0021-972X/00/$03.00/0
The Journal of Clinical Endocrinology & Metabolism
Copyright © 2000 by The Endocrine Society
Vol. 85, No. 8
Printed in U.S.A.
Twenty-Four-Hour Leptin Levels Respond to Cumulative
Short-Term Energy Imbalance and Predict
Subsequent Intake*
CATHERINE CHIN-CHANCE, KENNETH S. POLONSKY,
AND
DALE A. SCHOELLER
Department of Medicine (C.C.-C., K.S.P.), University of Chicago, Chicago, Illinois 60637; and
Department of Nutritional Sciences (D.A.S.), University of Wisconsin–Madison,
Madison, Wisconsin 53706
ABSTRACT
Leptin plays a vital role in the regulation of energy balance in
rodent models of obesity. However, less information is available about
its homeostatic role in humans. The aim of this study was to determine whether leptin serves as an indicator of short-term energy balance by measuring acute effects of small manipulations in energy
intake on leptin levels in normal individuals. The 12-day study was
composed of four consecutive dietary-treatment periods of 3 days
each. Baseline (BASE) [100% total energy expenditure (TEE)] feeding, followed by random crossover periods of overfeeding (130% TEE)
or underfeeding (70% TEE) separated by a eucaloric (100% TEE)
washout (WASH) period. The study participants were six healthy,
nonobese subjects. Leptin levels serially measured throughout the
study period allowed a daily profile for each treatment period to be
constructed and a 24-h average to be calculated; ad libitum intake
during breakfast “buffet” following each treatment period was also
T
HE HORMONE leptin plays a vital role in the regulation
of energy balance in rodent models of obesity (1–3). In
normal animals, leptin is secreted by adipocytes and seems
to indicate the size of fat stores to the hypothalamus, perhaps
acting as an adipostat (4). While extensively studied in animal models, less is known about this homeostatic role of
leptin in humans. Although severe obesity in humans has
been associated with mutations blocking leptin production
or leptin receptor expression, such mutations are rare (5, 6).
Instead, it has been consistently documented that leptin levels are increased in most obese individuals because there is
a strong positive correlation between fasting leptin concentrations and energy status, most notably the measures percent body fat or body mass index (BMI), suggesting that
deficient leptin expression is not the primary cause of the
majority of observed obesity (7). Based on this and other data,
Flier (8) proposes that leptin acts as an integrator of neuroendocrine function, signaling energy deficiency, rather
than an antiobesity hormone.
As of yet, it is not fully understood how human leptin
levels are regulated and which metabolic functions leptin
Received December 30, 1999. Revision received April 10, 2000. Accepted May 14, 2000.
Address correspondence and requests for reprints to: Dale A.
Schoeller, Nutritional Sciences, University of Wisconsin–Madison,
1415 Linden Drive, Madison, Wisconsin 53706. E-mail: dschoell@
nutrisci.wisc.edu.
* Supported in part by a USDA graduate training fellowship, NIH
Grants 5-P30-DK26678-18 and RO1-DK30031, and a Home Health Care
grant.
measured. Average changes in mesor leptin levels during WASH,
which were sensitive to energy balance effected during the prior
period, were observed. After underfeeding, leptin levels during WASH
were 88 ⫾ 16% of those during BASE compared with 135 ⫾ 22%
following overfeeding (P ⫽ 0.03). Leptin levels did not return to BASE
during WASH when intake returned to 100% TEE, but instead were
restored (104 ⫾ 21% and 106 ⫾ 16%; not significant) only after subjects crossed-over to complementary dietary treatment that restored
cumulative energy balance. Changes in ad libitum intake from BASE
correlated with changes in leptin levels (r2 ⫽ 0.40; P ⫽ 0.01). Leptin
levels are acutely responsive to modest changes in energy balance.
Because leptin levels returned to BASE only after completion of a
complementary feeding period and restoration of cumulative energy
balance, leptin levels reflect short-term cumulative energy balance.
Leptin seems to maintain cumulative energy balance by modulating
energy intake. (J Clin Endocrinol Metab 85: 2685–2691, 2000)
modulates. It does not seem that leptin modulates energy
expenditure because leptin levels correlate neither with resting metabolic rate (9) nor total energy expenditure (TEE) (10)
in humans. However, leptin is modulated by energy balance.
Protracted extreme caloric restriction or excess to induce
changes in weight have demonstrated that leptin levels increase and decrease accordingly and that the observed
changes exceed those expected from changes in fat mass (11,
12). Furthermore, these changes in leptin preceded changes
in body weight. To date, only one study has been performed
that investigated the effects of typical day-to-day variations
in energy balance (13). Although the amplitude of the nocturnal rise in leptin reflected energy balance, changes in
average daily levels were not detected.
Studies in animal models indicate that leptin regulates
intake (1–3), and this has been confirmed in humans lacking
functional leptin (14) or its cognate receptor (6). However,
attempts to correlate physiologic changes in leptin have not
demonstrated any association with subsequent energy intake
(15) or hunger (16). These studies did not characterize the
subjects according to restrained eating, whereas previous
studies of ingestive behavior have indicated that it may be
important to differentiate between restrained and nonrestrained eaters because the former may dissociate energy
intake from physiologic hunger and satiety signals (17).
It is also not evident whether short-term changes in leptin
levels reflect current energy status and, thus, will return to
normal once the individual consumes a diet equal to expen-
2685
2686
CHIN-CHANCE ET AL.
diture; or if they indicate cumulative energy balance and,
thus, will not return to normal until the subject consumes a
diet that repays the acute energy deficit or surfeit. We hypothesized that leptin levels acutely respond to caloric excess
or insufficiency, thus reflecting the body’s current energy
state in nonobese, nonrestrained eaters. By signaling energy
balance, leptin plays a role in regulating hunger and satiety,
enabling the maintenance of normal weight. To test this
hypothesis, 24-h leptin levels were measured as individuals
underwent a series of 3-day periods of eucaloric feeding
(100% TEE), moderate underfeeding (70% TEE), and overfeeding (130% TEE). In addition, changes in ad libitum intake were
compared to changes in leptin levels in nonrestrained eaters.
Subjects and Methods
JCE & M • 2000
Vol 85 • No 8
FIG. 1. Diagrammatical representation of feeding study protocol. The
12-day study consisted of four consecutive 3-day treatment periods of
varying caloric intake in a crossover design. Eucaloric feeding (100%
TEE) took place during the first and third periods, whereas intake
was altered by ⫾30% TEE during the second and fourth periods.
Subjects were randomized to either treatment order.
Subjects
We studied six healthy, nonobese (BMI, 18 –25 kg/m2) males between
the ages of 18 and 39 yr. Their clinical characteristics are shown in Table
1. Subjects were normolipemic (cholesterol ⬍2 g/L and triglycerides
⬍1.55 g/L), had normal thyroid function as determined by thyroidstimulating hormone level (0.4 –3.6 mU/L), and had scores ⱕ6 on the
restriction scale on the Eating Attitudes Test (17). Because of the volume
of blood to be drawn over the duration of the study, inclusion criteria
also included normal iron stores, as determined by serum iron (0.5–1.6
mg/L) and serum ferritin (15–200 ␮g/L), and normal total iron binding
capacity (2.5– 4.0 mg/L), in conjunction with normal hemoglobin (135–
175 g/L). We excluded individuals who had a history of metabolic
disease such as diabetes, hypertension, or liver disease, or were taking
prescription medications or over-the-counter substances such as melatonin. Subjects who exercised regularly or who did not eat three meals
a day including breakfast (an early morning meal) were also excluded.
The study protocol was approved by the Institutional Review Board at
the University of Chicago, and all subjects provided written informed
consent. The study was performed at the Clinical Research Center (CRC)
at the University of Chicago Hospital.
Experimental procedure
The subjects’ energy requirements were individually determined by
measuring TEE using the doubly labeled water method (18) during a
2-week maintenance period (no biologically significant changes in
weight) 4 –10 weeks before the feeding study. This value defined the
eucaloric state/diet for each individual. The average TEE was 12.5 ⫾ 1.7
MJ/day (2980 ⫾ 410 kcal/day).
The 12-day study consisted of four consecutive treatment periods of
3 days each with varying caloric intakes; a diagrammatical representation of the study protocol is shown in Fig. 1. During the first treatment
period, subjects were fed a eucaloric diet equal to 100% of their baseline
TEE. During the second 3-day period subjects were fed either 70% or
130% of their energy requirements. During the third treatment period
between the overfeeding and underfeeding periods, the subjects’ intake
was returned to 100% of their energy requirement. During the fourth
treatment period subjects received the complementary dietary treatment
from period 2. The order of overfeeding and underfeeding during the
TABLE 1. Subject characteristics
(n ⫽ 6)
Mean
SD
Age (yr)
Height (m)
Weight (kg)
BMI (kg/m2)
RMR (MJ/day)
Fat mass (kg)
% Body fat
TEE (MJ/day)
Leptin (ng/mL)
24
1.8
71.8
22.3
7.0
17.4
24.0%
12.5
2.8
3
0.1
7.5
1.6
0.6
5.7
5.9%
1.7
1.8
RMR, Resting metabolic rate.
FIG. 2. Diagrammatical representation of cumulative energy status
during feeding study. Subjects remain at baseline energy balance
after the first period. Cumulative energy status is affected by ⫾30%
TEE variance in caloric intake of the second treatment period. Subjects remain at the altered cumulative energy status after eucaloric
feeding of the third period, but return to cumulative energy balance
at completion of the study due to counter-balanced feeding treatment
of the fourth period.
second and fourth periods was randomized in a crossover design. In
other words, the three subjects who received dietary intakes of 70% TEE
during the second treatment period (days 4 – 6) received dietary intakes
increased to 130% TEE during the fourth treatment period (days 10 –12).
Meanwhile, the other three subjects (who received dietary energy intakes increased to 130% TEE during days 4 – 6) received dietary energy
intakes decreased to 70% TEE during days 10 –12. As shown in Fig. 2,
this treatment protocol yielded a counterbalanced study such that the
deficit or surfeit energy balance that was established during the second
period was not restored until completion of the fourth period.
Subjects were seen as outpatients, except for the 3rd day of each
dietary treatment period during which they were inpatients from 1800 –
0830 h. During the 12 study days all meals were prepared and served
by the dietary staff of the University of Chicago Hospital’s CRC. Subjects
were required to consume all their meals in the CRC, with the exception
of a packaged late-evening snack, which was to be eaten at a prescribed
time of the night. Daily energy intake was divided over three meals and
one snack as: breakfast, 20% (except during breakfast “buffet;” see below); lunch, 30%; dinner, 35%; and snack, 15%; and comprised 50%
carbohydrates, 35% fat, and 15% protein. Breakfast was served at 0805 h,
lunch at 1305 h, dinner at 1805 h, and the snack at 2100 h. The first meal
after each 3-day treatment period was a breakfast provided in a buffet
format. Foods were preweighed and postweighed to calculate energy
intake. Subjects were instructed to eat ad libitum, removing their desired
portions to their own platter (e.g. only the desired amount of cereal was
wetted with milk to obtain an accurate weight of the uneaten cereal. The
foods available in the breakfast buffet were the same as those normally
provided during the fixed breakfast meals: sugar-frosted corn flakes,
bananas, 2% fat milk, scrambled eggs, pork sausage patty, hash browns,
LEPTIN RESPONDS TO SHORT-TERM ENERGY CHANGES
raisin bagel, and margarine. The energy content of the two remaining
meals of that day (days 4, 7, and 10) were the amounts that would
normally be given during that treatment period.
Subjects were instructed to continue all habitual activities and to
maintain a consistent activity level throughout the study, avoiding any
prolonged vigorous activities (i.e. exercise). Subjects were also instructed
to go to bed at 2300 h. Intravenous cannulas were placed in the forearm
vein for blood withdrawal. Blood draws were conducted prior to every
meal, with the exception of a packaged late-evening snack. On every 3rd
day of each treatment period (days 3, 6, 9, and 12) additional blood
samples were drawn after every meal and subjects were admitted to the
CRC for an overnight visit. During this inpatient stay, hourly blood
samples were drawn from 1800 h to 0800 h the following morning. From
2200 – 0700 h, blood samples were drawn using a 15-foot catheter
through an opening in the wall to allow nighttime sampling without
interruption of sleep.
Experimental measurements
Energy requirements for eucaloric feeding were determined by measuring TEE with doubly labeled water. Water containing 0.2 g O-18
labeled water and 0.14 g deuterium labeled water per kilogram estimated total body water (TBW) was administered by mouth. Spot urine
specimens were collected before and 2, 3, and 6 h and 14 days and 14
days ⫹ 2 h after the dose. The specimens were decolorized with dry
carbon black, and the stable isotope abundances were measured by
isotope ratio mass spectrometry, as described previously (19). The isotope dilution spaces and elimination rates were determined and the
energy expenditure was calculated, as described previously (18). The
respiratory ratio was assumed to be 0.86. The TBW was calculated as the
average of the deuterium dilution space/1.041 and the O-18 dilution
space/1.007 (20). Percent body fat was calculated as: %BF ⫽ 100*(WTBW/0.73)/W, where W is body weight.
At all time points described above, leptin levels were measured in
serum in duplicate by RIA (Linco HL-81K, modified—a double antibody
technique; Linco Research, Inc., St. Charles, MO). Results from the 3rd
day of each dietary treatment period were subjected to analysis of
diurnal variation, as described below. In addition, every 3rd day sample
was analyzed for insulin, as well. Insulin levels were measured in serum
by a double antibody technique (21). The lower limit of detection was
20 pmol/L, and the average intra-assay coefficient of variation was 6%.
All 0800-h fasting blood samples were tested for triglyceride levels. The
0800-h fasting blood samples from the day after each dietary period
(days 4, 7, 10, and 13) were analyzed for T3 levels.
Data analysis
Chronobiological parameters. The daily variation in each individual leptin profile was quantitatively evaluated by building a best-fit curve, using a program that repeatedly calculates periodograms method based on repeated periodogram
calculations described in detail previously (22). Briefly, the
periodogram method consists of fitting a sum of sinusoidal
components on the series of data and of selecting those that
contribute significantly to the observed variations. The significant components in the low frequency range are summed
to render the best-fit curve for the determination of the standard chronobiology parameters. This procedure allowed the
objective comparisons of the parameters and their timing.
The zenith is defined as the time of occurrence and maximum
in the best-fit curve. Although the nadir (minimum) of the
leptin profile has been reported to take place during the
afternoon (23), our sampling protocol could not include
hourly leptin levels during the daytime and, therefore, we
were unable to accurately identify the nadir. Daytime sampling was limited to minimize alterations in the subjects’
normal daily activity and, thus, maintain TEE. The leptin
mesor (average) was calculated from the weighted mean of
2687
the leptin levels on the 3rd day of each treatment period at
0800, 0900, 1300, and 1400 h and hourly from 1800 h to 0800 h
the following morning. The amplitude was calculated by
subtracting the mesor from the zenith. The insulin mesor was
calculated using the same method as stated above for the
leptin mesor. All results are expressed as means ⫾ sd.
Statistical analysis. To test the primary hypothesis that leptin
levels reflect the current day’s energy balance, ANOVA with
repeated measures was performed on the mesor of the 3rd
day of each period. As a second test of the primary hypothesis, we compared the difference between the two eucaloric
period mesors (periods 1 and 3), as well as the differences
between the first and last period (period 4) mesors using a
paired two-tailed t test. If the primary hypothesis was correct, then the mesors of the two eucaloric periods should not
differ at all, whereas the changes in the mesors between
periods 1 and 4 should differ with respect to treatment order.
These analyses were repeated for the zenith and amplitude.
For the second test, results are expressed as the percentage
of the baseline value measured on the 3rd day of the first
eucaloric period.
Because leptin mesors did indicate an effect of treatment
order, we also tested whether within-subject leptin levels
increased or decreased progressively from days 1, 2, and 3
within the overfeeding, underfeeding, and eucaloric periods.
For this comparison, the three daily premeal levels (0800,
1300, and 1800 h) were available for analysis. We compared
leptin levels within each treatment period (before each meal
and also the average of all three premeal values) using
ANOVA to test for changes with time within each dietary
treatment period.
Linear correlation was calculated as the Pierson product.
Individual leptin levels for each time point were plotted
against the corresponding insulin level to find any correlation between these two putative energy indicators. We also
compared changes in average leptin levels to changes in ad
libitum intake during breakfast buffet for each dietary treatment period to determine whether there was any correlation
between this putative energy indicator and a quantitative
measure of hunger. The statistical significance of differences
was assessed at the 5% level.
Results
Chronobiological parameters
Leptin levels were measured on the 3rd day of each of four
treatment periods. The diurnal variation in leptin levels was
significant (P ⬍ 0.01) in 24 of the 24 test periods (four periods
each in six subjects). During the initial eucaloric period, the
serum leptin mesor averaged 2.8 ⫾ 1.8 ng/mL. The zenith
occurred at 0200 h ⫾ 2:05 h and averaged 3.7 ⫾ 2.3 ng/mL,
and the amplitude averaged 31 ⫾ 8%. The leptin mesor
during this first eucaloric period was correlated with percent
body fat (r ⫽ 0.90; P ⫽ 0.02) and trended with fat mass (r ⫽
0.79; P ⫽ 0.06), as demonstrated previously by Considine (7).
The zenith of the leptin profiles behaved in a similar manner as the mesors (Table 2). When the participants returned
to eucaloric intake levels during period 3, the leptin zeniths
differed depending on treatment order. Those who were
2688
TABLE 2. Zenith leptin levelsa (ng/mL): comparison of zenith
leptin levels before and after each treatment period
Subject
UO1c
UO2
UO3
OU1
OU2
OU3
Mean
SD
JCE & M • 2000
Vol 85 • No 8
CHIN-CHANCE ET AL.
Underfed
Overfed
TABLE 3. Amplitude of leptin levelsa (fraction above mesor):
comparison of amplitude of leptin levels before and after treatment
period
Eucaloricb
Before
After
Before
After
Before
After
2.8 Id
6.5 I
2.0 I
3.9 III
2.5 III
8.7 III
4.4
2.6
3.1 II
4.1 II
1.3 II
2.7 IV
1.8 IV
6.3 IV
3.2e
1.8
3.5 III
4.8 III
1.6 III
2.4 I
1.8 I
6.9 I
3.5
2.1
3.9 IV
6.3 IV
1.8 IV
3.3 II
2.3 II
7.7 II
4.2e
2.3
3.1 II
4.1 II
1.3 II
3.3 II
2.3 II
7.7 II
3.6
2.2
3.5 III
4.8 III
1.6 III
3.9 III
2.5 III
8.7 III
4.1e
2.5
a
Measured on the 3rd day of each dietary treatment period.
Before eucaloric is underfeeding in UO and overfeeding in OU
subjects.
c
UO indicates subjects who were underfed to 70% TEE during the
second treatment period and overfed to 130% TEE during the fourth
treatment period. OU indicates opposite treatment order.
d
Roman numerals indicate the chronological ordering of the treatment period (see Fig. 1).
e
P ⬍ 0.05 vs. prior period by paired t test.
b
underfed earlier had leptin zeniths that averaged 93 ⫾ 26%
of the first eucaloric period, and those who were overfed
earlier had leptin zeniths that averaged 142 ⫾ 20% of the first
eucaloric period (P ⫽ 0.07 with respect to feeding order). At
the end of period 4, when the participants had finally returned to cumulative energy balance, the zenith of those who
were first underfed averaged 110 ⫾ 28% of the baseline
eucaloric period [not significant (n.s.)], and those who were
first overfed averaged 102 ⫾ 11% of the baseline eucaloric
period (n.s.)
The amplitude of the leptin profiles showed a trend similar
to that of the mesor and zenith (Table 3), however, the effects
of feeding order did not reach significance at the end of the
second period, perhaps because one of the subjects (who
were initially underfed) demonstrated an increase in amplitude during the decreased intake treatment period (first underfed ⫽ 27 ⫾ 11% vs. overfed first ⫽ 39 ⫾ 10%; n.s.).
Underfed
Before
a
UO1
UO2
UO3
OU1
OU2
OU3
Mean
SD
d
0.3 I
0.3 I
0.4 I
0.4 III
0.2 III
0.5 III
0.3
0.1
Overfed
Eucaloricb
After
Before
After
Before
After
0.4 II
0.3 II
0.3 II
0.2 IV
0.3 IV
0.3 IV
0.3
0.1
0.5 III
0.2 III
0.4 III
0.3 I
0.2 I
0.3 I
0.3
0.1
0.5 IV
0.3 IV
0.3 IV
0.4 II
0.3 II
0.3 II
0.3
0.1
0.4 II
0.3 II
0.3 II
0.4 II
0.3 II
0.3 II
0.3
0.0
0.5 III
0.2 III
0.4 III
0.4 III
0.2 III
0.5 III
0.4
0.1
a
Measured on the 3rd day of each dietary treatment period.
Before eucaloric is underfeeding in UO and overfeeding in OU
subjects.
c
UO indicates subjects who were underfed to 70% TEE during the
second treatment period and overfed to 130% TEE during the fourth
treatment period. OU indicates opposite treatment order.
d
Roman numerals indicate the chronological ordering of the treatment period (see Fig. 1); all differences are not significant.
b
TABLE 4. Mesor leptin levelsa (ng/mL): comparison of mesor
leptin levels before and after each treatment period
Subject
UO1c
UO2
UO3
OU1
OU2
OU3
Mean
SD
Underfed
Overfed
Eucaloricb
Before
After
Before
After
Before
After
2.1 Id
5.1 I
1.4 I
2.8 III
2.0 III
5.8 III
3.2
1.8
2.2 II
3.1 II
1.0 II
2.3 IV
1.4 IV
4.7 IV
2.5e
1.3
2.3 III
3.9 III
1.1 III
1.8 I
1.5 I
5.1 I
2.6
1.6
2.6 IV
4.8 IV
1.4 IV
2.4 II
1.8 II
5.9 II
3.2e
1.8
2.2 II
3.1 II
1.0 II
2.4 II
1.8 II
5.9 II
2.7
1.7
2.3 III
3.9 III
1.1 III
2.8 III
2.0 III
5.8 III
3.0
1.7
a
Leptin levels and energy balance
Measured on the 3rd day of each dietary treatment period.
Before eucaloric is underfeeding in UO and overfeeding in OU
subjects.
c
UO indicates subjects who were underfed to 70% TEE during the
second treatment period and overfed to 130% TEE during the fourth
treatment period. OU indicates opposite treatment order.
d
Roman numerals indicate the chronological ordering of the treatment period (see Fig. 1).
e
P ⬍ 0.05 vs. prior period by paired t test.
The order of the treatments had a significant effect on the
leptin mesor (Table 4 and Fig. 3, A and B). Rather than return
to baseline levels when energy intake was returned to the
eucaloric level in period 3 as hypothesized, the leptin mesor
remained unchanged from the previous period (average
change, ⫺0.26 ⫾ 0.32 ng/mL; n.s.). Expressed as a percentage
of the baseline leptin mesor value, the leptin levels on the 3rd
day of returning to eucaloric intake levels (period 3) differed
with respect to dietary treatment order (⫹35 ⫾ 22% after
overfeeding vs. ⫺12 ⫾ 16% after underfeeding; P ⫽ 0.03). The
effect of feeding order, however, disappeared by the 3rd day
of period 4 when the energy deficit or surfeit effected during
period 2 was repaid by the complementary dietary treatment
during period 4 (Fig. 2). Expressed as a percentage of the
baseline mesor, the leptin levels on the 3rd day of period 4
averaged 104 ⫾ 21% (n.s.) of baseline for those who were
initially overfed vs. 106 ⫾ 16% (n.s.) for those who were
initially underfed.
Because leptin levels seemed to respond to cumulative
energy deficit or surfeit, we reanalyzed the underfeeding and
overfeeding data with respect to changes in leptin levels
between successive treatment periods. In this way, it was
possible to combine the data from all six subjects to test for
the effects of overfeeding or underfeeding relative to the
eucaloric period immediately preceding the treatment (Table
2). Decreasing intakes by 30% of the TEE decreased the mesor
by 0.75 ⫾ 0.71 ng/mL (⫺26 ⫾ 17%; P ⫽ 0.01), and increasing
intakes by 30% of the TEE increased mesor by 0.53 ⫾ 0.24
ng/mL (⫹21 ⫾ 8%; P ⫽ 0.001). Again, the other chronobiological parameters behaved in a similar manner (Tables 3 and
4). The leptin zenith exhibited an average decrease of 1.2 ⫾
1.1 ng/mL (⫺30 ⫾ 21%; P ⫽ 0.02) after decreasing intake by
30% of the TEE and an average increase of 0.72 ⫾ 0.45 ng/mL
(21 ⫾ 12%; P ⫽ 0.01) after increasing intake by 30% of the
TEE. The amplitude demonstrated little trend. The time of
the zenith did not change with underfeeding or overfeeding.
It was observed, on average, at 0200 h ⫾ 2:05 h during the
first eucaloric period; 0100 h ⫾ 1:15 h during the second
eucaloric period; 0110 h ⫾ 1:28 h during underfeeding; and
2350 h ⫾ 1:10 h during overfeeding.
b
LEPTIN RESPONDS TO SHORT-TERM ENERGY CHANGES
2689
Comparison to energy balance indicators
The responses in body weight, triglyceride, triiodothyronine, and insulin throughout the study are shown in Table
5. Changes in daily body weight as well as fasting triglyceride and triiodothyronine levels trended with changes in
energy balance, but only weight loss during underfeeding
reached statistical significance. On the other hand, mesor
insulin levels decreased during underfeeding (average
change, ⫺45 pmol/L; P ⫽ 0.02), increased during overfeeding (average change, 81 pmol/L; P ⫽ 0.02), and returned to
baseline levels when energy intake was returned to the eucaloric level in period 3 (average change, 11 pmol/L; P ⫽
0.47). These changes in mesor insulin levels were not affected
by feeding order (and subsequently cumulative energy balance) as observed with the changes in leptin levels, but were
instead the result of changes in daily caloric intake. Furthermore, hourly insulin levels (average insulin profiles are
shown in Fig. 4, A and B) did not correlate with hourly leptin
levels (data not shown), nor did changes in insulin between
treatments correlate with changes in leptin levels.
Ad libitum breakfast buffet intake averaged 4.14 ⫾ 0.99 MJ
at baseline, 5.01 ⫾ 1.79 MJ after underfeeding, 4.68 ⫾ 1.26 MJ
after overfeeding, and 4.86 ⫾ 2.73 MJ after the washout
period. When the changes from baseline were calculated for
each individual after overfeeding, underfeeding, and eucaloric feeding, it was found to correlate with the change in
mesor leptin levels from baseline (r2 ⫽ 0.40; P ⬍ 0.01; Fig. 5).
Discussion
FIG. 3. Twenty-four-hour leptin profiles of subjects underfed then
overfed (A) and overfed then underfed (B) for each of the four treatment periods: 1, baseline (E); 2, underfed (Œ); 3, eucaloric (䉫); 4,
overfed (f). Leptin levels decreased after underfeeding, increased
after overfeeding, and did not change significantly after eucaloric
feeding relative to the preceding treatment period. Data are the mean
of the three subjects who underwent each treatment order protocol.
In addition to the frequent blood sampling on the 3rd day
of each dietary period, blood was collected before each meal
on all 3 days of each treatment period. The daily specimens
were analyzed for leptin to determine whether daily leptin
levels changed progressively during the treatment. When
just the morning fasting values (0800 h) were compared,
leptin levels trended with the direction of the dietary treatment, but these differences did not reach statistical significance. Expressed as a percentage of the 3rd day of the previous eucaloric mesor, fasting leptin levels averaged 92%,
88%, and 80% on the 1st, 2nd, and 3rd days of underfeeding,
and 89%, 98%, and 102% on these respective days of overfeeding. When all three of the daytime leptin levels (0800,
1300, and 1800 h) were averaged for each participant and
then compared in a similar manner, daily leptin levels during
underfeeding decreased systematically (104 ⫾ 22%, 84 ⫾
20%, and 79 ⫾ 21%; P ⫽ 0.03). The leptin levels did not change
systematically during the 3 successive days of overfeeding
(98 ⫾ 15%, 105 ⫾ 25%, and 96 ⫾ 18%; n.s.).
Our data demonstrate that plasma leptin levels respond to
small changes in cumulative energy balance and changes in
leptin levels correlate with changes in ad libitum intake in
healthy, male, nonobese, nonrestrained eaters. Specifically,
we find that that the altered leptin levels resulting from
overfeeding and underfeeding were not restored to baseline
when the participants were returned to a eucaloric diet, an
effect not previously reported. Instead, leptin levels returned
to baseline only after the participants crossed over to the
complementary dietary treatment and repaid the deficit or
surfeit in cumulative energy balance. This is illustrated by the
average leptin profiles in Fig. 3, A and B, where the fourth
period profiles are superimposable with the first period profiles, regardless of treatment order. These findings extend the
observations of others (11, 12) who reported that 24-h average leptin levels decreased or increased from baseline levels
after participants were exposed to either fasting or ⬃8 MJ/
day overfeeding and those of van Aggel-Leijssen et al. (13),
who demonstrated that leptin levels also respond to modest
changes in energy intake.
In our study, the leptin response was detected in the 24-h
average (mesor) and the zenith. We could not detect this
response in the morning fasting level, however, this could be
a type 2 error. Both the mesor and zenith include a significant
averaging of multiple leptin levels, and, thus, variability due
to assay or pulsatile secretion is reduced. When averaged
over the three blood specimens drawn within each day, we
did detect a systematic daytime change in leptin levels during underfeeding, suggesting that the daytime levels also
responded to cumulative energy balance; however, this was
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JCE & M • 2000
Vol 85 • No 8
CHIN-CHANCE ET AL.
TABLE 5. Average changes in energy balance biomarker: comparison of energy balance indicator to baseline levels
Changes from previous period
Biomarker
Baseline levels
baselinea
Underfedb
Overfedc
Washoutd
Changes from
baseline ende
Body weight (kg)
Triglycerides (mg/L)
Triiodothyronine (ng/L)
Insulin (pmol*h/L)
70.9 (8.1)
840 (353)
960 (131)
3971 (1056)
⫺0.4 (0.3)f
⫺173 (250)
36 (174)
⫺1017 (904)f
0.6 (0.8)
185 (204)
13 (65)
1835 (1262)f
0.1 (0.6)
80 (179)
22 (160)
⫺281 (1669)
0.3 (0.9)
92 (204)
98 (19)f
537 (2117)
Results expressed as mean ⫾ SD.
a
Levels on the 3rd day of the first eucaloric period (3 days of feeding 100% eucaloric TEE).
b
Changes from the previous eucaloric period after 3 days of underfeeding to 70% of eucaloric TEE.
c
Changes from the previous eucaloric period after 3 days of overfeeding to 130% of eucaloric TEE.
d
Changes from the previous treatment period after 3 days of feeding to 100% eucaloric TEE.
e
Changes from baseline to end of study.
f
A significant difference from baseline (P ⬍ 0.05).
FIG. 5. Comparison of changes from baseline in mesor leptin levels
and ad libitum intake during the breakfast buffet. A significant negative correlation exists between changes in ad libitum intake and
changes in mesor leptin levels, suggesting a consistent relationship
between these two variables in nonobese, nonrestrained males.
FIG. 4. Twenty-four-hour insulin profiles of subjects underfed then
overfed (A) and overfed then underfed (B) for each of the four treatment periods: 1, baseline (E); 2, underfed (Œ); 3, eucaloric (䉫); 4,
overfed (f). Insulin levels decreased after underfeeding, increased
after overfeeding, and did not change significantly after eucaloric
feeding relative to the baseline treatment period. Data are the mean
of the three subjects who underwent each treatment order protocol.
not detected during overfeeding. This may still be a type 2
error, but it is consistent with the suggestion by Flier (8) that
leptin levels may be more responsive to negative energy
balance than positive energy balance.
The response of leptin to positive and negative cumulative
energy balance in the present study is consistent with a recent
report (13) that also measured 24-h leptin patterns with
roughly 30% changes in energy balance, resulting from exercising and manipulations in diet. The authors, however,
reported significant changes in the amplitude of the 24-h
leptin profile, but only trends in the mesors. In contrast, we
found that the mesors were significantly different, whereas
the amplitudes only trended. The 24-h patterns and absolute
changes in leptin levels, however, are qualitatively quite
similar between the studies. Thus, it is likely that that these
differences are type 2 errors because both studies involved
a modest number of subjects.
With the exception of insulin, the comparison of other
energy balance indicators to leptin levels was inconclusive.
Nonetheless, we do not assume this to indicate that the
subjects were either breaching protocol or that the dietary
intake and energy expenditure calculations were not accurate. There are a number of probable reasons as to why there
was poor correlation between these energy balance indicators and leptin levels. For example, triglyceride and triiodothyronine results did not vary with changes in energy intake,
perhaps due to the infrequency of sampling.
We were unable to replicate the observations of others that
there is a high degree of correlation between peripheral insulin and leptin levels (24). Changes in mesor insulin levels
LEPTIN RESPONDS TO SHORT-TERM ENERGY CHANGES
were affected by changes in daily caloric intake, whereas
leptin levels seemed to reflect cumulative energy balance.
Although this is not inconsistent with a regulatory control of
leptin by insulin, it does indicate that under conditions of
changing energy intake, the simple correlation of leptin and
insulin is lost. Leptin is known to affect energy intake in
rodent models (1–3) and beagles (25). It has also been shown
to have decrease energy intake in nonhuman primates, but only
when directly administered into the hypothalamic region (26).
The role of peripheral leptin in the acute regulation of
human energy intake has been previously thought to be
minor (8). We found, however, that changes in intake during
the ad libitum breakfast negatively correlated with changes in
24-h average leptin levels, indicating a possible relationship
between acute changes in leptin levels and hunger as first
hypothesized on leptin’s discovery. When leptin levels increased, ad libitum intake decreased, and when leptin levels
decreased, ad libitum intake increased. Although this correlation in and of itself does not prove causality between leptin
levels and intake, it does indicate a consistent relationship
between these two variables in male nonobese, nonrestrained eaters. More studies are needed to determine
whether this relationship is limited to nonobese, nonrestrained eaters.
The physiologic role of the diurnal variation in circulating
leptin is still not fully understood. In a previous study, we
demonstrated that the nocturnal rise was entrained to the
meal pattern during the day (27). The current study and the
study by van Aggel-Leijssen et al. (13) demonstrates that
leptin levels throughout the entire day are altered by changes
in energy balance. We speculate that the nocturnal rise may
be important in leptin role’s action on energy intake. Thus,
in future human studies involving leptin administration to
moderate energy intake, it may be important to provide
leptin in the evening as opposed to the morning.
In cross-sectional studies, it is clear that body fat significantly contributes to leptin levels (7). In the present study, we
demonstrate that the average 24-h leptin level and zenith
increase and decrease in response to moderate changes in
dietary energy intake. These observed changes, however, are
larger than would be predicted by increases and decreases in
body fat. Even if the entire excess (or deficit) in energy intake
were stored (or used) as body fat during the overfeeding (or
underfeeding) period, the change in fat would be only 0.3 kg
(11.3 MJ/39.8 MJ⬘kg). This is ⬍2% of the baseline body fat,
yet the average leptin levels increased by 17% during overfeeding and decreased by 24% during underfeeding. Thus,
we interpret this observation as an indication that leptin
levels are responding to the cumulative energy balance in
addition to body fat. The mechanisms underpinning this
action are uncertain at this date.
In summary, the results of this study add to the increasing
body of evidence that leptin levels reflect cumulative energy
balance and also demonstrate an association between leptin
levels and hunger. Taken together, these results suggest that
leptin provides a signal that could be used by the metabolic
regulatory center to maintain energy homeostasis by modulating intake in healthy nonobese, nonrestrained individuals.
Acknowledgments
We greatly appreciate the technical assistance of the Clinical Research
Center (Grant MO1-RR00055) and the Diabetes Research and Training
2691
Center (Grant DK20595). We thank Jacqueline Imperial, R.N., and other
members of the nursing and dietary staff of the Clinical Research Center
for the expert care of the subjects who participated in the study. We also
thank Paul Rue and Kimberly Biskup of the Diabetes Research and
Training Center and John Lukens of the Lipid Core Research for their
excellent technical assistance. We also thank Dr. Robert F. Kushner for
conducting physicals on the enrolled subjects and Dr. E. Van Cauter for
kindly providing the periodogram program and training in its use.
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