Departments of 1 Internal Medicine and 2 Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75390-9135
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ABSTRACT |
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To assess the importance of the sympathetic nervous system in regulating body weight during prolonged leptin infusion, we evaluated food intake, body weight, and physical activity in conscious, unrestrained rats. Initial studies illustrated that prolonged intracerebroventricular (ICV) infusion of leptin enhanced substrate oxidation so that adipose tissue lipid stores were completely ablated, and muscle triglyceride and liver glycogen stores were depleted. After neonatal chemical sympathectomy, changes in weight and food intake were compared in groups of sympathectomized (SYM) and control (CON) adult animals during ICV infusion of leptin. CON animals lost 60 ± 9 g over 10 days vs. 25 ± 3 g in the SYM animals when food intake was matched between the two groups. Greater weight loss despite similar energy intake points to an important role of the sympathetic nervous system in stimulating energy expenditure during ICV leptin infusion by increasing the resting metabolic rate, since no differences in physical activity were observed between CON and SYM groups. In conclusion, activation of the SNS by leptin increases energy expenditure by augmenting the resting metabolic rate.
lipid oxidation; sympathetic nervous system; intracerebroventricular
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INTRODUCTION |
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ADMINISTRATION OF LEPTIN in normal rodents activates the ventromedial hypothalamus, resulting in both anorexia and increased sympathetic stimulation of peripheral tissues (7, 19, 42). Seminal studies indicate that leptin infusion enhances daily energy expenditure in food-restricted animals by preventing circadian decreases in resting energy expenditure that are normally observed during the first few hours of the light cycle (14, 22, 39, 51, 52). It is postulated that this effect is mediated via disinhibition of sympathetically mediated thermogenesis (14). On the other hand, fatty (fa/fa) rats with a mutation in the gene encoding the long form of the leptin receptor that disrupts intracellular signaling events (11, 24, 25, 44, 55, 56) develop profound obesity that is linked to impaired sympathetic regulation of peripheral metabolism (33, 34, 37).
Despite the straightforward results obtained in the animal models just cited, questions remain regarding the role that sympathetic activation plays in regulating body weight. Decreased sympathetic activity is associated with weight gain in rodent models of obesity, but muscle sympathetic nerve activity measured by direct recordings obtained from the peroneal nerve is increased in obese human subjects (49, 50). Furthermore, peripheral sympathetic nerve terminals have been destroyed in rats by triggering an immune-mediated response with repeated injections of guanethidine or 6-hydroxydopamine (27-29). Surprisingly, chemically sympathectomized rats do not gain more weight than untreated littermates, even when challenged with a high-fat diet (27, 29, 35, 45). Given the complex nature of the multiple processes controlling energy balance, it is likely that additional mechanisms are triggered that compensate for the loss of peripheral sympathetic nerves. If a second factor is introduced, such as anorexia induced by leptin, the metabolic consequences of chemical sympathectomy may be unmasked. The following experiments test the hypothesis that chemical sympathectomy blunts metabolic responses and limits weight loss during prolonged intracerebroventricular (ICV) leptin infusion.
ICV infusion of leptin places the hormone directly in the central nervous system (CNS), where it can stimulate the ventromedial hypothalamus without achieving circulating concentrations that could directly affect peripheral target tissues. Although ICV infusion is an important technique for distinguishing CNS effects mediated through autonomic signals from peripheral actions of leptin, it is important to carefully characterize the response to this relatively nonphysiological mode of hormone administration for comparison with other models of hyperleptinemia. Therefore, a separate important goal of these studies is to measure the time course for metabolic and hormonal changes during prolonged ICV leptin infusion in normal rats.
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MATERIALS AND METHODS |
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Animal care.
Male Sprague-Dawley rats weighing 250-300 g were obtained from
Harlan Sprague Dawley, Indianapolis, IN. They were housed in approved
animal facilities at a temperature of 22°C and were maintained on a
12:12-h light-dark cycle (lights on from 10 AM to 10 PM) with ad
libitum access to water and powdered standard (4% fat) rat chow
prepared by Harlan Teklad, Madison, WI. Studies were performed after
the rats had been acclimated to the new facilities for 3 days, and
all protocols were approved by the University of Texas-Southwestern
Medical Center at Dallas Institutional Animal Care and Research
Advisory Committee.
Surgical procedures.
The rats were anesthetized with methoxyflurane and an intraperitoneal
injection of pentobarbital sodium at 75 mg/kg body wt before they were
placed on a Stoelting stereotaxic device. A midline incision of the
scalp exposed the periosteum, which was scraped away to reveal the
skull. A hole was drilled in the skull at a point centered 2.2 mm
posterior to bregma to visualize the superior sagittal sinus, and two
smaller holes were then drilled posterolaterally for the fixation of
1/8-in. 0-80 machine screws (Small Parts, Miami Lakes, FL). The dorsal
third ventricle was cannulated at stereotaxic coordinates CV = bregma 2.2 mm and AP = bregma
4.5 mm using a
28-gauge, 6-mm-long osmotic pump connector cannula (Plastics One,
Wallingford, CT). The apparatus was cemented into place with dental
cement, and the ICV cannula was connected to an osmotic minipump (model
2002, Durect, Cupertino, CA) by a PE-60 catheter. The pump was placed
in a subcutaneous pouch made in the interscapular region, and the
incision was closed. Surgery was performed using aseptic technique, and
the overall success rate of the procedure was 85-90%. In initial
investigations, correct placement of the catheter was confirmed by
autopsy to determine the ventricular distribution of methylene blue dye
injected via the infusion catheter.
ICV leptin administration and fat-free mass.
We obtained highly purified recombinant leptin from Novo Nordisk
Pharmaceutical, prepared as previously described (57). The
stock was diluted to the desired concentration in phosphate-buffered saline (pH = 7.4) containing 0.2% bovine serum albumin.
Preliminary experiments revealed that continuous ICV leptin infusion at
rates ranging from 5 to 500 ng/h (0.12-12 mg/day) significantly
reduced food intake and body weight in normal rats. A dose of 50 ng/h was chosen for all subsequent experimental protocols, because it was
the smallest dose to yield consistent results in all animals studied.
Twelve rats were acclimated to metabolic cages (Nalge Nunc
International, Rochester, NY) for 3 days before any surgery was
performed. They received either ICV leptin (n = 4; L)
or the dilution vehicle (n = 8) for 10 days. One-half
of the animals receiving vehicle were fed ad libitum (V), and the other
one-half were pair fed to the leptin group (PF). Body weight, food
intake, water ingestion, and urinary nitrogen excretion were monitored daily. Food spillage was accounted for by weighing any food that fell
through the floor of the cage into the collecting apparatus. On
days 3, 6, and 10, the animals were transported
to the NMR facility and briefly anesthetized with methoxyflurane.
1H spectra of the entire animal were collected with a 4.7-T
OMEGA system by use of established pulse sequences (13, 17, 23, 40, 53), and the resulting water and fat resonances were
quantified after line-fitting with NUTS software (ACORNNMR, Fremont,
CA). Fat mass and fat-free mass were computed from the ratio of fat and
water signal areas and known ratios of proton densities of fatty acid
chains and water, with the assumption that water comprises 72% of
fat-free mass (48). Fat-free mass = body mass (1 + fat/water ratio × 0.72), and fat mass = body
mass
fat-free mass. The NMR technique was validated by previous
investigators, with the correlation coefficient between total body
lipid and water content measured by whole body proton magnetic
resonance spectroscopy (HMRS), and classical carcass analysis being
0.97 (40).
ICV leptin administration and substrate oxidation. Separate experiments characterized key components of glucose and lipid metabolism in a single set of animals. Three groups of rats were studied: 8 animals receiving leptin, 10 pair-fed control rats, and 8 ad libitum-fed control rats receiving the dilution vehicle only. All animals received ICV and arterial catheters, as we have described, and were infused with either leptin or the dilution vehicle. The animals remained in metabolic cages, and food intake and weight were recorded daily for 5 days. Urine was collected daily for determination of nitrogen excretion, and blood was sampled daily between 10 AM and 12 noon for measurement of plasma glucose, insulin, free fatty acid (FFA), and triglyceride (TG) concentrations. Between 12 noon and 5 PM, the animals were placed in random order in an airtight glass container for 1 h for indirect calorimetry. Room air warmed to 28°C was pumped through the container at 400 ml/min, and changes in O2 and CO2 content were measured in the effluent while the animals were calm and rested. On the 6th postoperative day, the animals were anesthetized for HMRS measurements and killed to obtain the liver and gastrocnemius and soleus muscles.
Chemical sympathectomy.
Normal male and female Sprague-Dawley rats were bred in our animal
facility. Male pups were identified on day 5 after birth and
received subcutaneous injections of guanethidine (50 mg · kg1 · day
1)
prepared daily in phosphate-buffered saline (pH = 7-7.4) at a
concentration of either 5 or 10 mg/ml. A total of 15 injections were
given 5 days a week, as originally described by Johnson and colleagues
(27, 29). This treatment protocol results in a permanent
sympathectomy that has been verified by numerous investigators (27, 29, 35, 45-47), and we validated the functional
integrity of postganglionic sympathetic nerve responses in
representative adult animals from our treated group by measuring the
increment in blood pressure after a 250 µg/kg intravenous injection
of tyramine. Arterial and venous catheters were inserted, and the rats
had 5-7 days to recover before experiments were performed in
conscious, unrestrained animals. The arterial line was connected to a
continuous blood pressure transducer for 30 min before the first
tyramine bolus was administered, and cardiovascular responses were
monitored. We performed 2-3 trials in each animal separated by
10-15 min. Untreated littermates served as controls.
Analytical procedures and materials. Plasma glucose concentrations were measured by the glucose oxidase method on a Beckman Glucose Analyzer II machine purchased from Beckman Industries, Fullerton, CA. Plasma FFA concentrations were determined by a colorimetric assay incorporating fatty acyl-CoA synthetase, acyl-CoA oxidase, and a peroxidase-linked reagent (Boehringer Mannheim, Indianapolis, IN). Plasma TG concentrations were measured using a baseline correction for glycerol (Sigma Diagnostics). Urinary nitrogen content was calculated as the sum of urine ammonia, uric acid, and creatinine content measured using kits obtained from Sigma Diagnostics. Insulin and leptin levels in plasma were determined by radioimmunoassays with kits specific for rat insulin and leptin purchased from Linco Research, St. Charles, MO. The interassay coefficients of variation were <9 and 6% for insulin and leptin, respectively.
Statistical analyses. Experimental results were analyzed using multiple analysis of variance, with a probability for type I error set at P < 0.05%. Procedures for repeated measures were incorporated when appropriate. All calculations were made with SigmaStat software for Windows (SPSS, Chicago, IL).
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RESULTS |
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Time course of leptin's effect in dissipating adipose tissue.
The initial objective of these investigations was to characterize
closely the time course for the metabolic actions of ICV leptin in
normal rats maintained in metabolic cages. Figure
1 illustrates the changes in food intake,
body mass, and percent body fat that were observed as leptin-treated
(L), vehicle-treated (V), and pair-fed (PF) rats were followed for 10 days. Treatment with leptin inhibited food intake, but the anorexic
effect waned from days 6 to 10 compared with the
response noted earlier in the course of treatment. The control rats
receiving the dilution vehicle had a stable body weight compared with a
loss of 50 ± 5 g in PF and 73 ± 5 g in L animals.
Total body fat mass as measured by HMRS fell to essentially
undetectable levels at days 6 and 10 in animals
receiving leptin, whereas it was easily detected in the other two
groups. As body mass continued to fall from day 6 to
day 10 in the L group without any additional fall in body fat, estimated fat-free mass decreased significantly by 21 g in L
vs. PF animals. The urinary nitrogen excretion provided additional evidence for diminished fat-free mass in L animals. It was not significantly different in the groups from days 1-5,
but it increased with leptin infusion from days 7 to
9 (277 ± 11 vs. 215 ± 11 mg/72 h in L and PF
rats, respectively).
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Leptin enhances substrate oxidation and depletes intramyocellular
lipids.
These experiments directly measured key components of glucose and lipid
metabolism in a single set of animals as adipose tissue fat stores were
depleted over the initial 5 days of leptin treatment. Again, three
groups of animals were studied. Rats receiving ICV leptin at 50 ng/h
(L) were compared with littermates receiving the dilution vehicle that
were either eating ad libitum (V) or were pair fed (PF) to the L group.
Figure 2 shows the unique effect of
chronic ICV leptin administration on the respiratory quotient (RQ) and
resting metabolic rate (RMR) in normal rats. RQ in L, V, and PF rats
was initially quite similar. The RQ in the V rats steadily climbed to
expected levels as the animals recovered from surgical placement of ICV
and arterial catheters, but it remained equally suppressed in the PF
and L groups. As predicted by the aforementioned body composition and
caloric balance calculations, O2 consumption fell in the PF
rats to levels below values observed in the L or V rats having ad
libitum access to food.
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ICV leptin administration in chemically sympathectomized rats. We also examined the extent to which leptin alters food intake and body weight after the interruption of sympathetic efferent activity in rats treated with guanethidine as neonates. As previous investigators have noted, the weights of the pups receiving guanethidine were reduced by 10-15% during the injection period, but the rate of growth returned to normal after the injections concluded. Also, other distinct physical features were observed, including a reversible ptosis that peaked at 4-6 wk of age, and some modest diarrhea in the treated animals (27, 29, 35, 45). We validated the completeness of sympathectomy in guanethidine-treated animals by measuring the peak blood pressure responses to bolus injections of tyramine. Treatment with guanethidine during the neonatal period suppressed the response to central stimulation of vasoconstrictor mechanisms by 65 ± 5% (range 42-88%).
Figure 5 shows the changes in body weight and food intake that occurred in sympathectomized (SYM) or control (CON) rats that received continuous ICV infusions of leptin or the dilution vehicle under circumstances in which additional experimental procedures were avoided to minimize anxiety experienced by the animals. The two groups of rats behaved similarly when the dilution vehicle was administered. The weight gain over 10 days was 45 ± 5 and 47 ± 6 g in the CON and SYM groups, respectively, while the average daily food intake was 26.0 ± 1.2 vs. 26.8 ± 0.7 g. The interruption of sympathetic efferent activity had no discernible impact on weight loss during ICV leptin infusion, but there was an unexpected difference in food intake. Figures 5 and 6 show that the SYM animals lost 25 ± 4 g over 10 days vs. 29 ± 3 g in the CON group, but they also ate significantly less food (149 ± 5 vs. 171 ± 5 g). Metabolic efficiency, calculated as the total change in weight divided by the decrease of food intake from baseline, was significantly different between the two groups (
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DISCUSSION |
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The current experiments were designed to characterize the time course for the metabolic actions of ICV leptin in normal rats and to assess the importance of the SNS in regulating body weight during prolonged leptin infusion. The key finding of the initial characterization study was that leptin-treated animals failed to downregulate resting energy utilization during the early hours of the light cycle to compensate for diminished food intake. The end result was excessive depletion of tissue lipid stores and hepatic glycogen in leptin-treated animals. The experiments in rats after chemical ablation of peripheral sympathetic nerves revealed that an intact SNS is critical for regulating body weight and peripheral metabolism during prolonged ICV leptin infusion. When sympathectomized rats were treated with ICV leptin for 10 days, their weight loss was similar to that of nonsympathectomized, leptin-treated littermates, even though they ate significantly less food. Restricting rats with intact sympathetic responses to the same food intake as the sympathectomized animals resulted in additional weight loss.
The current report focuses on a scenario in which leptin activates
neurons in hypothalamic nuclei and is believed to stimulate peripheral
substrate utilization via an increase in sympathetic activity. ICV
leptin infusion was chosen because this method of hormone
administration stimulates the ventromedial hypothalamus without
achieving circulating concentrations that could directly affect
peripheral target tissues. In our hands, ICV leptin infusion in normal
rats prevented the decrease in resting energy expenditure that is
normally observed in food-restricted animals during the first few hours
of the light cycle. Similar alterations in the regulation of energy
expenditure have been observed by other investigators in mice after
subcutaneous (14), intraperitoneal (51, 52), and ICV (39) administration of leptin. Our experiments
extended previous reports by demonstrating that leptin-induced changes in carbohydrate and lipid metabolism were associated with depletion of
tissue energy stores and a progressive decline in plasma insulin, glucose, FFA, and TG concentrations. It is widely accepted that chronic
hyperleptinemia diminishes body weight and adipose tissue content as
well as plasma concentrations of insulin, glucose, and lipid substrates
below levels observed in pair-fed animals (1, 6, 18, 32).
Although smaller effects of leptin are seen when low physiological
levels of plasma leptin are achieved (2), large metabolic
perturbations appear when supraphysiological doses of peripheral leptin
are given or when the hormone is delivered directly to the CNS via an
ICV catheter (1, 18, 32, 43). The metabolic responses in
our experiments, particularly the loss of fat-free mass in the initial
characterization studies, were generally more dramatic than those
observed by most other investigators. Part of the explanation lies in
the exquisite sensitivity to centrally administered leptin vs.
peripheral infusion (18), but ICV leptin infusion also
increases interleukin-1 generation in the hypothalamus, triggering a
secondary neuroimmune response that may augment the effect of central
leptin administration beyond that which can be achieved with peripheral
hormone injections (36).
The physiological stress of multiple experimental procedures is a more basic complicating factor. Maintaining animals in metabolic cages, drawing daily blood samples from indwelling catheters, administering anesthesia, and transporting animals between buildings were all necessary components of the experiments, but the rigors associated with these procedures accentuated weight loss. Notably, when chemical sympathectomy experiments were performed under conditions that minimized stress, leptin-induced weight loss was approximately equal to the estimated total body fat mass. Therefore, it is important to emphasize that, although the magnitude of the responses in this ICV leptin infusion model may not have matched all previous results, the qualitative differences between leptin-treated and pair-fed rats undergoing identical procedures were consistent with other models of leptin administration.
After the model of ICV leptin infusion was adequately established, we progressed to elucidating the importance of the SNS in regulating body weight during chronic ICV leptin infusion. Because chemical sympathectomy with guanethidine should not result in any structural changes in the CNS or the adrenal gland (27-29), it was anticipated that the central anorexic effect of leptin would be similar in treated and untreated animals. Surprisingly, the decline in food intake for the sympathectomized group was 2.2 g/day (22%) greater than that of the control group. Additional evidence supporting the significance of this small difference in food intake was supplied by an experiment in which the food intake of control and sympathectomized rats receiving ICV leptin was matched. These data indicated that chemical sympathectomy modulated the anorexic effect of ICV leptin. Additional experiments are required to delineate whether this action occurs directly at the level of the hypothalamus or is indirectly mediated via other factors such as plasma insulin, glucose, or FFA concentrations.
Despite the influence of sympathectomy on food intake in this study, we found that intact peripheral sympathetic nerve responses were necessary for the normal metabolic response to leptin. The data provided two ways to estimate what portion of weight loss during ICV leptin infusion was mediated by sympathetic stimulation of peripheral metabolism. First, metabolic efficiency, calculated as the total change in weight over 10 days divided by the total decrease of food intake from baseline, was increased by 34% in the sympathectomized rats. Also, when control rats receiving ICV leptin were food restricted to match the intake of guanethidine-treated littermates, their weight dropped a net 108 g over 10 days compared with vehicle-treated control animals. The net difference in the guanethidine-treated groups was 70 g over 10 days, so chemical sympathectomy, independent of changes in food intake, again limited weight loss by about one-third. These values likely provide a minimal estimate of the importance of the SNS activity during leptin infusion due to nonuniformity of tissue responsivity and because functional studies indicated that the guanethidine protocol did not completely ablate sympathetic responses (46). Other investigators observed that tyrosine hydroxylase activity in the superior cervical ganglion was virtually absent in adult rats treated with guanethidine as neonates (27, 29, 45), whereas norepinephrine content was reduced in brown adipose tissue and soleus, gastrocnemius, and heart muscle by 65-95% (35). It is possible that some of the disparity between norepinephrine release and vasomotor responses resulted from a compensatory increase of the adrenergic receptor number in target tissues.
Two general views prevail regarding the physiological mechanisms through which leptin regulates body weight. The most prevalent viewpoint states that the effect of leptin is mediated by activation of neurons within the hypothalamus. Information is then transmitted to peripheral tissues via changes in food intake and nutrient fluxes or by modulating neuroendocrine and autonomic nervous system signals (30). Sympathetic nerve terminals directly innervate brown and white adipose tissue and skeletal muscle (3-5, 26), and intravenous infusion of leptin has been shown to increase sympathetic nerve traffic to brown adipose tissue, kidney, adrenal medulla, and the hindlimb of rats (10, 21). Although measures of increased sympathetic activity are consistent with an important role of the SNS in mediating the effects of leptin on energy utilization in peripheral tissues, they are not sufficient to establish that sympathetic nerve signals are necessary for normal leptin action. Previous experiments performed in normal rats after chemical sympathectomy with either guanethidine or 6-hydroxydopamine raise a note of caution. The treated rats did not gain more weight or gain weight at a faster rate than untreated littermates, even when high-fat diets were provided. The conclusion of the historical experiments was that the presence of a fully competent SNS was not required for weight maintenance under the majority of conditions (35).
An alternative viewpoint holds that leptin acts directly on -cells,
liver, muscle,and fat. Leptin receptors (OB-Rb) have been detected in
brown and white adipocytes by RT-PCR,and leptin treatment of cultured
adipocytes activates the JAK/STAT pathway (15). Incubated
soleus muscle also responds to high doses of leptin by increasing the
oxidation of fatty acids by 42% (41). Moreover, studies
with cultured C2C12 myotubules indicate that leptin can activate JAK-2,which induces tyrosine phosphorylation of
IRS-2 leading to activation of phosphatidylinositol 3-kinase (PI
3-kinase) in this skeletal muscle model system. Finally, human hepatocellular carcinoma cell lines also express OB-Ra, and these cells
have increased IRS-1-associated PI 3-kinase after exposure to
physiological concentrations of leptin (9).
The current results and other recent studies favor the view that leptin
stimulation of sympathetic activity is a key element promoting
substrate utilization in tissues. Leptin has been implicated in the
stimulation of the 2 catalytic subunit of the 5'AMP-activated protein kinase (
2 AMPK) in skeletal muscle (38).
Intravenous leptin injection (1 mg/kg body wt) stimulated
2 AMPK
activity in mouse soleus muscle and increased phosphorylation of
acetyl-CoA carboxylase, suppressing its ability to form malonyl-CoA,
the endogenous inhibitor of carnitine palmitoyltransferase I. Prolonged activation of
2 AMPK 6 h after intravenous leptin
administration was ablated in soleus muscle that had been denervated by
cutting the soleus, femoral, and obturator nerves (38).
Other acute denervation experiments delineated specific effects of
nerve activity on leptin action in peripheral tissues. ICV leptin
administration stimulated glucose uptake in the extensor digitorum
longus and soleus muscles, but this response was decreased after the
femoral nerve was surgically interrupted (31). Similarly,
local denervation of interscapular brown adipose tissue impaired the
ability of intrahypothalamic injections of leptin to potentiate
insulin-stimulated glucose uptake in that tissue (20).
The current study is unique, because peripheral sympathetic nerve terminals were specifically destroyed without the negative impact on motor function that would be expected from surgical nerve disruption. These techniques permitted close characterization of the importance of the SNS in regulating weight loss during prolonged leptin infusion. Specific destruction of peripheral sympathetic nerves impaired leptin acceleration of energy expenditure and limited weight loss when food intake was matched between control and sympathectomized rats. When animals had free access to food, compensatory mechanisms modulated food intake during prolonged ICV leptin infusion and minimized weight loss. Therefore, it is evident that the SNS played a critical role in increasing energy expenditure and promoting weight loss in response to leptin, but even this effect was difficult to unmask unless additional factors curbed changes in food intake. In the same way, weight loss interventions that only target energy expenditure will yield disappointing results unless compensatory mechanisms that regulate energy intake are subverted as well.
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ACKNOWLEDGEMENTS |
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We appreciate the excellent technical assistance provided by Nelda Parker, Ben Alexander, and Courtney Halbrooks.
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FOOTNOTES |
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The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (DK-57558), the Donald W. Reynolds Cardiovascular Clinical Research Center, and a Career Development Award from the American Diabetes Association supported this work. Additional support was received from NIDDK Grant DK-18573 and the Forrest C. Lattner Foundation.
Address for reprint requests and other correspondence: R. L. Dobbins, Univ. of Texas Southwestern Medical Center, Dallas, TX 75390-9135 (E-mail: robert.dobbins{at}utsouthwestern.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published December 27, 2002;10.1152/ajpendo.00128.2002
Received 22 March 2002; accepted in final form 13 December 2002.
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