The obese gene protein product leptin (LEP) is secreted
from adipocytes and acts primarily on the hypothalamus reg-
ulating energy expenditure, food intake, and body weight (16).
Although the intestine is not a classic target tissue for LEP,
extensive studies in various experimental models have estab-
lished that LEP determines important physiologic effects on
intestinal growth, cell maturation, and differentiation (17,18).
In our previous work, we have shown that LEP stimulates
intestinal adaptation after massive small bowel resection in a
rat (19). However, the mechanisms of this positive effect
remain poorly understood.
The purpose of this study was to evaluate the effects of LEP
on enterocyte turnover (proliferation and apoptosis) in con-
junction with LEP receptor (LEPr) expression along the vil-
lus-crypt axis after massive small bowel resection in the rat.
MATERIALS AND METHODS
Animals. Rappaport Faculty of Medicine (Technion, Haifa, Israel) Insti-
tutional Animal Care and Use Committee approved the animal facilities and
protocols. Adult Sprague-Dawley male rats weighing 240 to 260 g were kept
in individual stainless steel cages at constant temperature and humidity, and
a 12-h light-dark cycle was maintained. Rats were put on fasts 12 h before the
experiment with free access to water. General anesthesia was induced with
ketamine (i.p. 90 mg/kg) and xylasine (IP 15 mg/kg).
Experimental design. Forty rats were randomly assigned to one of three
groups: group A, Sham rats underwent bowel transection (Sham, n ⫽ 14);
group B, SBS animals underwent bowel resection (SBS, n ⫽ 13); and group
C, SBS-LEP rats underwent bowel resection (SBS-LEP, n ⫽ 13) and were
treated with LEP given i.p. at a dose of 50 mg/kg fromd4tod14.
Surgical procedure. Rats underwent one of two surgical procedures:
bowel transection followed by reanastomosis or 75% bowel resection. Using
sterile techniques, the abdomen was opened using a midline incision. In Sham
rats, the mid-small bowel was transected and reanastomosed without bowel
resection. In SBS animals, a 75% mid-small bowel resection was performed
similar to that previously described. This consisted of a resection of the bowel
between 5 cm distal to the ligament of Treitz and 10 cm proximal to the
ileocecal valve. Bowel continuity was restored by end-to-end anastomosis
using 6-0 absorbable suture (Vicryl, Ethicon Corporation, USA). For all
operations, the abdominal cavity was closed in two layers with a running
suture of 3/0 Vicryl (Ethicon Corporation, USA). Postoperative rats were
allowed ad libitum water and a liquid diet. The rats were killed on d 15 by i.p.
injection of pentobarbital (75 mg/kg).
Intestinal adaptation parameters. The small bowel was rapidly removed,
rinsed with cold isotonic saline, and divided into two segments: jejunum
proximal to anastomosis and terminal ileum. The intestine was split on the
antimesenteric border, washed with cold saline, dried, and each segment was
weighed. The mucosa was scraped from the underlying tissue using a spatula
(Sigma Chemical Co., Israel). Mucosal samples were homogenized with
TRIzol reagent. DNA and protein were extracted by Chomczynski method
(20) and were expressed as micrograms per centimeter of bowel per 100 g of
Histologic examination. Histologic sections were prepared from the prox-
imal jejunum, distal ileum, and comparable sites in the control animals.
Segments of small bowel were ﬁxed for 24 h in 10% formalin and processed
into standard parafﬁn blocks. Five-micron tissue slices were stained with
hematoxylin and eosin. The villus height and crypt depth were measured
using Image Pro Plus 4 image analysis software (Media Cybernetics, Balti-
more, MD). Ten villi and crypts in each section were measured, and the mean
reading was recorded in microns.
Laser-capture microdissection and RNA preparation. Fifteen-micron-
thick sections were mounted onto special RNase-free and UV-treated mem-
brane-covered slides (PALM Technologies, Bernried, Germany) and imme-
diately ﬁxed in ice-cold 70% ethanol for 2 min. After incubation for 60 s in
1% cresyl violet acetate, the sections were then dehydrated in an ethanol
series (70 and 100% on ice) and left to air-dry brieﬂy. Slides were stored at
⫺80°C until microdissection. Sections were laser dissected within 3 d. Slides
were observed using a Zeiss Axiovert 200 M inverted laser-capture micro-
scope and visualized on a monitor using the PALM Robo-Software. Villus
tips, lateral villi, and crypts were separated using the laser. RNA was
extracted using the RNeasy Microkit (Qiagen) and microdissection protocol.
All samples were put at ⫺80°C for long-term storage.
RT-PCR. The quality of RNA was evaluated using the Experion auto-
mated electrophoresis system (BioRad). Five micrograms RNA were reverse
transcribed to cDNA at 37°C using 200
M deoxynucleotides (Sigma Chem-
ical Co., St. Louis, MO), 5
M random hexamers (Amersham Pharmacia
Biotech, Piscataway, NJ), 20 U RNAguard (Amersham Pharmacia Biotech),
and 200 U/
L Moloney murine leukemia virus-reverse transcriptase (US
Biochemicals, Cleveland, OH). Thermal cycler settings were optimized to
ensure products were in the linear phase of production.
Real-time PCR. Expression of long form of the LEPr levels was deter-
mined by quantitative real-time PCR on the cDNA samples using TaqMan
assay-on demand kit (ABsolute Blue QPCR ROX Mix (ROX Dye) from
ABgene, Epsom, UK) with the ABI-PRISM 7000 (Applied Biosystems,
Foster City, CA). Single-exon primers (from PrimerDesing Ltd, UK) with
distance from 3⬘UTR-1064 bp (sense primer, GCAGGGCTGTATGTCATT-
GTA; anti-sense primer, GAACATGGTCCCAAAACAACTT) were de-
signed to produce an amplicon 109 bp. 18S (5⬘AGGAATTGACG-
GAAGGGCAC, 3⬘GTGCAGCCCCGGACATCTAAG) was used to access
equal cDNA loading for each compartment. The primers are speciﬁc for the
long form of the LEPr and they do not reside in the same exon; otherwise,
ampliﬁcation from contaminating DNA cannot be distinguished from ampli-
ﬁcation of cDNA.
Crypt cell proliferation and enterocyte apoptosis. Rats were injected with
standard 5-bromodeoxyuridine (5-BrdU) labeling reagent (Zymed Lab, Inc,
CA) at a dose of 1 mL per 100 g body weight, 2 h before sacriﬁce. Tissue
m) were deparafﬁnized with xylene, rehydrated with graded
alcohol, and stained with a biotinylated monoclonal anti-BrdU antibody
system using BrdU Staining Kit (Zymed Lab, Inc, CA). An index of prolif-
eration was determined as the ratio of crypt cells staining positively for BrdU
per 10 crypts.
m thick sections were prepared to establish the degree of
enterocyte apoptosis. Immunohistochemistry for Caspase-3 (Caspase-3
cleaved concentrated polyclonal antibody; dilution 1:100; Biocare Medical,
Walnut Creek, CA) was performed to identify apoptotic cells using a com-
bination of streptovidin-biotin-peroxidase method according to manufactur-
ers’ protocols. Expression of epithelial cell apoptosis is expressed as the total
number of apoptotic cells along this axis per 10 villi and 100 crypts. In some
cases, a more detailed analysis of the location of apoptosis was performed
using previously established techniques (21). For this, apoptosis along the
villi were differentiated between the lower one-third of the villi (lateral villi)
and upper one-third of the villi (villi tips). Apoptosis was recorded as the
number of apoptotic cells per 10 villi. A qualiﬁed pathologist blinded as to the
source of intestinal tissue performed all measurements.
Expression of Bax and Bcl-2 genes. Total RNA was isolated from frozen
mucosal samples (proximal jejunum and distal ileum) using TRIzol reagent
(GIBCO BRL, USA), as described by Chomczynski (20). A portion of total
g in a total volume of 25
L) was reverse transcribed using
Moloney murine leukemia virus (MMLV) First strand cDNA Synthesis Kit
(Gene Choice, Inc. Frederick, MD). After PCR, the ampliﬁed product (5
was run on a 2% agarose gel stained with ethidium bromide and photo-
graphed. The level of Bax and Bcl-2 gene expression was expressed as the
ratio of the gray density of the objective gene over the gray density of 18S at
densitometry. The sequences for the speciﬁc genes were as follows: Bax 5⬘
Bcl-2 5⬘TGAGGCCCTGTCTGCTTCTG, 3⬘AGGCTCCCGGGGCAGT-
CATGA (all primers were purchased from Sigma Chemical Co., Sigma
Chemical Co.-Aldrich Biotechnology LP). Because 18S RNA is expressed at
much higher levels than most mRNAs, the 18S rRNA primers was diluted
1:10 to bring the RT-PCR products within the same exponential range of
Statistical analysis. The data are expressed as the mean ⫾ SEM. A
one-way ANOVA test followed by Bonferroni post hoc test was used for
statistical analysis with p value ⬍ 0.05 considered statistically signiﬁcant.
Parameters of intestinal adaptation. Massive small bowel
resection resulted in a signiﬁcant decrease in body weight.
SBS rats (group B) had a signiﬁcantly lower ﬁnal body weight
compared with Sham rats (p ⬍ 0.001, Table 1). Treatment
with LEP resulted in a small but signiﬁcant increase in ﬁnal
body weight compared with SBS rats (p ⬍ 0.05). SBS rats
(group B) demonstrated a signiﬁcant increase in overall bowel
649LEPTIN AND ENTEROCYTE TURNOVER IN SBS