Regional Di e ences in T anspo , Lipid Composi ion, and Fluidi y o Apical
Memb anes o Small In es ine o Chicken
C. Ga iga,* C. M. Va
´zquez,† V. Ruiz-Gu ie
´ ez,‡ and J. M. Planas*
,1
*Depa amen de Fisiologia-Di isio
´IV, Facul a de Fa ma
`cia, Uni e si a de Ba celona, Spain;
†Depa amen o de Fisiologı
´a y Biologı
´a Animal, Facul ad de Fa macia, Uni e sidad de Se illa, Spain;
and ‡Ins i u o de la G asa y sus de i ados,
CSIC, Se illa, Spain
ABSTRACT Na
+
-dependen D-glucose anspo was
s udied in b ush-bo de memb ane esicles om duode-
num, jejunum, and ileum o 5- o 6-wk-old chickens. Re-
gional di e ences we e ound, and bo h ini ial a es and
accumula ion a io o D-glucose we e highe in he p oxi-
mal pa o he small in es ine han in he ileum. To es ab-
lish he mechanism(s) unde lying hese di e ences we
ha e s udied he densi y o Na
+
-dependen D-glucose
co anspo e (SGLT1) as well as lipid composi ion and
luidi y. Phlo izin-speci ic binding and Wes e n blo anal-
ysis indica ed a dec ease in he amoun o SGLT1 in he
(Key wo ds: b ush-bo de memb ane esicles, sodium-glucose anspo e iso o m 1, phospholipid,
a y acid, choles e ol)
2002 Poul y Science 81:537–545
INTRODUCTION
The small in es ine is a highly specialized o gan wi h
egional di e ences in s uc u e and unc ion. Changes
in he in es inal anspo o hexoses by he Na
+
-depen-
den D-glucose co anspo e along he small in es ine
ha e been desc ibed o many species (Budding on and
Diamond, 1989). In mammals, glucose up ake has been
demons a ed o be highe in he apical memb ane om
he p oximal (F eeman and Quamme, 1986) o mid-in es-
ine (Diamond and Ka aso , 1984; Thomson, 1984), de-
pending on he animal. In chickens, we ha e p e iously
shown ha capaci y o anspo suga s dec eased om
he p oximal o dis al egions o he small in es ine (Fe e
e al., 1994; Ro i a e al., 1994). Howe e he mechanism
esponsible o hese egional di e ences is no ye well
es ablished. The aim o he p esen wo k was o s udy he
D-glucose up ake using b ush-bo de memb ane esicles
(BBMV) om chicken duodenum, jejunum, and ileum
2002 Poul y Science Associa ion, Inc.
Recei ed o publica ion July 20, 2001.
Accep ed o publica ion No embe 19, 2001.
1
To whom co espondence should be add essed: jplanas@ a macia.
a .ub.es.
2
Gibe , Ta agona, Spain.
537
ileum when compa ed o he duodenum and jejunum.
The dis al pa o he small in es ine also showed a de-
c ease in ee choles e ol con en and sa u a ed- o-unsa u-
a ed a y acid a io oge he wi h an inc ease in lipid
con en and phospha idylcholine- o-sphingomyelin a io.
These esul s we e associa ed wi h a dec ease in he di-
phenylhex iene luo escence pola iza ion ound in
b ush-bo de memb anes o he ileum. We can conclude
ha he dec ease in he apical D-glucose anspo ound
in he ileum is p ima ily due o a educ ion in he amoun
o SGLT1 p esen in he b ush-bo de memb ane a he
han he di e ences in he lipid composi ion and luidi y.
and o es ablish he ela i e amoun o he Na
+
-dependen
D-glucose co anspo e . In addi ion, we ha e simul ane-
ously analyzed he lipid composi ion and luidi y o hese
p epa a ions conside ing ha many unc ions o biologi-
cal memb anes such as anspo and enzyme ac i i ies
a e in luenced by hei composi ion and physical s a e
(Le G imellec e al., 1992) and also ha he lipid composi-
ion and luidi y o he in es inal memb anes o mammals
p esen egional di e ences (Heubi and Fellows, 1985;
Schwa z e al., 1985; Ib ahim and Balasub amanian, 1995).
The ela ionships among he egional changes in suga
anspo , densi y o Na
+
-dependen D-glucose co ans-
po e , and memb ane composi ion and luidi y a e
also discussed.
MATERIALS AND METHODS
Bi ds
Male whi e Legho n chickens we e ob ained om a
comme cial a m
2
on he day o ha ch and b ough o he
Abb e ia ion Key: BBMV =b ush-bo de memb ane esicle; DPH
=1,6-diphenyl-1,3,5-hexa iene; FAMES = a y acid me hyl es e s an-
da ds; HEPES =N-2-hyd oxye hylpipe azine-N′-2-e hanesul onic acid;
PBS-T =PBS con aining 0.05% Tween 20; PL =phospholipids; SGLT1
=sodium-glucose anspo e iso o m 1.
GARRIGA ET AL.538
Facul a de Fa ma
`cia. They we e main ained in s anda d-
ized humidi y condi ions wi h a 12L:12D pho ope iod.
Tempe a u e was kep a 34 C du ing he i s week a e
ha ch and a 25 C he ea e . F om he day o ha ch, bi ds
had ee access o wa e and a comme cial die con aining
(g/kg die ): 218 c ude p o ein, 33.5 lipid, 375 ca bohy-
d a e, 33 c ude ibe , 11 lysine, 4 me hionine, 10 calcium,
7 phospho us, 5.4 sodium chlo ide, and 14,500 UI i amin
A/kg, 2,900 UI D
3
/kg, 29 UI i amin E/kg. The me abo-
lizable ene gy con en was 12.2 MJ/kg. Manipula ion and
expe imen al p ocedu es a e in acco dance wi h he Span-
ish egula ions o he use and handling o expe imen-
al animals.
Expe imen s we e ca ied ou a 5 o 6 wk o age. Bi ds
we e killed in he mo ning, wi hou p e ious s a a ion,
by ce ical disloca ion ollowed by exsanguina ion. Duo-
denum (panc ea ic loop), jejunum ( om he end o he
duodenal loop o Meckel’s di e iculum), and ileum
( om Meckel’s di e iculum o he ileocecal junc ion)
we e emo ed and immedia ely lushed wi h ice-cold
saline in p esence o phenyl me hanesulphonyl luo ide
(0.2 mM), LiN
3
(0.41 µM), and benzamidine (0.1 mM).
Segmen s we e opened leng hwise, ozen in liquid N
2
,
and s o ed a −80 C.
BBMV P epa a ion
The BBMV we e p epa ed by MgCl
2
p ecipi a ion, as
p e iously desc ibed (Va
´zquez e al., 1997). A e succes-
si e cen i uga ions, he inal pelle con aining pu i ied
BBMV was esuspended in a medium con aining: 300
mMmanni ol, 0.1 mMMgSO
4
, 0.41 µMLiN
3
, and 20 mM
N-2-hyd oxye hylpipe azine-N′-2-e hanesul onic acid
(HEPES)- is(hyd oxyme hyl)aminome hane (T is) (pH
7.4) o a inal p o ein concen a ion o 10 o 20 mg/mL.
The esicles we e ozen, s o ed in liquid N
2
in 150-mL
aliquo s, and used du ing a pe iod o 15 d. Du ing his
ime, he in eg i y and unc ion o he esicles we e no
modi ied ( esul s no shown).
Enzyme and P o ein De e mina ions
The b ush-bo de ma ke enzyme, suc ase (α-D-gluco-
hyd olase, EC 3.2.1.48), was ou inely assayed by he
me hod o Dahlquis (1964). The basola e al memb ane
ma ke enzyme, ouabain-sensi i e K
+
-ac i a ed phospha-
ase (EC 3.6.1.3), was assayed by he me hod desc ibed
by Colas and Ma oux (1980). P o ein was e alua ed by a
Coomassie B illian Blue p o ein assay wi h bo ine γ-
globulin as a s anda d (B ad o d, 1976).
Assay o D-glucose T anspo
The up ake o D-glucose was measu ed by a apid
il a ion echnique as p e iously desc ibed (Ga iga e
3
Millipo e (www.millipo e.com).
4
Sha lau, Ba celona, Spain.
al., 1999a). B ie ly, BBMV we e p eincuba ed o 30 min
a 37 C in a medium con aining K
+
in o de o pe o m he
up ake expe imen s in sho -ci cui ed condi ions. Loaded
BBMV had a inal concen a ion o 200 mMmanni ol, 50
mMKCl, 0.1 mMMgSO
4
, 0.41 µMLiN
3
, and 20 mM
HEPES/T is (pH 7.4). Fo ime-cou se s udies, esicles
we e incuba ed be ween 5 s and 30 min a 37 C wi h a
medium con aining 100 mMmanni ol, 100 mMNaCl, 0.1
mMMgSO
4
, 0.41 µMLiN
3
,20mMHEPES/T is (pH 7.4),
45 µM alinomycin, 0.1 mMD-glucose, and an aliquo
o D-[
14
C]-glucose. A selec ed imes, he up ake was
quenched by adding 1 mL o an ice-cold s op solu ion
con aining 300 mMmanni ol, 0.41 µMLiN
3
, and 20 mM
HEPES/T is (pH 7.4). The dilu ed samples we e apidly
il e ed unde nega i e p essu e by placing 0.9 mL o he
eac ion mix u e on a p ewe ed and chilled cellulose
ni a e il e (0.22-µm po e il e ).
3
The il e was insed
wi h 5 mL ice-cold s op solu ion.
The il e was dissol ed in a Biog een-6 cock ail,
4
and
he adioac i i y e ained was measu ed wi h a scin illa-
ion coun e . Non-speci ic adioac i i y binding o he
il e s was ob ained by adding he s op solu ion o eac-
ion ubes immedia ely a e addi ion o he esicles. This
non-speci ic binding was sub ac ed om he o al adio-
ac i i y o each sample. All expe imen s we e pe o med
in iplica e.
O ien a ion o Vesicles
Memb ane o ien a ion was s udied om suc ase ac i -
i y acco ding o Del Cas illo and Robinson (1982). Suc ase
ac i i y was de e mined in in ac esicles and in esicles
incuba ed o 30 min wi h a mix u e o 3 mMdeoxycho-
la e and 15 mMEDTA.
Phlo izin Binding Measu emen s
S eady-s a e phlo izin binding was assayed a 37 C by
he me hod desc ibed by Ga iga e al. (1999a). BBMV
suspensions we e apidly mixed wi h he incuba ion me-
dium con aining 100 mM manni ol, 100 mMNaCl o KCl,
0.1 mMMgSO
4
,20mMHEPES/T is (pH 7.4), 0.41 µM
LiN
3
,50µMphlo izin, and an aliquo o [
3
H]phlo izin.
A 5 s, he binding p ocess was s opped by addi ion o
1 mL o an ice-cold s op solu ion con aining 300 mM
manni ol, 0.1 mMMgSO
4
,20mMHEPES/T is (pH 7.4),
and 0.41 µMLiN
3
. The esul ing suspension was apidly
il e ed unde nega i e p essu e h ough 0.22-µm cellu-
lose ni a e il e s and washed wi h 5 mL o s op solu ion.
The adioac i i y emaining in he il e was de e mined
as desc ibed ea lie . Speci ic [
3
H]phlo izin binding was
calcula ed by sub ac ing he non-speci ic binding (in he
p esence o K
+
) om o al phlo izin binding (assayed in
he p esence o Na
+
). The densi y o phlo izin-binding
si es was exp essed as picomoles o phlo izin bound pe
millig am o p o ein wi h 50 µMphlo izin (B
50
).
Wes e n Blo Analysis o SGLT1
Measu emen s o sodium-glucose anspo e iso o m
1 (SGLT1) p o ein abundance in BBMV o chicken small
REGIONAL PROPERTIES OF THE SMALL INTESTINE 539
in es ine we e pe o med using Wes e n-blo analysis, as
p e iously desc ibed (Ga iga e al., 1999b). The BBMV
we e solubilized by boiling in he p esence o SDS and
2-me cap oe hanol. Aliquo s (30 mg o p o ein) we e sub-
jec ed o SDS-PAGE wi h an 8% linea polyac ylamide
gel. A e elec opho esis, he p o eins we e elec opho-
e ically ans e ed om he uns ained gel o ni ocellu-
lose memb anes in a ans e bu e con aining 20 mM
T is, 150 mMglycine, and 20% me hanol; ans e was
o 60 min a 4 C and a cons an ol age o 100 V using
a T ans-Blo appa a us.
5
Non-speci ic binding si es we e
i s blocked wi h PBS con aining 0.05% Tween 20 (PBS-
T) and 3% BSA.
Blo s we e incuba ed o e nigh a 4 C wi h a abbi
polyclonal an ibody (kindly dona ed by M. Kasaha a, Tei-
kyo Uni e si y, Tokyo, Japan) aised agains a syn he ic
pep ide co esponding o amino acids 564 o 575 o he
deduced amino acid sequence o abbi in es inal SGLT1
(Hedige e al., 1987) dilu ed o 1:5,000. In pa allel expe i-
men s, ni ocellulose memb anes we e incuba ed wi h he
same an ibody ha had i s been adso bed wi h he pep-
ide (1 mg/mL) agains which he an ibody was aised
(kindly p o ided by E. M. W igh , UCLA, Los Angeles,
CA). The memb anes we e washed (8 ×4 min) wi h PBS-
T. Then, an i-SGLT1 an ibody was de ec ed by ECL chem-
iluminiscence
6
by using a pe oxidase-conjuga ed an i-
abbi IgG
7
as a second monoclonal an ibody (1:3,000).
A e de ec ion, he samples we e measu ed by scan-
ning densi ome y.
Lipid Analysis
To al lipids we e ex ac ed om he b ush-bo de
memb ane by he me hod o Rose and Oklande (1965),
using 0.02% (w / ol) bu yla ed hyd oxy oluene as an i-
oxidan . The lipid ex ac was quan i ied g a ime ically
and kep in a s oppe ed essel unde ni ogen a mosphe e
a −30 C un il he assays. Lipid and phospholipid compo-
si ions we e ob ained by means o he Ia oscan hin-laye
ch oma og aphy/ lame ioniza ion de ec o echnique (De
Sch ij e and Ve meulen, 1991). Ia oscan MK-5 was used
in combina ion wi h Ch oma ods S, which ha e a p e-
coa ed ac i e silica hin laye . Ch oma ods S we e ou-
inely s o ed in 4.5 M H
2
SO
4
. P io o use, he ods we e
washed wi h dis illed wa e , d ied a 110 C o 15 min,
and ac i a ed by being passed h ough he lame o he
Ia oscan de ec o . Each od was spo ed wi h a 10-mL
Hamil on sy inge con aining 3 mL o o al lipids o phos-
pholipids (PL). To sepa a e o al lipids, ods we e de el-
oped in hexane-die hyl e he -ace ic acid (80:20:1, ol/
ol/ ol). The PL we e esol ed in wo s eps, s a ing wi h
an ini ial de elopmen o ods in chlo o o m-me hanol-
ace ic acid-wa e (201:84:9:6, ol/ ol/ ol), d ying a 70
C o 10 min, and a second de elopmen in hexane-die hy-
le he -ace ic acid (80:20:1, ol/ ol/ ol).
5
Bio-Rad, He cules, CA.
6
Ame sham In e na ional, Buckinghamshi e, UK.
7
Sigma Chemical Company, S . Louis, MO.
Rods we e scanned unde he ollowing condi ions: 150
mL/min hyd ogen low, 1,750 mL/min ai low, 47 mm/
s scanning speed, and 42 mm/min cha speed. A Ia o-
co de TC-11 in eg a o was used o eco d and in eg a e
he a ea.
Fa y Acid Analysis
Fa y acids o he o al lipid ex ac we e analyzed by
gas ch oma og aphy. The samples we e saponi ied by
hea ing o 5 min wi h 5 mL o 0.2 Msodium me hyla e
and hea ing again a 80 C o 5 min wi h 6% (w / ol)
H
2
SO
4
in anhyd ous me hanol. The a y acid me hyl es-
e s hus o med we e elu ed wi h hexane and analyzed
in a Hewle -Packa d 5890 se ies II gas ch oma og aph
equipped wi h a lame ioniza ion de ec o and using an
Omegawax 320 used silica capilla y column (30 m ×0.32
mm i.d., 0.25 mm ilm). The ini ial column empe a u e
was 200 C, which was main ained o 10 min, hen p o-
g ammed o inc ease om 200 o 230 C by 2 C/min. The
injec ion and de ec o empe a u es we e 250 and 269 C,
espec i ely. The low a e o helium was 2 mL/min,
he column head p essu e was 250 kPa, and he de ec o
auxilia y low a e was 25 mL/min. Peak a eas we e cal-
cula ed by a Hewle -Packa d 3990A eco ding in eg a o .
Indi idual a y acid me hyl es e s we e iden i ied on
iso he mal uns by compa ison o hei e en ion ime
agains hose o s anda ds. Fa y acid me hyl es e s we e
quan i ied by in e nal s anda iza ion ( icosanoic me hyl
es e , 23:0) by using peak-a ea in eg a ion.
Fluo escence Measu emen
The s eady-s a e luo escence pola iza ion and luo es-
cence aniso opy we e de e mined as p e iously de-
sc ibed (Va
´zquez e al., 1997), using he lipid-soluble lu-
o escen p obe 1,6-diphenyl-1,3,5-hexa iene (DPH). A
s ock solu ion o 2 mMDPH in e ahyd o u an was p e-
pa ed and s o ed in he da k a −30 C. BBMV equi alen
o 100 mg p o ein was incuba ed a 25 C o 1 h in 2 mL
o bu e ed saline con aining 250 mMsuc ose, 10 mM
T is/HCl (pH 7.4), and 2 mMDPH. Measu emen s we e
aken using a luo escence spec opho ome e equipped
wi h a pola izing il e . The exci a ion and emission wa e-
leng hs we e 365 and 430 nm, espec i ely.
The s eady-s a e luo escence pola iza ion (p) was cal-
cula ed as ollows:
p=(I
−I
h
/I
+I
h
)
whe e I
and I
h
a e obse ed in ensi ies measu ed wi h
pola ize s espec i ely pa allel o and pe pendicula o
he e ically o ien ed pola ize exci ing beam. The
s eady-s a e luo escence aniso opy (
s
) was calcula ed
om he ollowing a io:
s
=2p/(3-p).
Co ec ions o ligh sca e ing we e ou inely made by
sub ac ing he signal ob ained om iden ical bu unla-
GARRIGA ET AL.540
TABLE 1. Suc ase ac i i y o b ush-bo de memb anes om duodenum, jejunum, and ileum
1
Homogena e
speci ic BBMV-speci ic ac i i y Reco e y
2
In es inal egion ac i i y n (nka /mg p o ein) En ichmen (%)
Duodenum 0.249 ±0.019
c
4 4.132 ±0.58 14 ±3.0 45 ±3.5
Jejunum 1.193 ±0.070
a
13 11.39 ±0.91 11 ±0.5 42 ±5.2
Ileum 0.365 ±0.066
b
5 4.164 ±0.40 14 ±3.6 41 ±7.4
a-c
Values in each column wi h no common supe sc ip di e signi ican ly (P<0.05).
1
Values a e means ±SEM; n =numbe o sepa a e isola ions.
2
En ichmen is a io o he speci ic ac i i ies in inal b ush-bo de memb ane esicles p epa a ion (BBMV)
and ini ial homogena e. Reco e y shows o al ac i i y in inal b ush-bo de p epa a ion exp essed as a pe cen age
o ac i i y in o iginal homogena e.
beled samples and con ibu ed <3% o he luo escence
o he comple e sys em.
Chemicals
All unlabeled eagen s o he anspo s udies we e
ob ained om Sigma Chemical Company.
7
Measu e-
men s o he enzyme ac i i y we e ca ied ou using pu -
chased eagen s
8
and he enhanced chemiluminiscence
we e om Ame sham In e na ional.
6
D-[U-
14
C]-glucose
(speci ic ac i i y 251 mCi/mmol) and [
3
H]phlo izin (spe-
ci ic ac i i y 46.4 Ci/mmol) we e pu chased.
9
The inal
ac i i y o labeled subs a es in he incuba ion medium
was 0.5 o 2 mCi/mL. In he lipid composi ion s udies,
a y acid me hyl es e s anda ds (FAMES) we e ob ained
om La odan Fine Chemicals.
10
The in e nal s anda d
solu ions we e p epa ed by dissol ing 200 mg o icosa-
noid acid me hyl es e in 100 mL o hexane. Calib a ion
solu ions we e p epa ed by dissol ing speci ic amoun s
o FAMES in hexane con aining 2,6-di e bu yl-p-c esol
(bu yla ed hyd oxy- oluene) ob ained om Sigma Chem-
ical Company.
7
O he chemicals we e analy ical g ade.
11
S a is ical Analysis
S a is ical di e ences be ween in es inal egions we e
es ablished by ANOVA and Snedeco ’s F es . P<0.05
was aken as signi ican .
RESULTS
Pu i y o he BBMV P epa a ion
Table 1 shows he speci ic ac i i ies o suc ase in ho-
mogena es o duodenum, jejunum, and ileum. The spe-
ci ic ac i i y o suc ase in he jejunum was signi ican ly
highe han ha ob ained in he mucosa om duodenum
and ileum. The concen a ion o suc ase ac i i y in he
inal b ush-bo de memb ane pelle om di e en e-
gions was uni o mly 11- o 14- old g ea e han in he
8
Boeh inge , Manheim, Ge many.
9
New England Nuclea Resea ch P oduc s, D eieich, Ge many.
10
Malmo
¨, Sweden.
11
Me ck, Da ms ad , Ge many.
s a ing homogena e, and he eco e ies o suc ase we e
om 41 o 45%. The e was no signi ican di e ence in
en ichmen and eco e y be ween segmen s. B ush-bo -
de memb ane ac ions p esen ed low ac i i y o oua-
bain-sensi i e K
+
-ac i a ed phospha ase. The ac i i y o
his enzyme was educed du ing he isola ion p ocedu e
in any segmen s udied ( esul s no shown), indica ing
li le basola e al con amina ion. The memb ane o ien a-
ion s udies indica e ha 92 ±2% (duodenum), 93 ±5%
(jejunum), and 93 ±3% (ileum) o he esicle popula ion
we e ou side-ou o ien ed.
T anspo o D-Glucose Ac oss BBMV
Figu e 1 shows he ime cou se o D-glucose anspo
in o he BBMV along he h ee egions o he chicken small
in es ine. In he p esence o a 100 mMex a esicula - o-
in a esicula Na
+
g adien , D-glucose up ake exhibi ed
a ypical ansien inc ease in he in a esicula concen a-
ion o suga in h ee segmen s, a 5 s o incuba ion.
The o e shoo disappea ed when he Na
+
g adien was
eplaced by a K
+
g adien . Up ake o D-glucose a equilib-
ium (30 min) was iden ical in he p esence o absence
o Na
+
g adien and was no changed in any o he seg-
men s. The mean alue o he esicula olume was 0.56
±0.09 µL/mg o p o ein o he duodenum, 0.59 ±0.08
µL/mg o p o ein o he jejunum, and 0.61 ±0.07 µL/
mg o p o ein o he ileum. The accumula ion a ios we e
3.43 in he duodenum, 3.21 in he jejunum, and 2.16 in
he ileum.
Speci ic Phlo izin Binding
Figu e 2 shows ha he densi y o phlo izin-binding
si es was signi ican ly lowe in he ileum (34.5 ±3.7 pmol
o phlo izin bound/mg p o ein) when compa ed o he
duodenum and jejunum (50.1 ±2.7 and 51.2 ±1.9 pmol
phlo izin/mg p o ein, espec i ely). No di e ences we e
ound be ween duodenum and jejunum.
Immunoblo s
Figu e 3A shows a ypical Wes e n blo wi h abbi
polyclonal an ibody aised agains he 564-575 amino acid
sequence o abbi SGLT1. The an ibody ecognized a
single band o 75 kDa, which was blocked by p eab-
REGIONAL PROPERTIES OF THE SMALL INTESTINE 541
FIGURE 1. Time cou se o D-glucose up ake in he b ush-bo de
memb ane esicle o duodenum (D), jejunum (J) and ileum (I) om
chicken. Vesicles we e incuba ed (5, 10, and 30 s and 1, 2, and 30 min)
wi h 0.1 mMD-glucose, in he p esence o a Na
+
g adien ( illed symbols)
o in absence o a Na
+
g adien (emp y symbols). The ini ial a es (ex-
p essed as pmol D-glucose/(mg o p o ein pe s) we e 38.4 ±0.9, 37.9
±0.9, and 26.3 ±0.7, o D, J, and I, espec i ely). Resul s a e exp essed
as means ±SEM o h ee o ou sepa a e memb ane p epa a ions
pe segmen .
FIGURE 2. Speci ic phlo izin binding o he b ush-bo de memb ane
esicle o duodenum (D), jejunum (J), and ileum (I) om chicken. Values
a e means ±SEM o i e sepa a e expe imen s, exp essed as picomoles
o phlo izin bound pe millig am o p o ein a a phlo izin concen a ion
o 50 µM(B
50
). The B
50
alues (in pmol/mg o p o ein) we e: 50.1 ±2.7
(D), 51.2 ±1.9 (J), and 34.5 ±3.7 (I). *Signi ican di e ences (S uden ’s
- es ; P<0.05).
FIGURE 3. (Panel A) Wes e n blo analysis o sodium-glucose ans-
po e iso o m 1 (SGLT1) in he b ush-bo de memb ane esicle (BBMV)
o abbi small in es ine (Lane 1) and in BBMV om duodenum (D;
Lane 2), jejunum (J; Lane 3) and ileum (I; Lane 4) o chicken. Samples
(30 mg o p o ein/lane) we e blo ed using a abbi polyclonal an ibody
aised agains syn he ic pep ide co esponding o amino acids 564 o
575 o he deduced amino acid sequence o abbi in es inal SGLT1.
(Panel B) Rela i e abundance measu ed by op ical densi ome y. Resul s
show he means ±SEM o h ee di e en expe imen s. Molecula mass
s anda d is shown on he le . *Signi ican di e ences (S uden ’s - es ;
P<0.05).
so bing i wi h he an igenic pep ide. Figu e 3B shows
he densi ome ic analysis o h ee sepa a e assays ha
indica ed he abundance o SGLT1 in ileum was 30% less
when compa ed wi h duodenum and jejunum, wi hou
di e ences be ween hese p oximal segmen s.
Co ela ion Be ween Maximal T anspo
Ra es, Speci ic Phlo izin Binding, and
Densi ome ic Analysis o SGLT1
Immunoblo s
The e was a posi i e co ela ion be ween he ini ial
a es and bo h he speci ic phlo izin binding and he op i-
cal densi y de e mined by Wes e n blo analysis. These
co ela ions a e de ined by he equa ions y =0.73x +1.17
( =0.9954) and y =0.75x +1.03 ( =0.9967), espec i ely.
The ela i e alues o he h ee pa ame e s in duodenum,
jejunum, and ileum a e ep esen ed in he his og am o
Figu e 4. Iden ical pa e ns we e ound among he h ee
pa ame e s along he small in es ine. In all cases, he e
a e signi ican dec eases in he ileum in compa ison wi h
he p oximal segmen s.

GARRIGA ET AL.542
FIGURE 4. Rela i e alues o ini ial a es o D-glucose, speci ic bind-
ing using 50 µMphlo izin (Phz; B
50
), and op ical densi y de e mined by
Wes e n blo analysis. Resul s show he means ±SEM o h ee di e en
expe imen s. *Signi ican di e ences (S uden ’s - es ; P<0.05.
Lipid Composi ion o BBMV
B ush-bo de memb ane lipid composi ion o all seg-
men s s udied is shown in Table 2. The e was a signi ican
egional inc ease in he lipid/p o ein a io, wi h no di e -
ences be ween duodenum and jejunum, and a high a io
in ileum. The con en s o PL, ee a y acids, iglyce ides,
and choles e ol es e we e no modi ied. Highe alues
in he mola a ios o ee choles e ol/PL we e ound in
he duodenum and jejunum when compa ed o he ileum,
as a esul o an inc ease in ee choles e ol le els o hese
segmen s. No di e ences we e obse ed be ween he du-
odenum and jejunum.
The PL composi ion is shown in Table 3. The majo
PL in all s udied segmen s we e phospha idylcholine,
phospha idylse ine, and phospha idyle hanolamine.
Phospha idylinosi ol and sphingomyelin we e ound a
lesse concen a ions. Among he indi idual PL, duode-
num and jejunum had lowe phospha idylcholine le els,
which esul ed in lowe phospha idylcholine/sphingo-
myelin a io o hese segmen s when compa ed o he
ileum. No di e ences we e no ed o he o he phospho-
lipids o be ween duodenum and jejunum.
The a y acid composi ion in b ush-bo de memb anes
o di e en segmen s is gi en in Table 4. The majo a y
acids in all hese segmen s we e palmi ic (16:0), s ea ic
(18:0), oleic (18:1, n-9), and linoleic (18:2, n-6). A achidonic
acid was p esen al hough a low concen a ion. A de-
c ease in he le el o sa u a ed acids (mainly s ea ic acid)
oge he wi h an inc ease in he le el o unsa u a ed acids
TABLE 2. B ush-bo de memb ane lipid composi ion in duodenum, jejunum, and ileum o chicken
1
Lipid (% w /w ) Duodenum Jejunum Ileum
Lipid/p o ein (w/w) 0.64 ±0.07
b
0.65 ±0.03
b
1.8 ±0.4
a
To al phospholipid (PL) 74.2 ±2.3 64.2 ±4.7 69.0 ±3.0
F ee choles e ol (FC) 27.3 ±1.5
a
28.0 ±1.1
a
20.0 ±1.2
b
F ee a y acids 6.7 ±1.0 6.4 ±1.8 6.8 ±0.5
Choles e ol es e 4.7 ±0.2 3.4 ±0.3 5.5 ±1.0
T iglyce ides 3.9 ±0.7 3.4 ±0.8 2.5 ±0.6
FC/PL 0.38 ±0.01
a
0.44 ±0.03
a
0.28 ±0.02
b
a,b
Values in each ow wi h no common supe sc ip di e signi ican ly (P<0.05).
1
Values ep esen means ±SEM om a leas i e sepa a e memb ane p epa a ions.
(mainly oleic acid) was ound in he dis al segmen s.
These changes esul ed in a dec ease in he a io sa u-
a ed/unsa u a ed a y acids in ileal lipids.
Memb ane Fluidi y
The s eady-s a e luo escence pola iza ion (p) and lu-
o escence aniso opy (
s
) da a o DPH-labeled b ush-
bo de memb ane p epa a ions om all segmen s s udied
a e gi en in Table 5. In he duodenum and he jejunum,
bo h a iables we e highe han hose ound in he ileum,
indica ing ha he memb anes om p oximal small in es-
ine we e less luid. No di e ences we e obse ed be-
ween he duodenum and he jejunum.
DISCUSSION
The aim o his s udy was o es ablish he egional
di e ences in he ac i i y o SGLT1 in he small in es ine
o chickens and o analyze which mechanisms a e espon-
sible o hese changes. Fo his pu pose, we ha e mea-
su ed Na
+
-dependen D-glucose up ake, es ima ed he
numbe o SGLT1 anspo e s by he speci ic binding o
phlo idzin and Wes e n blo analysis, and s udied he
memb ane luidi y/lipid composi ion in BBMV isola ed
om chicken duodenum, jejunum, and ileum. Ou da a
indica e ha Na
+
-dependen D-glucose anspo is de-
c eased in he ileum when compa ed o he duodenum
and he jejunum. The ime cou se and o al up akes o
Na
+
-D-glucose co anspo we e s udied using sho -ci -
cui ed BBMV o a oid possible e ec s o egional changes
in he pe meabili y o elec oly es. The h ee in es inal
segmen s s udied showed a ansien o e shoo o D-
glucose. The ini ial a es and accumula ion a ios in he
ileum we e a ound 30% lowe han hose in he duode-
num and he jejunum (Figu e 1). These egional di e -
ences in he ac i i y o SGLT1 canno be a ibu ed o
a ia ions in esicle p epa a ions, as he pu i ica ion and
size o BBMV, as measu ed om he en ichmen o su-
c ase and Na
+
-dependen D-glucose up ake a equilib-
ium, espec i ely, we e simila in all h ee in es inal e-
gions (Table 1).
Simila esul s ha e been obse ed in he dis al small
in es ine om chicken (Fe e e al., 1994; Ro i a e al.,
1994), a s (F eeman and Quamme, 1986), monkey
(Ib ahim and Balasub amanian, 1995), mouse (Diamond
and Ka aso , 1984), and abbi (Thomson, 1984). How-
REGIONAL PROPERTIES OF THE SMALL INTESTINE 543
TABLE 3. B ush-bo de memb ane phospholipid composi ion in duodenum, jejunum,
and ileum o chicken
1
Phospholipid
2
(% w /w ) Duodenum Jejunum Ileum
PC 29.0 ±0.9
b
26.9 ±3.4
b
35.8 ±4.5
a
PS 29.3 ±2.9 33.7 ±2.4 29.0 ±6.7
PE 29.3 ±2.6 31.1 ±1.8 29.2 ±4.3
PI 8.0 ±0.6 7.5 ±1.4 6.7 ±2.3
SM 3.1 ±0.7 4.4 ±0.7 3.0 ±0.6
PC/SM 5.8 ±0.1
b
5.9 ±0.7
b
9.4 ±1.2
a
a,b
Values in each ow wi h no common supe sc ip di e signi ican ly (P<0.05).
1
Values ep esen means ±SEM om a leas i e sepa a e memb ane p epa a ions.
2
PC =phospha idylcholine; PS =phospha idylse ine; PE =phospha idyle hanolamine; PI =phospha idylinosi-
ol; SM =sphingomyelin.
e e , in ducklings, he maximal capaci y o anspo sug-
a s is ound in he ileum (Thomas e al., 1996).
To in es iga e whe he he egional changes in he
suga anspo we e co ela ed wi h changes in he den-
si y o SGLT1 p o ein along he chicken small in es ine,
he speci ic binding o phlo izin and Wes e n blo analysis
we e pe o med. When phlo izin was used as a compe i-
i e inhibi o o D-glucose up ake in he in es inal b ush-
bo de memb ane (Ga iga e al., 1999a), he speci ic
phlo izin binding in he ileum was lowe han hose in
he duodenum and he jejunum (a ound 30%), wi h no
signi ican di e ences be ween hese wo p oximal seg-
men s. When he speci ic phlo izin binding (B
50
) was plo -
ed agains he ini ial a e, a highly linea co ela ion was
ound, indica ing ha he obse ed changes in he Na
+
-
dependen D-glucose anspo along he small in es ine
a e di ec ly co ela ed wi h modi ica ions in he amoun s
o SGLT1 anspo e . Wi h adul chickens, Ga iga e
al. (1999a) desc ibed di e ences in he speci ic phlo izin
binding be ween jejunum, ileum, and ec um ha ully
accoun o he changes in he suga anspo a es ob-
se ed in chicken adap ed o die s con aining a ying
amoun s o Na
+
.
S udies on he densi y o SGLT1 p o ein by Wes e n
blo analysis showed a single band o 75-kDa immuno e-
ac i e p o ein in all in es inal egions analyzed. Resul s
o blo densi ome y showed egional changes in he
abundance o SGLT1 p o ein, wi h a dec ease in he
TABLE 4. B ush-bo de memb ane a y acid composi ion in duodenum, jejunum, and ileum o chicken
1
Fa y acid
(% w /w ) Duodenum Jejunum Ileum
14:0 1.2 ±0.3 1.9 ±0.2 1.6 ±0.3
16:0 20.9 ±0.7 23.1 ±1.2 21.2 ±0.7
16:1(n-7) 2.2 ±1.4 2.0 ±0.4 2.2 ±0.2
18:0 26.6 ±0.7
a
21.3 ±0.7
b
18.3 ±0.2
c
18:1(n-9) 13.1 ±0.2
c
16.0 ±0.4
b
18.2 ±0.7
a
18:2(n-6) 24.6 ±1.7 22.7 ±2.1 23.7 ±0.9
20:4(n-6) 7.3 ±0.9 6.7 ±0.4 8.5 ±0.5
Sa u a ed 48.7 ±1.7
a
46.7 ±0.3
a
41.3 ±0.7
b
Monounsa u a ed 16.7 ±0.5
b
19.7 ±1.3
a,b
21.9 ±0.6
a
Polyunsa u ed 51.3 ±1.7
b
53.5 ±0.4
b
58.9 ±0.6
a
Sa u a ed/unsa u a ed 0.96 ±0.07
a
0.88 ±0.02
a
0.70 ±0.02
b
a-c
Values in each ow wi h no common supe sc ip di e signi ican ly (P<0.05).
1
Values ep esen means ±SEM om a leas ou sepa a e memb ane p epa a ions.
b ush-bo de memb anes om he ileum when compa ed
o he duodenum and he jejunum, wi h no di e ences
be ween hese p oximal segmen s (Figu e 3). All hese
esul s show a clea co ela ion be ween SGLT1 p o ein
abundance and he SGLT1-media ed glucose anspo
along he chicken small in es ine.
In p e ious s udies (Ga iga e al., 1999a,b), we epo ed
no di e ences in he speci ic phlo izin binding and he
amoun s o SGLT1 p o ein de e mined by Wes e n blo
analysis be ween jejunal and ileal BBMV. In hese s udies,
we used 12-wk-old chickens adap ed o a die ha con-
sis ed o a mix u e o whea and ba ley (1:1) wi h a ca bo-
hyd a e con en o 626 g/kg die . The composi ion o his
die is signi ican ly di e en om he comme cial die
supplied o he 5-wk-old chickens used in he p esen
s udy. The comme cial die con ained hal o he amoun
o ca bohyd a e, 375 g/kg. This ma ked di e ence migh
explain an enhanced exp ession o hexose anspo e s in
he in es ine o animals ed wi h a die con aining highe
amoun s o suga s (Diamond and Ka aso , 1984).
Modi ica ions in he memb ane lipid composi ion and,
consequen ly, in memb ane mic o iscosi y could a ec
he unc ion o many memb ane-bound p o eins, such as
Na
+
-dependen D-glucose co anspo e (B asi us e al.,
1979; Ca ie
` e and Le G imellec, 1980; Fe na
´ndez e al.,
1984). The highes changes in memb ane lipid composi-
ion we e obse ed in apical memb anes om he ileum.
Dis al b ush-bo de memb anes we e shown o ha e
GARRIGA ET AL.544
TABLE 5. The s eady-s a e luo escence pola iza ion (pDPH) and s eady-s a e luo escence aniso opy (
s
)
in duodenum, jejunum, and ileum o chicken
1
Duodenum Jejunum Ileum
P
DPH
0.356 ±0.002
a
0.362 ±0.005
a
0.342 ±0.004
b
s
0.260 ±0.001
a
0.264 ±0.004
a
0.244 ±0.003
b
a-b
Values in each ow wi h no common supe sc ip di e signi ican ly (P<0.05).
1
Values ep esen means ±SEM om a leas eigh sepa a e memb ane p epa a ions.
highe mola a ios o lipid- o-p o ein, phospha idylcho-
line-sphingomyelin, PL- ee choles e ol, and unsa u-
a ed-sa u a ed han b ush-bo de memb anes om he
duodenum and he jejunum (Tables 3 o 5). These esul s
a e consis en wi h s udies o DPH luo escence pola iza-
ion ound in he ileum, which indica ed a dec eased
memb ane mic o iscosi y in he apical memb anes iso-
la ed om he ileum when compa ed o hose om he
duodenum and he jejunum. These esul s a e consis en
wi h he s udy o Ib ahim and Balasub amanian (1995)
in monkey small in es ine and Dudeja e al. (1989) in
human small in es ine, which showed ha he dis al po -
ion o he small in es ine was mo e luid mainly due o
egional changes in choles e ol, phospha idylcholine and
unsa u a ed a y acid con en . Howe e , s udies in a
(Heubi and Fellows, 1985) and abbi (Schwa z e al.,
1984) showed ha b ush-bo de memb anes isola ed
om ileum a e less luid han hose om duodenum and
jejunum, mainly due o inc eased con en o choles e ol
and a g ea e deg ee o sa u a ion in he a y acid compo-
si ion (Schwa z e al., 1985). These di e en esul s migh
be explained by di e ences in he animal species and by
he e ec s o di e en die s.
The highes memb ane luidi y obse ed in he ileum
om chicken in es ine migh indica e ha he SGLT1
u no e numbe in he ileum is highe han in he duode-
num and he jejunum. Howe e , when he u no e num-
be o SGLT1 p o ein was calcula ed as he ela ionship
be ween he ini ial a es o 0.1 mMD-glucose anspo
and he numbe o speci ic phlo izin binding si es mea-
su ed a a phlo izin concen a ion o 50 µM(Ga iga e
al., 1999a), no signi ican di e ences we e ound be ween
he h ee in es inal egions s udied indica ing ha he
highe luidi y o b ush-bo de memb anes isola ed om
he chicken ileum did no inc ease he u no e numbe
o he SGLT1 co anspo e in his segmen . Howe e ,
a ia ions in luidi y eco ded along he small in es ine
migh co espond o changes in localized domains o he
b ush-bo de memb ane and migh ha e li le e ec on
he luid en i onmen o he Na
+
-D-glucose co anspo e
(Le G imellec e al., 1992).
Al hough he e a e p e ious s udies demons a ing a
posi i e co ela ion be ween memb ane luidi y and Na
+
-
dependen D-glucose anspo in a (B asi us e al., 1979;
Heubi and Fellows, 1985; F eeman and Quamme, 1986)
o abbi (Schwa z e al., 1984; Thomson, 1984), he e is
a lack o ag eemen in he li e a u e and o he au ho s
demons a ed an in e se ela ionship be ween luidi y
and D-glucose anspo in in es inal o kidney b ush-
bo de memb anes (Ca ie
` e and Le G imellec, 1980; Fe -
na
´ndez e al., 1984).
In conclusion, we ound egional di e ences in he api-
cal Na
+
-dependen D-glucose up ake in BBMV ha a e
due o a ia ions in he exp ession o Na
+
-dependen D-
glucose anspo e , because he e was a dec ease in he
abundance o he co anspo e in he dis al pa o he
small in es ine. In addi ion, changes in he lipid composi-
ion and an inc ease in he luidi y o ileal BBMV we e
ound wi hou di ec e idence o co ela ion be ween
hese modi ica ions and Na
+
-glucose anspo . The e-
gional changes o he abundance o he co anspo e indi-
ca ed he p esence o molecula mechanisms ha egu-
la ed he exp ession o Na
+
-D-glucose co anspo e . Fu -
he s udies using speci ic cDNA o he Na
+
-D-glucose
co anspo e may p o ide a be e unde s anding o
his egula ion.
ACKNOWLEDGMENTS
The abbi polyclonal an ibody aised agains he syn-
he ic pep ide co esponding o amino acids 564 o 575
o he abbi in es inal SGLT1 sequence and he an igenic
pep ide we e kindly p o ided by M. Kasaha a (Teikyo
Uni e si y, Tokyo, Japan) and E. M. W igh (UCLA Uni-
e si y, Los Angeles, CA), espec i ely. We a e indeb ed
o M. Ga cı
´a-Rocha (Depa amen de Bioquı
´mica i Bio-
logia Molecula , Uni e si a de Ba celona, Ba celona,
Spain) o aluable echnical ad ice in Wes e n blo expe -
imen s. C. Ga iga was a ecipien o a Fo macio
´d’In es i-
gado s g an om Gene ali a de Ca alunya. This wo k
was suppo ed by g an s PB96/1255 om he Minis e io
de Educacio
´n y Cul u a and 1999-SGR-00271 om he
Gene ali a de Ca alunya (Spain).
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