Institut National de la Santé et de la Recherche Médicale U.539, Centre de Recherche en Nutrition Humaine, 44093 Nantes, France
Submitted 25 October 2002 ; accepted in final form 27 February 2003
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ABSTRACT |
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stable isotopes; leucine; small intestine; gut trophicity; human enterocyte
Because of inconsistent reports in literature, we do not, however, know whether nutrients and/or route of nutritional support regulate intestinal protein synthesis. As a matter of fact, several studies using pig model suggest the supply of nutrients through the gastrointestinal tract is critical to maintaining adequate rates of gut protein synthesis (14, 43). Similarly, the luminal perfusion of high glucose loads increases the protein synthesis rate in rat small intestinal mucosa (47). On the other hand, Adegoke et al. (1) suggested that luminal amino acids supply may paradoxically decrease gut protein synthesis in porcine intestinal loops, and Bouteloup-Demange et al. (7) observed unaltered rates of protein synthesis in the duodenum of healthy humans, whether they were in a fed or fasting state. The effect of nutrition per se on human gut protein synthesis therefore remains controversial.
Glutamine, a conditionally essential amino acid, appears to be a key nutrient for the gut, because it is a prominent source of energy for enterocytes (48), a major nitrogen donor for nucleotide synthesis (32), and a precursor for glutathione synthesis (10). Furthermore, in vitro studies demonstrated the role of glutamine in proliferation and differentiation of intestinal cells (13, 34), and glutamine supplementation was shown to reduce both villous atrophy and bacterial translocation during parenteral nutrition in animals (27, 44). Studies addressing the effects of glutamine on intestinal protein synthesis have, however, yielded conflicting results. In humans, although enteral glutamine enhanced whole body protein synthesis in healthy human volunteers (19), the putative protein anabolic effect and the effect of glutamine on human intestinal mucosal mass and permeability remains controversial (15). Regarding the small intestine, glutamine failed to promote duodenal protein synthesis in healthy dogs (29), and the trend toward an increased rate of duodenal protein synthesis failed to reach statistical significance in healthy humans receiving glucocorticoids (8) and in dogs submitted to dexamethasone and protein restriction (21). Because the gut wall is a complex tissue comprised not only of enterocytes but of numerous other cell types (e.g., immune cells, neurons, endocrine or muscle cells, etc.), in vivo studies may not yield the answer to whether glutamine regulates protein synthesis in enterocytes.
Although rodent intestinal cells have been used to address this issue in vitro, the model used, i.e., isolated enterocytes (20), may not mimic the conditions prevailing in the intact human intestinal epithelium. Using Caco-2 cells as model of human enterocytes in culture, we recently demonstrated that depletion of intracellular glutamine pool decreased protein synthesis (26). Yet in our earlier study, we used Caco-2 cells grown on plastic dishes, whereas the human intestinal epithelium is a polarized tissue in which enterocytes have access to both an apical and a basolateral nutrient supply from the intestinal lumen and the serosal circulation, respectively.
Although the Caco-2 cell line was originally derived from a colon carcinoma, Caco-2 cells differentiate as enterocytes in culture, and can be grown as a polarized monolayer when seeded on porous filters on Transwell chambers. The first aim of the present study was therefore to set up an experimental model that would allow access to both sides of the bipolar intestinal epithelium. This would enable us to use nutrient deprivation on the apical side of the epithelium to mimic the conditions prevailing during fasting in vivo. We used this model to address the following specific questions: 1) Does nutrient deprivation on the apical side, a situation mimicking in vivo fasting conditions, affect protein fractional synthesis and intracellular glutathione pool in our model of human enterocytes? 2) If so, can glutamine supplementation attenuate the effects of fasting? And 3) are the effects of glutamine dependent on its route (apical vs. basolateral) of delivery?
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MATERIAL AND METHODS |
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Cell culture. Intestinal Caco-2 cells were obtained from the American Type Culture Collection (ATCC; Manassas, VA). Under normal culture conditions, these cells differentiate into enterocyte-like cells. They form a polarized epithelial monolayer expressing several differentiation markers such as brush-border membrane, sucrase isomaltase (11), and transepithelial transport (16). After establishment of confluency (79 days), the process of differentiation occurs and is maximal within 2 wk. Cells were seeded onto six Transwell clear treated polyester membrane (Merck Eurolab; Strasbourg, France) plates at a density of 100,000 cells/cm2 in DMEM containing 2 mM glutamine, 20% (vol/vol) heat-inactivated FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin and were incubated at 37°C in 5% CO2-95% humidity. This type of plate allows an access to both apical and basolateral sides of the epithelial monolayer. Culture medium was replaced every other day for 20 days.
Experimental design. On the day of experiment, cells were incubated for 24 h with DMEM prepared with a fourfold dilution with 0.9% NaCl to obtain glucose and amino acid concentrations in the physiological range observed in plasma and without FBS. Cell luminal "fasting" was achieved by replacing the medium on the apical side with 0.9% saline buffer, whereas the basolateral medium was kept unchanged. At the same time, cells were either supplemented or not with glutamine alone applied either to the apical or the basolateral side (Fig. 1) and incubated with or without 5 mM DON, an inhibitor of glutaminase for 24 h. Cells were then incubated for 4 h with 2 µmol of [2H3]leucine added to each well apical side without changing the medium. After the experiment, medium was immediately aspirated, and each plate was washed two times with cold PBS and frozen until analytical procedures to retrieve the intracellular protein and cytosol.
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Analytical procedures. Six nanomoles L-[2-15N]glutamine and 24 nmol L-homoglutamate were added to each well as internal standards and cells were detached mechanically from dishes by scraping in 500 µl PBS. Suspended cells were then lyzed by ultrasound before protein was precipitated with 50 µl 5% (wt/vol) sulfosalicylic acid (SSA). Cell extracts were then centrifuged, and the supernatants were decanted, neutralized with 1 mM potassium hydroxide, and frozen until the day of analysis.
Intracellular glutamine, glutamate, and leucine analysis. Glutamine and glutamate in intracellular media were analyzed by using a modification of the procedure proposed by Reeds et al. (40) and Humbert et al. (22). Samples were dried, then incubated for 2 h with 1-propanol/acetylchloride mixture (5:1, vol/vol) at room temperature, dried under nitrogen, and finally incubated for 30 min at 60°C with heptafluorobutyric anhydride (HFBA) to convert glutamine and glutamate to their respective heptafluorobutyryl acetyl ester derivatives, which were finally dried under nitrogen and dissolved in ethylacetate.
To determine [2H3]leucine enrichments in protein-bound leucine, cell protein was precipitated by using 5% SSA and the cell protein pellets were hydrolyzed for 24 h in 6 M hydrochloric acid. The free leucine in the intracellular amino acid pool was extracted from the SSA soluble supernatant. Both free leucine and leucine resulting from protein hydrolysis were then extracted by cation-exchange chromatography on microcolumns containing AG50 resin as described (30), and derivatized to HFBA-leucine (28), which was then measured by GCMS. The isotopic enrichments in free and protein-bound leucine, glutamine, and glutamate concentrations were determined by using methods described previously (26).
Measurement of protein synthesis rate. Cell protein fractional synthesis rate (FSR; %/day) was calculated as FSR = (Ebound x 24 x 100)/(Efree x t), where t is the duration of incubation with labeled leucine, Ebound is the enrichment in protein-bound leucine at time t, and Efree is the steady-state isotopic enrichment in intracellular free leucine, the precursor amino acid pool.
Gluthatione analysis. Cells were harvested by scraping filters into PBS with a rubber policeman and sonicated at 0°C. The cell homogenate was incubated for 15 min in 5% SSA (wt/vol) and centrifuged. The supernatant was analyzed for reduced (GSH) and oxidized (GSSG) gluthatione content by the GSH disulfide reductase 5,5'-dithio-bis(2-nitrobenzoic acid) recycling method by using the procedure described in the gluthatione assay kit from Cayman Chemicals (17). The protein pellet was solubilized in 1 M NaOH and protein assay was performed by using the Bradford method (Bio-Rad, Hercules, CA).
Measurement of transepithelial resistance and permeability. Transepithelial resistance (TER) was measured by using Millicell-ERS from Millipore (Bedford, MA). This device measures membrane potential and resistance of epithelial cells in culture. It qualitatively measures cell monolayer health and cell confluency. For permeability quantification, apical medium was replaced by medium containing 1 mg/ml FITC-dextran 4 kDa and incubated for 2 h. Basolateral medium aliquots were then measured by fluorometry.
Statistical analysis. Results are reported as means ± SD for n independent cell cultures. Unpaired Student's t-test was used to determine differences between samples, and significance was established at P < 0.05.
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RESULTS |
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A steady state [CV = (100 x SD/mean) < 10%] was achieved in the
isotopic enrichment of intracellular free leucine within 30 min of incubation
with labeled leucine (data not shown). Incubating cells with saline on the
apical side and regular DMEM on the basolateral side, a situation we called
"fasting state," was associated with an 20% decline in cell
protein synthesis from 24.5 ± 2.3 to 20.2 ± 2.8%/day (P
< 0.01) (Fig. 2).
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Finally, the absence of nutrients on the apical side increased transepithelial permeability as measured by FITC-dextran fluxes (Fig. 3). There was no evidence for cell death as estimated by the extracellular lactate dehydrogenase (LDH) to total (extracellular + intracellular) LDH ratio [2 ± 2% in control vs. 4 ± 1% in fasting cells, not significant (NS)].
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Effects of glutamine supplementation in the fasting state. Supplementation of the medium with graded doses of glutamine applied either to the apical or basolateral side of the monolayer maintained protein synthesis at its control fed value (Fig. 2). Regardless of the site of supplementation, the maximal effect was obtained for a glutamine concentration of 0.6 mM, i.e., within the physiological range of plasma glutamine. Higher glutamine doses did not enhance protein synthesis beyond control fed values. Supplementation of the medium with glutamine alone did not restore the glutathione pool even with glutamine concentrations 4.4 mM (Table 2), but was associated with a dose-dependent increase of TER, which was completely restored with a 4.4 mM glutamine supplementation. Glutamine supplementation also decreased transepithelial permeability (Fig. 3).
Glutamate supplementation was either as effective or more effective than glutamine in restoring protein synthesis (Fig. 4) depending on doses and side of supplementation. This implies that glutamine needs to be deamidated through glutaminase into glutamate to exert its stimulatory effect. Like glutamine, 4.4 mM glutamate was ineffective in restoring glutathione pool (GSH: 11.0 ± 1.2 vs. 12.5 ± 2.3 nmol/mg protein, NS; GSSG: 0.8 ± 0.2 vs. 0.9 ± 0.8 nmol/mg protein, NS), but reduced paracellular permeability to dextran (0.14 ± 0.01 vs. 0.34 ± 0.03 µg · cm2 · h1, P < 0.05) during fasting. In a second set of experiments, fasted cells were incubated simultaneously with glutamine and 5 mM DON. In preliminary experiments, we found that dose of 10 mM DON was sufficient to induce a 75% decrease in L-[U-14C]glutamine oxidation (Fig. 5). The addition of this glutaminase inhibitor prevented the stimulatory effect of glutamine on protein synthesis (Fig. 5) in the fasting state. Yet the addition of DON to cells in the fed (control) state did not alter baseline rates of protein synthesis.
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Glutamine supplementation failed to enhance intracellular glutamine and glutamate concentrations in fasting cells under baseline conditions, except for the higher glutamine dose (1.4 mM) on the basolateral side (Table 1). DON treatment was associated with the accumulation of glutamine and depletion of glutamate (Table 1), underscoring the high rate of utilization of these nutrients in our model of human enterocytes in culture.
To determine whether other amino acids could mimic the effect of glutamine, we carried out experiments in which cells submitted to apical fasting were supplemented with L-leucine (1.4 mM) or L-glycine (2.8 mM) to provide the same amount of oxidizable carbons. Compared with glutamine, glycine supplementation failed to restore FSR to its control value (P < 0.01), whereas leucine supplementation decreased FSR (P < 0.01) (Table 3, experiment 1). On the other hand, glucose supplementation (1.4 mM) restored FSR as effi-ciently as glutamine (P < 0.01) (Table 3, experiment 2).
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DISCUSSION |
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The first aim of the present study was to establish a model to produce some
of the effects of fasting on a polarized human intestinal epithelium in vitro.
Studies (1,
14,
43) using animal models have
indeed yielded conflicting data on the regulation of gut protein synthesis by
nutrients and route of nutrient supply, and only few studies
(7) have investigated this
issue in the human gut. Besides, as the gut wall is comprised of many
different cell types, little is known on the effects of nutrients on
enterocytes per se. The 20% decline in enterocyte protein synthesis
observed in the present study is consistent with previous studies
(33,
41,
43) performed in the whole
intestinal wall in animals, and suggests that although the small intestine can
draw its energy from both circulating blood and intestinal lumen, the supply
of nutrients through the gastrointestinal tract may play a key role in
maintaining gut protein synthesis.
In a previous study (26), we observed that Caco-2 cells were able to proliferate and maintain normal rates of intracellular glutamine and cell protein synthesis for 20 days even when grown in the absence of glutamine in the medium because of upregulation of glutamine synthetase activity. The latter study also underlined the major role of glutamine in regulating protein synthesis because glutamine depletion induced by MSO, a glutamine synthetase inhibitor, induced a dramatic drop in protein synthesis. In the present study, "apical fasting" was accompanied by a 38% drop in intracellular free glutamine, although glutamine was present (0.6 mM) in the basolateral medium. This finding suggests that during apical fasting, enterocytes are unable to meet the demand for glutamine, presumably because glutamine synthesis is not upregulated within 24 h of fasting. Glutamine concentration in our cells was lower than in duodenal biopsy samples obtained in healthy humans in our laboratory (12.8 ± 1.6 µmol/g protein) but in the same range as the 5.1 and 4.8 µmol/g protein observed by others (3, 36). Extrapolation of these results to the in vivo situation should, however, be cautious, because very few studies have assessed the alterations in mucosal glutamine content in human gut in vivo (2, 45), and unaltered glutamine and glutamate concentrations were observed in human gut biopsies after a 3-day fast in one study (2).
Supplementation of the medium with glutamine either on the apical or the basolateral side restored protein synthesis rates to control levels with a maximal effect within the physiological range of plasma glutamine concentrations (Fig. 2) regardless of the route of glutamine administration. Glutamate was able to mimic the effects of glutamine and restore protein synthesis under fasting conditions as well (Fig. 4). Moreover, when DON was added to the medium, the effect of glutamine was completely abolished (Fig. 5). Taken together, these results strongly suggest that 1) glutamine needs to be deamidated into glutamic acid to exert its effect on protein synthesis, and 2) glutamate may be as efficient as glutamine in promoting protein accretion. Because DON treatment results in a 75% decrease in the production of 14CO2 during incubation with [U-14C]glutamine (Fig. 5), we speculate that the effect of glutamine on intestinal protein synthesis may be mediated by its role as a source of energy in our model of human enterocytes.
Energy supply may indeed be of paramount importance for sustaining normal rates of protein synthesis. Consistent with this view, supplementation with glucose, another fuel in enterocytes (35), restored FSR, whereas glycine and leucine were ineffective (Table 3). Taken together, these results suggest that only substrates that can be used as fuels by enterocytes (e.g., glutamine, glutamate, glucose) may enhance protein synthesis in enterocytes. Accordingly, Higashiguchi et al. (20) demonstrated that the effect of glutamine on the incorporation of [3H]phenylalanine into proteins was probably mediated by its role as a fuel for rodent intestinal cells. Although DON abolished the effect of glutamine in fasted cells, DON treatment failed to alter baseline rates of protein synthesis (Fig. 5) when Caco-2 cells had access to a complete medium containing amino acids and glucose on the apical side. This suggests that these human intestinal cells may use substrates other than glutamine to sustain normal rates of protein synthesis, provided these nutrients are supplied via the enteral route.
The data in Fig. 4 show that glutamate was either as effective or more effective than glutamine in restoring protein synthesis during fasting, depending on the route and dose of supplementation. The difference, however, only reaches statistical significance with the high dose of glutamate (1.4 mM) supplied on the apical side, and the low dose of glutamate (0.6 mM) supplied on the basolateral side. Several studies suggest that glutamate may be more efficiently extracted and oxidized than glutamine by the gut. As a matter of fact, when [13C]glutamate was administered via the enteral route in vivo in humans, 96% of the infused glutamate was retained in the first pass (5), compared with 64% of an equivalent dose of [13C]glutamine (18). Moreover, 78% of the infused 13C was recovered as exhaled CO2 when [13C]glutamate was infused, vs. 73% on enteral infusion of [13C]glutamine. The fraction of enterally delivered tracer that was oxidized specifically on first pass by the splanchnic bed was 78% for glutamate (5) vs. 53% for glutamine (18). If this holds true for Caco-2 cells as well, then glutamate would supply cells more energy than an equimolar amount of glutamine, and assuming glutamate and glutamine affect protein synthesis via their roles as a source of energy, would account for the more powerful effect of glutamate. Yet, because we did not measure glutamate oxidation by Caco-2 cells in our experimental setting, this remains speculative.
Maintenance of gut barrier function is a major goal in the supportive care of critically ill patients, because multiple trauma increased intestinal permeability (38). Nevertheless, patients admitted to intensive care units often receive most of their nutritional support via the parenteral route, which does not contain glutamine. This route is associated with increased permeability of the intestine (9), gut atrophy (39), and bacterial translocation (4) in animal models. In our study, we observed that a 24-h apical nutrient deprivation resulted in greater permeability as measured by FITC-dextran flux through the Caco-2 cell monolayer (Fig. 3), whereas apical or basolateral glutamine supplementation decreased permeability. Because of its role in the preservation of gut integrity (46, 49), it was proposed that altered glutamine metabolism and loss of gut barrier function were related to disturbed proliferation or differentiation of enterocytes (12, 42). Others postulated that fasting (23) and glutamine deprivation (37) induce apoptosis in intestinal epithelial cells. In the present study, the rise in transepithelial permeability associated with fasting was accompanied by a decrease in intracellular glutamine concentration (Table 1), but no rise in cell death, as estimated by the LDH ratio. This suggests that the loss of gut barrier function during fasting was mediated by functional alterations in the tight-junction complex rather than by permanent cell damage. In additional experiments using immunohistochemistry techniques on ZO-1, a specific protein of tight junctions, we observed no detectable change in the intensity of fluorescence or in the localization of ZO-1 with fasting or glutamine supplementation during fasting (data not shown).
Because there is little, if any, transport of intact glutathione from plasma to intestinal mucosa (31), maintenance of the gut glutathione pool depends on in situ glutathione synthesis, and, consequently, on the supply of adequate precursors. Depletion of systemic and tissue glutathione content has been reported during starvation. In particular, Kelly et al. observed that a 24-h food restriction decreased glutathione concentration in rat liver and small intestine (24). In our in vitro study, fasted cells had significantly lower levels of both reduced and oxidized glutathione (Table 2). Glutamine supplementation, whether on the apical or the basolateral side, was ineffective in restoring the GSH and GSSG pool, whereas it has been demonstrated to increase gut glutathione production in fed rats (10). This discrepancy may largely be explained by differences in experimental design, because fasting may have decreased the availability of cysteine as well, another building block of glutathione. Regardless of its mechanism, the depletion of cell glutathione may expose intestinal cells to oxidative stress and promote cellular damage such as atrophy, mitochondria degeneration, and vacuolization (31).
In summary, the results of this study strongly suggest that nutrient supply from the luminal surface is essential to maintaining the trophic state of enterocytes in a model of polarized human enterocytes in culture, because deprivation of luminal nutrients decreases cell protein synthesis, glutathione, glutamine and glutamate contents, and impairs epithelial barrier function. They further demonstrate that supplementation with free glutamine, regardless of its route of delivery, restores cell protein synthesis and diminishes permeability, but is ineffective in restoring the glutathione pool. The results are consistent with the view that glutamine's effects require its deamidation into glutamate. The specific mechanism of action of glutamate remains to be elucidated. It may work as a source of energy, or alternatively, act through signaling pathways, because recent studies pointed to a role of amino acids in the regulation of 5'-TOP mRNA synthesis and ribosomal biogenesis though p70S6 kinase (25), which may influence the rate of global protein synthesis. Because the in vitro model of intestinal fasting reported here reproduces some of the alterations observed during food deprivation and prolonged total parenteral nutrition, it may be useful to further investigate mechanisms implied in the maintenance of the barrier function of human gut, a function that may be critical for survival in critically ill humans.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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