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Am J Physiol Gastrointest Liver Physiol 294: G155-G164, 2008. First published October 25, 2007; doi:10.1152/ajpgi.00139.2007
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INFLAMMATION/IMMUNITY/MEDIATORS

Protein kinases, TNF-{alpha}, and proteasome contribute in the inhibition of fructose intestinal transport by sepsis in vivo

Josefina García-Herrera,1 M. Carmen Marca,2 Edith Brot-Laroche,3,4,5 Natalia Guillén,6 Sergio Acin,6 M. Angeles Navarro,6 Jesús Osada,6 and M. Jesús Rodríguez-Yoldi1

1Physiology Unit, Department of Pharmacology and Physiology and 2Internal Medicine Unit, Department of Animal Pathology, Veterinary Faculty, University of Zaragoza, Zaragoza, Spain; 3Institut National de la Santé et de la Recherche Médicale, Unité Mixte de Recherche S 872, 4Université Pierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers; 5Université Paris Descartes-Paris 5, UMR S 872, Paris, France; and 6Biochemistry Unit, Department of Biochemistry and Molecular Biology, Veterinary Faculty, University of Zaragoza, Zaragoza, Spain

Submitted 28 March 2007 ; accepted in final form 4 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Lipopolysaccharide (LPS) endotoxin is a causative agent of sepsis. The aim of this study was to examine LPS effects on intestinal fructose absorption and to decipher mechanisms. Sepsis was induced by intravenous injection of LPS in rabbits. The ultrastructural study and DNA fragmentation patterns were identical in the intestine of LPS and sham animals. LPS treatment reduced fructose absorption altering both mucosal-to-serosal transepithelial fluxes and uptake into brush border membrane vesicles (BBMVs). Cytochalasin B was ineffective on fructose uptake, indicating that GLUT5, but not GLUT2, transport activity was targeted. GLUT5 protein levels in BBMvs were lower in LPS than in sham-injected rabbits. Thus lower fructose transport resulted from lower levels of GLUT5 protein. LPS treatment decreased GLUT5 levels by proteasome-dependent degradation. Specific inhibitors of PKC, PKA, and MAP kinases (p38MAPK, JNK, MEK1/2) protected fructose uptake from adverse LPS effect. Moreover, a TNF-{alpha} antagonist blocked LPS action on fructose uptake. We conclude that intestinal fructose transport inhibition by LPS is associated with diminished GLUT5 numbers in the brush border membrane of enterocytes triggered by activation of several interrelated signaling cascades and proteasome degradation.

GLUT5; small intestine; LPS; intracellular signals; rabbit

THE PRIMARY FUNCTION OF THE gastrointestinal tract is to absorb water, electrolytes, and nutrients. The gastrointestinal mucosa also prevents environmental noxious agents like bacteria or bacterial products and antigenic macromolecules from spreading into nonintestinal organs (47). Lipopolysaccharide (LPS) or endotoxin is an integral component of the outer membrane of all gram-negative bacteria. Circulating LPS has been detected and implicated in a variety of septic states and can evoke pathophysiological responses when injected into animals (35). LPS stimulates cytokine production and activates opiate and neuroendocrine pathways, which in turn will modulate the inflammatory process. LPS strongly increases corticosterone and glucocorticoid levels with a moderate catecholaminergic influence (38), and there is a positive correlation between adrenocorticotropic hormone (ACTH) and tumor necrosis factor-{alpha} (TNF-{alpha}) levels after LPS injection in rat (55) and piglets (30). Intravenous LPS injection in rats also promotes a rapid increase of glycemia (42).

LPS binds to specific receptors in the plasma membranes and activates several protein kinase signaling pathways including protein kinase C (PKC), protein kinase A (PKA), protein tyrosine kinase, mitogen-activated protein kinase (MAPK), and proline-directed protein kinases. These signaling cascades generate proinflammatory cytokines (IL-1β, IL-6, TNF-{alpha}) and other mediators (nitric oxide) that will trigger secondary signaling pathways in target cells (29). These secondary cascades include activation of phospholipases and subsequent release of lipid mediators to activate other protein kinase pathways via diacylglycerol, sphingomyelinase, ceramide, and nuclear factor-{kappa}B (NF-{kappa}B) activation leading to cytotoxicity (49).

The transcription factor NF-{kappa}B is activated by a variety of physiological and pathological signals including inflammatory cytokines and bacterial LPS, as well as oxidative and fluid mechanical stress. Upon this activation, I{kappa}B (inhibitor of NF-{kappa}B) is phosphorylated and subsequently ubiquitinated to be degraded by the proteasome, thus leading to the nuclear translocation of NF-{kappa}B (9). Nuclear NF-{kappa}B regulates the expression of genes involved in immune and inflammatory responses, i.e., inflammatory cytokines and adhesion molecules (36). Protein kinases such PKC, PKA, and cAMP-responsive kinase are able to phosphorylate I{kappa}B in vitro (9, 48). However, neither phosphorylation alone nor ubiquitination alone is sufficient to release NF-{kappa}B from I{kappa}B (36).

The proteasome is a multicatalytic proteinase complex responsible for most of protein degradation and it has been implicated in systemic responses to infection or inflammatory stimuli (39). MAPKs and NF-{kappa}B are also activated in inflammatory bowel disease (44, 58). Thus MAPK and NF-{kappa}B signaling are distinct but interrelated transduction pathways triggered in response to inflammation (50). There are reports indicating that c-Jun-NH2-terminal kinase (JNK) and p38 kinase become activated in response to TNF-{alpha}, interleukin-1β (IL-1β), LPS, and UV light (46). JNK and p38 are important transducers of stress-related signals by phosphorylating intracellular enzymes and transcription factors involved in cell survival (43), apoptosis, and inflammatory cytokine production (33).

Infectious agents can cause intestinal dysfunction. Indeed, nonlethal doses of endotoxin modify intestinal water and electrolyte absorption (22) and alter sugar and amino acid transport (19, 51, 52). Studies in our laboratory have shown that incubation of intestinal tissue with LPS inhibits L-leucine (2) and D-fructose transport (17) across the mucosa of rabbit jejunum in vitro and in vivo upon LPS intravenous injection (1, 4).

In the intestine, D-fructose is transported from the lumen into enterocytes by GLUT5, a facilitative transporter of the SLC2A gene family. Fructose flows down its concentration gradient in an energy-independent manner (10, 37). We found that LPS exposure of rabbit jejunum tissue in vitro inhibits D-fructose uptake by a mechanism involving PKC and calmodulin (17). The aim of this study was to determine by what mechanisms sepsis induced by intravenous LPS administration in vivo will affect D-fructose transport in the jejunum.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials

LPS from Escherichia coli serotype 0111:B4, D-fructose, D-mannitol, HEPES, Tris (hydroxymethyl) amino-methane, sucrase, bovine albumin, ATP, protein kinase inhibitor (IP20), cytochalasin B, and anti-actin were from Sigma (Madrid, Spain). Bisindolylmaleimide I, hydrochloride (GF-109203X), and carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132) were from Calbiochem (Darmstadt, Germany). 4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine hydrochloride (SB-203580 hydrochloride), anthra [1-9-cd]pyrazol-6(2H)-one (SP-600125), and 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio] butadiene (U-0126) were from Tocris (Bristol, UK). The TNF-{alpha} receptor peptide antagonist was from Bachem (Bubendorf, Switzerland). Polyethylene glycol (PEG) was from Merck (Barcelona, Spain). D-[U-14C]fructose, [14C]PEG, anti-rabbit IgG peroxidase, and Biodegradable Counting Liquid Scintillation were obtained from GE Healthcare Life Sciences (Madrid, Spain). The membrane filters were from Millipore (Barcelona, Spain). The reagents used in Western blot analysis were from Bio-Rad (Barcelona, Spain), Sigma (Madrid, Spain), and Serva (Barcelona, Spain).

Animals, Preparation of Blood Samples, and Intestinal Tissue

Animal handling was in accordance with the European Council Legislation 86/609/EEC for experimental animal protection. The experimental animals were housed, handled, and euthanized according to European Union legislation (86/609/EEC). All experimental protocols were approved by the Ethical Committee of the University of Zarazoga (Spain). Male New Zealand rabbits weighing 1.8–2.0 kg were maintained at constant room temperature (24°C) with free access to water and standard rabbit fodder (25.1% proteins, 3.8% fat, 18.05% cellulose). One group of rabbits received an intravenous solution of 200 µl LPS at different concentrations (LPS-treated animals), and sham animals received 200 µl saline. Rabbits were killed 90 min after injection, following previously published protocols including ours (1, 56), and intestinal samples were taken. Inhibitor injections were made either in saline solution (IP20, GF-109203X, SB-203580) or DMSO (SP-600125, U-0126, MG-132). Vehicle administration alone was harmless (data not shown). These inhibitors were injected 15 min before LPS, and the dose was calculated from published EC50 in relation to body weight. It was administered in a final volume of 200 µl to avoid any changes in blood volume.

After euthanasia, the proximal jejunum was removed and rinsed with ice-cold Ringer's solution: (in mM) 140 NaCl, 10 KHCO3, 0.4 KH2PO4, 2.4 K2HPO4, 1.2 CaCl2 and 1.2 MgCl2, pH 7.4.

Blood and Serum Parameter Assays

To evaluate the toxicity of LPS injection and the possible development of an endotoxic shock, several hematological, biochemical, and endocrine parameters were measured. Blood samples were collected from the ear central vein before and 90 min after LPS injection. Whole blood was processed immediately and sera were stored at –20°C until analysis.

A complete hematological study was performed by a semiautomatic hematology analyzer (Sysmex F-800 Roche Diagnostics, Barcelona, Spain) that included white blood cell, red blood cell, hemoglobin, and hematocrit values. Differential white blood cell counts were analyzed visually from stained blood film. Glucose was measured with a commercial reagent and a multichannel analyzer (Technicon RA-500, Bayer, Barcelona, Spain). Finally, a commercial chemiluminescent assay (Immulite, DPC, Madrid, Spain) was used to quantify serum cortisol.

Cell Water Determinations

Rings of everted jejunum were incubated in Ringer's solution at 37°C containing 0.02 µCi/ml [14C]PEG 4000 for 15 min and continuously gassed with 95% O2-5% CO2. After incubation, tissue pieces were gently blotted on moist filter paper, weighed, and incubated overnight in 0.5 ml of 0.1 M HNO3 at 4°C to extract the labeled PEG from tissue. Aliquots of 200 µl from extract and bathing solutions were counted in 2 ml of scintillation fluid. Following extraction, tissues were dried at 80°C for 12 h and reweighed. Total tissue water was calculated as the difference between wet and dry weight. Extracellular water was estimated from PEG tissue counts. Intracellular water was calculated as the difference between total and extracellular tissue water.

Sugar Uptake Measurements

Tissue uptake. Rings of everted jejunum weighing about 100 mg were continuously gassed with 95% O2-5% CO2. Tissue rings were incubated for 3 min in Ringer's solution supplemented with 0.01 µCi/ml D-[U-14C]fructose and 5 mM fructose substrate. At the end of the experiment, rings were washed with three gentle shakes in ice-cold Ringer's solution and blotted carefully on both sides to remove excess of solution. Tissue was then processed as described for water content estimates. Data are expressed as micromoles of D-fructose per milliliter of tissue water.

Transepithelial flux measurements. Jejunal mucosa were stripped off serosal and muscle layers and mounted as flat sheets in Ussing-type chambers. The bathing solutions containing 5 mM D-fructose at the mucosal and serosal surfaces of tissue were maintained for a 40-min preincubation at 37°C using a circulating water bath. Mucosal-to-serosal fluxes (Jm-s) and serosal to mucosal fluxes (Js-m) were then measured by adding 0.04 µCi/ml D-[U-14C]fructose to the mucosal or serosal chamber respectively. Samples were removed from the opposite chamber at 20-min intervals for 60 min. Starting radioactivity at time zero and timely samples were counted with a liquid scintillation counter. Results are expressed as micromoles D-fructose per square centimeter per hour.

BBMV uptake assays. Brush border membrane (BBM) vesicles (BBMVs) were prepared from fresh tissue by the Mg2+ EGTA precipitation method (8). Freshly prepared BBMVs containing 300 mM mannitol and 10 mM HEPES-Tris pH 7.4 buffer were used for transport studies. Protein content was measured with the Bradford method using bovine serum albumin as standard. BBMV purity was determined by measuring sucrase activity enrichments and levels of basolateral plasma membranes were assayed by measuring Na+/K+ ATPase activity.

Fructose uptake was measured at 5, 10, 40 s; 1, 2, 10 min; and 1, 2, 3 h to reach equilibrium yielding an estimate of vesicular volume. Incubations were performed at room temperature (25°C) and started by adding 5 µl (200 µg) of BBMV to 45 µl of medium containing 10 mM HEPES-Tris, 100 mM NaCl, 0.01 µCi/ml D-[U-14C]fructose tracer, 5 mM unlabeled fructose, and D-mannitol to compensate for osmolarity (300 mosmol/l). D-Fructose uptakes are in picomoles per milligram protein.

Western Blotting

Similar amount of BBMV protein (10 µg) from sham and treated animals were solubilized in Laemmli buffer and resolved by 10% SDS-PAGE. Proteins were transferred onto PVDF membranes by use of a semidry transblot transfer apparatus (Bio-Rad). The protein transfer efficiency was visualized with Ponceau S and by the transfer of Rainbow molecular weight markers (Sigma). GLUT5 was detected by using a rabbit polyclonal anti-GLUT5 antibody (L1666/9; 1:2,000) antibody targeting the COOH-terminal human sequence. Equal loading were confirmed by with an anti-actin antibody (Sigma Chemical, Madrid, Spain). Detection was carried out using an anti-rabbit IgG conjugated to horseradish peroxidase (Sigma) (1:6,000 dilution) and ECL chemiluminescence (GE Healthcare, Madrid, Spain). Membranes were exposed to ECL films (GE Healthcare) for several time periods to achieve signal intensity within the dynamic range of quantitative detection, and films were scanned at a 600-dpi resolution (via AGFA Arcus II). Intensity of bands for each condition, taken as volume of pixels per square millimeter, was calculated with Quantity One (version 4.5) software (Bio-Rad, Madrid, Spain) and normalized to that corresponding to the actin signal. Membranes were stripped in 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris·HCl pH 6.7 for 30 min at 50°C before being reblotted with a rabbit anti-actin (1:150) antibody used as an internal loading standard to normalize GLUT5 expression accordingly.

Northern Blotting

At the moment of death, the jejunum was quickly frozen in liquid nitrogen. RNA was isolated by using TRI Reagent (MRC, Cincinnati, OH) following the manufacturer's instructions. Total RNA was subjected to Northern blot analysis (11). The cDNA probe phJHT5/hGLUT5 (1.9-kb insert) was generously provided by Dr. G. I. Bell. A mouse actin Kpn I/Xba I fragment of 250 pb was used as internal standard for loading. Probes were labeled using [{alpha}-32P]-dCTP and Rediprime. Filters were exposed to Biomax film (Kodak, Amersham) and signals were analyzed with a laser LKB 2202 densitometer (Amersham-Pharmacia).

DNA Fragmentation Assay

DNA was prepared according to a standard method. The jejunum was pulverized in liquid nitrogen with a mortar and suspended in 200 µl of 50 mM Tris·HCl pH 7.5, 100 mM EDTA, 100 mM NaCl, 1% SDS, and 100 µg/ml proteinase K buffer. Proteolytic digestion was carried out at 65°C for 12 h. Digestion was mixed with 50 µl of 5 M NaCl, and detritus was removed by centrifugation. DNA was precipitated by addition of 200 µl of absolute ethanol. DNA (1 µg) was loaded onto a 2% agarose gel containing 100 µg/ml ethidium bromide. DNA was visualized under UV light and photographed.

Histology

Jejunal tissue samples from sham and LPS rabbit were fixed in 10% buffered formaldehyde and embedded in paraffin. Sections of 4 µm were stained with hematoxylin-eosin. The Electron Microscopy Core Facility of the University of Zaragoza analyzed the ultrastructure of the intestines. Images were captured and digitized by use of a Nikon microscope equipped with Cannon digital camera.

Statistical Analysis

All results are expressed as means ± SE. Statistical analyses were carried out by one-way ANOVA and Tukey-Kramer multiple comparisons test. Fisher's protected least significant difference test was used to compare data between groups with the level of significance set at P < 0.05 or P < 0.01.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Sepsis Evoked by LPS Intravenous Administration

To evaluate the septic state induced by LPS, we performed hematological and biochemical analyses of animals. Table 1 summarizes results obtained before and 90 min after injection of saline or LPS solutions (sham and treated groups, respectively). Significant differences were found in LPS-treated group related to the number of white blood cells but not red blood cell content as indicated by red blood cell number, hemoglobin concentrations, and hematocrit values. Indeed, a strong decrease of total white blood cell number (neutrophils, lymphocytes, and monocytes) was induced in LPS compared with sham-injected rabbits. Glucose and cortisol levels were increased in both animal groups, but higher increases were reported in the LPS group. LPS at doses of 0.02, 0.2, 2, and 20 µg/kg body wt also increased rectal temperatures significantly by an average 1.4 ± 0.09°C (Fig. 1). All together these results indicate that LPS injection induced a septic state in rabbits.


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  		Table 1. Results of hematological and biochemical analyses

 

Figure 1
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  		Fig. 1. Effect of LPS on body temperature. Rectal temperature was measured every 30 min for 90 min. The results are from 5 animals at each condition. *P < 0.05 LPS vs. sham animals.

 
LPS Does Not Produce Apoptotic Patterns in Epithelial Cell

Histological examination of the jejunum showed that the epithelial structure and the basement membrane were similar in sham and LPS-treated animals (data not shown). Electron microscopy analysis of the integrity of intestinal membranes (Fig. 2) were showed unaltered after LPS treatment.


Figure 2
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  		Fig. 2. Electron microscopy images of the small intestine taken from treated rabbits with 2 µg/kg LPS for 90 min. A: jejunum (microvilli and parietal cells) at x8,000 magnification. Bars corresponds to 2 µm. B: jejunal cells (parietal and goblet cells) at x6,000 magnification. Bar = 2 µm.

 
We further looked for an apoptotic pattern of DNA fragmentation induced by LPS injection in rabbit jejunum. The DNA laddering pattern in enterocytes from sham and LPS (2 µg/kg body wt)-treated animals (Fig. 3) were identical confirming the absence of induction of apoptosis in the jejunum by systemic LPS.


Figure 3
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  		Fig. 3. Electrophoretic patterns of DNA fragments in the small intestine from sham and 2 µg/kg LPS-treated rabbits for 90 min. MGW, molecular weight markers.

 
LPS Inhibits Intestinal D-Fructose Transport

A dose response of the effect of intravenous injection of LPS on intestinal D-fructose uptake was performed using 0.2, 2, and 20 µg/kg body wt LPS for 90 min. Because changes in apparent sugar uptake might result from tissue water differences, we calculated water contents in rings from sham and LPS-treated animals. LPS decreased tissue water slightly but significantly in LPS compared with sham tissue (0.64 ± 0.01 vs. 0.72 ± 0.01, P < 0.05). These alterations were taken into account when calculating initial D-fructose uptake measured for 3 min in intestinal tissue rings (Fig. 4A). LPS (2 and 20 µg/kg) inhibited 20% of 5 mM D-fructose uptake. From then on, experiments were made with the 2 µg/kg body wt LPS dose to minimize possible side effects.


Figure 4
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  		Fig. 4. Effect of LPS on intestinal D-fructose transport. A: uptake of 5 mM D-fructose was measured for 3 min in everted jejunal rings taken from sham or LPS rabbits. Animals were injected intravenously (iv) with either saline (sham) or 0.2, 2, and 20 µg/kg LPS for 90 min before euthanasia. Results expressed as µmol D-fructose/ml tissue water represent the average 9 determinations per animal, with 5 animals. B: effect of 2 µg/kg LPS on 5 mM D-fructose mucosal-to-serosal (Jm-s) and serosal-to-mucosal (Js-m) fluxes. Results are expressed as µmol D-fructose·cm–2·h–1, average of 8 determinations per point from 5 animals (sham and LPS). C: time course of 5 mM D-fructose uptake in brush border membrane vesicle (BBMV) from sham and 2 µg/kg LPS-treated animals. D: effect of 0.1 mM cytochalasin B (CB) on 5 mM D-fructose uptake for 10 min in jejunal BBMVs from sham and 2 µg/kg LPS-treated animals. Uptakes, in pmol/mg of membrane protein ± SE, represent the average value of 3 determinations per point for 5 animals. *P < 0.05 compares results obtained in LPS with sham.

 
We then tested the origin of the LPS sensitive part of 5 mM D-fructose transport by measuring Jm-s and Js-m. Jm-s decreased in LPS-treated animals by 30% whereas Js-m were unchanged (Fig. 4B), indicating that LPS most probably affected BBM transport activity.

Intestinal fructose transport can occur via GLUT2 and GLUT5 facilitative transporters and depends on the sugar concentration gradient across the membrane. Changes in fructose uptake might therefore result from changes in transporter abundance in the plasma membrane and/or from metabolic alterations by increasing the slope of sugar gradients. We performed fructose uptakes in purified BBMV, i.e., in the absence of metabolic intervention. Fructose uptake was decreased by 30% in purified BBMVs prepared from LPS (Fig. 4C) compared with sham animals. Thus fructose transporters are likely the level of regulation that is altered in LPS-treated animals.

To decipher the respective contribution of GLUT2 and GLUT5 to fructose transport, BBMVs were preincubated for 10 min and incubated with 0.1 mM cytochalasin B, a competitive inhibitor of GLUT2 but not GLUT5 (10) (Fig. 4D). Fructose uptake in sham nor LPS BBMVs were changed by cytochalasin B, indicating the absence of GLUT2 contribution. All together these results indicate that LPS-sensitive fructose transport is by GLUT5 at the level of the BBM excluding any effect of metabolism.

Downstream Effectors of LPS Effect on Intestinal D-Fructose Transport

Kinases. All MAPK cascades cooperate in the orchestration of inflammatory responses, and extensive cross-talk to other inflammatory pathways, such as NF-{kappa}B and Janus kinases/STAT signaling, have been described (34). TNF-{alpha} is one of the best characterized agonists of the p38 and JNK pathways and is itself regulated by p38 and JNKs (28). To establish whether kinase pathways might be involved in LPS effects on intestinal D-fructose transport, specific kinase inhibitors were used. Intravenous injection of the PKC inhibitor GF-109203X (Fig. 5A) for 15 min before LPS treatment (53) also protected D-fructose uptake (88% at the 500 ng/kg body wt dose). Furthermore, the intravenous injection of 0.155 mg/kg body wt of IP20, a PKA inhibitor (13), protected fructose uptake from LPS adverse effects (Fig. 5B). Thus PKA and PKC pathways are involved in the inhibition of fructose uptake mediated by LPS.


Figure 5
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  		Fig. 5. Contribution of protein kinase pathways on LPS inhibition of fructose uptake. Intestinal rings were taken from sham and 2 µg/kg LPS-treated animals and transport assays were measured for 3 min as described in Fig. 4. A: inhibition by GF-109203X (GF) of PKC activity restores fructose uptake in LPS-treated animals. Increasing concentrations of GF, (25, 50, and 500 ng/kg body wt) were injected intravenously 15 min before LPS injection. B: inhibition of PKA activity by injection of 0.155 mg/kg body wt of protein kinase inhibitor (IP20) 15 min prior to LPS injection protected fructose uptake activity. Results are expressed as µmol D-fructose/ml tissue water. Results were obtained from 5 animals at each condition with 9 determinations per animal. *P < 0.05 LPS vs. sham animals.

 
Further investigation of LPS effects were performed to test MAP kinase pathways. Different doses of SB-203580 (5, 15 and 30 µg/kg body wt) a selective inhibitor of p38 MAPK (27) were injected intravenously, 15 min prior to LPS treatment. At least 79% of LPS inhibition on D-fructose transport was alleviated by SB-203580 (Fig. 6A). Applying a similar protocol, SP-600125 (12.5 and 25 µg/kg body wt) was used to test the contribution of JNK to LPS effect (6). Both concentrations of SP-600125 antagonized LPS inhibition by 58% (Fig. 6B). Finally, two doses (13.5 and 27 µg/kg body wt) of U-0126, an inhibitor of MEK1 and MEK2, were used (5), which both protected fructose uptake from LPS inhibition by 67% (Fig. 6C). None of these inhibitors had effect on D-fructose uptake in sham animals.


Figure 6
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  		Fig. 6. MAP kinase inhibitors protect D-fructose uptake from 2 µg/kg LPS inhibition. The inhibitors were administered iv 15 min before LPS treatment. A: SB-203580 (SB; p38 kinase inhibitor) was used at 5, 15, and 30 µg/kg. B: SP-600125 (SP; JNK inhibitor) was used at 12.5 and 25 µg/kg. C: U-0126 (U; MEK1/2 inhibitor) was used at 13.5 and 27 µg/kg. The uptake of 5 mM D-fructose was measured for 3 min in everted intestinal rings from sham and LPS-treated animals. Results, expressed as µmol D-fructose/ml tissue water, represent 9 determinations from 5 animals per group (sham and LPS treated). *P < 0.05 LPS vs. sham animals.

 
TNF-{alpha}. To demonstrate the possible implication of TNF-{alpha} in LPS inhibition on D-fructose intestinal uptake, we used a synthetic TNF-{alpha} receptor fragment as a TNF-{alpha} antagonist at 20 µg/kg body wt (54). The TNF-{alpha} antagonist completely blocked LPS inhibition (Fig. 7) but did not alter sugar absorption in sham animals. This protective effect indicates that TNF-{alpha} mediates LPS effect


Figure 7
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  		Fig. 7. TNF-{alpha} antagonist (antag. TNF) protects D-fructose uptake by 2 µg/kg LPS for 90 min. The antagonist (20 µg/kg) was injected iv 15 min before LPS treatment. D-Fructose uptakes were measured as described in Fig. 6. Results are expressed as µmol D-fructose/ml tissue water. Results represent 9 determinations made in 5 animals per group (sham and LPS-treated). *P < 0.05 LPS vs. sham animals.

 
Proteasome. To test the role of the proteasome in the decrease of BBM GLUT5 protein levels of LPS-treated rabbit jejunum, we made a dose-response assay of MG-132, a potent cell-permeable proteasome inhibitor (50, 125, and 250 µg/kg body wt) (23). MG-132 was intravenously injected to the animals prior to LPS injection. Proteasome inhibition by MG-132 completely protected intestinal fructose transport from LPS inhibition at all concentrations (Fig. 8), indicating that LPS activated the proteasome degradation pathway, resulting in decreased GLUT5 levels in the BBM of enterocytes.


Figure 8
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  		Fig. 8. Proteasome inhibition by MG-132 (MG) prevents D-fructose uptake inhibition by 2 µg/kg LPS for 90 min. MG-132 was injected iv 15 min before LPS treatment at doses of 50, 125, and 250 µg/kg. Transport results, expressed as µmol D-fructose/ml tissue water, represent 9 determinations made in 5 animals per group (sham and LPS treated). *P < 0.05 LPS vs. sham animals.

 
LPS Decreases GLUT5 Levels in the BBM

A Western blot analysis was performed to measure GLUT5 levels in BBMV from sham and LPS (2 µg/kg)-treated animals. The GLUT5 antibody recognized a single 49-kDa band in sham and LPS BBMs (Fig. 9). The densitometric analysis for GLUT5 normalized to actin levels was performed in five separate experiments, indicating an average 35% reduction of GLUT5 protein in LPS membranes. Thus LPS treatment inhibited fructose transport by reducing BBM GLUT5 protein (Fig. 9).


Figure 9
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  		Fig. 9. Effect of 2 µg/kg LPS and several inhibitors on GLUT5 protein expression in the brush border membrane (BBM) jejunum. Representative Western blot analysis of BBM GLUT5 prepared from sham and LPS (± inhibitors)-treated animals after 90 min. The immunoreactive protein weighs ~49 kDa. The results represent data obtained by densitometric analysis of immunoblotted signals for proteins normalized to those of β-actin on the same gels. Representative blots and data expressed as percent of control values (means ± SE) are given. The preparations of intestinal vesicles per animal of each group (n = 5) were prepared and analyzed in duplicate. S, sham; IP, IP20. Statistical analyses were carried out by 1-way ANOVA and Tukey-Kramer multiple comparisons test as post hoc test. *P < 0.001 control vs. LPS; #P < 0.05 vs. LPS.

 
We further examined the effect of specific inhibitors of the kinase, TNF-{alpha}, and proteasome pathways that were efficient to restore fructose transport. Interestingly, all these inhibitors could restore apical GLUT5 levels to control levels, indicating that effects were due to protein abundance in the apical membrane (Fig. 9).

To our surprise, GLUT5 mRNA abundance from LPS and sham intestines was significantly increased within 90 min of LPS treatment, a time when GLUT5 protein expression in the BBM and fructose transport were reduced (Fig. 10). Therefore BBM GLUT5 protein levels were regulated at a posttranscriptional level.


Figure 10
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  		Fig. 10. Effect of 2 µg/kg LPS on GLUT5 mRNA expression in the brush border membrane jejunum. Representative Northern blot analysis of GLUT5 mRNA in the jejunum of sham and LPS-treated animals after 90 min. GLUT5 mRNA expression was normalized to housekeeping β-actin. Relative mRNA abundances were normalized to the β-actin signal and expressed as LPS to (100%) control values. Results average 5 separate experiments of each animal group. *P < 0.05 compares LPS to sham animals.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study, we showed that sepsis achieved by intravenous administration of LPS inhibits D-fructose transport across the BBM of enterocytes and affects GLUT5 protein levels in the BBM. A proteasome-dependent degradation of the GLUT5 transporter could be involved. Several transduction pathways are activated during the inflammatory processes, and they affect the function of the small intestine. The PKC, PKA, and MAPK transduction pathways contributed to the regulation of fructose transport in the sepsis state induced by LPS. We also showed that TNF-{alpha} was implicated in mediating the effect.

LPS-injected rabbits displayed sepsis as indicated by hematological parameters with a significant and abrupt decrease in total white blood cells and in neutrophils, lymphocytes, and monocytes. Consistent with earlier studies (7), serum glucose increased 90 min after LPS administration, possibly in relation to the early phases of endotoxic shock although a small increase was also reported in the sham group probably because of handling stress. Similarly, increased serum cortisol levels in sham and LPS-treated groups could be explained by both LPS-induced endotoxemia and stress effects consistent with increases in plasma cortisol and ACTH blood levels during endotoxic condition measured in piglets (30).

Fever is a systemic inflammatory response, and several endogenous pyrogens have been identified, including proinflammatory cytokines like TNF-{alpha} (15). The injection of LPS to rabbits increased their body temperature significantly after 30 min (+1.4 ± 0.09°C). In rats, a monophasic febrile response was found with low doses of LPS (1.25 µg/kg), whereas higher doses (12.5 and 180 µg/kg) induced an initial hypothermic response to LPS (24). We did not observe the initial hypothermia at higher doses of LPS, suggesting that fever responses depend on animal species.

Our results exclude any impact of LPS through the induction of apoptosis. Morphologically, cells undergoing apoptosis show alterations of cell volume, cytoplasmic organelles, and microvilli and exhibit a regular pattern of DNA fragmentation on gel electrophoresis. LPS treatment in this study had none of the effects mentioned above on the structure of intestinal mucosa compared with sham rabbits.

Functional alterations of the gut were induced by LPS injection in rabbits. D-Fructose transport was significantly inhibited in the small intestine after LPS injection of low (2 µg/kg body wt) and high (20 µg/kg body wt) doses of LPS. The endotoxin inhibited Jm-s and sugar uptake across purified BBM vesicles indicating that LPS affected the capacity of transport at the luminal, food-facing, side of the cell. The fructose and glucose transporter GLUT2 is involved in the exit of sugars at the basolateral membrane of the cells, and GLUT2 was reported to traffic into the BBM of enterocytes in response to several physiological signals, including sugar consumption, fasting, and feeding, and to AMPK as well as PKCβ2 and P38 MAPK activation (32). In the BBM, the presence of GLUT2 can increase fructose and glucose transport (20). LPS treatment does not seem to alter basolateral membrane transport in the jejunum. Indeed the Js-m were unchanged by LPS treatment. Furthermore, in the present experiments, the BBM-associated transport is insensitive to cytochalasin B, an inhibitor of GLUT2 but not GLUT5. Explanation for the lack of GLUT2 related uptake can also come from the composition of fodder that delivers very low amounts of simple sugars in the lumen of the intestine and limits to a minimum level the priming of the enterocytes for GLUT2 insertion. Thus, in the absence of any GLUT2 in the apical membrane, the results presented in this paper show that the different inhibitors induce changes in GLUT5 protein abundance and transport activity.

Before sepsis was produced, a pretreatment of rabbits with MG-132, a specific inhibitor of the proteasome pathway, seems to abolish endotoxin effects on sugar transport. From our results, GLUT5 are controlled by the proteasome that might degrade the transport protein itself or, by indirect mechanisms, i.e., through NF-{kappa}B activation, would alter GLUT5 trafficking or activity. Interestingly, at the same time GLUT5 mRNA expression increases in LPS-treated intestine, suggesting a mechanism for the rescue of function.

We found TNF-{alpha}-dependent inhibition of intestinal absorption of sugars (3, 18). TNF-{alpha} is an endogenous substance that can be synthesized and released under a variety of stimuli, such as LPS, and has a central role in many pathophysiological processes. Injection of TNF-{alpha} antagonist prior to LPS administration revealed that LPS action on intestinal D-fructose absorption apparently involves TNF-{alpha} signaling. Therefore, systemic LPS could affect D-fructose intestinal absorption by TNF-{alpha}.

The regulation of cell function by ligands on membrane receptors (such as LPS) generate intracellular signals and second messenger signaling. Activation of PKC altered electrolyte transport in vivo in rat distal colon (25), fructose transport across the jejunum brush border in rat (26), and L-arginine, L-alanine, and sodium-glucose cotransport in cell lines (40, 41, 57). PKC inhibition by GF-109203X protected intestinal D-fructose transport from LPS-induced inhibition, suggesting that part of LPS's effect is under PKC control. These results are in accordance with other reports showing PKC-mediated endotoxin effects (14, 31).

PKA inhibition by IP20 partly reversed LPS inhibition of fructose transport suggesting a possible role for PKA. Cyclic AMP is a known second messenger involved in fluid and electrolyte secretion in the small intestine (16). Moreover, cAMP and the PKA pathway were shown to alter the GLUT5 mRNA abundance and translation in the human enterocytic cell line Caco-2/TC7 cells (21, 37). In addition, Wright et al. (59) concluded that PKA and PKC regulate rabbit SGLT1 activity by modulating the number of cotransporters in the plasma membrane and that this occurs through regulation of exocytosis and endocytosis. Thus PKA-mediated LPS effects might have antagonist effects by increasing the biosynthesis of GLUT5 and inhibiting its insertion in the BBM in the acute phase of sepsis. Indeed, the decrease of GLUT5 protein in the BBM is not paralleled by a similar decrease of GLUT5 mRNA levels, which were higher in LPS-treated compared with sham-treated animals. Increased GLUT5 mRNA abundance can result from increased stability and or transcription of the gene (21) consistent with the tenfold variation of effect of the half-lives of many mRNAs in response to cytokines, calcium, hormones, starvation, hypoxia, or viral infection (45).

The inhibition of sugar uptake after LPS treatment is significantly reduced by the MAPKs inhibitors (SB-203580, SP-600125, U-0126) indicating that this pathway could be involved in the action of the endotoxin on fructose absorption. Helliwell et al. (27) concluded that there is extensive cross-talk between the ERK, p38, and PI3-kinase pathways in the control of brush-border fructose transport by modulation of both the levels and intrinsic activities of GLUT5 and GLUT2. In mammals, MAPK signaling cascades regulate gene expression through a posttranscriptional mechanism involving cytoplasmic targets (12). Activation of p38 MAPK, p42/p44 MAPK, and JNK has been described in response to a variety of inflammatory agents, indicating that they control many cellular responses to inflammation. As a consequence, inhibitors of these kinases have been proposed as anti-inflammatory therapy (56). Therefore, the results show that, in sepsis state induced by LPS, kinases (PKC, PKA, and p38 MAPK) could be involved in the regulation of GLUT5 protein levels.

The absorptive function of the small intestine, including sugar and amino acid transport, is sensitive to LPS toxicity. This study provides a better understanding of the underlying mechanisms of endotoxin effects on the absorptive function of the small intestine. These advances might help construct therapeutic strategies to protect and improve nutrient absorption during sepsis.


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 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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This work was supported by grants from Ministerio de Ciencia y Tecnología AGL 2003-04497/GAN (PGE+FEDER) and by Departamento de Ciencia, Tecnología y Universidad del Gobierno de Aragón (Spain) A-32. The group is member of the Network for Cooperative Research on Membrane Transport Proteins (REIT), cofunded by the "Ministerio de Educación y Ciencia," Spain and the European Regional Development Fund (ERDF) (Grant BFU2005-24983-E/BFI).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. Rodriguez-Yoldi, Physiology, Veterinary Faculty, Zaragoza Univ., Miguel Servet 177, 50013 Zaragoza, Spain (e-mail: mjrodyol{at}unizar.es)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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