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Rapid Publication
A Preprandial Rise in Plasma Ghrelin Levels Suggests a
Role in Meal Initiation in Humans
David E. Cummings,
1
Jonathan Q. Purnell,
2
R. Scott Frayo,
1
Karin Schmidova,
1
Brent E. Wisse,
1
and David S. Weigle
1
The recently discovered orexigenic peptide ghrelin is
produced primarily by the stomach and circulates in
blood at levels that increase during prolonged fasting in
rats. When administered to rodents at supraphysiologi-
cal doses, ghrelin activates hypothalamic neuropeptide
Y/agouti gene–related protein neurons and increases
food intake and body weight. These findings suggest
that ghrelin may participate in meal initiation. As a first
step to investigate this hypothesis, we sought to deter-
mine whether circulating ghrelin levels are elevated
before the consumption of individual meals in humans.
Ghrelin, insulin, and leptin were measured by radio-
immunoassay in plasma samples drawn 38 times through-
out a 24-h period in 10 healthy subjects provided meals
on a fixed schedule. Plasma ghrelin levels increased
nearly twofold immediately before each meal and fell to
trough levels within 1 h after eating, a pattern recipro-
cal to that of insulin. Intermeal ghrelin levels displayed
a diurnal rhythm that was exactly in phase with that of
leptin, with both hormones rising throughout the day to
a zenith at 0100, then falling overnight to a nadir at
0900. Ghrelin levels sampled during the troughs before
and after breakfast correlated strongly with 24-h inte-
grated area under the curve values (rⴝ0.873 and 0.954,
respectively), suggesting that these convenient, single
measurements might serve as surrogates for 24-h pro-
files to estimate overall ghrelin levels. Circulating ghre-
lin also correlated positively with age (rⴝ0.701). The
clear preprandial rise and postprandial fall in plasma
ghrelin levels support the hypothesis that ghrelin plays
a physiological role in meal initiation in humans.
Diabetes 50:1714 –1719, 2001
Ghrelin, the endogenous ligand for the growth
hormone secretagogue (GHS) receptor (1), has
recently been implicated in the control of food
intake and energy balance. This highly con-
served acylated peptide is produced primarily by cells in
the oxyntic glands of the stomach as well as in the
intestine (2), and it is secreted into the bloodstream. When
administered either peripherally or centrally to rodents,
ghrelin rapidly increases food intake and body weight
(3– 6) in addition to stimulating gastric motility and acid
secretion (6,7). Ghrelin is a more potent stimulant of
short-term feeding than any known peptide except neu-
ropeptide Y (NPY), with which it has approximately equal
potency (5,6). Its orexigenic effects are independent of
growth hormone (GH) stimulation (3,4) and appear to be
mediated at least in part through activation of NPY/agouti
gene–related protein (AGRP) neurons in the hypothalamic
arcuate nucleus, 94% of which express ghrelin receptors
(8). Activation of the ghrelin receptor stimulates c-fos in
these cells (4,9,10) and also increases hypothalamic NPY
and AGRP expression (4,6,11,12). A role for NPY and AGRP
as mediators of ghrelin’s feeding effects is suggested by
studies in which antagonism of either NPY or AGRP sig-
naling in the brain was shown to attenuate the orexigenic
potency of injected ghrelin (4,6,12). In rodents, ghrelin ex-
pression increases with prolonged fasting (3,6), and fast-
ing blood levels are suppressed by refeeding or by infusion
of nutrients (but not water) into the stomach (3). Based on
these findings, it has been proposed that ghrelin is a hor-
mone that contributes to the initiation of individual meals.
Because the above studies used pharmacological doses
of exogenous ghrelin or measured serum levels after
prolonged fasting, they did not address whether ghrelin
plays a physiological role in initiating meals. If circulating
ghrelin does perform such a function, its levels would be
expected to rise before each meal and fall shortly after
food is consumed. Human plasma ghrelin levels were
reported in one recent study (13), but only fasting values
were examined. We sought to determine the daily pattern
of ghrelin secretion in normal humans as well as to assess
any potential diurnal variation. Plasma ghrelin levels were
measured 38 times during a 24-h period in healthy subjects
given three meals per day at specified times. These values
were compared with 24-h profiles of insulin and leptin. Our
results demonstrate a dramatic preprandial rise and post-
From the
1
University of Washington, VA Puget Sound Health Care System and
Harborview Medical Center, Seattle, Washington; and the
2
Oregon Health
Sciences University, Portland, Oregon.
Address correspondence and reprint requests to David E. Cummings,
Assistant Professor of Medicine, University of Washington, VA Puget Sound
Health Care System, Seattle Division, 1660 S. Columbian Way, S-111-Endo,
Seattle, WA 98108. E-mail: davidec@u.washington.edu.
Received for publication 25 April 2001 and accepted in revised form 30 May
2001. Posted on the World Wide Web at www.diabetes.org/diabetes on 29 June
2001.
AGRP, agouti gene–related protein; AUC, area under the curve; CV, coeffi-
cient of variation; GH, GH; GHS, GH secretagogue; NPY, neuropeptide Y; RIA,
radioimmunoassay.
1714 DIABETES, VOL. 50, AUGUST 2001
prandial fall in circulating ghrelin levels, a pattern that is
consistent with the hypothesis that ghrelin is a physiolog-
ical meal initiator.
RESEARCH DESIGN AND METHODS
A total of 10 apparently healthy subjects (9 women and 1 man) were recruited
through local newspaper advertising. Subjects were ⬎18 years old, weight-
stable for at least 3 months preceding the study, and at their lifetime maximal
weight. Exclusion criteria were as follows: BMI ⬎30 kg/m
2
, diabetes, chronic
medical illness, pregnancy, use of tobacco products, regular intense exercise
(⬎30 min of aerobics 3 times per week), and alcohol consumption of ⬎2
drinks per day. None of the subjects had undergone gastrointestinal surgery.
The age range was 29.1– 63.7 years, and the BMI range was 22.0–30.0 kg/m
2
.
After giving informed consent, eligible subjects were enrolled into the study.
All procedures and protocols took place at the General Clinical Research
Center (GCRC) and were approved by the Human Subjects Review Committee
at the University of Washington.
Diet and GCRC protocols. Before blood sampling, subjects were placed for
2 weeks on an outpatient diet prepared by the metabolic kitchen of the GCRC
at the University of Washington. The diet consisted of 35% fat, 45% carbohy-
drate, and 20% protein, a macronutrient content that approximates the
average American diet (14). During this time, subjects were seen and weighed
by GCRC dietitians twice weekly, and total ingested calories were adjusted to
maintain weight stability. At the end of the 2-week feeding period, subjects
were admitted to the GCRC, where they were given the same diet adminis-
tered as breakfast, lunch, and dinner at 0800, 1200, and 1730, respectively. An
intravenous catheter was placed in the subject, and blood was drawn into
EDTA tubes at 30-min intervals from 0800 –2100, then hourly until 0800 the
next morning (24 h total). Samples were stored at 4°C during the collection
period and then centrifuged. The plasma was separated into four aliquots and
stored at ⫺70°C.
Hormone assays. Plasma immunoreactive ghrelin levels were measured in
duplicate using a commercial radioimmunoassay (RIA) that uses
125
I-labeled
bioactive ghrelin as a tracer and a rabbit polyclonal antibody raised against
full-length octanoylated human ghrelin (Phoenix Pharmaceuticals, Belmont,
CA). In our study, the lower and upper limits of detection for this assay were
80 and 2,500 pg/ml, respectively. All assays included 12 plasma control
samples from common stocks that were transferred to aliquots and frozen at
the beginning of the study, used to normalize each test for interassay vari-
ability. Based on these controls, the intra-assay coefficient of variation (CV)
was 8.7% and the interassay CV was 14.6% (n⫽10). No cross-reactivity was
seen with human leptin, which was assessed at doubling dilutions from 100 to
1 ng/ml.
Plasma insulin was measured in duplicate using a modification of a
double-antibody RIA (15). The lower and upper limits of detection were 13 and
1,680 pmol/l, and the intra-assay CV was ⬍10%. Leptin was measured with a
commercial RIA kit that uses the double-antibody/polyethylene glycol tech-
nique (Linco Research, St. Charles, MO). The lower and upper limits of
detection were 0.5 and 100 ng/ml. The intra- and interassay CVs were 5.0 and
5.5%, respectively. For all three hormones, all samples from a single individual
were run in duplicate in the same assay.
Statistical analysis. Plasma hormone concentrations are expressed as the
means ⫾SE. Total 24-h integrated area under the curve (AUC) values for
plasma ghrelin were calculated using the trapezoidal rule, and integrated 24-h
averages were determined by dividing the AUC by 24 h. Linear regression was
used to determine the correlation between 24-h integrated AUC ghrelin values
and either the 0600 or 0930 levels as well as the correlation between age and
ghrelin levels. Multivariate regression analysis was used to determine the
correlation between ghrelin values (dependent variable) and age, BMI, and
total calories consumed in 24 h (independent variables). As a test for diurnal
variation, the 24-h profile of average plasma leptin concentrations was fit to a
cosine function using the NLREG program. The cosine equation used was [y⫽
20.27 ⫹3.23cos(0.27x)], where yis plasma leptin concentration, xis clock
time, 20.27 is the offset, 3.23 is the amplitude, and 0.27 is the frequency.
RESULTS
Plasma ghrelin levels rose by an average of 78% 1–2 h
before the onset of each meal and fell to trough levels with-
in 1 h after food was first consumed (Fig. 1A). Although
there was considerable variation in the range of ghrelin
values among individuals, all 10 subjects displayed pre-
prandial ghrelin surges, the only exception being the oc-
casional absence of a surge before breakfast. The 24-h
plasma ghrelin profiles for two subjects are shown in Fig.
2, illustrating the interindividual variability in the range
of ghrelin values and the heterogeneity in prebreakfast
surges.
With regard to its temporal relationship to meals, the
24-h time course of plasma ghrelin was reciprocal to that
of insulin (Fig. 1B). While ghrelin levels rose sharply be-
fore each designated meal time and declined precipitously
to trough values within 60 min after meal ingestion, insulin
levels increased from premeal trough values by 3.0- to
7.3-fold within 30 – 60 min after meal consumption. Simi-
larly, during the intermeal interval, ghrelin levels gradually
rose toward their next premeal peak, whereas insulin
levels decreased toward their basal value.
Plasma leptin levels did not change acutely before
meals, but they displayed clear diurnal variation, with a
daily nadir at 0900 and a zenith at 0100 (Fig. 1C). Levels at
FIG. 1. Average plasma ghrelin (A), insulin (B), and leptin (C)
concentrations during a 24-h period in 10 human subjects consuming
breakfast (B), lunch (L), and dinner (D) at the times indicated (0800,
1200, and 1730, respectively).
D.E. CUMMINGS AND ASSOCIATES
DIABETES, VOL. 50, AUGUST 2001 1715
the nadir were 23% lower than the integrated 24-h average
value. Consistent with a diurnal variation in leptin levels,
the 24-h profile correlated significantly with a cosine
function (r⫽0.906, P⬍0.00001). Superimposed on this
diurnal pattern, small (8 –9%) drops in circulating leptin
occurred within 1 h after the beginning of each meal.
Ghrelin levels between meals rose progressively through-
out the day, reaching a zenith at 0100, then gradually fell
overnight to a trough at 0600, before the prebreakfast
surge (Fig. 1A). This diurnal pattern resembled that of
plasma leptin, for which daily nadir and zenith values both
occurred at the same hours as did those for intermeal
ghrelin values (Fig. 3). Levels of both hormones also fell
after each meal, although the amplitude of this oscillation
was far greater for ghrelin than for leptin. No cross-
reactivity was seen in the ghrelin RIA, with human leptin
measured at concentrations as high as 100 ng/ml, which is
71 times greater than the highest measured plasma ghrelin
level. Because ghrelin intermeal trough values steadily
increased while the height of preprandial surges remained
constant, the amplitude of the postprandial fall in ghrelin
levels diminished with each successive meal. The magni-
tude of the average decrease in plasma ghrelin from peak
to trough was 54% after breakfast, 40% after lunch, and 34%
after dinner.
We sought to determine whether a single, conveniently
obtainable plasma ghrelin value could serve as a surrogate
for the integrated 24-h AUC ghrelin value in human sub-
jects. Accordingly, linear regression analysis was per-
formed between each subject’s 24-h integrated AUC
ghrelin value and either the 0930 level (from the trough
after breakfast) or the 0600 level (from the trough occur-
ring just before the prebreakfast surge after an overnight
fast) (Fig. 1A). As shown in Fig. 4A, postprandial ghrelin
levels measured at 0930 correlated very strongly with 24-h
AUC values (r⫽0.954, P⬍0.0001). This correlation was
seen even though the size of breakfast preceding the 0930
sampling was not standardized, and it varied among sub-
jects from 520 to 768 cal. Overnight fasting ghrelin levels at
0600 also correlated significantly with 24-h AUC values
(r⫽0.873, P⫽0.0004) (Fig. 4B).
Both of these associations remained statistically signif-
icant regardless of whether a single low-ghrelin outlier
was omitted from the analyses. The 24-h ghrelin profiles
were measured twice on this subject at times spaced 2
weeks apart. On both occasions, her 24-h AUC values were
substantially below the group mean (3,073 and 4,121 pg-
day/ml for the low-ghrelin subject vs. an average of 14,222
pg-day/ml for all other subjects). The low-ghrelin subject’s
serum was found to be positive for anti-parietal cell anti-
bodies at a 1:20 dilution and showed a mildly low B
12
level
of 205 pg/ml (normal ⬎224 pg/ml).
The 24-h AUC values of plasma ghrelin showed a signif-
icant positive correlation with age (r⫽0.701, univariate
P⫽0.022). In a multivariate regression analysis with 24-h
AUC ghrelin as the dependent variable and with age, BMI,
and total calories ingested over 24 h as the independent
variables, age was the only variable that correlated signif-
icantly with ghrelin levels (P⫽0.045). Similar results were
derived using 0600 or 0930 ghrelin levels as the dependent
variables compared with the same three independent
variables (multivariate P⫽0.038 and 0.046, respectively,
for ghrelin vs. age).
DISCUSSION
In this study, human plasma ghrelin levels were shown to
rise nearly twofold shortly before each meal and fall to
trough levels within 1 h after eating, a profile that is
consistent with a physiological role for ghrelin in initiating
FIG. 2. Representative 24-h profiles of plasma ghrelin levels from two
subjects (Aand B) consuming breakfast (B), lunch (L), and dinner (D)
at the times indicated. Note the wide range of circulating ghrelin values
between individuals and the heterogeneity of the surge before break-
fast.
FIG. 3. Overlaid average plasma ghrelin (F) and leptin (E) concentra-
tions during a 24-h period in 10 human subjects consuming breakfast
(B), lunch (L), and dinner (D) at the times indicated.
PREPRANDIAL SURGES IN HUMAN GHRELIN
1716 DIABETES, VOL. 50, AUGUST 2001
individual meals. The temporal patterns of ghrelin and
insulin surges were reciprocal, occurring just before and
after the designated meal times, respectively. Because it is
well established that insulin surges are postprandial, these
data confirm that meals were consumed at the times
planned in the study protocol and that increases of circu-
lating ghrelin occurred before each meal. Intermeal ghre-
lin levels rose progressively throughout the day, peaking at
0100, then decreased steadily until shortly before break-
fast. Plasma leptin displayed a very similar diurnal varia-
tion, as has been shown by others (16), with daily nadir
and zenith levels occurring at the same hours as those for
intermeal ghrelin levels. Both hormones also decreased
after meals, although the amplitude of this decline was
greater for ghrelin than for leptin. Ghrelin levels measured
at the 0600 and 0930 troughs before and after breakfast
correlated significantly with individual 24-h integrated
AUC ghrelin values. Circulating ghrelin levels correlated
positively with age.
Several observations from rodent studies support the
hypothesis that ghrelin is a physiological meal initiator.
First, ghrelin is synthesized primarily by the stomach (1),
an organ that is well positioned to sense short-term fluxes
in energy balance. Second, despite being produced periph-
erally, ghrelin acts centrally to stimulate food intake (3– 6).
Third, ghrelin affects feeding rapidly, increasing both food
intake (6) and gastric acid secretion (7) within 20 min of
intraperitoneal injection, a time course that is consistent
with a role in meal initiation. Fourth, exogenous ghrelin
triggers eating in rodents during the day (4 – 6), a time when
food intake is usually nominal. Finally, ghrelin activates
hypothalamic NPY/AGRP neurons and increases AGRP
gene expression (vide supra). AGRP has been implicated
as a central mediator of meal initiation because mRNA lev-
els in the hypothalamus rise shortly before the onset of
maximal daily food intake in ad libitum–fed rats, whereas
levels of other neuropeptides involved in energy balance
are stable throughout the day (17).
Together with our findings of a large preprandial rise
and postprandial fall in plasma ghrelin levels in humans,
these observations support a model in which ghrelin acts
as a physiological meal initiator. Because subjects in our
study were provided food at specified times, however, we
cannot conclude that the observed preprandial surges in
circulating ghrelin levels actually contributed to meal ini-
tiation. It is possible that the surges occurred as an antic-
ipatory response to meals because the subjects knew
when food was to be provided. Additional studies to dis-
tinguish between anticipatory responses and true meal
initiation are now warranted.
FIG. 4. Correlation between integrated 24-h AUC ghrelin values and plasma ghrelin levels measured at either 0930 (rⴝ0.954, P<0.0001), during
the trough after breakfast (A), or 0600 (rⴝ0.873, P<0.0004), during the trough before breakfast after an overnight fast (B).
D.E. CUMMINGS AND ASSOCIATES
DIABETES, VOL. 50, AUGUST 2001 1717
Ingested nutrients are the most likely mediator of the
rapid postprandial fall in circulating ghrelin levels. This
contention is supported by the finding that fasting serum
ghrelin levels are decreased in rats by filling the stomach
with a 50% dextrose solution but not with an equal vol-
ume of water (3). Neither this experiment nor our data
distinguish whether ingested nutrients suppress ghrelin
production directly or indirectly (e.g., through insulin), a
possibility that is consistent with the reciprocal 24-h
profiles of these hormones (Fig. 1).
The factors that dictate the relatively wide range of
ghrelin levels among individuals remain to be determined.
It has recently been reported that fasting plasma ghrelin
concentrations are negatively correlated with percent body
fat and are decreased in obesity (13). If ghrelin is involved
in regulating overall energy balance, then an ineffective
compensatory modulation of ghrelin levels in obesity,
rather than a causative role of ghrelin levels, is suggested.
Among our subjects, 24-h AUC ghrelin values tended to be
lower with increasing BMI (r⫽⫺0.519, P⫽0.128),
although the narrow range of BMI in our study cohort
(22.0 –30.0 kg/m
2
) limited our ability to detect a statisti-
cally significant relationship between these parameters.
One subject’s plasma ghrelin levels were significantly
lower than those of all other subjects on two separate 24-h
measurements, although on both occasions she did display
meal-related ghrelin oscillation. Because she has hypothy-
roidism, presumed to arise from an autoimmune etiology,
we evaluated the possibility that her ghrelin levels might
be low because of autoimmune gastrointestinal disease.
Her serum proved to have anti-parietal cell antibodies and
a low B
12
level. Although these findings were not dramatic
in magnitude, they are unusual for a young woman (29
years old) and suggest an evolving gastric autoimmune
process. This may be the cause of her low ghrelin levels, a
possibility that seems plausible in view of the close
juxtaposition of parietal and ghrelin cells within gastric
oxyntic glands (2).
The observation that leptin and intermeal ghrelin levels
display diurnal rhythms that are in phase with one another
suggests that the two may be coordinately regulated. The
diurnal pattern of leptin has been shown to be entrained to
meal timing (18), and the nocturnal rise in circulating
levels is thought to reflect the overall accumulation of
ingested calories throughout the day (19,20). Thus, it is
possible that in addition to being acutely negatively regu-
lated by the ingestion of individual meals, circulating
ghrelin is also positively regulated by fluxes in overall
energy balance. This would explain both the gradual rise
of basal levels throughout the day and evening and the fall
at night, during which times humans experience states of
positive energy balance followed by negative balance. If
ghrelin is a physiologically important circulating orexigen,
its effects may be counterbalanced during the late night by
high leptin levels.
In view of these in-phase diurnal rhythms, it is conceiv-
able that leptin directly stimulates ghrelin secretion. There
are conflicting data from rodent studies that show both
positive (21) and negative (6) regulation of ghrelin by
leptin. Alternatively, ghrelin might induce leptin, as ghrelin
receptors are expressed in adipose tissue (1) and the stom-
ach (22), the principal sites of leptin synthesis (23,24), and
this possibility warrants further investigation. The subtle
postprandial drop in leptin levels that we detected has been
observed, though not commented upon, by others (18,25).
It is conceivable that this reflects meal-related regulation
of gastric leptin.
The 24-h AUC ghrelin values correlated significantly
with levels at 0600 after an overnight fast as well as with
0930 levels measured 90 min after consumption of a non-
standardized breakfast. If our findings can be replicated in
larger studies, they indicate that these convenient single
measurements may serve as indexes of 24-h ghrelin con-
centrations in situations where 24-h blood sampling is
precluded. This has important implications for scientific
studies and for possible future uses of plasma ghrelin
levels in a clinical context.
Circulating ghrelin, assessed as 24-h AUC, 0600, or 0930
values, correlated positively with age. Although the valid-
ity of this observation is limited because of our small
sample size, the possibility may now be entertained that
rising ghrelin levels could play a role in the gradual
increase of body fat content that occurs throughout the
breadth of adult life in humans (26).
If ghrelin is proven to be a physiologically important
meal initiator, the medical implications would be consid-
erable. Bioactive ghrelin and orally bioavailable ghrelin
agonists have been synthesized (27) and can be tested as
remedies for the pathological anorexia that can accom-
pany cancer, AIDS, tuberculosis, and aging. In this regard,
it is noteworthy that although ghrelin is implicated in
triggering individual meals, chronic administration causes
significant weight gain in rodents (3,4). We have recently
shown that central administration of ghrelin increases
food intake and body weight in anorexic rats bearing
prostate adenocarcinoma (28). Ghrelin has been delivered
to humans in two small trials designed to determine its
effects on GH secretion (29,30). Although neither of these
studies examined food intake, one of them noted that
three of four subjects reported feeling hungry after receiv-
ing intravenous ghrelin (29). Whether ghrelin antagonism
could reduce food intake and be developed as a treatment
for obesity is an important question for future studies.
ACKNOWLEDGMENTS
These experiments were supported by a Burroughs Well-
come Fund Career Award (no. 233 to D.E.C.), the National
Institutes of Health (RO1-DK-55460 to D.S.W. and K23-DK-
02689 to J.Q.P.), a General Clinical Research Center grant
(MO1RR00037), a Diabetes Endocrine Research Center
grant (P3ODK17047), and the Medical Research Service of
the Department of Veterans Affairs.
We thank Patricia Breen, Pamela Yang, Colleen Matthys,
Holly Edelbrock, and Holly Callahan for their outstanding
work with subjects in the GCRC. We are also very grateful
to Dr. George Merriam for his vital intellectual input in this
research and Drs. Michael Schwartz and Steven Kahn for
their insightful reviews of the manuscript.
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