We previously demonstrated that glucose and glutamine, solutes metabolized by the gut, replenish ATP and enhance gut function compared with alanine, a solute not metabolized by the gut, following mesenteric ischemia/reperfusion (I/R). The purpose of the present study was to determine if the nonmetabolizable solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R. At laparotomy, rats had jejunal sacs filled with 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mm, osmotic control) followed by superior mesenteric artery clamping for 60 min and 30 min of reperfusion or sham laparotomy. Jejunum was harvested for evaluation by deconvolution microscopy, fluorescent measurement of F:G actin ratio, or mounted in an Ussing chamber for determination of intestinal permeability. Deconvolution microscopy revealed that the actin cytoskeleton was preserved by enteral glutamine, comparable to shams, but disrupted by enteral alanine. Glucose and controls resulted in comparable disruption, which was less than that with alanine. The F:G actin ratio was highest for glutamine and lowest for alanine; glucose was comparable to controls. Intestinal permeability was highest for alanine and lowest for glutamine, which was comparable to shams. Permeability following glucose and controls was higher than that following glutamine but lower than that following alanine. The nonmetabolizable solute alanine resulted in disruption of the actin cytoskeleton and enhanced intestinal permeability under conditions of mesenteric I/R. The metabolizable solute glutamine was protective under these conditions, whereas glucose exerted minimal effect on the integrity of the cytoskeleton and intestinal permeability. The individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications when administered to critically injured patients.
Departments of Surgery, Integrative Biology and Pharmacology, and Pathology, University of Texas-Houston School of Medicine, Houston, Texas 77030
Received 19 Dec 2003;
accepted in final form 5 Feb 2004
first review completed 25 Dec 2003;
Supported by the National Institutes of Health (grant KO8 GM62975 to Dr. Kozar).
Address reprint requests to Rosemary Kozar, MD, PhD, University of Texas-Houston School of Medicine, 6431 Fannin, Suite 4.284, Houston, Texas 77030 (e-mail: Rosemary.A.Kozar@uth.tmc.edu).
Enteral nutrition has been shown to be beneficial when administered early after severe injury. Following shock, however, the small intestine may remain metabolically compromised because of persistent splanchnic vasoconstriction and recurrent ischemic/reperfusion (I/R) insults. The administration of enteral solutes could prove harmful if they induce a metabolic stress. We have previously demonstrated that during mesenteric I/R, enteral solutes differentially modulate the metabolic demand of the small intestine (1). Alanine, a solute not metabolized by the gut, reduced tissue ATP and impaired gut function, whereas the metabolizable solutes glucose and glutamine maintained both cellular energetics and epithelial cell function.
An intact intestinal epithelium, while permitting the absorption of solutes, also serves as an effective barrier that prevents both the egress of intraluminal enteric bacteria and toxins and the consequent release of inflammatory cytokines. Paracellular permeability is a normal and regulated process that occurs in all epithelium to varying degrees. Enhanced paracellular permeability represents a type of barrier dysfunction that allows increased passage between viable cells and may induce an inflammatory cascade. The major components of the epithelial barrier are tight junctions, which bind cells together and serve as the gateways to the underlying paracellular spaces. The integrity of the tight junctions is modulated by the actin cytoskeleton, comprised of both F and G actin in a dynamic equilibrium. Under normal physiologic conditions, globular-actin (G-actin) polymerizes to form filamentous-actin (F-actin) with the concomitant hydrolysis of ATP. The dynamic balance between these two forms of actin is determined by the ambient ATP concentration (2–4). Thus, the actin cytoskeleton in particular is vulnerable to I/R-mediated injury. In cell culture models, a reduction in cellular ATP has been shown to cause disruption of the actin cytoskeleton network with an associated increase in endothelial and epithelial permeability (5–7). We have previously demonstrated in our mesenteric I/R model that glutamine and glucose resulted in significantly less decrease in ATP compared with alanine following mesenteric I/R (1). The purpose, therefore, of the present study was to determine if the nonmetabolizable enteral solute alanine differentially modulates cytoskeletal organization and paracellular small intestinal permeability compared with the metabolizable solutes glucose and glutamine following mesenteric I/R.
MATERIALS AND METHODS
Sprague-Dawley rats weighing 250–300 g were used after a period of acclimatization. Rats were fasted overnight but allowed free access to water. Anesthesia was induced and maintained for the duration of the experiment with 2% isoflurane, and body temperature was maintained at 37°C by use of a warming blanket. At the conclusion of the experiment, cardiac puncture and exsanguination were used to achieve euthanasia. All procedures performed were under approved protocols by the Animal Welfare Committee of the University of Texas-Houston School of Medicine. The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals.
An upper midline laparotomy was performed, and the jejunum identified 5 cm distal to the ligament of Treitz. The jejunum was used for experiments because it is the primary site for nutrient absorption. An 8-cm intestinal sac was created in each animal by occluding the lumen of the bowel with 3-0 silk ligatures. Sacs were filled with either 10 mM glucose, glutamine, alanine, or magnesium sulfate (5 mM, nonabsorbable osmotic control). The superior mesenteric artery was then isolated at its origin and clamped for 60 min. Following release of the clamp and restoration of pulsatile flow for 30 min, the jejunum was harvested for the following assays. Sham animals underwent superior mesenteric artery isolation without arterial clamping.
Samples of jejunum from within each sac were frozen in embedding media containing 10.24% polyvinyl alcohol, 4.2% polyethylene glycol, and 85.5% sucrose (O.C.T. Compound Tissue-Tek, Torrance, CA). Tissue blocks were sectioned at a thickness of 10 μm with a diamond knife at 4°C in a Reichert HistoSTAT cryotome.
Sample preparation for microscopy—
Samples were prepared as previously described (8). In brief, sectioned tissue is floated onto an 18-mm glass cover slip that had been coated with poly-l-lysine, placed in 3.7% paraformaldehyde for 5 min, and then treated with the appropriate probes: FITC-conjugated phallacidin for F-actin, Texas Red conjugated DNase for G-actin, and DAPI for nuclei (Molecular Probes, Eugene, OR). Sections were covered with Elvanol (an antifade) and a cover slip. Samples were scanned with an Applied Precision DeltaVision Scanning Microscope (Issaquah, WA) fitted with an Olympus IX70 microscope (Olympus America, Melville, NY). This machine is a wide-field sectioning microscope that employs fluorescent light and is equipped with a point spread function for better image quality (deconvolution). A 100-watt mercury arc lamp is used for illumination, and excitation/emission wavelengths are produced by filters specific for each probe (Chroma Technology Corp., Brattleboro, VT). The filter set combination for the low-emission probe is a 490-nm excitation filter with a bandpass width of 20 nm and an emission filter of 518 nm with a bandpass of 38 nm. The filter combination for the high-emission probe is a 555-nm excitation filter with a bandpass of 20 nm and emission filter of 617 nm with a 73-nm bandpass width. Image scans for each emission probe are acquired in series at a step-size thickness of 0.2 μm by a Photometrics (Tucson, AZ) PXL CCD camera. At least 30 sections are scanned per sample for each probe (i.e., yielding 60 total images when 2 probes are used). To avoid the complications arising from the heterogeneous structure of the villus, the villus core was removed by digital subtraction.
Image analysis was performed by transferring each data set to a Silicon Graphics workstation for deconvolution using SoftWoRxTM software from Applied Precision using an algorithm based on the convolution of a point spread function to differentiate and reduce extraneous or scattered light captured by the camera. All data sets are subjected to five deconvolution iterations and are then used for image reconstructions and modeling. Baseline subtraction of background fluorescence and change of intensity gain are optimally set for each emission and consistent for each analysis.
F:G actin ratio
Tissue samples are fixed in 3.7% formaldehyde for 5 min and then rinsed 5 times with phosphate-buffered saline. Stock 20 μM F-actin phallocidin probe is diluted 1:100, and stock 161 μM G-actin DNase1 probe is diluted 1:500 in DAPI (nucleus stain in phosphate-buffered saline, 100 mM final concentration) and incubated with the samples for 10 min at room temperature. Samples are rinsed with Tween 20 in phosphate-buffered saline, then covered with Elvanol antifade and a cover slip. Fluorescence was measured using Corel Paint 10 (Ottawak, Ontario) and Sigma Scan Pro 5.0 (SPSS, Chicago, IL).
Jejunal sacs were harvested, and the bowel was incised along its mesenteric border. Full-thickness jejunum was mounted between two halves of an Ussing chamber exposing 1.3 cm2 tissue to mucosal and serosal bathing solutions. Tissues were bathed at 37°C with oxygenated (95% O2, 5% CO2) Krebs buffer (without glucose) at a pH of 7.4. Following a 20-min stabilization period, 1 μCi of [14C]mannitol (approximately 2.2 × 108 counts per milliliter) (Sigma Chemical, St. Louis, MO) was added to the serosal bathing solution, and the appearance of radiolabeled tracer in the mucosal solution was determined at 20-min intervals. Samples were counted in a Packard Science Tri-Carb 2100TR liquid scintillation counter. Tissue viability was confirmed by the addition of glucose (10 mM) followed by theophylline (50 mM) to the mucosal solution at the conclusion of the experiment. [14C]Mannitol was dialyzed overnight at 4°C to ensure that the label did not detach. Changes in permeability were evaluated by measuring the unidirectional flux (serosa to mucosa) of mannitol (μmol/cm2/h) determined by dividing the rate of appearance of radioisotope in the mucosal solution (cpm/cm2/hr) by the specific activity of the radioisotope in the serosal solution (cpm/μmol) (9).
F:G actin ratios were analyzed by one-way ANOVA followed by post hoc testing with Tukey’s test (Sigma Stat 2.0 statistical software) and permeability by two-way repeated-measures analysis (SAS 8.02 statistical software). Results are expressed as mean ± SEM; significance was set at P < 0.05.
To assess the effect of enteral solutes on actin cytoskeleton organization, jejunal segments were fluorescently stained using FITC-conjugated phallacidin for F-actin and Texas red conjugated DNase for G-actin following mesenteric I/R or sham laparotomy. F-actin is predominantly located in the terminal web and along the lateral and basal membranes, forming the cortical cytoskeleton (Fig. 1A, sham). Glutamine demonstrates a relatively normal cytoskeleton architecture and preservation of F-actin staining (Fig. 1B). Alanine, on the other hand, resulted in disorganization of the terminal web with breakdown of the cytoskeleton and a concomitant decrease in F-actin staining (Fig. 1C). Glucose and controls revealed injury that was less than that with alanine but greater than that with glutamine (not shown).
F:G actin ratio
To further investigate the organization of the cytoskeleton, the F-actin to G-actin distribution ratio was calculated following digital subtraction of the villus core and is displayed in Figure 2. Glutamine resulted in the highest F:G actin ratio (1.3 ± 0.18), comparable to shams (1.6 ± 0.45). Alanine caused a significant decrease (0.48 ± 0.07) compared with glucose (0.77 ± 0.06) and controls (0.70 ± 0.10) as well as glutamine and shams.
As an index of gut barrier function, permeability was assessed in the Ussing chamber by measuring the flux of radiolabeled mannitol (Fig. 3). There were significant differences both between groups (P < 0.0001) and over time (P < 0.0001). Permeability was significantly higher with alanine and lower with glutamine, which was comparable to shams. Permeability following glucose and controls was significantly higher than that with glutamine and shams but lower than that with alanine.
Shock-induced mesenteric ischemia/reperfusion has been invoked to be a pivotal mechanism in the pathogenesis of multiple organ failure (10, 11). Increased intestinal permeability is one manifestation of mesenteric I/R injury that has been documented in high-risk patients after burns, sepsis, and shock (12–14). Additionally, barrier dysfunction has been suggested as a means by which inflammatory cytokines can lead to the systemic inflammatory response syndrome and multiple organ failure (15–18). The provision of early enteral nutrition to promote normal gut function following severe injury is believed to lessen the likelihood of development of multiple organ failure. One proposed mechanism is by reduction of intestinal permeability (19, 20). Permeability between epithelial cells is regulated by tight junctions, important determinants of barrier function in intestinal epithelial cells (21, 22). Tight junctions affiliate with the subcortical actin cytoskeleton via the zonula occludens (23, 24) and provide a mechanism by which the cytoskeleton regulates their function (25). Metabolic stress induced by ischemia/reperfusion can disrupt the actin cytoskeleton with consequent opening of the tight junctions and loss of the integrity of the permeability barrier (26). A number of investigators have demonstrated in cell culture models that a reduction of cellular ATP by metabolic inhibition (hypoxia or chemically) can result in disruption and/or remodeling of the actin cytoskeleton and an increase in endothelial or epithelial permeability (5–7). A reduction in ATP was also associated with an increase in intestinal permeability in one rodent model of mesenteric I/R and hemorrhagic shock (27).
The results of the current study demonstrate that under conditions of mesenteric ischemia/ reperfusion, alanine, which is not metabolized by epithelial cells, resulted in breakdown of the intestinal barrier by disrupting the actin cytoskeleton, led to a decreased F:G actin ratio (proportion of mature actin), and thereby increased epithelial permeability. The metabolizable solute glutamine maintained barrier function by preserving cytoskeleton integrity, facilitating incorporation of G-actin into F-actin, and enhancing intestinal permeability. Glucose, also a metabolizable solute, neither impaired nor improved barrier function. We propose that these solute-specific differences are based in part on cellular energetics because ATP supplies are decreased by alanine over that by gut I/R alone, whereas glucose and glutamine do not contribute to further depletion in ATP (1). However, both Hinshaw et al. (5) in an endothelial line and Molitoris et al. (28) in an epithelial cell line have demonstrated a paradoxical increase in F-actin and decrease in G-actin during ATP depletion. The current study did not validate these findings in that a decrease in F:G actin ratio was found when ATP was further diminished by alanine. There are several possibilities that may explain this discrepancy. First, both the techniques used to measure F- and G-actin and the models differ between studies. The previous studies used a DNase assay in a cell culture model, whereas the current study used a fluorometric technique for direct measurement of both F- and G-actin protein in an animal model. Additionally, the current study measured F- and G-actin during early reperfusion, as opposed to ischemia (metabolic inhibition) as used by other investigators.
The current study also suggests that enteral glutamine may have some role in preservation of barrier function in addition to its effect on enhancing tissue ATP levels. Both glutamine and glucose (8.5 ± 0.96 and 8.0 ± 1.40 nmol ATP/mg protein, respectively) resulted in similar increases in ATP in contrast to alanine (4.1 ± 0.40 nmol ATP/mg protein) during mesenteric I/R but differently modulated intestinal barrier function (1). Glutamine was able to both maintain integrity of the actin cytoskeleton and preserve permeability comparable to that in shams. Glucose, on the other hand, demonstrated effects on the cytoskeleton and permeability that were enhanced compared with alanine but diminished compared with glutamine. Additional mechanisms may be present. It is possible that there is differential modulation of inflammatory mediators (either pro- or anti-inflammatory) invoked by the enteral nutrients following mesenteric I/R that have not yet been identified. In the laboratory, mesenteric I/R can be studied using a variety of models including SMAO, controlled and uncontrolled hemorrhage. We have demonstrated that SMAO is a clinically relevant model that closely resembles hemorrhage models, particularly uncontrolled hemorrhage, yet avoids the confounding effects of a systemic insult (29). It is possible, though, that there could exist different degrees of modulation of inflammatory mediators between I/R models.
Clinically, both intravenous and enterally administered glutamine have been reported to decrease infectious morbidity (30–32), possibly by decreasing intestinal permeability (33, 34). However, enteral studies are limited and have not yet yielded results as significant as parenteral trials. Additionally, the optimal dose and timing of administration have not been well established. Patients are traditionally not begun on enteral diets for several days postinjury or postoperatively, which may limit the effectiveness of enteral glutamine. In the current study, individual nutrients were administered at the time of ischemia, not standard clinical practice but, as suggested by the glutamine data, one that may be safe and potentially beneficial. Early administration of an isolated nutrient such as glutamine may be applicable to patients during or immediately after shock resuscitation, during periods of enteral intolerance, or when medications (such as vasoactive drugs or paralytics) prohibit traditional feeding regimens (35). Additionally, this would avoid administration of potentially harmful nutrients, such as alanine, during periods of high risk.
In conclusion, under conditions of mesenteric I/R the nonmetabolizable enteral solute, alanine, disrupted the actin cytoskeleton, decreased F:G actin ratio, and enhanced intestinal permeability, whereas the metabolizable enteral solute, glutamine, maintained integrity of the cytoskeleton, increased F:G actin ratio, and decreased permeability. These results suggest that the individual components of enteral diets may differentially modulate intestinal barrier function, which could have important implications in the development of multiple organ failure when administered to critically injured patients.
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