Leptin plasma levels as a marker of sparing- energy ... - Nature

International Journal of Obesity (1999) 23, 470±475 ß 1999 Stockton Press All rights reserved 0307±0565/99 $12.00 http://www.stockton-press.co.uk/ijo

Leptin plasma levels as a marker of sparingenergy mechanisms in obese women E Bobbioni-Harsch*,1, F Assimacopoulos-Jeannet2, T Lehmann1, R MuÈnger1, A-F Allaz3 and A Golay1 1

Teaching Diabetic Division, Geneva University Hospital, Geneva, Switzerland; 2Medical Biochemistry Department, Geneva Medical School, Geneva, Switzerland and 3Department of Medicine, Geneva University Hospital, Geneva, Switzerland

OBJECTIVE: To investigate possible relationships between leptin and energy expenditure (EE), both in the condition of stable body weight and during weight loss. SUBJECTS: Seventy four Caucasian, adult obese women with stable body weight (including 10 obese women studied before and during a body weight-reducing program). MEASUREMENTS: Resting EE (REE) and substrate oxidation rates by indirect calorimetry; plasma leptin concentrations by radioimmunoassay (RIA). RESULTS: In conditions of stable body weight, leptin values showed a signi®cant, negative relationship with REE, as expressed in absolute values (P 0.030) and as adjusted for the variation in lean body mass (LBM) (P 0.017). This negative relationship was independent of both LBM and fat mass (FM). Linear regression analysis was used to obtain the equation linking REE and LBM; then both predicted REE and the percent deviation from predicted REE were calculated for each subject. Leptin values were negatively related (P < 0.0001) to the deviation from predicted REE. During active body weight loss, the modi®cations of both REE (D REE) and lipid oxidation (D lipid oxidation) were signi®cantly negatively related to leptin concentrations, which were measured before the dieting period (P < 0.03 for both). CONCLUSION: In obese women, high plasma leptin concentrations are associated with a low rate of REE, when body weight is stable, and with a reduction of REE and lipid oxidation, in response to a hypocaloric diet. This suggests that, in severely obese women, leptin is a marker of sparing energy mechanisms operating in both basal and reducing weight conditions. Keywords: resting energy expenditure (REE); lipid oxidation; lean body mass (LBM); fat mass (FM); plasma leptin

Introduction The three main components of energy expenditure (EE), that is, resting metabolic rate (RMR), diet induced thermogenesis1 ± 3 and energy spent for physical activity,4 have been largely investigated in obese subjects searching for one or more defects that could explain excessive accumulation of energy in the form of fat. Resting EE (REE), which represents 60 ± 80% of the daily energy needs,5 is mainly determined by lean body mass (LBM)6 and also by several other factors such as, fat mass (FM),7,8 thyroid9,10 or sex hormones10 or catecholamines.9 These factors could explain the individual variability of REE and could also be involved in the development and=or in the relapse of obesity. For instance, in a normal body weight population, subjects with a low rate of REE, adjusted for LBM, have more risks to gain weight than subjects with higher rates of REE.11 In obese people, the decrease in REE, observed in some subjects subjected to a hypocaloric diet, has been attributed to the effect of adaptative, energy-sparing mechanisms *Correspondence: Dr Elisabetta Bobbioni-Harsch, Teaching Diabetic Division, Geneva University Hospital, 1211 Geneva 14, Switzerland. Received 27 April 1998; revised 2 November 1998; accepted 2 December 1998

elicited by the restriction of energy intake (EI).12 ± 14 The existence of these mechanisms is strongly indicated by several reports,15 ± 17 but has to be de®nitely proved in humans. The presence, among obese subjects, of predictive factors for a reduction of REE during hypocaloric diet, would further support the existence of such mechanisms. Leptin, the adipose tissue-derived hormone, has received great interest, because of its possible involvement in the regulation of energy balance.18 However, the relationship between leptin and EE in human obesity, remains unclear, since studies in this ®eld have led to contrasting results, perhaps due to the differences in ethnicity,19 age,20 or gender 21 of the subjects and=or to the different experimental conditions. For this reason, the present study was aimed at investigating the relationship between plasma leptin concentrations and REE in adult, Caucasian, obese women, both in conditions of stable body weight and during active body weight reduction.

Material and methods The ®rst part of the study was conducted on 74 obese women, with a body mass index (BMI) between

Leptin and energy sparing E Bobbioni-Harsch et al

26 ± 59 kg=m2, whose characteristics are reported in Table 1. After an overnight fast, the subjects were admitted to the Hospital and the anthropometric measurements were performed. Body composition was obtained by the skinfold thickness method.22 Urine and blood samples were collected, and then the subjects were placed in a recumbent position and gas exchanges were measured, as described by JeÂquier and Felber23 (for a review, see Ref.24), by placing the head of the patients in a ventilated hood (Deltatrac, Datex Corp, Helsinki, Finland). After a 10 min equilibration period, VO2 and VCO2 were continuously measured for 30 min and used to calculate the respiratory quotient (RQ) and glucose and lipid oxidation rates, according to Lusk.25 Protein oxidation was calculated as 6.235 N, where N is nitrogen excretion (mg=min) in urine. REE was calculated from the rates of glucose, lipid and protein oxidation. A group of 10 lean and 10 obese subjects had indirect calorimetry and plasma leptin measurements made three times, with an interval of one week between each. This permitted the following within individual coef®cients of variation to be determined: REE 2.4%; RQ 1.3%; fat oxidation 7.4%; leptin 6.1%. REE was adjusted by the variations of LBM as suggested by Ravussin and Bogardus.26 Predicted REE was calculated according the equation obtained from the linear regression analysis linking, in our study group, REE and LBM: Predicted REEkJ=24 h 2533 19:4 LBM The LBM-adjusted REE was then calculated as: LBM-adjusted REE mean REE measured REE ÿpredicted REE where mean REE was the mean REE value obtained in our study group and measured REE was the individual values measured in our patients. The percent variation from predicted REE was calculated, for each patient, as: measured REE7 predicted REE 100 predicted REE Among the ®rst study group, 10 obese women (mean age 50.0 17 y), who applied for a body weight reducing program in hospital, participated in the

Table 1

General characteristics of the study group (n 74)

Age (y) Body weight (kg) BMI (kg=m2) LBM (kg) FM (kg) REE (MJ=24h) Predicted REE (MJ=24 h) LBM-adjusted REE (MJ=24 h) Plasma leptin (ng=ml)

47.8 1.6 100.7 3.2 38.4 0.9 58.5 1.5 42.2 1.4 7.3 0.2 7.3 0.1 7.3 0.1 48.6 3.1

Values are expressed as mean s.e.m. BMI body mass index; LBM lean body mass; FM fat mass; REE resting energy expenditure

second part of the study. These patients were submitted to the previously described measurements twice, that is, before and after a four-week period of hypocaloric diet. The daily energy requirement was calculated for each patient as: daily EI 1:25 REE where REE was the one measured before the beginning of the diet, that is, in conditions of stationary body weight and unrestricted EI. A hypocaloric diet was then established as: Hypocaloric diet 1:25 REE ÿ 4:18 MJ The hypocaloric diet, which was composed of 45% carbohydrates, 25% fat and 30% protein, was followed during a four-week period that the patient spent at the hospital. During the hospitalization, in addition to the hypocaloric diet, the patients participated in a multidisciplinary body weight reducing program, described in detail elsewhere,27 that included physical activity, nutritional education and standard behavioural techniques. After four weeks, and while the patients were still under caloric restriction, both anthropometric and calorimetric measurements were performed a second time. The individual caloric de®cit was calculated as: Daily EI ÿ 1:25 REE 100 1:25 REE Plasma leptin was determined by radioimmunoassay (RIA), using a commercial kit (Linco Research Inc., St Charles, MO). Relationships between different parameters, measured in our study group, were evaluated by multiple or single regression analysis; non parametric (Wilcoxon signed rank test), ANOVA was used when comparing basal vs post-diet values.

Results Leptin and EE in obese women of stable body weight

Table 1 summarizes the characteristics of the 74 obese women who participated in the ®rst part of the study. As expected and shown in Figure 1, a signi®cant, positive relationship linked plasma leptin values to FM (r2 0.55, P < 0.0001). As illustrated in Table 2, circulating leptin was signi®cantly (P 0.030), negatively related to REE, when expressed in absolute values, independent of both LBM and FM. When REE was adjusted by the variations in LBM, plasma leptin concentrations were signi®cantly (P 0.017) negatively related to adjusted REE, independent of FM. As mentioned in the material and methods section, predicted REE and the percent variation from the predicted REE were calculated for each patient. As shown in Figure 2, plasma leptin concentrations were signi®cantly negatively related to the individual

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Figure 1 Regression plot of fat mass (FM, kg) vs plasma leptin concentrations (ng=ml). r2 0.55, P < 0.0001.

Table 2 Multiple regression analysis with, in 2a, resting energy expenditure (REE, expressed in absolute values) and, in 2b, lean body mass (LBM) adjusted REE as dependent variables 2a. Dependent variable: REE (MJ=24 h) Independent variable Regression coefficient LBM (kg) FM (kg) Leptin (ng=ml) Age (y)

15.64 3.1 8.5 3.2 7 2.75 1.19 7 2.18 1.9

2b. Dependent variable: LBM-Adjusted REE (MJ=24 h) Independent variable Regression coefficient FM (kg) Leptin (ng=ml) Age (y)

6.33 2.5 7 2.94 1.19 7 0.91 1.56

P value < 0.0001 0.009 0.025 0.26 P value 0.014 0.016 0.56

FM fat mass.

percent variations from the predicted REE (r2 0.28, P < 0.0001). Leptin and EE in obese women during body weight loss

Table 3 reports the parameters measured in a group of 10 patients before (Basal, Table 3) and after four weeks of hypocaloric diet (Diet, Table 3). The dietinduced modi®cations of these parameters (D, Table 3) were calculated as the difference between the values measured while dieting and the ones measured before caloric restriction. The hypocaloric diet induced a signi®cant (P 0.005) decrease of BW, BMI, FM and leptin, while no signi®cant changes were observed in lipid oxidation and REE. Figure 3 illustrates the variability of the individual modi®cations of REE after hypocaloric diet. Plasma leptin concentrations, measured before the beginning of the diet, were signi®cantly, negatively related to both modi®cations of REE (r2 0.50, P 0.021, Figure 4) and of lipid oxidation (r2 0.49, P 0.025, Figure 5). Multiple regression analysis showed that the negative relationship between plasma leptin and

Figure 2 Regression plot of plasma leptin concentrations (ng=ml) vs percent deviation from predicted resting energy expenditure (r2 0.25, P < 0.0001.

the modi®cations of both REE and lipid oxidation, remained signi®cant (regression coef®cient: 7 7.99 3.58, P 0.039 for D REE and regression coef®cient: 7 1.58, P 0.036 for D lipid oxidation) independent of the initial FM, the caloric de®cit and the modi®cations to body weight. Basal leptin concentrations were not signi®cantly (r2 0.40, P 0.06) related to the modi®cations of glucose or protein oxidation (r2 0.09, P 0.4). A signi®cant (r2 0.71, P 0.002), negative relationship linked the modi®cations in REE to the ones of the respiratory quotient (RQ).

Discussion In the female obese population examined in our study, circulating leptin concentrations, measured in conditions of stable body weight, are negatively related to REE, either expressed in absolute values or adjusted for the variations in LBM. It is well established that, in conditions of stable body weight, circulating leptin concentrations are strictly related to the amount of fat mass.28,29 On the other hand, our results indicate that subjects with the highest leptin plasma concentrations and, therefore, the highest degree of obesity, are the ones whose REE is the lowest, relative to their LBM. Thus, a low rate of REE could favour the development of obesity;11 it could also contribute to determine the degree of obesity, as indicated by our results. These results could also be the consequence of leptin resistance, a characteristic of human obesity, as suggested by Niskanen et al,30 who reported a similar ®nding. Furthermore, the individual variations from the predicted REE were signi®cantly negatively related to circulating leptin: in other words, high circulating leptin concentrations are associated with a REE lower

Leptin and energy sparing E Bobbioni-Harsch et al Table 3 Characteristics of a group of 10 obese women, measured in conditions of stable body weight (basal) and during body weight loss (diet)

Body weight (kg) BMI (kg=m2) Fat mass (kg) LBM (kg) REE (MJ=24 h) Respiratory quotient Lipid oxidation (mg=min) Plasma leptin (ng=ml)

Basal

Diet

D

P

118.6 10.6 45.8 3.4 53.0 5.3 65.5 5.5 7.49 0.46 0.78 0.02 93.8 10.7 78.3 9.9

113.9 10.7 44.0 3.5 47.9 5.0 66.0 5.8 7.25 0.39 0.75 0.01 100.5 8.3 63.6 10.4

7 4.7 0.3 7 1.8 0.1 7 5.2 0.5 0.5 0.5 7 0.24 0.35 7 0.03 0.03 6.7 15.5 7 14.7 4.0

0.005 0.005 0.005 0.34, NS 0.58, NS 0.39, NS 0.64, NS 0.005

D indicates the differences between Diet and Basal measurements. P indicates the statistical signi®cance as obtained by non-parametric (Wilcockson signed rank test) ANOVA. BMI body mass index; LBM lean body mass; REE resting energy expenditure; NS not statistically signi®cant.

Figure 3 Individual variations of resting energy expenditure (REE, MJ=24 h) after a four week hypocaloric diet.

Figure 4 Regression plot of plasma leptin concentrations (ng=ml) vs the modi®cations of resting energy expenditure (D REE MJ=24 h), calculated by substracting the value of REE measured in basal to the one measured after four weeks hypocaloric diet. r2 0.50, P 0.022.

Figure 5 Regression plot of plasma leptin concentrations (ng=ml) vs the modi®cations in lipid oxidation (D lipid oxidation mg=min), calculated by substracting the value of lipid oxidation measured in basal to the one measured after four weeks hypocaloric diet. r2 0.49, P 0.025.

than the one predicted on the basis of LBM. Kennedy et al21 have reported no correlation between leptin and REE, when the latter is corrected by LBM. It is possible that these contrasting results are due to the differences in the populations studied: in fact, in Kennedy's study group, at least one third of the subjects had a BMI < 26 kg=m2 and, therefore, could not be considered as obese, while our study group was composed exclusively of obese subjects. Finally, other authors20 have found a positive relationship between leptin and REE (expressed in absolute values); however, this relationship is dependent upon FM and, therefore it simply indicates an increased REE with increasing obesity. It should be observed that in this study, as well as in the one reported by Kennedy, the groups were composed of both obese and lean subjects (as indicated by the BMI range). If one assumes that lean and obese humans have a different sensitivity to leptin, this could explain the lack of signi®cant relationship between leptin and REE, reported by Nagy et al.20

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The second part of our study was conducted in a group of patients who were submitted to an energy restriction of 4.18 MJ=24 h, established on the basis of each patient's REE measurement. After four weeks of reduced EI, no major changes in LBM could be detected. This was probably due to multiple reasons: on one hand, the relative innacuracy of the skinfold thickness method did not allow a precise quanti®cation of small variations in LBM; on the other hand, the high protein content (30%) of the diet and the physical exercise the patients performed during the hospitalization period have largely limited the loss in LBM, as previously demonstrated.27 Finally, when considering that the mean body weight loss averaged 4 kg, the loss in LBM, although underestimated, should not have played a major role in the modi®cations of REE. As illustrated in Figure 3, the modi®cations of REE showed a large individual variability, ranging from an increase to a marked reduction. This result is consistent with data already reported in the literature.17,31 On the bases of our results, we could demonstrate that the individual modi®cations in REE were not randomly distributed, but they were signi®cantly, negatively related to the basal plasma leptin values, measured before the hypocaloric diet. We cannot exclude that the degree of physical activity performed during the hospitalization period or the diet-induced modi®cations in IR have contributed to the modi®cations of REE. However, multiple regression analysis showed that the negative relationship between basal leptin plasma concentrations and the modi®cations of REE was independent of the initial FM, of the degree of caloric de®cit as well as of the diet-induced changes in body weight. To our knowledge, this is the ®rst time that the variations in REE, occurring during hypocaloric diet, have been demonstrated to be linked to a parameter measured in steady state conditions. This result strongly suggests that, among obese subjects, some individuals are able to reduce their REE, that is, to develop energy-sparing mechanisms, in response to a hypocaloric diet; on the other hand, it demonstrates the role of plasma leptin as a predictor of the modi®cations in REE subsequent to a reduced EI. If one accepts that plasma leptin values are a marker of the degree of obesity, this result suggests that the subjects with the highest degree of obesity are the ones who most counteract the reducing-weight effect of a hypocaloric diet, by decreasing their REE. This hypothesis is strongly supported by the recent report by van Gemert et al 33 who demonstrated a signi®cant decrease of sleeping metabolic rate (SMR) in severely obese patients, after vertical banded gastroplasty. Furthermore, the modi®cations in REE are signi®cantly, negatively related to the modi®cations in RQ, indicating that the reduction in REE was mostly due to a decrease in fat oxidation. This suggests that the energy-sparing mechanisms aim mainly at an economy of fat reserves. Rosenbaum et al32 and Havel et al,34 have reported no correlation between the changes in leptin plasma concentrations

and the changes in REE, during body weight loss. These results are not comparable to ours, since the changes in leptin concentrations (not the basal absolute values) were investigated as a function of the modi®cations in REE. It is possible that during hypocaloric diet, other factors, such as the decrease in FM and in circulating insulin,34 contribute to the changes in leptin concentrations. This is not incompatible with the predictive role of basal leptin demonstrated in this paper.

Conclusions Among obese women, the subjects with the highest concentrations of circulating leptin are the ones who show the lowest REE, in conditions of stable body weight. These subjects show a reduction of both REE and fat oxidation, in response to a restricted EI. We, therefore, suggest that high leptin concentrations in obese women are a marker of energy-sparing mechanisms which could contribute to both the development and the maintenance of obesity. Future strategy should be aimed at elucidating the nature of both the signals and the mechanisms leading to REE reduction. A better knowledge of these elements could improve the ef®cacy of reducing-weight therapies. Acknowledgements

We are grateful to Ms Nuria Flores for excellent secretarial assistance and to Mrs Francine Califano for her skilled technical work. This study has been partially funded by Grant Number 32-45957.95 of the Swiss National Science Foundation. References

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