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Nutrition and Disease

Arginine Activates Intestinal p70^S6k and Protein Synthesis in Piglet Rotavirus Enteritis^1,2

³ Department of Animal Sciences, North Carolina State University and ⁴ College of Veterinary Medicine, Raleigh, NC 27695; ⁵ Department of Pediatrics, Ochsner Clinic Foundation, New Orleans, LA; and ⁶ Department of Pediatrics, Division of Gastroenterology, University of Texas at Houston, Houston, TX 77030

^* To whom correspondence should be addressed. Email: j.marc.rhoads{at}uth.tmc.edu.

	ABSTRACT

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
We previously showed that phosphorylation of p70 S6 kinase (p70^S6k) in the intestine is increased during viral enteritis. In this study, we hypothesized that during rotavirus infection, oral Arg, which stimulates p70^S6k activation, will further stimulate intestinal protein synthesis and mucosal recovery, whereas the p70^S6k inhibitor rapamycin (Rapa) will inhibit mucosal recovery. Newborn piglets were fed a standard milk replacer diet supplemented with Arg (0.4 g · kg^–1 · d^–1, twice daily by gavage), Rapa (2 mg · m^–2 · d^–1), Arg + Rapa, or saline (controls). They were infected on d 6 of life with porcine rotavirus. Three days postinoculation, we measured the piglets' body weight, fecal rotavirus excretion, villus-crypt morphology, epithelial electrical resistance in Ussing chambers, and p70^S6k activation by Western blotting and immunohistochemistry. We previously showed a 2-fold increase in jejunal protein synthesis during rotavirus diarrhea. In this experiment, Arg stimulated jejunal protein synthesis 1.3-fold above standard medium, and the Arg stimulation was partially inhibited by Rapa. Small bowel stimulation of p70^S6k phosphorylation and p70^S6k levels were inhibited >80% by Rapa. Immunohistochemistry revealed a major increase of p70^S6k and ribosomal protein S6 phosphorylation in the crypt and lower villus of the infected piglets. However, in Arg-treated piglets, p70^S6k activation occurred over the entire villus. Jejunal villi of the Rapa-treated group showed inactivation of p70^S6k and a decrease in mucosal resistance (reflecting increased permeability), the latter of which was reversed by Arg. We conclude that, early in rotavirus enteritis, Arg has no impact on diarrhea but augments intestinal protein synthesis in part by p70^S6k stimulation, while improving intestinal permeability via a mammalian target of rapamycin/p70^S6k-independent mechanism.

	Introduction

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

During rotavirus infection, the mammalian target of rapamycin (mTOR) target ribosomal p70^S6k is strongly activated in the intestinal crypt, whereas p70^S6k is inactivated (unphosphorylated) in striated muscle (4). Our current studies were prompted by a desire to find a nutritional treatment that might enhance intestinal repair during diarrheal diseases. Arginine (Arg) and Leucine (Leu) were recently found to be the best amino acid stimulators of mTOR in cultured intestinal cells (5). Low plasma Arg is a nutritional deficiency associated with neonatal necrotizing enterocolitis, raising the possibility that supplemental Arg may be beneficial to hosts with intestinal injury (6,7). We tested the hypothesis that oral Arg, via stimulation of intestinal p70^S6k and protein synthesis, would facilitate mucosal restitution and villus regrowth. We used a well-established model of viral enteritis: rotavirus enteritis in formula-fed piglets (4,8).

	Materials and Methods

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Materials. Rabbit anti-p70 S6 kinase antibody (A300–510) was purchased from Bethyl Laboratories. Rabbit phospho-p70 S6 kinase (Thr421/Ser424) antibody (9204) and rabbit anti-ribosomal protein S6 (rpS6) antibody (2212), as well as rabbit phospho-rpS6 (Ser235/236) antibody (2211) were from Cell Signaling Technology. Anti-actin antibody (A2066) was from Sigma. Alexa Fluor–conjugated donkey anti-rabbit IgG, anti-goat IgG, 4',6-diamidino-2-phenylindole (DAPI), and Prolong Gold Antifade Reagent were all from Molecular Probes. Immun-Blot polyvinylidene difluoride membrane was from Bio-Rad. Enhanced Chemiluminescence Plus Western Blotting Detection System (ECL Plus) was purchased from Amersham Pharmacia Biotech. Protease inhibitor cocktail and phosphatase inhibitor cocktail I and II were from Sigma. ³H-Phe was purchased from American Radiolabeled Chemicals.

    Animals. We studied 35 artificially fed crossbred piglets. The protocol was approved by the Institutional Animal Care and Use Committee of North Carolina State University. All piglets received porcine rotavirus, 10⁷ particles orally on d 6 of life, as previously described. They were housed, raised, and killed as described previously (4), with the exception that none of the piglets had diet restrictions. Animal weights and feed intakes were measured daily. The mean weight for piglets on d 0 was 2086 ± 32 g. Piglets were rectally swabbed with a cotton-tipped applicator daily for the detection of rotavirus shedding, a commercial latex agglutination assay was used (Virogen Rotatest, Wampole Laboratories).

Piglets were fed a milk diet via a gravity-flow feeding system. The diet contained 0.92% Arg and provided 0.4 g · kg^–1 · d^–1. We chose the dose of Arg based on a previous prospective study in human premature infants by Amin et al. (1), in which the infants receiving 1.5 mmol · kg^–1 · d^–1 (0.26 g · kg^–1 · d^–1) had an 80% reduction in the incidence of necrotizing enterocolitis, compared with a placebo group. Wu et al. (9) found that dietary supplementation of a lower dose of 0.4% Arg administered to 7- to 21-d-old piglets enhanced the plasma Arg concentration (61%), reduced the plasma ammonia concentration (35%), and increased weight gain (66%). On the day of infection, piglets were orally gavaged with 1 of 4 treatments: with or without Arg and with or without Rapamycin (Rapa), according to a 2 x 2 factorial design. Arg was supplemented at 0.4 g · kg^–1 · d^–1 for the first 2 litters [dosing was based on previous human trials of Arg in newborn infants (10)]. Piglets not receiving Arg or Rapa were sham-gavaged with an equal volume of saline solution. A third litter was gavaged 1.5 g · kg^–1 · d^–1 of Arg; because the results were virtually identical to the other 2 litters, the results for Arg were pooled. Piglets were gavaged with Rapa (Rapamune) at a dose of 4 mg · m^–2 on the day of infection and 2 mg · m^–2 · d^–1 on subsequent days. This dose was based on compiled safety and efficacy data in humans (11). After piglets were killed, intestinal loops were removed for sampling, as previously described (4).

    In vitro protein synthesis by intestinal segments. Protein synthesis was measured in intestinal segments cultured with ³H-Phe, as previously described (4). In an initial experiment, segments of intestine from fully fed piglets not infected or infected with rotavirus were used. Mucosal explants were prepared to determine the in vitro effects of amino acid treatments. Mucosa was carefully removed from the lumen of the intestine lengths and used to prepare explants (30 mg). Basal medium Eagle (BME) was used as the platform medium for creating amino acid treatment media. Amino acids were added to create the following treatments: nonessential amino acids (NEAA), 4 mmol/L; BCAA, 4 mmol/L; twice the normal concentration of amino acids (2x AA); Arg, 4 mmol/L. Rapa (20 µmol/L) was added to additional 2x AA and Arg treatment flasks, producing 2x AA + Rapa and Arg + Rapa treatments.

    Intestinal histology and lactase-specific activity. Measurements were determined by previously published methods and performed in a blinded manner (8).

    Jejunal tissue permeability characteristics in vitro. We determined electrical resistance (1 per conductance) in vitro, by mounting seromuscular-stripped mucosa in Ussing chambers, as previously published (12).

    Western blotting. Levels of p70^S6k and phosphorylated p70^S6k were measured as previously described (4). In short, intestinal mucosa was prepared in a radioimmunopreciptation buffer containing protease and phosphatase inhibitors; protein concentration was determined and 40 µg of protein in an equal volume of 2x Laemmli sample buffer were combined, boiled, and electrophoretically separated on 7.5% SDS-PAGE. The proteins were then transferred to a polyvinylidene difluoride membrane and subsequently incubated with p70^S6k or phosphorylated p70^S6k antibodies (1:3000). Bands were detected with an enhanced chemiluminescence detection kit (ECL Plus, Amersham Biosciences), and semiquantitative data were obtained using a computer densitometer (Quantity One, Bio-Rad). Phosphorylated and total p70^S6k measurements were normalized to β-actin immunoreactivity.

    Immunohistochemistry. Tissues fixed in 4% formaldehyde were frozen-sectioned (5-µm sections), and nonspecific binding was blocked by incubation of the slides with 5% normal donkey serum in 1% bovine serum albumin in PBS for 15 min at room temperature. After washing with PBS, the slides were incubated for 90 min in 1% bovine serum albumin in PBS containing primary antibodies at a 1:200 dilution. Slides were washed 3 times for 5 min each with PBS and then labeled with 1:500 dilutions of combined conjugated Alexa Fluor 488 donkey anti-rabbit IgG for 30 min at room temperature with light shielding. The slides were then stained with DAPI at a 1:5000 dilution in PBS for 2 min and subsequently mounted with Prolong Gold Antifade Reagent. The fluorescent images were captured with a Zeiss deconvoluting microscope under 10x and 60x immersion objectives and by using Slidebook from Intelligent Imaging Innovations Software (13).

    Statistical analysis. Data for in vitro amino acid treatments were analyzed using SAS (SAS Institute). The model included treatment media, and comparisons were made within infection status using Dunnett's procedure for comparing treatments with a control [basal medium (BME)]. In vivo data were analyzed with a 2 x 2 factorial design using SAS. The model included the effects of Arg and Rapa treatment as well as the Arg x Rapa interaction. If the ANOVA F-test was significant, differences between means were assessed using Tukey's multiple comparison test and declared significant when P < 0.05. Data from uninfected piglets (4,12) are included in tables and figures for reference but were not included in statistical comparisons.

	Results

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    In vitro protein synthesis: response to amino acids. Mucosal explants were cultured in a BME with or without supplemental amino acids. Amino acid composition of the in vitro treatment media are shown in Table 1. We hypothesized that the addition of Arg would enhance the protein synthetic rate because it is a powerful activator of mTOR/p70^S6k in intestinal cells (3). The mucosa from uninfected piglets did not respond to Arg with an increase in protein synthesis (Fig. 1). It is likely that protein synthesis in the uninfected mucosa was limited by the absence of Gly and Ser, which are essential to the newborn gut (14). However, the rate of ³H-Phe incorporation in the infected tissue increased 25–38% when explants were cultured with Arg (4 mmol/L) or 2x AA compared with BME (Fig. 1, P < 0.05). For Arg and 2x AA, we added Rapa (20 µmol/L) to the media in paired samples to determine whether a portion of the protein synthetic response to Arg was mTOR/p70^S6k-dependent. Rapa nullified the increase in protein synthesis produced by 2x AA or Arg (P < 0.05).

View larger version (14K): [in this window] [in a new window]   FIGURE 1  In vitro protein synthesis of small intestine explants from fully fed piglets infected (n = 4) or uninfected (n = 4) with rotavirus on d 3 of infection and incubated in quadruplicate with amino acid treatments in vitro. Values are means ± SEM, n = 16. Means within infection status not sharing a common letter differ, P < 0.05.

View larger version (33K): [in this window] [in a new window]   FIGURE 2  In vitro levels of p70S6k, phosphorylated p70S6k, and actin from rotavirus-infected piglet jejunal segments. Small intestine explants from fully fed infected piglets on d 3 of infection were incubated with amino acid treatments. For each blot, the ratio of phosphorylated p70S6k:actin for tissue in BME was defined as 1. The upper panel is a typical blot. In the lower panel, densitometric values are presented. Values are means ± SEM, n = 4. *Different from BME, P < 0.05.

View larger version (115K): [in this window] [in a new window]   FIGURE 3  Immunohistochemical appearance of phosphorylated rpS6 (A) and total rpS6 (B) in the rotavirus-infected jejunal mucosa of piglets fed diets with and without Arg and Rapa. Representatives of 2 observations are shown. Antibodies to rpS6 and phospho-rpS6 yield a green color; DAPI staining for nuclei is blue, showing full-length villi.

During rotavirus and other small intestinal infections, gut permeability to normally nonabsorbable markers, such as lactulose and Cr-EDTA, is increased (15). Because intestinal permeability tests could reflect effects on either the small or large intestine, we performed in vitro testing using Ussing chambers to examine jejunal permeability. Feeding Arg to the piglets produced an increase in jejunal mucosal resistance, a sensitive indicator of intestinal integrity [P < 0.05; Table 2 (16)].

	Discussion

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 
    Rotavirus infection increases intestinal protein synthesis. A key finding from our previous investigations was that rotavirus infection produced a 2-fold increase in jejunal protein synthesis (4). There is abundant literature supporting increased mitogenesis within the crypt during rotavirus and similar experimental viral infections of the intestine (17,18). This evidence includes reports of crypt deepening [reflecting expansion of the proliferative compartment (19)], increased thymidine kinase activity (8), and increased proliferating cell nuclear antigen staining (18). However, we are unaware of previous studies that focused on gut protein synthesis. Because our previous work found that protein-energy malnourished piglets had a prolonged and more severe course of rotaviral diarrhea (2), we hypothesized that protein synthesis would be increased in the gut in a condition where enterocytes were proliferating, migrating up the villus, and differentiating. This differentiation process included the insertion of newly synthesized digestive enzymes and electrolyte and nutrient transporters into the apical membrane, as well as sodium-potassium-ATPase synthesis. Previous results from our group demonstrated that a diet supplemented with a high-quality protein source (plasma proteins) can prevent the development of diarrhea in rotavirus infected piglets (20).

    Effect of Arg and Rapa on clinical course of rotavirus enteritis. Before infection, all groups were gaining 100 g of body weight daily, but no groups gained weight during the first 2 d of infection. Control infected piglets and piglets receiving Arg had slight positive gains, but the Rapa-treated piglets lost weight over the 3-d interval and had significantly lower weight changes than the groups that did not receive Rapa. The study was terminated at the peak of infection, but our data suggest that in piglets with this relatively mild infection, clinical recovery was not improved by Arg. The one possible benefit would be an earlier improvement in gut permeability and possible subsequent (allergic) consequences of this increase.

    Effect of Arg on protein synthesis. Our studies were designed to determine whether dietary Arg supplementation would increase recovery of the intestine in rotavirus enteritis. In vitro "screening" studies examined responses to individual and combined amino acids in cultured intestinal cells. In previous cultured intestinal cell studies, Arg was found to be 1 of the 2 optimal amino acids to enhance the activation (by phosphorylation) of the protein synthesis regulator p70^S6k (5). In our studies, Arg again had optimal effects on protein synthesis. NEAA produced an effect that was intermediate to those of the BME control and Arg, but that did not differ significantly from the control (Fig. 1).

We also found that 0.1 mmol/L Arg (present in each of the media) was not enough to stimulate protein synthesis. Interestingly, BCAA without supplemental Arg and without proline, although adequate to stimulate phosphorylation of p70^S6k (Fig. 2), was not sufficient to stimulate intestinal protein synthesis. On the other hand, Arg at 4 mmol/L was sufficient to stimulate protein synthesis, even without proline, in the infected jejunum. As a possible mechanism, Arg may enhance protein synthesis via regulation of the mTOR/p70^S6k pathway. In addition, Arg may inhibit protein degradation, thereby increasing the net rate of protein synthesis (21).

Although Arg by itself was sufficient to stimulate protein synthesis in the infected piglet intestine, it was insufficient to stimulate protein synthesis in the jejunum from uninfected piglets. The basal medium to which Arg was added contained no Ser or Gly (Table 1). This is in contrast to the media with NEAA and 2x AA, to which the uninfected intestine responded with increased protein synthesis. We hypothesize that Ser and Gly, which are normally essential in the newborn piglet gut (14), were required for protein synthesis under control conditions. Enterocytes in the infected intestine may have been undergoing autophagic proteolysis, a process that provides amino acids for protein synthesis (22), and may have yielded sufficient quantities of free Ser and Gly to allow Arg stimulation of protein synthesis.

In the intestinal segments that we studied, only about half the stimulation of protein synthesis by 2x AA or Arg was inhibitable by Rapa (Fig. 1), demonstrating that the mTOR/p70^S6k pathway is only partially responsible for the protein-synthetic response to Arg. The addition of 4 mmol/L Arg to the culture medium was expected to yield physiological levels of nitric oxide (NO) by the jejunal explant because similar concentrations of Arg were present in the jejunal lumen of the milk-fed neonatal piglets (23). Pervin et al. (24) showed that NO upregulates the cyclin D1 level and ornithine decarboxylase, along with the mTOR and phosphorylation of p70^S6k. Therefore, both NO synthesis and mTOR/p70^S6k regulation may contribute to Arg-increased protein synthesis in jejunal explants.

    Arg enhances tissue resistance in rotavirus enteritis. Previous investigations by Isolauri et al. (25) and by Keljo et al. (26) found that jejunal permeability to a macromolecule is increased 9-fold in rat rotavirus and the very similar piglet transmissible gastroenteritis. This increase in permeability, while transient, is felt to contribute to the development of food allergies in humans following rotaviral diarrhea (27). At a cellular level, the virus disrupts tight junctions, reduces cellular ATP levels, and creates a redistribution of the tight junction proteins claudin-1, occludin, and ZO-1 (28). Our studies found that rotavirus-infected intestinal epithelial resistance (under short-circuit conditions) was lower than in the normal piglet intestine (Table 2). Our studies revealed a significant increase in tissue permeability after Arg treatment. Even changes as small as 5% in intestinal resistance, for example, in vitro in ovalbumin-sensitized intestine after ovalbumin challenge resulted in 3- to 4-fold increases in antigen flux (29). Further studies will be required to quantify the physiological importance of the Arg effect.

In our study, Rapa inhibited the Arg-mediated increase in protein synthesis but did not inhibit the Arg-mediated increase in the transepithelial resistance of the jejunum. One must hypothesize a mechanism of Arg independent of mTOR signaling for this effect. The most likely mechanism is NO signaling, consistent with work by Gookin et al. (16), which found that, in permeabilized intestine following bile salt injury, treatment with Arg plus serum produced an increase in piglet jejunal resistance. This return to normal transepithelial electrical resistance correlated well with histological restitution and was blocked by both nonspecific and specific inducible NO synthase inhibitors (16). In an infection model, the same investigators demonstrated that NO serves as a proximal mediator of prostaglandin E2 synthesis and barrier function in Cryptosporidium parvum enteritis (30). A similar activation of NO production by rotavirus infection (and specifically nonstructural protein 4) has been reported in human rotavirus infection (31).

In summary, we found a beneficial effect of Arg in stimulating protein synthesis via a p70^S6k-dependent mechanism and a beneficial effect in reducing transepithelial permeability by a p70^S6k-independent mechanism. In our ongoing studies, we have found that optimal intestinal cell migration in response to Arg required both p70^S6k stimulation (3) and NO formation (32). We have found that the BCAA Leu stimulated p70^S6k activation but not cell migration, whereas the NO donor deta NONOate was additive with Leu in stimulating migration (J. M. Rhoads and X. Niu, unpublished observations). In this study, although Rapa partially inhibited the stimulation by Arg, the effect of this inhibitor on overall protein synthesis was small, with no impact on gut morphology. We suggest that at least one other important intracellular regulator of protein synthesis (in addition to mTOR) is activated in viral diarrhea in the intestine. Possibilities include cyclin-dependent kinase-1/cyclin B (33), protein kinase B/Akt (34), and AMP-activated protein kinase (35). Alternatively, an inhibitory kinase, such as elongation initiation factor-2 kinase, could be deactivated (36). Arg or a precursor of Arg may be helpful in preserving intestinal resistance during viral diarrhea. Further time course studies are required to test this hypothesis.

	FOOTNOTES

 
¹ Supported by a grant (award no. 2003-35204-13262) from the USDA-NRI, CSREES.

² Author disclosures: B. A. Corl, J. Odle, X. M. Niu, A. J. Moeser, L. A. Gatlin, O. T. Phillips, A. T. Blikslager, and J. M. Rhoads, no conflicts of interest.

⁷ Abbreviations used: 2x AA, twice the normal concentration of amino acids; BME, basal medium Eagle; DAPI, 4',6-diamidino-2-phenylindole; mTOR, mammalian target of rapamycin; NEAA, nonessential amino acids; nitric oxide, NO; p70^S6k, p70 S6 kinase; Rapa, rapamycin; rpS6, ribosomal protein S6.

Manuscript received 6 June 2007. Initial review completed 19 June 2007. Revision accepted 12 September 2007.

	LITERATURE CITED

TOP ABSTRACT Introduction Materials and Methods Results Discussion LITERATURE CITED

 

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