Peptides, Vol. 17, No. 4, pp.

609-614, 1996
Copyright 0 1996 Elsevier Science Inc.
Printed in the USA. All rights reserved
0196.9781/96 $15.00 + ..Xl

PI1 SO196-9781( 96)00017-4

ELSEVIER

Enterostatin in Gut Endocrine CellsI[mmunocytochemical Evidence

M. SijRHEDE,

Department

* C . ERLANSON-ALBERTSSON,
J. MEI,
AND F. SUNDLERt

T. NEVALAINEN,$

of Cell and Molecular

Biology, *Section for Molecular Signal&g and fDepartment

Research, University of Lund, Lund, Sweden
$Department of Pathology, University of Turku, Finland
Received

SORHEDE,

2 1 November

A. AHO$

ofMedical Cell

1995

M., C. ERLANSON-ALBERTSSON,

J. MEI, T. NEVALAINEN.
A. AH0 AND F. SUNDLER. Enterosfufin in gut
17(4) 609-614,
1996.-The
presence of enterostatin, a pcntapeptide
acting as a potential satiety signal in rats, was investigated in rat intestine by immunocytochemical
methods. Using antibodies
directed against the C-terminal part of enterostatin, the peptide was identified in endocrine cells in the antral part of the stomach
and in the small intestine of rat. The immunoreactive
cells were more frequent in the antrum and duodenum and became gradually
fewer towards the distal small intestine. In some of the labeled endocrine cells, a coexistence of enterostatin with serotonin was
revealed by immunocytochemical
double staining, implying that the cells were enterochromaffin
cells. In the pancreas, no enterostatin-immunoreactlve
cells were detected, indicating enterostatin to be included in its parent molecule, procolipase. In addition,
the existence of procolipase in the gastrointestinal
tract, including the pancreas, was investigated.
Procolipase immunoreactivity
was also identified, except in the pancreas, in chief cells in the fundus region of the stomach. The number of labeled cells declined
distally in the stomach, finally being absent in the intestine. Immunoreactive
enterostatin was measured with a specific ELISA
method. Intestinal content and serum were found to average 540 ? 70 and 50 t 4 nM, respectively.
Pancreatic duct ligation
strongly reduced the levels of enterostatin in intestinal content to 5.4 ? 1.5 nM (p < 0.001 ), and also reduced the serum enterostatin
level to 35 i 5 nM (p < 0.05). It is concluded that the peptide enterostatin in the rat is produced both in the exocrine pancreas,
as part of pancreatic procolipase, and in gut endocrine cells, both sources of peptide being important for the circulating enterostatin.
endocrine cells-inlmunocytochemical

Pancreatic

procolipase

Serotonin

evidence. PEPTlDES

Endocrine

Immunoreactivity

is a pentapeptide,
first identified as the activation peptide of pancreatic
procolipase
(6,7 ), being released through the action of trypsin during fat digestion in the
intestine. Colipase is known to act as a cofactor for pancreatic
lipase, but since procolipase
and colipase were found to activate pancreatic lipase with similar kinetics (17) interest was
focused on the enterostatin
molecule released. It was then
found that enterostatin
inhibited food intake in the rat when
given peripherally
(6,7) or centrally (25 ) , inducing early satiety ( 19). Given a choice of macronutrients,
enterostatin
furthermore specifically decreased fat intake or high-fat food intake in the rat (21).
Immunoreactive
enterostatin
has been determined
and
identified, using a specific ELISA method, in intestinal content
in rats (20) and humans. (4,8), as well as in human serum
after a satiating meal (4). Studies on the absorption of enterostatin through the intestine showed a transepithelial
passage
of intact peptide, but onlly to a low extent ( 14). It was thus
deemed to be of interest to investigate whether the enterostatin
found in the circulating
blood originated from sources other
than the pancreas.
to Maria Siirhede,

Gut hormone

In the present study, the existence of enterostatin/procolipase

in the gastrointestinal
tract, including the pancreas, was investigated using immunocytochemistry.

ENTEROSTATIN

I Requests for reprints shotild be addressed

Box 94, S-221 00 Lund, Sweden.

cells

METHOD

Production and Characterization of the Antisera

The antisera against enterostatin (4A-873) and procolipase
(3BI-16) were raised in rabbit. Synthetic enterostatin (APGPR)
was conjugated to thyroglobulin using MBS (m-maleimidobenzoyl-N-hydroxysuccinimide
ester, Pierce) as cross-linker (3).
The purity of the procolipase used for immunization was established by SDS-PAGE analysis followed by silver staining of the
gel. Only one band, corresponding
to procolipase, with a molecular weight of 10 kDa was revealed. Rabbits were immunized
with the enterostatin conjugate or procolipase in Freuds complete adjuvant. Booster injections with the antigen dissolved in
Freuds incomplete adjuvant were given at 3-week intervals.
The antisera 4A-873, R5181 (antienterostatin)
and 3BI-16
(antiprocolipase)
were tested for cross-reactivity
with other proteins using Western blot technique. Proteins (100 pglsample)

Department

609

of Cell and Molecular

Biology,

Section for Molecular

Signalling,

P.O.

610

SGRHEDE

from gastric and intestinal mucosa, intestinal content, and pancress were separated on SDS-PAGE and then blotted onto a nylon membrane ( ProBlott, Applied Biosystems, USA ) . The membranes were then immunostained
according to the method of
Wong et al. (29). Briefly, membranes were blocked with nonfat
dry milk and then incubated with the primary antibody. After
washing, the membrane was incubated with the peroxidase-conjugated secondary antibody and then developed by the addition
of DAB-H202. Pig procolipase and an RSA-enterostatin
conjugate were used as positive controls for the antiprocolipase
and
antienterostatin
antibodies, respectively.
The blotted proteins
from gastric and intestinal mucosa, intestinal content, and pancreas showed that neither 4A-873, R.5 18 1 (antienterostatin)
, nor
3BI-16 (antiprocolipase)
showed any cross-reaction
with other
proteins in the tissues examined.
The enterostatin antiserum 4A-873 was tested for cross-reactivity with various enterostatin peptides and fragments (Table 1) .
Competition studies showed that the antibody reacted specifically
with the C-terminal part of enterostatin. Peptide fragments missing the C-terminal arginine did not react with the antiserum. Because only VPGPR and CAPGPR were recognized by the antibodies and not GPR, it could be inferred that the structure PGPR
is required for cross-reactivity.
In data base searches, no other
peptides or proteins with PGPR at the C-terminal end could be
identified.

TABLE
CROSS-REACTIVITY
ANTISERUM
ENTEROSTATIN

OF
WITH

ET AL.

1
ENTEROSTATIN
SYNTHETIC

FRAGMENTS

AND

ANALOGS

Peptide Sequence

Cross-Reactivity (96)

APGPR

100

VPGPR

30

VPDPR

<I

APGP

<l

AP

<1

CAPGPR

100

APGPRC

<l

PGP

<l

VPGP

<l

GPR

<I

VP

<l

VG

<l

hematoxylin.
For controls, the primary
with preimmune rabbit serum.

antibody

was replaced

Animals
Immunocytochemistry
Double Immunostaining for the Identi$cation of Enterostatin.
Tissue specimens were collected from the stomach (fundus and
antrum) and from the duodenum, jejunum, ileum, colon, and the
pancreas of six rats. The specimens were fixed by immersion in
Bouin solution overnight, dehydrated, and embedded in paraffin.
Altemativelv. , thev were frozen in liauid urooane. cooled to the
temperature of liquid nitrogen, freeze-died,
vapor-fixed in diethyl pyrocarbonate
(DEPC),
and embedded
in paraffin in
vacua. Sections were cut at 6 pm and, after removal of the embedding medium, subjected to immunocytochemistry
using indirect immunofluorescence.
The rabbit enterostatin antiserum R5181 (3) was used at a
dilution of 1:320. A monoclonal antibody against serotonin (code
No. YC5/45, dilution 1:80, Seralab, Oxford, UK) and a guinea
pig antiserum against gastrin and CCK (code No. 8818, dilution
1:640, Milab, Malmii, Sweden) were used for double immunostaining. Simultaneous double immunofluorescence
requires primary antibodies raised in two different species and secondary
antibodies labeled with two different fluorophores. In this study,
the secondary antibodies used were FITC-labeled
(fluorescein
isothiocyanate)
goat anti-rabbit IgG and TRITC-labeled
(tetramethyl-rhodamine-isothiocyanate)
goat anti-mouse or goat antiguinea pig IgG. Controls in the double immunostaining
were run
to exclude inappropriate binding of the second antibodies. The
specificity of the enterostatin immunostaining
was tested by applying antiserum preabsorbed with the antigen in excess ( 10 mg
of synthetic peptide per ml diluted antiserum).
Identi$cation of Procolipase and Enterostatin With Vectastain ABC-AP. Tissues from the gastrointestinal
tract, including
pancreas, were fixed in 10% phosphate-buffered
formalin and
embedded in paraffin. Sections were collected on polylysinecoated slides, reacted with polyclonal rabbit antiprocolipase
(3BI-16) and antienterostatin <4k-873) antibodies, and the primary immunoreaction
was localized using a Vectastain ABC-AP
kit (avidin-biotin complex alkaline phosphatase) according to the
manufacturers
instruction (Vector Laboratories,
Burlingame,
CA).
The slides were then counterstained
slightly
with

Female Sprague-Dawley
rats (230-280
g) were obtained
from B&K U&ersal
(Sollentuna,
Sweden).
The rats were
housed individually in cages in a temperature-regulated
room (22
2 1C) with a 12-h light-dark cycle. The rats were fed standard
pellets (R3, Lactamin AB, Sweden) containing 20.4% casein,
5 1.5% starch, and 5.2% corn oil by weight and had free access
to tap water.

Animal Experiments
For ligation of the pancreatic duct, rats were anesthetized with
an IP injection of pentobarbital (20 mg/kg) and diazepam (3.5
mg/kg). A midline abdominal incision was made and the pancreas and duodenum exposed. The common bile duct was ligated
close to the duodenal papilla and a cannula (0.28 mm id. x 0.61
mm o.d., medical grade polyethylene tube, Portex, England) inserted into the proximal common duct. The other end of the cannula was inserted into the duodenum, distal to the ampulla of
Vater, enabling the reintroduction of the bile into the duodenum
while occluding pancreatic secretion. After 24 h, the rats were
sacrificed and the intestinal content, intestinal mucosa, and blood
were collected. In a control group, rats were sham operated and
sacrificed after 24 h.
Preparation

of Samples

Immediately after sacrificing the rats, 15 cm of the proximal

small intestine was removed and rinsed with 4 ml iced distilled
water. The intestinal content (2 ml) was boiled for 10 min and
centrifuged at 3000 X g for 5 min at 4C. The supematant was
stored at -80C and assayed for enterostatin immunoreactivity
the next day. The remaining unboiled intestinal content was frozen at -80C and assayed for pancreatic enzyme activity within
3 days.
Determination

of Pancreatic

Enzyme Activity

Determination of pancreatic lipase and colipase activity was

performed with pH stat titration (Mettler components DK 10,
DK 11, and DV 11) using tributyrin as substrate (2.16). Amylase

ENTEROSTATlN

IN GIJT ENDOCRINE

611

CELLS

sensitivity of the assay was 4 nM, the intra-assay coefficient

variation 3-5%, and the interassay coefficient &lo%.

of

Statistics
Values are presented as means + SEM. Differences between
individual data were compared with Students unpaired t-test.
Significance was considered if p < 0.05.
RESULTS

Enterostatin-Immunoreactive

Endocrine

Cells in the Gut

Enterostatin-immunoreactive
cells were seen in the antral part
of the stomach and in the small intestine, in cells having the
characteristic morphology of endocrine cells. The immunoreactive cells were most frequent in the antrum, where they occurred
basally in the glands [Fig. 1 (a)]. They were less numerous, but
regularly seen in the small intestine [Fig. 1 (b)] . The immunoreactive cells became gradually fewer towards the distal small
intestine, and were virtually absent from the terminal ileum and
the large intestine. Both methods of fixation described in the
Method section were found to be useful for the demonstration of
enterostatin-immunoreactive
cells, and the density and distribution of cells were similar.
The presence of enterostatin in endocrine cells was further
demonstrated using another rabbit antienterostatin antibody (4A873). The antibody stained endocrine cells in the intestinal epithelium and in the stomach mucosa (Fig. 2). There was no stain-

FIG. 1. Sections from gastric antrum (a), duodenum (b), jejunum (c),
and terminal ileum (d) immunostained for enterostatin with the antienterostatin antibody R5 18I. Enterostatin-immunofluorescent cells are
quite numerous in the antrum, moderate in number in the duodenum,
few in the jejunum, and totally absent in the terminal ileum. x 118.

activity was measured according to the method of Ceska et al.

(5 ) using the Phadebas reagent (Pharmacia, Uppsala, Sweden).
Trypsin activity was measured using Na-benzoyl-DL-arginine-pnitroanilide as substrate ( 10). The determination of all enzyme
samples was carried out in duplicate.
ELBA Assay of Enterostatin
The concentration of enterostatin in serum and intestinal content was determined by an ELISA technique as described by Mei
et al. (20), a modification after the original method (3), using
antiserum R5 18 1 (obtained from J. Hermon-Taylor,
St Georges
Hospital Medical School, London, UK). The specificity of the
antibodies was tested by competitive assays in which various
peptides were used to inhibit the binding to RSA-CGG-APGPRcoated plates (3). Significant inhibition was observed with the
enterostatin peptides APGPR and VPGPR, and with the fragment
PGPR. There was a complete absence of immunoreactivity
with
unhydrolyzed procolipase.
In the ELISA
assay, synthetic
enterostatin
(APGPR,
Biomedical Unit, University of Lund, Lund, Sweden) was used
as standard. The values Iof unknown samples were calculated
from the standard curve with a Softmax computer program (Softmax 2.3, Molecular Devices Corporation Menlo Park, CA). The

FIG. 2. Enterostatin-immunoreactive endocrine cells in rat stomach (A)

and jejunum (B) stained with the antibody 4A-873. ~12.

SCjRHEDE

612

ET AL.

intestinal content in rats fasted for 4 h. In serum, the immunoreactive enterostatin was found to be 50 ? 4 nM.
After ligation of the pancreatic duct for 24 h, there being no
detectable pancreatic enzyme activity in the intestinal content
(Table 2 ) , intestinal enterostatin was strongly reduced, from 540
? 72 to 5.4 2 1.5 nM (p < 0.001). At the same time, serum
enterostatin was reduced from 50 5 4 to 35 ? 5 nA4 (p < 0.05).
In contrast, serum amylase was significantly increased from 117
5 17 peat/l in normal, sham-operated
rats to 201 t 11 peat/l
(p < 0.05) following pancreatic duct ligation.
DISCUSSION

These experiments demonstrate three things: 1) the presence

of procolipase immunoreactivity
(IR) in exocrine cells of stomach and pancreas, 2) the presence of enterostatin IR in endocrine
cells of the gastrointestinal
tract, predominantly
in the antrum
and upper small intestine, and 3) the coexistence of enterostatin
IR with serotonin IR in endocrine cells.
Immunoreactive
enterostatin was not found in the endocrine
or the exocrine parts of the pancreas. The lack of immunoreactive
enterostatin in the exocrine pancreas, containing high amounts of
the precursor molecule procolipase, is due to the fact that the
specific antibodies used to identify enterostatin (R5 18 1 and 4A873) were directed against the C-terminal end of enterostatin,
which is not exposed in procolipase (3 ). In the pancreas, procolipase was identified with the antiprocolipase
serum (3BI-16)

FIG. 3. Sections from gastric antrum (a, b) and duodenum (c, d). Double
immunostaining
for enterostatin (a, c) and for serotonin (b, d). Enterostatin-immunoreactive
cells are identical to some serotonin-immunoreactive enterochromaffin
(EC) cells. A few single enterostatin-immunoreactive cells do not store serotonin [indicated with an arrow in (a)].

ing in the exocrine

or in the endocrine
part of pancreas
(data not
shown).
Double immunostaining
for enterostatin
and serotonin revealed the presence of immunoreactive
enterostatin in a subpopulation of serotonin-immunoreactive
enterochromaffin
cells
(Fig. 3). In both the antrum and the small intestine a few enterostatin-immunoreactive
cells seemed to lack serotonin. None of
the enterostatin-immunoreactive
cells was identical to gastrin or
CCK cells. No enterostatin-immunoreactive
cells could be detected in the pancreas.
Procolipase/Colipase
Pancreas

Imrnunoreactivity

in the Gut and

Procolipase-immunoreactive
cells were identified with the antiprocolipase antibody (3BI-16) in exocrine cells of the pancreas.
There was no staining in the islets of Langerhans [Fig. 4(A)].
3BI-16 also stained basal cells, possibly chief cells, in the fundus
region of the stomach [Fig. 4 (B )] . The immunoreactive
cells
declined distally in the stomach and were absent in the intestinal
epithelium.
Effect of Pancreatic Duct Ligation on Enterostatin
Intestinal Content and in Serum

Level in

Immunoreactive
enterostatin in the intestinal content of normal Sprague-Dawley
rats was found to be 540 ? 72 nM in

FIG. 4. Identification
of procolipase immunoreactivity
in rat pancreas
(A) and stomach (B). The antibody 3BI-16-stained
exocrine cells of pancreas and chief cells of fundic epithelium. X 12.

ENTEROSTATIN

IN GIJT ENDOCRINE

TABLE

613

CELLS

EFFECT OF PANCREATIC DUCT LIGATION ON LIPASE, COLIPASE,

TRYPSIN, AND ENTEROSTATIN IN INTESTINAL CONTENT
COIltd

(II = 8)

Lipase (U/ml)
Colipase (U/ml)
Trypsin (&ml)
Enterostatin (t&f)
Values are expressed

74 -+ 14
522 11
130 r 14
549 & 72

Ligation of the Pancreatic Duct

(n = 8)

N.D.
N.D.
N.D.
5.4 2 1.4

as means 2 SEM. N.D. = not detectable.

only in the exocrine cells [Fig. 4(A)], thereby demonstrating the

antibody to be specific against procolipase. With this antibody,
procolipase immunoreactivity
was identified in chief cells in the
fundus region of the stomach [Fig. 4( B )] . Chief cells are secretory cells and would be expected to secrete procolipase into the
gastric juice.
Enterostatin
immunoreactivity
was identified in endocrine
cells of the antrum and duodenum. The presence of enterostatin
in the gastrointestinal
mucosa was further substantiated by purification and sequencing of the peptide. Procolipase could not
be identified in these cells, using immunocytochemistry
or in situ
hybridization (26). However, a Northern blot analysis identified
procolipase mRNA in the duodenum of the rat (23). Thus, it is
possible that enterostatin is produced intracellularly by the hydrolysis of procolipase. Another possibility may be that enterostatin is produced from another molecule, independent of procolipase.
The production of enterostatin in both gut endocrine cells and
the exocrine pancreas, as part of procolipase, was further substantiated by the finding that pancreatic duct ligation, which totally inhibited the secretion of pancreatic procolipase into the
duodenum and hence enterostatin formation in the intestinal lumen, still gave a significant enterostatin concentration in serum,
although being slightly but significantly
reduced (Table 2).
Thus, the serum enterostatin is derived not only from pancreas
but also from the enterostatin-containing
endocrine cells of the
gut or from other, yet unidentified, sources.

The finding that after pancreatic duct ligation the pancreatic

amylase activity in serum was increased is due to a well-established phenomenon of back-flow
of enzymes into the serum
following duct ligation. The absence of an increase in serum
enterostatin in this situation further supports the release of enterostatin from pancreatic procolipase only following secretion into
the intestine where, after tryptic activation, the peptide is released
and absorbed ( 14).
A third finding in this study is the coexistence of the peptide
enterostatin with serotonin (Fig. 3). Such a coexistence of neurohormonal peptides and classical neurotransmitters
is common
( l&13,27). It may be mentioned in this context that, so far, no
peptide partner for serotonin has been identified in these parts of
the rat gut. Enterostatin may thus turn out to be the first peptide
hormone candidate identified in gastroduodenal
enterochromaffin cells in the rat.
The importance of the coexistence of enterostatin and serotonin is, at the present time, unknown. Serotonin is known to
specifically reduce carbohydrate intake ( 18), whereas enterostatin reduces fat intake (9,21,24). It can thus be speculated that
the two substances may have complementary
effects on appetite
regulation. Dexfenfluramine,
known to release serotonin, has
been shown in some recent studies to decrease fat intake (1,15).
This could be interpreted as a simultaneous release of enterostatin
under certain conditions.
Previous studies have shown an inverse relationship between
fat intake and pancreatic procolipase (22), suggesting that the
enterostatin derived from pancreatic procolipase is important for
the regulation of fat intake. The presence of enterostatin in endocrine cells of the intestine is potentially interesting because
intestinal infusion of fat in an isolated rat intestinal segment has
been shown to release enterostatin (28), and this could be a
mechanism for the satiating effect of fat ( 11) .

ACKNOWLEDGEMENTS

Professor John E. Bhmdell, University of Leeds, UK is thanked for

stimulating discussions. This work was funded by The Swedish Medical
Research Council (B95-03X-07904-09A),
A. P&hlssons Foundation,
The Swedish Society for Medical Research, and The Swedish Nutrition
Foundation.

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