Appetite (2001) 37, 9±14
doi:10.1006/appe.2001.0407, available online at http://www.idealibrary.com on
1
Original Article
Reduced gastrin releasing peptide in
cerebrospinal fluid after recovery from
bulimia nervosa
G.K. Franka,W.H. Kayea, E.E. Ladenheimb and C. McConahaa
a
b
School of Medicine, Department of Psychiatry,Western Psychiatric Institute and Clinic,University of Pittsburgh;
School of Medicine, Department of Psychiatry and Behavioral Sciences, The Johns Hopkins University, Baltimore
(Received 20 September 2000, revision11April 2001, accepted in revised form 20 April 2001)
People with anorexia (AN) and bulimia nervosa (BN) have altered patterns of eating. It is possible that alterations of
the neuropeptide gastrin releasing peptide (GRP), a bombesin (BBS) -like peptide with potent central anorexigenic
activity, could contribute to disturbed eating behavior. To avoid the confounding effects of pathologic eating
behavior, we measured cerebrospinal fluid (CSF) GRP concentrations in women who were long-term recovered ( >1
year, normal weight, and regular menstrual cycles, no bingeing or purging) from AN (REC AN, N ˆ 12) or BN
(REC BN, N ˆ 21) compared to healthy control women (NC, N ˆ 15). CSF GRP was significantly lower
(2 ˆ 941(3), p < 001) in REC BN (96 31 pg/ml) compared to NC (134 55 pg/ml) and REC AN (116 29 pg/
ml). Persistent GRP abnormalities after recovery from BN raise the possibility that this alteration might be traitrelated and contribute to episodic hyperphagia in BN.
# 2001 Academic Press
Introduction
The eating disorders (EDs) anorexia (AN) and bulimia
nervosa (BN) are characterized by disturbed eating
behaviors and comorbid disturbances of mood, impulse
control, and temperament. Women with AN show a
pattern of food restriction and emaciation; however,
some AN are pure food restrictors, whereas others also
show binge-purging type behaviors. Women with BN are
usually at a normal body weight, and engage in a pattern
Supported in part by grants from NIMH #2 R01 MH
42984-09 ``The Neurobiology of Feeding Behavior in
Bulimia''; Children's Hospital Clinical Research Center,
Pittsburgh, PA (#5M01 RR00084), and NIMH #2 R01
MH 46001-07A1 ``Serotonin: A Trait Disturbance in
Anorexia Nervosa''.
Address correspondence to: Walter H. Kaye, M.D.,
University of Pittsburgh, Western Psychiatric Institute and
Clinic, Room E-724, 3811 O'Hara Street, Pittsburgh, PA
15213, U. S. A. Fax: ‡1 412 624 6618.
Email: kayewh@msx.upmc.edu
0195±6663/01/040009+06 $35.00/0
of food restriction alternating with regular binge-purge
episodes. The etiology of EDs is not known (American
Psychiatric Association, 1994). Recent studies suggest
that EDs are familial and that a genetic predisposition
might contribute to their etiology (Lilenfeld et al., 1998;
Strober et al., 2000).
A number of neuropeptides contribute to the modulation of appetite regulation (Kalra et al., 1999). It is
theoretically possible that a disturbance of the modulation of one or more neuropeptide systems plays a role
in disturbed eating behavior in humans. One neuropeptide system of interest is human gastrin releasing
peptide (GRP). GRP is a 27 amino acid peptide that
shares a similar decapeptide with bombesin (BBS;
Brown et al., 1980), and belongs to the family of
``bombesin-like peptides.'' BBS was first isolated from
amphibian skin (Anastasi et al., 1971) and showed
anorexigenic effects when administered peripherally or
centrally in mammals (Gibbs and Smith, 1988). GRP is
not as potent as BBS, but otherwise has similar
effects (Morley et al., 1985). Peripheral and central
# 2001 Academic Press
10
G. K. Frank et al.
administration of GRP attenuates food intake in
mammals and humans (Bray 1995; Flynn, 1994). Peripherally, BBS-like receptors are distributed throughout
the gastrointestinal (GI) tract, with receptors that have
specifically high affinity for GRP (von Schrenck et al.,
1990). They take part in the regulation of GI hormone
and enzyme secretion, cell growth, smooth muscle
activity and blood glucose levels (Dietrich, 1994; Walsh
et al., 1981). In the central nervous system (CNS), distinct BBS-like receptor subtypes have been identified in
brain tissue such as the bed nucleus of the stria terminalis, the olfactory tubercle, the putamen and neocortex,
with a neuromedin B and a GRP preferring subtype
(Ladenheim et al., 1992; Wolf & Moody, 1985). Both
subtypes have been implicated in the modulation of
BBS-like peptide induced food suppression (Ladenheim
et al., 1996). In addition, central GRP also modulates
temperature regulation and grooming behavior in animals (Cowan, 1988). Disturbances of neuropeptides
could be a consequence of disturbed eating behavior or
malnutrition, or pre-morbid traits that contribute to a
vulnerability to develop an ED. Determining whether
abnormalities are a consequence or a potential antecedent of pathological feeding behavior is a major
question in the study of eating disorders. It is impractical
to study people with ED prospectively due to the young
age of onset and difficulty in pre-morbid identification
of people who will develop an ED. Therefore, we decided to study women who had recovered for more than a
year from AN or BN. Any persistent psychobiological
abnormalities might be trait-related and potentially
have contributed to the pathogenesis of this disorder. In
this study we measured cerebrospinal fluid (CSF) values
of human GRP, a human BBS-like peptide. We hypothesized that AN would have increased and BN
decreased CSF values of GRP.
Materials and methods
Thirty-three women who had previously met DSM-IV
criteria for an ED were recruited: 21 women recovered
from normal weight BN (REC BN), and 12 subjects
recovered from binge eating/purging (eight) or restricting type (four) AN (REC AN). The REC BN women
had never had AN. Recovered ED subjects were
compared to 15 female normal controls (NC), who
were recruited through advertisements in local newspapers and who were without lifetime psychiatric or
major medical illnesses. In the year before the study, all
participating recovered subjects had to maintain a
body weight of > 90% average body weight (ABW;
Metropolitan Life Insurance, 1959); have regular
menstrual cycles; have not binged, purged (i.e. not
engaged in self-induced vomiting, the use of laxatives
or diuretics), excessively exercised, or engaged in
restrictive eating patterns; and have not met criteria
for substance-use related disorders. Subjects were free
of medication for 6 weeks prior to the study and gave
informed consent. The control women (NC) had no
history of an eating disorder or any psychiatric,
medical, or neurologic illness. They had normal
menstrual cycles, and had been within a normal weight
range since menarche. They also had no first degree
relatives with an eating disorder. Methods and
psychological assessment data are described in detail
elsewhere (Kaye et al., 1998a, 1999). For many of the
subjects, CSF 5-hydroxyindole acetic acid had been
measured previously.
A lumbar puncture (LP) for obtaining CSF samples
was performed during the first 10 days of the follicular
phase of the menstrual cycle. Subjects were admitted to a
research laboratory on the Eating Disorders Unit of
Western Psychiatric Institute and Clinic at 9 a.m. on the
day prior to the LP for adaptation to the laboratory and
for psychological assessments. Subjects fasted overnight, and the LP was performed the next day between 9
a.m. and 9:30 a.m. LP samples were frozen in liquid
nitrogen and stored at ÿ80 C. For the GRP assay a
commercial kit for radio immuno assay (RAI) from
Peninsula Laboratories, Inc., San Carlos, CA, was used.
The antibody in that kit shows 100% cross reactivity
with human GRP, as well as with BBS. No cross reactivity is reported for human gastrin I, human big gastrin1 peptide, gastric inhibitory peptide, or substance P. The
variability of the assay between runs was 4 3%
(mean SD). Plasma -hydroxybutyrate (BHBA), a
ketone body, was assessed as an index of starvation.
BHBA was measured by the enzymatic method of
Williamson et al. (1962).
Statistical analyses were performed using the SPSS
software package (SPSS, Chicago, IL; Barcikowski
1984). Data exploration assessed whether data were
normally distributed (Shapiro-Wilk test). When the
assumption of normality was met, One-Way ANOVA
assessed whether group means were similar, and
Scheffe's post hoc tests compared individual group differences. All variables aside from age and CSF GRP
were normally distributed. When the assumption of
normality was not met, Kruskal-Wallis test assessed
three group comparisons, and Mann-Whitney U tests
assessed individual group differences. Correlations were
investigated using Pearson Correlations for normally
distributed and Spearman Correlation Coefficients for
non-normally distributed variables. All data are
expressed as mean standard deviation (SD). Group
differences were considered significant if p < 005.
Gastrin releasing peptide and bulimia nervosa 11
Results
Age was similar between groups (Table 1). However,
% ABW at the time of the study was higher in REC
BN compared with NC and REC AN, and in NC
35
30
CSF GRP (pmol/ml)
25
20
15
compared with REC AN. Lifetime high % ABW was
higher in REC BN compared with NC and REC AN.
Lifetime low % ABW was lower in REC AN
compared with NC and REC BN. BHBA was similar
between groups. There was no difference between
length of recovery between groups.
One CSF GRP value for the NC group was excluded
as a non-true outlier (195 pg/ml). There was a significant
difference between group CSF GRP levels (Table 1).
Figure 1 shows a scatterplot with the distribution of the
GRP values across groups. The CSF GRP in REC BN
showed some overlap in distribution with NC and AN.
However, the majority of CSF GRP data points in REC
BN women was below 10 pmol/ml (15/21), whereas the
majority of data points for NC (12/15) and REC AN (9/
12) was above 10 pmol/ml. Separate comparisons
showed lower CSF GRP values in REC BN compared to
NC (p ˆ 0003; when applying Bonferroni correction
p < 0009), and REC AN (p < 005, p ˆ 01 with
Bonferroni correction), and similar values when comparing NC and REC AN (p ˆ 04). There was no significant relationship between CSF GRP and age, body
weight, or duration of recovery.
Discussion
10
5
0
NC
(N = 15)
REC AN
(N = 12)
REC BN
(N = 21)
Figure 1. Scatterplot comparing the distribution of cerebrospinal fluid (CSF) gastrin releasing peptide (GRP)
between groups. NC, normal controls; REC AN, recovered
anorexic women; REC BN; recovered bulimic women.
Consistent with a portion of our hypothesis, REC BN
women had reduced CSF GRP levels although no
abnormalities were found in REC AN subjects. To our
knowledge, BBS-like immunoreactivity has not been
previously assessed in people recovered from an ED.
However, subjects who were studied while ill with AN
had normal CSF BBS. In addition, schizophrenics had
lower CSF BBS, whereas CSF BBS was not related to
anxiety, depression, or mania (Gerner & Yamada,
1982; Gerner et al., 1985; Saiz Ruiz et al., 1992).
The finding of reduced CSF GRP levels after recovery from BN raises the question of whether this
Table 1. Clinical and biological data on subjects
NC (N ˆ 15)
Age (years)
Length of recovery (months)
% ABW
High % ABW
Low % ABW
BHBA (mmol/ml)
CSF GRP (pg/ml)
222 31
Ð
103 8a,c
108 7c
95 7c
1328 967
134 55c
REC AN (N ˆ 12)
239 33
33 21
94 7b,c
105 13c
68 8d
2014 1505
116 29
REC BN (N ˆ 21)
267 64
39 25
112 10d
126 15d
96 8c
1131 1074
96 31d
p <
2
ˆ 59
F ˆ 05
F ˆ 181
F ˆ 146
F ˆ 556
F ˆ 18
2 ˆ 94
ns
ns
000
000
000
ns
001
NC, Normal control women; REC AN, women recovered from anorexia nervosa; REC BN women recovered from bulimia nervosa; % ABW
average body weight; BHBA; -hydroxybutyric acid; EDI (numbers in parentheses indicate available number of subjects data), Eating
Disorders Inventory-2. All data are expressed as mean SD; One way ANOVA (F, df ˆ 2) for normally distributed values with Scheffe's test
for post hoc comparisons; Kruskall-Wallis test (2df ˆ 2) for 3-group comparison of non-normal distributed values with Mann-Whitney U test
for post hoc comparison using Bonferroni correction; p < 005 for a vs. b, p < 001 for c vs. d.
12
G. K. Frank et al.
neuropeptide is involved in the pathophysiology, or
even the etiology, of BN. GRP is an anorexigenic agent.
Recent animal literature suggests alterations of BBS
activity in response to feeding that is localized to the
hypothalamus and nucleus accumbens (Plamondon
et al., 1997). Mice lacking the BBS receptor subtype-3,
though with low affinity for BBS, developed obesity, a
reduced metabolic rate, and hyperphagia (OhkiHamazaki et al., 1997). One interpretation of these data
is that BN women have reduced satiety signaling. In
support of this possibility is that women with BN often
have increased lifetime high body weight compared to
NC. In addition, elevated taste pleasantness for sweetness in BN (Franko et al., 1994) could be related to
alterations in BBS-like peptide activity (Yamada et al.,
1999). Based on the study design, we can only speculate
about the source of CSF GRP measured. Peripheral
BBS-like peptides may not be able to readily cross the
blood brain barrier, but still might be able to access the
brain (Merali et al., 1999). In animals, however, GRPlike peptides have been shown to be produced in central
nerve fibers, and high GRP and GRP-like expression
was found in the hypothalamus, the nucleus of the
solitary tract, and in the trigeminal nucleus (Flynn, 1994;
Moody et al., 1980). Thus, it is possible that the CSF
GRP measured in this study was mainly released by
centrally located nerve fibers. However, we cannot
exclude that CSF GRP from peripheral organs, such as
the lungs (Jaramillo et al., 1995), or the acid secreting
glands of the stomach (Sjovall et al., 1990) contributed
to the CSF GRP levels.
Values of a number of neuropeptide systems have been
found to be abnormal in ill AN and BN subjects such as
corticotropin releasing hormone (CRH), opioids or proopiomelanocortin (POMC), leptin, neuropeptide Y
(NPY), and peptide YY (PYY). However, values of these
systems normalize after recovery (Gendall et al., 1999).
Still, REC BN subjects have been found to have
abnormalities of CNS serotonin (5-HT). BBS-like
receptors are associated with the 5-HT system
(Woodruff et al., 1996), and GRP has been found to excite
5-HT sensitive mesencephalic neurons in the dorsal raphe
of rats in vitro (Pinnock & Woodruff, 1991; Pinnock et al.,
1994). However, it has yet to be determined what functional implications such an interaction might have.
GRP activity may be irreversibly attenuated in
response to alcohol consumption (Madeira & PaulaBarbosa, 1999). On the other hand, the BBS peptides act
as potent inhibitors of not just food but also alcohol
ingestion in the rat (Kulkosky et al., 1985, 1993). It is
well known that there is a high comorbidity of BN and
alcohol abuse or dependence (Lilenfeld et al., 1998).
We therefore divided REC BN women in to two
groups: subjects with history of alcohol abuse (AA) or
dependence (AD) (N ˆ 10), and REC BN women without such history (N ˆ 11). REC BN with a history of AA
or AD (89 41) tended to have lower CSF GRP than
REC BN without alcohol history (105 19), however,
this was not statistically significant. In addition, we
compared the REC BN without a history of AA or AD
with the NC women. We still found CSF GRP in REC
BN women significantly lower than in the NC group
(p ˆ 003).
In terms of limitations, this is a relatively small
number of subjects. Only four of the REC AN women
were pure food restrictors. The remaining REC AN
subjects were binge eating-purging type AN. Even in the
absence of symptoms according to the DSM-IV
(American Psychiatric Association, 1994), elevated
scores on measures for ED related pathology, depression, anxiety, and obsessionality occur in both recovered
AN and BN women (Kaye et al., 1998a; Pollice et al.,
1997; Srinivasagam et al., 1995). The REC BN women in
this study had persistent symptoms that were mild to
moderate in severity compared to the ill state; those data
have been published previously (Kaye et al., 1998a). Our
experience suggests that these are traits that persist after
recovery (Kaye et al., 1998b). We did not find a relationship between CSF GRP and subtypes of AN, but the
very small sample size makes such analyses difficult. In
addition, future studies should investigate whether CSF
values actually reflect activity of this peptide system in
the brain, or other factors, such as peptide metabolism.
Finally, it is possible that persistent disturbances of CSF
GRP could be a secondary effect of years of malnutrition
or other state-related factors. The design of this study,
however, does not permit answering this question.
Taken together, these data suggest that CSF GRP
is reduced in CSF of REC BN. Lower CSF GRP in
this group could be a trait-related disturbance
that might add to hyperphagic behavior, and thus to
the pathophysiology of this illness. However, this finding has to be replicated in a larger subject group. In
addition, a possible interaction of BN and alcohol abuse
on CSF GRP has to be investigated further. CSF GRP
was not significantly reduced in REC AN. This supports
that AN and BN may have, at least in part, different
pathophysiologies.
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