Leptin Levels Are Associated with Fat Oxidation ... - Wiley Online Library

Leptin Levels Are Associated with Fat Oxidation and Dietary-Induced Weight Loss in Obesity Camilla Verdich,* Søren Toubro,* Benjamin Buemann,* Jens Juul Holst,† Jens Bu¨low,‡ Lene Simonsen,‡ Susanne Bonnichsen Søndergaard,§ Niels Juel Christensen,§ and Arne Astrup*

Abstract VERDICH, CAMILLA, SØREN TOUBRO, BENJAMIN ¨ LOW, LENE BUEMANN, JENS JUUL HOLST, JENS BU SIMONSEN, SUSANNE BONNICHSEN SØNDERGAARD, NIELS JUEL CHRISTENSEN, AND ARNE ASTRUP. Leptin levels are associated with fat oxidation and dietary induced weight loss in obesity. Obes Res. 2001;9:452– 461. Objective: To examine the relationship between fasting plasma leptin and 24-hour energy expenditure (EE), substrate oxidation, and spontaneous physical activity (SPA) in obese subjects before and after a major weight reduction compared with normal weight controls. To test fasting plasma leptin, substrate oxidations, and SPA as predictive markers of success during a standardized weight loss intervention. Research Methods and Procedures: Twenty-one nondiabetic obese (body mass index: 33.9 to 43.8 kg/m2) and 13 lean (body mass index: 20.4 to 24.7 kg/m2) men matched for age and height were included in the study. All obese subjects were reexamined after a mean weight loss of 19.2 kg (95% confidence interval: 15.1–23.4 kg) achieved by 16 weeks of dietary intervention followed by 8 weeks of weight stability. Twenty-four-hour EE and substrate oxidations were measured by whole-body indirect calorimetry. SPA was assessed by microwave radar. Results: In lean subjects, leptin adjusted for fat mass (FM) was correlated to 24-hour EE before (r ⫽ ⫺0.56, p ⬍ 0.05)

Submitted for publication December 6, 2000. Accepted for publication in final form May 17, 2001. *Research Department of Human Nutrition, Centre for Food Research, The Royal Veterinary and Agricultural University, DK-1958 Frederiksberg C, Denmark; †Department of Medical Physiology, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N, Denmark; ‡Department of Clinical Physiology, Bispebjerg Hospital, Copenhagen, Denmark; and §Division of Endocrinology, Medical Department E, Herlev Hospital, University. Address correspondence to Dr. Arne Astrup, Research Department of Human Nutrition, The Royal Veterinary and Agricultural University, Rolighedsvej 30, DK-1958 Frederiksberg C, Denmark. E-mail: [email protected] Copyright © 2001 NAASO

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but not after adjustment for fat free mass. In obese subjects, leptin correlated inversely with 24-hour and resting nonprotein respiratory quotient (r ⫽ ⫺0.47, p ⬍ 0.05 and r ⫽ ⫺0.50, p ⬍ 0.05) both before and after adjustments for energy balance. Baseline plasma leptin concentration, adjusted for differences in FM, was inversely related to the size of weight loss after 8 weeks (r ⫽ ⫺0.41, p ⫽ 0.07), 16 weeks (r ⫽ ⫺0.51, p ⬍ 0.05), and 24 weeks (r ⫽ ⫺0.50, p ⬍ 0.05). Discussion: The present study suggests that leptin may have a stimulating effect on fat oxidation in obese subjects. A low leptin level for a given FM was associated with a greater weight loss, suggesting that obese subjects with greater leptin sensitivities are more successful in reducing weight. Key words: energy expenditure, spontaneous physical activity, sympathetic nervous system

Introduction Since the early 1970s, it has been known that rodents homozygous for certain mutations will become extremely obese early in life (1). In 1994, leptin, a hormone primarily produced in fat cells, was described for the first time by Zhang and colleagues (2). A lack of production of leptin in its active form and a defect in the leptin receptor are the causes behind the ob/ob mouse and the db/db mouse, respectively (2,3). In humans mutations causing a lack of production of functional leptin or a defect in the leptin receptor have been shown to cause severe obesity (4,5). However, such mutations are rare, and obesity in humans is characterized in general by high plasma leptin concentrations (6 – 8), which suggests a central leptin resistance in obesity. Administration of leptin induces weight loss in ob/ob mice and in their lean littermates through inhibition of food intake and stimulation of energy expenditure (EE) (9 –11). The reduction in energy intake is apparently mediated

Leptin, Fat Oxidation, and Weight Loss, Verdich et al.

through numerous pathways, involving inhibition of hypothalamic production of neuropeptide-Y and agouti-related protein, and stimulation of production of proopiomelanocortin and cocaine/amphetamine-regulated transcript (12). The sympathetic nervous system has been proposed to be a final mediator of the effect of leptin on EE (13–15). However, recent studies have suggested that the sympathetic system exerts an inhibitory effect on leptin secretion, suggesting a negative feedback that reduces leptin secretion independently of the reduction in fat mass (FM) (16). Findings from some previous studies in humans have supported the hypothesis of a role of leptin in stimulating EE (7,17–21), whereas others have failed to find such relationship (22–25). Furthermore, inverse correlations between fasting plasma leptin and resting metabolic rate have been reported in obese subjects, in contrast to lean individuals, supporting the hypothesis of leptin-resistance in obesity (26 –28). The purpose of the present study was to investigate the change of leptin during and after a major diet-induced weight loss, the role of leptin as a determinant of 24-hour EE, basal metabolic rate (BMR) and substrate oxidation, and the possible association between leptin and sympathetic activity.

Research Methods and Procedures Subjects Thirty-seven healthy men between the ages of 18 and 50 years were recruited through advertising in newspapers, on television, or by personal communication. Twenty-four subjects with grade I [body mass index (BMI), 30.0 to 34.9

kg/m2], grade II (BMI, 35.0 to 39.9 kg/m2), or grade III (BMI ⱖ 40 kg/m2) obesity (29) were compared with 13 normal weight controls. Three obese subjects did not complete the study, and data are presented only for the 21 subjects who completed the study (Table 1). All subjects were white, nondiabetic, nonsmokers, and none were competitive athletes. The two groups were matched for height and age and the obese subjects were controlled for weight stability every second week throughout a run-in period of a minimum of 6 weeks. None of the subjects used medication, had proteinuria, hematuria, or glucosuria as tested by sticks (Ecure-test; Boehringer Mannheim, Mannheim, Germany). Fasting plasma triglycerides, total cholesterol, high density lipoprotein, alanine aminotransferase, aspartate aminotransferase, and glucose were within the normal range for lean subjects. As expected, these values tended to be slightly increased in obese subjects. Weight Loss Program The obese subjects were reexamined following a mean maintained weight reduction of 19.2 kg (range: 5.3 to 32.7 kg) or 15.4% (range: 4.0% to 28%) of their initial body weight. The weight-reducing diet program, which is described in details elsewhere (30), consisted of 8 weeks on a strictly controlled, 1000-kcal/d, low-calorie formula diet (Gerline´a; Wasabrød A/S, Skovlunde, Denmark) followed by 8 weeks on an energy-restricted diet providing 1500 kcal/d. The weight reduction was followed by an 8-week weight maintenance diet also assisted by the counter system. Fasting body weight and waist-to-hip ratio were recorded at

Table 1. Anthropometrical data and leptin concentrations Obese (n ⴝ 21) Baseline After weight loss

Lean (n ⴝ 13) Body weight (kg) Height (m) Body mass index (kg/m2) Age (yr) FFM (kg)* FM (kg)* Fat percentage Waist: hip ratio Preperitoneal-subcutaneous abdominal fat ratio Fasting plasma leptin (ng/mL)

75.2 181 22.9 33.8 60.4 13.1 17.7 0.87 1.11 2.8

(71.4–79.0) (176–186) (22.1–23.7) (28.5–39.2) (56.5–64.3) (9.9–16.3) (13.7–21.7) (0.83–0.91) (0.530–1.69) (1.7–3.8)

126.6 181 38.6 35.4 74.7 49.9 39.8 1.01 0.945 20.2

(120.4–132.9)* (178–185) (37.2–40.0)* (31.0–39.9) (71.5–77.9)* (45.5–54.4)* (37.5–42.2)* (0.98–1.04)* (0.826–1.06) (15.1–25.3)*

107.4 (99.7–115.2)‡¶ 32.7 (30.6–34.8)‡ 72.7 34.5 31.2 0.93 0.875 12.6

(69.7–75.7)‡§ (28.4–40.7)‡¶ (27.5–35.0)‡¶ (0.89–0.96)†¶ (0.645–1.11) (7.5–17.8)‡¶

Mean (95% confidence interval). * Measured by DXA-scan. p ⬍ 0.001 vs. lean subjects. † p ⬍ 0.05, ‡ p ⬍ 0.001 vs. lean subjects. § p ⬍ 0.01, ¶ p ⬍ 0.001 vs. obese subjects.


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4, 8, 16, and 24 weeks. Body weight was measured to the nearest 0.1 kg by a Lindeltronic 8000 scale (Copenhagen, Denmark). All measurements of waist and hip circumferences were performed by the same person. Dropouts One obese subject dropped out of the study after a weight loss of 25 kg during the first 8 weeks, because he was not able to adhere to the 1500-kcal/d diet as he began to regain weight. Two left the study immediately before the reexamination. The three dropouts did not differ from the 21 subjects who completed the study with respect to initial anthropometric measures or weight loss. Standardization On the day before and the day after the stay in the respiratory chamber, all subjects adhered to a standardized isocaloric conventional diet, collected at the department and eaten at home (30). Estimated energy requirements were based on the equation of Klausen et al. (31), using body composition (fat free mass [FFM]) estimated by the bioelectrical impedance method using an Animeter (HTSEngineering Inc., Odense, Denmark). FFM was estimated from the equation of Heitmann based on the following parameters: gender, age, height, body weight, and bioelectrical impedance (32). Energy content of the provided diet was within 0.5 MJ of estimated requirement and had an energy composition of 15% protein, 50% carbohydrate, and 35% fat. Energy intake was distributed with 20% at breakfast, 30% at lunch, and 50% at afternoon snack and dinner. Respiratory Chamber The 24-hour EE was measured in two open-circuit respiratory chambers as described elsewhere (33,34). The measurement started at 9:00 AM and continued for 24 hours. BMR was measured during the last hour of the stay. The subjects arrived at the department at 10:00 PM on the evening before the examination and stayed in the chamber overnight. This protocol was chosen to familiarize the subject with the chamber, thereby minimizing stress during the 24-hour measurement period. During the 24-hour stay, subjects followed a standard protocol including three periods of 10 minutes of cycling on an exercise bicycle (75 watt) and two periods of walking back and forth in the chamber (with a total distance of 182.5 m each time). Three standard meals were served in amounts corresponding to the individual estimated energy requirement (31). Diet composition and energy distribution throughout the day was identical to the free-living standard diet. Subjects were requested to eat all of the food served and nothing else. They were only allowed to drink tap water, decaffeinated coffee, and tea ad libitum. The percentage of time engaged in spontaneous physical activity (SPA) was assessed by two microwave radar detectors (Sisor Mini-Radar; Statistic Input System SA, Lau454


sanne, Switzerland) that continuously emit and receive signals. When the radar detects a moving object, a signal is generated and received by the transceiver. The SPA measurements indicate the percentage of time in which the subjects are active to a detectable degree. Room temperature was kept at 24 °C during daytime hours (9:00 AM to 11:00 PM) and at 18 °C during the night and during assessment of BMR. Sleeping EE (SEE) was assessed from 1:00 AM to 6:00 AM in the morning. At the end of the chamber stay, anthropometric measures were assessed. Leptin Fasting plasma leptin concentration was measured at baseline, after 4 weeks of weight loss intervention, and at week 24, after ending the weight loss and weight stability interventions. The subjects arrived at the department at 8:00 AM, having used the least strenuous means of transportation. An indwelling cannula was placed in the antecubital vein and the subjects rested in the supine position for 45 minutes. Blood was drawn without stasis into plain tubes, centrifuged at 3000g and 4 °C and the plasma was stored at ⫺20 °C. Leptin was analyzed by radioimmunoassay using a DRG human leptin radioimmunoassay kit (DRG Instruments, Marburg, Germany). The intraserial coefficient of variation was 4.4% and the interserial coefficient of variation was 4.2%. At baseline and at week 24, subjects consumed a standardized isocaloric conventional diet for 3 days before blood sampling, whereas after 4 weeks of weight loss intervention, they had consumed the low-calorie formula diet. Sympathetic Activity In 22 subjects, 12 obese and 10 lean, plasma and thrombocytic concentrations of adrenaline and noradrenaline were determined, as a measure of sympathetic activity as described previously (35). These subjects did not differ from the whole group with respect to age, height, weight, body composition, 24-hour EE, or fasting plasma leptin. Blood samples were taken in the fasting state together with the leptin samples. DXA Scan Body composition was assessed initially by DXA scan in all subjects and also after 24 weeks for the obese subject. For this examination, a Lunar DPX-IQ Image Densiometer (DPX; Lunar Radiation Corporation, Madison, WI) was used. All scans were performed in the morning on fasting subjects. Lean subjects were generally scanned in the fast mode and obese subjects were scanned mostly in slow mode at both examinations. The following outcome values were chosen: total region percentage of fat (body fat %), which expresses total FM in percentage of total body mass; trunk region percentage of fat (trunk fat %), which expresses trunk FM in percentage of total mass of the trunk and FM.


Finally, FFM was calculated from adding together total bone mineral content and lean tissue mass. Abdominal Wall Fat Index Abdominal wall fat index was estimated by ultrasonography, as described by Suzuki et al. (36). Ultrasonography was performed using a Siemens Elegra System (Siemens Ultrasonics, Seattle, WA) with a 5-MHz and 7.5-MHz linear probe. The maximum thickness of the preperitoneal fat and the minimum thickness of the subcutaneous fat in the middle of the abdomen were measured and the ratio was calculated. Ethics Written informed consent was obtained from all subjects after the experimental protocol had been described to them in writing and orally. The study was approved by the ethical committees of Copenhagen, Frederiksberg and Zealand in accordance with the Helsinki II Declaration. Statistical Analyses SPSS, Version 10.0 for windows (SPSS, Chicago, IL) was used for all analyses. Anthropometric measures and data on EE were compared by nonpaired t test (lean vs. obese) and paired t test (obese before and after weight loss). Group differences in concentrations of leptin and catecholamines were tested nonparametrically, because the data were not normally distributed. Partial correlations analyses were used when controlling for the effect of body weight, FFM, and FM on parameters of EE. Because leptin was found to increase exponentially with increasing FM, residuals for the exponential function were saved and used in place of leptin. This correction was made to distinguish between a possible true effect of leptin and the confounding effect of FM, which is likely to be a determinant of both leptin level, EE, and respiratory quotient (RQ). Thus, this adjustment was made to minimize the risk of a type I error when analyzing the relationship between leptin and measures of EE. The variable leptin adjusted for FM was calculated by adding the residuals from the estimate exponential relationship between leptin and FM to the mean leptin value. This was performed separately for lean, obese, and reduced obese subjects. Leptin was not normally distributed in the overall dataset or in the three groups analyzed separately. However, the adjusted leptin values were normally distributed, and this derived variable was used instead of the raw data in all correlation and regression analyses for testing the relationship between leptin and the different aspects of metabolism and the sympathoadrenal system, thus, avoiding a logarithmic transformation of the leptin data. Finally, the hypothesis that total weight loss decreases gradually from the first to the fourth quartile baseline leptin concentration was tested by the nonparametric Jonckheere–Terpstra test.

Results Leptin An exponential relationship was seen in all groups between leptin and FM (p ⱕ 0.001) and between leptin and body fat percent (p ⬍ 0.001). The relationship between leptin and body mass index was likewise found to be exponential (p ⫽ 0.06 for lean; p ⫽ 0.004 for obese; p ⬍ 0.001 for reduced obese). Waist-to-hip ratio, the ration between preperitoneal and subcutaneous abdominal fat, and truncal fat % did not correlate with fasting plasma leptin concentration after controlling for total FM. Before the weight loss, fasting plasma leptin was 7-fold higher in obese than in lean subjects (p ⬍ 0.001; Table 1). Four weeks of intake of a hypocaloric diet, resulting in an 11.0-kg weight loss (95% confidence interval: 10.1–11.9), induced a 60% reduction in fasting plasma leptin from 20.2 ng/mL to 8.1 ng/mL (p ⬍ 0.001). After a total stabilized weight loss of 19.3 kg, fasting plasma leptin was reduced by 38% compared with baseline (p ⬍ 0.001) but was still ⬃4-fold higher than in lean subjects. It was also higher than the concentration measured after 4 weeks (p ⫽ 0.003). Nine lean subjects were followed for the whole 24-week period to observe possible seasonal variations in body weight and leptin. No changes in body weight or leptin were observed in these subjects. Leptin and Weight Loss No relationship was found between changes in fasting plasma leptin and changes in body weight from baseline to week 4. However, changes in plasma leptin from baseline to week 24 correlated with concurrent changes in both body weight (r ⫽ 0.57, p ⫽ 0.007) and FM (r ⫽ 0.53, p ⫽ 0.01). Baseline leptin level adjusted for differences in FM (leptinadjFM) correlated with the amount of the weight loss after 4 and 8 weeks (r ⫽ ⫺0.41, p ⫽ 0.07 for both), after 16 weeks (r ⫽ ⫺0.51, p ⫽ 0.02) (Figure 1), and with the overall numerical weight change from baseline to the end of the weight maintenance phase at week 24 (r ⫽ ⫺0.50, p ⫽ 0.02). Similar results were achieved when applying a stepwise regression analysis. With total weight loss after 16 or 24 weeks as the dependent variable, leptinadjFM was the only independent predictor, whereas baseline weight, baseline FFM, 24-hour EE, and resting nonprotein respiratory quotient (RQnp) at baseline did not contribute significantly. Finally, weight loss after 24 weeks was twice as high for subjects in the lowest quartile of leptinadjFM compared with subjects in the highest quartile (p ⬍ 0.01; Figure 2), and weight loss was gradually decreased from the lowest to the highest quartile as tested by the Jonckheere–Terpstra test (p ⬍ 0.03). Baseline weight did not differ between the quartiles of leptinadjFM. EE and SPA Twenty-four hour EE, BMR, and SEE were significantly higher in obese compared with lean subjects, both before OBESITY RESEARCH Vol. 9 No. 8 August 2001

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Figure 1: Relationship between leptinadjFM at baseline and weight loss after 16 weeks of hypocaloric diet in 21 obese subjects.

and after the weight loss (Table 2). Twenty-four-hour RQnp was significantly higher in reduced obese than in both lean and obese subjects. Energy balance was established during the stay in the respiratory chamber for the obese subjects, whereas both lean and reduced obese subjects were in a slightly positive energy balance (Table 2). After the weight loss 24-hour EE, BMR, and SEE were all reduced compared with baseline values. The frequency of SPA during the chamber stay was similar in lean and obese subjects. However, in obese subjects, an 11% drop in the frequency of SPA was seen from the first to the second examination (p ⬍ 0.01). Leptin and EE An inverse correlation between leptinadjFM and 24-hour EE was seen in lean subjects (r ⫽ ⫺0.57, p ⫽ 0.048). A similar relationship was not seen for obese subjects before or after weight loss, and no relationship was seen between leptinadjFM and BMR. However, an inverse correlation was found between leptinadjFM and SEE in both lean and reduced obese subjects (r ⫽ ⫺0.63, p ⫽ 0.022 for lean; r ⫽ ⫺0.44, p ⫽ 0.048 for reduced obese). Stepwise regression analysis including FFM, FM, SPA, and leptinadjFM as independent variables, showed FFM to be a significant determinant of 24-hour EE in all groups (r ⫽ 0.79, p ⫽ 0.001 for lean; r ⫽ 0.82, p ⬍ 0.001 for obese; r ⫽ 0.78, p ⬍ 0.001 for reduced obese). In addition, FM was significant in reduced obese (p ⫽ 0.001), explaining an additional 18% of the variation in 24-hour EE, whereas leptinadjFM was not a significant determinant of 24-hour EE, BMR, or SEE in any group. Finally, in obese subjects, differences in SPA were found to explain an additional 4% of the variation in 24-hour EE before (p ⫽ 0.036), but not after, weight loss (Table 3). 456


Figure 2: Weight changes (mean ⫾ SEM) for subjects in quartiles of baseline leptin concentration adjusted for differences in FM. Lowest quartile ⫽ square, second lowest quartile ⫽ star, second highest quartile ⫽ circle, and highest quartile ⫽ triangle. After 4 weeks of intervention, subjects in the lowest quartile had lost 12.1 kg, whereas subjects in the highest quartile had lost only 10.0 kg (p ⫽ 0.06). After 8 weeks, subjects in the lowest quartile had lost 18.1 kg and subjects in the highest quartile had lost 14.7 kg (p ⬍ 0.01). After 16 and 24 weeks, the total weight reduction was 25.1 kg and 25.7 kg for subjects in the lowest quartile vs. 15.2 kg and 12.9 kg for subjects in the highest quartile (p ⬍ 0.01), respectively.

A positive correlation was seen between the percentage reduction in fasting leptin concentration from before to after the stabilized weight loss and the concomitant absolute reduction in 24-hour EE (r ⫽ 0.45, p ⫽ 0.04), BMR (r ⫽ 0.50, p ⫽ 0.02), and SEE (r ⫽ 0.48, p ⫽ 0.03) as well as the percentage reduction in 24-hour EE (r ⫽ 0.52, p ⫽ 0.02), BMR (r ⫽ 0.52, p ⫽ 0.02), and SEE (r ⫽ 0.56, p ⫽ 0.008). However, neither of these associations persisted after controlling for the size of the weight loss. Furthermore, using a stepwise regression procedure, the change in body weight and FFM were the only independent determinants of the change in 24-hour EE (r ⫽ 0.85, p ⬍ 0.001 for the final model) and changes in SEE (r ⫽ 0.74, p ⫽ 0.01 for the final model), whereas change in body weight was the only independent determinant in BMR (r ⫽ 0.52, p ⫽ 0.015). The changes in leptin level, body weight, FM, and FFM were not significant determinants for the change in SPA. Thus, neither absolute nor percentage changes in fasting leptin concentration were independent determinants for the changes in EE. Leptin and Substrate Oxidation In obese subjects before weight reduction, leptinadjFM was found to be inversely correlated to 24-hour RQnp (r ⫽


Table 2. Energy expenditure

24-hour EE (kcal/24-h) BMR (kcal/h) SEE (kcal/h) RQnp 24-hour SPA 24 hour (% of time) Energy balance (EI-EE) (kcal/24-h)

Obese (n ⴝ 21) After weight loss

Lean (n ⴝ 13)

Baseline

2290 (2182–2400) 80.5 (75.8–85.6) 71 (68–74) 0.868 (0.844–0.882) 7.91 (7.04–8.78) 256 (189–323)

3095 (2959–3231)* 107 (101–113)* 98 (92–103)* 0.859 (0.851–0.866) 8.48 (7.80–9.15) 2.4 (⫺53–98)

2729 (2603–2856)‡¶ 99 (92–106)‡§ 87 (83–91)‡¶ 0.889 (0.877–0.902)†¶ 7.57 (6.84–8.30)§ 217 (136–301)

* p ⬍ 0.001 vs. lean subjects. † p ⬍ 0.05, ‡ p ⬍ 0.001 vs. lean subjects. § p ⬍ 0.01, ¶ p ⬍ 0.001 vs. obese subjects.

⫺0.47, p ⫽ 0.03; Figure 3) and basal RQnp (r ⫽ ⫺0.50, p ⫽ 0.02). These correlations remained after controlling for energy balance (r ⫽ ⫺0.43, p ⫽ 0.06 for 24-hour RQnp and r ⫽ ⫺0.46, p ⫽ 0.04 for basal RQnp), whereas similar correlations could not be found for lean and reduced obese subjects. The changes in leptin level, body weight, FM, FFM, SPA, or energy balance were not significant determinants for the change in RQ in response to weight loss. Catecholamines Plasma concentrations of adrenaline, noradrenaline, and the thrombocytic concentrations of catecholamines (tA and tNA) did not differ between lean and obese subjects, whereas the plasma noradrenaline concentration was reduced after weight loss (p ⬍ 0.02; Table 3). In all groups tA and tNA were strongly correlated (r ⫽ 0.92, p ⬍ 0.001 for lean, r ⫽ 0.85, p ⬍ 0.001 for obese, r ⫽ 0.92, p ⬍ 0.001 for reduced obese). In obese subjects, tA and tNA were inversely correlated to FM (r ⫽ ⫺0.68, p ⬍ 0.02 and r ⫽ ⫺0.77, p ⬍ 0.01, respectively). No correlations were seen between catecholamines and EE or substrate oxidation, or between adjusted leptin concentrations and catecholamines.

Discussion In the present study, we found a positive exponential association between fasting plasma leptin and FM, which is in agreement with previous findings (6,18,28). Waist-to-hip ratio, the ratio between preperitoneal and subcutaneous abdominal fat, and truncal fat percentage were not independently related to fasting plasma leptin concentration. Because the number of subjects in the present experiment and the intersubject variation in fat distribution is limited, the negative findings in the present study might be explained by lack of statistical power. However, our findings are in agreement with previous findings from studies on 150 to 200 subjects (37,38). In contrast, a relationship between leptin and body fat distribution has been described in a number of studies (28,39,40). These conflicting findings might be due to differences in methods used to assess fat distribution as well as to differences in subject characteristics and intersubject variation in body composition. However, fat distribution does not seem to be a major determinant of leptin concentration. Leptin levels were found to be markedly reduced after 4 weeks of hypocaloric diet, which induced a mean weight

Table 3. Catecholamines Obese (n ⴝ 12) Baseline After weight loss

Lean (n ⴝ 10) Plasma adrenaline (nM) Plasma noradrenaline (nM) Thrombocytic adrenaline (pg pr. 108) Thrombocytic noradrenaline (pg pr. 108)

0.077 0.57 3.3 41

(0.049–0.10) (0.41–0.73) (1.9–4.7) (30–52)

0.068 0.85 3.5 51

(0.042–0.094) (0.53–1.2) (2.2–4.9) (35–67)

0.045 0.65 4.5 58

(0.021–0.070) (0.42–0.88)* (2.9–6.1) (39–77)

* p ⬍ 0.02 for differences between the first and the second examination of obese subjects.


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Figure 3: Relationship between fasting plasma leptinadjFM and 24-hour RQnp in 21 obese subjects before weight reduction.

loss of 11.0 kg. After an additional 12-week weight loss phase, followed by 8 weeks of weight stability, a mean weight loss of 19.2 kg was achieved. At that time leptin level had increased compared with the 4-week level but was significantly lower compared with the level at baseline. This time-course relationship in response to weight loss and subsequent weight stabilization is in agreement with previous studies (6,41,42). No correlation was found between 4 weeks reduction in weight and the concomitant reduction in leptin, whereas 32% of the variation in the change in leptin after 24 weeks could be accounted for by variation in total weight loss. These findings support the notion that plasma leptin level is a marker of nutritional status, reflecting both the size of FM per se and current energy balance (43,44). Although it is well-established that exogenously administrated leptin increases EE and physical activity in rodents, there seems to be some difficulty in proving a role of endogenous leptin in the regulation of EE in humans, and findings are not consistent. In the present study, an inverse relationship between leptin adjusted for FM and 24-hour EE could be found for lean subjects. Furthermore, the change in EE in response to weight reduction was found to be positively associated with the concomitant percentage change in plasma leptin level. However, none of these association persisted when body weight and body composition were taken into account. Recent studies have shown a positive association between the reduction in plasma leptin level after a weight loss intervention and the concomitant reduction in resting EE and fat oxidation (7,45). However, these correlations were found to be independent of changes in body composition only in the study of Doucet et al. (45). A positive association between leptin and EE has been described previously in cross-sectional studies pertaining to humans (17–21). In one study, however, leptin was not 458


controlled for differences in FM (18). However, in a group of lean and obese 5-year-old Pima Indian children, Salbe et al. (17) found that fasting leptin concentration was correlated positively and independently with both total EE assessed by doubly labeled water and with physical activity level. In another study, a nearly significant positive relationship was seen between leptin and EE under free-living conditions in women, after adjusting for differences in body fat percentage (19). Recently Jørgensen and colleagues (21) showed that, in men, leptin is a stronger positive determinant of resting metabolic rate than is FM, whereas the opposite is seen in women. In addition, the increase in leptin concentration induced by euglycemic hyperinsulinemia has been shown to correlate with the concomitant increase in EE, only when an increase in EE and fat oxidation had already been induced by running a marathon on the day before the experiment (20). Taken together, these findings suggest some role for leptin in stimulating EE in humans. In obese subjects, however, an inverse relationship between leptin and resting metabolic rate (27,28), RQ, and carbohydrate oxidation has been shown (26). These findings suggest that severe obesity and high leptin level might be associated with a resistance toward the stimulatory effect of leptin on overall EE, whereas in the obese state, a stimulatory effect on fat oxidation becomes evident. In our present study, we found an inverse correlation between RQ and leptin in obese subjects, indicating that leptin may stimulate fat oxidation in obese subjects but not in reduced obese or lean subjects. In the study by Tuominen et al. (20) leptin was found to correlate positively with EE and to correlate negatively with triglyceride concentration during euglycaemic hyperinsulinemia, but only on the day after running a marathon. Tuominen et al. (20) suggested that these findings might indicate a stimulatory effect of leptin on fat oxidation, provided that lipolysis is already stimulated, which could either occur as an acute response to vigorous exercise or as a long-term result of obesity. In the present study, only fasting leptin concentrations were measured. The plasma leptin level is known to vary somewhat throughout the day (46,47), and, apparently, the amplitude of the diurnal variations is influenced by factors, such as diet composition, physical activity, insulin levels, and energy balance (46,48,49). Therefore, fasting leptin concentration may not reflect the mean leptin concentration during the day. Furthermore, it is possible that peak leptin concentration or the diurnal excursions in leptin level are independently involved in the regulation of energy balance (50). The role of leptin in the regulation of EE is still not fully clear. However, a stimulation of the sympathetic nervous system has been proposed as a mediator of this effect (13–15). In the present study, we were not able to show any relationship between leptin and the sympathoadrenal system, as reflected by the fasting concentration of cat-


echolamines in plasma and thrombocytes, or by SPA. In rodents, leptin has been shown to stimulate sympathetic outflow to brown adipose tissue (14) and increased expression of uncoupling proteins, thereby increasing the EE and thermogenesis in this tissue (11). The relative contribution of brown and white adipose tissue in the overall stimulation of EE and fat oxidation in rodents is not known (51). It is tempting to hypothesize that sparse amounts of brown adipose tissue might reduce the stimulatory effect of leptin on EE and fat oxidation in adult humans (52). This hypothesis is supported tenuously by the lack of response in EE to intracerebroventricular administration of leptin in rhesus monkeys (53). However, in this study, leptin administration was associated by a marked reduction in food intake, and, therefore, the lack of changes in EE in response to leptin can be taken as evidence for a stimulatory effect of leptin on EE, counteracting the reduction in EE due to reduced dietary induced thermogenesis (53). Considering the proposed role of leptin in regulation energy balance, one might expect initial leptin level or reduction in leptin during the first part of the weight loss phase to be predictive of the weight loss achieved during a standardized weight loss intervention. Low plasma leptin, adjusted for body fat, has been reported to predict high spontaneous weight gain in Pima Indians (54). In contrast, low baseline leptin concentration and high reduction in leptin levels during the initial weight loss phase have been shown to predict a greater long-term weight loss (42). The latter finding is consistent with our finding of an inverse correlation between baseline leptin and weight loss at all time points during the weight loss intervention and weight maintenance. However, we were not able to show any relationship between the decline in leptin during the initial weight loss phase and subsequent weight loss or weight maintenance. The findings in this study do not support the concept that lowering the leptin level serves as a starvation signal, which stimulates food intake and suppresses EE to defend energy stores. It might be hypothesized that a low leptin level for a given FM among obese subjects may reflect preserved leptin sensitivity. Accordingly, high leptin levels could indicate a more pronounced leptin resistance. Another possibility is that low leptin levels reflect the catecholamine sensitivity of intraabdominal fat cells. Previously, it has been observed that intraabdominal fat cells are more responsive to the lipolytic effect of catecholamines compared with subcutaneous fat cells (55). There is some evidence that catecholamines might inhibit leptin secretion (16) and, as mentioned above, the rate of leptin secretion might be lower in intraabdominal fat tissue than in subcutaneous fat tissue (28,39). Clearly additional studies are needed to complete our understanding of the mechanism. The finding of an inverse correlation between thrombocytic catecholamines and FM in obese subjects might, as dis-

cussed previously (35), reflect a reduction in the sympathoadrenal activity with increasing body mass index. In conclusion, we found only sparse direct relationships between leptin on one side and EE, substrate oxidation, and sympathetic activity on the other side. However, the inverse relationship between baseline leptin concentration and subsequent diet-induced weight loss suggests some role of leptin in the regulation of energy metabolism. Due to the close interrelationship between FM and leptin and the obvious need to control for the role of FM when investigating the relationship between leptin and EE, it is our belief that the role of leptin in the regulation of EE can only be firmly established through studies involving exogenous administration of leptin or neutralization of endogenous leptin.

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