Published online before print April 7, 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
in the postischemic gutDepartment of Surgery, University of Texas Health Science Center of Houston, Houston, Texas, USA
1 Correspondence: Department of Surgery, University of Texas Health Science Center of Houston, 6431 Fannin, MSB 4.284, Houston, TX 77030, USA. E-mail: rosemary.a.kozar{at}uth.tmc.edu
ABSTRACT
Early enteral nutrition supplemented with glutamine, arginine, omega-3 fatty acids, and nucleotides has been shown to decrease infection complications in critically injured patients. Concern has been raised, however, that under conditions of hyperinflammation, these diets may be injurious through the induction of inducible NO synthase by enteral arginine. In a rodent model of gut ischemia/reperfusion, inflammation and injury are intensified by enteral arginine and abrogated by glutamine. These findings correlate with the degree of metabolic stress imposed upon the gut by hypoperfusion. Glutamine is metabolized by the gut and therefore, can contribute back energy in the form of ATP, whereas arginine is a nonmetabolizable nutrient, using but not contributing energy. Recent data suggest that one of the molecular mechanisms responsible for the gut-protective effects of enteral glutamine is the activation of peroxisome proliferator-activated receptor 
. This anti-inflammatory transcription factor belongs to the family of nuclear receptors, plays a key role in adipocyte development and glucose homeostasis, and has been recognized as an endogenous regulator of intestinal inflammation. Preliminary clinical studies support the use of enteral glutamine in patients with gut hypoperfusion.
Key Words: gut ischemia/reperfusion • IEC-6 cells • arginine • peroxisome proliferator response element
BENEFITS OF EARLY ENTERAL NUTRITION IN THE CRITICALLY INJURED
Traumatic injuries are a leading cause of death for all ages. Although early deaths are secondary to hemorrhage, late deaths are attributed to septic-related, multiple organ failure [1 ]. Early enteral nutrition decreases septic morbidity and therefore lessens the incidence of late, post-injury multiple organ failure [2 ]. To further improve outcomes, standard enteral diets have been supplemented with glutamine, arginine, omega-3-fatty acids, and/or nucleotides [immune-enhancing nutrients (IENs)]. These IENs, when added to standard enteral diets, enhance immune responsiveness, independent of their presumed nutritional effects [3 ]. Prospective, randomized clinical trials have indeed demonstrated a reduction in infectious complications, in most but not all studies [4 ]. Additionally, their use in critically ill patients is questioned, as mortality may actually be increased in septic Intensive Care Unit patients [5 ]. Arginine, as a substrate for NO, has been hypothesized to enhance systemic inflammation through the induction of inducible NO synthase (iNOS), although this has not been well investigated [6 ].
The mechanism by which each of the IENs exerts its effect, particularly under conditions of gut hypoperfusion, is unclear. Glutamine is the preferred fuel for the enterocyte, hence, its inclusion as an IEN. A meta-analysis that included 14 randomized trials demonstrated that relatively high doses (>0.25 g/kg) of glutamine supplementation are beneficial, although at lower doses, this may not be true [7 ]. Four prospective, randomized trials tested enteral glutamine as an isolated IEN added to standard enteral diets, and all demonstrated decreased infectious complications [8 9 10 11 ]. A recent review about intestinal permeability and systemic infections in critically ill patients concluded that glutamine (i.v. and enteral administration) reduced the frequency of systemic infections, presumably by maintaining intestinal barrier function [12 ]. In addition to glutamine’s gut protective effects, glutamine is important in nucleotide synthesis, is anti-catabolic, has anti-oxidant properties via metabolism to glutathione, and may enhance immune responsiveness.
DIFFERENTIAL MODULATION OF METABOLIC STRESS BY ENTERAL NUTRIENTS IN THE POSTISCHEMIC GUT
The inappropriate administration of enteral nutrients to the postischemic gut may lead to nonocclusive bowel necrosis, a frequently fatal condition in which there is compromised bowel viability at sites of contact with enteral nutrients. The reported incidence ranges from less than 1% to as high as 8.5%, and mortality is up to 100% in some series [13 14 15 ]. Systemic hypoperfusion is believed to precede gut hypoperfusion, although overt hypotension does not typically occur until late. Symptoms are unfortunately nonspecific, but significant abdominal distention should prompt a search for the diagnosis of nonocclusive bowel necrosis [16 ]. Computed tomography most commonly shows pneumotosis intestinalis, although abdominal-free fluid and thickened loops of small bowel may also be seen. Laparotomy is usually required, unless the diagnosis was made very early in the course of the disease. Metabolic stress, dysmotility, and bacterial colonization have all been implicated in its pathogenesis. With progressive ileus, distension may lead to compromised perfusion of the bowel wall. Additionally, bacterial overgrowth is encouraged in a dysmotile gut. Metabolic stress imposed by enteral nutrients can be explained by the metabolic fate of administered nutrients. As most enteral nutrients are absorbed across the intestinal epithelium via sodium-coupled, active transport, energy expenditure occurs. Under normal conditions, this poses no stress to the gut. However, during times of hypoperfusion, the gut’s energy reserves may be depleted to a point that additional requirements outstrip supply with consequent gut ischemia. This phenomenon has been equated to a "stress test" of the gut [17 ]. There are several enteral nutrients, glucose and glutamine, in particular, which are metabolized by the gut and therefore, may preserve energy, even under conditions of stress. In a rodent model of gut ischemia/reperfusion, glucose and glutamine maintained gut absorption and protected against mucosal injury compared with the nonmetabolizable nutrient, alanine [18]. Interestingly, these changes paralleled those seen in ATP. When mucosal flow was measured in a similar model, flow was enhanced by glucose (glutamine was not tested) during ischemia and reperfusion compared with alanine [19 ]. Glutamine has also been shown to enhance mucosal blood flow [20 ]. In general, glucose and other carbohydrates induce the greatest degree of postprandial hyperemia with amino acids causing the least. Glutamine, though, has been shown to cause intestinal vasodilation. Matheson et al. [21] have postulated an adenosine–NO-mediated pathway as the etiology. Further work revealed that glutamine, but not alanine, maintained small bowel barrier function in the postischemic gut, and glucose was comparable with controls [22]. Deconvolution microscopy demonstrated a relatively normal cytoskeleton architecture and preservation of F-actin after glutamine. Alanine, on the other hand, resulted in disorganization of the terminal web with breakdown of the cytoskeleton and a concomitant decrease in F-actin. Only in the presence of adequate ATP supplies can immature G-actin form mature F-actin. Therefore, with ATP depletion, such as occurs in the hypoperfused gut with alanine administration, less F-actin was formed, and cytoskeleton integrity was compromised. As arginine is one of the identified, immune-enhancing nutrients and is also not metabolized by the gut, changes similar to those of alanine with compromised gut barrier function would be anticipated [23 ].
In summary, the differential metabolic stress imposed on the gut by metabolizable versus nonmetabolizable nutrients suggests that gut function may be maintained by metabolizable nutrients, even when administered to the postischemic gut (Table 1 ).
|
View this table: [in a new window] |
Table 1. Glutamine in the Hypoperfused Gut
|
Laboratory and recent clinical data demonstrated that glutamine administered i.v. enhanced heat shock protein (HSP) expression and correlated with improved outcomes [25
] (Table 1)
. Surprisingly, this same mechanism of protection is not true for glutamine when administered via the enteral route. Neither HSP70 nor heme oxygenase-1 (HO-1) was differentially modulated by enteral nutrients when delivered to the hypoperfused gut [24
]. HO-1, a member of the HSP family, is increased by gut ischemia/reperfusion and further increased by gut-protective strategies such as hypertonic saline and gut hypothermia [28
29
]. Based on previous observations that PPAR was preferentially induced by cyclooxygenase 2 (COX-2) inhibition in the postischemic gut, the effect of nutrients on PPAR DNA-binding activity was investigated [30
]. PPARs are transcription factors that belong to the family of nuclear receptors and play a key role in adipocyte development and glucose homeostasis. The natural ligand of PPAR is 15-deoxy-
12,14-PGJ2, and a new class of antidiabetic agents, the thiazolidinediones, are synthetic ligands [31
32
33
]. Lastly, PPAR has recently been recognized as an endogenous regulator of intestinal inflammation [34
35
].
When PPAR DNA-binding activity was measured in the postischemic gut following administration of enteral nutrients, glutamine significantly increased PPAR beyond that of ischemia/reperfusion alone, and arginine was comparable with shams [24
] (Table 1)
. The increased PPAR DNA-binding activity seen after glutamine was associated with gut mucosal preservation and abrogation of inflammation. To further explore this novel finding, the specific and irreversible antagonist of PPAR, GW9662, was tested, and findings suggested that glutamine was acting as a PPAR agonist. GW9662 abrogated the increase in PPAR-binding activity by glutamine and enhanced gut injury and inflammation. PPAR controls expression of genes implicated in the inflammatory response via negative interference with proinflammatory pathways such as NF-
B, AP-1, STAT1, and NFAT [36
]. Specifically, inhibition of NF-B and AP-1 occurred via a ligand-dependent transrepression [37
]. This pathway of inflammation is similar to that seen in the postischemic gut. Tissue ischemia and oxygen stress activate protein kinases, such as MAPKs, which converge on AP-1 and NF-B to regulate the expression of proinflammatory genes. The resulting gene products include enzymes, such as iNOS and COX-2, as well as adhesion molecules, such as ICAM-1 [30
]. These initiate local inflammation, which is amplified further by recruitment of circulating leukocytes, appearing to be key effector cells in causing tissue injury and subsequent dysfunction. Although glutamine does abrogate inflammation, its effect on leukocyte recruitment has not been investigated.
NF-B and AP-1 are increased in the postischemic gut but with a variable response to enteral nutrients. Surprisingly, only AP-1, but not NF-B, was differentially modulated by nutrients [38
]. Although NF-B-binding activity was increased following gut ischemia/reperfusion, there was no change in activity after glutamine or arginine. In contrast, arginine increased, and glutamine decreased AP-1-binding activity. Supershift revealed the JNK pathway, via c-jun, was responsible. The selective activation of AP-1 has not been well described. Typically, NF-B and AP-1 cotranslocate into the nucleus and activate transcription of their target gene, such as iNOS. Recently, AP-1, but not NFB, was shown to be targeted by the NO-mediated protein kinase G (PKG) pathway, which feeds back from NO to AP-1 [39
]. As arginine increased iNOS expression and AP-1 activity beyond that of ischemia/reperfusion alone, activation of this pathway is a feasible but unproven explanation (Fig. 1
). Consistent with repression of AP-1 by glutamine, iNOS was significantly less compared with ischemia/reperfusion [24
]. The mechanism by which glutamine represses iNOS is not known but may occur via ligand-dependent small ubiquitin-related modifier (SUMO)ylation of PPAR [40
]. In the basal state, corepressor complexes are present on inflammatory genes such as iNOS. Once activated by LPS or other proinflammatory mediators, the corepressor complexes are cleared, and a switch from active repression to transcriptional activation occurs. However, when a PPAR agonist activates PPAR, transrepression may be initiated by SUMOylation, which targets PPAR to the corepressor complexes associated with the promoter of iNOS, preventing their clearance and maintaining iNOS in a repressed state. This is hypothesized, but not proven, to be the mechanism by which glutamine repressed iNOS expression.
 View larger version (21K): [in a new window] |
Figure 1. Overview of the proposed mechanisms for differential modulation of gut function by enteral glutamine and arginine in the hypoperfused gut. Following gut ischemia/reperfusion (Gut IR), the proinflammatory transcription factor, AP-1, is increased, as is the downstream, proinflammatory enzyme iNOS. Both are associated with gut inflammation, mucosal injury, and dysfunction. When the enteral nutrient, arginine, is added to the hypoperfused gut, iNOS expression and AP-1 DNA-binding activity are increased further. Arginine is a known precursor to iNOS, but the mechanism by which arginine increases AP-1 is unknown. There is a recently described, NO-mediated PKG pathway that may explain the increase in AP-1 by arginine. PPAR is believed to inhibit the proinflammatory pathway at the level of AP-1, protecting the hypoperfused gut. Glutamine increases PPAR DNA-binding activity and may be a novel PPAR agonist. When PPAR is activated, it heterodimerizes with the retinoic acid receptor (RXR) and then binds to the peroxisome proliferator response element (PPRE), resulting in target gene expression.
|
can activate transcription via ligand-independent and -dependent mechanisms. For example, insulin and C-peptide are ligand-independent activators, and arachidonate metabolites and thiazolidinediones are ligand-dependent activators [31
41
]. Preliminary work done in an intestinal-epithelial cell 6 (IEC-6) culture model of oxidant stress suggests that glutamine may function as a ligand-dependent activator of PPAR (unpublished data). When a PPRE–luciferase construct was transfected into IEC-6 cells, a concentration-dependent increase in luciferase expression was demonstrated, which was abrogated by the specific PPAR inhibitor GW9662. Once activated by agonists, PPAR heterodimerizes with the RXR and binds to a PPRE in the promoter of target genes, acting as a transcriptional regulator [37
]. Further studies are needed to determine if indeed glutamine is a specific ligand for PPAR or whether glutamine administration causes the release of an intracellular ligand. Taking these laboratory findings to the bedside (Table 1) , a recent clinical trial demonstrated that enteral glutamine was safe and enhanced enteral tolerance when administered to critically injured patients undergoing active shock resuscitation [26 ]. A large Canadian study, REDOX, is now in progress to investigate the effects of parenteral and enteral glutamine and antioxidant supplementation in critically ill patients with evidence of hypoperfusion [27 ]. Future areas of clinical investigation will likely be focused on the use of gut-specific nutrients, antioxidants, and macro- and microbiotics, all of which may possess unique benefits to the critically ill. As translational research gains momentum (and funding), clinical studies can merge with mechanistic studies to aid in examining gut-specific therapies. For example, changes in serum cytokines have recently been shown to provide early markers for the onset of multiple organ failure [42 ]. Modulation of cytokines by gut-specific therapies can easily be studied. The use of Bioplex technology allows for the measurement of large numbers of cytokines simultaneously, making patient sampling less onerous for patients and investigators. Mechanistic studies are limited though to gut therapies with systemic effects. Glutamine, for example, is believed to act at the level of the gut, whereas arginine acts systemically. Clinical indicators of tolerance and gut function as well as infectious complications can be studied in patients receiving enteral glutamine, whereas molecular mechanisms are currently limited to animal and cell culture data.
In conclusion, although the use of immune-enhancing enteral diets in critically injured patients provides benefit, administration to the hypoperfused gut should be done with caution. The metabolizable nutrient, glutamine, demonstrated protection by a significant reduction in mucosal injury, inflammation, and preservation of gut barrier function compared with the nonmetabolizable, immune-enhancing nutrient arginine (Fig. 1)
. Recent data suggest that arginine may increase not only iNOS but also AP-1, whereas transcriptional activation of PPAR may be the molecular mechanism responsible for gut protection by glutamine. Further investigations into these novel findings are warranted to ensure the safe and effective administration of enteral nutrients to the hypoperfused gut.
ACKNOWLEDGEMENTS
This work was supported by the National Institutes of Health RO1 GM077282. This paper was presented in part at the 40th Annual Meeting of the Society of Leukocyte Biology (Cambridge, MA, USA), October 13, 2007.
Received November 18, 2007; revised February 5, 2008; accepted March 6, 2008.
REFERENCES
} by luminal glutamine and iNOS by luminal arginine in the rodent post ischemic small bowel Am. J. Physiol. Gastrointest. Liver Physiol. 290,G616-G62312,14-prostaglandin J2 is a ligand for the adipocyte determination factor PPAR Cell 83,803-812[CrossRef][Medline] agonist rosiglitazone Circulation 116,163-173 is required for normal skeletal muscle and hepatic insulin sensitivity and full antidiabetic effects of thiazolidinediones J. Clin. Invest. 117,1658-1659[CrossRef][Medline] is a new therapeutic target in sepsis and inflammation Shock 23,393-399[CrossRef][Medline] is a negative regulator of macrophage activation Nature 391,79-82[CrossRef][Medline] Nature 437,759-763[CrossRef][Medline] by insulin and C-peptide in kidney proximal tubular cells J. Biol. Chem. 279,49747-49754This article has been cited by other articles:
 |
|
 |
|
  P. E. Wischmeyer Glutamine in acute lung injury: the experimental model matters Am J Physiol Lung Cell Mol Physiol, March 1, 2009; 296(3): L286 - L287. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
