| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Original Studies |
Departments of Medicine (J.A.L., N.L.E., M.D.J.) and Biochemistry and Molecular Biology (N.L.E.), Endocrine Research Unit, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905
Address all correspondence and requests for reprints to: Michael Jensen, M.D., Endocrine Research Unit, Mayo Clinic, 200 1st Street SW, Rochester, Minnesota 55905. E-mail: jensen.michael{at}mayo.edu
 |
Abstract |
|---|
 Top Abstract IntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
The importance of changes in nonexercise physical activity to energy balance in humans was recently underscored by the observation that activation of nonexercise activity thermogenesis (NEAT) largely mediated resistance to weight gain during overfeeding (7). We found that changes in NEAT accounted for more than half of the 10-fold variance in fat storage that occurred with overfeeding (r = -0.77; P < 0.001).
Could the leptin response to overeating mediate the activation of NEAT in humans? To address this question we examined adipocyte leptin gene expression and plasma leptin concentrations before, during, and after overfeeding in this same group of volunteers (7).
|
Subjects and Methods |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Sixteen (12 men and four women) healthy volunteers, 25–36 yr old, provided informed written consent. Subjects were excluded if they used any medication at the time of the study or within 6 months of the study, exercised more than twice each week, smoked, used alcohol, were pregnant, or reported unstable body weight.
Study design
Details of the study design have been published previously (7). Subjects remained sedentary throughout the study and were provided with foods from the metabolic kitchen at the General Clinical Research Center, Mayo Clinic for 10 weeks. Foods were weighed and analyzed, and meals were supervised. For the first 2 weeks, volunteers were fed so as to establish weight maintenance dietary intake. For the remaining 8 weeks, each subject received 1000 Cal in addition to weight maintenance requirements. Hence, each volunteer, regardless of basal dietary intake, received 56,000 Cal in excess of that required to maintain body weight. Dietary composition remained constant throughout the study at 40% carbohydrate, 40% fat, and 20% protein. The Mayo Institutional review board approved the study.
Measurements of body composition
Each volunteer was weighed daily using the same validated scale while lightly clothed and without shoes. Body fat was measured in duplicate using dual energy x-ray absorptiometry after baseline feeding and after completion of 4 and 8 weeks of overfeeding. All measurements were performed on the same instrument, which was calibrated against tissue phantoms as well as nontissue standards provided with the equipment. The SD for duplicate measurements was less than 2%. Fat-free mass was calculated from the difference in body weight and fat mass. Additional validation experiments have been reported previously (7).
Measurements of energy expenditure
The components of energy expenditure and total daily energy expenditure were measured after baseline feeding and after 8 weeks of overfeeding. The basal metabolic rate was measured using a calibrated, indirect hood flow-over calorimeter (Deltatrac, SensorMedics, Yorba Linda, CA) on 2 consecutive mornings at 0630 h in fasted subjects who had slept the previous nights at the General Clinical Research Center. The thermic effect of food (TEF) was measured by providing subjects with a meal that provided one third of their daily intake (40% carbohydrate, 40% fat, and 20% protein). Energy expenditure was measured using the indirect calorimeter for 15 of every 30 min (to prevent agitation) until values within 4 Cal/h of resting energy expenditure were recorded for two consecutive measurements (mean duration of measurement ± SD, 414 ± 39 min). Areas under the curves for time (x-axis) and energy expenditure (y-axis) were used to determine TEF. Daily TEF was calculated by tripling this value. Total daily energy expenditure was measured using doubly labeled water and the slope/intercept equations and propagation of error analysis described by Coward and colleagues (8, 9, 10). Changes in NEAT were determined by subtracting values for activity-related thermogenesis before overfeeding from values measured after overfeeding. This calculation represented the change in NEAT, because the amount of volitional exercise and the thermic efficiency to exercise were unchanged (7).
Plasma leptin assay
Plasma samples were drawn from volunteers in the overnight postabsorptive state on 2 consecutive mornings at 0700 h after baseline feeding and after 4 and 8 weeks of overfeeding. Blood was placed on ice after collection, immediately separated from whole blood by centrifugation while maintained at 4 C, and then stored at -70 C. Plasma leptin concentrations were measured using RIA (Linco Research, Inc., St. Louis, MO). The coefficient of variation for measurements averaging 3.8 ng/mL was 6.3%. All samples for the study were run in duplicate in a single batch.
Leptin gene expression
Subcutaneous abdominal and gluteal adipose tissues were sampled
by percutaneous suction biopsy under overnight postabsorptive
conditions (11) after baseline feeding and 8 weeks of overfeeding.
Adipocytes were isolated (12) by incubating the biopsy aspirate for 20
min at 37 C in a solution of 0.05% collagenase, 4% nuclease-free BSA,
and 0.5 mmol/L glucose diluted in phosphate-buffered saline.
Collagenase digestion was followed by two steps of centrifugation
(200 x g, 2 min), floatation, and resuspension of the
adipocytes in phosphate-buffered saline/glucose. Total ribonucleic acid
(RNA) was isolated by homogenization (30 s), isovolemic chloroform
extraction, and application of the spun (10,000 x g,
10 min) supernatant to RNAeasy columns. The presence and integrity of
RNA were confirmed on denaturing 1.5% agarose gels (13) and quantified
by absorbance at 260 nmol/L. Samples were treated with
deoxyribonuclease (Life Technologies, Inc., Gaithersburg,
MD) and RNASEin (Roche Molecular Biochemicals,
Indianapolis, IN). Total adipocyte RNA (2.5 µg) from each subject was
applied to a single positively charged nylon membrane using a slot blot
apparatus. The membrane was hybridized for 10 h at 65 C with an
antisense, psoralen-labeled RNA probe to human leptin messenger RNA
(mRNA; the probe was also used for Northern blot analysis to detect a
single mRNA species 
4.5 kb in size). After development and exposure
to x-ray film, the probe was stripped by immersing the membrane twice
in 0.1% SDS in diethylpyrocarbonate water for 20 min at 121 C in
an autoclave. The membrane was redeveloped and exposed to x-ray film to
ensure that the probe was effectively stripped. The membrane was then
hybridized for 4 h at 65 C with an antisense, psoralen-labeled RNA
probe to 28S ribosomal RNA, developed, and exposed to x-ray film.
Optical densitometry was performed on the x-ray films obtained after
hybridization with the leptin and 28S ribosomal RNA probes. Leptin gene
expression was defined as the intensity of the signal obtained using
the leptin probe divided by the intensity of the signal obtained using
the 28S probe. Leptin sense and RNA controls in serial dilutions and
RNA diluent controls were included.
Statistical analysis
All values are presented as the mean ± SD. Comparisons among the 16 subjects before and after overfeeding were made using paired t tests. Linear regression analyses were performed where appropriate. Statistical significance was defined as P < 0.05.
|
Results |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
Body fat increased significantly from 16.6 ± 7.1% at baseline to 17.7 ± 6.3% (P < 0.01) after 4 weeks of overfeeding and 19.0 ± 7.1% (P < 0.005) after 8 weeks of overfeeding. Mean plasma leptin levels also increased with overfeeding from 4.4 ± 4.1 ng/mL at baseline to 5.4 ± 7.4 ng/mL (P = NS) after 4 weeks and 6.6 ± 6.3 ng/mL (P < 0.05) after 8 weeks of overfeeding. Overall, after 8 weeks of overfeeding, mean weight gain was 4.7 ± 1.8 kg, fat gain was 2.39 ± 1.15 kg, and fat-free mass gain was 2.35 ± 1.18 kg.
Adipocyte leptin mRNA levels increased after overfeeding (Fig. 1
). Changes in abdominal leptin mRNA
correlated with changes in gluteal adipose tissue leptin mRNA (r =
0.57; P = 0.022). Also, the degree of leptin mRNA
up-regulation was positively related to the changes in body fat for
both abdominal (r = 0.62; P = 0.01) and gluteal
(r = 0.58; P = 0.02) adipose tissue.
|
). Also, the change in body fat was
directly related to the change in leptin (r = 0.69;
P = 0.003; Fig. 3). There
was no association however, between the change in leptin and changes in
NEAT (Fig. 3).
|
|
|
Discussion |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
We must acknowledge that because we did not infuse leptin into our volunteers to assess their NEAT responses, these data should be considered indirect evidence against an important role of leptin in NEAT activation. Nevertheless, the lack of any relationship between plasma leptin concentrations or adipocyte leptin mRNA and the change in NEAT in response to prolonged overfeeding appears to argue strongly against increasing leptin production as an important regulator of spontaneous physical activity in humans. Although we did not measure the diurnal variation (15, 16) of leptin in this study, the overnight postabsorptive plasma leptin concentrations appear to predict the concentrations later in the day (15, 16); this pattern would have to be completely disrupted for changes in leptin to predict changes in NEAT. It might be argued that our study did not have sufficient power to detect a correlation between changes in leptin and NEAT. However, as we did demonstrate a significant positive correlation between leptin and change in fat and a significant negative correlation between change in fat and change in NEAT, it seems unlikely that this study is subject to type II error.
Our data contrast the finding that low levels of leptin are somewhat predictive of excess future weight gain in Pima Indians (6, 17). It is possible that the Pima Indian characteristics are relatively unique, however, because studies of other populations have not found that low levels of leptin predict future excess weight gain (18).
In summary, leptin production increases proportionately to body fat with overfeeding. Increases in leptin, however, are not predictive of increases in NEAT. We conclude that leptin is unlikely to activate NEAT in humans.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Received April 14, 1999.
Accepted May 10, 1999.
|
References |
|---|
|
TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
|---|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
