Chronic hypoxia alters glucose utilization during
GSH-dependent detoxication in rat small intestine
Terry S.
Legrand and
Tak Yee
Aw
Department of Molecular and Cellular Physiology, Louisiana State
University Medical Center, Shreveport, Louisiana 71130
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ABSTRACT |
We showed that hypoxia alters glutathione
(GSH)-dependent detoxication and induces mucosal metabolic instability.
To determine the impact of these changes and the role of reductant
supply in intestinal lipid peroxide disposition, pair-fed (16 g/day)
Sprague-Dawley rats were exposed to air (20.9%
O2;
n = 6) or 10%
O2
(n = 6) for 10 days. Jejunal and ileal
everted sacs were exposed to 75 µM peroxidized fish oil with or
without 10 mM glucose or 1 mM GSH. Peroxide transport was determined as
the abluminal recovery of thiobarbituric acid-reactive substances.
Peroxide recovery in hypoxic intestine was twice that in normoxic
intestine. Addition of GSH and glucose did not affect peroxide
recovery, indicating reduced intracellular GSH-dependent metabolism and
enhanced output by the hypoxic intestine. Glucose uptake by normoxic
and hypoxic intestine is similar, whereas its utilization for
detoxication is decreased in hypoxic cells. Determination of NADPH
supply indicates that decreased glucose availability for NADPH
production during hypoxia impairs GSH disulfide reduction, compromises
hydroperoxide metabolism, and increases peroxide output from hypoxic
intestine.
hypermetabolism; oxidative stress; glutathione redox cycle
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INTRODUCTION |
CHRONIC O2 deficiency is
a common clinical condition in disease states such as obstructive lung
disease and cardiovascular insufficiency (10, 31). Chronic hypoxia has
been associated with a decrease in total plasma protein content (16),
as well as an overall reduction in protein synthesis (13). The
intestine and other tissues rely on an adequate
O2 supply for optimal function (2,
17, 19). Previous studies have shown that chronic
O2 deficiency impairs renal
function (18), alters nutrient (23) and drug absorption (4), and
decreases gastric emptying, which may impact pharmacodynamics in drug
therapy regimens (33). In recent studies, Bai and Jones (7) showed that
glutathione (GSH) transport by intestinal cells is inhibited by chronic
hypoxia, and the rate of peroxidized methyl linoleate uptake is
decreased in hypoxic compared with normoxic intestine. We (21, 22) have shown in our laboratory that chronic hypoxia promotes intestinal oxidative stress, induces mucosal metabolic instability, and
compromises GSH-dependent detoxication in the intestine. Because GSH is
an important component of peroxide metabolism in the intestine (3), these findings suggest that disposition of toxic peroxides may be
compromised in the hypoxic intestine.
Lipid peroxides are present in our diets to varying degrees. One source
of lipid peroxides in the diet is foods that are high in oxidizable
polyunsaturated fats (1). Fats comprise up to 40% of the calories in
the American diet (15, 20), an appreciable amount of which is oxidized
(29); thus the gastrointestinal tract must be equipped with an
efficient detoxication system to maintain its function as an interface
between ingested substances and the body. An important peroxide
detoxication pathway in the intestine is the GSH redox system (Fig.
1). In this system GSH peroxidase reduces
peroxides at the expense of GSH, with concomitant production of
glutathione disulfide (GSSG). GSSG is converted to its reduced form by
GSSG reductase, utilizing NADPH as a reductant. A major source of NADPH
in the intestine is the pentose phosphate pathway (35), which is
regulated by glucose flux (3).
We previously found that luminal uptake (absorption) and contraluminal
output (transport into lymph) of lipid peroxides by the small intestine
is dependent on function of the GSH redox cycle (5, 6). Our hypothesis
is conceptually illustrated in Fig. 2. In the
GSH-sufficient state, the intracellular catabolism of hydroperoxides by
the GSH redox cycle drives the absorption of peroxides from the lumen
via a large lumen-to-cell gradient, a form of metabolic trapping. The
result is decreased hydroperoxide retention in the lumen as well as
decreased output into lymph. The GSH-deficient state, in which
intracellular peroxide catabolism is impaired, results in diminished
peroxide absorption from the gut lumen and enhanced peroxide output
into lymph as a result of decreased intracellular peroxide metabolism.
Given that chronic hypoxia compromises GSH-dependent pathways, it is
likely that luminal lipid peroxide disposition and peroxide transport
are impaired in the hypoxic intestine. Elevated plasma peroxides may have implications for systemic pathologies, such as atherogenesis (8,
27). The purpose of the current study is to determine the impact of
prolonged O2 deficiency on
intestinal lipid peroxide metabolism and disposition and to define the
contribution of glucose and reductant supply (GSH and NADPH) to
peroxide detoxication in the hypoxic intestine.
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METHODS |
Induction of chronic hypoxia.
Male Sprague-Dawley rats weighing 300-350 g were exposed to either
normoxia (20.9% O2) or hypoxia
(10% O2) in plastic cages for
10 days as previously described (4). This protocol induces moderately
severe, but not life-threatening, hypoxia. Briefly, the desired
PO2 was
achieved by using air (normoxia) or by combining air and nitrogen in a
Matheson gas mixer. The
PO2 in the
chambers was monitored with a Clark-type
O2 electrode inserted through a
small opening in the cover. Because hypoxic animals exhibit decreased
food intake, normoxic controls were fed equivalent amounts (16 g/day),
in contrast to the 25 g/day typically consumed by ad libitum-fed
normoxic animals. Water was freely available at all
times.

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Fig. 1.
Glutathione (GSH) redox cycle in hydroperoxide metabolism. GSSG,
glutathione disulfide; ROH, lipid hydroxide; ROOH, lipid
hydroperoxide.
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Fig. 2.
Intestinal lipid peroxide absorption and transport under GSH-sufficient
and GSH-deficient conditions.
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Preparation and incubation of everted intestinal sacs.
Tissue segments from both jejunum and ileum were used. Jejunum and
ileum were removed and everted using a glass rod according to the
method of Wilson and Wiseman (34). Everted sacs were filled with
modified Krebs-Henseleit buffer, pH 7.4, warmed to 37°C for 20 min,
and exposed to 75 µM peroxidized Menhaden fish oil
without or with added 10 mM glucose and/or 1 mM GSH for 30 min.
Segments were removed at designated time points, and peroxide transport
was determined as the contraluminal recovery of thiobarbituric acid-reactive substances (TBARS). Contraluminal hydroperoxide contents
were determined spectrophotometrically by the thiobarbituric acid (TBA)
assay (9). The interaction of malondialdehyde, a breakdown product of
lipid hydroperoxides and/or cyclic endoperoxides, with TBA was
measured at 532 nm. It has been validated by Aw et al. (6) with the use
of the iodometric method (9) that the TBA assay predominantly measures
lipid hydroperoxides under these experimental conditions. The
iodometric method involves the specific reduction by iodide of
hydroperoxides, but not endoperoxides, and correlates well with values
obtained with the TBA assay, indicating that this assay can be used to
quantify lipid hydroperoxides in the current study. Furthermore,
high-performance liquid chromatography (HPLC) analyses revealed that
the lipid hydroperoxides are predominantly those of
hydroperoxyeicosapentaenoic (20:5) and docosahexaenoic (22:6) acids
(11). A 20% homogenate was prepared from intestinal tissue after
exposure to peroxidized fish oil, and samples were taken for
determinations of GSH and protein thiol concentrations.
Cell isolation and measurement of glucose uptake.
Jejunal and ileal enterocytes were isolated from rat intestine
according to the method of Masola and Evered (24). Briefly, rats were
killed under halothane anesthesia, and the first 5 cm of intestine
distal to the stomach (duodenum) were discarded. The remaining small
intestine was divided, with the proximal portion taken as jejunum and
the distal portion as ileum. The lumen was washed with cold 0.9%
saline solution to remove particulate matter. The lumen was then filled
with Krebs-Henseleit buffer, pH 7.4, containing 10 mM dithiothreitol
(DTT), and incubated for 10 min at 37°C to remove excess mucus. The
lumen was refilled with buffer containing 5 mM ethylene
glycol-bis(
-aminoethyl
ether)-N,N,N',N'-tetraacetic acid (EGTA) and 10 mM DTT and incubated for an additional 15 min at
37°C. Segments were massaged gently to release enterocytes, which
were filtered through two layers of gauze and washed in buffer
containing 10 mM DTT and 0.25% bovine serum albumin (BSA). Cells were
resuspended in DTT- and BSA-free buffer at a concentration of 5.0 × 106 cells/ml. Hypoxic
enterocytes were exposed to atmospheric
O2 tension during these
experiments, but we have previously shown that acute exposure of
hypoxic cells to environmental O2
tension does not cause metabolic properties of the cells to revert to those of normal cells for at least 48 h (4). Cells were incubated with
10 mM [14C]glucose for
30 min. At designated times, samples were taken and added to ice-cold
enterocyte isolation buffer and filtered through a 47-mm, 5-µm
polycarbonate filter. Cell-associated
[14C]glucose was
measured using a Wallac model 1409 liquid scintillation counter.
Glucose uptake is expressed as nanomoles per
106 cells.
Diamide infusion into enterocyte suspensions.
Enterocytes (4-5 × 106/ml) were incubated in 5-ml
rotating round-bottom flasks at 37°C, without or with added 10 mM
glucose. Diamide-containing solutions at different concentrations were infused at a rate of 1 ml/h into the cell suspensions with the use of a
peristaltic pump (Pharmacia LKB Biotechnology, Piscataway, NJ),
equipped with 2-mm ID tubing as previously described (32). Diamide
concentrations were varied to give infusion rates of 0.01-0.8 nmol · min
1 · 106
cells
1. Incubations were
performed for 15 min, with samples taken at various time points. Our
laboratory has previously shown that there is no difference in cell
viability at the different diamide doses, and at the low infusion rate
the maximum dilution of cell suspensions was 3% (3). At designated
time points, 0.5 ml of cell suspension was removed and the cells were
separated from the incubation mixture by centrifugation. The acid
supernatants were assayed for GSH and GSSG.
Biochemical assays.
TBARS were measured using the method of Buege and Aust (9).
Determination of lipid hydroperoxides using the TBARS method was
correlated with that using the iodometric method (9) and HPLC (11). GSH
concentration was determined spectrophotometrically by the method of
Owens and Belcher (26), and by HPLC according to Reed et al. (28),
which allowed measurement of GSH and GSSG. Protein thiols were measured
according to the method of Ellman and Lysko (14).
Statistical analysis.
Data are expressed as means ± SE. Analysis of variance and
Student's t-test were used to
determine significance of differences. P < 0.05 was considered significant.
Materials.
DTT,
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid, EGTA, BSA, TBA, GSH, taurocholic acid, trichloroacetic acid,
2,4-dinitrofluorobenzene (Sanger's reagent),
5,5'-dithio-bis(2-nitrobenzoic acid) (Ellman's reagent),
N-ethylmaleimide, and Triton X-100
were purchased from Sigma Chemical (St. Louis, MO).
[14C]glucose was
purchased from ICN Pharmaceuticals (Costa Mesa, CA). All other
chemicals were of reagent grade and were available locally.
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RESULTS |
Effect of chronic hypoxia on intestinal transport of
peroxide. Figure 3 shows peroxide
transport by normoxic and hypoxic ileum over 30 min. A previous study
in our laboratory showed a decreasing proximal-to-distal gradient of
intestinal GSH redox cycle enzymes (21), suggesting more pronounced
impairment of detoxication capacity in ileal intestine. Data from the
ileal intestine are shown graphically, whereas those from the jejunum
are given in tables. The results in Fig. 3 show that in ad libitum-fed
and pair-fed normoxic controls, ileal peroxide transport over the experimental period was minimal. Although hypoxic ileal enterocytes initially transported negligible amounts of peroxide as well, after
10-min exposure to peroxidized lipids, hypoxic enterocytes began to
transport increasing quantities of peroxide into the contraluminal
compartment. At 20 and 30 min, hypoxic cells had transported
significantly more peroxide than pair-fed or ad libitum-fed normoxic
controls. When hydroperoxide transport over 30 min is expressed as a
percentage of that made available to intestinal segments in the
incubation medium (1,125 nmol in 15 ml volume), intestine from ad
libitum-fed and pair-fed normoxic rats transported about 5% of the
available hydroperoxides per gram of tissue, whereas hypoxic intestine
transported about 13% per gram of tissue. Results in ad libitum- and
pair-fed normoxic controls are similar in this and subsequent studies;
therefore, data are limited to results from pair-fed normoxic and
chronically hypoxic animals. Response of jejunal enterocytes to
hydroperoxide exposure was similar to that of ileal enterocytes (Table
1).

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Fig. 3.
Effect of chronic hypoxia on transport of peroxidized lipids by ileal
enterocytes. Everted intestinal sacs containing Krebs-Henseleit buffer
were incubated for 0-30 min at 37°C in 75 µM peroxidized
lipid solution. At designated times, intestinal sacs were removed and
peroxide transport was determined as the contraluminal recovery of
thiobarbituric acid-reactive substances (TBARS). Data are means ± SE; n = 3 everted sac preparations for
ad libitum-fed ( ) and pair-fed normoxic controls ( ), and
n = 4 everted sac preparations for
chronically hypoxic animals ( ).
* P < 0.05 vs. ad libitum-fed
and pair-fed normoxic controls.
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Effect of exogenous glucose and GSH on peroxide transport and tissue
peroxide retention.
Figure 4A
shows peroxide transport by normoxic and hypoxic ileal intestine in the
absence or presence of 10 mM glucose and/or 1 mM GSH over 30 min. Before exposure to peroxidized lipids, peroxide transport by
normoxic and hypoxic ileum is minimal. In the absence of substrates
(glucose and/or GSH), contraluminal peroxide recovery in
hypoxic ileum was significantly higher than in normoxic ileum. Transport of peroxide is attenuated in the presence of exogenous glucose and GSH in normoxic conditions, whereas addition of these substrates did not affect abluminal peroxide recovery in hypoxic intestine. This finding signifies the presence of reduced intracellular peroxide metabolism and enhanced output from the hypoxic intestine. Peroxide transport data from normoxic and hypoxic jejunum are shown in
Table 1. Contraluminal peroxide recovery in hypoxic jejunum was
significantly higher than in normoxic jejunum at 20 and 30 min and was
not attenuated by exogenous glucose or GSH.

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Fig. 4.
Effect of glucose and GSH supplementation on transport
(A) and tissue retention
(B) of lipid peroxides by ileal
intestine under normoxic and hypoxic conditions. Everted intestinal
sacs were incubated for 30 min at 37°C in 75 µM peroxidized lipid
solution in absence or presence of 10 mM glucose and/or 1 mM
GSH, and peroxide transport was determined as the contraluminal
recovery of TBARS. Data are means ± SE;
n = 3 everted sac preparations for
pair-fed normoxic controls (solid bars);
n = 4 everted sac preparations for
chronically hypoxic animals (hatched bars). Conditions:
1, control (baseline);
2, lipid hydroperoxide exposure only;
3, lipid hydroperoxide + glucose;
4, lipid hydroperoxide + GSH;
5, lipid hydroperoxide + glucose + GSH. + P < 0.05 vs. pair-fed
normoxic condition 2;
* P < 0.05 vs. pair-fed
normoxic controls under same condition.
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Figure 4B shows retention of peroxide
in ileal tissue exposed to lipid hydroperoxides over 30 min. The data
show that even before exposure to peroxidized lipids, ileal intestinal
tissue from hypoxic rats exhibits significantly higher endogenous
peroxide production compared with normoxic intestine, correlating with a previously described hypoxia-induced oxidative stress in rat small
intestine (21, 22). After 30-min exposure to lipid hydroperoxides, hypoxic ileal intestine continues to exhibit significantly greater levels of peroxide retention than normoxic tissue, whether in the
presence or absence of exogenously supplied glucose or GSH. Data from
normoxic and hypoxic jejunum are shown in Table 1. As with the hypoxic
ileum, tissue peroxide concentration in the hypoxic jejunum was
significantly higher than normoxic tissue before lipid hydroperoxide
exposure (control) and exhibited a trend similar to that observed in
hypoxic ileum under the other conditions, although only the
hydroperoxide plus glucose condition achieved statistical significance.
Effect of exposure to peroxidized lipids on mucosal thiol/disulfide
status and protein thiol concentration.
Figure 5A
shows mucosal GSH concentration after 30-min exposure to lipid
hydroperoxides. Before exposure to peroxidized lipids, GSH levels in
ileal tissue were similar in normoxic and hypoxic intestine. Control
GSH levels appear lower than those reported in previous studies (6),
but this difference may be due to GSH efflux (25) during eversion of
intestinal sacs and/or during a 20-min prewarming period before
incubation with peroxidized lipids. Thus absolute GSH values may be
underestimates, but comparison of trends between experimental
conditions should be unaffected because all tissue segments were
treated similarly. After 30-min exposure in the absence of added
substrates, GSH concentration in both conditions was significantly
decreased. This decrease was attenuated in normoxic intestine in the
presence of exogenous glucose or GSH, but addition of substrates did
not preserve GSH concentration in hypoxic tissue, corroborating our
previous finding that hypoxic enterocytes lack the ability to utilize
glucose to maintain constant cell GSH (22). Although GSH levels were
greatly reduced by peroxidized lipid exposure, the ratio of GSH to GSSG did not change (data not shown), possibly due to efflux of GSSG from
the cells under these conditions or to the interaction of GSSG with
protein thiols.

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Fig. 5.
Effect of lipid hydroperoxide exposure on GSH and protein thiol
concentration in ileal tissue under normoxic and hypoxic conditions.
After 30-min exposure of everted intestinal sacs to peroxidized lipid
solution at 37°C in absence or presence of 10 mM glucose
and/or 1 mM GSH, a 20% homogenate was prepared, and samples
were taken for determination of GSH
(A) and protein thiols
(B). Data are means ± SE;
n = 3 everted sac preparations for
pair-fed normoxic controls (solid bars);
n = 4 everted sac preparations for
chronically hypoxic animals (hatched bars). Conditions as in Fig. 4. + P < 0.05 vs. respective
condition 1;
* P < 0.05 vs. pair-fed
normoxic controls under same condition.
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An important function of cellular GSH is preservation of the reduced
state of cellular protein thiols (protein-bound sulfhydryl groups),
which are critical to the function of many enzymes and other proteins
in the cell. Figure 5B shows that
before exposure to peroxidized lipids, baseline protein thiol
concentration in ileal mucosa was similar in normoxic and hypoxic
intestine. After exposure to peroxidized lipids, protein thiol
concentration in hypoxic intestine was significantly decreased compared
with normoxic tissue and was not restored by the addition of glucose or
GSH. Protein thiol concentration in normoxic intestine, on the other hand, was preserved in the presence of exogenous glucose or GSH during
exposure to peroxidized lipids. Data from normoxic and hypoxic jejunum
are shown in Table 2. Incubation of
normoxic jejunum with lipid peroxides caused modest decreases in tissue GSH and protein thiols that were preserved by exogenous glucose and
GSH. In contrast, exposure of hypoxic jejunum to lipid peroxides resulted in significant decreases in GSH and protein thiol levels that
were not restored by glucose or GSH.
Effect of chronic hypoxia on glucose uptake by enterocytes.
Because hypoxic intestinal cells appear to be unable to utilize
exogenous glucose to enhance detoxication capacity, it is important to
determine if glucose is gaining access to the cells. To determine
glucose uptake, isolated enterocytes were incubated for 30 min with 10 mM [14C]glucose.
Figure 6 shows that there is no difference
in glucose uptake between normoxic and hypoxic cells, indicating that
the lack of glucose dependence for peroxide elimination in hypoxic intestine is not due to decreased uptake. Glucose uptake was similar in
normoxic and hypoxic jejunum (data not shown).

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Fig. 6.
Effect of chronic hypoxia on glucose uptake by ileal enterocytes.
Enterocytes (5 × 106
cells/ml) were incubated for 0-30 min at 37°C with 10 mM
[14C]glucose. At
designated times, samples were taken and added to ice-cold enterocyte
isolation buffer and filtered through a 47-mm, 5-µm polycarbonate
filter. Glucose uptake was measured as cell-associated
[14C]glucose. Data are
means ± SE; n = 4 cell
preparations for pair-fed normoxic controls ( ) and chronically
hypoxic animals ( ).
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Diamide infusion and determination of NADPH supply rate.
An alternate explanation for the inability of hypoxic enterocytes to
utilize glucose for augmentation of peroxide elimination is that
glucose is being diverted from the pentose phosphate pathway and
therefore from production of NADPH for GSSG reduction. To test this
suggestion, the cellular rate of NADPH supply was measured using an
approach that was previously established for hepatocytes (32). This
method utilizes controlled infusions of diamide, a thiol oxidant, into
cell suspensions and subsequent quantification of the cells' ability
to maintain a constant GSH level. As shown in Fig. 1, the rate of NADPH
supply for hydroperoxide elimination can be determined by the rate of
GSSG reduction to maintain a steady-state GSH pool within the cell.
Based on this reasoning, the rate of GSH oxidation by diamide at which
the cell becomes unable to maintain steady-state GSH levels is equal to
the maximal NADPH supply rate. The diamide infusion rate at this
"break point" is termed the critical infusion rate (3, 32). Thus
determination of critical diamide infusion rates in normoxic and
hypoxic enterocytes provides a reasonable estimate of the NADPH supply
rate for GSH regeneration from GSSG under our experimental conditions.
In the absence of diamide, cell GSH was maintained equally well in
normoxic and hypoxic cells for the 15-min experimental period (Fig.
7). Infusion of diamide into suspensions of
ileal cells from normoxic rats had little effect on these cells'
ability to maintain a constant GSH pool (Fig.
7A). Under hypoxic conditions, however, diamide infusion caused a dose- and time-dependent decrease in
cell GSH (Fig. 7B). Constant cell
GSH in hypoxic enterocytes was maintained at lower infusion rates, up
to 0.07 nmol
diamide · min
1 · 106
cells
1, but the GSH pool
progressively decreased over time and as diamide concentration
increased. For example, at 15 min there was a significant decrease in
the cellular GSH pool at a diamide infusion rate of 0.4 nmol · min
1 · 106
cells
1, and at the highest
infusion rate of 0.8 nmol · min
1 · 106
cells
1 significant GSH
decrease occurred at 5 min (Fig.
7B). This failure of hypoxic cells
to maintain GSH with increasing diamide concentration indicates that
the rate of GSH oxidation exceeded that of GSSG reduction by NADPH at
these higher concentrations of the thiol oxidant.

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Fig. 7.
Diamide-induced depletion of cell GSH in ileal cells under normoxic
(A) and hypoxic
(B) conditions. Enterocytes were
isolated from intestines of fed rats. Cells (4-5 × 106 cells/ml) were infused with
varying doses of diamide (0.01-0.8
nmol · min 1 · 106
cells 1) as described in
METHODS. Cells were separated from the
incubation medium by centrifugation, and the pellet was treated with
10% trichloroacetic acid (TCA). Acid supernatants were assayed for GSH
(25). Data are means ± SE; n = 6 cell preparations for pair-fed normoxic controls and chronically
hypoxic animals. Diamide infusion rates
(nmol · min 1 · 106
cells 1): , control (no
diamide); , 0.01; , 0.07; , 0.4; , 0.8. * P < 0.05 vs. control at same
time point.
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To determine the maximal NADPH supply rate, data from Fig. 7 were
replotted to show decreases in GSH (Fig.
8A) and
the ratio of GSH to GSSG (Fig. 8B)
as a function of the diamide infusion rate. From this relationship, we
estimated the critical diamide infusion rate (the rate at which cell
GSH fell), which occurred at >0.8
nmol · min
1 · 106
cells
1 for normoxic
enterocytes and at 0.4 nmol · min
1 · 106
cells
1 for hypoxic
enterocytes. Addition of 10 mM glucose to the incubation medium
resulted in preservation of the cellular GSH pool at all doses of
diamide in normoxic as well as hypoxic cells (Fig.
8A). Changes in the ratio of GSH to
GSSG (Fig. 8B) were similar to those
for GSH alone. Diamide infusion (0.4 and 0.8 nmol · min
1 · 106
cells
1) caused
significant oxidative stress in hypoxic enterocytes, which was
abrogated by glucose addition, consistent with a greater vulnerability
of the hypoxic intestine to oxidant injury. GSH data from jejunum are
shown in Table 3. Diamide infusion caused no significant decrease in GSH in normoxic enterocytes, but cell GSH
was significantly decreased in hypoxic cells. The loss of cell GSH in
both conditions was largely prevented by glucose addition. Changes in
the jejunal GSH-to-GSSG ratio were similar to those in ileal tissue
during diamide infusion at 0.8 nmol · min
1 · 106
cells
1 without added
glucose, causing a significant oxidative stress (normoxia, 7.1 ± 2.1 vs. 0.4 ± 0.1 in hypoxic jejunum).

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Fig. 8.
Diamide-induced changes in cell GSH
(A) and ratio of thiol to disulfide
(GSH:GSSG) (B) in ileal cells under
normoxic and hypoxic conditions as a function of diamide infusion rate.
Enterocyte suspensions from intestines of fed rats were infused with 0, 0.4, or 0.8 nmol
diamide · min 1 · 106
cells 1 for 15 min in
absence or presence of 10 mM glucose. Cells were separated from
incubation medium by centrifugation, and pellet was treated with 10%
TCA. Acid supernatants were assayed for GSH and GSSG. Data are means ± SE; n = 4 cell preparations for
pair-fed normoxic controls (solid and cross-hatched bars, without and
with glucose, respectively) and chronically hypoxic animals (open and
hatched bars, without and with glucose, respectively).
* P < 0.05 vs. same condition
at diamide infusion rate of 0 nmol · min 1 · 106
cells 1.
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DISCUSSION |
Our previous studies have shown that GSH availability is a key
determinant of metabolism and elimination of toxic hydroperoxides by
the intestine (5, 6). In view of the fact that GSH uptake by the
intestine is impaired by chronic hypoxia (7), a critical source of
intracellular GSH for peroxide detoxication in the hypoxic intestine is
from the reduction of GSSG by the GSH redox system. Our laboratory has
shown that glucose availability is an important factor regulating
production of the reductant NADPH for GSH regeneration by this redox
system (3). Glucose flux through the pentose phosphate pathway is
responsible for much of the NADPH production in enterocytes (3). As
shown in Fig. 1, hydroperoxides are reduced at the expense of GSH, and
regeneration of GSSG by NADPH maintains the thiol redox balance within
the cell. If glucose flux is diverted from this pathway, decreased
availability of NADPH compromises cell GSH concentration. In the
current study, we have shown that enterocytes from hypoxic intestine
are substrate limited by the amount of glucose available for use by the
pentose phosphate pathway and NADPH production.
We have shown previously that a variety of metabolic aberrations are
present in the hypoxic intestine (22). For example, the initial rate of
hydroperoxide metabolism by chronically hypoxic enterocytes is greatly
exaggerated compared with that of normoxic cells. We also found that
exogenously supplied glucose significantly increases hydroperoxide
metabolism in normoxic cells but has little effect on augmentation of
peroxide elimination in hypoxic cells. Wide swings in mitochondrial
O2 consumption during substrate
(glucose) or oxidant (tert-butyl
hydroperoxide) challenge led us to conclude that hypoxic enterocytes
exhibit loss of mitochondrial regulation and thus inherent metabolic
instability, appearing to operate in a "hyperdynamic" state in
which they continuously metabolize not optimally but maximally. It has
been reported (12) that patients with an unresolved focus of stress
exhibit a prolonged hypermetabolic response, predisposing them to
development of progressive metabolic dysregulation. Thus prolonged
O2 deficiency appears to impose an
ongoing stress on tissues, similar to that accompanying burn, trauma,
or sepsis, all of which are associated with the development of a
hypermetabolic state (30), whereby metabolism may be pushed to
increasingly higher levels by a systemic insult. Our current findings
suggest that continually elevated metabolism in the intestine could
lead to failure of intestinal function in the face of a secondary
challenge, such as exposure to luminal lipid hydroperoxides.
In this study peroxide transport into the contraluminal compartment of
everted intestinal sacs represents transport of peroxides into lymph,
where they gain access to the systemic circulation. Both jejunum and
ileum were used in our experiments, because functions such as nutrient
absorption and active transport of bile salts are often specific to a
given segment of intestine. Thus differences in experimental results
between segments would be expected to impact intestinal function
differently. Our data demonstrate that initial lipid hydroperoxide
transport into the contraluminal compartment by hypoxic and normoxic
intestine is low, suggesting that the hypoxic intestine can handle
peroxide elimination during short-term peroxide exposure. However, the
detoxication capacity in hypoxic intestine is decreased with continual
peroxide stress, as evidenced by an increase in contraluminal transport
of hydroperoxides. This finding is consistent with our hypothesis
depicted in Fig. 2 and previous studies (5, 6), wherein increased
transport into lymph is consistent with impaired hydroperoxide
metabolism by the GSH redox system in the cell. The data are also
consistent with our contention that the GSH-deficient state is the
predominant one under hypoxic conditions. This suggestion is supported
by the finding that endogenous hydroperoxide production in intestinal tissue before hydroperoxide exposure is significantly higher in the
hypoxic state. These results corroborate our earlier study in which the
hypoxic state was associated with elevation of TBARS in the urine of
rats and a compromised thiol-to-disulfide ratio in the intestine (21).
Our current data show that contraluminal transport of hydroperoxides by
normoxic, but not hypoxic, enterocytes is attenuated by exogenous GSH
or glucose, consistent with decreased utilization of GSH and glucose to
support GSH redox function in peroxide metabolism in hypoxic intestine.
This finding is not surprising in view of the fact that GSH uptake is
impaired in the hypoxic intestine (7), and previous studies in our
laboratory have shown that glucose supplementation does not enhance
hydroperoxide elimination in hypoxic cells (22). Importantly, the
enhanced oxidative stress consequent to decreased peroxide detoxication
in the hypoxic intestine causes significant oxidation of protein
thiols. Because preservation of thiols is critical to the function of
cellular enzymes and other proteins, substantial protein thiol
oxidation would have important consequences for cell integrity.
Moreover, the added inability of the hypoxic intestine to restore cell
GSH and redox homeostasis with exogenous glucose and GSH suggests that
this organ is highly susceptible to oxidant-induced injury. These
findings strongly support our contention that hypoxic intestine,
already in a state of oxidative stress, is incapable of maintaining its functional integrity in the face of additional peroxide challenge (21,
22).
Previous studies have shown that both acute (23) and chronic hypoxia
(7) were associated with decreased intestinal nutrient absorption. It
is interesting that in our study we found that the hypoxic intestine is
just as capable of glucose uptake as the normoxic intestine. Thus the
inability of glucose to stimulate peroxide detoxication in the hypoxic
intestine cannot be explained on the basis of decreased glucose uptake
by enterocytes. Rather, our data are consistent with a reduction of
glucose flux through the pentose phosphate shunt to support NADPH
production for GSSG reduction. Our previous studies have shown that
exogenous glucose significantly increases mitochondrial respiration
(22). These results suggest that both endogenous and exogenous glucose
may preferentially be diverted to support primary metabolic demands, e.g., mitochondrial function, in the hypoxic intestine, thereby decreasing glucose utilization by secondary metabolic pathways such as
the pentose phosphate pathway to support peroxide detoxication. Consequently, the hyperdynamic metabolic state associated with chronic
hypoxia leaves the hypoxic intestine with little critical reserve to
deal with additional challenges, such as the presence of luminal lipid
hydroperoxides. The compromised state of the hypoxic intestine could
result in local damage to the mucosa, as well as in enhanced transport
of peroxidized lipid into the circulation via lymph. These
oxidant-induced changes have important implications for the genesis of
gut pathologies and atherosclerosis, respectively.
 |
ACKNOWLEDGEMENTS |
This study was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-44510. T. Y. Aw is a recipient
of an American Heart Association Established Investigatorship Award.
 |
FOOTNOTES |
Address for reprint requests: T. Y. Aw, LSU Medical Center, Dept. of
Molecular and Cellular Physiology, 1501 Kings Hwy., PO Box 33932, Shreveport, LA 71130-3932.
Received 28 July 1997; accepted in final form 6 November 1997.
 |
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