University of Groningen
HEPATIC-PORTAL AND CARDIAC INFUSION OF CCK-8 AND GLUCAGON INDUCE
DIFFERENT EFFECTS ON FEEDING
STRUBBE, JH; WOLSINK, JG; SCHUTTE, AM; BALKAN, B; PRINS, AJA
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Physiology & Behavior
DOI:
10.1016/0031-9384(89)90345-4
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STRUBBE, J. H., WOLSINK, J. G., SCHUTTE, A. M., BALKAN, B., & PRINS, A. J. A. (1989). HEPATICPORTAL AND CARDIAC INFUSION OF CCK-8 AND GLUCAGON INDUCE DIFFERENT EFFECTS ON
FEEDING. Physiology & Behavior, 46(4), 643-646. DOI: 10.1016/0031-9384(89)90345-4
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Physiology & Behavior. Vol. 46, pp. 643~46. ©Pergamon Press ptc, 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Hepatic-Portal and Cardiac Infusion
of CCK-8 and Glucagon Induce
Different Effects on Feeding
J. H. S T R U B B E , J. G. W O L S I N K , A. M . S C H U T T E ,
B. B A L K A N A N D A. J. A. P R I N S
Department o f Animal Physiology, University o f Groningen, P.O. Box 14
9750 AA Haren, The Netherlands
R e c e i v e d 4 M a y 1989
STRUBBE, J. H., J. G. WOLSINK, A. M. SCHUTTE, B. BALKAN AND A. J. A. PRINS. Hepatic-portal and cardiac infusion
of CCK-8 and glucagon induce different effects on feeding. PHYSIOL BEHAV 46(4) 643~:r46, 1989.--In order to compare effects
of circulating CCK-8 and glucagon on food intake, rats were provided with a permanently implanted catheter in the right atrium.
Another cannula was implanted into the hepatic-portal vein by a new technique. After a standard fasting period graded loads of CCK-8
and glucogon were infused via these catheters during refeeding. Intracardiac glucagon and CCK loads dose-dependently suppressed
meal size. Intraportal infusion of glucagon caused similar suppression compared to intracardiac administration. This may indicate a
minor role of the liver as a target for the suppression of feeding by glucagon. In contrast, intraportal infusion of CCK-8 did not reduce
food intake. The results indicate that CCK-8 is removed or inactivated by the liver. It is suggested that CCK-8 acts locally on vagat
nerve endings to exert its suppressive action on food intake.
Food intake
Satiety
CCK-8
Glucagon
Liver
Hepatic portal vein
CHOLECYSTOKININ and glucagon are proposed to be satiety
signals in the control of food intake (5, 10, 11, 25). Since CCK is
secreted by the intestines and glucagon mainly by the pancreas
they reach the liver via the hepatic-portal vein. To test the satiety
hypothesis, CCK and glucagon should depress food intake when
they are infused into the portal vein. Recently some doubt raised
whether CCK acts via this route, as intraportal infusion of CCK-8
caused very little suppression of food intake in rats (7,8). In many
studies in this field glucagon and CCK-8 were administered by
intraperitoneal injection (4, 5, 10, 11). Although after intraperitoneal injection these hormones are taken up rapidly into the portal
circulation they may exert their suppressive effects by a direct
influence on the serosal side of the intestines (7,18). Some
evidence exists that in rats intraportally injected glucagon is
effective in suppressing food intake (12,25), probably via afferent
vagal pathways (13).
However, the comparison with infusion in the general circulation is needed to shed more light on the satiety effects of CCK-8
and glucagon and the possible site of action. Therefore graded
loads of glucagon and CCK-8 were infused during feeding after
moderate fasting periods via permanently implanted cannulas into
the right atrium and hepatic portal vein in rats.
room on a light-dark regime (LD 12:12). For practical reasons
lights were on from 0 - 1 2 hr and off from 12-24 hr. The rats were
housed individually in perspex cages. Food pellets of rat chow
(Hope Farms, The Netherlands) were available ad lib unless
otherwise stated. Room temperature was thermostatically controlled at 21 _0.2°C.
Cannulation Technique
The rats were provided with a jugular vein catheter with the tip
in the right atrium (20). During the same surgery procedure, the
rats were provided with a permanent cannula for infusion into the
portal vein. During the last decade we developed this new surgical
technique. This technique has three main advantages over other
techniques (16, 17, 23, 24) described in the past: 1) disturbances
to intestinal blood flow are minimal because only a very small part
of the cannula is inserted in the widest part of the portal vein, 2)
cannula longevity is enhanced such that cannulas can be used for
at least one month for blood sampling and for at least 2 months for
infusion, 3) no special stitch techniques as used in microsurgery
are required. In the following paragraphs this technique will be
described shortly.
The preparation of the cannula. A silastic medical-grade
tubing (Dow Coming, Midland, MI, Silastic 602-135, i.d. 0.51
mm, o.d. 0.94 mm) of 22 cm is beveled at one end to a point. At
a distance of l mm from the tip a hole was cut with a sharp cut 20
ga needle. Two rings of silicon glue were placed at l0 mm and at
METHOD
Animals and Housing
Male Wistar rats (350-400 g) were kept in a sound attenuated
643
STRUBBE ET AL.
644
40 mm from the cannula tip. Cannulas were sterilized before
surgery.
Surgery
The sterilized cannula was filled with heparin solution in saline
(500 U/ml). Under ether anesthesia the ventral abdominal wall was
exposed. The hairs at the place of the incision, i.e., 2-4 cm caudal
to the xyphoid process, were removed. The skin was sterilized
with a chlorhexidine solution. A midline incision of about 3 cm
was made and the duodenal loop was turned, so that the portal vein
was exposed. The venous part of the superior mesenteric vein
between the entrance of the gastroduodenal and superior pancreatico-duodenal vein was clamped very shortly (1-2 min). With a
20 ga needle a hole was made in the middle of this venous
compartment. Immediately thereafter the cannula tip was inserted
and pushed downstream till the silicon ring touched the vein. The
clamps were removed and the cannula tip was situated in the main
stream of the portal vein. A piece of silk thread (7-0, Ethycon
Perma Hand Seide) was fixed just behind the silicon ring. The
loose ends of the thread were slipped under the vein and tied in
such a way that the blood stream was not hindered. The second
silicon ring in the cannula was fixed onto the abdominal wall. This
ring prevents tension on the just implanted cannula. The cannula is
inserted under the skin and drawn to the skull where it is connected
to a stainless steel tubing (20 ga). Subsequently, the cannula is
anchored onto the skull with dental cement and filled with 50%
w/v polyvenylpyrrolidon (mol.wt. 25,000, Merck) in a heparine
solution 500 IU/ml as described earlier (20). The rats were allowed
to recover for about a week from surgery before they were used in
the experiments.
Feeding Behavior
Rats were enabled to gnaw food through vertical stainless steel
bars situated in front of a food hopper. Spillage was collected in an
undertray attached to the food hopper. Each food hopper's weight
was sampled by a programmed microprocessor every tenth of a
second. Every second the mean weight was calculated and stored
on a magnetic cassette tape. From this a computer retrieved the
size and timing of meals. An Esterline Angus event recorder was
used to monitor feeding behavior directly, thereby obtaining a
visual check on the computer data. Feeding sequences of at least
1 minute which were separated from each other by nonfeeding
intervals longer than 15 minutes were considered as separate
meals.
Experimental Procedures
Access to food was restricted by means of an automatic
horizontally sliding door situated in front of a food hopper. The
rats were deprived of food over the light phase and in addition over
the first 2 hr of the dark phase. Immediately after opening of the
doors they ate a large meal of fairly constant size and duration of
about 20 minutes. The effect of CCK and glucagon infusions was
tested on this meal. Previous work has shown that at this time there
is minimal interaction with the circadian control of feeding
behavior (22). Moreover, this method leaves the meal pattern in
the other part of the night unaffected. Therefore, this method is
less disturbing for a rat than the often used overnight fast.
Infusions
In order to prevent disturbances in the experiment by handling
the animals or by entering the experimental room, the rats were
connected with the pump 4 hr before the end of the deprivation.
This connection occurred by attaching a long polyethylene tubing
on the catheters ending on the skull. The tubing was kept tight by
a counterweight. A small swivel joint prevented torsion of the
tubing and a stainless steel spring around the tubing protected it
against biting (21). The door in front of the food hopper and the
infusion pump were activated simultaneously. The rats were
habituated to the infusion procedure for about a week with saline
infusions (0.9% NaC1 w/v) until they ate a constant amount of
food during the first meal after deprivation. Intraportal and
intracardiac infusions of glucagon and saline were randomly given
to the same animals (n = 6). Thereafter, the same approach was
used for CCK and saline (n = 6) in the same animals.
Solutions. The hormones were dissolved in saline 0.9% NaC1
w/v. In order to prevent binding of the hormones to the walls of the
infusion system, we added 0.1% w/v bovine serum albumin to the
solution. The rate of infusion was 0.075 ml/min. Glucagon
(NOVO) infusions of 75, 150 and 300 p~g/kg/hr were given
intravenously and 30, 75, 150 and 300 p.g/kg/hr via intraportal
routes. Intravenous infusions of CCK-8 (sulphated, Sigma) of 1.0,
1.5, 3.0, 9.0 and 15 Ixg/kg/hr and portal infusions at a rate of 3.0,
9.0, and 15 Ixg/kg/hr were given. All loads were tested twice.
After these experiments the rats were subjected to another
surgery. A small incision in the abdominal wall was made in order
to localize the portal vein cannula. At that time we had a visual
check on the proper functioning of the portal vein cannula. By
giving a saline injection into all cannulas we had a check on
possible leakage of these cannulas. In the experimental group no
leakage was observed. In some cases blood could be withdrawn.
This cannula was cut and the part connected with the portal vein
was tied off, so that the remaining part was suitable for intraperitoneal infusion. After recovery from surgery intraperitoneal infusions of saline and CCK (9 p.g/kg/hr) were given.
Data Analysis
MANOVA was applied to all data with dose as covariant. Dose
response relations were tested by a Spearman rank correlation. As
individual differences contributed substantially to the total variance, statistics on the drug and dose effects are applied to the
percentages of the control values, i.e., food consumed during
infusion of saline. Spearman Rank correlation and paired t-test
two-sided were applied to the means of the two tests for all
animals, doses and routes tested.
RESULTS
MANOVA on the complete dataset revealed a high variance in
individual intakes and interactions between animal and route of
administration as well as the hormone given. For that reason all
intakes were recalculated as percentages of the mean of two
control meals (i.e., during saline infusion) taken by each individual animal nearest in time to that particular experiment. Mean food
intake during saline infusions of the glucagon test amounted to
4.9 + 0 . 2 g and 5.3 -+0.2 g (n = 12) for intracardiac and intraportal
administration, respectively. For the CCK-8 experiment these
values amounted to 6.1 -+0.4 and 5.0-+0.3 g (n= 12), respectively. During glucagon infusion all doses caused significant
suppression of food intake [MANOVA: F(1,69)=72, p<0.001]
(Fig. 1). Spearman rank correlation between dose and percentage
of the food taken relative to that taken during saline infusion was
significant for both infusion routes via the jugular vein (n = 24,
CCK AND GLUCAGON EFFECTS ON FOOD INTAKE
645
120
12ol
100
100
80
80
60
E 60
-(3
0
0
q~
0
40
o 40
20
20
0
[
0
[
I
100
200
glucagon ( p g / k g / h r )
I
300
FIG, 1. Satiety effect of graded loads of glucagon expressed as percentage
of control intake when infused into the right atrium (O) or into the portal
vein (©) (mean+SEM, n=6).
r = - . 7 , p<0.005) and portal vein (n= 30, r = - . 7 , p<0.005).
There was a dose-dependent suppression of food intake when
CCK-8 was administered into the right atrium. The correlation
between CCK-8 dose and percentage of food intake was significant ( n = 3 6 , r = - . 8 ,
p<0.001) (Fig. 2). In contrast, when
CCK-8 was infused into the portal vein no dose-response effect
was found (n = 24, r = .2, p = 0.27) nor was any significant effect
seen of the drug with a paired t-test between intake in grams during
saline infusion and portal infusion of the higher doses (Fig. 2),
i.e., for 9 Ixg/kg/hr ( n = 6 , p =0.27) and for 15 ixg/kg/hr ( n = 6 ,
p = 0 . 7 ) , or computed for relative intakes with the same doses: 9
txg/kg/hr (n = 6, p = 0.6) and 15 ~g/kg/hr (n = 6, p = 0.80). There
was a significant suppression of food intake after intraperitoneal
administration of 9 txg/kg/hr CCK-8, resulting in 52--- 6% of the
intake during saline infusion ( n = 6 , p<0.01). Total daily food
intake was not affected by any of the treatments. The reason for
this is probably the short half-life of the hormones. During the rest
of the night-time there is enough time to compensate for the
relatively small reductions of food intake.
DISCUSSION
These experiments clearly show that glucagon caused similar
suppression during intraportal and intracardiac infusion, whereas
CCK-8 only reduced food intake when administered into the
general circulation or into the abdominal cavity. For obvious
reasons the liver will sense much higher plasma glucagon concentrations when glucagon is administered in the portal vein compared
with infusion into the general circulation. Yet there is no difference in effect. This may indicate that a functional target for the
suppressive effects of glucagon could be outside the liver. In this
respect it has been reported that intraportal glucagon is a potent
stimulus for insulin secretion (26). Since insulin may have satiety
effects (15), it could be a potential mediator of glucagon-induced
inhibition of feeding (26). There is also evidence that vagal
afferents are involved in glucagon-induced suppression of food
intake in chickens (9) and rats (13). It has also been reported that
0
I
0
I
I
5
10
CCK-8 (IJg/kg/hr)
I
15
FIG. 2. Satiety effect of graded loads of CCK-8 expre~ed as percentage of
control intake when infused into the right atrium (O) or irtto the portal vein
(©) (mean ---SEM, n = 6).
suppression of food intake by intraperitoneal glucagon injections
was blocked by selective denervation of the liver, suggesting that
the target for the suppressive effects of glucagon on food intake is
located in the liver (4). Further experiments are needed using
blood sampling and plasma assaying of glucagon to establish
whether glucagon reaches physiological levels during the infusions.
The absence of any reduction when CCK-8 was intraportally
infused suggests that intraportal CCK-8 may not suppress feeding
under normal physiological conditions. In contrast, the present
study also shows that intraperitoneal administration caused strong
suppressions of food intake, thereby confirming others (5, 7, 8).
This may indicate that CCK-8 given via this route may act on
intraabdominal receptors or probably on the serosal side of the
intestines or stomach. There is indeed much evidence that CCK-8
inhibits stomach emptying rate (1,18) and activates intestinal
motility (18). Moreover, cutting branches of the vagus nerve to the
stomach decreased CCK-8 induced anorexia (19). It is very likely
that intravenously given CCK-8 acts on the same place although
other areas like the brain and vagus nerve are also potential
candidates (14, 28, 29). But how does CCK-8 act when it is not
passing the liver in an active form? One possibility is a local
paracrine action as suggested by others (7,8). It is also possible
that CCK-8 is released as a hormone to the portal circuit and
reaches by way of diffusion or via arteriovenous connections in the
intestines the target areas for CCK. There is some evidence that
the liver clearance of the larger hormone CCK-33 is slower than
that of CCK-8 (2,6), which may result in low peripheral levels of
CCK-8. Therefore, intraportal CCK-33 may be more effective
than the octapeptide. CCK-8 has been found in significant quantities in plasma (3), but the precise origin is difficult to ascertain
(27). Considering the available evidence the most likely possibility
is that CCK-8 acts locally on vagal sensory nerve endings to exert
its suppressive action on food intake.
ACKNOWLEDGEMENTS
The authors wish to thank Dr. A. B. Steffens, J. E. Bruggink and Mrs.
J. Poelstra for their helpful comments and assistance.
646
S T R U B B E E T AL.
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