Vol. 283, Issue 6, G1336-G1342, December 2002
Hypoxia differentially regulates nutrient transport in rat
jejunum regardless of luminal nutrient present
K. A.
Kles1 and
K. A.
Tappenden1,2
1 Division of Nutritional Sciences and 2 Department
of Food Science and Human Nutrition, University of Illinois at
Urbana-Champaign, Urbana, Illinois 61801
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ABSTRACT |
Aggressive enteral nutrition and
poor intestinal perfusion are hypothesized to play an important
pathogenic role in nonocclusive small bowel necrosis. This study tests
the hypothesis that glucose and glutamine transport are differentially
regulated during hypoxia regardless of the luminal nutrient present.
Sprague-Dawley rats (247 ± 3 g; n = 16) were
randomized to receive 1 h of intestinal hypoxia or serve as
normoxic controls. During this hour, jejunal loops were randomized to
receive in situ perfusions of mannitol, glucose, or glutamine. When
compared with normoxic groups, glucose but not glutamine transport was
impaired (P < 0.001) during hypoxia. Messenger RNA
abundance of the sodium glucose cotransporter sodium-dependent glucose
cotransporter-1 (SGLT-1) and neutral basic amino acid transporter
Bo did not differ with hypoxia or nutrient perfused.
Jejunal brush-border SGLT-1 abundance was decreased (P = 0.039) with hypoxia; however, total cellular SGLT-1 protein abundance
did not differ among treatment groups. These data indicate that SGLT-1
activity is regulated during hypoxia at the posttranslational level.
Additional information regarding the mechanisms regulating nutrient
transport in the hypoperfused intestine is critical for optimizing the
composition of enteral nutrient formulas.
sodium-dependent glucose cotransporter-1; nutrient absorption; small intestine
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INTRODUCTION |
THE INCIDENCE OF
NONOCCLUSIVE small bowel necrosis is significant in traumatically
injured patients, despite adequate systemic resuscitation. Currently,
the causative mechanism remains unknown; however, enteral nutrition has
been provided in ~90% of cases (21), suggesting that
the inappropriate administration of specific nutrients into a poorly
perfused small bowel may play a pathogenic role (12, 21, 23,
25). During states of low blood flow to the intestine, enteral
nutrients may increase oxygen demand beyond that available, potentially
increasing intestinal hypoxia and impairing intestinal function.
Gastrointestinal function has been shown to be impaired during
hypoperfusion or decreased oxygenation of the intestine
(28). Previous in vivo rat studies from our lab indicate
that, during hypoperfusion, impaired barrier function, increased
lactate concentration, decreased ATP concentration, and altered
nutrient transport are characteristic (15). The detrimental effects of hypoperfusion may be minimized with an enteral
formula composed of nutrients easily processed by the hypoperfused intestine.
The purpose of the current study was to determine how nutrient
perfusion alters sodium-dependent glucose and glutamine transport activity and their regulation at the cellular level during
hypoperfusion. Glutamine transport occurs via system Bo,
which is part of a family of transporters for neutral amino acids
(14). Glucose transport occurs via the sodium-glucose cotransporter [sodium-dependent glucose cotransporter-1
(SGLT-1)]. We previously reported that, during hypoxia,
brush-border glucose transport is impaired, but glutamine
transport remains unaltered following luminal perfusion of hexoses
(15). It is possible that glutamine transport was
maintained in this previous study, because the jejunum had not been
exposed to luminal glutamine. Therefore, the current study was designed
to examine the effect of luminal glucose or glutamine on jejunal
nutrient transport during hypoxia. We hypothesize that glucose and
glutamine transport are differentially regulated at the cellular level
during hypoxia regardless of the luminal nutrient present.
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METHODS |
Animals.
Sixteen male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis,
IN) weighing 247 ± 3 g were acclimatized and housed in a
temperature- and humidity-controlled facility with a 12:12-h light-dark
cycle in individual cages. Animals were maintained on a standard rodent
diet and were given free access to water. The University of Illinois at
Urbana-Champaign Laboratory Animal Care Advisory Committee approved all
procedures described in accordance with the Guide for the Care and Use
of Laboratory Animals.
Experimental model.
After a 24-h food restriction, surgical plane was achieved using
ketamine (87 mg/kg im) and xylazine (13 mg/kg im) anesthesia, and a
laparotomy was performed. Procedures were performed at the same time
each day to account for diurnal variations of SGLT-1 activity
(24). Two centimeters distal to the ligament of Treitz, three 8-cm in situ jejunal loops were cannulated with flexible Silastic
tubing (3.2 mm OD × 2.0 mm ID; Fisher Scientific, Itasca, IL) and
secured with 4-0 silk suture (Ethicon, Somerville, NJ). After the
preparation of jejunal loops, rats were randomized to one of two groups
for 1 h: 1) hypoxia (created by clamping the superior
mesenteric artery with a microbulldog clip (Roboz Surgical, Rockville,
MD) or 2) normoxia control (with unrestricted small bowel
perfusion). Sterile instruments and aseptic technique were used at all times.
Within each animal, in situ jejunal loops were randomized to receive
luminal perfusions with one of three nutrients: 1) mannitol (an osmotic control), 2) glucose (absorbed by active
transport via SGLT-1), or 3) glutamine (actively transported
via Bo). All of the nutrients were perfused for 60 min at a
concentration of 120 mM in a modified Krebs solution [(in mM) 141 Na,
117.6 Cl, 26 HCO3, 1.2 Mg, 1.2 Ca, 5.2 K, 2.4 HPO4, and 0.4 H2PO4, pH 7.4]
maintained at 38°C throughout the experiment. Fresh nutrient infusion
solutions were continuously perfused during the 60 min. Body
temperature was maintained at 38°C with a heating element. At the end
of the experimental protocol, anesthetized animals were euthanized by
cardiac puncture.
Tissue preparation.
After 60 min of luminal nutrient perfusions, the perfused segments were
rapidly excised and the mesentery was removed. Two 2-cm sections were
removed for electrophysiological analysis in modified Ussing chambers.
A 1-cm section was snap-frozen and stored at 
80°C for RNA isolation
and determination of relative mRNA abundance of SGLT-1 and
Bo. A 1-cm section was snap frozen and stored at 20°C
for determination of ATP, lactate, pyruvate, and protein concentration.
A 1.5-cm section was frozen for quantification of total cellular and
brush-border SGLT-1 protein abundance.
Ion and nutrient transport.
Techniques assessing gastrointestinal function through measurement of
ion flux in modified Ussing chambers have been previously described
(3, 7, 14, 19). Jejunal sections were cut longitudinally
along the mesentery and mounted in modified Ussing chambers
(Physiologic Instruments, San Diego, CA) to expose 0.5 cm2
of the brush border and serosal sides. The tissue was bathed in 8 ml of
oxygenated (95% O2-5% CO2) modified Krebs
buffer solution maintained at 37°C with a circulating water bath
(Fisher Scientific). Basal transmural short-circuit current,
resistance, and potential difference were measured using established
techniques after a 20- to 30-min equilibration period. Sodium-dependent
nutrient transport was determined by measuring the changes in
short-circuit current induced by addition of either 10 mM glucose or
glutamine to the medium in the mucosal reservoir. The modified Ussing
chambers were connected to dual-channel voltage/current clamps (VCC
MC2, Physiologic Instruments) with a computer interface allowing for real-time data acquisition and analysis (Acquire & Analyze software, Physiologic Instruments).
Jejunal ATP/ADP ratio.
To ensure that jejunal mucosa exhibited metabolic indicators of
oxygen deprivation, jejunal ATP/ADP ratio was measured. Frozen tissue
sections were homogenized (model PCU-11, Brinkman Instruments, Westbury, NY) and sonicated for 30 s at setting 3 (model 450, Branson Sonifier, Danbury, CT) in a modified Krebs buffer for determination of jejunal ATP levels using the luciferin/luciferase method (30). One hundred microliters (20 mg/ml) of
luciferin/luciferase (Sigma Chemical, St. Louis, MO) were added to 100 µl of sample and counted for 30 s on a scintillation counter
(Beckman LS 6500, Fullerton, CA) using chemiluminescence parameters.
Sample ATP concentrations were then calculated based on an ATP standard
curve (Sigma Chemical). Additionally, ADP concentration was measured on
a fluorometer (F-2000; Hitachi Instruments, Chicago, IL) by reduction
of NADH and read at excitation of 340 nm and emission of 460 nm.
Samples and standards were placed in a buffer of NADH (20 µM) and
phosphoenolpyruvate (40 µM), and ADP reduction occurred following
addition of pyruvate kinase (0.30 U/ml) (20). The sample
ADP concentration was calculated based on an ADP standard curve.
Jejunal lactate concentration.
As a marker of anaerobic metabolism, jejunal lactate concentration was
measured in the prepared tissue homogenate using the Sigma Diagnostics
Lactate kit (procedure no. 735) at 540 nm using an Elx800
plate reader (Bio-Tek, Winooski, VT). Sample lactate concentrations
were determined using a lactate standard curve (Sigma Chemical). To
normalize for differences in glycolytic substrate available between
treatment groups, data were expressed as (micromoles lactate/micromoles
pyruvate)/milligram protein. From the prepared homogenate, jejunal
pyruvate concentration was quantified on a fluorometer (F-2000) based
on a pyruvate standard curve. Ten microliters of each sample were
placed in 1 ml NADH buffer (0.95 µM NADH; 0.5 M
NaH2PO4; 0.5 M K2HPO4)
read at excitation of 340 nm and emission of 460 nm. The sample was
then reduced following addition of 5 U of lactate dehydrogenase (LDH)
and read at excitation of 340 nm and emission of 460 nm. The
differences in sample emission readings (initial reading minus reading
following LDH addition) were determined and compared with a pyruvate
standard curve (Sigma Chemical) (20, 30).
Relative RT-PCR mRNA abundance of nutrient transporters.
Total cellular RNA was isolated from the snap-frozen jejunal samples
using the guanidium isothiocyanate phenol-chloroform method of
Chomczynski (8). Total RNA was quantified by using a
spectrophotometer (U-2000, Hitachi) at OD260. RT-PCR was
performed in a Gene Mate Genius thermocycler (ISC Biosexpress,
Kaysville, UT) in a 20-µl total volume containing 3 µg RNA, 2.5 µM random decamers, 0.5 mM deoxynucleotide triphosphate
(dNTP) mix of the four dNTPs, 1× first strand buffer, 10 mM DTT, and
200 U Superscript II RT (GIBCO-BRL, Rockville, MD; Ambion, Austin,
Texas). Samples were then stored at 20°C.
The oligonucleotide primers used for the detection of cDNA
specific to rat SGLT-1 and Bo mRNA were synthesized by
GIBCO-BRL. Primer sequences for Bo determined using
Primer3
(http://www-genome.wi.mit.edu/cgi-bin/primer/ primer3_www.cgi) and
GeneBlast (http://www.ncbi.nlm.nih. gov/BLAST/) were: (forward)
5'-ATG GCT CTG GGA GAC AGA GA-3' and (reverse) 5'-GGA GAA ATG GAC TGG
GTG TG-3'. SGLT-1 mRNA primers were based on a report by Scholtka
et al. (26). PCR mixtures for amplification of cDNA were
performed in a 50-µl total volume containing 0.2 mM dNTPs, 1×
PCR buffer without MgCl2, 1.5 mM MgCl2,
template cDNA, forward and reverse primers, 2.5 U Taq
polymerase (GIBCO-BRL), and primers and competimers for 18S (4:6 ratio;
Ambion). The SGLT-1 reaction mixture underwent 25 cycles of denaturing
at 94°C for 45 s, annealing at 48°C for 30 s, and
extension at 72°C for 50 s, followed by 72°C for 10 min. The
Bo reaction mixture underwent 33 cycles of denaturing at
94°C for 45 s, annealing at 55°C for 30 s, and extension
at 72°C for 50 s, followed by 72°C for 10 min. Tris-borate
ethylenediaminetetraacetic acid (TBE) agarose gels (1.2%) were stained
with ethidium bromide and photographed using the FOTO/Analyst
image-analysis system (Fotodyne, Hartland, WI). Densitometry of the 18S
ribosomal product and the nutrient transporter of interest was
performed using Collage image-analysis software 4.0 (Hartland, WI).
Preliminary trials determined the appropriate level of 18S
competimers/primer ratio and temperature cycles necessary to ensure
both the gene of interest and 18S bands produced were well within the
linear range of ethidium bromide detection.
SGLT-1 protein abundance.
SGLT-1 protein abundance was determined in tissue homogenate in
modified Krebs solution and brush border isolated by the procedure described by Tappenden and McBurney (29) and confirmed by
ensuring sucrase-isomaltase enrichment with the brush-border fraction. Total and brush-border protein concentrations were determined using the
Bio-Rad Protein Assay (Bio-Rad, Hercules, CA) with a BSA standard
(1). Next, the protein was denatured by boiling for 4 min,
and proteins were separated by size using 12.5% SDS-PAGE and were
transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad)
using a semidry transfer apparatus (Bio-Rad). Before analyzing
experimental samples, a linear range from 0 to 30 µg was established
as the appropriate amount of protein per well to ensure that the bands
produced fell within the linear range of the colorimetric detection
system. Western blot analysis for SGLT-1 protein was performed using
polyclonal antibody with known rat reactivity (Chemicon, Temecula, CA).
In addition, actin monoclonal antibody with known rat reactivity
(Chemicon) was added to the membrane to allow for SGLT-1 normalization
to a constitutively expressed protein. PVDF membranes were developed
using the Opti-4CN kit (Bio-Rad). According to the method established
by Tappenden and McBurney (29), PVDF membranes were
blocked using 5% nonfat dry milk in Tris-buffered saline (20 mM Tris,
137 mM NaCl, pH 7.6, 0.1% Tween) for 3 h at room temperature on a
metabolic shaker. The membrane was washed for 5 min in
phosphate-buffered saline with Tween 20 (PBST; 4.3 mM
Na2PO4, 1.4 mM KH2PO4,
2.7 mM KCl, 137 mM NaCl, 0.1% Tween 20, pH 7.3) at room temperature.
The SGLT-1 primary and actin primary antibodies were diluted 1:5,000
and 1:2,000, respectively, in PBST and 0.01% BSA. The primary
antibodies were coincubated for 4.5 h at room temperature and
washed at room temperature (3 × 10 min). Secondary antibodies
with horseradish peroxidase conjugated to goat anti-rabbit IgG (for the
SGLT-1 primary antibody) and goat anti-mouse IgG (for the actin primary antibody) were added at room temperature for 30 min (1:10,000 dilution
in PBST and 0.01% BSA; Bio-Rad) and washed with PBST (3 × 10 min). Colorimetric detection followed addition of Opti-4CN diluent
solution. Photographs of gels were taken using the FOTO/Analyst image-analysis system. Densitometry of SGLT-1 protein abundance was
performed using Collage image-analysis software 4.0.
Statistical analysis.
The effects of hypoxia and nutrient perfusions on the outcome
parameters were determined using a two-way ANOVA. The sources of
variation were hypoxia (h = 2), nutrient perfused (p = 3), and hypoxia interacted with nutrient perfused. For analysis of relative
transporter mRNA and protein abundance, the effect of gel was
calculated using a randomized block ANOVA and was significant due to
expected experimental variation. Therefore, a completely randomized
block ANOVA was performed for these analyses (block = gel; main
effects = hypoxia and nutrient perfused; interaction = hypoxia × nutrient perfused). When a significant effect existed, comparisons were completed using Tukey's post hoc analysis.
Computations were performed using SAS (Version 8.1, SAS Institute,
Cary, NC). Statistical significance was defined as P 
0.05.
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RESULTS |
Indicators of jejunal hypoxia.
A pilot trial established the presence of hypoxia by determining a
reduction of the partial pressure of oxygen in the mesenteric venous
effluent using the protocol described herein [normoxia (n = 3; PO2 = 75.6 ± 1.5 mmHg; %O2 saturation = 96.0 ± 1.7); hypoxia rats (n = 3; PO2 = 44.0 ± 10.9 mmHg; %O2 saturation = 79.6 ± 7.6)]. In addition, indirect physiological responses to hypoxia were evaluated (basal transmural resistance, lactate concentration, and
ATP/ADP). Consistent with previous reports (19), basal
transmural resistance was significantly lower (P = 0.005) in the hypoxia groups, indicating increased permeability (Fig.
1). Additionally, glucose perfusion
resulted in significantly higher resistance than glutamine perfusion
(P = 0.002), regardless of hypoxia. However, jejunal
lactate concentration was higher (P < 0.001) during
hypoxia with glucose perfusion (Fig. 2).
This outcome was normalized to jejunal pyruvate concentration to
account for flux through the glycolytic pathway and therefore indicated
that luminal glucose alone increases the level of anaerobic metabolism.
The ATP/ADP ratio was measured as a metabolic indicator of energy
stores. ATP/ADP ratio was significantly lower (P < 0.001) in the hypoxia groups compared with the normoxia control groups
(Fig. 3). Furthermore, glucose perfusion
significantly increased ATP/ADP ratio compared with mannitol
(P = 0.01) and glutamine (P = 0.055)
both in normoxia and hypoxia groups.
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Fig. 1.
Effect of jejunal nutrient perfusion during hypoxia on
basal transmural resistance in rats. As expected, during oxygen
deprivation, transmural resistance was significantly lower
(P = 0.005) compared with control normoxia.
Additionally, glucose perfusion resulted in significantly higher
resistance than glutamine or mannitol perfusion (P = 0.002), irrespective of oxygenation status. Data are reported as pooled
means ± SE. Means with different letters are significantly
different from each other.
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Fig. 2.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal lactate concentration following hypoxia in rats.
Jejunal lactate concentration was significantly higher
(P < 0.001) in the hypoxia group perfused with glucose
than all other groups. Data are reported as means ± SE. Means
with different letters are significantly different from each other.
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Fig. 3.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal ATP/ADP ratio following hypoxia in rats. ATP
concentration was significantly lower (P < 0.001) in
the hypoxia groups compared with the normoxia control groups,
irrespective of nutrient perfused. In addition, glucose perfusion
significantly increased (P = 0.006) ATP/ADP ratio
compared with mannitol and glutamine. Data are reported as means ± SE. Means with different letters are significantly different from
each other.
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Jejunal sodium-dependent nutrient transport.
The effect of hypoxia on sodium-dependent glucose and glutamine
transport activities was assessed. Compared with normoxia controls,
glucose transport was significantly impaired (P < 0.001) in the hypoxia groups (Fig. 4).
However, glucose transport activity did not vary among nutrient
perfused (Fig. 4). In contrast, glutamine transport activity was not
affected by hypoxia (Fig. 5) and was significantly higher following luminal perfusion of glutamine compared
with glucose or mannitol (P = 0.02), regardless of
oxygenation state (Fig. 5).
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Fig. 4.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal sodium-dependent glucose transport (SGLT1)
following hypoxia in rats. SGLT1 was significantly lower
(P < 0.001) in the hypoxia groups than the normoxia
controls, irrespective of nutrient perfused. Data are reported as
means ± SE. Means with different letters are significantly
different from each other.  , change; Isc,
short-circuit current.
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Fig. 5.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal SGLT following hypoxia in rats. SGLT was not
altered by hypoxia compared with control normoxia levels. SGLT was
increased during glutamine perfusion regardless of oxygenation compared
with glucose perfused (P = 0.02). Data are reported as
means ± SE. Means with different letters are significantly
different from each other as pooled means (by hypoxia).
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Jejunal sodium-dependent nutrient transporter mRNA abundance.
Relative RT-PCR was performed for SGLT-1 and Bo to
discern whether observed functional alterations were regulated at the
level of mRNA abundance. The relative mRNA abundance of Bo
did not differ following hypoxia (Fig.
6), consistent with the functional data
(Fig. 5). However, the changes in Bo function following
glutamine perfusion were not observed at the mRNA level, indicating
that the level of nutrient regulation for this transporter may be
posttranscriptional. SGLT-1 mRNA abundance did not differ following
hypoxia or nutrient perfused (Fig. 7). However, SGLT-1 activity was functionally impaired following hypoxia. These data necessitated further investigation into the regulation of
SGLT-1 at the level of protein abundance.
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Fig. 6.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal relative Bo mRNA abundance following
hypoxia in rats. Relative Bo mRNA abundance did not differ
with hypoxia or nutrient perfused. Data are reported as means ± SE.
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Fig. 7.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal SGLT-1 relative mRNA abundance following hypoxia
in rats. Relative SGLT-1 mRNA abundance did not differ with hypoxia or
nutrient perfused. Data reported as means ± SE.
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Jejunal sodium-dependent glucose cotransporter protein abundance.
To discern the location of the SGLT-1 protein during hypoxia, SGLT-1
protein abundance was determined in both brush-border membrane and
total cellular fractions. Brush-border SGLT-1 protein abundance was
significantly lower during hypoxia (P = 0.039) compared with normoxia controls, regardless of nutrient perfused (Fig. 8). Consistent with the functional
observations, these data indicate that there is indeed less SGLT-1
protein with its functional location at the brush-border membrane
following hypoxia. Total cellular SGLT-1 protein abundance was not
altered by either hypoxia or nutrient perfusion (Fig.
9), indicating possible translocation of
the SGLT-1 protein into intracellular pools during hypoxia rather than
a targeted proteolytic degradation of this protein.
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Fig. 8.
Effect of luminal perfusion of mannitol, glucose, or
glutamine on jejunal SGLT-1 brush-border protein abundance following
hypoxia in rats. Brush-border SGLT-1 protein abundance was
significantly lower during hypoxia (P = 0.039),
irrespective of nutrient perfused. Data are reported as pooled means
(by nutrient perfused) ± SE. Means with different letters are
significantly different from each other.
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Fig. 9.
Effect of luminal infusion of mannitol, glucose, or
glutamine on jejunal SGLT-1 total protein abundance following hypoxia
in rats. SGLT-1 protein abundance did not differ with hypoxia or
nutrient perfusion. Data reported as means ± SE.
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DISCUSSION |
Multiple studies have demonstrated the importance of minimal
enteral nutrition to improve outcomes in traumatically injured adults
(16, 19, 22). However, information regarding the appropriate nutrient composition for these patients is needed, because
relatively few data exist regarding the functional and cellular
responses to the provision of specific luminal nutrients into the
hypoperfused small intestine. The results from this study support the
hypothesis that, during hypoxia, glucose and glutamine transport are
differentially regulated regardless of the luminal nutrient present.
Kles et al. (15) previously reported that, although
glucose transport is impaired, glutamine transport is maintained during
hypoxia regardless of luminal carbohydrate present. However, it
is possible that glutamine transport was maintained in this previous
trial, because the jejunum had not been exposed to luminal glutamine.
Therefore, the current study was designed with luminal perfusions of
both glucose and glutamine to determine whether or not, during hypoxia,
the provision of a specific nutrient inhibits the brush-border
transport capacity of that particular nutrient. The results of the
current study indicate that, during hypoxia, SGLT-1 activity is
impaired but there is no effect on glutamine transport. This
differential effect of hypoxia on nutrient transport activity was not
impacted by the luminal nutrient present.
The hypoxia model used in the current study was associated with changes
in three physiological indexes: transmural resistance, lactate, and
ATP/ADP ratio. Regardless of nutrient provision, hypoperfusion reduced
transmural resistance. Decreased resistance in this hypoxia model
mimics that of a traumatic injury with increased septic morbidity,
which may play a pathogenic role in the development of nonocclusive
small bowel necrosis. (16) In addition, lactate concentration was increased in the current study due to anaerobic metabolism of glucose, which further increased lactate concentration beyond mannitol and glutamine levels during hypoxia. These results support the hypothesis that carbohydrates, such as glucose, could exacerbate intestinal injury during hypoperfusion. A decrease in ATP
may represent decreased synthesis or increased utilization of ATP
during hypoxia. Glucose perfusion significantly increased ATP/ADP ratio
compared with mannitol and glutamine, indicating that there is
increased synthesis of ATP/ADP during glucose perfusion regardless of
oxygenation. In conclusion, decreased ATP/ADP ratio, increased lactate
concentration, and decreased resistance occurred in response to the
reduced jejunal oxygenation obtained with this model.
Evidence suggests that, when glucose and glutamine are provided to the
small intestine, glutamine is preferentially oxidized (10). Therefore, we hypothesized that perhaps this
specific alteration in brush-border nutrient transport during hypoxia, a condition of considerable stress, represents strategic sampling of
luminal nutrients based on specific metabolic preferences and/or cellular requirements. In addition to the maintenance of glutamine transport during hypoxia, glutamine perfusion increases glutamine transport regardless of oxygenation status. This observation is consistent with those of other nutrient transporters, wherein the
provision of the nutrient specific to the transporter will increase
that transporter's maximal transport rate (27). These data illustrate a differential regulation of both glucose and glutamine
transporters during hypoxia. Previously, nutrient transport was
investigated in an intriguing study in which rats underwent atmospheric
hypoxia, not superior mesenteric artery occlusion, in which labeled
carbohydrate and amino acid uptake was decreased (18). The
authors suggested that nutrient transport is depressed due to decreased
Na+-K+-ATPase activity. However, Iannoli and et
al. (13) measured both glucose and glutamine uptake in
rabbits following superceliac aortic occlusion and did not report a
statistically significant effect of hypoxia. Another study investigated
the enzymatic analysis of glucose and 3-O-methylglucose
uptake into tissue; the authors demonstrated decreased uptake as
PO2 decreased in everted sacs (2).
Although this previous report used semistarved and fed rats, it
supports the hypothesis that decreasing levels of oxygen lead to lower
glucose transport.
The impairment of glucose but not glutamine transport activity warrants
further investigation to determine the differential regulation of
transporters during hypoxia. The regulation of Bo appears
to be posttranscriptional. However, a commercial antibody is not
available to obtain data related to protein abundance. The regulation
of SGLT-1 activity was measured at three levels: relative mRNA
expression, brush-border protein abundance, and total cellular protein
abundance. In our study, the relative mRNA abundance of SGLT-1
indicated that the SGLT-1 activity is not regulated at the level of
mRNA abundance. Therefore, we measured SGLT-1 protein abundance to
determine whether hypoxia downregulates this protein
posttranslationally. Previous reports indicate that SGLT-1 protein is
posttranslationally regulated (11, 17). One mechanism of
posttranslational modification is that inactive stores of SGLT-1
protein are located within intracellular pools (4-6,
9). The data obtained in this study indicate that the rapid
decline in SGLT-1 activity during hypoxia may be due to trafficking of
functional SGLT-1 protein from the brush-border membrane to
intracellular pools.
Additional information regarding the regulation of nutrient transport
into the hypoperfused intestine is critical so that the composition of
enteral nutrients provided to at-risk patient populations can be
optimized to correspond with gastrointestinal function. Although
relative SGLT-1 mRNA abundance and total cellular SGLT-1 protein
abundance did not differ with hypoxia or nutrient perfusion,
brush-border SGLT-1 protein abundance was impaired during hypoxia.
Further investigation is necessary, because these results indicate that
caution should be used in administering glucose to the hypoperfused
jejunum due to impaired brush-border SGLT-1 activity.
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FOOTNOTES |
Address for reprint requests and other correspondence: K. A. Tappenden, 443 Bevier Hall, 905 South Goodwin Ave., Dept. of Food
Science and Human Nutrition, Univ. of Illinois at Urbana-Champaign, Urbana, IL 61801 (E-mail: tappende{at}uiuc.edu).
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.
July 25, 2002;10.1152/ajpgi.00055.2002
Received 24 May 2002; accepted in final form 12 August 2002.
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