Spray-dried porcine plasma reduces the effects of staphylococcal enterotoxin B on glucose transport in rat intestine.
ABSTRACT We investigated the intestinal transport of D-glucose (D-Glc) and 3 essential amino acids in a model of intestinal inflammation, and the effects of dietary supplementation with animal plasma proteins on this function. Wistar Lewis rats were fed a diet containing an isonitrogenous amount of milk protein (control group) or a diet supplemented with either spray-dried animal plasma (SDAP) or immunoglobulin concentrate (IC) from porcine plasma, from d 21 of life (weaning) until d 35. On d 30 and 33, rats were challenged intraperitoneally with Staphylococcus aureus enterotoxin B (SEB; groups SEB, SEB-SDAP, and SEB-IC) and on d 35, brush border membrane vesicles (BBMVs) were prepared and used for transport and binding studies. Administration of SEB reduced D-Glc transport across sodium glucose transporter 1 [SGLT1; 20% reduction in maximal transport rate (Vmax); P < 0.05], without affecting the Michaelis constant (Km). The results from specific phlorizin binding, Western blot, and immunohistochemistry supported the view that the effects of SEB are due to reduced expression of D-Glc transporters in the apical membrane. SEB increased the passive diffusion constant (Kd) for D-Glc 3-fold (P < 0.05). SEB did not affect mediated or passive amino acid fluxes of L-leucine, L-methionine, or L-lysine. Dietary SDAP increased the D-Glc Vmax in the SEB group without affecting the passive component. Changes in d-Glc Vmax due to SEB and to the dietary treatments were correlated with changes in the number of SGLT1 transporters present in the BBMVs (r = 0.9468; P < 0.05). Dietary IC had no observed effect. We estimate that, in rats challenged with SEB, SDAP supplementation can increase glucose absorption by 8-9% during the interdigestive periods.
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ABSTRACT: The immune system acts to protect the host from infectious agents that exist in the environment (bacteria, viruses, fungi, parasites) and from other noxious insults. The immune system is constantly active, acting to discriminate 'non-self' from 'self'. The immune system has two functional divisions: the innate and the acquired. Both components involve various blood-borne factors (complement, antibodies, cytokines) and cells. A number of methodologies exist to assess aspects of immune function; many of these rely upon studying cells in culture ex vivo. There are large inter-individual variations in many immune functions even among the healthy. Genetics, age, gender, smoking habits, habitual levels of exercise, alcohol consumption, diet, stage in the female menstrual cycle, stress, history of infections and vaccinations, and early life experiences are likely to be important contributors to the observed variation. While it is clear that individuals with immune responses significantly below 'normal' are more susceptible to infectious agents and exhibit increased infectious morbidity and mortality, it is not clear how the variation in immune function among healthy individuals relates to variation in susceptibility to infection. Nutrient status is an important factor contributing to immune competence: undernutrition impairs the immune system, suppressing immune functions that are fundamental to host protection. Undernutrition leading to impairment of immune function can be due to insufficient intake of energy and macronutrients and/or due to deficiencies in specific micronutrients. Often these occur in combination. Nutrients that have been demonstrated (in either animal or human studies) to be required for the immune system to function efficiently include essential amino acids, the essential fatty acid linoleic acid, vitamin A, folic acid, vitamin B6, vitamin B12, vitamin C, vitamin E, Zn, Cu, Fe and Se. Practically all forms of immunity may be affected by deficiencies in one or more of these nutrients. Animal and human studies have demonstrated that adding the deficient nutrient back to the diet can restore immune function and resistance to infection. Among the nutrients studied most in this regard are vitamin E and Zn. Increasing intakes of some nutrients above habitual and recommended levels can enhance some aspects of immune function. However, excess amounts of some nutrients also impair immune function. There is increasing evidence that probiotic bacteria improve host immune function. The effect of enhancing immune function on host resistance to infection in healthy individuals is not clear.British Journal Of Nutrition 12/2002; 88 Suppl 2:S165-77. · 3.30 Impact Factor
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ABSTRACT: In the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyer's patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells.Life Sciences 02/1996; 59(15):1227-36. · 2.56 Impact Factor
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ABSTRACT: Two experiments were conducted in order to evaluate spray dried animal plasma (SDAP) as an alternative to antimicrobial prophylactic medication with colistin in weaned pigs. Experiment 1 was conducted with 96 piglets (21-day-old) and Experiment 2 was conducted with 128 piglets, (64 weaned at 22 days of age and 64 at 32 days of age). In both experiments the effects of the use of SDAP and/or colistin were compared. Four dietary treatments were used in a factorial arrangement: negative control (N), diet containing SDAP (P), diet containing colistin (C) and diet containing both SDAP and colistin (PC). SDAP was added to diets P and PC at 50gkg−1 between days 0–14 of the trial and at 20gkg−1 from day 15 to the end of the trial. In Experiment 1, SDAP improved average daily gain (ADG; +42g per day; PAnimal Feed Science and Technology - ANIM FEED SCI TECH. 01/2002; 99(1):119-129.
Biochemical and Molecular Actions of Nutrients
Spray-Dried Porcine Plasma Reduces the Effects of Staphylococcal
Enterotoxin B on Glucose Transport in Rat Intestine1,2
Carles Garriga, Anna Pe ´rez-Bosque, Concepcio ´ Amat, Joy M. Campbell,* Louis Russell,*
Javier Polo,†Joana M. Planas, and Miquel Moreto ´3
Departament de Fisiologia, Facultat de Farma `cia, Universitat de Barcelona, Barcelona, Spain; *APC Inc.,
Ankeny, IA 50021; and†APC Europe, S.A., Granollers, Spain
of intestinal inflammation, and the effects of dietary supplementation with animal plasma proteins on this function.
Wistar Lewis rats were fed a diet containing an isonitrogenous amount of milk protein (control group) or a diet
supplemented with either spray-dried animal plasma (SDAP) or immunoglobulin concentrate (IC) from porcine
plasma, from d 21 of life (weaning) until d 35. On d 30 and 33, rats were challenged intraperitoneally with
Staphylococcus aureus enterotoxin B (SEB; groups SEB, SEB-SDAP, and SEB-IC) and on d 35, brush border
membrane vesicles (BBMVs) were prepared and used for transport and binding studies. Administration of SEB
reduced D-Glc transport across sodium glucose transporter 1 [SGLT1; 20% reduction in maximal transport rate
(Vmax); P ? 0.05], without affecting the Michaelis constant (Km). The results from specific phlorizin binding, Western
blot, and immunohistochemistry supported the view that the effects of SEB are due to reduced expression of D-Glc
transporters in the apical membrane. SEB increased the passive diffusion constant (Kd) for D-Glc 3-fold (P ? 0.05).
SEB did not affect mediated or passive amino acid fluxes of L-leucine, L-methionine, or L-lysine. Dietary SDAP
increased the D-Glc Vmaxin the SEB group without affecting the passive component. Changes in D-Glc Vmaxdue
to SEB and to the dietary treatments were correlated with changes in the number of SGLT1 transporters present
in the BBMVs (r ? 0.9468; P ? 0.05). Dietary IC had no observed effect. We estimate that, in rats challenged with
SEB, SDAP supplementation can increase glucose absorption by 8–9% during the interdigestive periods.
Nutr. 135: 1653–1658, 2005.
We investigated the intestinal transport of D-glucose (D-Glc) and 3 essential amino acids in a model
● spray-dried porcine plasma ● immunoglobulin concentrate
● Staphylococcus aureus enterotoxin B ● intestinal inflammation ● glucose transport
Farm animals at weaning are exposed to many stresses (e.g.,
changing from a liquid to a solid diet) and frequently suffer
infections, mainly caused by enterotoxigenic pathogens. En-
teric infections may cause intestinal inflammation, villous
atrophy, maldigestion, and malabsorption, which contribute to
the high rates of mortality (1,2). Rats and mice can also
develop intestinal inflammation at weaning, showing in-
creased plasma cytokine concentrations (3) and increased
numbers of T-helper secreting lymphocytes (4). In humans,
dietary nutrient deficiencies (5) and acute bacterial infections
also induce local immunoinflammatory reactions that may
impair the absorptive and barrier functions of the intestine
(6). Furthermore, infants may be sensitized to dietary antigens
even during breast-feeding, which can also alter the barrier
function of the gut (7) and eventually have a negative effect
In weanling pigs, dietary supplementation with spray-dried
animal plasma (SDAP)4has shown beneficial effects in growth
and performance (8,9). In calves infected with Cryptosporidium
parvum, bovine serum concentrate reduced fecal losses and
returned villous surface area to normal values (10). SDAP has
been proposed as an alternative to antimicrobial medication
(11,12). In humans, studies to assess the acceptability and
safety of SDAP supplementation show that it may increase the
fractional absorption of dietary lipid and of total energy in
malnourished children (13). We have recently studied a rat
model of mild intestinal inflammation to investigate the ef-
fects of dietary supplements on the pathophysiology of bacte-
rial enterotoxins. With this model we demonstrated that di-
1Presented in part at the 19th Meeting of the European Intestinal Transport
Group, Guilford, UK, 2004 [Garriga, C., Pe ´rez-Bosque, A., Amat, C., Campbell,
J. M., Quigley, J. D., Polo, J. & Moreto ´, M.
mented with animal plasma and immunoglobulin concentrate on intestinal trans-
port of glucose and amino acids in rats challenged with the Staphylococcus
aureus enterotoxin B. J. Physiol. Biochem. 60:164 (abs.)].
2Supported by the programs PROFIT (FIT-010000–2003-164) and Eureka
Euroagri (E!2452) and by grant 2001SGR0142 (Generalitat de Catalunya, Spain).
3To whom correspondence should be addressed. E-mail: email@example.com.
(2004) Effects of diets supple-
4Abbreviations used: BBMV, brush border membrane vesicle; D-Glc, D-glu-
cose; GLUT2, glucose transporter type 2; IC, immunoglobulin concentrate; i.p.,
intraperitoneal; Kd, diffusion constant; Km, Michaelis constant; MPO, myeloper-
oxidase; SDAP, spray-dried animal plasma; SEB, Staphylococcus aureus entero-
toxin B; SEB-IC, Staphylococcus aureus enterotoxin B plus immunoglobulin
concentrate; SEB-SDAP, Staphylococcus aureus enterotoxin B plus spray-dried
animal plasma; SGLT1, sodium glucose transporter 1; UMPO, unit of myeloper-
oxidase activity; Vmax, maximal transport rate.
0022-3166/05 $8.00 © 2005 American Society for Nutritional Sciences.
Manuscript received 8 February 2005. Initial review completed 4 March 2005. Revision accepted 27 April 2005.
by guest on June 1, 2013
etary SDAP can modulate the immune response of gut-
associated lymphoid tissue and prevent Staphylococcus aureus
enterotoxin B (SEB)-induced intestinal water secretion (14).
The gastrointestinal tract, particularly the small intestine
and its mucosal epithelium, is capable of rapid functional and
morphological adaptations in response to evolutionary, ge-
netic, and ontogenetic development and demands (15) as well
as to environmental and nutritional challenges (16). Intestinal
inflammation represents a pathological situation that can alter
gastrointestinal function and morphology.
During intestinal inflammation, the barrier function of the
epithelium is altered, and the absorption of nutrients may be
impaired. For example, in rabbits with chronic ileal inflam-
mation, both the affinity of apical sodium glucose transporter
1 (SGLT1) for D-glucose (D-Glc) and the activity of basolat-
eral Na?/K?-ATPase are reduced (17). In C. parvum–infected
rats there is also impairment of Na?-glucose cotransport, al-
though the effect may be ascribed to a concomitant reduction
in villous height (18). Absorption of amino acids is also
impaired in a rabbit model (19) and in a rat model (20) of
intestinal inflammation. In the present study, we investigated
the effects of dietary supplementation with SDAP or immu-
noglobulin concentrates on the intestinal transport of D-Glc
and amino acids in rats challenged with SEB.
Animals and protocol. Male Wistar Lewis rats (n ? 48; Harlan
Ibe ´rica) were used. Rats were kept under stable temperature and
humidity conditions, with a 12-h light:dark cycle. Intestinal inflam-
mation was induced by the intraperitoneal (i.p.) administration of
SEB (Toxin Technologies) dissolved in PBS (3 mmol/L KCl, 137
mmol/L NaCl, 1.5 mmol/L KH2PO4, and 17 mmol/L Na2HPO4). The
protocol consisted of the administration of 2 SEB doses (0.5 mg/kg
body wt) on d 30 and 33 (14).
At d 21 after birth, rats were weaned, randomly distributed into 4
groups, and fed the experimental diets until d 35. Rats in the control
group were fed a control diet (without plasma proteins but containing
an isonitrogenous amount of dried milk protein); rats in the SEB
group were fed the control diet and were treated with SEB; rats in the
SEB-SDAP group were fed a diet supplemented with 80 g SDAP/kg
diet and were treated with SEB; and rats in the SEB-IC group were
fed a diet supplemented with 22.7 g porcine immunoglobulin con-
centrate (IC)/kg diet and were treated with SEB. The SDAP and IC
supplements were obtained from the same batch of fresh porcine
blood (APC Europe) and the full composition of the diets was
identical to that used in a previous study (14).
All protocols used in this study were approved by the ethical
committees of the Universitat de Barcelona and of the regional
government (Departament d’Agricultura, Ramadaria i Pesca, Gener-
alitat de Catalunya) for the use and handling of experimental ani-
Brush border membrane vesicle preparation.
membrane vesicles (BBMVs) were prepared using the MgCl2precip-
itation method (21). For each BBMV preparation, the small intestine
(except the proximal 5 cm) of 2 rats was used. After successive
centrifugations, the final pellet was resuspended in a medium con-
taining 300 mmol/L mannitol, 0.1 mmol/L MgSO4, 0.41 ?mol/L
LiN3, and 20 mmol/L Hepes/Tris (pH 7.4), with a protein concen-
tration of 15–20 g/L. The purity of BBMV preparations was assessed
from the enrichment factor of marker enzymes. Sucrase (?-D-glucohy-
drolase, EC 18.104.22.168) activity, the marker of brush border membrane,
was determined according to Garriga et al. (22). The activity of the
ouabain-sensitive Na?/K?-ATPase (EC 22.214.171.124) was routinely as-
sayed as a marker of the basolateral membrane as previously described
(22). The overall recovery of enzymatic activities was calculated as
the sum of recoveries of all fractions. In addition, membrane orien-
tation was studied according to Garriga et al. (22).
Transport and kinetic studies.
amino acids were measured at 25°C by a rapid filtration technique, as
The uptakes of D-Glc and L-
described elsewhere (22). For the studies of the effect of specific
glucose transporter type 2 (GLUT2) inhibitors on initial rates of
D-Glc transport, the BBMVs were incubated in a medium in which
the NaCl was replaced by KCl to prevent transport across SGLT1.
The kinetic analysis of D-Glc uptake was carried out by nonlinear
regression analysis of total D-Glc fluxes from 6 independent experi-
ments, using the Biosoft EnzFitter program (Biosoft). Because the
errors associated with experimental fluxes were roughly proportional
to their values, the data were proportionally weighted.
The initial rates of L-leucine, L-methionine, and L-lysine uptake
were measured by incubating BBMVs for 3 s in a medium containing
an aliquot of radiolabeled L-amino acid (L-[3H]-leucine, L-[14C]-me-
thionine, or L-[14C]-lysine, New England Nuclear Research Products;
final activity: 1.1–2.0 mCi/L), 100 mmol/L mannitol, 100 mmol/L
NaCl, 20 mmol/L Hepes/Tris (pH 7.4), 0.1 mmol/L MgSO4, and 0.41
?mol/L LiN3. Initial rates were determined at initial concentrations
of the substrates that were either low (0.1 mmol/L) or relatively high
(10 mmol/L for L-leucine and L-lysine and 1 mmol/L for L-methio-
nine, because of its lower water solubility).
Phlorizin binding. The specific steady-state phlorizin binding to
SGLT1 was assayed to determine the correlation between D-Glc
transport rate and the number of SGLT1 transporters present in the
BBMVs, using the method described by Garriga et al. (22). Each
determination was carried out in triplicate using 4 different BBMV
preparations. The specific phlorizin binding was expressed as pmol
bound phlorizin/mg protein at a phlorizin concentration of 50 ?mol/L
Western blot analysis of SGLT1.
protein abundance in BBMVs of rat small intestine were performed
using Western-blot analysis, as previously described (23). Blots were
incubated with a rabbit polyclonal antibody raised against the syn-
thetic peptide corresponding to amino acids 564–575 of the deduced
amino acid sequence of rabbit intestinal SGLT1 at a 1:5000 dilution
for 16 h at 4°C. In simultaneous experiments, nitrocellulose mem-
branes were incubated with the same antibody previously preadsorbed
with the antigenic peptide (1 g/L). Hybridization bands were quan-
tified by scanning densitometry.
Determination of myeloperoxidase activity.
(MPO) activity was measured as an indicator of neutrophil infiltra-
tion in mucosal samples from the jejunum and was determined as
described previously (14). One unit of MPO activity (UMPO) is
defined as the amount of enzyme that degrades 1 ?mol H2O2/min at
Immunohistochemistry. Jejunum fragments (0.5 cm) were fixed
with IHC Zinc Fixative®(Becton Dickinson) at 20°C for 24 h, then
embedded in OCT compound medium (Miles). Cryostat sections (10
?m; ?30°C) were prepared, transferred onto glass slides, and dried
overnight at room temperature. Sections were postfixed with acetone
at ?20°C for 10 min, then permeabilized with Triton®X-100
(Sigma) 0.1% and blocked with 1% bovine serum albumin (v:v;
Sigma) at room temperature for 30 min. Sections were then incu-
bated with the same rabbit polyclonal anti-SGLT1 used for Western
blot analysis (1:500 dilution in PBS) in a humidified chamber over-
night at 4°C. Sections were washed with PBS and incubated with
secondary antibody, Alexa 548 conjugated goat anti-rabbit antibody
(Molecular Probes; 1:300 dilution in PBS), for 1 h at room temper-
ature. Sections were rinsed in PBS and mounted in Mowiol®-488
Image acquisition and processing.
scanned with a confocal scanning laser microscope (CLSM SPII,
Leica) in a blinded protocol. The captured images were analyzed
using the ImageJ program (24) to quantify the fluorescence from the
antibody. The fluorescence intensity was quantified and expressed as
the mean gray level per pixel of the stained area.
Statistical analyses. Results were expressed as means ? SEM.
The effects of SEB were tested by comparing the SEB and control
groups by ANOVA using SPSS-10.0 software (SPSS). To analyze the
effects of dietary supplementation, the SEB-SDAP and SEB-IC
groups were compared with the SEB group by ANOVA followed by
Scheffe ´’s post-hoc test. Differences were considered significant at P
Measurements of SGLT1
Sections (4 per rat) were
GARRIGA ET AL.
by guest on June 1, 2013
Mucosal enzyme activities.
mucosal MPO activity compared to the control group (SEB
group, 3.1 ? 0.2 UMPO/g mucosa; control group, 2.2 ? 0.2
UMPO/g mucosa; P ? 0.05). Dietary supplementation with
SDAP or IC did not affect the SEB-induced MPO activity
(SEB-SDAP group, 2.9 ? 0.2 UMPO/g mucosa; SEB-IC
group, 3.2 ? 0.4 UMPO/g mucosa). Neither SEB treatment
nor dietary supplements affected the activities of the mucosal
marker enzymes, sucrase and Na?/K?-ATPase (Table 1).
Characterization of the membrane vesicles. In the final
BBMV preparations, sucrase activity was highly enriched and
showed a high overall recovery. The low final Na?/K?-
ATPase activity indicated that the BBMVs were not contam-
inated with basolateral membrane. Neither SEB treatment nor
dietary supplements modified enrichment and overall recovery
(Table 1). The intravesicular volume, calculated at equilib-
rium conditions as 0.1 mmol/L D-Glc, did not differ between
the SEB group and the other groups (0.74 ? 0.09 ?L/mg
protein; n ? 12). The membrane orientation of the vesicle
population was 91 ? 5% right side out (n ? 12).
Transport of D-Glu across BBMVs.
duced the maximal transport rate (Vmax) across the apical
SGLT1 by 20% (P ? 0.05) and increased passive permeability
(Kd) 3-fold (P ? 0.05) (Table 2). The Michaelis constant
(Km) did not differ between the SEB group and the other
groups (Table 2). The SDAP-supplemented diet increased the
D-Glc Vmaxby 10% compared to the SEB group, without
affecting the passive component (Table 2). The IC-supple-
mented diet did not affect the D-Glc kinetic constants.
The initial rate of D-Glc uptake of 25 mmol/L was measured
in BBMVs in control and SEB-treated rats. The results, ex-
pressed in pmol D-Glc/(mg protein ? s) (n ? 6) were as follows:
control group: 126 ? 6 (without inhibitor), 122 ? 5 (with
SEB treatment stimulated
SEB treatment re-
cytochalasin B), and 130 ? 4 (with glucosamine); SEB group:
601 ? 11 (without inhibitor), 610 ? 14 (with cytochalasin B),
and 607 ? 18 (with glucosamine). The lack of effect of the
inhibitors on D-Glc uptake indicates that GLUT2 is not in-
volved in the change in Kdand that the increased passive
permeability must be due to an increase in the simple diffusion
of D-Glc across the membrane.
Specific phlorizin binding measurements.
linear correlation between D-Glc Vmaxand specific binding of
50 ?mol/L phlorizin (Fig. 1), indicating that the changes in
D-Glc Vmaxwere due to changes in the density of SGLT1
transporters in the membrane. This linear correlation between
D-Glc Vmaxand B50is defined by the equation y ? 3.63x
? 107.5 (r ? 0.9468; P ? 0.05). The slope of the line is the
turnover number for the phlorizin binding site; that is, the
number of cycles SGLT1/s. The value obtained in the present
experiments (3.63 cycles/s) is in the range of values previously
reported for chicken (22) and rabbit intestine (25).
The antibody recognized a single
band of 75 kDa in BBMVs from all experimental groups that
could be blocked by preadsorption with antigenic peptide (Fig.
2). The densitometric analysis indicated that SEB treatment
decreased SGLT1 abundance to 83.9% of the control group
value (P ? 0.05). Dietary supplementation with SDAP in-
creased the amount of SGLT1 compared to the SEB group by
11% (P ? 0.05), whereas supplementation with IC had no
SGLT1 immunohistochemistry. The villous apex in con-
trol rats had a fluorescence intensity of 94.8 ? 2.7 arbitrary
units (Fig. 3, Table 3), and SEB treatment reduced this value
by 31% (P ? 0.05). The SDAP diet increased SGLT1 expres-
sion in the villous apex by 21% compared to the SEB group (P
? 0.05), whereas values in the SEB-IC rats were similar to
those in the SEB rats. In mid and base villous regions, SEB and
There was a
Epithelial enzymes and characterization of BBMVs in control, SEB, SEB-DAP, and SEB-IC rats1
EnzymeGroup Homogenate specific activityEnrichment factor Overall recovery
1.10 ? 0.11
1.14 ? 0.09
0.98 ? 0.10
1.11 ? 0.10
0.18 ? 0.02
0.14 ? 0.03
0.13 ? 0.05
0.17 ? 0.03
10.7 ? 1.9
9.9 ? 1.7
10.0 ? 2.0
10.5 ? 1.7
0.85 ? 0.07
0.83 ? 0.11
0.82 ? 0.08
0.90 ? 0.10
2.5 ? 0.3
2.0 ? 0.2
1.8 ? 0.4
2.2 ? 0.6
1Values are means ? SEM, n ? 6.
Kinetic constants of D-Glc uptake by BBMVs of small intestine in control, SEB, SEB-SDAP, and SEB-IC rats1
Control SEBSEB-SDAP SEB-IC
Vmax, pmol/(mg protein ? s)
Kd, nL/(mg protein ? s)
100.6 ? 1.3
0.24 ? 0.02
6.0 ? 0.2
79.7 ? 1.3*
0.26 ? 0.02
25.9 ? 0.4*
87.7 ? 1.6†
0.30 ? 0.02
25.3 ? 1.1
82.5 ? 0.5
0.27 ? 0.03
24.8 ? 0.4
1Values are means ? SEM, n ? 6. * Different from control group, P ? 0.05.†Different from SEB group, P ? 0.05.
DIETARY PLASMA PROTEIN AND NUTRIENT TRANSPORT
by guest on June 1, 2013
the dietary supplements did not affect the fluorescence asso-
ciated with SGLT1.
Uptake of L-leucine, L-methionine, and L-lysine across
Two different substrate concentrations were used
for each L-amino acid, and all the incubations were performed
in the presence of an Na?gradient to guarantee the contri-
bution of all amino acid transport systems present in the brush
border membrane. In control rats (n ? 3), the initial rates of
apical transport, expressed in pmol/(mg protein ? s) were as
follows: L-leucine, 28.4 ? 3.0 (0.1 mmol/L) and 132 ? 7 (10
mmol/L); L-methionine, 128 ? 6 (0.1 mmol/L) and 458 ? 45
(1 mmol/L); and L-lysine, 16.7 ? 0.9 (0.1 mmol/L) and 256
? 11 (10 mmol/L). Values in the SEB group did not differ
from the control group, and SDAP and IC treatments had no
effect (data not shown).
We have recently characterized a rat model of intestinal
inflammation induced by the i.p. administration of SEB. The
model shows increased MPO activity, increased water content
in feces, and activation of lymphoid populations of Peyer’s
patches, without affecting blood variables, food intake, or body
weight (14). In the present study, we demonstrated that ad-
ministration of SEB also affects the intestinal absorption of
nutrients by reducing the capacity of the jejunum to transport
D-Glc through the apical SGLT1, without changing the affin-
ity constant of the transporter for the substrate. The hypoth-
esis that this effect of SEB is due to a reduction in the number
of D-Glc transporters in the brush border membrane was fur-
ther supported by Western blot results and by immunohisto-
binding to BBMVs in control, SEB, SEB-SDAP, and SEB-IC rats. Values
of D-Glc Vmaxare means ? SEM, n ? 6; see Table 2. B50,specific
phlorizin binding to BBMVs at a phlorizin concentration of 50 ?mol/L;
Phz, phlorizin. There is a linear correlation between D-Glc Vmaxand B50,
defined by the equation y ? 3.63x ? 107.5 (r ? 0.9468; P ? 0.05).
Correlation between D-Glc Vmaxand specific phlorizin
SEB, SEB-SDAP, and SEB-IC rats. Representative Western blots of
BBMVs of small intestine from control (lanes 1 and 5), SEB (lanes 2 and
6), SEB-SDAP (lanes 3 and 7), and SEB-IC rats (lanes 4 and 8) blotted
with anti-SGLT1 antibody in the presence (lanes 1 to 4) and in the
absence (lanes 5 to 8) of the antigenic peptide. Each lane contained 30
?g of protein. Molecular mass standard is shown on the left.
Western blot analysis of SGLT1 in BBMVs in control,
SEB, SEB-SDAP, and SEB-IC rats. Representative images of indirect
immunofluorescence of SGLT1 in the jejunum, showing the typical
localization of SGLT1 on the apical membrane of enterocytes and the
differing degree of SGLT1 expression among the experimental groups.
Immunohistochemical localization of SGLT1 in control,
Immunohistochemical localization of SGLT1 in the small
intestine in control, SEB, SEB-SDAP, and SEB-IC rats1,2
94.8 ? 2.7
72.7 ? 1.0
67.8 ? 1.0
65.2 ? 2.7*
66.6 ? 3.7
70.2 ? 0.9
78.8 ? 2.5†
76.6 ? 3.3
74.6 ? 2.1
75.2 ? 3.0
70.1 ? 1.7
70.0 ? 2.2
1Data are arbitrary units of pixel intensity.
2Values are means ? SEM, n ? 3. * Different from control group, P
? 0.05;†Different from SEB group, P ? 0.05.
GARRIGA ET AL.
by guest on June 1, 2013
chemical localization of SGLT1 along the villus. Sundaram et
al. (26) also observed a specific reduction of Na?-glucose
cotransport during chronic ileitis due to inhibition of SGLT1
expression in the apical membrane, in a model based on the
administration of Eimeria magna oocytes to rabbits; Sekikawa
et al. (27) observed a strong reduction in SGLT1 expression in
the jejunum without changes in SGLT1 mRNA transcription
in rats infected with the nematode Nippostrongylus brasiliensis.
The recent demonstration that IFN? downregulates Na?-
coupled D-Glc transport in T84 cells (28) and that TNF?
inhibits D-fructose uptake in rabbit intestine (29) supports the
view that cytokines produced during inflammatory processes
may be involved in the control of nutrient transporter expres-
Our results from immune staining SGLT1 showed that the
protein is present in absorptive epithelial cells along the crypt-
villus axis, with higher expression in the upper villous region
than in the mid and basal regions, confirming the results of
Ferraris et al. (30). The findings that the integrity of the
mucosa and the size of the villi were not affected by SEB
administration support the view that the effects of SEB on
mucosal morphology are small, if any. Therefore, the changes
in D-Glc transport kinetics observed in rats challenged with
SEB are due to specific effects on the function of SGLT1. The
kinetic analysis of D-Glc transport also showed that SEB in-
creased D-Glc Kd?3-fold. This observation raised the possi-
bility that SEB affected the passive flux of D-Glc across a low
affinity GLUT-type facilitated mechanism located in the api-
cal membrane (31). However, the use of specific inhibitors
demonstrated that our brush border vesicles lacked any facil-
itated D-Glc transport and confirmed that the rise in the
transmembrane D-Glc flux during the enterotoxin-induced
mucosal inflammation was the result of increased D-Glc simple
SEB administration had no observable effect on amino acid
uptake rates in the present study. This differs from the results
of Topouchian et al. (20), who reported a marked decrease in
leucine and glutamate fluxes across the mucosa of rats infected
with C. parvum, an effect that was attributed to a decrease in
the Na?electrochemical gradient (18). We observed no effect
of SEB administration on Na?/K?-ATPase in the mucosal
homogenates either, indicating that the absorption of nutri-
ents whose uptake depends on the maintenance of Na?and
electrical gradients across the membrane will not be affected.
In rats challenged with SEB, SDAP increased SGLT1 max-
imal transport capacity. No changes occurred in the SEB-IC
group. This effect of SDAP was confirmed by Western and
immunohistochemistry analyses of the mucosa. SDAP, a com-
plex mixture of serum proteins used as food additives in farm
animal production, improves growth and performance in pigs
(8) and has been suggested as an alternative to antimicrobial
medication (11). Although the mechanism of action of SDAP
is still controversial, there is evidence that it prevents patho-
gen infectivity by improving immunocompetence and reduc-
ing bacterial adhesion to the mucosa (32). Recently, Bosi et al.
(9) showed that SDAP can also reduce proinflammatory cy-
tokine expression in the gut of early-weaned piglets challenged
with enterotoxigenic Escherichia coli K88, and Pe ´rez-Bosque et
al. (14) have observed that SDAP brings SEB-induced cyto-
toxic populations back to values close to those in healthy rats.
These findings agree with the lower intravillous lamina propria
cell density observed by Jiang et al. (33) and are consistent
with the view that SDAP can limit the production of cyto-
kines by modulating the immune response.
The nutritional consequences of increased passive perme-
ability and reduced mediated uptake on total D-Glc absorption
are difficult to predict. After a meal, the luminal concentration
of D-Glc is expected to be high, and in this condition absorp-
tion of D-Glc in vivo by apical transporters and by simple
diffusion will also be high. The effects of SEB on passive
permeability would enhance D-Glc absorption by the nonme-
diated transcellular pathway, fully compensating for the small
reduction in SGLT1 Vmax. We calculated that in control rats,
at a luminal D-Glc concentration of 50 mmol/L, 25% of total
uptake would take place by the mediated mechanism and 75%
by simple diffusion, whereas in SEB rats, these figures would be
6 and 94%, respectively. At low luminal D-Glc concentrations,
however, the contribution of enhanced Kdwould be low, while
the mediated pathway would contribute substantially. We
estimated that at a luminal
mmol/L, a level typically found during interdigestive periods
(34), SDAP would increase the intestinal capacity to absorb
D-Glc by 8–9%. This small but significant effect may contrib-
ute to the increase in growth and performance observed in
farm animals fed SDAP supplements (8,9).
D-Glc concentration of 0.2
The rabbit polyclonal antibody raised against the synthetic pep-
tide corresponding to amino acids 564–575 of the rabbit intestinal
SGLT1 sequence and the antigenic peptide were generously provided
by Prof. M. Kasahara. The authors gratefully acknowledge the tech-
nical assistance of Ms. Sandra Rubio.
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