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*
Laboratoire de Biochimie, Biologie Moléculaire et Nutrition EA 2416, Faculté de Pharmacie et Centre de Recherche en Nutrition Humaine, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France,
IRCAD CJF INSERM 95-09, 1 place de l'Hôpital Civil, BP 426, 67091 Strasbourg, France,
INSERM U458, Hôpital Robert Debré, 48 boulevard Sérurier, 75935 Paris cedex 19, France, and
§
Laboratoire de Bactériologie, Faculté de Pharmacie, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France
2 To whom correspondence should be addressed.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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0.05)
enhanced plantaris protein content by 18% compared to the
Cas-LPS rats and reduced the plasma phenylalanine-to-tyrosine ratio
(1.32 ± 0.05 vs. 1.54 ± 0.10, respectively,
P 0.01). Gut translocation and histomorphology were
unaffected by diet. However, Gln pretreatment reduced the fall in
sucrase and glucoamylase activities in the ileum, respectively, by
55 and 63% vs. Cas supplementation (P 0.05). In a
second study, after endotoxin challenge, healthy 24-mo-old rats were
then food-deprived for 2 d (from d0 to d2), received a
nonpurified diet for 4 d (from d2 to d6), and then Cas or
L-Gln-supplemented diet for 7 d (from d6 to d13). No
beneficial effects of Gln supplementation were observed except an
increase of 50 and 56% in sucrase and glucoamylase activities in the
ileum of Gln-treated rats, (P 0.01 vs. healthy
rats). In conclusion, the effects of L-Gln supplementation
in aged endotoxemic rats were limited.
KEY WORDS: • aged rats • Gln-supplemented diet • endotoxemia • amino acids • muscles • small bowel
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Muscles form a massive dynamic reservoir
of proteins and free amino acids especially glutamine
(Gln).3
The metabolic importance of Gln is evident during stress, when it
becomes a conditionally indispensable amino acid because endogenous de
novo synthesis fails to meet increased demand (Hall et al. 1996
). The loss of skeletal muscle mass and protein depletion
may predipose to a Gln deficiency which could contribute to the high
sensitivity of elderly people to catabolic situations. Indeed,
metabolic (Jeevanandam et al. 1990
) and immunological
(Lesourd 1997
) responses to trauma are blunted in the
elderly. However, this fact may be questioned considering the
unexpected finding that the muscle Gln depletion was significantly less
in aged, stressed rats than in young adult rats (Farges et al. 1994
, Minet-Quinard et al. 1999
) in spite of a
similar increase in muscle Gln synthetase activity
(Meynial-Denis et al. 1996
, Minet et al. 1999
). A possible explanation for these unexpected results is
that the relative maintenance of the muscle Gln pool in aged rats could
be due to a decrease in muscle Gln efflux, as previously reported by
Parry-Billings et al. (1991)
in soleus from healthy aged
rats, perhaps secondary to impaired hormonal control (Grizard et al. 1995
). This would imply that in aged, stressed rodents,
muscle is unable to supply Gln in sufficient amounts to other tissues.
Several experimental and clinical studies documented the nutritional
efficiency of Gln-enriched nutrition support in the prevention and
treatment of critical illness (Fürst and Stehle 1995
, Neu et al. 1996
). To our knowledge, no
data are available for aged, stressed animals. Thus, the purpose of the
present study was to test whether metabolic disorders induced by
endotoxemia could be prevented or reversed by providing exogenous Gln.
The experiments focused on the small bowel (a major site of Gln uptake
and utilization) and skeletal muscle (the main tissue of Gln storage
and production).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male Sprague-Dawley rats aged 24-mo purchased from Iffa Credo (l'Arbresle, France) were used as old rats. They were individually housed at an ambient temperature of 21–23°C with a 12-h light/dark cycle (dark from 0800 h to 2000h) and were allowed free access to nonpurified diet and water. The nonpurified diet [AO4; Usine d'Alimentation Rationnelle (UAR), France) contained proteins (17%), carbohydrates (59%), fats (3%), and minerals, moistures, fibers, vitamins and water (21%). All the rats were adapted to their new environment for 15 d [from d-15 (d-15) to d 0 (d0)]. The experiments complied with the National Research Council Guide for the care and use of laboratory animals. One of us (LC) is authorized (N° 005226) by the French Ministry of Agriculture and Forestry to use this experimental model.
Experimental designs
Experiment 1: prophylactic use of a Gln-enriched diet.
At the end of the acclimation period (d0), old rats (n
= 14) weighed 709 ± 8 g and ate 23.8 ± 0.5 g
daily. They were then randomly assigned to receive for 7 d (from
d0 to d7) the nonpurified diet (chow), as described above, supplemented
with either Gln (1.0 g/kg bw/day; Gln lypopolysaccharide (LPS) group;
n = 7) (Fluka, Germany), or casein (Cas isonitrogenous
to Gln, 1.4 g/kg bw/day; Cas-LPS group; n = 7)
(UAR). Gln and Cas were mixed daily with the nonpurified diet to obtain
homogeneous powders, which were given in conical ceramic troughs to
avoid spillage. The intake was limited to 90% of that recorded during
the second week of acclimation in order to be certain that the rats ate
all the food given. Taking into account the amount of Gln contained in
AO4 and in Cas and estimating that Cas contains roughly
10% Gln (Swaisgood 1982
), Gln intake was 0.62 g/kg/day
in the Cas-LPS group and 1.49 g/kg/day in the Gln-LPS group. Rats
that ate less than the food given were excluded from the study. On d7,
the two groups of rats received an i.p. injection of
chromatographically purified phenol-extracted LPS
(Escherichia coli, serotype O127:B8; Sigma, France) at 3
mg/kg dissolved in 9 g/L NaCl to a final concentration of 1 mg/mL. This
dose was previously shown to induce a catabolic state in aged rats
(Farges et al. 1996
). Subsequently, the rats were
food-deprived for 24 h (d7 to d8) because we previously
observed that old endotoxemic rats do not eat during this period
(Farges et al. 1994
, Farges et al. 1996
) and because this food deprivation period contributes
greatly to the catabolic state induced by LPS (Lasnier et al. 1996
). This was also verified in a preliminary set of
experiments where LPS- and fasted-treated rats were compared to
saline-injected rats (data not shown), all the rats having been
previously fed chow ad libitum. The rats were then killed (n
= 5 in Cas-LPS group and n = 6 in Gln-LPS
group). The results were compared with those obtained in an absolute
control group (no treatment) fed the chow ad libitum and common to
experiments 1 and 2 (see below).
Experiment 2: Effects of a Gln-enriched diet administered
after LPS challenge.
On day 0 (d0), the rats (n = 27) weighed 718 ± 15 g and ate 26.9 ± 0.9 g daily. They were given an
i.p. injection of LPS (E. coli serotype O127:B8) at 1.5
mg/kg. The rats were then fasted for 48 h (from d0 to d2) since
they do not eat anyway during this period (Farges et al. 1996
), and fasting increases catabolic response to stress.
Because of the marked anorexia, they were subsequently refed with free
access to the chow for 4 d (from d2 to d6). During this time, they
gradually returned to a food intake similar to that measured during the
week preceding the LPS challenge. Also, this wash-out period
prevented uncontrolled variation due to heterogeneous intake. At d4
post-LPS challenge, food intake was 10.0 ± 2.7 and 24.5
± 1.4 g in LPS and healthy rats, respectively (P
0.05 vs. healthy). On d6, food intake was normalized in
LPS-treated rats compared to controls (22.7 ± 2.0 g vs.
24.2 ± 0.8 g, P > 0.05). At this time,
LPS-treated rats (n = 13) were randomly allocated
to receive daily during 7 d (up to d13) the chow-supplemented
Gln or Cas (LPS-Gln group, n = 7 and LPS-Cas group,
n = 6) in the same amounts and conditions as described
in experiment 1. On d13, the rats were killed and measurements
performed (n = 6 in each group). This experiment was
conducted twice. Since the two replicates yielded similar results
(Student's t-test, P > 0.05), these were pooled.
For experiments 1 and 2, a common control group of healthy 24-mo-old
rats with free access to the chow was used (n = 5). For
the two experiments, food consumption and total body weight were
recorded daily. The cause of mortality in LPS-treated rats was not
explored. The rats concerned were lethargic before death and did not
exhibit any sign of pain. The experimental designs are summarized in
Figure 1.
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At the end the experimental periods, all rats were in the postabsorptive state or starved and were killed by decapitation after general anesthesia with diethylether (Prolabo, Fontenay-sous-Bois, France). Blood samples, collected in heparinized tubes, were rapidly centrifuged and the plasma deproteinized with sulfosalicylic acid (50 g/L). The supernatants were stored at -80°C until analysis. In the first experiment only, the mesenteric lymph nodes (MLN) were removed under sterile conditions.
The small intestine extending from the ligament of Treitz to the ileocaecal junction was promptly resected. Its total length was determined by vertical suspension with an attached 3 g weight, and the intestine was cut at its mid-point, resulting in two portions, jejunum and ileum. Pieces 1 cm long were removed in proximal parts of the jejunum and the ileum for histomorphometric examination. The following 20 cm of the jejunum and the ileum was divided into two segments used for hydrolase activity determination and free amino acid (AA) content analysis, respectively. Each segment was cut off, flushed with ice-cold saline buffer, everted, and the mucosa scraped, weighed and frozen in liquid nitrogen.
Finally the soleus, a muscle composed of type I fibers, the extensor digitorum longus (EDL) and the plantaris, muscles composed of a majority of type II fibers, were promptly removed, weighed and frozen in liquid nitrogen. All samples were stored at -80°C until analysis.
Analytical methods
Plasma AA. Plasma samples were analyzed for amino acid (AA) concentrations by ion-exchange chromatography with ninhydrin detection on an AA analyzer (model 6300; Beckman Instruments, Palo Alto, CA). Our laboratory participates in the European Control Quality Scheme (ERNDIM, Brussels, Belgium), and our results indicate the accuracy and reproducibility of the measurements with a between-batch reproducibility ranging from 2 to 8% for various AA.
Tissue AA and protein contents.
Frozen tissues were pulverized and homogenized in ice-cold 10%
trichloroacetic acid containing 0.5 mmol/L EDTA (1 mL/100 mg of
tissue). The acid-soluble fraction containing free AA was separated
by centrifugation from precipitated proteins. AA concentrations were
determined in the supernatant as described above. Fat was removed from
the protein precipitate with alcohol/ether (50/50). The homogenate was
dissolved in 1 mol/L of NaOH (4 mL/100 mg of tissue) at 40°C for
12 h and protein content was assayed by Gornall's method as
described previously (Gornall et al. 1949
).
MLN culture. Mesenteric lymph nodes were cultured on a Muller Hinton agar and on deoxycholate medium for the isolation of Gram-negative enteric bacilli (Diagnostic Pasteur, France). The plates were examined after 24 h incubation at 37°C.
Histology. After fixation in Bouin's solution, the samples were dehydrated and embedded in paraffin. Tissue cross sections (5 µm) were stained with hematoxylin and eosin. Villus height and crypt depth were measured with a semi-automatic image analyzer (Biocom®, Lyon, France). Only villi cut from their base to the top with a single epithelial layer at their tip and crypts with a visible lumen along their entire depth were kept for analysis.
Hydrolase activity determinations.
Enzyme assays were performed on frozen mucosa homogenized in 4 vol (4x
the mucosal weight) of 0.01 mol/L NaH2PO4,
K2HPO4 buffer, pH 6.1. An aliquot was used to
determine protein content using albumin as standard (Gornall et al. 1949
). Sucrase and glucoamylase activities were determined
by a modification of Dahlquist's technique (Cézard et al. 1979
). Leucine aminopeptidase was measured as described
previously (Ahnen et al. 1982
).
Statistical analysis.
All results are expressed as mean ± SEM. Statistical
significance was determined by one way ANOVA followed by the
Newman-Keüls test or by Student's t-test using PCSM software
(Programme Conversationnel de Statistiques pour les Sciences et le
Marketing, Version 5.0, Deltasoft, Grenoble-Meylan, France).
Differences were considered significant when P 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weight, food intake and tissue weight.
At the time the rats received Gln-enriched or isonitrogenous
Cas-enriched diets, food intake did not differ between the two
groups. They consumed 97 and 93% of the food given, respectively. Thus
any differences between these two groups did not originate from
different intakes. However, during this period, these groups had lower
food intakes than the control group fed ad libitum (21.0 ± 1
and 20.9 ± 0.5 g for Gln-LPS and Cas-LPS groups,
respectively, vs. 27.9 ± 1.5 g in healthy rats, P
0.01). Soon after endotoxemia developed, the rats became
lethargic and displayed symptoms of illness (piloerection,
chromodacryorrhea). One and two rats died in the Gln-LPS and in the
Cas-LPS group, respectively, (P > 0.05).
Post-LPS body weight losses were 7 and 5%, respectively. One
healthy rat died during the experiment (for an undetermined reason).
Whatever the dietary pretreatment (chow, Gln- or Cas-supplemented
chow) before the LPS challenge, tissue (muscles, jejunum, ileum)
weights at the time of killing were not significantly affected (data
not shown).
Plasma AA concentrations.
Plasma serine and glycine concentrations were lower in both Gln-LPS
and Cas-LPS groups (P < 0.01 vs. Healthy)
(Table 1
) and in LPA rats (P 0.05 vs.
NaCl, data not
shown).Whatever the nutritional treatment, the LPS challenge induced
hyperphenylalaninemia. The phenylalanine-to-tyrosine ratio, reflecting
the degree of catabolism during infection (Wannemarcher et al. 1976
), was significantly greater in all LPS-treated groups
compared with their respective control group (P 0.01), but less in the Gln-LPS group than in the Cas-LPS group
(P 0.01) (Table 1)
. LPS-treatment induced
hypoglutaminemia (data not shown), while no significant effect was
observed in either Gln-LPS or Cas-LPS groups compared with
their controls. Glutamate concentrations were markedly lower in
LPS-treated rats except in those receiving Gln.
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0.05).
Tissue free AA concentrations.
The combined effect of LPS challenge and 24 h food
deprivation induced a muscle Gln depletion in both the EDL and the
plantaris (data not shown). Similar results were observed
for the prophylactic study: Gln- or Cas-enriched diets did not
prevent the muscle Gln depletion (EDL: -47%, plantaris:
-38%) (Table 2
). Whatever the diet, Gln concentration in the soleus was not
altered by endotoxemia (P > 0.05). Glutamate
concentrations were not significantly reduced in LPS-treated rats
except in the EDL of LPS rats compared with NaCl-treated rats (data
not shown) and of Cas-LPS rats compared with healthy controls. The
concentrations of some other nonessential AA (histidine, arginine,
glycine, serine) were significantly lower (P 0.01) in the muscles of all the LPS-treated groups, whereas levels
of essential AA (phenylalanine, branched-chain AA, methionine)
tended to be or were greater in LPS, Gln-LPS or Cas-LPS groups
compared with their respective controls.
|
). Jejunal free serine, glycine and alanine concentrations were reduced in
the Gln-LPS and Cas-LPS groups (P 0.01 vs.
healthy rats). The citrulline level was significantly lower in the
Cas-LPS group compared with both control and Gln-LPS rats. In
the ileum, glycine concentrations were reduced in the two endotoxemic
groups (P 0.05 vs. healthy control rats) (Table 3)
.
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Small bowel morphometry.
In comparison with healthy rats, LPS treatment and starvation resulted
in lower total height of the jejunum and total and villus heights of
the ileum from old rats fed the Cas-enriched chow diet. Gln supply
did not prevent small bowel atrophy (Fig. 2
). Crypt depth in both intestinal segments was unaffected by the
nutritional treatment and the LPS challenge. The total villus height
showed a significant marked reduction in both the jejunum and the ileum
of Gln-LPS and Cas-LPS groups (P 0.01 and
P 0.05 vs. healthy group).
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0.01 vs. healthy rats) (Fig. 3
). Gln supplementation did not prevent or limit the fall in enzyme
activities. In the ileum, hydrolase activities were also affected by
the stress, but sucrase and glucoamylase activities were greater in the
rats receiving Gln (P 0.05 vs. Cas-LPS group)
(Fig. 3)
.
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Body weight, food intake and tissue weight.
Soon after endotoxemia developed, the rats became lethargic and
displayed symptoms of severe illness. The LPS challenge induced a
catabolic state with a reduced food intake. During the wash-out
period (from d2 to d6), on d4 with free access to the rat chow the
endotoxemic rats ate only 37% of d0 intake (P 0.05
vs. healthy group), but on d6 when the nutritional supplementation
started they had normalized their food intake compared to the healthy
group. During the supplementation period (from d6 to d13), the daily
food intakes for the three groups did not differ (22.8 ± 1.0 g; 22.4 ± 0.8 g; 25.0 ± 0.9 g in LPS-Gln,
LPS-Cas and healthy control rats, respectively). Endotoxemia associated
with the reduced food intake led to a body weight loss of 10% on d2
compared with their d0 body weight. When the feeding was resumed (from
d2 to d13), body weight changes were similar for both endotoxemic
groups, but body weight remained significantly lower compared with
controls (P 0.01, data not shown). Before the
beginning of supplementation, the mortality rate was about 52%;
afterward one Gln-treated rat died.
Skeletal muscle weights were not different across the three groups (data not shown).
Plasma AA concentrations.
Irrespective of the supplementation employed, plasma concentrations
(µmol/L) of four essential AA and of tyrosine were significantly
lower in the LPS-Cas and LPS-Gln rats compared with healthy ad
libitum rats: methionine: 40 ± 2, 36 ± 2 vs. 49
± 5, P 0.05; isoleucine: 66 ± 6, 65 ± 2
vs. 87 ± 7, P 0.05; leucine: 95 ± 8, 93
± 3 vs. 129 ± 9, P 0.05; phenylalanine:
54 ± 3, 54 ± 3 vs. 73 ± 6, P 0.01
and tyrosine 61 ± 4, 57 ± 5 vs. 86 ± 10, P
0.05. No differences among the three groups were noted for
other AA. The Phe/Tyr ratio and the total plasma AA concentration did
not differ in the three groups (data not shown).
Tissue protein contents.
EDL plantaris and soleus protein concentrations did not
differ but in contrast, the soleus protein content was
significantly lower in LPS-Gln and LPS-Cas rats than in the
healthy group (42 ± 3 mg/muscle; 38 ± 5 mg/muscle
vs. 50 ± 3 mg/muscle, P 0.01).
Tissue free AA concentrations.
For the free intramuscular AA, Gln supplementation had no effect on the
EDL-free AA pattern compared with Cas-supplemented and ad
libitum-fed rats (data not shown); plantaris
concentrations of asparagine, ornithine and histidine were
significantly lower in both the LPS-Gln and Cas-LPS groups
compared with healthy rats (Table 4
). In the soleus, besides the lower levels of asparagine and
histidine in the two LPS-treated groups, threonine, lysine and
arginine concentrations were specifically reduced in the LPS-Gln
group. Most striking was the depletion of Gln levels in the
LPS-Gln-treated rats compared with the two other groups (P
0.01) (Table 4)
.
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Small bowel histology and hydrolase activities.
For the morphometry, no variation occurred in either the jejunal or
ileal segments (data not shown). Total hydrolase activities were not
modified by the supplemented diet in the jejunum. In contrast, in the
ileum, sucrase and glucoamylase activities were significantly higher in
the rats fed the Gln-enriched diet than in the healthy ad
libitum group (P 0.01 vs. healthy) (Fig. 4)
.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We examined the effects of a Gln-enriched diet provided before or
after inducing endotoxemia in old rats. Endotoxemia induced with
E. coli LPS can be used as a reliable model of infection
since it reproduces many of the metabolic disturbances that occur in
clinical sepsis (Glauser et al. 1991
). LPS is the
primary pathogenic factor associated with Gram-negative bacteria.
The rationale for the use of such a model is that the incidence of
bacterial sepsis, which increases with age, and mortality due to sepsis
remain high, particularly for the elderly (Gardner et al. 1980
). The underlying mechanism of the vulnerability to
infections was ascribed to the breakdown of anatomic barriers, to
underlying host diseases and to the declining capacity of the immune
system (Gardner et al. 1980
, Lesourd 1997
). It became clear that LPS does not injure host tissues
directly but rather, through the action of a variety of inflammatory
mediators such as cytokines. The great sensitivity of aged animals to
sepsis, mortality up to 100% in some studies, was related to an
excessive tumor necrosis factor-
, interferon-
and nitric oxide
production (Chorinchath et al. 1996
, Tateda et al. 1996
, Kuschnaroff et al. 1997
), resulting
from perturbed endogenous glucocorticoid control of cytokine production
(Chorinchath et al. 1996
, Tateda et al. 1996
). The prevalence of malnutrition is high in septic elderly
patients (Naber et al. 1997
). The nutritional status,
the occurrence of complications, and the underlying diseases constitute
a triangle in which the original cause is unclear. Because anorexia
induced by endotoxin shock is long-lasting in aged rats as we
previously reported (Farges et al. 1996
) and the
nutritional depletion is usually caused by the joint action of an
underlying disease and a dietary deficiency, our experimental model
combined both the LPS challenge and 24–48 h fasting. These endotoxemic
rats were compared to a control group of healthy, freely fed, aged
rats.
Prophylactic administration of a Gln-supplemented diet.
Gln, a nonessential AA, is attracting widespread attention because of
its relevance to numerous metabolic processes and its potential role in
the prevention and treatment of critical illness (Fürst and Stehle 1995
, Neu et al. 1996
). However, to
date all the available data are derived from studies in young adult
animals. Gln supplementation before LPS challenge did not exert a
protective effect on the muscle Gln depletion either in the EDL or
the plantaris but increased protein concentration in the
plantaris. The relationship between Gln content and protein
synthesis is controversial. A striking correlation between the size of
muscle-free Gln pool and protein turnover rates was found in some
studies (Ardawi and Majzoub, 1991
, McLennan et al. 1987
, Millward et al. 1988
). However, no
direct relationship between these variables was found in other
catabolic states such as burn injury (Le Boucher et al. 1997
) or sepsis induced by cecum ligation and puncture
(Fang et al. 1995
).
In our model, aged endotoxemic rats were catabolic as shown by muscle
branched-chain AA and phenylalanine accumulation as the result of
muscle protein breakdown (Jeevanandam 1995
). In
agreement with the plasma AA pattern previously described in old
stressed rats (Milakofsky et al. 1996
) and in geriatric
trauma patients (Jeevanandam et al. 1990
), the decrease
in nonessential AA such as serine, Gln, glycine and arginine suggests a
central utilization greater than their peripheral release. Gln
supplementation contributed to a smaller decrease in the plasma
phenylalanine to tyrosine ratio, suggesting a limitation of protein
wasting (Wannemacher et al. 1976
).
In the small bowel, Gln appears to be essential for the integrity of
the mucosal barrier as well as the maintenance of its metabolic and
immune function (Souba et al. 1990
). Consequently, when
muscle de novo synthesis is not sufficient, the needs of enterocytes
and immune cells are no longer met, leading to immune and gut
dysfunction. There is considerable experimental evidence linking the
failure of the gut barrier to multiple organ failure syndrome
(Soeters 1996
). However, the ability of Gln to prevent
bacterial translocation is controversial (Bark et al. 1995
, Gianotti et al. 1995
, Klimberg et al. 1990
). Although our model is different from others in the
literature because it focuses on the aged rat thus rendering
comparisons difficult, notably we found no significant difference in
survival rate, mucosal atrophy or bacterial translocation between the
two groups supplemented and then LPS treated.
The response of the gut to sepsis is characterized by a marked
reduction in gut Gln utilization associated with impaired Gln transport
and a decrease in glutaminase activity (Salloum et al. 1991
). Since glutaminase is preferentially located in the
proximal intestine, this may induce a high Gln availability to the
ileum and therefore explain the elevated ileal-free Gln
concentration that we observed. At the functional level, whereas Gln
supplementation did not exert a protective effect against the stress in
the jejunum, it preserved the functional capacities of the ileum. Our
results confirm the reduced adaptative capacities of the jejunum of
senescent rats (Reville et al. 1991
). The increase in
disaccharidase activities in the ileum of rats previously fed the
Gln-supplemented diet might be related to the presence of luminal
stimuli and especially the presence of AA that enhance brush border
hydrolase activities and are normally hydrolyzed and absorbed upstream
by the proximal intestine. These results are also consistent with the
fact that 90% of the brush border enzymes are synthesized from luminal
AA (Babst et al. 1993
, Poullain et al. 1991
) and with the anabolic properties of Gln
(Higashiguchi et al. 1995
). By comparison, the low
enzyme activity observed for LPS-Cas rats might result from the
enhanced degradation by pancreatic proteases previously stimulated by
the casein supply (Goda et al. 1988
). The lack of
variation in aminopeptidase activity might be related to its slow
turnover compared with disaccharidase turnover and also to a defect in
dietary protein hydrolysis by pancreatic enzymes leading to a lower
intraluminal formation of oligopeptides, which stimulate brush border
aminopeptidase activity (Raul et al. 1987
). In addition,
it was reported (Greenberg and Holt 1986
) that
pancreatic function is impaired during aging.
Effects of a Gln-supplemented diet administered after LPS challenge.
As discussed above, aged stressed rats characteristically display a
long-lasting marked anorexia followed by a period of very
heterogeneous intake (Farges et al. 1996
). For this
reason, in the experiment described here, a long period was allowed
after LPS challenge before starting the supplementation. Therefore, at
the time of killing, the effects observed were probably more
representative of chronic malnutrition than of LPS treatment, per se,
as shown by the low plasma Phe/Tyr ratio. In addition, considering the
decreased plasma levels of most essential AA, the general nitrogenous
homeostasis remained impaired. Clearly, the rats were no longer
catabolic but malnourished (Cynober et al. 1992
). We
performed another experiment starting the supplementation earlier
(Bérard et al., unpublished data) in which the
rats were starved for 24 h after endotoxemia and thereafter fed by
continuous intragastric enteral nutrition supplemented either with Gln
or with Cas. Unfortunately, all the rats died during the first 48 h. The LPS injection after the gastrostomy tube implantation may have
been too stressful for old rats. The polymeric diet may have been
poorly tolerated and use of semi-elementary diet should be
considered. Finally the flow rate of the enteral mixture administered
may have been too high. Since this type of nutrition is
well-tolerated in young adult rodents (Raul et al. 1995
), this mortality underlines the extreme sensitivity of old
rats and the difficulty in managing models of old stressed rats. The
single significant effect of Gln was as in experiment 1, an improvement
of the hydrolase activities in the distal part of the small bowel.
Many studies involving young adult stressed rats indicate the
beneficial effects of Gln supplementation (Ardawi 1991
,
Fürst and Stehle 1995
, Neu et al. 1996
). Using aged stressed rats, we were unable to reproduce
these results. However, none of the models described in the literature
is exactly the same as those used in this study, and the effect of Gln
supplementation may depend on the type and extent of stress in
experimental models. It would therefore be interesting to compare the
response of aged rats to that of young adult rats with the same
experimental design. Hence, we must be cautious in assuming that the
age of animals is solely responsible for the weak effects of
Gln-supplemented diet.
In conclusion, administration of a Gln-enriched diet before endotoxemia challenge did not prevent muscle Gln depletion and small bowel atrophy in aged rats; it only preserved the intestinal function in the ileum. When administered after LPS challenge, Gln had only a marginal effect. Contrary to numerous experimental or clinical studies suggesting anabolic effects of Gln-supplemented diets, the effects of Gln administration may be limited in old stressed rats.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 AA, amino acid; Cas, casein; EDL, extensor
digitorum longus; Gln, glutamine; LPS, lipopolysaccharide; MLN,
mesenteric lymph node; UAR, Usine d'Alimentation Rationnelle
Manuscript received July 15, 1998. Initial review completed February 12, 1999. Revision accepted July 1, 1999.
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