Department of Agricultural, Food and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5
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ABSTRACT |
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To validate a system to study acute regulation of protein synthesis in intestinal mucosa by luminal nutrients, we compared the fractional rate of protein synthesis (Ks) in jejunal mucosa using the intravenous flooding dose technique with the administration of a comparable concentration and specific activity of tracer in a luminal perfusate. Routes of tracer administration and surgery and perfusion trauma had no effect on mucosal Ks. Furthermore, four 10-cm jejunal segments (within a piglet) simultaneously but separately perfused with a luminal flooding dose had similar Ks values (mean, 43 ± 2%/day; P > 0.05). Nutrient solutions perfused through four intestinal segments within an animal did not affect plasma levels of most amino acids or glucose. Because cellular hydration is important in regulating metabolism, the effects of physiological variation in luminal osmolarity were studied. Luminal osmolarities between 250 and 380 mosM did not affect mucosal Ks. The system described allows multiple comparisons within an animal and provides a robust model to study acute modulation of protein synthesis in intestinal mucosa by luminal stimuli.
intestine; perfusion; specific radioactivity; cellular hydration
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INTRODUCTION |
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THE SMALL INTESTINE IS AN important component of whole body protein metabolism, accounting for up to 12% of total protein synthesized (31). Although the intestinal mucosa is routinely presented with luminal nutrients, previous studies have focused only on long-term feeding trials and therefore do not allow distinction between the effects of nutrients and those of growth factors and hormones released in response to feeding. Thus acute regulation of intestinal mucosal protein synthesis by luminal influences is largely unknown. Although use of isolated enterocytes has been employed (16), this system does not allow distinction of luminal vs. basolateral influences. This is an important factor because the brush-border and basolateral membranes of epithelial cells of the intestinal mucosa are exposed to two different environments and regulation of metabolism can be effected through either of the two membranes.
Small intestinal mucosa can incorporate amino acids from either the luminal or basolateral side into protein (2, 3). Recent studies (24, 35) on the effects of route of tracer administration showed that intravenous and luminal routes resulted in similar rates of protein synthesis. However, those data were obtained using a continuous infusion of a trace amount of labeled amino acid for 1-4 h. This condition may be less than ideal for tissues such as the small intestine in which protein turnover is rapid (12) and secretory proteins are produced (32). The features of the flooding dose technique (12, 13) make it a better approach for studying intestinal mucosal protein synthesis. If a flooding dose can be delivered luminally, this will be distinctively advantageous in that requirements for radioactively labeled amino acid can be reduced by up to 90%. Furthermore, because intestinal tissue does not metabolize phenylalanine (14, 18), luminal delivery of tracer phenylalanine also saves cost and analytic time since it is not necessary to separate the tracer from its metabolites that might have become labeled due to metabolism. Finally, simultaneous but independent perfusion of multiple jejunal segments within an animal would permit compound observations within an animal.
Change in cellular hydration state, resulting from exposure to anisosmotic environments, nutrients, or hormones, is one of the mechanisms involved in regulation of cellular metabolism (6, 15). For example, exposure of hepatocytes to a hypoosmotic environment induces variations in hydration states. Such variations trigger an anabolic cascade of events, including alkalinization of the lysozomes, leading to decreased proteolysis and increased protein synthesis (6, 15). Although the intestinal mucosa is exposed to variability in luminal osmolarity (11), the effect of modest changes in luminal osmolarity on intestinal protein metabolism is not known.
Our objective was to validate a system that allows systematic study of the acute effect of luminal nutrients on intestinal mucosal protein synthesis, involving luminal perfusion of four intestinal segments within a piglet. Apart from the advantages of an in vivo system, variability is avoided as the effects of four luminal treatments can be tested within the same animal. We examined the suitability of the luminal flooding dose technique to measure jejunal mucosal protein synthesis and determined the effects of surgical and perfusion procedures and associated trauma on protein synthesis in jejunal mucosa. We compared rates of protein synthesis in multiple jejunal segments within an animal using the luminal flooding dose technique. It is essential that this system be functionally a first-pass system for it to be useful. Therefore, the effects of luminal perfusion with different nutrient solutions on systemic parameters were also determined. A final objective was to examine the effects of physiological variations in luminal osmolarity on jejunal mucosal protein synthesis.
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MATERIALS AND METHODS |
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Chemicals
L-[2,6-3H]phenylalanine was purchased from Amersham International (Amersham Place, Little Chalfont Bucks, United Kingdom). L-Tyrosine decarboxylase, pyridoxal 5-phosphate, phenylalanine, andAnimals and Surgical and Perfusion Procedures
All experiments were performed in accordance with the Canadian Council on Animal Care Guidelines. Male Sprague-Dawley rats (100-170 g) and 6-wk-old piglets were obtained from the University of Alberta Health Sciences Laboratory Animal Services. The piglets were weaned at 4 wk and maintained on a standard starter diet (crude protein, 20.5%; digestible energy, 3.55 kcal/kg), while rats were offered laboratory chow. Before experiments, animals were fasted overnight, but water was made available at all times.Rats were maintained under anesthesia with 1.5-2.5% halothane
delivered with oxygen. A 10-cm segment of the jejunum was cannulated at
both ends with polyethylene tubing (ID, in.; OD,
in.). Feed remnants in the segment were cleaned with warm PBS (144.6 mM
NaCl, 15.9 mM
Na2HPO4,
and 1.2 mM
NaH2PO4 · H2O,
pH 7.4, 37°C).
Anesthesia was induced in piglets with a mixture of Torbugesic
(0.2 mg/kg), ketamine (11 mg/kg), Rompun (2.2 mg/kg), and
glycopyrrolate (0.01 mg/kg) and maintained with 2% halothane delivered
with oxygen. Four 10-cm jejunal segments were cannulated at both ends
with polyethylene tubing (inlet cannula: and
in.
for ID and OD, respectively; outlet cannula:
and
in. for ID and OD, respectively). The inlet
cannula for the first segment was inserted 15 cm from the ligament of
Treitz, whereas successive sections were separated by 50 cm of
intestine. Segments were rinsed of digesta remnants with warm PBS. A
similar preparation was used to study glutamine metabolism in
endotoxin-treated piglets (10).
Experiments
Comparison of conventional and luminal flooding dose techniques. For measurement of protein synthesis with luminal delivery of tracer the perfusion solution was 2 mM [2,6-3H]phenylalanine with a specific radioactivity (SR) of 730 dpm/nmol. This concentration was chosen because a preliminary study showed that, under intravenous flooding conditions in rats, plasma concentrations of phenylalanine did not rise beyond 2 mM. Also, when a 10-cm jejunal segment from a pig or rat was perfused (recirculated) with 2 mM phenylalanine for up to 30 min, the tracer concentration did not change appreciably (data not shown). This was because large volumes, relative to the volume of the segments, were perfused [volume of perfused solutions, 10 ml (rat) and 50 ml (pig); volume of intestinal segments, 0.7 ml (rat) and 5 ml (pig)].
To examine the effect of the route of tracer delivery on protein synthesis, we luminally perfused (1 ml/min) one group of rats with tracer solution for 2 or 10 min (luminal-flood group; n = 6-7 for each time point). A second group of rats (intravenous-flood group; n = 5 for each time point) received only a standard intravenous flooding dose [150 mM L-[2,6-3H]phenylalanine; SR, 730 dpm/nmol; 1 ml/100 g body wt (Ref. 13)]. To examine the effects of perfusion procedures on protein synthesis, a third group of rats (intravenous plus perfusion group; n = 7-8 for each time point) underwent surgery and intestinal cannulation and perfusion with PBS. These rats also received an intravenous flooding dose of tracer as done for the intravenous-flood group rats. Tissues were harvested after 2 or 10 min. Mucosal samples were frozen in liquid N2 and stored atMucosal protein synthesis in multiple jejunal segments and effects of cannulation and perfusion procedures. Four jejunal segments within each piglet (n = 7) cannulated as described above were individually but separately perfused with a luminal flooding dose as described for rats but for 15 min at 6 ml/min. Mucosa from these segments was stored frozen as described for rats. To examine the effect of extended perfusion time on the basal rate of protein synthesis, we separately but simultaneously perfused two intestinal segments within the same piglet (n = 2) with PBS for 1.5 h. Afterward, animals were injected via the jugular vein with a flooding dose of tracer (150 mM L-[2,6-3H]phenylalanine; SR, 730 dpm/nmol at 8 ml/kg body wt). Mucosal samples from perfused and adjacent unperfused segments were collected 15 min after isotope injection and stored frozen.
Systemic effects. For a system employing multiple segments within an animal to be useful, the absence of systemic alterations after application of luminal treatments must be documented. If luminal perfusion of multiple jejunal segments with nutrient solutions did not raise systemic levels of perfused nutrients or hormones, the treatment could then be considered to consist of first-pass nutrients. The presence of systemic effects was examined in two ways. First, in animals administered tracer luminally, the only source of radioactivity in adjacent, unperfused segments is the absorbed tracer that has been redistributed systemically. Therefore, determination of phenylalanine SR in these segments is useful in assessing the effects of luminal perfusion on systemic parameters. Second, animals were tested for changes in blood concentrations, after luminal perfusion of nutrient-rich solutions. One of three intestinal segments within overnight-fasted piglets (n = 3) was independently but simultaneously perfused for 1 h with 30 mM glutamine, while two other intestinal segments were perfused with 30 mM amino acid mixture plus 50 mM glucose. The fourth segment in this experiment was perfused with PBS. The amino acid mixture contained (in mM) 0.67 aspartate, 2.03 serine, 2.34 glutamate, 1.84 glutamine, 3.15 proline, 3.75 glycine, 2 alanine, 0.3 cysteine, 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 phenylalanine, 1.58 lysine, and 0.19 tryptophan. These concentrations were based on observed luminal total free amino acid and glucose levels after a meal (1, 11, 20).
Blood samples were taken before and at the end of perfusion. Plasma was stored frozen atEffects of luminal osmolarity on protein synthesis in intestinal mucosa. One of four jejunal segments within the same piglet (n = 4) was perfused with PBS, pH 7.4, containing 250, 300, 339, and 380 mosM for 1.5 h. Segments were then emptied and perfused for 15 min with the same solution but containing 2 mM L-[2,6-3H]phenylalanine (SR, 730 dpm/nmol). Mucosal samples were collected and treated as described above.
Sample processing and analysis.
Determinations of phenylalanine SR in plasma and in mucosal free and
protein-bound fractions were done by conversion of phenylalanine to
-phenylethylamine as described previously (13, 28). This is because
with intravenous tracer administration phenylalanine radioactivity can
be transferred to other metabolites, especially tyrosine, due to
hepatic and renal metabolism of the tracer (14, 18). This will lead to
overestimation of phenylalanine SR if the tracer is not separated from
its metabolites.
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RESULTS |
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Comparison of Conventional and Luminal Flooding Dose Techniques
Plasma and mucosal free phenylalanine SR were lower at 10 min compared with the values at 2 min with intravenous tracer administration (Fig. 1, P < 0.05). Although free phenylalanine SR in mucosa of luminally flooded rats was slightly lower than that observed in the intravenous groups, it is noteworthy that mucosal free phenylalanine SR in the luminal-flood group remained steady at the two time points studied. Routes of flooding or perfusion procedures had no effect on mucosal Ks (Fig. 2).
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Mucosal Protein Synthesis in Multiple Segments of Jejunum
In multiple segments constructed within the same piglet, mucosal free phenylalanine SR and Ks were similar (Fig. 3, P > 0.05). Because terminal intracellular free phenylalanine SR was used in the calculation of Ks, it was necessary to examine changes in free phenylalanine SR over the period of measurement. One piglet was luminally perfused with labeled phenylalanine for 13, 23, 31, and 40 min. Similar to rat data (Fig. 1), free phenylalanine SR stayed constant throughout, being 492, 494, 486, and 497 dpm/nmol, respectively, at the four time points. To examine the amount of protein secreted into perfused segments during the time of label incorporation, the entire perfusate was collected after 15 min of perfusion. Protein secretion (cell-associated and soluble proteins) was minimal, being only 3% of total mucosal protein in perfused segments.
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Effect of Cannulation and Extended Perfusion Time on Mucosal Protein Synthesis
After segments of intestine were perfused with PBS for 1.5 h, mucosal Ks, measured with the intravenous flooding dose technique, was identical to that obtained in mucosa of adjacent jejunal segments that were not perfused (Fig. 4, P > 0.05). Because we did not correct for any decline in plasma free phenylalanine SR over the labeling period, the rate of protein synthesis obtained in these two piglets might have been overestimated. This however would not affect the conclusion drawn from the experiment since comparison is made within piglets. Additionally, the Ks values obtained are comparable to those shown in Fig. 3. Thus, as in rat experiments, the route of flooding did not affect piglet jejunal mucosal Ks.
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Systemic Effects
Compared with segments luminally perfused with radioactive tracer, free phenylalanine SR in plasma (28 ± 6 dpm/nmol) and in mucosa of adjacent jejunal tissue not perfused with isotope (36 ± 7 dpm/nmol) were negligible, being only 5-7% of free phenylalanine SR in mucosa of luminally flooded segments (516 ± 6 dpm/nmol; n = 4, P = 0.0002). Also, protein-bound phenylalanine SR in mucosa of adjacent unperfused jejunal tissue (0.17 ± 0.15 dpm/nmol) was only 5% of the corresponding values in luminally flooded segments (3.18 ± 0.15 dpm/nmol; n = 4, P = 0.0002). Plasma phenylalanine concentrations before and after perfusing four jejunal segments within a piglet with 2 mM phenylalanine were identical (0.133 ± 0.01 vs. 0.143 ± 0.01 µmol/ml; n = 2, P > 0.05). Thus luminal flooding with phenylalanine in up to four intestinal segments had minimal effects on tracer plasma concentration and on its SR in plasma and adjacent unperfused intestinal segments.To further test for systemic changes resulting from luminal nutrient
perfusion, we examined plasma variables (Table
1) in animals in which all segments were
perfused with PBS vs. animals with segments perfused with high
concentrations of glucose (50 mM) and amino acids (30 mM) or glutamine
(30 mM). Plasma glucose concentrations were similar before and at the
end of perfusion (Table 1). Insulin levels at both times were below
detection limit (4 µU/ml). This is not unusual as the piglets were
fasted for 16 h before the experiment. Plasma concentrations of
individual amino acids were also not affected by luminal nutrient
perfusion. The exceptions to these are the concentrations of valine,
glycine, and alanine, which were 33-40% higher in the amino
acid-perfused group compared with the saline-perfused piglets (Table
1). Thus, under the conditions of the experiments, luminal perfusion
with glucose and/or an amino acid-rich mixture had minimal
effects on circulating levels of glucose, insulin, and most amino
acids.
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Osmolarity and Protein Synthesis in Jejunal Mucosa
Luminal osmolarity in the range of 250-380 mosM did not affect tissue free phenylalanine SR (618 ± 15, 611 ± 15, 591 ± 11, 594 ± 6 dpm/nmol; P > 0.05) or Ks (Fig. 5, P > 0.05).
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DISCUSSION |
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We have described and validated a system for studying the acute effects of luminal nutrients on intestinal mucosal protein synthesis. Mucosal Ks values determined after luminal or intravenous tracer administration were identical. Using luminal administration of a flooding dose of tracer, we found that multiple jejunal segments constructed within the same animal had similar mucosal free phenylalanine SR and Ks. This implies that each of the four intestinal segments constructed within a piglet can serve as an experimental unit so that the effects of different luminal treatments on mucosal protein synthesis can be tested, in vivo, on the same piglet. In vivo variations of mucosal Ks in response to dietary perturbations (such as fed vs. fasted state) are of the order of 16-18% (8). Our system should be adequate to detect changes of this magnitude given the error term and our ability to make within-animal comparisons.
We also confirmed that when the intestinal lumen is the sole source of tracer, administered tracer radioactivity was associated mainly with phenylalanine, indicating that the tracer was not metabolized to other compounds by the intestinal tissue. Thus cost, time, and analytic error, associated with isolation of phenylalanine from any other metabolites that might have become labeled, are removed.
As to be expected from the percentage of intestine perfused with 3H tracer, labeling of plasma phenylalanine was very limited. In piglets of the size used in these experiments, the small intestine is at least 10-m long. Because we perfused a total length of 40 cm, only 4% of the small intestine was exposed to radioactive tracer. For comparison with rat studies, it is important to note that the 10-cm segment perfused constitutes ~13% of small intestinal length. Our observations do not imply that absorption of phenylalanine (or any other amino acids) is impaired. Rather, because intestinal surface area perfused in the piglet was small relative to the total absorptive capacity, the relatively small amount of absorbed amino acids does not affect plasma concentration and SR appreciably. Other factors that might have contributed to low plasma SR include loss of radioactivity on phenylalanine due to metabolism of phenylalanine to tyrosine in the liver and dilution of absorbed tracer by unlabeled phenylalanine resulting from high protein turnover of portal drained visceral tissues (24).
In luminally flooded rats, we also observed that intracellular free phenylalanine SR was lower at 2 min compared with the values in intravenously flooded groups. One reason for this might be the unstirred water layer effect (23), a factor that would make phenylalanine concentration at the brush-border membrane lower than that present luminally, therefore reducing the extent of the flooding. Apart from the reason given in MATERIALS AND METHODS for the choice of a phenylalanine concentration of 2 mM, we did not wish to use an unphysiological concentration, as this is one of the main criticisms of the intravenous flooding dose technique (27). Two millimolar is close to the luminal phenylalanine concentration after a protein meal (1, 23). A second possible reason for the observed lower free phenylalanine SR in luminally flooded intestine may relate to the fate of absorbed amino acids in the intestinal mucosa. Amino acids absorbed basolaterally can be either incorporated into protein or metabolized to other compounds. For amino acids absorbed luminally, however, a third "sink" would be the release of absorbed amino acids into the portal circulation. Finally, differences in the Michaelis constant (Km) for phenylalanine transport at the brush-border and basolateral membranes might also have contributed to this observation (Km for basolateral and brush-border membrane is 0.18 and 2.7-4.7 mM, respectively; see Ref. 17). Because of these reasons, it might be easier to flood through the basolateral than the brush-border membranes. Our observation that phenylalanine SR in perfused segments reached 500 dpm/nmol within 2 min of onset of perfusion (Fig. 1) clearly demonstrated that absorption was not impaired in the studied segments.
It was necessary to test whether luminal perfusion with nutrient solutions affected systemic levels of the perfused nutrients and whether the surgical and perfusion procedures affected intestinal mucosal protein synthesis. Because the perfused segments constitute only ~4% of the absorptive surface area of the small intestine and because perfusion lasted only 60 min, luminal perfusion of jejunal segments with nutrient solution did not alter plasma concentrations of the perfused glucose, amino acids, or insulin. Only a small increase in the plasma concentrations of alanine, glycine, and valine was attributable to luminal perfusion. There was, however, an effect of surgery and anesthesia per se on plasma amino acid levels. Previous studies (7, 19, 25) have shown that surgery and anesthesia increased plasma concentrations of essential amino acids by up to 45% when measurements were made during and immediately (2-12 h) postsurgery. Levels of nonessential amino acids, especially of alanine, glutamine, and asparagine, were either unchanged or increased by 20-30%. Thus surgery- and anesthesia-induced increases in plasma amino acid concentrations seen in this study are consistent with the literature. It is not clear why only plasma concentrations of valine, glycine, and alanine were increased by the luminal perfusate, more so when the concentration of leucine in the perfusate was higher than that of valine. Because 20 cm of intestine was perfused with 30 mM amino acid mixture for 1 h, it is possible that these few and small effects could be further reduced by perfusing only one intestinal segment (10 cm) for a shorter time.
Similar to what was observed for plasma nutrient concentrations, luminal nutrient perfusion did not affect plasma insulin concentration. Insulin-like growth factor I and proglucagon-derived peptides such as glucagon-like peptide-1 (GLP-1) and GLP-2 are some of the gut-derived hormones implicated in the regulation of gut growth (9). We did not examine the effects of luminal nutrient perfusion on gut hormone release. However, GLP-1 is insulinotropic and the anticatabolic effect of the peptide is insulin dependent (30). Therefore, a lack of effect of luminal nutrient perfusion on plasma insulin level would seem to imply that the amount, if any, of proglucagon-derived peptides produced is insufficient to affect systemic levels of these peptides and therefore of insulin.
Surgery does not affect whole jejunal protein synthesis (4, 26) or suppresses jejunal seromuscular (22) protein synthesis, whether measurements were made immediately or 2 to 7 days after surgery and anesthesia. However, the effect of surgical procedure on mucosal protein synthesis has not been previously reported. Our results indicate that trauma associated with intestinal cannulation and perfusion for up to 2 h had no effect on jejunal mucosal protein synthesis compared with unperfused segments in situ.
Because perfusion of multiple intestinal segments within an animal had minimal effects on circulating levels of the perfused nutrients and insulin, this model permits controlled studies of the effects of nutrients in the first pass (i.e., apical but not basolateral exposure to nutrients) on intestinal protein metabolism. Moreover, because it is an in vivo system, many of the complications associated with incubation of isolated epithelia cells (34) are also avoided. Weber et al. (35) used a similar preparation in the rat to study the effect of luminal jejunal perfusion with 56 mM glucose for 1.75 h on mucosal protein synthesis. A major improvement of the system described here is that, because of the size of the perfused segments relative to the total absorptive capacity of the animal, systemic levels of perfused nutrients and plasma levels of hormone were not modified. Therefore, observations made could only be attributed to first-pass nutrients. The same may not be true if similar studies are conducted in smaller animals such as rats, since the perfused segment would constitute a greater proportion of the small intestine. Perfusion of a 10-cm length of rat jejunum with 56 mM glucose for 1.75 h, as done by Weber et al. (35), would likely raise plasma glucose and insulin levels and therefore make interpretation of luminal vs. basolateral as well as nutrient vs. hormone effects difficult.
Cellular hydration state participates in the regulation of cell function (6, 15). In hepatocytes, cell swelling increased protein synthesis and suppressed proteolysis while cell shrinking had opposite effects (6). Furthermore, the effects of hormones and amino acids on hepatic protein synthesis and proteolysis can be mimicked quantitatively when the cell volume changes that occur in response to these effectors are induced to the same degree by anisosmotic exposure (15). Following from the work of Ferraris et al. (11), which showed that rat small intestinal luminal osmolality fluctuated between 316 and 384 mosmol/kg, we examined the effects of physiological changes (250-380 mosM) in luminal osmolarity on intestinal mucosal protein synthesis. Free phenylalanine SR and protein synthesis were identical in all osmolar treatment groups. This contrasts with a previous work (21) in which 30 mM short-chain fatty acids and isosmolar (NaCl) solution suppressed protein synthesis in isolated rat colonocytes. The differences in the design of the two experiments (species, cell, and tissue types) do not allow easy comparison. Also in the study of Marsman and McBurney (21), isolated epithelial cells were used in contrast to the present experiment in which epithelial cells retained a basolateral and brush-border sidedness and the intestinal architecture was intact.
In conclusion, this work validated the luminal flooding dose technique for study of intestinal mucosal protein synthesis. When combined with the multiple segments perfusion system within an animal, the model offers a unique technique to study acute luminal nutrient modulation of protein metabolism in small intestinal mucosa. It allows systematic study of luminal vs. basolateral effect, avoids interanimal variability, and reduces cost of experimentation. We have also used the model to show that, unlike hepatocytes (6, 15), the intestinal mucosa is resistant to physiological variations in luminal osmolarity with respect to protein synthesis. Ongoing studies in our laboratory are using the model described here to examine the effects of first-pass (luminal) nutrients, as well as route of delivery of nutrients (enteral vs. parenteral), on intestinal mucosal protein metabolism. A related study (unpublished observations) contains a description of changes in mucosal protein synthesis and expression of proteolytic genes in response to luminal and basolateral stimuli (including energy substrates and amino acids).
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ACKNOWLEDGEMENTS |
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We are grateful to Jinzeng Yang for technical assistance and to Jody Aldrich for animal care and surgical assistance.
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FOOTNOTES |
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This study was supported by the Alberta Agricultural Research Institute, the Natural Sciences and Engineering Research Council of Canada, and the Swine Endowment Fund, University of Alberta. O. A. J. Adegoke is a University of Alberta PhD and Keith Gilmore Scholar and also received financial assistance from Halchemix Canada Inc.
A portion of this work was presented at the Experimental Biology meeting in New Orleans, LA, in April 1997.
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. §1734 solely to indicate this fact.
Address for reprint requests: V. E. Baracos, Dept. of Agricultural, Food and Nutritional Science, 4-10 Agriculture-Forestry Center, Univ. of Alberta, Edmonton, Alberta, Canada T6G 2P5.
Received 9 April 1998; accepted in final form 2 September 1998.
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