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604 Huin, Carriveau, Bianchi, Keller, Collet, Krémarik-Bauillaud, Damenjaud, Bécuwe, Schahn, Ménard, Dauça
et al. 1999). Severallines of evidence suggest an early
role of the fetal digestive tract in fat digestion. We
have reported that peroxisomes with fatty acid f3-oxidation
capacity are present in the fetal human gut (Dauça
et al. 1996). The latter exhibits functional mechanisms
to synthesize al! the lipid classes and to secrete them in
the form of lipoproteins (Levy et al. 1992; Basque et
al. 1998). Thus far, no studies have been devoted to
the expression of PPARs in the developing human fetal
digestive tract. Because the localization of these receptors
may help to clarify the physiological roles of
PPARs, this analysis was undertaken by immunohistochemistry
in esophagus, stomach, smal! intestine, and
colon of human fetuses from Week 7 to Week 23 of
development (WD). In this study we demonstrated a
spatial and temporal distribution of PPARs in developing
digestive tract that suggests a differential role
for these receptors during morphogenesis and cel! differentiation.
Materials and Methods
Tissue Specimens
Samples of esophagus, stomach, intestine, and colon from
23 fetuses ranging from 7 to 22 weeks of age were obtained
from normal elective pregnancy terminations. The project
was performed in accordance with the requirements of the
Institutional Human Subject Review Board (University of
Sherbrooke) for the use of human tissues. The latter were
embedded in Polyfreeze Tissue Freezing Medium (Polysciences;
Warrington, PA) and frozen in Iiquid nitrogen.
Production of Anti-PPAR Antibodies
As shown in Figure 1, the anti-PPARCl' antibody was raised
against the amino acid sequence 4sSSGSFGFTEYQYs6 of
human PPARCl' (Sher et al. 1993). The sequence 24EGAPE­
LNGGPQHAL37 of human NUCI (Schmidt et al. 1992) was
used to produce the anti-PPAR13 polyclonal antibody. The
anti-PPAR~/Y2 antiserum was raised against the amino acid
sequence EMPFWPTNFGISSVD common to PPAR)'l and
PPAR)'2 [amino acids 5-19 of mouse PPAR)'l (Zhu et al.
1993); 35-49 of PPAR)'2 (Tontonoz et al. 1994)]. The sequence
is weIl conserved in the two human PPAR)' isoforms
(Elbrecht et al. 1996; Fajas et al. 1997). Taking advantage of
the fact that the PPAR)'2 differs from the PPAR)'l by an additional
specific N-terminal amino acid region, the sequence
of the hapten used to produce the anti-human PPAR)'2 antibody
was mapped at that region and corresponded to
2GETLGDSPIDPESDS,6 of human PPAR)'2 (Elbrecht et al.
1996; Fajas et al. 1997). The synthetic peptides were coupIed
to keyhole limpet hemocyanin as a carrier according to
the glutaraldehyde method (Avrameas 1969). Polyclonal antibodies
were raised by SC injections into rabbits using standard
procedures. In addition, we used a commercial anti­
PPAR)' antiserum (Interchim; Montluçon, France) directed
against the amino acid sequence MMGEDKIKFKHITPL common
to PPAR)'l and PPAR)'2 (amino acids 256-270 of human
PPAR)'h 284-298 of human PPAR)'2).
Characterization of the Antibodies
The polyclonal antibodies produced were characterized by
immunoprecipitation and Western blotting assays. In vitro
transcription and translation of mouse PPARCl'/pSG5, PPAR13/
pSG5, PPAR)'l/pSG5, and human PPAR)'2/pBluescript IIKS+
plasmids (gift of Prof. W. WahIi; University of Lausanne, Switzerland)
were performed using reticulocyte lysate (Promega;
Charbonnières, France) and L-[35SJ-methionine. Translated
products were either immunoprecipitated with the antibodies
and analyzed by SDS-PAGE, followed by autoradiography,
or directly submitted to Western blotting and enhanced
chemiluminescence (ECL) in crossreaction assays.
Adult and fetal colon mucosae were homogenized in 25
mM Hepes buffer, pH 704, containing 004 M KCI, 1 mM
EDTA, 2 mM dithiothreitol, and a cocktail of protease inhibitors
(Complete; Roche, Mannheim, Germany). The homogenates
were centrifuged at 15,000 X g for 20 min (4C).
The protein concentration of the supernatant was determined
(Bradford 1976). Samples were analyzed by Western
blotting and ECL according to the manufacturer's protocol
(Boehringer Mannheim Biochemica; Mannheim, Germany)
using the different antibodies.
Nuclease Protection Assay
Partial human PPAR)'2 cDNA corresponding to the 5'UTR
sequence (Fajas et al. 1997) was obtained by standard RT­
PCR using total RNA extracted from human adipocytes
(Chomzynski and Sacchi 1987) and primers up (5'-CCCATC­
TCTCCCAAATATTT-3') and down (5'-GGGCCAGAAT­
GCGATCTCTGTG-3'). The resulting fragment (282 bp)
was cloned into the pBSIIKS+ plasmid (Stratagene; La Jolla,
CA), giving rise to the pBIIKS+/hPPAR)'2 vector. A pBIIKS+/
hG3PDH clone containing a 380-bp DNA fragment of the
human G3PDH encoding sequence (Tso et al. 1985) was
produced using the same protocol and primers up (S'-CCC­
ATCACCATCTTCCGA-3') and down (5'-CTACAGGCC­
ACAGTTTCC-3'). Nuclease protection assay was carried out
CIE 1
t
SSGSFGFTEYQY
102 166 281 468
LBO
1 PPAR.
74 138 253 441
LBO 1 NUCI
CIE
t
EGAPELNGGPQHAl
[] ~
1 ID' 173 2B8 478
t
EMPFWPTNFGISSVD
t
MMGEDKIKFKHITPl
0 ~
L80 1 PPARyl
1
+ 13' 203 t 318 506
t
GETlGDSPIDPESDS
LBD 1 PPAR"
Figure 1 Schematic comparison of the different human PPAR subtypes
vs PPARa. The positions of the peptides used ta produce the
polyclanal antibadies are mapped. The DNA (DBD) and ligand
(LBD) binding damains are represented.