Glutamine has been the focus of intensive research for almost two decades, and much has been learned. Hundreds of studies are published each year evaluating its roles in normal and pathologic states. We know that it is the most abundant circulating amino acid, the major substrate for inter-organ nitrogen and carbon transfer, and the principal metabolic fuel (1) for the small intestine. Currently, L-glutamine is approved by the Food and Drug Administration as a protein supplement and is available in health food stores. It is mainly used by body builders for anabolic purposes. What effects does exogenous glutamine (GLN) have on intestinal physiology and does it have defined therapeutic roles? Certain aspects of glutamine physiology have become clear from the research of the past two decades.
Glutamine stimulates intestinal salt and water absorption.
My laboratory and that of Robert Argenzio at North Carolina School of Veterinary Medicine have studied the effects of glutamine on intestinal fluid and electrolyte absorption in pigs and calves for many years. Our studies and those of others have consistently shown that glutamine stimulates Na+ absorption by both by electrogenic or coupled Na+ absorption similar to glucose-Na+ co-transport and by electroneutral NaCl absorption. GLN stimulates absorption in the jejunum or ileum of every mammalian species studied including humans and is effective in newborn as well as older animals (2). GLN stimulates absorption in rotaviral (3) and cryptosporidial enteritis (4), augments glucose in promoting Na+ absorption (5), and has the unusual effect of stimulating NaCl absorption even when administered to the serosal surface of the intestine (5). Interestingly, in cryptosporidial enteritis, in which intestinal glucose-Na+ cotransport is diminished by infection, there is an up-regulation of a glutamine transporter (ASCT1) in the brush border membrane (6).
An objective of therapy in diarrhea is to maximally enhance Na+ absorption with the expectation that Cl− and water absorption will follow passively. Dechelotte et al. (7) first showed that GLN and glucose have additive effects on Na+ absorption in rabbit ileum and we have found that this additivity persists even in intestines infected with cryptosporidium and rotavirus (3,4). Desjeux et al. (8) showed that GLN augments the impact of glucose on absorption in a rabbit model of enteroadherent Escherichia coli enteritis. Islam et al. in Bangladesh (9) and Silva et al. in Charlottesville (10), have found that GLN stimulates more Na+ absorption than glucose in normal and cholera toxin-exposed rabbit intestine. In human volunteers, Van Loon et al. showed that GLN and glucose had comparable proabsorptive effects on intestinal Na+ and water absorption (11). However, a clinical trial in Brazil comparing GLN supplemented oral rehydration solution (ORS) with standard ORS in infants with diarrhea showed no difference in efficacy of the two preparations (12). Two criticisms of this well-designed study are that the ORS supplemented with GLN was hypertonic and that study subjects were only about 2% dehydrated at admission. Subsequent studies have shown that hypo-osmolar ORS is more efficacious than standard ORS. In view of the studies in multiple species, I think it is quite likely that additive effects on absorption will be shown in human studies at some time. However, a clinical trial should target subjects with moderate or severe dehydration and the solutions tested should be iso- or hyptonic.
Glutamine stimulates crypt cell proliferation.
We have found that when cultured rat crypt cells (IEC-6) or piglet enterocytes (IPEC-J2) are removed from GLN and then re-exposed to GLN for 24 hours, 3H-thymidine incorporation into DNA increases by more than 20-fold. This effect could be attributable in part to conversion of GLN to pyrimidines but an additional mitogenic effect is suggested by the magnitude of the response. When cells preincubated in media containing higher-than-physiological concentrations of GLN are treated with an additional bolus of 10 mM GLN, there is a significant increase in 3H-thymidine incorporation (13). In fact, these cell lines respond as well to GLN as they do to therapeutic doses of the hormones epidermal growth factor (EGF) or insulin like growth factor-1 (IGF-1).
Byrne et al. (14) first proposed that growth factors and GLN (as a nucleotide substrate) might have additive effects in the intestine. We have found that there are indeed additive effects of the growth factors (EGF, IGF-1) and GLN on crypt cell proliferation (13). Ko et al. (15) showed that the enhancement of proliferation in the crypt cell line IEC-6 was absolutely contingent on the presence of GLN in the media.
GLN is not just a substrate for intestinal cell proliferation; it is also a mitogenic signal. Exposure of intestinal cells to GLN within minutes activates a signaling pathway mediated by mitogen-activated protein kinases, both the extracellular signal-related kinases (ERKs) and the nuclear kinase, c-Jun (16). MAPKs transmit signals from membrane-bound insulin receptor and growth factor receptors to downstream targets, including nuclear proto-oncogenes that can initiate cell migration, promote entry into the S-phase of mitosis and other biologic effects. The activation of MEK, ERKs, and nuclear Elk-1 phosphorylation by GLN results in the immediate transcription of an activating protein-1–dependent luciferase reporter gene (16). Metabolism of GLN is required for these effects in intestinal cells suggesting that GLN may have a unique function as a primitive growth factor.
In vitro studies of cultured biopsy explants show that a glutamine dipeptide stimulates crypt cell proliferation in the human ileum and colon (17). We were unable to demonstrate in piglet rotavirus enteritis that GLN-supplemented ORS produced a more rapid recovery of the villus surface or mucosal digestive enzymes (18). However, in a more severe intestinal injury produced by ischemia/reperfusion of the calf ileum, Blikslager et al. (19) showed that inclusion of GLN plus TGF-alpha, a growth factor present in human milk, greatly enhanced villus regrowth with restoration of villus surface area 72 hours more rapidly than control tissues exposed only to glucose. Neither GLN nor TGF-alpha stimulated villus regrowth when given alone (19). MAP kinase activation by GLN plus TGF-alpha was twice as effective as activation by either GLN or TGF-alpha alone. No study has yet addressed the question of whether GLN administration to humans with acute enteritis or other enteropathies facilitates villus regrowth.
The “score card” for GLN in the literature includes both positive and negatives. On the positive side, GLN seems to be associated with decreased gut permeability in patients receiving total parenteral nutrition; decreased muscle catabolism in Duchenne muscular dystrophy; decreased infection rates in burned patients and those with bone marrow transplantation; better survival of small bowel allografts; decreased severity of acetic acid-induced colitis in rats; and decreased endotoxin-induced permeability in pig and rat intestine. GLN also may reduce the infection rate, intestinal feeding intolerance, and hospital costs in premature infants (20).
In the current issue, Yalcin et al. report that powdered glutamine given as a medication (not as ORS with GLN) to Turkish infants with acute diarrhea (0.1 g/kg orally three times daily) in a prospective, double-masked, placebo-controlled trial promoted recovery from diarrhea about 29 hours earlier was observed in controls. At days 3 to 5 after enrollment, about 20% fewer children had ongoing diarrhea in the GLN group than in the control group (both findings were significant). Weight gain at 1 month was 36% better in the group that received GLN (P = 0.05). A significant negative finding was that serum IL-8 and salivary IgA levels did not change with GLN treatment, suggesting that GLN had more effect on the intestinal epithelium than on the immune system. No convincing evidence was found that GLN reduced the risk of persistent diarrhea.
The strengths of this investigation are the study design, the large number of subjects, the success of randomization, the excellent monitoring of compliance, and the significant positive effect on the primary outcome variable. The importance of the study is heightened by the potential therapeutic application of the medication. Although probiotics may shorten the duration of acute diarrhea, clinicians today have very few safe and effective antidiarrheal drugs for children. GLN, if effective would be a welcome addition to our therapeutic choices. Weaknesses of this study are the lack of identification of the pathogens producing diarrhea and the lack of quantitative data on stool weight. The dose of GLN used (0.3 g/kg/day) seems low compared with doses in adult studies which range from 0.63 to 1.25 g/kg/d. As in most other clinical studies of GLN, there were no significant side effects.
Glutamine originally seemed to be the cure-all for many intestinal diseases. However, the field of “glutaminology” has recently been peppered with negative clinical studies and pessimistic editorials questioning its clinical usefulness (21,22). Several well-designed recent studies have concluded that GLN is ineffective at the doses studied in reducing disease activity in pediatric patients with Crohn disease; in enhancing small bowel adaptation in rats and humans with short bowel syndrome; and in reducing bacterial translocation in experimental models of gut injury. Preliminary data from a multicenter trial of oral GLN prophylaxis for infants reportedly indicates no decrease in infection rate among premature infants treated with GLN. Better enteral feeding tolerance and decreased gastric residuals were noted in treated patients (J. Neu, personal communication, February 18, 2003).
The physician can envision the influence of GLN on intestinal cell physiology by thinking of the small intestinal enterocyte as an automobile. Glutamine keeps the car fueled so that it can absorb sodium and maintain its tight junctions. Glutamine will not convert a Ford Escort into a Ferrari; however, if in the presence of GLN, salt and water absorption increase, crypt cells undergo mitosis, the enterocyte becomes more resistant to injury via heat shock protein induction (23), and the intestinal barrier becomes more resistant to endotoxin (24), then when growth factors such as EGF or TGF-alpha are co-administered, the car will “shift into second gear.” If GLN is reduced below an as-yet-undefined critical level, the intestine will become vulnerable to infection, necrotizing enterocolitis (25), or other pathologic conditions. In this analogy, a low serum GLN concentration or a low tissue level is equivalent to taking one's foot off the accelerator. Under these conditions, the car stalls, and loses all function which, in the intestinal cell translates into apoptosis (programmed cell death) (26).
Despite a lack of corporate sponsors, GLN research has not undergone apoptosis. This study by Yalcin et al. may mark the shift of pediatric clinical trials of glutamine into high gear. Indeed, a recent meta-analysis showed that (in adults with multiorgan system failure) intravenous, high-dose GLN significantly reduces mortality. The meta-analysis also concluded that in postoperative surgical patients, intravenous GLN reduces infection rate and hospital stay (27). Studies such as the one by Yalcin et al. may lead to the acceptance of GLN as a component of therapy for diarrheal diseases. I am convinced that the large body of GLN research involving animals and humans will not ultimately be judged fruitless. Studies such as the one by Yalcin et al. will lead to the acceptance of GLN as a component of therapy for diarrheal diseases.
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