Modulation of intestinal protein synthesis and protease mRNA by luminal and systemic nutrients

Olasunkanmi A. J. Adegoke1, Michael I. McBurney1, Susan E. Samuels2, and Vickie E. Baracos1

1 Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, T6G 2P5; and 2 Food, Nutrition and Health, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z4


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Route of nutrient supply is important in regulation of intestinal protein metabolism, because total parenteral nutrition, compared with enteral feeding, leads to profound atrophy. Participation of the fractional rate of protein synthesis (Ks), their degradation in regulation of gut protein balance, and their possible modulation by specific nutrients are the focus of our work. We developed an in situ experimental system that allows controlled exposure of intestinal mucosa to nutrients systemically, luminally, or both. We examined the effects of systemic glucose and amino acid (AA) infusion in overnight-fasted piglets. Jejunal segments within each piglet were simultaneously, luminally perfused with solutions containing various AAs or glucose. Intravenous infusion of glucose increased mucosal Ks by 16% (P < 0.05), whereas intravenous infusion of AA had no effect on Ks. Systemic glucose infusion had no effect on mRNA levels for components of the ubiquitin-proteasome proteolytic pathway. However, levels of these mRNA were reduced by intravenous or luminal AA supply. This effect was greatest (-50%) when highest tissue concentrations of AAs were achieved by the simultaneous infusion of AA by both routes (P < 0.05). Our findings suggest that not only is the modulation of protein balance in the intestine in response to nutrients in part attributable to anabolic stimulation of protein synthesis initiated by the systemic appearance of glucose, but a fall in protein degradation is also a likely contributor. AAs appear to be a key factor required to reduce expression of genes connected with proteolysis.

amino acids; intestine; protein breakdown; glutamine; ubiquitin


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

FEEDING, FASTING, AND SPECIFIC nutrients participate in regulating small intestinal protein metabolism and mass. Mucosal protein synthesis and mass increase after feeding and decrease during deprivation of food (8, 18, 22). The route of nutrient supply is also important because total parenteral nutrition (TPN) compared with enteral feeding leads to profound atrophy and decreased protein synthesis rates (13, 20, 23). Although this response may be attenuated by the addition of specific nutrients, such as glutamine or short-chain fatty acids, to parenteral nutrition, full restoration of intestinal protein mass is not achieved (20, 23).

We had hypothesized that luminal nutrients per se may be needed to maintain intestinal protein mass by activation of protein synthesis and/or suppression of protein degradation. To test the specific roles of individual luminal nutrients in regulating intestinal protein turnover, we developed an in situ experimental system that allows exposure of mucosa to nutrients on the apical side without exerting a systemic effect (1). By using jejunal segments subjected to acute (40-90 min) luminal infusion (2), we showed that neither energy fuels, such as glucose or short-chain fatty acids, nor a mixture of amino acids or glutamine reproduced the rise in protein synthesis associated with oral feeding (8, 18). The difference between oral feeding and our luminal perfusion approach is the appearance of nutrients and hormones in the systemic circulation. In the study reported here, we test the hypothesis that the regulation of intestinal protein metabolism is also dependent on systemic signals reflecting overall nutritional status and that such signals would permit elevations of protein synthesis as seen after feeding in vivo. We selected two potential candidates, amino acids and glucose, as the systemic nutrient regulators. The plasma concentrations of these nutrients rise after feeding (8, 12, 19, 24). These were infused intravenously, with or without simultaneous perfusion with luminal nutrients to additionally test for interaction between luminal and systemic treatments.

Regulation of intestinal proteolysis by feeding and in response to specific nutrients is considerably less well known compared with protein synthesis. Although no direct method for estimating intestinal protein catabolism is presently available, we measured mRNA levels of components of different proteolytic systems to examine the potential regulation of proteolysis at this level (22). We (2) previously observed that mucosal mRNA levels for several elements of the ubiquitin-proteasome-dependent proteolytic system decreased after luminal exposure to amino acids, suggesting that protein degradation may be an important determinant of intestinal protein mass. Thus a second objective of this study was to assess gene expression within the ubiquitin-proteasome system in response to the systemic and luminal treatments.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Study Design

Our prior work focused exclusively on the provision of luminal nutrients in the absence of systemic effects (1, 2). In the present study, we examined intravenously administered nutrients. In study 1, intravenous infusion of glucose was compared with saline; in study 2, intravenous infusion of amino acids was compared with saline. Within both of these main experiments, we examined several luminal nutrient treatments (saline, 30 mM amino acids ± 50 mM glucose, 30 mM glutamine), which we had used in our prior studies to determine whether there were interactions between the luminal and systemic provision of nutrients.

Chemicals

Sterile 75% glucose and physiological saline were purchased from Baxter (Deerfield, IL). L-[2,6-3H]phenylalanine (2.15 TBq/mmol; radiochemical purity: 99.6%) was purchased from Amersham International (Little Chalfont, UK). Other chemicals were from Sigma (St. Louis, MO).

Animals and Surgical and Perfusion Procedures

All studies were performed in accordance with the Canadian Council on Animal Care Guidelines and were authorized by the institutional Animal Policy and Welfare Committee. Six-week-old male piglets, weighing ~12 kg, (Camborough × Canabrid pig improvement crosses) were obtained through the University of Alberta Health Sciences Laboratory Animal Services. Piglets were weaned at 4 wk of age and were maintained on a wheat/oatgroat-soybean/whey powder starter diet (crude protein 205 g/kg; digestible energy 15.07 kJ/g). Animals were food deprived overnight before experimentation, but water was available at all times.

Piglets were anesthetized as previously described (1). Catheters were placed in the right and left jugular veins for intravenous infusion and blood sampling, respectively. Catheters were filled with heparinized saline while the intestinal cannulation was done. Insertion of cannulae and luminal perfusion of 6-cm intestinal segments were done as described previously (1, 2).

Treatment Groups

Study 1: Intravenous glucose infusion. Piglets were randomly allocated to receive an intravenous infusion of either glucose or saline (n = 6/treatment). Piglets were injected with 5 ml of saline or 5 ml of a 75% glucose solution through the infusion catheter and then infused with saline or 75% glucose solution at a rate of 2.4 ml · kg body wt-1 · h-1 for 75 min. The amount of glucose infused was chosen to substantially increase plasma glucose levels to test our hypothesis that systemic signals are required to stimulate protein synthesis. A preliminary experiment, using this rate of glucose infusion, showed a plateau in glucose and insulin concentrations between 40 and 60 min of the start of infusion (data not shown). Two piglets were studied each day, one infused with glucose and the other with saline.

Four jejunal segments within each animal in the glucose or saline-infused piglets were independently but simultaneously perfused for 75 min with either PBS (in mM: 126 NaCl, 14.1 Na2HPO4, 1 NaH2PO4 · H2O, pH 7.4) or different nutrient solutions, including 1) 30 mM amino acid mixture + 50 mM glucose, 2) 30 mM amino acid mixture, or 3) 30 mM glutamine. The amino acid mixture, with or without 50 mM glucose, was chosen to simulate some of the components of a meal and was formulated on the basis of published composition of jejunal digesta (3, 14, 16). This mixture contained (in mM) 0.67 aspartate, 2.03 serine, 2.34 glutamate, 1.84 glutamine, 3.15 proline, 3.75 glycine, 2.00 alanine, 0.30 cystine, 0.79 tyrosine, 0.59 histidine, 1.29 arginine, 0.73 asparagine, 1.34 threonine, 1.66 valine, 0.55 methionine, 1.14 isoleucine, 2.09 leucine, 2.00 phenylalanine, 1.58 lysine, and 0.19 tryptophan. A treatment with glutamine was included, because this amino acid is a preferred fuel of intestinal mucosal cells (28). All perfusates were made isoosmotic to a level typical of jejunal digesta in the piglet (300 mosM) in a PBS buffer.

Study 2: Intravenous amino acid infusion. This study focused specifically on systemic and luminal effects of amino acids. Piglets were randomly allocated to receive an intravenous infusion of either saline or amino acids (see Table 1). The infusion was intended to deliver a bolus of amino acids approximating 1/3 of daily protein requirements (i.e., a large protein meal). This was specifically intended to substantially increase plasma amino acid concentrations to test our hypothesis that systemic signals modulate intestinal protein metabolism. The infusate was on the basis of the complete amino acid mixture for feeding piglets orally or intravenously used by Bertolo et al. (6) to which we also added glutamine (Table 1). Piglets (n = 6/treatment) were injected with 5 ml of saline or 5 ml of amino acid solution (119 g/l) (Table 1) through the infusion catheter and were then infused with saline or the amino acid solution at a rate of 7.5 ml · kg body wt-1 · h-1 for 90 min. Two piglets were studied each day, one infused with amino acids and the other with saline. Within each intravenous infusion group, two intestinal segments within each piglet were luminally perfused with PBS or 30 mM amino acid mixture, as described above. The overall duration of intravenous and luminal treatments was 90 min.

                              
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Table 1.   Amino acid composition of intravenous infusate

Measurement of protein synthesis and tissue sampling. During the last 15 min of perfusion, protein synthesis was measured by using the luminal flooding dose technique (1). We validated this experimental system and showed that: 1) the rate of protein synthesis was the same for luminal and intravenous flooding, 2) the phenylalanine free specific radioactivity was constant over the labeling period, 3) the tissue free phenylalanine specific radioactivity rapidly rose to a level indistinguishable from that of the perfusate, and 4) the tissue free specific radioactivity in adjacent jejunal tissue not perfused with isotope was insignificant. We also showed that luminal perfusion of 30 mM amino acid mixtures ± 50 mM glucose did not affect free phenylalanine specific radioactivity compared with saline perfusion (2).

Tissue phenylalanine concentrations and specific activity were determined in every segment in which protein synthesis was determined. Under conditions of luminal and/or intravenous amino acid perfusion phenylalanine specific activity was not different among treatments and furthermore was not different from that of the perfusate (Table 2).

                              
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Table 2.   Mucosal free phenylalanine specific radioactivity after intravenous and luminal amino acid treatments

In the present study, intestinal segments were emptied and perfused with the test solutions containing 2 mM L-[2,6-3H]phenylalanine (specific activity = 42 kBq/nmol) for 15 min. In all experiments the final phenylalanine concentration was 2 mM. Segments were then rapidly removed, emptied, and flushed with three changes of ice-cold saline. Mucosa was scraped on an ice-cold surface, frozen in liquid N2, and stored at -80°C until it was analyzed. Animals were then killed by cardiac injection of Euthanyl (1 ml/kg body wt; MTC Pharmaceuticals, Cambridge, ON, Canada).

Sample processing for the measurement of protein synthesis was as described in Ref. 1. Fractional rate of protein synthesis (Ks), expressed as percent per day, was calculated according to McNurlan and Garlick (18): Ks = 100 Sb/Sf t, where Sb is the specific radioactivity of protein-bound phenylalanine, t is the duration of isotope perfusion in days, and Sf is the intracellular free phenylalanine specific radioactivity in tissue samples.

Northern Hybridization

We examined the effects of the various treatments on mRNA levels of critical components of the ubiquitin-proteasome and the calcium-activated proteolytic systems. Northern hybridization was done for m-calpain (calcium-activated system) and for ubiquitin, the ubiquitin-conjugating enzyme 14-kDa E2, known to be involved in ubiquitin conjugation of substrates in skeletal muscles before proteolysis, and for the C8 and C9 subunits of the 20S proteasome, the proteolytic core of the 26S proteasome (4). mRNA levels for various components of proteolytic systems have been used as an indirect approach to study proteolysis in rodent muscle (27, 31) and intestine (22). In skeletal muscle, these mRNA levels correlate with rates of protein degradation measured in vitro (27, 29). Because there are no in vivo techniques available to measure mucosal proteolysis, the rate of this process in the mucosa remains unknown. Although relative changes in mRNA do not necessarily imply changes in the levels of proteins encoded by those mRNA, treatment differences may suggest directional changes in protein degradation as well as indicate possible transcriptional regulation.

Total RNA was isolated from mucosal samples with TRIzol reagent (Life Technologies, Burlington, ON, Canada) according to the manufacturer's instructions. Total RNA (15 µg) were electrophoresed in 1% agarose-formaldehyde gels containing ethidium bromide for 5 h at 100 V. RNA was checked visually for integrity of 28S and 18S ribosomal RNA and was transferred to nylon membranes (GeneScreen; New England Nuclear, Boston, MA) by capillary transfer and cross-linked to membranes under ultraviolet light using Stratalinker (Stratagene, La Jolla, CA). Membranes were hybridized with cDNA probes derived from the genes mentioned above (2, 22). Because the results from study 1 showed that mRNA for ubiquitin and 14-kDa E2 were consistently suppressed by luminal amino acids, in study 2, hybridization was carried out only with probes for these two genes. Hybridization signals were quantified with a phosphorimager (Molecular Dynamics, Sunnyvale, CA). Differences in RNA loading were corrected for by stripping membranes and reprobing with 32P-labeled cDNA probe for GAPDH (22). GAPDH mRNA levels did not differ among treatments (P > 0.05).

Other Analyses

Plasma glucose concentration was measured by using glucose Trinider kits (Sigma, St. Louis, MO). Plasma insulin concentration was determined by using the Enzymum-T (Boehringer-Mannheim Immunodiagnostics, Laval, PQ, Canada). Plasma and tissue amino acid concentrations were determined by using high performance liquid chromatography (1).

Statistical Analyses

Data are expressed as means ± SE or pooled SE. Data were analyzed by using a two-way ANOVA (SAS version 6.02; SAS Institute, Cary, NC) with luminal and intravenous treatments as independent variables. Intravenous treatments served as the blocks. When no significant interaction between luminal and intravenous treatments was present, statistical comparisons were only made for main effect parameters (luminal and intravenous treatments) and data were pooled. Significant differences (P < 0.05) between means were examined by using Fisher's protected least significant difference test.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Study 1: Intravenous Glucose Infusion

Plasma glucose and insulin levels. Preinfusion plasma glucose and insulin levels did not differ significantly (glucose: 6.0 ± 0.9 vs. 5.0 ± 0.3 mM; insulin: 8.5 ± 0.7 vs. 6.5 ± 1.14 µU/ml, for glucose- and saline-infused piglets, respectively). Both plasma insulin and glucose levels were increased by 300% by the end of the infusion (P < 0.05, Fig. 1). Increase in glucose was rapid and plasma glucose remained constant during the last 30 min of infusion, whereas the increase in insulin did not occur until the last 15 min of infusion. As expected, peak plasma glucose concentrations were higher than in fed piglets (~7.5 mM) (25). Plasma insulin concentration at this time (20 ± 2 µU/ml) was lower than in fed 4-wk-old piglets (36 µU/ml) (8).


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Fig. 1.   Intravenous glucose infusion increased plasma glucose and insulin concentrations. Piglets were intravenously infused with a 75% glucose solution (2.4 ml · kg body wt-1 · h-1; n = 6) or saline for 90 min. Blood samples collected before and at 15-min intervals during infusion were analyzed. Glucose concentrations with different letters (a-c) are significantly different from one another (P < 0.05). * Insulin concentration at this time point is significantly different from the preinfusion value (P < 0.05).

Mucosal protein synthesis. Analysis of variance revealed no interactions between the effects of intravenous and luminal treatments (P > 0.05). Pooled data for mucosal protein synthesis in response to intravenous treatments and in response to luminal treatments are shown in Fig. 2. Intravenous glucose infusion increased mucosal protein synthesis by 16% (P < 0.05, n = 6). Irrespective of intravenous treatments, the different luminal nutrient solutions (30 mM amino acids with or without 50 mM glucose or 30 mM glutamine) suppressed mucosal protein synthesis by 10% relative to segments perfused with PBS (P < 0.05, n = 12, Fig. 2). These data suggest that the systemic supply of glucose increases the rate of mucosal protein synthesis independent of luminal nutrients.


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Fig. 2.   Intravenous glucose infusion increases mucosal fractional rate of protein synthesis (Ks) independently of the effects of luminal treatments. Piglets were intravenously infused with a 75% glucose solution (2.4 ml · kg body wt-1 · h-1) or saline for 75 min. Within each infusion group, 1 of 4 intestinal segments within each piglet was perfused with PBS, pH 7.4, 30 mM amino acid (AA) mixture (with or without 50 mM glucose), or 30 mM glutamine for 75 min. Protein synthesis was then measured in the last 15 min of perfusion. Glucose infusion increased the Ks (P < 0.05, n = 6), independently of intravenous treatments. Luminal nutrients modestly, but significantly, suppressed mucosal protein synthesis relative to control PBS, independently of glucose infusions. P < 0.05, n = 12; Gln, glutamine.

mRNA levels for elements of proteolytic systems. We measured the mRNA levels of components of ATP-ubiquitin-proteasome and calcium-activated proteolytic systems. Analysis of variance revealed no interactions between the effects of intravenous and luminal treatments (P > 0.05). Intravenous glucose infusion exerted no effects on the expression of mRNA for m-calpain, ubiquitin, 14-kDa E2, or proteasome subunits C8 and C9 (P > 0.05; data not shown). Perfusion of intestinal segments (Fig. 3) with 30 mM amino acid mixture or 30 mM glutamine suppressed mRNA levels of total ubiquitin, the 1.2-kb transcript of 14-kDa E2, and the proteasome subunit C9 by ~30% (P < 0.05) relative to PBS. Although there are two bands in the 14-kDa E2 Northern blot, only the 1.2-kDa band was suppressed; this transcript has been shown to be regulated by different nutritional manipulations in skeletal muscle (29, 30). However, the suppression of mRNA was not seen in segments perfused with 30 mM amino acids plus glucose. There were no effects of luminal treatments on mRNA for proteasome subunit C8 or m-calpain (P > 0.05; data not shown).


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Fig. 3.   Luminal amino acids decrease mRNA levels for components of the ubiquitin-proteasome proteolytic system. Treatments were as described in the legend to Fig. 2. Northern hybridizations were carried out for ubiquitin, 14-kDa ubiquitin-conjugating enzyme (E2), and C9 subunit of the proteasome. Data are expressed in arbitrary units after phosphorimage analysis. * Significant decrease in mRNA levels after luminal treatments with amino acids or glutamine alone (P < 0.05; n = 12).

Plasma and mucosal free amino acid concentrations. To examine the possible mechanisms by which the different treatments regulate mucosal Ks and gene expression, we measured the effects of the intravenous glucose and luminal treatments on plasma and mucosal tissue free amino acid concentrations. Intravenous glucose infusion had no effect on plasma amino acid concentrations compared with saline-infused piglets (data not shown). Intravenous glucose infusion had no effect on mucosal free amino acid concentrations compared with saline-infused piglets. Therefore, these data were pooled with saline-infused piglets (Table 3). The effects of luminal treatments on mucosal free amino acids (Table 3) may be summarized as follows. First, when 30 mM amino acids or 30 mM glutamine was luminally perfused, the concentrations of most amino acids in tissue were significantly elevated in a manner that reflected their concentrations in the perfusates. Second, the presence of 50 mM glucose plus 30 mM amino acids in perfusates significantly reduced intracellular levels of most amino acids when compared with perfusion of 30 mM amino acids alone. Finally, 30 mM glutamine perfusion alone increased concentrations of aspartate, glutamate, arginine, alanine, and ornithine, relative to PBS perfusion.

                              
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Table 3.   Effects of luminal nutrient treatments on tissue free amino acid concentrations in jejunal mucosa tissue

We observed lower mucosal free phenylalanine concentrations in segments perfused with 30 mM amino acids compared with segments perfused with saline containing 2 mM phenylalanine. This was likely due to increased competition for amino acid uptake. This, however, would not be expected to interfere with the estimation of protein synthesis because free phenylalanine specific radioactivity was not different among luminal treatments.

Study 2: Intravenous Amino Acid Infusion

Plasma and mucosal free amino acid levels. With the exception of citrulline and asparagine, plasma concentrations of all measured amino acids increased significantly (P < 0.05) during infusion of the complete amino acid mixture (Fig. 4). Plasma amino acid levels were, on average, about twice as high as in continuously intragastrically fed piglets as expected (6). Intravenous amino acid infusion resulted in a significant increase in plasma and tissue free glutamine (+29%; P < 0.05); this increase paralleled a 28% increase in concentration of tissue total free amino acids (P < 0.05; Table 4). Tissue levels of most amino acids were elevated (P < 0.05) after intravenous amino acid infusion compared with saline infusion; only levels of glycine, aspartate, and glutamate did not change (Table 4). Total tissue free amino acids increased by 25% during intravenous infusion, by 52% during luminal perfusion, and by 72% when both intravenous and luminal amino acids were supplied together.


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Fig. 4.   Intravenous amino acid infusion raises plasma concentrations of amino acids. Piglets were infused intravenously with saline or a mixture of amino acids (Table 1) for 90 min. Except for Asn and Cit, intravenous amino acid infusion increased the plasma concentrations of all amino acids (P < 0.05; n = 6).


                              
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Table 4.   Intravenous and luminal amino acid perfusions raise free amino acid concentrations in jejunal mucosa

Mucosal protein synthesis and mRNA levels for ubiquitin-proteasome proteolytic system. By contrast to intravenous glucose infusion, intravenous amino acid infusion did not affect mucosal protein synthesis, although tissue free amino acid levels were significantly increased by this treatment (Fig. 5). Intravenous and luminal amino acid infusion independently decreased levels of mRNA encoding ubiquitin and the 1.2-kb transcript of the 14-kDa E2 (P < 0.05). When amino acids were delivered simultaneously through both routes, mRNA level decreased further (Fig. 6). The decrease in mRNA levels paralleled the increase in total mucosal free amino acid concentrations (see Fig. 5 and Table 4). Thus when mucosal amino acids were elevated to the greatest extent by simultaneous luminal and intravenous amino acid delivery, the effect on mRNA levels was the greatest.


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Fig. 5.   Intravenous amino acids raise tissue amino acid levels but do not activate protein synthesis. Piglets were intravenously infused with saline or a complete amino acid mixture (n = 6). Data are means ± SE. Within each infusion group, intestinal segments were perfused luminally with a complete amino acid mixture (Table 1) or PBS. Mucosa samples harvested from perfused segments were used to determine Ks and free amino acid concentrations. * Significant increase in amino acid levels (P < 0.05; n = 6).



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Fig. 6.   Luminal and systemic amino acids additively suppressed the expression of ubiquitin (A) and 14-kDa E2 (B) in a manner dependent on the total tissue free amino acids (C). Piglets were intravenously infused with saline or a complete amino acid mixture (n = 6). Data are means ± SE. Within each infusion group, intestinal segments were perfused luminally with a complete 30 mM amino acid mixture or PBS as described in MATERIALS AND METHODS. RNA from mucosa samples was analyzed for the expression of ubiquitin and the 14-kDa E2. C: changes in intracellular total free amino acids. a,b,cSignificant differences (P < 0.05; n = 6).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Our experimental system was developed to allow a clear discrimination between the effects of nutrients applied luminally and intravenously on intestinal mucosal protein metabolism. Because the size of the perfused segments is small, luminal perfusion is not associated with any detectable systemic accumulation of perfused substrate (1). This is therefore a unique "first pass" system, which affords the opportunity to identify any direct influence of luminal nutrients on metabolism.

Results of this study point to a fascinating degree of multiplicity in the regulation of intestinal protein turnover. We demonstrated that the effects of glucose and amino acids on mucosal protein synthesis and mRNA levels for proteolytic systems were rapid, nutrient-specific, and dependent on route of delivery. We observed surprisingly few effects of luminal nutrients, which led us to speculate that the appearance of systemic nutrients could acutely modulate mucosal protein metabolism. Intravenous glucose stimulated mucosal protein synthesis. However, when delivered luminally by itself (our previous observations, Ref. 2) or with amino acids (present study), glucose had no effect on protein synthesis, suggesting that its effect might be indirect. An intravenous amino acid mixture, on the other hand, had no effect on protein synthesis but decreased mRNA levels for components of the ubiquitin-proteasome system, an effect that was augmented when luminal amino acids were also provided. This suggests that amino acids may play an important and direct role in reducing mucosal proteolysis. Collectively, our data indicate a cooperative role for glucose and amino acids in regulating intestinal protein balance.

Regulation of Intestinal Mucosal Protein Turnover by Glucose

The stimulatory effect of feeding on tissue (such as skeletal muscle) and whole body protein mass in young growing animals is well documented (8, 12). However, the precise role of nutrients in regulating intestinal protein synthesis and catabolism is both complex and poorly understood. Our novel finding that luminal energy fuels or amino acids did not reproduce the effect of oral feeding on protein synthesis (2) led us to examine whether additional (systemic) factors are needed to reproduce the feeding-induced stimulation of protein synthesis. Our observation that intravenous glucose infusion increased protein synthesis, whether or not nutrients were present in the lumen, is consistent with the effects of feeding on protein synthesis and with the known anabolic effects of glucose in other tissues (19, 24). Given that feeding is associated with an increase in mucosal protein synthesis of ~18% (8), this study suggests that the elevated systemic level of glucose is a major contributor to augmented mucosal protein synthesis and, therefore, the anabolic effect of feeding.

The systemic effects of glucose may be mediated by the increased plasma insulin levels in the glucose-infused piglets, a hypothesis consistent with increased jejunal protein synthesis and plasma insulin levels observed during refeeding (8). The regulation of intestinal protein metabolism via systemic nutrients is complex. By contrast to the highly controlled conditions of luminal exposure, intravenous infusion of nutrients unleashes a host of metabolic changes, including secretion of hormones and growth factors, and changes in substrate concentrations secondary to their appearance, making it difficult to explain how systemic nutrients precisely regulate intestinal protein metabolism. Experiments specifically designed to manipulate hormone and substrate concentrations (i.e., clamp experiments) could be employed to resolve the identity of the individual factors that may act systemically. With the use of clamp experiments, elevation of plasma insulin concentrations, in the absence of increased plasma glucose or amino acid concentrations, failed to affect protein synthesis in fasted piglets (9, 10), suggesting that the effect of systemic glucose is not mediated by insulin and/or that the presence of both glucose and insulin are required. IGF-1 also failed to stimulate jejunal protein synthesis under these conditions (11). Further research is required to ascertain the mechanism by which systemic glucose increases mucosal protein synthesis.

The contribution of proteolysis to intestinal protein balance has not been extensively studied. However, two recent studies examined the effect of somatotropin or GLP-2 on intestinal protein metabolism and concluded that proteolysis may be a major determinant of intestinal protein balance, because protein balance changed without alteration in the rates of protein synthesis (7, 26). In our investigation, the intravenous supply of glucose had no effect on mucosal mRNA levels for proteolytic systems, suggesting that glucose (and/or the associated elevated plasma insulin and hormone concentrations) may not influence mucosal proteolysis. Larbaud et al. (15) showed that a 6-h infusion of insulin had no effect on the mRNA levels for various proteolytic systems in caprine jejunum; this is consistent with our results.

Regulation of Intestinal Mucosal Protein Turnover by Amino Acids

Amino acids may also be mediators of the feeding-induced upregulation of protein synthesis and net anabolism in the intestine, because plasma and tissue amino acid levels are increased with feeding (8, 19, 22). Intravenous amino acid infusion did not stimulate mucosal protein synthesis. This lack of effect was not due to suboptimal levels of systemic amino acids, because the plasma concentrations achieved exceeded those seen after intragastric feeding (6). Davis et al. (10) showed that intravenous infusion of amino acids (euinsulinemic) or amino acids plus insulin failed to stimulate jejunal protein synthesis in euglycemic-fasted piglets. Intravenous-infused glutamine also does not stimulate intestinal protein synthesis in dogs (17). These results are consistent with ours. However, luminal provision of amino acids modestly but consistently reduced protein synthesis (2). This result was surprising. This was not due to methodological problems, because we extensively validated our experimental system (1). In the present experiment, the intracellular phenylalanine free specific radioactivity was not different between all luminal and systemic nutrient treatments including saline and was indistinguishable from that of the perfusate. Circumstances under which decreased protein synthesis was observed is a model first pass system in which luminal influences can be studied without systemic alterations in concentrations of the same nutrients. Consequently, it is impossible to compare our results with any previous studies, because observations of this kind have never been made before. This may not correspond directly to any specific event in the cycles of feeding and fasting other than the first few moments of refeeding. Regardless, amino acids do not directly stimulate mucosal protein synthesis.

Irrespective of the route of delivery, amino acids were effective in suppressing mRNA levels for the ubiquitin-proteasome proteolytic system, the effect being maximal when amino acids were given both intravenously and luminally. This effect is not due to a nonspecific regulation of gene expression, because we did not observe any changes in mRNA levels of GADPH in response to luminal amino acid perfusion. The ability of luminal amino acids to decrease gene expression implies a direct mechanism, because our perfusion is without apparent systemic accumulation of free amino acids or changes in hormone levels. Furthermore, the effect of amino acids on gene expression could not be ascribed to any one single amino acid. A consistent observation was that the higher the concentration of mucosal intracellular amino acids, the lower the mRNA levels. This may explain why TPN fails to maintain intestinal protein balance, because TPN is associated with modest rises in plasma and tissue free amino acid concentrations compared with oral feeding (5, 6). If protease gene expression in mucosa is a function of amino acid concentration achieved in the tissue, it may be that intravenous feeding simply cannot sufficiently raise amino acid concentrations to suppress the expression of genes involved in degradative processes.

The luminal coperfusion of glucose and amino acids blunted the effects of amino acids on the expression of components of the ubiquitin-proteasome proteolytic system. Total mucosal free amino acid concentrations were significantly lower in intestinal segments perfused with a mixture of both amino acids and glucose compared with segments perfused with the amino acid mixture alone. This is consistent with our supposition that high mucosal amino acid concentrations are required to decrease protease gene expression. The reduced intracellular amino acid levels in intestinal segments perfused with both glucose and amino acids may be due to a reduced amino acid uptake. This hypothesis is supported by the fact that 1) mucosal intracellular concentrations of citrulline and ornithine, amino acids not included in the perfusates but which the intestine can synthesize (28), were not affected; and 2) the presence of luminal sugars may limit the inward transport of amino acids (21). Taken together, the coordinated suppression of mRNA of components of ubiquitin-proteasome proteolytic pathway by systemic and luminal amino acids suggests that amino acids, irrespective of route of delivery, may directly decrease gene expression and play a role in controlling intestinal protein mass.

Significance

The regulation of jejunal protein synthesis and gene expression depends on direct effects of nutrients and on systemic signals reflecting the overall nutritional status. In the fed state, with elevated circulating nutrients, hormones and growth factors, the intestine is capable of responding to systemic anabolic stimuli; systemic glucose may initiate this response. Amino acids appear to directly reduce expression of genes connected with proteolysis. Observed changes in gene expression, if accompanied by alterations in protein degradation of a similar magnitude, may be of considerable physiological importance in regulation of gut protein mass. A disruption in the mechanism by which nutrients regulate protein synthesis and catabolism could explain the wasting observed in the small intestine during many disease states and with intravenous feeding.


    ACKNOWLEDGEMENTS

We thank Dr. Chantal Farges for technical assistance and Jody Aldrich, Abha Dunichand-Hoedl, and the staff of Edmonton Research Station, Metabolic Swine Unit, for animal care and surgical assistance. We also thank Dr. Simon Wing (McGill University, Montreal, PQ, Canada) for generous provision of plasmids containing cDNA sequences encoding rat 14-kDa E2 ubiquitin-conjugating enzyme and Dr. Keiji Tanaka (Tokyo Institute for Medical Research, Tokyo, Japan) for the gift of plasmids containing the cDNA sequence of the rat C8 and C9 proteasome subunits. We thank Dr. Robert Hardin for valuable advice regarding statistical analysis.


    FOOTNOTES

This research was supported by grants from the Alberta Agricultural Research Institute (to V. E. Baracos) and from the Natural Sciences and Engineering Research Council of Canada to (to V. E. Baracos and S. E. Samuels).

Present addresses: O. A. J. Adegoke, Polypeptide Hormone Laboratory, Department of Medicine, McGill University, 3640 University St., Montreal, PQ, Canada H3A 2B2; M. I. McBurney, W. K. Kellogg Institute for Food and Nutrition Research, 2 Hamblin Ave. East, Battle Creek, MI 49016-3232.

Address for reprint requests and other correspondence: V. E. Baracos, Dept. of Agricultural, Food and Nutritional Science, 4-10 Agriculture-Forestry Centre, University of Alberta, Edmonton, AB, Canada T6G 2P5 (E-mail: vickie.baracos{at}ualberta.ca).

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.

First published February 5, 2002;10.1152/ajpgi.00402.2002

Received 18 September 2002; accepted in final form 22 January 2003.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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