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Nestlé Research Center, Vers-chez-les-blanc, 1000 Lausanne, Switzerland
1To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • starvation • refeeding • rats • intestinal permeability • glutamine stores
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Nùñez et al.1996
, Ortega et al. 1996
). Moreover,
increased intestinal permeability to macromolecules facilitates the
movement of antigens into the bloodstream, thus increasing the risk of
provoking local or systemic immune sensitization (Boza et al. 1995 and 1996
, Uhnoo et al. 1990
). Similarly,
this increased permeability alters the plasma amino acid profile
(Roosouw and Pettifor, 1990
). Depletion of systemic
glutathione levels has been reported in a number of stress conditions,
including short-term food deprivation and chronic dietary protein
deficiency (Grimble et al. 1992
, Hum et al. 1991
). The fall in glutathione levels under these conditions
implies that a persistent oxidative load leads to the net consumption
of reduced glutathione in excess of the body's ability to resynthesize
the molecule (Jahoor et al. 1995
).
Refeeding rapidly restores the morphology and function of the intestine
(repair of gut atrophy and normalization of intestinal permeability) in
rats. The extent of the changes depends on the amount of food consumed
and, in particular, on the amount and quality (amino acid profile and
molecular form) of the dietary nitrogen (Poullain et al. 1989
and 1991
).
The objective of this work was to determine the effects of severe starvation and refeeding on growth, nutritional recovery and intestinal repair. The variables assessed were as follows: nutritional and growth variables (body weight gain and nitrogen balance during recovery); metabolic variables (muscle glutamine concentration, tissue protein content, glutathione levels); and intestinal barrier integrity (intestinal permeability tests and measurement of the apoptotic index in the epithelium and lamina propria).
The final goal of the study was to establish the severely starved rat as an experimental animal model for comparing the nutritional and functional performances of enteral diets in a model closer to clinical situations.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The semipurified diet (Soy + Met) was composed and designated as
follows (g/kg): soy protein isolate, 220; sucrose, 120; glucose, 50;
cellulose, 50; corn oil, 100; mineral mix AIN 76, 35 (AIN 1977
);
vitamin mix AIN76, 10 (AIN 1977
); L-methionine, 3.5;
choline bitartrate, 2; inositol, 0.25; cornstarch, 409.25. The protein
content of the formula was 19.02% and the energy density, 18.8 kJ/g.
The amino acid composition of the diet is given in Table 1
.
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All experiments were approved by the Ethical Committee of Nestlé Research Center and by the Service Vétérinaire Cantonal (Lausanne, Switzerland).
Male Wistar rats (n = 24), weighing 200 g, were obtained from Iffa–Credo (Lyon, France). Rats were housed in Macrolon cages at 23°C with a 12-h light period (0700–1900 h). They had free access to the Soy + Met diet for 3 d. On d 4, a fixed amount of the Soy + Met diet was offered at 1600 h every day for the following 3 d (269 kJ/d). On d 7, rats were put into metabolic cages and divided into groups with equalized mean body weights. Eight rats continued to receive the Soy + Met diet for the rest of the study (Control), whereas the rest were food-deprived for 72 h with free access to water only. On d 10, 8 rats were killed (Starved). The rest of the rats were transferred to new metabolic cages and refed the Soy + Met diet for 3 d (Refed). A fixed amount of diet was offered (269 kJ/d). Previous experiments had confirmed that the rats would consume all of the feed offered. Body weights were recorded daily at 1600 h. During this period (d 10–13), feces and urine from all rats were collected to determine N balance. On d 13, lactulose and mannitol were used as markers of intestinal permeability as follows: 1 mL of a solution containing 100 mg lactulose and 50 mg of mannitol in water was given to the rats by gavage; urine was collected for the next 5 h in a tube that contained chlorhexidine as preservative. Urine was stored at –20°C until lactulose and mannitol analysis. During the test, rats had access only to water. On d 14, rats were fed 1 mL of ovalbumin solution (500 g/L) by gastric gavage. Rats were anesthetized with the use of isoflurane 90 min later and total blood from dorsal aorta collected into heparinized tubes. The blood was immediately centrifuged for 6 min at 2000 x g, 4°C and stored at –20°C until required for the determination of the plasma ovalbumin concentration. The liver, muscle tibialis and the small intestine were collected. Jejunum samples, frozen in liquid nitrogen, were prepared for histologic and immunologic examination. Another sample of jejunum was scraped with a glass slide under ice. A part of the mucosa obtained (100 mg) and a liver sample (100 mg) were immediately homogenized in 2 mL of cold perchloric acid (50 g/L) solution and centrifuged at 13000 x g for 20 min at 4°C. Supernatants were frozen in liquid nitrogen and stored at -80°C until required for glutathione determination.
Nitrogen utilization test.
The indices used to estimate the nutritional quality of the protein
source were determined during the refeeding period after starvation as
follows (Boza et al. 1994
): body weight gain (g/3 d);
apparent digestibility, i.e., absorbed N/ingested N; apparent
biological value, i.e., retained N/absorbed N; apparent net protein
utilization, i.e., retained N/ingested N; protein efficiency ratio,
i.e., body weight gain (g)/intake of protein (g). Body weight gain was
calculated from weight at the end of the starvation period and after
72 h of refeeding (for control animals, body weights were recorded
at the same time points). Rats were weighed daily at 1600 h, just
before the food was offered.
Muscle glutamine.
A 100-mg muscle sample was mixed with 2 mL of an ice-cold solution
of trichloroacetic acid (100 g/L) and homogenized with the use of a
Polytron at 10,000 rpm for 1 min, after which samples were centrifuged
at 13,000 x g for 10 min at 4°C. After addition
of D-glucosaminic acid as internal standard, samples were
stored at –80°C until analysis. The analyses were performed in a
Beckman 6300 amino acid analyzer (Fullerton, CA). To avoid glutamine
degradation, samples were kept at 10°C before analysis. Glutamine
concentrations (µmol/g muscle) were calculated from
individual peak area, the external standard and the internal standard
area (Jeevanandam et al. 1995
).
Tissue protein.
A 100-mg tissue sample (liver, muscle or gut mucosa) was suspended in 2
mL water and homogenized under ice at 15,000 rpm for 30 s with the
use of a Polytron. An aliquot of the homogenate was assayed for protein
content by using the bicinchoninic acid method from Pierce (Rockford,
IL) (Smith et al. 1985
).
Glutathione determination in liver and gut mucosa.
Reduced glutathione (GSH) and oxidized glutathione (GSSG)
concentrations were determined by HPLC (Waters, Milford, MA), using
fluorometric detection, according to the method of Martin and White (1991)
. GSH and GSSG concentrations were calculated from
individual peak area, external standard and internal standard area, and
expressed in nmol/mg protein.
Intestinal permeability to ovalbumin.
Circulating ovalbumin (OVA) was determined in the plasma of rats by an ELISA inhibition method. Briefly, microtitration plates were coated with OVA (0.15 mL at 50 mg/L) for 24 h at 4°C. Free uncoated sites were blocked with fish gelatin. In separate plates, 1 part OVA dilution (standard) or test sample was incubated for 1 h at room temperature with 1 part rabbit anti-OVA antibody (diluted 1:50,000). After incubation, 0.1 mL of this mixture was added to the OVA-coated plates and incubated for 2 h at room temperature. A goat anti-rabbit peroxidase labeled conjugate (1:20,000) followed by enzyme substrate were then added and optical density measured at 492 nm.
Intestinal permeability to lactulose and mannitol.
Urine samples were prepared as follows: samples were diluted 20-fold
with deionized water. Diluted urine (1 mL) was desalted with 0.25 g of a washed ion-exchange resin (Amberlite IR120 and IRA400),
mixed and filtered through 0.2-µm (pore size) filters.
HPLC analysis was performed on a 250 x 40 mm Dionex CarboPac PA1
ion exchange guard and analytical column (Dionex, Sunnyvale, CA). The
detection was done amperometrically. The quantification was performed
by peak-height analysis and peak-height ratios with internal
standardization (Fleming 1990
).
Histochemical detection and quantification of apoptosis.
Histochemical detection and quantification of apoptosis in intestinal
cells was based on the method of Gavrieli et al. (1992)
,
which uses terminal deoxynucleotidyl transferase (TDT) to incorporate
biotinylated deoxyuridine at sites of DNA fragmentation. This process
is detected by conventional histochemistry after incubation with
avidin-biotin.
Tissue samples were fixed in 40 g/L formalin and embedded in paraffin. Deparaffinization of 4-µm sections was done by heating the sections for 10 min at 70°C. Hydration was done by transferring the slides through the following sequence: twice to xylene for 5 min and then at room temperature for 3 min to 100% ethanol, 95% ethanol, 80% ethanol, 60% ethanol, 40% ethanol and double-distilled water. Nuclei in the tissue sections were stripped from proteins by incubations with 10 mg/L proteinase K (Sigma, St. Louis, MO) in 10 mmol/L Tris-HCl (pH 7.4–8.0) for 20 min at room temperature. Sites were then washed twice in double-distilled water (four times for 2 min each) and incubated with 20 µL of 1 mg/L of Avidin D (Vector Labs, Burlingame, CA) at room temperature for 30 min before washing twice in PBS and blocking of endogenous peroxidase activity. This was done by incubating the sections for 5 min with 30 g/L H2O2 plus 10 mg/L biotin (Sigma) in PBS. The slides were then rinsed in PBS and incubated for 60 min at 37°C in a moist atmosphere with TDT buffer containing 25 mmol/L cobalt chloride, TDT enzyme (3 x 105 U/L), and 1 mmol/L biotinylated-16-dUTP (Boehringer Mannheim, Rotkruez, Switzerland). After the reaction was stopped and sections rinsed with double-distilled water, the sections were covered with 20 g/L bovine serum albumin for 10 min at room temperature, rinsed again in double-distilled water, then PBS, and then incubated with avidin-biotin horseradish peroxidase (Vector Labs) for 45 min before incubation with the substrate 3-amino-9-ethyl-carbazole (Sigma) for ~10 min at room temperature.
The total numbers of intestinal epithelial cells (IEC) and apoptotic IEC in proximal and distal intestinal tissues were determined by counting the number of cells in well-oriented, intact villi. Between 12 and 37 villi per tissue section were examined. Cells were scored as apoptotic if the nucleus showed an intense brown staining. The results were expressed as the percentage of stained IEC in each villus. The lamina propria is an anatomically ill-defined compartment, making identification and quantification of apoptotic cells difficult. Nevertheless, differences in the apoptotic index were estimated by expressing the number of villi with positive staining as a percentage of the total number of villi examined for each treatment group.
Statistical analysis.
Data are expressed as means ± SEM One-way ANOVA and post-hoc Bonferroni tests were used to determine mean differences among the groups for all the variables studied. A difference was considered significant at P < 0.05. In cases in which variances were not equal, a nonparametric ANOVA (Kruskal-Wallis test) was used to confirm significant differences.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Starvation for 72 h produced an average weight loss of 14.5%.
Rats gradually recovered their weight during refeeding. In fact, rats
gained 28.6 ± 1.4 g body weight during the 3-d refeeding
period, whereas non-starved control rats fed the same diet gained
only 13.4 ± 0.8 g in the same period (Fig. 1
). Digestibility was significantly higher (P < 0.05) in
refed rats compared with controls, whereas the fecal output was
significantly lower (P < 0.05) in refed rats than in
controls. Rats refed the experimental diet did not differ in net
protein utilization from control rats, but energy conversion efficiency
and the protein efficiency ratios of the refed rats were greater than
those of control rats (Table 2
).
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Three days of starvation resulted in muscle glutamine concentrations
that were 35% lower than those in the control group (Fig. 2
). Refeeding rats for 3 d raised muscle glutamine to a
concentration that was not different from controls.
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After 72-h of food deprivation, the specific protein concentration of
gut mucosa (mg protein/g tissue) was 19% lower than that of controls
(P < 0.05), but that of refed rats was not different
from that of controls (Table 3
). The specific protein concentration of liver was significantly higher
in starved rats than in refed rats. The liver weight of the starved
rats (5.2 ± 0.1 g) was significantly (P < 0.05) lower than that of controls (7.5 ± 0.1 g) or refed
rats (6.6 ± 0.2 g). Total liver protein content of control
rats (1.01 ± 0.03 g) was significantly (P < 0.05) greater than that of starved (0.76 ±0.01 g) or refed rats (0.82
± 0.05 g).
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Starved rats had significantly greater levels of oxidized glutathione
(GSSG) in liver and gut mucosa than did control or refed rats. In
contrast, food deprivation did not affect GSH levels in gut mucosa or
liver (Table 4
).
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In rats starved for 72 h, intestinal permeability to ovalbumin was
greater than that of control rats (Fig. 3
). However, this variable was normalized in refed rats, such that no
significant difference was observed between them and the control rats.
There were no significant differences in the lactulose/mannitol
concentration ratios found in the urine of the three groups (0.79
± 0.05, 0.72 ± 0.06, 0.88 ± 0.07 for the control
group, starved rats and refed rats, respectively).
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Control rats had very few apoptotic IEC which, when present, were
located at the extreme tips of the villi. On the other hand, starved
and refed rats had higher percentages of apoptotic IEC per villus than
did controls (Table 5
). Identification and quantification of apoptotic cells in the lamina
propria is difficult. Nevertheless, a difference in the degree of
staining among the different treatment groups was observed. Few villi
in control rats had apoptotic cells in the lamina propria compartment,
especially in distal intestine. In starved rats, a significantly
greater percentage of villi in the distal intestine showed staining
(Fig. 4B
and Table 5
). However, there was no major difference in the pattern of
staining between tissues from the refed rats and those from control
rats at either intestinal location.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and an increase in the
intestinal permeability to macromolecules (Bjarnason et al. 1983
) in humans. Starvation for 72 h produced an average
weight loss of 14.5%. These data confirm previous studies by
Poullain et al. (1989)
who observed an average weight
loss of 13% in adult rats after 3 d of starvation. Body weight
gain during the refeeding period was significantly higher (P
< 0.05). Nevertheless, control and refed rats did not differ in
net protein utilization or biological value (Table 2)
. Thus, even after
starvation, when there is a huge demand for protein for cell mass
recovery, especially in the gut, increasing the protein content to 19%
of the total diet (Soy + Met diet) did not lead to a comparably greater
use of the nitrogen source for protein synthesis in the refed group.
Because body weight changes were not due to a higher net protein
utilization or biological value, they probably resulted from cell
hydration. Indeed, we observed higher specific protein concentration in
the liver of starved rats than in refed animals. Moreover, starvation
produced a fall in liver weight of 30%, compared with a decrease of
14.5% in total body weight. This effect of food deprivation in rat
hepatocyte cell volume has been reported by Blommart et al. (1995)
who described it as a short-term physiologic
stimulus for protein catabolism. According to these authors,
circulating insulin levels and cell volume also decrease total liver
protein content. In our case, the hepatocellular protein content
decreased to a lesser extent than cell volume. This fact could explain
why specific liver protein concentration was greater in starved rats
than in refed rats. Björntorp and Yang (1982)
,
using rats starved for 65 h, explained the body weight loss during
the starvation period as mainly the result of water losses. As in this
study, total body weight gain was greater in refed rats than in control
rats with no difference in food intake. Body weight gain in that study
was accomplished by a fivefold greater energy efficiency in the refed
rats. This was explained in part by a decrease in thermogenesis in the
refed rats, allowing less energy to be lost as heat and more
accumulated as protein, fat and glycogen. This energy conservation due
to a decreased energy requirement for maintenance in food-deprived
rats was also observed by Hill et al. (1984)
in 300-g
rats that were starved for 3 d and then refed for 17 d. This
energy conservation mechanism would be present during food restriction
and for some time during early refeeding. Taking into account the daily
body weight changes shown in Figure 1
, the highest growth rate during
refeeding was observed during the first 24 h after refeeding.
Therefore, data on body weight gain in the refed rats were affected by
undigested food in the intestinal contents. Fecal output during the
refeeding period was measured and was found to be significantly higher
in the control group than in the refed group. Therefore, energy
conversion and protein efficiencies were overestimated because
intestinal contents cannot be considered as growth.
The lower protein content we observed in the gut mucosa of rats starved
for 72 h might be due to the gut atrophy that developed in these
rats. However, this variable was normalized in refed rats, such that
there was no significant difference between these rats and those of the
control group. Starvation resulted in a higher specific protein
concentration of liver and muscle (mg protein/g tissue) compared with
that of control and refed rats, mainly due to water loss and depletion
of the levels of glycogen in both tissues. Blommart et al. (1995)
reported that overnight food deprivation produces the
virtual disappearance of hepatic glycogen stores in rats.
Depletion of systemic glutathione levels (whole blood) has been
reported in a number of stress conditions, including short-term
food deprivation (Hum et al. 1991
) and chronic dietary
protein deficiency (Grimble et al. 1992
). A fall in
reduced glutathione level when these conditions are present implies
that a persistent oxidative load leads to the net consumption of
reduced glutathione, exceeding the rate of synthesis of the molecule.
In this study, starved rats had significantly higher levels of oxidized
glutathione in liver and gut mucosa than did control rats. Refeeding
led to a normalization in GSSG concentrations in both tissues. In this
study, we found little difference among groups (Table 4)
in GSH.
Starvation for 3 d did not alter liver GSH concentration
(expressed in nmol/mg protein). Our results are in agreement with those
reported by other investigators (Hunter and Grimble 1997
, Jahoor et al. 1995
, Malmezat 1997
) who observed that 24–48 h after stress induction, rats
and pigs had plasma and tissue GSH concentrations similar to or even
higher than those of nonstressed controls. They suggested that a
minimum time is necessary to equilibrate the oxidant/antioxidant
changes that the stress has produced. GSH synthesis in rats would be
enhanced to compensate for the increased conversion to GSSG that
results from starvation-induced oxidative stress. If the starvation
period had been longer, we would probably have observed depleted liver
and mucosa GSH concentrations, especially because of the lack of
cysteine availability for GSH synthesis. Nevertheless, we have shown
that the GSH/GSSG ratio was significantly lower in liver and gut mucosa
of starved rats. This decreased ratio has been associated with an
acceleration of proteolysis in the skeletal muscle, an increased
susceptibility to hepatotoxicity, an impairment in
nutrient-stimulated insulin release in pancreatic islets and an
increased risk of developing hyperoxic damage in lungs (Godin and Wohaieb 1988
). Therefore, the GSH/GSSG ratio in tissues is
a clear marker of oxidative stress. Under conditions of metabolic
stress, the rate of formation of GSSG exceeds the capacity of the cell
to regenerate GSH from GSSG.
The intestinal permeability confirmed previous results obtained in
starved rats at weaning (Boza et al. 1995 and 1996
),
which showed that a 3-d starvation period produced an increase in the
serum concentration of ovalbumin after force-feeding (10-fold
higher concentration in malnourished rats compared with rats at
weaning). In the previous studies, refeeding for 96 h was enough
to normalize permeability. In this study, refeeding also led to the
normalization of the intestinal permeability after starvation. However,
this starvation effect on mucosal integrity (which is also observed in
critically ill and postoperative patients) may provoke a massive
translocation of microbes, microbial products or microbial residues.
This could initiate or maintain clinical sepsis that could ultimately
lead to multiple organ failure (Bengmark and Jeppsson 1995
).
There were no significant differences in the lactulose/mannitol ratios
in the urine of rats fed the control diet and the urine of starved
rats. In spite of the small difference in molecular weight between
mannitol (MW: 182) and lactulose (MW: 342), the former can pass through
the enterocyte cell membrane by the transcellular route, whereas the
latter may follow a paracellular route. The lactulose molecule is much
smaller than most biological macromolecules; however, there is evidence
that once a critical size has been exceeded, similar patterns may be
demonstrated for larger molecules (Wheeler et al. 1978
).
Elia et al. (1987)
showed that a 4-d starvation period
in humans decreased excretion of mannitol but had no effect on
lactulose excretion, thus leading to an increase in the
lactulose/mannitol ratio. This was not due to an expected increase of
the paracellular absorption of lactulose, which is the case in patients
suffering from diseases of the small intestinal(Bjarnason et al. 1983
). It seems that the decrease observed in mannitol
absorption after starvation is due to a reduction in the functioning
mucosal mass. Perhaps this affects the lactulose/mannitol ratio to such
an extent that, for example, we do not see differences between control
and starved rats. However, circulating ovalbumin levels showed that
starved rats had a greater intestinal permeability compared with
controls.
Programmed cell death or apoptosis is the most common form of eukaryotic cell death and is the way in which the body eliminates unwanted cells and helps maintain homeostasis. Under normal situations, this process is not accompanied by an inflammatory response, but dysregulation may lead to pathologic conditions. The turnover of intestinal epithelial cells is a well-regulated process with immature crypt cells moving up to the villus to become terminally differentiated cells at the villous tips 2–3 d later. These differentiated cells ultimately undergo apoptosis and are either extruded from the tips or phagocytosed by cells within the villus. Thus, the number of cells undergoing apoptosis at any time will determine the level of mature, differentiated cells on the crypt-villus axis and will give an indication of repair mechanisms after injury or stress. It is for this reason that the number of apoptotic intestinal cells was examined in the different treatment groups. Under normal conditions, apoptotic cells are very few and are located at the very tips of intestinal villi and, occasionally, in the crypts. Animals in the non-starved control groups showed this pattern. Although starvation led to an increase in the number of apoptotic cells, this was normalized by refeeding.
There is increasing evidence that the function and integrity of the
intestinal mucosa are important for the outcome in postoperative trauma
patients, patients suffering from malabsorption syndromes or septic
patients, for example. By stimulating the mucosal cell proliferation,
enteral feeding may provide great benefit to critically ill patients.
Enteral diets produce enterothrophic effects that reverse gut atrophy,
maintain mucosal integrity, thus reducing the possibility of bacterial
translocation. Little is known about the molecular mechanisms that
mediate the enterothrophic actions of specific nutrients
(Jenkins and Thompson, 1994
). Further research on the
benefits of enterothrophic dietary regimens in clinical settings is
required. In so doing, the formulation of diets containing specific
nutrients to promote protection and healing of the gut mucosa may be
possible (Raul and Schleiffer, 1996
).
In conclusion, using severely starved rats, we have established a reversible experimental animal model of malnutrition, which describes modifications of key metabolic variables and might prove useful in comparing enteral diets for clinical nutrition.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received September 24, 1998. Initial review completed October 26, 1998. Revision accepted April 12, 1999.
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