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MUCOSAL BIOLOGY
1Department of Pediatrics, Ochsner Clinic Foundation and Ochsner Children's Research Institute, New Orleans, Louisiana; 2Department of Animal Sciences and 3College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina; 4Faculty of Nutrition, Texas A&M University, College Station; and 5Department of Pediatrics, University of Texas, Houston, Texas
Submitted 10 October 2006 ; accepted in final form 23 November 2006
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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mammalian target of rapamycin; diarrhea; virus; malnutrition
$10 million annually (36). In pigs, after acute viral injury to the surface epithelium of the villi, rapid restoration of epithelial continuity depends on the migration of uninjured cells to cover denuded sections of the basement membrane. At the cellular level, enterocyte migration is a proliferation-independent process that generally is completed within 6–12 h and is followed by crypt cell proliferation to regenerate the villi. Previous studies from our laboratories and others have shown that migration requires nitric oxide (NO), prostaglandins, and polyamines (7, 17, 40–42, 57). Furthermore, migration was stimulated by the amino acid L-arginine by a process requiring protein synthesis and NO formation from L-arginine (42). Migration was impaired by amino acid deprivation and serum starvation, modeling a condition called protracted diarrhea of infancy in the malnourished host (19, 34). Acute regulation of protein synthesis is achieved through changes in the rate of translation of mRNA via alterations in peptide chain initiation. A central mediator of this process is the mammalian target of rapamycin (mTOR). mTOR has been called a "nutrient sensor," capable of integrating amino acid and hormone levels with an organism's ability to survive (24, 48). mTOR activation results in the parallel phosphorylation and activation of two key mediators, p70 S6 kinase (p70S6K; which phosphorylates ribosomal protein p70) and 4E-binding protein-1 (4E-BP1). Peterson et al. (39) reported that mTOR enhances p70S6K phosphorylation indirectly by restraining a phosphatase. Phosphorylation of protein phosphatase 2A (PP2A) by mTOR in response to amino acids prevents the dephosphorylation of p70S6K, placing p70S6K in the activated state and activating the transcription of mRNAs with an oligopyrimidine tract that regulates ribosomal biogenesis (39).
mTOR is inhibited by individual and global amino acid starvation, and the macrolide rapamycin blocks p70S6K reactivation by amino acids (54). The current experiments were designed to determine if during piglet rotavirus enteritis, the jejunal protein synthetic rate and jejunal mTOR signaling via p70S6K would increase and if malnutrition would reduce the level of activation of p70S6K.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Rabbit anti-p70 S6 kinase antibody (A300-510) was purchased from Bethyl Laboratories (Montgomery, TX). Rabbit phospho-p70S6K (Thr421/Ser424) antibody (no. 9204) and rabbit S6 ribosomal protein antibody (no. 2212) as well as rabbit phospho-S6 ribosomal protein (Ser235/236) antibody (no. 2211) were from Cell Signaling Technology (Beverly, MA). Anti-actin antibody (A2066), protease inhibitor cocktail (P8340), and phosphatase inhibitor cocktails I (P2850) and II (P5726) were from Sigma (St. Louis, MO). Fluorophuor-conjugated donkey Alexa Fluor anti-rabbit IgG, anti-goat IgG (A-11057), 4',6-diamidino-2-phenylindole (DAPI; D1360), and Prolong Gold Anti-fade Reagent (P36930 [GenBank] ) were all from Molecular Probes (Eugene, OR). Immunoblot polyvinylidene difluoride (PVDF) membranes were from Bio-Rad (catalog no. 162-0174, Hercules, CA). The Enhanced Chemiluminescence plus Western Blotting Detection System (ECL Plus) was purchased from Amersham Pharmacia Biotech (RPN2132, Piscataway, NJ). [3H]phenylalanine was purchased from American Radiolabeled Chemicals (St. Louis, MO).
Animals and Experimental Design
All protocols were approved by the Institutional Animal Care and Use Committee of North Carolina State University. Pregnant sows were injected with 12.5 mg PGF2
(Dinoprost, Pharmacia Upjohn, Kalamazoo, MI) on day 112 of gestation to induce parturition. Piglets were collected directly from the birth canal, immediately cleaned with a 70% ethanol solution, and transported to a biosecure rearing facility. Piglets were housed individually within cages (length: 0.5 m; width: 0.3 m; and height: 0.4 m) contained in two rooms equipped with radiant heat sources to provide zonal temperatures of 32°C. Pigs were offered a milk diet via a gravity flow feeding system adapted from Oliver et al. (38). The composition of the full-strength formula exceeded piglet nutrient requirements (National Research Council, 1998) and was composed of commercial ingredients (Milk Specialties, Dundee, IL) as follows (in g/l): 93 dried skim milk, 45 Fatpack 80 (80% ether extract), and 12 vitamins/minerals. The dry matter content of the full-strength formula was 15% (vs. 7.5% for the malnourished groups; see below), and the calculated analysis (DM basis) was as follows: 31.1% crude protein, 36% lactose, 25% ether extract, and 4.6 Mcal/kg total energy. For the first 24 h, a liquid colostrum diet (LaBelle Associates, Bellingham, WA) was fed to provide passive immunity. Details of feeding rates were as reported by Zijlstra et al. (61). The fresh milk formula was offered 4 times/day (0800, 1300, 1800, and 2300 hour), and the milk reservoir and feeding apparatus for each pig were sanitized every 12 h. After 4 days of adaptation to the housing and feeding system, pigs were allocated to experimental treatments.
Extending our previously established malnutrition-rotavirus model (61, 62), in experiment 1, we used a total of 40 piglets distributed over 2 treatment regimens as follows: 1) control noninfected animals fed full-strength formula or 2) rotavirus-infected animals malnourished by feeding formula diluted 50% with electrolyte solution (1.91 g/l KCl, 0.15 g/l NaCl, and 1.65 g/l Na citrate). Intestinal samples were collected on days 0, 1, 3, 5, and 11 postinfection.
In experiment 2, we further compared well-nourished and malnourished piglets (n = 16) that were allocated to a 2 x 2 factorial treatment scheme examining the effects of malnutrition and viral infection as follows: 1) full-strength formula, noninfected (positive control); 2) full-strength formula, rotavirus infected; 3) malnourished (half-strength formula), noninfected; or 4) malnourished, rotavirus infected (negative control). Based on the results from experiment 1, intestinal samples from experiment 2 were collected only on day 3 postinfection.
Rotavirus Inoculation and Clinical Measurements
The rotavirus inoculum, originally isolated by Lecce et al. (27), was passaged through colostrum-deprived sentinel pigs and prepared as a bacteria-free intestinal supernatant. Rotavirus (107 particles) and sham inoculants were suspended in 20 ml of full-strength milk formula, and piglets were gastrically intubated at 1000 hours on day 4. Previous investigations used 2 x 108 particles, but this dose resulted in a high mortality rate in preliminary experiments. Therefore, in a pilot experiment, we lowered the dose down to 107 particles, which gave consistent infections with negligible mortality.
Animal weights and feed intakes were measured daily. Feces were given a consistency score of 0, 1, 2, or 3, indicating firm, soft but formed, runny, or severe watery diarrhea, respectively. Pigs were rectally swabbed with a cotton-tipped applicator daily for the detection of rotavirus shedding using a commercial latex agglutination assay (Virogen Rotatest, Wampole Laboratories, Cranbury, NJ).
Intestinal Sampling
On designated sampling days, pigs were anesthetized with isoflurane and killed by American Veterinary Medical Association-approved electrocution followed by exsanguination. A heparinized blood sample was collected at this time, and plasma was stored at –20°C for amino acid analysis (59). Intestinal samples from the midjejunum area were collected, snap-frozen, and stored at –70°C until analysis by Western blotting. Intestinal segments were collected and fixed for histological analysis of intestinal morphology and immunohistochemical staining (38). Additional segments for in vitro measurements of protein synthesis were collected and used immediately. The mucosa was scraped, homogenized in PBS (pH 7.4), and stored at –20°C until assayed. The midjejunum was chosen because our previous studies identified this site as the region of maximal rotaviral injury in this model.
Analytical Procedures
Intestinal morphology and lactase specific activity were measured according to the procedures previously described (38).
Protein Synthesis
Protein synthesis was measured in vitro using intestinal explants prepared fresh from harvested tissue collected in experiments 1 and 2. Explants (totaling 50 mg) were incubated in 25-ml Erlenmeyer flasks with 3 ml medium 199 containing 0.5 µCi [3H]phenylalanine for 2 h and maintained at 37°C in a shaking water bath. Following incubation, the tissue was removed from the flasks and immediately transferred to a tube containing 3 ml of 0.2 M perchloric acid. Explants were homogenized and centrifuged at 3,000 g for 20 min to separate proteins from free amino acids. The pellet was washed and centrifuged twice with 3 ml of 0.2 M perchloric acid and then incubated at 37°C for 90 min in 1.5 ml of 0.3 M sodium hydroxide to solubilize the protein pellet. Radioactivity was quantified by liquid scintillation counting, and protein synthesis calculated based on the specific activity of the radiolabeled phenylalanine in the media and the quantity found in the protein pellet.
Western Blot Analysis
The intestinal mucosa and muscle homogenates were prepared in RIPA buffer containing protease and phosphatase inhibitors. The protein concentration was determined using the Bio-Rad DC Protein Assay Kit (Bio-Bad) with BSA as the standard.
Determination of Phosphorylated p70S6K and Total p70S6K
Protein (40 µg) and an equal volume of 2x Laemmli sample buffer were combined; subsequently, samples were boiled for 5 min and electrophoretically separated by 7.5% SDS-PAGE. Protein was then transferred to a PVDF membrane with a semidry transfer cell. Membranes were incubated in blocking buffer (5% nonfat dry milk) in Tris-buffered saline (TBS; pH 7.6) plus 0.05% (vol/vol) Tween 20 (TBST) for 1 h at room temperature, followed by an incubation with p70S6K phosphoralyted antibodies (1:3,000) in blocking buffer overnight at 4°C. After being washed with TBST, blots were incubated with the appropriate peroxidase-conjugated secondary antibody for 1 h at room temperature, and visualization of the protein was done with ECL Plus. The membrane was stripped for 30 min at 55°C. After being blocked for 1 h, total p70S6K (1:3,000) and anti-actin were probed. Bands were detected with an enhanced chemiluminescence detection kit (ECL Plus, Amersham Biosciences). Quantitative data were obtained using a computer densitometer (Quantity One, Bio-Rad). Phosphorylated and total p70S6K measurements were normalized to 
-actin immunoreactivity.
Determination of Phosphorylated and Total Ribosomal Protein S6
We resolved proteins on a 15% acrylamide gel, followed by application of the appropriate primary antibody at 1:3,000 dilution, as described above. Results were normalized for -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% BSA in PBS for 15 min at room temperature. After being washed with PBS, slides were incubated for 90 min in 1% BSA in PBS containing primary antibodies at a 1:200 dilution. Slides were washed three 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. Slides were then stained with DAPI at 1:5,000 dilution in PBS for 2 min and subsequently mounted with Prolong Gold Antifade Reagent. Fluorescent images were captured with a Zeiss deconvoluting microscope at x10 magnification with a x60 immersion objective and with the use of Slidebook from Intellingent Imaging Innovations software (www.intelligent-imaging.com).
Statistical Analysis
Statistical comparisons were performed using ANOVA and t-tests for unpaired data (two-tailed) analysis (Statview4, Abacus Concepts, Berkely, CA) and using NCSS (www.ncss.com) for Wilcoxon signed-rank tests for paired analyses. Results are expressed as means ± SE, and P 
0.05 was considered statistically significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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The initial studies were designed to determine the effects of rotavirus enteritis on gut protein synthesis and mTOR activation in a worst-case scenario, when malnourished piglets were infected with live rotavirus particles in the first week of life. Zijlstra et al. (61) previously showed that this design produces a more prolonged and severe illness in piglets, lasting 16 days. Thus, malnourished subjects were pair fed 50% of the per kilogram diet of their fully fed counterparts by formula dilution with electrolyte solution (see METHODS). Forty piglets, in groups of eight piglets across five time points, pair-matched infected-malnourished and noninfected pigs, were studied at each time point.
Jejunal in vitro protein synthesis. Over a 2-h assay period, we compared the in vitro protein synthetic rate. We found that the infected jejunum had a significant increase in the protein synthesis rate, ranging from 50% to 200% higher than that in the jejunum from uninfected pigs (Fig. 1). Furthermore, despite the malnutrition, there was a steady increase in the infected jejunal protein synthetic rate, which peaked at day 5 postinoculation.
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68 kDa (Fig. 2A). There was considerable variability in the total p70S6K level with postnatal age in uninfected pigs (with more immunoreactivity at day 5 than earlier or later in the blot shown, reflecting the first litter of piglets). However, when averaged out with day 1 uninfected pig jejunal p70S6K normalized to a densitometric value of 1.0, there was a small but statistically insignificant decrease in signal up to day 11 in the noninfected pig jejunum. The most marked changes were increases in the jejunal levels of p70S6K and phosphorylated p70S6K at the peak of infection (days 3 and 5). Significant increases in p70S6K levels were seen on days 3 and 5 postinoculation (2- and 2.5-fold increases in level). These results were confirmed in duplicate and were also confirmed by immunohistochemistry (see below).
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Immunolocalization of p70S6K and phospho-p70S6K in normal and rotavirus-infected piglet jejunum. Viral enteritis has markedly different effects along the villus-crypt axis. For example, in piglets, villus tip cells are infected by the virus, whereas crypt cells undergo mitogenesis to regenerate the damaged villus tips (13, 22). Previous investigations of mTOR activation in the intestine used Western blot techniques (14). We are unaware of studies determining the histological site of local activation of mTOR in the diseased intestine. Using polyclonal antibodies to rat p70S6K, we found that the normal uninfected villus epithelium had very little immunoreactivity, whereas some immunoreactivity was found in the crypt epithelium and lamina propria of the villus core (subepithelial portion of the villus; Fig. 3A). We also found strong crypt cell immunoreactivity of the infected jejunal epithelium, and there was a highly significant increase in immunoreactivity of the infected tissue compared with control tissue (Fig. 3A). With respect to phospho-p70S6K, only scattered sites of crypt and villus core immunoreactivity were seen in uninfected jejunal tissues (Fig. 3B), whereas very strong staining was seen throughout the crypt epithelium and in small "patches" along the sides of some of the regenerating villi in infected tissues.
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Diarrhea and fecal rotavirus scores. We examined our clinical data to determine if the time of peak activation of mTOR coincided with the peak of the clinical illness. Compared with uninoculated piglets, which had soft stools (fecal scores of <1) throughout the study (Table 1), rotavirus-inoculated animals had diarrhea scores of 2–3, lasting from days 2 to 9 after inoculation, indicating loose-watery diarrhea. Diarrhea scores peaked on days 3 and 5 postinoculation, which corresponded to the peak activation of p70S6K by phosphorylation. Similarly, fecal rotavirus antigen scores peaked at days 3–5 postinoculation.
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1.5 kg over the 11-day study period, or an increase of >75% in body weight, without any change in p70S6K phosphorylation. The malnourished-infected group had a plateau in body weight after inoculation and dietary restriction (Table 1). Although none of the rotavirus-infected pigs died, the oral energy intake was reduced to 35% of normal in infected dilute milk-fed piglets (data not shown) for 1–2 days. Despite the reduced protein and calorie intake, the intestinal mTOR activation described above occurred in these malnourished piglets. Jejunal mucosal histological features and lactase activity. Although the studies above confirmed peak clinical symptoms at the time of protein synthesis activation in the infected gut, it was important to verify that peak intestinal epithelial damage occurred during the same interval. Histological examination of jejunal tissues showed the most severe villus blunting and altered villus-to-crypt ratio at days 3–5 (Table 2). Previously, our studies identified lactase as the most prominent disaccharidase marker of jejunal rotavirus infection, especially in the midjejunum (44). In the infected piglet jejunum, the current study showed a >50% decrease in jejunal lactase activity (P < 0.05; Table 2), with maximal blunting at day 5. Notably, these abnormalities persisted during the entire study period.
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Effect of Malnutrition Versus Full Feeding on Rotavirus Activation of mTOR: Experiment 2
Clinical response to infection. To separate the effects of malnutrition and infection on mTOR pathway activation during viral enteritis, we studied a separate group of piglets in a 2 x 2 factorial analysis. Control pigs fed full-strength formula with and without rotavirus infection were compared with 50% pair-fed malnourished piglets with and without rotavirus infection. Pigs were sampled on day 3 postinfection based on the results of experiment 1. Infected animals weighed significantly less than uninfected controls (Table 3). Diarrhea scores, rotavirus scores, and the percentage body weight reduction during infection were not significantly different when well-nourished infected and malnourished-infected subjects were compared (Table 3). Infected piglets on the control diet (see below) had a reduction to 50% of normal intake on the day of infection, which returned to normal values on day 6.
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Serum amino acid levels. We initially hypothesized that serum levels of branched-chain and other amino acids might be reduced in malnourished-infected animals and that the reduced circulating levels might correspond to the reduced level of activation in the infected bowel. When we found activation of the mTOR pathway by viral enteritis, we predicted that serum levels would not be significantly reduced in the malnourished-infected group. In infected-malnourished piglets, serum levels of certain amino acids were significantly increased. This finding pertained to five amino acids (serine, phenylalanine, glutamin, tryptophan, and alanine); only citrulline was decreased (Table 4). The total amino acid concentration increased only 29% in infected-malnourished pigs. We conclude that the increased activation of mTOR/p70S6K in the infected bowel was unlikely to be due to this minor change in the total circulating amino acid level.
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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Previous investigations of rotavirus enteritis have shown virus invasion and apoptosis at the tips of infected villi (9, 28). In the piglet model, we (44) have shown that the site of maximal injury is the midjejunum. At this site, rapid crypt cell proliferation characterized by increased thymidine kinase activity and crypt cell mitosis has been demonstrated (13). During this reparative phase of rotavirus infection, we are unaware of previous investigations of protein synthesis in the intestine. However, diarrheal disease has been shown to coexist with reduced protein synthesis in other organs, such as the heart (20). Our investigation showed a 50–100% increase in radioactive phenylalanine incorporation into intestinal protein at days 1, 3, and 5 postinoculation. By days 3–5 postinoculation, when the diarrhea was relatively severe, villus-to-crypt ratios were markedly altered to 2.5, or 50% of the normal ratio. Corresponding to the increase in protein synthesis was an increase in cell proliferation and expansion of the crypt. Our previous study (44) showed a sevenfold elevation in the intestinal level of thymidine kinase during the first 8 days of rotavirus inoculation, which decreased after this time point. Even in uninfected piglets, there was a less severe (50%) reduction in villus-to-crypt ratio during early postnatal life in uninfected piglets. Control piglets were highly unlikely to have had intestinal infection because they were without diarrhea and were raised artificially in our barrier-intact facility (43). Also supporting the concept that rotavirus infection led to a "proliferative" epithelium, villi of the infected piglet jejunum were populated by less-differentiated enterocytes, as demonstrated by the severe reduction in lactase specific activity (<10% of normal).
Intestinal p70S6K Is Activated During Rotavirus Enteritis
In this infected epithelium, there was a marked (>3-fold) increase in both p70S6K level and mTOR activation as determined by both the phosphorylation of p70S6K and of its downstream target ribosomal protein S6. The activation was most evident in immunohistochemical stains as a "light up" of phospho-p70S6K staining in epithelial cells lining the lower 20–25% of the villi. An increase in p70S6K abundance and activity occurred in the face of a significantly decreased mucosal mass, as evidenced by histological examination (Table 2) as well as by a reduced circulating level of citrulline, an amino acid that is synthesized exclusively in the intestine and serves as a marker of intestinal mass (Table 4) (46).
mTOR has been shown to regulate dietary effects on liver and muscle protein synthesis (1, 24, 24). In the intestine, increased activation of mTOR has been observed in colon cancer (16, 23). We believe that this is the first study to describe in vivo activation of this pathway in the gut during an infectious disease. In distinction to oncogenesis, the activation of this pathway in the infected intestine appears to be transient and tightly regulated, with activation for at least 5 days and a complete return to baseline by day 11 after infection. Although we have not investigated the mechanism(s) by which rotavirus activates mTOR signaling via p70S6K, there are three possiblities consistent with the published literature. First, rotavirus infection evokes a mobilization of intracellular calcium (36), a stimulator of intracellular p70S6K activation. A calcium increase during rotavirus infection is known to contribute to enterocyte chloride secretion via elaboration of nonstructural protein 4. An example is angiotensin II activation of p70S6K via the calcium-dependent tyrosine kinase PYK-2 in rat liver epithelial cells (18). A second possibility is that intracellular rotavirus particles produce a "stress signal" similar to ultraviolet irradiation, which activates p70S6K along with JNK and ERK1 (60). Similar stress signaling has been reported with NO donors that modulate cell motility (5). Finally, when infected villus tip cells are sloughed, the migration process itself could trigger p70S6K activation. This has been shown during cell spreading across the mammalian lectin galectin-8 (30), and p70S6K has been shown to colocalize at the edge of migrating cells with phosphatidylinositol 3-kinase, actin, Akt1, and activated mTOR (6). It should be noted that, regardless of the mechanism, the reparative response to infection had a dominant effect compared with the level of feeding. In fact, rotaviral activation of p70S6K appeared equal in malnourished compared with well-nourished piglets. However, activation by infection is probably not maximal. In unpublished studies, we were able to show in intestinal explants that p70S6K from infected pigs can be activated even further by arginine or high-dose amino acids.
One interesting observation is that some viruses, such as adenovirus and rotavirus, are capable of producing proteins that mimic components of the mTOR signaling pathway and "hijack" the translational machinery (37, 56). However, rotavirus' main site of infection is the villus tip cells (10, 29), whereas we found that mTOR is activated primarily in the crypt. Furthermore, viral shedding was still present at day 11, even though p70S6K was not phosphorylated. This finding rules against viral hijacking of the translational apparatus as the major etiology activation of mTOR that was seen at days 3 and 5 postinoculation, because the virus was still being excreted at a time point (day 11) when intestinal p70S6K was not phosphorylated. It should also be noted that the latex agglutination assay we used is likely to underestimate the amount of viral excretion.
Activation of this pathway appears predominant in the crypt region of the regenerating intestine. The crypt is the site of epithelial progenitor cell mitosis, a necessary event in villus cell regeneration (Fig. 3) (9). High-power microscopy of this site revealed a cytoplasmic distribution of p70S6K. Activation by phosphorylation in the autoinhibitory loop (Thr421/Ser424) was seen in the nucleus of cells on shortened villi. We (45) have previously found nuclear phosphorylation in arginine-treated intestinal cells in culture. Although only recently published, nucleocytoplasmic shuttling has been observed for the -isoform of p70S6K(one of two S6K isoforms) after phosphorylation by PKC (55). Strong evidence for similar shuttling of mTOR has been presented, with evidence that directed mutation of the nuclear import sequence impaired activation of p70S6K, whereas the structure of mTOR was not changed (3). The direct mediator of p70S6K activation might be S6K-1-associated REF-like target, a nuclear protein that binds p70S6K, is sensitive to rapamycin, impacts cell size, and is a member of the REF family involved in mRNA export (20, 47).
Our findings are not consistent with rotavirus-activated intestinal mTOR signaling resulting from an increase in circulating amino acid levels. We observed a marginal (29%, P = 0.05) increase in total amino acids that was not associated with any significant change in the two major amino acids that activate mTOR/p70S6K in intestinal cells: branched-chain amino acids and L-arginine (4).
Viral Enteritis Increases Serum Glutamine and Gluconeogenic Amino Acid Levels
The small intestine is a major site for the catabolism of glutamine and the exclusive organ for its conversion into citrulline in the piglet (58). Thus, in rotavirus-infected piglets, a marked increase in the plasma glutamine level and a concomitant decrease in the citrulline level indicates reduced intestinal utilization of glutamine due to a compromised number of villus cells. In these piglets, an increase in plasma levels of all three major gluconeogenic amino acids (serine, alanine, and glutamine) suggests an increase in their release from skeletal muscle to synthesize glucose so as to meet the increased need for glucose by cells of the immune system, including lymphocytes and macrophages (15). Finally, an increase in plasma concentrations of both aromatic amino acids (tryptophan and phenylalanine), whose catabolism requires the same unique biopterin cofactor (53), suggests a relative deficiency of tetrahydrobiopterin for their utilization, likely due to an increase in its utilization for NO synthesis.
Relative Effects on p70S6K Activity in the Muscle and Intestine
To determine if there was differential regulation of the mTOR pathway in the muscle and intestine during the catabolic state induced by rotavirus infection, we performed Western blot analysis using antibodies to total and phosphorylated ribosomal protein S6 (Fig. 5). We showed a dramatic effect of the infection in upregulating intestinal ribosomal protein S6 phosphorylation while downregulating skeletal muscle ribosomal protein S6 phosphorylation. A steady decrease in the muscle ribosomal protein S6 level (in all piglets, whether uninfected and infected) was seen beginning at day 5 (corresponding to day 10 of life), which may correspond to a decline in the very rapid protein synthesis rate seen in the first week of life (14). The observed heavy phosphorylation of muscle ribosomal protein S6 was severely inhibited during rotavirus infection (Fig. 5). We hypothesize that rotavirus infection markedly inhibits this process.
Both acute and chronic bacterial septicemia inhibit muscle protein synthesis and enhance muscle protein breakdown, concomitant with increased intestinal and liver protein synthesis (11). Furthermore, endotoxemia markedly alters mTOR pathway signaling and translation initiation in pigs (25) and rats (26). During viral enteritis, hormonal or other signals from the gut to muscle have not been investigated but could include cytokines such as IL-6, IL-1, TNF-, IL-12, IL-10, and IFN-
(2, 21); reactive oxygen species; glucocorticoid hormones [which are among the best-established inhibitors of mTOR (50, 51)]; and the virus itself (8, 12). Among these, the best-recognized inhibitors of protein synthesis and mTOR in inflammatory diarrheas are glucocorticoid hormones (32, 48, 49, 52), which have been shown to inhibit the translational pathway below the level of Akt (most likely at mTOR itself) (31, 33).
In summary, our investigation demonstrated that infectious diarrhea produced an increase in intestinal protein synthesis early after viral inoculation. The increased protein synthesis appears to have preceded intestinal p70S6K activation by 1–2 days. We suggest that p70S6K is not a sensitive marker of level of nutrient intake (because it is not affected by formula restriction) but represents a part of the proliferative response to rotavirus enteritis. We suggest that the mTOR/p70S6K pathway is markedly activated in viral enteritis, as it is in other conditions reflecting increased mitotic activity, e.g., oncogenesis. In a recent microarray study (35), gene expression profiling of the intestinal epithelial cells along the crypt-villus axis was performed. Genes regulating ribosomal biogenesis and RNA processing were upregulated in the crypt and downregulated at the villus tip. The activation of mTOR signaling to p70S6K is not generalized in the infected host, as evidenced by muscle p70S6K inactivation at the same time that jejunal p70S6K is activated. Our observations lead to the testable hypothesis that mTOR signaling is mechanistically important to coordinate the reciprocal regulation of muscle and intestinal protein synthesis during diarrheal disease. Clinically, the findings may be relevant to the nutritional therapy of diarrheal disease. Specifically, during acute viral diarrhea, supplementation with high levels of one or more amino acids could facilitate bowel repair and/or mitigate muscle wasting.
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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