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Original Research Communications |
1 From the National Institute of Nutrition, Rome, and the Departments of Physiopathology and Neuropsychiatry, University La Sapienza, Rome.
2 Supported in part by the National Research Council, Rome. 3 Address reprint requests to A Polito, National Institute of Nutrition, Via Ardeatina, 546 00178 Rome, Italy. E-mail: polito{at}inn.ingrm.it.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: The objective was to evaluate the relation between energy turnover, body composition, and plasma leptin concentrations in anorexia nervosa (AN).
Design: Sixteen weight-stable women with AN were compared with 22 control subjects and 14 rehabilitated AN patients (R-AN). Basal metabolic rate (BMR) was measured by indirect calorimetry; fat-free mass (FFM) and fat mass (FM) were calculated according to a 4-compartment model. Plasma leptin was determined by radioimmunoassay.
Results: The BMR of AN patients (2.73 ± 0.37 kJ/min) was significantly lower than that of control subjects (3.45 ± 0.34 kJ/min) (P < 0.001), even after adjustment for FFM (2.92 ± 0.33 kJ/min in AN patients and 3.30 ± 0.26 kJ/min in control subjects; P < 0.004). Plasma leptin concentrations in AN patients were 76% lower than in control subjects, even after body fat was controlled for. In R-AN patients, BMR was not significantly different from that of control subjects and leptin concentrations were generally close to normal. Plasma leptin concentrations correlated significantly with FM (r2 = 0.53, P < 0.0000) and BMR, even after adjustment for FFM (r2 = 0.21, P < 0.0003).
Conclusions: BMR and plasma leptin concentrations are depressed in patients with AN; this is not explained by body-composition changes. The relation between leptin and BMR suggests that leptin plays a role in the energy sparing response to exposure to chronic energy deficiency. The return of BMR to normal and the significant increase in leptin concentrations in R-AN patients suggests a full reversibility of this adaptation mechanism.
Key Words: Basal metabolic rate • leptin • body composition • fat-free mass • anorexia nervosa • Italy • women
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Several physiologic mechanisms have been proposed to explain these changes in energy expenditure. Factors such as hormonal concentrations and substrate oxidation rates may operate and interact to influence metabolic processes. Energy deficit reduces activity of the sympathetic nervous system, alters peripheral thyroid metabolism, and lowers insulin secretion (16). Several studies have indicated that leptin might play a role in the complex mechanism that regulates energy balance. Leptin (also known as the ob gene product) is a protein whose expression is mainly localized in adipose tissue, from which it is secreted into the circulation and transported to the hypothalamic area, where it is presumed to act as a lipostatic mechanism through modulation of satiety signals and sympathetic nervous system–mediated energy expenditure (17). Plasma leptin concentrations increase in the fed state and decrease rapidly during nutritional deprivation (18–19). Long-term leptin administration was shown to markedly decrease food consumption, decrease body weight and fat mass, and increase energy expenditure in ob/ob mice (20–22). In humans, circulating leptin concentrations appear to be highly correlated with adipose tissue mass (19).
Several studies showed that plasma leptin concentrations are elevated in most obese people, which implies that obesity might be associated with leptin insensitivity due to several mechanisms, such as a leptin-receptor defect (23), impaired postreceptor signal transduction, and alteration of transport capacity across the blood-brain barrier (24). On the other hand, a significant decrease in plasma leptin concentration has been shown to occur in AN (25, 26) and during dietary treatment of obesity (27, 28). It has been suggested that this fall in leptin might be involved in the neuroendocrine adaptation to starvation (29). An inverse association was found between leptin concentrations and BMR in obese subjects (30, 31); however, this relation was not observed in other studies (32, 33). A return to normal BMR after refeeding was shown in adults (8, 9), in an undernourished population (34), and in subjects with AN (35–38). Similarly, leptin concentrations were reported to increase with weight gain (26, 39). However few studies addressed the role of leptin in the regulation of energy expenditure in humans. The aim of the present study was to examine the effect of chronic energy undernutrition on energy turnover and plasma leptin concentrations in patients with AN and to explore the relation between body composition, BMR, and plasma leptin.
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SUBJECTS AND METHODS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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3 mo before the study, as assessed by clinical report. Eight of these subjects had AN of the restricting type and the other 8 had the binge-eating or purging type of AN. The duration of AN ranged from 6 mo to 12 y (
± SD: 4.5 ± 4.2 y). A second group consisted of 14 recently rehabilitated AN patients (R-AN; BMI > 18.5 for 1 y). This group had a history of AN ( ± SD: 5.3 ± 3.2 y) and a mean BMI, at their lowest weight, of 14.3 ± 2.4, as assessed by clinical report. A last group of 22 women of normal weight (BMI: 18.5–24.9), who were free of a history of eating disorders, served as control subjects and were recruited from the staff and students of the National Institute of Nutrition. All subjects were measured in the fasting state early in the morning. BMR and body-composition measurements were conducted on the same day to minimize within-subject biological variability. Dual-energy X-ray absorptiometry (DXA) and blood collection for leptin assay were performed, within the next 3 d, in the Department of Medical Physiopathology of the University of Rome, La Sapienza. Control subjects and R-AN patients were studied between the 6th and 12th days of their menstrual cycles; all the AN patients were amenorrheic. The study protocol was reviewed and approved by the ethics committee of the University of Rome, La Sapienza. All subjects gave written, informed consent to participate in the study.
Basal metabolic rate
BMR was measured in triplicate (CV <2%) by open-circuit indirect calorimetry under standardized conditions with Douglas bags. The measurements were made at the National Institute of Nutrition at 0800 after 10–12 h of fasting and 30 min of resting, in absolute quietness and at thermic neutrality (the temperature inside the room was 22–25°C). Expired air was collected for 10 min. The volume of expired air was measured on a calibrated wet gas meter (SIM Brunt, Milan, Italy), analyzed for oxygen content with Servomex 1100 A (Taylor Instrument Analytics Ltd, Sussex, United Kingdom), and analyzed for carbon dioxide content with a Morgan infrared analyzer (PK Morgan Ltd, Chatham, Kent, United Kingdom). The gas analyzers were calibrated daily with certified gas mixtures (pure nitrogen and atmospheric air for Servomex and a mixture of 6.60% carbon dioxide in nitrogen for the carbon dioxide analyzer). The metabolic rate was calculated by Weir's equation (41).
Body composition
Body fat was derived according to a 4-compartment model that requires measurements of body volume, total body water (TBW), total-body bone mineral mass, and body weight. We used the equation proposed by Fuller et al (42), with body volume measured by underwater weighing. TBW was derived by impedance analysis with use of Kushner's equation (43), which was validated in patients with AN (44), and total-body bone mineral mass was measured by DXA. The propagated measurement error for this method is 3.0% of fat weight (45).
Underwater weighing
Body density was determined by using underwater weighing as described by Durnin and Rahaman (46), with simultaneous measurement of residual lung volume by using the nitrogen dilution technique (47). The subjects were measured in the fasting state after voiding, having refrained from intense exercise and diuretics over the previous 24 h. To reduce gastrointestinal gas, the subjects were asked to consume a low-fiber, meat-free diet during the 4 d before the test and took an antiflatulent (160 mg activated dimethyl-polysiloxanes/d; Warner Lambert Consumer Healthcare S Com pA, Milan, Italy). Body density measurements were repeated until a difference of ±1.5 g/L between any 3 replicates was obtained (corresponding to 
0.4% of fat).
Bioelectrical impedance
Whole-body impedance was measured with an impedance analyzer (Human Im Scan, Dietosystem, Milan, Italy). Signal and detecting electrodes were positioned according to the recommended protocol on each subject's right wrist and right ankle (48). The measurements were made 10 min after the subjects lay supine on a nonconductive surface with their legs slightly divaricated and their arms next to, but not touching, their trunks. Calibration of the analyzer was checked daily by using a standard resistor. TBW was estimated by using Kushner's equation (43). SEs of repeated measurements for 10 subjects were 7 
for impedance.
Dual-energy X-ray absorptiometry
DXA measurements were made by using a total body scanner (model QDR-4500W; Hologic, Walthman, MA) according to a previously published procedure (49). Total-body bone mineral mass was derived according to the computer algorithms provided by the manufacturer. The within-subject day-to-day CV for total-body bone mineral mass is <0.1%.
Anthropometric measurements
Height and weight were measured according to the standard procedure (50). Height was measured to the nearest 0.1 cm with a wall-mounted Holtain stadiometer (Holtain Ltd, Crosswell, Crymych, United Kingdom). Body weight was recorded to the nearest 0.01 kg by using a calibrated computerized digital balance (K-Tron P1-SR; K-Tron SA, Hasler Division, Colombier, Switzerland); each subject was barefoot and wore a light bathing suit.
Leptin
After the subjects fasted overnight, blood samples were collected for leptin assay. Plasma leptin was measured by using a commercially available radioimmunoassay kit (Linco Research, St Charles, MO), as described elsewhere (51). The intra- and interassay CVs were 3.5% and 4.2%, respectively. All measurements were performed in duplicate.
Statistical analysis
Statistical analyses were performed by using COMPLETE STATISTICAL SYSTEM (StatSoft, Inc, Tulsa, OK). The data were first tested for normal distribution by using the Shapiro-Wilk test for normality. Leptin concentrations were not normally distributed, so a Kruskal-Wallis test was used to compare leptin concentrations among groups. The logarithm of leptin (log leptin) was normally distributed and was used for regression analyses. Relations among log leptin concentration and body composition or BMR were determined by using regression analyses. Analysis of variance (ANOVA) and analysis of covariance (ANCOVA) were used to determine intergroup differences in body composition and BMR (52). Results are presented as group means and SDs. A level of significance of P < 0.05 was used for all data analyses.
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RESULTS |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The mean ages and heights of the 3 study groups were similar. The mean BMI of the AN patients (15.5 ± 1.2) indicates the severe grade of these patients' undernutrition. The fat mass of the AN patients (6.8 ± 3.3 kg) was 58% and 50% lower, respectively, than the fat mass of the control subjects (15.7 ± 3.7 kg) and of the AN-R group (13.6 ± 4.8 kg). The FFM of AN patients (34.7 ± 3.5 kg) was 15% less than that of the control subjects. The R-AN patients had a fat mass that was 16% less and a FFM (37.9 ± 3.8 kg) that was 7% less than that of the control subjects.
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. The mean absolute BMR (in kJ/min) was significantly lower (by 21%) in AN patients than in control subjects. R-AN patients had an intermediate BMR that was significantly higher than that of AN patients (by 17%) but only 7% lower than that of the control subjects. BMR was significantly correlated with FFM and body weight. The regression equation showed that 62% of the variance in BMR was attributed to differences in body weight (Figure 1). When BMR was regressed against FFM, 48% of the variance was explained by FFM (Figure 1). There was no significant difference between regression lines (BMR against body weight and FFM) of different groups (data not shown). When BMR was expressed per unit of body weight or per unit of FFM, no significant differences between the 3 groups were observed. Adjustment by ANCOVA for FFM reduced the differences but did not eliminate them (AN group, -10%; R-AN group, -3%). There were no significant differences in BMR within the same analysis with body weight as the covariate. Respiratory quotients were similar among the 3 groups, indicating no significant difference in substrate utilization.
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. Leptin concentrations were lowest in the AN patients (1.9 ± 1.5 µg/L), the maximum difference being 76%. R-AN patients appeared to have the highest absolute concentrations (8.4 ± 10.5 µg/L). The difference between groups persisted when leptin concentrations were adjusted for fat mass. The logarithm of leptin concentration was significantly correlated across groups with body fat mass (Figure 2). Although the slope for AN patients was not significantly different from that for control subjects and R-AN patients, the intercept was slightly significantly different (P < 0.04). A positive correlation was found also between the logarithm of leptin concentration and BMR (Figure 3, top). The slope and intercept of the regression line for AN patients were not significantly different from those for control subjects and R-AN patients, possibly because of the wide scatter of the points and the relatively small number of subjects in each group. The correlation with BMR persisted after statistical adjustment for FFM and the slight apparent individual regression lines of the 3 groups disappeared (Figure 3, bottom). In a stepwise regression analysis, with BMR as the dependent variable and FFM and leptin as the independent variables, the final model included both terms as significant variables, which together accounted for 58% of the variation in BMR (Table 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONSUBJECTS AND METHODSRESULTSDISCUSSIONREFERENCES |
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16%) in a group of women with AN who were similar in age and BMI to the women in the present study. In adolescent girls with AN, Vaisman et al (4) showed that BMR was 28% lower than that of control subjects even when expressed per unit of lean body mass.
There are several possible explanations for these discrepancies in findings. First, note that the studies that did not find a decrease in BMR did not use ANCOVA. It is well known that the normalization procedure of dividing energy expenditure measurements by body weight or FFM is mathematically biased (52). In the present study, although BMR did not differ significantly among groups when expressed per kilogram of body weight or per kilogram of FFM, we found a significantly lower BMR after adjustment, by ANCOVA, for FFM differences. The FFM compartment is composed of tissues and organs that differ profoundly in their metabolic activity. Elia (54) estimated that 40% of the body weight of a man is represented by muscle, which contributes only 22% of BMR, whereas visceral organs account for >60% of BMR. Organ mass has been shown to be inversely related to FFM (55); therefore, it is to be expected that BMR reflects this diversity of proportion (56). The relative proportion of the various components of FFM changes in relation to the intensity and duration of energy deprivation (57). In mild-to-moderate energy deficiency, muscle mass is more likely to be lower than normal than is nonmuscle or visceral mass, whereas in the more severe forms of energy deficiency, such as found in AN patients with low BMIs (<16), mobilization of tissues from visceral mass may dominate. Thus, it is possible that the lower FFM-adjusted BMR of the AN patients in the present study might reflect a prevalent loss of visceral tissue. Weight stability of AN patients is also an important variable in the study of energy metabolism. Although the subjects in the present study were weight stable at a low BMI (<17) 1 y before the study, the other studies did not find lower BMRs and did not report on the stability of their subjects.
AN patients were found to have plasma leptin concentrations that were 76% lower than those of control subjects. These results agree with the findings of other investigators (25, 26, 58), who found that AN patients of similar age and BMI had plasma leptin concentrations that were 70%, 77%, and 70% lower, respectively, than those of control subjects. Plasma leptin concentrations in the present study remained lower even when adjusted for fat mass, suggesting that leptin secretion might not depend entirely on the size of the fat mass. Both insulin and glucocorticoids have been proposed as having a stimulatory effect on the expression of the ob gene (59). In the subjects in the present study, leptin was positively correlated with BMR. To our knowledge, there are no reports in the literature on the relations between BMR and leptin in AN patients because most results were obtained in obese people. Moreover, conflicting results were obtained. In some of these studies, there was a positive relation between leptin and BMR (60, 61), others found a negative relation (30, 31), and others (32, 33) found no relation at all. In animal models, leptin appears to stimulate energy expenditure through mechanisms that may involve suppression of neuropeptide Y secretion and thus activation of the sympathetic nervous system (62). Leptin is also known to determine changes in the expression of uncoupling proteins, which have been implicated in mitochondrial oxidative phosphorylation (63).
Rehabilitation is an important phase of AN because it is during this phase that the lost functions and tissues are restored to normal or near-normal conditions. Recovery appears to be influenced by several factors, such as age, duration of AN, and the refeeding protocol used. Ideally, the recovery process should be studied with a longitudinal design. Despite the cross-sectional nature of the present study, the findings show that the BMR and leptin concentrations of the subjects who had anthropometrically recovered for 1 y returned to near normal, suggesting full reversibility of the changes produced by AN. It is interesting to note that there was a high interindividual variability in leptin concentrations in the R-AN patients in our study; in some cases, these concentrations exceeded the concentrations of control subjects. The direct causes of this remain to be identified. As pointed out by other authors, large increases in leptin concentrations during weight restoration could be due to disproportionate accumulation of fat during this phase (64). Moreover, a disregulation of leptin after weight gain in patients with AN was supposed by Eckert et al (26) with the hypothesis that plasma leptin concentrations during nutritional rehabilitation could be related to neuroendocrine abnormalities during weight loss.
In conclusion, this study showed that there is a relation between BMR and leptin concentration, both of which are lower in AN patients than in healthy control subjects. The study also showed that the decrease in BMR observed in AN patients cannot be fully explained by a modification in the composition of FFM, a residual 30% being explained by plasma leptin concentrations. In animal models, mechanisms have been shown to exist (such as suppression of neuropeptite Y secretion and uncoupling mitochondrial oxidative phosporylation) that might be involved in the modulation of the relation between BMR and leptin in AN subjects.
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