Aust, J. Exp. Biol. Med. Sci., 61 (Pt. 5) 581-587 (1983)
© STARVATION IN THE RAT:
EFFECT ON PEPTIDES OF THE GUT AND BRAIN
by A. SHULKES*, Y. CAUSSIGNAC, C. B. LAMERS, T. E. SOLOMON,
T. YAMADA AND J. H. WALSH
(From the UCLA Health Sciences Centre and Centre for Ulcer Research and
Education, Los Angeles, CA, U.S.A.)
(Accepted for publication May 16,1983.)
Summary. The effects of starvation on the tissue concehtrations of some peptides
common to the gastrointestinal tract and the central nervous system have been
examined. Groups of 6 rats were either fed ad libitum or starved for up to 4 days
and killed by decapitation. Antrum, fundus, duodenum, jejunum, ileum, colon,
pancreas and brain were dissected, weighed and then frozen on dry ice. The
tissues were extracted sequentially in boiling water and 3% acetic acid, centrifuged
and the supernatants radioimmunoassayed for gastrin, cholecystQkinin (CCK),
vasoactive intestinal peptide (VIP), gastric inhibitory peptide (GIP) and somatostatin. Each peptide was not assayed in each tissue. Starvation had no effect on
the concentrations of peptides measured in the fundus (somatostatin and VIP),
ileum (somatostatin, GIP, VIP) and colon (somatostatin, GIP, VIP). VIP
concentration was increased in the jejunum and GIP was increased in both the
duodenum and jejunum. Antral gastrin was the only peptide in the gastrointestinal
tract to be decreased by food deprivation. Somatostatin concentration was approximately doubled in the antrum, duodenum, jejunum and pancreas. Brain VIP
was unchanged. Brain somatostatin and CCK were significantly reduced by
starvation. We conclude that starvation results in organ-specific and hormonespecific alterations in tissue concentrations of peptides of the gastrointestinal tract
and the central nervous system.
INTRODUCTION
Starvation produces a variety of compensatory metabolic and endocrine
changes (Palmblad et ah, 1977). Since many of the gastrointestinal peptides
are involved in the secretory and absorbtive response to food intake (Walsh,
1981), starvation may alter synthesis or release of these peptides. However,
the only gastrointestinal hormone which has been studied in detail in relation
to starvation is gastrin. Lichtenberger, Lechago and Johnson (1975)
reported that gastrin is decreased in plasma and antral tissue in rats fasted
Abbreviations used in this paper: VIP, Vasoactive intestinal peptide; GIP, Gastric inhibitory peptide; CCK, Cholecystokinin.
* Present address: Department of Surgery, University of Melbourne, Austin Hospital,
Melbourne, Victoria, Australia.
582
A. SHULKES ET AL,
for 4 days and postulated that food in the gastrointestinal tract is necessary
for the maintenance of normal plasma and antral gastrin. In contrast, pancreatic somatostatin was reported in a separate study to be increased following starvation (Shapiro et ai., 1979).
We have extended these studies by examining in the one study the effects
of starvation on the concentrations of various peptides in the entire gastrointestinal tract, some of which are released by a meal into the circulation
(gastrin, cholecystokinin, GIP) and others which do not circulate peripherally
(VIP, somatostatin) but act as either paracrine agents or neurotransmitters
(Walsh, 1981; Grossman, 1979). We report the tissue concentrations of
gastrin, somatostatin, VIP, GIP and CCK in the gastrointestinal tract and
brain of fed rats and rats starved for up to 4 days.
MATERIALS AND METHODS
Animals
Twenty-four male rats weighing between 168-217 g were divided into four groups of 6.
The body weight of each group expressed as mean ± SE (range) were 191±4(182-206),
191±6(168-208), 190±4( 179-206) and 198±5(181-217) g.
At the end of the fasting periods the rats were decapitated. The stomach was dissected
free from the fore-stomach and the remaining red oxyntic tissue (fundus) was separated
from the yellow tissue (antrum) surrounding the pyloric opening. The portion of the intestine
between the pylorus and the bile duct was considered to be duodenum. The remainder of the
small intestine was quartered and the proximal quarter was considered representative of
jejunum and the distal quarter, representative of ileum. The colon was taken in its entirety
including the caecum. The pancreas and brain, including the cerebellum and medulla, were
also collected. The tissues were washed in saline, blotted dry, weighed and quickly frozen
and stored at —20° until extraction 2 weeks later.
Tissue extraction
A standard method of sequential boiling water and boiling acid was used (Rehfeld,
1978; Lamers et al., 1980). Five parts of distilled water were added to the frozen tissue and
heated for 10 min in a boiling water bath. The tissue was then centrifuged for 15 min at
3000 rev./min and the supernatant (water extract) stored at —20° for up to 2 months. This
step would destroy the proteolytic enzymes and extract the acidic peptides such as gastrin. To
extract basic and neutral peptides, 3% acetic acid was then added to the pellet (5 ml/gm
tissue) and homogenized with a Polytron (Beckman) at maximum speed for 1 min. The
mixture was heated for 10 min in a boiling water bath, centrifuged at 3000 rev./min for
15 min and the supernatant (acid extract) stored at —20° for up to 2 months. The recoveries
of peptides through this extraction procedure has been reported previously from this (Yamada
et al., 1980) and other laboratories and generally exceeds 80% (Bryant et al., 1983;
Chayvialle et al., 1980; Rehfeld, 1978).
Radioimmunoassay
All extracts were prediluted at least 1:10 in 0 03 M sodium phosphate (pH 7-4), 0 07 M
NaCI to minimize non-specific effects. Final dilution in each assay tube was at least 1:100.
Appropriate dilutions were made so that values were Tead near the 50% inhibition dose
(ID50) of the different standard curves. The acid and water extracts were assayed separately
and the results combined. Details of the radioimmunoassays have been published. Gastrin
was measured using antiserum 1611 which recognizes gastrin peptides containing 10 or
more residues and a crossreactivity of 0 6% or less with CCK peptides (Walsh, 1974; Walsh,
Lamers and Valenzuela, 1982). CCK was measured using antiserum 5135 which crossreacts
PEPTIDES AND STARVATION
583
with the carboxy terminus of both gastrin-17 and CCK on an approximately equimolar basis
(Lamers et al., 1980; Walsh et al., 1982). Somatostatin was measured with antiserum 7812
which is specific for the ring portion of somatostatin (Yamada et al., 1980). The GIP antiserum was specific for GIP with no crossreactivity with other gastrointestinal peptides including
the structurally related peptides secretin, glucagon and VIP (Maxwell et al, 1980). VIP
antiserum 6 was directed towards the amino terminus of the molecule (Krejs et al., 1980;
Dimaline, Vaillant and Dockray, 1980).
Statistical analysis
The Student's t-test for unpaired values was used. Initial analysis showed that there was
no significant difference (p > 0 1 ) between the 1, 2 and 4 day starved groups, so these values
have been grouped.
RESULTS
The results are expressed as pmol per g wet weight. GIP was measured
only in the duodenum, jejunum and ileum. Gastrin was measured only in the
antrum and CCK only in the brain.
Antrum: Starvation resulted in a significant decrease in gastrin from 1330 ±
140 to 920 ± 130 (mean ± SEM) (p <0 05) (Fig. 1). Somatostatin more
than doubled from 268 dr 61 to 592 ± 45 (p <0 01) (Fig. 1). The antrum
had the highest concentration of somatostatin both in the control and the
starved group (Table 1). There was no significant change in VIP.
ANTRUM
VIP
SS
GASTRIN
* *
1500
*
^1000
o
T
J
600
NS
I
24
16
400
T
Q.
8
200
500
FED
ST
FED
ST
FED
ST
Fig. 1. Antral gastrin, somatostatin (SS) and vasoactive intestinal peptide (VIP) concentrations in fed (n = 6) and starved rats (ST) (n = 18). Results are expressed as mean ±
SEM.
NA Not significantly different frorn fed rats.
• p < 0 05
• • p < 0 01
584
A. SHULKES ET AL.
TABLE 1
Tissue somatostatin, gastric inhibitory peptide (GIP) and vasoactive intestinal peptide (VIP)
concentrations in fed (n = 6) and starved rats (n = 18). Results expressed as pmol/g,
mean ± SEM.
SOMATOSTATIN
GIP
FED
STARVED
STARVED
Antrum
268±61
592±45**
Fundus
232±30
270±24
Duodenum 56±6
79±8*
28±5
43±4*
Jejunum
16±2
35±2**
16 3±2 7
26 7 ±2 9*
Ileum
42±8
45±5
17 7 ± 5 0
17-7±20
Colon
89±18
106±8
ll±0-6
1 3±0 2
—
Pancreas 225±32
456±71*
* p <0 05 compared to fed rats
**p <0-01 compared to fed rats.
FED
VIP
FED
160+1 4
41±0 9
20-3±3-4
31±1.0
6 7±1 9
12.8±2-5
STARVED
20 8±4 .4
6 0±0 9
21±5 -6
7-4±0 .9*
10 6±1 •2
13-5 + 1 0
Fundus: Somatostatin and VIP concentrations were not changed (Table 1).
Duodenum: Somatostatin increased from 56 =t 6 to 79 ± 8 (p < 0 - 0 5 ) . VIP
was unchanged, while GIP increased from 28 ± 5 to 43 ± 4 (p <0-05)
(Table 1). These were the highest concentrations of GIP in both the control
and starved groups. Similarly, the highest concentration of VIP, 20 to 25
pmol/g, was in the duodenum (Table 1).
Jejunum: Somatostatin increased from 16 ±: 2 to 35 + 2 (p <0-01).
Starvation also increased VIP and GIP (p <0-05) (Table 1).
Ileum: There was no significant change in somatostatin, VIP and GIP
(Table 1).
Colon: There was no significant change in somatostatin, VIP and GIP
(Table 1).
Pancreas: Starvation increased somatostatin from 225 ± 32 to 456 ± 71
(p < 0 05) (Table 1). Only trace amounts of VIP were detected in the
control and starved groups.
Brain: Starvation decreased somatostatin from 165 ± 24 to 117 ± 12
(p < 0 05), and CCK from 10-9 ± 2 to 7-2 ± 0 - 5 (p <0-05) (Fig. 2 ) . VIP
was not changed.
DISCUSSION
Our study shows that starvation is associated with organ-specific and
hormone-specific alterations in tissue concentrations of the gut peptides. The
decrease in antral gastrin reported previously (Lichtenberger et ah, 1975)
has been confirmed, but this was the only hormone in the gastrointestinal
tract which decreased during starvation.
A consistent finding was the increase in somatostatin concentration.
Increases were seen in the antrum, small bowel and pancreas. This contrasts
with Shapiro and colleagues (1979), who reported that somatostatin was
increased only in the pancreas after starvation, and that of Voyles et al.
(1982) who observed increases only in the stomach. Changes in tissue
concentrations are not necessarily paralleled by changes in circulating levels.
PEPTIDES AND STARVATION
585
BRAIN
SS
VIP
150
12
- 100
o
8
CCK
12 -
E
T
*
O)
NS
8
T
Q.
50
•
FED
ST
FED
ST
FED
ST
Fig. 2. Somatostatin, VIP and cholecystokinin concentrations in the brain. Symbols as in
Fig. 1.
However, somatostatin has only a minor role as a circulating hormone.
Somatostatin is released into the portal circulation, but may act only as a
local endocrine agent, since very little enters the general circulation
(Berelowitz et al, 1978). Its major mode of action may be as a paracrine
agent or neuro transmitter (Costa et al, 1977; Vale, Rivier and Brown,
1977). Schusdziarra et al (1980) has suggested that somatostatin reduces
the rate at which ingested nutrients enter the circulation—-a physiological
regulator of nutrient homeostatis. The increase in gastrointestinal and pancreatic somatostatin in the starved group is consistent with somatostatin
having a role in the regulation of nutrient flux. However, the measurement of
tissue levels cannot distinguish between increased synthesis or decreased
release.
In contrast to somatostatin, starvation decreased antral gastrin concentration. It has previously been proposed that the release of gastrin (e.g. by
food) is necessary to maintain normal antral gastrin levels (Lichtenberger
et al, 1975). The inverse relationship between antral gastrin and somatostatin reported in the present study has also been shown with circulating
gastrin and somatostatin using in vivo (Gustavsson and Lundquist, 1978)
and in vitro models (SafEouri et al, 1979). For instance, antral perfusion
with alkali increases antral vein gastrin and decreases antral vein somatostatin
(Gustavsson and Lundquist, 1978).
Basal plasma GIP increases during starvation in man (Willms, Ebert
and Creutzfeldt, 1978). This has been explained in part by the simultaneous
586
A. SHULKBS ET AL.
fall in insulin reducing the feedback inhibition of GIP (Willms et al, 1978).
Thus, the increased concentration of GIP in the duodenum and jejunum in
the present study may reflect an increased synthesis. VIP in the small intestine is localized to enteric nerves (Dimaline and Dockray, 1979). There was
a general tendency for VIP to be increased by starvation, but this was only
significant in the jejunum. The meanings of these changes are unclear.
The brain was the only tissue in which somatostatin was decreased.
Brain CCK was also decreased. It has been proposed that CCK and somatostatin are satiety factors since infusions of these peptides reduce food intake
(Woods et al, 1981). Genetically obese mice have been reported to have
less brain CCK (Straus and Yalow, 1979), although this has not been confirmed (Hansky and Ho, 1979), and obese Zucker rats to have less hypothalamic somatostatin (Voyles et al, 1982). Production of a satiety factor
during food deprivation is inappropriate. Thus, the reduced brain CCK and
somatostatin in the starved rats are consistent with the proposal. Although
the whole brain was assayed, the majority of the measured CCK would
originate from the cortex, so the changes observed reflect primarily changes
in cortical CCK (Rehfeld, 1978).
Starvation is associated with compensatory endocrine changes which
act to maintain an adequate energy supply. For instance, increases in adrenal
and decreases in thyroid hormones serve to mobilize glucose and reduce
metabolic rate (Palmblad et al, 1977). The present study shows that starvation also produces significant changes in the gastrointestinal peptides. How
these changes influence the metabolic adaptation to starvation is unclear.
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