Effects of glutamine deprivation on protein synthesis in a model of human enterocytes in culture

Olivier Le Bacquer, Hassan Nazih, Hervé Blottière, Dominique Meynial-Denis, Christian Laboisse, and Dominique Darmaun

INSERM U.539, Centre de Recherche en Nutrition Humaine, 44093 Nantes, France


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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).


    MATERIAL AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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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Table 1.   Effect of glutamine deprivation on mixed cell protein FSR and intracellular free glutamine and glutamate concentrations

Similarly, cell protein synthesis rate (FSR), as measured from the incorporation of labeled leucine into cell protein, remained unaltered in cells grown in glutamine-free medium (Table 1) even after 20 days of glutamine deprivation: 19.7 ± 1.4 vs. 21.0 ± 0.3%/day (controls vs. 20-day deprived cells).

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-1 · mg protein-1. Glutamine synthetase activity was enhanced (to 231 ± 28 nmol · h-1 · mg protein-1; P < 0.05) by growth for 20 days in a glutamine-free medium, where the only glutamine supplied arose from fetal calf serum (measured glutamine concentration 0.1 mM). In contrast, glutamine synthetase activity was undetectable in extracts from Caco-2 cells that had been exposed to MSO for 24 h.

To verify whether MSO inhibited glutamate conversion to glutamine in living Caco-2 cells as well, Caco-2 cells were incubated for 24 h in DMEM-free glutamine medium spiked with 1 mM L-15N-labeled glutamic acid, and the appearance of L-2-15N-labeled glutamine in the intracellular free amino acid pool was used as an index of glutamate conversion to glutamine. The amount of glutamine arising from de novo glutamine synthesis ([Glnde novo], µmol/g protein) was estimated by [Glnde novo] = (EGln/EGlu) × [Gln], where [Gln] is intracellular free glutamine concentration (µmol/g protein) and EGln and EGlu are the 15N enrichments (mole % excess) in intracellular free glutamine and glutamate, respectively. We observed a significant decrease of glutamine coming from de novo synthesis when cells were treated with MSO (Table 2).

                              
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Table 2.   Effect of a 24-h incubation with 10 mM MSO on intracellular glutamine concentration and the conversion of 15N-labeled glutamate to 15N-labeled glutamine

As expected, MSO treatment resulted in a significant approx 20% reduction in glutamine concentration (5.6 ± 0.1 vs. 4.7 ± 0.1 µmol/g protein; P < 0.001) along with a approx 60% rise in glutamate concentration (79.6 ± 4.5 vs. 128.3 ± 10.5 µmol/g protein; P < 0.001; Fig. 1A).


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Fig. 1.   Effect of methionine sulfoxide (MSO) on intracellular glutamine and glutamate levels and cell protein fractional synthesis rate (FSR). Caco-2 cells underwent a 24-h incubation with 10 mM MSO either alone or followed by supplementation with 0.25-1 mM glutamine for 4 h. Values are means ± SD. Significant difference (*P < 0.05; **P < 0.01) by unpaired t-test: MSO vs. control and MSO + glutamine vs. MSO. A: Caco-2 cells grown in DMEM medium with 0.6 mM glutamine were subsequently submitted to MSO and eventually supplemented with 2 mM glutamine. B: Caco-2 cells grown with DMEM containing either 0.1, 0.6, or 5 mM glutamine were then incubated for 24 h with 10 mM MSO before being supplemented with 2 mM glutamine for 4 h. [Gln], intracellular free glutamine concentration.

A dramatic approx 75% drop in cell protein FSR (Fig. 1B) accompanied the reduction in intracellular glutamine concentration. The effect of MSO on cell protein FSR was dose dependent (Fig. 2), and 10 mM produced maximal inhibition of cell protein synthesis.


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Fig. 2.   Dose-dependent response of cell protein FSR to MSO. Cells were grown on 0.6 mM glutamine DMEM medium and incubated for the last 24 h with graded concentrations of MSO in glutamine-free DMEM medium. Values are means ± SD. Significant difference (**P < 0.01) by unpaired t-test, MSO vs. control.

"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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Fig. 3.   Dose dependence and time course of the effect of glutamine on protein FSR in MSO-treated cells. A: Caco-2 cells were grown in 0.6 mM glutamine DMEM medium and incubated for the last 24 h with 10 mM MSO before being supplemented for 4 h with graded glutamine concentrations. Comparisons are MSO vs. control (*) and MSO vs. MSO + glutamine (open circle ). B: in the same way, cells were incubated 24 h with 10 mM MSO before being supplemented with 2 mM glutamine for incubations of various durations. Statistics are control vs. time <135 min (*) and time >135 min vs. time <135 min (open circle ). NS, not significant.


                              
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Table 3.   Effect of supplemental amino acids on cell protein FSR after a 24-h incubation with 10 mM MSO

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 approx 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 approx 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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Table 4.   Effect of a 24-h incubation with 10 mM MSO, with or without subsequent supplementation with 2 mM glutamine for 4-24 h, on cell protein content and secreted apolipoprotein A-IV


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 approx 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 approx 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 (approx 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 approx 60 and approx 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., approx 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 alpha -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 approx 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 approx 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 approx 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.


    ACKNOWLEDGEMENTS

O. Le Bacquer was supported by a fellowship grant from the Ministère de la Recherche et de la Technologie, Paris, France.


    FOOTNOTES

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).

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Received 27 October 2000; accepted in final form 30 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIAL AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Abdulrahman, A, Ashy M, and Ardawi S. Glucose, glutamine, and ketone-body metabolism in human enterocytes. Metabolism 37: 602-609, 1988[ISI][Medline].

2.   Adegoke, OA, McBurney MI, and Baracos VE. Jejunal mucosal protein synthesis: validation of luminal flooding dose method and effect of luminal osmolarity. Am J Physiol Gastrointest Liver Physiol 276: G14-G20, 1999[Abstract/Free Full Text].

3.   Ahlman, B, Ljungqvist O, Persson B, Bindsley L, and Wernerman J. Intestinal amino acid content in critically ill patients. JPEN J Parenter Enteral Nutr 19: 272-278, 1995[Abstract].

4.   Chantret, I, Barbat A, Dussaulx E, Brattain M, and Zweibaum A. Epithelial polarity, villin expression, and enterocytic differentiation of cultured human colon carcinoma cells: a survey of twenty cell lines. Cancer Res 48: 1936-1942, 1988[Abstract].

5.   Dudley, M, Wykes L, Dudley A, Burrin D, Nichols B, Rosenberg J, Jahoor F, Heird W, and Reeds P. Parenteral nutrition selectively decreases protein synthesis in the small intestine. Am J Physiol Gastrointest Liver Physiol 274: G131-G137, 1998[Abstract/Free Full Text].

6.   Dugan, M, and McBurney M. Luminal glutamine perfusion alters endotoxin-related changes in ileal permeability of the piglet. JPEN J Parenter Enteral Nutr 19: 83-87, 1995[Abstract].

7.   Elshourbagy, N, Walker D, Paik Y, Boguski M, Freeman M, Gordon J, and Tayor J. Structure and expression of the human apolipoprotein A-IV gene. J Biol Chem 262: 7973-7981, 1987[Abstract/Free Full Text].

8.   Grasset, E, Bernabeu J, and Pinto M. Epithelial properties of human colonic carcinoma cell line Caco-2: effect of secretagogues. Am J Physiol Cell Physiol 248: C410-C418, 1985[Abstract].

9.   Griffith, O, and Meister A. Differential inhibition of glutamine and gamma -glutamylcysteine synthetase by alpha -alkyl analogs of methionine sulfoximine that induce convulsions. J Biol Chem 253: 2333-2338, 1978[Abstract].

10.   Hankard, R, Darmaun D, Sager B, D'Amore D, Parsons W, and Haymond M. Response of glutamine metabolism to exogenous glutamine in humans. Am J Physiol Endocrinol Metab 269: E663-E670, 1995[Abstract/Free Full Text].

11.   Hankard, R, Haymond M, and Darmaun D. Effect of glutamine on leucine metabolism in humans. Am J Physiol Endocrinol Metab 271: E748-E754, 1996[Abstract/Free Full Text].

12.   Higashiguchi, T, Hasselgren P, Wagner K, and Fischer J. Effect of glutamine on protein synthesis in isolated intestinal epithelial cells. JPEN J Parenter Enteral Nutr 17: 307-314, 1993[Abstract].

13.   Hiramatsu, T, Cortiella J, Marchini J, Chapman T, and Young V. Source and amount of dietary nonspecific nitrogen in relation to whole-body leucine, phenylalanine, and tyrosine kinetics in young men. Am J Clin Nutr 59: 1347-1355, 1994[Abstract].

14.   James, LA, Lunn PG, and Elia M. Distrbution of glutaminase and glutamine synthetase activities in the human gastrointestinal tract. Clin Sci 94: 313-319, 1998[ISI][Medline].

15.   Jepson, M, Bates P, Broadbent P, Pell J, and Millward D. Relationship between glutamine concentration and protein synthesis in rat skeletal muscle. Am J Physiol Endocrinol Metab 255: E166-E172, 1988[Abstract/Free Full Text].

16.   Lowry, O, Rosebrough N, Farr A, and Randall R. Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275, 1951[Free Full Text].

17.   March, J. A modified technique for the quantitative analysis of amino acids by gas chromatography using heptafluorobutyric n-propyl derivatives. Anal Biochem 69: 420-442, 1974.

18.   Marchini, J, Guyen PN, Deschamps J, Maugère P, Krempf M, and Darmaun D. Effect of intravenous glutamine on duodenal mucosa protein synthesis in healthy growing dogs. Am J Physiol Endocrinol Metab 276: E747-E753, 1999[Abstract/Free Full Text].

19.   Matthews, D, Ben-Galim E, and Bier D. Determination of stable isotopic enrichment in individual plasma amino acids by chemical ionization mass spectrometry. Anal Chem 51: 80-84, 1979[ISI][Medline].

20.   Matthews, D, Marano M, and Campbell R. Splanchnic bed utilization of glutamine and glutamic acid in humans. Am J Physiol Endocrinol Metab 264: E848-E854, 1993[Abstract/Free Full Text].

21.   Max, SR, Mill J, Mearow K, Koanagaya M, Konagaya Y, Thomas JW, Banner C, and Vitkovic L. Dexamethasone regulates glutamine synthetase expression in rat skeletal muscles. Am J Physiol Endocrinol Metab 255: E397-402, 1988[Abstract/Free Full Text].

22.   McCauley, R, Kong S, and Hall J. Glutamine and nucleotide metabolism within enterocytes. JPEN J Parenter Enteral Nutr 22: 105-111, 1998[Abstract].

23.   Meynial-Denis, D, Mignon M, Miri A, Imbert J, Aurousseau E, Taillandier D, Attaix D, Arnal M, and Grizard J. Glutamine synthetase induction by glucocorticoids is preserved in skeletal muscle of aged rats. Am J Physiol Endocrinol Metab 271: E1061-1066, 1996[Abstract/Free Full Text].

24.   Nakshabendi, I, McKee R, Downie S, Russell R, and Rennie M. Rates of small intestinal mucosal protein synthesis in human jejunum and ileum. Am J Physiol Endocrinol Metab 277: E1028-E1031, 1999[Abstract/Free Full Text].

25.   Nazih, H, Nazih-Sanderson F, Krempf M, Huvelin JM, Mercier S, and Bard JM. Butyrate stimulates apo-AIV-containing lipoprotein secertion in differentiated Caco-2 cells: role in cholesterol efflux. J Cell Biochem 83: 230-238, 2001[ISI][Medline].

26.   Neu, J, Shenoy V, and Chakrabarti R. Glutamine nutrition and metabolism: where do we go from here? FASEB J 10: 829-837, 1996[Abstract/Free Full Text].

27.   Ollenschlager, G, Langer K, Steffen HM, Schrappe-Bacher M, Schmitt H, Allolio B, and Roth E. Intracellular free amino acid patterns in duodenal and colonic mucosa. Clin Chem. 36: 378-381, 1990[Abstract/Free Full Text].

28.   Parry-Billings, M, Evans J, Calder PC, and Newsholme EA. Does glutamine contribute to immunosuppression after major burns? Lancet 336: 523-525, 1990[ISI][Medline].

29.   Reeds, P, Burrin D, Jahoor F, Wykes L, Henry J, and Frazer M. Enteral glutamine is almost completely metabolized in first pass by the gastrointestinal tract of infant pigs. Am J Physiol Endocrinol Metab 270: E413-E418, 1996[Abstract/Free Full Text].

30.   Ronzio, R, Rowe W, and Meister A. Studies on the mechanism of inhibition of glutamine synthetase by methionine sulfoximine. Biochemistry 8: 1066-1075, 1969[ISI][Medline].

31.   Stehle, P, Zander J, Mertes N, Albers S, Puchstein C, Lawin P, and Fürst P. Effect of parenteral glutamine peptide supplements on muscle glutamine loss and nitrogen balance after major surgery. Lancet 4: 231-233, 1989.

32.   Stoll, B, Gerok W, Lang F, and Haussinger D. Liver cell volume and protein synthesis. Biochem J 287: 217-222, 1992[ISI][Medline].

33.   Turowski, G, Rashid Z, Hong F, Madri J, and Basson M. Glutamine modulates phenotype and stimulates proliferation in human colon cancer cell lines. Cancer Res 54: 5974-5980, 1994[Abstract].

34.   Van der Hulst, RRWJ, Deutz NEP, Von Meyenfeldt MF, Elbers JMH, Stockbrügger RW, and Soeters PB. Decrease of mucosal glutamine concentration in the nutritionally depleted patient. Clin Nutr 13: 228-233, 1994[ISI].

35.   Van Der Hulst, R, Van Krell B, Von Meyenfeldt M, Brummer R, Arends J, Deutz N, and Soeters P. Glutamine and the preservation of gut integrity. Lancet 334: 1363-1365, 1993.

36.   Van Der Hulst, R, Von Meyenfeldt M, Deutz N, and Soeters P. The effect of glutamine administration on intestinal glutamine content. J Surg Res 61: 30-34, 1996[ISI][Medline].

37.   Weiss, M, DeMarco V, Strauss D, Samuelson D, Lane M, and Neu J. Glutamine synthetase: a key enzyme for intestinal epithelial differentiation? JPEN J Parenter Enteral Nutr 23: 140-146, 1999[Abstract].

38.   Windmueller, H, and Spaeth A. Uptake and metabolism of plasma glutamine by the small intestine. J Biol Chem 249: 5070-5079, 1974[Abstract/Free Full Text].

39.   Wykes, L, Fiorotto M, Burrin D, Del Rosario M, Frazer M, Pond W, and Jahoor F. Chronic low protein intake reduces tissue protein synthesis in a pig model of protein malnutrition. J Nutr 126: 1481-1488, 1996[ISI][Medline].

40.   Yoshida, S, Leskiw M, Schluter M, Bush K, Nagele R, Lanza-Jacoby S, and Stein T. Effect of total parenteral nutrition, systemic sepsis, and glutamine on gut mucosa in rats. Am J Physiol Endocrinol Metab 263: E368-E373, 1992[Abstract/Free Full Text].

41.   Yoshida, S, Matsui M, Shirouzu Y, Fujita H, Yamana H, and Shirouzu K. Effects of glutamine supplements and radiochemotherapy on systemic immune and gut barrier function in patients with advanced oesophageal cancer. Ann Surg 227: 485-491, 1998[ISI][Medline].


Am J Physiol Gastrointest Liver Physiol 281(6):G1340-G1347
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