Effect of a satiating meal on the concentrations of procolipase propeptide in the serum and urine of normal and morbidly obese subjects - Gut
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1520 Gut 1993; 34: 1520-1525
Effect of a satiating meal on the concentrations of
procolipase propeptide in the serum and urine of
normal and morbidly obese subjects
R C Bowyer, W M Rowston, A M T Jehanli, J H Lacey, J Hermon-Taylor
Abstract profound hyperphagia, have a 60% reduction in
The effect of a satiating meal on the serum and their fasting pancreatic procolipase content com-
urinary concentrations of procolipase propep- pared with normal rats lead to the suggestion that
tide (Ala-Pro-Gly-Pro-Arg, APGPR) immuno- the peptide might have a possible physiological
reactivity, as measured by enzyme linked role in appetite regulation.'5
immunosorbent assay (ELISA) specific for Further studies'5 in Sprague-Dawley rats
free APGPR, has been studied in normal showed that intraperitoneal or intravenous injec-
and morbidly obese human subjects. The tion of VPDPR changed the pattern but not the
normal subjects displayed a biphasic response overall consumption of food with a reduced
with coordinate increases in both serum intake at the beginning and increased intake at
and urine APGPR immunoreactivity both the end of a four hour test period. This early
occurring within the first two hours after the 'satiating effect' was dose dependent and most
meal. In two of three of the morbidly obese marked at a dose of 40 ,ug per animal, about
subjects, this early rise in APGPR concentra- equimolar to procolipase secretion during
tion in urine was not seen but was foliowed by a maximal stimulation with secretin and chole-
slow rise in urinary APGPR immunoreactivity cystokinin (CCK). Addition of purified porcine
at four to six hours. In both the normal and procolipase, corresponding to 2 [ig/g VPDPR
obese groups, the urinary immunoreactive after tryptic proteolysis, to the feed pellets of
signal was found to coelute with synthetic these rats lead to the loss of normal weight gain
APGPR on gel chromatography. In rats, pro- and maintenance of body weight. Once the
colipase propeptide (Val-Pro-Asp-Pro-Arg, normal diet was resumed, pellet consumption
VPDPR) specifically inhibits fat intake early in increased and weight gain returned to normal.
the postprandial period when given peripher- Further studies showed that this effect on
ally or centrally. This study suggests that animal weight and pellet consumption was not
in humans APGPR reaches the circulation seen with the addition of porcine colipase alone
shortly after feeding and is excreted in the and that there was a significant reduction in total
urine. These findings are consistent with the weight of food consumed at six hours when 10
hypothesis that human procolipase propeptide ig synthetic VPDPR was given intraperitone-
may also act as a satiety signal. In addition the ally.'6 This second finding was confirmed by
late appearance of the peptide in some of the Shargill et all7 although total consumption at 24
morbidly obese patients could be associated hours was unchanged with an intraperitoneal
with perturbation of appetite control in these dose of 19-6 ,ug.
subjects. Infusion of synthetic VPDPR (16-5-500 ng)
(Gut 1993; 34: 1520-1525) into the third ventricle of conscious Sprague-
Dawley rats over five minutes significantly
reduced food intake from 30 minutes to six
Intestinal fat digestion occurs as a result of the hours. At an effective dose of 16-5 ng (33 pM),
combined action of lipase and colipase.' Colipase VPDPR is one of the more potent agents to
is a heat stable, water soluble, non-enzymatic inhibit food intake after injection into the third
protein of molecular weight of about 10 kilo ventricle. 'I
Daltons (kDa).23 This cofactor is required for A significant dose dependent reduction in fat
lipase activity in the presence of bile salts above consumption after intraperitoneal VPDPR
the critical micelle concentration4 and acts by administration was shown in Osborne-Mendel
Departments of Surgery anchoring lipase in a one to one molar ratio to the rats, a species susceptible to obesity on high fat
R C Bowyer
A M T Jehanli bile salt emulsified triglyceride substrate.56 diets but not low fat high carbohydrate diets.
J Hermon-Taylor Colipase is secreted by the exocrine pancreas as This effect lasted up to four hours with a dose of
the precursor, procolipase and its activation 20 [tg per animal but was prolonged for up to six
and Mental Health
Sciences, St George's involves the tryptic cleavage of the Arg5-Gly6 hours with a dose of 40-80 [ig per animal. A
Hospital Medical School, bond releasing an N-terminal pentapeptide.7 compensatory increase in fat intake was seen
London This procolipase propeptide has an amino acid after six hours and at 12 hours the total fat intake
W M Rowston was the same as in the control group. 8 A
J H Lacey
sequence that seems to be highly conserved in
Correspondence to: nature with the sequence Val-Pro-Asp-Pro-Arg significant dose dependent reduction in fat
Professor J Hermon-Taylor, (VPDPR) in pig,8 ox,9, rat,'0 and horse" and intake was also shown with infusion of 100-400
Department of Surgery, Ala-Pro-Gly-Pro-Arg (APGPR) in man'2 and ng into the right lateral ventricle of Sprague-
Jenner Wing, St George's
Hospital Medical School, chicken.'3 The sequence VPGPR has also been Dawley rats. '9
Cranmer Terrace, London identified in the rat. 14 This homology along with The hypothetical action of VPDPR is that it is
SW17 ORE.
Accepted for publication the finding that the genetically obese Zucker rat released from procolipase in the duodenum
16 March 1993 (fa/fa), in which obesity is characterised by during digestion, is actively or passivelyEffect ofa satiating meal on the concentrations ofprocolipase propeptide in the serum and urine ofnormal and morbidly obese subjects 1521
70-
Meal absorbed, passes through the circulation to act
on a receptor site in the central nervous system,
and so inhibits, in the short term, further
60- consumption of food, specifically that food
c
C
c which contains fat. At present, free VPDPR has
4-
0 50- not yet been shown to be absorbed into the
o circulation nor specific receptors in the central
a) 40- nervous system identified.
The effect of APGPR on satiation is more
LC difficult to study in humans and has not so far
0~ been investigated. It is known, however, that
E synthetic APGPR has a very short half life (1522 Bowyer, Rowston,Jehanli, Lacey, Hermon-Taylor
consisted of 300 g 'Special Recipe' Muesli acetate, allowed to clot, and within 2 5 hours of
(W Jordan Ltd, Biggleswade, UK) and 300 g collection, centrifuged at 3000 g for 20 minutes,
fresh pasteurised whole cows milk, the whole the separated serum mixed with an equal volume
meal containing a total of 1191 kcal, 43-3 g of ELISA immunoassay buffer containing 50
protein, 170-4 g carbohydrate, 41-7 g fat, 39 g mM TRIS/HCL, 0-05% (wt/vol) casein, 3-1 mM
fibre, and 305 g water. The meal was consumed NaN3, 10 mM ethylenediaminetetra-acetic acid,
as rapidly as was comfortable within 15 minutes 0 05% (wt/vol) Tween 20 at pH 7X2-7*4, and
or stopped when satiation occurred. The mean suspended in a boiling bath for 10 minutes. Each
mass of meal consumed was 557 g for the normal sample was then centrifuged for five minutes at
and 308 g for the morbidly obese subjects. At 30 10 000 g and the supernatant labelled and stored
and 0 minutes before and at 15, 30, 45, 60, 75, at -20°C until assayed.
90, 120, 180, 240, 300, 360, and 420 minutes The stored aliquots of urine and serum were
after the onset of the meal, the subjects emptied later thawed at room temperature, thoroughly
their bladders, the volume of urine voided was whirlnixed, centrifuged for five minutes at
recorded and 10 ml urine immediately made 20 10 000 g, and the supernatant assayed using the
mM with zinc acetate. To minimise loss of APGPR ELISA for free APGPR that we have
APGPR immunoreactivity on storage, each previously described.20 All samples were assayed
urine sample was centrifuged at 3000 g for 20 at a 50% dilution in immunoassay buffer and
minutes, the supernatant suspended in a boiling corrected for dilution after subtraction of the
bath for 10 minutes, centrifuged for five minutes assay detection limit. Urinary APGPR excretion
at 10000 g, and the supernatant aliquoted and was calculated as a product of the urinary
stored at -20°C until assayed. Each subject had concentration for a given sample and the volume/
5 ml of blood sampled from a forearm vein hour of urine output over that sampling period.
immediately before and every 10 minutes for two Two 7 5 ml urine samples from one male
hours after the onset of the meal. Each blood subject at 75 and 90 minutes after the onset of the
sample was immediately made 20 mM with zinc meal, collected and stored as previously des-
cribed, were thawed at room temperature,
pooled, lyophilised, and then reconstituted by
Meal mixing with 6 ml distilled water. This pooled
2
2 20-
25-
A
n~ concentrated urine was then centrifuged at
10 000 g for five minutes and 1 ml of supernatant
chromatographed on a Sephadex G-25 column
(0 9x43 cm) in TRIS buffered saline (TBS)
containing 50 mM TRIS/HCl, 0-15 M NaCl,
+-
0 3*1 mM NaN3 at pH 7*3 at a flow rate of 39 5 ml/
2? 15- hour and 1 056 ml fractions collected and
0
x * assayed using the APGPR ELISA. Similarly,
0D three 8 ml urine samples collected at 240, 300,
r-
10- and 360 minutes after the onset of the meal from
0DO the third morbidly obese subject were pooled,
lyophilised, and reconstituted as before with
co 5- 6 ml distilled water. The resultant pooled con-
.
centrated urine was centrifuged and 1 ml
chromatographed and assayed as before. The
'I chromatography column was calibrated using
u
5 180 I 2 4
-61 )-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420
synthetic APGRP in 50 mM TRIS buffered
saline and the fractions assayed for APGPR.
Meal
STATISTICAL ANALYSIS
-* Mol
The results of sequential APGPR propeptide
2 2(
M02 assay of urine and serum were analysed usinig the
c --
M03 Wilcoxon matched pairs signed ranks test and
c
4-
0 the urinary APGPR concentrations and excre-
Co 1!9
(D tion from normal and morbidly obese groups
0
x analysed using the Kruskal-Wallis one way
a)
c: analysis of variance (ANOVA).
CL 1
Results
.D
APGPR CONCENTRATION IN SERUM
In the normal subjects the fasted mean serum
APGPR concentration was at the detection limit
60 90 120 150 180 210 240 270 300 330 360 390 420
-60 -30 0 30
of the assay but postprandially two peaks of
Mean time of sampling after meal (min) APGPR immunoreactivity were seen in the first
Figure 3: (A) Urinary APGPR excretion from normal healthy subjects (n=6) before and after and second hours after the start of the meal
a satiating meal expressed as concentration v midpoint in time of sampling period showing mean (Fig 1). Only the peak at 90 minutes, however,
values (SEM) with samples (*) differing significantly (pEffect ofa satiating meal on the concentrations ofprocolipase propeptide in the serum and urine of normal and morbidly obese subjects 1523
-M-
MOl
the APGPR urine concentrations between the
Meal normal subjects and MO 1 and M03 emphasising
Vn M03
-'-
-o- Normal
the differing appearance of the immunoreactive
signal between these two groups. The significant
difference at 45 and 360 minutes postprandially
between the results in the normal and these two
morbidly obese subjects cannot be accounted for
by a difference in the urine output (one way
ANOVA, p>0 05). A similar difference in
urinary APGPR excretion postprandially was
seen between the normal subjects and MOI and
cc
M03 and was significant at 15, 30, and 45
C) minutes after the start of the meal (data not
a)
shown).
.7
CHROMATOGRAPHY OF URINARY APGPR
Assay offractions ofpooled and lyophilised urine
from the second APGPR immunoreactivity peak
from one of the normal male subjects clearly
showed that the immunoreactive signal coeluted
-30 0 30 60 90 120 150 180 210 240 270 300 330 360 390 420 with synthetic APGPR suggesting that the
Time of sampling after meal (min)
immunoreactive peak was principally a result of
the native peptide (Fig 5A). Finally chromato-
Figure 4: APGPR nM in urine from normal healthy subjects (n=6) (mean (SEM)) and from graphy and assay of the lyophilised late pooled
two morbidly obese subjects (MOI, M03) before and after a satiating meal (*) differing
significantly (p1524 Bowyer, Rowston,Jehanli, Lacey, Hermon-Taylor
350 A Vo Vs concentration at four to six hours. If APGPR
were a satiety signal and the urine concentrations
mirrored circulating blood concentrations, then
300- the late appearance of APGPR in the circulation
could explain the loss of appetite control in these
250-v : morbidly obese patients. Whether this abnormal
250 | - * APGPR calibration response to the meal in these morbidly obese
2200- 11 [oPooled
-6- normal urine peak subjects is as a result of an intrinsic abnormality
or is a consequence of pancreatic hypofunction
seen in the morbidly obese30 is unclear. Larger
cL 150- numbers of subjects need to be studied to
determine the significance of these preliminary
100-
findings.
This research was supported by grants from the Medical Research
50- Committee of St George's Hospital and the South West Thames
Regional Health Authority.
The data describing detection of APGPR postprandially in
normal subjects have previously been published in abstract form
10 20 30 40 in Digestion 1991;49: 10-1.
50
100- B Vo Vs
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