INSERM U.539, Centre de Recherche en Nutrition Humaine, 44093 Nantes, France
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ABSTRACT |
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To assess the effect of glutamine availability on rates of protein synthesis in human enterocytes, Caco-2 cells were grown until differentiation and then submitted to glutamine deprivation produced by exposure to glutamine-free medium or methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methylsulfoximine (MSO)], a glutamine synthetase inhibitor. Cells were then incubated with 2H3-labeled leucine with or without glutamine, and the fractional synthesis rate (FSR) of total cell protein was determined from 2H3-labeled enrichments in protein-bound and intracellular free leucine measured by gas chromatography-mass spectrometry. Both protein FSR (28 ± 1.5%/day) and intracellular glutamine concentration (6.1 ± 0.6 µmol/g protein) remained unaltered when cells were grown in glutamine-free medium. In contrast, MSO treatment resulted in a dramatic reduction in protein synthesis (4.6 ± 0.6 vs. 20.2 ± 0.8%/day, P < 0.01). Supplementation with 0.5-2 mM glutamine for 4 h after MSO incubation, but not with glycine nor glutamate, restored protein FSR to control values (24 ± 1%/day). These results demonstrate that in Caco-2 cells, 1) de novo glutamine synthesis is highly active, since it can maintain intracellular glutamine pool during glutamine deprivation, 2) inhibition of glutamine synthesis is associated with reduced protein synthesis, and 3) when glutamine synthesis is depressed, exogenous glutamine restores normal intestinal FSR. Due to the limitations intrinsic to the use of a cell line as an experimental model, the physiological relevance of these findings for the human intestine in vivo remains to be determined.
stable isotopes; leucine; nutrition
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INTRODUCTION |
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AS GLUTAMINE IS SYNTHESIZED de novo from glutamate and ammonia in a wide variety of tissues containing glutamine synthetase, it is considered a nonessential amino acid. Over the last two decades, evidence has, however, accumulated to suggest that glutamine may play a key role in the regulation of protein homeostasis (26). A striking correlation between free glutamine concentration and the rate of protein synthesis in skeletal muscle was observed in rats submitted to endotoxin treatment or protein restriction (15). Other workers, however, failed to observe this correlation in other experimental models (39). In humans, supplementation of total parenteral nutrition (TPN) with glutamine enhanced nitrogen balance in patients undergoing major surgery (31), and a doubling in plasma glutamine concentration enhanced nonoxidative leucine disposal, an index of whole body protein synthesis, in healthy fasting volunteers (11). Yet in another study, dietary glutamine supplementation failed to alter leucine and phenylalanine kinetics in healthy volunteers in the fed state (13). The role of glutamine as an anabolic agent in humans, therefore, remains controversial.
The tissue site of glutamine's putative anabolic effect is debated as well. The small intestine is a prominent site of glutamine uptake in many species. Although glutamine was first proven to serve as a respiratory fuel for enterocytes (38), more recent evidence suggests that glutamine may increase gut trophicity as well. Glutamine indeed mitigated the intestinal mucosa atrophy induced by infection (40) or experimental induced enterocolitis in rats (6). In addition, Higashiguchi et al. (12) recently showed that glutamine stimulated protein synthesis in rat isolated enterocytes. Although largely circumstantial, there is evidence that glutamine may affect trophicity of the human gut as well. Indeed, glutamine is extensively extracted in the human small intestine (20) and may prevent the small decline observed in intestinal villus height or improve the absorptive function of the gut in patients receiving TPN (35) and ameliorate gut barrier functions in patients submitted to anticancer chemotherapy (35, 41).
Together, these studies prompted us to examine whether glutamine availability affects protein synthesis in human enterocytes. The Caco-2 cell line was deemed a reasonable experimental model because 1) the cell line is of human origin; 2) although originally derived from a colon carcinoma, Caco-2 cells undergo an enterocytic differentiation in vitro and share many characteristics of normal human enterocytes, including the oxidation of glutamine to CO2 and the secretion of complex specific protein particles that are highly specific for enterocytes, such as high-density lipoprotein-bound apolipoprotein A-IV (apo A-IV) (25); and 3) the use of a cell line allows for the study of a homogenous population of epithelial cells as opposed to a heterogeneous mixture of cell population found in biopsy samples. To delineate the relationships between glutamine availability and protein synthesis, Caco-2 were submitted to glutamine deprivation either by maintaining cells in glutamine-free medium alone or along with cell treatment with methionine sulfoximine [L-S-[3-amino-3-carboxypropyl]-S-methylsulfoximine (MSO)], a specific inhibitor of glutamine synthetase (9).
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MATERIAL AND METHODS |
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Chemicals. Trypsin, Ham's F-12/DMEM mixture, fetal bovine serum (FBS), antibiotics, MSO, and all other culture reagents were from Sigma Chemicals (Saint Quentin Fallavier, France). L-2-15N-labeled glutamine (99% 15N), L-15N-labeled glutamate (99% 15N), and L-3,3,3-2H3-labeled leucine (99% 2H3) were purchased from Cambridge Isotope Laboratories (Andover, MA). Ion exchange resins were obtained from Touzart et Matignon (Temex 1X8, 100-200 mesh, hydrogen form; Courtaboeuf, France) and Aldrich (Dowex 50WX8-200; Saint Quentin Fallavier, France). All chemicals and isotopic purities were verified by gas chromatography-mass spectrometry (GCMS).
Cell culture. Caco-2 cells were incubated at 37°C in 5% CO2 and 95% humidity. These cells have been used by several groups as a model of differentiated human enterocytes. This is because under normal culture conditions, Caco-2 cells rapidly divide to form an adherent monolayer of undifferentiated cells and subsequently, after establishment of confluence (7-9 days), differentiate like normal enterocytes to form a polarized epithelial layer with a brush border membrane that contains hydrolases such as sucrase-isomaltase (4). When grown on plastic, they form domes typical of normal transporting epithelial cells (8). The process of differentiation occurs and is maximal within 2 wk and can be easily monitored by visually checking the formation of domes. Cells were seeded onto six-well tissue-culture plates at a density of 30,000 cells/cm2 in Ham's F-12/DMEM nutrient mixture (without glutamine) containing 20% (vol/vol) FBS, 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Culture medium, containing various glutamine concentrations ranging from 5 to 0.1 mM (in this case, glutamine only derived from FBS), was replaced every other day for 20 days.
Experimental design. On the day of experiment, cells were incubated for 24 h with 2 ml Ham's F-12/DMEM nutrient mixture without FBS or glutamine and 10 mM MSO to inhibit glutamine synthetase activity. The MSO concentration used was similar to that inducing complete irreversible inhibition of sheep brain glutamine synthetase (30). During the following 4 h, 2 µmol of 2H3-labeled leucine were added to each well without changing the medium. After various times of incubation, the incubation 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.
Analytical procedures. Six nanomoles of L-2-15N-labeled glutamine or L-U-13C5-labeled glutamine and 24 nmol of L-homoglutamic acid were added into each well as internal standards, and cells were detached mechanically from dishes by scraping in 300 µl PBS. Suspended cells were then lysed by ultrasounds before proteins were precipitated with 50 µl 5% (wt/vol) sulfosalicylic acid (SSA). Deproteinized cell extracts were centrifuged, and the supernatants were decanted, neutralized with 1 M potassium hydroxide, and frozen until the day of analysis.
Intracellular glutamine and glutamate analysis. The glutamine and glutamate in intracellular media were analyzed using a modification of the procedure proposed by Reeds et al. (29). Samples were dried, and glutamine and glutamate were then converted to their respective heptafluorobutyryl acetyl ester (HFBA) derivatives by a 2-h incubation with 1-propanol/acetylchloride mixture (5:1 vol/vol) at room temperature dried under nitrogen before being incubated for 30 min at 60°C with heptafluorobutyric anhydride. Samples were finally dried under nitrogen and dissolved in ethylacetate. This procedure essentially prevents glutamine degradation to glutamate during sample processing and derivatization.
Leucine assay. To determine 2H3-labeled enrichments in protein-bound leucine, cell proteins were precipitated using 5% SSA and the cell protein pellet. The free leucine in the intracellular amino acid pool was extracted from the SSA-soluble supernatant. Both free leucine and the leucine resulting from protein hydrolysis were then extracted by cation-exchange chromatography on microcolumns containing AG50 resin, as described (19), and derivatized to HFBA leucine (17), which was then measured by GCMS.
GCMS assays. The isotopic enrichments in free and protein-bound leucine were determined using electron-ionization (EI) GCMS on a 5890 series II gas chromatograph coupled with a 5971A mass selective detector (Hewlett-Packard) equipped with a capillary column (DB-1, 30 m × 0.25 mm ID, 0.25 mm film thickness; J & W Scientific). Selected ion monitoring (SIM) was used on ions of mass-to-charge ratio (m/z) 282 and 285, representing natural and 2H3-labeled leucine, respectively. Ions at m/z 279, 280, and 283, representing natural glutamine, 15N-labeled glutamine, and 13C5-labeled glutamine, respectively, were also selectively monitored. For glutamate, ions at m/z 252 and 253, representing natural and 15N-labeled glutamate, were monitored, as well as ion at m/z = 252 for the homoglutamate internal standard.
Measurement of protein synthesis rate. Cell protein fractional synthesis rate [FSR (%/day)] was calculated as FSR = (Ebound × 24 × 100)/(Efree × t), where t is the duration of the 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.
Total cell protein assay. The cell layer protein was assayed by Lowry's method (16).
Apo A-IV assay. Secreted apo A-IV was quantified using a sandwich ELISA with rabbit anti-human apo A-IV polyclonal antibodies tagged with peroxydase (Pasteur Institute, Lille, France). Plates were treated with 10-µg/well antibodies overnight at 4°C and incubated in succession with BSA (1 h, 37°C), standard serum (SEBIA, Issyles-moulineaux, France), and polyclonal anti-apo A-IV (1/4,000). Plates were washed four times between each incubation. Absorbance at 405 nm was determined by using a spectramax 190 microplate reader (Molecular Device).
Measurement of glutamine synthetase activity. Glutamine synthetase activity was measured on Caco-2 cell extracts obtained using by freeze-thawing, as described earlier (21, 23).
Statistical analysis. Results are reported as means ± SD for n independent cell cultures. 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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Glutamine-deprivation experiments.
An isotopic enrichment plateau [CV = (100 × SD/mean)
<10%] was achieved within 30 min of incubation with labeled leucine
(data not shown). In this series of experiments, Caco-2 cells were
incubated in glutamine-free DMEM, with FBS as the only glutamine
supply, yielding a measured final glutamine concentration of 0.1 mM in the well. Baseline intracellular free glutamine and glutamate concentrations were 6.1 ± 0.6 and 65.5 ± 12.6 µmol/g
protein, respectively, and intracellular free glutamine concentration
failed to decline despite the lack of glutamine in the culture medium (Table 1).
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Effects of methionine sulfoximine on glutamine synthetase activity, cell glutamine concentration, and cell protein synthesis. In this second series of experiments, cells were grown at either low (0.1 mM), physiological (0.6 mM), or high (5 mM) glutamine concentrations.
In preliminary experiments, we verified that MSO was able to cross the cell membrane by following the appearance of a substrate with the characteristic spectrum of MSO in intracellular milieu (data not shown). In another series of experiments, the effects of glutamine deprivation and MSO on glutamine synthetase activity were measured on extracts from Caco-2 cells using a reference method based on the conversion of U-14C-labeled glutamate to U-14C-labeled glutamine. When cells had been grown under control conditions (2 days with a DMEM medium containing 0.6 m mM glutamine), glutamine synthetase activity was 151 ± 30 nmol · h
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"Rescue effect" of glutamine on protein FSR.
After MSO treatment, supplementation of the medium with 1 mM of
glutamine for 4 h restored [gln] to 5.5 ± 0.5 µmol/g
protein (Fig. 1A) as well as cell protein FSR (Fig.
1B). In another set of experiments (depicted in Fig.
3, top), MSO-treated cells
were exposed to graded levels of extracellular glutamine (0.25-2
mM); the maximal effect of glutamine was obtained for extracellular glutamine concentrations between 0.5 and 1 mM, i.e., within the physiological range of plasma glutamine. No further stimulation of
protein synthesis was observed with higher concentrations (Fig. 3A). As shown in Fig. 3B, the effect of glutamine
on cell protein FSR was detectable after 2 h of exposure to
glutamine. In contrast, neither a 4-h exposure to an isonitrogenous
glycine solution (4 mM) nor 24-h incubation with glutamate (2 mM) was
able to restore protein FSR to its baseline value (Table
3).
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Effect of MSO on cell protein content and on secreted apo A-IV.
The amount of total cell protein in Caco-2 cells cultured with 0.6 mM
glutamine was ~1.15 ± 0.05 mg/well. Protein content was reduced
by 13% under MSO treatment (P < 0.01). This
reduction was, in part, corrected by glutamine supplementation (Table
4). Absolute protein synthesis rate (cell
protein content × FSR) was 0.23 and 0.05 mg protein/24 h under
baseline conditions and MSO treatment, respectively. The
0.18-mg
protein/24 h reduction in cell protein synthesis can thus account for
the 0.15-mg protein/24 h loss in cell protein content measured by the
Lowry method, implying that MSO did not have a major impact on rates of
protein breakdown. The effect of MSO was not restricted to
intracellular proteins. The concentration of apo A-IV, an
apolipoprotein secreted by differentiated enterocytes in extracellular
medium, indeed declined sharply after MSO and was restored by glutamine
(Table 4).
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DISCUSSION |
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The aim of this study was to determine whether glutamine depletion alters protein synthesis in a model of human enterocytes in culture. We observed that 1) de novo glutamine synthesis is highly active in differentiated Caco-2 enterocyte-like cells, since it allows the maintenance of intracellular glutamine concentration in intestinal cells and of normal rates of cell protein synthesis, even in a glutamine-free medium, and 2) inhibition of glutamine synthesis slows down cell protein synthesis, and a supply of glutamine under these conditions acutely restores protein synthesis in a dose-dependent fashion. These findings suggest that maintenance of intracellular glutamine plays a significant physiological role in the control of protein synthesis in a cell line of human origin that exhibits an enterocytic differentiation in vitro. Due to the limitations inherent in the use of a cell line as an experimental model, further studies would be needed to determine the relevance of these findings in human small intestine in vivo.
Little is known about glutamine metabolism in Caco-2 cells, and to our
knowledge, this study is the first to report glutamine and glutamate
concentrations in the intracellular milieu in this widely used cell
line. We found intracellular glutamine concentrations to be 6.4
µmol/g protein in Caco-2 cells (Table 1), i.e., slightly, albeit
significantly (P < 0.001), lower than in duodenal
biopsy samples obtained in healthy human volunteers in our laboratory (12.8 ± 1.6 µmol/g protein; mean ± SD of 3 subjects,
unpublished preliminary data) but in the same range as the 5.1 and 4.8 µmol/g protein observed by others in human duodenum mucosa (3,
27). Besides, in recent preliminary experiments (data not shown)
carried out with U-14C-labeled glutamine, we verified that
Caco-2 cells actively oxidize glutamine to CO2. This
suggests that Caco-2 cells share some metabolic characteristics with
normal human enterocytes.
Although conventional culture media contain high glutamine
concentrations (2-5 mM) and although glutamine is required for the
proliferation and the differentiation of intestinal cell lines in
culture (33, 37), the current study shows that these cells can be maintained in culture with as little as 0.1 mM glutamine without
compromising an important physiological function, i.e., cell protein
synthesis. The ability to maintain normal glutamine concentration when
grown in a glutamine-free medium is a clear indication that glutamine
synthetase activity produces enough glutamine to accommodate the
cell's requirements. Growth in a glutamine-free medium indeed
upregulates glutamine synthesis, as attested by the 53% rise in
glutamine synthetase activity found in cells grown for 20 days in a
glutamine-free medium, compared with controls (see
RESULTS). Moreover, upregulation of glutamine synthetase by
the glutamine-poor medium had a slight "protective" effect against
the subsequent reduction in protein FSR associated with MSO treatment.
Indeed, although baseline protein FSR was identical regardless of the
prevailing extracellular glutamine concentration (21.0, 20.6, and
19.6%/day for 0.1, 0.6, and 5 mM glutamine in the incubation medium,
respectively; Fig. 1B), the decline in protein FSR
associated with subsequent MSO treatment was less dramatic for the
cells that had previously been grown in the 0.1 mM glutamine incubation
medium (from 21.0 to 7.1%/day) compared with cells previously grown in
5 mM glutamine (from 19.6 to 4.7%/day), i.e., a 64% vs. a 76%
relative reduction (P < 0.0001; Fig. 1B).
In humans, apo A-IV is exclusively synthesized in the small intestine (7). The fact that Caco-2 cells produce and secrete significant amounts of apo A-IV clearly demonstrates the high level of differentiation of these cells under our experimental conditions (25). In addition, the decline in secreted apo A-IV associated with MSO and its restoration with glutamine suggest that glutamine regulates the synthesis rate not only of intracellular protein but of other exported proteins as well.
The protein synthesis rate values measured using the incorporation of
2H3-labeled leucine into mixed cell protein in
the current studies (20-30%/day under control conditions) are
of the same order of magnitude as the 28%/day FSR reported for human
ileum in vivo and lower than the
60 and
50%/day rates observed
in human duodenum and jejunum, respectively (24), or the
60-100%/day FSR documented in rat or dog duodenum using stable
isotope infusions in vivo (5, 18). Many factors can
contribute to this discrepancy. 1) From the outset, it
should be kept in mind that the Caco-2 cell line was originally derived
from a tumor, not from normal enterocytes, and therefore may only be
construed as an imperfect model of normal enterocytes. 2)
Caco-2 cells grown on plastic dishes may not reflect the protein
synthesis rates of enterocytes that have access to both intestinal
lumen (through their apical brush border) and circulating blood
(through their basolateral membrane) in vivo. Moreover, the
concentration of most nutrients in the commonly used DMEM culture
medium far exceeds the physiological range, because leucine and glucose
concentrations, for instance, reach 800 µM and 450 mg/dl,
respectively, i.e.,
5 times their respective concentrations in human
plasma. We therefore carried out additional experiments (data not
shown) in Caco-2 cells grown not on plastic dishes but on Transwell
chambers, a setup in which cells are exposed to a basolateral chamber
and an apical chamber, in which separate media can be placed. In
addition, these cells were exposed to an incubation medium that was
made of a 1:4 dilution of DMEM with normal saline (0.9% NaCl) to mimic
the glucose and amino acid concentrations encountered in arterial
plasma. Under these conditions, the values for cell protein FSR were
nevertheless identical to those measured under "regular"
conditions: 28.7 ± 0.2 and 28.7 ± 9.4%/day on cells grown
in plastic dishes with regular DMEM and Transwell filters with the 1:4
DMEM dilution, respectively; not significant). 3) Mucosa
samples obtained from gut biopsies include a mixture of cells from both
the bottom of the crypts as well as cells that have completed their
migration to the top of the villi. Because we assessed protein FSR in
Caco-2 cells after 20 days of culture at a time when they resemble
highly differentiated enterocytes, these cells may more closely mimic enterocytes from the top of intestinal villi. 4) Finally,
duodenum and jejunum are known to have higher protein turnover rates
than ileum (24), and Caco-2 cells may be closer to ileal
than duodenal cells in their metabolic characteristics.
The current findings suggest that the observed fall in cell protein FSR
under MSO treatment is due to intracellular glutamine depletion per se.
As a matter of fact, although the drop in protein synthesis may, in
theory, reflect a nonspecific cytotoxic effect of MSO or a decreased
cell number, this is unlikely because glutamine supply acutely
"rescued" rates of protein synthesis in MSO-treated cells within
<1 h. The mechanisms by which glutamine depletion affects rates
of protein synthesis remain to be elucidated. As glutamine is a major
source of energy for the small intestine (38),
Higashigushi et al. (12) suggested that glutamine
stimulates protein synthesis in rat enterocytes by energy provision. In
the current study, the drop in protein synthesis observed under
conditions of glutamine depletion is, however, unlikely to result from
lack of energy, because our Caco-2 cells were exposed to high
concentrations of glucose (17.5 mM), and glucose is readily usable as a
fuel in enterocytes (1). Furthermore, the use of glutamine
as a fuel implies glutamine deamidation to glutamate and ammonia (via mitochondria glutaminase) followed by glutamate transamination to
-ketoglutarate, which enters the tricarboxylic acid cycle to yield
energy. The lack of effect of glutamate on protein synthesis in
MSO-treated cells suggests that, in the current study, glutamine's effect may not be mediated by its role as a source of energy. Similarly, the drop in protein synthesis associated with MSO treatment could not be accounted for by nitrogen deficiency, because ample amounts of all amino acids (except glutamine) were supplied by the DMEM
culture medium and the effect of MSO on protein synthesis was not
corrected by supplementation with other amino acids. These results
collectively suggest that glutamine depletion per se was the cause of
the drop in protein FSR observed with MSO treatment.
Studies by others have shown that intracellular duodenal glutamine
concentration can be altered by malnutrition and/or infection in vivo.
For instance, intracellular glutamine concentration was 17% lower
(5.1 vs. 6.1 µmmol/g protein) in malnourished patients with a body
weight at 87 ± 7% of ideal body weight compared with well-nourished patients (34). Conversely, intracellular
glutamine was 29% higher in critically ill patients compared with
controls (6.6 vs. 5.1 µmol/g protein) (3) and 119%
higher in patients infected with human immunodeficiency virus
compared with healthy controls (10.4 vs. 4.8 µmol/g protein)
(27). Together, these data suggest that intracellular
glutamine concentration does vary in human small intestine in vivo. The
20% decline observed on MSO treatment in Caco-2 cells in the
current study is of the same magnitude as the 17% decline in
intestinal glutamine concentration observed in undernourished patients
in vivo (34, 36).
Extrapolation to the human small intestine in vivo must, however, be
made with caution for several reasons. The purpose of using MSO in the
current studies was to create a rather extreme model of glutamine
depletion in Caco-2 cells (to a degree that may not occur in vivo), not
to determine the physiological role of glutamine synthetase in
enterocytes. As a matter of fact, human enterocytes can usually draw
glutamine from two main sources: exogenous glutamine from the enteral
lumen, via their apical brush border, and endogenous glutamine from
circulating blood, via their basolateral membrane. Although James et
al. (14) recently showed glutamine synthetase to be
present throughout human gut, the activity of glutamine synthetase
(estimated at 16 µmol/min for the entire gut) is 152 times lower
than that of glutaminase (2,463 µmol/min) in the same tissue. In situ
glutamine synthetase activity therefore is unable to meet the demand
for glutamine in the gut in vivo. Only in rare situations when plasma
glutamine would decline as a consequence of stress-induced protein
wasting, such as severe burns (28), and when
glutamine-free enteral regimen would be supplied could the activity of
glutamine synthetase in the gut be of potential physiological
relevance. Yet this remains to be examined in vivo.
Recently, Stoll et al. (32) proposed that cell swelling might mediate an anabolic effect of glutamine in hepatocytes. Cell swelling is, however, unlikely to account for the relationship between glutamine availability and protein synthesis in MSO-treated Caco-2, because 1) when we intentionally altered the osmolarity of the incubation between 320 and 420 mosM (additional preliminary experiments; data not shown), we failed to observe any change in cell protein FSR and 2) Adegoke et al. (2) recently reported unaltered rates of jejunal protein synthesis rate during luminal infusion of solutions ranging from 250 to 380 mosM in vivo in piglets. Alternatively, glutamine could affect the repair and/or proliferation of enterocytes through its role as a major nitrogen donor for the synthesis of nucleotides (22). This, however, seems unlikely because glutamine supply was able to restore rates of protein synthesis within <4 h after MSO treatment. Finally, the observation that a 20% drop in intracellular glutamine concentration resulted in a 75% fall in total protein synthesis is intriguing, because it remains to be determined whether this reduction in cell glutamine content affected other functions among the many parameters believed to depend on glutamine. Does the enterocyte cope with the lack of glutamine in the medium by reducing its protein synthesis to maintain intracellular glutamine concentration at a level sufficient for other functions, such as the synthesis of nucleotides or glutathione, considered of prime importance for its survival? Further experiments using stable isotope-labeled glutamine could address this hypothesis by following the appearance of 15N in purines and pyrimidines on incubation with 15N-labeled glutamine under conditions of glutamine deprivation.
In summary, the current findings clearly suggest that 1) glutamine de novo synthesis plays a major role in the maintenance of intracellular glutamine pools in Caco-2 cells and 2) glutamine availability affects rates of protein synthesis in this model. Although Caco-2 cells share several metabolic characteristics with normal human enterocytes, extrapolation to the human small intestine in vivo must be made with caution in view of the limitations inherent in the use of a cell line as an experimental model.
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ACKNOWLEDGEMENTS |
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O. Le Bacquer was supported by a fellowship grant from the Ministère de la Recherche et de la Technologie, Paris, France.
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. Darmaun, INSERM U.539, Centre de Recherche en Nutrition Humaine, Hôtel-Dieu, 3ème étage aile nord, Nantes Cedex 1, France (E-mail: ddarmaun{at}nantes.inserm.fr).
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.
Received 27 October 2000; accepted in final form 30 August 2001.
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