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Article

Luminal Amino Acids Acutely Decrease Intestinal Mucosal Protein Synthesis and Protease mRNA in Piglets^{1 ,2}

Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, AB, T6G 2P5 Canada and ^* Faculty of Agricultural Sciences, The University of British Columbia, Vancouver, BC, V6T 1Z4 Canada

⁴To whom correspondence and reprint request should be addressed.

	ABSTRACT

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Because parenteral feeding is associated with negative N balance and reduced rates of protein synthesis in intestinal mucosa, we hypothesized that luminal exposure to specific amino acids or energy fuels would stimulate intestinal protein synthesis. We studied the acute effects of luminal nutrients on mucosal protein synthesis in the absence of systemic influences. Multiple jejunal segments constructed in piglets deprived of food overnight (n = 6) were randomly assigned to luminal perfusion with saline, 30 mmol/L amino acid mixture with or without 50 mmol/L glucose, or 30 mmol/L glutamine for 90 min. Protein synthesis was then measured by luminal perfusion with L-[2,6-³H]-phenylalanine. Energy substrates (glucose, short-chain fatty acids or ß-hydroxybutyrate) had no effect on mucosal protein synthesis. Relative to saline, a 30 mmol/L amino acid mixture or 30 mmol/L glutamine suppressed mucosal protein synthesis by 20–25% (P < 0.05). On the basis of these surprising results, we speculated that a coordinate reduction of proteolytic processes would be required to maintain positive intestinal N balance. Although intestinal protein catabolism cannot be assessed directly, the 30 mmol/L amino acid mixture acutely suppressed mucosal levels of mRNA encoding ubiquitin, 14-kDa ubiquitin conjugating enzyme and the C9 subunit of the proteasome by 20–30% (P < 0.05), demonstrating the sensitivity of components of the ATP-ubiquitin proteolytic pathway to acute regulation by nutrients. The suppression of protein synthesis by luminal amino acids in the absorptive state might lower intestinal utilization of amino acids to ensure efficient allocation of absorbed nutrients to nonintestinal tissues.

KEY WORDS: • pigs • intestinal mucosa • protein synthesis

	INTRODUCTION

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The small intestine is an important organ, contributing 9–12% of daily whole-body protein synthesis (Attaix and Arnal 1987 , Simon et al. 1982 ). Intestinal protein synthesis responds to feeding and to protein in the diet. Deprivation of food for 10 h to 5 d decreased mucosal or whole intestinal fractional protein synthesis rate (Ks)⁵ by 20–30% relative to the fed state (Burrin et al. 1991 , McNurlan et al. 1979 , Samuels et al. 1996 ). Others have shown that feeding protein-restricted diets for 1–8 wk led to a fall in mucosal Ks (McNurlan and Garlick 1981 , Wykes et al. 1996 ).

A unique aspect of intestinal protein synthesis is that it is sensitive to the route of nutrient supply. Total parenteral nutrition (vs. enteral feeding) is associated with negative N balance in the intestine, as a result of suppressed fractional and absolute rates of protein synthesis (-30 to -40%) (Dudley et al. 1998 ). Although variations in the composition of total parenteral nutrition such as the addition of glutamine have been shown to improve intestinal weight, DNA content and protein synthesis, these are not restored to levels seen in enterally fed animals (O'Dwyer et al. 1989 , Stein et al. 1994 ). These results suggest that protein synthesis in the intestinal mucosa might be sensitive to luminal exposure with specific nutrients, as distinct from the same nutrients appearing systemically as a consequence of absorption or intravenous administration. We hypothesized that amino acids would stimulate protein synthesis on luminal delivery, in a manner that would account for the difference in protein synthesis seen in enterally vs. parenterally fed animals. Because protein synthesis is an energy-consuming process, we further hypothesized that glucose, glutamine, ketone bodies and short-chain fatty acids (SCFA), the energy fuels of mucosal cells (Windmueller and Spaeth 1980 ), might also stimulate protein synthesis.

Earlier studies of intestinal protein synthesis were generally feeding trials that did not distinguish the direct effects of luminal nutrient exposure from the contribution of nutrients, hormones and growth factors released systemically consequent to nutrient consumption. Feeding results in increased plasma concentrations of the fed substrates, of metabolites thereof and of hormones such as insulin, insulin-like growth factor 1 and glucagon-like peptides. We recently developed and validated a system that allows study of the effects of luminal nutrients in the absence of systemic influences (Adegoke et al. 1999 ). In this experimental system, luminal perfusion of short intestinal segments comprising <4% of total absorptive surface area did not alter plasma concentrations of perfused nutrients or insulin. We have now used this system to study the direct effects of luminal nutrients on mucosal protein synthesis.

These studies generated the surprising observation that protein synthesis in the jejunal mucosa was significantly inhibited by luminal exposure to amino acids. In these circumstances, a coordinate reduction of proteolytic processes would be required to maintain positive intestinal N balance. To test this hypothesis would require a direct method of estimating intestinal proteolysis; unfortunately, none is available. However, in a recent study with rats, mRNA levels of m-calpain, of lysosomal cathepsin B, and of components of the ATP-ubiquitin-proteasome proteolytic system were seen to increase in parallel with the elevated protein loss observed in intestinal tissue after 1- or 5-d food deprivation (Samuels et al. 1996 ). We also used this indirect approach involving measurement of mRNA encoding proteases and other elements of intestinal proteolytic systems. Thus, we examined the effects of luminal amino acid perfusion on the mRNA levels of m-calpain, ubiquitin, 14-kDa ubiquitin conjugating enzyme (E2), and the C8 and C9 subunits of the proteasome.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS Experiments RESULTS DISCUSSION REFERENCES

 
Chemicals.

L-[2,6-³H]Phenylalanine (2.15 TBq/mmol; radiochemical purity, 99.6%) was purchased from Amersham International (Amersham Place, Little Chalfont, Bucks, UK). Other chemicals were from Sigma Chemical (St. Louis, MO). Tubing for intestinal cannulation was from Fisher Scientific (Pittsburgh, PA).

Experimental system.

We have described and validated a system for studying the effects of acute luminal nutrient exposure on intestinal mucosal protein synthesis [described in detail in Adegoke et al. (1999) ]. Mucosal Ks values determined after intravenous or luminal administration of a flooding dose of ³H phenylalanine were identical. With the use of luminal administration of tracer, we found that four jejunal segments constructed within an animal (Fig. 1 ) had similar mucosal specific radioactivities of free phenylalanine and fractional rates of protein synthesis. Because the four segments in total comprised <4% of the absorptive surface of the small intestine, luminal perfusion with amino acids and glucose did not raise plasma levels of these perfused nutrients or of insulin. These results support the idea that each of the four segments within an animal can serve as an experimental unit so that the effects of different luminal treatments on mucosal protein synthesis can be tested.

View larger version (19K): [in this window] [in a new window]   Figure 1. Schematic representation of perfusion of multiple jejunal segments from piglets deprived of food overnight. Four 6-cm jejunal segments were cannulated in situ at both ends (inlet at the pyloric end, outlet at the ileal end) with polyethylene tubing. The inlet cannula for the first segment was inserted 15 cm from the ligament of Treitz, and successive segments were separated by 50 cm of intestine. Each segment was independently perfused at a flow rate of 3 mL/min. Each segment received a control or test perfusate, followed by a 15-min perfusion of the same composition also containing 3H-phenylalanine for determination of protein synthesis.

All experiments were performed in accordance with the Canadian Council on Animal Care Guidelines and were authorized by the institutional Animal Policy and Welfare Committee. Male piglets (6 wk old; Camborough x Canabrid Pig Improvement Company crosses) were obtained through the University of Alberta Health Sciences Laboratory Animal Services. Piglets were weaned at 4 wk and maintained on a wheat/oatgroat-soybean/whey powder starter diet (crude protein 205g/kg; digestible energy 15.07 kJ/g). Animals were food deprived overnight before experimentation but water was available at all times.

Intestinal perfusion was done as described previously (Adegoke et al. 1999 ). Briefly, under halothane anesthesia, a midline incision on the abdomen was made and four 6-cm jejunal segments were cannulated at both ends (inlet at the pyloric end, outlet at the ileal end) with polyethylene tubing. The inlet cannula for the first segment was inserted 15 cm from the ligament of Treitz, and successive segments were separated by 50 cm of intestine (Fig. 1) . Cannulated segments were rinsed of digesta remnants with warm PBS (126 mmol/L NaCl, 14.1 mmol/L Na₂HPO₄ 1.0 mmol/L NaH₂PO₄8729 · H₂O, pH 7.4, 300 mosmol/L). Intestinal segments were kept moist by spraying with warm PBS; they were covered with PBS-soaked gauze and transparent polyethylene to reduce evaporation. Solutions, at 37.5 ± 0.5°C, were perfused at 3 mL/min.

In all experiments, the four segments within a piglet were perfused independently but simultaneously (Fig. 1) . Experimental treatments were randomized to the four intestinal segments within each animal. Statistical analysis by ANOVA with segment position as an independent variable revealed no significant effect of segment position (P = 0.68) in any experiment. Four to six piglets were used in each experiment (see legends to Tables and Figures).

	Experiments

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Effects of different luminal nutrients on intestinal mucosal protein synthesis.

The four intestinal segments within a piglet were perfused for the indicated times with PBS, of the composition given above, or different nutrient solutions, including 30 mmol/L glutamine or 30 mmol/L amino acids with or without 50 mmol/L glucose. The pH of all perfusates was 7.4. All perfusates were made isoosmotic to a level typical of jejunal digesta in piglets (300 mosmol/L). The PBS was 300 mosmol/L; when nutrients were added to perfusates, the osmolarity contributed by the PBS was reduced correspondingly such that the total was 300 mosmol/L. The amino acid mixture, with or without 50 mmol/L glucose, was chosen to simulate some of the components of a meal, and was formulated on the basis of published composition of jejunal digesta (Adibi and Mercer 1973 , Ferraris et al. 1990 , Low 1979 ). This amino acid mixture contained (mmol/L) aspartate (0.67), serine (2.03), glutamate (2.34), glutamine (1.84), proline (3.15), glycine (3.75), alanine (2), cystine (0.3), tyrosine (0.79), histidine (0.59), arginine (1.29), asparagine (0.73), threonine (1.34), valine (1.66), methionine (0.55), isoleucine (1.14), leucine (2.09), phenylalanine (2), lysine (1.58) and tryptophan (0.19). A treatment with glutamine was included because this amino acid is a preferred fuel of intestinal mucosal cells (Windmueller and Spaeth 1980 ), and studies have shown the importance of glutamine in stimulating intestinal growth and protein synthesis under different pathophysiologic conditions (O'Dwyer et al. 1989 , Stein et al. 1994 ).

In Experiment 2, the effects of energy substrates were investigated. Four intestinal segments within each piglet were perfused with PBS, 50 mmol/L glucose, 50 mmol/L mixture of SCFA (in the ratio 60:12.5:7.5, for acetate, propionate and butyrate, respectively) or 20 mmol/L ß-hydroxybutyrate (ß-OH-butyrate). We selected glucose, glutamine, SCFA and ß-OH-butyrate as energy substrates on the basis of the work of Windmueller and Spaeth (1978). Concentrations of substrates in piglet digesta including glucose (50 mmol/L)(Ferraris et al. 1990 ), glutamine (30 mmol/L)(Weber et al. 1982 ) and SCFA concentrations (30 mmol/L) (Bergman, 1990 ) have been reported, and these were used as a basis for selection of perfusate concentrations. We selected a concentration of ß-OH-butyrate within this range.

In Experiment 3, we examined whether the effects of amino acids were concentration dependent. Four intestinal segments within a piglet were perfused with PBS, or with the amino acid solution described above diluted 2x (15 mmol/L), 4x (7.5 mmol/L) or 8x (3.75 mmol/L). Perfusion was for 40 min because results from Experiment 1 showed that the effect of amino acids was already maximal at 40 min.

On the basis of previous work showing the importance of glutamine in intestinal metabolism, Experiment 4 was conducted to examine whether glutamine alone could account for the observed effects of the amino acid mixture. To do this, protein synthesis in intestinal segments perfused with 1.8 mmol/L glutamine (corresponding to the concentration of glutamine in the complete amino acid mixture) was compared with that in segments perfused with PBS, or a 30 mmol/L mixture of the complete amino acid solution. A further comparison involved segments perfused with an amino acid mixture in which glutamine and its potential metabolites (glutamate, arginine and proline)(Wu 1998 ) had been deleted. To keep the concentration of this mixture at 30 mmol/L, we raised the concentrations of glycine (6.62 mmol/L), alanine (4.88 mmol/L) and serine (4.90 mmol/L). We selected those amino acids that seemed unlikely to elicit modifications of intestinal protein synthesis. As seen in Table 1 and Figure 4 , when tissue alanine concentrations were doubled by the addition of 1.8 mmol/L glutamine to the perfusate, there was no effect on protein synthesis. Serine and glycine are metabolized to a limited extent in intestinal tissue to N⁵,N¹⁰-methylene tetrahydrofolate and glutathione (Wu 1998 ), two factors whose expected effect, if any, would be to raise rather than suppress protein synthesis.

View larger version (33K): [in this window] [in a new window]   Figure 4. Amino acid mixtures and glutamine modify mucosal fractional protein synthetic rates. Piglet jejunal segments were luminally perfused with one of four solutions as follows: PBS, 30 mmol/L amino acid mixture lacking glutamine, arginine, glutamate and proline (amino acids without glutamine), 1.84 mmol/L glutamine (the concentration of the amino acid in the complete amino acid mixture), or 30 mmol/L amino acid mixture (amino acids with glutamine). After perfusion for 40 min, protein synthesis was measured as described in Materials and Methods. Ks in control (PBS) segment was 51 ± 4%/d. Values are means and pooled SEM, n = 6. Bars with different letters are significantly different (P < 0.05).

After perfusion of intestinal segments with desired substrates for the stated times, protein synthesis was measured by the luminal flooding dose (Adegoke et al. 1999 ). Briefly, the intestinal segments were emptied and refilled with the test solutions also containing 2 mmol/L L-[2,6-³H]phenylalanine (specific activity, 42 kBq/nmol). Perfusion continued for another 15 min. Segments were then removed, emptied, flushed with ice-cold saline and rinsed in two changes of cold saline. Mucosa was scraped onto an ice-cold surface, frozen in liquid N₂ and stored frozen at -50°C until analyzed. In the experiment in which Northern hybridizations were done, samples were stored at -80°C. After the removal of perfused intestinal segments, piglets were killed by cardiac injection of Euthanyl (pig: 1 mL/kg body weight; MTC Pharmaceuticals, Cambridge, Canada).

Sample processing and analysis.

Approximately 300 mg of mucosa were powdered in liquid N₂ and then homogenized in 3 mL of ice-cold 2% perchloric acid. Samples were then centrifuged at 2800 x g for 15 min. Supernatants were collected and neutralized with a half-volume of saturated potassium citrate and stored frozen until ready for analysis. Pellets were washed four times with 8 mL ice-cold 2% perchloric acid, followed by centrifugation at 3000 x g. Washed pellets were hydrolyzed in 5 mL of 6 mol/L HCl at 110°C for 24 h. Excess HCl was dried off under vacuum.

In the conventional intravenous flooding dose technique, injected ³H-phenyalanine is metabolized to other compounds; to prevent overestimation of specific radioactivities, phenylalanine is converted to ß-phenethylamine before analysis. In agreement with previous observations that catabolism of phenylalanine by intestinal tissue is minimal (Goodwin 1979 ), we confirmed that when the intestinal lumen was the source of tracer, there was no detectable conversion of ³H-phenylalanine to other metabolites within either the mucosal free or protein-bound phenylalanine pools (Adegoke et al. 1999 ). The conversion to ß-phenethylamine was therefore omitted. Phenylalanine concentration was determined by reversed-phase HPLC using precolumn derivation with o-phthaldehyde as described previously (Samuels and Baracos 1995 ). [³H]-Phenylalanine radioactivity was counted using a Beckman LS 5801 scintillation counter (Beckman Instruments, Mississauga, Canada). Fractional rate of protein synthesis (Ks), expressed as percentage per day, was calculated according to McNurlan et al. (1979) as follows:

where S_b is the specific radioactivity of protein-bound phenylalanine, t is the duration of isotope perfusion, S_f is the intracellular free phenylalanine specific radioactivity in tissue samples.

Northern hybridization.

Total RNA was isolated from mucosal samples with Trizol Reagent (Life Technologies, Burlington, Canada) according to the manufacturer's instructions. Total RNA (15 µg) was electrophoresed in 1% agarose-formaldehyde gels containing ethidium bromide. Gels were run at 100 V for 5 h. RNA was checked visually for integrity of 28S and 18S ribosomal RNA. RNA was transferred to nylon membranes (GeneScreen, NEN, Boston, MA) by capillary transfer and cross-linked to membranes under UV light at 1200 mJ using Stratalinker (Stratagene, La Jolla, CA).

Membranes were hybridized with a cDNA sequence encoding rat 14-kDa ubiquitin carrier protein E2 (14-kDa E2) (Wing and Banville 1994 ), polyubiquitin (Agell et al. 1988 ), C8 and C9 proteasome subunits (Kumotori et al. 1990 , Tanaka et al. 1990 ) and m-calpain (Imajoh et al. 1988 ). Membranes were prehybridized at 65°C for 2 h and then hybridized overnight at 65°C with ³²P-labeled cDNA probes prepared by the random-priming method as previously described (Medina et al. 1995 ). After hybridization, membranes were washed four times for 15 min in 0.1% SDS; the stringency of the washes was varied between 1X and 2X SSC, depending on the probe. For 14-kDa E2 hybridization, membranes were autoradiographed for 24–48 h at -70°C with intensifying screens on X-OMAT-AR film (Kodak, Rochester, NY). Blots were quantified with BioRad Imaging Densitometer or Phosphorylimager (Molecular Dynamics, Sunnyvale, CA). Northern hybridization analysis for ubiquitin, the C8 and C9 subunits of the proteasome and m-calpain was carried out as described previously (Samuels et al. 1996 ). Differences in RNA loading were corrected for by quantifying the 18S RNA band using a Bio-Rad Imaging Densitometer (Bio-Rad, Hercules, CA). All membranes were stripped and reprobed with ³²P-labeled cDNA probe for glyceraldehydephosphate dehydrogenase (GAPDH) (Medina et al. 1995 ).

Protein synthesis data (means ± SEM) are expressed as percentages of the rate of protein synthesis in segments perfused with PBS and were analyzed using a two-way ANOVA with treatment and perfusion segment as independent variables (SAS, Version 6.02, SAS Institute, Cary, NC). There was no significant effect of perfusion segment in any experiment. The effect of amino acid mixture (vs. PBS) on the levels of mRNA encoding elements of proteolytic systems was examined using two-way ANOVA with piglets as the blocks. Significant differences (P < 0.05) among means were examined using Fisher's protected least significant difference test.

	RESULTS

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Luminal nutrients and mucosal protein synthesis.

Free phenylalanine specific radioactivity did not differ among segments, regardless of the luminal perfusate applied (PBS, amino acids plus glucose, glutamine; Fig. 2 A). The different nutrient solutions suppressed mucosal protein synthesis by 20–25% relative to PBS (Fig. 2 B, P < 0.05). This was observed whether the amino acid mixture was perfused for 40 or 90 min. Lower concentrations of the 30 mmol/L amino acid mixture did not significantly affect mucosal Ks (relative to PBS, Ks were 91 ± 2, 89 ± 2 and 97 ± 2% in segments perfused with 3.75, 7.5 and 15 mmol/L amino acid mixture, respectively, n = 4, P > 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 2. Mucosal free phenylalanine specific radioactivity (Panel A) and Ks (Panel B), after luminal perfusion of piglet intestinal segments with nutrient solutions. Data are expressed as percentages of control (PBS) solution. The four intestinal segments within each piglet were perfused with PBS or 30 mmol/L amino acid mixture + 50 mmol/L glucose (amino acids + glucose) or 30 mmol/L glutamine for the indicated times. Mucosal protein synthesis in perfused segments was then measured as described in Materials and Methods. Ks in control (PBS) segment was 42 ± 2%/d. Values are means ± pooled SEM, n = 6. Bars with different letters are significantly different (P < 0.05).

To examine the contributions of energy substrates to the observed effects of the amino acid-glucose mixture on mucosal Ks, the effects of 50 mmol/L glucose or SCFA, or 20 mmol/L ketone bodies were studied. The various energy substrates had no significant effect on mucosal free phenylalanine specific radioactivity (not shown), or Ks (Fig. 3 ).

View larger version (18K): [in this window] [in a new window]   Figure 3. Luminal perfusion of energy substrates into multiple jejunal segments of piglets does not influence mucosal Ks. Data are expressed as percentages of control (PBS) solution. The four intestinal segments within each piglet were perfused with PBS, glucose (50 mmol/L), short-chain fatty acids (SCFA; 50 mmol/L) or ß-hydroxybutyrate (ß-OH-butyrate; 20 mmol/L). After perfusion for 40 min, protein synthesis was measured as described in Materials and Methods. Ks in control (PBS) segment was 39 ± 2%/d. Values are means and pooled SEM, n = 4.

The results of Experiment 1 showed that, compared with PBS, 30 mmol/L glutamine perfusion significantly (P < 0.05) increased mucosal free intracellular concentrations of glutamine (310 vs. 10548 nmol/g, pooled SEM = 644), glutamate (1849 vs. 4372 nmol/g, pooled SEM = 200), and arginine (126 vs. 207 nmol/g, pooled SEM = 24). Perfusion experiments were conducted to examine whether the observed effects of glutamine or the amino acid mixture perfusion on mucosal protein synthesis were due to these changes. When the amino acid mixture lacking glutamine, glutamate, proline and arginine was perfused, changes in the intracellular concentrations of these amino acids induced by perfusion with the complete amino acid mixture or glutamine alone were prevented (Table 1) . Changes in mucosal amino acid concentrations induced by perfusion with the different amino acid solutions, however, did not correlate with changes in mucosal protein synthesis. The suppressive effect of amino acids on protein synthesis was seen whether glutamine (along with glutamate, arginine and proline) was or was not included in the amino acid mixture (Figure 4 ). Furthermore, although glutamine alone at its concentration in the complete amino acid mixture (1.8 mmol/L) increased free intracellular levels of glutamate and glutamine, it had no effect on protein synthesis relative to saline (Table 1 and Fig. 4 ).

Perfusion of intestinal segments with the 30 mmol/L amino acid mixture increased tissue ammonia content and decreased Ks by 26% (Table 2 ). The addition of ammonium chloride alone to the perfusate at 0.5 or 1.0 mmol/L increased tissue ammonia concentrations to the levels seen with the amino acid perfusion, but did not significantly influence Ks.

Our finding that luminal amino acids suppressed protein synthesis relative to a saline control was surprising. We speculated that this change must be associated with a parallel reduction in proteolytic processes for N balance to be maintained. Therefore, the effects of the complete amino acid mixture (30 mmol/L) or PBS perfusion on mucosal mRNA levels of m-calpain, and of components of the ATP-ubiquitin-proteasome proteolytic pathway were examined. Northern hybridization analyses were done for m-calpain, ubiquitin, 14-kDa E2, known to be involved in ubiquitin conjugation of substrates for proteolysis (Wing and Banville 1994 ), and for the C8 and C9 subunits of the 20S proteasome, the proteolytic core of 26S proteasome (Attaix et al. 1998 ).

As previously reported for rat intestine (Samuels et al. 1996 ), two ubiquitin transcripts of 2.6 and 1.2 kb were detected. Densitometric analysis on both bands showed that intestinal mucosal total ubiquitin mRNA level was decreased 28% by luminal amino acid perfusion (P < 0.05, Fig. 5 ). Luminal nutrient perfusion also significantly decreased the expression of the 1.2-kb transcript of the 14-kDa ubiquitin conjugating enzyme by 20% (Fig. 5 , P < 0.05). This is the transcript whose expression has been shown by others to be responsive to nutritional and endocrine regulations (Wing and Banville 1994 ). In parallel to mRNA levels of ubiquitin and 14 kDa E2, expression of the C9 subunit of the proteasome was also decreased by 30% by luminal amino acids (Fig. 5 , P < 0.05). There were no effects of luminal treatments on mRNA levels of GAPDH or the C8 subunit of the 20S proteasome (6.5 ± 1 and 7.8 ± 1 arbitrary densitometric units for PBS and amino acid solution, respectively, n = 8, P > 0.05). Finally, luminal PBS or amino acid perfusion had no effects on mucosal m-calpain expression (10.7 ± 1 and 10.8 ± 1 arbitrary densitometric units for PBS and 30 mmol/L amino acid solution, respectively, n = 8, P > 0.05).

View larger version (22K): [in this window] [in a new window]   Figure 5. Luminal amino acids decrease mucosal levels of mRNA encoding components of the ATP-ubiquitin-proteasome proteolytic pathway. One of two intestinal segments within each piglet was perfused with PBS or 30 mmol/L mixture of the 20 amino acids. Total mucosal RNA was separated on agarose gels, transferred to nylon membrane and hybridized with 32P-labeled cDNA encoding ubiquitin, 14-kDa ubiquitin carrier protein E2 (14-kDa E2), the C9 subunit of 20S proteasome and GAPDH. Data refer to densitometric signals of all transcripts (ubiquitin), 1.2-kb transcript (14-kDa E2) and 1.3-kb transcript (C9). Values are means and pooled SEM, n = 8. Bars with different letters are significantly different (P < 0.05). Representative Northern blots are shown.

	DISCUSSION

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Our finding on the suppressive effects of luminal amino acids on intestinal protein synthesis is contrary to what one might expect. However, our results do not contradict any previous data because this is the first time that luminal exposure has been studied in the absence of systemic stimuli. Data from feeding trials show stimulatory effects of proteins and amino acids on intestinal Ks and protein mass; however, feeding is inevitably associated with systemic effects. In terms of the design and methodology, the only study remotely similar to this one is that of Weber et al. (1989) . These authors luminally perfused a 10-cm segment of jejunum in the rat for 2 h with 56 mmol/L glucose and showed that glucose perfusion increased mucosal protein synthesis by 20–37%. Plasma glucose and hormone levels were not measured in that study. Perfusion of a 10-cm segment of the jejunum in rats of the size they used would expose ~30% of the jejunum to a concentrated glucose solution. Such a situation would likely increase plasma glucose, glucagon-like peptide 1 and insulin concentrations. Although the effects of glucagon-like peptides and insulin on intestinal protein synthesis are not yet clear, a recent study demonstrated the stimulatory effects of insulin-like growth factor-1 on protein synthesis in both mucosal and serosal layers of small intestine of parenterally fed rats (Lo and Ney 1996 ). Therefore a direct comparison of our study with that of Weber et al. (1989) is not possible because we demonstrated previously that luminal nutrient perfusion, as done in this study, did not induce systemic changes (Adegoke et al. 1998).

Suppression of protein synthesis by luminal amino acids cannot be fully understood without a consideration of protein catabolism. The intestinal mucosa could maintain positive protein balance by a parallel decrease in proteolytic rate. The absence of adequate methods for direct determination of intestinal protein catabolism imposes an important roadblock in our understanding of intestinal protein metabolism. Although there are no direct methods for estimating intestinal proteolysis, an indirect method involves the measurement of mRNA of the different proteolytic systems. The three well-characterized intracellular proteolytic systems, ATP-ubiquitin-proteasome, lysosomal and Ca²⁺-dependent proteolytic pathways are present in the intestine (Hubbard and Carne 1994 , Kishibuchi et al. 1995 , Samuels et al. 1996 ). Although the ATP-ubiquitin-proteasome pathway is thought to be responsible for much of intracellular proteolysis in skeletal muscle (Attaix et al. 1998 ), the quantitative importance of any of the proteolytic pathways in the intestine is not known. In recent studies with food-deprived rats, ubiquitin level (Hubbard and Carne 1994 ) and mRNA levels of m-calpain and of components of the ATP-ubiquitin-proteasome proteolytic pathway (Samuels et al. 1996 ) varied in parallel with the loss of intestinal protein. We therefore examined the effects of the 30 mmol/L amino acid mixture on proteolytic gene expression. The 30 mmol/L amino acid mixture had no effects on m-calpain expression but suppressed mucosal expression of ubiquitin, 14 kDa E2 and the C9 proteasome subunit in a coordinated manner. No changes in the expression of C8 proteasome subunit were observed. Because the importance of the different steps in regulating the ATP-ubiquitin-proteasome pathway is not known (Attaix et al. 1998 , Coux et al. 1996 ), the implication of lack of changes in mRNA levels of C8 in the presence of changes in the expression of the other components of the pathway is unknown. In spite of this, our observation of coordinated regulation of ubiquitin, 14 kDa E2 and C9 expression in the absence of any decrease in expression of GAPDH likely indicates a specific down-regulation of the ATP-ubiquitin-proteasome proteolytic pathway.

With the model used in this study, we have shown that perfusion of intestinal segments with different nutrients had no effect on plasma glucose, amino acid and insulin concentrations (Adegoke et al. 1999 ). However, in the absorptive state, there are elevated blood concentrations of absorbed nutrients as well as anabolic hormones and growth factors such as insulin, insulin-like growth factor-1, growth hormone and glucagon-like peptides-1 and -2. Therefore, it is also possible that the rise in protein synthesis seen after a meal is dependent on elevated levels of systemic factors. In a related study, we observed that intravenous glucose infusion increased mucosal protein synthesis by 16% (Adegoke et al., unpublished observations).

We sought to examine the specificity of amino acids involved in the observed effects of a luminal amino acids mixture. We could not attribute the effects of 30 mmol/L luminal amino acids on mucosal protein synthesis to any specific amino acid. Because of the importance of glutamine in regulating intestinal metabolism (O'Dwyer et al. 1989 , Stein et al. 1994 ), we had hypothesized that this amino acid and/or its metabolites (glutamate, arginine, proline, ornithine, citrulline) might be involved in the observed luminal amino acid–induced suppression of mucosal protein synthesis. Although luminal perfusion with an amino acid mixture lacking glutamine and its related amino acids prevented the rise in concentrations of these amino acids, suppression of mucosal protein synthesis was still seen (Table 1 and Fig. 4 ). Moreover, luminal perfusion of 1.84 mmol/L glutamine alone restored the intracellular concentrations of glutamine and glutamate to that seen in intestinal segments perfused with the complete amino acid mixture but did not suppress mucosal protein synthesis. Taken together, these data indicate that glutamine (at its concentration in the complete amino acid mixture) was not responsible for the observed effects of amino acids. Because the effects of amino acids were seen with 30 mmol/L amino acid mixture (irrespective of composition) or 30 mmol/L glutamine alone but not at lower concentrations of either, our data also imply a threshold level of amino acids for stimulation of protein synthesis. Because the total amino acid concentration of jejunal digesta in the fasted state is ~20 mmol/L (Adibi and Mercer 1973 ), this would explain why we observed stimulation at 30 mmol/L but not at lower concentrations.

The effects of amino acids on mucosal protein synthesis could be mediated by other metabolites. A feature of intestinal amino acid metabolism is the production of ammonia, whose concentration in the portal blood can account for up to 18% of amino acid nitrogen intake (Stoll et al. 1998 , van Berlo et al. 1988 , Wu 1998 ). We hypothesized that ammonia produced in the absorptive state might serve as a signal involved in the amino acid–induced regulation of mucosal Ks. However our results clearly show that elevated levels of ammonia per se have no influence on Ks. Physiologic concentrations of NH₄Cl induce cell hypertrophy in cultured renal epithelial cells by mechanisms involving alkalinization of the lysosome and reduced protein degradation (Franch and Preisig 1996 , Ling et al. 1996 ). It remains to be determined whether ammonia is also used by intestinal epithelial cells as a signaling molecule in the regulation of protein catabolism.

Changes in proteolytic gene expression observed in this study were quite rapid, i.e., they were noticed in <1.5 h. Other workers have also shown rapid regulation of intestinal epithelial cell expression of ornithine decarboxylase mRNA in response to glutamine, and of intestinal explant expression of sucrase-isomaltase mRNA in response to insulin after 0.3 and 5 h of incubation, respectively (Kandil et al. 1995 , Takenoshita et al. 1998 ). However, in the study of Kandil et al. (1995) , the cells were starved of serum for 4 h before the experiments; such treatment would make the cells quiescent and thus represent a high degree of challenge to the cells. The rapid decrease in mRNA encoding elements of the ATP-ubiquitin-proteasome system (in <1.5 h) seen in this study appears to be one of the most rapid nutrient-induced changes in intestinal gene expression in vivo.

It is tempting to speculate on the biological importance of the suppression of mucosal protein synthesis and degradation by luminal amino acids. Protein synthesis is an energetically expensive process and the formation of a single peptide bond at the translational stage alone involves the consumption of at least five molecules of ATP (Newsholme and Leech 1985 ). Net protein synthesis also requires substrate amino acids. Because of costly inputs, it would appear advantageous for the intestine to suppress protein synthesis as a way of limiting its energy and substrate utilization, thereby increasing the efficiency with which absorbed nutrients are delivered to the systemic circulation. A coordinate down-regulation of protein catabolism would be required to enable the intestine to be in positive N balance during the absorptive state.

The results presented here concern several major types of macronutrients including amino acids and energy substrates. These data demonstrate that protein synthesis in the jejunal mucosa responds selectively to different nutrients. The system employed here can be used further to test the effects of other macro- and micronutrients on intestinal protein synthesis and gene expression, and to explore the full range of interactions among nutrients, and between nutrients and physiologic states.

	ACKNOWLEDGMENTS

 
We are grateful to Chantal Farges for technical assistance and to Jody Aldrich and the staff of Edmonton Research Station, Metabolic Swine Unit, for animal care and surgical assistance.

	FOOTNOTES

 
¹ Presented in part at Experimental Biology 97, April 1997, New Orleans, LA [Jimoh, O. A., McBurney, M. I. & Baracos, V. E. (1997) Protein synthesis measurements by luminal flooding dose technique in multiple segments of jejunum. FASEB J. 11: A365 (abs.)].

² Supported by grants from the Alberta Agricultural Research Institute to V.E.B. and from the Natural Sciences and Engineering Research Council of Canada to V.E.B. and to S.E.S.

³ Current address: W. K. Kellogg Institute for Food and Nutrition Research, 2 Hamblin Avenue East, Battle Creek MI 49016-3232.

⁵ ß-OH-butyrate, ß-hydroxybutyrate; 14-kDa E2, 14-kDa ubiquitin carrier protein E2; Ks, fractional rate of protein synthesis, GAPDH, glyceraldehyde phosphate dehydrogenase; S_b specific radioactivity of protein-bound phenylalanine; SCFA, short-chain fatty acids; S_f is the intracellular free phenylalanine specific radioactivity in tissue samples.

Manuscript received November 12, 1998. Initial review completed January 18, 1999. Revision accepted June 11, 1999.

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