The Journal of Nutrition Vol. 128 No. 8 August 1998, pp. 1296-1301

Glutamine Reduces Heat Shock-Induced Cell Death in Rat Intestinal Epithelial Cells¹

Alice Chow² and Rongping Zhang

Division of Digestive Diseases, Emory University School of Medicine, Atlanta, GA 30322

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Glutamine supplementation is beneficial for preventing intestinal atrophy and maintaining mucosal functions in metabolically stressed patients. The mechanisms by which glutamine prevents mucosal atrophy remain unclear. In particular, the role of glutamine in the survival of cells under stress is unknown. Intestinal epithelial cells (IEC-6) were cultured in media with or without supplementation of L-glutamine. A low concentration of L-glutamine (1.0 mmol/L) was sufficient to minimize the percentage of floating cells under basal conditions. Heat shock at 43°C for 90 min decreased (P < 0.001) the number of attached cells, while increasing (P < 0.001) the number of floating cells, which is a measurement of the extent of cell death in these cultures. Glutamine enhanced attached cell count and diminished heat shock-induced cell death in a dose-dependent manner. Of note, 2 mmol/L was suboptimal in both respects, thus indicating that heat-shocked cells require higher concentrations of glutamine for optimal cell survival. Maximal effect was achieved with 8 mmol/L glutamine, which increased (P < 0.001) cell growth (indicated by the number of attached cells) and diminished (P < 0.001) cell death (indicated by the number of floating cells). Further increase of L-glutamine concentration to 12 or 20 mmol/L did not provide additional benefit in minimizing cell death. Heat shock protein 70 (hsp 70) mRNA was induced by heat shock only in cultures supplemented with L-glutamine, and the induction was more consistent and greater in cultures containing higher concentrations of glutamine. Thus, glutamine supplementation reduced heat shock-induced cell death. This effect, together with the maintenance of cell growth, may play a key role in the prevention of intestinal mucosal atrophy.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

Glutamine is traditionally classified as a nonessential amino acid. However, it is the preferred oxidative substrate for intestinal epithelial cells (Fleming et al. 1991), and it is heavily utilized by replicating cells (Reitzer et al. 1979). During injuries, glucocorticoid administration, catabolic illnesses or cell proliferation, glutamine utilization may exceed its production, resulting in a decreased plasma glutamine level and a depletion of glutamine stores (Souba et al. 1985, Wilmore et al. 1988). Thus, under such conditions, glutamine becomes conditionally essential (Souba et al. 1990).

In individuals sustained by parenteral nutrition, mucosal atrophy may develop readily (O'Dwyer et al. 1989). Dietary supplementation with glutamine may maintain mucosal mass and prevent malabsorption and bacterial translocation across the intestinal mucosa (Fox et al. 1988), thus averting nutritional deprivation or predisposition towards sepsis (Souba et al. 1990). Similarly, mucosal atrophy is attenuated by including glutamine in total parenteral nutrition solutions (Grant and Snyder 1988). Parenteral supplementation with glutamine improves nitrogen balance and decreases bacterial translocation (Hammargvist et al. 1989). Thus, supplementing glutamine in either enteral or parenteral nutrition solutions (Souba et al. 1990) may benefit patients under severe catabolic stress or with intestinal dysfunction.

View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig 1. Effects of low concentrations of L-glutamine on intestinal epithelial cells (IEC-6) under basal (nonheat-shocked) and heat shock conditions. Cells were grown in six-well plates in Dulbecco's modification of Eagle's minimum essential medium containing 2 mmol/L of L-glutamine until confluence. Cells were then cultured in glutamine-free media for 24 h. Individual wells in the six-well plates were then assigned to the 0, 0.1, 0.5, 1, and 2 mmol/L treatment groups with corresponding levels of glutamine supplementation. Twenty-four hours later, plates belonging to the heat shock groups were immersed in a 43°C water bath for 90 min and then returned to the 37°C incubator for 24 h. Cells were then harvested and counted. Values are means of a representative experiment; error bars represent SEM, n = 5. The experiment was performed twice. (A) Total attached cell count per well in nonheat-shocked (squares) and heat-shocked (triangles) groups. There were significant glutamine (P < 0.001), heat shock (P < 0.001) and interaction effects (P < 0.01). (B) Total floating cell count per well of the same samples represented in Figure 1A. Total floating cell count was significantly increased by heat shock (P < 0.001) and decreased by glutamine (P < 0.001). (C) The percentage of floating cells was <2% in control cultures supplemented with >0.5 mmol/L L-glutamine. However, this concentration did not reduce the percentage of cell deaths due to heat shock.

Although glutamine is needed by most types of cultured cells (Reitzer et al. 1979), the basis for this requirement is not well understood. Nucleotide supplements enhance the growth and maturation of intestinal epithelial cells (IEC-6)³ (He et al. 1993) as well as obviate the need for exogenous glutamine, suggesting that glutamine may affect the availability of nucleotides. Most previous studies emphasized the growth-promoting effects of glutamine as being important for the prevention of mucosal atrophy. Because the maintenance of mucosal mass reflects the balance between proliferation and cell death, we hypothesize that the ability of glutamine to prevent mucosal atrophy may be due in part to reduction of epithelial cell death.

Many conditions of stress such as ischemia or metabolic stress in which glutamine supplementation is beneficial induce heat shock or stress responses (Welch 1992). Glutamine may enhance cell survival under stressful conditions by enhancing the stress response (Klimberg and Souba 1990). To address this hypothesis, we used an in vitro cell culture system to determine the effects of glutamine under basal and stressful (heat shock) conditions.

Heat shock is a prototypic stress that induces heat shock proteins (hsp) or stress proteins; these constitute, evolutionarily, a highly conserved system that confers protective effects on cells under stress. A low level of heat shock can induce thermotolerance for subsequent stress (Morimoto et al. 1990). However, heat stress itself may cause cell death. In a study of mastocytomas (Harmon et al. 1990), mild heat shock (42-44°C) caused apoptosis, whereas severe heat shock (46-47°C) caused necrosis. Because heat shock is a universal injury and its effect is dose dependent (Harmon et al. 1990), it was used to induce cell death in our experiments.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  All chemicals, except for those specified, were from Sigma Chemical (St. Louis, MO). IEC-6 cells (passage 13; ATCC, CRL 1592) were acquired from American Type Culture Collection (Rockville, MD). Culture media and fetal bovine sera were from Gibco BRL (Bethesda, MD). Tissue culture disposables were from Sarstedt (Newton, NC). Deoxycytidine-5'[³²P]-triphosphates (dCTP) were obtained from Amersham (Arlington Heights, IL).

Cell culture.  IEC-6 cells were originally established from jejunal epithelial cells of neonatal rats (Quaroni et al. 1979). IEC-6 cells were routinely cultured in Dulbecco's modification of Eagle's minimum essential medium (DMEM) containing high glucose, 5% heat-inactivated fetal bovine sera, 10 g/L insulin-transferrin-selenium and 1% antibiotics-antimycotics under 10% CO₂ at 37°C. IEC-6 cells were tested to be free of mycoplasma by the Hochest 33258 staining method (Freshney 1994) with confirmation by an enzymatic test (MycoTect, Gibco BRL) using 3T6 cells (ATCC CCL 96) as the indicator cell line. Floating cells were collected from culture media, and attached cells were harvested from the monolayers by trypsinization. Cell viability was determined according to trypan blue exclusion (Freshney 1994).

Glutamine and heat shock treatments.  To minimize the effects of intracellular glutamine, IEC-6 cells were cultured in glutamine-free media for 24 h before assignment of groups. Cells were then cultured with serum-free media containing 10 g/L insulin-transferrin-selenium and supplementation of L-glutamine at various concentrations.

View larger version (19K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   View larger version (17K): [in this window] [in a new window]   Fig 2. Effects of higher concentrations of glutamine on intestinal epithelial cells (IEC-6) under basal and heat shock conditions. The experimental conditions were identical to those described in Figure 1, except that higher concentrations of glutamine were used for supplementation. Values are means of a representative experiment; error bars represent SEM, n = 6. The experiment was performed three times. (A) Total attached cell count per well in nonheat-shocked (squares) and heat-shocked (triangles) groups. There were significant glutamine (P < 0.001), heat shock (P < 0.001) and interaction effects (P < 0.05). (B) Total floating cell count per well in the same samples represented in Figure 1A. Total floating cell count was significantly increased by heat shock (P < 0.001) and decreased by glutamine (P < 0.001). Glutamine at concentrations of 12 and 20 mmol/L glutamine was not toxic because the floating cell counts were not different from that at 8 mmol/L. (C) Glutamine, heat shock and interaction effects were all significant (P < 0.001). The percentage of floating cells was minimized by 8 mmol/L under heat shock conditions, in contrast to nonheat-shocked cultures, which required only 0.5 mmol/L to minimize the percentage of floating cells (see Fig. 1C). The percentage of floating cells in the 12 and 20 mmol/L groups was not different from that of the 8 mmol/L group.

Northern hybridizations using the pH2.3 probe.  The pH 2.3 (human hsp 70; ATCC #57495), is a 2.3-kb Bam HI/Hind III fragment cloned in the pAT153 vector. The 2.3-kb insert was labeled with [³²P]-dCTP by random-primer-extension to a specific activity of 1.6 GBq/g. Total RNA was isolated from IEC-6 cells with the guanidinium-isothiocyanate method (Puissant and Houdebine 1990). Total RNA (20 µg) was electrophoresed in 0.66 mol/L formaldehyde gels with 10 g/L agarose and then transferred to GeneScreen (Dupont NEN, Boston, MA) nylon membranes. Northern blots were prehybridized in 30% formamide, 1 mol/L NaCl, 100 g/L dextran sulfate and 10 g/L SDS at 37°C for 4 h. Hybridization was performed at 37°C for 6 h in the same buffer containing 100 g/L denatured salmon sperm DNA and 0.16 GBq/L ³²P-labeled PH 2.3 cDNA probes. Filters were washed at room temperature in 2× standard sodium citrate (NaCl, 150 mmol/L; sodium citrate, 15 mmol/L, pH 7.0) and then in 0.2× standard sodium citrate containing 5 g/L SDS at 65°C for 30 min. Filters were exposed to Kodak X-Omat AR film at 70°C.

Statistics.  Data analyses were performed by using the Statistical Analysis System (SAS/STAT Version 5, SAS Institute, Cary, NC). Treatment effects were compared by using the general linear model with a single well as the experimental unit. One sample from each treatment group was represented on each six-well plate. Permutation was used to assign well position of samples in replicate six-well plates.

	RESULTS

Abstract Introduction Methods Results Discussion References

Most cell types require L-glutamine supplementation in culture media, which typically contain 2 mmol/L L-glutamine. The effects of low concentrations (2 mmol/L) of L-glutamine were examined under basal (nonheat-shock) conditions. Supplementation of 1 mmol/L glutamine increased mean attached cell count by 38%, and further increase of glutamine concentration to 2 mmol/L did not augment the attached cell counts (Fig. 1A). We confirmed that all floating IEC-6 cells could not exclude trypan blue and that >95% of attached cells excluded trypan blue. Therefore, the number of floating cells is a good indicator of the extent of cell death. Under basal conditions, the percentage of dead cells in nonsupplemented cultures was 11.8, decreasing to <3% in cultures supplemented with 0.5 mmol/L L-glutamine (Fig. 1C). Thus, supplementation at 0.5 mmol/L was sufficient to minimize the number of floating cells at 37°C (Fig. 1B).

View larger version (49K): [in this window] [in a new window]   Fig 3. Northern hybridization of intestinal epithelial cell (IEC-6) RNA to PH2.3 cDNA (human hsp 70) probe. Total cellular RNA was harvested from cultures 24 h after heat shock. RNA (20 µg) was loaded in each lane of the RNA gel. Groups with 0, 2 or 8 mmol/L glutamine supplementation are designated 0, 2 and 8, respectively. Upper panel: Hsp 70 was undetectable in nonheat-shocked cells, but was induced in heat-shocked cells that had been supplemented with 8 mmol/L L-glutamine; 28S and 18S mark the locations of respective ribosomal RNA. Lower panel: Ethidium bromide staining of agarose gel to show equal loading of RNA samples.

Heat shock at 43°C led to a general reduction in attached cell number in nonsupplemented and supplemented cultures (P < 0.001). Attached cell counts decreased by >90% in cultures without glutamine. In the presence of 2 mmol/L glutamine, mean attached cell counts still amounted to only ~20% of the nonstressed counterparts (Fig. 1A).

Heat shock at 43°C induced cell death, as reflected by large increases in floating cell count (Fig. 1B). In the absence of glutamine supplementation, the percentage of floating cells reached 90% (Fig. 1C). The minimal effective concentration for decreasing heat shock-induced cell death was ~1 mmol/L because 0.5 mmol/L had no significant effect in reducing the percentage of floating cells (Fig. 1C). Supplementation of glutamine at concentrations >1 mmol/L caused a dose-dependent decrease in the percentage of floating cells (Fig. 1C). However, the percentage of floating cells in cultures supplemented with 2 mmol/L glutamine remained substantial (Fig. 1C). Thus, cells under heat stress apparently require a higher level of glutamine supplementation for cell survival.

In contrast to the modest effect of 2 mmol/L L-glutamine, supplementation with 5, 8, 12 and 20 mmol/L glutamine caused more marked, though incomplete, protection from heat shock-induced cell death (Fig. 2). The effects of glutamine on increasing attached cell count and decreasing floating cell count were dose dependent up to 8 mmol/L. Increasing the level of supplementation to 12 or 20 mmol/L did not provide additional benefit in either effect. Supplementation of 8 mmol/L L-glutamine was unlikely to be toxic because there was no increase in the percentage of floating cells even at 20 mmol/L (Fig. 2C).

To determine if the protective effect of glutamine is associated with induction of hsp, we extracted RNA from control and heat-shocked cultures that were in 0, 2 or 8 mmol/L glutamine. Northern analyses (Figs. 3 and 4) indicated that hsp 70 mRNA was consistently induced in heat-shocked cells supplemented with 8 mmol/L of glutamine. Induction of hsp 70 at 2 mmol/L was inconsistent and detectable only at a lower level in some experiments (Fig. 4). Expression of hsp 70 at 5 mmol/L was intermediate between 2 and 8 mmol/L (Fig. 4). This suggested that glutamine supplementation is necessary for the induction of hsp 70 by heat shock under our experimental conditions.

View larger version (50K): [in this window] [in a new window]   Fig 4. Additional assay of heat shock protein (hsp) 70 induction in intestinal epithelial cells (IEC-6) at intermediate glutamine concentrations. Upper panel: Northern hybridization of IEC-6 RNA to the pH2.3 (human hsp 70) cDNA probe shows induction of hsp 70 mRNA in cultures supplemented with 2, 5, and 8 mmol/L L-glutamine. Hsp 70 mRNA was undetectable in nonheat-shocked cultures. Lower panel: Ethidium bromide staining of the agarose gel showing equal loading of RNA. The two prominent bands are 28S and 18S ribosomal RNAs.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

In both control and heat-shocked IEC-6 cells, glutamine supplementation increased the number of cells remaining attached in the monolayer and diminished the number of floating cells. Thus, glutamine enhanced cell survival under basal conditions, but it especially protected against cell death under stressful conditions.

The recommended L-glutamine concentration in most culture media is 2 mmol/L. Glutamine is essential for in vitro intestinal cell proliferation (Ko et al. 1993). Addition of 0.5 mmol/L glutamine was found to be sufficient for stimulating epidermal growth factor-induced DNA synthesis. The growth stimulatory effects of glutamine may be related to its ability to cause activation of mitogen-activated protein kinases, such as the extracellular signal-regulated kinases and Jun nuclear kinases (Rhoads et al. 1997). The above study indicated that maximal stimulation of proliferation required ~2.5 mmol/L L-glutamine. Our finding that 2 mmol/L glutamine is adequate for supporting optimal culture under nonheat-stressed conditions is in keeping with these findings. Together, these studies provide justification for using 2 mmol/L L-glutamine for general culture conditions.

In contrast to the proliferative effects of glutamine, the impairment of cell survival in the absence of glutamine has not been examined thoroughly. Wischmeyer et al. (1997) reported that glutamine supplementation provided cytoprotection, as indicated by a decrease in ⁵¹crominum release from cells injured by heat shock. Our study demonstrates directly, for the first time, that glutamine actually protects cells from cell death. It also shows that prevention of cell death is an important and specific reason for the increase in glutamine requirement during stress, which is a well-recognized clinical phenomenon. In our in vitro system, 2 mmol/L was more than sufficient for cells under basal conditions, whereas a much higher level of supplementation (8 mmol/L) was required for optimal survival of cells under stressful conditions.

The cell death induced by heat shock does not represent apoptosis. Apoptosis occurs in a small percentage of IEC-6 cells in high density cultures (Zhou and Chow 1994). Apoptotic IEC-6 cells remain attached to the monolayer. In contrast, the heat shock-induced cell deaths observed in this study were marked by increases in floating cells. Furthermore, heat shock-induced cell death was not associated with markers of apoptosis, such as DNA fragmentation (data not shown).

There are many candidate mechanisms by which glutamine may promote cell proliferation and cell survival. In addition to stimulation of mitogen-activated protein kinases, glutamine is also believed to favor cell proliferation by enhancing nucleotide biosynthesis (Salloum et al. 1993), increasing intracellular pH via the production of CO₂ and HCO₃ from oxidative metabolism of glutamine (Rhoads et al. 1992) and stabilizing ornithine decarboxylase in rat hepatoma cells (Hogan and Murden 1974). In the promotion of cell survival, a consistently reported effect of glutamine is the induction of hsp. Glutamine induces hsp expression in a wide variety of heat-shocked cells, including Chinese hamster ovary cells (Cai et al. 1991), opossum kidney cells (Nissim et al. 1993), intestinal epithelial cells (Wischmeyer et al. 1997) and myotubes (Zhou and Thompson 1997). Heat shock responses are well documented in prokaryotes and throughout the evolutionary spectrum. Glutamine has been shown to induce hsp in Drosophila Kc cells (Sanders and Kon 1991) and even in the fungus Achlya klebsiana (LeJohn et al. 1994). Thus it appears that the role of glutamine in inducing heat shock response may be a well-conserved pathway among eukaryotes. Enhancement of heat shock response is generally thought to favor survival during stress (Welch 1992). Our observations corroborate this suggestion in that optimal cell survival under stress and a consistently and large induction of hsp 70 both required a high concentration of glutamine (8 mmol/L).

Previously, the effectiveness of glutamine in maintaining mucosal mass was attributed to direct or indirect support of proliferation. We demonstrated in this study that glutamine supplementation diminished cell death. Mucosal mass and homeostasis are critically dependent on the balance of cell growth and death; therefore, the effect of glutamine on cell death is potentially at least as important as its effect on proliferation in maintaining intestinal mucosal mass. In summary, the findings of this study may explain the increase in glutamine requirement under conditions of stress, especially situations in which cell survival is in jeopardy. Our findings may also provide an additional rationale for glutamine supplementation in severely stressed patients.

	FOOTNOTES

Manuscript received 16 March 1998. Initial reviews completed 30 March 1998. Revision accepted 13 April 1998.

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