline) in A. ***P ⬍ 0.001 vs. pig primary IEC in C.
ARGININE AND IEC PROTEIN SYNTHESIS
E907
(8). In addition, recent studies suggest that mTORC1 exhibits
rapamycin-sensitive and -insensitive activities (10, 33). Taken
together, these findings suggest that the “partial effect” of
rapamycin on arginine-induced protein synthesis may be due to
the fact that rapamycin does not completely inhibit the
mTORC1 signaling pathway. Several novel active-site mTOR
kinase inhibitors, which inhibit mTORC1 and mTORC2, were
recently developed (8). Nevertheless, the rapamycin-sensitive
effect on arginine-dependent cell survival reinforces the point
that mTOR signaling is vital for IEC cycle function.
In summary, our results from a porcine IEC line (IPEC-J2)
demonstrate that arginine is required for cell survival and
protein synthesis and that the maximal response occurs in the
physiological concentration range. In addition, arginine increased the activation of mTOR, p70 S6 kinase, and 4E-BP1 in
a time- and dose-dependent manner. Moreover, the arginineinduced protein synthesis response was not affected by NOS
inhibitors, such as L-NAME and aminoguanidine, or the presAJP-Endocrinol Metab • VOL
ence of the NO donor SNAP. We also determined that IPEC-J2
cells, in contrast to primary pig IEC, have a lower capacity to
synthesize arginine, partly due to a markedly higher arginase
activity, and this likely explains why it is required for IPEC-J2
cell survival. Furthermore, rapamycin, which is not a complete
inhibitor of mTOR, partially reduced the arginine-induced
protein synthesis response. Thus we conclude that arginineinduced stimulation of protein synthesis and survival in
IPEC-J2 cells is mediated by mTOR but does not involve NO
or insulin. The intracellular signals that mediate the effect of
arginine on the activation of mTOR, protein synthesis, and cell
survival in IEC warrant further study.
GRANTS
This work is a publication of the US Department of Agriculture/Agricultural Research Service Children’s Nutrition Research Center, Department of
Pediatrics, Baylor College of Medicine and Texas Children’s Hospital. This
work was supported by a grant from the Ajinomoto Amino Acid Research
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Fig. 7. A: protein synthesis response to rapamycin (mTOR inhibitor) in IPEC-J2 cells incubated for 4 h in arginine-free DMEM in the presence or absence of
arginine. Protein synthesis was determined by [3H]phenylalanine incorporation into protein (dpm/␮g protein). Values (means ⫾ SE) are expressed as percentage
of control (0 nM rapamycin). B: cell growth response, i.e., cell count, to arginine and/or rapamycin in IPEC-J2 cells incubated for 2 days in arginine-free DMEM
in the presence or absence of 0.5 mM arginine and 10 nM rapamycin. C: activation response of mTOR, p70 S6 kinase, and 4E-BP1 to arginine and/or rapamycin
in IPEC-J2 cells incubated for 1 h in arginine-free DMEM in the presence or absence of 0.5 mM arginine or 10 nM rapamycin, as determined by Western blot
analysis of phosphorylated (Ser2448) mTOR, phosphorylated (Thr421/Ser424) p70 S6 kinase, and phosphorylated (Thr70) 4E-BP1 expression relative to their
respective total protein expression. Values (means ⫾ SE) are expressed as percentage of control (no arginine-no rapamycin) in A and C. *P ⬍ 0.001 vs. no
arginine-no rapamycin, † P ⬍ 0.001 vs. 0.5 mM arginine-no rapamycin; #P ⬍ 0.05 vs. 0.5 mM arginine-0.1 nM rapamycin in A. **P ⬍ 0.01, ***P ⬍ 0.001
vs. T0 in B. *P ⬍ 0.05 vs. no arginine-no rapamycin in C. Main effect of rapamycin and arginine ⫻ rapamycin interaction for mTOR (P ⬍ 0.05) and 4E-BP1
(P ⬍ 0.05) based on 2-way ANOVA.
E908
ARGININE AND IEC PROTEIN SYNTHESIS
Program and by federal funds from the US Department of Agriculture,
Agricultural Research Service under Cooperative Agreement 58-6250-6-001.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
DISCLAIMER
The contents of this publication do not necessarily reflect the views or
policies of the US Department of Agriculture, nor does mention of trade
names, commercial products, or organizations imply endorsement by the US
Government.
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